Aversive experiences can lead to complex behavioral adaptations including increased levels of anxiety and fear generalization. The neuronal mechanisms underlying such maladaptive behavioral changes, however, are poorly understood. Here, using a combination of behavioral, physiological and optogenetic approaches in mouse, we identify a specific subpopulation of central amygdala neurons expressing protein kinase C δ (PKCδ) as key elements of the neuronal circuitry controlling anxiety. Moreover, we show that aversive experiences induce anxiety and fear generalization by regulating the activity of PKCδ+ neurons via extrasynaptic inhibition mediated by α5 subunit-containing GABAA receptors. Our findings reveal that the neuronal circuits that mediate fear and anxiety overlap at the level of defined subpopulations of central amygdala neurons and demonstrate that persistent changes in the excitability of a single cell type can orchestrate complex behavioral changes.

Hippocampal CA1 pyramidal cells, which receive γ-aminobutyric acid (GABA)ergic input from at least 18 types of presynaptic neuron, express 14 subunits of the pentameric GABA A receptor. The relative contribution of any subunit to synaptic and extrasynaptic receptors influences the dynamics of GABA and drug actions. Synaptic receptors mediate phasic GABA-evoked conductance and extrasynaptic receptors contribute to a tonic conductance. We used freezefracture replica-immunogold labelling, a sensitive quantitative immunocytochemical method, to detect synaptic and extrasynaptic pools of the alpha1, alpha2 and beta3 subunits. Antibodies to the cytoplasmic loop of the subunits showed immunogold particles concentrated on distinct clusters of intramembrane particles (IMPs) on the cytoplasmic face of the plasma membrane on the somata, dendrites and axon initial segments, with an abrupt decrease in labelling at the edge of the IMP cluster. Neuroligin-2, a GABAergic synapse-specific adhesion molecule, co-labels all beta3 subunit-rich IMP clusters, therefore we considered them synapses. Double-labelling for two subunits showed that virtually all somatic synapses contain the alpha1, alpha2 and beta3 subunits. The extrasynaptic plasma membrane of the somata, dendrites and dendritic spines showed low- Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts density immunolabelling. Synaptic labelling densities on somata for the alpha1, alpha2 and beta3 subunits were 78-132, 94 and 79 times higher than on the extrasynaptic membranes, respectively. As GABAergic synapses occupy 0.72% of the soma surface, the fraction of synaptic labelling was 33-48 (alpha1), 40 (alpha2) and 36 (beta3)% of the total somatic surface immunolabelling. Assuming similar antibody access to all receptors, about 60% of these subunits are in extrasynaptic receptors.

G-protein-coupled inwardly rectifying Kϩ channels (Kir3 channels) coupled to metabotropic GABA B receptors are essential for the control of neuronal excitation. To determine the distribution of Kir3 channels and their spatial relationship to GABA B receptors on hippocampal pyramidal cells, we used a high-resolution immunocytochemical approach. Immunoreactivity for the Kir3.2 subunit was most abundant postsynaptically and localized to the extrasynaptic plasma membrane of dendritic shafts and spines of principal cells. Quantitative analysis of immunogold particles for Kir3.2 revealed an enrichment of the protein around putative glutamatergic synapses on dendritic spines, similar to that of GABA B1 . Consistent with this observation, a high degree of coclustering of Kir3.2 and GABA B1 was revealed around excitatory synapses by the highly sensitive SDS-digested freeze-fracture replica immunolabeling. In contrast, in dendritic shafts receptors and channels were found to be mainly segregated. These results suggest that Kir3.2-containing K ϩ channels on dendritic spines preferentially mediate the effect of GABA, whereas channels on dendritic shafts are likely to be activated by other neurotransmitters as well. Thus, Kir3 channels, localized to different subcellular compartments of hippocampal principal cells, appear to be differentially involved in synaptic integration in pyramidal cell dendrites.