Currents In Cerebellar Granule Cells Research Articles

Mechanisms of GABAB receptor enhancement of extrasynaptic GABAA receptor currents in cerebellar granule cells

Many neurons, including cerebellar granule cells, exhibit a tonic GABA current mediated by extrasynaptic GABAA receptors. This current is a critical regulator of firing and the target of many clinically relevant compounds. Using a combination of patch clamp electrophysiology and photolytic uncaging of RuBi-GABA we show that GABAB receptors are tonically active and enhance extrasynaptic GABAA receptor currents in cerebellar granule cells. This enhancement is not associated with meaningful changes in GABAA receptor potency, mean channel open-time, open probability, or single-channel current. However, there was a significant (~40%) decrease in the number of channels participating in the GABA uncaging current and an increase in receptor desensitization. Furthermore, we find that adenylate cyclase, PKA, CaMKII, and release of Ca2+ from intracellular stores are necessary for modulation of GABAA receptors. Overall, this work reveals crosstalk between postsynaptic GABAA and GABAB receptors and identifies the signaling pathways and mechanisms involved.

Altered Sodium and Potassium, but not Calcium Currents in Cerebellar Granule Cells in an In Vitro Model of Neuronal Injury.

Acute injury of central nervous system (CNS) starts a cascade of morphological, molecular, and functional changes including formation of a fibrotic scar, expression of transforming growth factor beta 1 (TGF-β1), and expression of extracellular matrix proteins leading to arrested neurite outgrowth and failed regeneration. We assessed alteration of electrophysiological properties of cerebellar granule cells (CGCs) in two in vitro models of neuronal injury: (i) model of fibrotic scar created from coculture of meningeal fibroblasts and cerebral astrocytes with addition of TGF-β1; (ii) a simplified model based on administration of TGF-β1 to CGCs culture. Both models reproduced suppression of neurite outgrowth caused by neuronal injury, which was equally restored by chondroitinase ABC (ChABC), a key disruptor of fibrotic scar formation. Voltage-dependent calcium current was not affected in either injury model. However, intracellular calcium concentration could be altered as an expression of inositol trisphosphate receptor type 1 was suppressed by TGF-β1 and restored by ChABC. Voltage-dependent sodium current was significantly suppressed in CGCs cultured on a model of fibrotic scar and was only partly restored by ChABC. Administration of TGF-β1 significantly shifted current-voltage relation of sodium current toward more positive membrane potential without change to maximal current amplitude. Both transient and sustained potassium currents were significantly suppressed on a fibrotic scar and restored by ChABC to their control amplitudes. In contrast, TGF-β1 itself significantly upregulated transient and did not change sustained potassium current. Observed changes of voltage-dependent ion currents may contribute to known morphological and functional changes in injured CNS.

Alcohol Suppresses Tonic GABAA Receptor Currents in Cerebellar Granule Cells in the Prairie Vole: A Neural Signature of High-Alcohol-Consuming Genotypes.

Evidence indicates that the cerebellum plays a role in genetic predilection to excessive alcohol (ethanol [EtOH]) consumption in rodents and humans, but the molecular mechanisms mediating such predilection are not understood. We recently determined that EtOH has opposite actions (enhancement or suppression) on tonic GABAA receptor (GABAA R) currents in cerebellar granule cells (GCs) in low- and high-EtOH-consuming rodents, respectively, and proposed that variation in GC tonic GABAA R current responses to EtOH contributes to genetic variation in EtOH consumption phenotype. Voltage-clamp recordings of GCs in acutely prepared slices of cerebellum were used to evaluate the effect of EtOH on GC tonic GABAA R currents in another high-EtOH-consuming rodent, prairie voles (PVs). EtOH (52mM) suppressed the magnitude of the tonic GABAA R current in 57% of cells, had no effect in 38% of cells, and enhanced the tonic GABAA R current in 5% of cells. This result is similar to GCs from high-EtOH-consuming C57BL/6J (B6) mice, but it differs from the enhancement of tonic GABAA R currents by EtOH in low-EtOH-consuming DBA/2J (D2) mice and Sprague Dawley (SD) rats. EtOH suppression of tonic GABAA R currents was not affected by the sodium channel blocker, tetrodotoxin (500nM), and was independent of the frequency of phasic GABAA R-mediated currents, suggesting that suppression is mediated by postsynaptic actions on GABAA Rs, rather than a reduction of GABA release. Finally, immunohistochemical analysis of neuronal nitric oxide synthase (nNOS; which can mediate EtOH enhancement of GABA release) demonstrated that nNOS expression in the GC layer of PV cerebellum was similar to the levels seen in B6 mice, both being significantly reduced relative to D2 mice and SD rats. Combined, these data highlight the GC GABAA R response to EtOH in another species, the high-EtOH-consuming PV, which correlates with EtOH consumption phenotype and further implicates the GC GABAA R system as a contributing mechanism to high EtOH consumption.

FGF14 Regulates Presynaptic Ca2+ Channels and Synaptic Transmission

Fibroblast growth factor homologous factors (FHFs) are not growth factors, but instead bind to voltage-gated Na+ channels (NaV) and regulate their function. Mutations in FGF14, an FHF that is the locus for spinocerebellar ataxia 27 (SCA27), are believed to be pathogenic because of a dominant-negative reduction of NaV currents in cerebellar granule cells. Here, we demonstrate that FGF14 also regulates members of the presynaptic CaV2 Ca2+ channel family. Knockdown of FGF14 in granule cells reduced Ca2+ currents and diminished vesicular recycling, a marker for presynaptic Ca2+ influx. As a consequence, excitatory postsynaptic currents (EPSCs) at the granule cell to Purkinje cell synapse were markedly diminished. Expression of the SCA27-causing FGF14 mutant in granule cells exerted a dominant-negative reduction in Ca2+ currents, vesicular recycling, and the resultant EPSCs in Purkinje cells. Thus, FHFs are multimodal, regulating several discrete neuronal signaling events. SCA27 most likely results at least in part from dysregulation of Ca2+ channel function.

Bestrophin1 Channels are Insensitive to Ethanol and Do not Mediate Tonic GABAergic Currents in Cerebellar Granule Cells

The granule cell layer of the cerebellum functions in spatio-temporal encoding of information. Granule cells (GCs) are tonically inhibited by spillover of GABA released from Golgi cells and this tonic inhibition is facilitated by acute ethanol. Recently, it was demonstrated that a specialized Ca2+-activated anion-channel, bestrophin1 (Best1), found on glial cells, can release GABA that contributes up to 50–75% of the tonic GABAergic current. However, it is unknown if ethanol has any actions on Best1 function. Using whole-cell electrophysiology, we found that recombinant Best1 channels expressed in HEK-293 cells were insensitive to 40 and 80 mM ethanol. We attempted to measure the Best1-mediated component of the tonic current in slices using 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We confirmed that this agent blocks recombinant Best1 channels. Unexpectedly, we found that NPPB significantly potentiated the tonic current and the area and decay of GABAA-mediated spontaneous inhibitory post-synaptic currents (IPSCs) in GCs in rodent slices under two different recording conditions. To better isolate the Best1-dependent tonic current component, we blocked the Golgi cell component of the tonic current with tetrodotoxin and found that NPPB similarly and significantly potentiated the tonic current amplitude and decay time of miniature IPSCs. Two other Cl−-channel blockers were also tested: 4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS) showed no effect on GABAergic transmission, while niflumic acid (NFA) significantly suppressed the tonic current noise, as well as the mIPSC frequency, amplitude, and area. These data suggest that acute ethanol exposure does not modulate Best1 channels and these findings serve to challenge recent data indicating that these channels participate in the generation of tonic GABAergic currents in cerebellar GCs.

Effects of Ethanol on Tonic GABA Currents in Cerebellar Granule Cells and Mammalian Cells Recombinantly Expressing GABAAReceptors

The effects of ethanol on the GABA(A) receptors, which are regarded as one of the most important target sites of ethanol, are very controversial, ranging from potentiation to no effect. The delta subunit-containing GABA(A) receptors expressed in Xenopus oocytes were recently reported to be potently augmented by ethanol. We performed patch-clamp experiments using the cerebellar granule cells and mammalian cells expressing recombinant GABA(A) receptors. In granule cells, the sensitivity to GABA increased from 7 to 11 days in vitro. Furosemide, an antagonist of alpha6-containing GABA(A) receptors, inhibited GABA-induced currents more potently at 11 to 14 days than at 7 days. Ethanol at 30 mM had either no effect or an inhibitory effect on currents induced by low concentrations of GABA in granule cells. On alpha4beta2delta, alpha6beta2delta, or alpha6beta3deltaGABA(A) receptors expressed in Chinese hamster ovary cells, ethanol at 10, 30, and 100 mM had either no effect or an inhibitory effect on GABA currents. Ethanol inhibition of GABA(A) receptor was observed in all of the subunit combinations examined. In contrast, the perforated patch-clamp method to record the GABA currents revealed ethanol effects on the alpha6beta2delta subunits ranging from slight potentiation to slight inhibition. Ethanol seems to exert a dual action on the GABA(A) receptors and the potentiating action may depend on intracellular milieu. Thus, the differences between the GABA(A) receptors expressed in mammalian host cells and those in Xenopus oocytes in the response to ethanol might be due to changes in intracellular components under patch-clamp conditions.

Inhibition of constitutive inward rectifier currents in cerebellar granule cells by pharmacological and synaptic activation of GABAB receptors

gamma-Aminobutyric acid (GABA)(B) receptors are known to enhance activation of Kir3 channels generating G-protein-dependent inward rectifier K(+)-currents (GIRK). In some neurons, GABA(B) receptors either cause a tonic GIRK activation or generate a late K(+)-dependent inhibitory postsynaptic current component. However, other neurons express Kir2 channels, which generate a constitutive inward rectifier K(+)-current (CIRK) without requiring G-protein activation. The functional coupling of CIRK with GABA(B) receptors remained unexplored so far. About 50% of rat cerebellar granule cells in the internal granular layer of P19-26 rats showed a sizeable CIRK current. Here, we have investigated CIRK current regulation by GABA(B) receptors in cerebellar granule cells, which undergo GABAergic inhibition through Golgi cells. By using patch-clamp recording techniques and single-cell reverse transcriptase-polymerase chain reaction in acute cerebellar slices, we show that granule cells co-express Kir2 channels and GABA(B) receptors. CIRK current biophysical properties were compatible with Kir2 but not Kir3 channels, and could be inhibited by the GABA(B) receptor agonist baclofen. The action of baclofen was prevented by the GABA(B) receptor blocker CGP35348, involved a pertussis toxin-insensitive G-protein-mediated pathway, and required protein phosphatases inhibited by okadaic acid. GABA(B) receptor-dependent CIRK current inhibition could also be induced by repetitive GABAergic transmission at frequencies higher than the basal autorhythmic discharge of Golgi cells. These results suggest therefore that GABA(B) receptors can exert an inhibitory control over CIRK currents mediated by Kir2 channels. CIRK inhibition was associated with an increased input resistance around rest and caused a approximately 5 mV membrane depolarization. The pro-excitatory action of these effects at an inhibitory synapse may have an homeostatic role re-establishing granule cell readiness under conditions of strong inhibition.

Contributions of the GABAAReceptor α6 Subunit to Phasic and Tonic Inhibition Revealed by a Naturally Occurring Polymorphism in the α6 Gene

GABAA receptors (GABARs) are heteromultimeric proteins composed of five subunits. The specific subunit composition determines critical properties of a GABAR such as pharmacological sensitivities and whether the receptor contributes to synaptic or extrasynaptic forms of inhibition. Classically, synaptic but not extrasynaptic GABARs are thought to respond to benzodiazepines, whereas the reverse has been suggested for ethanol. To examine the effects of subunit composition on GABAR function in situ, we took advantage of two naturally occurring alleles of the rat gene for GABAR subunit alpha6 (Gabra6(100R) and Gabra6(100Q)). Depending on their subunit partners, these two variants of alpha6 can lead to differential sensitivities to benzodiazepines and ethanol. An examination of synaptic and extrasynaptic GABA-mediated currents in cerebellar granule cells from Gabra6(100R/100R) and Gabra6(100Q/100Q) rats uncovered marked allele-dependent differences in benzodiazepine sensitivity. Unexpectedly, we found that the benzodiazepines flunitrazepam and diazepam enhanced extrasynaptic inhibition mediated by delta subunit-containing GABARs in Gabra6(100Q/100Q) rats. Complementary experiments on recombinant GABARs confirmed that, at subsaturating [GABA], flunitrazepam potentiates alpha6/delta subunit-containing GABARs. Based on data and a simple theoretical analysis, we estimate that the average extrasynaptic [GABA] is approximately 160 nm in perfused slices. These results (1) demonstrate contributions of alpha6 subunits to both synaptic and extrasynaptic GABA responses, (2) establish that delta subunit-containing GABARs are benzodiazepine sensitive at subsaturating [GABA] and, (3) provide an empirical estimate of extrasynaptic [GABA] in slices.

Anticonvulsant phenytoin affects voltage-gated potassium currents in cerebellar granule cells

The anticonvulsant drug phenytoin (diphenylhydantoin, DPH) was examined for its action on potassium currents in cerebellar granule cells using the whole-cell patch-clamp technique. Granular cells expressed two main types of voltage-dependent potassium currents: the first, sensitive to Tetraethylammonium ion (TEA), resembles a delayed rectifier K + channel ( I d); the second shows biophysical and pharmacological properties similar to an I A-type potassium current. Phenytoin blocks the I A current in a dose-dependent manner, with an apparent dissociation constant K d of (73±7) μM. The drug shifts the steady-state inactivation curves towards a more negative potential, stabilizing the inactivated state, while the activation kinetics remain unaffected. The estimated K d when the cell is held to −100 mV (closed state of the channel) is 145±8 μM which decreases to 35±10 μM at −80 mV holding potential (partial inactivation of the channel). Phenytoin shows a discriminant behaviour between the two different types of potassium channels because at high concentration the effect of the drug on the delayed rectifier K + channel is negligible.

Ablation of P/Q-type Ca(2+) channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the alpha(1A)-subunit.

The Ca(2+) channel alpha(1A)-subunit is a voltage-gated, pore-forming membrane protein positioned at the intersection of two important lines of research: one exploring the diversity of Ca(2+) channels and their physiological roles, and the other pursuing mechanisms of ataxia, dystonia, epilepsy, and migraine. alpha(1A)-Subunits are thought to support both P- and Q-type Ca(2+) channel currents, but the most direct test, a null mutant, has not been described, nor is it known which changes in neurotransmission might arise from elimination of the predominant Ca(2+) delivery system at excitatory nerve terminals. We generated alpha(1A)-deficient mice (alpha(1A)(-/-)) and found that they developed a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying approximately 3-4 weeks after birth. P-type currents in Purkinje neurons and P- and Q-type currents in cerebellar granule cells were eliminated completely whereas other Ca(2+) channel types, including those involved in triggering transmitter release, also underwent concomitant changes in density. Synaptic transmission in alpha(1A)(-/-) hippocampal slices persisted despite the lack of P/Q-type channels but showed enhanced reliance on N-type and R-type Ca(2+) entry. The alpha(1A)(-/-) mice provide a starting point for unraveling neuropathological mechanisms of human diseases generated by mutations in alpha(1A).

Antisense oligonucleotides against alpha1E reduce R-type calcium currents in cerebellar granule cells.

Many neurons of the central nervous system display multiple high voltage-activated Ca2+ currents, pharmacologically classified as L-, N-, P-, Q-, and R-type. Of these current types, the R-type is the least understood. The leading candidate for the molecular correlate of R-type currents in cerebellar granule cells is the alpha1E subunit, which yields Ca2+ currents very similar to the R-type when expressed in heterologous systems. As a complementary approach, we tested whether antisense oligonucleotides against alpha1E could decrease the expression of R-type current in rat cerebellar granule neurons in culture. Cells were supplemented with either antisense or sense oligonucleotides and whole-cell patch clamp recordings were obtained after 6-8 days in vitro. Incubation with alpha1E antisense oligonucleotide caused a 52.5% decrease in the peak R-type current density, from -10 +/- 0.6 picoamperes/picofarad (pA/pF) (n = 6) in the untreated controls to -4.8 +/- 0.8 pA/pF (n = 11) (P < 0.01). In contrast, no significant changes in the current expression were seen in sense oligonucleotide-treated cells (-11.3 +/- 3.2 pA/pF). The specificity of the alpha1E antisense oligonucleotides was supported by the lack of change in estimates of the P/Q current amplitude. Furthermore, antisense and sense oligonucleotides against alpha1A did not affect R-type current expression (-11.5 +/- 1.7 and -11.7 +/- 1.7 pA/pF, respectively), whereas the alpha1A antisense oligonucleotide significantly reduced whole cell currents under conditions in which P/Q current is dominant. Our results support the hypothesis that members of the E class of alpha1 subunits support the high voltage-activated R-type current in cerebellar granule cells.

Suppression of Neuronal and Cardiac Transient Outward Currents by Viral Gene Transfer of Dominant-Negative Kv4.2 Constructs

To probe the molecular identity of transient outward (A-type) potassium currents, we expressed a truncated version of Kv4.2 in heart cells and neurons. The rat Kv4.2-coding sequence was truncated at a position just past the first transmembrane segment and subcloned into an adenoviral shuttle vector downstream of a cytomegalovirus promoter (pE1Kv4.2ST). We hypothesized that this construct would act as a dominant-negative suppressor of currents encoded by the Kv4 family by analogy to Kv1 channels. Cotransfection of wild-type Kv4.2 with a beta-galactosidase expression vector in Chinese hamster ovary (CHO)-K1 cells produced robust transient outward currents (Ito) after two days (14.0 pA/pF at 50 mV, n = 5). Cotransfection with pE1Kv4.2ST markedly suppressed the Kv4.2 currents (0.8 pA/pF, n = 6, p < 0.02; cDNA ratio of 2:1 Kv4.2ST:wild type), but in parallel experiments, it did not alter the current density of coexpressed Kv1.4 or Kv1.5 channels. Kv4.2ST also effectively suppressed rat Kv4.3 current when coexpressed in CHO-K1 cells. We then engineered a recombinant adenovirus (AdKv4.2ST) designed to overexpress Kv4.2ST in infected cells. A-type currents in rat cerebellar granule cells were decreased two days after AdKv4. 2ST infection as compared with those infected by a beta-galactosidase reporter virus (116.0 pA/pF versus 281.4 pA/pF in Ad beta-galactosidase cells, n = 8 each group, p < 0.001). Likewise, Ito in adult rat ventricular myocytes was suppressed by AdKv4.2ST but not by Adbeta-galactosidase (8.8 pA/pF versus 21.4 pA/pF in beta-galactosidase cells, n = 6 each group, p < 0.05). Expression of a GFP-Kv4.2ST fusion construct enabled imaging of subcellular protein localization by confocal microscopy. The protein was distributed throughout the surface membrane and intracellular membrane systems. We conclude that genes from the Kv4 family are the predominant contributors to the A-type currents in cerebellar granule cells and Ito in rat ventricle. Overexpression of dominant-negative constructs may be of general utility in dissecting the contributions of various ion channel genes to excitability.

Spontaneous GABA-mediated synaptic currents in cerebellar granule cells in culture.

The whole-cell configuration of the patch clamp technique was used to characterize the electrophysiological properties of spontaneous GABA-mediated synaptic currents in cerebellar granule cells grown in a low (5 mM) potassium medium. In the presence of kynurenic acid (1 mM), to block the excitatory drive, bicuculline-sensitive synaptic events were recorded. Their amplitude distribution could be fitted by several Gaussians having the same interpeak distance. In tetrodotoxin (TTX, 1 microM) spontaneous miniature events occurred at a lower frequency. Spontaneous currents reversed polarity at 8.17 +/- 0.63 mV, a potential close to ECl; the decay phase could be fitted with a single exponential having at -60 mV a time constant of 48.7 +/- 1.5 ms. In low noise recordings, channel closing could be resolved during the decay phase of miniature events. It appeared that a single quantum of GABA opened few channels on the postsynaptic membrane.

The effect of intracellular Ca2+ on GABA-activated currents in cerebellar granule cells in culture

The patch clamp technique was used to study the effects of intracellular free calcium ([Ca2+]i) on GABAA-evoked whole-cell and single channel currents of cultured cerebellar granule cells. Changes in [Ca2+]i were obtained by adding to the extracellular solution the calcium ionophore A23187 (2 microM). The relationship between [Ca2+]i and [Ca2+]o in the presence or absence of A23187 was assessed using fluorimetric measurements from Fura-2 loaded cells. In 2 mM [Ca2+]o and A23187, [Ca2+]i was about 1.5 microM, whereas in the absence of A23187 it was about 250 nM. In whole-cell experiments (symmetrical chloride concentrations) at -50 mV, GABA (0.5 microM) evoked inward currents that did not desensitize. Bath application of A23187 significantly reduced the steady-state amplitude of GABA currents by 37 +/- 6%. Single channel currents activated by GABA (0.5 microM) were also recorded in the outside-out configuration of the patch clamp technique. Kinetic analysis of single channel events revealed that A23187 significantly increased the long closed time constant (tau c3) without affecting the open time constants (tau o1 and tau o2) or the short and medium closed time constants (tau c1 and tau c2). Moreover, application of A23187 induced a significant reduction of burst duration (tau b). We conclude that a rise in [Ca2+]i by A23187 may decrease the binding affinity of GABA for the GABAA receptor.

Properties of the transient potassium currents in cerebellar granule cells.

Macroscopic potassium currents were studied in cell-attached and inside-out patches from rat cerebellar granule cells. They were related with transient IA type potassium channels. Currents activated rapidly at potentials higher than -40 mV and did not inactivate completely. The magnitude of the current diminished when the membrane patches were excised. No differences in the activation and inactivation properties were found between patches in the integral cells and cell free membrane patches. A biophysical description of the currents is presented.

The relationship between synaptogenesis and expression of voltage-dependent currents in cerebellar granule cells in situ

In this work we consider the ontogenetic changes of membrane currents and their relationship with synaptogenesis in cerebellar granule cells. Recordings were performed in whole-cell patch-clamp configuration from cerebellar slices obtained from 4 to 31-day-old rats. Granule cells in the external granular layer, and non-connected granule cells in the internal granular layer expressed outward currents, and inconstantly also small Ca 2+ currents, but no fast Na + currents. Most connected granule cells expressed Ca 2+ and Na + currents. These data indicate that Ca 2+ and Na + current development occurs after synapse formation, while outward (K +) currents begin their development before. Mixed NMDA/non-NMDA synaptic currents were observed at all stages, while synaptic currents with a prominent NMDA component were observed exclusively at immature stages. At P4, ie 1–2 days after the arrival of the first granule cells in the internal granular layers, some granule cells already expressed mature synaptic and voltage-dependent currents, suggesting that establishment of mossy fibre synapses and development of membrane properties takes just 1 – 2 days to complete. Starting at P4, the probability of activating mossy fibre currents, and sizeable Ca 2+ and Na + currents increased at a similar rate, attaining a plateau level around P20. Average amplitude of Na + and outward currents decreased until P10 and then increased attaining plateau soon beyond P20. Average amplitude of Ca 2+ currents increased monotonically. The time courses of probability and average current amplitude curves are likely explained by changes in the rate of accumulation of migrating granule cells in the internal granular layer, and by changes in granule cell membrane surface extension. These data suggest a relevant role for the process of synapse formation in inducing the expression of new channels in the developing granule cells, which may involve Ca 2+ influx through the NMDA channel.