Does modulation of the endocannabinoid system have potential therapeutic utility in cerebellar ataxia?

Cerebellar ataxias represent a spectrum of disorders which are, however, linked by common symptoms of motor incoordination and are typically associated with deficient in Purkinje cell firing activity and, often, degeneration. Cerebellar ataxias currently lack a curative agent. The endocannabinoid (eCB) system includes eCB compounds and their associated metabolic enzymes, together with cannabinoid receptors, predominantly the cannabinoid CB1 receptor (CB1R) in the cerebellum; activation of this system in the cerebellar cortex is associated with deficits in motor coordination characteristic of ataxia, effects which can be prevented by CB1R antagonists. Of further interest are various findings that CB1R deficits may also induce a progressive ataxic phenotype. Together these studies suggest that motor coordination is reliant on maintaining the correct balance in eCB system signalling. Recent work also demonstrates deficient cannabinoid signalling in the mouse ‘ducky2J’ model of ataxia. In light of these points, the potential mechanisms whereby cannabinoids may modulate the eCB system to ameliorate dysfunction associated with cerebellar ataxias are considered.

The du2J mouse model of ataxia and absence epilepsy has deficient cannabinoid CB1 receptor-mediated signalling

Key point summary • Cerebellar ataxias are progressive debilitating diseases with no known treatment and are associated with defective motor function and, in particular, abnormalities to Purkinje cells. • Mutant mice with deficits in Ca2+ channel auxiliary α2δ-2 subunits are used as models of cerebellar ataxia. • Our data in the du2J mouse model shows an association between the ataxic phenotype exhibited by homozygous du2J/du2J mice and increased irregularity of Purkinje cell firing. • We show that both heterozygous +/du2J and homozygous du2J/du2J mice completely lack the strong presynaptic modulation of neuronal firing by cannabinoid CB1 receptors which is exhibited by litter-matched control mice. • These results show that the du2J ataxia model is associated with deficits in CB1 receptor signalling in the cerebellar cortex, putatively linked with compromised Ca2+ channel activity due to reduced α2δ-2 subunit expression. Knowledge of such deficits may help design therapeutic agents to combat ataxias. Abstract Cerebellar ataxias are a group of progressive, debilitating diseases often associated with abnormal Purkinje cell (PC) firing and/or degeneration. Many animal models of cerebellar ataxia display abnormalities in Ca2+ channel function. The ‘ducky’ du2J mouse model of ataxia and absence epilepsy represents a clean knock-out of the auxiliary Ca2+ channel subunit, α2δ-2, and has been associated with deficient Ca2+ channel function in the cerebellar cortex. Here, we investigate effects of du2J mutation on PC layer (PCL) and granule cell (GC) layer (GCL) neuronal spiking activity and, also, inhibitory neurotransmission at interneurone-Purkinje cell(IN-PC) synapses. Increased neuronal firing irregularity was seen in the PCL and, to a less marked extent, in the GCL in du2J/du2J, but not +/du2J, mice; these data suggest that the ataxic phenotype is associated with lack of precision of PC firing, that may also impinge on GC activity and requires expression of two du2J alleles to manifest fully. du2J mutation had no clear effect on spontaneous inhibitory postsynaptic current (sIPSC) frequency at IN-PC synapses, but was associated with increased sIPSC amplitudes. du2J mutation ablated cannabinoid CB1 receptor (CB1R)-mediated modulation of spontaneous neuronal spike firing and CB1Rmediated presynaptic inhibition of synaptic transmission at IN-PC synapses in both +/du2J and du2J/du2J mutants; effects that occurred in the absence of changes in CB1R expression. These results demonstrate that the du2J ataxia model is associated with deficient CB1R signalling in the cerebellar cortex, putatively linked with compromised Ca2+ channel activity and the ataxic phenotype.

NG2 cells differentiate into astrocytes in cerebellar slices

NG2-glia are an abundant population of glial cells that have been considered by many to be oligodendrocyte progenitor cells (OPCs). However, growing evidence suggests that NG2-glia may also be capable of differentiating into astrocytes and neurons under certain conditions. Here, we have examined NG2-glia in cerebellar slices, using transgenic mice in which the astroglial marker glial specific protein (GFAP) drives expression of the reporter gene enhanced green fluorescent protein (EGFP). Immunolabelling for NG2 shows that NG2-glia and GFAP-EGFP astroglia are separate populations in most areas of the brain, although a substantial population of NG2-glia in the pons also express the GFAP-EGFP reporter. In the cerebellum, NG2-glia did not express EGFP, either at postnatal day (P)12 or P29-30. We developed an organotypic culture of P12 cerebellar slices that maintain cytoarchitectural integrity of Purkinje neurons and Bergmann glia. In these cultures, BrdU labelling indicates that the majority of NG2-glia enter the cell cycle within 2 days in vitro (DIV), suggesting that NG2-glia undergo a [`]reactive' response in cerebellar cultures. After 2 DIV NG2-glia began to express the astroglial reporter EGFP and in some cases the respective GFAP protein. However, NG2-glia did not acquire phenotypic markers of neural stem cells or neurons. The results suggest that NG2-glia are not lineage restricted OPCs and are a potential source of astrocytes in the cerebellum.

Evidence that GABA rho subunits contribute to functional ionotropic GABA receptors in mouse cerebellar Purkinje cells

Ionotropic gamma-amino butyric acid (GABA) receptors composed of heterogeneous molecular subunits are major mediators of inhibitory responses in the adult CNS. Here, we describe a novel ionotropic GABA receptor in mouse cerebellar Purkinje cells (PCs) using agents reported to have increased affinity for rho subunit-containing GABA(C) over other GABA receptors. Exogenous application of the GABA(C)-preferring agonist cis-4-aminocrotonic acid (CACA) evoked whole-cell currents in PCs, whilst equimolar concentrations of GABA evoked larger currents. CACA-evoked currents had a greater sensitivity to the selective GABA(C) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) than GABA-evoked currents. Focal application of agonists produced a differential response profile; CACA-evoked currents displayed a much more pronounced attenuation with increasing distance from the PC soma, displayed a slower time-to-peak and exhibited less desensitization than GABA-evoked currents. However, CACA-evoked currents were also completely blocked by bicuculline, a selective agent for GABA(A) receptors. Thus, we describe a population of ionotropic GABA receptors with a mixed GABA(A)/GABA(C) pharmacology. TPMPA reduced inhibitory synaptic transmission at interneurone-Purkinje cell (IN-PC) synapses, causing clear reductions in miniature inhibitory postsynaptic current (mIPSC) amplitude and frequency. Combined application of NO-711 (a selective GABA transporter subtype 1 (GAT-1) antagonist) and SNAP-5114 (a GAT-(2)/3/4 antagonist) induced a tonic GABA conductance in PCs; however, TPMPA had no effect on this current. Immunohistochemical studies suggest that rho subunits are expressed predominantly in PC soma and proximal dendritic compartments with a lower level of expression in more distal dendrites; this selective immunoreactivity contrasted with a more uniform distribution of GABA(A) alpha 1 subunits in PCs. Finally, co-immunoprecipitation studies suggest that rho subunits can form complexes with GABA(A) receptor alpha 1 subunits in the cerebellar cortex. Overall, these data suggest that rho subunits contribute to functional ionotropic receptors that mediate a component of phasic inhibitory GABAergic transmission at IN-PC synapses in the cerebellum.

Abnormal functional activation and maturation of fronto-striato-temporal and cerebellar regions during sustained attention in autism spectrum disorder

Objective Sustained attention problems are common in people with autism spectrum disorder (ASD) and may have significant implications for the diagnosis and management of ASD and associated comorbidities. Furthermore, ASD has been associated with atypical structural brain development. The authors used functional MRI to investigate the functional brain maturation of attention between childhood and adulthood in people with ASD. Method Using a parametrically modulated sustained attention/vigilance task, the authors examined brain activation and its linear correlation with age between childhood and adulthood in 46 healthy male adolescents and adults (ages 11–35 years) with ASD and 44 age- and IQ-matched typically developing comparison subjects. Results Relative to the comparison group, the ASD group had significantly poorer task performance and significantly lower activation in inferior prefrontal cortical, medial prefrontal cortical, striato-thalamic, and lateral cerebellar regions. A conjunction analysis of this analysis with group differences in brain-age correlations showed that the comparison group, but not the ASD group, had significantly progressively increased activation with age in these regions between childhood and adulthood, suggesting abnormal functional brain maturation in ASD. Several regions that showed both abnormal activation and functional maturation were associated with poorer task performance and clinical measures of ASD and inattention. Conclusions The results provide first evidence that abnormalities in sustained attention networks in individuals with ASD are associated with underlying abnormalities in the functional brain maturation of these networks between late childhood and adulthood.

Effects of Delta9-tetrahydrocannabivarin on [35S]GTPgammaS binding in mouse brain cerebellum and piriform cortex membranes

BACKGROUND AND PURPOSE: We have recently shown that the phytocannabinoid Delta9-tetrahydrocannabivarin (Delta9-THCV) and the CB1 receptor antagonist AM251 increase inhibitory neurotransmission in mouse cerebellum and also exhibit anticonvulsant activity in a rat piriform cortical (PC) model of epilepsy. Possible mechanisms underlying cannabinoid actions in the CNS include CB1 receptor antagonism (by displacing endocannabinergic tone) or inverse agonism at constitutively active CB1 receptors. Here, we investigate the mode of cannabinoid action in [35S]GTPgammaS binding assays. EXPERIMENTAL APPROACH: Effects of Delta9-THCV and AM251 were tested either alone or against WIN55,212-2-induced increases in [35S]GTPgammaS binding in mouse cerebellar and PC membranes. Effects on non-CB receptor expressing CHO-D2 cell membranes were also investigated. KEY RESULTS :Delta9-THCV and AM251 both acted as potent antagonists of WIN55,212-2-induced increases in [35S]GTPgammaS binding in cerebellar and PC membranes (Delta9-THCV: pA2=7.62 and 7.44 respectively; AM251: pA2=9.93 and 9.88 respectively). At micromolar concentrations, Delta9-THCV or AM251 alone caused significant decreases in [35S]GTPgammaS binding; Delta9-THCV caused larger decreases than AM251. When applied alone in CHO-D2 membranes, Delta9-THCV and AM251 also caused concentration-related decreases in G protein activity. CONCLUSIONS AND IMPLICATIONS: Delta9-THCV and AM251 act as CB1 receptors antagonists in the cerebellum and PC, with AM251 being more potent than Delta9-THCV in both brain regions. Individually, Delta9-THCV or AM251 exhibited similar potency at CB1 receptors in the cerebellum and the PC. At micromolar concentrations, Delta9-THCV and AM251 caused a non-CB receptor-mediated depression of basal [35S]GTPgammaS binding.

The phytocannabinoid Delta(9)-tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum

Background and purposeThe phytocannabinoid Delta(9)-tetrahydrocannabivarin (Delta(9)-THCV) has been reported to exhibit a diverse pharmacology; here, we investigate functional effects of Delta(9)-THCV, extracted from Cannabis sativa, using electrophysiological techniques to define its mechanism of action in the CNS.Experimental approachEffects of Delta(9)-THCV and synthetic cannabinoid agents on inhibitory neurotransmission at interneurone-Purkinje cell (IN-PC) synapses were correlated with effects on spontaneous PC output using single-cell and multi-electrode array (MEA) electrophysiological recordings respectively, in mouse cerebellar brain slices in vitro.Key resultsThe cannabinoid receptor agonist WIN 55,212-2 (WIN55) decreased miniature inhibitory postsynaptic current (mIPSC) frequency at IN-PC synapses. WIN55-induced inhibition was reversed by Delta(9)-THCV, and also by the CB(1) receptor antagonist AM251; Delta(9)-THCV or AM251 acted to increase mIPSC frequency beyond basal values. When applied alone, Delta(9)-THCV, AM251 or rimonabant increased mIPSC frequency. Pre-incubation with Delta(9)-THCV blocked WIN55-induced inhibition. In MEA recordings, WIN55 increased PC spike firing rate; Delta(9)-THCV and AM251 acted in the opposite direction to decrease spike firing. The effects of Delta(9)-THCV and WIN55 were attenuated by the GABA(A) receptor antagonist bicuculline methiodide.Conclusions and implicationsWe show for the first time that Delta(9)-THCV acts as a functional CB(1) receptor antagonist in the CNS to modulate inhibitory neurotransmission at IN-PC synapses and spontaneous PC output. Delta(9)-THCV- and AM251-induced increases in mIPSC frequency beyond basal levels were consistent with basal CB(1) receptor activity. WIN55-induced increases in PC spike firing rate were consistent with synaptic disinhibition; whilst Delta(9)-THCV- and AM251-induced decreases in spike firing suggest a mechanism of PC inhibition.British Journal of Pharmacology advance online publication, 3 March 2008; doi:10.1038/bjp.2008.57.

Impulsiveness as a timing disturbance: neurocognitive abnormalities in attention-deficit hyperactivity disorder during temporal processes and normalization with methylphenidate.

We argue that impulsiveness is characterized by compromised timing functions such as premature motor timing, decreased tolerance to delays, poor temporal foresight and steeper temporal discounting. A model illustration for the association between impulsiveness and timing deficits is the impulsiveness disorder of attention-deficit hyperactivity disorder (ADHD). Children with ADHD have deficits in timing processes of several temporal domains and the neural substrates of these compromised timing functions are strikingly similar to the neuropathology of ADHD. We review our published and present novel functional magnetic resonance imaging data to demonstrate that ADHD children show dysfunctions in key timing regions of prefrontal, cingulate, striatal and cerebellar location during temporal processes of several time domains including time discrimination of milliseconds, motor timing to seconds and temporal discounting of longer time intervals. Given that impulsiveness, timing abnormalities and more specifically ADHD have been related to dopamine dysregulation, we tested for and demonstrated a normalization effect of all brain dysfunctions in ADHD children during time discrimination with the dopamine agonist and treatment of choice, methylphenidate. This review together with the new empirical findings demonstrates that neurocognitive dysfunctions in temporal processes are crucial to the impulsiveness disorder of ADHD and provides first evidence for normalization with a dopamine reuptake inhibitor.

Inhibition of cellular proliferation by the genistein metabolite 5,7,3',4'-tetrahydroxyisoflavone is mediated by DNA damage and activation of the ATR signalling pathway

The cellular actions of genistein, and its in vivo metabolites, are believed to mediate the decreased risk of breast cancer associated with high soy consumption. The genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone (THIF), induced G2-M cell cycle arrest in T47D tumorigenic breast epithelial cells via a mechanism involving the activation of ataxia telangiectasia and Rad3-related kinase (ATR) via its phosphorylation at Ser(428). This activation of ATR appeared to result from THIF-induced increases in intracellular oxidative stress, a depletion of cellular GSH and an increase in DNA strand breakage. THIF treatment also led to an inhibition of cdc2, which was accompanied by the phosphorylation of both p53 (Ser(15)) and Chk1 (Ser(296)) and the de-activation of cdc25C phosphatase. We suggest the anti-proliferative actions of THIF may be mediated by initial oxidative DNA damage, activation of ATR and downstream regulation of the p53 and Chk1 pathways leading to cell cycle arrest in G2-M. This may represent one mechanism by which genistein exerts its cellular activity in vivo. (c) 2007 Elsevier Inc. All rights reserved.

The phytocannabinoid Δ9-tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum

Background and purpose: The phytocannabinoid Delta(9)-tetrahydrocannabivarin (Delta(9)-THCV) has been reported to exhibit a diverse pharmacology; here, we investigate functional effects of Delta(9)-THCV, extracted from Cannabis sativa, using electrophysiological techniques to define its mechanism of action in the CNS. Experimental approach: Effects of Delta(9)-THCV and synthetic cannabinoid agents on inhibitory neurotransmission at interneurone-Purkinje cell (IN-PC) synapses were correlated with effects on spontaneous PC output using single-cell and multi-electrode array (MEA) electrophysiological recordings respectively, in mouse cerebellar brain slices in vitro. Key results: The cannabinoid receptor agonist WIN 55,212-2 (WIN55) decreased miniature inhibitory postsynaptic current (mIPSC) frequency at IN-PC synapses. WIN55-induced inhibition was reversed by Delta(9)-THCV, and also by the CB1 receptor antagonist AM251; Delta(9)-THCV or AM251 acted to increase mIPSC frequency beyond basal values. When applied alone, Delta(9)-THCV, AM251 or rimonabant increased mIPSC frequency. Pre-incubation with Delta(9)-THCV blocked WIN55-induced inhibition. In MEA recordings, WIN55 increased PC spike firing rate; Delta(9)-THCV and AM251 acted in the opposite direction to decrease spike firing. The effects of Delta(9)-THCV and WIN55 were attenuated by the GABA(A) receptor antagonist bicuculline methiodide. Conclusions and implications: We show for the first time that Delta(9)-THCV acts as a functional CB1 receptor antagonist in the CNS to modulate inhibitory neurotransmission at IN-PC synapses and spontaneous PC output. Delta(9)-THCV- and AM251-induced increases in mIPSC frequency beyond basal levels were consistent with basal CB1 receptor activity. WIN55-induced increases in PC spike firing rate were consistent with synaptic disinhibition; whilst Delta(9)-THCV-and AM251-induced decreases in spike firing suggest a mechanism of PC inhibition.

Generation, description and storage of dendritic morphology data

It is generally assumed that the variability of neuronal morphology has an important effect on both the connectivity and the activity of the nervous system, but this effect has not been thoroughly investigated. Neuroanatomical archives represent a crucial tool to explore structure–function relationships in the brain. We are developing computational tools to describe, generate, store and render large sets of three–dimensional neuronal structures in a format that is compact, quantitative, accurate and readily accessible to the neuroscientist. Single–cell neuroanatomy can be characterized quantitatively at several levels. In computer–aided neuronal tracing files, a dendritic tree is described as a series of cylinders, each represented by diameter, spatial coordinates and the connectivity to other cylinders in the tree. This ‘Cartesian’ description constitutes a completely accurate mapping of dendritic morphology but it bears little intuitive information for the neuroscientist. In contrast, a classical neuroanatomical analysis characterizes neuronal dendrites on the basis of the statistical distributions of morphological parameters, e.g. maximum branching order or bifurcation asymmetry. This description is intuitively more accessible, but it only yields information on the collective anatomy of a group of dendrites, i.e. it is not complete enough to provide a precise ‘blueprint’ of the original data. We are adopting a third, intermediate level of description, which consists of the algorithmic generation of neuronal structures within a certain morphological class based on a set of ‘fundamental’, measured parameters. This description is as intuitive as a classical neuroanatomical analysis (parameters have an intuitive interpretation), and as complete as a Cartesian file (the algorithms generate and display complete neurons). The advantages of the algorithmic description of neuronal structure are immense. If an algorithm can measure the values of a handful of parameters from an experimental database and generate virtual neurons whose anatomy is statistically indistinguishable from that of their real counterparts, a great deal of data compression and amplification can be achieved. Data compression results from the quantitative and complete description of thousands of neurons with a handful of statistical distributions of parameters. Data amplification is possible because, from a set of experimental neurons, many more virtual analogues can be generated. This approach could allow one, in principle, to create and store a neuroanatomical database containing data for an entire human brain in a personal computer. We are using two programs, L–NEURON and ARBORVITAE, to investigate systematically the potential of several different algorithms for the generation of virtual neurons. Using these programs, we have generated anatomically plausible virtual neurons for several morphological classes, including guinea pig cerebellar Purkinje cells and cat spinal cord motor neurons. These virtual neurons are stored in an online electronic archive of dendritic morphology. This process highlights the potential and the limitations of the ‘computational neuroanatomy’ strategy for neuroscience databases.

Effects of the Allosteric Antagonist 1-(4-Chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea (PSNCBAM-1) on CB1 Receptor Modulation in the Cerebellum

PSNCBAM-1 has recently been described as a cannabinoid CB1 receptor allosteric antagonist associated with hypophagic effects in vivo; however, PSNCBAM-1 effects on CB1 ligand-mediated modulation of neuronal excitability remain unknown. Here, we investigate PSNCBAM-1 actions on CB1 receptor-stimulated [35S]GTPγS binding in cerebellar membranes and on CB1 ligand modulation of presynaptic CB1 receptors at inhibitory interneurone-Purkinje cell (IN-PC) synapses in the cerebellum using whole-cell electrophysiology. PSNCBAM-1 caused non-competitive antagonism in [35S]GTPγS binding studies, with higher potency against the CB receptor agonist CP55940 than for WIN55,212-2 (WIN55). In electrophysiological studies, WIN55 and CP55940 reduced miniature inhibitory postsynaptic currents (mIPSCs) frequency, but not amplitude. PSNCBAM-1 application alone had no effect on mIPSCs; however, PSNCBAM-1 pre-treatment revealed agonist-dependent functional antagonism, abolishing CP55940-induced reductions in mIPSC frequency, but having no clear effect on WIN55 actions. The CB1 antagonist/inverse agonist AM251 increased mIPSC frequency beyond control, this effect was reversed by PSNCBAM-1. PSNCBAM-1 pre-treatment also attenuated AM251 effects. Thus, PSNCBAM-1 reduced CB1 receptor ligand functional efficacy in the cerebellum. The differential effect of PSNCBAM-1 on CP55940 versus WIN55 actions in [35S]GTPγS binding and electrophysiological studies and the attenuation of AM251 effects are consistent with the ligand-dependency associated with allosteric modulation. These data provide the first description of functional PSNCBAM-1 allosteric antagonist effects on neuronal excitability in the mammalian CNS. PSNCBAM-1 allosteric antagonism may provide viable therapeutic alternatives to orthosteric CB1 antagonists/inverse agonists in the treatment of CNS disease.

A comparative study of multilayered and single layered neural network based predictive controllers

The authors compare the performance of two types of controllers one based on the multilayered network and the other based on the single layered CMAC network (cerebellar model articulator controller). The neurons (information processing units) in the multi-layered network use Gaussian activation functions. The control scheme which is considered is a predictive control algorithm, along the lines used by Willis et al. (1991), Kambhampati and Warwick (1991). The process selected as a test bed is a continuous stirred tank reactor. The reaction taking place is an irreversible exothermic reaction in a constant volume reactor cooled by a single coolant stream. This reactor is a simplified version of the first tank in the two tank system given by Henson and Seborg (1989).

Disorder-specific functional abnormalities during sustained attention in youth with Attention Deficit Hyperactivity Disorder (ADHD) and with Autism

Attention Deficit Hyperactivity Disorder (ADHD) and Autism Spectrum Disorder (ASD) are often comorbid and share behavioural-cognitive abnormalities in sustained attention. A key question is whether this shared cognitive phenotype is based on common or different underlying pathophysiologies. To elucidate this question, we compared 20 boys with ADHD to 20 age and IQ matched ASD and 20 healthy boys using functional magnetic resonance imaging (fMRI) during a parametrically modulated vigilance task with a progressively increasing load of sustained attention. ADHD and ASD boys had significantly reduced activation relative to controls in bilateral striato–thalamic regions, left dorsolateral prefrontal cortex (DLPFC) and superior parietal cortex. Both groups also displayed significantly increased precuneus activation relative to controls. Precuneus was negatively correlated with the DLPFC activation, and progressively more deactivated with increasing attention load in controls, but not patients, suggesting problems with deactivation of a task-related default mode network in both disorders. However, left DLPFC underactivation was significantly more pronounced in ADHD relative to ASD boys, which furthermore was associated with sustained performance measures that were only impaired in ADHD patients. ASD boys, on the other hand, had disorder-specific enhanced cerebellar activation relative to both ADHD and control boys, presumably reflecting compensation. The findings show that ADHD and ASD boys have both shared and disorder-specific abnormalities in brain function during sustained attention. Shared deficits were in fronto–striato–parietal activation and default mode suppression. Differences were a more severe DLPFC dysfunction in ADHD and a disorder-specific fronto–striato–cerebellar dysregulation in ASD.

Localization and function of the Kv3.1b subunit in the rat medulla oblongata: focus on the nucleus tractus solitarii

The voltage-gated potassium channel subunit Kv3.1 confers fast firing characteristics to neurones. Kv3.1b subunit immunoreactivity (Kv3.1b-IR) was widespread throughout the medulla oblongata, with labelled neurones in the gracile, cuneate and spinal trigeminal nuclei. In the nucleus of the solitary tract (NTS), Kv3.1b-IR neurones were predominantly located close to the tractus solitarius (TS) and could be GABAergic or glutamatergic. Ultrastructurally, Kv3.1b-IR was detected in NTS terminals, some of which were vagal afferents. Whole-cell current-clamp recordings from neurones near the TS revealed electrophysiological characteristics consistent with the presence of Kv3.1b subunits: short duration action potentials (4.2 +/- 1.4 ms) and high firing frequencies (68.9 +/- 5.3 Hz), both sensitive to application of TEA (0.5 mm) and 4-aminopyridine (4-AP; 30 mum). Intracellular dialysis of an anti-Kv3.1b antibody mimicked and occluded the effects of TEA and 4-AP in NTS and dorsal column nuclei neurones, but not in dorsal vagal nucleus or cerebellar Purkinje cells (which express other Kv3 subunits, but not Kv3.1b). Voltage-clamp recordings from outside-out patches from NTS neurones revealed an outward K(+) current with the basic characteristics of that carried by Kv3 channels. In NTS neurones, electrical stimulation of the TS evoked EPSPs and IPSPs, and TEA and 4-AP increased the average amplitude and decreased the paired pulse ratio, consistent with a presynaptic site of action. Synaptic inputs evoked by stimulation of a region lacking Kv3.1b-IR neurones were not affected, correlating the presence of Kv3.1b in the TS with the pharmacological effects.