Interactions between prefrontal cortex and cerebellum revealed by trace eyelid conditioning

Eyelid conditioning has proven useful for analysis of learning and computation in the cerebellum. Two variants, delay and trace conditioning, differ only by the relative timing of the training stimuli. Despite the subtlety of this difference, trace eyelid conditioning is prevented by lesions of the cerebellum, hippocampus, or medial prefrontal cortex (mPFC), whereas delay eyelid conditioning is prevented by cerebellar lesions and is largely unaffected by forebrain lesions. Here we test whether these lesion results can be explained by two assertions: (1) Cerebellar learning requires temporal overlap between the mossy fiber inputs activated by the tone conditioned stimulus (CS) and the climbing fiber inputs activated by the reinforcing unconditioned stimulus (US), and therefore (2) trace conditioning requires activity that outlasts the presentation of the CS in a subset of mossy fibers separate from those activated directly by the CS. By use of electrical stimulation of mossy fibers as a CS, we show that cerebellar learning during trace eyelid conditioning requires an input that persists during the stimulus-free trace interval. By use of reversible inactivation experiments, we provide evidence that this input arises from the mPFC and arrives at the cerebellum via a previously unidentified site in the pontine nuclei. In light of previous PFC recordings in various species, we suggest that trace eyelid conditioning involves an interaction between the persistent activity of delay cells in mPFC-a putative mechanism of working memory-and motor learning in the cerebellum.Eyelid conditioning is a form of associative learning that has proven useful for mechanistic studies of learning (Thompson 1986). All variants of eyelid conditioning involve pairing a conditioned stimulus (CS, typically a tone) with a reinforcing unconditioned stimulus (US, mild electrical stimulation near the eye) to promote learned eyelid closure in response to the CS (also known as a conditioned response). Delay eyelid conditioning, where the CS and US overlap in time (Fig. 1A , left), is largely unaffected by forebrain lesions (Solomon et al. 1986; Mauk and Thompson 1987; Kronforst-Collins and Disterhoft 1998; Weible et al. 2000; Powell et al. 2001; McLaughlin et al. 2002) and engages the cerebellum relatively directly (but see Halverson and Freeman 2006). Presentation of the tone and the US are conveyed to the cerebellum via activation of mossy fibers and climbing fibers, respectively (Fig. 1B; Mauk et al. 1986; Steinmetz et al. 1987, 1989; Sears and Steinmetz 1991; Hesslow 1994; Hesslow et al. 1999). In addition, output via a cerebellar deep nucleus is required for the expression of conditioned responses (McCormick and Thompson 1984). This relatively direct mapping of stimuli onto inputs and of output onto behavior makes delay eyelid conditioning a powerful tool for the analysis of cerebellar learning and computation (Mauk and Donegan 1997; Medina and Mauk 2000; Medina et al. 2000, 2002; Hansel et al. 2001; Ohyama et al. 2003).Open in a separate windowFigure 1.The procedures, neural pathways, and putative signals involved in delay and trace eyelid conditioning. (A) Stimulus timing for delay (left) and trace (right) training trials. For delay conditioning, the US overlaps in time with the tone CS. In this and subsequent figures, green is used to indicate the presentation of the CS for delay conditioning. For trace conditioning, the US is presented after CS offset, and “trace interval” refers to the period between CS offset and US onset. For convenience, we used red and maroon regions to represent the CS and trace interval, respectively. Sample conditioned eyelid responses are shown below, for which an upward deflection indicates closure of the eyelid. (B) Schematic representation of the pathways engaged by delay conditioning. The CS and US, respectively, engage mossy fibers and climbing fibers relatively directly, and forebrain input is not required for normal learning. (C) The signals hypothesized to engage the cerebellum during trace conditioning. The activity of mossy fibers directly activated by the tone CS does not significantly outlast the stimulus. Thus, a forebrain structure is thought to provide an input that overlaps in time with the US and is necessary to produce cerebellar learning.Trace eyelid conditioning, where the US is presented after tone offset (Fig. 1A, right), has attracted interest for its potential to reveal the nature of interactions between the forebrain and cerebellum as well as the learning mechanisms within these systems. This potential stems from the sensitivity of trace conditioning not only to lesions of cerebellum but also to lesions of hippocampus, medial prefrontal cortex (mPFC), or mediodorsal thalamic nucleus (Woodruff-Pak et al. 1985; Moyer Jr. et al. 1990; Kronforst-Collins and Disterhoft 1998; Weible et al. 2000; Powell et al. 2001; McLaughlin et al. 2002; Powell and Churchwell 2002; Simon et al. 2005). Given the general inability of forebrain lesions to affect delay conditioning, these results have promoted the general interpretation that the forebrain and cerebellum interact to mediate trace conditioning (Weiss and Disterhoft 1996; Clark and Squire 1998; Clark et al. 2002).Here we test the specific hypotheses that (Fig. 1C) (1) cerebellar learning requires that mossy fiber and climbing fiber inputs overlap in time (or nearly so) and (2) that cerebellar learning in trace conditioning occurs in response to a forebrain-driven mossy fiber input that outlasts the CS to overlap with the US rather than the inputs activated by the tone CS (Clark et al. 2002). The data provide direct support for both assertions and, together with recent anatomical studies (Buchanan et al. 1994; Weible et al. 2007), reveal a pathway between the mPFC and cerebellum that is necessary for the expression of trace eyelid responses. When combined with previous recordings from PFC in primates and rodents (Funahashi et al. 1989; Bodner et al. 1996; Fuster et al. 2000; Narayanan and Laubach 2006), these data support the hypothesis that trace eyelid conditioning is mediated by interactions between working memory-related persistent activity in mPFC and motor learning mechanisms in the cerebellum.     

Post-training reversible inactivation of the hippocampus enhances novel object recognition memory

Research on the role of the hippocampus in object recognition memory has produced conflicting results. Previous studies have used permanent hippocampal lesions to assess the requirement for the hippocampus in the object recognition task. However, permanent hippocampal lesions may impact performance through effects on processes besides memory consolidation including acquisition, retrieval, and performance. To overcome this limitation, we used an intrahippocampal injection of the GABA agonist muscimol to reversibly inactivate the hippocampus immediately after training mice in two versions of an object recognition task. We found that the inactivation of the dorsal hippocampus after training impairs object-place recognition memory but enhances novel object recognition (NOR) memory. However, inactivation of the dorsal hippocampus after repeated exposure to the training context did not affect object recognition memory. Our findings suggest that object recognition memory formation does not require the hippocampus and, moreover, that activity in the hippocampus can interfere with the consolidation of object recognition memory when object information encoding occurs in an unfamiliar environment.The medial temporal lobe plays an important role in recognition memory formation, as damage to this brain structure in humans, monkeys, and rodents impairs performance in recognition memory tasks (for review, see Squire et al. 2007). Within the medial temporal lobe, studies have consistently demonstrated that the perirhinal cortex is involved in this form of memory (Brown and Aggleton 2001; Winters and Bussey 2005; Winters et al. 2007, 2008; Balderas et al. 2008). In contrast, the role of the hippocampus in object recognition memory remains a source of debate. Some studies have reported novel object recognition (NOR) impairments in animals with hippocampal lesions (Clark et al. 2000; Broadbent et al. 2004, 2010), yet others have reported no impairments (Winters et al. 2004; Good et al. 2007). Differences in hippocampal lesion size and behavioral procedures among the different studies have been implicated as the source of discrepancy in these findings (Ainge et al. 2006), but previous studies have not examined the consequences of environment familiarity on the hippocampus dependence of object recognition memory.Previous studies addressing the role of the hippocampus in recognition memory relied on permanent, pre-training lesions (Clark et al. 2000; Broadbent et al. 2004; Winters et al. 2004; Good et al. 2007). Permanent lesions inactivate the hippocampus not only during the consolidation phase, but also during habituation, acquisition, and memory retrieval, potentially confounding interpretation of the results. Furthermore, permanent lesion studies require long surgery recovery times during which extrahippocampal changes may emerge to mask or compensate for the loss of hippocampal function. To overcome these problems, we reversibly inactivated the dorsal hippocampus after training mice in two versions of the object recognition task. We infused muscimol, a γ-aminobutyric acid (GABA) receptor type A agonist, into the dorsal hippocampus immediately after training in an object-place recognition task or immediately following training in a NOR task. Consistent with previous studies (Save et al. 1992; Galani et al. 1998; Mumby et al. 2002; Stupien et al. 2003; Aggleton and Brown 2005), we observed that hippocampal inactivation impairs object-place recognition memory. Interestingly, we observed that the degree of contextual familiarity can influence NOR memory formation. We found that when shorter periods of habituation to the experimental environment were used, hippocampal inactivation enhances long-term NOR memory. In contrast, after extended periods of contextual habituation, long-term recognition memory was unaltered by hippocampal inactivation. Together these results suggest that if familiarization with objects occurs at a stage in which the contextual environment is relatively novel, the hippocampus plays an inhibitory role on the consolidation of object recognition memory. Supporting this view, we observed that object recognition memory is unaffected by hippocampal inactivation when initial exploration of the objects occurred in a familiar environment.     

Posterior parietal cortex and episodic retrieval: Convergent and divergent effects of attention and memory

Functional neuroimaging studies of humans engaged in retrieval from episodic memory have revealed a surprisingly consistent pattern of retrieval-related activity in lateral posterior parietal cortex (PPC). Given the well-established role of lateral PPC in subserving goal-directed and reflexive attention, it has been hypothesized that PPC activation during retrieval reflects the recruitment of parietal attention mechanisms during remembering. Here, we evaluate this hypothesis by considering the anatomical overlap of retrieval and attention effects in lateral PPC. We begin by briefly reviewing the literature implicating dorsal PPC in goal-directed attention and ventral PPC in reflexive attention. We then discuss the pattern of dorsal and ventral PPC activation during episodic retrieval, and conclude with consideration of the degree of anatomical convergence across the two domains. This assessment revealed that predominantly divergent subregions of lateral PPC are engaged during acts of episodic retrieval and during goal-directed and reflexive attention, suggesting that PPC retrieval effects reflect functionally distinct mechanisms from these forms of attention. Although attention must play a role in aspects of retrieval, the data reviewed here suggest that further investigation into the relationship between processes of attention and memory, as well as alternative accounts of PPC contributions to retrieval, is warranted.Episodic memory—declarative memory for events—has long been known to depend on the medial temporal lobe and, to a lesser extent, the prefrontal cortex (Squire 1992; Shimamura 1995; Wheeler et al. 1995; Gabrieli 1998; Eichenbaum and Cohen 2001; Squire et al. 2004). Recently, an explosion of functional neuroimaging studies has revealed that episodic retrieval is also consistently associated with activity in lateral posterior parietal cortex (PPC), including in the intraparietal sulcus and inferior parietal lobule (Figs. 1, ​,2;2; for detailed review, see Wagner et al. 2005; Cabeza 2008; Cabeza et al. 2008; Ciaramelli et al. 2008; Vilberg and Rugg 2008b; Olson and Berryhill 2009). This unexpected finding raises the possibility that parietal mechanisms may be more central to episodic retrieval than previously thought.Open in a separate windowFigure 1.Anatomy of posterior parietal cortex (PPC). A posterior-lateral view of human PPC is depicted, with PPC separated into dorsal and ventral portions by the intraparietal sulcus (IPS). Dorsal PPC includes the superior parietal lobule (SPL) and IPS. Ventral PPC includes inferior parietal lobule (IPL) and its subregions: supramarginal gyrus (SMG), temporoparietal junction (TPJ), and angular gyrus (AnG).Open in a separate windowFigure 2.Left lateral PPC activity during episodic retrieval. (A) A comparison of hits relative to correct rejections reported by Kahn et al. (2004) revealed “old/new” effects in dorsal PPC, inclusive of IPS. Average signal change within IPS was greater for items perceived as old (hits and false alarms) vs. those believed to be new (misses and correct rejections). (B) A comparison of successful, relative to unsuccessful, cued recall by Kuhl et al. (2007) revealed greater activity in AnG, compatible with the broader literature on recollection success effects (see Fig. 4). In addition, effects were observed in more anterior aspects of ventral PPC (SMG), as well as in dorsal PPC (principally SPL) (see Discussion). (C) Orienting to memory in attempts to recollect, independent of recollection success, is often associated with activity in dorsal PPC. For example, comparison of temporal recency judgments to novelty-based decisions elicited greater IPS activity (Dudukovic and Wagner 2007).At the neuropsychological level, human lesion evidence regarding the necessity of lateral PPC mechanisms for episodic retrieval is limited and mixed (Berryhill et al. 2007; Davidson et al. 2008; Haramati et al. 2008; Simons et al. 2008). By contrast, other neuropsychological data indicate that lateral PPC is unambiguously associated with another cognitive domain—attention (Posner et al. 1984; Mesulam 1999; Parton et al. 2004). This latter lesion literature is further complemented by rich functional neuroimaging evidence implicating dorsal and ventral PPC in goal-directed and reflexive attention, respectively (for review, see Corbetta and Shulman 2002; Corbetta et al. 2008).Drawing from the rich literature linking attention to lateral PPC, memory researchers have recently proposed that lateral PPC activity during episodic retrieval tasks reflects the engagement of attention mechanisms during remembering (Cabeza 2008; Cabeza et al. 2008; Ciaramelli et al. 2008; Olson and Berryhill 2009). Specifically, it has been hypothesized that: (1) Dorsal PPC activity during retrieval may reflect the recruitment of goal-directed attention in service of performing retrieval tasks and (2) ventral PPC engagement during retrieval may mark the reflexive capture of attention by mnemonic representations. While prior comprehensive reviews of the neuroimaging literature on parietal correlates of episodic retrieval have documented functional dissociations along the dorsal/ventral axis of lateral PPC, which qualitatively parallel those seen in the attention literature, evaluation of the hypothesis that PPC retrieval activity reflects attention mechanisms further requires an assessment of the degree to which attention and retrieval effects co-localize. Here we review lateral PPC correlates of both episodic retrieval and attention, with the goal of directly assessing to the degree of anatomic overlap.It should be noted from the outset that the aim of the present review is to evaluate the hypothesis that lateral PPC episodic retrieval effects can be explained in terms of goal-directed and reflexive attention mechanisms. As such, we a priori imposed three constraints that served to focus our treatment of these two substantial literatures. First, while both the dual-attention and memory retrieval literatures focus on effects on the lateral parietal surface, retrieval effects are predominantly left lateralized. Thus, we constrained our analysis of attention and retrieval findings to left lateral PPC.⁵ Second, because prior retrieval reviews focused theoretical discussion on dual-attention accounts, here we similarly constrained our treatment of the extensive attention literature to include only those effects relevant to dual-attention theory. Finally, because the preponderance of evidence offered in support of dual-attention theory''s proposed dorsal attention network derives from studies of visual attention, the present review of the dorsal network is also confined to visual attention. As such, the present review should not be viewed as a comprehensive review of the entire attention literature.We first survey the functional neuroimaging literature on parietal correlates of goal-directed and reflexive attention, and then discuss how these correlates converge and diverge with the patterns of lateral PPC activity present during episodic retrieval. We conclude by considering theoretical frameworks that focus on the role of attention in episodic retrieval, as well as nonattention-based accounts of PPC activity during retrieval, and we highlight open questions that await further investigation.     

Theta bursts in the olfactory nerve paired with β-adrenoceptor activation induce calcium elevation in mitral cells: A mechanism for odor preference learning in the neonate rat

Odor preference learning in the neonate rat follows pairing of odor input and noradrenergic activation of β-adrenoceptors. Odor learning is hypothesized to be supported by enhanced mitral cell activation. Here a mechanism for enhanced mitral cell signaling is described. Theta bursts in the olfactory nerve (ON) produce long-term potentiation (LTP) of glomerular excitatory postsynaptic potentials (EPSPs) and of excitatory postsynaptic currents (EPSCs) in the periglomerular (PG) and external tufted (ET) cells. Theta bursts paired with β-adrenoceptor activation significantly elevate mitral cell (MC) calcium. Juxtaglomerular inhibitory network depression by β-adrenoceptor activation appears to increase calcium in MCs in response to theta burst stimulation.Early odor preference learning provides us with a model in which the necessary and sufficient inputs for learning can be localized to a relatively simple cortical structure, the olfactory bulb (Sullivan et al. 2000). The critical changes for this natural form of learning occur in the olfactory bulb network (Coopersmith and Leon 1986, 1987, 1995; Wilson et al. 1987; Woo et al. 1987; Wilson and Leon 1988; Wilson and Leon 1991; Guthrie et al. 1993; Johnson et al. 1995; Yuan et al. 2002). Odor preference learning is induced by the pairing of odor with activation of the locus coeruleus noradrenergic system, a component of our arousal circuitry (Harley 1987; Berridge and Waterhouse 2003; Berridge 2008), which is critically involved in other forms of memory (Harley 1987; Berridge and Waterhouse 2003) and compromised in diseases of memory, such as Alzheimer''s (Palmer and DeKosky 1993; Weinshenker 2008). The unconditioned stimulus for early odor preference learning is mediated by β-adrenoceptor activation in the olfactory bulb (Sullivan et al. 1991, 1992; Wilson and Sullivan 1991, 1994; Wilson et al. 1994; Harley et al. 2006). β-Adrenoceptor agonists and antagonists infused in the olfactory bulb can induce or block learning, respectively (Sullivan et al. 1989, 2000).Despite the fact that the behavioral model for early odor preference learning has been established for decades (Sullivan et al. 1988, 1989), and despite the fact that the olfactory bulb circuit contains synapses that are essential for the formation of a localizable long-term memory that is easily indexed in behavior (Coopersmith and Leon 1986; Wilson et al. 1987; Woo et al. 1987; Wilson and Leon 1988; Woo and Leon 1991; Johnson et al. 1995; McLean et al. 1999; Yuan et al. 2002), the circuitry and the synapses in the olfactory bulb that mediate learning are not well understood. Long-term potentiation (LTP), the putative synaptic model for associative learning in other brain regions (Bliss and Lomo 1973; Brown et al. 1988; Barnes 1995; Malenka 1994), has not been demonstrated compellingly in the rat olfactory bulb. The lack of evidence for a synaptic locus and a mechanism to support odor preference learning is partly due to the dissociation of the neural changes previously observed following early odor learning (seen at the glomerular input level) (Coopersmith and Leon 1986; Wilson et al. 1987; Woo et al. 1987; Wilson and Leon 1988; Woo and Leon 1991; Johnson et al. 1995; Yuan et al. 2002) and the innervation pattern of noradrenergic fibers in the olfactory bulb (seen mostly in the deep layers of the olfactory bulb, but sparse in the glomerular layer) (McLean et al. 1989; McLean and Shipley 1991).McLean and colleagues have proposed, based on physiological and anatomical evidence, that early odor preference learning leads to a long-term facilitation of the olfactory nerve (ON) inputs to mitral cells (MCs, the main output cell of the olfactory bulb) (McLean et al. 1999; Yuan et al. 2003b; McLean and Harley 2004). In the present study, odor input is mimicked in vitro by theta burst stimulation (TBS) of the ON, and the modulation of glomerular and MC responses to theta bursts alone and in conjunction with bath application of the β-adrenoceptor agonist, isoproterenol, is assessed. The results support the McLean glomerular/MC hypothesis of early odor preference learning.In the first set of experiments (Fig. 1A–D), the effects of theta burst ON input on the field glomerular excitatory postsynaptic potential (EPSP) were tested. The ON was stimulated by a single test stimulus (20–100 μA) every 20 sec in horizontal olfactory bulb slices from postnatal 6–14-d-old Sprague–Dawley rats (Fig. 1A). TBS (10 bursts of high frequency stimulation at 5 Hz, each burst containing five pulses at 100 Hz, same stimulation intensity as test stimuli) that mimics the sniffing cycles in the ON (Kepecs et al. 2006) was given after a baseline was taken. All the experiments were done in aCSF containing (in millimolars) 119 NaCl, 2.5 KCl, 1.3 MgSO₄, 1 NaH₂PO₄, 26.2 NaHCO₃, 22 mM glucose, and 2.5 CaCl₂, equilibrated with 95% O₂/5% CO₂. Field recording pipettes were filled with aCSF. All recordings were acquired at 30°C–32°C. Data are presented as mean ± SEM. Student''s t-test was used to determine statistical significance.Open in a separate windowFigure 1.Theta LTP can be induced in the olfactory bulb first synapses. (A) Stimulation and recording configuration. A bipolar stimulation pipette was placed on a bundle of ON fibers that innervated the recorded glomerulus. ON, olfactory nerve; GL, glomerular layer; MC, mitral cell. (B–D) LTP of glomerular field EPSPs induced by ON theta burst stimulation (TBS). (B) Time course of glomerular field EPSPs (upper: single example; lower: average traces from N = 34 recordings). Note field EPSP was potentiated following TBS. (C) Paired-pulse ratio (PPR, N = 7) of ON field EPSPs (interval 50 msec) was depressed following TBS. (D) LTP of glomerular field EPSPs was D-APV independent (N = 5). (E–G) TBS of ON induced LTP in postsynaptic external tufted (ET) cells. (E) Time course of EPSCs recorded from ET cells (N = 6). (F) PPR of EPSCs (N = 4). (G) LTP of ET cell EPSCs are D-APV independent (N = 3). (H,I) TBS of ON induced potentiation of periglomerular (PG) cell EPSCs. (H) Time course of EPSCs (N = 8). (I) PPR of EPSCs (N = 4).There was on average a 14.5 ± 2.5% increase in the field EPSP peak amplitude at 30 min post TBS induction (N = 34, t = 5.82, P < 0.001, Fig. 1B). Bath application of D-APV (50 μM), an NMDAR antagonist, did not eliminate TBS potentiation; the EPSP peak is 115.6 ± 5.4% of baseline, 30 min post-induction (N = 5, t = 2.90, P = 0.022, Fig. 1D). This result suggests that plasticity occurs at the first step of odor processing (Ennis et al. 1998; Mutoh et al. 2005; Tyler et al. 2007; Dong et al. 2008; Jones et al. 2008). Hence, the synapses between the ON and its postsynaptic neurons are potential targets for learning-dependent plasticity as suggested by the long-lasting metabolic and anatomical changes observed at the olfactory bulb glomerular level following early odor preference training (Coopersmith and Leon 1986; Wilson et al. 1987; Woo et al. 1987; Wilson and Leon 1988; Woo and Leon 1991; Johnson et al. 1995; Yuan et al. 2002). Paired stimuli given to the ON using a 50-msec interval were used to test presynaptic changes to TBS. Paired-pulse ratios, an indicator of changes in presynaptic release (Murphy et al. 2004), decreased following TBS (91.5 ± 2.8% of control, N = 7, t = 3.05, P = 0.011, Fig. 1C). The decrease in paired-pulse ratio suggests TBS potentiation is presynaptically mediated. The glomerular potentiation seen here is consistent with a recent adult mouse model showing an increase in odor-specific glomeruli and olfactory sensory neurons following odor learning (Jones et al. 2008).In the second set of experiments, glomerular excitatory postsynaptic currents (EPSCs) in postsynaptic juxtaglomerular (JG) cells were recorded in voltage-clamp mode (membrane potential held at −60 mV). The effects of TBS on JG cell EPSCs were tested. Patch pipettes were filled with an internal solution containing (in millimolars) 114 K-gluconate, 17.5 KCl, 4 NaCl, 4 MgCl₂, 10 HEPES, 0.2 EGTA, 3 Mg₂ATP, and 0.3 Na₂GTP. There are two main populations of JG cells in the glomeruli, periglomerular (PG) cells, and external tufted (ET) cells. PG cells are inhibitory on MCs (Murphy et al. 2005), while ET cells are excitatory neurons, which receive monosynaptic ON input and then excite PG interneurons (Hayar et al. 2004). PG cells form two functionally distinct populations: ∼30% are driven by monosynaptic ON input, while the remaining population receive their input mainly through ET cells (ON–ET–PG circuit) (Shao et al. 2009). These JG cells form a glomerular inhibitory network in which inhibitory PG cells (activated by either ON input or ET cells) provide both feed-forward and feedback inhibition to MCs of the olfactory bulb through dendrodendritic synapses (Hayar et al. 2004; Murphy et al. 2005). ET cells can be distinguished from PG cells by their morphology, location, and characteristic spontaneous rhythmic spike bursting pattern recorded extracellularly before switching to whole-cell voltage-clamp mode (Hayar et al. 2004; Dong et al. 2007). ET cells were significantly potentiated by TBS while the TBS effect on PG cells was more moderate and more variable (ET: 124.1 ± 11.4% of control at 30 min post-induction, N = 6, t = 2.12, P = 0.043, Fig. 1E; PG: 115.7 ± 15.5%, N = 8, t = 1.01, P = 0.172, Fig. 1H). It should be noted that there is a stronger dialysis effect in small cells, such as the PG cells, which is suggested to account for the weaker and more variable potentiation in this subgroup. Three PG cells showed compelling potentiation (164 ± 13.5% of control at 30 min post-induction, t = 4.73, P = 0.021). The paired-pulse ratio of the EPSCs was reduced following TBS induction (ET: 81.6 ± 4.6% of control, N = 4, t = 4.01, P = 0.014, Fig. 1F; PG: 80.1 ± 10.5%, t = 1.90, P = 0.070, Fig. 1I), consistent with a presynaptic expression mechanism. Furthermore, ET cell LTP was NMDA-receptor independent when tested with APV (161.9 ± 23.3% of control, 15 min post-induction, N = 3, t = 2.66, P = 0.028, Fig. 1G).The role of theta LTP in learning is of particular interest because this pattern of activation is likely to occur in the ON. Since theta is the frequency associated with sniffing in the olfactory bulb (Kepecs et al. 2006), it is straightforward to hypothesize that it has a role in the learning of odor associations. Yet odor preference learning does not occur with odor exposure per se. An interaction between a theta paced odor input and the arousal modulator, norepinephrine (NE), is critical.In the third set of experiments, calcium imaging was used to examine the network of MC responses in each slice. Conventionally, it has been assumed that the glomerular field EPSP mostly reflects MC activity (Mori 1987; Shipley et al. 1996). But recent studies demonstrate the contribution of other cell types to the glomerular field EPSP such as the JG cells (ET and PG cells) (Karnup et al. 2006) monitored here. Therefore, the glomerular field EPSP is not a simple summation of synchronized MC activity. Moreover, synaptic activities at MC dendritic tufts in glomeruli may not propagate efficiently enough to the soma to change MC spiking rate due to the significant length of MC apical dendrites (Karnup et al. 2006). Therefore, this set of experiments used direct monitoring of MC activity through calcium imaging to characterize activity-dependent changes in the olfactory bulb output map. Calcium imaging permits the examination of a large population of neurons with single-cell resolution (Egger 2007).A calcium indicator dye, Oregon Green BAPTA-1, was pressure-injected into the MC layer to stain populations of MCs (Fig. 2A,B). Calcium transients were imaged at 494 nm excitation (15–20 Hz, 2 × 2 binning). Regions of interest (∼20 μm diameter) centered over MC somata were used for kinetic analysis. By selecting 8–12 cells per slice (including both strongly and weakly activated cells), and measuring single MC somatic calcium transients (ΔF/F, average over six to eight trials, background subtracted from an immediate adjacent area next to the somata), changes were observed in the MC responses to ON stimulation (40–100 μA) 30 min following TBS (the same time point at which the magnitude of theta LTP in the glomerular layer was measured). The overall MC calcium responses to ON stimulation were not significantly affected by TBS (113.0 ± 10.5% of control, N = 74 cells from nine slices, t = 1.24, P = 0.220, Fig. 2 panel C1). The olfactory bulb network is, as noted, functionally complex with both feed-forward and feedback inhibition modifying the MC responses to ON/odor stimulation (Murphy et al. 2005). Given the increase in coupling to inhibitory interneurons seen in the second set of experiments, it may be that enhanced feed-forward inhibition from the inhibitory JG network prevented an increase in MC responding. However, it should be noted that increases in MC responses related to TBS could be masked due to the limitation of the in vitro methodology (e.g., selected cells may include those projecting to glomeruli further away from the stimulation site and those deeper in the tissue which have weaker signal-to-noise ratios). However, as subsequent experiments, described below, reveal significant MC calcium responses with the same in vitro methodology, it is likely that the response to TBS alone is relatively minor.Open in a separate windowFigure 2.Pairing of TBS and isoproterenol (ISO) increased MC calcium responses. (A) ΔF/F calcium image (20× objective) of a slice stained with a calcium indicator dye, Oregon Green BAPTA-1 AM. The MC layer is labeled by white dashed lines. Scale bar, 50 μm. (B) An example of ΔF/F calcium imaging showing enhanced calcium responses following TBS+ISO in one MC (white asterisk). Lower traces are calcium transients recorded from the soma of the MC. (C) Averaged calcium transient changes in MCs following TBS, ISO, and TBS+ISO (normalized to control), measured at 30 min post-manipulations. **P < 0.01. (C1) Single-cell calcium transient changes before and after TBS. Dashed black line indicates no change. Cells above the dashed line show increased calcium responses, whereas those below the dashed line show decreased calcium responses. (C2) MC calcium responses to paired TBS and ISO. Note that the majority of MCs showed increased calcium responses following TBS+ISO. (C3) MC calcium responses to ISO only.Since TBS itself was not sufficient to produce significant MC calcium responses, the combined effect of TBS and the β-adrenoceptor agonist, isoproterenol, were examined. Isoproterenol was applied to the bath solution 5–10 min before the TBS and washed out after the TBS induction. The pairing of TBS and isoproterenol (10 μM) increased MC calcium responses in most of the cells measured (137.0 ± 11.0% of control, N = 55 from five slices, t = 3.27, P < 0.001, Fig. 2 panel C2). Five to ten min application of isoproterenol alone to the bath solution did not alter the MC responses observed 30 min after isoproterenol washout (102.5 ± 8.8% of control, N = 47 from four slices, t = 0.28, P = 0.781, Fig. 2 panel C3). Thus, the potentiated MC calcium response was only seen with paired isoproterenol and TBS. As a caveat it should be noted that MCs fire action potentials spontaneously in vivo at ∼3 Hz (Cang and Isaacson 2003) and in vitro at ∼15 Hz (up to 75 Hz) (Griff et al. 2008). A change in ΔF/F calcium response reflects a change in the ratio of the evoked response over the baseline spontaneous response. Changes in MC spontaneous firing, therefore, can affect the ΔF/F calcium signal measured from the MC. However, if pairing TBS with isoproterenol increased spontaneous firing, the increase in the evoked firing rate of MCs had to occur to a greater degree.This result, that only the pairing of TBS with isoproterenol enhanced MC calcium responses, correlates with the behavioral studies (Langdon et al. 1997; Sullivan and Leon 1987; Sullivan et al. 2000) showing that only the pairing of an odor and isoproterenol produces associative learning, while either odor alone or isoproterenol alone does not lead to associative learning. It supports the view that at the level of physiological mechanism, classical conditioning is the interaction of arousal modulation and theta modulation while either alone does not create the necessary associative conditions. Consistent with previous work showing enhanced CREB phosphorylation in MCs following learning (McLean et al. 1999; Yuan et al. 2000, 2003a), the present calcium imaging results support the hypothesis that odor learning results in increased firing in odor encoding MCs. Increased firing in MCs is also consistent with recent work by Gire and Schoppa showing that the pairing of NE with TBS induces an enhancement of MC long-lasting depolarization and gamma frequency oscillation (Gire and Schoppa 2008). Interestingly, MC firing is mainly suppressed to a familiar odor in neonatal rats (Wilson et al. 1985). TBS potentiation of the inhibitory JG circuitry may contribute to the odor habituation observed behaviorally.In the fourth set of experiments, the effects of isoproterenol on JG cell activity were examined. Previous research using slice physiology to identify a synaptic site of NE action was constrained by the known distribution of noradrenergic input to the olfactory bulb. Noradrenergic fibers project heavily to the subglomerular layers; they terminate densely in the internal plexiform and the granule cell (GC) layers, and moderately in the external plexiform and the MC layers (McLean et al. 1989). Based on this anatomical evidence, it was assumed that either GCs or MCs were the potential targets for β-adrenoceptor action. However, the Ennis group showed that the β-adrenoceptor agonist isoproterenol has no direct effect on MC excitability in acute rat olfactory bulb slices (Hayar et al. 2001). Although isoproterenol caused an inward current in MCs in voltage-clamp mode, this inward current was blocked by synaptic transmission blockers, suggesting an indirect effect of isoproterenol, possibly through interneurons. NE could disinhibit MCs through suppressing GC activity as reported in the turtle and dissociated rat olfactory bulb cultures (Jahr and Nicoll 1982; Trombley and Shepherd 1992; Trombley 1994). However, this effect was attributed to an α2-, but not β-adrenoceptor, mediated presynaptic inhibition of GC and/or MC dendrites (Trombley 1992, 1994; Trombley and Shepherd 1992). Since isoproterenol here altered MC calcium responses to ON theta burst input, it was possible that JG cells were potential targets for NE action. Studies using immunocytochemistry (Yuan et al. 2003b) and receptor autoradiography (Woo and Leon 1995) show that β-adrenoceptors are expressed in JG cells (Woo and Leon 1995; Yuan et al. 2003b), as well as in MCs (Yuan et al. 2003b) and GCs (Woo and Leon 1995).With the application of isoproterenol the JG cell EPSCs to ON stimulation were suppressed (ET: 86.6 ± 6.0% of control, N = 6, t = 2.25, P = 0.037, Fig. 3A; PG: 87.3 ± 3.6%, t = 3.55, P = 0.006, Fig. 3A). Isoproterenol was applied for 5 min in the bath solution and then washed out. The effect of isoproterenol was reversed after washout (94.8 ± 6.7%, N = 3, t = 0.78, P = 0.259, Fig. 3A). Similar results were found with calcium imaging using the averaged cell calcium transients from 8–20 cells per slice (Fig. 3B). The averaged response from each slice was counted as one experiment. JG cell calcium transients were suppressed in the presence of isoproterenol (83.7 ± 3.0% of control, N = 7, t = 5.40, P = 0.002, Fig. 3B). The effect of isoproterenol was reversed 30 min following washout (100.6 ± 1.8% of control, N = 3, t = 0.36, P = 0.754). In two experiments, recordings were made from slices that had a cut between the MC and the glomerular layer to isolate the potential secondary effect from MCs onto JG cells. In this case, isoproterenol still suppressed JG cell calcium transients (Fig. 3B, dashed circles).Open in a separate windowFigure 3.The effects of ISO on JG cells (PG+ET cells). (A) ISO (10 μM) application suppressed EPSCs of ET cells (N = 6) and PG cells (N = 7), which was reversed following ISO washout (N = 3). *P < 0.05, **P < 0.01. (B) ISO application suppressed ON-evoked calcium transients in JG cells (N = 7 slices). The ISO effect was reversed 30 min after washout (N = 3). N = 2 experiments were recorded from slices of a cut that was made between the glomerular layer and the MCs layer. **P < 0.01Given the results of both whole-cell recording and calcium imaging, it was reasonable to hypothesize that isoproterenol would decrease JG cell activity, reducing GABA release onto MCs and causing MC disinhibition. Indeed, it has been shown NE application caused a reduction of inhibitory postsynaptic currents (IPSCs) in MCs (Gire and Schoppa 2008). Taken together these findings support the enhanced MC excitation model for early odor preference learning. While the present experiments were conducted with the β_1/2 agonist, isoproterenol, associative odor preference learning depends on β₁, not β₂, receptors (Harley et al. 2006). β₁ adrenoceptors are also expressed in JG cells (Yuan et al. 2003b). The role of specific β-adrenoceptor subtypes in mediating MC disinhibition will be tested in the future studies.The present results argue that potentiation of MC calcium responses occurs only when theta frequency activity is paired with β-adrenoceptor activation. They also suggest that one critical role of NE activation via β-adrenoceptors in the olfactory bulb is to suppress the inhibitory JG network, subsequently transiently disinhibiting MCs and providing the conditions for strengthening ON–MC connections in selected glomeruli. This mechanism likely operates in concert with changes promoted by patterned cAMP waves in MCs (Cui et al. 2007).     

The basolateral amygdala and nucleus accumbens core mediate dissociable aspects of drug memory reconsolidation

A distributed limbic-corticostriatal circuitry is implicated in cue-induced drug craving and relapse. Exposure to drug-paired cues not only precipitates relapse, but also triggers the reactivation and reconsolidation of the cue-drug memory. However, the limbic cortical-striatal circuitry underlying drug memory reconsolidation is unclear. The aim of this study was to investigate the involvement of the nucleus accumbens core and the basolateral amygdala in the reconsolidation of a cocaine-conditioned stimulus-evoked memory. Antisense oligodeoxynucleotides (ASO) were infused into each structure to knock down the expression of the immediate-early gene zif268, which is known to be required for memory reconsolidation. Control infusions used missense oligodeoxynucleotides (MSO). The effects of zif268 knockdown were measured in two complementary paradigms widely used to assess the impact of drug-paired CSs upon drug seeking: the acquisition of a new instrumental response with conditioned reinforcement and conditioned place preference. The results show that both intranucleus accumbens core and intrabasolateral amygdala zif268 ASO infusions at memory reactivation impaired the reconsolidation of the memory underlying a cocaine-conditioned place preference. However, knockdown of zif268 in the nucleus accumbens at memory reactivation had no effect on the memory underlying the conditioned reinforcing properties of the cocaine-paired CS measured subsequently, and this is in contrast to the marked impairment observed previously following intrabasolateral amygdala zif268 ASO infusions. These results suggest that both the basolateral amygdala and nucleus accumbens core are key structures within limbic cortical-striatal circuitry where reconsolidation of a cue-drug memory occurs. However reconsolidation of memory representations formed during Pavlovian conditioning are differentially localized in each site.Through Pavlovian association with the effects of addictive drugs, a conditioned stimulus (CS) acquires both general motivational and sensory-specific conditioned reinforcing properties (Everitt et al. 2000). These associations contribute to the high likelihood of relapse in addicted individuals, yet the extinction of drug CSs by nonreinforced exposure has proved to be of limited therapeutic utility (Conklin and Tiffany 2002). In abstinent humans, drug CSs evoke salient and persistent memories of drug-taking experiences, inducing craving and relapse (Childress et al. 1988; O''Brien et al. 1992), while in animals they also precipitate relapse to, or reinstatement of, drug-seeking behavior (de Wit and Stewart 1981; Meil and See 1996; Fuchs et al. 1998; Weiss 2000). Thus, disrupting drug-related memories might significantly diminish relapse propensity on subsequent exposure to drug-paired CSs, and thereby promote abstinence.Exposure to a drug-associated CS also triggers a process of memory reconsolidation, which restabilizes the reactivated and labile memory (Nader 2003). While reconsolidation may adaptively update memories (Dudai 2006; Hupbach et al. 2007; Rossato et al. 2007; Lee 2009), its disruption may reduce the impact of intrusive or aberrant memories on behavior subsequently (Lee et al. 2005, 2006; Brunet et al. 2008; Kindt et al. 2009; Taubenfeld et al. 2009). The reconsolidation of CS–cocaine memories has been shown to depend upon protein synthesis and expression of the plasticity-associated immediate-early gene, zif268, in the basolateral amygdala (BLA), since zif268 knockdown at memory reactivation disrupted the acquired conditioned reinforcing properties of the CS measured in drug-seeking tasks days or weeks later (Lee et al. 2005, 2006).Although the BLA has an established role in CS-drug memory reconsolidation, it remains unclear whether other sites within limbic cortical-ventral striatal circuitry participate in this process. The nucleus accumbens core (AcbC) is a primary candidate, as zif268 is up-regulated in the AcbC as well as in the BLA following exposure to cocaine CSs (Thomas et al. 2003). Furthermore, the AcbC, which is strongly implicated in Pavlovian influences on drug seeking and relapse (Cardinal et al. 2002; Kalivas and McFarland 2003), has been shown to be a site where the reconsolidation of a drug conditioned place preference (CPP) memory can be disrupted (Miller and Marshall 2005).Given the evidence of increased zif268 expression in the AcbC following CS-drug memory reactivation, we investigated its requirement in the reconsolidation of cocaine-associated memories. To address this issue, we employed two different but complementary paradigms widely used to measure the conditioned effects of CSs associated with drugs of abuse: the acquisition of a new instrumental response with conditioned reinforcement (ANR) and CPP. These procedures have been used successfully to investigate the mechanisms underlying the reconsolidation of appetitive Pavlovian memories, but it is likely that they depend upon different associative mechanisms (Everitt et al. 1991; White and McDonald 1993) that in turn depend upon different neural loci within limbic cortical-striatal circuitry (Cardinal et al. 2002). Therefore, to enable a full comparison with the functional involvement of the BLA, we investigated the necessity for BLA zif268 expression in drug memory reconsolidation as assessed in the CPP paradigm.     

New behavioral protocols to extend our knowledge of rodent object recognition memory

Animals often show an innate preference for novelty. This preference facilitates spontaneous exploration tasks of novelty discrimination (recognition memory). In response to limitations with standard spontaneous object recognition procedures for rodents, a new task (“bow-tie maze”) was devised. This task combines features of delayed nonmatching-to-sample with spontaneous exploration. The present study explored aspects of object recognition in the bow-tie maze not amenable to standard procedures. Two rat strains (Lister Hooded, Dark Agouti) displayed very reliable object recognition in both the light and dark, with the Lister Hooded strain showing superior performance (Experiment 1). These findings reveal the potential contribution of tactile and odor cues in object recognition. As the bow-tie maze task permits multiple trials within a session, it was possible to derive forgetting curves both within-session and between-sessions (Experiment 1). In Experiment 2, rats with hippocampal or fornix lesions performed at normal levels on the basic version of the recognition task, contrasting with the marked deficits previously seen after perirhinal cortex lesions. Next, the training protocol was adapted (Experiment 3), and this modified version was used successfully with mice (Experiment 4). The overall findings demonstrate the efficacy of this new behavioral task and advance our understanding of object recognition.Understanding the neural basis of recognition memory, the ability to discriminate whether a stimulus is novel or familiar, is heavily reliant on animal research. Here, advances have been closely tied to the introduction of new behavioral tests. The preeminent example concerns one-trial tests of recognition memory for monkeys using delayed nonmatching-to-sample (Mishkin and Delacour 1975). These tasks reward the natural preference that monkeys have for selecting novel items and permit multiple recognition trials within a single session. These features make the task relatively easy to train and then maximize findings from small group sizes. Although rat tasks closely based on delayed nonmatching-to-sample have been devised (Aggleton 1985; Mumby et al. 1990; Steckler et al. 1998; Prusky et al. 2004), they are very rarely employed as they are difficult to train and performance levels are unreliable.Almost all studies of rodent recognition memory now employ the spontaneous object recognition test and its direct variants (Ennaceur and Delacour 1988; Dix and Aggleton 1999; Winters et al. 2008). These tasks again take advantage of an innate preference for novel items, but this preference is displayed by spending more time exploring novel than familiar stimuli. In the standard version of the task, a rodent (rat or mouse) is placed in an arena containing two identical objects and then freely allowed to explore these objects for several minutes (“sample” phase). After a delay, the rodent is placed back in the arena (“test” phase), which now contains one familiar object (a copy of the sample phase objects) and a novel object. Recognition is signified by greater exploration of the novel object. Because the task measures spontaneous behavior, it requires minimal pretraining, but for the same reason it is prone to considerable variance. Unlike delayed nonmatching-to-sample, each trial (sample plus test phase) takes many minutes, and so only one recognition trial is normally given per session. Advantages are that proactive interference between objects is minimized, and the one-trial design lends itself to episodic-like tests of memory (Dere et al. 2005; Good et al. 2007). Disadvantages include the fact that data accumulation, with appropriate counterbalancing, is slow.To address these limitations, a new object recognition test using a “bow-tie maze” (Fig. 1A) has been developed for rats (Albasser et al. 2010). This test combines features of delayed nonmatching-to-sample with spontaneous object preference: It permits multiple trials per session, but the measure of recognition comes from the preferential exploration of novelty. The rat is first placed in one end of a bow-tie–shaped maze that contains a single object (object A; Fig. 1B). After a minute, the rat is allowed to run to the other end of the maze where there are two dissimilar objects (A and B; Fig. 1B). Object A is familiar as it is identical to the object previously explored, while object B is novel. Consequently, a rat will typically prefer to explore object B. On the next trial, a minute later, the rat shuttles back to the initial start point, but this time encounters objects B and C. Object B is now familiar, while object C is novel. The next trial, 1 min later, is between object C (now familiar) and object D (novel), and so on. A food reward placed under every object promotes shuttling back and forth within the maze, and encourages interaction with the objects. Unlike delayed nonmatching-to-sample, the food reward is not contingent on first selecting the novel object.Open in a separate windowFigure 1.(A) Schematic of the bow-tie maze. A sliding door separates the two ends of the maze in which two objects are placed. (B) General procedure showing the presentation order of the objects in the standard object recognition task. All objects are rewarded (+). (Arrow) Rat movements. (Black print) Novel objects, (gray print) familiar objects.The present study used the bow-tie maze to explore recognition memory on four fronts. In Experiment 1, recognition in the light and recognition in the dark were compared to help determine the cues available to detect object familiarity. It is known that rats can perform recognition tasks when solely reliant on visual cues (Aggleton 1985; Bartko et al. 2007; Winters and Reid 2010), but object recognition based on other modalities remains largely unexplored (Winters and Reid 2010). Potential cues for recognition include odor differences and tactile information. It is known that rats can discriminate novel from familiar olfactory cues (Otto and Eichenbaum 1992; Kesner et al. 2002; Fortin et al. 2004; Wolff et al. 2006), while tactile (e.g., vibrissae) cues can be used to distinguish surfaces (e.g., Birrell and Brown 2000). The bow-tie maze is ideal for studying object recognition in the dark as the rats are rewarded for visiting items in set locations (to receive food rewards), and so should readily approach the objects. In contrast, running the standard spontaneous object recognition test (in an arena) in the dark would be problematic as it is not clear how the rats would first appreciate the presence of the to-be-discriminated objects.An additional goal of Experiment 1 was to determine how readily the bow-tie maze could be used to compile within-session and between-session forgetting curves. A limitation with the standard spontaneous object task is that with only one trial per session it can be very time consuming to create forgetting curves, while within-session forgetting curves for individual animals are not feasible. These shortcomings create limitations when examining manipulations thought to affect memory. The possibility of deriving within-session forgetting curves is particularly appealing as: (1) it minimized the impact of those factors that introduce variance when performance is compared across sessions, and (2) the animal need not be removed from the maze, which could additionally disrupt performance, e.g., by increasing stress. A further component of Experiment 1 manipulated object memory strength by presenting objects either once (“single”) or six times (“repeated”). Recognition was tested after a 3-h delay with the twin goals of determining whether repeated presentation would aid performance and whether these performance levels would be sufficiently above chance so that they could be used to examine factors involved in longer term memory.At the same time, Experiment 1 provided the opportunity to compare two rat strains, Dark Agouti (DA) and Lister Hooded (LH). Previous studies suggest that the Dark Agouti strain might be particularly good at visual recognition tasks (Aggleton 1996), though others have argued that this strain has aberrant behavioral properties, including higher anxiety and higher levels of inappropriate nonspatial behaviors in spatial learning tasks (Mechan et al. 2002; Harker and Whishaw 2004; but see Aggleton and Vann 2004).Experiment 2 examined the ability of rats with either hippocampal or fornix lesions to perform object recognition in the bow-tie maze. There is a longstanding debate over the impact of hippocampal damage on recognition memory, with mixed findings coming from spontaneous object recognition tests (Clark et al. 2000; Mumby 2001; Gaskin et al. 2003; Winters et al. 2008). The bow-tie maze should prove informative as numerous trials can be run to assess the impact of selective brain lesions. Although in this initial study only short retention delays were examined, these same delays and conditions are highly sensitive to perirhinal cortex lesions (Aggleton et al. 2010; Horne et al. 2010), a brain region regarded as vital for recognition memory (Brown and Aggleton 2001). In Experiment 3 the training protocol changed so that objects did not cover food rewards. Rather, a single food reward was always placed between the test objects. This modification was examined because: (1) it would preclude any exploration scores that were simply derived from attempts to move the test objects in order to uncover the food reward, and (2) it would introduce a task variant that might be amenable to small rodents not able to move objects. Accordingly, Experiment 4 examined the performance of mice (strain C57Bl/6) on a test of object recognition based on the modified version of the bow-tie maze from Experiment 3.     

Making context memories independent of the hippocampus

We present evidence that certain learning parameters can make a memory, even a very recent one, become independent of the hippocampus. We confirm earlier findings that damage to the hippocampus causes severe retrograde amnesia for context memories, but we show that repeated learning sessions create a context memory that is not vulnerable to the damage. The findings demonstrate that memories normally dependent on the hippocampus are incrementally strengthened in other memory networks with additional learning. The latter provides a new account for patterns of hippocampal retrograde amnesia and how memories may become independent of the hippocampus.Contextual fear conditioning can be supported by two neural systems, one that contains the hippocampus (HPC), and one that does not. Evidence for this assertion comes from studies in which the HPC, in rats, is damaged either before or after the contextual fear conditioning. Extensive damage to the HPC before conditioning has little effect on contextual fear conditioning (Maren et al. 1997; Frankland et al. 1998; Wiltgen et al. 2006). This result can only mean that there is a non-HPC memory system that can support fear of context. In contrast, there is unequivocal evidence that moderate to extensive damage to the HPC soon after learning severely impairs the ability of the conditioning context to evoke fear, suggesting that the HPC normally makes a major contribution to this type of memory (Kim and Fanselow 1992; Maren et al. 1997; Frankland et al. 1998; Anagnostaras et al. 1999; Debiec et al. 2002; Lehmann et al. 2007b; Sutherland et al. 2008; Wang et al. 2009).The dissociable effects of pre- and post-training HPC damage on contextual fear conditioning have been interpreted as suggesting that: (1) When the HPC is intact during learning it interferes with other systems and prevents them from acquiring an independent contextual fear conditioning memory, and (2) when the HPC is absent, these other systems are released from this interference and are able to rapidly acquire an independent memory (Maren et al. 1997; Frankland et al. 1998; Fanselow and Poulos 2004; Driscoll et al. 2005; Lehmann et al. 2006; Sutherland et al. 2006). The latter interference from the HPC on the other memory systems has been termed overshadowing. Supplemental Figure S1 depicts data from our laboratory demonstrating the overshadowing phenomenon and the dissociable effects of HPC damage induced before and after contextual fear conditioning.Very little, however, is known about the parameters determining the extent to which the HPC system interferes with the non-HPC system for control over contextual fear. The purpose of the current study is to provide some insight into this issue. Typically, contextual fear conditioning in rats is conducted in a single conditioning session in which a configuration of static background cues is paired with several footshocks. When returned to the conditioning context, rats display several species-specific defensive responses including freezing (i.e., absence of movement except for breathing). Several theorists have proposed that non-HPC systems are more likely to be recruited when there are multiple experiences with similar events, which, in turn, would mitigate the necessity of the HPC for memory expression (O''Keefe and Nadel 1978; Sherry and Schacter 1987; McClelland et al. 1995; O''Reilly and Rudy 2001; White and McDonald 2002). Accordingly, we hypothesized that repeated contextual fear conditioning sessions separated by hours and days would overcome the HPC interference or overshadowing effect. In other words, with repeated learning sessions, enough information would be incrementally captured by the non-HPC system to support a contextual fear memory that would survive complete damage to the HPC.Adult male rats received 11 fear-conditioning sessions across 6 d. In each session, they were placed in a context and received mild footshocks (Shock Context). Concurrently, the rats were exposed 10 times to another context in which they never received shock (No-Shock Context). The No-Shock Context served as a control condition to measure whether the rats simply showed generalized fear or could show context-specific memory. Within 72 h following the last conditioning session, rats either received sham surgery or complete lesions of the HPC using the neurotoxin N-methyl-d-aspartic acid (NMDA) (Lehmann et al. 2007a). Rats were then tested for retention in both the Shock and No-Shock Contexts in a counterbalanced order. In addition, in a single learning episode, another group of rats received a matching number of shocks (i.e., 12 shocks) and context exposure (i.e., 17 min), and then received surgery 7–10 d after conditioning. The latter interval is identical to the interval between the initial conditioning session and surgery in the repeated learning condition. Figure 1 illustrates and describes the design of the experiments.Open in a separate windowFigure 1.Illustration of the experimental design used in (A) the single conditioning session and (B) repeated conditioning session experiments. In A the rats were initially placed in the conditioning chamber for 17 min and received the first of 12 footshocks (1 mA/2 sec) at the 300-sec mark, and then one every following 58 sec after shock offset. Seven to 10 d later, the rats either received sham or HPC damage (Sx). Approximately 10 d after, the rats were returned to the chamber to assess freezing over a 5-min retention test. In B the rats were placed initially in the conditioning chamber for 1 min and received a shock at the 45-sec mark (Shock Context). Approximately 45 min later, the rats were placed in a different chamber for 1 min and did not receive shock (No-Shock Context). The procedure was repeated twice daily for five consecutive days, and the Shock and No-Shock chamber order was counterbalanced according to the principles of a Latin Square design. The rats then received sham or HPC damage 1–3 d later. The rats'' retention was assessed in both contexts ∼10 d after surgery in both the Shock and No-Shock Context in a counterbalanced order with a 24-h span between tests. Importantly, the number of shocks, context exposure time, and interval between initial learning and surgery were matched between both experiments.When all shocks were delivered in a single session, HPC damage caused profound retrograde amnesia. As illustrated in Figure 2A, the HPC rats displayed significantly less freezing than control rats during the retention test (t₍₈₎ = 23.895, P < 0.001). This result replicates all previous studies in which the HPC was damaged days after a single contextual fear conditioning training session (Kim and Fanselow 1992; Maren et al. 1997; Frankland et al. 1998; Anagnostaras et al. 1999; Debiec et al. 2002; Lehmann et al. 2007b; Sutherland et al. 2008).Open in a separate windowFigure 2.Mean (± SEM) percent time freezing by Sham and HPC rats during the retention test of the (A) single conditioning (12 shocks) experiment and (B) repeated conditioning session experiment. In A the HPC rats showed significantly less freezing (P < 0.001) than the Sham rats, suggesting that the damage caused profound retrograde amnesia for contextual fear conditioning learned in a single session 7–10 d before surgery. In B the performance of the HPC rats did not significantly differ from the Sham rats, and they exhibited significantly more freezing in the Shock Context than the No-Shock Context (P < 0.001). Consequently, repeated conditioning sessions prevented the retrograde amnesic effects normally observed in contextual fear conditioning following HPC damage, suggesting that other neural networks were now able to support the memory.In striking contrast, memory for contextual fear conditioning was spared when the HPC was damaged after repeated conditioning sessions. Figure 2B shows the percent time spent freezing during the retention test in the Shock and No-Shock Contexts. An ANOVA with between-group factor (Lesion: Sham and HPC) and within-group factor (Context: Shock and No-Shock) revealed a significant main effect of Context (F_(1,14) = 84.731, P < 0.001), indicating that the rats displayed higher levels of freezing in the Shock than in the No-Shock Context. The effect of Lesion (F_(1,14) = 4.280, P = 0.058) was not significant, nor was the Lesion × Context interaction (F_(1,14) = 0.877, P = 0.369), suggesting that extensive HPC damage did not impair memory. The tendency for an effect of Lesion is due to the HPC rats freezing less than the Sham rats in the No-Shock Context (P = 0.06) rather than freezing less in the Shock Context (P = 0.457).The repeated conditioning sessions clearly enabled a contextual fear representation to be established in non-HPC memory systems. However, it is surprising that the HPC damage did not impair the ability to discriminate between the Shock and No-Shock Context, because evidence suggests that context discrimination is dependent on the HPC (see Moscovitch et al. 2006). Indeed, studies of rats with HPC damage induced before learning have shown that contextual fear conditioning is acquired quickly by non-HPC systems in a single session, but the ability to discriminate between the training context and a new context is lost (Frankland et al. 1998; Antoniadis and McDonald 2000; Winocur et al. 2007). Hence, it is significant in the present study that the HPC damage did not impair context discrimination abilities in the rats that received repeated learning episodes. The latter appear to have established a context representation, outside of the HPC, that was not bereft of details. Yet, one should consider that the rats in the repeated sessions experiment received experience in both the Shock and No-Shock Contexts prior to surgery, and this discrimination training procedure may have established two different non-HPC representations. It remains possible that HPC damage would impair the ability to discriminate the Shock Context from a new context, which is what is found in anterograde amnesia studies (Frankland et al. 1998; Antoniadis and McDonald 2000; Winocur et al. 2007). To address this possibility, a new experiment examined whether HPC-damaged rats could discriminate the Shock Context from a Novel Context. Rats were trained with the same repeated learning protocol as described earlier, with the exception that the rats were never placed in the No-Shock Context prior to surgery. One to 3 d following learning, the rats either received Sham or complete HPC damage. They were then tested for retention in the Shock and the Novel (i.e., No-Shock) Context in a counterbalanced order. Figure 3 shows the percent time spent freezing during the retention test in the Shock and Novel Contexts. An ANOVA with between-group factor (Lesion: Sham and HPC) and within-group factor (Context: Shock and Novel) revealed that the rats froze significantly more in the Shock than the Novel Context (F_(1,10) = 57.393, P < 0.001). However, no significant difference was found between the HPC and Sham groups (F_(1,10) = 0.597, P = 0.458) and the Lesion × Context interaction did not reach significance (F_(1,10) = 0.123, P = 0.733). Thus, as in the previous repeated sessions experiment, the HPC damage did not cause retrograde amnesia for contextual fear conditioning and, more importantly, the HPC damage did not impair the ability to discriminate between the original context and new context.Open in a separate windowFigure 3.Mean (± SEM) percent time freezing by Sham and HPC rats in the Shock and Novel Contexts during the retention tests of the discrimination experiment. The rats exhibited significantly more freezing in the Shock than the Novel Context (P < 0.001), and the HPC rats did not significantly differ from the Sham rats, suggesting that the HPC-damaged rats remembered the specific meaning of the Shock Context as well as control rats. Hence, repeated conditioning sessions established a context-rich representation in non-HPC systems, which supports successful context discriminations.The absence of amnesia for contextual fear conditioning in the current study is not due to insufficient damage to the HPC. We calculated (see Lehmann et al. 2007b) that an average of 83% of the HPC was damaged across rats (smallest: 64%; largest: 90%) in the repeated learning experiments (see Supplemental material for more histological details). The amount of HPC damage is substantially more than that found in most studies reporting impairments for contextual fear conditioning following HPC damage (Kim and Fanselow 1992; Maren et al. 1997; Frankland et al. 1998; Anagnostaras et al. 1999; Debiec et al. 2002) and more than for the single-session experiment (average 76%) in which we currently report amnesia. Therefore, the amount of HPC damage inflicted in the rats in this study is certainly sufficient to disrupt HPC-dependent memories.Like others (Kim and Fanselow 1992; Maren et al. 1997; Frankland et al. 1998; Anagnostaras et al. 1999; Debiec et al. 2002; Lehmann et al. 2007b; Sutherland et al. 2008), we found that damage to the HPC after a single contextual fear-conditioning session involving multiple shocks produces profound retrograde amnesia for contextual fear conditioning. However, in two separate experiments, distributing shock across multiple conditioning sessions prevented this amnesia. In one case, the rats experienced Context–Shock pairings in one context and no shock in another context. Following this training, rats with damage to the HPC did not differ from control rats in the absolute amount of freezing in the training context nor in their ability to discriminate between the two contexts. In the second case, rats only received the multiple Context–Shock sessions. Rats with damage to the HPC could not be distinguished from control rats during the test in the training context or in their responses to a novel context. These findings provide new support for the general idea that contextual fear conditioning can be supported by both HPC and non-HPC systems. This conclusion is supported by (1) the finding that damage to the HPC following a single conditioning session virtually eliminates freezing during the test, implying the importance of the HPC system, and (2) that following multiple conditioning sessions, damage to the HPC has no effect on either contextual fear displayed in the training context or their ability to discriminate the training context from other contexts, suggesting the existence of non-HPC systems that can support contextual fear. The findings also reveal that the overshadowing or interference by the HPC over the non-HPC memory systems for control over contextual fear is not absolute. Following a single conditioning session, removal of the HPC produced a devastating retrograde amnesia, illustrating substantial overshadowing. However, distributing conditioning across several sessions completely attenuated the effects of damage to the HPC, revealing that non-HPC systems can support contextual fear conditioning despite the HPC, and revealed the importance of multiple sessions for this to occur.The overshadowing by the HPC is based on the familiar idea in associative learning at the behavioral level, where through a competitive process some of the cues that redundantly predict a reinforcer acquire the ability to generate strong conditioned responding, while other equally predictive, but less salient cues do not (Stout et al. 2003). Conditioning to the less potent cues proceeds more effectively if the more potent competitors are absent. Following the same principle, if the HPC representation is active, then learning in the non-HPC systems suffers strong interference. In contrast, in the absence of the HPC representation, learning in non-HPC systems is released from this interfering effect of the HPC. Thus, the learning rate in non-HPC networks is potently lowered by the activity of the HPC. However, with repeated learning, other structures, which are overshadowed by the HPC, may cumulatively build a representation that achieves HPC independence. The current findings clearly support this hypothesis, whereby repeated learning episodes incrementally established a contextual fear-conditioning representation outside of the HPC that mitigated the usual retrograde amnesic effects of HPC damage.One important question is where does the HPC interference occur? Biedenkapp and Rudy (2009) recently reported that the HPC competes with the basolateral region of the amygdala during fear conditioning. Previously, Guarraci et al. (1999) found that the amount of conditioned fear produced by training could be increased if the dopamine D1 receptor agonist {"type":"entrez-protein","attrs":{"text":"SKF82958","term_id":"1156217255","term_text":"SKF82958"}}SKF82958 was injected into the basolateral region. Biedenkapp and Rudy (2009) reasoned that if this is the area where the HPC interferes with non-HPC systems for the association with shock, then a local infusion of {"type":"entrez-protein","attrs":{"text":"SKF82958","term_id":"1156217255","term_text":"SKF82958"}}SKF82958 before a single session of contextual fear conditioning should attenuate the interference and allow the non-HPC system to gain more control over contextual fear. Their data supported this hypothesis, which leads to the possibility that with multiple conditioning sessions, the non-HPC system gradually gains association with these fear-supporting neurons in this region of the brain.Patients with bilateral damage to the HPC often exhibit temporally graded retrograde amnesia, such that recently acquired memories are lost, whereas remote memories, especially those acquired years before the damage, are more likely to be spared (Scoville and Milner 1957; Rempel-Clower et al. 1996). This pattern of amnesia is taken as evidence for temporally based systems consolidation, whereby over time the essential support for memories is “switched” from dependence on the HPC to neocortical networks (McClelland et al. 1995; Squire and Alvarez 1995; Anagnostaras et al. 2001; Meeter and Murre 2004; Squire et al. 2004; Wiltgen et al. 2004; Frankland and Bontempi 2005). Our research, however, points to another process for becoming independent of the HPC, a change in the strength of the representation in non-HPC systems during learning rather than a consolidation process linked to the passage of time since the learning episode. A study of a former London taxi driver with bilateral HPC damage alludes to this possibility (Maguire et al. 2006). This amnesic patient showed greater retrograde amnesia for roads that he used less commonly than the major arteries that he used regularly. Hence, greater exposure to the major arteries established memories in non-HPC systems, whereas roads with less exposure remained dependent on the HPC regardless of the age of the memory. Our findings add support to this view, because studies examining the effects of complete HPC damage after a single conditioning episode suggest that the HPC is permanently involved in contextual fear conditioning (Lehmann et al. 2007b; Sutherland et al. 2008); yet, with repeated learning episodes we clearly demonstrated that the memory rapidly becomes independent of the HPC. The latter is important because the process for memories becoming independent of the HPC need not require systems consolidation.In conclusion, this is the first example of intact contextual fear memories following complete HPC damage induced soon after learning. Importantly, repetition of the learning episode underlies the change in memory from HPC dependent to HPC independent. We argue that each learning episode incrementally establishes a representation in non-HPC memory systems—a representation that ultimately becomes sufficiently strong to support memory expression without the HPC. The current findings also demonstrate the critical need to consider learning parameters when discussing patterns of retrograde amnesia and the role of the HPC in memory.     

Trace and contextual fear conditioning require neural activity and NMDA receptor-dependent transmission in the medial prefrontal cortex

The contribution of the medial prefrontal cortex (mPFC) to the formation of memory is a subject of considerable recent interest. Notably, the mechanisms supporting memory acquisition in this structure are poorly understood. The mPFC has been implicated in the acquisition of trace fear conditioning, a task that requires the association of a conditional stimulus (CS) and an aversive unconditional stimulus (UCS) across a temporal gap. In both rat and human subjects, frontal regions show increased activity during the trace interval separating the CS and UCS. We investigated the contribution of prefrontal neural activity in the rat to the acquisition of trace fear conditioning using microinfusions of the γ-aminobutyric acid type A (GABA_A) receptor agonist muscimol. We also investigated the role of prefrontal N-methyl-d-aspartate (NMDA) receptor-mediated signaling in trace fear conditioning using the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV). Temporary inactivation of prefrontal activity with muscimol or blockade of NMDA receptor-dependent transmission in mPFC impaired the acquisition of trace, but not delay, conditional fear responses. Simultaneously acquired contextual fear responses were also impaired in drug-treated rats exposed to trace or delay, but not unpaired, training protocols. Our results support the idea that synaptic plasticity within the mPFC is critical for the long-term storage of memory in trace fear conditioning.The prefrontal cortex participates in a wide range of complex cognitive functions including working memory, attention, and behavioral inhibition (Fuster 2001). In recent years, the known functions of the prefrontal cortex have been extended to include a role in long-term memory encoding and retrieval (Blumenfeld and Ranganath 2006; Jung et al. 2008). The prefrontal cortex may be involved in the acquisition, expression, extinction, and systems consolidation of memory (Frankland et al. 2004; Santini et al. 2004; Takehara-Nishiuchi et al. 2005; Corcoran and Quirk 2007; Jung et al. 2008). Of these processes, the mechanisms supporting the acquisition of memory may be the least understood. Recently, the medial prefrontal cortex (mPFC) has been shown to be important for trace fear conditioning (Runyan et al. 2004; Gilmartin and McEchron 2005), which provides a powerful model system for studying the neurobiological basis of prefrontal contributions to memory. Trace fear conditioning is a variant of standard “delay” fear conditioning in which a neutral conditional stimulus (CS) is paired with an aversive unconditional stimulus (UCS). Trace conditioning differs from delay conditioning by the addition of a stimulus-free “trace” interval of several seconds separating the CS and UCS. Learning the CS–UCS association across this interval requires forebrain structures such as the hippocampus and mPFC. Importantly, the mPFC and hippocampus are only necessary for learning when a trace interval separates the stimuli (Solomon et al. 1986; Kronforst-Collins and Disterhoft 1998; McEchron et al. 1998; Takehara-Nishiuchi et al. 2005). This forebrain dependence has led to the hypothesis that neural activity in these structures is necessary to bridge the CS–UCS temporal gap. In support of this hypothesis, single neurons recorded from the prelimbic area of the rat mPFC exhibit sustained increases in firing during the CS and trace interval in trace fear conditioning (Baeg et al. 2001; Gilmartin and McEchron 2005). Similar sustained responses are not observed following the CS in delay conditioned animals or unpaired control animals. This pattern of activity is consistent with a working memory or “bridging” role for mPFC in trace fear conditioning, but it is not clear whether this activity is actually necessary for learning. We address this issue here using the γ-aminobutyric acid type A (GABA_A) receptor agonist muscimol to temporarily inactivate cellular activity in the prelimbic mPFC during the acquisition of trace fear conditioning.The contribution of mPFC to the long-term storage (i.e., 24 h or more) of trace fear conditioning, as opposed to a strictly working memory role (i.e., seconds to minutes), is a matter of some debate. Recent reports suggest that intact prefrontal activity at the time of testing is required for the recall of trace fear conditioning 2 d after training (Blum et al. 2006a), while mPFC lesions performed 1 d after training fail to disrupt the memory (Quinn et al. 2008). The findings from the former study may reflect a role for prelimbic mPFC in the expression of conditional fear rather than memory storage per se (Corcoran and Quirk 2007). However, blockade of the intracellular mitogen-activated protein kinase (MAPK) cascade during training impairs the subsequent retention of trace fear conditioning 48 h later (Runyan et al. 2004). Activation of the MAPK signaling cascade can result in the synthesis of proteins necessary for synaptic strengthening, providing a potential mechanism by which mPFC may participate in memory storage. To better understand the nature of the prefrontal contribution to long-term memory, more information is needed about fundamental plasticity mechanisms in this structure. Dependence on N-methyl-d-aspartate receptors (NMDAR) is a key feature of many forms of long-term memory, both in vitro and in vivo. The induction of long-term potentiation (LTP) in the hippocampus, a cellular model of long-term plasticity and information storage, requires NMDAR activation (Reymann et al. 1989). Genetic knockdown or pharmacological blockade of NMDAR-mediated neurotransmission in the hippocampus impairs several forms of hippocampus-dependent memory, including trace fear conditioning (Tonegawa et al. 1996; Huerta et al. 2000; Quinn et al. 2005), but it is unknown if activation of these receptors is necessary in the mPFC for the acquisition of trace fear conditioning. Data from in vivo electrophysiology studies have shown that stimulation of ventral hippocampal inputs to prelimbic neurons in mPFC produces LTP, and the induction of prefrontal LTP depends upon functional NMDARs (Laroche et al. 1990; Jay et al. 1995). If the role of mPFC in trace fear conditioning goes beyond simply maintaining CS information in working memory, then activation of NMDAR may be critical to memory formation. We test this hypothesis by reversibly blocking NMDAR neurotransmission with 2-amino-5-phosphonovaleric acid (APV) during training to examine the role of prefrontal NMDAR to the acquisition of trace fear conditioning.Another important question is whether mPFC contributes to the formation of contextual fear memories. Fear to the training context is acquired simultaneously with fear to the auditory CS in both trace and delay fear conditioning. Conflicting reports in the literature suggest the role of mPFC in contextual fear conditioning is unclear. Damage to ventral areas of mPFC prior to delay fear conditioning has failed to impair context fear acquisition (Morgan et al. 1993). Prefrontal lesions incorporating dorsal mPFC have in some cases been reported to augment fear responses to the context (Morgan and LeDoux 1995), while blockade of NMDAR transmission has impaired contextual fear conditioning (Zhao et al. 2005). Post-training lesions of mPFC impair context fear retention (Quinn et al. 2008) in trace and delay conditioning. Contextual fear responses were assessed in this study to determine the contribution of neuronal activity and NMDAR-mediated signaling in mPFC to the acquisition of contextual fear conditioning.     

Distinct roles for dorsal CA3 and CA1 in memory for sequential nonspatial events

Previous studies have suggested that dorsal hippocampal areas CA3 and CA1 are both involved in representing sequences of events that compose unique episodes. However, it is uncertain whether the contribution of CA3 is restricted to spatial information, and it is unclear whether CA1 encodes order per se or contributes by an active maintenance of memories of sequential events. Here, we developed a new behavioral task that examines memory for the order of sequential nonspatial events presented as trial-unique odor pairings. When the interval between odors within a studied pair was brief (3 sec), bilateral dorsal CA3 lesions severely disrupted memory for their order, whereas dorsal CA1 lesions did not affect performance. However, when the inter-item interval was extended to 10 sec, CA1 lesions, as well as CA3 lesions, severely disrupted performance. These findings suggest that the role of CA3 in sequence memory is not limited to spatial information, but rather appears to be a fundamental property of CA3 function. In contrast, CA1 becomes involved when memories for events must be held or sequenced over long intervals. Thus, CA3 and CA1 are both involved in memory for sequential nonspatial events that compose unique experiences, and these areas play different roles that are distinguished by the duration of time that must be bridged between key events.Episodic memory involves the ability to encode and retrieve the order of events in individual experiences (Tulving 1983). Recent evidence in both animals and humans indicates that the hippocampus plays a critical role in this capacity. In animals, damage to the hippocampus impairs memory for the order of associated elements that compose an episode (Fortin et al. 2002; Kesner et al. 2002), and hippocampal neuronal activity reflects processing of the order of events in both spatial (Dragoi and Buzsáki 2006; Foster and Wilson 2007) and nonspatial episodes (Manns et al. 2007). In humans, hippocampal activation has also been related to memory for the order of elements (Kumaran and Maguire 2006; Lehn et al. 2009; Ross et al. 2009).Within the hippocampal circuitry, contributions of the CA3 and CA1 fields are probably most extensively studied, but this work has not yet clarified the distinct roles of these areas in sequence memory. Computational models suggest that the recurrent connections of CA3 cells operate as an attractor network that computes associations between elements (Norman and O''Reilly 2003; Rolls 2007) and is suitable for representing sequences of events in episodic memories (Jensen and Lisman 1996; Levy 1996; Lisman 1999). Studies on the effects of selective damage within the hippocampus have shown that CA3 is critical for remembering sequences of spatial locations (Hunsaker et al. 2008a), but not sequences of nonspatial events (Hoge and Kesner 2007). It is, therefore, uncertain whether CA3 is critical for sequence memory per se, rather than other aspects of spatial processing. Other observations suggest that CA1 may be involved in memory for the order of both spatial (Hunsaker et al. 2008a) and nonspatial stimuli (Hoge and Kesner 2007; Manns et al. 2007). However, it is not clear whether the contribution of CA1 involves integrating sequential elements of a memory or instead participates by active maintenance of event memories that underlies bridging sequential events in an episode (Kesner et al. 2005).To shed light on these issues, we compared the effects of selective damage to CA3 and CA1 on memory for the order of nonspatial events that occurred in unique episodes. We designed a task, based on the delayed-nonmatching-to-sample test, wherein subjects were required to remember the order of two sequentially presented stimuli in trial–unique-paired associations (Fig. 1).Open in a separate windowFigure 1.Test of memory for the order of stimuli in trial-unique odor pairs. At study, animals were presented with 10 odor-paired associates and odors in a pair were presented one at a time. At test, animals were presented with the same 10 odor pairs and were required to distinguish pairs where the odors within a pair were presented in the same order as during study (“old”) from pairs where the odors were presented in the reverse order (“new”). Old and new order test pairs were presented in a pseudorandom order. The first odor in each test pair acted as a cue to the ordering of the odors within a test pair; the animal was required to place its nose over the cup, but no digging response was required or rewarded. When the second cup was presented, the animal could dig to retrieve a reward if the order was new. If the order was old, the animal was required to approach an empty cup in the back of the home cage to obtain reward.     

Cocaine self-administration alters the relative effectiveness of multiple memory systems during extinction

Rats were trained to run a straight-alley maze for an oral cocaine or sucrose vehicle solution reward, followed by either response or latent extinction training procedures that engage neuroanatomically dissociable “habit” and “cognitive” memory systems, respectively. In the response extinction condition, rats performed a runway approach response to an empty fluid well. In the latent extinction condition, rats were placed at the empty fluid well without performing a runway approach response. Rats trained with the sucrose solution displayed normal extinction behavior in both conditions. In contrast, rats trained with the cocaine solution showed normal response extinction but impaired latent extinction. The selective impairment of latent extinction indicates that oral cocaine self-administration alters the relative effectiveness of multiple memory systems during subsequent extinction training.Understanding the psychological and neural mechanisms underlying the acquisition and extinction of drug-seeking behavior has important implications for therapies targeting drug addiction. A better understanding of the neurobiology of extinction can potentially allow for the development of treatments to produce more effective and persistent extinction learning. Dissociable hippocampus-dependent “cognitive” and dorsal striatal-dependent “habit” memory systems are engaged during the initial acquisition of learned behavior (for reviews, see Packard and Knowlton 2002; White and McDonald 2002; Squire 2004). Interestingly, recent evidence indicates that multiple memory systems can also be engaged during the new learning that occurs during behavioral extinction (Gabriele and Packard 2006). For example, the behavior of a rat trained to traverse a straight-alley runway for a food reward can be extinguished using either habit/response or cognitive/latent extinction training procedures. During response extinction, rats are allowed to perform the runway approach response to an empty food cup. In contrast, during latent extinction, rats are placed at the empty food cup without performing the runway approach response. Consistent with evidence indicating a selective role for the hippocampus in cognitive memory, neural inactivation of this brain structure impairs latent extinction and spares response extinction (Gabriele and Packard 2006). Moreover, consistent with evidence that the dorsal striatum selectively mediates habit memory (for review, see Packard and Knowlton 2002), neural inactivation of this brain region impairs response extinction and spares latent extinction (A. Gabriele and M.G. Packard, unpubl.).The transition from initial drug use to eventual addiction may involve, at least in part, the development of compulsive drug-seeking and drug-taking behaviors that are increasingly guided by dorsal striatal-dependent habit learning mechanisms (for reviews, see White 1996; Everitt et al. 2001; Everitt and Robbins 2005; Belin et al. 2008). This hypothesis raises the possibility that once “habit-like” drug-seeking behaviors are firmly acquired, the extinction of such behaviors may be differentially influenced by engaging habit and cognitive memory systems. In the present study, we examined this idea by comparing the relative effectiveness of response and latent extinction training procedures in rats trained to run a straight-alley maze for an oral cocaine reward. Consistent with criteria considered important for demonstrating drug dependence, oral cocaine self-administration produces withdrawal following forced abstinence (Barros and Miczek 1996) and additionally is resistant to reinforcer devaluation (Miles et al. 2003), indicating that this behavior becomes divorced from its consequences in a manner similar to the dorsal striatum-mediated compulsive drug-seeking behavior that may characterize addiction (for reviews, see White 1996; Everitt et al. 2001; Everitt and Robbins 2005; Belin et al. 2008).The apparatus was an elevated (86.4 cm) straight-alley maze with a black Plexiglas floor and clear Plexiglas sides (117.8 cm long, 11.4 cm wide, and 20.3 cm tall). A fluid cup (2.5-cm diameter) was located at the goal end of the maze. The maze was located in a room containing several extra-maze cues.Subjects were 32 adult male Long-Evans rats (275–300 g). Rats were individually housed on a 12:12-h light–dark cycle, with lights on from 8:00 a.m.–8:00 p.m. All animals received food ad libitum.During all behavioral procedures, water bottles were removed from home cages 24 h prior to training, and animals received 15 min/day access to water following each day''s procedures. Training began with 3 d of habituation to the solution to be used during training (cocaine–sucrose [0.1% cocaine HCl/20% sucrose in ddH₂0] or sucrose [20% in ddH₂0] alone). Each habituation day involved presentations of 0.5 mL of the solution in a novel environment consisting of a half-white, half-black box (41.9 cm long, 31.8 cm wide, 35.6 tall) with the fluid cup located in the center of the black side. The number of presentations increased with each habituation day (1, 2, and 4). Each individual presentation had a maximum time of 20 min, and rats were removed when the solution was consumed. Volume consumed and amount of time to consume the solution were recorded for each rat. Each sucrose rat was matched to a cocaine rat to ensure that there were no differences between groups in terms of volume of solution consumed prior to training. For each matched pair, the volume consumed by the rat receiving the cocaine solution during each presentation was measured, and an identical amount was made available to the matched sucrose animal. If, during any given presentation, the cocaine animal did not consume any solution, then the matched sucrose animal received 20 min in the habituation environment with no solution present.Behavioral procedures were similar to those of our previous study using food reward (Gabriele and Packard 2006). During maze training, animals received either the cocaine–sucrose solution or sucrose vehicle solution reward. On days 1–10 of solution-rewarded maze training (six trials per day), rats were placed in the start end and allowed to traverse the maze and drink the available reward solution (0.5 mL). Upon consuming the solution, rats were removed from the maze and placed in an opaque holding box adjacent to the maze for a 30-sec intertrial interval. On each trial, the latency (in seconds) to reach the fluid cup was recorded and used as the measure of task acquisition. If a rat failed to reach the fluid cup within 60 sec, it was removed for the intertrial interval and a latency of 60 sec was recorded.Twenty-four hours following the completion of training (i.e., day 11), rats were assigned to one of two extinction conditions; latent extinction (n = 18, 10 cocaine and eight sucrose) or response extinction (n = 14, seven cocaine and seven sucrose). For both the latent and response conditions, extinction training was administered over 3 d (six trials per day, 30-sec intertrial interval) with no reward solution present. In the latent extinction condition, rats were placed facing the empty fluid cup in the goal end of the maze and were confined for 60 sec by placement of a clear Plexiglas barrier 20 cm from the rear wall of the goal end of the maze. Following confinement, rats were removed from the maze and placed in the holding box for the 30-sec intertrial interval. In the response extinction condition, rats were placed into the start end of the maze as during training and allowed to run to an empty fluid cup at the goal end of the maze. Upon reaching the empty fluid cup and being allowed to discover its emptiness (or after 60 sec if the rat did not reach the reward cup), rats were removed from the maze and placed in the holding box for the 30-sec intertrial interval. Latency to reach the fluid cup was recorded and used as the measure of extinction behavior. On day 3 of extinction, 90 min following the sixth daily extinction trial, all rats were given an additional four extinction “probe” trials in which they were placed in the start end of the maze and latency to reach the empty fluid cup was recorded. These four trials allowed for an assessment of the effectiveness of each extinction procedure.Data from the runway acquisition sessions are presented in Figure 1. A two-way one-repeated-measure ANOVA (Group [cocaine vs. sucrose] × Session) comparing the latencies to reach the fluid cup during acquisition in rats that subsequently received latent extinction revealed a significant effect of Session (F_(9,16) = 61.03, P < 0.001), indicating that latency to reach the fluid cup during acquisition decreased across sessions. However, the absence of a main effect of Group (F_(1,16) = 1.94, n.s.) or interaction between Group and Session (F_(9,16) = 0.53, n.s.) indicates that rats trained to run for cocaine and sucrose acquired the task at similar rates (Fig. 1A). Similar results were observed in rats that subsequently received response extinction (Fig. 1B) in that there was a main effect of Session (F_(9,12) = 13.11, P < 0.001) but no main effect (F_(1,12) = 0.44, n.s.) or interaction (F_(9,12) = 1.50, n.s.) involving drug Group.Open in a separate windowFigure 1.Acquisition of maze runway behavior. (A) Acquisition of maze runway behavior by rats that subsequently received latent extinction. (B) Acquisition of maze runway behavior by rats that subsequently received response extinction. Mean ± SEM of latency (in seconds) to reach the solution cup over training days. For both extinction conditions, there were no group differences in the initial acquisition of runway behavior.The effects of oral cocaine self-administration on latent and response extinction are shown in Figure 2. A two-way ANOVA (Group × Extinction condition) comparing mean runway latencies (collapsed across the four probe trials) for each group revealed a significant main effect of Extinction condition (F_(1,28) = 32.440, P < 0.001), indicating that the response extinction procedures produced greater extinction of the runway response, and a significant interaction effect between Extinction condition and Group (F_(1,28) = 4.813, P < 0.05) but no effect of Group (F_(1,28) = 0.96, n.s.). Simple effects tests showed a significant effect of Group within the latent extinction condition (F_(1,16) = 5.688, P < 0.05) but not the response extinction condition (F_(1,12) = 0.663, n.s.), indicating that oral cocaine self-administration selectively impaired latent but not response extinction. Additionally, a two-way one-repeated-measure ANOVA (Group × Trial) computed on the latencies to reach the fluid cup during response extinction training revealed a main effect of Trial (F_(2,12) = 16.44, P < 0.001), but no significant main effect (F_(1,12) = 2.27, n.s.) or interaction (F_(2,12) = 0.88, n.s.) involving Group, further indicating that oral cocaine did not impair response extinction.Open in a separate windowFigure 2.Effects of oral cocaine self-administration on extinction. The effect of oral cocaine self-administration on runway latent and response extinction. Mean ± SEM latency (in seconds) to reach the fluid cup is shown over the four extinction probe trials. Oral cocaine self-administration impaired latent extinction, but did not impair response extinction.The present experiments investigated the effect of oral cocaine self-administration on response and latent extinction in a straight-alley maze. Following training, rats in the response extinction condition performed the approach response to an empty goal box, whereas rats in the latent extinction condition were placed in the goal box with no reward present. Consistent with previous studies using food reward (e.g., Seward and Levy 1949; Gabriele and Packard 2006), rats rewarded with a sucrose solution were able to extinguish the approach response following both response and latent extinction procedures. In contrast, rats rewarded with a cocaine solution displayed normal response extinction (see also Schoenbaum and Setlow 2005) but impaired latent extinction. The selective impairing effect of oral cocaine self-administration on latent extinction indicates that the drug does not impair processes that contribute to general maze behavior (e.g., motivational, motor, or sensory processes), as any such influence would also likely produce a deficit in response extinction.Previous findings indicate that latent extinction of runway behavior is hippocampus dependent, whereas response extinction is dorsal striatal dependent (Gabriele and Packard 2006; A. Gabriele and M.G. Packard, unpubl.). In view of evidence that the hippocampus and dorsal striatum mediate cognitive and habit learning mechanisms, respectively (for reviews, see Packard and Knowlton 2002; White and McDonald 2002; Squire 2004), the findings suggest that oral cocaine self-administration can affect the relative use of multiple memory systems during extinction learning. The medial prefrontal cortex and basolateral amygdala have been implicated in extinction of several forms of learned behavior, and prior cocaine exposure can impair some forms of extinction learning (Burke et al. 2006; Peters et al. 2008; Quirk and Mueller 2008). However, neural inactivation of medial prefrontal cortex or basolateral amygdala does not affect latent extinction of maze runway behavior (A. Gabriele and M.G. Packard, unpubl.), suggesting that cocaine-induced dysfunction of these structures does not account for the results observed here.One explanation of the cocaine-induced impairment of latent extinction is that the approach response acquired during task acquisition is guided by a supra-normal stimulus-response habit, thereby rendering cognitive learning mechanisms ineffectual during latent extinction training. Consistent with this possibility, drug-seeking behaviors underlying addiction may involve, at least in part, a transition from goal-directed behaviors to habitual behaviors that characterize the function of the dorsal striatal memory system (e.g., Tiffany 1990; White 1996; Packard 1999; Everitt et al. 2001; Porrino et al. 2004; Everitt and Robbins 2005; Belin et al. 2008). Indeed, recent evidence implicates the dorsal striatum in habitual drug-seeking behaviors. For example, intradorsal striatum administration of dopamine antagonists impairs cocaine seeking (Vanderschuren et al. 2005), and inactivation of the dorsal striatum attenuates drug seeking, following both abstinence and extinction (Fuchs et al. 2006; See et al. 2007). Interestingly, disconnection between the ventral and dorsolateral striatum impairs cocaine-seeking behavior (Belin and Everitt 2008), and extended cocaine use enhanced cue-selective firing in the dorsal striatum and reduced cue-selective firing in the ventral striatum in go/no go discrimination learning, indicating an accelerated shift to dorsolateral striatal control (Takahashi et al. 2007). In addition, dopamine release increases in the dorsal striatum of rats following presentation of a response-contingent cue associated with cocaine (Ito et al. 2002). Similar results from fMRI and PET studies of human cocaine addicts showed increased activation in the dorsal striatum (Garavan et al. 2000) and an increase in dopamine release within the dorsal striatum (Volkow et al. 2006) following cue-induced cravings.A second explanation of the cocaine-induced impairment in latent extinction is that drug intake during task acquisition may have affected hippocampal physiology in a manner that negatively impacted the hippocampus-dependent learning that subsequently mediates latent extinction. Consistent with this possibility, chronic cocaine exposure impairs subsequent performance of hippocampus-dependent tasks such as the Morris water maze and the win-shift radial arm maze task (Melnick et al. 2001; Quirk et al. 2001; Mendez et al. 2008). However, it should be noted that the impairments observed in the latter studies were observed following exposure to cocaine doses considerably higher than those used in the present oral self-administration study. Since the current experiments do not explicitly examine the potential neurobiological progression underlying the acquisition of runway responding, further research is necessary to determine whether the cocaine-induced impairment of latent extinction involves the interfering effect of a supra-normal response habit, or a direct impairing effect on hippocampal physiology. It should also be noted that both oral cocaine self-administration and a passive cocaine administration regimen produce results analogous to those presented here, in that they impair “cognitive” representations of rewards (Miles et al. 2003; Schoenbaum and Setlow 2005). However, the relationship between this type of cognitive reward representation (mediated by interactions between basolateral amygdala and orbitofrontal cortex) (Pickens et al. 2003) and cognitive representations in latent extinction mediated by the hippocampus (Gabriele and Packard 2006) is currently unclear.Finally, the selective impairing effect of cocaine self-administration on latent extinction may have implications for understanding the persistent ability of drug-predictive cues and contexts to compel drug-seeking behavior and relapse. Specifically, if the ability to use cognitive learning mechanisms to extinguish drug-seeking behaviors is impaired following the transition from initial to habitual and compulsive drug use, then contextual/relational cues might be expected to maintain greater control over behavior following extinction training. This in turn might suggest that incorporation of response extinction procedures into treatment strategies might provide greater therapeutic efficacy.     

Intrahippocampal infusions of anisomycin produce amnesia: Contribution of increased release of norepinephrine,dopamine, and acetylcholine

Intra-amygdala injections of anisomycin produce large increases in the release of norepinephrine (NE), dopamine (DA), and serotonin in the amygdala. Pretreatment with intra-amygdala injections of the β-adrenergic receptor antagonist propranolol attenuates anisomycin-induced amnesia without reversing the inhibition of protein synthesis, and injections of NE alone produce amnesia. These findings suggest that abnormal neurotransmitter responses may be the basis for amnesia produced by inhibition of protein synthesis. The present experiment extends these findings to the hippocampus and adds acetylcholine (ACh) to the list of neurotransmitters affected by anisomycin. Using in vivo microdialysis at the site of injection, release of NE, DA, and ACh was measured before and after injections of anisomycin into the hippocampus. Anisomycin impaired inhibitory avoidance memory when rats were tested 48 h after training and also produced substantial increases in local release of NE, DA, and ACh. In an additional experiment, pretreatment with intrahippocampal injections of propranolol prior to anisomycin and training significantly attenuated anisomycin-induced amnesia. The disruption of neurotransmitter release patterns at the site of injection appears to contribute significantly to the mechanisms underlying amnesia produced by protein synthesis inhibitors, calling into question the dominant interpretation that the amnesia reflects loss of training-initiated protein synthesis necessary for memory formation. Instead, the findings suggest that proteins needed for memory formation are available prior to an experience, and that post-translational modifications of these proteins may be sufficient to enable the formation of new memories.A dominant view of the molecular basis for memory is that the formation of long-term memory for an experience depends on de novo protein synthesis initiated by that experience (Davis and Squire 1984; Frey and Morris 1998; Kandel 2001; Dudai 2002; Nader 2003; Alberini 2008). This view is supported by numerous studies showing that drugs that interfere with protein synthesis by inhibiting translational processes near the time of training produce later amnesia.Despite the centrality of experience-induced protein synthesis in contemporary models of memory formation, the necessity of protein synthesis for memory consolidation and long-term potentiation (LTP) stabilization has been questioned since the beginning of experiments of this type (e.g., Flexner and Goodman 1975; Barraco and Stettner 1976; Flood et al. 1978; Martinez et al. 1981), and continues to be questioned in several recent reviews (Routtenberg and Rekart 2005; Gold 2006, 2008; Radulovic and Tronson 2008; Routtenberg 2008; Rudy 2008). There are many instances of intact memories formed in the presence of extensive inhibition of protein synthesis, and a wide range of behavioral and pharmacological manipulations can rescue memory impaired by protein synthesis inhibitors. For example, amnesia is attenuated in a graded manner by increasing the training trials and foot shock intensity in avoidance tasks (Flood et al. 1975, 1978). Moreover, a wide range of stimulants, such as amphetamine, strychnine, corticosteroids, and caffeine, block amnesia induced by anisomycin (Flood et al. 1978). Like memory, LTP is sometimes insensitive to protein synthesis inhibitors. Simultaneous inhibition of both protein synthesis and degradation does not interfere with induction and maintenance of LTP (Fonseca et al. 2006a). Also, the specific schedule and frequency of test pulses after induction of LTP determine the vulnerability of LTP to anisomycin-induced impairment; anisomycin treatment does not impair LTP unless test pulses at a rate of 1/10 sec were administered during the anisomycin exposure (Fonseca et al. 2006b).Findings that memory and LTP can survive the inhibition of protein synthesis challenge the necessity of specific training- or stimulation-initiated protein synthesis for memory formation and synaptic plasticity. Several actions of protein synthesis inhibitors offer alternative accounts for amnesia produced by these drugs. These include cell sickness (Rudy et al. 2006; Rudy 2008), activation of protein kinases and superinduction of immediate early genes (Radulovic and Tronson 2008), abnormal neural electrical activity (Agnihotri et al. 2004; Xu et al. 2005), and intrusion of neural “noise” that masks the primary changes representing memory formation (Gold 2006). Neural responses to inhibition of protein synthesis such as these may impair memory either secondary to or independent of interference with protein synthesis.Another example of the mechanisms by which inhibition of protein synthesis might impair memory is by altering neurotransmitter functions. This possibility was suggested in early studies (e.g., Flexner and Goodman 1975; Quartermain et al. 1977) and has recently been supported by studies of neurotransmitter release at the site of intra-amygdala injections of anisomycin (Canal et al. 2007). In addition to impairing later memory after inhibitory avoidance training, pretraining injections of anisomycin into the amygdala produced rapid and dramatic increases in release of norepinephrine (NE), dopamine (DA), and serotonin (5-HT) at the sites of injection. The release of NE and DA then plummeted below baselines from 2 to 6 h after anisomycin injections, recovering within 48 h after anisomycin injection. The possibility that these neurochemical changes contribute to anisomycin-induced amnesia was supported by studies showing attenuation of amnesia in rats pretreated with intra-amygdala injections of the β-adrenergic receptor antagonist propranolol, apparently acting to blunt the effects of the large increases in release of NE after anisomycin injection. In addition, amnesia was produced by injections of high doses of norepinephrine into the amygdala.In addition to amnesias produced by anisomycin injections into the amygdala, as above, anisomycin also impairs memory when administered to other memory systems, including the hippocampus, where anisomycin impairs inhibitory avoidance memory (Quevedo et al. 1999; Debiec et al. 2002; Milekic et al. 2006). The present study extends the prior findings (Canal et al. 2007) in several respects. Experiments presented here determine whether anisomycin injections into the hippocampus result in changes in release of the catecholamines, NE and DA, at the site of injection, as seen previously in the amygdala. Additionally, the present experiments determine whether intrahippocampal injections of anisomycin result in increased release of acetylcholine, a neurotransmitter not examined in the previous study. To examine parallels with earlier amygdala findings, a further experiment determines whether intrahippocampal pretreatment with propranolol is effective in attenuating anisomycin-induced amnesia.     

Perirhinal cortex is necessary for acquiring,but not for retrieving object–place paired association

Remembering events frequently involves associating objects and their associated locations in space, and it has been implicated that the areas associated with the hippocampus are important in this function. The current study examined the role of the perirhinal cortex in retrieving familiar object–place paired associates, as well as in acquiring novel ones. Rats were required to visit one of two locations of a radial-arm maze and choose one of the objects (from a pair of different toy objects) exclusively associated with a given arm. Excitotoxic lesions of the perirhinal cortex initially impaired the normal retrieval of object–place paired-associative memories that had been learned presurgically, but the animals relearned gradually to the level of controls. In contrast, when required to associate a novel pair of objects with the same locations of the maze, the same lesioned rats were severely impaired with minimal learning, if any, taking place throughout an extensive testing period. However, the lesioned rats were normal in discriminating two different objects presented in a fixed arm in the maze. The results suggest that the perirhinal cortex is indispensable to forming discrete representations for object–place paired associates. Its role, however, may be compensated for by other structures when familiar object–place paired associative memories need to be retrieved.Remembering an event in space often requires associating objects and their locations. Associating object and place information into a unitary event representation is believed to be a foundation of episodic memory (Cahusac et al. 1989; Gaffan 1994; Davachi 2006). It has been suggested that the hippocampus and its associated regions in the medial temporal lobe (MTL) are essential in this cognitive process, and amnesic patients with damage in the MTL structures exhibit severe deficits in associating object and place information (Smith and Milner 1981; Vargha-Khadem et al. 1997; Stepankova et al. 2004). Animal models produced by localized lesions in the hippocampus and other MTL structures also support the idea by showing that the lesioned animals are impaired in associating objects and places (Parkinson et al. 1988; Gaffan and Parker 1996; Sziklas et al. 1998; Bussey et al. 2001; Gilbert and Kesner 2003, 2004; Malkova and Mishkin 2003; Lee et al. 2005; Bachevalier and Nemanic 2008; Kesner et al. 2008; Lee and Solivan 2008). Although the theoretical importance of the MTL structures in object–place association has been well acknowledged, specific contributions of the MTL structures in object–place associative memory are poorly understood. The current study examined the role of the perirhinal cortex, one of the extra hippocampal regions in the MTL, using a behavioral paradigm previously shown to be dependent on the intact hippocampus (Lee and Solivan 2008).The literature suggests that the role of the hippocampus in the object–place paired-associate task is to put together object and place information into a unified and distinct event representation. It has been suggested that spatial information and nonspatial information (such as object information) may be streamed into the hippocampus in a relatively segregated fashion, the former information mostly fed through the medial entorhinal cortex to the hippocampus via the postrhinal cortex and the latter being fed through the lateral entorhinal cortex via the perirhinal cortex (Mishkin et al. 1997; Suzuki et al. 1997; Burwell 2000; Fyhn et al. 2004; Witter and Amaral 2004; Hafting et al. 2005; Hargreaves et al. 2005; Furtak et al. 2007; Kerr et al. 2007). In our previous study (Lee and Solivan 2008) in which rats were required to discriminate rewarding versus nonrewarding pairs of similar object–place paired associates, the hippocampal lesioned rats demonstrated severe and irrecoverable deficits. The results from the study not only corroborate the long-held view that the hippocampus associates object and place information, but also demonstrate that the hippocampus is critical for disambiguating similar object–place paired associates. However, it requires examining functions of other upstream structures of the hippocampus to conclusively assign the role of associating object and place information to the hippocampus. If, for example, lesions produced in the perirhinal cortex produce similar deficits, it would be premature to conclude that the association between object and place information uniquely occurs in the hippocampus.To elucidate the relative contributions of the MTL structures in the hippocampal-dependent object–place paired-associate task (Fig. 1), we manipulated the perirhinal cortex in the current study, one of the regions implicated as an object-information provider to the hippocampus (Knierim et al. 2006; Eichenbaum and Lipton 2008). Here we tested whether the perirhinal cortex was involved in the acquisition of new object–place paired associations. Importantly, we also tested the perirhinal cortical contributions to retrieving learned paired associates between objects and places. In the current study, the rats needed to pay attention to both object and place information. Therefore, if the perirhinal cortex is unique in its function for providing object information to the hippocampus, it is predicted that lesions in the perirhinal cortex will produce severe deficits as seen in the hippocampal lesioned animals in our previous study. A simple object-discrimination task that did not require spatial information was also employed to further examine the role of the perirhinal cortex only in specific conditions.Open in a separate windowFigure 1.Illustration of the radial arm maze and behavioral paradigms. (A) Phase 1: Two objects (Spider-Man and LEGO block) were presented on arms 3 and 5 in gray color. Only one of the objects was rewarded in arm 3 (Spider-Man) and arm 5 (LEGO block) irrespective of its locations in the choice platform. Possible configuration of objects and appropriate choices are provided for both arms. In each trial, only one arm was open in the maze and objects were available in that open arm. (B) Phase 2: For acquisition of novel object–place paired associations, a pair of new objects (Barney and Girl) was presented on arms 3 and 5. Possible locations of the objects are shown as in A. Each object was rewarded only in a particular arm (Barney in arm 3 and Girl in arm 5) irrespective of its location in the choice platform. (C) Phase 3: Illustration of the task using only one arm (arm 4) in the maze. Two new objects (Mr. Potatohead and Cylinder) were used and the Mr. Potatohead choice was rewarded regardless of its location in the choice platform.     

D-cycloserine potentiates the reconsolidation of cocaine-associated memories

Conditioned cue-induced relapse to drug seeking is a major challenge to the treatment of drug addiction. It has been proposed that D-cycloserine might be useful in the prevention of relapse by reducing the conditioned reinforcing properties of drug-associated stimuli through facilitation of extinction. Here we show that intrabasolateral amygdala infusions of D-cycloserine in fact potentiate the reconsolidation of stimulus–cocaine memories to increase cue-induced relapse to drug seeking in rats with an extensive drug self-administration history. This elevation of cocaine seeking was correlated with an increase in the expression of the reconsolidation-associated gene zif268.Drug addiction is often described as a chronic relapsing disorder, in which craving and relapse to drug seeking occur even after prolonged abstinence (Gawin and Kleber 1986; Lu et al. 2004). A major contributor to relapse is exposure to environmental stimuli that have previously been associated regularly with the effects of self-administered drugs of abuse. Such drug-associated stimuli induce craving in abstinent addicts, and precipitate relapse to drug seeking (Gawin and Kleber 1986; Ehrman et al. 1992; O''Brien et al. 1998; Childress et al. 1999). In experimental animals, a conditioned stimulus (CS) paired repeatedly with self-administered cocaine similarly induces relapse to drug seeking (de Wit and Stewart 1981; Fuchs et al. 1998; Grimm et al. 2001).The basolateral amygdala (BLA) is a primary locus of CS–US (unconditioned stimulus) associations (LeDoux 2000; Everitt et al. 2003), and is critical for the control of goal-directed behavior by conditioned reinforcers (Burns et al. 1993; Corbit and Balleine 2005). Furthermore, lesions of the BLA disrupt conditioned cue-induced reinstatement of drug seeking (Meil and See 1997) and the acquisition of drug seeking under a second-order schedule of reinforcement (Whitelaw et al. 1996). The infusion of antisense oligodeoxynucleotides targeting the immediate-early gene zif268 (also known as EGR1, Krox24, and NGFI-A) into the BLA impairs the reconsolidation of CS–cocaine associations and thereby reduces cue-induced cocaine seeking and relapse (Lee et al. 2005, 2006a). Memory reconsolidation is the process that is hypothesized to restabilize memories following their reactivation through stimulus re-exposure (Nader 2003), the disruption of which both results in amnesia and has been proposed to be a potential treatment strategy for neuropsychiatric disorders, such as post-traumatic stress and drug addiction in which persistent maladaptive memories play an important role (Lee et al. 2005; Brunet et al. 2007).An alternative therapeutic approach for such disorders is to use cognitive enhancement strategies to potentiate the extinction of the maladaptive memories (Ressler et al. 2004; Richardson et al. 2004). The partial N-methyl-D-aspartate (NMDA) receptor agonist D-cycloserine (DCS) potentiates extinction in aversive and appetitive tasks when administered both systemically and directly into the BLA (Walker et al. 2002; Ledgerwood et al. 2003; Botreau et al. 2006). However, under certain conditions intra-BLA DCS can increase, rather than reduce, subsequent fear memory expression (Lee et al. 2006b). This finding is consistent with a potentiation of fear memory reconsolidation, and predicts that DCS might also enhance drug memories, which would be a potentially major limitation in a therapeutic setting. Therefore, using conditions that have been previously demonstrated to engage drug memory reconsolidation in a translational model of cocaine seeking (Lee et al. 2006a); we investigated whether or not DCS infusion into the BLA would enhance memory reconsolidation to increase cocaine seeking. Also investigated was the expression of zif268 in the amygdala following DCS treatment and CS re-exposure since this plasticity gene provides a cellular correlate of memory reconsolidation (Lee et al. 2005).The subjects were 45 adult male Lister hooded rats, weighing 280–350 g. They were housed in pairs prior to surgery, and singly thereafter, in holding rooms maintained at 21°C on a reversed-light cycle (12 h light:12 h dark; lights on at 19:00). After recovery from surgery, food was restricted to 20 g/day. Water was freely available throughout the experiment. All procedures were conducted in accordance with the UK 1986 Animals (Scientific Procedures) Act (Project License PPL 80/1767). Rats were implanted with a single catheter in the right jugular vein, and also with bilateral chronic indwelling guide cannulae targeting the BLA. The coordinates for cannulae implantation were (relative to bregma): AP − 2.6; ML ± 4.5; DV − 5.6 (from dura). Details of the intravenous and stereotaxic surgical procedures are described elsewhere (Di Ciano and Everitt 2001). A minimum of 7 d was allowed before behavioral training and testing began.All behavioral procedures were carried out in operant chambers (Med Associates) as previously described (Di Ciano and Everitt 2001), and were based on previous experiments (Lee et al. 2006a). Rats underwent 10 d of cocaine self-administration training under a fixed-ratio-1 (FR1) schedule of reinforcement. At the start of each self-administration session, two levers were inserted into the operant chamber and the house light was illuminated. Each response on the active lever (counterbalanced left or right) was reinforced with a single infusion of i.v. cocaine (0.25 mg in 0.1 mL over 5 s per infusion), accompanied by a 20-s illumination of the CS light located above the active lever, during which both levers were retracted and the house light was extinguished. Responses on the other, inactive, lever had no programmed consequence. To prevent accidental overdose, rats were limited to 30 infusions per hour in the 3-h sessions. In the event of 30 infusions being received, the levers were retracted and the house light extinguished until an hour had elapsed, after which the levers were again extended and the house light illuminated.The memory for the CS–cocaine association was reactivated in a single 30-min session through 30 noncontingent presentations of the CS light (20 s; 40-s ISI). No levers were present during the reactivation session, which took place 3 d after the completion of self-administration training, and 20 min following infusion of DCS (Sigma, UK; 20 μg/μL; 0.5 μL/side; 0.25 μL/min) or phosphate buffered saline (PBS) into the BLA as described previously (Lee et al. 2006b). The absence of the levers should decrease the probability of substantial reactivation of the instrumental memory. Nonreactivated groups were infused with DCS and then returned immediately to the home cage without a behavioral test session.Testing, which took place 6 d after self-administration training, involved the levers again being extended into the operant chamber and assessed the impact of the CS upon instrumental responding after 6-d abstinence as a model of cue-induced relapse. A response on the active lever was reinforced by a 1-s presentation of the CS light, during which the house light was extinguished, and a response on the inactive lever again had no programmed consequence. There were no cocaine infusions, and the number of lever presses was recorded in a 60-min session. The rats were subsequently retested on the following day.After completion of behavioral testing, the rats were perfused and their brains cut to produce 60 μm coronal sections, which were stained with cresyl violet. Assessment was conducted under light microscopy, and subjects were only included in the statistical analysis if the injectors were located bilaterally within the BLA, and there was no bilateral damage to the amygdala or any other area of the brain (5 rats were excluded on this basis). Fourteen rats were killed by carbon dioxide inhalation 2 h after memory reactivation (or at the same time point following infusions and nonreactivation). Their brains were extracted, the BLA microdissected bilaterally, and the samples frozen on dry ice and stored at –80°C prior to the quantification of zif268 protein levels through Western blot analysis as described previously (Lee et al. 2005).All rats included in the behavioral analyses had cannulae placed bilaterally in the BLA (Fig. 1). There was no difference between any of the groups during cocaine self-administration training and on average rats received 505 pairings of the light CS with an infusion of cocaine (data not shown; Condition × DCS: F < 1).Open in a separate windowFigure 1.Location of injectors within the BLA. Schematic representation of the brain at three rostro-caudal levels (−2.30, −2.56, and −2.80 mm from bregma). All rats included in the statistical analyses had injectors placed bilaterally in the BLA (× = PBS reactivated; • = DCS reactivated; □ = PBS nonreactivated; △ = DCS nonreactivated).Infusion of DCS into the BLA before drug cue re-exposure elevated subsequent active lever responding in a reactivation-dependent manner (Fig. 2). For the first test, ANOVA revealed a significant DCS × Reactivation × Lever interaction (F _(1,22) = 8.62; P < 0.01), as well as a significant DCS × Reactivation interaction (F _(1,22) = 4.71; P < 0.05). The effect was specific to the reactivated condition, as DCS infusion had no impact upon the nonreactivated condition (Fs < 1). This was confirmed by a significant DCS × Lever interaction (F _(1,13) = 13.12; P < 0.01) for the reactivated condition, and a significant main effect of DCS (F _(1,13) = 9.66; P < 0.01), but no effect of DCS on inactive lever presses (F < 1). The DCS-induced elevation of responding was again observed 24 h later in test 2 (DCS × Reactivation × Lever: F _(1,22) = 4.44; P < 0.05), indicating a persistent potentiation of responding, and an overall analysis revealed no difference in the effect of DCS between tests 1 and 2 (DCS × Reactivation × Lever: F _(1,13) = 7.41; P < 0.02; DCS × Reactivation × Lever × Test: F _(1,22) = 1.66; P > 0.21).Open in a separate windowFigure 2.Effect of prereactivation intra-BLA DCS on cocaine seeking. The number of active and inactive lever presses during the 60-min tests are presented for rats infused 3 d and tested 6 d after the end of self-administration training (A) in both the reactivated (PBS n = 8, DCS n = 7) and nonreactivated (PBS n = 6, DCS n = 5) conditions. DCS increased subsequent active lever responding across both bins in a reactivation-dependent manner, and the elevation was observed in a further test 24 h later (B). Data presented as mean + SEM. An asterisk (*) represents a significant DCS × Lever interaction, P < 0.05.Infusion of DCS into the BLA before drug cue re-exposure potentiated the subsequent expression in the BLA of zif268 protein levels 2 h after the reactivation session as measured by Western blotting analysis (Fig. 3). ANOVA revealed a significant effect of DCS when infused prior to memory reactivation (F _(1,6) = 9.42; P < 0.03). However, there was no effect of DCS treatment when infused in the absence of memory reactivation (F _(1,4) = 1.41; P > 0.30).Open in a separate windowFigure 3.Effect of prereactivation intra-BLA DCS on zif268 protein levels in the BLA. The images of the gels were quantified and normalized to give a measure of zif268 protein relative to control PBS-infused rats. DCS increased the levels of zif268 protein 2 h after memory reactivation [(A); n = 4 per group], but not at the equivalent time point in the nonreactivated condition [(B); n = 3 per group]. Data presented as mean + SEM.The present results demonstrate that infusion of the partial NMDA receptor agonist DCS into the BLA shortly before re-exposure to a cocaine-associated CS increased subsequent cocaine seeking behavior maintained by that CS. Moreover, the expression of the reconsolidation-associated gene zif268 in the amygdala was also elevated by DCS infusion coupled with CS re-exposure. Both of these effects were critically dependent upon rats being re-exposed to the cocaine CS shortly after DCS infusion. We have previously demonstrated both that paired presentations of CS and reward are necessary for the acquisition of conditioned reinforcing properties (Parkinson et al. 2005), and that re-exposure to contextual and other stimuli are insufficient to reactivate the CS–cocaine memory (Lee et al. 2006a). Therefore, the present results most likely reflect an effect of DCS to potentiate the reconsolidation of the CS–cocaine memory, thereby enhancing the appetitive properties of the CS and increasing cue-induced cocaine seeking.DCS has been shown to potentiate a number of memory plasticity processes, including initial memory acquisition/consolidation (Monahan et al. 1989; Land and Riccio 1999; Kalisch et al. 2008), memory extinction (Walker et al. 2002; Ledgerwood et al. 2003), and memory reconsolidation (Lee et al. 2006b). Moreover, the effect of DCS here was memory reactivation dependent and hence not a result of an acute effect upon behavior. Therefore, the elevation of subsequent cue-induced cocaine seeking reflects an enhancement of CS–cocaine memory expression. The delay of 3 d between the end of self-administration training and DCS infusion ensured that initial consolidation processes were complete, and hence the effect of DCS is more likely be related to memory extinction or reconsolidation, of which only a potentiation of memory reconsolidation can account for the present results. Therefore, the behavioral evidence strongly indicates that DCS infusion into the BLA can potentiate drug memory reconsolidation to elevate subsequent drug seeking, at least under certain circumstances.The cellular data obtained in the present study provides further evidence that DCS elevation of NMDA receptor-mediated glutamate transmission enhances cocaine seeking through the potentiation of drug memory reconsolidation. The expression of the immediate-early gene zif268 has been shown in several settings to be a critical and causal mechanism in memory reconsolidation. The expression of zif268 at both the mRNA and protein levels is upregulated by stimulus re-exposure that induces the reconsolidation of aversive contextual fear (Hall et al. 2001; Lee et al. 2004), discrete cue fear (Hall et al. 2001), and conditioned withdrawal memories (Hellemans et al. 2006), as well as appetitive CS–cocaine associations (Thomas et al. 2003). Moreover, functional reduction of zif268 expression in transgenic mice or through the local intracerebral infusion of zif268 antisense oligodeoxynucleotides impairs the reconsolidation of several types of memory (Bozon et al. 2003; Lee et al. 2004, 2005, 2006a; Hellemans et al. 2006). Of special importance is the observation that zif268 expression in the BLA is correlated with, and necessary for, the reconsolidation of CS–cocaine memories (Thomas et al. 2003; Lee et al. 2005, 2006a), and hence zif268 protein levels in the BLA are a cellular marker for drug memory reconsolidation. Here zif268 protein levels in the BLA were greatly increased by intra-BLA DCS infusion in conjunction with memory reactivation, strongly suggesting that DCS acts to enhance the cellular mechanisms of drug memory reconsolidation, resulting in the observed elevation of drug seeking behavior at a later test. Importantly, this potentiation of zif268 expression was not observed when DCS was infused in the absence of a memory reactivation session, thus demonstrating that the impact of DCS conforms to the reactivation-dependent criterion of memory reconsolidation effects (Lewis 1979; Dudai 2004).Previous studies have demonstrated that treatment with DCS in conjunction with nonreinforced CS re-exposure results in a subsequent decrease in drug-related behavior, consistent with an enhancement of drug memory extinction, leading to the suggestion that DCS might be used in conjunction with cue exposure therapy as a treatment strategy for addiction (Botreau et al. 2006; Kelley et al. 2007). However, these studies used a drug conditioned place preference procedure, involving only four experimenter-administered intraperitoneal injections of cocaine. The present study uses a more translationally relevant model of chronic cocaine self-administration with hundreds of pairings of the CS with intravenous cocaine. Therefore, the effect of DCS to increase subsequent cue-induced cocaine seeking may reflect more accurately the likely outcome of a DCS-based treatment strategy, given the chronic nature of drug exposure characteristic of addiction. Moreover, it may be the case that DCS combined with cue exposure may not be effective in prolonging abstinence and preventing relapse, as this approach may, in fact, further strengthen the detrimental impact of exposure to drug-associated stimuli, making relapse more likely.The different levels of drug memory strength between the conditioned place preference and self-administration studies can explain the contrasting outcomes observed with DCS. The strength of conditioning is an important factor in determining whether memory-modulating treatments impact upon reconsolidation or extinction, with stronger training biasing toward memory reconsolidation (Eisenberg et al. 2003). Therefore, it is not surprising that DCS enhanced drug memory reconsolidation here, while potentiating extinction in the more weakly conditioned place preference studies, especially given that we have previously shown DCS to have bidirectional mnemonic effects in a fear conditioning procedure (Lee et al. 2006b). Future studies will be required to clarify parametrically the impact of memory strength and also the extent of cue exposure upon the effects of DCS. In addition, while the present study focused upon the Pavlovian conditioned reinforcing effects of drug-associated stimuli, due to their importance in relapse, the effect of DCS upon instrumental responding is also of interest. Any impact of DCS upon instrumental memories is likely to be mediated by neural loci beyond the amygdala, and so it will be important to establish how systemic, rather than localized intracerebral, injections of DCS affect the many drug-related memories that contribute to drug seeking behavior. The present results do not invalidate the potential application of DCS in the future treatment of drug addiction. However, they demonstrate clearly that its use must be carefully controlled, as there is the distinct likelihood that memory reconsolidation, rather than extinction, processes will be potentiated, leading to the deleterious consequence of promoting the conditioned elicitation of drug seeking behavior and relapse.     

Pre-exposure to context affects learning strategy selection in mice

The multiple memory systems hypothesis proposes that different types of learning strategies are mediated by distinct neural systems in the brain. Male and female mice were tested on a water plus-maze task that could be solved by either a place or response strategy. One group of mice was pre-exposed to the same context as training and testing (PTC) and the other group was pre-exposed to a different context (PDC). Our results show that the PTC condition biased mice to place strategy use in males, but this bias was dependent on the presence of ovarian hormones in females.The participation of different brain areas in place and response learning strategies has been studied extensively (White and McDonald 2002; Gold 2004; Mizumori et al. 2004). Place strategy is an allocentric navigation strategy that depends on extramaze cues. Response strategy is an egocentric navigation strategy based on proprioceptive cues. Inactivation of the hippocampus biased animals to response strategy use, and inactivation of the striatum biased animals to place strategy use (Packard and McGaugh 1996; Lee et al. 2008). Furthermore, glutamate infusion into the hippocampus strengthened place strategy use and, conversely, glutamate infusion into the striatum enhanced response strategy use (Packard 1999). These studies suggest that the hippocampus system mediates place strategy, while the striatum system mediates response strategy.Various factors can modulate learning strategy use, including training intensity (Packard and McGaugh 1996; Martel et al. 2007). A recent study investigated the influence of training on strategy use on a probe trial conducted 1 h after training (Martel et al. 2007). Male mice displayed enhanced place strategy use when trained on 12 or 22 trials compared with four trials, suggesting an effect of training intensity on strategy choice (Martel et al. 2007). This study further investigated the effect of pre-exposure to the training and testing context (PTC). Pre-exposure enhanced place strategy use in male mice after only four trials relative to animals pre-exposed to a different context (PDC). These results suggest that a sufficient exposure to the training and testing context promotes place strategy use in mice.The type of strategy used by rats is affected by both biological sex and gonadal steroids. Male rats typically employ a place strategy, especially during the early phase of training, on both land and water T-mazes (Packard and McGaugh 1992, 1996; Packard and Teather 1997). However, strategy use by female rats depends on hormonal conditions (Dohanich 2002; Dohanich et al. 2009). Place strategy is preferred by intact female rats on the day of proestrus when estradiol levels are elevated, and by ovariectomized rats treated with estradiol (Korol and Kolo 2002; Korol 2004; Korol et al. 2004). In contrast, response strategy is more often displayed by intact females on diestrus, and by ovariectomized females that did not receive estradiol replacement (Korol and Kolo 2002; Korol et al. 2004). To date, the effects of biological sex and gonadal steroids on learning strategy have not been studied in mice.In this study, we developed a modified version of the dual-solution water plus-maze task to further investigate the role of PTC compared with PDC in male and female mice. We hypothesized that strategy choice in both sexes would be dependent on context pre-exposure, and ovarian hormones would influence strategy choice in females. Our results show that PTC significantly enhanced place strategy use in male mice. Although there was no significant difference between PTC and PDC female mice, ovariectomy significantly reduced place strategy use in the PTC females, suggesting that ovarian hormones play a significant role in strategy use in female mice.Sixteen male and 39 female 129/Sve strain mice were obtained at 2–3 mo of age from Charles River Laboratories (Boston, MA). Mice were housed in groups of four on a 12/12 light/dark cycle (lights on at 07:00 h) with free access to food and water. All protocols followed the guidelines from a protocol approved by the Animal Care and Use Committee of Tulane University in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals.Mice were pre-exposed for 5 min to the dry plus-maze either in the context of the subsequent training and testing (PTC), or in a different context in a different room (PDC), 30 min prior to the first training trial. The maze consisted of four clear Plexiglas arms (40 cm in length, 10 cm in width, and 40 cm in height). During the pre-exposure, mice were able to visit three arms of the maze. The rooms had different visual cues surrounding the maze. No extramaze cues were placed directly at the end of any arm. After the pre-exposure, the animal was placed in its home cage. The maze was wiped clean with 70% ethanol between trials.After pre-exposures, the maze was filled to 1.5 cm above the Plexiglas escape platform (15 cm in height) with room-temperature water colored opaque with white nontoxic tempera paint. Mice were trained in the water plus-maze task (Fig. 1A). The training was ended when the animals made six correct choices or reached nine trials. The animals that made fewer than four correct choices during training were not included in the study. Trials were continued until the mouse reached the platform or a maximum of 1 min. Each trial was separated by an intertrial interval of 4 min. Throughout the training trials, one arm (north) was blocked off by a white Plexiglas shield, creating a T-shaped maze. Mice were placed in the start arm of the maze (south) and were allowed to swim to the escape platform, which was consistently located in one arm of the maze for each animal and alternated between animals (east or west). Entry of the entire animal into the maze arm that contained the escape platform was scored as a correct response during the training trials, and entry of the entire animal into the maze arm that did not contain the escape platform was scored as an incorrect response. Mice were allowed to remain on the escape platform for 15 sec before being returned to their cages. Mice that failed to find the escape platform within 60 sec were manually guided to the platform. The water was distributed across all arms of the maze and the maze walls were wiped down to reduce intramaze cues between training and probe trials. One hour after training, mice were tested on a probe trial (Fig. 1B) in order to determine their relative use of “place” and “response” strategy. On the probe trial, mice were placed into the start arm 180° opposite the start arm used during training (i.e., end of the north arm) and were allowed to make an entry into either the east or west maze arm. The white Plexiglas shield blocked the south arm during the probe trial. Mice were designated as using place or response strategy based on the probe trial. Place strategy was designated as entry of the entire animal into the arm with the platform, and response strategy was designated as entry of the entire animal into the opposite arm.Open in a separate windowFigure 1.The effects of pre-exposure to the training and testing context (PTC) or to a different context (PDC) on strategy selection of male mice. (A) Schematic diagram of the water plus-maze. Mice were released from the south arm during training trials and from the north arm during the probe test. (B) More male mice used place strategy than response strategy when pre-exposed to the same context prior to training and testing (PTC, n = 5) compared with male mice pre-exposed to a different context (PDC, n = 7, P < 0.05). (C) Latency curves show the actual latency to escape to the platform. Two-way ANOVA (non-repeated measures) revealed no significant difference across training trials in escape latencies between PDC and PTC mice (P > 0.5), although a significant effect of trial indicated that mice reduced their escape latencies across trials (P < 0.001). Values represent mean ± S.E.M.Sixteen male mice were randomly divided into two groups based on pre-exposure context, PTC or PDC. Four of the 16 males were not included in the study for failure to reach criterion (four correct out of nine trials) or failure to escape to the platform due to floating, which is a behavior commonly seen in this strain (Wolfer et al. 1997). On the probe trial PTC males used the place strategy significantly more often than PDC males (P < 0.05, χ² = 5.182, Fig. 1B). Four of five PTC males used place strategy, whereas only one of seven PDC males used place strategy. Pre-exposure of animals to the same or different context prior to training did not affect the latency to escape the platform during training. Latency to find the platform during training trials revealed a significant effect of trial (F_(8,89) = 3.830, P = 0.0007, non-repeated measures two-way ANOVA) but not pre-exposure condition (F_(1,89) = 0.103, P = 0.75, non-repeated measures two-way ANOVA; Fig. 1C). Moreover, the average swim speed of PDC male mice (6 ± 1.6 cm/sec, n = 7) was not significantly different than the average swim speed of PTC male mice (6 ± 2.5 cm/sec, n = 5; P = 0.34, t = 0.9 [t-test]). Together, these data suggest that the pre-exposure condition did not influence learning during the training period, but PTC did enhance place strategy use in the probe trial in male mice.Female mice at 3 mo of age were randomly divided into two groups: mice that would receive ovariectomy (Ovx), and a sham surgery group (Sh). Mice were anesthetized with a ketamine (80 mg/kg) and xylazine (8 mg/kg) mixture. The first group of mice (n = 20) received ovariectomy using a dorsolateral approach. The other group (n = 19) of female mice received sham surgery, which consisted of ovary exposure only. Animals were injected with the pain reliever, buprenorphine (5 mg/kg), immediately after the surgery. One week after the surgery, vaginal smears were collected from all females, including Ovx as handled controls, at the same time each morning by lavage to track their estrus cycles (Marcondes et al. 2002). After two regular cycles, Sh animals were trained and tested on the day of proestrus (high estradiol).Ovariectomy has been reported to affect anxiety levels (Walf et al. 2006), and anxiety levels may alter performance on water maze tasks. To assay possible anxiety differences between Sh and Ovx, female mice were tested on open field and elevated plus-maze (EPM) 2 wk after the surgery in a room different from the rooms used in water maze tasks. A single mouse was placed in the center of a white, Plexiglas chamber measuring 43 cm in length × 43 cm in width × 18 cm in height. The animal explored the novel environment for 15 min, and movements were monitored by a camera interfaced with a tracking system (US HVS Image). The area was divided into 16 virtual squares (10.75 × 10.75 cm) by the program, and the middle four squares were defined as the center area. The Plexiglas chamber was wiped clean with 70% ethanol between trials. The EPM consisted of four arms (5 cm in width × 30 cm in length) arranged perpendicularly in a plus shape and elevated 38 cm above the floor. Two arms were enclosed by 15.5-cm dark Plexiglas walls and two arms were open. Each animal was placed in the center of the EPM facing a closed arm and allowed to move freely for 5 min. Behavior was monitored by a camera interfaced with the tracking system.Animals with high anxiety levels tend to spend less time in the open arms of the EPM and in the center of the open field. The percent time spent in the open arms of the EPM by Ovx mice (37.9% ± 7.5%, n = 14) was not significantly different than the percent of time spent in the open arms by Sh mice (27.8% ± 6.2%, n = 15; P = 0.30, t = 1.1). The percent time spent in the center of the open field by Ovx mice (35.1% ± 7.1%, n = 14) was not significantly different from Sh mice (29.0% ± 7.1%, n = 15; P = 0.55, t = 0.61). These results indicate that ovarian hormones did not have a significant effect on the anxiety levels of the female mice tested in this study.Two weeks after the anxiety tests, the Ovx and Sh groups were divided randomly into two groups based on the pre-exposure context: Ovx PTC, Ovx PDC, Sh PTC, Sh PDC. Sh females with regular estrus cycles were trained and tested on the day of proestrus. Five Ovx and seven Sh animals were not included in the study because of floating, failing to reach criterion (four correct out of nine trials), or exhibiting irregular estrus cycles. Five of eight Sh PTC and only one of six Sh PDC females used place strategy; however, this difference was not significant (P > 0.05, χ² = 2.94, Fig. 2A). Therefore, the pre-exposure condition did not significantly affect strategy use in females at proestrus.Open in a separate windowFigure 2.The effects of ovarian hormone status and pre-exposure to the training and testing (PTC) or to a different context (PDC) on strategy selection of female mice. (A) When pre-exposed to the same context prior to training and testing (PTC), more gonadally intact female mice at proestrus (Sh, n = 8) used place strategy than response strategy compared with ovariectomized female mice (Ovx, n = 8, P < 0.05). When pre-exposed to a context different than the training and testing context (PDC), gonadally intact female mice at proestrus (Sh, n = 6) and ovariectomized mice (Ovx, n = 6) used response strategy rather than place strategy. (B) Latency curves show the actual latency to escape to the platform. Two-way ANOVA (non-repeated measures) revealed no significant differences across training trials in escape latencies between sham and ovariectomized PTC and PDC mice (P > 0.5), although a significant effect of trial indicated that mice reduced their escape latencies across trials (P < 0.0001). Values represent mean ± S.E.M.Interestingly, ovariectomy did significantly affect strategy use in PTC females. Five of eight Sh PTC and only one of eight Ovx PTC females used place strategy (P < 0.05, χ² = 4.267, Fig. 2A). One of six Sh PDC females and zero of the six Ovx PDC animals used place strategy (Fig. 2A). Therefore, both Sh and Ovx PDC females used response strategy, and ovarian hormones did not enhance place strategy use in PDC females (P > 0.05, χ² = 1.09, Fig. 2A). Ovarian hormones did enhance place strategy use in PTC females. Furthermore, PTC did not enhance place strategy use in Ovx animals. Similar to males, there was a significant effect of training trial on latency to find the platform in female animals (F_(8,189) = 10.32, P < 0.0001, Fig. 2B). Ovarian hormones or pre-exposure to either context also did not affect escape latency during training in PTC or PDC females (F_(3,189) = 0.33, P = 0.80, Fig. 2B). In addition, there was no significant difference in the average swim speed between groups (F_(3,14) = 0.15, P = 0.93, one-way ANOVA). The average swim speed for each group was as follows: Ovx PTC (5 ± 1.5 cm/sec, n = 5), Ovx PDC (5 ± 1.0 cm/sec, n = 4), Sh PTC (5 ± 1.8 cm/sec, n = 5), Sh PDC (6 ± 1.5 cm/sec, n = 4). The numbers of animals are lower because in some cases, speed was not measured. Together, these data suggest that ovarian hormones and pre-exposure condition did not influence learning during the training period, but ovarian hormones did enhance place strategy use in the probe trial in only PTC mice.Consistent with previous literature (Martel et al. 2007), we found that ∼80% of PTC males favored the use of place strategy. In addition, 63% of PTC females on proestrus also used place strategy. Ovx female mice used response strategy regardless of the pre-exposure condition. These results confirm that pre-exposure to the training and testing context significantly increased the use of place strategy or reduced response strategy in male mice, while female mice on proestrus were not significantly different than chance. Ovariectomy diminished the use of place strategy and enhanced response strategy use in our study, implicating ovarian hormones in strategy choice.Male rats rely initially on a hippocampus-dependent place strategy, and then switch to a striatum-based response strategy over training (Packard and McGaugh 1996; Packard 1999). This suggests that response strategy is incrementally learned with repeated exposure to the same task. However, a sufficient amount of time to explore the extramaze cues during or before training increased place strategy use in male mice (Martel et al. 2007). In addition, it has been proposed that the presence of an increased number of salient extramaze cues favors place strategy use in rats (Restle 1957). Therefore, it is possible that pre-exposing mice to the learning environment allowed them to build a cognitive map that facilitated the use of a spatial place strategy. Another possible advantage of pre-exposure for place strategy use is that it may reduce the impact of non-mnemonic factors, such as anxiety, on performance (Cain 1998). Indeed, it was shown that peripheral injection and infusion of anxiogenic drugs into the basolateral amygdala biased rats toward the use of response strategy (Packard 1999; Wingard and Packard 2008; Packard and Gabriele 2009).While PTC female mice were not significantly different than PDC female mice, ovariectomy did reduce place strategy choice in the PTC mice. An emerging theory proposes that estradiol modulates cognitive performance via shifts in learning strategy (Korol and Kolo 2002; Daniel and Lee 2004; Korol 2004; McElroy and Korol 2005; Zurkovsky et al. 2007). Shifts in strategy use occurred across the estrus cycle in rats such that the hippocampus-dependent strategy was favored when estradiol levels were high (Korol et al. 2004). Similarly, estradiol treatment in ovariectomized rats increased hippocampus-dependent place strategy and impaired response strategy use compared with nontreated ovariectomized females (Korol and Kolo 2002). Our results showing that the lack of ovarian hormones reduced place strategy and increased response strategy use in PTC mice are consistent with these studies.In summary, we present a new design to a traditional dual-solution land plus-maze. One issue with the land maze version of the task is that it requires food deprivation. The possible increase in the appetite as a result of ovariectomy (Wade 1975) or disruption in the estrus cycle in response to food deprivation (Daniel et al. 1999) could confound the results in females in tasks that present food reward. In order to avoid these confounds, we used a modified version of a water-escape plus-maze (Packard and Wingard 2004). In this design, compared with the water-escape plus-maze, the clear Plexiglas maze itself is filled with water, instead of placing the plus-maze into a water maze, allowing a better view of extramaze visual cues. However, unlike rats, mice tend to be prey animals when in the water; therefore they are highly motivated to escape the water (Francis et al. 1995; Van Dam et al. 2006). Consequently, the stressful nature of the task prevents mice from utilizing the spatial cues as efficiently (Frick et al. 2000). Therefore, we pre-exposed the mice to the maze while it was dry, allowing them to build a cognitive map before they were released in water. The water plus-maze is important not only for the design of future studies, but also for the evaluation of previous studies that investigated learning strategies using tasks dependent on food deprivation.     

The NMDA antagonist MK-801 disrupts reconsolidation of a cocaine-associated memory for conditioned place preference but not for self-administration in rats

Recent research suggests that drug-related memories are reactivated after exposure to environmental cues and may undergo reconsolidation, a process that can strengthen memories. Conversely, reconsolidation may be disrupted by certain pharmacological agents such that the drug-associated memory is weakened. Several studies have demonstrated disruption of memory reconsolidation using a drug-induced conditioned place preference (CPP) task, but no studies have explored whether cocaine-associated memories can be similarly disrupted in cocaine self-administering animals after a cocaine priming injection, which powerfully reinstates drug-seeking behavior. Here we used cocaine-induced CPP and cocaine self-administration to investigate whether the N-methyl-D-aspartate receptor antagonist (+)-5methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) given just prior to reactivation sessions would suppress subsequent cocaine-primed reinstatement (disruption of reconsolidation). Systemic injection of MK-801 (0.05 or 0.20 mg/kg administered intraperitoneally) in rats just prior to reactivation of the cocaine-associated memory in the CPP context attenuated subsequent cocaine-primed reinstatement, while no disruption occurred in rats that did not receive reactivation in the CPP context. However, in rats trained to self-administer cocaine, systemic administration of MK-801 just prior to either of two different types of reactivation sessions had no effect on subsequent cocaine-primed reinstatement of lever-pressing behavior. Thus, systemic administration of MK-801 disrupted the reconsolidation of a cocaine-associated memory for CPP but not for self-administration. These findings suggest that cocaine-CPP and self-administration do not use similar neurochemical processes to disrupt reconsolidation or that cocaine-associated memories in self-administering rats do not undergo reconsolidation, as assessed by lever-pressing behavior under cocaine reinstatement conditions.The ability to disrupt previously consolidated memories in a reactivation-dependent manner is thought to be due to the disruption of a memory reconsolidation process. This disruption of reconsolidation has been observed in a wide variety of tasks and species (Nader et al. 2000b; Sara 2000; Alberini 2005; Riccio et al. 2006). Early reconsolidation experiments primarily focused on aversive learning paradigms, with an emphasis on disruption of reconsolidation as a potential treatment for posttraumatic stress disorder (Misanin et al. 1968; Nader et al. 2000a; Debiec and Ledoux 2004; Brunet et al. 2008). Only more recently have investigators demonstrated that appetitive memories also undergo reconsolidation; most, but not all (Yim et al. 2006), studies found a disruption of expression for the drug-associated memory, suggesting the potential to target the reconsolidation process as a treatment for drug addiction (Lee et al. 2005; Miller and Marshall 2005; Milekic et al. 2006; Valjent et al. 2006; Brown et al. 2007; Kelley et al. 2007; Sadler et al. 2007; Fricks-Gleason and Marshall 2008; Milton et al. 2008a, b).Miller and Marshall (2005) showed that reconsolidation of cocaine conditioned place preference (CPP) in the rat could be disrupted by either pre- or post-treatment of a phosphorylation inhibitor of extracellular signal-regulated kinase (1/2) (ERK) in a reactivation-dependent manner. Other studies have shown that protein synthesis inhibitors (Milekic et al. 2006), a matrix metalloproteinase (MMP) inhibitor (Brown et al. 2007), a β-noradrenergic receptor antagonist (Bernardi et al. 2006; Robinson and Franklin 2007a; Fricks-Gleason and Marshall 2008), and an N-methyl-D-aspartate (NMDA) receptor antagonist (Kelley et al. 2007; Sadler et al. 2007) can also disrupt the reconsolidation of drug-associated CPP memories. Studies by Lee and colleagues have shown that Zif268 antisense oligodeoxynucleotide infused into the basolateral amygdala prior to reactivation of memory for a cocaine-associated cue (the conditioned stimulus or CS) disrupts the ability of cocaine-associated cues to establish subsequent acquisition of a new instrumental response (Lee et al. 2005), and the ability of a drug-associated cue to induce relapse under a second-order schedule (Lee et al. 2006a). Thus, cocaine-associated memories appear to undergo reconsolidation in both Pavlovian and operant conditioning paradigms.Relapse to drug-seeking or drug-taking behavior can occur after re-exposure to three types of stimuli: the drug itself, drug-associated contextual and discrete cues, and stress; and all of these may promote relapse in humans (for review, see Epstein et al. 2006). Only a few CPP studies (Valjent et al. 2006; Brown et al. 2007) and no self-administration studies to our knowledge have tested whether the drug-associated memory can be rendered susceptible to disruption by pharmacological agents such that subsequent cocaine-primed reinstatement is suppressed. This drug-primed effect is observed in humans, producing relapse (Ludwig et al. 1974; Jaffe et al. 1989), and in rats, producing robust reinstatement of drug-seeking behavior in both CPP and self-administration tasks (McFarland and Ettenberg 1997; McFarland and Kalivas 2001; Sanchez and Sorg 2001; Kalivas and McFarland 2003). The development of a treatment strategy that makes use of the reconsolidation process will ultimately need to be powerful enough to diminish drug-seeking behavior in the presence of sizable doses of the drug itself. Therefore, the primary goal of this study was to determine whether drug-primed reinstatement could be suppressed in rats that have the memory reactivated in the presence of a pharmacological agent in cocaine self-administering rats. Since we previously have demonstrated the ability to disrupt cocaine-primed reinstatement only in animals in which the memory was reactivated using cocaine-induced CPP, we also tested the extent to which the same parameters used to disrupt reconsolidation in a cocaine-induced CPP task would disrupt reconsolidation in a cocaine self-administration task under conditions of drug-induced reinstatement.To examine this question, we chose the noncompetitive NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801). MK-801 has been shown to disrupt reconsolidation of spatial tasks (Przybyslawski and Sara 1997), fear tasks (Lee et al. 2006b), amphetamine-induced CPP (Sadler et al. 2007), cocaine-induced CPP (Kelley et al. 2007), and sucrose self-administration (Lee and Everitt 2008). Importantly, the two studies examining CPP using MK-801 did not explore whether MK-801 suppressed drug-seeking behavior in a manner that was dependent on whether the memory was reactivated, leaving open the possibility that it was not a reconsolidation process that was disrupted by MK-801.Here we demonstrate that MK-801 injected prior to cocaine-primed reinstatement of CPP disrupted subsequent cocaine-primed reinstatement of CPP, and this disruption was dependent on CPP contextual reactivation since injection of MK-801 and cocaine in the home cage did not disrupt subsequent cocaine-primed reinstatement of CPP. However, drug-seeking behavior in animals trained for cocaine self-administration was not disrupted when rats were reactivated under the same parameters that disrupted cocaine-induced CPP or when rats were given a reactivation session identical to their self-administration sessions. We thus demonstrate for the first time that memories associated with cocaine-induced CPP and cocaine self-administration are not similarly susceptible to disruption by MK-801.     

Genetic inactivation of D-amino acid oxidase enhances extinction and reversal learning in mice

Activation of the N-methyl-d-aspartate receptor (NMDAR) glycine site has been shown to accelerate adaptive forms of learning that may benefit psychopathologies involving cognitive and perseverative disturbances. In this study, the effects of increasing the brain levels of the endogenous NMDAR glycine site agonist D-serine, through the genetic inactivation of its catabolic enzyme D-amino acid oxidase (DAO), were examined in behavioral tests of learning and memory. In the Morris water maze task (MWM), mice carrying the hypofunctional Dao1^G181R mutation demonstrated normal acquisition of a single platform location but had substantially improved memory for a new target location in the subsequent reversal phase. Furthermore, Dao1^G181R mutant animals exhibited an increased rate of extinction in the MWM that was similarly observed following pharmacological administration of D-serine (600 mg/kg) in wild-type C57BL/6J mice. In contextual and cued fear conditioning, no alterations were found in initial associative memory recall; however, extinction of the contextual fear memory was facilitated in mutant animals. Thus, an augmented level of D-serine resulting from reduced DAO activity promotes adaptive learning in response to changing conditions. The NMDAR glycine site and DAO may be promising therapeutic targets to improve cognitive flexibility and inhibitory learning in psychiatric disorders such as schizophrenia and anxiety syndromes.The N-methyl-d-aspartate receptor (NMDAR) has an important role in excitatory neurotransmission and contributes to numerous brain processes, including synaptic plasticity, learning, and memory formation (Nicoll 2003). Activation of NMDARs requires membrane depolarization in addition to concurrent binding of glutamate to NMDAR2 (NR2) and glycine to the NMDAR1 (NR1) subunit (Johnson and Ascher 1987; Clements and Westbrook 1991). D-serine has also been shown to be an endogenous co-agonist for the NR1 glycine site, acting with high selectivity and a potency similar to or greater than that of glycine (Matsui et al. 1995). In the brain, the localization of D-serine closely resembles that of NMDARs (Schell et al. 1997), and D-serine has been reported to be the predominant physiologic co-agonist for the maintenance of NMDAR-mediated currents in the hippocampus, retina, and hypothalamus (Mothet et al. 2000; Yang et al. 2003). Moreover, in vivo studies have demonstrated that the NMDAR glycine site is not saturated at the synapses of several brain regions (Fuchs et al. 2005). Consequently, increasing D-serine levels may modulate neurotransmission and behavioral responses reliant on NMDAR activity.The NMDAR glycine site has been implicated in the pathophysiology and treatment of a number of psychiatric conditions (Coyle and Tsai 2004; Millan 2005). Blockade of the NMDAR with noncompetitive antagonists like phencyclidine results in the production and exacerbation of schizophrenic-like symptoms in humans and animals (Javitt and Zukin 1991; Krystal et al. 1994). Genetic studies have associated genes that mediate D-serine synthesis and degradation with a vulnerability to schizophrenia, and levels of D-serine are decreased in the CSF and serum of schizophrenic patients (Chumakov et al. 2002; Hashimoto et al. 2003, 2005; Schumacher et al. 2004; Morita et al. 2007). These observations prompted clinical trials with direct and indirect activators of the NMDAR glycine site, including D-serine, and improvements were revealed when these compounds were added to conventional antipsychotic regimes, particularly with the negative and cognitive symptoms of schizophrenia (Tsai et al. 1998; Coyle and Tsai 2004; Heresco-Levy et al. 2005). Furthermore, altered NMDAR activation has also been shown to affect extinction, a learning process that may be of benefit in anxiety illnesses, such as post-traumatic stress syndrome and obsessive-compulsive disorder (Davis et al. 2006). In rodents, extinction was shown to be impaired following inhibition of NMDARs in contextual fear conditioning, inhibitory avoidance, and eyeblink conditioning tasks (Kehoe et al. 1996; Lee and Kim 1998; Szapiro et al. 2003). In contrast, the partial NMDAR agonist D-cycloserine facilitated the extinction of fear memories in rodents and individuals with phobias and other anxiety disorders (Ressler et al. 2004; Ledgerwood et al. 2005; Norberg et al. 2008). Thus, the NMDAR glycine site and its related modulatory proteins may be important targets for the amelioration of psychopathologies involving cognitive dysfunction and maladaptive behaviors.Endogenous levels of D-serine in the brain are regulated by its catabolic enzyme, D-amino acid oxidase (DAO); by the D-serine synthesis enzyme, serine racemase (Srr); and by neuronal and glial transporters (Foltyn et al. 2005; Martineau et al. 2006). Agents targeting such proteins may prove to be an effective method of increasing cerebral D-serine and occupancy of the NMDAR glycine site, which could overcome the difficulties D-serine and similar compounds have with penetrating the blood-brain barrier (Coyle and Tsai 2004; Bauer et al. 2005). Inhibiting DAO function in the brain is of particular interest as it would circumvent any nephrotoxicity associated with high levels of systemic D-serine (Maekawa et al. 2005a). DAO is a peroxisomal flavoprotein that at physiological pH is highly selective for D-serine, and in the brain, DAO is located predominantly in astrocytes (Mothet et al. 2000). An inverse correlation between the brain distribution of DAO and D-serine evinces the efficacy of this enzyme, with the most abundant DAO expression located in the D-serine-sparse hindbrain and cerebellum (Schell et al. 1995; Moreno et al. 1999). To study the effects of limiting DAO function, we tested a line of mice carrying a single point mutation (G181R) that results in a complete lack of DAO activity and consequently augmented D-serine in serum and brain (Sasaki et al. 1992; Hashimoto et al. 1993). These mice have previously been shown to exhibit an in vitro increase in NMDAR-mediated excitatory postsynaptic currents in dorsal horn neurons of the spinal cord and an in vivo elevation of cGMP that is indicative of augmented NMDAR activity (Wake et al. 2001; Almond et al. 2006). This demonstrates that reduced DAO function is capable of augmenting NMDAR activation, and it may follow that cognitive and extinction processes influenced by NMDARs are enhanced in Dao1^G181R mutant mice. To investigate this possibility, we assessed the effects of the Dao1^G181R mutation on learning, memory, and extinction in Morris water maze (MWM) and in contextual and cued fear conditioning paradigms.