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Background Although extinction-based therapies are effective treatments for anxiety disorders, the neural bases of fear extinction remain still largely unclear. Recent evidence suggests that the intercalated cell masses of the amygdala (ITCs) are critical structures for fear expression and extinction. They consist of clusters of densely packed medium spiny GABAergic neurons surrounding the basolateral amygdaloid complex (BLA). Five percent of ITC neurons are large cells mostly present near the cluster borders. So far, no information is available regarding the neurochemical features, afferents and efferents of large ITC cells, preventing any elucidation of their functional role. Only recently we discovered that large ITC neurons display immunoreactivity for either neurokinin 1 or metabotropic glutamate 1a (mGlu1a) receptors. We also found that dendrites of these neurons receive inhibitory inputs from medial capsular projecting ITC cells [1]. The aim of our study consists in the characterization of the morphological features, as well as the afferent and efferent connectivity, of large ITC neurons in order to further clarify their potential participation in the neuronal processes underlying fear extinction.

Background Previous work has suggested that alterations in GABAergic function within the amygdala underlie fear learning. In particular, it has been shown that Pavlovian fear conditioning induces a downregulation of benzodiazepine binding sites as well as transcripts for gephyrin and some GABAA receptor subunits in the basal nucleus of the amygdala (BA), which were restored to control levels after fear extinction.

Background In Pavlovian fear conditioning, in which initially a conditioned stimulus (CS) is repeatedly paired with an aversive stimulus (US), animals learn to associate CS with US, hence attaching emotional significance to sensory stimuli. Conditioned fear can be suppressed when the CS is repeatedly presented alone, a phenomenon known as fear extinction. Previous studies suggest that the mechanisms underlying fear conditioning involve inhibitory neurotransmission through GABAA receptors. GABAA receptors are generally composed of two a, two b and one g subunits. Sixteen GABAA receptor subunits have been identified in mammals, which indicates that these receptors may have multiple biophysical and pharmacological properties. Recent work showed that fear conditioning induces a dramatic downregulation of benzodiazepine binding sites and transcripts for gephyrin and some GABAA receptor subunits in the basal nucleus of the amygdala (BA), which were restored to control levels after fear extinction.

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.

Calcium-activated potassium channels have been shown to be critically involved in neuronal function but an elucidation of their detailed roles awaits identification of the subcellular domains and microdomains where they are located. This study was undertaken to unravel the precise subcellular distribution of the big-conductance calcium-activated potassium channels (called BK, KCa1.1 or Slo1) in the somato-dendritic compartment of cerebellar Purkinje cells by means of postembedding immunogold cytochemistry and SDS-digested freeze-fracture replica labeling (SDS-FRL). We found BK channels to be unevenly distributed over the Purkinje cell plasma membrane and localized to specific subcellular domains. At distal dendritic compartments, BK channels were scattered over the plasma membrane of shafts and spines, but absent from postsynaptic densities. At the soma and proximal dendrites, BK channels formed two distinct pools. One pool was scattered over the plasma membrane, the other pool was clustered in plasma membrane domains overlying subsurface membrane cisterns, also called hypolemmal cisternae. These subcompartments of the endoplasmic reticulum likely represent calciosomes that unload and refill Ca2+ independently. Purkinje cell subsurface cisterns are enriched in inositol 1,4,5-triphosphate receptors that mediate the effects of several neurotransmitters, hormones and growth factors by releasing Ca2+ into the cytosol, generating local Ca2+ sparks. Such increases in cytosolic [Ca2+] may be sufficient for BK channel activation. Clustered BK channels in the plasma membrane may thus participate in building a functional unit (plasmerosome) with the underlying calciosome that contributes significantly to local signaling in Purkinje cells.