Activity of voltage-operated calcium channels in rat cerebellar granule neurons and neuronal survival

Neuronal activity and Ca2+ channel activation play important roles in neuronal survival and development. In cerebellar granule neurons, the culture conditions can induce differential expression of various membrane receptor proteins. To test the hypothesis that culture conditions might affect the activity of voltage-operated Ca2+ channels, the present study analysed the differences in Ca2+ signalling between granule neurons grown in the presence of normal (5 mM) or high (25 mM) KCl. The Ca2+ transients evoked by 50 mM KCl developed similarly in both cultures, as a function of age. In contrast, when compared with neurons grown in 25 mM KCl, a proportion of the neurons grown in normal KCl showed, between days in vitro 4 and 6, a higher Ca2+ transient in response to 12.5 mM KCl. These neurons were less sensitive to the effect of 10 microM nifedipine and, conversely, more sensitive to the effects of 10 microM omega-conotoxin MVIIC when stimulated with 50 mM KCl, indicating that they express preferentially, at this stage, the N- and/or Q-type Ca2+ channels. This period of maximal activity of the N/Q-type Ca2+ channels was associated with a significant increase in the rate of neuronal apoptosis. The present study also shows, by comparing the rates of neuronal apoptosis, that the long-term maintenance in 25 mM KCl appears to "synchronize" and sensitize the neuronal population to the apoptotic process. These results illustrate the differential effect the culture conditions can have on the expression and activity of Ca2+ channels, which, in turn, can modulate neuronal survival.     

Cell intrinsic mechanisms regulate mouse cerebellar granule neuron differentiation

Cerebellar granule cells isolated from postnatal day 7 mice, and cultured in minimal medium containing only insulin-like growth factor-I (IGF-I), both survive and differentiate. This differentiation is marked by neurite growth and expression of genes associated with terminal differentiation, the myocyte-specific enhancer factor 2A (MEF2A) and the α6 subunit of the γ-aminobutyric acid_A receptor (GABA_Aα6). Percoll gradient purified granule cells maintained without IGF-I, in minimal medium alone or in medium containing the antioxidant N-acetylcysteine (NAC), also express MEF2A and GABA_Aα6. Thus, cultured granule neurons can differentiate to some extent cell-autonomously and IGF-I may not be a critical factor for this process.     

N-methyl-D-aspartate promotes the survival of cerebellar granule cells in culture

Our previous studies on the survival-promoting influence of elevated concentrations of extracellular K+ ([K+]e) on cultured cerebellar granule cells led to the proposal that depolarization in vitro mimics the effect of the earliest afferent inputs received by the granule cells in vivo. This, in turn, might be mediated through the stimulation of excitatory amino acid receptors, in particular the N-methyl-D-aspartate-preferring subtype gating ion channels which are also permeable to Ca2+. Here we report that N-methyl-D-aspartate indeed has a dramatic effect on the survival in culture of cells derived from dissociated cerebella of 7-8-day-old rats and cultured in media containing 'low' [K+]e (5-15 mM). In addition to the visual inspection of the cultures, the effect of N-methyl-D-aspartate was quantitatively evaluated, using estimates related to the number of viable cells (determination of DNA and of reduction rate of a tetrazolium salt). Furthermore, proteins which are relatively enriched in either nerve cells (neuronal cell adhesion molecule, D3-protein and synaptin) or in glia (glutamine synthetase) were also measured. The findings showed that the rescue of cells by N-methyl-D-aspartate involved primarily nerve cells and that the survival requirement for N-methyl-D-aspartate, as for high K+, developed between 2 and 4 days in vitro. The effect depended on both the concentration of N-methyl-D-aspartate and the degree of depolarization of the cells: both the potency and the efficacy of N-methyl-D-aspartate were increased as [K+]e was raised from 5 to 15 mM, at which range K+ on its own has little if any influence on granule cell survival. These characteristics are consistent with the voltage-dependence of ion conductance through the N-methyl-D-aspartate receptor-linked channel. The most pronounced effect of N-methyl-D-aspartate was obtained in the presence of 15 mM K+, when cell survival approached that obtained in 'control' cultures (grown in 25 mM K+-containing media without N-methyl-D-aspartate), and the potency of N-methyl-D-aspartate (half-maximal effective concentration, EC50, about 20 microM) was similar to its known affinity in binding to cerebral membranes. The effect of N-methyl-D-aspartate was blocked by the specific receptor antagonist 2-amino-5-phosphonovalerate, which also reduced the limited survival of cells in cultures grown in 'low' K+ in the absence of N-methyl-D-aspartate.(ABSTRACT TRUNCATED AT 400 WORDS)     

TRPC channels as signal transducers

The study of the TRPC cation channels as signal transducers in sensory neurons is in its infancy. Mechanoreceptors that monitor arterial pressure are prime candidates for the involvement of TRPC channels as either primary mechanical transducers or as modulators of the transduction process. Their activity patterns can be regulated by growth factors such as BDNF and by a variety of ligands that activate Gq-coupled receptors, mechanisms that have been shown in heterologous expression systems to activate TRPC channels. We investigated the distribution of TRPC1 and TRPC3–7 in nodose sensory neurons and in their peripheral axons that terminate as mechanosensitive receptors in the aortic arch of the rat. Using immunocytochemical techniques we identified these six TRPC proteins in the soma of the nodose neurons but only TRPC1 and TRPC3–5 were found to distribute to the peripheral axons and the mechanosensory terminals. TRPC1 and TRPC3 extended into the low threshold complex sensory endings with very strong labeling. In contrast, TRPC4 and TRPC5 were found primarily in major branches of the receptor but immunoreactivity was weak in the region where mechanotransduction is presumed to occur. Terminals arising from unmyelinated fibers also expressed TRPC1 and TRPC3–5 but not all fibers expressed all of the channels suggesting that specific TRPC protein may be aligned with previously described subclasses of the unmyelinated C-fibers.     

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.     

Differential targeting and functional specialization of sodium channels in cultured cerebellar granule cells

The ion channel dynamics that underlie the complex firing patterns of cerebellar granule (CG) cells are still largely unknown. Here, we have characterized the subcellular localization and functional properties of Na⁺ channels that regulate the excitability of CG cells in culture. As evidenced by RT-PCR and immunocytochemical analysis, morphologically differentiated CG cells expressed Nav1.2 and Nav1.6, though both subunits appeared to be differentially regulated. Nav1.2 was localized at most axon initial segments (AIS) of CG cells from 8 days in vitro DIV 8 to DIV 15. At DIV 8, Nav1.6 was found uniformly throughout somata, dendrites and axons with occasional clustering in a subset of AIS. Accumulation of Nav1.6 at most AIS was evident by DIV 13–14, suggesting it is developmentally regulated at AIS. The specific contribution of these differentially distributed Na⁺ channels has been assessed using a combination of methods that allowed discrimination between functionally compartmentalized Na⁺ currents. In agreement with immunolocalization, we found that fast activating–fully inactivating Na⁺ currents predominate at the AIS membrane and in the somatic plasma membrane.     

The role of cell density in the survival of cultured cerebellar granule neurons

The dependence for survival of cerebellar granule neurons on the cell density was examined both experimentally and theoretically. The results of batch experiments revealed that the cell survival index (CSI) was inappreciable, if cell density was below a critical level. If cell density exceeded this critical value, CSI increased with the increase in cell density. In addition, CSI was significantly increased by using a conditioned medium from the dense cultures. This suggests that not only cell density promotes survival of neurons, but also an increased concentration of growth factors produced by neurons has a direct effect on the survival of the neurons. A quantitative model describing the distribution of the growth factor at different cell densities was proposed to investigate the role of cell density in the survival of the neurons. We showed the existence of a critical level for cell density, and good agreement in the improvement of CSI was found between the theoretical prediction and the experimental result. Finally, the average concentration of growth factor necessary for cell survival based on our model was in a reasonable range compared to the practice of the addition of neurotrophic factors to the medium of cultured cerebellar granule neurons.     

Modulation of voltage-dependent calcium channels by glutamate in rat cerebellar granule cells in culture

Voltage-dependent calcium channels of cerebellar granule cells maintained in a Ca²⁺-free depolarising solution were recorded using the cell-attached configuration of the patch-clamp technique. An increase in the maximum open probability of calcium channels and a shift in their activation curve toward more hyperpolarising potentials were found in the presence of glutamate, a natural, excitatory amino acid. Such an increase in the activity of calcium channels was not due to ionic fluxes activated by glutamate, and was probably produced by a second messenger pathway triggered by the binding of glutamate to its receptor.     

Involvement of endogenous PACAP expression in the activity-dependent survival of mouse cerebellar granule cells

Membrane depolarization causes Ca2+ influx through L-type voltage-dependent calcium channels (L-VDCC), which promotes the activity-dependent survival of mouse cerebellar granule cells (CGCs). Although exogenously added pituitary adenylate cyclase activating polypeptide (PACAP) is effective in promoting the survival of CGCs, it is unknown whether PACAP is synthesized in CGCs and involved in the activity-dependent survival of CGCs. In this study, we found that the PACAP gene was activated in depolarized CGCs cultured at 25 mM KCl (high K+), independently of de novo protein synthesis. In addition, the PACAP immunoreactivity increased through the activation of L-VDCC in depolarized CGCs, indicating that PACAP is concomitantly produced with PACAP mRNA in an activity-dependent manner. Exogenously added PACAP attenuated the apoptosis of CGCs through a specific interaction with PACAP receptors. Furthermore, a PACAP receptor antagonist, PACAP(6-38), reduced the survival of CGCs at high K+. These findings indicate that endogenous PACAP production induced by Ca2+ signals exerts a survival effect on CGCs via PACAP receptors, which, at least in part, accounts for the activity-dependent survival of CGCs.     

Differentiation and developmental origin of cerebellar granule neuron ectopia in protein O-mannose UDP-N-acetylglucosaminyl transferase 1 knockout mice

The cerebellar cortex of protein O-mannose UDP-N-acetylglucosaminyl transferase 1 (POMGnT1) knockout mice contains discrete clusters of granule neurons that fail to migrate from the external germinal layer (EGL) to the internal granule cell layer (IGL). To test the hypothesis that the breaches in the pial basement membrane and glia limitans contribute to the formation of such heterotopias, POMGnT1 deficient mice were used to examine the mechanisms underlying these migration defects. The basement membrane, glia limitans, and granule neuron development were assessed with protein markers and immunofluorescent microscopy. Further, the integrity of the pial basement membrane, and granule neuron differentiation state were assessed by electron microscopy. Localized breaches in pial basement membrane and disruptions in the glia limitans were strongly associated with ectopia of EGL cells. In such ectopias, Bergmann glia fibers were retracted and disorganized with very few protruded into the ectopic area. Thus, migration failure was correlated with a compromised Bergmann glia scaffold. Nevertheless, the ectopic EGL cells showed characteristics of differentiated granule neurons and formed synapses with mossy fibers. Altogether, these results suggest that pial basement membrane breaches and glia limitans disruptions are the underlying causes of cerebellar granule neuron ectopia in POMGnT1 knockout mice. Moreover, migration into the IGL is not required for granule cell acquisition of certain differentiated characteristics.     

Schwann cells genetically modified to express neurotrophins promote spiral ganglion neuron survival in vitro

The intracochlear infusion of neurotrophic factors via a mini-osmotic pump has been shown to prevent deafness-induced spiral ganglion neuron (SGN) degeneration; however, the use of pumps may increase the incidence of infection within the cochlea, making this technique unsuitable for neurotrophin administration in a clinical setting. Cell- and gene-based therapies are potential therapeutic options. This study investigated whether Schwann cells which were genetically modified to over-express the neurotrophins brain-derived neurotrophic factor (BDNF) or neurotrophin 3 (Ntf3, formerly NT-3) could support SGN survival in an in vitro model of deafness. Co-culture of either BDNF over-expressing Schwann cells or Ntf3 over-expressing Schwann cells with SGNs from early postnatal rats significantly enhanced neuronal survival in comparison to both control Schwann cells and conventional recombinant neurotrophin proteins. Transplantation of neurotrophin over-expressing Schwann cells into the cochlea may provide an alternative means of delivering neurotrophic factors to the deaf cochlea for therapeutic purposes.     

Comparative study of survival signal withdrawal- and 4-hydroxynonenal-induced cell death in cerebellar granule cells

The lipid peroxidation product, 4-hydroxynonenal (HNE), has been shown to induce apoptosis in PC12 cells and hippocampal neurons. We compared the degree of cell death induced by survival signal withdrawal (K+ and serum deprivation) with that induced by HNE, and investigated whether agents that block survival signal withdrawal-induced apoptosis could also prevent HNE-induced cell death in cultured cerebellar granule cells. Cell death induced by K+ and serum deprivation was inhibited by cycloheximide, a CPP 32-like protease inhibitor (Ac-DEVD-CHO) and a pituitary adenylate cyclase-activating polypeptide (PACAP)-38. In addition, nuclear cyclic AMP responsive element (CRE)- and activator protein 1 (AP-1) DNA-binding activities were increased 2 h after K+ and serum withdrawal, and these increases were inhibited by cycloheximide, Ac-DEVD-CHO and PACAP 38. Although these agents also blocked HNE-induced cell death, consistent with their efficacy in preventing survival signal withdrawal-induced cells death, CRE and AP-1 DNA-binding activities were decreased in a time-dependent manner during HNE-induced cell death. These results suggest that mechanistic differences exist between apoptosis induced by HNE and that induced by withdrawal of survival signals in cerebellar granule neurons.     

Extracellular signal-regulated kinases are not involved in activity-dependent survival or apoptosis in cerebellar granule neurons

Cerebellar granule neurons (CGNs) depend on potassium depolarization for survival and undergo apoptosis when deprived of depolarizing concentration of potassium. Extracellular signal-regulated kinases (ERK1/2) are thought to be activated in response to potassium depolarization and responsible for the activity-dependent survival in CGNs, but one recent study has revealed that ERK1/2 is activated by potassium deprivation and is required for apoptosis of CGNs. In this study we showed that ERK1/2 was inactivated, rather than activated, by potassium deprivation, indicating a lack of ERK1/2 involvement in potassium deprivation-induced apoptosis. Furthermore, suppression of potassium depolarization-induced activation of ERK1/2 with chemical inhibitor U0126 or PD98059 had no influence on the pro-survival effect of potassium depolarisation. Thus, ERK1/2 was not required for potassium depolarization-dependent survival of CGNs. Taken together, our findings suggest that ERK1/2 is not involved in activity-dependent survival or apoptosis of CGNs.     

Nitric oxide lacks direct effect on TRPC5 channels but suppresses endogenous TRPC5-containing channels in endothelial cells

TRPC5 is a member of the canonical transient receptor potential (TRPC) family of proteins that forms cationic channels either through homomultimeric assembly or heteromultimeric coordination with other TRPC proteins. It is expressed in a variety of cells including central neurones and endothelial cells and has susceptibility to stimulation by multiple factors. Here we investigated if TRPC5 is sensitive to nitric oxide. Mouse TRPC5 or human TRPC5 was over-expressed in HEK293 cells, and TRPC5 activity was determined by measuring the cytosolic Ca²⁺ concentration with an indicator dye or by recording membrane current under voltage clamp. TRPC5 activity could be evoked by carbachol acting at muscarinic receptors, lanthanum, or a reducing agent. However, S-nitroso-N-acetylpenicillamine (SNAP) and diethylamine NONOate (DEA-NONOate) failed to stimulate or inhibit TRPC5 at concentrations that generated nitric oxide, caused vasorelaxation, or suppressed activity of TRPC6 via protein kinase G. At high concentrations, SNAP (but not DEA-NONOate) occasionally stimulated TRPC5 but the effect was confounded by background TRPC5-independent Ca²⁺ signals. Endogenous Ca²⁺-entry in bovine aortic endothelial cells (BAECs) was suppressed by SNAP; TRPC5 blocking antibody or dominant-negative mutant TRPC5 suppressed this Ca²⁺ entry and occluded the effect of SNAP. The data suggest that nitric oxide is not a direct modulator of homomeric TRPC5 channels but may inhibit endogenous BAEC channels that contain TRPC5.     

TRPC1 channels regulate directionality of migrating cells

Cell migration depends on the generation of structural asymmetry and on different steps: protrusion and adhesion at the front and traction and detachment at the rear part of the cell. The activity of Ca²⁺ channels coordinate these steps by arranging intracellular Ca²⁺ signals along the axis of movement. Here, we investigated the role of the putative mechanosensitive canonical transient receptor potential channel 1 (TRPC1) in cell migration. We analyzed its function in transformed renal epithelial (Madin–Darby canine kidney-focus) cells with variation of TRPC1 expression. As shown by time lapse video microscopy, TRPC1 knockdown cells have partially lost their polarity and the ability to persistently migrate into a given direction. This failure is linked to the suppression of a local Ca²⁺ gradient at the front of migrating TRPC1 knockdown cells, whereas TRPC1 overexpression leads to steeper Ca²⁺ gradients. We propose that the Ca²⁺ signaling events regulated by TRPC1 within the lamellipodium determine polarity and directed cell migration. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.