SPECIES,TEPEES, SCOTTIES,AND JOCKEYS: SELECTED BY CONSEQUENCES

“Ideas are like species: they must evolve.” This claim forms the conceptual core of an engaging book by Jonnie Hughes (2011), On the Origin of Tepees. Hughes asks: If evolution by natural selection explains the origin of the human species, then does selection by consequences also explain the origin of what we humans make and do? This question prompts consideration of three important analogies: between natural selection and artificial selection, between the law of natural selection and the law of effect, and between biological evolution and cultural evolution. These analogies in turn stimulate examination of the notions of purpose, design, and agency. Finally, discussion moves to the selectionism of Darwin and Skinner; although still controversial, this view remains the best way for natural science to understand the origins of adaptive behavior.     

Evaluating Merger and Intersection of Equivalence Classes with One Member in Common

Sidman (1994) noted that the existence of a member that is common to more than one class may produce either class merger (union) or class intersection. A multiple-selection, matching-to-sample test was developed to examine the conditions under which these outcomes occur. Test trials each required three conditional discriminations involving selection or rejection of comparison stimuli under control of samples representing two categories. Test results obtained from an initial group of typical adults using familiar stimuli (DOG and BIRD, pictures of dogs and birds and relevant printed breed names (e.g., DALMATIAN, RETRIEVER) showed the conditional stimulus control best described as intersection. For example, the word DALMATIAN provided the context for selecting the dalmatian but not the retriever picture. However, these results may have depended on the participants'' verbal history as English speakers. Would conditional-discrimination training with overlapping sets of laboratory-generated stimuli also result in intersection? Naïve typical adults were assigned to one of three different training conditions. Like the participants tested with familiar stimuli, these participants demonstrated highly reliable test outcomes best described as showing class intersection, regardless of training condition. These findings begin to elucidate the necessary and sufficient conditions for establishing complex category-like classes of stimuli.     

WITHIN‐SUBJECT REVERSIBILITY OF DISCRIMINATIVE FUNCTION IN THE COMPOSITE‐STIMULUS CONTROL OF BEHAVIOR

According to the composite-stimulus control model (Weiss, 1969, 1972b), an individual discriminative stimulus (S^D) is composed of that S^D''s on-state plus the off-states of all other relevant S^Ds. The present experiment investigated the reversibility of composite-stimulus control. Separate groups of rats were trained to lever-press for food whenever a tone or a light S^D was present. For one group, the nonreinforced S^Δ condition was tone-and-light absence ($\bar{T}$+$\bar{L}$). Tone-plus-light (T+L) was S^Δ in the other group. On a “stimulus compounding” test that recombined composite elements, maximum responding occurred to that composite consisting only of elements occasioning response increase. That was T+L for the group trained with $\bar{T}$+$\bar{L}$ as S^Δ and $\bar{T}$+$\bar{L}$ for the group trained with T+L as S^Δ. The S^Δ composite was next reversed over groups in Phase 2. In Phase 2 tests, maximum responding that was comparable in magnitude to that of Phase 1 was again controlled by the composite consisting only of elements most recently occasioning response increase—whether T+L or $\bar{T}$+$\bar{L}$. The inhibitory conditioning history of both composite-elements currently occasioning responding did not weaken the summative effect. These results confirm and extend Weiss''s composite-stimulus control model, and demonstrate that such control is fully reversible. We discuss how translating conditions of the stimulus-compounding paradigm to a composite continuum creates a functional and logical connection to intradimensional control measured through stimulus generalization, reducing the number of different behavioral phenomena requiring unique explanations.     

The Influence of Bloomfield's Linguistics on Skinner

Bloomfield''s “Linguistics as a Science” (1930/1970), Language (1933/1961), and “Language or Ideas?” (1936a/1970), and Skinner''s Verbal Behavior (1957) and Science and Human Behavior (1953) were analyzed in regard to their respective perspectives on science and scientific method, the verbal episode, meaning, and subject matter. Similarities between the two authors were found. In particular both asserted that (a) the study of language must be carried out through the methods of science; (b) the main function of language is to produce practical effects on the world through the mediation of a listener; and (c) a physicalist conception of meaning. Their differences concern the subject matter of their disciplines and their use of different models for the analysis of behavior. Bloomfield''s linguistics and Skinner''s functional analysis of verbal behavior are complementary approaches to language.     

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.     

Generalization of conditioned fear along a dimension of increasing fear intensity

The present study investigated the extent to which fear generalization in humans is determined by the amount of fear intensity in nonconditioned stimuli relative to a perceptually similar conditioned stimulus. Stimuli consisted of graded emotionally expressive faces of the same identity morphed between neutral and fearful endpoints. Two experimental groups underwent discriminative fear conditioning between a face stimulus of 55% fear intensity (conditioned stimulus, CS+), reinforced with an electric shock, and a second stimulus that was unreinforced (CS−). In Experiment 1 the CS− was a relatively neutral face stimulus, while in Experiment 2 the CS− was the most fear-intense stimulus. Before and following fear conditioning, skin conductance responses (SCR) were recorded to different morph values along the neutral-to-fear dimension. Both experimental groups showed gradients of generalization following fear conditioning that increased with the fear intensity of the stimulus. In Experiment 1 a peak shift in SCRs extended to the most fear-intense stimulus. In contrast, generalization to the most fear-intense stimulus was reduced in Experiment 2, suggesting that discriminative fear learning procedures can attenuate fear generalization. Together, the findings indicate that fear generalization is broadly tuned and sensitive to the amount of fear intensity in nonconditioned stimuli, but that fear generalization can come under stimulus control. These results reveal a novel form of fear generalization in humans that is not merely based on physical similarity to a conditioned exemplar, and may have implications for understanding generalization processes in anxiety disorders characterized by heightened sensitivity to nonthreatening stimuli.Fear generalization occurs when a fear response acquired to a particular stimulus transfers to another stimulus. Generalization is often an adaptive function that allows an organism to rapidly respond to novel stimuli that are related in some way to a previously learned stimulus. Fear generalization, however, can be maladaptive when nonthreatening stimuli are inappropriately treated as harmful, based on similarity to a known threat. For example, an individual may acquire fear of all dogs after an aversive experience with a single vicious dog. In this case, recognizing that a novel animal is related to a feared (or fear-conditioned) animal is made possible in part by shared physical features to the fear exemplar, such as four legs and a tail. On the other hand, fear generalization may be selective for those features that are associated with natural categories of threat; a harmless dog may not pose a threat, but possesses naturally threatening features common to other threatening animals, such as sharp teeth and claws. Moreover, the degree to which an individual fearful of dogs responds with fear may be related to either the physical similarity to the originally feared animal (e.g., from a threatening black dog to another black dog), or the intensity of those threatening features relative to the originally feared animal (e.g., sharp teeth from one animal to sharp teeth of another animal). Therefore, fear generalization based on perceptual information may occur via two routes—similarity to a learned fear exemplar along nonthreatening physical dimensions or along dimensions of fear relevance. Given that fear generalization often emerges as a consequence of conditioning or observational learning, it is important to determine which characteristics of novel stimuli facilitate fear generalization and the extent to which generalization processes can be controlled.Early explanations of stimulus generalization emphasized that an organism''s ability to generalize to nonconditioned stimuli is related to both the similarity and discriminability to a previously conditioned stimulus (CS) (Hull 1943; Lashley and Wade 1946). While Lashley and Wade (1946) argued that generalization was simply a failure of discriminating between a nonconditioned stimulus (CS−) and the reinforced CS (CS+), contemporary views contend that generalization enables learning to extend to stimuli that are readily perceptually distinguished from the CS (Pearce 1987; Shepard 1987; McLaren and Mackintosh 2002). This latter view has been supported by empirical studies of stimulus generalization in laboratory animals (Guttman and Kalish 1956; Honig and Urcuioli 1981). In these studies, animals were reinforced for responding to a CS of a specific physical quality such as color, and then tested with several different values along the same stimulus dimension as the CS (e.g., at various wavelengths along the color spectrum). Orderly gradients of responses are often reported that peak at or near the reinforced value and decrease as a function of physical similarity to the CS along the stimulus dimension (Honig and Urcuioli 1981). Further generalization was shown to extend from the CS+ to discriminable nonconditioned stimuli, suggesting that generalization is not bound to the organism''s ability to discriminate stimuli (Guttman and Kalish 1956, 1958; Shepard 1987).Interestingly, when animals learn to distinguish between a CS+ and a CS−, the peak of behavioral responses often shift to a new value along the dimension that is further away from the CS− (Hanson 1959). For instance, when being trained to discriminate a green CS+ and an orange CS−, pigeons will key peck more to a greenish-blue color than the actual CS+ hue. Intradimensional generalization of this sort is reduced when animals are trained to discriminate between two or more stimulus values that are relatively close during conditioning (e.g., discriminating a green-yellow CS+ from a green-blue CS−), suggesting that the extent of generalization can come under stimulus control through reinforcement learning (Jenkins and Harrison 1962). Spence (1937) described the transposition of response magnitude as an effect of interacting gradients of excitation and inhibition formed around the CS+ and CS−, respectively, which summate to shift responses to values further from the inhibitory CS− gradient. In all, early theoretical and empirical treatments of stimulus generalization in nonhuman animals revealed that behavior transfers to stimuli that are physically similar, but can be discriminated from a CS, and that differential reinforcement training can both sharpen the stimulus gradient and shift the peak of responses to a nonreinforced value.Although this rich literature has revealed principles of generalization in nonhuman animals, few studies of fear generalization have been conducted in humans (for review, see Honig and Urcuioli 1981; Ghirlanda and Enquist 2003). Moreover, the existing human studies have yet to consider the second route through which fear responses may generalize—via gradients of fear relevance. While a wide range of neutral stimuli, such as tones or geometric figures, can acquire fear relevance through conditioning processes, other stimuli, such as threatening faces or spiders, are biologically prepared to be fear relevant (Lanzetta and Orr 1980; Dimberg and Öhman 1996; Whalen et al. 1998; Öhman and Mineka 2001). Compared with fear-irrelevant CSs, biologically prepared stimuli capture attention (Öhman et al. 2001), are conditioned without awareness (Öhman et al. 1995; Öhman and Soares 1998), increase brain activity in visual and emotional processing regions (Sabatinelli et al. 2005), and become more resistant to extinction when paired with an aversive unconditioned stimulus (US) (Öhman et al. 1975). Although the qualitative nature of the CS influences acquisition and expression of conditioned fear, it is unknown how generalization proceeds along a gradient of natural threat. For instance, human studies to date have all tested variations of a CS along physically neutral stimulus dimensions, such as tone frequency (Hovland 1937), geometric shape (Vervliet et al. 2006), and physical size (Lissek et al. 2008). These investigations implicitly assume that the generalization gradient is independent of the conditioned value (equipotentiality principle). In other words, since the stimuli are all equally neutral prior to fear learning, fear generalization operates solely as a function of similarity along the reinforced physical dimension. However, since fear learning is predisposed toward fear-relevant stimuli, generalization may be selective to those shared features between a CS+ and CS− that are associated with natural categories of threat. Examining generalization using fear-relevant stimuli is thus important to gain better ecological validity and to develop a model system for studying maladaptive fear generalization in individuals who may express exaggerated fear responses to nonthreatening stimuli following a highly charged aversive experience (i.e., post-traumatic stress disorder or specific phobias).To address this issue, the present study examined generalization to fearful faces along an intradimensional gradient of fear intensity. A fearful face is considered a biologically prepared stimulus that recruits sensory systems automatically for rapid motor responses (Öhman and Mineka 2001), and detecting fearful faces may be evolutionarily selected as an adaptive response to social signals of impending danger (Lanzetta and Orr 1980; Dimberg and Öhman 1996). During conditioning, an ambiguous face containing 55% fear intensity (CS+) was paired with an electric shock US, while a relatively neutral face (11% fear intensity) was explicitly unreinforced (CS−) (Experiment 1). Skin conductance responses (SCR) were recorded as a dependent measure of fear conditioning. Before and following fear conditioning, SCRs were recorded in response to face morphs of the same actor expressing several values of increasing fear intensity (from 11% to 100%; see Fig. 1). A total of five values along the continuum were used: 11% fear/88% neutral, 33% fear/66% neutral, 55% fear/44% neutral, 77% fear/22% neutral, and 100% fear. For clarity, these stimuli are herein after labeled as S1, S2, S3, S4, and S5, respectively.Open in a separate windowFigure 1.Experimental design. (A) Pre-conditioning included six presentations of all five stimulus values without the US. (B) Fear conditioning involved discriminative fear learning between the S3, paired with the US (CS+), and either the unreinforced S1 (Experiment 1) or the unreinforced S5 (Experiment 2) (CS−). (C) The generalization test included nine presentations of all five stimuli (45 total), with three out of nine S3 trials reinforced with the US. Stimuli are not drawn to scale.Testing generalization along an intradimensional gradient of emotional expression intensity allows for an examination of the relative contributions of fear intensity and physical similarity on the magnitude of generalized fear responses. If fear generalization is determined purely by the perceptual overlap between the CS+ and other morph values, without regard to fear intensity, then we would expect a bell-shaped generalization function with the maximum SCR centered on the reinforced (intermediate) CS+ value (S3), less responding to the directly adjacent, but most perceptually similar values (S2 and S4), and the least amount of responding to the most distal and least perceptually similar morph values (S1 and S5). This finding would be in line with stimulus generalization reported along fear-irrelevant dimensions (Lissek et al. 2008) and in stimulus generalization studies using appetitive instrumental learning procedures (Guttman and Kalish 1956). If, however, fear generalization is biased toward nonconditioned stimuli of high fear intensity, then an asymmetric generalization function should result with maximal responding to the most fear-intense nonconditioned stimuli. This finding would suggest that fear generalization is selective to the degree of fear intensity in stimuli, similar to studies of physical intensity generalization gradients in nonhuman animals (Ghirlanda and Enquist 2003). We predicted that the latter effect would be observed, such that the magnitude of SCRs will disproportionately generalize to stimuli possessing a greater degree of fear intensity than the CS+ (Experiment 1). A secondary goal was to determine whether fear generalization to nonconditioned stimuli can be reduced through discriminative fear learning processes. Therefore, a second group of participants was run for whom the CS− was the 100% fearful face (Experiment 2). In this case, we predicted that discriminative fear conditioning between the CS+ (55% intensity) and the most fear-intense nonconditioned stimulus would sharpen the generalization gradient around the reinforced CS+ value, and that responses to the most fear-intense stimulus would decrease relative to Experiment 1. Moreover, this discriminative fear-learning process may provide evidence that fear generalization is influenced by associative learning processes and is not exclusively driven by selective sensitization to stimuli of high fear relevance (Lovibond et al. 1993). Finally, we were interested to discover whether generalization processes would yield subsequent false memory for the intensity of the CS+ in a post-experimental retrospective report. In sum, the present study has implications for understanding how fear generalization is related to the degree of fear intensity of a nonconditioned stimulus, the extent to which discrimination training efforts can thwart the generalization process, and how fear generalization affects stimulus recognition.     

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.     

Cannabinoid and cholinergic systems interact during performance of a short-term memory task in the rat

It is now well established that cannabinoid agonists such as Δ⁹–tetrahydrocannabinol (THC), anandamide, and WIN 55,212-2 (WIN-2) produce potent and specific deficits in working memory (WM)/short-term memory (STM) tasks in rodents. Although mediated through activation of CB1 receptors located in memory-related brain regions such as the hippocampus and prefrontal cortex, these may, in part, be due to a reduction in acetylcholine release (i.e., cholinergic hypofunction). To determine the interaction between cannabinoid and cholinergic systems, we exposed rats treated with WIN-2 or cholinergic drugs to a hippocampal-dependent delayed nonmatch to sample (DNMS) task to study STM, and recorded hippocampal single-unit activity in vivo. WIN-2 induced significant deficits in DNMS performance and reduced the average firing and bursting rates of hippocampal principal cells through a CB1 receptor-mediated mechanism. Rivastigmine, an acetylcholinesterase inhibitor, reversed these STM deficits and normalized hippocampal discharge rates. Effects were specific to 1 mg/kg WIN-2 as rivastigmine failed to reverse the behavioral and physiological deficits that were observed in the presence of MK-801, an NMDA receptor antagonist. This supports the notion that cannabinoid-modulated cholinergic activity is a mechanism underlying the performance deficits in DNMS. Whether deficits are due to reduced nicotinic or muscarinic receptor activation, or both, awaits further analysis.Administration of both synthetic and phytocannabinoids, including Δ⁹–tetrahydrocannabinol (Δ⁹–THC), WIN 55,212-2 (WIN-2), and CP 55,940, impair working memory (WM) and short-term memory (STM) through a CB1 receptor-mediated mechanism in rats (Lichtman et al. 1995; Lichtman and Martin 1996; Hampson and Deadwyler 1998, 1999, 2000; Braida and Sala 2000; Egashira et al. 2002). This suggestive evidence for endocannabinoid involvement in memory formation was confirmed by Terranova and coworkers (1996), who demonstrated that the CB1 receptor antagonist rimonabant facilitated short-term olfactory memory, and this was partially reversed by the muscarinic receptor antagonist scopolamine. This suggests an interaction between cannabinoid and cholinergic systems such that endocannabinoid tone suppresses cholinergic transmission. Consequently, rats pretreated with eptastigmine, a second-generation cholinesterase inhibitor remained unaffected by the full CB1 receptor agonist CP 55,940 when tested in an eight arm radial maze (Braida and Sala 2000). And more recent evidence from Mishima and coworkers (2002) suggests that a block of cholinesterase with physostigmine and tetrahydroaminoacridine protects against WM impairments induced by Δ⁹–THC. These findings further support a potential role of the cholinergic system in cannabinoid-induced memory impairments.The exact mechanisms for this interaction still remain elusive, although cholinergic projection neurons from medial septum to hippocampus are likely to play an important role (Harkany et al. 2003, 2005; Fitz et al. 2008). However, the neuromodulatory action of pharmacologically active cannabinoids on septo-hippocampal cholinergic activity in vivo remains unexplored. Within the hippocampus, cannabinoids presynaptically inhibit the release of acetylcholine, possibly through the activation of CB1 receptors located on cholinergic nerve terminals given that these effects were blocked by rimonabant (Gifford and Ashby Jr. 1996; Gifford et al. 1997a, 2000; Kathmann et al. 2001a). Direct in vivo microdialysis studies in awake rats also showed cannabinoid-induced decreases in acetylcholine release in the hippocampus through a CB1 receptor-mediated mechanism (Gessa et al. 1997; Carta et al. 1998). High doses of rimonabant alone increase the amount of acetylcholine release in the hippocampus (Gessa et al. 1997, 1998) either by blocking the tonic inhibitory influence of endocannabinoids and/or through its inverse agonism at CB1 receptors. Such actions are in agreement with a 100% greater increase in electrically evoked hippocampal acetylcholine release in CB1^−/− mice (Kathmann et al. 2001b).In contrast, low doses of Δ⁹–THC (0.01–0.15 mg/kg), WIN-2 (0.01–0.5 mg/kg), and HU-210 (0.001–0.004 mg/kg) have been shown to enhance acetylcholine release (Acquas et al. 2000, 2001), indicating that cannabinoid modulation of acetylcholine release in the hippocampus is “biphasic.” This has been further supported by the work carried out by Tzavara and coworkers (2003), who demonstrated that low (0.5 mg/kg, intraperitoneally [i.p.]) and high (5 mg/kg, i.p.) doses of WIN-2 induce transient stimulation and prolonged inhibition of hippocampal acetylcholine efflux, respectively. This demonstrates that the dose of cannabinoids plays a key role in determining how much acetylcholine is released in the hippocampus.Such an interaction is likely to play an important role during the performance of a delayed nonmatch to sample (DNMS) task but has not been explored. Hence, a comprehensive pharmacological assessment was carried out here to (1) reveal the existence of such an interaction in terms of DNMS performance and (2) assess a possible cannabinoid-acetylcholine cross-talk on burst characteristics of hippocampal principal cells in CA3 and CA1.     

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

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

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

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

DHPG activation of group 1 mGluRs in BLA enhances fear conditioning

Group 1 metabotropic glutamate receptors are known to play an important role in both synaptic plasticity and memory. We show that activating these receptors prior to fear conditioning by infusing the group 1 mGluR agonist, (R.S.)-3,5-dihydroxyphenylglycine (DHPG), into the basolateral region of the amygdala (BLA) of adult Sprague–Dawley rats enhances freezing normally supported by a weak footshock. This effect of DHPG was blocked when it was co-infused with either the general group 1 mGluR1 antagonist, (R,S)-1-aminoindan-1,5 dicarboxylic acid (AIDA), or with the selective mGluR5 antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP). These results support previous findings by Rodrigues and colleagues that mGluR5s in the lateral region of the amygdala make an import contribution to fear conditioning. More importantly, they support the general ideas embedded in the concept of metaplasticity, as per Abraham, and the synaptic-tagging hypothesis per Frey and Morris—that the processes that specify the content of experience can be experimentally separated from those needed to acquire the memory.The last decade has seen an increased appreciation of the view that the plasticity state of neurons—their ability to be modified by experience—is not fixed. Instead, the effect of experience depends on the physiological or biochemical state of the neurons or synapses that receive and store information contained in the experience, and this state is variable. This perspective is captured by the concept called “metaplasticity” (Abraham and Bear 1996; Abraham and Tate 1997; Abraham 2008), which recognizes that prior events can change the general plasticity or modifiability of neurons and synapses that will potentially store information contained in a subsequent target experience. This idea is also embedded in the synaptic-tagging hypothesis (Frey and Morris 1997, 1998) that will be described in some detail in the Discussion section. This view is important because it recognizes that the processes that specify the content of experience can be experimentally separated from those that are needed to store the memory for the experience.As reviewed by Abraham (2008), the range of prior events that can potentially determine a neuron''s state of plasticity is quite large and can be mediated by their effects on many components of the machinery that supports changes in synaptic strength. They include modification in NMDA-receptor function, AMPA-receptor trafficking, neuronal excitability, and epigenetic modifications.One way that the potential for plasticity can be altered is by activation of group 1 metabotropic glutamate receptors (mGluR1s and mGlur5s) (Abraham 2008). A number of reports based on the in vitro long-term potentiation (LTP) methodology suggest that activating this class of receptors prior to the delivery of a relatively weak inducing stimulus can transform a normally short-lasting form of LTP into a more persistent form (Cohen and Abraham 1996; Cohen et al. 1998; Raymond et al. 2000). For example, an infusion of (R.S.)-3,5-dihydroxyphenylglycine (DHPG), a group 1 mGluR agonist, into the bathing medium 30 min prior to the delivery of a weak inducing stimulus can significantly enhance the persistence of the resulting LTP, while having no effect on the baseline response (Cohen et al. 1998; Raymond et al. 2000). This effect of DHPG, however, is blocked (Raymond et al. 2000) when its administration is accompanied by an infusion of the group 1 mGluR antagonist, (R,S)-1-aminoindan-1,5 dicarboxylic acid (AIDA).Group 1 mGluRs also have been implicated in fear conditioning. For example, Rodrigues et al. (2002) reported that one subtype, mGluR5, is localized in dendrites and spines in neurons in the lateral nucleus of the amygdala, and fear conditioning can be significantly impaired if the mGluR5 antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), is infused into the lateral nucleus prior to conditioning.These findings, from the studies of synaptic plasticity and fear conditioning, provide the empirical basis for the hypothesis that motivates the experiments reported here—that the plasticity potential of neurons in the amygdala that support fear conditioning can be increased by activating group 1 mGluRs prior to the conditioning experience.To evaluate this idea, we followed a strategy similar to that used to study the role of mGluRs in LTP (Cohen and Abraham 1996; Cohen et al. 1998; Raymond et al. 2000). In these in vitro LTP experiments, a relatively weak inducing stimulus was used to generate a short-lasting LTP. Infusing the group 1 agonist DHPG prior to the delivery of the inducing stimulus transformed this short-lasting LTP into a more persistent form. Their results suggest that the activation of group 1 mGluR1 receptors independently initiates processes that make an important contribution to long-lasting LTP. To apply this strategy to fear conditioning, we used a very low level of shock—one that by itself produced an almost undetectable level of conditioned fear (as measured by freezing, the innate defensive response of rodents). We then determined if the activation of group 1 mGluRs prior to the conditioning experience would transform this outcome and produce a stronger level of freezing. DPHG was infused into the basolateral region of the amygdala (BLA) to activate the mGluRs.     

Early extinction after fear conditioning yields a context-independent and short-term suppression of conditional freezing in rats

Extinction of Pavlovian fear conditioning in rats is a useful model for therapeutic interventions in humans with anxiety disorders. Recently, we found that delivering extinction trials soon (15 min) after fear conditioning yields a short-term suppression of fear, but little long-term extinction. Here, we explored the possible mechanisms underlying this deficit by assessing the suppression of fear to a CS immediately after extinction training (Experiment 1) and the context specificity of fear after both immediate and delayed extinction training (Experiment 2). We also examined the time course of the immediate extinction deficit (Experiment 3). Our results indicate that immediate extinction produces a short-lived and context-independent suppression of conditional freezing. Deficits in long-term extinction were apparent even when the extinction trials were given up to 6 h after conditioning. Moreover, this deficit was not due to different retention intervals that might have influenced the degree of spontaneous recovery after immediate and delayed extinction (Experiment 4). These results suggest that fear suppression under immediate extinction may be due to a short-term, context-independent habituation process, rather than extinction per se. Long-term extinction memory only develops when extinction training occurs at least six hours after conditioning.Pavlovian fear conditioning and extinction are important behavioral models for studying the brain mechanisms underlying the acquisition, storage, retrieval, and suppression of traumatic fear (LeDoux 2000; Maren 2001, 2005; Kim and Jung 2005). In this procedure, an emotionally neutral stimulus, such as a tone, is paired with an aversive stimulus (US), such as an electric foot shock. After a few tone–foot shock pairings, the previous neutral tone becomes a potent conditioned stimulus (CS) and acquires the ability to elicit fear responses, such as freezing (CR). However, with repeated presentations of the CS-alone, the previously acquired CR gradually subsides, a process called extinction (Davis et al. 2003; Maren and Quirk 2004; Kim and Jung 2005; Myers and Davis 2007). The behavioral processes and the underlying neural mechanisms of extinction have attracted extensive attention in contemporary research of learning and memory (Bouton et al. 2006). Indeed, it has been suggested that failure to extinguish fear may contribute to post-traumatic stress disorder (PTSD) (Bouton et al. 2001; Rothbaum and Davis 2003). To avoid the possible long-term consequences and costs of PTSD or other anxiety disorders, clinical interventions are essential. While early interventions may manage the stress response to trauma, their efficacy has been challenged, because the acute intense stress of the traumatic experience might actually exacerbate relapse of fear (McNally 2003; Rothbaum and Davis 2003; Gray and Litz 2005). Thus, it is essential to learn when these interventions generate the best long-term extinction of fear responses.In a recent study, we demonstrated that delivering extinction trials shortly after fear conditioning yields poor long-term fear reduction (Maren and Chang 2006; but, see Myers et al. 2006). We observed that conditional freezing decreased during extinction training, but recovered completely 24 h later. This was true even when we gave 225 massed extinction trials 15 min after fear conditioning. However, in these experiments the within-session decrease in fear in rats that underwent extinction was similar to that in rats that were not exposed to extinction trials. Thus, it is unclear to what extent the short-term fear suppression we observed was due to a loss of fear to the context, the auditory CS, or both. It is also not clear whether fear suppression was due to extinction or, alternatively, another learning process such as habituation.To examine these issues further, in the present study we first assessed fear suppression to the auditory CS after immediate extinction by probing CS fear 15 min after extinction training. In a second experiment, we examined whether short-term fear suppression to the CS is renewed outside of the extinction context, as context specificity is one of the hallmarks of extinction (Bouton 2002; Ji and Maren 2007). In the third and fourth experiments, we examined the temporal delay necessary between conditioning and extinction to yield long-term suppression of fear. In our previous work (Maren and Chang 2006), all phases of training were conducted in the same context. Therefore, fear to the context decreased conditional freezing to the tone, particularly when extinction occurred shortly after conditioning, a time at which sensitized context fear was high. In an effort to isolate fear to the tone CS during extinction, we conducted extinction and test sessions in a context that was different from the conditioning context (i.e., an ABB procedure, where each letter denotes the context used for conditioning, extinction, and test, respectively). Our results reveal that delivering CS-alone trials shortly after fear conditioning produces a short-lived and context-independent suppression of freezing. This fear suppression may be due to a short-term, context-independent habituation process, rather than extinction. Furthermore, poor long-term extinction occurs even when the extinction trials were administered up to 6 h after conditioning.     

Impairments in fear conditioning in mice lacking the nNOS gene

The fear conditioning paradigm is used to investigate the roles of various genes, neurotransmitters, and substrates in the formation of fear learning related to contextual and auditory cues. In the brain, nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) functions as a retrograde neuronal messenger that facilitates synaptic plasticity, including the late phase of long-term potentiation (LTP) and formation of long-term memory (LTM). Evidence has implicated NO signaling in synaptic plasticity and LTM formation following fear conditioning, yet little is known about the role of the nNOS gene in fear learning. Using knockout (KO) mice with targeted mutation of the nNOS gene and their wild-type (WT) counterparts, the role of NO signaling in fear conditioning was investigated. Plasma levels of the stress hormone corticosterone were measured to determine the relationship between physiological and behavioral response to fear conditioning. Contextual fear learning was severely impaired in male and female nNOS KO mice compared with WT counterparts; cued fear learning was slightly impaired in nNOS KO mice. Sex-dependent differences in both contextual and cued fear learning were not observed in either genotype. Deficits in contextual fear learning in nNOS KO mice were partially overcome by multiple trainings. A relationship between increase in plasma corticosterone levels following footshock administration and the magnitude of contextual, but not cued freezing was also observed. Results suggest that the nNOS gene contributes more to optimal contextual fear learning than to cued fear learning, and therefore, inhibition of the nNOS enzyme may ameliorate context-dependent fear response.Anxiety disorders, such as post-traumatic stress disorder (PTSD), constitute the most prevalent mental illnesses in the United States, costing nearly one-third of the country''s total health bill (Greenberg et al. 1999). The treatment of these disorders requires overcoming complications such as reluctance to seek mental health treatment and an extremely high comorbidity rate with other affective disorders, reaching 80% (Brady 1997; Solomon and Davidson 1997). Emerging evidence suggests that dysfunctions underlying acquired anxiety and PTSD include an abnormal reaction to stress, which is mediated by specific neurochemical and neuroanatomical substrates (Yehuda and McFarlane 1995; Adamec 1997). Pharmacotherapies that target neuronal signaling molecules, such as nitric oxide (NO), may play a role in the treatment of these disorders.In the brain, N-methyl-d-aspartate receptor (NMDAR) activation and calcium influx into the cell activates the neuronal nitric oxide synthase (nNOS) enzyme to produce NO, which has the role of retrograde messenger (Snyder 1992). NO is involved in memory formation and synaptic plastic events such as late-phase long-term potentiation (LTP) (Lu et al. 1999; Arancio et al. 2001; Puzzo et al. 2006). Behavioral evidence in invertebrates (Lewin and Walters 1999; Muller 2000; Kemenes et al. 2002; Matsumoto et al. 2006) and vertebrates (Medina and Izquierdo 1995; Rickard et al. 1998; Ota et al. 2008) suggest that NO has a major role in consolidation of long-term memory (LTM). Recently, studies have shown that site-specific pharmacological blockade of NO signaling in rats impairs contextual (Resstel et al. 2008) and cued (Schafe et al. 2005) fear learning. However, the role of the nNOS gene in fear conditioning has not been investigated.In the present study, fear conditioning was investigated in homozygous nNOS knockout (KO) and wild-type (WT) mice. In the fear-conditioning paradigm, the association of a footshock (unconditioned stimulus; US), with a specific context and a neutral stimulus (auditory cue) results in learned fear. Re-exposure to the conditioning context and to the previously neutral auditory cue (conditioned stimulus; CS) elicits a freezing response in the absence of the aversive US. Thus, the fear-conditioning paradigm includes both contextual and cued fear learning components, which can be measured in separate tests. Fear conditioning recruits both the amygdala (emotional cue learning) and the hippocampus (spatial/contextual learning) (Phillips and LeDoux 1992; Goosens and Maren 2004; Mei et al. 2005). The involvement of these brain regions in fear learning and anxiety has been confirmed by animal and human imaging studies (LeDoux 1998; Rauch et al. 2006).We report that nNOS KO mice showed a severe deficiency in contextual fear learning and a less marked deficit in cued fear learning compared with WT mice after a single fear-conditioning session. This deficiency was partially improved by multiple (four) fear-conditioning sessions. In addition, we observed that plasma levels of corticosterone, the primary stress hormone in rodents, are related to contextual fear learning ability.     

Extended Analysis of Empirical Citations with Skinner's Verbal Behavior: 1984-2004

The present paper comments on and extends the citation analysis of verbal operant publications based on Skinner''s Verbal Behavior (1957) by Dymond, O''Hora, Whelan, and O''Donovan (2006). Variations in population parameters were evaluated for only those studies that Dymond et al. categorized as empirical. Preliminary results indicate that the majority of empirical research in the area of verbal behavior has been conducted with the younger developmentally disabled population and has focused on verbal operants from the introductory chapters of Skinner''s book. It is clear that Verbal Behavior has influenced empirical research over the past 50 years. We believe, however, that there are many underdeveloped research areas originating from Verbal Behavior that have not yet been addressed. Suggestions for extended areas of research are provided.     

Lifelong reductions of PKMζ in ventral hippocampus of nonhuman primates exposed to early-life adversity due to unpredictable maternal care

Protein kinase Mζ (PKMζ) maintains long-term potentiation (LTP) and long-term memory through persistent increases in kinase expression. Early-life adversity is a precursor to adult mood and anxiety disorders, in part, through persistent disruption of emotional memory throughout life. Here we subjected 10- to 16-wk-old male bonnet macaques to adversity by a maternal variable-foraging demand paradigm. We then examined PKMζ expression in their ventral hippocampi as 7- to 12-yr-old adults. Quantitative immunohistochemistry reveals decreased PKMζ in dentate gyrus, CA1, and subiculum of subjects who had experienced early-life adversity due to the unpredictability of maternal care. Adult animals with persistent decrements of PKMζ in ventral hippocampus express timid rather than confrontational responses to a human intruder. Persistent down-regulation of PKMζ in the ventral hippocampus might reduce the capacity for emotional memory maintenance and contribute to the long-lasting emotional effects of early-life adversity.

Early-life adversity is associated with an increased vulnerability to stress-related disorders that is maintained into adulthood, suggesting a very long-lived effect on emotional memory by the early-life event (Coplan et al. 1996). Although several structural and neurochemical sequelae of early-life adversity have been reported (Teicher et al. 2003; Jackowski et al. 2011), the direct effects of early-life adversity on the molecular substrates maintaining long-term memory storage have not been explored.Accumulating evidence supports a crucial role for the autonomously active, atypical protein kinase C (PKC) isoform protein kinase Mζ (PKMζ) in maintaining synaptic long-term potentiation (LTP), a putative physical substrate for memory, and long-term memory storage (Ling et al. 2002; Pastalkova et al. 2006; Glanzman 2013; Sacktor and Fenton 2018). The autonomous activity of PKMζ is due to its unusual structure that differs from other PKC isoforms (Sacktor et al. 1993). Most PKCs consist of two domains: a catalytic domain and an autoinhibitory regulatory domain that suppresses the catalytic domain. Therefore, most PKCs are inactive until second messengers bind to the regulatory domain and induce a conformational change that releases the autoinhibition. Because second messengers that activate PKCs such as Ca²⁺ or diacylglycerol have short half-lives, most PKCs are only transiently activated.PKMζ, in contrast, consists of an independent PKCζ catalytic domain, and the absence of an autoinhibitory regulatory domain results in autonomous and thus persistent activity once the kinase is synthesized. PKMζ mRNA is transcribed from an internal promoter within the PKCζ/PKMζ gene that is active only in neural tissue (Hernandez et al. 2003). The mRNA is translationally repressed and transported to dendrites of neurons (Muslimov et al. 2004). High-frequency afferent synaptic activity during LTP induction or learning derepresses PKMζ mRNA translation, triggering new synthesis of PKMζ protein (Osten et al. 1996; Hernandez et al. 2003; Tsokas et al. 2016; Hsieh et al. 2017).Once increased, the steady-state amount of PKMζ remains elevated during LTP or long-term memory maintenance. Recent work with quantitative immunohistochemistry (IHC) shows that spatial conditioning induces persistent increases of PKMζ in somatic and selective dendritic compartments of dorsal hippocampal CA1 pyramidal cells that can last at least 1 mo (Hsieh et al. 2021). The persistent increases are preferentially expressed in CA1 pyramidal cells that were activated during the formation of the memory, specifically at the termination zone of the Schaffer collateral/commissural inputs from subfield CA3. In contrast, persistent PKMζ increases are not evident in stratum lacunosum-moleculare, the termination zone that originates in entorhinal cortex that nonetheless is capable of expressing PKMζ. Postsynaptic domain-specific PKMζ expression patterns hint at distinct circuit-specific modifications of cortical–hippocampal synaptic function by maturational and experiential factors.Persistent changes in PKMζ expression are also associated with changes in the capacity for learning and memory across the life span of animals. Decreased memory ability in aged rats is associated with decreased training-induced, persistent PKMζ expression in prelimbic cortex, and increases in PKMζ are crucial for the cognition-enhancing effects of environmental enrichment in the aged animals (Chen et al. 2016). Hara et al. extended the connection between PKMζ and cognitive function to nonhuman primates (NHPs), showing that levels of PKMζ expression in dentate gyrus (DG) axospinous synapses correlate with successful performance on cognitive tasks in young and aged monkeys (Hara et al. 2012). These studies suggest that persistent down-regulation of PKMζ may comprise an important pathophysiological mechanism for cognitive impairment.Here we used a validated NHP model of early-life adversity, maternal variable-foraging demand (VFD), to explore the links between adversity in infancy and PKMζ expression in adulthood (Coplan et al. 1996; Jackowski et al. 2011). Previous studies of the VFD paradigm have revealed that both infants and their mothers exposed to VFD show significant cerebrospinal fluid (CSF) elevations of the stress neuropeptide, corticotropin-releasing factor (CRF). Moreover, the magnitude of CRF change in mothers and infants are positively correlated, suggesting synchronization of maternal–infant stress responses to the VFD stressor (Coplan et al. 2005). From a behavioral standpoint, maternal social rank plays a negligible role in determining an aggregate score of maternal–infant proximity, suggesting preferential attention of mothers to their infants. During the VFD condition, maternal social rank predicts >80% of the variance of maternal–infant proximity, suggesting mothering patterns are interrupted by preferential orientations to social rank; the latter determines food accessibility (Coplan et al. 2015). Dominant females show relative increases in maternal–infant proximity, whereas subordinate females show relative reductions in maternal–infant proximity. Neither pattern of attachment ameliorates an abnormal association between CSF oxytocin concentrations and hypothalamic-pituitary-adrenal (HPA) axis activity (Coplan et al. 2015). Offspring exposed to VFD rearing assessed both as juveniles and as full adults demonstrate persistent increases in CSF CRF concentrations in comparison with controls reared under non-VFD conditions (Coplan et al. 1996, 2001).Our prior neurohistological studies pointed to the DG as a region particularly vulnerable to VFD exposure, as shown by reduced trophic signaling and neurogenesis (Jackowski et al. 2011; Perera et al. 2011; Schoenfeld et al. 2021). We therefore hypothesized that early-life adversity due to unpredictable maternal care (for brevity, subsequently referred to as “early-life adversity”) reduces the persistent expression of PKMζ within the DG of ventral intrahippocampal neurocircuitry that mediates affective memory processing (Fanselow and Dong 2010). We used PKMζ antisera validated by the lack of immunostaining in PKMζ-null mice (Hsieh et al. 2021) to examine PKMζ expression in ventral hippocampus (NHP anterior hippocampus) in both DG granule cell layer and the stratum moleculare of the suprapyramidal blade that receives direct input from entorhinal cortex, as well as other regions encompassing the hippocampal formation, including the hilus, CA3, CA1, and subiculum.To assess behavioral correlates of hippocampal PKMζ expression, we used a stress-inducing paradigm designed specifically for singly housed bonnet macaque male NHPs, which we refer to as the “human exposure response” (Jackowski et al. 2011; Hamel et al. 2017), which is a variation of the paradigm used in human exposure studies by Kalin et al. in rhesus macaques (Kalin and Shelton 1989). On exposure to a direct human presence, singly housed adult male bonnet macaques react with a dichotomy of responses—confrontational versus timid (see the Materials and Methods) (Jackowski et al. 2011). In our macaque colony, groups of fully adult males are necessarily housed individually to prevent injury sustained during male agonistic encounters, whereas adult females and/or juveniles are safely housed in social groups. Because group housing of nursing females and/or juveniles of both sexes elicits a range of behaviors intrinsic to the species’ social repertoire (Rosenblum et al. 2001; Coplan et al. 2015) that complicates behavioral analyses to human exposure, we restricted our current study to male macaques.     

Entorhinal cortical Island cells regulate temporal association learning with long trace period

Temporal association learning (TAL) allows for the linkage of distinct, nonsynchronous events across a period of time. This function is driven by neural interactions in the entorhinal cortical–hippocampal network, especially the neural input from the pyramidal cells in layer III of medial entorhinal cortex (MECIII) to hippocampal CA1 is crucial for TAL. Successful TAL depends on the strength of event stimuli and the duration of the temporal gap between events. Whereas it has been demonstrated that the neural input from pyramidal cells in layer II of MEC, referred to as Island cells, to inhibitory neurons in dorsal hippocampal CA1 controls TAL when the strength of event stimuli is weak, it remains unknown whether Island cells regulate TAL with long trace periods as well. To understand the role of Island cells in regulating the duration of the learnable trace period in TAL, we used Pavlovian trace fear conditioning (TFC) with a 60-sec long trace period (long trace fear conditioning [L-TFC]) coupled with optogenetic and chemogenetic neural activity manipulations as well as cell type-specific neural ablation. We found that ablation of Island cells in MECII partially increases L-TFC performance. Chemogenetic manipulation of Island cells causes differential effectiveness in Island cell activity and leads to a circuit imbalance that disrupts L-TFC. However, optogenetic terminal inhibition of Island cell input to dorsal hippocampal CA1 during the temporal association period allows for long trace intervals to be learned in TFC. These results demonstrate that Island cells have a critical role in regulating the duration of time bridgeable between associated events in TAL.

The linkage of temporally discontiguous events, called temporal association learning (TAL), is an essential function for episodic memory formation; for animals, when an event took place, and in what order a series of events occurred is directly linked to adaptation to continuous changes in the environment (Eichenbaum 2000; Tulving 2002a,b; Kitamura et al. 2015a; Kitamura 2017; Pilkiw and Takehara-Nishiuchi 2018). The entorhinal cortical–hippocampal (EC-HPC) network in particular is currently considered to bridge the temporal discontinuity between events (Solomon et al. 1986; Moyer et al. 1990; Wallenstein et al. 1998; McEchron et al. 1999; Eichenbaum 2000; Huerta et al. 2000; Ryou et al. 2001; Takehara et al. 2003; Chowdhury et al. 2005; Esclassan et al. 2009; Morrissey et al. 2012; Suter et al. 2013; Sellami et al. 2017; Wilmot et al. 2019).Two major excitatory inputs to HPC arise from the superficial layers of the EC (Fig. 1A), forming the direct (monosynaptic), and indirect (trisynaptic) pathways (Amaral and Witter 1989; Amaral and Lavenex 2007; Kitamura 2017; Kitamura et al. 2017). While pyramidal cells in EC layer III (ECIII cells) project directly to CA1 (Kohara et al. 2014; Kitamura et al. 2015b), the trisynaptic pathway originates from excitatory Reelin⁺ stellate cells in EC layer II (ECII) projecting directly to DG, CA3, and CA2 (Fig. 1B; Tamamaki and Nojyo 1993; Varga et al. 2010). CalbindinD-28K⁺/Wolfram syndrome 1 (Wfs1)⁺ pyramidal cells, another excitatory neural population in EC layer II called “Island cells,” form cell clusters along the ECII/ECI border (Alonso and Klink 1993; Fujimaru and Kosaka 1996; Klink and Alonso 1997; Kawano et al. 2009; Varga et al. 2010; Kitamura et al. 2014; Ray et al. 2014) and directly project to the GABAergic interneurons of stratum lacunosum (SL-INs) in HPC CA1 and drive feedforward inhibition to HPC CA1 pyramidal cells (Fig. 1B; Kitamura et al. 2014; Surmeli et al. 2016; Kitamura 2017; Ohara et al. 2018; Yang et al. 2018; Zutshi et al. 2018).Open in a separate windowFigure 1.Circuit schematic diagram of the medial entorhinal cortex (MEC)–hippocampal (HPC) circuit. (A) Major projections in the entorhinal cortical (EC)-HPC network. ECIII neurons (green) project directly to CA1. ECII Ocean cells (ECIIo, purple) project to the dentate gyrus (DG) (light blue)/CA3 (pink) initiating the trisynaptic pathway. ECII Island cells (ECIIi, blue) project directly into CA1. (B) ECIII projections (green) excite the distal portions of CA1 pyramidal cell (yellow) dendrites in the stratum moleculare. Island cells (ECIIi, blue) excite the interneurons of stratum lacunosum (SL-INs, red), which in turn inhibit the distal dendrites of CA1 pyramidal cells in SL.Trace fear conditioning (TFC) has been established as one suitable animal model for TAL (Fendt and Fanselow 1999; Maren 2001; Kim and Jung 2006) that can be also used as a translational bridge between animal and human learning (Clark and Squire 1998; Buchel and Dolan 2000; Delgado et al. 2006). Lesion, pharmacological, molecular, and optogenetic manipulation, as well as disease models in medial entorhinal cortex (MEC), demonstrate that MEC is crucial for TFC and temporal learning (Ryou et al. 2001; Woodruff-Pak 2001; Runyan et al. 2004; Esclassan et al. 2009; Gilmartin and Helmstetter 2010; Suh et al. 2011; Morrissey et al. 2012; Shu et al. 2016; Hales et al. 2018; Yang et al. 2018; Heys et al. 2020). Specifically, MECIII inputs into the HPC CA1 pyramidal cells are essential for the formation of TFC (Yoshida et al. 2008; Suh et al. 2011; Kitamura et al. 2014; Kitamura 2017). However, the temporal association function driven by MECIII neurons must be regulated for optimal adaptive memory formation, as too strong an association of a particular pair of events may interfere with associations of other useful pairs, whereas too weak an association for a given pair of events, in terms of weaker impact of events or longer duration of temporal gap between events, would not result in an effective memory (Kitamura et al. 2015a; Marks et al. 2020). In a naturalistic context, this would mean that more distant/quieter sounds, less intense somatic sensations (e.g., pain), or increased temporal distance between any two events would signal that the events are less likely to be causally associated, therefore less relevant, and less likely to be stored and recalled. In fact, successful TFC depends on the strength of event stimuli and duration of temporal gap between events (Stiedl and Spiess 1997; Misane et al. 2005; Kitamura et al. 2014; Kitamura 2017). However, the underlying regulatory mechanism for TAL remains hidden. Previously we demonstrated that feedforward inhibition by Island cells acts as a gating controller for the MECIII inputs to the distal dendrites of HPC CA1 pyramidal cells in stratum moleculare (SM) (Kitamura et al. 2014) to control TFC when weaker (in this case diminished footshock intensity) unconditioned stimuli were delivered for TFC, indicating that Island cell activity controls the temporal association when the strength of two discontinuous events are relatively weaker. However, the way in which the EC-HPC network regulates TFC with a longer trace period still remains unknown. Because the activation of Island cells would result in a net inhibitory effect on the local network in CA1, imposing a tight and specific regulation on associations of events across the temporal gap in TAL (Crestani et al. 2002; Moore et al. 2010; Kitamura et al. 2014, 2015b), we hypothesized that the length of the temporal gap between events would also be modulated by this mechanism. In this study, we examined the role of the regulatory input to this circuit arising specifically from the Island cells in the MECII using apoptotic elimination of Island cells, chemogenetic neural inhibition, and optogenetic terminal inhibition methods within an L-TFC protocol to give a thorough and complete assessment of the circuit involvement while considering each technique''s unique features.     

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.     

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

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

The 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.