Bruce E. McKay

Physiological and morphological development of the rat cerebellar Purkinje cell

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.

Ca(V)3 T-type calcium channel isoforms differentially distribute to somatic and dendritic compartments in rat central neurons.

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 low‐voltage‐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‐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.

Kv3 K+ channels enable burst output in rat cerebellar Purkinje cells

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 Ca2+ spike discharge or their ability to regulate Ca2+ spike‐dependent burst output. Here we identify the role of Kv3 K+ channels in the regulation of Na+ and Ca2+ 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 ∼ 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 Ca2+ 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 Ca2+‐activated K+ channels to ensure an effective coupling between Ca2+ and Na+ spike discharge by preventing Na+ spike inactivation. By contributing significantly to the repolarization of Na+ and especially Ca2+ spikes, our data reveal a novel function for Kv3 K+ channels in the maintenance of high‐frequency burst output for cerebellar Purkinje cells.

Kv1 K+ Channels Control Purkinje Cell Output to Facilitate Postsynaptic Rebound Discharge in Deep Cerebellar Neurons

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.

Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells

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 Ca2+-activated K+ channels are known to control spike frequency in central neurons, Ca2+-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 Ca2+ channels at the nanodomain level to support a previously undescribed transient voltage- and Ca2+-dependent current. As a result, subthreshold parallel fiber excitatory postsynaptic potentials (EPSPs) activate Cav3 Ca2+ 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.

TEMPERATURE DEPENDENCE OF T-TYPE CALCIUM CHANNEL GATING

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.

Biotin is endogenously expressed in select regions of the rat central nervous system

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. J. Comp. Neurol. 473:86–96, 2004.

IKCa-Cav3 complex creates a high pass filter for parallel fiber input in 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.