Kv1.2-containing K+ channels regulate subthreshold excitability of striatal medium spiny neurons

A slowly inactivating, low-threshold K(+) current has been implicated in the regulation of state transitions and repetitive activity in striatal medium spiny neurons. However, the molecular identity of the channels underlying this current and their biophysical properties remain to be clearly determined. Because previous work had suggested this current arose from Kv1 family channels, high-affinity toxins for this family were tested for their ability to block whole cell K(+) currents activated by depolarization of acutely isolated neurons. alpha-Dendrotoxin, which blocks channels containing Kv1.1, Kv1.2, or Kv1.6 subunits, decreased currents evoked by depolarization. Three other Kv1 family toxins that lack a high affinity for Kv1.2 subunits, r-agitoxin-2, dendrotoxin-K, and r-margatoxin, failed to significantly reduce currents, implicating channels with Kv1.2 subunits. RT-PCR results confirmed the expression of Kv1.2 mRNA in identified medium spiny neurons. Currents attributable to Kv1.2 channels activated rapidly, inactivated slowly, and recovered from inactivation slowly. In the subthreshold range (ca. -60 mV), these currents accounted for as much as 50% of the depolarization-activated K(+) current. Moreover, their rapid activation and relatively slow deactivation suggested that they contribute to spike afterpotentials regulating repetitive discharge. This inference was confirmed in current-clamp recordings from medium spiny neurons in the slice preparation where Kv1.2 blockade reduced first-spike latency and increased discharge frequency evoked from hyperpolarized membrane potentials resembling the "down-state" found in vivo. These studies establish a clear functional role for somato-dendritic Kv1.2 channels in the regulation of state transitions and repetitive discharge in striatal medium spiny neurons.     

Multiple neuropeptide Y receptors regulate K+ and Ca2+ channels in acutely isolated neurons from the rat arcuate nucleus

We examined the effects of neuropeptide Y (NPY) and related peptides on Ca2+ and K+ currents in acutely isolated neurons from the arcuate nucleus of the rat. NPY analogues that activated all of the known NPY receptors (Y1-Y5), produced voltage-dependent inhibition of Ca2+ currents and activation of inwardly rectifying K+ currents in arcuate neurons. Both of these effects could occur simultaneously in the same cells. In some cells, activation of Y4 NPY receptors also caused oscillations in [Ca2+]i. NPY hyperpolarized arcuate neurons through the activation of a K+ conductance and increased the spike threshold. Molecular biological studies indicated that arcuate neurons possessed all of the previously cloned NPY receptor types (Y1, Y2, Y4, and Y5). Thus activation of multiple types NPY receptors on arcuate neurons can regulate both Ca2+ and K+ conductances leading to a reduction in neuronal excitability and a suppression of neurotransmitter release.     

Large-conductance calcium-dependent potassium channels prevent dendritic excitability in neocortical pyramidal neurons

Large-conductance calcium-dependent potassium channels (BK channels) are homogeneously distributed along the somatodendritic axis of layer 5 pyramidal neurons of the rat somatosensory cortex. The relevance of this conductance for dendritic calcium electrogenesis was studied in acute brain slices using somatodendritic patch clamp recordings and calcium imaging. BK channel activation reduces the occurrence of dendritic calcium spikes. This is reflected in an increased critical frequency of somatic spikes necessary to activate the distal initiation zone. Whilst BK channels repolarise the somatic spike, they dampen it only in the distal dendrite. Their activation reduces dendritic calcium influx via glutamate receptors. Furthermore, they prevent dendritic calcium electrogenesis and subsequent somatic burst discharges. However, the time window for coincident somatic action potential and dendritic input to elicit dendritic calcium events is not influenced by BK channels. Thus, BK channel activation in layer 5 pyramidal neurons affects cellular excitability primarily by establishing a high threshold at the distal action potential initiation zone. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ternary Kv4.2 channels recapitulate voltage-dependent inactivation kinetics of A-type K+ channels in cerebellar granule neurons

Kv4 channels mediate most of the somatodendritic subthreshold operating A-type current (I(SA)) in neurons. This current plays essential roles in the regulation of spike timing, repetitive firing, dendritic integration and plasticity. Neuronal Kv4 channels are thought to be ternary complexes of Kv4 pore-forming subunits and two types of accessory proteins, Kv channel interacting proteins (KChIPs) and the dipeptidyl-peptidase-like proteins (DPPLs) DPPX (DPP6) and DPP10. In heterologous cells, ternary Kv4 channels exhibit inactivation that slows down with increasing depolarization. Here, we compared the voltage dependence of the inactivation rate of channels expressed in heterologous mammalian cells by Kv4.2 proteins with that of channels containing Kv4.2 and KChIP1, Kv4.2 and DPPX-S, or Kv4.2, KChIP1 and DPPX-S, and found that the relation between inactivation rate and membrane potential is distinct for these four conditions. Moreover, recordings from native neurons showed that the inactivation kinetics of the I(SA) in cerebellar granule neurons has voltage dependence that is remarkably similar to that of ternary Kv4 channels containing KChIP1 and DPPX-S proteins in heterologous cells. The fact that this complex and unique behaviour (among A-type K(+) currents) is observed in both the native current and the current expressed in heterologous cells by the ternary complex containing Kv4, DPPX and KChIP proteins supports the hypothesis that somatically recorded native Kv4 channels in neurons include both types of accessory protein. Furthermore, quantitative global kinetic modelling showed that preferential closed-state inactivation and a weakly voltage-dependent opening step can explain the slowing of the inactivation rate with increasing depolarization. Therefore, it is likely that preferential closed-state inactivation is the physiological mechanism that regulates the activity of both ternary Kv4 channel complexes and native I(SA)-mediating channels.     

Membrane excitability and fear conditioning in cerebellar Purkinje cell

In a previous study it has been demonstrated that fear conditioning is associated with a long-lasting potentiation of parallel fiber to Purkinje cell synaptic transmission in vermal lobules V and VI. Since modifications of intrinsic membrane properties have been suggested to mediate some forms of memory processes, we investigated possible changes of Purkinje cell intrinsic properties following the same learning paradigm and in the same cerebellar region. By means of the patch clamp technique, Purkinje cell passive and active membrane properties were evaluated in slices prepared from rats 10 min or 24 h after fear conditioning and in slices from control naïve animals. None of the evaluated parameters (input resistance, inward rectification, maximal firing frequency and the first inter-spike interval, post-burst afterhyperpolarization, action potential threshold and amplitude, action potential afterhyperpolarization) was significantly different between the three studied groups also in those cells where parallel fiber–Purkinje cell synapse was potentiated. Our results show that fear learning does not affect the intrinsic membrane properties involved in Purkinje cell firing. Therefore, at the level of Purkinje cell the plastic change associated with fear conditioning is specifically restricted to synaptic efficacy.     

Cholinergic modulation of Kir2 channels selectively elevates dendritic excitability in striatopallidal neurons

Dopamine-depleting lesions of the striatum that mimic Parkinson's disease induce a profound pruning of spines and glutamatergic synapses in striatopallidal medium spiny neurons, leaving striatonigral medium spiny neurons intact. The mechanisms that underlie this cell type-specific loss of connectivity are poorly understood. The Kir2 K(+) channel is an important determinant of dendritic excitability in these cells. Here we show that opening of these channels is potently reduced by signaling through M1 muscarinic receptors in striatopallidal neurons, but not in striatonigral neurons. This asymmetry could be attributed to differences in the subunit composition of Kir2 channels. Dopamine depletion alters the subunit composition further, rendering Kir2 channels in striatopallidal neurons even more susceptible to modulation. Reduced opening of Kir2 channels enhances dendritic excitability and synaptic integration. This cell type-specific enhancement of dendritic excitability is an essential trigger for synaptic pruning after dopamine depletion, as pruning was prevented by genetic deletion of M1 muscarinic receptors.     

Hyperforin modulates gating of P-type Ca2+ current in cerebellar Purkinje neurons

Whole-cell, patch-clamp recordings from acutely isolated cerebellar Purkinje neurons demonstrate a two-stage modulation of P-type high-voltage-activated (HVA) Ca2+ current by a constituent of St. John's wort, hyperforin (0.04-0.8 microM). The first stage of modulation was voltage dependent and reversible. It comprised slow-down of the activation kinetics and a shift in the voltage dependence of P-current to more negative voltages. Hyperforin (0.8 microM) shifted the maximum of the current/voltage (I/V) relationship by -8+/-2 mV. The second, voltage-independent stage of modulation was manifested as a slowly developing inhibition of P-current that could not be reversed within the period of study. Neither form of modulation was abolished by intracellular guanosine 5'-O-(2-thiodiphosphate) (GDPPS) or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) or by strong depolarising pre-pulses, indicating that modulation via guanine nucleotide-binding proteins (G proteins) is not involved in the observed phenomenon. Calmidazolium (0.5 microM), an antagonist of the intracellular Ca2+-binding protein calmodulin significantly inhibited the hyperforin-induced shift of the IIV curve maximum and the slow-down of the activation kinetics. It did not, however, affect the delayed inhibition of P-current, indicating that the two stages of modulation are mediated by separate mechanisms.     

GDNF acutely modulates excitability and A-type K(+) channels in midbrain dopaminergic neurons

Glial cell line-derived neurotrophic factor (GDNF) prevents lesion-induced death of midbrain dopaminergic neurons, but its function in normal brain remains uncertain. Here we show that GDNF acutely and reversibly potentiated the excitability of cultured midbrain neurons by inhibiting transient A-type K(+) channels. The effects of GDNF were limited to large, tyrosine hydroxylase (TH)-positive dopaminergic neurons, and were mediated by mitogen associated protein (MAP) kinase. Application of GDNF also elicited a MAP kinase-dependent enhancement of the excitability in dopaminergic neurons in midbrain slice. These results demonstrate an acute regulation of GDNF on ion channels and its underlying signaling mechanism, and reveal an unexpected role of GDNF in normal midbrain dopaminergic neurons.     

Post-hatch development of dendritic arborization in cerebellar Purkinje neurons of quail chicks: a morphometric study

To study development of the cerebellum in precocial birds during the early post-hatch period, dendritic arborization of Purkinje neurons was analyzed by intracellular Lucifer-yellow filling in fixed slices. Purkinje neurons were sampled from parasagittal slices of the cerebellar vermis of quail chicks (from 1 day pre-hatch to 14 days post-hatch). Confocal images revealed that the dendritic arborization expanded two-fold during the initial 3 days post-hatch, whereas the growth of cell bodies was much smaller. The dendritic expansion was accompanied by significant elongation of distal dendritic segments (5th and more distal segments), while the length of proximal dendrites (1st-4th segments) and the number of dendritic branches remained unchanged. Active synaptogenesis may occur selectively in distal dendrites during the early post-hatch period.     

Incomplete inactivation and rapid recovery of voltage-dependent sodium channels during high-frequency firing in cerebellar Purkinje neurons

The ability of individuals to adapt locomotion to constraints associated with the complex environments normally encountered in everyday life is paramount for survival. Here, we tested the ability of 24 healthy young adults to adapt to a rightward prism shift (～11.3°) while either walking and stepping to targets (i.e., precision stepping task) or stepping over an obstacle (i.e., obstacle avoidance task). We subsequently tested for generalization to the other locomotor task. In the precision stepping task, we determined the lateral end-point error of foot placement from the targets. In the obstacle avoidance task, we determined toe clearance and lateral foot placement distance from the obstacle before and after stepping over the obstacle. We found large, rightward deviations in foot placement on initial exposure to prisms in both tasks. The majority of measures demonstrated adaptation over repeated trials, and adaptation rates were dependent mainly on the task. On removal of the prisms, we observed negative aftereffects for measures of both tasks. Additionally, we found a unilateral symmetric generalization pattern in that the left, but not the right, lower limb indicated generalization across the 2 locomotor tasks. These results indicate that the nervous system is capable of rapidly adapting to a visuomotor mismatch during visually demanding locomotor tasks and that the prism-induced adaptation can, at least partially, generalize across these tasks. The results also support the notion that the nervous system utilizes an internal model for the control of visually guided locomotion.     

Low threshold calcium currents in rat cerebellar Purkinje cell dendritic spines are mediated by T-type calcium channels

The functional role of low voltage activated (LVA) calcium channels in the cerebellar Purkinje cell dendritic tree is not completely understood. Since the localization of these channels will influence their possible roles in dendritic integration and induction of plasticity, we set out to characterize the LVA calcium current in Purkinje cell dendrites in acute cerebellar slices of young rats. Using a combination of electrophysiological recordings and two-photon laser scanning microscopy, we show that LVA calcium current recorded at the soma can be correlated with voltage-dependent calcium transients in Purkinje cell dendritic spines. Blocking sodium and potassium conductances allowed us to isolate and characterize a fast inactivating inward current activated positive to −55 mV. Activation and steady-state inactivation kinetics, voltage-dependent deactivation kinetics, and pharmacological experiments (using ω-agatoxin-IVA, mibefradil and nickel) show that this current is carried by T-type calcium channels. Furthermore, the LVA calcium transient observed in the dendritic spines of the Purkinje cell is well correlated with the current recorded at the soma, suggesting that T-type calcium channels are the main component of the LVA calcium input in spines. The fast rising phase of the calcium transient in spines and the absence of delay between the onset in the spine and the parent dendrite show that T-type calcium channels are present both in spines and dendrites of the Purkinje cell.     

Subtypes of voltage-sensitive calcium channels in cultured rat brain neurons

Subtypes of voltage-sensitive calcium channels have been investigated in cultured rat brain neurons using two classes of specific probes, dihydropyridine compounds and omega-conotoxin. Membranes prepared from cultured neurons contain specific binding sites for [3H]PN200-110, a dihydropyridine antagonist, and for 125I-omega-conotoxin with a stoichiometry of about 1:1. A depolarization induced 45Ca2+ influx into intact brain neurons was partially inhibited by a dihydropyridine antagonist, nifedipine and stimulated by a dihydropyridine agonist, Bay K8644. This dihydropyridine sensitive 45Ca2+ flux was insensitive to omega-conotoxin at concentrations which saturate the specific toxin binding sites indicating that in cultured brain neurons, dihydropyridine-sensitive calcium channels are not sensitive to omega-conotoxin.     

Erg K+ currents modulate excitability in mouse mitral/tufted neurons

Different erg (ether-à-go-go-related gene; Kv11) K⁺ channel subunits are expressed throughout the brain. Especially mitral cells of the olfactory bulb are stained intensely by erg1a, erg1b, erg2, and erg3 antibodies. This led us to study the erg current in mitral/tufted (M/T) neurons from mouse olfactory bulb in primary culture. M/T neurons were identified by their morphology and presence of mGluR1 receptors, and RT-PCR demonstrated the expression of all erg subunits in cultured M/T neurons. Using an elevated external K⁺ concentration, a relatively uniform erg current was recorded in the majority of M/T cells and isolated with the erg channel blocker E-4031. With 4-s depolarizations, the erg current started to activate at ?65 mV and exhibited half maximal activation at ?51 mV. An increase in the external K⁺ concentration resulted in an increase in erg whole-cell conductance. The specific group 1 mGluR agonist, DHPG, which depolarizes mitral cells, reduced erg channel availability. DHPG accelerated erg current deactivation, reduced the maximum current amplitude, and shifted availability and activation curves to more depolarized potentials. A pharmacological block of erg channels depolarized the resting potential of M/T cells and clearly demonstrated the involvement of erg channels in the control of mitral cell excitability.     

Spontaneous Na+ and Ca2+ spike firing of cerebellar Purkinje neurons at high pressure

 The effects of high pressure (up to 10.1 MPa) on the spontaneous firing of Purkinje neurons in guinea-pig cerebellar slices were studied using the macropatch clamp technique. Pressure did not significantly alter the single somatic Na⁺ spike parameters or the frequency of regular Na⁺ spike firing. When Na⁺ currents were blocked by 0.5–1 μM tetrodotoxin (TTX), a pressure of 10.1 MPa slightly reduced the dendritic Ca²⁺ spike amplitude to 90.2±3.1% of its control value, and slowed its kinetics. The effects of pressure on the single Ca²⁺ spike were even less prominent when K⁺ currents were blocked by 5 mM 4-aminopyridine (4-AP). Pressure prolonged the active period of Ca²⁺ spike firing to 152.2±10.4% of the control value. Within the active period pressure increased the inter-spike interval to 164.9±8.7% and suppressed the typical firing of doublets. The latter changes were reversed by a high extracellular potassium concentration ([K⁺]_o) and 1 μM 4-AP, whereas in the presence of 5 mM 4-AP the pattern was insensitive to pressure. A high [Ca²⁺]_o reduced the firing frequency and suppressed doublet firing in a manner reminiscent of the pressure effect, but these changes could not be reversed by 4-AP. A low [Ca²⁺]_o slightly increased the firing of doublets. These results show that the single somatic Na⁺ spike is insensitive and the dendritic Ca²⁺ spike is only mildly sensitive to pressure. However, alterations in Ca²⁺ spike firing pattern suggest that modulation of dendritic K⁺ currents induce depression of dendritic excitability at pressure. Received: 19 May 1998 / Received after revision: 15 July 1998 / Accepted: 3 September 1998     

Large conductance calcium-activated potassium channels affect both spontaneous firing and intracellular calcium concentration in cerebellar Purkinje neurons

We investigated the contribution of large conductance calcium-activated potassium (BK) channels to spontaneous activity of cerebellar Purkinje neurons in mice and rats. In Purkinje neurons which fire tonically, block of BK channels increased the firing rate and caused the neurons to fire irregularly. In Purkinje neurons which exhibited a trimodal pattern of activity, present primarily in mature animals, block of BK channels had little effect on firing rate or regularity but shortened the single cycle duration of the trimodal pattern. The contribution of BK channels to the action potential waveform was also examined. BK channels contributed a brief afterhyperpolarization (AHP) of approximately 3 mV which followed each action potential, but made little contribution to action potential repolarization. The amplitude of the BK-dependent AHP did not change with age although there was an increase in the total AHP. The difference in the contribution of BK channels to the firing rate among the two populations of Purkinje neurons was the consequence of the decrease in the fractional contribution of BK channels to the AHP. We also found that block of BK channels increases intracellular calcium concentration during spontaneous firing. Thus, although BK channels do not affect action potential repolarization, they nevertheless control calcium entry with each action potential by contributing to the AHP.     

Endothelin-1 inhibits voltage-sensitive Ca2+ channels in cultured rat cerebellar granule neurones via the ET-A receptor

 In this study, we have investigated the effect of the vasoconstrictor peptide endothelin-1 (ET-1) on voltage-sensitive Ca²⁺ channels in rat cerebellar granule neurones using the patch-clamp technique. Using amphotericin B perforated-patch recording of whole-cell currents, the Ca²⁺ channel current was inhibited by 28.4±6.4% by 400 nM ET-1, but was unaffected when experiments were repeated using the whole-cell, ruptured-patch configuration. In cell-attached patches, 400 nM ET-1 inhibited unitary L-type Ca²⁺ channel currents (I _Ba) by 85±5%. ET-1 decreased the open probability (NP _o) and the frequency of channel opening and increased the mean closed time of channels. No effects on the mean open time or the time constants for channel opening or closure were observed. L-type Ca²⁺ channel inhibition was dose dependent with an IC₅₀ of 19 nM. The effect of ET-1 was prevented by the combined endothelin-A and -B receptor antagonist PD145065 (10 μM), indicating a receptor-mediated effect. The ET-A receptor antagonist BQ-123 (10 μM) prevented Ca²⁺ channel inhibition by ET-1, while the ET-B receptor agonist sarafotoxin 6c (500 nM) had no effect. The inhibition by ET-1 was not due to a change in the voltage of channel activation. Fura-2 Ca²⁺ imaging showed that no substantial rise in intracellular Ca²⁺ levels occurred during ET-1 application excluding a Ca²⁺-dependent inhibition of the channels. Thus in cultured rat cerebellar granule neurones, ET-1 inhibits L-type Ca²⁺ channels via activation of the ET-A receptor. Inhibition may be mediated by an as yet unidentified cytoplasmic second messenger. Received: 13 March 1998 / Received after revision and accepted: 14 May 1998     

Neurotoxic effects of homocysteine on cerebellar Purkinje neurons in vitro

Whilst a plethora of studies that describe the toxicity of homocysteine to CNS neurons have been published, the effects of homocysteine on the Purkinje neurons of the cerebellum that play a vital role in motor function remain wholly unexplored. We have therefore established cultures of embryonic cerebellar Purkinje neurons and exposed them to a range of concentrations of homocysteine and determined its effects on their survival. The experiments revealed that all concentrations of homocysteine studied, from 50 to 500microM, caused a significant decrease in cerebellar Purkinje neuron number. This loss could be counteracted by the pan-caspase inhibitor z-VAD-fmk in the first 24h following homocysteine exposure, revealing that the initial loss was apoptotic. However, z-VAD-fmk could not prevent homocysteine-mediated loss of cerebellar Purkinje neurons in the longer term, after 6 days in vitro. In addition to its effects on Purkinje neuron survival, homocysteine markedly reduced both the overall magnitude and the complexity of the neurite arbor extended by the cerebellar Purkinje neurons, following 6 days incubation with this agent in vitro. Taken together our data reveal that homocysteine is toxic to cerebellar Purkinje neurons in vitro, inhibiting both their survival and the outgrowth of neurites.     

Kappa-bungarotoxin blockade of nicotine electrophysiological actions in cerebellar Purkinje neurons

The agonistic actions of nicotine in cerebellum were selectively blocked by kappa-bungarotoxin depending on the cell type studied. Nicotine-induced Purkinje cell inhibitions were antagonized by the simultaneous application of this toxin. In contrast, nicotine-induced cerebellar interneuron excitations were unaltered. These findings suggest that kappa-bungarotoxin may be used as a selective pharmacological tool for the study of nicotine actions which are dependent on ganglionic-like receptors, which have been associated with Purkinje cells in previous studies.