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

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

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.     

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

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

Object recognition memory and the rodent hippocampus

In rodents, the novel object recognition task (NOR) has become a benchmark task for assessing recognition memory. Yet, despite its widespread use, a consensus has not developed about which brain structures are important for task performance. We assessed both the anterograde and retrograde effects of hippocampal lesions on performance in the NOR task. Rats received 12 5-min exposures to two identical objects and then received either bilateral lesions of the hippocampus or sham surgery 1 d, 4 wk, or 8 wk after the final exposure. On a retention test 2 wk after surgery, the 1-d and 4-wk hippocampal lesion groups exhibited impaired object recognition memory. In contrast, the 8-wk hippocampal lesion group performed similarly to controls, and both groups exhibited a preference for the novel object. These same rats were then given four postoperative tests using unique object pairs and a 3-h delay between the exposure phase and the test phase. Hippocampal lesions produced moderate and reliable memory impairment. The results suggest that the hippocampus is important for object recognition memory.Recognition memory refers to the ability to judge a previously encountered item as familiar and depends on the integrity of the medial temporal lobe (Squire et al. 2007). Tasks that assess recognition memory (and object recognition memory in particular) have become increasingly useful tools for basic and preclinical research investigating the neural basis of memory (Winters et al. 2008). Perhaps the best known of these tasks is the novel object recognition task (NOR) (also known as the visual paired-comparison task in studies with humans and monkeys).Studies of the NOR task in humans with hippocampal damage (McKee and Squire 1993; Pascalis et al. 2004) and in monkeys with selective damage to the hippocampus (Pascalis and Bachevalier 1999; Zola et al. 2000; Nemanic et al. 2004) have resulted in clear and consistent findings. Damage limited to the hippocampus is sufficient to produce impaired recognition memory (Squire et al. 2007, Box 1). In rats and mice, the NOR task has become particularly popular and is currently a benchmark task for assessing recognition memory. Yet despite its widespread use in rodents, the findings are rather mixed. For example, in the rat, although there is agreement that the perirhinal cortex is critically important for normal NOR performance, there is less agreement about the hippocampus (for review, see Winters et al. 2008). Although some of the discrepancies between studies may be attributed to differences in lesion size and in the length of the retention delay (Broadbent et al. 2004), these factors cannot account for all the findings (Squire et al. 2007).Whereas most studies have investigated the effects of hippocampal lesions on postoperative NOR performance, there is also interest in the effects of hippocampal lesions on memory for previously encountered objects. For a number of tasks, hippocampal lesions produce temporally graded retrograde amnesia, such that memory acquired recently is impaired and memory acquired more remotely is spared (for review, see Squire et al. 2004; Frankland and Bontempi 2005). In the case of the single study of retrograde memory that has involved the NOR task, recognition memory was impaired when a 5-wk interval intervened between training and hippocampal surgery (Gaskin et al. 2003). It remains possible that memory might be spared if a longer delay was imposed between training and surgery.The aim of the present study was to assess both the anterograde and retrograde effects of hippocampal lesions on recognition memory using the NOR task. To thoroughly assess the effects of hippocampal lesions we used (1) large groups of animals, (2) multiple tests of NOR memory, (3) a scoring method that allowed object preference to be determined on a second-by-second basis during the recognition tests, and (4) a novel training protocol that permitted the evaluation of recognition memory even after a retention interval as long as 10 wk.     

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.     

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.     

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.     

Physiological effects of enriched environment exposure and LTP induction in the hippocampus in vivo do not transfer faithfully to in vitro slices

A number of experimental paradigms use in vitro brain slices to test for changes in synaptic transmission and plasticity following a behavioral manipulation. For example, a number of previous studies have reported a variety of effects of environmental enrichment (EE) exposure on field potential responses in hippocampal slices, but in no study was is it known what changes had been elicited in vivo. In the present study, we recorded from the hippocampus in vivo while rats underwent a brief period of EE. There was no detectable EE-induced change in synaptic efficacy in the dentate gyrus in vivo, but there was an increase in cellular excitability. In slices prepared from the same animals, we failed to observe any evidence of the excitability increase. We next tested whether LTP induction in vivo was better preserved in vitro. However, when slices from these rats were examined, there was no observable change in perforant path synaptic strength, although there was a modest increase in excitability that correlated with the increased excitability observed in vivo. These findings suggest that synaptic changes induced in vivo either are not preserved faithfully or are difficult to detect in hippocampal slices, while changes in cellular excitability are better preserved.Exposure to an enriched environment (EE) can improve performance on a variety of hippocampus-dependent memory tasks in both normal (Kempermann et al. 1997; Duffy et al. 2001; Teather et al. 2002; Schrijver et al. 2004; Irvine and Abraham 2005) and disease model (Ohlsson and Johansson 1995; Young et al. 1999; Jankowsky et al. 2005; Lazarov et al. 2005; Nithianantharajah and Hannan 2006; Laviola et al. 2008) animals. Previous studies attempting to understand the physiological changes that mediate these effects have yielded mixed results, which may in part be due to the variability in enrichment paradigms used in different laboratories, but which may also be due to the method used to measure hippocampal physiology.Traditionally, researchers have studied the effects of EE using ex vivo brain slices. Such studies have sometimes reported an increase in synaptic strength following enrichment (Green and Greenough 1986; Foster et al. 1996; Foster and Dumas 2001), but a lack of a change has also been observed (Duffy et al. 2001; Feng et al. 2001; Parsley et al. 2007). The ex vivo approach is predicated on the assumption that EE (or other behavioral) treatment induces changes in neural function that are of sufficient magnitude and extent that they will still be present when the brain is removed and studied in vitro. However, there could be many hidden effects of slice preparation (Kirov et al. 2004) that change or obscure effects occurring in vivo.In a previous study, we were surprised to find few effects of a 3-mo EE treatment on hippocampal synaptic function and plasticity when assessed in vitro (Eckert et al. 2010), despite our having observed with in vivo recordings substantial effects with shorter periods of EE exposure (Irvine and Abraham 2005; Irvine et al. 2006). We therefore considered the possibility that effects measured electrophysiologically in vivo may not be readily detectable in vitro. Testing this hypothesis requires studying the same animals in vivo and in vitro, a control procedure we are not aware of having been reported previously in the literature. In the present study, we examined whether the effects of EE or LTP induction in vivo could be detected in hippocampal slices taken from the same animals. We failed to detect any of the in vivo changes, except for a modest increase in cellular excitability following LTP.     

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

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

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.     

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.     

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.     

The shift from a response strategy to object-in-place strategy during learning is accompanied by a matching shift in neural firing correlates in the hippocampus

Hippocampal-dependent tasks often involve specific associations among stimuli (including egocentric information), and such tasks are therefore prone to interference from irrelevant task strategies before a correct strategy is found. Using an object-place paired-associate task, we investigated changes in neural firing patterns in the hippocampus in association with a shift in strategy during learning. We used an object-place paired-associate task in which a pair of objects was presented in two different arms of a radial maze. Each object was associated with reward only in one of the arms, thus requiring the rats to consider both object identity and its location in the maze. Hippocampal neurons recorded in CA1 displayed a dynamic transition in their firing patterns during the acquisition of the task across days, and this corresponded to a shift in strategy manifested in behavioral data. Specifically, before the rats learned the task, they chose an object that maintained a particular egocentric relationship with their body (response strategy) irrespective of the object identity. However, as the animal acquired the task, it chose an object according to both its identity and the associated location in the maze (object-in-place strategy). We report that CA1 neurons in the hippocampus changed their prospective firing correlates according to the dominant strategy (i.e., response versus object-in-place strategy) employed at a given stage of learning. The results suggest that neural firing pattern in the hippocampus is heavily influenced by the task demand hypothesized by the animal and the firing pattern changes flexibly as the perceived task demand changes.An event often involves objects placed in particular locations. To form a cognitive representation of such an event, an animal needs to represent many heterogeneous pieces of information such as individual objects, their spatial locations, and also egocentric body movements associated with the navigation through the objects and locations. All the information then needs to be associated in a correct way (e.g., proper object-place association) for successful representation of the event. Processing only a subset of information successfully may lead to an error in identifying the correct event. It is believed that the hippocampus is essential for forming and retrieving such conjunctive representations among stimuli for processing events (O''Reilly and McClelland 1994).Due to its conjunctive information processing after receiving inputs from multiple sources (e.g., information for object recognition, egocentric memory, allocentric memory, etc.), the hippocampal system is prone to competition with other systems especially when a simpler strategy that requires information from a single source seems a viable option. Behavioral and pharmacological studies have supported this hypothesis (Packard and McGaugh 1996; Poldrack and Packard 2003). Physiologically, the influence of switching strategy on hippocampal neuronal firing has been documented in the literature and often tested in a plus maze. It has been shown, for example, that passing a common area of space using different task demands alters the firing properties of hippocampal neurons (Wood et al. 2000; Ferbinteanu and Shapiro 2003; Lee et al. 2006; Eschenko and Mizumori 2007). In most of these studies, however, rats were pre-trained before electrophysiological recordings were made and an experimenter enforced the change in task demand. Therefore, it has been difficult to investigate a naturally occurring competition between different strategies as learning proceeds and the resulting neural dynamics in the hippocampus especially when learning takes place for the first time.Using a hippocampal dependent task, we aimed to characterize such naturally occurring changes in hippocampal firing patterns as a wrong strategy is replaced by the correct strategy during learning. We also investigated the relationships between the changes in firing patterns and behavioral performance. We used a biconditional object-place paired-associate task for this purpose. We demonstrated previously that hippocampal lesioned rats were severely impaired in this task (Lee and Solivan 2008). In this task, two different objects are presented simultaneously as a pair, and rats are required to discriminate between the objects according to the location (an arm in a radial maze) in which they are presented. Once it enters an arm, the rat positions itself between two objects and must choose either the left or right direction toward either object to displace the object for obtaining a food reward. Since the relative object positions within an arm vary across trials, remembering the running direction in which a reward is found (i.e., response strategy) is meaningless and the rat must learn to pay attention to the object identity and its associated location in space (i.e., object-in-place strategy). In our previous studies (Lee and Solivan 2008, 2010; Jo and Lee 2010), we observed a strong competition in this task between a response strategy and object-in-place strategy. Normal rats used the response strategy before learning occurred, but exhibited a sharp transition to the object-in-place strategy in several days. The selective involvement of the hippocampus was strongly implicated in the task as the hippocampal lesioned rats show stereotypic egocentric responses (Lee and Solivan 2008). The current study aimed to find and characterize in detail matching neural correlates in the hippocampus in the object-place paired-associate task.     

Hedonic and nucleus accumbens neural responses to a natural reward are regulated by aversive conditioning

The nucleus accumbens (NAc) plays a role in hedonic reactivity to taste stimuli. Learning can alter the hedonic valence of a given stimulus, and it remains unclear how the NAc encodes this shift. The present study examined whether the population response of NAc neurons to a taste stimulus is plastic using a conditioned taste aversion (CTA) paradigm. Electrophysiological and electromyographic (EMG) responses to intraoral infusions of a sucrose (0.3 M) solution were made in naïve rats (Day 1). Immediately following the session, half of the rats (n = 6; Paired) received an injection of lithium chloride (0.15 M; i.p.) to induce malaise and establish a CTA while the other half (n = 6; Unpaired) received a saline injection. Days later (Day 5), NAc recordings during infusions of sucrose were again made. Electrophysiological and EMG responses to sucrose did not differ between groups on Day 1. For both groups, the majority of sucrose responsive neurons exhibited a decrease in firing rate (77% and 71% for Paired and Unpaired, respectively). Following conditioning, in Paired rats, EMG responses were indicative of aversion. Moreover, the majority of responsive NAc neurons now exhibited an increase in firing rate (69%). Responses in Unpaired rats were unchanged by the experience. Thus, the NAc differentially encodes the hedonic value of the same stimulus based on learned associations.Our search for sustenance and pleasurable stimuli is often balanced by our desire to avoid punishment and harm. Similarly, neural systems responsible for generating approach behavior must be countered by signals that suppress approach behavior under contexts where approach is dangerous or maladaptive (Hoebel et al. 2007). The nucleus accumbens (NAc) is acutely involved in food intake and goal-directed, approach behavior. Pharmacological manipulations of the NAc promote food intake even in sated rats (Maldonado-Irizarry and Kelley 1995; Stratford and Kelley 1997). Lesions or inactivation of the NAc impair conditioned approach behavior (Cardinal et al. 2002; Blaiss and Janak 2009). Interestingly, drugs that lead to inhibition of select regions of the NAc increase positive hedonic responses to palatable taste solutions (Pecina and Berridge 2005). Recordings from individual NAc neurons have mirrored these findings. We and others have shown that consumption of palatable food stimuli is associated with decreases in the firing rate of the majority of responsive NAc neurons (Nicola et al. 2004b; Roitman et al. 2005; Taha and Fields 2005; Wheeler et al. 2008). In addition, decreases in NAc neural activity are associated with bouts of licking behavior for palatable stimuli (Taha and Fields 2006), and disruption of these decreases halt feeding bouts (Krause et al. 2010). Finally, decreases in NAc neural activity are associated with preferred locations previously paired with drug reward (German and Fields 2007). Thus, decreases in NAc activity appear to be closely linked to positive hedonic stimuli, stimuli that have been explicitly paired with them and behavioral approach.The NAc is also responsive to aversive stimuli (Carlezon and Thomas 2009; Levita et al. 2009). The delivery of aversive taste stimuli to rats is associated with increases in the firing rate of the majority of responsive NAc neurons (Roitman et al. 2005; Wheeler et al. 2008). In addition to responding to primary appetitive and aversive taste stimuli, NAc neurons develop responses to predictors of reward and aversion. Individual NAc neurons selectively encode cues that predict either appetitive (Roitman et al. 2005; Day et al. 2006) or aversive (Roitman et al. 2005) stimuli following purely Pavlovian conditioning or a combination of instrumental and Pavlovian conditioning (Setlow et al. 2003; Nicola et al. 2004a). NAc neurons come to encode departure from locations not associated with reward with the majority response being that of excitation (German and Fields 2007). Thus, NAc neurons appear to encode aversive stimuli and withdrawal behavior with increases in activity. These and other findings have led to the recent postulation that reward and aversion are differentially encoded by the activity of NAc neurons (Carlezon and Thomas 2009).Data supportive of the activity hypothesis (Carlezon and Thomas 2009) have been generated by the use of different stimuli to serve as appetitive or aversive primary or predictive stimuli. Thus, selective encoding could be biased by the sensory properties of each stimulus rather than their hedonic valence. When a novel, palatable taste is paired with visceral malaise, a Pavlovian association is made and a conditioned taste aversion (CTA) is established. Subsequent exposure to the once palatable stimulus is met with avoidance or aversion and rejection (Garcia et al. 1974; Schafe et al. 1995). Thus, the same taste stimulus can either be appetitive or aversive, depending on Pavlovian associations. Here, individual NAc neurons were recorded in rats (Paired) before (Day 1) and after a CTA (Day 5) was established and compared with rats that received equal exposure to the same stimuli but in an unpaired manner (Unpaired), and hence no CTA developed. Simultaneously, oro-motor behavior was characterized to provide an index of the associative strength of the taste stimulus. Using this paradigm, we determined that the population response of the NAc does indeed encode hedonic valence.     

Impact of predatory threat on fear extinction in Lewis rats

Humans with post-traumatic stress disorder (PTSD) are deficient at extinguishing conditioned fear responses. A study of identical twins concluded that this extinction deficit does not predate trauma but develops as a result of trauma. The present study tested whether the Lewis rat model of PTSD reproduces these features of the human syndrome. Lewis rats were subjected to classical auditory fear conditioning before or after exposure to a predatory threat that mimics a type of traumatic stress that leads to PTSD in humans. Exploratory behavior on the elevated plus maze 1 wk after predatory threat exposure was used to distinguish resilient vs. PTSD-like rats. Properties of extinction varied depending on whether fear conditioning and extinction occurred before or after predatory threat. When fear conditioning was carried out after predatory threat, PTSD-like rats showed a marked extinction deficit compared with resilient rats. In contrast, no differences were seen between resilient and PTSD-like rats when fear conditioning and extinction occurred prior to predatory threat. These findings in Lewis rats closely match the results seen in humans with PTSD, thereby suggesting that studies comparing neuronal interactions in resilient vs. at-risk Lewis rats might shed light on the causes and pathophysiology of human PTSD.Following a severe traumatic event, some individuals manifest a syndrome, known as post-traumatic stress disorder (PTSD), characterized by repeated painful recollection of the trauma, avoidance of trauma reminders, intrusive thoughts, startle, hyperarousal, and disturbed sleep. Lifetime prevalence of PTSD ranges from 1.4% to 11.2% in representative samples (Afifi et al. 2010). Review of heritability studies indicate that there is a significant genetic component to PTSD (Nugent et al. 2008) as shared genes explain approximately 25%–38% of variability in PTSD symptom clusters and total symptoms (Afifi et al. 2010). Moreover, PTSD heritability coincides with that of other psychiatric conditions such as generalized anxiety, panic disorder, and depression (Chantarujikapong et al. 2001; Fu et al. 2007), suggesting that these disorders gain expression through common biological pathways.Although our understanding of PTSD has improved recently, we still have a limited grasp of the factors that predispose some to be at risk for PTSD, as well as those contributing to PTSD expression following trauma. In part, this situation results from the ethical limitations associated with human studies. For example, humans cannot be randomly assigned to trauma, and, importantly, the invasive techniques required to study the pathophysiology of PTSD can be used only in animals. Thus, a promising approach toward understanding the underlying pathophysiology of PTSD would be to study the disease in a valid animal model of the human syndrome.Fortunately, much work has already been performed to define an animal model of PTSD that reproduces the salient features of the human syndrome (see Adamec et al. 2006; Cohen et al. 2006a; Siegmund and Wotjak 2006). The most promising research has focused on the impact of exposing rodents to species-relevant threatening stimuli that mimic the kind of life-and-death circumstances that precipitates PTSD in humans. Indeed, rodents exposed to predators or their odor develop long-lasting (3 wk or more) manifestations of anxiety as seen in a variety of behavioral assays including the elevated plus maze (EPM), social interaction test, and acoustic startle (Adamec and Shallow 1993; Blanchard et al. 2003; Adamec et al. 2006). The inherent strength of this species-relevant stimulus was demonstrated in studies where predator odor served as an unconditioned stimulus to support cued or contextual fear conditioning (Blanchard et al. 2001; McGregor et al. 2002). As is the case with human PTSD, differential vulnerability to predatory threat was also observed in rodents. In one study, for instance, the propensity of different strains of rats to develop extreme behavioral manifestations of anxiety (EBMAs) as a result of predatory threat has been characterized, revealing that a much higher proportion (50%) of Lewis rats (an inbred strain) develops EBMAs as a result of an intense predatory threat compared with 10% of Fisher rats and 20% of Sprague–Dawley rats (Cohen et al. 2006b).Although these results are promising, it remains unclear whether Lewis rats also exhibit traits that parallel the pathophysiology of human PTSD. One such factor, thought to play a particularly critical role in the persistence of PTSD, is a compromised ability to extinguish fear memories (for review, see Quirk and Mueller 2008). Two main lines of evidence support this notion. First, in functional imaging studies, the brain structures that normally support fear expression and extinction (for review, see Pape and Pare 2010) show abnormal activity patterns in PTSD (Rauch et al. 2006; Shin et al. 2006; Bremner et al. 2008; Milad et al. 2009). Second, several studies have reported that individuals with PTSD are deficient at extinguishing classically conditioned fear responses (Orr et al. 2000; Peri et al. 2000; Blechert et al. 2007; Milad et al. 2008, 2009). Of particular interest, a study of identical twins discordant for trauma exposure has revealed that this extinction deficit was not a pre-existing condition but developed as a result of trauma (Milad et al. 2008). Given the possibility that an inability to extinguish fear might contribute to the maintenance of PTSD, we therefore tested whether Lewis rats reproduced the properties of extinction seen in human PTSD.     

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.     

The role of amygdala nuclei in the expression of auditory signaled two-way active avoidance in rats

Using a two-way signaled active avoidance (2-AA) learning procedure, where rats were trained in a shuttle box to avoid a footshock signaled by an auditory stimulus, we tested the contributions of the lateral (LA), basal (B), and central (CE) nuclei of the amygdala to the expression of instrumental active avoidance conditioned responses (CRs). Discrete or combined lesions of the LA and B, performed after the rats had reached an asymptotic level of avoidance performance, produced deficits in the CR, whereas CE lesions had minimal effect. Fiber-sparing excitotoxic lesions of the LA/B produced by infusions of N-methyl-d-aspartate (NMDA) also impaired avoidance performance, confirming that neurons in the LA/B are involved in mediating avoidance CRs. In a final series of experiments, bilateral electrolytic lesions of the CE were performed on a subgroup of animals that failed to acquire the avoidance CR after 3 d of training. CE lesions led to an immediate rescue of avoidance learning, suggesting that activity in CE was inhibiting the instrumental CR. Taken together, these results indicate that the LA and B are essential for the performance of a 2-AA response. The CE is not required, and may in fact constrain the instrumental avoidance response by mediating the generation of competing Pavlovian responses, such as freezing.Early studies of the neural basis of fear often employed avoidance conditioning procedures where fear was assessed by measuring instrumental responses that reduced exposure to aversive stimuli (e.g., Weiskrantz 1956; Goddard 1964; Sarter and Markowitsch 1985; Gabriel and Sparenborg 1986). Despite much research, studies of avoidance failed to yield a coherent view of the brain mechanisms of fear. In some studies, a region such as the amygdala would be found to be essential and in other studies would not. In contrast, rapid progress in understanding the neural basis of fear and fear learning was made when researchers turned to the use of Pavlovian fear conditioning (Kapp et al. 1984, 1992; LeDoux et al. 1984; Davis 1992; LeDoux 1992; Cain and Ledoux 2008a). It is now well established from such studies that specific nuclei and subnuclei of the amygdala are essential for the acquisition and storage of Pavlovian associative memories about threatening situations (LeDoux 2000; Fanselow and Gale 2003; Maren 2003; Maren and Quirk 2004; Schafe et al. 2005; Davis 2006).Several factors probably contributed to the fact that Pavlovian conditioning succeeded where avoidance conditioning struggled. First, avoidance conditioning has long been viewed as a two-stage learning process (Mowrer and Lamoreaux 1946; Miller 1948b; McAllister and McAllister 1971; Levis 1989; Cain and LeDoux 2008b). In avoidance learning, the subject initially undergoes Pavlovian conditioning and forms an association between the shock and cues in the apparatus. The shock is an unconditioned stimulus (US) and the cues are conditioned stimuli (CS). Subsequently, the subject learns the instrumental response to avoid the shock. Further, the “fear” aroused by the presence of the CS motivates learning of the instrumental response. Fear reduction associated with successful avoidance has even been proposed to be the event that reinforces avoidance learning (e.g., Miller 1948b; McAllister and McAllister 1971; Cain and LeDoux 2007). Given that Pavlovian conditioning is the initial stage of avoidance conditioning, as well as the source of the “fear” in this paradigm, it would be more constructive to study the brain mechanisms of fear through studies of Pavlovian conditioning rather than through paradigms where Pavlovian and instrumental conditioning are intermixed. Second, avoidance conditioning was studied in a variety of ways, but it was not as well appreciated at the time as it is today; that subtle differences in the way tasks are structured can have dramatic effects on the brain mechanisms required to perform the task. There was also less of an appreciation for the detailed organization of circuits in areas such as the amygdala. Thus, some avoidance studies examined the effects of removal of the entire amygdala or multiple subdivisions (for review, see Sarter and Markowitsch 1985). Finally, fear conditioning studies typically involved a discrete CS, usually a tone, which could be tracked from sensory processing areas of the auditory system to specific amygdala nuclei that process the CS, form the CS–US association, and control the expression of defense responses mediated by specific motor outputs. In contrast, studies of avoidance conditioning often involved diffuse cues, and the instrumental responses used to indirectly measure fear were complex and not easily mapped onto neural circuits.Despite the lack of progress in understanding the neural basis of avoidance responses, this behavioral paradigm has clinical relevance. For example, avoidance behaviors provide an effective means of dealing with fear in anticipation of a harmful event. When information is successfully used to avoid harm, not only is the harmful event prevented, but also the fear arousal, anxiety, and stress associated with such events; (Solomon and Wynne 1954; Kamin et al. 1963). Because avoidance is such a successful strategy to cope with danger, it is used extensively by patients with fear-related disorders to reduce their exposure to fear- or anxiety-provoking situations. Pathological avoidance is, in fact, a hallmark of anxiety disorders: In avoiding fear and anxiety, patients often fail to perform normal daily activities (Mineka and Zinbarg 2006).We are revisiting the circuits of avoidance conditioning from the perspective of having detailed knowledge of the circuit of the first stage of avoidance, Pavlovian conditioning. To most effectively take advantage of Pavlovian conditioning findings, we have designed an avoidance task that uses a tone and a shock. Rats were trained to shuttle back and forth in a runway in order to avoid shock under the direction of a tone. That is, the subjects could avoid a shock if they performed a shuttle response when the tone was on, but received a shock if they stayed in the same place (two-way signaled active avoidance, 2-AA). While the amygdala has been implicated in 2-AA (for review, see Sarter and Markowitsch 1985), the exact amygdala nuclei and their interrelation in a circuit are poorly understood.We focused on the role of amygdala areas that have been studied extensively in fear conditioning: the lateral (LA), basal (B), and central (CE) nuclei. The LA is widely thought to be the locus of plasticity and storage of the CS–US association, and is an essential part of the fear conditioning circuitry. The basal amygdala, which receives inputs from the LA (Pitkänen 2000), is not normally required for the acquisition and expression of fear conditioning (Amorapanth et al. 2000; Nader et al. 2001), although it may contribute under some circumstances (Goosens and Maren 2001; Anglada-Figueroa and Quirk 2005). The B is also required for the use of the CS in the motivation and reinforcement of responses in other aversive instrumental tasks (Killcross et al. 1997; Amorapanth et al. 2000). The CE, through connections to hypothalamic and brainstem areas (Pitkänen 2000), is required for the expression of Pavlovian fear responses (Kapp et al. 1979, 1992; LeDoux et al. 1988; Hitchcock and Davis 1991) but not for the motivation or reinforcement of aversive instrumental responses (Amorapanth et al. 2000; LeDoux et al. 2009). We thus hypothesized that damage to the LA or B, but not to the CE, would interfere with the performance of signaled active avoidance.