Ray W. Turner

Modulation of ion channels in rod photoreceptors by nitric oxide

Subcellular compartments in the outer retina of the larval tiger salamander were identified as likely sites of production of nitric oxide (NO), a recently recognized intercellular messenger. NADPH diaphorase histochemistry and NO synthase immunocytochemistry labeled photoreceptor ellipsoids and the distal regions of bipolar and glial cells apposing photoreceptor inner segments, suggesting a role for NO in visual processing in the outer retina. We investigated the actions of NO on several rod photoreceptor ion channels. Application of the NO-generating compound S-nitrosocysteine increased Ca2+ channel current and a voltage-independent conductance, but had no affect on voltage-gated K+ or nonspecific cation currents. Given the steep relation between voltage-dependent Ca2+ influx and photoreceptor synaptic output, these results indicate that NO could modulate transmission of the photoresponse to second order cells.

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.

Regulation of neuronal activity by Cav3-Kv4 channel signaling complexes

Kv4 low voltage–activated A-type potassium channels are widely expressed in excitable cells, where they control action potential firing, dendritic activity and synaptic integration. Kv4 channels exist as a complex that includes K+ channel–interacting proteins (KChIPs), which contain calcium-binding domains and therefore have the potential to confer calcium dependence on the Kv4 channel. We found that T-type calcium channels and Kv4 channels form a signaling complex in rat that efficiently couples calcium influx to KChIP3 to modulate Kv4 function. This interaction was critical for allowing Kv4 channels to function in the subthreshold membrane potential range to regulate neuronal firing properties. The widespread expression of these channels and accessory proteins indicates that the Cav3-Kv4 signaling complex is important for the function of a wide range of electrically excitable cells.

Deterministic Multiplicative Gain Control with Active Dendrites

Multiplicative gain control is a vital component of many theoretical analyses of neural computations, conferring the ability to scale neuronal firing rate in response to synaptic inputs. Many theories of gain control in single cells have used precisely balanced noisy inputs. Such noisy inputs can degrade signal processing. We demonstrate a deterministic method for the control of gain without the use of noise. We show that a depolarizing afterpotential (DAP), arising from active dendritic spike backpropagation, leads to a multiplicative increase in gain. Reduction of DAP amplitude by dendritic inhibition dilutes the multiplicative effect, allowing for divisive scaling of the firing rate. In contrast, somatic inhibition acts in a subtractive manner, allowing spatially distinct inhibitory inputs to perform distinct computations. The simplicity of this mechanism and the ubiquity of its elementary components suggest that many cell types have the potential to display a dendritic division of neuronal output.

Nitric oxide synthase-immunoreactive cells in the CNS and periphery of Lymnaea

The presence and distribution of nitric oxide synthase (NOS) in the CNS and peripheral organs (buccal muscles, oesophagus, salivary glands, foot, mantle and pneumostome) of the pulmonate mollusc, Lymnaea stagnalis were studied using an antiserum developed against rat cerebellar NOS. NOS-immunopositive neurones in Lymnaea were localized predominantly in the buccal ganglia as well as in distinct areas of the cerebral and suboesophageal ganglia. NOS-immunoreactive terminals were also found on the somata of some central neurones. In the periphery, NOS-immunostaining was detected only in a few neurones in the pneumostome area and in the osphradial ganglion. In addition, approximately 100 NOS-immunopositive cells have been found in the salivary glands. Our data supports other recent reports indicating that NO may be a signal molecule in the CNS of molluscs.

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.

Importance of K+-dependent Na+/Ca2+-exchanger 2, NCKX2, in Motor Learning and Memory

Plasma membrane Na+/Ca2+-exchangers play a predominant role in Ca2+ extrusion in brain. Neurons express several different Na+/Ca2+-exchangers belonging to both the K+-independent NCX family and the K+-dependent NCKX family. The unique contributions of each of these proteins to neuronal Ca2+ homeostasis and/or physiology remain largely unexplored. To address this question, we generated mice in which the gene encoding the abundant neuronal K+ -dependent Na+/Ca2+-exchanger protein, NCKX2, was knocked out. Analysis of these animals revealed a significant reduction in Ca2+ flux in cortical neurons, a profound loss of long term potentiation and an increase in long term depression at hippocampal Schaffer/CA1 synapses, and clear deficits in specific tests of motor learning and spatial working memory. Surprisingly, there was no obvious loss of photoreceptor function in cones, where expression of the NCKX2 protein had been reported previously. These data emphasize the critical and non-redundant role of NCKX2 in the local control of neuronal [Ca2+] that is essential for the development of synaptic plasticity associated with learning and memory.