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.     

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.     

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.     

Post-retrieval disruption of a cocaine conditioned place preference by systemic and intrabasolateral amygdala β2- and α1-adrenergic antagonists

Previous work has demonstrated post-retrieval impairment in associative learning paradigms, including those mediated by drugs of abuse, using nonspecific β-adrenergic receptor (β-AR) antagonists. Remarkably little is known about the role of the specific β-AR subtypes, or other adrenergic receptors, in these effects. The current study examined the effects of β₁ and β₂, as well as α₁-adrenergic receptor antagonism following retrieval of a cocaine conditioned place preference (CPP). We found that rats administered the β₂ antagonist ICI 118,551 (8 mg/kg intraperitoneal [IP]) or the α₁ antagonist prazosin (1 mg/kg IP) following a drug-free test for CPP showed attenuated preference during a subsequent test, while the β₁ antagonist betaxolol (5 or 10 mg/kg IP) and a lower dose of prazosin (0.3 mg/kg IP) had no effect. Furthermore, post-test microinfusion of ICI 118,551 (6 nmol/side) or prazosin (0.5 nmol/side) into the basolateral amygdala (BLA) also impaired a subsequent preference. Systemic or intra-BLA ICI 118,551 or prazosin administered to rats in their home cages, in the absence of a preference test, had no effect on CPP 24 h later. ICI 118,551 also attenuated the FOS response in the BLA induced by the CPP test. These results are the first to demonstrate a role for α₁- and β₂-specific adrenergic mechanisms in post-retrieval memory processes. These systemic and site-specific injections, as well as the FOS immunohistochemical analyses, implicate the importance of specific noradrenergic signaling mechanisms within the BLA in post-retrieval plasticity.Substantial evidence indicates that information acquired during a learning event is initially plastic, at which time memory retention can be disrupted, but is strengthened by a time-dependent consolidation process (McGaugh 2000). Recent work has focused on retrieval-induced plasticity, a process by which changes in the retention of previously acquired information are possible. The notion of reconsolidation, one theoretical mechanism by which such changes may occur, suggests that a retrieved memory enters a labile state and is vulnerable to disruption (Sara 2000; Nader 2003). Although the theoretical mechanisms underlying reconsolidation remain unclear, the behavioral effects have been demonstrated across many different learning paradigms using a variety of pharmacological manipulations (for review, see Tronson and Taylor 2007; Diergaarde et al. 2008). Studies with aversive and appetitive preparations, including drug reward-mediated learning, have demonstrated that the noradrenergic system is important for these post-retrieval memory processes (Przybyslawski et al. 1999; Debiec and Ledoux 2004; Bernardi et al. 2006; Diergaarde et al. 2006; Robinson and Franklin 2007; Abrari et al. 2008; Fricks-Gleason and Marshall 2008; Milton et al. 2008). For example, using an animal model of cocaine-conditioned behaviors, Bernardi et al. (2006) demonstrated that systemic post-retrieval administration of propranolol impaired a subsequent conditioned place preference (CPP), suggesting that β-adrenergic receptors (β-ARs) play an important role in processes occurring following drug memory retrieval.However, most of what is known about the noradrenergic system in the memory processes that follow cued reminder trials comes from studies that use nonspecific β-AR antagonists, such as propranolol. As a consequence, several issues regarding ARs and post-retrieval memory processes remain unresolved. First, because propranolol has affinity for both β₁- and β₂-AR subtypes, it is unclear which subtype mediates these effects. To date, no studies have examined reconsolidation-like impairments using subtype-specific β-AR antagonists, which is important because more specific medications may be equally efficacious with less adverse effects. Second, no studies to date have examined α-ARs regarding a potential role in reconsolidation-like effects. α-ARs—specifically α₁-ARs—have a demonstrated role in memory consolidation (Ferry et al. 1999a,b) and may also mediate post-retrieval processes. Third, although the BLA has had a demonstrated role in reconsolidation-like effects in numerous studies, the behavioral conditions during retrieval of drug-associated memories leading to gene expression within the basolateral amygdala (BLA) have not clearly been defined. Specifically, in the CPP paradigm used here, it is unclear whether exposure to a cocaine cue alone will induce gene expression or whether a preference for the drug-associated environment needs to be expressed for BLA involvement (Franklin and Druhan 2000; Miller and Marshall 2005).Understanding the role of specific adrenergic receptors in mediating post-retrieval memory processes is particularly important in drug-induced CPP. In humans, drug-associated stimuli can facilitate drug use (Gawin 1991; See 2005) or precipitate relapse following abstinence (O''Brien et al. 1992). Thus, pharmacotherapies targeting these memory processes would benefit from a clearer understanding of the specific receptors that mediate behavioral effects (Taylor et al. 2009).Here, we first examined the effects of systemic post-test β₁-, β₂-, and α₁-AR antagonism on cocaine CPP. We then focused on the BLA due to its involvement in reconsolidation-like effects in drug learning paradigms (e.g., Lee et al. 2005), employing microinfusions of AR antagonists and measuring FOS immunoreactivity (FOS-IR) to examine the BLA as a potential site of AR-mediated impairments.     

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.     

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).     

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.     

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.     

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

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

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.     

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.     

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

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

Recognition memory: Adding a response deadline eliminates recollection but spares familiarity

A current controversy in memory research concerns whether recognition is supported by distinct processes of familiarity and recollection, or instead by a single process wherein familiarity and recollection reflect weak and strong memories, respectively. Recent studies using receiver operating characteristic (ROC) analyses in an animal model have shown that manipulations of the memory demands can eliminate the contribution of familiarity while sparing recollection. Here it is shown that a different manipulation, specifically the addition of a response deadline in recognition testing, results in the opposite performance pattern, eliminating the contribution of recollection while sparing that of familiarity. This dissociation, combined with the earlier findings, demonstrates that familiarity and recollection are differentially sensitive to specific memory demands, strongly supporting the dual process view.Receiver operating characteristic (ROC) analysis holds the promise of dissecting the contributions to recognition memory of episodic recollection and familiarity (Yonelinas 2001), and this method can be applied equally well to examine these memory processes in animals as well as humans (Fortin et al. 2004; Sauvage et al. 2008). According to the dual process model, recollection is indexed by the asymmetry of the ROC function whereas familiarity is measured by the degree of curvilinearity of that function, and correspondingly, these two parameters can vary independently (Yonelinas 2001). However, there is controversy about this interpretation of ROC components. Some have argued that the asymmetry and curvilinearity of the ROC function both reflect the strength of memories mediated by a single process (Wixted 2007), and correspondingly, these components of the ROC increase or decrease together in stronger or weaker memories, respectively (Squire et al. 2007).A resolution of this controversy can be advanced by examining whether the ROC asymmetry and curvilinearity are independently influenced by task manipulations that favor either recollection or familiarity, consistent with dual process theory, or instead are similarly influenced by conditions that affect memory strength. Recent data from an animal model of recognition have shown that adding a demand for remembering associations between independent stimuli eliminates the ROC curvilinearity without affecting the asymmetry, consistent with the dual process view (Sauvage et al. 2008; for discussion of associative recognition, see Mayes et al. 2007). However, in order to provide compelling evidence of independence of the two ROC components, it is also critical to show that other memory demands that favor familiarity produce the opposite pattern, elimination of the ROC asymmetry while sparing its curvilinearity. Together these findings would constitute a double dissociation between the two parameters of the ROC function that cannot be explained by a single process theory.As originally conceived in models proposed in the 1970s, familiarity is characterized as a perceptually driven, pattern matching process that is completed rapidly, whereas recollection is characterized as a conceptually driven, organizational process that requires more time (Mandler 1972; Atkinson and Juola 1973, 1974; for reviews, see Yonelinas 2002; Mandler 2008). Consistent with this view, the results of several studies that employ response deadlines in the test phase report that familiarity is more rapid than recollection. For example, forcing people to make speeded recognition responses has little effect on simple yes–no recognition but strikingly reduces performance when subjects must remember where or when an item was studied (Yonelinas and Jacoby 1994; Gronlund et al. 1997; Hintzman et al. 1998). Other studies that require subjects to oppose familiarity and recollection reveal a two-component temporal function that includes a rapidly available familiarity process and a slower recollective process (Dosher 1984; Gronlund and Ratcliff 1989; Hintzman and Curran 1994; McElree et al. 1999). In addition, studies that measure brain evoked response potentials (ERPs) have revealed two distinct ERP modulations commonly observed during recognition: a mid-frontal negativity onsetting about 400 msec after stimulus onset that is associated with familiarity, and a parietally distributed positivity beginning about 500 msec after stimulus onset that is associated with recollection (Smith 1993; Duzel et al. 1997; Curran 2004; Duarte et al. 2006; Woodruff et al. 2006; but see Voss and Paller 2009).Dual process theory predicts that applying an appropriate early response deadline should allow sufficient time for contribution of familiarity but not that of recollection, and so should reduce the ROC asymmetry while sparing its curvilinearity, opposite to the already observed effects of associative memory demands that favor recollection (Sauvage et al. 2008). Confirmation of this prediction combined with the previous findings of the opposite effects in associative recognition would constitute a double dissociation between the features of recollection and familiarity. This result would therefore strongly support the conclusion that the asymmetry and curvilinearity are independent parameters of the ROC function that are differentially linked to features of recollection and familiarity, respectively.     

Behavioral deficits and subregion-specific suppression of LTP in mice expressing a population of mutant NMDA receptors throughout the hippocampus

The NMDA receptor (NMDAR) subunit GluN1 is an obligatory component of NMDARs without a known functional homolog and is expressed in almost every neuronal cell type. The NMDAR system is a coincidence detector with critical roles in spatial learning and synaptic plasticity. Its coincidence detection property is crucial for the induction of hippocampal long-term potentiation (LTP). We have generated a mutant mouse model expressing a hypomorph of the Grin1^N598R allele, which leads to a minority (about 10%) of coincidence detection-impaired NMDARs. Surprisingly, these animals revealed specific functional changes in the dentate gyrus (DG) of the hippocampal formation. Early LTP was expressed normally in area CA1 in vivo, but was completely suppressed at perforant path-granule cell synapses in the DG. In addition, there was a pronounced reduction in the amplitude of the evoked population spike in the DG. These specific changes were accompanied by behavioral impairments in spatial recognition, spatial learning, reversal learning, and retention. Our data show that minor changes in GluN1-dependent NMDAR physiology can cause dramatic consequences in synaptic signaling in a subregion-specific fashion despite the nonredundant nature of the GluN1 gene and its global expression.According to Hebb''s postulate, neurons require a molecular mechanism to detect synchronous activity in order to change the strength of synaptic connectivity (Hebb 1949). NMDA receptors (NMDARs) are molecular coincidence detectors, and selective NMDAR antagonists block the induction of long-term potentiation (LTP) in both the dentate gyrus (DG) and CA1 regions of the hippocampus (Bliss and Collingridge 1993; Martin et al. 2000). NMDARs have been long known for their role in spatial learning, but more recently have been implicated in other forms of cognitive function and dysfunction (Gruart et al. 2006; Whitlock et al. 2006; Castner and Williams 2007; Kristiansen et al. 2007; Wilson and Linster 2008).Neuronal NMDARs are hetero-tetrameric ligand-gated ion channels typically comprised of two types of subunits. Two copies of the mandatory GluN1 subunit (or NR1 subunit [Collingridge et al. 2009] encoded by Grin1) are associated with two copies from the GluN2 family, GluN2A–D (or NR2A–D). The GluN1 subunit is expressed ubiquitously both spatially and temporally throughout the developing and adult brain. Global knockout mice models of the GluN1 subunit are postnatally lethal within hours after birth (Forrest et al. 1994; Li et al. 1994), and cell-specific GluN1 mice knockouts (Tsien et al. 1996; Nakazawa et al. 2002; McHugh et al. 2007; Niewoehner et al. 2007) have provided insights on how specific synapses and regional neuronal networks are dependent on NMDAR function.The early postnatal lethality of the global GluN1 knockout is in contrast to the null mutants of the four AMPA receptor genes and other major synaptic proteins, such as αCaMKII (Silva et al. 1992a,b; Jia et al. 1996; Zamanillo et al. 1999; Meng et al. 2003). This can be at least partially explained by the absence of any close GluN1 homologs, which could functionally compensate for the absence of the GluN1 subunit. Recombinant expression studies defined the GluN1 subunit as a mandatory component of NMDARs. This constellation provides a specific opportunity to test whether different local neuronal subnetworks are affected differentially by mutant Grin1 alleles associated with subtle alterations of the functional properties of NMDARs.GluN1 subunits with the N598R point mutation (GluN1^R) yield functional NMDARs that are Mg²⁺ insensitive and Ca²⁺ impermeable (Burnashev et al. 1992; Mori et al. 1992). The Grin1^N598R allele that codes for GluN1^R subunits is a gain-of-function mutation that is dominant lethal, even in heterozygous and hemizygous lines (Single et al. 2000; Rudhard et al. 2003). NMDARs with GluN1^R subunits do not act as coincidence detectors and, interestingly, mice expressing exclusively the GluN1^R allele lack whisker-related pattern formation in the neonate brainstem (Rudhard et al. 2003).To investigate the functional importance of GluN1 subunits with the N598R point mutation, we took advantage of the generation of a variant mutant line of mice (GluN1^Rneo/+) expressing a minority (around 10%) of these mutant NMDARs. Even though the majority of the NMDARs are normal, all neurons expressing NMDARs will contain a subset of receptors carrying this mutation.Therefore, this mouse model is an ideal candidate to study the impact of subtle alterations of NMDAR function on different neuronal networks, such as those comprising the hippocampal formation.Studies examining region-specific targeted disruption of GluN1 expression in subregions of the hippocampus have revealed subtle yet important contributions of this NMDAR subunit in synaptic plasticity and spatial learning and memory. CA1-restricted knockout of GluN1 expression in the hippocampus caused impaired spatial learning and memory as well as reduced CA1-LTP (Tsien et al. 1996). In the case of the disruption of GluN1 expression in the DG region of the hippocampus, more subtle behavioral impairments were apparent, including the inability to discriminate between two similar contexts (pattern separation) and deficits in spatial working memory despite normal LTP in the CA1 region (McHugh et al. 2007; Niewoehner et al. 2007).Our GluN1^Rneo/+ mice differ from the region-specific GluN1 mutant mice in that they express the mutant hypomorph at the same level in different subregions of the hippocampus. Interestingly, we found that this allele leads to substantial differences in short- and long-term plasticity between area CA1 and the DG of the hippocampus. The specific impairment in the DG was accompanied by impaired spatial recognition, spatial learning, reversal learning, and retention. Our data establish the possibility of a circuit-specific phenotype caused by a mutant variant of a globally expressed major nonredundant synaptic protein.     

Prenatal choline availability alters the context sensitivity of Pavlovian conditioning in adult rats

The effects of prenatal choline availability on Pavlovian conditioning were assessed in adult male rats (3–4 mo). Neither supplementation nor deprivation of prenatal choline affected the acquisition and extinction of simple Pavlovian conditioned excitation, or the acquisition and retardation of conditioned inhibition. However, prenatal choline availability significantly altered the contextual control of these learned behaviors. Both control and choline-deprived rats exhibited context specificity of conditioned excitation as exhibited by a loss in responding when tested in an alternate context after conditioning; in contrast, choline-supplemented rats showed no such effect. When switched to a different context following extinction, however, both choline-supplemented and control rats showed substantial contextual control of responding, whereas choline-deficient rats did not. These data support the view that configural associations that rely on hippocampal function are selectively sensitive to prenatal manipulations of dietary choline during prenatal development.There is increasing evidence that variations in maternal dietary choline intake during the second half of pregnancy cause structural, biochemical, and physiological changes in basal forebrain neurons and their projections to the hippocampal complex as well as long-term cognitive changes in the offspring (e.g., Meck and Williams 2003; McCann et al. 2006; Meck et al. 2008). We know, for instance, that the adult offspring of pregnant rats supplemented with 4.5 times the amount of choline in the standard laboratory diet display improved memory capacity and precision on the radial-arm maze (e.g., Meck et al. 1988, 1989; Meck and Williams 1997b, 1999; Tees 1999a), Morris water maze (e.g., Tees 1999b; Tees and Mohammadi 1999; Yang et al. 2000; Brandner 2002), as well as facilitation of sustained attention and interval timing (e.g., Meck and Williams 1997a, c; Mohler et al. 2001; Cheng et al. 2006, 2008a, b; Cheng and Meck 2007) compared with offspring of dams fed a standard diet. Choline deficiency during the same developmental time frame, embryonic days (ED) 12–17, results in impaired performance on some, but not all, of these behavioral measures (e.g., Meck and Williams 1999, 2003). Furthermore, perinatal choline supplementation can alter behavior following a variety of developmental disorders, including the alleviation of abnormalities associated with fetal alcohol syndrome in rats (Thomas et al. 2000, 2004, 2007; Wagner and Hunt 2006), attenuation of some of the motor deficits observed in a Mecp21^lox mouse model of Rett syndrome (Nag and Berger-Sweeney 2007), and the improvement of sensory gating in a DBA/2 mouse model of schizophrenia that exhibits reduced numbers of hippocampal a7 nicotinic receptors (Stevens et al. 2008).These choline-induced alterations in cognitive function are accompanied by changes in the size and shape of basal forebrain cholinergic neurons (e.g., Williams et al. 1998; McKeon-O’Malley et al. 2003); modifications in acetylcholine turnover and choline transporter expression in the septum and hippocampus (Cermak et al. 1999; Mellott et al. 2007b); modulation of hippocampal neurogenesis, gene expression, phospholipase D activity, NGF levels, and MAPK and CREB activation (e.g., Holler et al. 1996; Sandstrom et al. 2002; Mellott et al. 2004, 2007a; Glenn et al. 2007); changes in dendritic fields and spine density in CA1 and dentate gyrus (DG) regions of the hippocampus (Meck et al. 2008); as well as modification of the neuropathological response to status epilepticus (e.g., Holmes et al. 2002; Wong-Goodrich et al. 2008a) and thresholds for eliciting long-term potentiation (LTP) in the hippocampus (Pyapali et al. 1998; Jones III et al. 1999). Together, these findings suggest that alterations in choline availability during early development may have specific impact on the ontogeny and later functioning of basal forebrain cholinergic neurons as well as efferent neurons involved in hippocampal LTP (Montoya et al. 2000). These findings also predict that behaviors that rely on the hippocampus are likely to be most affected by this dietary manipulation.Although choline is well known as the precursor for the neurotransmitter acetylcholine, it may be especially crucial to young or developing mammals for a number of other reasons (see Blusztajn and Wurtman 1983; Blusztajn 1998; Zeisel 2000, 2004, 2005). It is the precursor of certain phospholipids (e.g., phosphatidylcholine, sphingomyelin, and plasmenylcholine), which constitute the bulk of phospholipids in all biological membranes. Thus, there may be a particularly high demand for choline during prenatal and neonatal periods associated with rapid neurogenesis and synaptogenesis. Choline can also be enzymatically oxidized to betaine (mostly in peripheral tissues) and the methyl groups of betaine can then be used to resynthesize methionine from homocysteine. Changes in methionine availability alter the methylation of regulatory sequences of genes and of histones, leading to alterations in the patterns of gene expression (e.g., Waterland and Michels 2007; Nafee et al. 2008). Choline is also the precursor of two signaling molecules, platelet-activating factor, and sphingosylphosphorylcholine. Changes in choline availability may also alter membrane synthesis, methylation, and signaling broadly throughout the brain and periphery as well as more restricted effects on cholinergic neuronal pathways (e.g., Zeisel and Blusztajn 1994; Meck and Williams 2003).One common distinction in the Pavlovian-conditioning literature is between tasks that are sensitive to manipulation of the hippocampal formation from those that are not (e.g., Ross et al. 1984; Meck 1988; Schmajuk and Buhusi 1997; Holland et al. 1999). For example, simple excitatory Pavlovian conditioning is typically found to be unaffected by lesions of the hippocampus, while conditional discriminations in which animals must rely on combinations of predictive cues to respond correctly are disrupted by hippocampal damage (e.g., Jarrard and Davidson 1990, 1991). If prenatal choline availability is altering the development of cholinergic neurons in the basal forebrain that project to the hippocampus (see Meck and Williams 2003), our dietary manipulation might only be expected to affect conditioning tasks that require hippocampal involvement, not relatively simple tasks such as excitatory conditioning which do not rely on the hippocampus (e.g., Green and Woodruff-Pak 2000).In the current series of experiments, we examined the effects of prenatal choline supplementation and deficiency using a series of appetitive Pavlovian-conditioning tasks, all of which require associative learning. Our rationale was to determine whether variations in choline availability during prenatal development altered the learning of a simple association between the conditioned (CS) and unconditioned (US) stimuli (e.g., noise → food sequence), or if the dietary manipulation primarily affected conditioning tasks that require more complex relational processing and intact septal-hippocampal function (e.g., context A = tone → food; context B = noise → no food).In order to assess the importance of prenatal choline availability on associative learning, we investigated basic aspects of appetitive Pavlovian conditioning, i.e., conditioned excitation and extinction (e.g., Pavlov 1927) in experiment 1. In this paradigm, rats first receive repeated trials in which the CS occurs just before the presentation of the US, i.e., in a noise → food sequence. During this initial phase of training, the rat develops an increasing tendency to perform the conditioned response (CR) in the presence of the CS indicating that it expects the occurrence of the US. Typically, the probability of the CR increases in a negatively accelerating fashion until it reaches an asymptotic level. If the CS is then repeatedly presented in the absence of the reinforcing US (i.e., noise → no food), then the CR gradually declines; this is referred to as extinction of the CR (Gallistel and Gibbon 2000).One behavioral phenomenon that has been shown to be sensitive to hippocampal manipulation is the discriminative use of contextual cues to control conditioned responding (e.g., Holland and Bouton 1999). Typically, when CS-US pairings occur in one training environment or context, there is a small loss of responding to the CS if it is subsequently presented to the animal in the presence of a different set of contextual cues (e.g., Lovibond et al. 1984; Hall and Honey 1990; Honey et al. 1990; Kaye and Mackintosh 1990). However, this typical decrement in responding with a context switch is not observed in rats with electrolytic or aspiration lesions of the hippocampus (e.g., Good et al. 1997).In order to assess the effects of prenatal choline availability on contextual control of conditioned responding, we employed a renewal design (e.g., Bouton and Bolles 1979) in experiment 2. In this design, rats receive conditioning in one physical context (context A) prior to extinction in either the same context or a context different from that in which they received the initial CS-US pairings (context B). Finally, all of the rats are retested in the original conditioning context (i.e., context A). Bouton and colleagues (e.g., Bouton and Bolles 1979; Frohardt et al. 2000) have found that when rats that are conditioned in context A followed by extinction training in context B are later returned to the original training context for the final testing phase, they show a substantial recovery of the initial CR. Presumably, stimuli contained within the original training context act as reminder cues in this ABA condition, retrieving the memory for the initial acquisition phase (A) of the experiment during the final test phase as opposed to the more recent extinction phase (B). Rats in the AAA condition have no effective cues to discriminate the different phases of the experiment and as a consequence cannot selectively retrieve a specific memory from the sequence. In contrast, test session responding for the ABA condition should be more similar to the low levels observed at the end of the initial extinction phase due to the availability of differential contextual cues. This renewal design is particularly useful in that it provides for the potential to observe treatment effects in both the extinction and the renewal test phases of the experiment. Specifically, either the loss of responding with a context switch during extinction or the response recovery in the renewal test (or both) may be affected by prenatal choline availability. More importantly, those two effects may be due to either the same mechanism (e.g., processing of contextual stimuli) or two different mechanisms (e.g., context conditioning and memory retrieval)—potentially resulting in nonlinear effects of prenatal choline availability across the two experimental phases.A second basic type of associative learning, conditioned inhibition, in which the animal learns to predict the absence of an important event, was described by Pavlov (1927). A typical conditioned-inhibition task consists of training with two types of intermixed trials: On reinforced trials, one CS is followed by reinforcement (e.g., noise → food). On other trials, the same CS is paired with a second stimulus in the absence of the reinforcement (i.e., light/noise → no food). It is presumed that under these training conditions animals learn that the noise predicts the occurrence of the food, while light, the “conditioned inhibitor,” comes to predict the absence of food. That is, light “inhibits” the learned response to noise alone.A relatively small number of studies have examined the neural substrates of inhibitory learning. Aspiration lesions of the hippocampus, for example, impaired a relatively complex phenomenon called “blocking” of excitatory conditioning, but not the learning of conditioned inhibition (e.g., Solomon 1977; Chan et al. 2001). These data suggest that the hippocampal complex is not required for learning conditioned inhibition. Thus, in order to further assess whether prenatal choline availability affects basic associative learning, experiment 3 was designed to evaluate conditioned inhibition in supplemented (SUP), deficient (DEF), and control (CON) rats. In this experiment, rats were given randomly mixed presentations of reinforced and nonreinforced trial types. As training proceeds, the rats should learn to respond more on reinforced trials than on nonreinforced trials. After acquisition of the discrimination, the rats were presented with a retardation test phase in which the inhibitory light CS was paired with food. Rescorla (1969) described this retardation test as one of the critical measures of conditioned inhibition. Presumably, if the CS is a true inhibitor and predicts the absence of reinforcement at the outset of the retardation test, then acquisition of conditioned responding to the cue should be relatively slow during this phase of testing. Consequently, tests of conditioned inhibition should distinguish among prenatal choline treatment groups if inhibitory mechanisms are strengthened or weakened by prenatal choline availability.     

A cognitive map for object memory in the hippocampus

The hippocampus has been proposed to support a cognitive map, a mental representation of the spatial layout of an environment as well as the nonspatial items encountered in that environment. In the present study, we recorded simultaneously from 43 to 61 hippocampal pyramidal cells as rats performed an object recognition memory task in which novel and repeated objects were encountered in different locations on a circular track. Multivariate analyses of the neural data indicated that information about object identity was represented secondarily to the primary information dimension of object location. In addition, the neural data related to performance on the recognition memory task. The results suggested that objects were represented as points of interest on the hippocampal cognitive map and that this map was useful in remembering encounters with particular objects in specific locations.The hippocampus plays an important role in spatial memory for both humans and rodents (O''Keefe 1999; Burgess et al. 2002). Findings from many studies in rodents indicate that the hippocampus supports memory for locations referenced to external landmarks, a capacity that O''Keefe and Nadel (1978) described over 30 yr ago as a “cognitive map” (using a term they borrowed from Tolman 1948). In the time since that pioneering thesis, it has become clear that the rodent hippocampus is also important for nonspatial memory (Eichenbaum et al. 1999). Damage to the rat hippocampus (defined here as CA fields, dentate gyrus, and subiculum) leads to impairments on nonspatial tasks, including object recognition memory (Clark et al. 2000; Fortin et al. 2004), transitive odor associations (Bunsey and Eichenbaum 1996), memory for temporal order (Fortin et al. 2002; Kesner et al. 2002), and social transmission of food preference (Alvarez et al. 2001; Clark et al. 2002).The circuitry by which information arrives at and exits from the hippocampus is consistent with the idea that the hippocampus is important for both spatial and nonspatial memory. In both rats and macaques, detailed anatomical studies have indicated that spatial information arrives at the hippocampus via the postrhinal cortex (parahippocampal cortex in primates) and the medial entorhinal cortex, whereas nonspatial information takes a path largely through the perirhinal cortex and lateral entorhinal cortex (Witter and Amaral 1991; Suzuki and Amaral 1994; Witter et al. 2000). Thus, the hippocampus is ideally situated to combine spatial and nonspatial information in the service of remembering item–location associations (Manns and Eichenbaum 2006).Single-unit recording studies in the rat hippocampus have largely focused on the spatial correlates of hippocampal pyramidal neuron firing rates. Fewer studies have investigated nonspatial correlates of hippocampal activity during memory tasks for nonspatial items. However, in one such study, Wood et al. (1999) found that some individual hippocampal pyramidal neurons responded to particular odors and that others responded to particular odors in specific locations during an odor recognition memory task. Thus, activity of individual cells appeared to contain information about nonspatial items as well as spatial locations.An important question is how the activity of individual hippocampal neurons combine to represent item–location associations as a neural ensemble. In particular, how is an encounter with an object in a particular location represented in the pattern of spiking among many hippocampal pyramidal neurons? How might this representation relate to memory for the object or for the location? In the present study, we recorded simultaneously from 43 to 61 hippocampal pyramidal cells as rats performed an object recognition memory task in which novel and repeated objects were encountered in different locations on a circular track. Multivariate analyses of the neural data indicated that information about object identity was represented secondarily to the primary information dimension of object location. In addition, the analyses indicated that the neural data related to performance on the recognition memory task. The results suggest that objects were represented as points of interest on the hippocampal cognitive map and that this map was useful in remembering encounters with particular objects in specific locations.     

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).