Lateral Amygdaloid Research Articles

Downregulation of the GABAA receptor β2 subunit in a rat model of autism

Introduction: Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain, and GABA type A receptor (GABAA) activation mediates fast inhibitory actions. Numerous studies have shown that individual with autism spectrum disorder (ASD) exhibit abnormalities in the expression of GABAA receptors in various brain areas. Additionally, animal models of ASD have suggested alterations in GABAergic neurotransmission and a dysregulation in the balance between inhibitory and excitatory systems. Objective: We investigated the immunolabeling of the GABAA receptor β2 subunit (GARB2) in the hippocampus, the amygdala, and thalamus of infant rats prenatally exposed to valproic acid (VPA) as an ASD model. Methods: Pregnant females were injected with VPA (600mg/Kg, i.p.) during the twelfth embryonic day; control rats were injected with saline. On the fourteen-postnatal-day, rats from both experimental groups were anesthetized, transcardially perfused with 0.9% NaCl and 4% paraformaldehyde, and sequential coronal brain sections (40μm thickness) were obtained. Immunohistochemistry was performed to detect GARB2 and the relative optical density (OD) of expression was analyzed. Results: Our data showed a statistically significant downregulation of GARB2 in the lateral amygdaloid nucleus, as well as in the ventral and lateral thalamic nuclei when compared to control rats. No statistically significant differences were detected in the hippocampus. Discussion: Our findings suggest that prenatal exposure to VPA reduces GARB2 expression in limbic brain regions involved in social-emotional behaviors, like previous reports in individuals with ASD. Conclusion These results support for the involvement of the GABAergic system in the pathogenesis of ASD.

Mechanism of Chronic Stress-Induced Glutamatergic Neuronal Damage in the Basolateral Amygdaloid Nucleus.

Stress is a ubiquitous part of our life, while appropriate stress levels can help improve the body's adaptability to the environment. However, sustained and excessive levels of stress can lead to the occurrence of multiple devastating diseases. As an emotional center, the amygdala plays a key role in the regulation of stress-induced psycho-behavioral disorders. The structural changes in the amygdala have been shown to affect its functional characteristics. The amygdala-related neurotransmitter imbalance is closely related to psychobehavioral abnormalities. However, the mechanism of structural and functional changes of glutamatergic neurons in the amygdala induced by stress has not been fully elucidated. Here, we identified that chronic stress could lead to the degeneration and death of glutamatergic neurons in the lateral amygdaloid nucleus, resulting in neuroendocrine and psychobehavioral disorders. Therefore, our studies further suggest that the Protein Kinase R-like ER Kinase (PERK) pathway may be therapeutically targeted as one of the key mechanisms of stress-induced glutamatergic neuronal degeneration and death in the amygdala.

Neurochemical fingerprinting of amygdalostriatal and intra-amygdaloid projections: a tracing–immunofluorescence study in the rat

Amygdalostriatal and intra-amygdaloid fiber connectivity was studied in rats via injections of one of the tracers Phaseolus vulgaris leucoagglutinin (PHA-L) or biotinylated dextran amine (BDA) into various amygdaloid nuclei. To determine the neurotransmitter identity of labeled fibers we combined tracer detection with immunofluorescence staining, using antibodies against vesicular transporters (VTs) associated with glutamatergic (VGluT1, VGluT2) or GABAergic (VGAT) neurotransmission. High-magnification confocal laser scanning images were screened for overlap: occurrence inside tracer labeled fibers or axon terminals of immunofluorescence signal associated with one of the VTs.Labeled amygdalostriatal fibers were seen when tracer had been injected into the magnocellular and parvicellular portions of the basal amygdaloid nucleus and the lateral amygdaloid nucleus (nuclei belonging to 'cortical type' amygdaloid nuclei). Intra-amygdaloidal projection fibers were mostly found after tracer injections in the central and medial amygdaloid nuclei ('striatal type' amygdaloid nuclei).Terminals of tracer-labeled amygdalostriatal fibers contained immunofluorescence signal associated mostly with VGluT1 and to a lesser degree with VGluT2 or VGAT. Intra-amygdaloid labeled fibers showed colocalization mostly of VGluT1, followed by VGAT. VGluT2 co-occurred in a minority of intra-amygdaloid tracer-containing fiber terminals.We conclude from our observations that both amygdalostriatal and intra-amygdaloid projections, arising from, respectively, 'cortical type' and 'striatal type' amygdaloid nuclei contain strong glutamatergic and modest GABAergic components. The glutamatergic fibers express either VGluT1 or VGluT2. The absence in large numbers of tracer labeled fibers of expression of one of the selected VTs leads us to suspect that amygdalostriatal projection fibers may contain hitherto neglected neurotransmitters in these connections, e.g., aspartate.

The Klüver-Bucy Syndrome.

In 1937, Heinrich Klüver and Paul Bucy described a dramatic behavioral syndrome in monkeys after bilateral temporal lobectomy. The full Klüver-Bucy syndrome (KBS) - hyperorality, placidity, hypermetamorphosis, dietary changes, altered sexual behavior, and visual agnosia - is evident within 3 weeks following operation. Some KBS features (i.e., hyperorality, placidity, hypermetamorphosis) persist indefinitely, whereas others gradually resolve over several years. Klüver and Bucy were initially unaware of an earlier report of KBS by Sanger Brown and Edward Schäfer in 1888. Human cases were recognized in the 1950s, as surgeons employed bilateral temporal lobectomies to treat seizures. Various attempts were made to localize the component features to specific areas of the temporal lobe, with mixed success. Bilateral ventral temporal ablations and bilateral temporal lobectomies produced marked impairment in visual discrimination, whereas lateral resections or unilateral lesions did not. Discrete bilateral lesions of the lateral amygdaloid nucleus produced a permanent "hypersexed state." By the 1970s, it was clear that the major symptoms of KBS are produced by destroying either the temporal neocortex or the amygdala bilaterally. KBS is now thought to be caused by disturbances of temporal portions of limbic networks that interface with multiple cortical and subcortical circuits to modulate emotional behavior and affect. The clinical features of KBS in man are similar to those in monkeys, but the full syndrome is rarely seen, probably because the anterior temporal lobe dysfunction is usually less severe than that following total temporal lobe ablation in monkeys. Human KBS does not occur in isolation, but is typically part of a complex behavioral syndrome that almost always includes amnesia and aphasia, and that may also include dementia and seizures. The treatment of KBS is difficult and often unsatisfactory.

Amygdalar Auditory Neurons Contribute to Self-Other Distinction during Ultrasonic Social Vocalization in Rats.

Although, clinical studies reported hyperactivation of the auditory system and amygdala in patients with auditory hallucinations (hearing others' but not one's own voice, independent of any external stimulus), neural mechanisms of self/other attribution is not well understood. We recorded neuronal responses in the dorsal amygdala including the lateral amygdaloid nucleus to ultrasonic vocalization (USVs) emitted by subjects and conspecifics during free social interaction in 16 adult male rats. The animals emitting the USVs were identified by EMG recordings. One-quarter of the amygdalar neurons (15/60) responded to 50 kHz calls by the subject and/or conspecifics. Among the responsive neurons, most neurons (Type-Other neurons; 73%, 11/15) responded only to calls by conspecifics but not subjects. Two Type-Self neurons (13%, 2/15) responded to calls by the subject but not those by conspecifics, although their response selectivity to subjects vs. conspecifics was lower than that of Type-Other neurons. The remaining two neurons (13%) responded to calls by both the subject and conspecifics. Furthermore, population coding of the amygdalar neurons represented distinction of subject vs. conspecific calls. The present results provide the first neurophysiological evidence that the amygdala discriminately represents affective social calls by subject and conspecifics. These findings suggest that the amygdala is an important brain region for self/other attribution. Furthermore, pathological activation of the amygdala, where Type-Other neurons predominate, could induce external misattribution of percepts of vocalization.

The amygdala as a neurobiological target for ghrelin in rats: neuroanatomical, electrophysiological and behavioral evidence.

Here, we sought to demonstrate that the orexigenic circulating hormone, ghrelin, is able to exert neurobiological effects (including those linked to feeding control) at the level of the amygdala, involving neuroanatomical, electrophysiological and behavioural studies. We found that ghrelin receptors (GHS-R) are densely expressed in several subnuclei of the amygdala, notably in ventrolateral (LaVL) and ventromedial (LaVM) parts of the lateral amygdaloid nucleus. Using whole-cell patch clamp electrophysiology to record from cells in the lateral amygdaloid nucleus, we found that ghrelin reduced the frequency of mEPSCs recorded from large pyramidal-like neurons, an effect that could be blocked by co-application of a ghrelin receptor antagonist. In ad libitum fed rats, intra-amygdala administration of ghrelin produced a large orexigenic response that lasted throughout the 4 hr of testing. Conversely, in hungry, fasted rats ghrelin receptor blockade in the amygdala significantly reduced food intake. Finally, we investigated a possible interaction between ghrelin's effects on feeding control and emotional reactivity exerted at the level of the amygdala. In rats allowed to feed during a 1-hour period between ghrelin injection and anxiety testing (elevated plus maze and open field), intra-amygdala ghrelin had no effect on anxiety-like behavior. By contrast, if the rats were not given access to food during this 1-hour period, a decrease in anxiety-like behavior was observed in both tests. Collectively, these data indicate that the amygdala is a valid target brain area for ghrelin where its neurobiological effects are important for food intake and for the suppression of emotional (anxiety-like) behaviors if food is not available.

Subpopulations of neurokinin 1 receptor-expressing neurons in the rat lateral amygdala display a differential pattern of innervation from distinct glutamatergic afferents

Substance P by acting on its preferred receptor neurokinin 1 (NK1) in the amygdala appears to be critically involved in the modulation of fear and anxiety. The present study was undertaken to identify neurochemically specific subpopulations of neuron expressing NK1 receptors in the lateral amygdaloid nucleus (LA), a key site for regulating these behaviors. We also analyzed the sources of glutamatergic inputs to these neurons. Immunofluorescence analysis of the co-expression of NK1 with calcium binding proteins in LA revealed that ∼35% of NK1-containing neurons co-expressed parvalbumin (PV), whereas no co-localization was detected in the basal amygdaloid nucleus. We also show that neurons expressing NK1 receptors in LA did not contain detectable levels of calcium/calmodulin kinase IIα, thus suggesting that NK1 receptors are expressed by interneurons. By using a dual immunoperoxidase/immunogold-silver procedure at the ultrastructural level, we found that in LA ∼75% of glutamatergic synapses onto NK1-expressing neurons were labeled for the vesicular glutamate transporter 1 indicating that they most likely are of cortical, hippocampal, or intrinsic origin. The remaining ∼25% were immunoreactive for the vesicular glutamate transporter 2 (VGluT2), and may then originate from subcortical areas. On the other hand, we could not detect VGluT2-containing inputs onto NK1/PV immunopositive neurons. Our data add to previous localization studies by describing an unexpected variation between LA and basal nucleus of the amygdala (BA) in the neurochemical phenotype of NK1-expressing neurons and reveal the relative source of glutamatergic inputs that may activate these neurons, which in turn regulate fear and anxiety responses.

Amygdalotrigeminal projection in the rat: An anterograde tracing study

Previous neurophysiological studies have demonstrated that the amygdala has a direct influence upon trigeminal motoneuron activity. The existence of a direct amygdalotrigeminal pathway in rats was proved by anterograde tracing with the neuroanatomical tracer, biotinylated dextran amine (BDA). After ipsilateral BDA application to the central nucleus of the amygdala (AmCe), widespread ipsilateral projections emerging from its medial subnucleus were traced to the trigeminal brainstem nuclear complex, including the principal sensory (Pr5) and mesencephalic trigeminal nucleus (Me5), and their premotoneurons and interneurons, located in the supratrigeminal, intertrigeminal and peritrigeminal nuclei. Sparse BDA-labeled axons and their terminals were also distributed in the contralateral Pr5, interpolar and caudal subnuclei of the spinal trigeminal nucleus. The central lateral amygdaloid nucleus gives rise to a light ipsilateral projection to the pontine part of the Me5. The present data indicate that AmCe sends massive efferents to the trigeminal nuclei in the brainstem, wherein its medial subnucleus sends the major input to them. The medial amygdaloid nucleus sparsely innervates Me5 neurons, specifically those located in its mesencephalic portion, while basomedial and basolateral efferents do not target the trigeminal nuclear complex. These results suggest that the amygdaloid input may modulate the activity of trigeminal sensory and motor neurons and, thus, the amygdala is possibly involved in the control of masticatory behavior.

Demonstration of disturbed activity of the lateral amygdaloid nucleus projection neurons in depressed patients by the AgNOR staining method

Background The aim to find a morphological biomarker of disturbed activity of the lateral amygdaloid nucleus in depression was approached by a karyometric analysis of projection neurons. Methods The study was performed on paraffin-embedded brains from 19 depressed patients from both the major depressive disorder (MDD) and the bipolar disorder (BD) diagnostic groups, including 10 suicides, and 24 matched controls. The karyometric parameters of the lateral amygdaloid nucleus (La) projection neurons bilaterally were evaluated by the argyrophilic nucleolar organiser region (AgNOR) silver staining method. Results An increased AgNOR number was found in the right La in suicides compared to controls. The intra-group comparisons between the hemispheres suggest a disturbed amygdaloid lateralisation in depressed patients. The effects were independent from psychotropic medication. There was a strong positive correlation between the nuclear area in La projection neurons and prefrontal limbic areas pyramidal neurons in the right hemisphere specific for suicide and MDD. Limitations A major limitation of this study is the relatively small number of cases. A further limitation is given by the lack of data on drug exposure across the entire lifespan. Conclusion The results suggest that depressed patients from both the MDD and BD diagnostic groups exhibit an increased activity of the La output neurons specific for suicidal patients. The distinctness of the diagnostic groups of mood disorders was accentuated in the correlation analysis. This putative hyperactivity was specific for the right hemisphere and psychotropic medication most likely did not counteract it.

Differential pattern of co-expression between the substance P receptor NK1 and calcium-binding proteins in the lateral and basolateral amygdaloid nuclei

Increasing evidence suggests that substance P (SP) and its preferred receptor, namely the neurokinin 1 receptor (NK1-R), play an important role in the modulation of stress-related, affective and/or anxious behaviours. Both SP and NK1-R are expressed in brain regions critically involved in stress, fear and affective responses such as the amygdala, hippocampus and frontal cortex. In this study we aimed at identifying the types of NK1-R-immunoreactive neurones in the basolateral complex of the amygdala according to their content in calcium-binding proteins by dual or triple labelling immunofluorescence. The basolateral amygdaloid complex consists of the lateral (LA) and basolateral (BL) nuclei, which are believed to be cytoarchitectonically and cytochemically similar. Our study reveals that in adult male Sprague-Dawley rats, 38.7 ± 6.7% of NK1-R immunopositive LA neurones (124/331) co-express parvalbumin, representing 15.2 ± 3.4% (124/820) of parvalbumin-immunopositive neurones. Conversely, in the BL no coexistence between NK1-R (293 neurones counted) and parvalbumin (2,385 neurones counted) expressing neurones was detected. In addition, we found that 32.4 ± 3.8% of NK1-R-immunoposititive LA interneurones (33/104) co-express calbindin-D28k, that is 6.0 ± 1.7% (33/626) of the total number of calbindin-D28k-immunoreactive neurones. On the other hand, in the BL a large number (72.3 ± 6.9%) of NK1-R-immunopositive neurones (109/149) were found to co-express calbindin-D28k, representing 6.8 ± 1.3% of all calbindin-D28k-labelled neurones. We also found a lack of coexistence between NK1 and CR in both lateral and basolateral amygdala. These results suggest that interneurones in the LA and BL amygdala differentially express molecules involved in cell signalling and indicate a distinct organization in local interneurones. The BL resembled the hippocampal CA1 region, in which NK1-R-expressing neurones do not coexist with parvalbumin.

The distribution of motilin receptor in the amygdala of rats and its role in migrating myoelectric complex

Objective To investigate the distribution of the motilin receptor in the amygdala of rats and its role in regulating the duodenal migrating myoelectric complex (MMC). Methods The distribution of motilin receptor in the amygdala in adult SD rats was detected by immunohistochemistry methods, and the duodenal interdigestive MMC was recorded via the electrodes implanted in the duodenum and analyzed using a multichannel recorder. Results Motilin receptor was observed in the amygdala of rats. The great amount of motilin receptor was found in the medial amygdaloid nucleus, which was also abundant in the basolateral nucleus but less abundant in the basomedial amygdaloid nucleus, the central amygdaloid nucleus and the lateral amygdaloid nucleus. The shortening of the duodenal MMC cycle duration and the increase of the amplitude and the frequency of phase III were recorded after motilin receptors being bound with exogenous motilin in the amygdala. The effects could be completely blocked by the subdiaphragmatic vagotomy but not by the intravenous injections of atropine, phentolamine or propranolol. Anti-motilin serum could partially block these effects, and the destruction of the basolateral nucleus of the amygdala had no significant effects on the duodenal MMC. Conclusion Motilin receptor is present in all the subnuclei of the amygdala, with the greatest amount of motilin receptor present in the medial amygdaloid nucleus. Microinjections of motilin in the amygdala can shorten the duodenal MMC cycle duration and increase the amplitude and the frequency of phase III. These effects might be accomplished via the amygdala-hypothalamus-brainstem-vagus pathway, indicating the important role of the amygdala motilin receptor in the duodenal MMC regulation.

Nitric oxide synthase-containing neurons in the amygdaloid nuclear complex of the rat

The nitric oxide-producing neurons in the rat amygdala (Am) were studied, using reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. Almost all nuclei of the Am contained NADPHd-positive neurons and fibers, but the somatodendritic morphology and the intensity of staining of different subpopulations varied. The strongly stained neurons displayed labeling of the perikaryon and the dendritic tree with Golgi impregnation-like quality, whilst the dendrites of the lightly stained neurons were less successfully followed. Many strongly positive neurons were located in the external capsule and within the intraamygdaloid fiber bundles. A large number of small, strongly stained cells was present in the amygdalostriatal transition area. In the Am proper, a condensation of deeply stained cells occurred in the lateral amygdaloid nucleus. In the basolateral nucleus, the strongly NADPHd-positive neurons were few, and were located mainly along the lateral border of the nucleus. These cells clearly differed from the large, pyramidal, and efferent cells. The basomedial nucleus contained numerous positive cells but most of them were only lightly labeled. A moderate number of strongly stained neurons appeared in the medial division of the central nucleus, and a larger accumulation of strongly positive cells was present in the lateral and the capsular divisions. The medial amygdaloid nucleus contained numerous moderately stained neurons and displayed the strongest diffuse neuropil staining in Am. In the nucleus of the lateral olfactory tract, the first layer contained only NADPHd-stained axons, in the second layer, there were numerous moderately stained cells, and in the third layer, a few but deeply stained neurons. From the cortical nuclei, the most appreciable number of stained neurons was seen in the anterior cortical nucleus. The anterior amygdaloid area contained numerous NADPHd-positive neurons; in its dorsal part the majority of cells were only moderately stained, whereas in the ventral part the neurons were very strongly stained. The intercalated amygdaloid nucleus lacked NADPHd-positive neurons but an appreciable plexus of fine, tortuous axons was present. In the intra-amygdaloid part of the bed nucleus of the stria terminalis (st) some lightly stained cells were seen but along the entire course of st strongly stained neurons were observed. Some Am nuclei, and especially the central lateral nucleus and the intercalated nucleus, display considerable species differences when compared with the primate Am. The age-related changes of the nitrergic Am neurons, as well as their involvement in neurodegenerative diseases is discussed.

Exposure to high- and low-light conditions in an open-field test of anxiety increases c-Fos expression in specific subdivisions of the rat basolateral amygdaloid complex

Anxiety states and anxiety-related behaviors appear to be regulated by a distributed and highly interconnected system of forebrain structures including the basolateral amygdaloid complex (basolateral amygdala). Despite a wealth of research examining the role of the basolateral amygdala in anxiety-related behaviors and anxiety states, the specific subdivisions of the basolateral amygdala that are involved in responses to anxiogenic stimuli have not been examined. In this study, we investigated the effects of exposure to a novel open-field environment, with either low- or high-levels of illumination, on expression of the protein product of the immediate-early gene c-Fos in subdivisions of the rat basolateral amygdala. The subdivisions studied included the lateral, ventrolateral and ventromedial parts of the lateral amygdaloid nucleus, the anterior, posterior and ventral parts of the basolateral amygdaloid nucleus and the anterior and posterior part of the basomedial amygdaloid nucleus. Small increases in the number of c-Fos-immunoreactive cells were observed in several, but not all, of the subdivisions of the basolateral amygdala studied following exposure of rats to either the high- or low-light conditions, compared to home cage or handled control groups. Open-field exposure in both the high- and low-light conditions resulted in a marked increase in c-Fos expression in the anterior part of the basolateral amygdaloid nucleus compared to either home cage or handled control groups. These findings point toward anatomical and functional heterogeneity within the basolateral amygdaloid complex and an important role of the anterior part of the basolateral amygdaloid nucleus in the neural mechanisms underlying physiological or behavioral responses to this anxiety-related stimulus.

Localization of Id2 mRNA in the adult mouse brain

Id proteins are negative regulators of basic helix–loop–helix transcription factors and are involved in cellular differentiation and proliferation. Four members of the Id gene family exhibit closely related but distinct expression patterns in various mammalian organs of not only embryos but also adults. Among them, Id2 is known to be expressed in Purkinje cells and neurons in the cortical layers of the adult mouse brain, suggesting that Id2 is involved in some neural functions in the adult. To get insight into the role of Id2 in the nervous system, we investigated the localization of Id2 mRNA-expressing cells in the adult mouse brain in detail by in situ hybridization with the radiolabeled antisense probe and compared it with the localization of other Id gene family members. The results indicated that Id2 mRNA is detected in more varied brain regions than previously reported. These regions include the amygdaloid complex, caudate putamen, globus pallidus, substantia nigra pars reticulata, suprachiasmatic nucleus, and the anterior part of the subventricular zone. These results suggest the possibility that Id2 plays a role in the neural activity and cognitive functions. On the other hand, Id1 was barely detectable. Although moderate or low expression of Id3 was observed diffusely, high expression was observed in some specific regions including the molecular layer of the dentate gyrus and the external capsule. Id4 mRNA was detected in the regions such as the caudate putamen and the lateral amygdaloid nucleus. Thus, the expression pattern of Id2 is distinct from those of other Id gene family members.

Calcitonin gene-related peptide-containing pathways in the rat forebrain

The present study focuses on the topographical distribution of calcitonin gene-related peptide (CGRP)-containing cell bodies and fibers and their connections and pathways in the rat forebrain. We confirm previously reported CGRP projections from the perifornical area of the hypothalamus to the lateral septum, from the posterior thalamus to the caudate putamen and cerebral cortex, and from the parabrachial nuclei to the central extended amygdala, lateral hypothalamus, and ventromedial thalamus. Despite previous descriptions of CGRP in the central nervous system, important neuroanatomical aspects of the forebrain CGRP system remained obscure, which we addressed by using brain lesion techniques combined with modern immunohistology. We first report CGRP terminal fields in the olfactory-anterior septal region and also CGRP projections from the parabrachial nuclei to the olfactory-anterior septal region, the medial prefrontal cortex, the interstitial nucleus of the anterior commissure, the nucleus of the lateral olfactory tract, the anterior amygdaloid area, the posterolateral cortical amygdaloid nucleus, and the dorsolateral part of the lateral amygdaloid nucleus. In addition, we identified a CGRP cell group in the premamillary nuclei and showed that it projects to the medial CGRP layer of the lateral septum. CGRP fibers usually join other pathways rather than forming bundles. They run along the fornix from the hypothalamus, along the supraoptic decussations or the inferior thalamic peduncle-stria terminalis pathway from the posterior thalamus, and along the superior cerebellar peduncle, thalamic fasciculus, and ansa peduncularis from the parabrachial nuclei. This description of the forebrain CGRP system will facilitate investigation of its role in higher brain functions.

Auditory-conditioned-fear-dependent c-Fos expression is altered in the emotion-related brain structures of Fyn-deficient mice

Fyn-tyrosine-kinase-deficient mice exhibit increased fearfulness. To elucidate the neural mechanisms of their emotional defects, we compared fyn(−/−) and fyn(+/−) mice by behavioral analysis of conditioned fear and by functional neuroanatomical analysis of the distribution of highly responsive neurons associated with conditioned fear. The mice were exposed to the auditory conditioned stimulus paired with electric shock as the unconditioned stimulus. After the fear conditioning, auditory stimulus-induced freezing behavior was enhanced in fyn(−/−) mice. When the occurrence of c-Fos-immunoreactive neurons in the brain of fear-conditioned mice was examined following exposure to the auditory stimulus, a significant increase in immunoreactive neurons was found in the amygdala, hypothalamus, and midbrain of both genotypes. The occurrence of conditioned-fear-dependent c-Fos-immunoreactive neurons was enhanced in the central, medial, cortical, and basomedial amygdaloid subdivisions, the hypothalamic nuclei, and the midbrain periaqueductal gray of the fyn(−/−) mice in comparison with the fyn(+/−) mice. However, remarkably, the occurrence of conditioned-fear-dependent c-Fos-immunoreactive neurons was very low in the basolateral and lateral amygdaloid subdivisions of the fyn(−/−) mice, in striking contrast to a significant increase in c-Fos-immunoreactive neurons in these subdivisions in the fyn(+/−) mice. These findings suggest that the increased excitability of the specific amygdaloid subdivisions including the central nucleus, and of the projection targets such as the hypothalamus and midbrain in fyn(−/−) mice, is directly related to the enhanced fear response, and that the decreased excitability in the basolateral and lateral amygdaloid subdivisions is involved in the defective control of the neural circuit for emotional expression in this mutant.

EDI BOARD

Projections from the thalamic gustatory nucleus, i.e. the parvicellular part of the posteromedial ventral thalamic nucleus (VPMpc) to the forebrain regions were studied in the rat by the tract-tracing methods with anterograde tracer (biotinylated dextran amine, BDA) and anterograde/retrograde tracer (wheat-germ agglutinin-horseradish peroxidase, WGA-HRP). After BDA injection into the VPMpc, terminal labeling was observed in the insular cortex, amygdaloid complex, and fundus striati. The terminal labeling in the amygdaloid complex was distributed in dorsolateral area of the rostral part of the lateral amygdaloid nucleus and the rostral part of the lateral subdivision of the central amygdaloid nucleus. The terminal labeling in the central amygdaloid nucleus extended to the fundus striati. The retrograde tracing study with WGA-HRP revealed that the projection fibers from the VPMpc to the amygdaloid complex originated from the medial part of the VPMpc and also from the thalamic area medial to the VPMpc. In the rats injected with Fluoro-Gold and WGA-HRP, respectively into the insular cortex and amygdaloid complex, no double-labeled neuronal cell bodies were found in the VPMpc, although neurons labeled singly with Fluoro-Gold were intermingled with those singly labeled with WGA-HRP in the medial part of the VPMpc. The results indicated that VPMpc neurons projecting to the amygdaloid complex constituted a population different from VPMpc neurons projecting to the insular cortex.

Lateral amygdaloid nucleus expansion in adult rats is associated with exposure to prenatal stress

Anxiety disorders in humans have been associated with chronic activation of the hypothalamic–pituitary–adrenal axis and changes in the volume of the amygdala. Interest in the etiology of anxiety disorders has led us and others to investigate the effects of prenatal stress on the brain development of adult male rat offspring. Prenatally stressed rats represent a promising animal model for anxiety disorders in that they have already been characterized as having both upregulated corticotropin-releasing factor (CRF) brain biochemistry and altered, more fearful, behaviors. Consistent with this, there is now evidence that prenatal stress also has an impact on the development of CRFergic neurons in the hypothalamic paraventricular nucleus and neurogenesis in the hippocampus. At this time, little information about the impact of prenatal stress on amygdala anatomy has been presented. Here we asked whether prenatal stress also has an impact on the development of the amygdala, because this structure plays a direct role in the emotions of anxiety and fear. Stereological measures of well-defined subregions of amydgdaloid nuclei revealed significantly expanded dimensions of the lateral nucleus in prenatally stressed offspring, due, in part, to more neurons and glia. These data may have direct import for the effect of adverse early life experiences and the etiology of anxiety disorders in humans. They also imply that early experiences may not be “grown out of” with development; in fact, the opposite might be true—adverse early life experiences may set developmental events into motion in the brain that last a lifetime.

Inhibitory control of rat lateral amygdaloid projection cells

The amygdaloid complex has long been implicated in seizure disorders. Yet, projection cells of the lateral amygdaloid nucleus (LA) display little spontaneous activity suggesting that this seizure prone structure is normally controlled by strong inhibitory mechanisms. This control is achieved in part by local interneurons; however, a synaptically activated, Ca 2+-dependent K + (K Ca) conductance has recently been identified as a second major inhibitory mechanism. In the present study, we investigated which K Ca channels underlie this conductance, and their roles in the generation of the synaptic responses and spike adaptation of LA projection cells. Intracellular recordings were obtained from LA projection cells in barbiturate-anesthetized rats. In recordings with K-acetate pipettes, perirhinal stimulation evoked an initial excitatory postsynaptic potential (EPSP) followed by a prolonged monophasic hyperpolarization, similar to what was observed in cats under in vivo conditions, and distinct from the multiphasic hyperpolarization observed previously in rodents with in vitro recordings. This indicates that differences in the cellular environment, not interspecies differences, are responsible for the differing response profiles previously reported. In recordings with pipettes containing 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid or Cs-acetate the reversal potentials were significantly more positive than those recorded with K-acetate, consistent with a K Ca conductance contribution to the response. To investigate the K Ca channels underlying this conductance, intracellular recordings were obtained while perfusing the LA with Ringer's solution and then switching to a solution containing charybdotoxin, isoproterenol, or apamin. Charybdotoxin and isoproterenol produced positive shifts in the reversal potential, whereas apamin did not. By contrast, all three substances decreased adaptation during spike trains elicited by depolarizing current injections. These results suggest that intermediate (IK) and small (SK) conductance K Ca channels limit LA projection cell excitability, with IK channels involved in controlling both the synaptic response and intrinsic excitability of these neurons, and SK channels being involved only in the latter.

A statistical approach in using cDNA array analysis to determine modest changes in gene expression in several brain regions after neurotoxic insult.

Modest changes in gene expression of three-fold or less might be expected after mild to moderate neurotoxic exposure to classes of compounds, such as the substituted amphetamines, or at time points that are weeks after more severe neurotoxic exposures. When many genes appear to change expression by less than two-fold, it is crucial to run several pairs of arrays and use statistical analysis to determine which genes are really changing. This limits the number of genes that have to undergo the time consuming task of performing RT-PCR to validate change in expression levels. We describe here methods for statistically determining which genes are being expressed above background levels. These methods are used to compare expression differences among the striatum, parietal cortex, posterior lateral amygdaloid nucleus, and substantia nigra brain regions, all of which differ significantly in their gene expression profiles. In these comparisons, it was possible to distinguish differences among hundreds of genes with manageable estimated false discovery rates. The effect of amphetamine treatment on gene expression in posterior lateral amygdaloid nucleus was also evaluated. The expression data indicate that many genes have changed, but in this case it is more difficult to separate affected genes from false positives. The optimum list has 50 genes, of which 32% are expected to be false positives.