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Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

Medical university of innsbruck

G-protein-coupled inwardly rectifying K+ channels (Kir3 channels) coupled to metabotropic GABAB receptors are essential for the control of neuronal excitation. To determine the distribution of Kir3 channels and their spatial relationship to GABAB 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+ 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.

Compartment-dependent colocalization of Kir3.2-containing K+ channels and GABAB receptors in hippocampal pyramidal cells.

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 microdomains where they are located. This study was undertaken to unravel the precise subcellular distribution of the large-conductance calcium-activated potassium channels (called BK, KCa1.1, or Slo1) in the somatodendritic 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. At distal dendritic compartments, BK channels were scattered over the plasma membrane of dendritic 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, whereas the other pool was clustered in plasma membrane domains overlying subsurface cisterns. The labeling density ratio of clustered to scattered channels was about 60:1, established in SDS-FRL. Subsurface cisterns, also called hypolemmal cisterns, are subcompartments of the endoplasmic reticulum likely representing 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.

Large-conductance calcium-activated potassium channels in purkinje cell plasma membranes are clustered at sites of hypolemmal microdomains.

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 freeze-fracture 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-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.

Quantitative localisation of synaptic and extrasynaptic GABAA receptor subunits on hippocampal pyramidal cells by freeze-fracture replica immunolabelling.

In the amygdala, GABAergic neurons in the intercalated medial paracapsular cluster (Imp) have been suggested to play a key role in fear learning and extinction. These neurons project to the central (CE) amygdaloid nucleus and to other areas within and outside the amygdala. In addition, they give rise to local collaterals that innervate other neurons in the Imp. Several drugs, including benzodiazepines (BZ), are allosteric modulators of GABA(A) receptors. BZ has both anxiolytic and sedative actions, which are mediated through GABA(A) receptors containing α2/α3 and α1 subunits, respectively. To establish whether α1 or α2/α3 subunits are expressed at Imp cell synapses, we used paired recordings of anatomically identified Imp neurons and high resolution immunocytochemistry in the mouse. We observed that a selective α3 subunit agonist, TP003 (100 nM), significantly increased the decay time constant of the unitary IPSCs. A similar effect was also induced by zolpidem (10 μM) or by diazepam (1 μM). In contrast, lower doses of zolpidem (0.1-1 μM) did not significantly alter the kinetics of the unitary IPSCs. Accordingly, immunocytochemical experiments established that the α2 and α3, but not the α1 subunits of the GABA(A) receptors, were present at Imp cell synapses of the mouse amygdala. These results define, for the first time, some of the functional GABA(A) receptor subunits expressed at synapses of Imp cells. The data also provide an additional rationale to prompt the search of GABA(A) receptor α3 selective ligands as improved anxiolytic drugs.

Functional expression of the GABA(A) receptor α2 and α3 subunits at synapses between intercalated medial paracapsular neurons of mouse amygdala.

Type 1 metabotropic glutamate (mGlu1) receptors play a pivotal role in different forms of synaptic plasticity in the cerebellar cortex, e.g. long-term depression at glutamatergic synapses and rebound potentiation at GABAergic synapses. These various forms of plasticity might depend on the subsynaptic arrangement of the receptor in Purkinje cells that can be regulated by protein-protein interactions. This study investigated, by means of the freeze-fracture replica immunogold labelling method, the subcellular localization of mGlu1 receptors in the rodent cerebellum and whether Homer proteins regulate their subsynaptic distribution. We observed a widespread extrasynaptic localization of mGlu1 receptors and confirmed their peri-synaptic enrichment at glutamatergic synapses. Conversely, we detected mGlu1 receptors within the main body of GABAergic synapses onto Purkinje cell dendrites. Although Homer proteins are known to interact with the mGlu1 receptor C-terminus, we could not detect Homer3, the most abundant Homer protein in the cerebellar cortex, at GABAergic synapses by pre-embedding and post-embedding immunoelectron microscopy. We then hypothesized a critical role for Homer proteins in the peri-junctional localization of mGlu1 receptors at glutamatergic synapses. To disrupt Homer-associated protein complexes, mice were tail-vein injected with the membrane-permeable dominant-negative TAT-Homer1a. Freeze-fracture replica immunogold labelling analysis showed no significant alteration in the mGlu1 receptor distribution pattern at parallel fibre-Purkinje cell synapses, suggesting that other scaffolding proteins are involved in the peri-synaptic confinement. The identification of interactors that regulate the subsynaptic localization of the mGlu1 receptor at neurochemically distinct synapses may offer new insight into its trafficking and intracellular signalling.

Distinct subsynaptic localization of type 1 metabotropic glutamate receptors at glutamatergic and GABAergic synapses in the rodent cerebellar cortex.

γ-Aminobutyric acid (GABA)- and glycine-mediated hyperpolarizing inhibition is associated with a chloride influx that depends on the inwardly directed chloride electrochemical gradient. In neurons, the extrusion of chloride from the cytosol primarily depends on the expression of an isoform of potassium-chloride cotransporters (KCC2s). KCC2 is crucial in the regulation of the inhibitory tone of neural circuits, including pain processing neural assemblies. Thus we investigated the cellular distribution of KCC2 in neurons underlying pain processing in the superficial spinal dorsal horn of rats by using high-resolution immunocytochemical methods. We demonstrated that perikarya and dendrites widely expressed KCC2, but axon terminals proved to be negative for KCC2. In single ultrathin sections, silver deposits labeling KCC2 molecules showed different densities on the surface of dendritic profiles, some of which were negative for KCC2. In freeze fracture replicas and tissue sections double stained for the β3-subunit of GABAA receptors and KCC2, GABAA receptors were revealed on dendritic segments with high and also with low KCC2 densities. By measuring the distances between spots immunoreactive for gephyrin (a scaffolding protein of GABAA and glycine receptors) and KCC2 on the surface of neurokinin 1 (NK1) receptor-immunoreactive dendrites, we found that gephyrin-immunoreactive spots were located at various distances from KCC2 cotransporters; 5.7 % of them were recovered in the middle of 4-10-µm-long dendritic segments that were free of KCC2 immunostaining. The variable local densities of KCC2 may result in variable postsynaptic potentials evoked by the activation of GABAA and glycine receptors along the dendrites of spinal neurons.

Differential expression patterns of K(+) /Cl(-) cotransporter 2 in neurons within the superficial spinal dorsal horn of rats.

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.

Regulating anxiety with extrasynaptic inhibition.

The dynamic interactions between hippocampus and amygdala are critical for emotional memory. Theta synchrony between these structures occurs during fear memory retrieval and may facilitate synaptic plasticity, but the cellular mechanisms are unknown. We report that interneurons of the mouse basal amygdala are activated during theta network activity or optogenetic stimulation of ventral CA1 pyramidal cell axons, whereas principal neurons are inhibited. Interneurons provide feedforward inhibition that transiently hyperpolarizes principal neurons. However, synaptic inhibition attenuates during theta frequency stimulation of ventral CA1 fibers, and this broadens excitatory postsynaptic potentials. These effects are mediated by GABAB receptors and change in the Cl(-) driving force. Pairing theta frequency stimulation of ventral CA1 fibers with coincident stimuli of the lateral amygdala induces long-term potentiation of lateral-basal amygdala excitatory synapses. Hence, feedforward inhibition, known to enforce temporal fidelity of excitatory inputs, dominates hippocampus-amygdala interactions to gate heterosynaptic plasticity. VIDEO ABSTRACT.

Yu Kasugai

Department of Pharmacology

Medical university of innsbruck

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala