The amygdaloid complex consists of a number of distinct nuclear groups that process emotionally significant behaviours and cognitive functions. Of these, the medial nucleus of the amygdala (MeA) plays a key role in social behaviors by relaying chemosensory information to the reproductive and defensive nuclei of the hypothalamus. The MeA receives vomeronasal chemosensory inputs directly from the accessory olfactory bulb (AO). Olfactory information also reaches the MeA via afferents from the main olfactory bulb as well as olfactory-processing structures such as the cortical amygdala (CoA). MeA is a heterogeneous structure with four cytoarchitectonically and functionally distinct subnuclei. The posteroventral nucleus of the medial amygdala (MePV) processes both reproductive and defensive chemosensory information and is particularly involved in mediating innate fear responses. A thorough understanding of the MePV at a cellular and circuit level is therefore essential to understand the function of the MeA.
I have studied the properties of neurons and their synaptic inputs in the MePV using a transgenic mouse strain in which green fluorescent protein (GFP) is expressed under the control of the GAD67 promoter (GAD67-GFP). This promoter is specific to GABAergic cells, allowing differentiation between GABAergic (GFP+) and non-GABAergic (GFP-) neurons, and thus investigation of excitatory and inhibitory populations separately. All experiments in this thesis were performed on coronal brain slices obtained from male GAD67-GFP knock-in mice (p30-p50).
Initially, MePV neurons were characterized based on their electrophysiological and morphological properties as well as expression of GABAergic interneuron markers. While parvalbumin, CCK and VIP immunoreactive cells were sparse, largely non-overlapping populations of calbindin-, calretinin-, and somatostatin-immunoreactive cells were found. MePV neurons also showed diversity in their passive and active membrane properties, with many overlapping features between the GFP- and GFP+ cell types. Hyperpolarization voltage sag and firing frequency accommodation were the most reliable parameters to distinguish between GFP+ and GFP- cells. Using hierarchical cluster analysis, two classes of GFP- (type 1 and type 2) and four classes of GFP+ cells (regular firing, high frequency stuttering, accelerating, and irregular firing) were identified. Upon reconstruction of the recorded cells, most neurons were found to be multipolar with 3-6 spiny primary dendrites and modest dendritic arborisations. The two electrophysiologically identified types of GFP- cells also showed different dendritic branching, suggesting that they were both physiologically and morphologically distinct. Furthermore, within the GFP+ populations, the high-frequency stuttering cells were clearly distinct with their high intensity of GFP expression, neurogliaform-like morphology, short spine-sparse dendrites, and axonal projections that were largely restricted to the molecular layer of the MePV. The expression pattern of interneuron markers among recorded cells was multiform with calbindin being the most frequently observed marker across GFP+ clusters.
Synaptic inputs to MePV neurons were tested by the electrical stimulation of AO and CoA afferents, representing the vomeronasal and processed olfactory pathways, respectively. GFP+ and GFP- cells of all types received convergent synaptic inputs from these afferents. Evoked responses consist of a monosynaptic excitatory component, which was mediated by the AMPA and NMDA receptors, and often a disynaptic inhibitory component, which was mediated by the GABA-A receptors. Paired-recording experiments suggested that the disynaptic inhibition was most likely evoked by the high-frequency stuttering GFP+ cells, which were functioning as feed-forward local inhibitory interneurons. Finally, using a combination of electrophysiological and pharmacological techniques as well as two-photon calcium imaging, AO and CoA synapses were found to be spatially segregated on the dendritic arbour of GFP- cells (an also possibly GFP+ cells), and they showed distinct synaptic integration.
In summary, my data show that the MePV contains heterogeneous populations of excitatory and inhibitory cells that are involved in processing chemosensory information from both the accessory olfactory bulb and the olfactory CoA. These two inputs converge on individual MePV neurons at distinct dendritic compartments prompting differential synaptic integration. These findings provide novel knowledge to the cellular organization and connectivity of the MeA and further advance our understanding of amygdala circuits involved in chemosensory information processing and social behaviors.