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.     

Extinction circuits for fear and addiction overlap in prefrontal cortex

Extinction is a form of inhibitory learning that suppresses a previously conditioned response. Both fear and drug seeking are conditioned responses that can lead to maladaptive behavior when expressed inappropriately, manifesting as anxiety disorders and addiction, respectively. Recent evidence indicates that the medial prefrontal cortex (mPFC) is critical for the extinction of both fear and drug-seeking behaviors. Moreover, a dorsal-ventral distinction is apparent within the mPFC, such that the prelimbic (PL-mPFC) cortex drives the expression of fear and drug seeking, whereas the infralimbic (IL-mPFC) cortex suppresses these behaviors after extinction. For conditioned fear, the dorsal-ventral dichotomy is accomplished via divergent projections to different subregions of the amygdala, whereas for drug seeking, it is accomplished via divergent projections to the subregions of the nucleus accumbens. Given that the mPFC represents a common node in the extinction circuit for these behaviors, treatments that target this region may help alleviate symptoms of both anxiety and addictive disorders by enhancing extinction memory.Emotional memories, both in the aversive and appetitive domains, are important for guiding behavior. Regulating the expression of these memories is critical for mental health. Extinction of classical conditioning is one form of emotion regulation that is easily modeled in animals. In the aversive domain, a conditioned stimulus (CS) is typically paired with a shock, while in the appetitive domain, a CS is paired with the availability of food or drug reward. Repeated presentation of the CS in the absence of the reinforcer leads to extinction of conditioned fear or drug-seeking behaviors. In recent years, there have been great advances in our understanding of the neural circuitry responsible for this form of inhibitory learning (for reviews, see Cammarota et al. 2005; Maren 2005; Myers and Davis 2007; Quirk and Mueller 2008). The prefrontal cortex has been strongly implicated in fear expression (Powell et al. 2001; Vidal-Gonzalez et al. 2006; Corcoran and Quirk 2007) and fear extinction (Herry and Garcia 2002; Milad and Quirk 2002; Gonzalez-Lima and Bruchey 2004; Hugues et al. 2004; Burgos-Robles et al. 2007; Hikind and Maroun 2008; Lin et al. 2008; Mueller et al. 2008; Sotres-Bayon et al. 2008), and more recently, in expression of drug seeking after extinction (Peters et al. 2008a,b). These findings are consistent with a well-documented role of the prefrontal cortex in executive function and emotional regulation (Miller 2000; Fuster 2002; Quirk and Beer 2006; Sotres-Bayon et al. 2006).In this review, we propose that the medial prefrontal cortex (mPFC) regulates the expression of both fear and drug memories after extinction, through divergent projections to the amygdala and nucleus accumbens, respectively. Extinction failure in the aversive domain can lead to anxiety disorders (Delgado et al. 2006; Milad et al. 2006), while extinction failure in the appetitive domain can lead to relapse in addicted subjects (Kalivas et al. 2005; Garavan and Hester 2007). A common neural circuit for extinction of fear and drug memories would suggest shared mechanisms and treatment strategies across both domains.     

LTP in hippocampal area CA1 is induced by burst stimulation over a broad frequency range centered around delta

Long-term potentiation (LTP) is typically studied using either continuous high-frequency stimulation or theta burst stimulation. Previous studies emphasized the physiological relevance of theta frequency; however, synchronized hippocampal activity occurs over a broader frequency range. We therefore tested burst stimulation at intervals from 100 msec to 20 sec (10 Hz to 0.05 Hz). LTP at Schaffer collateral–CA1 synapses was obtained at intervals from 100 msec to 5 sec, with maximal LTP at 350–500 msec (2–3 Hz, delta frequency). In addition, a short-duration potentiation was present over the entire range of burst intervals. We found that N-methyl-d-aspartic acid (NMDA) receptors were more important for LTP induction by burst stimulation, but L-type calcium channels were more important for LTP induction by continuous high-frequency stimulation. NMDA receptors were even more critical for short-duration potentiation than they were for LTP. We also compared repeated burst stimulation with a single primed burst. In contrast to results from repeated burst stimulation, primed burst potentiation was greater when a 200-msec interval (theta frequency) was used, and a 500-msec interval was ineffective. Whole-cell recordings of postsynaptic membrane potential during burst stimulation revealed two factors that may determine the interval dependence of LTP. First, excitatory postsynaptic potentials facilitated across bursts at 500-msec intervals but not 200-msec or 1-sec intervals. Second, synaptic inhibition was suppressed by burst stimulation at intervals between 200 msec and 1 sec. Our data show that CA1 synapses are more broadly tuned for potentiation than previously appreciated.Long-term potentiation (LTP) is used as a model for studying synaptic events during learning and memory (Bliss and Collingridge 1993; Morris 2003; Lynch 2004). At most synapses, LTP is triggered by postsynaptic Ca²⁺ influx through N-methyl-d-aspartic acid (NMDA) glutamate receptors (Collingridge et al. 1983; Harris et al. 1984; Herron et al. 1986) and, under some conditions, through L-type voltage-gated Ca²⁺ channels (Grover and Teyler 1990, 1994; Morgan and Teyler 1999). LTP was discovered in the dentate gyrus (Bliss and Lomo 1973) following several seconds of 10–100 Hz stimulation of the perforant path. Since then, many LTP studies have used similar long, high-frequency stimulation (HFS) protocols, most typically 100 Hz, 1 sec (Bliss and Collingridge 1993). Although effective, HFS does not resemble physiological patterns of activity (Albensi et al. 2007). Patterned stimulation resembling physiological activity, in particular theta burst stimulation, is also effective for LTP induction (Larson et al. 1986; Staubli and Lynch 1987; Capocchi et al. 1992; Nguyen and Kandel 1997). Theta burst stimulation consists of short bursts (4–5 stimuli at 100 Hz) repeated at 5 Hz, which lies within the hippocampal theta frequency range (4–12 Hz) (Bland 1986; Buzsáki 2002). Primed burst stimulation, another form of patterned stimulation, involves delivery of a priming stimulus followed by a single short burst (Larson and Lynch 1986; Rose and Dunwiddie 1986). The temporal requirements for primed burst LTP are quite precise (Diamond et al. 1988; Greenstein et al. 1988; Larson and Lynch 1989): Intervals less than 140 msec or greater than 200 msec are ineffective.The mechanisms underlying theta frequency-dependent LTP have been studied primarily using the primed burst protocol (Larson and Lynch 1986, 1988, 1989; Pacelli et al. 1989; Davies and Collingridge 1996). Activation of GABA_B autoreceptors during the priming stimulus suppresses GABA release during the following burst (Davies et al. 1990; Lambert and Wilson 1994; Olpe et al. 1994), allowing greater postsynaptic depolarization (Larson and Lynch 1986; Pacelli et al. 1989) and more effective NMDA receptor activation (Davies and Collingridge 1996). Consequently, temporal requirements for primed burst potentiation match the time course of GABA_B autoreceptor-mediated suppression of GABA release (Davies et al. 1990; Davies and Collingridge 1993; Mott et al. 1993).Besides theta, hippocampal activity is observed at other frequencies, notably sharp waves (0.01–5 Hz) (Buzsáki 1986, 1989; Suzuki and Smith 1987) and low-frequency oscillations (≤1 Hz) (Wolansky et al. 2006; Moroni et al. 2007). These lower frequencies dominate during slow wave sleep (Buzsáki 1986; Suzuki and Smith 1987; Wolansky et al. 2006; Moroni et al. 2007), and contribute to hippocampal memory processing (Buzsáki 1989; Pennartz et al. 2002). While synchronized population activity over frequencies from <1 Hz to 12 Hz is associated with hippocampal memory function, previous LTP studies have focused on theta. We therefore investigated burst stimulation at frequencies from 0.05 Hz to 10 Hz. We found that CA1 synapses potentiate to some degree over this entire range and that maximal potentiation occurs around delta frequency rather than theta.     

The amygdala is not necessary for unconditioned stimulus inflation after Pavlovian fear conditioning in rats

The basolateral complex (BLA) and central nucleus (CEA) of the amygdala play critical roles in associative learning, including Pavlovian conditioning. However, the precise role for these structures in Pavlovian conditioning is not clear. Recent work in appetitive conditioning paradigms suggests that the amygdala, particularly the BLA, has an important role in representing the value of the unconditioned stimulus (US). It is not known whether the amygdala performs such a function in aversive paradigms, such as Pavlovian fear conditioning in rats. To address this issue, Experiments 1 and 2 used temporary pharmacological inactivation of the amygdala prior to a US inflation procedure to assess its role in revaluing shock USs after either overtraining (Experiment 1) or limited training (Experiment 2), respectively. Inactivation of the BLA or CEA during the inflation session did not affect subsequent increases in conditioned freezing observed to either the tone conditioned stimulus (CS) or the conditioning context in either experiment. In Experiment 3, NBQX infusions into the BLA impaired the acquisition of auditory fear conditioning with an inflation-magnitude US, indicating that the amygdala is required for associative learning with intense USs. Together, these results suggest that the amygdala is not required for revaluing an aversive US despite being required for the acquisition of fear to that US.Pavlovian fear conditioning in rats is a behavioral model used to investigate the neurobiology underlying the development and maintenance of fear learning and memory (Grillon et al. 1996; LeDoux 1998, 2000; Bouton et al. 2001; Maren 2001b, 2005; Kim and Jung 2006). In this model, an innocuous conditioned stimulus (CS), such as a tone, is paired with an aversive unconditioned stimulus (US), such as a footshock. After one or more pairings, the rat learns that the CS predicts the US. As a consequence, CS presentations alone elicit a conditioned fear response (CR), which includes increases in heart rate, arterial blood pressure, hypoalgesia, potentiated acoustic startle, stress hormone release, and freezing (somatomotor immobility).The amygdala has been identified as one of the major regions in which fear memories are encoded and stored. Within the amygdala, the basolateral complex of the amygdala (BLA; consisting of the lateral, basolateral, and basomedial nuclei) and the central nucleus of the amygdala (CEA) receive convergent CS and US information and are involved in the acquisition of fear memories (LeDoux 1998, 2000; Fendt and Fanselow 1999; Davis and Whalen 2001; Maren 2001b; Schafe et al. 2001; Fanselow and Gale 2003; Wilensky et al. 2006; Zimmerman et al. 2007). In addition, the CEA has an important role in the expression of fear CRs (Fendt and Fanselow 1999; LeDoux 2000; Davis and Whalen 2001; Maren 2001b; Fanselow and Gale 2003). In support of this, many studies have shown that either permanent or temporary lesions of the BLA or CEA prevent the acquisition and/or expression of fear memories (Helmstetter 1992; Helmstetter and Bellgowan 1994; Campeau and Davis 1995; Maren et al. 1996a,b; Killcross et al. 1997; Muller et al. 1997; Walker and Davis 1997; Cousens and Otto 1998; Maren 1998, 1999, 2001a,b; Wilensky et al. 1999, 2000, 2006; Goosens and Maren 2001, 2003; Nader et al. 2001; Fanselow and Gale 2003; Gale et al. 2004; Koo et al. 2004; Zimmerman et al. 2007).In addition to its role in encoding CS–US associations during conditioning, recent work suggests that the amygdala is also involved in representing properties of the US itself. For example, temporary or permanent lesions of the BLA reduce both decrements in conditioned responding after devaluation of a food US (Hatfield et al. 1996; Killcross et al. 1997; Blundell et al. 2001; Balleine et al. 2003; Everitt et al. 2003; Pickens et al. 2003; Holland 2004) and increments in conditional responding after inflation of a shock US (Fanselow and Gale 2003). Moreover, recent electrophysiological studies in primates indicate that amygdala neurons represent the value of both aversive and appetitive outcomes (Paton et al. 2006; Belova et al. 2007, 2008; Salzman et al. 2007). These studies suggest that one function of the BLA is to represent specific properties of biologically significant events, such as the food or shock USs that are typically used in Pavlovian conditioning paradigms. By this view, the BLA may represent specific sensory properties of USs that shape the nature of learned behavioral responses to the US (Balleine and Killcross 2006) and allow CSs to gain access to the incentive value of the US (Everitt et al. 2003).In contrast to this view, we recently reported that rats with neurotoxic BLA lesions exhibit normal US revaluation after Pavlovian fear conditioning (Rabinak and Maren 2008). In this study, auditory fear conditioning (75 CS–US trials) with a moderate footshock (1 mA) was followed by several exposures (five US-alone trials) to an intense footshock (3 mA) during an inflation session. Both intact rats and rats with BLA lesions exhibit a robust increase in conditional freezing to the auditory CS during a subsequent retention test (Rabinak and Maren 2008). Control experiments suggested that this was due to a revaluation of the US with which the CS was associated, rather than nonassociative sensitization of fear engendered by exposure to intense shock. These data reveal that the BLA may not be necessary for representing properties of shock USs during Pavlovian fear conditioning. To address these issues further, we have examined the consequence of reversible pharmacological manipulations of the amygdala during US inflation on conditional fear responses established with either extensive or limited training.     

Memory deficits are associated with impaired ability to modulate neuronal excitability in middle-aged mice

Normal aging disrupts hippocampal neuroplasticity and learning and memory. Aging deficits were exposed in a subset (30%) of middle-aged mice that performed below criterion on a hippocampal-dependent contextual fear conditioning task. Basal neuronal excitability was comparable in middle-aged and young mice, but learning-related modulation of the post-burst afterhyperpolarization (AHP)—a general mechanism engaged during learning—was impaired in CA1 neurons from middle-aged weak learners. Thus, modulation of neuronal excitability is critical for retention of context fear in middle-aged mice. Disruption of AHP plasticity may contribute to contextual fear deficits in middle-aged mice—a model of age-associated cognitive decline (AACD).Plasticity of intrinsic neuronal excitability increases the overall storage capacity of neurons and therefore likely plays a critical role in learning and memory (Zhang and Linden 2003). Increased neuronal excitability via reductions of the post-burst afterhyperpolarization (AHP) is hypothesized as a general mechanism underlying learning and memory tasks (Disterhoft et al. 1986; Disterhoft and Oh 2006). The AHP serves to limit subsequent firing in response to excitation (Madison and Nicoll 1984; Lancaster and Adams 1986; Storm 1990; Sah and Bekkers 1996). Generally speaking, the size of the AHP is inversely related to neuronal excitability, and the measurement of the AHP is routinely used as an index of neuronal excitability.Our laboratory and others have shown that AHP reductions are observed in hippocampal neurons from animals that learn hippocampal-dependent tasks including trace eyeblink conditioning in rabbit and rat (de Jonge et al. 1990; Moyer Jr et al. 1996, 2000; Kuo 2004) and spatial water maze in rat and mouse (Oh et al. 2003; Tombaugh et al. 2005; Ohno et al. 2006b). Learning-related reductions in the AHP have also been observed in cortical neurons following odor discrimination (Saar et al. 1998) and extinction learning (Santini et al. 2008). In vitro, activity-dependent plasticity of the AHP is induced using physiologically relevant stimuli (Kaczorowski et al. 2007). Because the AHP serves to limit subsequent firing, learning-related reductions in the AHP are poised to facilitate mechanisms crucial for information storage, such as long-term potentiation (LTP), synaptic integration (Sah and Bekkers 1996), metaplasticity (Le Ray et al. 2004), and spike-timing dependent plasticity (STDP) (Le Ray et al. 2004).Hippocampal neurons from naïve aged rodents and rabbits show a decrement in basal excitability evidenced by a robust enhancement of the AHP (Landfield and Pitler 1984; Moyer Jr et al. 1992, 2000; Oh et al. 1999; Kumar and Foster 2002, 2004; Power et al. 2002; Hemond and Jaffe 2005; Murphy et al. 2006b; Gant and Thibault 2008). Enhancement of the AHP in hippocampal neurons in aged animals correlates with impaired performance on learning paradigms that depend on a functional hippocampus, such as trace eyeblink and spatial water maze (Moyer Jr et al. 2000; Tombaugh et al. 2005; Murphy et al. 2006a). Pharmaceuticals aimed at reducing the AHP and increasing basal excitability (Moyer Jr et al. 1992; Moyer Jr and Disterhoft 1994) have been successful at restoring performance of aged rats on trace eyeblink conditioning (Deyo et al. 1989; Straube et al. 1990; Kowalska and Disterhoft 1994). Interestingly, AHPs from neurons recorded from aged learners are indistinguishable from young learners; both are reduced compared to that of aged weak-learners (Moyer Jr et al. 2000; Tombaugh et al. 2005). These data suggest that mechanisms that permit learning-related modulation of the AHP are also critical determinants of learning abilities in an aged population. To date, age-related impairments in hippocampal-dependent tasks and biophysical alterations in hippocampal neurons have largely focused on studies that compare animals at extreme ends of the aging spectrum.In an effort to better understand physiological changes that underlie the onset of early cognitive decline, the development of rodent models of “normal” age-associated cognitive decline (AACD), as well as mild cognitive impairment (MCI), is critical (Pepeu 2004). Therefore, we set out to characterize the development of age-related deficits indicative of hippocampal dysfunction in middle-aged C57Bl6/SJL mice and to examine the biophysical changes in hippocampal neurons that accompany such deficits.Recently, age-related deficits in contextual fear memory following trace fear conditioning were reported in a subset of middle-aged rats (Moyer Jr and Brown 2006). Because the dorsal hippocampus is critical for trace and contextual fear conditioning in mice and rats (McEchron et al. 1998; Chowdhury et al. 2005; Misane et al. 2005), trace fear conditioning is an ideal paradigm for exploring cellular mechanisms that underlie early-age-related cognitive decline.Here we investigate the effects of “early” aging on trace fear conditioning by comparing performance outcomes of young (2 mo, n = 7; and 4 mo, n = 8) and middle-aged (8 mo, n = 22) male C57/SJL F1 hybrid mice. Mice were trained and tested singly, and the experimenter was blind to the training and retention status of the mice. All animal procedures were approved by the Northwestern University Animal Care and Use Committee. Preliminary data were reported previously (Kaczorowski 2006).To assess hippocampal function with aging, young and middle-aged mice were trained on a trace fear conditioning task followed by retention tests of the auditory conditioned stimulus (CS) and contextual CS memory. The basic protocol for trace fear conditioning has been described previously (Ohno et al. 2006a). Mice were trained in a Plexiglas conditioning chamber with a stainless-steel floor grid used for shock delivery. After the baseline period (150 sec), mice received four pairings of the CS (tone; 15 sec, 3 kHz, 75 dB) and US (shock; 1 sec, 0.7 mA). The CS and unconditioned stimulus (US) were separated by a 30-sec empty trace interval. The intertrial interval was set at 210 ± 10 sec. The training chamber was wiped with 95% ethyl alcohol, illuminated with a 10-W bulb in an otherwise dark room, and provided with 65-dB white noise to make it distinct. During training on trace fear conditioning, no effect of age was observed on measures of baseline freezing (F_(2,34) = 2.0, P = 0.15), the expression of freezing during tone (F_(2,34) = 0.6, P = 0.6), or post-shock freezing (F_(2,34) = 0.2, P = 0.8), suggesting that middle-aged and young mice do not differ in measures of anxiolysis or expression of behavioral freezing (measured index of fear) (Fig. 1A).Open in a separate windowFigure 1.Onset of early aging deficits in 8-mo-old middle-aged mice. (A) Baseline (BL) freezing and auditory CS freezing during trace fear conditioning was similar between young (2 mo and 4 mo) and middle-aged (8 mo) mice. (B1) Mean baseline freezing and retention of the auditory CS memory (tones 1–4) were comparable in young (2 mo and 4 mo) and middle-aged (8 mo) mice. (B2) Middle-aged mice showed a significant decrease in freezing compared to young (2 mo and 4 mo) mice when exposed to the original context chamber where they had been trained 1 d earlier; (*) P < 0.05.Retention of the auditory CS:US memory was tested 24 h later in a novel context that differed in its location, size, scent, lighting, background noise, and flooring (bedding) compared to the training chamber. Data from three mice (one young, two middle-aged) were excluded because of video malfunction. Following a 150-sec baseline, mice received four presentations of the tone CS in the absence of footshock. Neither baseline freezing (F_(2,31) = 2.6, P = 0.1) nor conditional freezing in response to the tone CS (F_(2,31) = 1.6, P = 0.2) differed between young and middle-aged mice (Fig. 1B1). Thus, retention of the auditory CS following trace fear conditioning was intact in middle-aged compared to young mice. Although deficits in retention of auditory trace fear have been reported in aged mice and rats (Blank et al. 2003; McEchron et al. 2004; Villarreal et al. 2004), the results herein agree with report of intact trace fear memory in middle-aged rats (Moyer Jr and Brown 2006).One hour after this testing, retention of the contextual fear memory was assessed by placing mice in the original context (in the absence of the tone and footshock) and measuring freezing for 10 min. A subtle but significant difference in freezing was observed as a function of age (F_(2,31) = 4.3, P = 0.02; Fig. 1B ​2).2). A student''s post-hoc t-test revealed that mean freezing (collapsed across 10 min) of middle-aged mice was reduced compared to 2-mo (P < 0.05) and 4-mo (P < 0.05) young mice. Contextual fear memory deficits have been similarly reported in aged mice (Fukushima et al. 2008). Studies that failed to observe contextual fear deficits in aged (>18 mo) mice may result from a floor effect because young mice showed weak conditioning to the context (∼30% freezing) (Feiro and Gould 2005; Gould and Feiro 2005) or employment of delay (Corcoran et al. 2002; Feiro and Gould 2005; Gould and Feiro 2005) compared to trace procedures (Moyer Jr and Brown 2006). Although differences in experimental parameters are plausible, heterogeneity in the performance of aged mice may make detection of age-related impairments difficult owing to increased variability.Open in a separate windowFigure 2.Selective deficits on retention of contextual fear in middle-aged weak-learner mice. (A,B) Summary plot and histogram show young mice (100%, n = 6) at 2 mo of age showed robust recall of contextual fear memory (range 75%–99%) with mean and standard deviation (SD) of 91% ± 10%, whereas retention of middle-aged mice (n = 21) varied to a much greater extent (range 21%–95%; M, SD = 74% ± 19%). Distribution of middle-aged mice relative to their mean percent freezing shows two distinct populations. Middle-aged mice with freezing levels less than 3 SD from the mean freezing in young wild-type (WT) mice (61%, dashed line) were characterized as having weak contextual fear memory (weak learners) and those with freezing levels ≥62% as having strong contextual fear memory (learners). (C) Baseline (BL) freezing, expression of post-shock freezing and freezing during retention tests for auditory CS and trace CS memories, were comparable in both weak-learners and learners. (D) Selective deficits in retention of contextual fear memories were observed in middle-aged weak learners as compared to middle-aged learners; (*) P < 0.05.Previous studies in the rat report heterogeneity in spatial water maze and contextual fear conditioning in middle-aged and/or aged rats compared to young animals (Fischer et al. 1992; Wyss et al. 2000; Moyer Jr and Brown 2006). Therefore, we determined if middle-aged impairments of context fear (Fig. 2A) were driven by a subset of impaired mice. The degree of age-related impairment in each middle-aged mouse was determined by comparison to a reference group of young mice tested concurrently (shown in Fig. 1). The behavioral criterion for retention of contextual fear in middle-aged mice was set at 61%, which was 3 standard deviations (SD) below the mean freezing in young mice (mean and SD, 91% ± 10%; Fig. 1). A bimodal distribution of freezing of middle-aged mice was observed (Fig. 2B), where 70% of middle-aged mice performed above criterion and were labeled learners (n = 14), and 30% of middle-aged mice performed below criterion and were labeled as weak learners (n = 6). Comparison on measures of baseline freezing (F_(1,18) = 1.8, P = 0.2) and expression of post-shock freezing (F_(1,18) = 2.1, P = 0.2) revealed no differences between the groups during auditory trace fear training (Fig. 2C). Similarly, no differences in baseline freezing (F_(1,18) = 0.03, P = 0.9) or acquisition/recall of conditioned auditory trace fear (tone, F_(1,18) = 0.08, P = 0.8; trace, F_(1,18) = 2.4, P = 0.1) were observed 24 h later during retention tests. Thus, deficits ascribed to middle-aged weak learners were limited to contextual processing/retention, where middle-aged weak learners responded to the contextual CS with significantly lower levels of freezing compared to middle-aged learners (F_(1,18) = 47, P = 0.001; Fig. 2D). To summarize, we found that onset of cognitive decline in the C56Bl6/SJL mice was first apparent in a subset of middle-aged mice. Middle-aged weak learners showed a mild but specific deficit in hippocampal-dependent contextual learning/memory (spatial learning) but not hippocampal-dependent auditory trace learning/memory (temporal learning), assessed following trace fear conditioning.Given that contextual fear deficits occurred in a subset of middle-age mice, we were able to directly assess age-related alterations in excitability and AHP plasticity in CA1 neurons as they relate to learning abilities (learners vs. weak learners). Within 1 h of cessation of behavioral tests, middle-aged learners and weak learners were decapitated under deep halothane anesthesia and their brains quickly removed and placed into ice-cold artificial cerebral spinal fluid (aCSF): 125 mM NaCl, 25 mM glucose, 25 mM NaHCO₃, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 2 mM CaCl₂, 1 MgCl₂ (pH 7.5, bubbled with 95%O₂/5%CO₂). Naïve mice were removed from their home cage and underwent identical decapitation procedures. Slices (300 μm) of the dorsal hippocampus and adjacent cortex were made using a Leica vibratome. The slices were first incubated for 30 min at 34°C in bubbled aCSF, and held at room temperature in bubbled aCSF for 1–4 h before use. Recording electrodes prepared from thin-walled capillary glass were filled with potassium methylsulfate-based internal solution and had a resistance of 5–6 MΩ.Whole-cell current-clamp recordings were performed on CA1 hippocampal pyramidal neurons of middle-aged learners (n = 36, 14 mice) and weak-learners (n = 15, 6 mice), as well as middle-aged naïve mice (n = 35 cells, 18 mice). Neuronal excitability was compared by measuring the post-burst AHP generated by 25 action potentials at 50 Hz (Fig. 3A), a stimulus shown to reliably evoke an AHP of sizable—but not maximal—amplitude from hippocampal neurons of mice (Ohno et al. 2006b). A significant difference in the peak amplitude of the AHP from learners, weak learners, and naïve mice was observed (F_(2,83) = 5, P < 0.01). Because the peak AHP and sAHP amplitudes did not differ between neurons from weak-learners and naïve mice (Fig. 3B). No differences in membrane resistance (F_(2,83) = 1.6, P = 0.2) or action potential properties, elicited using a brief (2 msec) near threshold current step (pA), were observed (Open in a separate window^aP < 0.05 compared to Weak L.^bP < 0.05 compared to Naïve.^cP < 0.05 compared to Pooled.Open in a separate windowFigure 3.Learning-related AHP plasticity is impaired in middle-aged weak-learner mice. (A) Representative traces showing the sAHP is reduced in neurons from (black) middle-aged learners compared to (blue) weak-learner mice and (gray) naïve mice. (Inset) The medium AHP (mAHP) of neurons from (black) learner mice was decreased compared to (blue) weak-learner mice and (gray) naïve mice. (B) No differences in the AHP from naïve and weak learners were observed; therefore, their data were pooled, and mean AHP was plotted by time on a log scale. (Inset) The mean amplitude of the peak AHP (1 msec) and sAHP (600 msec) was significantly reduced in neurons from learners compared to AHPs from weak learners and naïve labeled control; (*)P < 0.05.The results presented here are important in two respects. First, we demonstrate that the successful acquisition and recall of trace fear conditioning results in a significant reduction in the AHP in CA1 hippocampal neurons from the mouse. Our data are similar to previous reports showing learning-related reductions of the AHP in hippocampal neurons following training on hippocampal-dependent tasks (Disterhoft and Oh 2007) and thus strengthen the case for neuronal excitability change as a general mechanism underlying hippocampal-dependent learning. Second, we demonstrate that the onset of age-related cognitive decline in the C56Bl6/SJL mouse (termed “weak learners”) first manifests as a specific deficit in spatial associative learning in a subset of middle-age mice. These data, combined with a previous report from middle-aged rats (Moyer Jr and Brown 2006), suggest that initiation of age-related hippocampal dysfunction results in specific spatial—as opposed to temporal—deficits in associative learning and memory during middle age. By combining trace fear conditioning with whole-cell patch-clamp recordings in middle-aged mice, we revealed that “early” age-related impairments in spatial associative learning—like those in the aged hippocampus (Tombaugh et al. 2005)—result in part from an impairment of AHP plasticity of hippocampal neurons. Because AHP reductions are poised to facilitate mechanisms crucial for information storage, it is interesting that trace fear conditioning facilitates the long-term potentiation (LTP) of field excitatory postsynaptic potentials in the CA1 region of the rat hippocampus (Song et al. 2008).Generally speaking, both LTP and activation of AHP currents (I_AHP and sI_AHP) are sensitive to changes in intracellular Ca²⁺ (Storm 1990; Sah 1996; Malenka and Nicoll 1999). Thus, dysregulation of Ca²⁺ homeostasis in the hippocampus of middle-aged rats via enhancement of Ca²⁺-induced Ca²⁺ release (CICR) is an important finding (Gant et al. 2006). Age-related enhancement of Ca²⁺-dependent AHPs has been shown to raise the threshold for induction of LTP (Kumar and Foster 2004). These data support our hypothesis that impairments in contextual fear reported herein, as well as deficits in spatial water maze reported in middle-aged rats (Frick et al. 1995; Markowska 1999; Kadish et al. 2009), result from dysfunction of AHP plasticity.Studies in middle-aged mice have important implications for the treatment of “normal” age-associated cognitive decline (AACD), as well as mild cognitive impairment (MCI) (Pepeu 2004). Further studies aim to examine alterations in cholinergic function in our middle-aged mouse model, as the cholinergic agonist carbachol suppressed the AHP in neurons from naïve middle-aged mice (Supplemental Fig. 1). Activation of cholinergic receptors shape neuronal excitability and synaptic throughput (Tai et al. 2006) through multiple Ca²⁺-dependent processes (Gahwiler and Brown 1987; Tai et al. 2006). Restoration of cholinergic function has been shown to rescue deficits on hippocampal-dependent tasks in aged rodent and mouse models of Alzheimer''s disease (AD) (Disterhoft and Oh 2006), as well as in human AD patients (Cummings et al. 1998; Morris et al. 1998; Pettigrew et al. 1998), and therefore is a potential target aimed at the rescue of early age-related cognitive decline.     

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.     

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.     

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.     

Stimulation of the lateral geniculate,superior colliculus,or visual cortex is sufficient for eyeblink conditioning in rats

The role of the cerebellum in eyeblink conditioning is well established. Less work has been done to identify the necessary conditioned stimulus (CS) pathways that project sensory information to the cerebellum. A possible visual CS pathway has been hypothesized that consists of parallel inputs to the pontine nuclei from the lateral geniculate nucleus (LGN), superior colliculus (SC), pretectal nuclei, and visual cortex (VCTX) as reported by Koutalidis and colleagues in an earlier paper. The following experiments examined whether electrical stimulation of neural structures in the putative visual CS pathway can serve as a sufficient CS for eyeblink conditioning in rats. Unilateral stimulation of the ventral LGN (Experiment 1), SC (Experiment 2), or VCTX (Experiment 3) was used as a CS paired with a periorbital shock unconditioned stimulus. Stimulation was delivered to the hemisphere contralateral to the conditioned eye. Rats in all experiments were given five 100-trial sessions of paired or unpaired eyeblink conditioning with the stimulation CS followed by three paired sessions with a light CS. Stimulation of each visual area when paired with the unconditioned stimulus supported acquisition of eyeblink conditioned responses (CRs) and substantial savings when switched to a light CS. The results provide evidence for a unilateral parallel visual CS pathway for eyeblink conditioning that includes the LGN, SC, and VCTX inputs to the pontine nuclei.Pavlovian eyeblink (eyelid closure and nictitating membrane movement) conditioning is established by pairing a conditioned stimulus (CS), usually a tone or light, with an unconditioned stimulus (US) that elicits the eyeblink reflex. The eyeblink conditioned response (CR) emerges over the course of paired training, occurs during the CS, and precedes the US (Gormezano et al. 1962; Schneiderman et al. 1962). Neurobiological investigations of Pavlovian eyeblink conditioning have primarily focused on the cerebellum, which is the site of memory formation and storage (Thompson 2005). The anterior interpositus nucleus is necessary for acquisition and retention of the eyeblink CR (Lavond et al. 1985; Krupa and Thompson 1997; Freeman Jr. et al. 2005; Thompson 2005; Ohyama et al. 2006). Lobule HVI and the anterior lobe of the cerebellar cortex (lobules I–V) contribute to acquisition, retention, and timing of the CR (McCormick and Thompson 1984; Perrett et al. 1993; Perrett and Mauk 1995; Attwell et al. 1999, 2001; Medina et al. 2000; Nolan and Freeman Jr. 2005; Nolan and Freeman 2006). The brainstem nuclei that comprise the proximal ends of the CS and US input pathways to the cerebellum have also been identified.The pontine nuclei (PN) and inferior olive (IO) receive CS and US information, respectively, and are the primary sensory relays into the interpositus nucleus and cerebellar cortex (Thompson 2005). Conditioned stimulus information converges in the PN, which receives projections from lower brainstem, thalamus, and cerebral cortex (Glickstein et al. 1980; Brodal 1981; Schmahmann and Pandya 1989; Knowlton et al. 1993; Campolattaro et al. 2007). The lateral pontine nuclei (LPN) are the sources of auditory CS information projected into the cerebellum. Lesions of the LPN block CR retention to a tone CS, but have no effect on CRs to a light CS (Steinmetz et al. 1987). Thus, CS inputs from different sensory modalities may be segregated at the level of the PN. Neurons in the PN project CS information into the contralateral cerebellum via mossy fibers in the middle cerebellar peduncle that synapse primarily on granule cells in the cerebellar cortex and on neurons in the deep nuclei (Bloedel and Courville 1981; Brodal 1981; Steinmetz and Sengelaub 1992). Stimulation of the PN acts as a supernormal CS supporting faster CR acquisition than conditioning with peripheral stimuli (Steinmetz et al. 1986, 1989; Rosen et al. 1989; Steinmetz 1990; Tracy et al. 1998; Freeman Jr. and Rabinak 2004). The primary focus of these experiments was to investigate the most proximal components of the CS pathway in eyeblink conditioning. There has been less emphasis on identifying the critical CS pathways that project information to the PN.Recent studies using lesions, inactivation, stimulation, and neural tract tracing have provided evidence that the auditory CS pathway that is necessary for acquisition and retention of eyeblink conditioning is comprised of converging inputs to the medial auditory thalamic nuclei (MATN), and a direct ipsilateral projection from the MATN to the PN (Halverson and Freeman 2006; Campolattaro et al. 2007; Freeman et al. 2007; Halverson et al. 2008). Unilateral lesions of the MATN, contralateral to the conditioned eye, block acquisition of eyeblink CRs to a tone CS but have no effect on conditioning with a light CS (Halverson and Freeman 2006). Inactivation of the MATN with muscimol blocks acquisition and retention of CRs to an auditory CS, and decreases metabolic activity in the PN (Halverson et al. 2008). The MATN has a direct projection to the PN and stimulation of the MATN supports rapid CR acquisition (Campolattaro et al. 2007). Our current model of the auditory CS pathway consists of converging inputs to the MATN, and direct unilateral thalamic input to the PN (Halverson et al. 2008).Less work has been done to identify the visual CS pathway necessary for eyeblink conditioning. A possible parallel visual CS pathway has been hypothesized, which includes parallel inputs to different areas of the PN from the lateral geniculate nucleus (LGN), superior colliculus (SC), visual cortex (VCTX), and pretectal nuclei (Koutalidis et al. 1988). In the Koutalidis et al. study, lesions of the LGN, SC, VCTX, or pretectal nuclei alone had only a partial effect on CR acquisition with a light CS. Lesions of any two of these structures together produced a more severe impairment on acquisition and combined lesions of all of these areas completely blocked CR acquisition to a light CS (Koutalidis et al. 1988). Each visual area investigated in the Koutalidis et al. study has a direct projection to the PN that could be important for eyeblink conditioning. The ventral LGN projects to the medial, and to a lesser extent, the lateral PN (Graybiel 1974; Wells et al. 1989). The superficial, intermediate, and deep layers of SC send projections to both the dorsomedial and dorsolateral PN (Redgrave et al. 1987; Wells et al. 1989). The VCTX has a direct projection to the rostral and lateral portions of the PN (Glickstein et al. 1972; Baker et al. 1976; Mower et al. 1980; Wells et al. 1989). The pretectal nuclei also have a direct projection to both the medial and lateral PN (Weber and Harting 1980; Wells et al. 1989). However, stimulation of the anterior pretectal nucleus is not an effective CS for eyeblink conditioning (Campolattaro et al. 2007). The failure to establish conditioning with stimulation of the anterior pretectal nucleus as a CS suggests that there may be differences in the efficacy of the various visual inputs to the PN for cerebellar learning. The following experiments investigated the sufficiency of stimulation of the LGN, SC, or primary VCTX as a CS for eyeblink conditioning in rats.     

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.     

Beta-adrenergic receptor activation during distinct patterns of stimulation critically modulates the PKA-dependence of LTP in the mouse hippocampus

Activation of β-adrenergic receptors (β-ARs) enhances hippocampal memory consolidation and long-term potentiation (LTP), a likely mechanism for memory storage. One signaling pathway linked to β-AR activation is the cAMP-PKA pathway. PKA is critical for the consolidation of hippocampal long-term memory and for the expression of some forms of long-lasting hippocampal LTP. How does β-AR activation affect the PKA-dependence, and persistence, of LTP elicited by distinct stimulation frequencies? Here, we use in vitro electrophysiology to show that patterns of stimulation determine the temporal phase of LTP affected by β-AR activation. In addition, only specific patterns of stimulation recruit PKA-dependent LTP following β-AR activation. Impairments of PKA-dependent LTP maintenance generated by pharmacologic or genetic deficiency of PKA activity are also abolished by concurrent activation of β-ARs. Taken together, our data show that, depending on patterns of synaptic stimulation, activation of β-ARs can gate the PKA-dependence and persistence of synaptic plasticity. We suggest that this may allow neuromodulatory receptors to fine-tune neural information processing to meet the demands imposed by numerous synaptic activity profiles. This is a form of “metaplasticity” that could control the efficacy of consolidation of hippocampal long-term memories.The hippocampus importantly contributes to memory function in the mammalian brain (Zola-Morgan et al. 1986; Eichenbaum et al. 1990; Otto and Eichenbaum 1992; Phillips and LeDoux 1992; Remondes and Schuman 2004). It has reciprocal connections with numerous cortical areas, including those responsible for high-level integration of spatial and contextual data from the external environment (Lavenex and Amaral 2000). As such, the hippocampus is well positioned to receive and survey a broad range of information and select behaviorally salient data for long-term storage. Activity-dependent enhancement of hippocampal synaptic strength can store information carried in patterns of afferent neural activity (Bliss and Collingridge 1993; Moser et al. 1998; Nathe and Frank 2003; Whitlock et al. 2006). Substantial evidence suggests that long-term potentiation (LTP) of synaptic strength plays important roles in the formation of long-term memory (LTM) (Doyere and Laroche 1992; Bourtchuladze et al. 1994; Abel and Lattal 2001; Genoux et al. 2002). As such, mechanistic studies of LTP have shed valuable light on how the mammalian brain stores new information.The hippocampus receives dense noradrenergic projections from the locus coeruleus, a brain structure that can influence many vital brain functions, including attention, sleep, arousal, mood regulation, learning, and memory (Berridge and Waterhouse 2003). Both α- and β-adrenergic receptor subtypes are present on hippocampal neurons (Morrison and Foote 1986; Berridge and Waterhouse 2003), and noradrenaline (NA) acts on hippocampal β-adrenergic receptors (β-ARs) to facilitate the retention and recall of memory (Izquierdo et al. 1998; Ji et al. 2003; Murchison et al. 2004). In humans, stimulation of the noradrenergic neuromodulatory system enhances memory for emotional stimuli, and inhibition of β-ARs prevents this memory enhancement (Cahill et al. 1994; van Stegeren et al. 1998; O’Carroll et al. 1999).Consistent with the notion that selective enhancement of LTM may occur following β-AR activation, stimulation of β-ARs can also facilitate the persistence of LTP. In areas CA3 and CA1, β-AR activation facilitates the induction of long-lasting LTP when paired with certain patterns of electrical stimulation (Huang and Kandel 1996; Gelinas and Nguyen 2005). However, the mechanisms by which different patterns of stimulation control synaptic responsiveness to β-AR activation are unclear.β-ARs couple to guanine-nucleotide-binding regulatory Gs proteins to stimulate adenylyl cyclase activity and increase intracellular cAMP (Seeds and Gilman 1971; Maguire et al. 1977). A main target of cAMP signaling is activation of cAMP-dependent protein kinase (PKA), a kinase that is required for some forms of long-lasting LTP and for consolidation of hippocampal LTM (Frey et al. 1993; Abel et al. 1997; Nguyen and Woo 2003). Interestingly, the PKA-dependence of hippocampal LTP displays plasticity: Specific temporal patterns of synaptic stimulation, such as repeated and temporally spaced 100-Hz stimulation, elicit LTP that requires PKA for its expression (Woo et al. 2003). Also, spatial “enrichment” can increase the PKA-dependence of LTP in mice, and this is correlated with improved hippocampal memory function (Duffy et al. 2001). However, it is unclear whether activation of β-ARs can critically gate the PKA-dependence of LTP. In this study, we examine the effects of β-AR activation on LTP generated by various patterns of afferent stimulation in area CA1 of the hippocampus, and we determine the role of PKA in these β-AR-modulated forms of LTP.     

Stress impairs optimal behavior in a water foraging choice task in rats

Stress is a biologically significant social–environmental factor that plays a pervasive role in influencing human and animal behaviors. While stress effects on various types of memory are well characterized, its effects on other cognitive functions are relatively unknown. Here, we investigated the effects of acute, uncontrollable stress on subsequent decision-making performance in rats, using a computer vision-based water foraging choice task. Experiencing stress significantly impaired the animals'' ability to progressively bias (but not maintain) their responses toward the larger reward when transitioning from equal to unequal reward quantities. Temporary inactivation of the amygdala during stress, however, blocked impairing effects on decision making.It is now well documented that exposure to uncontrollable stress can produce alterations in multiple brain–memory systems in humans and animals (McEwen and Sapolsky 1995; Kim and Diamond 2002; Joels et al. 2006; Shors 2006; Luethi et al. 2008). In humans, impairments in long-term, but not short-term, verbal recall tasks have been observed in people with post-traumatic stress disorder (PTSD) (Bremner et al. 1995) or Cushing''s disease (a hypercortisolemia condition) (Starkman et al. 1992) and in healthy individuals subjected to stress (Lupien et al. 1997) or exposed to stress levels of cortisol (Newcomer et al. 1994). In rodents, stress and corticosterone administration interfere with spatial and working memory (Diamond and Rose 1994; de Quervain et al. 1998; Kim et al. 2001) and potentiate aversive conditioning (Shors et al. 1992; Maier et al. 1995). Further, a number of stress-associated neurobiological changes have been identified (e.g., in hippocampus, medial prefrontal cortex, and amygdala) subserving different memory functions (Arnsten and Goldman-Rakic 1998; Kim and Yoon 1998; Vyas et al. 2003; Holmes and Wellman 2009).Although stress effects on memory have been well studied, far less is known about whether (and in what manner) stress influences other higher cognitive functions. The present study investigated the effects of acute, uncontrollable stress (60-min restraint + 60 intermittent tailshocks) on subsequent decision-making performance in rats. The stress procedure, in which animals learn that they can neither escape nor predict an aversive experience, was adapted from earlier studies (e.g., Maier and Seligman 1976; Kim et al. 1996). Decision making was assessed using an automated Figure-8-shaped maze on which rats were motivated to forage for water rewards in two different locations under equal and unequal quantity conditions (Fig. 1). In addition to behavioral stress, we examined the effects of corticosterone administration and inactivation of the amygdala during stress on decision making. Both corticosterone (a glucocorticoid hormone released in response to stress) and the amygdala (a structure crucial in defensive behavior) have been implicated in mediating neurocognitive effects of stress (McEwen and Sapolsky 1995; Kim and Diamond 2002).Open in a separate windowFigure 1.Decision-making task. Thirst-motivated rats were trained to forage for water on an automated Figure-8-shaped maze. A computer algorithm controlled the raising and lowering of four gates (represented by rectangles), the delivery of water (blue circles), and tracking of the animal''s location on the maze. During the baseline days, both left and right sides of the maze presented 0.04 mL of water at 80% probability (equal reward trials). Following each trial (a left or right loop), the animal returned to the center bridge to start the next trial (40 trials per day). During the bias test days, one side of the maze (counterbalanced) offered 0.12 mL of water at 80% probability, while the other side continued to present 0.04 mL of water at 80% probability (unequal reward trials). Animals received either stress, CORT injections, or neither (for group details, see text).Experimentally naïve male Charles River Sprague–Dawley rats (initially weighing 275–300 gm) were singly housed and maintained on a reverse 12-h light-dark cycle (lights on at 19:00 h). After 7 d of acclimation and for the duration of the experiment, daily water access was restricted to maintain approximately 85% of the animal''s normal body weight. Food was available ad libitum throughout the experiment. All experiments were conducted during the dark phase of the cycle and in strict compliance with the University of Washington Institutional Animal Care and Use Committee guidelines. Under anesthesia (30 mg/kg ketamine and 2.5 mg/kg xylazine, i.p.) amygdala (AMYG) animals were chronically implanted with 26-gauge guide cannulae (Plastics One) bilaterally into the amygdala (from bregma: anteroposterior, −2.3 mm; mediolateral, ±5 mm; dorsoventral, −7.7 to 8.0 mm). During 10–15 d of postoperative recovery, each dummy cannula was removed and replaced with a clean one.Muscimol free base (Sigma-Aldrich), dissolved in artificial cerebrospinal fluid (10 mM at pH ∼7.4), was microinfused into the amygdala (bilaterally) via 33-gauge infusion cannulae that protruded 1 mm beyond the guide cannulae (cf. Kim et al. 2005). An infusion volume of 0.3 μL (per side) was delivered using a Harvard PHD2000 syringe pump (Harvard Apparatus) over the course of 3 min. Animals were returned to their home cages for 30 min before undergoing the stress procedure. We based the timing of inactivation on previous findings that pre-stress but not immediate post-stress inactivation of the amygdala interferes with stress effects on hippocampal long-term potentiation (LTP) and spatial memory (Kim et al. 2005). Based on studies that examined ³H-muscimol spreading (Krupa et al. 1993; Arikan et al. 2002) in the cerebellum, in which 1 μL diffused a radius of 1.6–2.0 mm, it was estimated that 0.3 μL of muscimol would spread within a radius of approximately 0.5–0.7 mm from the infusion needle tip. Hence, it is likely that infused muscimol would have diffused to the central, lateral, and basal nuclei of the amygdala and possibly to portions of adjacent neighboring structures. The BODIPY TMR-X muscimol conjugate (Invitrogen) was infused in the same manner as muscimol free base (cf. Allen et al. 2008) to image the spread of reversible amygdalar inactivation.Corticosterone (CORT) animals received three daily subcutaneous injections of 3 mg/kg corticosterone (suspended in sesame oil; Sigma-Aldrich) 30 min prior to bias testing.Rats undergoing stress were restrained in Plexiglas tubes and presented with 60 tailshocks (1-mA intensity, 1-sec duration, 5- to 115-sec variable intershock interval) for 60 min (Kim et al. 2005). Animals were divided into four groups: control, stress, amygdalar inactivation plus stress (AMYG), and daily corticosterone (CORT).Following 2 d of habituation (to transport, maze, and room ambiance), all animals underwent successive shaping and testing phases. The dimensions and automatic features of the Figure-8 maze have been described previously (Pedigo et al. 2006; Yoon et al. 2008); for details, see Supplemental material. During shaping, each rat is placed into the center runway with all four gates in the up position (Supplemental Fig. S1). After 3 sec, the front and one of the side gates drop, until the rat on its own volition moves out of the center and onto the open side runway. At this point, the lowered front and side gates rise (to prevent the rat from going backward), water is delivered to both the open arm and the center spout, and the back gate drops. The rat consumes water from the open arm, and a new trial begins when he returns to and consumes water from the center arm. There was a 3-sec delay between each trial. During shaping, left and right choices were thus forced choices and were presented in a pseudorandom pattern, such that there was an equal number of both in a complete session (40 laps). Rats underwent shaping once daily until they met predetermined criteria: completion of 40 laps in less than 30 min, and less than five back edge errors (i.e., after making a choice, running up the opposite arm instead of going back to the center arm). The automated program controlled the gates and water delivery, according to the rat''s position on the maze.During baseline testing (Supplemental Fig. S2), the rewards dispensed on left and right arms were equal in both volume (0.04 mL) and probability (0.8). Each rat remained on baseline testing until he demonstrated a stable left/right choice pattern across three consecutive days. If a slight preference to one side was present, the opposite arm would be the increased reward side when bias testing commenced.Stress and AMYG rats were exposed to restraint + tailshock stress 1 d before their first bias test, and CORT rats were given corticosterone injections 30 min prior to each bias test. During bias testing (Supplemental Fig. S3), the reward value on one side was tripled in volume (0.12 mL) while the value for the other side remained constant (0.04 mL). Control, AMYG, and CORT animals were given three consecutive days of bias tests, while stress animals underwent six consecutive days of bias tests.At the completion of behavioral testing, animals were overdosed with urethane and perfused intracardially with 0.9% saline followed by 10% buffered formalin. The brains were removed and stored in 10% formalin overnight and then kept in 30% sucrose solution until they sank. Transverse sections (50 μm) were taken through the extent of the cannulae tract, mounted on gelatin-coated slides, and stained with cresyl violet to verify cannulae placements.The visit number to the left (or right) side of the maze for baseline and bias days were normalized to the mean left (or right) visits across the three baseline days. Statistical comparisons between groups were examined using ANOVA. For a significant difference (P < 0.05), post-hoc comparisons were performed using Tukey''s honestly significant difference test.Thirst-motivated rats readily learned to forage for water on the maze, and when left and right sides of the maze provided the same quantity (0.04 mL) and probability (80%) of water, the animals made comparable numbers of left and right visits (during 40 laps daily) that were stable across three baseline days (Fig. 2A). The 80% probability (a partial reinforcement schedule) was used so that animals frequently explored both sides of the maze. After animals demonstrated stable baseline choices, the volume of water on one side was tripled (0.12 mL at 80%), while the other side remained constant (0.04 mL at 80%). Overall, bias performance (choice of the larger reward) increased across the first three bias test days (repeated-measures ANOVA; main effect of day, F_(2,54) = 78.16, P < 0.001). However, the four groups differentially increased their bias across days (group × day interaction, F_(6,54) = 4.14, P < 0.01). Specifically, stressed rats displayed a significantly slower rate of bias toward the larger reward than did the controls (P < 0.01, Tukey). The stressed rats did ultimately develop a bias compared with their baseline choices (F_(6,62) = 8.44, P < 0.001). This bias was first significant on the fourth day (P < 0.05, Dunnett t-test). However, even after 6 d of bias testing, their bias (127 ± 5.7%, mean ± SEM) did not reach the level of controls'' third day bias (182 ± 12.2%). Unlike the behavioral stress group, however, animals that received three daily corticosterone injections (3 mg/kg, subcutaneously) prior to testing chose the larger reward side more frequently (173 ± 6.7%, bias day 3) and did not differ from the control group (P > 0.7, Tukey). When the amygdalae were inactivated during stress (Fig. 3), these animals behaved like controls (P > 0.7, Tukey) and increased their visit frequency toward the larger reward side of the maze (174 ± 13.0%, bias day 3) (Fig. 2A). Although control, CORT, and AMYG animals developed strong bias responses, they did not exclusively visit the larger reward side of the maze because on 20% of trials they did not receive a reward.Open in a separate windowFigure 2.Stress effects on decision making. (A) All groups of animals showed comparable visits to left and right sides of the maze during the three baseline days. When transitioning from equal to unequal reward trials, stressed rats (n = 7) displayed an impaired ability to bias their responses toward the larger reward side compared with control (n = 10), AMYG (n = 7), and CORT (n = 7) rats (P = 0.002, group × bias day interaction). (B) Example visit maps of a control rat during baseline and bias days (40 laps each).Open in a separate windowFigure 3.(Top) Histological reconstruction of injection cannulae placement tips in the amygdala. (Bottom) A photomicrograph of fluorophore-conjugated muscimol (0.3 μL over 3 min) spread in the amygdala. The red fluorescence is overlaid with a dark field image.We then examined whether stress might have produced alterations in motor, motivation, and reference memory performances that hindered the animals'' ability to bias their responses toward the larger reward. The latency to complete 40 laps of the first bias test session (Supplemental Fig. S4A) showed a trend of stress animals completing the bias test faster than the other three groups, but this group × day interaction was not significant (F_(6,54) = 1.89, P > 0.05). Stress also did not impair the animals'' reference memory of the maze (Supplemental Fig. S4B). That is, after making a left or right visit, stressed animals readily re-entered the center runway to start the next trial (one-way ANOVA; average baseline and first three bias days, F_(3,41) = 0.20, P > 0.8), whereas control, CORT, and AMYG animals displayed an increased propensity to investigate the other side before re-entering the center, particularly as bias testing progressed (repeated-measures ANOVA; main effect of day, F_(2,54) = 4.84, P < 0.05).Our results indicate that rats clearly demonstrate the capacity to change their foraging behavior to acquire a larger water reward when transitioning from equal to unequal quantities, and that such behavioral flexibility is vulnerable to acute, uncontrollable stress. Specifically, rats that experienced 1 h of restraint stress + 60 intermittent tailshocks were significantly impaired in biasing their responses toward the side of the maze with a larger quantity of water. This effect on bias was not due to any lingering post-stress motivational or motor effects, as stress did not increase the latency to complete the bias test. Daily corticosterone injections did not interfere with this task, indicating that corticosterone elevation per se cannot reproduce behavioral stress effects on behavioral flexibility. However, similar to previous stress–memory studies (Kim et al. 2001; Waddell et al. 2008), amygdalar inactivation during stress effectively blocked this effect. This suggests that the amygdala plays a crucial role in mediating stress effects across different cognitive domains.Although stress altered the rats'' behavior in our choice-based task, it remains unclear precisely which neural and cognitive systems were affected. For instance, the impairment of behavioral flexibility might be an indirect consequence of stress effects on hippocampal memory functioning. The stress paradigm used here is known to impede LTP in the CA1 hippocampus and hinder spatial memory (Foy et al. 1987; Kim et al. 2001). However, corticosterone, which also impedes LTP (McEwen and Sapolsky 1995) and spatial memory when administered 30 min before testing (de Quervain et al. 1998), did not impair behavioral flexibility. The impairment to choose the larger reward may be due to stress effects on working memory, such that the rats cannot remember (and thus learn) that one reward is larger. However, if true, the stress-induced memory impairment is unusually persistent in our task: Rats were affected through at least 6 d beyond the stress exposure. Because acquisition and retrieval of information are integral components of decision making, the contribution of stress-associated changes in learning and memory cannot be fully excluded. Another possibility is that stressed rats are more likely to use habitual rather than flexible strategies, even when a change in behavior may be optimal. Consistent with this explanation are recent findings that chronic stress exposure increases habit-based responding, with corresponding atrophy and hypertrophy of goal-directed and habit-based neural circuitry, respectively (Dias-Ferreira et al. 2009). The reliance on habit memory is also increased following anxiogenic drug infusions into the amygdala (Elliott and Packard 2008), which further implicates amygdalar modulatory activity during and after stress exposure in decreasing flexible behavior. If the stress experience did increase perseverative choice behavior, this may partially explain previously observed associations between distress and perseveration in humans (Robinson et al. 2006). Alternately, stress may have disrupted the reward circuitry and impaired the ability to discriminate between the two reward values from the two side arms, in which case stress effects on a dopamine-related reward circuit (Schultz et al. 1997) should be explored. However, this cannot be the sole explanation because post-bias stress did not affect the animals'' bias behavior toward the larger reward (Supplemental Table S1), and nonstressed rats resume equal arm visits when rewards are returned to baseline values (data not shown).The present findings reveal that a single exposure to an acutely stressful experience is enough to affect an animal''s behavior on a simple forging task for several days. There is accumulating evidence that exposure to stress increases amygdala and decreases prefrontal activity in both humans and animals (for review, see Arnsten 2009) and that even a single stress exposure alters cellular morphology in prefrontal cortex (Izquierdo et al. 2006). This shift in neural activity may promote the use of one cognitive strategy over another (e.g., habitual versus flexible). To address this, future studies need to investigate brain structures implicated in decision making, including the prefrontal and the parietal cortices (Gold and Shadlen 2007; Lee 2008), for their susceptibility to stress. Regardless, the present findings, to our knowledge, provide the first direct evidence that acute uncontrollable stress persistently impairs decision-making performance in animals and that this effect is dependent upon amygdalar activity during stress.     

Ventral lateral geniculate input to the medial pons is necessary for visual eyeblink conditioning in rats

The conditioned stimulus (CS) pathway that is necessary for visual delay eyeblink conditioning was investigated in the current study. Rats were initially given eyeblink conditioning with stimulation of the ventral nucleus of the lateral geniculate (LGNv) as the CS followed by conditioning with light and tone CSs in separate training phases. Muscimol was infused into the medial pontine nuclei (MPN) after each training phase to examine conditioned response (CR) retention to each CS. The spread of muscimol infusions targeting the MPN was examined with fluorescent muscimol. Muscimol infusions into the MPN resulted in a severe impairment in retention of CRs with the LGNv stimulation and light CSs. A less severe impairment was observed with the tone CS. The results suggest that CS information from the LGNv and light CSs is relayed to the cerebellum through the MPN. Retrograde tracing with fluoro-gold (FG) showed that the LGNv and nucleus of the optic tract have ipsilateral projections to the MPN. Unilateral inputs to the MPN from the LGNv and nucleus of the optic tract may be part of the visual CS pathway that is necessary for visual eyeblink conditioning.The neural substrates of associative motor learning have been studied extensively using eyeblink conditioning (Christian and Thompson 2003; Thompson 2005). Eyeblink conditioning is typically established by pairing a tone or light conditioned stimulus (CS) with an unconditioned stimulus (US) that elicits the eyeblink reflex. An eyeblink conditioned response (CR) emerges over the course of paired training, and the peak of eyelid closure occurs at the onset time of the US. Results from experiments using temporary lesions of the cerebellar deep nuclei or cerebellar cortex indicate that the anterior interpositus nucleus and cerebellar cortex are necessary for acquisition, expression, and extinction of eyeblink conditioning (Krupa et al. 1993; Hardiman et al. 1996; Krupa and Thompson 1997; Garcia and Mauk 1998; Medina et al. 2001; Bao et al. 2002; Freeman et al. 2005a). Blocking cerebellar output with inactivation of the superior cerebellar peduncle, red nucleus, or brainstem motor nuclei selectively blocks CR expression but not acquisition, providing further evidence that learning occurs in the cerebellum (Chapman et al. 1990; Krupa et al. 1993, 1996; Krupa and Thompson 1995).Sensory stimuli from every modality are sent to the pontine nuclei (PN), which receive projections from the lower brainstem, thalamus, and cerebral cortex (Glickstein et al. 1980; Brodal 1981; Mihailoff et al. 1989; Schmahmann and Pandya 1989; Wells et al. 1989; Knowlton et al. 1993; Campolattaro et al. 2007). Neurons in the PN project CS information to the cerebellum via mossy fibers in the middle cerebellar peduncle that synapse on granule cells in the cerebellar cortex and on neurons in the interpositus nucleus (Bloedel and Courville 1981; Brodal 1981; Steinmetz and Sengelaub 1992; Mihailoff 1993). Lesions of the middle cerebellar peduncle impair eyeblink conditioning with auditory, somatosensory, and visual CSs (Lewis et al. 1987). Bilateral electrolytic lesions of the dorsolateral and lateral pontine nuclei (LPN) block retention of CRs to an auditory CS but have no effect on light-elicited CRs (Steinmetz et al. 1987). Inactivation of the contralateral LPN blocks CRs to a tone CS but not to lateral reticular nucleus stimulation in rabbits (Bao et al. 2000). Moreover, stimulation of the LPN or middle cerebellar peduncle is a sufficient CS for eyeblink conditioning (Steinmetz et al. 1986, 1987; Tracy et al. 1998; Bao et al. 2000; Freeman and Rabinak 2004; Freeman et al. 2005b; Campolattaro and Freeman 2008). The findings from the lesion, inactivation, and stimulation studies provide evidence that the PN is the proximal part of the CS pathway for cerebellar learning. These studies also indicate that the LPN is the primary source of auditory CS input to the cerebellum.Only a few studies have examined the visual CS pathway necessary for eyeblink conditioning. The dorsal and ventral divisions of the lateral geniculate nucleus of the thalamus (LGNd, LGNv), pretectal nuclei, visual cortex (VCTX), and superior colliculus (SC) comprise a hypothesized parallel visual CS pathway for eyeblink conditioning (Koutalidis et al. 1988). Combined lesions of all of these visual areas completely block acquisition, lesions of two visual areas produce a partial impairment, and lesions in one visual area do not impair CR acquisition (Koutalidis et al. 1988). Stimulation of the VCTX, SC, and LGNv support eyeblink conditioning, and each of these structures has a direct unilateral projection to the PN that could be important for eyeblink conditioning (Halverson et al. 2009). The lesion and stimulation studies provide evidence that structures in the hypothesized visual CS pathway are independently capable of supporting conditioning. An important aspect of the visual CS pathway proposed in the Koutalidis et al. (1988) study is distributed projections of each visual area to different regions of the PN. The important projections were hypothesized to be from the VCTX to the rostral portion of the PN, from both the SC and pretectal nuclei to the dorsolateral PN, and the LGNv projection to the medial pontine nuclei (MPN) (Koutalidis et al. 1988). Lesions of the VCTX were substituted for LGN lesions in the Koutalidis et al. (1988) study due to technical problems with animal survival. The LGNv projection to the MPN was therefore not examined in their combined lesion group. Stimulation of the anterior pretectal nucleus is not a sufficient CS to support eyeblink conditioning (Campolattaro et al. 2007). The direct PN projection from the VCTX is not necessary for CR retention to a light CS, as lesions do not prevent eyeblink conditioning to a light CS in dogs or monkeys (Hilgard and Marquis 1935, 1936). Moreover, lesions of the entire cerebral cortex do not prevent acquisition or retention of delay eyeblink conditioning to a tone or light CS in rabbits (Oakley and Russell 1972, 1977). The LGNv and SC, therefore, are likely sources of visual input to the PN that is necessary for eyeblink conditioning.The current experiment investigated whether information from the LGNv and a visual CS (light) share similar inputs into the MPN and whether those inputs are different from an auditory CS. The visual projections to the MPN were also investigated with the retrograde tracer fluoro-gold (FG) to identify structures that may be involved with the relay of CS information during eyeblink conditioning. In the conditioning experiment, rats received three phases of training, with each phase consisting of three acquisition sessions followed by a muscimol infusion into the MPN, and then a saline recovery session. Each rat received unilateral stimulation of the LGNv (contralateral to the trained eye) during phase 1 of training followed by either a tone or light CS in phases 2 and 3 (order of stimulus presentation was counterbalanced). One group received LGNv stimulation in phase 1 followed by a light CS in phase 2, and a tone CS in phase 3 (SLT). The other group received the tone CS in phase 2, and light CS in phase 3 (STL).     

Environmental novelty is associated with a selective increase in Fos expression in the output elements of the hippocampal formation and the perirhinal cortex

If the hippocampus plays a role in the detection of novel environmental features, then novelty should be associated with altered hippocampal neural activity and perhaps also measures of neuroplasticity. We examined Fos protein expression within subregions of rat hippocampal formation as an indicator of recent increases in neuronal excitation and cellular processes that support neuroplasticity. Environmental novelty, but not environmental complexity, led to a selective increase of Fos induction in the final “output” subregion of the dorsal hippocampal trisynaptic circuit (CA1) and a primary projection site (layer five of the lateral entorhinal cortex, ERC), as well as in the perirhinal cortex. There was no selective effect of novelty on Fos expression within “input” elements of the trisynaptic circuit (ERC layer two, the dentate gyrus or CA3) or other comparison brain regions that may be responsive to overall motor-sensory activity or anxiety levels (primary somatosensory and motor cortex or hypothalamic paraventricular nucleus). Test session ambulatory behavior increased with both novelty and environmental complexity and was not significantly correlated with Fos expression patterns in any of the brain regions examined. In contrast, the extent of manipulated environmental novelty was strongly correlated with Fos expression in CA1. These results support the prospect that a novelty-associated signal is generated within hippocampal neurocircuitry, is relayed to cortical projection sites, and specifically up-regulates neuroplasticity-supporting processes with dorsal hippocampal CA1 and ERC layer five. Whether novelty-dependent Fos induction in perirhinal cortex depends on this hippocampal output or reflects an independent process remains to be determined.The hippocampus appears to play an essential role in the encoding of configural and temporal relationships between experiential elements thereby supporting memory for environmental contexts and discrete episodes (Rudy and Sutherland 1995). A related hypothesis is that the hippocampus serves as a functional comparator of present and past (stored) experience, and consequently directs attention and mnemonic processes to the novel aspects of present experience (Margulies 1985; Otto and Eichenbaum 1992; Knight 1996; Mizumori et al. 1999; Moser and Paulsen 2001; Vinogradova 2001; Fyhn et al. 2002; Norman and O''Reilly 2003). A comparator capability of the hippocampus seems plausible given the converging parallel neural pathways by which multimodal sensory information is presented to the hippocampus. The entorhinal cortex serves as an anatomical gateway through which the majority of cortically processed information is presented to the hippocampus. This cortical information is relayed directly (via monosynaptic connections) to CA1 neurons (originating primarily from layer three of the entorhinal cortex) or to CA3 neurons (originating primarily from layer two of the entorhinal cortex) (Steward and Scoville 1976; Remondes and Schuman 2004; Witter and Amaral 2004). In addition, CA1 neurons are presented with cortical information (originating primarily from layer two of the entorhinal cortex) that has first been processed by the dentate gyrus and CA3, via the serial connections of the hippocampal formation trisynaptic circuit (Andersen et al. 1971). Although both CA1 and CA3 neurons receive direct and indirect neural input from entorhinal cortex, several hippocampal-circuit models propose that CA1 neurons have unique access to both past (stored) and ongoing experiential neural patterns (Hasselmo and Schnell 1994; Moser and Paulsen 2001; Norman and O''Reilly 2003). Alternatively, other models posit an important role of CA3 neurons (Mizumori et al. 1999; Vinogradova 2001; Lee et al. 2005a) and/or dentate gyrus granule cells (Meeter et al. 2004; Lee et al. 2005a) in the detection of novel features of experience.Implicit in these models of hippocampal function is the assumption that the hippocampus is engaged differently when presented with novel versus familiar stimuli patterns. There is some evidence for experience-dependent differences in rodent hippocampal activity that are manifest by electrophysiological differences in individual or ensemble neuronal activity patterns (Otto and Eichenbaum 1992; Fyhn et al. 2002; Nitz and McNaughton 2004). Neuroimaging studies in humans have detected increased fMRI activity in the hippocampal region during encoding of novel visual stimuli (Stern et al. 1996; Johnson et al. 2008). Moreover, humans with hippocampal damage exhibit altered event-related potentials in response to novel stimuli (Knight 1996).Hippocampal activity that varies with the novelty of an experience may be important for guiding ongoing behavior (e.g., exploratory behavior and vigilance), and if so, should also produce detectable differences in activity of hippocampal efferents. In addition, detection of novelty may be important for altering neuroplastic processes within components of the hippocampus. The goal of our study was to examine across hippocampal formation subregions the levels of a cellular marker of neural activity and neuroplasticity (Fos expression) associated with environmental experiences that vary in novelty and complexity. The expression of the protein product, Fos, of the immediate early gene, c-fos, may be a good molecular indicator of recent increases in general molecular changes that contribute to neuroplasticity. Expression of Fos reflects an intracellular state of cells that varies primarily as a result of recent activation by intercellular signals (e.g., neurotransmitters, hormones, paracrine factors, and adhesion molecules) (Herdegen and Leah 1998). Hippocampal Fos expression is associated with recent increases in neuronal firing, although apparently in a complex fashion (Labiner et al. 1993). Increases in hippocampal Fos is also believed to be an important mediator of activity-dependent neuroplasticity (Sheng and Greenberg 1990).In our study we examined the number of Fos immunopositive cells in the dentate gyrus, subregions of the hippocampus (CA1, CA2, CA3, and CA4), and layers two and five of the lateral entorhinal cortex. In addition, we examined Fos immunoreactivity in the perirhinal cortex. There is accumulating support for this brain region to play a role in the detection of novel stimuli in a configuration independent manner (Brown and Aggleton 2001; Kumaran and Maguire 2007). For comparison purposes, we also examined Fos expression patterns in primary somatosensory cortex, primary motor cortex, and the hypothalamic paraventricular nucleus (PVN). Fos expression levels in the somatosensory and motor cortex may reflect the varying amounts of somatosensation and motor activity present during the experimental test-day experiences. Fos expression levels in the PVN may reflect the varying amounts of test-day stress and anxiety associated with the different treatment conditions.Several other rat studies have examined the relationship between stimuli novelty (e.g., visual images, extramaze environmental cues, or new learning tasks) and Fos expression in the hippocampus (Hess et al. 1995a; Wan et al. 1999; Vann et al. 2000). Whereas those other studies utilized tasks that had a training phase and operant reward component, our study examined Fos expression in rats placed in a novel or familiar environment with no training components or operant contingencies. The pattern of Fos expression associated with unrewarded exploratory behavior may better reflect the extent to which novelty and complexity differentially and automatically engage the hippocampus than does the pattern of Fos expression associated with various learning regimens and their particular task demands (Kumaran and Maguire 2007).     

Reciprocal patterns of c-Fos expression in the medial prefrontal cortex and amygdala after extinction and renewal of conditioned fear

After extinction of conditioned fear, memory for the conditioning and extinction experiences becomes context dependent. Fear is suppressed in the extinction context, but renews in other contexts. This study characterizes the neural circuitry underlying the context-dependent retrieval of extinguished fear memories using c-Fos immunohistochemistry. After fear conditioning and extinction to an auditory conditioned stimulus (CS), rats were presented with the extinguished CS in either the extinction context or a second context, and then sacrificed. Presentation of the CS in the extinction context yielded low levels of conditioned freezing and induced c-Fos expression in the infralimbic division of the medial prefrontal cortex, the intercalated nuclei of the amygdala, and the dentate gyrus (DG). In contrast, presentation of the CS outside of the extinction context yielded high levels of conditioned freezing and induced c-Fos expression in the prelimbic division of the medial prefrontal cortex, the lateral and basolateral nuclei of the amygdala, and the medial division of the central nucleus of the amygdala. Hippocampal areas CA1 and CA3 exhibited c-Fos expression when the CS was presented in either context. These data suggest that the context specificity of extinction is mediated by prefrontal modulation of amygdala activity, and that the hippocampus has a fundamental role in contextual memory retrieval.Considerable interest has emerged in recent years in the neural mechanisms underlying the associative extinction of learned fear (Maren and Quirk 2004; Myers et al. 2006; Quirk and Mueller 2008). Notably, extinction is a useful model for important aspects of exposure-based therapies for the treatment of human anxiety disorders such as panic disorder and post-traumatic stress disorder (PTSD) (Bouton et al. 2001, 2006). During extinction, a conditioned stimulus (CS) is repeatedly presented in the absence of the unconditioned stimulus (US), a procedure that greatly reduces the magnitude and probability of the conditioned response (CR). After the extinction of fear, there is substantial evidence that extinction does not erase the original fear memory, but results in a transient inhibition of fear. For example, extinguished fear responses return after the mere passage of time (i.e., spontaneous recovery) or after a change in context (i.e., renewal) (Bouton et al. 2006; Ji and Maren 2007). In other words, extinguished fear is context specific. The return of fear after extinction is a considerable challenge for maintaining long-lasting fear suppression after exposure-based therapies (Rodriguez et al. 1999; Hermans et al. 2006; Effting and Kindt 2007; Quirk and Mueller 2008).In the last several years, considerable progress has been made in understanding the neural mechanisms underlying the context specificity of fear extinction. For example, lesions or inactivation of the hippocampus prevent the renewal of fear when an extinguished CS is presented outside of the extinction context (Corcoran and Maren 2001, 2004; Corcoran et al. 2005; Ji and Maren 2005, 2008; Hobin et al. 2006). In addition, neurons in the basolateral complex of the amygdala exhibit context-specific spike firing to extinguished CSs (Hobin et al. 2003; Herry et al. 2008), and this requires hippocampal input (Maren and Hobin 2007). Indeed, amygdala neurons that fire more to extinguished CSs outside of the extinction context are monosynaptically excited by hippocampal stimulation (Herry et al. 2008). In contrast, neurons that responded preferentially to extinguished CSs in the extinction context receive synaptic input from the medial prefrontal cortex (Herry et al. 2008).The prevalent theory of the interactions between the prefrontal cortex, hippocampus, and amygdala that lead to regulation of fear by context assumes that when animals experience an extinguished CS in the extinction context, the hippocampus drives prefrontal cortex inhibition of the amygdala to suppress fear (Hobin et al. 2003; Maren and Quirk 2004; Maren 2005). When animals encounter an extinguished CS outside of the extinction context, the hippocampus is posited to inhibit the prefrontal cortex and thereby promote amygdala activity required to renew fear. The hippocampus may also drive fear renewal through its direct projections to the basolateral amygdala (Herry et al. 2008). Although this model accounts for much of the extant literature on the context specificity of extinction, it is not known whether the nodes of this hypothesized neural network are coactive during the retrieval of fear and extinction memories. As a first step in addressing this issue, we used ex vivo c-Fos immunohistochemistry (e.g., Knapska et al. 2007) to generate a functional map of the neural circuits involved in the contextual retrieval of fear memory after extinction. Our results reveal reciprocal activity in prefrontal-amygdala circuits involved in extinction and renewal and implicate the hippocampus in hierarchical control of contextual memory retrieval within these circuits.     

Odor-specific habituation arises from interaction of afferent synaptic adaptation and intrinsic synaptic potentiation in olfactory cortex

Segmentation of target odorants from background odorants is a fundamental computational requirement for the olfactory system and is thought to be behaviorally mediated by olfactory habituation memory. Data from our laboratory have shown that odor-specific adaptation in piriform neurons, mediated at least partially by synaptic adaptation between the olfactory bulb outputs and piriform cortex pyramidal cells, is highly odor specific, while that observed at the synaptic level is specific only to certain odor features. Behavioral data show that odor habituation memory at short time constants corresponding to synaptic adaptation is also highly odor specific and is blocked by the same pharmacological agents as synaptic adaptation. Using previously developed computational models of the olfactory system we show here how synaptic adaptation and potentiation interact to create the observed specificity of response adaptation. The model analyzes the mechanisms underlying the odor specificity of habituation, the dependence on functioning cholinergic modulation, and makes predictions about connectivity to and within the piriform neural network. Predictions made by the model for the role of cholinergic modulation are supported by behavioral results.Filtering sensory input is critical for information processing tasks such as background segmentation, and shifting processing power away from redundant, stable, or repetitive stimuli toward dynamic, novel stimuli. A critical aspect of this filtering however, is stimulus specificity. Under most circumstances it may be most beneficial to selectively filter the redundant stimulus, while maintaining responsiveness to different, though perhaps highly similar stimuli.In the olfactory system, short-term habituation to stable or repeated odorants involves a metabotropic glutamate receptor (mGluR)-dependent depression of afferent synapses to the piriform cortex (Best and Wilson 2004). Blockade of group III mGluR receptors prevents cortical adaptation odors (Best and Wilson 2004), and reduces short-term habituation of odor-evoked reflexes (Best et al. 2005) and odor investigation (Yadon and Wilson 2005; Bell et al. 2008; McNamara et al. 2008). This short-term habituation is highly odor specific, with minimal cross-adaptation of piriform cortical single-unit responses or cross-habituation of behavioral responses to similar odors, including between mixtures and their components (Wilson 2000; Cleland et al. 2002). Interestingly, there is an experience-dependent component to short-term habituation odor specificity. The odor specificity is most pronounced for familiar odors, with very brief (<20 sec) exposure to odors producing more generalization, and longer exposures (>50 sec) sufficient to permit strong odor specificity in cortex adaptation (Wilson 2003).The homosynaptic nature of afferent synaptic depression underlying cortical adaptation (Wilson 1998; Best and Wilson 2004) may contribute to this odor specificity. However, the experience dependence suggests that there may be an additional process involved. In fact, theoretical views of piriform cortical function suggest that the cortex learns previous patterns of input via potentiation of intracortical association fiber synapses (Hasselmo et al. 1990; Barkai et al. 1994; Haberly 2001; Linster et al. 2003). This autoassociative process essentially creates a template of previous network activity, against which new input patterns can be compared, allowing enhanced discrimination between similar patterns, as well as completion of degraded patterns (Barkai et al. 1994; Barnes et al. 2008). In support of this hypothesis, previous work has demonstrated that disruption of normal synaptic potentiation in association fiber synapses through blockade of cholinergic muscarinic receptors (Patil et al. 1998; Linster et al. 2003), reduces odor specificity of cortical adaptation (Wilson 2001b), prevents the effects of odor experience on subsequent behavioral cross-habituation (Fletcher and Wilson 2002), and disrupts odor discrimination (Linster et al. 2001).The present series of studies further explored the role of combined afferent synaptic depression and intracortical association fiber synaptic potentiation on the specificity of cortical adaptation and odor habituation. Using a computational model of the olfactory system (Linster et al. 2007), the results suggest that activity-dependent association fiber plasticity is necessary to account for the specificity of odor habituation. Furthermore, in behavioral experiments blockade of cholinergic muscarinic receptors during habituation enhances generalization of odor habituation, consistent with the modeling and with previous electrophysiological results.     

Nuclear disconnection within the amygdala reveals a direct pathway to fear

It is widely believed that a descending serial circuit consisting of neural projections from the basolateral complex (BLA) to the central nucleus (CEA) of the amygdala mediates fear expression. Here we directly test this hypothesis and show that disconnecting the BLA and CEA with asymmetric neurotoxic lesions after Pavlovian fear conditioning in rats completely abolishes the expression of conditional freezing. These results demonstrate that neural projections from the BLA to CEA are essential for the expression of learned fear responses.Long-standing anatomical models of the brain circuitry underlying learned fear posit serial flow of information through the amygdala to engage the expression of fear responses (LeDoux 2000; Maren 2001). Specifically, conditioning-induced plasticity in the lateral nucleus of the amygdala (LA) is thought to drive learned fear through excitatory axonal projections to the basal nuclei of the amygdala (BA; basolateral and basomedial nuclei), which in turn send unidirectional and excitatory synaptic projections to the medial division of the central nucleus of the amygdala (CEm). Neurons in the CEm project to brain structures involved in the production of a variety of fear responses (Pitkänen et al. 1997; Swanson and Petrovich 1998). Alternatively, neurons in LA might excite neurons in CEm by limiting inhibitory input from the intercalated cell masses (ITC) interposed between the basolateral complex (BLA; lateral and basal nuclei) and the central nucleus of the amygdala (CEA) (Paré et al. 2004). In either case, the BLA is well positioned to drive learned fear responses via anatomical connections with the CEA.Although an extensive literature demonstrates the importance of both the BLA and CEA in the acquisition and expression of fear (LeDoux et al. 1990; Lee et al. 1996; Amorapanth et al. 2000; Goosens and Maren 2001), it is not known whether a functional connection between the two structures is essential for the expression of learned fear. Indeed, the BLA and CEA make independent contributions to aversively motivated learning under some conditions (Killcross et al. 1997; Amorapanth et al. 2000). Moreover, recent work in appetitive conditioning paradigms challenges the necessity of serial circuits in the amygdala for associative learning processes (Holland and Gallagher 1999; Everitt et al. 2003; Balleine and Killcross 2006). It is therefore essential to determine whether serial connections between the BLA and CEA are involved in the expression of fear memories as widely assumed in the literature.To address this issue, we made asymmetric neurotoxic lesions of the BLA and CEA after Pavlovian fear conditioning in rats. That is, we placed BLA lesions in one hemisphere and CEA lesions in the contralateral hemisphere, thereby producing a functional disconnection of the two brain regions. Control animals received neurotoxic BLA and CEA lesions in the same hemisphere, thereby leaving both structures and their connections intact in one hemisphere. This disconnection strategy (e.g., Olton et al. 1982) capitalizes on the fact that projections from the BLA to CEA are both ipsilateral and unidirectional (Pitkänen et al. 1997). It has been used by several groups to assess the contribution of connections between brain areas to learning and memory processes, including conditioned stimulus processing (Han et al. 1999) and appetitive spatial learning (Ito et al. 2008), for example.Fear conditioning was conducted in standard observation chambers (see Supplemental Methods) and consisted of five pairings of an auditory conditional stimulus (CS) (2 kHz, 10 sec, 80 dB) with a footshock unconditioned stimulus (US) (2 sec, 1 mA); the intertrial interval (ITI) was 1 min. Freezing behavior served as the measure of conditional fear. Twenty-four hours after fear conditioning, the rats were deeply anesthetized, and amygdala lesions were made by infusing N-methyl-d-aspartate (NMDA; 20 mg/mL in 100 mM phosphate buffered saline with a pH 7.4; Sigma) into the CEA and BLA through a 28-g injection cannula attached to a Hamilton syringe via polyethylene tubing. Control rats received unilateral lesions of the BLA and CEA in the same hemisphere (IPSI, n = 13) or sham surgery (SHAM, n = 16), whereas experimental rats received the unilateral BLA and CEA lesions in opposite hemispheres (CONTRA, n = 11). Hence, functional connectivity between the BLA and CEA was left intact in one hemisphere among rats in the IPSI group, whereas the asymmetric lesions in rats in the CONTRA group eliminated this connection. Retention tests assessing fear to the conditioning context and tone CS were conducted in separate sessions one week after recovery from surgery. Histological examination of coronal brain sections obtained from the subjects after the experiment revealed selective CEA and BLA lesions in each group; representative lesions are illustrated in Figure 1, A and B. The lesions spared fibers of passage (Fig. 1C).Open in a separate windowFigure 1.Disconnection of the BLA and CEA impairs the expression of conditional fear. (A) Schematic representation of a typical BLA (shaded) and CEA (black) lesions among rats with unilateral lesions placed in the same hemisphere (IPSI) or in opposite hemispheres (CONTRA). (B) Photomicrograph of a thionin-stained coronal section from a representative rat in the CONTRA group. The image has been cropped to include only the left and right amygdale. Broken lines encircle CEA and BLA lesions in the left and right hemispheres, respectively. (C, left) Photomicrograph of a thionin-stained coronal section from a representative rat in the IPSI group. The image has been cropped to focus on the lesion in the right amygdala. Broken lines encircle the CEA and BLA lesions. (C, right) Photomicrograph of an AuCl-stained coronal section adjacent to that shown at left indicates that NMDA infusions into the amygdala did not damage myelinated axons in the vicinity of the lesion. (D) Mean percentage of freezing (± SEM) during the conditioning session (collapsed across CSs and ITIs) and retention tests in rats in each of the three groups. The expression of fear to the conditioning context and tone CS was severely impaired by disconnection of the BLA and CEA in the CONTRA group. *P < 0.05.As shown in Figure 1D (left panel), all rats acquired similar levels of conditional freezing (collapsed across the CS and ITI for each trial) by the end of the presurgical fear conditioning session (main effect of trial, F_(5,185) = 42.1, P < 0.0001); there was neither a main effect of group (F < 1) nor a group × trial interaction (F < 1.3). One week after recovery from surgery, conditional freezing to the conditioning context and the auditory CS was assessed in separate retention tests. Importantly, functional disconnection of the BLA and the CEA after fear conditioning severely impaired the expression of conditioned freezing to both the conditioning context and the auditory CS (Fig. 1D, right). Indeed, the expression of fear to the auditory CS was essentially abolished. Freezing among rats in the CONTRA group was significantly lower than that in both the SHAM and IPSI groups during both retention tests (main effect of group, F_(2,37) = 9.98, P < 0.001), and this difference did not interact with the nature of the retention test (F < 1). Hence, functional disconnection of the BLA and CEA produced an impairment in the expression of conditional fear that was much greater than that produced by comparable lesions that spared this connection.The logic of the disconnection procedure requires that focal CEA and BLA lesions in each hemisphere do not damage the adjacent BLA or CEA, respectively. For instance, if CEA lesions produce retrograde degeneration in the neighboring BLA, rats in the CONTRA group would effectively have bilateral lesions in the BLA. Such an outcome would be expected to yield the massive deficits in conditional freezing that we have observed. Inspection of thionin-stained coronal sections suggested that, as in previous studies (Goosens and Maren 2001), our lesions were indeed selective for the targeted areas (Fig. 1B). However, to increase our confidence that the BLA adjacent to a CEA lesion was in fact functional, we performed c-fos immunohistochemistry on brain sections obtained from a separate group of rats trained and tested as previously described (SHAM, CONTRA, and IPSI groups; n = 8 per group). In addition to these rats, we included a group of rats that were not conditioned (NO-SHOCK, n = 8) to quantify fear-induced increases in c-fos expression. All rats were sacrificed 90 min after the retention test to the auditory CS. Amygdaloid c-fos expression in the SHAM rats and the intact hemisphere of the IPSI rats did not differ, and these groups were therefore collapsed into a single SHAM group.As shown in Figure 2, SHAM rats exhibited a greater density of c-fos positive nuclei in both the CEA and BLA relative to nonshocked controls. Quantification of these data confirmed this observation and revealed significant differences in c-fos expression among the groups in both the CEA (Fig. 2, left; F_(2,40) = 5.59, P < 0.01) and the BLA (Fig. 2, right; F_(2,40) = 5.07, P < 0.01). Importantly, BLA c-fos expression adjacent to CEA lesions in the CONTRA group was similar to that in intact SHAM rats, and significantly greater than that in nonshocked controls (Fig. 2B, right). This suggests that the failure of CONTRA rats to express conditional fear responses was not due to a failure to engage the intact BLA but rather to the functional disconnection of BLA activity from CEA output. Indeed, the CEA adjacent to a BLA lesion exhibited a marked reduction in c-fos expression relative to SHAM controls (Fig. 2, left), indicating that BLA lesions failed to drive ipsilateral CEA neurons important for the expression of learned fear. It is possible that the absence of c-fos expression in the CEA, in this case, is due to encroachment of the adjacent BLA lesion. However, thionin-stained sections revealed that BLA lesions were selective. Moreover, we observed intact BLA c-fos expression in rats with adjacent CEA lesions suggesting that nearby lesions per se do not disrupt c-fos expression.Open in a separate windowFigure 2.Functional disconnection of the BLA and CEA revealed by c-fos expression. Mean density (± SEM) of c-fos positive nuclei in the CEA (left) and BLA (right) in rats from each of the three groups. Disconnection of the BLA and CEA eliminated fear-related increases in c-fos expression in the CEA (left), but not the BLA (right). *P < 0.05.These results reveal that a functional connection between the BLA and CEA is required for the expression of learned fear. Considering that BLA projections to the CEA are largely unidirectional, our data reveal that a serial circuit from the BLA to the CEA mediates the expression of conditional fear responses. Consistent with this view, there are numerous reports that permanent lesions or reversible inactivation of either the BLA or CEA prevent the expression of conditioned fear (Lee et al. 1996; Maren 1999; Zimmerman et al. 2007). It is now apparent that the necessity for both the BLA and CEA in fear expression arises from the functional connectivity between them. Anatomically, this connection might involve a direct excitatory projection from BA to CEm (Paré et al. 1999) or indirect projections from LA to ITC and the lateral division of the central nucleus (CEl), both of which project to CEm (Smith and Paré 1994; Paré et al. 2004). Recent data reveal, however, that selective immunotoxic lesions of the ITC do not impair the expression of conditioned freezing (Likhtik et al. 2008). Moreover, CEl projections to CEm are inhibitory, making it unlikely that that an LA-CEl projection drives learned fear responses via CEm (Paré et al. 2004). Hence, it appears that the most likely route by which fear CSs drive learned fear involves projections from BA to CEm. Consistent with this, selective BA lesions disrupt the expression of conditioned freezing when made either before (Goosens and Maren 2001) or after (Anglada-Figueroa and Quirk 2005) fear conditioning.The dependence of conditional fear on a serial circuit between the BLA and CEA stands in contrast to the independent roles these areas have been proposed to play in appetitive conditioning paradigms (Holland and Gallagher 1999; Everitt et al. 2003; Balleine and Killcross 2006). For example, CEA, but not BLA, lesions have been reported to produce deficits in the acquisition and expression of autoshaped conditioned responses (CRs), indicating that the CEA has an independent contribution to CR expression for food-motivated responses (Parkinson et al. 2000). Moreover, the BLA has a role in the attribution of incentive salience to rewarding stimuli independent of the generation of CRs to those stimuli (Hatfield et al. 1996). However, in aversive conditioning, it appears that the BLA may not encode the motivational properties of the shock US (Rabinak and Maren 2008), but rather CS–US associations that are essential for organizing conditional fear responses by the CEA. Indeed, there is an emerging body of data suggesting that these associations may be established not only in sensory afferents in the BLA but also in the CEA (Wilensky et al. 2006; Zimmerman et al. 2007). Synaptic plasticity in projections from BA to CEm may be the essential substrate underlying the functional connectivity between these structures that is essential for the expression of fear memory.