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Toluene is a psychoactive chemical found in many household products including adhesives and thinners. Inhalation of these vapors can cause euphoria and impairments in motor control and neurological functioning. Misuse and abuse of toluene is most common in children, which may in part be due to an age-dependent neurobehavioral sensitivity to toluene. Here we assessed the effects of acute binge-like toluene inhalations (15 or 30 min; ∼5,000 ppm) on tasks that examine locomotion, exploration, balance, gait, and neurological functioning for adolescent (1 month), young adult (2–3 months), adult (5–6 months), and older adult (10–12 months) rats. Both motor and neurological functions were impaired following acute toluene inhalation at all ages. However, only the duration to recover from deficits in motor functions differed among age groups, with adolescent and young adult rats requiring notably longer recovery times than older rats. Our results are suggestive of an age-dependent vulnerability to the intoxicating effects of toluene. © 2013 Wiley Periodicals, Inc. Dev Psychobiol 56: 657–673, 2014.

Binge inhalation of toluene, a psychoactive chemical found in many household and industrial products, leads acutely to intoxication with comorbid impairments in motor function and cognitive abilities that appear to recover quickly. Recent evidence, however, indicates that the administration of toluene results in marked changes in neurons of the medial prefrontal cortex that persist for relatively longer periods compared with other brain regions. To elucidate the potential toluene-induced (∼5000 ppm) cognitive dysfunctions that continue following the recovery of locomotor abilities, rats were entered into a series of water maze tasks. Following acute toluene intoxication, rats were initially severely impaired in their swimming ability and in their ability to learn and perform a visible platform task. After about 20 min, swimming behavior mostly returned to normal, although cognitive impairments were still evident. Whereas rats with extensive toluene-free training in the maze were able to show normal spatial recall following toluene intoxication, the same acute toluene exposure severely impaired reversal learning, with the rats showing a marked perseveration for the previously learned platform location. Our results indicate that toluene inhalation results in specific cognitive dysfunctions that outlast major impairments in motor abilities, which may be related to impairments in medial prefrontal cortex activity.

Toluene, a psychoactive volatile solvent found in adhesives and other products, is inhaled for its euphoric and intoxicating effects. Toluene inhalation additionally results in cognitive disturbances including impairments in select types of spatial and non-spatial memory, which converging evidence suggests may involve neurons of the dentate gyrus. In the present study we examined the effects of acute binge-like (~ 5000 ppm) toluene inhalation on dentate gyrus granule cell output and perforant path synaptic transmission, using extracellular field potential recordings in anesthetized adult rats in vivo. We found that toluene rapidly and reversibly increased or decreased the amplitudes of evoked population spikes from granule cells over a wide range of stimulation intensities. These changes in granule cell output could not be accounted for by changes in perforant path action potential discharge or presynaptic neurotransmitter release. A marked decrease in the power of the theta rhythm measured within the dentate gyrus was additionally noted. Overall our results suggest that inhalation of abuse-relevant concentrations of toluene changes the readout of perforant path inputs by dentate gyrus granule cells, putatively through a postsynaptic mechanism, and may contribute to explanations for specific learning and memory deficits associated with toluene inhalation.

Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central nervous system and participate in a variety of physiological functions. Recent advances have revealed roles of nAChRs in the regulation of synaptic transmission and synaptic plasticity, particularly in the hippocampus and midbrain dopamine centers. In general, activation of nAChRs causes membrane depolarization and directly and indirectly increases the intracellular calcium concentration. Thus, when nAChRs are expressed on presynaptic membranes their activation generally increases the probability of neurotransmitter release. When expressed on postsynaptic membranes, nAChR-initiated calcium signals and depolarization activate intracellular signaling mechanisms and gene transcription. Together, the presynaptic and postsynaptic effects of nAChRs generate and facilitate the induction of long-term changes in synaptic transmission. The direction of hippocampal nAChR-mediated synaptic plasticity - either potentiation or depression - depends on the timing of nAChR activation relative to coincident presynaptic and postsynaptic electrical activity, and also depends on the location of cholinergic stimulation within the local network. Therapeutic activation of nAChRs may prove efficacious in the treatment of neuropathologies where synaptic transmission is compromised, as in Alzheimer's or Parkinson's disease.

Spike output in many neuronal cell types is affected by low-voltage-activated T-type calcium currents arising from the Cav3.1, Cav3.2 and Cav3.3 channel subtypes and their splice isoforms. The contributions of T-type current to cell output is often proposed to reflect a differential distribution of channels to somatic and dendritic compartments, but the subcellular distribution of the various rat T-type channel isoforms has not been fully determined. We used subtype-specific Cav3 polyclonal antibodies to determine their distribution in key regions of adult Sprague‐Dawley rat brain thought to exhibit T-type channel expression, and in particular, dendritic lowvoltage-activated responses. We found a selective subcellular distribution of Cav3 channel proteins in cell types of the neocortex and hippocampus, thalamus, and cerebellar input and output neurons. In general, the Cav3.1 T-type channel immunolabel is prominent in the soma ⁄proximal dendritic region and Cav3.2 immunolabel in the soma and proximal-mid dendrites. Cav3.3 channels are distinct in distributing to the soma and over extended lengths of the dendritic arbor of particular cell types. Cav3 distribution overlaps with cell types previously established to exhibit rebound burst discharge as well as those not recognized for this activity. Additional immunolabel in the region of the nucleus in particular cell types was verified as corresponding to Cav3 antigen through analysis of isolated protein fractions. These results provide evidence that different Cav3 channel isoforms may contribute to low-voltageactivated calcium-dependent responses at the somatic and dendritic level, and the potential for T-type calcium channels to contribute to multiple aspects of neuronal activity.

T-type calcium channel isoforms are expressed in a multitude of tissues and have a key role in a variety of physiological processes. To fully appreciate the physiological role of distinct channel isoforms it is essential to determine their kinetic properties under physiologically relevant conditions. We therefore characterized the gating behavior of expressed rat voltage-dependent calcium channels (Ca(v)) 3.1, Ca(v)3.2, and Ca(v)3.3, as well as human Ca(v)3.3 at 21 degrees C and 37 degrees C in saline that approximates physiological conditions. Exposure to 37 degrees C caused significant increases in the rates of activation, inactivation, and recovery from inactivation, increased the current amplitudes, and induced a hyperpolarizing shift of half-activation for Ca(v)3.1 and Ca(v)3.2. At 37 degrees C the half-inactivation showed a hyperpolarizing shift for Ca(v)3.1 and Ca(v)3.2 and human Ca(v)3.3, but not rat Ca(v)3.3. The observed changes in the kinetics were significant but not identical for the three isoforms, showing that the ability of T-type channels to conduct calcium varies with both channel isoform and temperature.

Cerebellar Purkinje cells integrate multimodal afferent inputs and, as the only projection neurones of the cerebellar cortex, are key to the coordination of a variety of motor- and learning-related behaviours. In the neonatal rat the cerebellum is undeveloped, but over the first few postnatal weeks both the structure of the cerebellum and cerebellar-dependent behaviours mature rapidly. Maturation of Purkinje cell physiology is expected to contribute significantly to the development of cerebellar output. However, the ontogeny of the electrophysiological properties of the Purkinje cell and its relationship to maturation of cell morphology is incompletely understood. To address this problem we performed a detailed in vitro electrophysiological analysis of the spontaneous and intracellularly evoked intrinsic properties of Purkinje cells obtained from postnatal rats (P0 to P90) using whole-cell patch clamp recordings. Cells were filled with neurobiotin to enable subsequent morphological comparisons. Three stages of physiological and structural development were identified. During the early postnatal period (P0 to ∼P9) Purkinje cells were characterized by an immature pattern of Na+-spike discharge, and possessed only short multipolar dendrites. This was followed by a period of rapid maturation (from ∼P12 to ∼P18), consisting of changes in Na+-spike discharge, emergence of repetitive bursts of Na+ spikes terminated by Ca2+ spikes (Ca2+–Na+ bursts), generation of the trimodal pattern, and a significant expansion of the dendritic tree. During the final stage (> P18 to P90) there were minor refinements of cell output and a plateau in dendritic area. Our results reveal a rapid transition of the Purkinje cell from morphological and physiological immaturity to adult characteristics over a short developmental window, with a close correspondence between changes in cell output and dendritic growth. The development of Purkinje cell intrinsic electrophysiological properties further matches the time course of other measures of cerebellar structural and functional maturation.

Purkinje cells (PCs) generate the sole output of the cerebellar cortex and govern the timing of action potential discharge from neurons of the deep cerebellar nuclei (DCN). Here, we examine how voltage-gated Kv1 K+channels shape intrinsically generated and synaptically controlled behaviors of PCs and address how the timing of DCN neuron output is modulated by manipulating PC Kv1 channels. Kv1 channels were studied in cerebellar slices at physiological temperatures with Kv1-specific toxins. Outside-out voltage-clamp recordings indicated that Kv1 channels are present in both somatic and dendritic membranes and are activated by Na+spike-clamp commands. Whole-cell current-clamp recordings revealed that Kv1 K+channels maintain low frequencies of Na+spike and Ca-Na burst output, regulate the duration of plateau potentials, and set the threshold for Ca2+spike discharge. Kv1 channels shaped the characteristics of climbing fiber (CF) responses evoked by extracellular stimulation or intracellular simulated EPSCs. In the presence of Kv1 toxins, CFs discharged spontaneously at ∼1 Hz. Finally, “Kv1-intact” and “Kv1-deficient” PC tonic and burst outputs were converted to stimulus protocols and used as patterns to stimulate PC axons and synaptically activate DCN neurons. We found that the Kv1-intact patterns facilitated short-latency and high-frequency DCN neuron rebound discharges, whereas DCN neuron output timing was markedly disrupted by the Kv1-deficient stimulus protocols. Our results suggest that Kv1 K+channels are critical for regulating the excitability of PCs and CFs and optimize the timing of PC outputs to generate appropriate discharge patterns in postsynaptic DCN neurons.

The vitamin biotin is an endogenous molecule that acts as an important cofactor for several carboxylases in the citric acid cycle. Disorders of biotin metabolism produce neurological symptoms that range from ataxia to sensory loss, suggesting the presence of biotin in specific functional systems of the CNS. Although biotin has been described in some cells of nonmammalian nervous systems, the distribution of biotin in mammalian CNS is virtually unknown. We report the presence of biotin in select regions of rat CNS, as revealed with a monoclonal antibody directed against biotin and with avidin- and streptavidin-conjugated labels. Detectable levels of biotin were primarily found caudal to the diencephalon, with greatest expression in the cerebellar motor system and several brainstem auditory nuclei. Biotin was found as a somatic label in cerebellar Purkinje cells, in cell bodies and proximal dendrites of cerebellar deep nuclear neurons, and in red nuclear neurons. Biotin was detected in cells of the spiral ganglion, somata and proximal dendrites of cells in the cochlear nuclei, superior olivary nuclei, medial nucleus of the trapezoid body, and nucleus of the lateral lemniscus. Biotin was further found in pontine nuclei and fiber tracts, the substantia nigra pars reticulata, lateral mammillary nucleus, and a small number of hippocampal interneurons. Biotin was detected in glial cells of major tract systems throughout the brain but was most prominent in tracts of the hindbrain. Biotin is thus expressed in select regions of rat CNS with a distribution that correlates to the known clinical sequelae associated with biotin deficiencies.

Toluene belongs to a class of psychoactive drugs known as inhalants. Found in common household products such as adhesives, paint products, and aerosols, toluene is inhaled for its intoxicating and euphoric properties. Additionally, exposure to toluene disrupts motor behaviors in a manner consistent with impairments to cerebellar function. Previous work has suggested a role of GABA in mediating toluene's neurobehavioral effects, but how this manifests in the cerebellar cortex is not yet understood. In the present study, we examined the effects of toluene on cerebellar Purkinje cell action potential output and inhibitory synaptic transmission onto Purkinje cells using patch clamp electrophysiology in acute rat cerebellar slices. Toluene (1mM) reduced the frequency of Purkinje cell action potential output without affecting input resistance. Furthermore, toluene dose-dependently enhanced inhibitory synaptic transmission onto Purkinje cells, increasing the amplitude and frequency of inhibitory postsynaptic currents; no change in the frequency of action potentials from molecular layer interneurons was noted. The observed decreases in Purkinje cell action potential output could contribute to toluene-evoked impairments in cerebellar and motor functions.

Toluene is a psychoactive chemical found in many household products including adhesives and thinners. Inhalation of these vapors can cause euphoria and impairments in motor control and neurological functioning. Misuse and abuse of toluene is most common in children, which may in part be due to an age-dependent neurobehavioral sensitivity to toluene. Here we assessed the effects of acute binge-like toluene inhalations (15 or 30 min; ∼5,000 ppm) on tasks that examine locomotion, exploration, balance, gait, and neurological functioning for adolescent (1 month), young adult (2-3 months), adult (5-6 months), and older adult (10-12 months) rats. Both motor and neurological functions were impaired following acute toluene inhalation at all ages. However, only the duration to recover from deficits in motor functions differed among age groups, with adolescent and young adult rats requiring notably longer recovery times than older rats. Our results are suggestive of an age-dependent vulnerability to the intoxicating effects of toluene.

Encoding sensory input requires the expression of postsynaptic ion channels to transform key features of afferent input to an appropriate pattern of spike output. Although Ca 2+ -activated K + channels are known to control spike frequency in central neurons, Ca 2+ -activated K + channels of intermediate conductance (KCa3.1) are believed to be restricted to peripheral neurons. We now report that cerebellar Purkinje cells express KCa3.1 channels, as evidenced through single-cell RT-PCR, immunocytochemistry, pharmacology, and single-channel recordings. Furthermore, KCa3.1 channels coimmunoprecipitate and interact with low voltage-activated Cav3.2 Ca 2+ channels at the nanodomain level to support a previously undescribed transient voltage- and Ca 2+ -dependent current. As a result, subthreshold parallel fiber excitatory postsynaptic potentials (EPSPs) activate Cav3 Ca 2+ influx to trigger a KCa3.1-mediated regulation of the EPSP and subsequent after-hyperpolarization. The Cav3-KCa3.1 complex provides powerful control over temporal summation of EPSPs, effectively suppressing low frequencies of parallel fiber input. KCa3.1 channels thus contribute to a high-pass filter that allows Purkinje cells to respond preferentially to high-frequency parallel fiber bursts characteristic of sensory input.

Purkinje cells of the cerebellar cortex and neurons in the deep cerebellar nuclei (DCN) were among the first central neurons to be studied extensively through the use of in vitro preparations. Yet, the degree to which the patterns of action potential (“spike”) output recorded in these cells in vitro match those recorded in vivo has been a matter of debate and uncertainty. We have identified relatively simple steps that can be applied to the preparation of cerebellar tissue slices or to recording conditions that increase the probability of recording spike output in vitro that more closely matches that found in the live animal. Of particular importance are considerations of the rapid development of Purkinje cell structure and spike output properties during the period used for patch-clamp recordings, reintroducing key synaptic inputs to the in vitro preparation and establishing appropriate reversal potentials for ion species. We also report that storing water to be used for preparing medium in polyethylene carboys introduces a contaminant that decreases the amplitude of T-type Ca 2+ currents and the ability to generate rebound burst discharge in DCN cells. Controlling for these factors restores spontaneous tonic firing, increases the gain to parallel fiber input, uncovers bistable rhythmic behavior in Purkinje cells, and increases the probability for generating rebound discharge in DCN cells.

Inhalation of vapors from toluene-containing products results in euphoria accompanied by a variety of cognitive impairments and motor dysfunctions. The profound behavioral changes observed during and following toluene inhalation suggest changes in the activity of cells in potentially many brain regions; however, a comprehensive assessment of the neuroanatomical structures activated by toluene vapor has not been completed. Thus in the present study we systematically mapped in over 140 brain structures the distribution of c-Fos immunoreactivity (c-Fos IR), a proxy for neural activation, following exposure to an abuse-like concentration (~ 5000 ppm) of toluene vapor for 0, 5, 10 or 30 min. Quantitative analyses revealed increases in c-Fos IR in about one-third of the brain structures examined, with most of these structures significantly activated only after prolonged toluene exposure. The majority of brain structures activated by toluene were found in the forebrain and midbrain, with particularly pronounced activation in nuclei implicated in the processing of rewarding, emotional, and olfactory stimuli, and those controlling motor output. These structures included the ventral tegmental area, nucleus accumbens, select regions of the amygdala and hypothalamus, cingulate cortex, olfactory nuclei, piriform cortex, secondary motor cortex and caudate-putamen. In contrast, all subregions of the hippocampus and most thalamic nuclei were not significantly activated by toluene vapor. In the brainstem, effects of toluene vapor were restricted to select nuclei in the pons. The pattern of c-Fos IR evoked by inhalation of toluene vapor appears distinct from other psychoactive substances, consistent with the unique and complex behavioral outcomes associated with acute toluene inhalation.

The interaction between avidin and biotin or streptavidin and biotin forms the basis of several widely used immunohistochemical techniques. An assumption inherent to these techniques is that endogenous biotin is not present in the tissue in detectable quantities, as neither avidin nor streptavidin can discriminate between endogenous biotin and biotinylated antibodies. However, biotin is a known cofactor for numerous carboxylases required in oxidative metabolism, raising the possibility for potential false-positive results in many tissues. This issue has been appreciated in liver and kidney tissue, but has received very little attention in nervous tissue. To address this concern, we examined the distribution of biotin throughout the rat central nervous system using avidin- and streptavidin-based detection systems, as well as a monoclonal antibody raised against biotin. Significant levels of endogenous biotin were identified within specific neuronal types, particularly in neurons associated with the cerebellar motor system and the brainstem auditory system. Non-specific (non-biotin) interactions of avidin and streptavidin conjugates with rat brain tissue were further identified and were most pronounced in the lower brainstem. The binding of avidin- and streptavidin-conjugated markers to endogenous biotin and other non-specific interactions with neural tissue were overcome by several methods including the use of blocking kits, prolonged post-fixation of the tissue in paraformaldehyde, or omission of Triton X-100 from the working solution. Without these measures, a reliable estimate of immunolabel may only be achieved in certain brain regions with markers conjugated directly to secondary antibodies.

The interaction between avidin and biotin or streptavidin and biotin forms the basis of several widely used immunohistochemical techniques. An assumption inherent to these techniques is that endogenous biotin is not present in the tissue in detectable quantities, as neither avidin nor streptavidin can discriminate between endogenous biotin and biotinylated antibodies. However, biotin is a known cofactor for numerous carboxylases required in oxidative metabolism, raising the possibility for potential false-positive results in many tissues. This issue has been appreciated in liver and kidney tissue, but has received very little attention in nervous tissue. To address this concern, we examined the distribution of biotin throughout the rat central nervous system using avidin- and streptavidin-based detection systems, as well as a monoclonal antibody raised against biotin. Significant levels of endogenous biotin were identified within specific neuronal types, particularly in neurons associated with the cerebellar motor system and the brainstem auditory system. Non-specific (non-biotin) interactions of avidin and streptavidin conjugates with rat brain tissue were further identified and were most pronounced in the lower brainstem. The binding of avidin- and streptavidin-conjugated markers to endogenous biotin and other non-specific interactions with neural tissue were overcome by several methods including the use of blocking kits, prolonged post-fixation of the tissue in paraformaldehyde, or omission of Triton X-100 from the working solution. Without these measures, a reliable estimate of immunolabel may only be achieved in certain brain regions with markers conjugated directly to secondary antibodies.

The contribution of Purkinje cells to cerebellar motor coordination and learning is determined in part by the chronic and acute effects of climbing fiber (CF) afferents. Whereas the chronic effects of CF discharge, such as the depression of conjunctive parallel fiber (PF) inputs, are well established, the acute cellular functions of CF discharge remain incompletely understood. In rat cerebellar slices, we show that CF discharge presented at physiological frequencies substantially modifies the frequency and pattern of Purkinje cell spike output in vitro. Repetitive CF discharge converts a spontaneous trimodal pattern of output characteristic of Purkinje cells in vitro to a more naturalistic nonbursting pattern consisting of spike trains interrupted by short CF-evoked pauses or longer pauses associated with state transitions. All effects of CF discharge could be reproduced in the presence of synaptic blockers by using current injections to simulate complex spike depolarizations, revealing that CF-evoked changes in Purkinje cell output can occur independently of network activation. Rather postsynaptic changes are sufficient to account for the CF-evoked block of trimodal activity and include at least the activation of Ca2+-dependent K+ channels. Furthermore by controlling the frequency of Purkinje cell spike output over three discrete firing levels, CF discharge modulates the gain of Purkinje cell responsiveness to PF inputs in vitro through postsynaptic mechanisms triggered by the complex spike depolarization. The ability for CF discharge to acutely modulate diverse aspects of Purkinje cell output provides important insights into the probable cellular factors contributing to motor disturbances following CF denervation.

Spike output in many neuronal cell types is affected by low-voltage-activated T-type calcium currents arising from the Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 channel subtypes and their splice isoforms. The contributions of T-type current to cell output is often proposed to reflect a differential distribution of channels to somatic and dendritic compartments, but the subcellular distribution of the various rat T-type channel isoforms has not been fully determined. We used subtype-specific Ca(v)3 polyclonal antibodies to determine their distribution in key regions of adult Sprague-Dawley rat brain thought to exhibit T-type channel expression, and in particular, dendritic low-voltage-activated responses. We found a selective subcellular distribution of Ca(v)3 channel proteins in cell types of the neocortex and hippocampus, thalamus, and cerebellar input and output neurons. In general, the Ca(v)3.1 T-type channel immunolabel is prominent in the soma/proximal dendritic region and Ca(v)3.2 immunolabel in the soma and proximal-mid dendrites. Ca(v)3.3 channels are distinct in distributing to the soma and over extended lengths of the dendritic arbor of particular cell types. Ca(v)3 distribution overlaps with cell types previously established to exhibit rebound burst discharge as well as those not recognized for this activity. Additional immunolabel in the region of the nucleus in particular cell types was verified as corresponding to Ca(v)3 antigen through analysis of isolated protein fractions. These results provide evidence that different Ca(v)3 channel isoforms may contribute to low-voltage-activated calcium-dependent responses at the somatic and dendritic level, and the potential for T-type calcium channels to contribute to multiple aspects of neuronal activity.

T-type calcium channels are thought to transform neuronal output to a burst mode by generating low voltage-activated (LVA) calcium currents and rebound burst discharge. In this study we assess the expression pattern of the three different T-type channel isoforms (Ca v 3.1, Ca v 3.2, and Ca v 3.3) in cerebellar neurons and focus on their potential role in generating LVA spikes and rebound discharge in deep cerebellar nuclear (DCN) neurons. We detected expression of one or more Ca v 3 channel isoforms in a wide range of cerebellar neurons and selective expression of different isoforms in DCN cells. We further identify two classes of large-diameter DCN neurons that exhibit either a strong or weak capability for rebound discharge, despite the ability to generate LVA spikes when calcium currents are pharmacologically isolated. By correlating the Ca v 3 channel expression pattern with the electrophysiological profile of identified DCN cells, we show that Ca v 3.1 channels are expressed in isolation in DCN-burst cells, whereas Ca v 3.3 is expressed in DCN-weak burst cells. Ca v 3.1-expressing DCN cells correspond to excitatory or GABAergic neurons, whereas Ca v 3.3-expressing cells are non-GABAergic. The Ca v 3 class of LVA calcium channels is thus expressed in specific combinations in a wide range of cerebellar neurons but contributes to rebound burst discharge in only a select number of cell classes.

The ability of cells to generate an appropriate spike output depends on a balance between membrane depolarizations and the repolarizing actions of K(+) currents. The high-voltage-activated Kv3 class of K(+) channels repolarizes Na(+) spikes to maintain high frequencies of discharge. However, little is known of the ability for these K(+) channels to shape Ca(2+) spike discharge or their ability to regulate Ca(2+) spike-dependent burst output. Here we identify the role of Kv3 K(+) channels in the regulation of Na(+) and Ca(2+) spike discharge, as well as burst output, using somatic and dendritic recordings in rat cerebellar Purkinje cells. Kv3 currents pharmacologically isolated in outside-out somatic membrane patches accounted for approximately 40% of the total K(+) current, were very fast and high voltage activating, and required more than 1 s to fully inactivate. Kv3 currents were differentiated from other tetraethylammonium-sensitive currents to establish their role in Purkinje cells under physiological conditions with current-clamp recordings. Dual somatic-dendritic recordings indicated that Kv3 channels repolarize Na(+) and Ca(2+) spikes, enabling high-frequency discharge for both types of cell output. We further show that during burst output Kv3 channels act together with large-conductance Ca(2+)-activated K(+) channels to ensure an effective coupling between Ca(2+) and Na(+) spike discharge by preventing Na(+) spike inactivation. By contributing significantly to the repolarization of Na(+) and especially Ca(2+) spikes, our data reveal a novel function for Kv3 K(+) channels in the maintenance of high-frequency burst output for cerebellar Purkinje cells.

Cerebellar Purkinje cells are contacted by up to ~150,000 parallel fibers from granule cells, of which only a subset will convey sensory information at any given time. Purkinje cells must then possess the means to respond effectively to meaningful parallel fiber input over background noise. Previous work has shown that parallel fiber excitatory postsynaptic potential (EPSP) summation can be shaped by feedforward synaptic inhibition and the hyperpolarization-activated current IH[1,2]. We now report that parallel fiber EPSPs activate T-type calcium channels that are linked to intermediate conductance calcium-activated potassium (IKCa) channels in Purkinje cells. This novel complex exerts a frequency-dependent suppression of temporal summation, such that only high frequency parallel fiber inputs undergoing presynaptic facilitation can elicit spike output from Purkinje cells. Cerebellar slices were prepared from P18-30 rats and patch recordings obtained from PC somata at 32-35°C. PFs were activated using a monopolar stimulating electrode in the molecular layer or granule cell layer. Alternatively, the role of postsynaptic PC ion channels were selectively tested by injecting simulated EPSCs to evoke PF simulated EPSPs (simEPSPs) at the soma. PF EPSPs below threshold for spike discharge were followed by an after hyperpolarization (AHP) of up to 2.5 mV and 250 ms. Application of blockers against high voltage activated Ca2+ channels (Cd2+, Agatoxin IVA), SK channels (apamin), or BK channels (TEA, iberiotoxin, paxilline) did not significantly affect the rate of simEPSP decay. However, T-type Ca2+ channel blockers (Ni2+, Mibefradil, kurtoxin) caused a ~35% decrease in the rate of simEPSP decay. Moreover, these effects were reproduced by application of IKCa channel blockers (TRAM-34, charybdotoxin). Immunofluorescent labeling for IKCa protein confirmed its expression in Purkinje cells somata and dendrites. Ni2+ and TRAM-34 sensitive outward currents were found in outside-out patches from PC somata, confirming current clamp data showing a functional link between Cav3 and IKCa channels. The outward current was further blocked by BAPTA (10 mM) but not EGTA (10 mM) in the internal patch solution, indicating that the Ca2+-IKca channel interaction resides within a nanodomain. To examine the effect of this interaction on temporal summation, PFs were stimulated at varying frequencies. For frequencies up to 25 Hz, no temporal summation was observed in control conditions. However, blocking either Cav3 or IKca channels caused significant summation for 25 Hz stimulations. This effect was seen in both the presence and absence of feedforward-inhibition. Application of TRAM-34 greatly altered the frequency response of PC to 50 and 100 Hz PF stimulation during tonic firing. Finally, the Cav3-IKCa complex selectively suppresses non-facilitating inputs while allowing smaller-amplitude, facilitating inputs to generate output. Our current work is the first to demonstrate the expression of IKCa channels in central neurons, its association with Cav3 channels and the role of this Cav3-IKCa complex in controlling the response of PCs to PF inputs. The Cav3-IKCa complex creates a high pass filter that reduces the effectiveness of background activity and allows Purkinje cells to respond preferentially to parallel fiber input indicative of sensory input carried by mossy fibers.