Wen Jie Song

Somatodendritic depolarization-activated potassium currents in rat neostriatal cholinergic interneurons are predominantly of the A type and attributable to coexpression of Kv4.2 and Kv4.1 subunits.

Unlike other neostriatal neurons, cholinergic interneurons exhibit spontaneous, low-frequency, repetitive firing. To gain an understanding of the K+ channels regulating this behavior, acutely isolated adult rat cholinergic interneurons were studied using whole-cell voltage-clamp and single-cell reverse transcription-PCR techniques. Cholinergic interneurons were identified by the presence of choline acetyltransferase (ChAT) mRNA. Depolarization-activated potassium currents in cholinergic interneurons were dominated by a rapidly inactivating, K+-selective A current that became active at subthreshold potentials. Depolarizing prepulses inactivated this component of the current, leaving a delayed, rectifier-like current. Micromolar concentrations of Cd2+ dramatically shifted the voltage dependence of the A current without significantly affecting the delayed rectifier. The A-channel antagonist 4-aminopyridine (4-AP) produced a voltage-dependent block (IC50, ∼1 mm) with a prominent crossover at millimolar concentrations. On the other hand, TEA preferentially blocked the sustained current component at concentrations <10 mm. Single-cell mRNA profiling of subunits known to give rise to rapidly inactivating K+ currents revealed the coexpression of Kv4.1, Kv4.2, and Kv1.4 mRNAs but low or undetectable levels of Kv4.3 and Kv3.4 mRNAs. Kv1.1, β1, and β2 subunit mRNAs, but not β3, were also commonly detected. The inactivation recovery kinetics of the A-type current were found to match those of Kv4.2 and 4.1 channels and not those of Kv1.4 or Kv1.1 and β1 channels. Immunocytochemical analysis confirmed the presence of Kv4.2 but not Kv1.4 subunits in the somatodendritic membrane of ChAT-immunoreactive neurons. These results argue that the depolarization-activated somatodendritic K+ currents in cholinergic interneurons are dominated by Kv4.2- and Kv4.1-containing channels. The properties of these channels are consistent with their playing a prominent role in governing the slow, repetitive discharge of interneurons seen in vivo.

Genes responsible for native depolarization-activated K+ currents in neurons.

Depolarization-activated, Ca2+-independent K+ currents can be largely divided into delayed rectifiers and transient A-type currents. In mammals, each of these subtypes exhibits large variations in voltage dependence and kinetics according to cell types. At the molecular level, the principal subunits of depolarization-activated K+ channels are thought to be coded by genes from nine subfamilies, Kv1 through Kv9, of which members within each of the Kv1-Kv4 subfamilies can form either homomeric or heteromeric, functional tetrameric channels. The variations in current properties and the large number of genes make it difficult to identify genes responsible for native K(+) channels in mammalian neurons. Nevertheless, progress has been made in recent years, in which the single cell/reverse transcription/polymerase chain reaction (scRT-PCR) protocol combined with patch clamp recording played important roles. With this technique, it has been shown in a number of neuronal phenotypes that mammalian neurons create diversity of channel function by coexpression of members of different Kv subfamilies, coexpression of multiple members of a Kv subfamily, and coexpression of multiple principal and auxiliary subunits. Some genes appear to be expressed at higher levels than others. In the somatodendritic domain, evidence is accumulating that Kv4 subfamily is a major contributor for the typical A-type current, while delayed rectifiers are often attributable to Kv2 and Kv3 subfamily genes. It thus appears that mammalian neurons express some particular Kv genes at higher levels while coexpress multiple genes for the composition of depolarization-activated K+ channels. In addition to the evolution of a large number of K+ channel genes, coexpression of multiple members of the genes in a single neuron also appears to be a strategy for mammalian neurons to create channel diversity.

Evidence for the preferential localization of Glutamate Receptor-1 subunits of AMPA receptors to the dendritic spines of medium spiny neurons in rat striatum

Although immunohistochemical studies have typically found the perikarya of striatal projection neurons to be devoid of immunohistochemical labelling for the GluR1 AMPA type glutamate receptor subunit, the striatal neuropil is rich in GluR1 immunolabelling and in situ hybridization histochemistry has indicated the presence of GluR1 message in many striatal neurons. To explore the possibility that GluR1 subunits may be synthesized by many striatal projection neurons, but selectively localized to their dendrites, we have used light-microscopic and electron-microscopic immunohistochemistry in combination with single-cell reverse transcription-polymerase chain reaction. Light-microscopic immunohistochemical studies confirmed the presence of abundant GluR1 immunoreactivity in the striatal neuropil in rats. Perikaryal labelling was restricted to neurons previously identified as parvalbuminergic neurons. Single-cell reverse transcription-polymerase chain reaction for individual striatal neurons in rats confirmed that most striatal projection neurons (i.e. containing either or both substance P message or enkephalin message) make GluR1 message. For example, 94% of enkephalin-containing neurons, 75% of substance P-containing neurons, and 87% of enkephalin and substance P co-containing neurons expressed GluR1 messenger RNA. Electron-microscopic immunohistochemistry revealed that GluR1 immunolabelling was prominent in 61% of dendritic spines and 53% of dendritic shafts. While prominent perikaryal GluR1 immunolabelling was observed only in a small population of interneurons, sparse perikaryal GluR1 immunolabelling was found associated with the rough endoplasmic reticulum, the Golgi apparatus, the outer membranes of the mitochondria, and the outer envelope of the nucleus of about 30% of striatal projection neurons (identified by their non-indented nuclei). These results indicate that striatal projection neurons selectively target GluR1 subunits to their spines and dendritic shafts. Our finding has implications for the functioning of striatal projection neurons and for the general issue of whether neurons can control the subcellular localization of glutamate receptors.

Inwardly Rectifying Potassium (IRK) Currents Are Correlated with IRK Subunit Expression in Rat Nucleus Accumbens Medium Spiny Neurons

Inwardly rectifying K+ (IRK) channels are critical for shaping cell excitability. Whole-cell patch-clamp and single-cell RT-PCR techniques were used to characterize the inwardly rectifying K+ currents found in projection neurons of the rat nucleus accumbens. Inwardly rectifying currents were highly selective for K+ and blocked by low millimolar concentrations of Cs+ or Ba2+. In a subset of neurons, the inwardly rectifying current appeared to inactivate at hyperpolarized membrane potentials. In an attempt to identify this subset, neurons were profiled using single-cell RT-PCR. Neurons expressing substance P mRNA exhibited noninactivating inward rectifier currents, whereas neurons expressing enkephalin mRNA exhibited inactivating inward rectifier currents. The inactivation of the inward rectifier was correlated with the expression of IRK1 mRNA. These results demonstrate a clear physiological difference in the properties of medium spiny neurons and suggest that this difference could influence active state transitions driven by cortical and hippocampal excitatory input.

Mitochondrial Dysfunction and Increased Reactive Oxygen Species Impair Insulin Secretion in Sphingomyelin Synthase 1-null Mice

Sphingomyelin synthase 1 (SMS1) catalyzes the conversion of ceramide to sphingomyelin. Here, we generated and analyzed SMS1-null mice. SMS1-null mice exhibited moderate neonatal lethality, reduced body weight, and loss of fat tissues mass, suggesting that they might have metabolic abnormality. Indeed, analysis on glucose metabolism revealed that they showed severe deficiencies in insulin secretion. Isolated mutant islets exhibited severely impaired ability to release insulin, dependent on glucose stimuli. Further analysis indicated that mitochondria in mutant islet cells cannot up-regulate ATP production in response to glucose. We also observed additional mitochondrial abnormalities, such as hyperpolarized membrane potential and increased levels of reactive oxygen species (ROS) in mutant islets. Finally, when SMS1-null mice were treated with the anti-oxidant N-acetyl cysteine, we observed partial recovery of insulin secretion, indicating that ROS overproduction underlies pancreatic β-cell dysfunction in SMS1-null mice. Altogether, our data suggest that SMS1 is important for controlling ROS generation, and that SMS1 is required for normal mitochondrial function and insulin secretion in pancreatic β-cells.

Nucleocytoplasmic translocation of HDAC9 regulates gene expression and dendritic growth in developing cortical neurons

Transcriptional regulation of gene expression is thought to play a pivotal role in activity‐dependent neuronal differentiation and circuit formation. Here, we investigated the role of histone deacetylase 9 (HDAC9), which regulates transcription by histone modification, in the development of neocortical neurons. The translocation of HDAC9 from nucleus to cytoplasm was induced by an increase of spontaneous firing activity in cultured mouse cortical neurons. This nucleocytoplasmic translocation was also observed in postnatal development in vivo. The translocation‐induced gene expression and cellular morphology was further examined by introducing an HDAC9 mutant that disrupts the nucleocytoplasmic translocation. Expression of c‐fos, an immediately‐early gene, was suppressed in the mutant‐transfected cells regardless of neural activity. Moreover, the introduction of the mutant decreased the total length of dendritic branches, whereas knockdown of HDAC9 promoted dendritic growth. These findings indicate that chromatin remodeling with nucleocytoplasmic translocation of HDAC9 regulates activity‐dependent gene expression and dendritic growth in developing cortical neurons.

Nigral GABAergic inhibition upon cholinergic neurons in the rat pedunculopontine tegmental nucleus

We investigated, in a midbrain parasagittal slice preparation of Wistar rats (postnatal day 9–17), the synaptic inhibition of neurons in the pedunculopontine tegmental nucleus (PPN), which was mediated by gamma (γ)‐amino‐butyric acid (GABA). Whole‐cell patch‐clamp recording was used, in combination with a single‐cell reverse transcription‐polymerase chain reaction amplification technique, to record synaptic potentials and to identify the phenotype of the recorded PPN neuron. In the presence of the ionotropic glutamate receptor antagonists, 6‐cyano‐2, 3‐dihydroxy‐7‐nitro‐quinoxaline‐2, 3, dione, and dl‐2‐amino‐5‐phosphonovaleric acid, single electrical stimuli were applied to the substantia nigra pars reticulata (SNr), one of the basal ganglia output nuclei. Stimulation of the SNr evoked inhibitory postsynaptic potentials (IPSPs) in 73 of the 104 neurons in the PPN. The IPSPs were abolished with a GABAA receptor antagonist, bicuculline. Inhibitory postsynaptic currents of the neurons were reversed in polarity at approximately −93.5 mV, which was close to the value of the equilibrium potential for chloride ions of −88.4 mV. Single‐cell reverse transcription‐polymerase chain reactions revealed that approximately 30% (9/32) of the PPN neurons that received inhibition from the SNr expressed detectable levels of choline acetyltransferase mRNA. These findings show that output from the SNr regulates the activity of cholinergic PPN neurons through GABAA receptors.

Plasticity of neuronal connections in developing brains of mammals

Although mature nervous systems show substantial malleability following various surgical or environmental manipulations, developing brains show far more prominent plasticity, particularly in terms of morphological features. Neuronal circuits, for example, can be dramatically rewired following neonatal but not adult brain lesions. It remains unknown why neuronal circuits in developing brains show such remarkable plasticity. A number of anatomical and physiological studies suggest that there are transient projections in developing brains and they are eliminated by cell death and/or collateral elimination as development proceeds. This raises a possibility that aberrant projections observed following various surgical or environmental manipulations such as partial denervation, results from retention or stabilization of transient projections. However, evidence suggests that cell death does not play an important role in developmental fine-tuning of neuronal projections. Furthermore, although the elimination of axon collaterals takes place, individual neurons appear to elaborate axonal arbors in appropriate target areas, resulting in a net increase in the size of axonal arbor emerging from individual neurons. In accord with these observations, the number of synapses appear to increase during the period when axonal elimination proceeds. Taken together, reinforcement of appropriate projections rather than elimination of excessive connections plays a major role in developmental specification of neuronal connections. Appearance of aberrant projections after partial denervation may not be a consequence of disordered axonal growth, since they form topographic maps which precisely mirrors those for normal projections. They may be induced due to reinforcement of pre-existing neuronal connections rather than to construction of novel pathways. Observations of axonal morphology in denervated areas indicate that lesion-induced enlargement of projections is due to transformation of axonal morphology, from simple and poorly branched to multiply branched. Perhaps such simple and poorly branched axons in inappropriate target areas may represent ones in the course of elimination but they may serve as a source of sprouting when denervated. In other words, after total elimination of axons any surgical or environmental manipulation cannot induce enlargement of projections. The mechanisms underlying such modifiability of neuronal connections remains unclarified but possible participation of an activity-dependent competitive mechanism is discussed.

Developing corticorubral axons of the cat form synapses on filopodial dendritic protrusions

Developing neurons transiently grow numerous spine- or filopodium-like dendritic protrusions (SLDPs). Electron microscopy on identified input and intracellular staining of postsynaptic cells were performed to gain insight into their significance. Newborn kitten-corticorubral axons, labelled with biocytin, commonly made synapses on SLDP, often multiply invaginated by the SLDPs. Correspondingly, intracellularly labelled kitten rubrospinal cells had numerous SLDPs. Taking into account that corticorubral synapses are largely formed on dendritic shafts in adult cats, it is likely that the SLDPs play some important role in the development of corticorubral synapses. We hypothesize that rubrospinal cells elongate SLDPs searching for corticorubral axons to form synapses.

Voltage-gated K+ channel KCNQ1 regulates insulin secretion in MIN6 β-cell line

KCNQ1, located on 11p15.5, encodes a voltage-gated K(+) channel with six transmembrane regions, and loss-of-function mutations in the KCNQ1 gene cause hereditary long QT syndrome. Recent genetic studies have identified that single nucleotide polymorphisms located in intron 15 of the KCNQ1 gene are strongly associated with type 2 diabetes and impaired insulin secretion. In order to understand the role of KCNQ1 in insulin secretion, we introduced KCNQ1 into the MIN6 mouse β-cell line using a retrovirus-mediated gene transfer system. In KCNQ1 transferred MIN6 cells, both the density of the KCNQ1 current and the density of the total K(+) current were significantly increased. In addition, insulin secretion by glucose, pyruvate, or tolbutamide was significantly impaired by KCNQ1-overexpressing MIN6 cells. These results suggest that increased KCNQ1 protein expression limits insulin secretion from pancreatic β-cells by regulating the potassium channel current.