Properties of interneurones in the intermediolateral cell column of the rat spinal cord: role of the potassium channel subunit Kv3.1

Sympathetic preganglionic neurones located in the intermediolateral cell column (IML) are subject to inputs descending from higher brain regions, as well as strong influences from local interneurones. Since interneurones in the IML have been rarely studied directly we examined their electrophysiological and anatomical properties. Whole cell patch clamp recordings were made from neurones in the IML of 250 microM slices of the thoracic spinal cord of the rat at room temperature. Action potential durations of interneurones (4.2+/-0.1 ms) were strikingly shorter than those of sympathetic preganglionic neurones (9.4+/-0.2 ms) due to a more rapid repolarisation phase. Low concentrations of tetraethylammonium chloride (TEA) (0.5 mM) or 4-aminopyridine (4-AP) (30 microM) affected interneurones but not sympathetic preganglionic neurones by prolonging the action potential repolarisation as well as decreasing both the afterhypolarisation amplitude and firing frequency. Following recordings, neurones sensitive to TEA and 4-AP were confirmed histologically as interneurones with axons that ramified extensively in the spinal cord, including the IML and other autonomic regions. In contrast, all cells that were insensitive to TEA and 4-AP were confirmed as sympathetic preganglionic neurones. Both electrophysiological and morphological data are therefore consistent with the presence of the voltage-gated potassium channel subunit Kv3.1 in interneurones, but not sympathetic preganglionic neurones. Testing this proposal immunohistochemically revealed that Kv3.1b was localised in low numbers of neurones within the IML but in higher numbers of neurones on the periphery of the IML. Kv3.1b-expressing neurones were not sympathetic preganglionic neurones since they were not retrogradely labelled following intraperitoneal injections of Fluorogold. Since Kv3.2 plays a similar role to Kv3.1 we also tested for the presence of Kv3.2 using immunohistochemistry, but failed to detect it in neuronal somata in the spinal cord. These studies provide electrophysiological and morphological data on interneurones in the IML and indicate that the channels containing the Kv3.1 subunit are important in setting the firing pattern of these neurones.     

Association of potassium channel Kv3.4 subunits with pre- and post-synaptic structures in brainstem and spinal cord

Voltage-gated K+ channels (Kv) are divided into eight subfamilies (Kv1-8) and play a major role in determining the excitability of neurones. Members of the Kv3 subfamily are highly abundant in the CNS, with each Kv3 gene (Kv3.1-Kv3.4) exhibiting a unique pattern of expression, although single neurones can express more than one subtype. Of the Kv3 subunits relatively little is known of the Kv3.4 subunit distribution in the nervous system, particularly in the brainstem and spinal cord of the rat. We performed immunohistochemistry to determine both the cellular and sub-cellular distribution of the Kv3.4 subunit in these areas. Kv3.4 subunit immunoreactivity (Kv3.4-IR) was widespread, with dense, punctate staining in many regions including the intermediolateral cell column (IML) and the dorsal vagal nucleus (DVN), nucleus ambiguus (NA) and nucleus tractus solitarius (NTS). In the ventral horn a presynaptic location was confirmed by co-localization of Kv3.4-IR with the synaptic vesicle protein, SV2 and also with the glutamate vesicle markers vesicular glutamate transporter (VGluT) 1, VGluT2 or the glycine transporter GlyT2, suggesting a role for the channel in both excitatory and inhibitory neurotransmission. Electron microscopy confirmed a presynaptic terminal location of Kv3.4-IR in the VH, IML, DVN, NA and NTS. Interestingly however, patches of Kv3.4-IR were also revealed postsynaptically in dendritic and somatic structures throughout these areas. This staining was striking due to its localization at synaptic junctions at terminals with morphological features consistent with excitatory functions, suggesting an association with the postsynaptic density. Therefore the pre and postsynaptic localization of Kv3.4-IR suggests a role both in the control of transmitter release and in regulating neuronal excitability.     

High-threshold, Kv3-like potassium currents in magnocellular neurosecretory neurons and their role in spike repolarization

We identified Kv3-like high-threshold K+ currents in hypothalamic supraoptic neurons using whole cell recordings in hypothalamic slices and in acutely dissociated neurons. Tetraethylammonium (TEA)-sensitive currents (< 1 mM TEA) evoked from -50 mV were characterized by a large component that inactivated in 10-30 ms, and a smaller, persistent component that inactivated in 1-2 s. I/V relations in dissociated neurons revealed TEA-subtracted currents with a slope and voltage dependency consistent with the presence of Kv3-like channels. In slices, tests with 0.01-0.7 mM TEA produced an IC50 of 200-300 nM for both fast and persistent currents. The fast transient current was similar to currents associated with the expression of Kv3.4 subunits, given that it was sensitive to BDS-I (100 nM). The persistent TEA-sensitive current appeared similar to those attributed to Kv3.1/3.2 subunits. Although qualitatively similar, oxytocin (OT) and vasopressin (VP) neurons in slices differed in the stronger presence of persistent current in VP neurons. In both cell types, the IC50 for TEA-induced spike broadening was similar to that observed for current suppression in voltage clamp. However, TEA had a greater effect on the spike width of VP neurons than of OT neurons. Immunochemical studies revealed a stronger expression of the Kv3.1b alpha-subunit in VP neurons, which may be related to the greater importance of this current type in VP spike repolarization. Because OT and VP neurons are not considered fast firing, but do exhibit frequency- and calcium-dependent spike broadening, Kv3-like currents may be important for maintaining spike width and calcium influx within acceptable limits during repetitive firing.     

Kv3.1b and Kv3.3 channel subunit expression in murine spinal dorsal horn GABAergic interneurones

GABAergic interneurones, including those within spinal dorsal horn, contain one of the two isoforms of the synthesizing enzyme glutamate decarboxylase (GAD), either GAD65 or GAD67. The physiological significance of these two GABAergic phenotypes is unknown but a more detailed anatomical and functional characterization may help resolve this issue. In this study, two transgenic Green Fluorescent Protein (GFP) knock-in murine lines, namely GAD65-GFP and GAD67-GFP (Δneo) mice, were used to profile expression of Shaw-related Kv3.1b and Kv3.3 K⁺-channel subunits in dorsal horn interneurones. Neuronal expression of these subunits confers specific biophysical characteristic referred to as ‘fast-spiking’. Immuno-labelling for Kv3.1b or Kv3.3 revealed the presence of both of these subunits across the dorsal horn, most abundantly in laminae I-III. Co-localization studies in transgenic mice indicated that Kv3.1b but not Kv3.3 was associated with GAD65-GFP and GAD67-GFP immunopositive neurones. For comparison the distributions of Kv4.2 and Kv4.3 K⁺-channel subunits which are linked to an excitatory neuronal phenotype were characterized. No co-localization was found between GAD-GFP +ve neurones and Kv4.2 or Kv4.3. In functional studies to evaluate whether either GABAergic population is activated by noxious stimulation, hindpaw intradermal injection of capsaicin followed by c-fos quantification in dorsal horn revealed co-expression c-fos and GAD65-GFP (quantified as 20-30% of GFP +ve population). Co-expression was also detected for GAD67-GFP +ve neurones and capsaicin-induced c-fos but at a much reduced level of 4-5%. These data suggest that whilst both GAD65-GFP and GAD67-GFP +ve neurones express Kv3.1b and therefore may share certain biophysical traits, their responses to peripheral noxious stimulation are distinct.     

Functional role of A-type potassium currents in rat presympathetic PVN neurones

Despite the fact that paraventricular nucleus (PVN) neurones innervating the rostral ventrolateral medulla (RVLM) play important roles in the control of sympathetic function both in physiological and pathological conditions, the precise mechanisms controlling their activity are still incompletely understood. In the present study, we evaluated whether the transient outward potassium current I _A is expressed in PVN-RVLM neurones, characterized its biophysical and pharmacological properties, and determined its role in shaping action potentials and firing discharge in these neurones. Patch-clamp recordings obtained from retrogradely labelled, PVN-RVLM neurones indicate that a 4-AP sensitive, TEA insensitive current, with biophysical properties consistent with I _A, is present in these neurones. Pharmacological blockade of I _A depolarized resting V _m and prolonged Na⁺ action potential duration, by increasing its width and by slowing down its decay time course. Interestingly, blockade of I _A either increased or decreased the firing activity of PVN-RVLM neurones, supporting the presence of subsets of PVN-RVLM neurones differentially modulated by I _A. In all cases, the effects of I _A on firing activity were prevented by a broad spectrum Ca²⁺ channel blocker. Immunohistochemical studies suggest that I _A in PVN-RVLM neurons is mediated by Kv1.4 and/or Kv4.3 channel subunits. Overall, our results demonstrate the presence of I _A in PVN-RVLM neurones, which actively modulates their action potential waveform and firing activity. These studies support I _A as an important intrinsic mechanism controlling neuronal excitability in this central presympathetic neuronal population.     

A-type voltage-gated K+ currents influence firing properties of isolectin B4-positive but not isolectin B4-negative primary sensory neurons

Voltage-gated K+ channels (Kv) in primary sensory neurons are important for regulation of neuronal excitability. The dorsal root ganglion (DRG) neurons are heterogeneous, and the types of native Kv currents in different groups of nociceptive DRG neurons are not fully known. In this study, we determined the difference in the A-type Kv current and its influence on the firing properties between isolectin B4 (IB4)-positive and -negative DRG neurons. Whole cell voltage- and current-clamp recordings were performed on acutely dissociated small DRG neurons of rats. The total Kv current density was significantly higher in IB+-positive than that in IB(4)-negative neurons. Also, 4-aminopyridine (4-AP) produced a significantly greater reduction in Kv currents in IB4-positive than in IB4-negative neurons. In contrast, IB4-negative neurons exhibited a larger proportion of tetraethylammonium-sensitive Kv currents. Furthermore, IB4-positive neurons showed a longer latency of firing and required a significantly larger amount of current injection to evoke action potentials. 4-AP significantly decreased the latency of firing and increased the firing frequency in IB4-positive but not in IB4-negative neurons. Additionally, IB4-positive neurons are immunoreactive to Kv1.4 but not to Kv1.1 and Kv1.2 subunits. Collectively, this study provides new information that 4-AP-sensitive A-type Kv currents are mainly present in IB4-positive DRG neurons and preferentially dampen the initiation of action potentials of this subpopulation of nociceptors. The difference in the density of A-type Kv currents contributes to the distinct electrophysiological properties of IB4-positive and -negative DRG neurons.     

Neuronal responses to transient hypoglycaemia in the dorsal vagal complex of the rat brainstem

Several regions of the mammalian brain contain glucosensing neurones. In vivo studies have suggested that those located in the hypothalamus and lower brainstem are involved in glucoprivic feeding and homeostatic control of blood glucose. We have identified and characterized hypoglycaemia-sensitive neurones in the dorsal vagal complex of the brainstem using in situ hybridization, single-cell RT-PCR and whole-cell patch-clamp recordings from rat brainstem slices. Approximately 80% of neurones did not respond to hypoglycaemia (changing artificial cerebrospinal fluid (ACSF) glucose from 10 m m to 0 m m ) within 5 min (non-responsive: NR). Another 10% depolarized within 155 ± 31 s (mean ± s.e.m. ) of glucose removal (glucose-inhibited: GI), and the remaining neurones hyperpolarized within 53 ± 7 s (glucose-excited: GE). The hyperpolarization was reversed by the K_ATP channel blocker tolbutamide. Single-cell RT-PCR revealed that GI and GE, but not NR, cells expressed glucokinase (GLK). In contrast, SUR1, a K_ATP channel subunit, was expressed in GE and some NR cells. In situ hybridization with biotin-labelled riboprobes in the dorsal vagal complex revealed ubiquitous expression of SUR1, and widespread, but sparse, expression of GLK. Identification of astrocytes using a GFAP (glial fibrillary acidic protein) antibody showed that GLK and GFAP were not colocalized. In summary, we have demonstrated that GI and GE neurones exist in the brainstem and that GLK is essential for their function. It seems likely that GE neurones work in a way analogous to pancreatic β-cells in that they require both GLK and K_ATP channels.     

Cannabinoids suppress synaptic input to neurones of the rat dorsal motor nucleus of the vagus nerve

Cannabinoids bind central type 1 receptors (CB1R) and modify autonomic functions, including feeding and anti-emetic behaviours, when administered peripherally or into the dorsal vagal complex. Western blots and immunohistochemistry indicated the expression of CB1R in the rat dorsal vagal complex, and tissue polymerase chain reaction confirmed that CB1R message was made within the region. To identify a cellular substrate for the central autonomic effects of cannabinoids, whole-cell patch-clamp recordings were made in brainstem slices to determine the effects of CB1R activation on synaptic transmission to neurones of the dorsal motor nucleus of the vagus (DMV). A subset of these neurones was identified as gastric related after being labelled retrogradely from the stomach. The CB1R agonists WIN55,212-2 and anandamide decreased the frequency of spontaneous excitatory or inhibitory postsynaptic currents in a concentration-related fashion, an effect that persisted in the presence of tetrodotoxin. Paired pulse ratios of electrically evoked postsynaptic currents were also increased by WIN55,212-2. The effects of  WIN55,212-2 were sensitive to the selective CB1R antagonist AM251. Cannabinoid agonist effects on synaptic input originating from neurones in the nucleus tractus solitarius (NTS) were determined by evoking activity in the NTS with local glutamate application. Excitatory and inhibitory synaptic inputs arising from the NTS were attenuated by WIN55,212-2. Our results indicate that cannabinoids inhibit transfer of synaptic information to the DMV, including that arising from the NTS, in part by acting at receptors located on presynaptic terminals contacting DMV neurones. Inhibition of synaptic input to DMV neurones is likely to contribute to the suppression of visceral motor responses by cannabinoids.     

Effects of hypothalamic stimulation on activity of dorsomedial medulla neurons that respond to subdiaphragmatic vagal stimulation

1. Effects of hypothalamic stimulation on activity of dorsomedial medulla neurons that responded to subdiaphragmatic vagal stimulation were investigated in urethan-anesthetized rats. 2. Extracellular recordings were made from 231 neurons in the nucleus of the tractus solitarius (NTS) that fired repetitively in response to single-pulse subdiaphragmatic vagal stimulation and from 320 neurons in the dorsal motor nucleus of the vagal nerve (DMV) that responded antidromically to subdiaphragmatic vagal stimulation. The mean latencies of responses to subdiaphragmatic vagal stimulation were 90.3 +/- 17.1 ms (mean +/- SD) for NTS neurons, and 90.8 +/- 11.2 ms for DMV neurons. This indicated that both afferent and efferent subdiaphragmatic vagal fibers were thin and unmyelinated and had a conduction velocity of approximately 1 m/s. 3. In extracellular recordings from 320 DMV neurons, marked inhibition preceded the antidromic response and subdiaphragmatic vagal stimulation evoked orthodromic spikes in only a few neurons. 4. Intracellular recordings from 66 DMV neurons revealed inhibitory postsynaptic potentials (IPSPs) before the antidromic responses. These IPSPs suppressed spontaneous firing and prevented excitatory postsynaptic potentials (EPSPs) from generating action potentials. 5. Stimulation in all hypothalamic loci studied, the ventromedial hypothalamic nucleus (VMH), the lateral hypothalamic area (LHA), and the paraventricular nucleus (PVN), induced responses with similar characteristics of excitation alone or excitation followed by inhibition in most NTS and DMV neurons. 6. No reciprocal effect of VMH and LHA stimulation was observed on NTS and DMV neurons. 7. Intracellular recordings from DMV neurons revealed monosynaptic EPSPs in response to stimulation of the VMH, the LHA, and the PVN. 8. PVN stimulation evoked significantly more responses in NTS and DMV neurons than VMH stimulation and more responses in DMV neurons than LHA stimulation. This suggests a difference in the number of connections between each hypothalamic site and the dorsomedial medulla. 9. The same dorsomedial medulla neurons were tested with VMH and LHA stimulation. The respective mean latencies of the antidromic and the orthodromic NTS neuron responses were 37.3 +/- 3.2 and 39.6 +/- 12.9 ms for VMH stimulation and 29.8 +/- 5.3 and 31.8 +/- 8.7 ms for LHA stimulation. The mean latencies of the orthodromic DMV neuron responses were 39.4 +/- 8.3 ms for VMH stimulation and 31.1 +/- 5.2 ms for LHA stimulation. The estimated conduction velocity from the VMH to the dorsomedial medulla was approximately 0.25 m/s and from the LHA it was approximately 0.33 m/s, which was significantly faster.(ABSTRACT TRUNCATED AT 400 WORDS)     

Function of specific K(+) channels in sustained high-frequency firing of fast-spiking neocortical interneurons

Fast-spiking GABAergic interneurons of the neocortex and hippocampus fire high-frequency trains of brief action potentials with little spike-frequency adaptation. How these striking properties arise is unclear, although recent evidence suggests K(+) channels containing Kv3.1-Kv3.2 proteins play an important role. We investigated the role of these channels in the firing properties of fast-spiking neocortical interneurons from mouse somatosensory cortex using a pharmacological and modeling approach. Low tetraethylammonium (TEA) concentrations (相似文献   

Differential expression of Kv3.1b and Kv3.2 potassium channel subunits in interneurons of the basolateral amygdala

The expression of Kv3.1 and Kv3.2 voltage-gated potassium channel subunits appears to be critical for high-frequency firing of many neuronal populations. In the cortex these subunits are mainly associated with fast-firing GABAergic interneurons containing parvalbumin or somatostatin. Since the basolateral nuclear complex of the amygdala contains similar interneurons, it is of interest to determine if these potassium channel subunits are expressed in these same interneuronal subpopulations. To investigate this issue, peroxidase and dual-labeling fluorescence immunohistochemistry combined with confocal laser scanning microscopy was used to determine which interneuronal subpopulations in the basolateral nuclear complex of the rat amygdala express Kv3.1b and Kv3.2 subunits. Antibodies to parvalbumin, somatostatin, calretinin, and cholecystokinin were used to label separate subsets of basolateral amygdalar interneurons. Examination of immunoperoxidase preparations suggested that the expression of both channels was restricted to nonpyramidal interneurons in the basolateral amygdala. Somata and proximal dendrites were intensely-stained, and axon terminals arising from presumptive basket cells and chandelier cells were lightly stained. Immunofluorescence observations revealed that parvalbumin+ neurons were the main interneuronal subpopulation expressing the Kv3.1b potassium channel subunit in the basolateral amygdala. More than 92-96% of parvalbumin+ neurons were Kv3.1b+, depending on the nucleus. These parvalbumin+/Kv3.1b+ double-labeled cells constituted 90-99% of all Kv3.1b+ neurons. Parvalbumin+ neurons were also the main interneuronal subpopulation expressing the Kv3.2 potassium channel subunit. More than 67-78% of parvalbumin+ neurons were Kv3.2+, depending on the nucleus. However, these parvalbumin+/Kv3.2+ double-labeled cells constituted only 71-81% of all Kv3.2+ neurons. Most of the remaining neurons with significant levels of the Kv3.2 subunit were somatostatin+ interneurons. These Kv3.2-containing somatostatin+ interneurons constituted 27-50% of the somatostatin+ population, depending on the nucleus in question. These data suggest that both fast-firing and burst-firing parvalbumin+ interneurons in the basolateral amygdala express the Kv3.1b subunit. The significance of Kv3.2 expression in some parvalbumin+ and somatostatin+ interneurons remains to be determined.     

Subcellular localization of the voltage-gated potassium channels Kv3.1b and Kv3.3 in the cerebellar dentate nucleus of glutamic acid decarboxylase 67-green fluorescent protein transgenic mice

Deep cerebellar dentate nuclei are in a key position to control motor planning as a result of an integration of cerebropontine inputs and hemispheric Purkinje neurons signals, and their influence through synaptic outputs onto extracerebellar hubs. GABAergic dentate neurons exhibit broader action potentials and slower afterhyperpolarization than non-GABAergic (presumably glutamatergic) neurons. Specific potassium channels may be involved in these distinct firing profiles, particularly, Kv3.1 and Kv3.3 subunits which rapidly activate at relatively positive potentials to support the generation of fast action potentials. To investigate the subcellular localization of Kv3.1b and Kv3.3 in GAD- and GAD+ dentate neurons of glutamic acid decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice a preembedding immunocytochemical method for electron microscopy was used. Kv3.1b and Kv3.3 were in membranes of cell somata, dendrites, axons and synaptic terminals of both GAD- and GAD+ dentate neurons. The vast majority of GAD- somatodendritic membrane segments domains labeled for Kv3.1b and Kv3.3 (96.1% and 84.7%, respectively) whereas 56.2% and 69.8% of GAD- axonal membrane segments were immunopositive for these subunits. Furthermore, density of Kv3.1b immunoparticles was much higher in GAD- somatodendritic than axonal domains. As to GAD+ neurons, only 70.6% and 50% of somatodendritic membrane segments, and 53.3% and 59.5% of axonal membranes exhibited Kv3.1b and Kv3.3 labeling, respectively. In contrast to GAD- cells, GAD+ cells exhibited a higher density labeling for both Kv3 subunits at their axonal than at their somatodendritic membranes. Taken together, Kv3.1b and Kv3.3 potassium subunits are expressed in both GAD- and GAD+ cells, albeit at different densities and distribution. They likely contribute to the distinct biophysical properties of both GAD- and GAD+ neurons in the dentate nucleus.     

Initial segment Kv2.2 channels mediate a slow delayed rectifier and maintain high frequency action potential firing in medial nucleus of the trapezoid body neurons

The medial nucleus of the trapezoid body (MNTB) is specialized for high frequency firing by expression of Kv3 channels, which minimize action potential (AP) duration, and Kv1 channels, which suppress multiple AP firing, during each calyceal giant EPSC. However, the outward K⁺ current in MNTB neurons is dominated by another unidentified delayed rectifier. It has slow kinetics and a peak conductance of ∼37 nS; it is half-activated at −9.2 ± 2.1 mV and half-inactivated at −35.9 ± 1.5 mV. It is blocked by several non-specific potassium channel antagonists including quinine (100 μ m ) and high concentrations of extracellular tetraethylammonium (TEA; IC₅₀= 11.8 m m ), but no specific antagonists were found. These characteristics are similar to recombinant Kv2-mediated currents. Quantitative RT-PCR showed that Kv2.2 mRNA was much more prevalent than Kv2.1 in the MNTB. A Kv2.2 antibody showed specific staining and Western blots confirmed that it recognized a protein ∼110 kDa which was absent in brainstem tissue from a Kv2.2 knockout mouse. Confocal imaging showed that Kv2.2 was highly expressed in axon initial segments of MNTB neurons. In the absence of a specific antagonist, Hodgkin–Huxley modelling of voltage-gated conductances showed that Kv2.2 has a minor role during single APs (due to its slow activation) but assists recovery of voltage-gated sodium channels (Nav) from inactivation by hyperpolarizing interspike potentials during repetitive AP firing. Current-clamp recordings during high frequency firing and characterization of Nav inactivation confirmed this hypothesis. We conclude that Kv2.2-containing channels have a distinctive initial segment location and crucial function in maintaining AP amplitude by regulating the interspike potential during high frequency firing.     

Rapid inhibition of neural excitability in the nucleus tractus solitarii by leptin: implications for ingestive behaviour

The fat-derived peptide leptin regulates cellular activity in areas of the CNS related to feeding, and application of leptin to the fourth ventricle or the nucleus tractus solitarii (NTS) inhibits food intake and weight gain. The hypothesis that leptin modulates cellular activity in the NTS was tested using whole-cell patch-clamp recordings in brainstem slices. Leptin caused a rapid membrane hyperpolarization in 58% of rat NTS neurones, including neurones receiving tractus solitarius input (i.e. viscerosensory) and those involved in regulating output to the stomach, identified after gastric inoculation with a transneuronal retrograde viral label. The hyperpolarization was accompanied by a decrease in input resistance and cellular responsiveness, reversed near the K⁺ equilibrium potential, and was prevented by intracellular Cs⁺. Perfusion of tolbutamide (200 μ m ) or wortmannin (100–200 n m ) prevented the hyperpolarization, indicating activation of an ATP-sensitive K⁺ channel via a PI3 kinase-dependent mechanism. Constant latency tractus solitarius-evoked EPSCs were decreased in amplitude by leptin, and the paired-pulse ratio was increased, suggesting effects on evoked EPSCs involved activation of receptors on vagal afferent terminals. Leptin reduced the frequency of spontaneous and miniature EPSCs, whereas IPSCs were largely unaffected. Leptin's effects were observed in neurones from lean, but not obese, Zucker rats. Neurones that expressed enhanced green fluorescent protein (EGFP) in a subpopulation of putative GABAergic neurones in transgenic mice did not respond to leptin, whereas unlabelled murine neurones responded similarly to rat neurones. Leptin therefore directly and rapidly suppresses activity of excitatory NTS neurones likely to be involved in viscerosensory integration and/or premotor control of the stomach.     

Kv3-like potassium channels are required for sustained high-frequency firing in basal ganglia output neurons

The GABA projection neurons in the substantial nigra pars reticulata (SNr) are key output neurons of the basal ganglia motor control circuit. These neurons fire sustained high-frequency, short-duration spikes that provide a tonic inhibition to their targets and are critical to movement control. We hypothesized that a robust voltage-activated K(+) conductance that activates quickly and resists inactivation is essential to the remarkable fast-spiking capability in these neurons. Semi-quantitative RT-PCR (qRT-PCR) analysis on laser capture-microdissected nigral neurons indicated that mRNAs for Kv3.1 and Kv3.4, two key subunits for forming high activation threshold, fast-activating, slow-inactivating, 1 mM tetraethylammonium (TEA)-sensitive, fast delayed rectifier (I(DR-fast)) type Kv channels, are more abundant in fast-spiking SNr GABA neurons than in slow-spiking nigral dopamine neurons. Nucleated patch clamp recordings showed that SNr GABA neurons have a strong Kv3-like I(DR-fast) current sensitive to 1 mM TEA that activates quickly at depolarized membrane potentials and is resistant to inactivation. I(DR-fast) is smaller in nigral dopamine neurons. Pharmacological blockade of I(DR-fast) by 1 mM TEA impaired the high-frequency firing capability in SNr GABA neurons. Taken together, these results indicate that Kv3-like channels mediating fast-activating, inactivation-resistant I(DR-fast) current are critical to the sustained high-frequency firing in SNr GABA projection neurons and hence movement control.     

Persistence of the cardio-inhibitory response to brain stem ischaemia after destruction of the area postrema and the dorsal vagal nuclei

1. In anaesthetized cats, in which the carotid arterial bifurcation had been denervated and the spinal cord transected at the cervical level, reversible bradycardia mediated by the vagus nerves was elicited by temporary arrest of the cranial circulation. Methoxamine was infused intravenously to maintain peripheral vascular resistance, and artificial ventilation was given to avert systemic asphyxia.2. The bradycardia persisted in cats subjected to one or more of the following acute surgical procedures: left vagotomy, mid-collicular decerebration, decerebellation, bulbar transections at the acoustic striae and inferior fovea, and destruction of the area postrema and the underlying dorsal vagal nuclei. Ischaemia-induced bradycardia was invariably abolished after bilateral vagotomy or the administration of atropine.3. Bradycardia could not be elicited by electrical stimulation of the dorsal vagal nuclei, but was evoked by stimulation of deep structures in the vicinity of the nucleus ambiguus even after destruction of the dorsal vagal nuclei.4. Simultaneous application of ischaemia and electrical stimulation of the medullary cardio-decelerator locus produced convergent occlusion of the vagal response. The effect of ischaemia was inhibited by stimulation of a neighbouring region in the medial reticular formation. These interactions indicate that an interneuronal link is involved in the mechanism of ischaemia-induced bradycardia.5. It is concluded that the cardio-decelerator response to ischaemia is initiated upstream to the primary efferent vagal motor neurones and that the cardio-inhibitory fibres do not originate in the dorsal vagal nuclei.     

Resilient RTN fast spiking in Kv3.1 null mice suggests redundancy in the action potential repolarization mechanism

Fast spiking (FS), GABAergic neurons of the reticular thalamic nucleus (RTN) are capable of firing high-frequency trains of brief action potentials, with little adaptation. Studies in recombinant systems have shown that high-voltage-activated K(+) channels containing the Kv3.1 and/or Kv3.2 subunits display biophysical properties that may contribute to the FS phenotype. Given that RTN expresses high levels of Kv3.1, with little or no Kv3.2, we tested whether this subunit was required for the fast action potential repolarization mechanism essential to the FS phenotype. Single- and multiple-action potentials were recorded using whole-cell current clamp in RTN neurons from brain slices of wild-type and Kv3.1-deficient mice. At 23 degrees C, action potentials recorded from homozygous Kv3.1 deficient mice (Kv3.1(-/-)) compared with their wild-type (Kv3.1(+/+)) counterparts had reduced amplitudes (-6%) and fast after-hyperpolarizations (-16%). At 34 degrees C, action potentials in Kv3.1(-/-) mice had increased duration (21%) due to a reduced rate of repolarization (-30%) when compared with wild-type controls. Action potential trains in Kv3.1(-/-) were associated with a significantly greater spike decrement and broadening and a diminished firing frequency versus injected current relationship (F/I) at 34 degrees C. There was no change in either spike count or maximum instantaneous frequency during low-threshold Ca(2+) bursts in Kv3.1(-/-) RTN neurons at either temperature tested. Our findings show that Kv3.1 is not solely responsible for fast spikes or high-frequency firing in RTN neurons. This suggests genetic redundancy in the system, possibly in the form of other Kv3 members, which may suffice to maintain the FS phenotype in RTN neurons in the absence of Kv3.1.     

Presynaptic Rat Kv1.2 Channels Suppress Synaptic Terminal Hyperexcitability Following Action Potential Invasion

Voltage-gated K⁺ channels activating close to resting membrane potentials are widely expressed and differentially located in axons, presynaptic terminals and cell bodies. There is extensive evidence for localisation of Kv1 subunits at many central synaptic terminals but few clues to their presynaptic function. We have used the calyx of Held to investigate the role of presynaptic Kv1 channels in the rat by selectively blocking Kv1.1 and Kv1.2 containing channels with dendrotoxin-K (DTX-K) and tityustoxin-Kα (TsTX-Kα) respectively. We show that Kv1.2 homomers are responsible for two-thirds of presynaptic low threshold current, whilst Kv1.1/Kv1.2 heteromers contribute the remaining current. These channels are located in the transition zone between the axon and synaptic terminal, contrasting with the high threshold K⁺ channel subunit Kv3.1 which is located on the synaptic terminal itself. Kv1 homomers were absent from bushy cell somata (from which the calyx axons arise); instead somatic low threshold channels consisted of heteromers containing Kv1.1, Kv1.2 and Kv1.6 subunits. Current-clamp recording from the calyx showed that each presynaptic action potential (AP) was followed by a depolarising after-potential (DAP) lasting around 50 ms. Kv1.1/Kv1.2 heteromers had little influence on terminal excitability, since DTX-K did not alter AP firing. However TsTX-Kα increased DAP amplitude, bringing the terminal closer to threshold for generating an additional AP. Paired pre- and postsynaptic recordings confirmed that this aberrant AP evoked an excitatory postsynaptic current (EPSC). We conclude that Kv1.2 channels have a general presynaptic function in suppressing terminal hyperexcitability during the depolarising after-potential.     

Neurones in the dorsal motor vagal nucleus of the cat with non-myelinated axons projecting to the heart and lungs

Extracellular recordings were made from ninety-four single motoneurones in the dorsal motor vagal nucleus of chloralose-anaesthetized or decerebrate cats. Fifty-five neurones had axons in cardiac vagal branches and thirty-nine had axons in pulmonary vagal branches; the conduction velocities of the axons were in the C fibre range, i.e. the axons were non-myelinated. The neurons exhibited little or no spontaneous activity. Excitatory and inhibitory synaptic inputs were demonstrated by electrical stimulation of the vagus nerve or its branches. Twenty-four neurones were tested by carotid sinus distension but only one was excited. Iontophoretic excitation of neurones projecting to cardiac vagal branches had no effect on heart rate. The properties of these neurones and their possible functions are discussed and contrasted with those of cardiac and pulmonary vagal motoneurones in the nucleus ambiguus.