The family of mammalian genes related to the Drosophila Shaker gene, consisting of four subfamilies, is thought to encode subunits of tetrameric voltage-gated K+ channels. There is compelling evidence that subunits of the same subfamily, but not of different subfamilies, form heteromultimeric channels in vitro, and thus, each gene subfamily is postulated to encode components of an independent channel system. In order to identify cells with native channels containing subunits of one of these subfamilies (Shaw-related or ShIII), the cellular distribution of ShIII transcripts was examined by Northern blot analysis and in situ hybridization. Three of four ShIII genes (KV3.1, KV3.2, and KV3.3) are expressed mainly in the CNS. KV3.4 transcripts are also present in the CNS but are more abundant in skeletal muscle. In situ hybridization studies in the CNS reveal discrete and specific neuronal populations that prominently express ShIII mRNAs, both in projecting and in local circuit neurons. In the cerebral cortex, hippocampus, and caudate- putamen, subsets of neurons can be distinguished by the expression of specific ShIII mRNAs. Each ShIII gene exhibits a unique pattern of expression; however, many neuronal populations expressing KV3.1 transcripts also express KV3.3 mRNAs. Furthermore, KV3.4 transcripts are present, albeit at lower levels, in several of the neuronal populations that also express KV3.1 and/or KV3.3 mRNAs, revealing a high potential for heteromultimer formation between the products of three of the four genes. Expression of ShIII cRNAs in Xenopus oocytes was used to explore the functional consequences of heteromultimer formation between ShIII subunits. Small amounts of KV3.4 cRNA, which expresses small, fast-inactivating currents when injected alone, produced fast-inactivating currents that are severalfold larger when coinjected with an excess of KV3.1 or KV3.3 cRNA. This amplification is due to both an increase in single-channel conductance in the heteromultimeric channels and the observation that less than four, perhaps even a single KV3.4 subunit is sufficient to impart fast- inactivating properties to the channel. The oocyte experiments indicate that the apparently limited, low-level expression of KV3.4 in the CNS is potentially significant. The anatomical studies suggest that heteromultimer formation between ShIII proteins might be a common feature in the CNS. Moreover, the possibility that the subunit composition of heteromultimers varies in different neurons should be considered, since the ratios of overlapping signals change from one neuronal population to another. In order to proceed with functional analysis of native ShIII channels, it is important to known which subunit compositions might occur in vivo. The studies presented here provide important clues for the identification of native homo- and heteromultimeric ShIII channels in neurons.

Genome-encoded microRNAs (miRNAs) are potent regulators of gene expression. The significance of miRNAs in various biological processes has been suggested by studies showing an important role of these small RNAs in regulation of cell differentiation. However, the role of miRNAs in regulation of differentiated cell physiology is not well established. Mature neurons express a large number of distinct miRNAs, but the role of miRNAs in postmitotic neurons has not been examined. Here, we provide evidence for an essential role of miRNAs in survival of differentiated neurons. We show that conditional Purkinje cell–specific ablation of the key miRNA-generating enzyme Dicer leads to Purkinje cell death. Deficiency in Dicer is associated with progressive loss of miRNAs, followed by cerebellar degeneration and development of ataxia. The progressive neurodegeneration in the absence of Dicer raises the possibility of an involvement of miRNAs in neurodegenerative disorders.

Potassium channels play major roles in the regulation of many aspects of neuronal excitability. These channels are particularly well suited for such multiplicity of roles since there is a large diversity of channel types. This diversity contributes to the ability of specific neurons (and possibly different regions of the same neuron) to respond uniquely to a given input. Neuronal integration depends on the local response of spatially segregated inputs to the cell and the communication of these integration centers with the axon. Therefore, the functional implications of a given set of K+ channels varies depending on their precise location on the neuronal surface. Site-specific antibodies were utilized to characterize the distribution of KV3.1b, a subunit of voltage-gated K+ channels in CNS neurons. KV3.1b subunits are expressed in specific neuronal populations of the rat brain, such as cerebellar granule cells, projecting neurons of deep cerebellar nuclei, the substantia nigra pars-reticulata, the globus pallidus, and the ventral thalamus (reticular thalamic nucleus, ventral lateral geniculate and zona incerta). The KV3.1b protein is also present in various neuronal populations involved in the processing of auditory signals, including the inferior colliculus, the nuclei of the lateral lemniscus, the superior olive, and some parts of the cochlear nuclei; as well as in several other neuronal groups in the brainstem (e.g., in the oculomotor nucleus, the pontine nuclei, the reticulotegmental nucleus of the pons, trigeminal and vestibular nuclei, and the reticular formation) and subsets of neurons in the neocortex, the hippocampus and the caudate-putamen shown by double staining to correspond to neurons containing parvalbumin. KV3.1b subunits are localized predominantly in somatic and axonal membranes (particularly in axonal terminal fields) but are much less prominent in dendritic arborizations. This distribution is different than that of other subunits of voltage gated K+ channels and is consistent with a role in the modulation of action potentials. KV3.1b proteins have a cellular and subcellular distribution different than the related KV3.2 subunits which express in Xenopus oocytes currents similar to those expressed by KV3.1b.

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Specific chemical lesion of the rat inferior olive by intraperitoneal administration of 3-acetylpyridine prevents recuperation from motor abnormalities generated by unilateral labyrinthine lesion. Moreover, in animals that have recuperated from the balyrinthine lesion, 3-acetylpyridine produces a reversal of the symptoms within 2 hours of administration. These results indicate that the integrity of the olivo-cerebellar system is necessary for the acquisition and retention of this form of motor learning, but that the cerebellum itself is not the seat of such learning.

G-protein-gated inward rectifier potassium (GIRK) channels are coupled to numerous neurotransmitter receptors in the brain and can play important roles in modulating neuronal function, depending on their localization in a given neuron. Site-directed antibodies to the extreme C terminus of GIRK1 (or KGA1), a recently cloned component of GIRK channels, have been used to determine the relative expression levels and distribution of the protein in different regions of the rat brain by immunoblot and immunohistochemical techniques. We report that the GIRK1 protein is expressed prominently in the olfactory bulb, hippocampus, dentate gyrus, neocortex, thalamus, cerebellar cortex, and several brain stem nuclei. In addition to the expected localization in somas and dendrites, where GIRK channels may mediate postsynaptic inhibition, GIRK1 proteins were also found in axons and their terminal fields, suggesting that GIRK channels can also modulate presynaptic events. Furthermore, the distribution of the protein to either somatodendritic or axonal-terminal regions of neurons varied in different brain regions, which would imply distinct functions of these channels in different neuronal populations. Particularly prominent staining of the cortical barrels of layer IV of the neocortex, and the absence of this staining with unilateral kainate lesions of the thalamus, suggest that the GIRK1 protein is expressed in thalamocortical nerve terminals in which GIRK channels may mediate the actions of mu opiate receptors.

The distribution of the P-type calcium channel in the mammalian central nervous system has been demonstrated immunohistochemically by using a polydonal specific antibody. This antibody was generated after P-channel isolation via a fraction from funnel-web spider toxin (FIX) that blocks the voltage-gated P channels in cerebellar Purklije cells.In the cerebellar cortex, immunolabeling to the antibody appeared throughout the molecular layer, while all the other regions were negative. Intensely labeled patches of reactivity were seen on Purkije cell dendrites, especially at bifurcatlons; much weaker reactivity was present in the soma and stem segment. Electron microscopic loItion revealed labeled patches of plasma membrane on the soma, min_ dendrites, spiny branchlets, and spines; portions of the smooth endoplasmic reticulum were also labeled. Strong labeling was present in the periglomerular cells of the olfactory bulb and scattered neurons in the deep layer of the entorhinal and pyriform cortices. Neurons in the brainstem, habenula, nucleus of the trapezoid body and inferior olive and along the floor Of the fourth ventricle were also labeled intensely. Medium-intensity reactions were observed in layer H pyramidal cells of the frontal cortex, the CAl cells of the hippocampus, the lateral nucleus of the substantia nigra, lateral reticular nucleus,, and spinal fifth nucleus. Light labeling was seen in the neocortex, striatum, and in some brainstem neurons.Knowing the specific localization of voltage-gated ion channels on the soma-dendritic membrane of neurons is fundamental to understanding their intrinsic and integrative functions. The question of voltage-gated channel localiation has been of particular interest since the first report of dendritic action potentials in Purkinje cells over 2 decades ago (1, 2). Indeed, the existence of such electroresponsiveness was not readily accepted until intradendritic recordings were made from different points in the dendritic tree of Purkinje cells (3,4).The calcium-dependent nature of these potentials was initially shown in avians (5) and later in mammals (4,6). The recent availability of specific calcium-channel blockers has allowed a more precise identification of these conductances and the different types of calcium channels involved. Specifically, calcium-channel blockers such as the dihydropyridines (7, 8) and a-conotoxin (9, 10) were ineffective in Purkinje cells, while these responses were blocked by a funnel-web spider toxin (FTX) (11). The results obtained from the venom study were confirmed at both macroscopic current and single-channel levels for the calcium channels induced by rat brain mRNA injection into Xenopus oocytes (12, 13). The toxin (FTX) specifically responsible for this calcium-channel block was then isolated from the venom and a synthetic analog (sFTX) was made (14, 15). The calcium channel, called the P channel, was then isolated from bovine cerebella by using sFTX (16), and a polyclonal antibody was generated from this protein (17). In a more ...

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