Cheng-Chang Lien

Hyperpolarization‐activated cation channels in fast‐spiking interneurons of rat hippocampus

Hyperpolarization‐activated channels (Ih or HCN channels) are widely expressed in principal neurons in the central nervous system. However, Ih in inhibitory GABAergic interneurons is less well characterized. We examined the functional properties of Ih in fast‐spiking basket cells (BCs) of the dentate gyrus, using hippocampal slices from 17‐ to 21‐day‐old rats. Bath application of the Ih channel blocker ZD 7288 at a concentration of 30 μm induced a hyperpolarization of 5.7 ± 1.5 mV, an increase in input resistance and a correlated increase in apparent membrane time constant. ZD 7288 blocked a hyperpolarization‐activated current in a concentration‐dependent manner (IC50, 1.4 μm). The effects of ZD 7288 were mimicked by external Cs+. The reversal potential of Ih was −27.4 mV, corresponding to a Na+ to K+ permeability ratio (PNa/PK) of 0.36. The midpoint potential of the activation curve of Ih was −83.9 mV, and the activation time constant at −120 mV was 190 ms. Single‐cell expression analysis using reverse transcription followed by quantitative polymerase chain reaction revealed that BCs coexpress HCN1 and HCN2 subunit mRNA, suggesting the formation of heteromeric HCN1/2 channels. ZD 7288 increased the current threshold for evoking antidromic action potentials by extracellular stimulation, consistent with the expression of Ih in BC axons. Finally, ZD 7288 decreased the frequency of miniature inhibitory postsynaptic currents (mIPSCs) in hippocampal granule cells, the main target cells of BCs, to 70 ± 4% of the control value. In contrast, the amplitude of mIPSCs was unchanged, consistent with the presence of Ih in inhibitory terminals. In conclusion, our results suggest that Ih channels are expressed in the somatodendritic region, axon and presynaptic elements of fast‐spiking BCs in the hippocampus.

Gating, modulation and subunit composition of voltage‐gated K+ channels in dendritic inhibitory interneurones of rat hippocampus

GABAergic interneurones are diverse in their morphological and functional properties. Perisomatic inhibitory cells show fast spiking during sustained current injection, whereas dendritic inhibitory cells fire action potentials with lower frequency. We examined functional and molecular properties of K+ channels in interneurones with horizontal dendrites in stratum oriens‐alveus (OA) of the hippocampal CA1 region, which mainly comprise somatostatin‐positive dendritic inhibitory cells. Voltage‐gated K+ currents in nucleated patches isolated from OA interneurones consisted of three major components: a fast delayed rectifier K+ current component that was highly sensitive to external 4‐aminopyridine (4‐AP) and tetraethylammonium (TEA) (half‐maximal inhibitory concentrations < 0.1 mm for both blockers), a slow delayed rectifier K+ current component that was sensitive to high concentrations of TEA, but insensitive to 4‐AP, and a rapidly inactivating A‐type K+ current component that was blocked by high concentrations of 4‐AP, but resistant to TEA. The relative contributions of these components to the macroscopic K+ current were estimated as 57 ± 5, 25 ± 6, and 19 ± 2 %, respectively. Dendrotoxin, a selective blocker of Kv1 channels had only minimal effects on K+ currents in nucleated patches. Coapplication of the membrane‐permeant cAMP analogue 8‐(4‐chlorophenylthio)‐adenosine 3′:5′‐cyclic monophosphate (cpt‐cAMP) and the phosphodiesterase blocker isobutyl‐methylxanthine (IBMX) resulted in a selective inhibition of the fast delayed rectifier K+ current component. This inhibition was absent in the presence of the protein kinase A (PKA) inhibitor H‐89, implying the involvement of PKA‐mediated phosphorylation. Single‐cell reverse transcription‐polymerase chain reaction (RT‐PCR) analysis revealed a high abundance of Kv3.2 mRNA in OA interneurones, whereas the expression level of Kv3.1 mRNA was markedly lower. Similarly, RT‐PCR analysis showed a high abundance of Kv4.3 mRNA, whereas Kv4.2 mRNA was undetectable. This suggests that the fast delayed rectifier K+ current and the A‐type K+ current component are mediated predominantly by homomeric Kv3.2 and Kv4.3 channels. Selective modulation of Kv3.2 channels in OA interneurones by cAMP is likely to be an important factor regulating the activity of dendritic inhibitory cells in principal neurone‐interneurone microcircuits.

Visual stimuli-induced LTD of GABAergic synapses mediated by presynaptic NMDA receptors

Local GABA (γ-aminobutyric acid) circuits contribute to sensory experience–dependent refinement of neuronal connections in the developing nervous system, but whether GABAergic synapses themselves can be rapidly modified by sensory stimuli is largely unknown. Here we report that repetitive light stimuli or theta burst stimulation (TBS) of the optic nerve in the developing Xenopus retinotectal system induces long-term potentiation (LTP) of glutamatergic inputs but long-term depression (LTD) of GABAergic inputs to the same tectal neuron. The LTD is due to a reduction in presynaptic GABA release and requires activation of presynaptic NMDA (N-methyl-D-aspartate) receptors (NMDARs) and coincident high-level GABAergic activity. Thus, the presynaptic NMDAR may function as a coincidence detector for adjacent glutamatergic and GABAergic activities, leading to coordinated synaptic modification by sensory experience.

Mutations in KCND3 Cause Spinocerebellar Ataxia Type 22

To identify the causative gene in spinocerebellar ataxia (SCA) 22, an autosomal dominant cerebellar ataxia mapped to chromosome 1p21‐q23.

Cell Type-Specific Expression of Acid-Sensing Ion Channels in Hippocampal Interneurons

Acid-sensing ion channels (ASICs), a member of the degenerin/epithelial Na+ channel superfamily, are widely expressed in the mammalian CNS. Accumulating evidence suggests that ASIC current density is higher in GABAergic interneurons than that in glutamatergic pyramidal neurons (PNs) in the hippocampus. Such differential expression of ASICs in cortical networks is thought to be a key element for seizure termination. However, GABAergic interneurons are highly diverse; it is unclear whether the functional expression of ASICs differs in distinct GABAergic interneuron subtypes. Moreover, the subunit composition of ASICs in individual GABAergic interneurons remains unknown. By combining patch-clamp recording and single-cell reverse transcription (RT)-PCR analysis, we correlated ASIC currents with their gene expression in acute rat hippocampal slices. The results yielded several surprising findings. First, ASIC current density of oriens lacunosum-moleculare (O-LM) cells in the CA1 region, a classical type of dendrite-targeting interneuron, is 6 times greater than that of fast-spiking basket cells (BCs) in the dentate gyrus, a major class of soma-targeting interneuron. Second, the recovery of ASICs from desensitization is slowest in BCs, intermediate in PNs, and fastest in O-LM cells. Third, the tarantula venom psalmotoxin 1, the specific blocker for ASIC1a homomers, inhibits ASIC currents in BCs but not in O-LM cells. Finally, single-cell RT-PCR analysis reveals coexpression of ASIC1a and ASIC2 subunit transcripts in O-LM cells, whereas only ASIC1a subunit transcript is detected in most BCs. Thus, differential expression of ASICs in inhibitory microcircuits likely contributes to the distinct roles of GABAergic interneurons in normal physiology and pathophysiology.

Mirror-image pain is mediated by nerve growth factor produced from tumor necrosis factor alpha-activated satellite glia after peripheral nerve injury.

Summary After peripheral nerve injury, tumor necrosis factor &agr;‐activated satellite glia in the contralateral dorsal root ganglion produce excess nerve growth factor that, in turn, enhances nociceptor excitability and results in mirror‐image pain. ABSTRACT Mirror‐image pain is characterized by mechanical hypersensitivity on the uninjured mirror‐image side. Recent reports favor central mechanisms, but whether peripheral mechanisms are involved remains unclear. We used unilateral spinal nerve ligation (SNL) to induce mirror‐image pain in rats. On the mirror‐image (contralateral) side, we found that satellite glia in the dorsal root ganglion (DRG) were activated, whereas macrophages/Schwann cells in the DRG and astrocytes/oligodendrocytes/microglia in the dorsal spinal cord were not. Subsequently, an increase in nerve growth factor (NGF) was detected in the contralateral DRG, and NGF immunoreactivity was concentrated in activated satellite glia. These phenomena were abolished if fluorocitrate (a glial inhibitor) was intrathecally injected before SNL. Electrophysiological recordings in cultured small DRG neurons showed that exogenous NGF enhanced nociceptor excitability. Intrathecal injection of NGF into naive rats induced long‐lasting mechanical hypersensitivity, similar to SNL‐evoked mirror‐image pain. Anti‐NGF effectively relieved SNL‐evoked mirror‐image pain. In the contralateral DRG, the SNL‐evoked tumor necrosis factor alpha (TNF‐&agr;) increase, which started later than in the ipsilateral DRG and cerebrospinal fluid, occurred earlier than satellite glial activation and the NGF increase. Intrathecal injection of TNF‐&agr; into naive rats not only activated satellite glia to produce extra NGF in the DRG but also evoked mechanical hypersensitivity, which could be attenuated by anti‐NGF injection. These results suggest that after SNL, satellite glia in the contralateral DRG are activated by TNF‐&agr; that diffuses from the injured side via cerebrospinal fluid, which then activates satellite glia to produce extra NGF to enhance nociceptor excitability, which induces mirror‐image pain.

Acid-Sensing Ion Channel-1a Is Not Required for Normal Hippocampal LTP and Spatial Memory

Acid-sensing ion channel-1a (ASIC1a) is localized in brain regions with high synaptic density and is thought to contribute to synaptic plasticity, learning, and memory. A prominent hypothesis is that activation of postsynaptic ASICs promotes depolarization, thereby augmenting N-methyl-d-aspartate receptor function and contributing to the induction of long-term potentiation (LTP). However, evidence for activation of postsynaptic ASICs during neurotransmission has not been established. Here, we re-examined the role of ASIC1a in LTP in the hippocampus using pharmacological and genetic approaches. Our results showed that a tarantula peptide psalmotoxin, which profoundly blocked ASIC currents in the hippocampal neurons, had no effect on LTP. Similarly, normal LTP was robustly generated in ASIC1a-null mice. A further behavioral analysis showed that mice lacking ASIC1a had normal performance in hippocampus-dependent spatial memory. In summary, our results indicate that ASIC1a is not required for hippocampal LTP and spatial memory. We therefore propose that the role of ASIC1a in LTP and spatial learning should be reassessed.

GABA Is Depolarizing in Hippocampal Dentate Granule Cells of the Adolescent and Adult Rats

GABAergic signaling in hippocampal pyramidal neurons undergoes a switch from depolarizing to hyperpolarizing during early neuronal development. Whether such a transformation of GABAergic action occurs in dentate granule cells (DGCs), located at the first stage of the hippocampal trisynaptic circuit, is unclear. Here, we use noninvasive extracellular recording to monitor the effect of synaptically released GABA on the DGC population. We find that GABAergic responses in adolescent and adult rat DGCs are still depolarizing from rest. Using a morphologically realistic DGC model, we show that GABAergic action, depending on its precise timing and location, can have either an excitatory or inhibitory role in signal processing in the dentate gyrus.

High-density expression of Ca2+-permeable ASIC1a channels in NG2 glia of rat hippocampus.

NG2 cells, a fourth type of glial cell in the mammalian CNS, undergo reactive changes in response to a wide variety of brain insults. Recent studies have demonstrated that neuronally expressed acid-sensing ion channels (ASICs) are implicated in various neurological disorders including brain ischemia and seizures. Acidosis is a common feature of acute neurological conditions. It is postulated that a drop in pH may be the link between the pathological process and activation of NG2 cells. Such postulate immediately prompts the following questions: Do NG2 cells express ASICs? If so, what are their functional properties and subunit composition? Here, using a combination of electrophysiology, Ca2+ imaging and immunocytochemistry, we present evidence to demonstrate that NG2 cells of the rat hippocampus express high density of Ca2+-permeable ASIC1a channels compared with several types of hippocampal neurons. First, nucleated patch recordings from NG2 cells revealed high density of proton-activated currents. The magnitude of proton-activated current was pH dependent, with a pH for half-maximal activation of 6.3. Second, the current-voltage relationship showed a reversal close to the equilibrium potential for Na+. Third, psalmotoxin 1, a blocker specific for the ASIC1a channel, largely inhibited proton-activated currents. Fourth, Ca2+ imaging showed that activation of proton-activated channels led to an increase of [Ca2+]i. Finally, immunocytochemistry showed co-localization of ASIC1a and NG2 proteins in the hippocampus. Thus the acid chemosensor, the ASIC1a channel, may serve for inducing membrane depolarization and Ca2+ influx, thereby playing a crucial role in the NG2 cell response to injury following ischemia.

M1-like muscarinic acetylcholine receptors regulate fast-spiking interneuron excitability in rat dentate gyrus

Cholinergic transmission through muscarinic acetylcholine receptors (mAChRs) plays a key role in cortical oscillations. Although fast-spiking (FS), parvalbumin-expressing basket cells (BCs) are proposed to be the cellular substrates of gamma oscillations, previous studies reported that FS nonpyramidal cells in neocortical areas are unresponsive to cholinergic modulation. Dentate gyrus (DG) is an independent gamma oscillator in the hippocampal formation. However, in contrast to other cortical regions, the direct impact of mAChR activation on FS BC excitability in this area has not been investigated. Here, we show that bath-applied muscarine or carbachol, two mAChR agonists, depolarize DG BCs in the acute brain slices, leading to action potential firing in the theta-gamma bands in the presence of blockers of ionotropic glutamate and gamma-aminobutyric acid type A receptors at physiological temperatures. The depolarizing action persists in the presence of tetrodotoxin, a voltage-gated Na(+) channel blocker. In voltage-clamp recordings, muscarine markedly reduces background K(+) currents. These effects are mimicked by oxotremorine methiodide, an mAChR-specific agonist, and largely reversed by atropine, a non-selective mAChR antagonist, or pirenzepine, an M(1) receptor antagonist, but not by gallamine, an M(2/4) receptor antagonist. Interestingly, in contrast to M(1)-receptor-mediated depolarization, M(2) receptor activation by the specific agonist arecaidine but-2-ynyl ester tosylate down-regulates GABA release at BC axons-the effect is occluded by gallamine, an M(2) receptor antagonist. Overall, muscarinic activation results in a net increase in phasic inhibitory output to the target cells. Thus, cholinergic activation through M(1)-like receptor enhances BC activity and promotes the generation of nested theta and gamma rhythms, thereby enhancing hippocampal function and associated performance.