Yuri Gonchar

Multiple distinct subtypes of GABAergic neurons in mouse visual cortex identified by triple immunostaining

The majority of cortical interneurons use GABA (gamma amino butyric acid) as inhibitory neurotransmitter. GABAergic neurons are morphologically, connectionally, electrically and chemically heterogeneous. In rat cerebral cortex three distinct groups of GABAergic interneurons have been identified by the expression of parvalbumin (PV), calretinin (CR) and somatostatin (SOM). Recent studies in mouse cerebral cortex have revealed a different organization in which the CR and SOM populations are partially overlapping. Because CR and SOM neurons derive from different progenitors located in different embryonic structures, the coexpression of CR + SOM suggests that the chemical differentiation of interneurons is regulated postmitotically. Here, we have taken an important first step towards understanding this process by triple immunostaining mouse visual cortex with a panel of antibodies, which has been used extensively for classifying developing interneurons. We have found at least 13 distinct groups of GABAergic neurons which include PV, CR, SOM, CCK (cholecystokinin), CR + SOM, CR + NPY (neuropeptide Y), CR + VIP (vasointestinal polypeptide), SOM + NPY, SOM + VIP, VIP + ChAT (choline acetyltransferase), CCK + NPY, CR + SOM + NPY and CR + SOM + VIP expressing cells. Triple immunostaining with PV, CR and SOM antibodies during postnatal development further showed that PV is never colocalized with CR and SOM. Importantly, expression of SOM and CR + SOM developed after the percentage of CR cells that do not express SOM has reached the mature level, suggesting that the chemical differentiation of SOM and CR + SOM neurons is a postnatal event, which may be controlled by transcriptional regulation.

Differential subcellular localization of forward and feedback interareal inputs to parvalbumin expressing GABAergic neurons in rat visual cortex

In rat visual cortex, forward and feedback interareal pathways innervate both pyramidal and gamma‐aminobutyric acid (GABA)ergic (Johnson and Burkhalter [1996] J. Comp. Neurol. 368:383–398). GABAergic neurons consist of different cell types of which the largest group expresses parvalbumin (PV; Gonchar and Burkhalter [1997] Cereb. Cortex 4:347–358). Here, we report that PV neurons in layers 2/3 are synaptic targets of forward and feedback projections between area 17 and the lateromedial area (LM) of rat visual cortex.

Differential expression of IA channel subunits Kv4.2 and Kv4.3 in mouse visual cortical neurons and synapses

In cortical neurons, pore-forming α-subunits of the Kv4 subfamily underlie the fast transient outward K+ current (IA). Considerable evidence has accumulated demonstrating specific roles for IA channels in the generation of individual action potentials and in the regulation of repetitive firing. Although IA channels are thought to play a role in synaptic processing, little is known about the cell type- and synapse-specific distribution of these channels in cortical circuits. Here, we used immunolabeling with specific antibodies against Kv4.2 and Kv4.3, in combination with GABA immunogold staining, to determine the cellular, subcellular, and synaptic localization of Kv4 channels in the primary visual cortex of mice, in which subsets of pyramidal cells express yellow fluorescent protein. The results show that both Kv4.2 and Kv4.3 are concentrated in layer 1, the bottom of layer 2/3, and in layers 4 and 5/6. In all layers, clusters of Kv4.2 and Kv4.3 immunoreactivity are evident in the membranes of the somata, dendrites, and spines of pyramidal cells and GABAergic interneurons. Electron microscopic analyses revealed that Kv4.2 and Kv4.3 clusters in pyramidal cells and interneurons are excluded from putative excitatory synapses, whereas postsynaptic membranes at GABAergic synapses often contain Kv4.2 and Kv4.3. The presence of Kv4 channels at GABAergic synapses would be expected to weaken inhibition during dendritic depolarization by backpropagating action potentials. The extrasynaptic localization of Kv4 channels near excitatory synapses, in contrast, should stabilize synaptic excitation during dendritic depolarization. Thus, the synapse-specific distribution of Kv4 channels functions to optimize dendritic excitation and the association between presynaptic and postsynaptic activity.

Subcellular localization of GABAB receptor subunits in rat visual cortex

Although studies in the visual cortex have found γ‐aminobutyric acid B (GABAB) receptor‐mediated pre‐ and postsynaptic inhibitory effects on neurons, the subcellular localization of GABAB receptors in different types of cortical neurons and synapses has not been shown directly. To provide this information, we have used antibodies against the GABAB receptor (R)1a/b and GABABR2 subunits and have studied the localization of immunoreactivities in rat visual cortex. Light microscopic analyses have shown that both subunits are expressed in cell bodies and dendrites of 65–92% of corticocortically projecting pyramidal neurons and in 92–100% of parvalbumin (PV)‐, calretinin (CR)‐, and somatostatin (SOM)‐containing GABAergic neurons. Electron microscopic analyses of immunoperoxidase‐ and immunogold‐labeled tissue revealed staining in the nucleus, cytoplasm and cell surface membranes with both antibodies. Colocalization of both subunits was observed in all of these structures. GABABR1a/b and GABABR2 were concentrated in excitatory and inhibitory synapses and in extrasynaptic membranes. In GABAergic synapses, GABABR1a/b and GABABR2 were more strongly expressed postsynaptically on pyramidal and nonpyramidal cells than presynaptically. In type 1 synapses GABABR1a/b and GABABR2 was found in pre‐ and postsynaptic membranes. The nuclear localization of GABABR1 and GABABR2 subunits suggests a novel role for neurotransmitter receptors in controlling gene expression. The synaptic colocalization of GABABR1 and GABABR2 indicates that subunits form heteromeric assemblies of the functional receptor in inhibitory and excitatory synapses. Subunit coexpression in GABAergic synapses that include PV‐containing and PV‐deficient terminals suggests that pre‐ and postsynaptic GABAB receptor activation is provided by several different types of interneurons. The coexpression of both subunits in excitatory synapses suggests a role for GABAB receptors in the regulation of glutamate release and raises the question how these receptors are activated in the absence of pre‐or postsynaptic GABAergic synaptic inputs to excitatory synapses. J. Comp. Neurol. 431:182–197, 2001.

Axo-axonic synapses formed by somatostatin-expressing GABAergic neurons in rat and monkey visual cortex.

In cerebral cortex of rat and monkey, the neuropeptide somatostatin (SOM) marks a population of nonpyramidal cells (McDonald et al. [1982] J. Neurocytol. 11:809–824; Hendry et al. [1984] J. Neurosci. 4:2497:2517; Laemle and Feldman [1985] J. Comp. Neurol. 233:452–462; Meineke and Peters [1986] J. Neurocytol. 15:121–136; DeLima and Morrison [1989] J. Comp. Neurol. 283:212–227) that represent a distinct type of γ‐aminobutyric acid (GABA) ‐ergic neuron (Gonchar and Burkhalter [1997] Cereb. Cortex 7:347–358; Kawaguchi and Kubota [1997] Cereb. Cortex 7:476–486) whose synaptic connections are incompletely understood. The organization of inhibitory inputs to the axon initial segment are of particular interest because of their role in the suppression of action potentials (Miles et al. [1996] Neuron 16:815:823). Synapses on axon initial segments are morphologically heterogeneous (Peters and Harriman [1990] J. Neurocytol. 19:154–174), and some terminals lack parvalbumin (PV) and contain calbindin (Del Rio and DeFelipe [1997] J. Comp. Neurol. 342:389–408), that is also expressed by many SOM‐immunoreactive neurons (Kubota et al. [1994] Brain Res. 649:159–173; Gonchar and Burkhalter [1997] Cereb. Cortex 7:347–358). We studied the innervation of pyramidal neurons by SOM neurons in rat and monkey visual cortex and examined putative contacts by confocal microscopy and determined synaptic connections in the electron microscope. Through the confocal microscope, SOM‐positive boutons were observed to form close appositions with somata, dendrites, and spines of intracortically projecting pyramidal neurons of rat area 17 and pyramidal cells in monkey striate cortex. In addition, in rat and monkey, SOM boutons were found to be associated with axon initial segments of pyramidal neurons. SOM axon terminals that were apposed to axon initial segments of pyramidal neurons lacked PV, which was shown previously to label axo‐axonic terminals provided by chandelier cells (DeFelipe et al. [1989] Proc. Natl. Acad. Sci. USA 86:2093–2097; Gonchar and Burkhalter [1999a] J. Comp. Neurol. 406:346:360). Electron microscopic examination directly demonstrated that SOM axon terminals form symmetric synapses with the initial segments of pyramidal cells in supragranular layers of rat and monkey primary visual cortex. These SOM synapses differed ultrastructurally from the more numerous unlabeled symmetric synapses found on initial segments. Postembedding immunostaining revealed that all SOM axon terminals contained GABA. Unlike PV‐expressing chandelier cell axons that innervate exclusively initial segments of pyramidal cell axons, SOM‐immunoreactive neurons innervate somata, dendrites, spines, and initial segments, that are just one of their targets. Thus, SOM neurons may influence synaptic excitation of pyramidal neurons at the level of synaptic inputs to dendrites as well as at the initiation site of action potential output. J. Comp. Neurol. 443:1–14, 2002.

Experience-dependent development of feedforward and feedback circuits between lower and higher areas of mouse visual cortex.

Using whole cell recordings, we studied excitatory and inhibitory postsynaptic currents (EPSCs, IPSCs) in feedforward (FF) and feedback (FB) circuits between areas V1 and LM (lateromedial) in developing mouse visual cortex. We found that in mice reared with normal visual experience, FF and FB synapses onto layer 2/3 pyramidal neurons develop equal but submaximal strengths whose EPSCs are increased by monocular lid suture. In contrast, the development and experience-dependence of FF- and FB-IPSCs is pathway-specific. The difference develops during the critical period by strengthening FF-IPSCs, while keeping FB-IPSC amplitudes constant. Monocular lid suture increases FB-IPSCs but does not affect FF-IPSCs.

Rearrangement of Synaptic Connections with Inhibitory Neurons in Developing Mouse Visual Cortex

Cortical inhibition is determined in part by the organization of synaptic inputs to γ‐aminobutyric acidergic (GABAergic) neurons. In adult rat visual cortex, feedforward (FF) and feedback (FB) connections that link lower with higher areas provide ∼10% of inputs to parvalbumin (PV)‐expressing GABAergic neurons and ∼90% to non‐GABAergic cells (Gonchar and Burkhalter [1999] J. Comp. Neurol. 406:346–360). Although the proportions of these targets are similar in both pathways, FF synapses prefer larger PV dendrites than FB synapses, which may result in stronger inhibition in the FF than in the FB pathway (Gonchar and Burkhalter [1999] J. Comp. Neurol. 406:346–360). To determine when during postnatal (P) development FF and FB inputs to PV and non‐PV neurons acquire mature proportions, and whether the pathway‐specific distributions of FF and FB inputs to PV dendrites develop from a similar pattern, we studied FF and FB connections between area 17 and the higher order lateromedial area (LM) in visual cortex of P15–42 mice. We found that the innervation ratio of PV and non‐PV neurons is mature at P15. Furthermore, the size distributions of PV dendrites contacted by FF and FB synapses were similar at P15 but changed during the third to sixth postnatal weeks so that, by P36–42, FF inputs preferred thick dendrites and FB synapses favored thin PV dendrites. These results suggest that distinct FF and FB circuits develop after eye opening by rearranging the distribution of excitatory synaptic inputs on the dendritic tree of PV neurons. The purpose of this transformation may be to adjust differentially the strengths of inhibition in FF and FB circuits. J. Comp. Neurol. 464:426–437, 2003.