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Nencki Institute, Warsaw, Poland
ABSTRACT I. ORGANIZATION OF THE AMYGDALA A. An Outline of the Amygdala Anatomy B. The Major External Connections of the Amygdala C. Theories of the Functional Organization of the Amygdala II. GENE ACTIVITY AS A MAPPING TOOL: THEORETICAL AND TECHNICAL CONSIDERATIONS III. AMYGDALA ACTIVATION FOLLOWING BEHAVIORAL TRAINING A. Introduction B. Pattern of c-Fos Expression in the Amygdalar Nuclei in Aversively Motivated Standard Behavioral Tests C. Pattern of Expression of Other Activity Markers in the Amygdalar Nuclei in Aversively Motivated Standard Behavioral Tests D. Pattern of c-Fos Expression in the Amygdalar Nuclei in Aversively Motivated, Ethologically Relevant Behavioral Tests E. Pattern of Expression of Other c-fos Gene Activity Markers in the Amygdalar Nuclei in Aversively Motivated, Ethologically Relevant Behavioral Tests F. Pattern of c-Fos Expression in the Amygdalar Nuclei in Appetitively Motivated Standard Behavioral Tests G. Pattern of Expression of Other Activity Markers in the Amygdalar Nuclei in Appetitively Motivated Standard Behavioral Tests H. Pattern of c-fos Expression in the Amygdalar Nuclei in Appetitively Motivated, Ethologically Relevant Behavioral Tests 1. Sexual interaction/stimulation 2. Maternal/paternal interaction 3. Nonagonistic social interaction I. Pattern of Expression of Other Gene Activity Markers in the Amygdalar Nuclei in Appetitively Motivated, Ethologically Relevant Behavioral Tests J. The IntelliCage System: In-Between of Standard Tests and Ethologically Based Behavioral Paradigms K. Comparison of c-Fos Versus Zif268 Expression Patterns Following Behavioral Training IV. GENE ACTIVITY MARKERS AND DRUGS OF ABUSE A. Model Systems 1. Forced drug administration: acute and chronic 2. Instrumental intravenous drug self-administration 3. Conditioned place preference and avoidance 4. Voluntary alcohol drinking B. Cocaine-Evoked c-Fos Expression C. Cocaine and Other Gene Activity Markers D. Amphetamines and c-Fos Expression E. Amphetamines and Other Gene Activity Markers F. Methylphenidate and c-Fos Expression G. Nicotine and c-Fos Expression H. Nicotine and Other Than c-fos Gene Activity Markers I. Opioids, Morphine Derivatives (Morphine, Heroin), and c-Fos Expression J. Cannabinoids and c-Fos Expression K. Hallucinogens and c-Fos Expression L. Ethyl Alcohol and c-Fos Expression V. DIFFERENT BEHAVIORAL TASKS INVOLVE SPECIFIC SUBDIVISIONS OF THE AMYGDALA A. c-Fos in Neuronal Plasticity B. Basolateral Amygdala C. Central Amygdala D. Medial Amygdala E. Cortical Amygdala VI. CONCLUSIONS: THE FUNCTIONAL HETEROGENEITY OF THE AMYGDALA ACKNOWLEDGMENTS REFERENCES
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ABSTRACT |
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I. ORGANIZATION OF THE AMYGDALA |
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The amygdaloid complex is a small, spherical gray mass located, in most mammals, in the medial wall of the temporal lobes, close to the terminal part of the inferior horn of the lateral ventricle and adjacent to the hippocampus. This phylogenetically old, almond-shaped structure, like many other parts of the limbic system, has been considered for a long time as controlling various emotional and motivated behaviors. In recent years, the amygdala has emerged as the key forebrain structure mediating inborn and acquired emotional responses, as well as processing, interpreting, and integrating various aspects of biologically and/or emotionally important information. Since the amygdala was described for the first time in the human temporal cortex by Burdach in the early 19th century, many subdivisions of the amygdalar complex have been identified, and the classification of its subdivisions, the extent of its outer border, as well as the nomenclature and number of nuclei have remained controversial (see Ref. 381 for comments).
Different investigators have grouped the amygdaloid nuclei in various ways, using different criteria. One of the first anatomical descriptions was proposed by Johnston (201), who divided the amygdala into a two-part system based on a detailed analysis of comparative vertebrate material. He distinguished 1) a phylogenetically older corticomedial group of nuclei, composed of the central, medial, and cortical nuclei and the nucleus of the lateral olfactory tract and 2) a phylogenetically newer basolateral group, composed of the lateral, basal, and basomedial nuclei. It is worth noting that the nucleus of the lateral olfactory tract (see Ref. 483) and the cortical nuclei (141, 142) were sometimes excluded from the corticomedial part of the amygdaloid complex. Therefore, some authors used the term centromedial and/or dorsomedial (121) to designate Johnston's corticomedial division. On the basis of neuronal morphology (299), the amygdaloid nuclei were grouped into two major subdivisions: 1) the cortex-like nuclei that contain mostly pyramidal or modified pyramidal projection neurons and 2) the noncortex-like nuclei that do not have pyramidal-like neurons. According to McDonald (299), the cortex-like nuclei include the whole Jonston's basolateral group, as well as the cortical nuclei, whereas the noncortex-like nuclei include the central and medial nuclei.
Obviously, the nomenclatures that were set, especially in earlier studies (see Refs. 47, 77, 78, 123, 256), were based mostly on appearance of the tissue, without regard to organization of its connections with other brain structures. On the other hand, more recent anatomical descriptions combine examination of the intrinsic and extrinsic connections of separate amygdaloid nuclei with their cytoarchitectonic and histochemical characteristics. For instance, Price et al. (381) identified three distinct groups of nuclei, differently connected with other brain structures. The first, the basolateral group (called also the group of deep nuclei), is composed of the lateral, basal, and basomedial nuclei (the basomedial nucleus is also termed the accessory basal nucleus). This group is characterized by substantial interconnections with the neocortex. The second, the corticomedial or superficial group, is made up of the periamygdaloid cortex, the anterior and posterior cortical nuclei, the medial nucleus, and the nucleus of the lateral olfactory tract, which are directly connected with the olfactory and accessory olfactory system. The third group of nuclei is composed of the central nucleus and the anterior amygdaloid area, which are strongly interconnected with the autonomic control centers in the lateral hypothalamus and the brain stem structures.
Recently, McDonald (299) as well as Swanson and Petrovich (463) have further developed the parcellation introduced by Price et al. (381). Taking into consideration currently available cytoarchitectonic, chemoarchitectonic, and fiber connections data, they divided the amygdala into three parts: 1) the deep or basolateral group, which is constituted by a ventromedial extension of the deepest layer of the cortex (the lateral, basal, and basomedial nuclei); 2) the centromedial group, which is specialized ventromedial expansion of the striatum (the central and medial nuclei, as well as the amygdaloid part of the bed nucleus of stria terminalis); and 3) the superficial or cortex-like group being a part of the caudal olfactory cortex (the anterior and posterior cortical nuclei, the nucleus of the olfactory tract, and the periamygdaloid cortex). Several further subdivisions have been also distinguished. For instance, in the lateral nucleus, two components (the anterior and posterior) were described by Krettek and Price (245). On the other hand, Alheid et al. (5) divided the lateral nucleus into three regions (dorsal, ventrolateral, and ventromedial). According to Pitkanen et al. (373), the basal nucleus is comprised of three regions: the magnocellular, intermediate, and parvocellular subdivisions, and the basomedial nucleus is divided into the magnocellular and parvocellular subdivisions. Moreover, three regions were distinguished in the central nucleus: the capsular, lateral, and medial subdivisions. The medial nucleus was divided into the rostral, central, and caudal subdivisions, whereas in the periamygdaloid cortex the medial and sulcal regions were distinguished (see Ref. 373). Furthermore, Price et al. (381) and some other authors (see Refs. 10, 373, 374, 427) included to the amygdaloid complex the anterior amygdaloid and amygdalo-hippocampal areas, as well as groups of the intercalated nuclei. The present understanding of the internal organization of the amygdala is shown on the Figure 1.
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The amygdaloid complex is characterized by widespread and well-organized connections with many forebrain, midbrain, and hindbrain structures. The stria terminalis (ST) and the ventral amygdalofugal pathway (VAFP) are two major bundles of fibers connecting the amygdala with other areas of the brain. The ST, which arises mainly from the corticomedial group of nuclei, conveys fibers to several subcortical structures: the ventromedial hypothalamus as well as the septal and medial preoptic areas (93, 173, 270). The fibers of ST project also to the autonomic centers in the periaqueductal gray matter, parabrachial nucleus, and sympathetic preganglion neurons in the spinal cord. Moreover, the basal nucleus of the amygdala, which projects to the bed nucleus of the stria terminalis, influences a variety of hypothalamic, ventral, and dorsal striatum structures, as well as the brain stem areas. The latter also receive direct projections from the corticomedial amygdala. On the other hand, the VAFP is a rather diffuse fiber tract, which connects mostly the basolateral part of the amygdala with the thalamus, hypothalamus, septum, nucleus accumbens, and other structures of the ventral striatum, parahippocampal gyrus, and certain parts of the cingulum, piriform, and orbitofrontal cortices. The VAFP connects also the lateral and central amygdala with the lateral hypothalamus and with the dopaminergic neurons in the brain stem reticular formation.
From the functional point of view, all connections of the amygdaloid complex can be divided into three major systems (380). The first one provides sensory information to the amygdala, thus supporting amygdaloid modulation of sensory processing. It is composed mostly of the reciprocal projections and connects the amygdala with the olfactory cortex and ascending taste/visceral pathways. Moreover, it transfers sensory information from the posterior thalamus and sensory association cortex. The second system forms outputs of the amygdaloid complex to the hypothalamus and brain stem structures, which modulate visceral functions in relation to the emotional significance of internal and external stimuli. The last forebrain circuit is composed of the amygdala connections with the ventromedial frontal, rostral insular, and rostral temporal cortical areas, as well as with the medial thalamus and ventromedial basal ganglia. Through these connections the amygdala is able to be directly involved in regulation of emotional behavior and to influence several somatic motor responses.
It should be noted that each individual subdivision of the amygdala is characterized by a specific pattern of internal and external connections. An overview of those is presented in Figures 2–4. Their importance for understanding of the functional heterogeneity of the amygdala is discussed below.
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More than 100 years ago, Brown and Schafer (49) demonstrated in monkeys that after extensive bilateral damage to the temporal lobes, the animals became unnaturally fearless and tame and that this amazing transformation in the emotional behavior was not associated with apparent sensory changes. Similar emotional transformation was later described by Klüver and Bucy (225) after bilateral destruction of the amygdala and inferior temporal cortex. They observed that rhesus monkeys, earlier fierce and rather apprehensive, after the lesion approached fear-inducing stimuli with no display of anger or fear. The animals became emotionally dulled, and their facial expression and vocalization were less expressive. They were unable to recognize or use previously familiar objects, although their vision was not impaired, and they tended to explore every object. The animals also showed inappropriate sexual behavior, changes in food preferences, and disruption of social behavior evidenced by a loss in rank or even withdrawal from social life.
Later, the amygdala function was studied with the use of a variety of methods, including ablations and/or partial lesions, electrical brain stimulation, neurochemical intra-amygdala injections, and single-unit recordings. Notably, considering anatomical heterogeneity in the amygdala, the most significant conclusions resulting from these studies clearly indicated an advantage in performing subtotal amygdalar lesions over the total amygdalectomy (see Ref. 426).
One of the earliest concepts explaining the functional organization of the amygdala was proposed by Wutz and Olds (518), starting from the point that one can discriminate phylogenetically and morphologically two major subdivisions of the amygdalar complex: the dorsomedial and basolateral groups of nuclei (see above). Wutz and Olds, relying on the results of the self-stimulation studies, suggested the former as a rewarding and the latter as a punishing system. Thus they pointed at the importance of the valence of motivation in the functional descriptions of the amygdala. Although, for the next decades most of the studies were focused on the involvement of the amygdala in negative emotions, recent evidence supports a role of this structure in processing positive emotions, in addition to the negative ones (28, 216).
Nearly all studies on the functional organization within the amygdala have been focused on the role of the basolateral and the central nuclei, while the functions of the cortical and the medial nuclei remain more elusive. Countless studies of conditioned fear showed very clearly that the amygdala is involved in Pavlovian conditioning of aversive emotional responses (see below). Lesions of either the basolateral or central nuclei impaired freezing that indicated the conditioned state of fear (146, 258). According to the well-established model of fear conditioning, information flows to the lateral nucleus and is conveyed directly or via the basal nucleus to the central nucleus, which is primarily seen as the universal output of the amygdala, able to coordinate the autonomic, endocrine, and behavioral responses via brain stem arousal and response systems. The selective lateral nucleus damage demonstrated that it is a "sensory interface" of the amygdala in fear conditioning (259, 401), at least with respect to the auditory and contextual cues (146; but see Ref. 468). Moreover, the basolateral complex of the amygdala is seen as a place of formation of the conditioned stimulus (CS)-unconditioned stimulus (US) associations in fear conditioning (11, 259, 261, 288, 291, 292, 336, 401, 431, 494).
A widely held model, in which the basolateral complex (primarily the lateral nucleus) acts as the associative site for stimulus-outcome representations and the central nucleus provides the output pathway through which these associations gain access to appropriate responses, such as the conditioned freezing response, is known as a serial model of the basolateral/central amygdala function (401) (Fig. 5). This single, serially organized hierarchical lateral-to-central flow of information has been challenged by Killcross, Everitt, and others (see Refs. 22, 218, 219), who showed that the instrumental avoidance responses were impaired in the basolateral amygdala-lesioned animals, whereas Pavlovian conditioned suppression required intact central nucleus. Moreover, an analogous double dissociation using an appetitively motivated task was reported (179, 359). Thus Killcross et al. proposed the parallel model of information processing in the amygdala (22, 61, 218, 219, 335) (see Fig. 5).
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II. GENE ACTIVITY AS A MAPPING TOOL: THEORETICAL AND TECHNICAL CONSIDERATIONS |
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The fact that among IEGs those encoding transcription factors are the most often investigated in the brain, should be noted here, as it bears two very important consequences. Functionally, it suggests that we are just touching upon a tip of an iceberg of the genomic responses driven by those transcription factors, responses very poorly defined so far. It cannot, however, pass unnoticed that orchestrating such potentially very far-reaching responses is the only functional meaning of the phenomenon of the IEGs' expression. As a matter of fact, coordination of neuronal plasticity has not only been suggested as IEGs' function, but in a few cases has already been proven (consider, e.g., a pathway of extracellular matrix remodeling driven by AP-1; Refs. 197, 209, 338). Another important consequence of studies on IEGs/transcription factors is nuclear localization of the IEGs' protein products that greatly facilitates their immunostaining-based detection, because of excellent spatial resolution and rather simple way of numerical evaluation. The fact that extracellularly regulated gene expression was found to be often very transient has proven to be also very convenient, as it allows for a good temporal resolution of phenomena analyzed.
To help to appreciate the results obtained with IEG-based mapping, it is worthwhile to describe briefly a molecular scenario of their cellular expression pattern. Usually, their mRNA and protein levels are very low at basal conditions, i.e., in a variety of nonstimulated cells, including neurons. Increased neuronal expression results from stimulation of membrane receptors and subsequent rise in second messengers, including Ca2+, followed by kinase activation. The temporal pattern of this activation is quite uniform. An increase in mRNA levels is observed within a few minutes after the signal arrives at the cell membrane, and next the protein is accumulated, which occurs roughly between 30 and 90 min. Both mRNA and protein increases are transient. The transcription is soon shut off, and because of the aforementioned short half-lives of mRNA and protein, those gene products are gone within a couple of hours from the activated cells. Hence, if their expression is still observed in a group of neurons at later times, it suggests that it either results from novel stimuli that arrived later at the cell membrane, or we deal with a subset of late-activated cells. It is also of note that, especially in the case of c-fos, the gene expression in the brain is responsive to the first encounter to a novel stimulus. Repeated animal exposure to the same conditions gradually, within just a handful of sessions, diminishes c-Fos levels to virtual nothing. However, any novel element in otherwise very familiar set-up alerts the animal and results in c-fos activation (13, 343).
Two major experimental approaches to visualize IEGs' products expression are 1) in situ hybridization, detecting mRNA, and 2) immunocytochemistry for protein visualization. The former should provide unequivocal results, whereas the latter may be less specific, as different IEGs' proteins share structural similarities and there are antibodies available that do not differentiate among various Fos proteins. In situ hybridization may either be based on radioactive probe detection, and in this case, typically, provide resolution not allowing to visualize precisely single cells. This drawback has been circumvented recently with efficient nonradioactive probe labeling and detection. On the other hand, immunocytochemistry easily allows for a single-cell resolution at the levels of individual neuronal nuclei. Notably, whereas it has been shown that glia may express IEGs (see, e.g., Ref. 74), this apparently has not been a case in the brain in vivo in a context of neuronal plasticity, learning and memory, drugs of abuse, etc. It is also of note that excitatory neurons comprise the major population of IEG expressing neurons; however, inhibitory interneurons can also do so (65, 115).
In this analysis we have focused on the gene activity markers in the amygdala. These data are presented in a context of wealth of available information gathered on internal heterogeneity of the amygdala with other methods. One has to be, however, aware of the methodical limitations of the approaches employed. In all reported experiments, expression of the gene activity markers within amygdalar nuclei or their subdivisions was compared in the experimental groups with that of the control groups, and the statistically significant differences were determined. For such results to be valid, all the factors affecting the amygdala activation in the control groups should also be carefully taken into account. However, in many different experiments, nonequivalent procedures of handling or habituation to the experimental conditions in the control animals have been used (see also Refs. 98, 227). Furthermore, our recent study shows that in addition to the effects of the designed experimental treatment on the amygdala, one shall also consider information transfer among the animals that also activates this brain structure (228). These could seriously affect the conclusions of such studies leading to apparent discrepancies. Moreover, very often different statistical tests are used; therefore, the level of reliability is different.
The amygdaloid complex consists of several cytoarchitectonically well defined and internally distinguishable nuclei. However, furthermore, in concert with the anatomical data, there are also functional differences between various nuclei (see, e.g., Refs. 80, 111, 113, 162, 217, 229, 235, 282, 332, 385) or even subnuclei (see, e.g., Refs. 159, 385, 391). Therefore, it seems valuable to apply more precise dissection of the nuclei of the amygdala in analyzing the functions of the amygdala in a context of behavior. It is of note that this could be easily achievable with the use of activity markers immunolabeling approach that provides a mapping tool enabling a single-cell resolution. Unfortunately, this important approach has not routinely been employed.
Another obstacle in drawing the conclusions about the functional role of different amygdalar nuclei is also unavailability of the global picture of the amygdala activation for many experimental situations, namely, activation of the medial and cortical nuclei of the amygdala has predominantly been studied in sexual and social behaviors, whereas that of the basolateral and central amygdala has been studied in aversively motivated learning paradigms.
Finally, one additional, very poorly addressed important factor influencing the obtained results might be the amygdala lateralization. Notably, Holahan and White (181) as well as Scicli et al. (435) have recently shown the substantial differences in the c-Fos response between the left and right amygdala during contextual fear learning and memory retrieval.
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III. AMYGDALA ACTIVATION FOLLOWING BEHAVIORAL TRAINING |
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In this section we present the results of the studies that have documented the patterns of the amygdalar nuclei activation measured with various gene activity markers under different conditions of behavioral training. To systematize the existing data we have dissected them accordingly to either appetitive or aversive motivation, which the behavioral tests employ, as well as we distinguish a category of the behavioral tests with an ethological relevance.
The evolutionary foundation of the very complex system of emotional responses, that one can observe in mammals, has apparently a two-factor motivational organization, i.e., affects are organized by brain systems that adaptively respond to two basic kinds of stimulation, appetitive and aversive (see Ref. 255). Among the first to propose such a two-factorial system were Schneirla (433) and Konorski (240), who divided behavioral responses according to their adaptational and motivational significance into either approach versus withdrawal behaviors or preservative versus protective reflexes, respectively. Approach and/or preservative reactions included ingestion, copulation, or nurture of progeny, whereas withdrawal and/or protective ones, either escape from or rejection of noxious agents. Herein, we would like to compare the patterns of the amygdalar nuclei activation caused by appetitively versus aversively motivated behaviors.
An involvement of the amygdalar nuclei in emotionally charged behavior and memory is studied mainly in the well-established experimental models of conditioning, such as Pavlovian fear conditioning, active and inhibitory avoidance, or bar-pressing for food reinforcement. All of them employ artificial signaling stimuli and procedures designed for laboratory conditions. However, there is also recent interest in experimental paradigms that exploit more ethologically relevant approaches, using such stimuli as predators, sexual partners, or pups' presence. These paradigms employ evolutionarily and adaptively formed unconditioned emotional states, e.g., fear of predators (see Ref. 408). In this review, we compare the pattern of the amygdalar nuclei activation evoked by these two types of experimental conditions.
Thus far, the most frequently used neuronal activity marker has been c-fos mRNA and its protein (c-Fos). Therefore, we first analyze the data on c-fos activation and then compare them with the results on expression of other, less widely investigated gene activity markers, such as FosB, c-Jun, JunB, JunD, Zif268, NGFI-B, Arc, ICER, phospho-CREB, and brain-derived neurotrophic factor (BDNF).
B. Pattern of c-Fos Expression in the Amygdalar Nuclei in Aversively Motivated Standard Behavioral Tests
The majority of the studies on the expression of gene activity markers and behavioral training involved fear conditioning. Most often, two main training/testing procedures were employed: 1) contextual fear conditioning that is a training paradigm in which the animal is placed for a short time in a chamber, where it receives an inescapable foot shock or foot shocks (unconditioned stimulus, US). Then, it is brought back to the home cage and after some delay it is reexposed to the same apparatus and tested without the US. The memory of the situation is examined by measuring the freezing reaction (freezing or tonic immobility is a temporary state of profound motor inhibition induced by situations that supposedly generate intense fear, with the objective to protect the animal from attacks by predators) during the immediate post-shock period and the retention test. 2) The procedure of cued fear conditioning is similar, except that the original exposure to the US is accompanied by a clear sensory stimulus, e.g., a tone, light, or scent (conditioned stimulus, CS), and the testing is carried out in an experimental cage different from the training chamber, but in the presence of the CS. It is noteworthy that the animals exposed to a foot shock immediately upon entering a novel environment do not efficiently acquire a conditioned freezing reaction. The reduction of contextual conditioning can also be achieved by a preexposure to the training environment (termed as latent inhibition). These two phenomena are often used as control conditions. However, one should bear in mind that these conditions do not exclude learning as such. For instance, the latent inhibition relies on learning that the experimental set-up is initially safe, and this produces a difficulty in learning that it is unsafe later. Notably, recently, Levenson et al. (271) have shown that latent inhibition results in increased acetylation of histone H4 in the hippocampus that in turn may imply rather widespread changes in the gene expression.
Nearly all studies on the expression of gene activity markers within the amygdala during acquisition of conditioned fear have been focused on the basolateral amygdala (and its components, lateral and/or basal nuclei), as well as the central nucleus. Most of the results point to an involvement of the basolateral amygdala in both contextual and cued fear acquisition (for reference, see Table 1). In most cases, no differences in the pattern of c-Fos expression in the amygdalar nuclei between contextual and cued fear conditioning have been reported. However, Radulovic et al. (383) noted the increased c-Fos expression in the basolateral amygdala in cued but not in contextual fear conditioning. Notably, in most of the studies, the lack of activation of c-Fos in the central amygdala was observed (see Table 1).
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To sum up, it seems that the basolateral part and, less certainly, the medial nucleus of the amygdala are activated in both contextual and cued fear conditioning, whereas the level of c-Fos expression is not augmented in the central and cortical nuclei following such training.
During retrieval trials in the contextual fear conditioning, an animal is placed in the same experimental context as during the training but in an absence of the shock, whereas in cued fear conditioning, it is placed in a different experimental context and the CS cue is presented. In both conditions as a measure of the strength of the memory trace, the level of freezing response is evaluated (the other measures, however less frequently used, are suppression of previously learned licking behavior, ultravocalization, heart rate changes, and defecation frequency). Importantly, this procedure leads to an extinction of the training-evoked behavior, which results in a decrease of the fear evoked by the context, because the animal learns that the context no longer predicts the shock. It has been demonstrated that although the formerly established associations remain strong, the performance in the presence of the extinguished stimulus is diminished (44). Thus the extinction seems to be an active learning process that suppresses, rather than removes, the traces of former learning. Furthermore, the retrieval of the fear memory appears to involve an active, protein synthesis-sensitive component termed reconsolidation (337). Hence, one would expect gene-expression component to the retrieval sessions. Importantly, the gene expression changes observed following the retrieval session (without any foot shock) clearly argue against a notion that painful experience alone is responsible for those changes.
The basolateral part of the amygdala (and its parts, lateral and/or basal nuclei) were the most frequently described as activated, as far as c-Fos expression is concerned, in both contextual and cued fear memory retrieval (for reference, see Table 1). The activation of the basolateral part of the amygdala has also been shown in extinction of fear (176). In contrast to the original training (see above), in most of the studies activation of the central nucleus in both contextual and cued fear memory retrieval was observed (see Table 1). The c-Fos activation within the medial and cortical parts of the amygdala following fear memory retrieval was investigated less frequently, and the existing results are rather contradictory (see Table 1).
To summarize, it appears that the basolateral and central parts of the amygdala are active during contextual and cued fear memory retrieval, whereas the data on the activity of the medial and cortical nuclei of the amygdala are inconsistent.
The two-way (active) avoidance behavior is acquired in a shuttle-box apparatus that consists of two compartments. Both of them are equipped with a source of a CS and a gridded floor through which a US, a foot shock, can be delivered. The animal is originally placed in one of the two compartments, then the CS is presented and followed by the US within a few seconds. The animal is supposed to learn the signaling value of the CS and to avoid the US by moving to the opposite compartment. The training includes several sessions, which are usually composed of a number of trials.
There are reports of the activity of the basolateral, medial, and cortical nuclei, but not the central nucleus, of the amygdala after one session of two-way avoidance training (29, 99, 315, 428). Notably, Savonenko et al. (428) applied factor analysis to separate and group a number of behavioral components observed during the training. Surprisingly, no correlation was found between the level of c-Fos expression observed in any of the 13 amygdalar subdivisions that were analyzed and either avoidance reaction or sum of the shock received. On the other hand, c-Fos expression in the cortical amygdala correlated with grooming behavior (reflecting the lack of fear), whereas the c-Fos levels in the basolateral and medial amygdala correlated with anticipatory anxiety.
In the conditioned emotional response training, the animals that previously acquired an instrumental response, e.g., bar-pressing for a food reward, are subsequently trained to learn that an acoustic stimulus signals a foot shock. The suppression of the instrumental reaction is treated as a measure of acquired emotional response. The training is usually composed of four or five sessions, each including several trials.
The increased c-Fos expression after the first session of such training was observed in the basolateral and medial, but not in the cortical, nuclei of the amygdala (229, 455). The results on c-Fos expression in the central amygdala are inconsistent (see Table 1).
When consumption of a food with a novel taste is followed by exposure to a toxin, animals will avoid that taste in the future. This phenomenon is used in the conditioned taste aversion (CTA) test, in which learning is achieved in a single trial. The water-deprived animals learn to associate a novel taste (e.g., sucrose or saccharin) with the malaise caused by a toxin (e.g., lithium chloride, injected after the onset of drinking of sweetened water). Thus CTA is a form of classical conditioning in which animals avoid a taste (CS) that has previously been paired with a treatment that produces transient illness (US). However, CTA differs from most forms of classical conditioning in the speed and efficacy with which the animals acquire the conditioned response. CTA learning is rapidly gained, tolerates long delays between CS and US (they can reach even a few hours), and is very robust.
The involvement of the amygdala in conditioning of taste aversion measured by c-Fos expression was studied apparently only in the central and basolateral nuclei of the amygdala. The conditioning of taste aversion was shown to induce the elevated level of c-fos and its protein expression in the central amygdala (for reference, see Table 1). However, there are some noticeable discrepancies between the results of those studies. Navarro et al. (340), who compared the level of c-Fos expression in the animals in which saccharin was paired with lithium chloride either once or three times, observed activation of the central amygdala only when three pairings of the stimuli were applied. In this study, the control group consisted of animals in which lithium chloride was replaced by NaCl. Notably, the authors observed the behavioral effects, i.e., rejection of saccharin, following both the first and third trial. In contrast, Lamprecht and Dudai (254), Koh and Bernstein (231), and Wilkins and Bernstein (513) showed activation of the central nucleus after a single trial of taste aversion conditioning. However, Lamprecht and Dudai (254) did not observe the difference in the c-fos expression between CTA-trained animals and lithium chloride-injected rats. On the other hand, Koh and Bernstein (231) as well as Wilkins and Bernstein (513) showed much lower levels of c-Fos expression in the central amygdala following either the lithium chloride injection itself or pairings of the familiar taste with the lithium chloride injection (familiarity of the CS precluded learning of CTA) compared with the animals trained in the CTA.
In the above-mentioned studies, the distribution of the c-Fos-positive neurons within subdivisions of the central amygdala was not investigated. Interestingly, Yamamoto et al. (519) showed that the injection of the lithium chloride induced the elevated c-Fos expression mainly in the lateral part of the central nucleus of the amygdala. However, the influence of the injection itself was not controlled in this study. In contrast, drinking of sucrose elicited the increased c-Fos expression mainly in the medial part of the central nucleus. On the other side, the intraoral infusion of sucrose after CTA induced c-Fos expression evenly in the central amygdala (519).
The data on c-Fos activation in the basolateral amygdala following conditioning of taste aversion are not consistent (for reference, see Table 1). Interestingly, Wilkins and Bernstein (513) found the significant difference in the pattern of c-Fos expression following CTA established by exposing rats to a novel taste CS through a bottle or through intraoral infusion. Conditioning rats with the bottle method led to the increased c-Fos expression in the basolateral and central amygdala, whereas intraoral infusion induced the activation restricted to the central nucleus of the amygdala.
The injection of lithium chloride alone induced the elevated level of c-fos and its protein expression in the central nucleus of the amygdala, whereas the data on activation of the basolateral amygdala following the lithium chloride injection are less consistent (for reference, see Table 1).
It is known that rats rapidly become anorectic when eating an amino acid-imbalanced diet that induces a deficiency of an indispensable amino acid. Recognition of amino acid deficiency that leads to learned taste aversion was studied by Wang et al. (496). They found the increase in c-Fos expression in the central amygdala of rats following the introduction of a diet imbalanced in threonine.
In aggregate, it appears that the central and basolateral nuclei of the amygdala are activated during conditioned taste aversion learning and retrieval, whereas no central amygdala activation was noted during extinction (see Fig. 6). However, it is not clear, especially in the case of the central amygdala, if central amygdala activation is evoked by learning of associations between taste CS and US or coding information about taste CS and US themselves.
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The expression of other members of Fos/Jun family in the amygdala was studied following CTA acquisition, as well as after lithium chloride injection alone. The increased level of fosB expression was not observed in the central and basolateral amygdala following either CTA training or lithium chloride injection (254, 462). On the other hand, it was shown that c-Fos upregulation in the central nucleus after the injection of lithium chloride is accompanied by the increased expression of JunB (462).
Besides c-Fos, one of the most frequently applied gene activity markers is Zif268 (also known as NGFI-A, Egr-1, Krox-24, ZENK, and TIS-8; see Ref. 227). Almost all studies on the expression of Zif268 within the amygdala were carried out following fear conditioning, and they have been focused on the lateral nucleus of the basolateral amygdala and the central nucleus. Unfortunately, the results of the Zif268 expression following fear conditioning in the lateral amygdala are rather inconsistent. The increase in the zif268 mRNA expression was demonstrated following contextual fear conditioning (284–286, 410), as well as following cued fear acquisition (176). In contrast, Hall et al. (163) reported nonspecific induction of zif268 mRNA, i.e., the expression of zif268 was increased in the lateral nucleus of the amygdala not only in the experimental group but also in all control groups exposed to the training chamber compared with naive controls. Moreover, Weitemier and Ryabinin (504) have shown lack of differences in the Zif268 expression within the amygdala between fear-conditioned and naive mice after the training phase. In this study, both delayed conditioning (the US delivered simultaneously with the last period of CS application) and trace conditioning (the US delivered after a temporal gap after the CS) procedures were applied. In contrast to the lateral nucleus of the amygdala, none of the studies reported activation of the central nucleus following fear acquisition (163, 284, 285).
Similarly inconsistent and incomplete are the results on the expression of Zif268 in the lateral nucleus of the amygdala following fear memory retrieval (161, 176, 286, 410).
Taken together, the data on the Zif268 expression in the lateral nucleus of the amygdala following fear conditioning and memory retrieval are inconclusive. The observed discrepancies might have resulted from different procedures that were used by the authors (for extensive discussion on this issue, see Ref. 227). On the other hand, it appears that no elevated Zif268 expression occurs within the central amygdala following fear conditioning. Interestingly, the expression of another transcription factor protein related to Zif268, NGFI-B, was found to be augmented in the lateral nucleus of the amygdala following contextual fear conditioning, but not after contextual fear memory retrieval (286).
The expression of other immediate early genes, Arc and ICER, was investigated only following very few kinds of behavioral trainings. For instance, the increased arc mRNA expression was found in the basolateral and central nuclei of the amygdala after contextual memory retrieval (529). Furthermore, the expression of ICER was shown to be increased in the central, but not the basolateral, amygdala following both lithium chloride injection and learning of CTA (254, 456; see also Ref. 319).
Phosphorylated form of cAMP-responsive element binding transcription factor (P-CREB) is also believed to serve as a marker of neuronal activity. The increased expression of P-CREB was noted in the basolateral amygdala after both contextual and cued fear conditioning (457, 502). Moreover, the increased level of P-CREB expression was shown in the central amygdala following contextual fear conditioning (457). On the other hand, the elevated expression of P-CREB was observed in the basolateral amygdala, but not in the central nucleus of the amygdala, following cued fear memory retrieval (162). Furthermore, the expression of P-CREB in the basolateral, central, medial, and cortical nuclei of the amygdala was reported after one session of two-way avoidance training (420).
The elevated level of BDNF mRNA expression was seen in the basolateral amygdala after both visually and olfactory cued fear conditioning, but not in the medial amygdala following visually cued fear conditioning (390).
Summing up, the pattern of expression of the other gene activity markers in the amygdalar nuclei following different behavioral tests often, although not always, resembles the pattern seen with the use of c-Fos expression. For instance, fear conditioning induced the elevated level of expression of c-Fos and P-CREB in the basolateral part of the amygdala. Similarly, both injection of lithium chloride and learning of CTA caused the increased level of c-Fos, JunB, and Zif268 expression in the central amygdala. However, there are also clear discrepancies, e.g., on activation of the basolateral part of the amygdala measured with the c-Fos and Zif268 expression following fear conditioning and retrieval.
D. Pattern of c-Fos Expression in the Amygdalar Nuclei in Aversively Motivated, Ethologically Relevant Behavioral Tests
In addition to the standard behavioral tests, there are also paradigms that employ more natural stimuli, in a hope to evoke evolutionarily and adaptively formed unconditioned emotional states. Notably, they are often used and interpreted in different ways: in the standard tests predominantly learning processes are assessed, while in the more ethologically relevant behaviors mainly the evoked emotional states are considered. However, as in the standard tests, in the ethologically based behavioral paradigms both learning and emotions are involved and should be taken into account. For instance, the existence of learning processes during exposure to a predator is proven by the observation that rats develop conditioned fear to both context and cues that have been paired with, e.g., a cat odor (for review, see Ref. 97). Another piece of evidence is provided by the result of Figueiredo et al. (114), who observed a clear decrease of c-fos expression in the medial amygdala after the seventh day of habituation to the cat presence compared with the level of expression after the first session of such exposure. Another example, supporting the involvement of learning processes, is a phenomenon of avoiding a familiar winner by the subordinate animals 1 day after a fight (249). These data suggest that the animals learned to recognize the winner.
To evoke an innate fear, in a more ethologically relevant approach, exposure to a high, open, and/or highly illuminated space, a live predator, a predatory cue (e.g., an odor), an alarm signal (e.g., ultrasonic vocalization), or aggressive conspecifics are used. The elevated plus-maze and the elevated T-maze are considered as ethologically based animal models of anxiety. The plus-maze consists of four arms, which form a regular cross and are elevated over the ground. Two opposing arms are enclosed by side walls, and the remaining two are open. Due to the innate fear of height and open space, the animals remain longer in the enclosed arms. The more pronounced this innate fear is, the longer they stay in the enclosed arms, e.g., it is known that the animals receiving anxiolytic drugs enter more often and remain longer in the open arms. The elevated T-maze is derived from the elevated plus-maze and consists of three elevated arms, one enclosed and two open. An animal is supposed to perform two tasks in the apparatus: the one-way escape and the inhibitory avoidance. For the former task, an animal is placed in an open arm, from which it tends to escape. The time of escape latency is measured. This procedure is supposed to represent innate fear. For the inhibitory avoidance an animal is placed in the enclosed arm and the time to leave the arm is measured. Due to innate fear the animals will learn to avoid the open arms and stay longer in the enclosed arm; therefore, this procedure is supposed to represent conditioned fear. This test usually involves several trials.
The elevated c-Fos expression was observed in the basolateral, medial, and cortical, but not in the central, parts of the amygdala after exposure of rats to the elevated plus-maze (99, 450, but see Ref. 274 as well as Table 2 and Figure 7). The expression of c-Fos after exposure to the one-way escape task of the elevated T-maze was increased in the basolateral part of the amygdala, whereas after exposure to the inhibitory avoidance task, in the medial amygdala (451).
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In the predatory exposure procedure, in which rats were confined to a small box and exposed to a live cat, Figueiredo et al. (114) found the increased c-fos mRNA expression in the medial nucleus of the amygdala. Similarly, the exposure to a cat odor in rats was shown to induce an increased level of c-Fos expression in the medial, but not the basolateral and central nuclei of the amygdala (96, 308). Moreover, Dielenberg et al. (96) showed lack of activation of c-Fos in the cortical nuclei of the amygdala after exposure to the cat odor. In both studies the examined rats were presented to the odor in the arena equipped with the small chamber, which made it possible for the rats to hide.
Using a fox odor, Funk and Amir (131) showed the activation of the basolateral, but not the central, parts of the amygdala. In contrast, Day et al. (89) applying 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), an isolated chemical component from fox feces, observed elevated c-fos mRNA expression in the central and medial, but not basolateral and cortical, nuclei of the amygdala. In this study, the rats were presented to the fox odor in small cages without any place to hide.
Summing up, the predatory presence, or even its odor, seem to evoke the augmented level of c-Fos expression in the medial, but not cortical, nuclei of the amygdala. The data on the c-Fos expression in the basolateral and central amygdala are less consistent. The discrepancies might stem from different odors or odor concentrations that were used in the above-mentioned studies, but also from different experimental conditions, e.g., presence or absence of a place to hide.
Recently, we have described an experimental rat model of between-subject transfer of emotional information and effects of the transferred fear on activation of the amygdala. Briefly, the rats were kept in pairs, and one animal ("demonstrator") was treated either to a foot-shock reinforced context conditioning or exposed to a novel cage without any foot-shocks. Then the demonstrator rats returned to the home cages, to the other animals (called "observers"). We have found that by measuring startle reflex and exploration index we can distinguish between observers paired with demonstrators of both kinds. When we examined the influence of the demonstrators' presence on the activity of the amygdalar nuclei of the observers, we found that the observers had in the all amygdalar subdivisions the same levels of c-Fos expression as demonstrators, except for the central nucleus, where the observers displayed more c-Fos than the demonstrators (see Ref. 228; see also Fig. 8).
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20 kHz, which were able to induce defense behavior. Similarly, Mongeau et al. (327) observed the increase in c-fos mRNA expression in the basolateral, medial, and cortical nuclei of the amygdala in mice exposed to the ultrasonic vocalization. In contrast, they did not show the increased c-fos expression in the central nucleus of the amygdala. The exposure to aggressive conspecifics is the other behavioral paradigm used to evoke innate fear or aggression. In rodents, aggression against conspecifics is a form of social behavior in which adult animals fight to establish dominance relationships. This is used in the resident-intruder test, in which an unfamiliar animal, an "intruder," is placed into the home cage of the other animal, "resident," to provoke an encounter. Kollack-Walker and Newman (235) investigated the pattern of c-Fos expression in the male Syrian hamster amygdala in the resident-intruder test. They found activation of the medial and cortical, but not the basolateral, part of the amygdala in both dominant and subordinate animals. Furthermore, Kollack-Walker et al. (237) studied the c-fos mRNA expression in the hamster amygdala in similar conditions, except for much intense habituation to handling and to the novelty of another male's cage and odors. They found the increased c-fos expression in the medial, but not basolateral, central and cortical nuclei of the amygdala in the dominant males. In contrast, the animals that experienced social defeat had activated the central and medial, but not basolateral and cortical, nuclei of the amygdala. The activation of the central nucleus of the amygdala after experiencing social defeat was also observed in the study of Kollack-Walker et al. (234). Overall, in the dominant animals, the increased level of c-Fos expression was consistently seen in the medial nucleus of the amygdala (for reference, see Table 2), in both males and females, in hamster, mouse, and prairie vole. Moreover, the activation of the cortical nuclei was often shown in such conditions, whereas the results for the basolateral and central nuclei of the amygdala are less consistent (see Table 2).
Taken together, it appears that the aggressive encounter consistently induced the increased level of c-Fos expression within the medial amygdala (see Fig. 7), whereas the data on activation of the basolateral, central, and cortical nuclei of the amygdala are not consistent. These discrepancies might have been a result of using different experimental conditions, as well as various species of animals employed.
The obtained data also show that the pattern of c-Fos expression in the amygdala may depend on the result of aggressive encounter, being different for its losers and winners. It is known that after an aggressive encounter, a loser selectively avoids his own, familiar winner but does not avoid other males. This suggests that the losers are able to recognize and remember the familiar individual. The neural basis of this phenomenon was studied by Lai et al. (248). They showed activation of the basolateral, but not central and medial, parts of the amygdala in such conditions.
E. Pattern of Expression of Other c-fos Gene Activity Markers in the Amygdalar Nuclei in Aversively Motivated, Ethologically Relevant Behavioral Tests
There are very few pieces of data on the expression of gene activity markers other than c-fos in the amygdala following ethologically relevant behavioral tests. Moreover, they are often not consistent with the c-fos expression results. For instance, in the predatory exposure procedure, in which rats were exposed to a live cat, Rosen et al. (409) did not find the increased zif268 mRNA expression in the lateral nucleus of the amygdala. In this study the examined rats were presented to the cat in the large arena without separate compartments for both animals. The authors compared the expression of zif268 between rats either exposed to a live cat or just handled or confined to a small box. Furthermore, Lai et al. (248) studied Zif268 expression in a loser that selectively avoided his own, familiar winner but did not avoid other males. They investigated the basolateral, central, and medial parts of the amygdala and did not observe the elevated expression of Zif268 in any of the studied nuclei. Activation of the nuclei of the amygdala following aggressive encounter was studied also by Gammie and Nelson (137). Using P-CREB, they did not find the increased activation of the medial and cortical amygdala.
F. Pattern of c-Fos Expression in the Amygdalar Nuclei in Appetitively Motivated Standard Behavioral Tests
There is much less data on the pattern of c-fos expression in the amygdalar nuclei in the appetitively motivated behavioral tests than in the aversively motivated ones. In most of the studies, palatable food or sweet solutions were applied as reinforcements in classical and instrumental conditioning. The increased c-Fos expression in the central nucleus of the amygdala was a result of just drinking such sweet solutions as sucrose or saccharin (for reference, see Table 3). However, Lamprecht and Dudai (254) did not observe the elevated c-fos mRNA expression in the central amygdala following drinking of sweet solution; however, they used rather diluted, 0.1% saccharin. Interestingly, sucrose caused stronger activation of the central nucleus than saccharin. This effect was observed after both drinking and intragastric infusions (519). Furthermore, Koh et al. (232) showed that a novel taste of saccharin induced a greater c-Fos response in the central nucleus of the amygdala than a familiar taste. This effect was dependent on taste intensity, i.e., 0.5% solution evoked stronger response than 0.15% solution of saccharin. A similar effect of novelty was observed by Barot and Bernstein (23) for isotonic concentration of NaCl but not for a highly palatable polysaccharide preparation Polycose (for extensive discussion of possible causes of the observed discrepancies, see Ref. 23). Moreover, it was shown that the increased level of c-Fos expression in the central amygdala induced by drinking of sweet solutions was restricted to the medial part of the nucleus (519).
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Park and Carr (357) exposed rats to a palatable meal and the meal-paired environment. They did not observe the increased level of c-Fos expression in the central and basolateral parts of the amygdala. Noteworthy, the c-Fos expression was measured after the 11th of daily sessions; thus the animals could have been well habituated both to the cage and the food. Interestingly, Timofeeva et al. (472) observed the augmented level of c-fos expression in the central amygdala following the ingestion of a meal after 24 h of fasting (refeeding procedure). Summing up, the palatable food or sweet solutions induced the increased level of c-Fos expression in the central amygdala, but rather not in the basolateral amygdala.
In Pavlovian appetitive conditioning task, in which a signaling stimulus (CS) is paired with a reinforcement (US), animals acquire two distinct responses: an orienting response to the CS source and approaching the site of reinforcement delivery. Lee et al. (264) examined c-Fos expression in the central amygdala in a Pavlovian appetitive conditioning task, in which a visual CS was paired with food. They found activation of the medial, but not lateral subdivisions of the central nucleus in such training. Moreover, the more intense the training was, the more c-Fos expression in the central nucleus of the amygdala was observed.
In the experiment of Hess et al. (177), rats were trained to nose-poke for water reward. The authors found c-fos activation within the basolateral, medial, and central nuclei of the amygdala following such training, compared with the home-caged controls. However, the expression was studied after several training days; thus it could have been much less pronounced than after the first training session (see above).
Hess et al. (177) examined also the level of c-fos mRNA expression in the amygdalar nuclei in two-odor discrimination test. In this task, the animals learned to nose-poke at a positive odor (peppermint) port for a water reward and to avoid a negative odor (amyl acetate) port, which was punished with a brief strobe fla
