Mechanisms for Synchronous Calcium Oscillations in Cultured Rat Cerebellar Neurons

Removal of Mg²⁺ caused oscillations of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the membrane potential in cultured cerebellar granule neurons. Oscillations of [Ca²⁺]_i were synchronous in all the cells, and were restricted to the neurons (immunocytochemically identified) that responded to exogenous N -methyl-D-aspartate (NMDA). Oscillations were blocked by Ca²⁺ removal, nickel, NMDA receptor antagonists, ω-agatoxin IVA, tetrodotoxin, sodium removal and γ-aminobutyric acid, but not by dihydropyridines, ω-conotoxin M VIIA or by emptying the intracellular Ca²⁺ stores with thapsigargin or ionomycin. The upstroke of the [Ca²⁺]_i oscillations coincided in time with an increase in manganese permeability of the plasma membrane. Propagation of the [Ca²⁺]_i wave followed more than one pathway and the spatiotemporal pattern changed with time. Membrane potential oscillations consisted of transient slow depolarizations of ˜20 mV with faster phasic activity superimposed. We propose that the synchronous [Ca²⁺]_i oscillations are the expression of irradiation of random excitation through a neuronal network requiring generation of action potentials and functional glutamatergic synapses. Oscillations of [Ca²⁺]_i are due to cyclic Ca²⁺ entry through NMDA receptor channels activated by synaptic release of glutamate, which requires Ca²⁺ entry through P-type Ca²⁺ channels activated by action potentials at the presynaptic terminal.     

Microcalcification after excitotoxicity is enhanced in transgenic mice expressing parvalbumin in all neurones, may commence in neuronal mitochondria and undergoes structural modifications over time

Aims:  Parenchymal microcalcification in the brain coincides with neurodegenerative diseases, but is also frequently found in neurologically normal individuals. The origin and role of this process are still under debate. Parvalbumin (PV) is a protein acting as a Ca²⁺ buffer and Ca²⁺ shuttle towards intracellular Ca²⁺ sinks, like mitochondria and the endoplasmic reticulum. Constitutively, it is present in a subset of inhibitory neurones. In transgenic mice expressing pan-neuronal PV, the mitochondrial volume is reduced. We tested whether elevated levels of intraneuronal [Ca²⁺] and reduced mitochondrial volume in the neurone interfere with the generation of parenchymal microcalcification. Methods:  The striatum of wild type and transgenic mice was injected with the glutamate receptor agonist ibotenic acid (IBO), which is known to induce not only excitotoxic neurodegeneration, but also parenchymal calcification. Sections were studied by light and electron microscopy at various time points after IBO application. Results:  Morphometric analysis 2, 4 and 20 weeks after IBO application revealed microcalcification in transgenic and wild type mice; the calcification process, however, was enhanced and accelerated in the transgenic animals. Ultrastructural analyses suggest neuronal mitochondria as the nucleators of the deposits which consist of hydroxyapatite. The time-dependent changes in size and surface structure of the deposits indicate the presence of biological mechanisms in the brain promoting regression of bioapatites. Conclusions:  The overload of intraneuronal [Ca²⁺] in combination with impaired mitochondrial function activates neuronal microcalcification. It is hypothesized that this process is an alternative/adaptive mechanism of the neurone to reduce further brain damage.     

Complete brain-type creatine kinase deficiency in mice blocks seizure activity and affects intracellular calcium kinetics

Purpose:   Brain-type creatine kinase (CK-B) and ubiquitous mitochondrial creatine kinase (UbCKmit) act as components of local phosphocreatine ATP shuttles that help in the compartmentalization and maintenance of pools of high-energy phosphate molecules in both neurons and glial cells. We investigated the role of these brain-type creatine kinases during extreme energy-demanding conditions in vivo (generalized tonic–clonic seizures) and in vitro.
Methods:   The physiologic response of wild-types and mice lacking both CK-B and UbCKmit (CK--/--mice) to pentylenetetrazole (PTZ)–induced seizures was measured using electroencephalography (EEG) recordings and behavioral monitoring. In vitro intracellular Ca²⁺ kinetics in hippocampal granule neurons were monitored upon single and repetitive depolarizations.
Results:   PTZ induced in only a few CK--/-- mice PTZ seizure-like behavior, but in all wild-types a full-blown seizure. EEG analysis showed that preseizure jerking was associated with high-amplitude discharges. Wild-type EEG recordings showed continuous runs of rhythmic 4–6 Hz activity, whereas no rhythmic EEG activities were observed in the few CK--/-- mice that developed a behavioral seizure. All other CK--/-- mice displayed a sudden postictal depression without any development of a generalized seizure. Hippocampal granule neurons of CK--/-- mice displayed a higher Ca²⁺ removal speed following repetitive KCl-induced depolarizations.
Discussion:   Deficiency for creatine kinase is affecting brain energy metabolism and will likely contribute to the disturbance of seizure development. Because CK--/-- hippocampal neurons exhibited an increase in Ca²⁺ removal rate of elevated intracellular levels, we conclude that altered Ca²⁺ clearance in CK--/-- neurons could play a role in the abnormal EEG and seizure activity.     

Depolarization promotes GAD 65-mediated GABA synthesis by a post-translational mechanism in neural stem cell-derived neurons

Neuronal activity regulates neurogenesis and neuronal differentiation in the mammalian brain. The commencement of neurotransmitter expression establishes the neuronal phenotype and enables the formation of functional connectivity between neurons. In addition, release of neurotransmitters from differentiating neurons may modulate the behaviour of neural precursors. Here, we show that neuronal activity regulates γ-aminobutyric acid (GABA) expression in neurons generated from stem cells of the striatum and adult subventricular zone (SVZ). Differentiating neurons display spontaneous Ca²⁺ events, which are voltage-gated calcium channel (VGCC) dependent. Depolarization increases both the frequency of Ca²⁺ transients and the amount of Ca²⁺ influx in differentiating neurons. We show that depolarization-dependent GABA expression is regulated by the amplitude and not by the frequency of Ca²⁺ influx. Brief activation of VGCCs leads to Ca²⁺ influx that in turn promotes a rapid expression of GABA. Depolarization-dependent GABA expression does not require changes in gene expression. Instead, it involves cAMP-dependent protein kinase (PKA) and Ca²⁺ and phospholipid-dependent protein kinase (PKC) signalling. Activity increases the number of glutamic acid decarboxylase (GAD) 65-immunoreactive neurons in a PKA-dependent manner, without altering the expression of GAD 65, suggesting that depolarization promotes recruitment of GAD 65 by a post-translational mechanism. In line with this, depolarization does not permanently increase the expression of GABA in neurons derived from neural stem cells of the embryonic striatum, cortex and adult SVZ. Thus, neuronal activity does not merely accelerate neuronal differentiation but it may alter the mechanism of GABA synthesis in newly generated neurons.     

Muscarinic Regulation of Ca2+ Currents in Rat Sensory Neurons: Channel and Receptor Types, Dose - response Relationships and Cross-talk Pathways

We studied, in rat sensory neurons, the modulation of high voltage-activated Ca²⁺ currents (I_Ca mediated by the pertussis toxin-sensitive activation of muscarinic receptors, which were found to be of subtypes M₂, or M₄. Muscarine reversibly blocked somatic Ca²⁺ spikes but strong predepolarizations only partially relieved the inhibited Ca²⁺ current. On the other hand, the putative coupling messenger could not rapidly diffuse towards channels whose activity was recorded from a macro-patch. The perforated patch technique virtually prevented the response rundown present during whole-cell experiments. Both ω-conotoxin GVIA (ω-CgTx)-sensitive channels and ω-CgTx- and dihydropyridine-resistant channels are coupled to the muscarinic receptor, but not the L-channel. When measured in the same neuron, dose - response relationships for the first and subsequent agonist applications differed; maximal inhibition, the reciprocal of half-maximal concentration and the Hill coefficient were always highest in the first trial. Muscarine and oxotremorine exhibited monotone dose - response curves, but oxotremorine-M showed non-linear relationships which became monotonic when cells were intracellularly perfused with inhibitors of protein kinase A (PKA) and C (PKC), suggesting that either PKA or receptor-induced PKC could phosphorylate and thus inactivate G-proteins or other unknown proteins involved in inhibitory muscarinic actions on I_Ca. In summary, these data provide a preliminary pharmacological characterization of the muscarinic inhibition of the Ca²⁺ channels in sensory neurons, with implications about agonist specificity and the interplay between signalling pathways.     

Neuronal Ca2+ Channels and Nicotinic ACh Receptors as Functional Targets of the Nootropic Nefiracetam

Abstract: Piracetam-like nootropics (or cognitive enhancers) have been used for the treatment of various forms of dementia, including Alzheimer's disease. The underlying mechanisms of their actions, however, are largely unknown. Our recent studies have demonstrated that nefiracetam, a nootropic agent, can markedly enhance activities of neuronal L-and N-type (α_1B) Ca²⁺ channels as well as those of presynaptic nicotinic acetylcholine (ACh) receptors, thereby increasing neurotransmitter release. Aniracetam exerted a slight facilitatory effect on Ca²⁺ channels, but no effect on nicotinic ACh receptors. Piracetam and oxiracetam have no such actions on Ca²⁺ channels and nicotinic ACh receptors. It is suggested that inhibitory G-proteins (Go/Gi) and protein kinase A (PKA) mediate the nefiracetam action on Ca²⁺ channels, whereas protein kinase C (PKC) mediates the drug action on nicotinic ACh receptors. In the hippocampus of the rodent, nefiracetam induces a long-lasting (>4 h) facilitation of synaptic transmission. The 'LTP-like' facilitation appears to result from activation of presynaptic nicotinic ACh receptors (and Ca²⁺ channels as well) by nefiracetam. In conclusion, nefiracetam is distinguished from other nootropic agents for its preferential actions on both presynaptic Ca²⁺ channels and nicotinic ACh receptors, and could therefore be of great therapeutic importance to the neurotransmission failure that contributes to the symptoms of Alzheimer's disease and associated disorders.     

Effects of the HIV-1 Envelope Glycoprotein gp120 in Cerebellar Cultures. [Ca2+]i Increases in a Glial Cell Subpopulation

The various types of cells present in cultures prepared from the postnatal rat cerebellum, identified by their gross morphology and immunocytochemistry, were loaded with the specific dye fura-2 and analysed individually for [Ca²⁺]_i changes induced by the HIV-1 envelope glycoprotein gp120 and a variety of other treatments. In granule neurons [Ca²⁺]_i increases were induced by high KCl and glutamate (mainly through the NMDA receptor) while in type-1 astrocytes this effect was observed after serotonin, carbachol and also quisqualate. In contrast, administration of gp120 was always without effect in these cells. Type-2 astrocytes (an arborized cell type responsive to agonists targeted to the glutamatergic AMPA and cholinergic receptors) were also most often unresponsive to the viral glycoprotein. However, among the cells exhibiting the arborized phenotype, a subpopulation (-13%) responded to gp120 with conspicuous [Ca²⁺]_i increases sustained by both release from intracellular stores and influx across the plasma membrane. These responses to the viral protein did not involve activation of either voltage-gated Ca²⁺ channels or glutamatergic receptors. Although not yet conclusively identified by specific cytochemical markers, the gp120-responsive cells resemble type-2 astrocytes and differ from neurons and type-1 astrocytes both in gross phenotype and in a number of receptor/channel properties: positivity to AMPA and cholinergic agonists; negativity to NMDA, serotonin and high KCl. From these results it is concluded that a subpopulation of glial cells is affected by gp120. The role of these cells in HIV brain infection and damage requires further studies to be precisely established.     

HIV-1 Envelope Proteins gp120 and gp160 Potentiate NMDA-induced [Ca2+]i Increase, Alter [Ca2+]i Homeostasis and Induce Neurotoxicity in Human Embryonic Neurons

The envelope glycoprotein gp120 of the human immunodeficiency virus HIV-1 has been proposed to cause neuron death in developing murine hippocampal cultures and rat retinal ganglion cells. In the present study, cultured human embryonic cerebral and spinal neurons from 8- to 10-week-old embryos were used to study the neurotoxic effect of gp120 and gp160. Electrophysiological properties as well as N -methyl- d -aspartate (NMDA)-induced currents were recorded from neurons maintained in culture for 10–30 days. Neither voltage-activated sodium or calcium currents nor NMDA-induced currents were affected by exposure of neurons to 250 pM gp120 or gp160. In contrast, when neurons were subjected to photometric measurements using the calcium dye indo-1 to monitor the intracellular free Ca²⁺ concentration ([Ca²⁺]_i), gp120 and gp160 (20–250 pM) potentiated the large rises in [Ca²⁺]_i induced by 50 μM NMDA. The potentiation of NMDA-induced Ca²⁺ responses required the presence of Ca²⁺ in the medium, and was abolished by the NMDA antagonist d -2-amino-5-phosphonovalerate (AP5) and the voltage-gated Ca²⁺ channel inhibitor nifedipine. Moreover, exposure of a subpopulation of spinal neurons (25% of the cells tested) to 20–250 pM gp120 or gp160 resulted in an increase in [Ca²⁺]_i that followed three patterns: fluctuations not affected by AP5, a single peak, and the progressive and irreversible rise of [Ca²⁺]_i. The neurotoxicity of picomolar doses of gp120 and gp160 cultures was estimated by immuno-fluorescence and colorimetric assay. Treatment of cultures with AP5 or nifedipine reduced gp120-induced toxicity by 70 and 100% respectively.     

Rapid Action of Oestrogen in Luteinising Hormone-Releasing Hormone Neurones: The Role of GPR30

Previously, we have shown that 17β-oestradiol (E₂) induces an increase in firing activity and modifies the pattern of intracellular calcium ([Ca²⁺]_i) oscillations with a latency < 1 min in primate luteinising hormone-releasing hormone (LHRH) neurones. A recent study also indicates that E₂, the nuclear membrane impermeable oestrogen, oestrogen-dendrimer conjugate, and the plasma membrane impermeable oestrogen, E₂-BSA conjugate, all similarly stimulated LHRH release within 10 min of exposure in primate LHRH neurones, indicating that the rapid action of E₂ is caused by membrane signalling. The results from a series of studies further suggest that the rapid action of E₂ in primate LHRH neurones appears to be mediated by GPR30. Although the oestrogen receptor antagonist, ICI 182, 780, neither blocked the E₂-induced LHRH release nor the E₂-induced changes in [Ca²⁺]_i oscillations, E₂ application to cells treated with pertussis toxin failed to result in these changes in primate LHRH neurones. Moreover, knockdown of GPR30 in primate LHRH neurones by transfection with human small interference RNA for GPR30 completely abrogated the E₂-induced changes in [Ca²⁺]_i oscillations, whereas transfection with control siRNA did not. Finally, the GPR30 agonist, G1, resulted in changes in [Ca²⁺]_i oscillations similar to those observed with E₂. In this review, we discuss the possible role of G-protein coupled receptors in the rapid action of oestrogen in neuronal cells.     

Tuning the excitability of midbrain dopamine neurons by modulating the Ca2+ sensitivity of SK channels

Small conductance Ca²⁺ -activated K⁺ (SK) channels play a prominent role in modulating the spontaneous activity of dopamine (DA) neurons as well as their response to synaptically-released glutamate. SK channel gating is dependent on Ca²⁺ binding to constitutively bound calmodulin, which itself is subject to endogenous and exogenous modulation. In the present study, patch-clamp recording techniques were used to examine the relationship between the apparent Ca²⁺ affinity of cloned SK3 channels expressed in cultured human embryonic kidney 293 cells and the excitability of DA neurons in slices from rat substantia nigra using the positive and negative SK channel modulators, 6,7-dichloro-1 H -indole-2,3-dione-3-oxime and R- N -(benzimidazol-2-yl)-1,2,3,4-tetrohydro-1-naphtylamine. Increasing the apparent Ca²⁺ affinity of SK channels decreased the responsiveness of DA neurons to depolarizing current pulses, enhanced spike frequency adaptation and slowed spontaneous firing, effects attributable to an increase in the amplitude and duration of an apamin-sensitive afterhyperpolarization. In contrast, decreasing the apparent Ca²⁺ affinity of SK channels enhanced DA neuronal excitability and changed the firing pattern from a pacemaker to an irregular or bursting discharge. Both the reduction in apparent Ca²⁺ affinity and the bursting associated with negative SK channel modulation were gradually surmounted by co-application of the positive SK channel modulator. These results underscore the importance of SK channels in 'tuning' the excitability of DA neurons and demonstrate that gating modulation, in a manner analogous to physiological regulation of SK channels in vivo , represents a means of altering the response of DA neurons to membrane depolarization.     

Effect of Thiol Reagents on Phosphoinositide Hydrolysis in Rat Brain Synaptoneurosomes

Some divalent ions, such as Cd²⁺ and Zn²⁺, are able to stimulate phosphoinositide (PI) breakdown and to inhibit receptor-mediated PI metabolism. These ions are also known to react with the free – SH groups of proteins. This prompted us to investigate the effects of more potent sulphhydryl reagents, Hg²⁺ and p -chloromercuric benzosulphonic acid (PCMBS), on the inositol phosphate (IP) accumulation triggered by the neuroactive substances: glutamate, carbachol and K⁺, using synaptoneurosomes from 8-day-old rat forebrains. Hg²⁺ and PCMBS, depending on their concentration, had two distinct effects on IP accumulation: at low doses, Hg²⁺ (from 1 to 10 μM) and PCMBS (0.1 mM) by themselves stimulated PI breakdown, inhibited glutamate-elicited IP accumulation and had additive effects with respect to carbachol-induced IP stimulation. At higher doses, Hg²⁺ (from 0.01 to 1 mM) inhibited both basal and neuroactive substance-stimulated IP accumulation. PCMBS (1 mM), provoked only an inhibition of the agonist-stimulated IP formation. Monitoring membrane potential and intracellular Ca²⁺ with the fluorescent dyes diSC2(5) and fura2, respectively, indicated that these mercurials could strongly depolarize the synaptoneurosomal membrane and produce a Ca²⁺ influx dependent on extracellular Ca²⁺. The stimulatory effects of low concentrations of mercurials on PI turnover could be linked to the depolarization they provoke and the subsequent Ca²⁺ rise, which in turn is known to stimulate some phospholipase C enzymes. The inhibitory effects observed at high concentrations might be due to a loss of activity of proteins involved in PI breakdown, as all receptor-mediated IP accumulations were inhibited.     

GABAergic signaling induces divergent neuronal Ca2+ responses in the suprachiasmatic nucleus network

Intercellular communication between γ-aminobutyric acid (GABA)ergic suprachiasmatic nucleus (SCN) neurons facilitates light-induced phase changes and synchronization of individual neural oscillators within the SCN network. We used ratiometric Ca²⁺ imaging techniques to record changes in the intracellular calcium concentration ([Ca²⁺]_i) to study the role of GABA in interneuronal communication and the response of the SCN neuronal network to optic nerve stimulations that mimic entraining light signals. Stimulation of the retinohypothalamic tract (RHT) evoked divergent Ca²⁺ responses in neurons that varied regionally within the SCN with a pattern that correlated with those evoked by pharmacological GABA applications. GABA_A and GABA_B receptor agonists and antagonists were used to evaluate components of the GABA-induced changes in [Ca²⁺]_i. Application of the GABA_A receptor antagonist gabazine induced changes in baseline [Ca²⁺]_i in a direction opposite to that evoked by GABA, and similarly altered the RHT stimulation-induced Ca²⁺ response. GABA application induced Ca²⁺ responses varied in time and region within the SCN network. The NKCC1 cotransporter blocker, bumetanide, and L-type calcium channel blocker, nimodipine, attenuated the GABA-induced rise of [Ca²⁺]_i. These results suggest that physiological GABA induces opposing effects on [Ca²⁺]_i based on the chloride equilibrium potential, and may play an important role in neuronal Ca²⁺ balance, synchronization and modulation of light input signaling in the SCN network.     

Relationship between afterhyperpolarization profiles and the regularity of spontaneous firings in rat medial vestibular nucleus neurons

Our previous in vivo and in vitro whole-cell patch-clamp recording studies demonstrated that neurons in the medial vestibular nucleus (MVN) could be characterized on the basis of three electrophysiological properties: afterhyperpolarization (AHP) profile; firing pattern; and response pattern to hyperpolarizing current pulses. In the present study, to clarify which types of the classified MVN neurons correspond to neurons with regular or irregular firing, we investigated their spike discharge patterns using whole-cell patch-clamp recording in both in vivo and in vitro preparations. The discharge regularity was related to AHP profiles, and we found that: (i) the coefficient of variation (CV) of interspike intervals during spike discharges was smaller in neurons exhibiting AHP with a slow component [AHP(s+)] than in those without a slow component [AHP(s−)], or with a slow AHP component preceded by afterdepolarization (ADP) [AHP(s+) with ADP]; (ii) the blockade of Ca²⁺-dependent K⁺ channels by 100 n m apamin abolished the slow component and increased the CV in neurons exhibiting AHP(s+); and (iii) the modulation of firing (firing gain) in response to ramp current was larger in neurons exhibiting AHP(s−) than in the other two neuronal types. These results suggest that neurons exhibiting AHP(s+) are regularly discharging neurons with small firing gains to stimulus, neurons exhibiting AHP(s+) with ADP are irregularly discharging neurons with small firing gains, and neurons exhibiting AHP(s−) are irregularly discharging neurons with large firing gains. The regular firing of neurons exhibiting AHP(s+) is attributed to the activation of apamin-sensitive Ca²⁺-dependent K⁺ channels.     

Regulation of smooth muscle excitation and contraction

Abstract  Smooth muscle cells (SMC) make up the muscular portion of the gastrointestinal (GI) tract from the distal oesophagus to the internal anal sphincter. Coordinated contractions of these cells produce the motor patterns of GI motility. Considerable progress was made during the last 20 years to understand the basic mechanisms controlling excitation-contraction (E-C) coupling. The smooth muscle motor is now understood in great molecular detail, and much has been learned about the mechanisms that deliver and recover Ca²⁺ during contractions. The majority of Ca²⁺ that initiates contractions comes from the external solution and is supplied by voltage-dependent Ca²⁺ channels (VDCC). VDCC are regulated largely by the effects of K⁺ and non-selective cation conductances (NSCC) on cell membrane potential and excitability. Ca²⁺ entry is supplemented by release of Ca²⁺ from IP₃ receptor-operated stores and by mechanisms that alter the sensitivity of the contractile apparatus to changes in cytoplasmic Ca²⁺. Molecular studies of the regulation of smooth muscle have been complicated by the plasticity of SMC and difficulties in culturing these cells without dramatic phenotypic changes. Major questions remain to be resolved regarding the details of E-C coupling in human GI smooth muscles. New discoveries regarding molecular expression that give GI smooth muscle their unique properties, the phenotypic changes that occur in SMC in GI motor disorders, tissue engineering approaches to repair or replace defective muscular regions, and molecular manipulations of GI smooth muscles in animals models and in cell culture will be topics for exciting investigations in the future.     

GHRP6-Stimulated Hormone Secretion in Somatotrophs: Involvement of Intracellular and Extracellular Calcium Sources

GHRP6 is a synthetic hexapeptide which stimulates growth hormone (GH) secretion from the pituitary in vivo and in vitro . We have previously shown that in identified somatotrophs, GHRP6 induces a biphasic increase in cytosolic Ca²⁺ concentration ([Ca²⁺]i) consisting of an abrupt increase (first phase) followed by a sustained plateau of elevated [Ca²⁺]i (second phase). The first phase corresponds to mobilization of intracellular Ca²⁺ pools and the second phase to influx of extracellular Ca²⁺ ions through voltage-sensitive Ca²⁺ channels. In these experiments, we investigated the specific role of each of these two phases in the hormone response to GHRP6. We found that inhibition by thapsigargin of the intracellular Ca²⁺ mobilization phase significantly inhibited the hormone response to the peptide during 30  min incubations. Inhibition of the extracellular Ca²⁺ influx phase by nifedipine, a blocker of voltage-sensitive Ca²⁺ channels, resulted in a 53% reduction of the secretory response to 10⁻⁵  M GHRP6. Antagonism of PKC by phloretin, a flavonoid which prevents PKC activation, and PKC depletion induced by a 24  h treatment with 10⁻⁶  M PMA, completely inhibited the response to GHRP6. Somatostatin, which also inhibits the second phase of the Ca²⁺ response, suppressed the secretory response to GHRP6. We conclude that, Ca²⁺ is the main second messenger and both Ca²⁺ mobilization and Ca²⁺ entry play a role in the response to GHRP6. However, experiments with PKC depletion and SRIF suggest that other messengers are implicated in GHRP6 signalling in somatotrophs.     

ATP-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice

Abstract  The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro–glia interaction. The aim was to investigate neuro–glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca²⁺-imaging. Putative progenitor-like cells (expressing both PGP9.5 and S-100) differentiated over approximately 5 days into glia or neurons expressing typical cell-specific markers. The glia–neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca²⁺-response to either depolarization (high K⁺) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5-HT, ATP and electrical stimulation, while glia responded to ATP and ADPβs. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL-67156, an ecto-ATPase inhibitor. In this mouse enteric co-culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPβs. Activation of neuronal cells (DMPP, K⁺) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.     

Is Protein Kinase C Activity Required for the N-Methyl-d-Aspartate-evoked Rise in Cytosolic Ca2+ in Mouse Striatal Neurons?

The present study investigates the roles of protein kinase C (PKC) and A (PKA) activities in NMDA-mediated Ca²⁺ entry in primary cultures of mouse striatal neurons. Inhibitors of protein kinases, such as sphingosine, RO 31 – 8220 and staurosporine inhibited the NMDA- but also the KCI-induced rise in cytosolic Ca²⁺. However, the PKA antagonist Rp-adenosine-3',5'monophosphothioate (Rp-cAMPS) did not alter the NMDA + d -serine response, whereas it completely suppressed the KCI response. The NMDA + d -serine-evoked rise in cytosolic Ca²⁺, observed in the absence of external Mg²⁺, was potentiated by the PKC activator phorbol 12-myristate 13-acetate (PMA) only when submaximal effective concentrations of this agonist and co-agonist were used. In addition, the PKC activator did not alter the NMDA + d -serine-evoked response in the presence of varying concentrations of Mg²⁺. Confirming the dependence on PKC activity, desensitization of PKC resulting from long-term PMA treatment led to an impairment of the NMDA response, leaving the KCI-induced response intact. We therefore propose that PKC not only potentiates but is also required for the NMDA-evoked elevation in cytosolic Ca²⁺ in mouse striatal neurons.     

Development of glutamate receptors in auditory neurons from long-term organotypic cultures of the embryonic chick hindbrain

We used long-range organotypic cultures of auditory nuclei in the chick hindbrain to test the development of glutamate receptor activity in auditory neurons growing in a tissue environment that includes early deprivation of peripheral glutamatergic input, subsequent to removal of the otocyst. Cultures started at embryonic day (E)5, and lasted from 6 h to 15 days. Neuronal migration, clustering and axonal extension from the nucleus magnocellularis (NM) to the nucleus laminaris (NL) partially resembled events in vivo . However, the distinctive laminar organization of the NL was not observed. Glutamate receptor (GluR) activity was tested with optical recordings of intracellular Ca²⁺ in the NM. α-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)/kainate receptors had Ca²⁺ responses with a time course similar to that in control slices. Peak amplitude, however, was significantly lower. N -methyl- d -aspartate (NMDA)-mediated Ca²⁺ responses were higher in 2-day cultures (E5 + 2d) than in E7 explant controls, returning later to control values. Metabotropic GluRs did not elicit Ca²⁺ responses at standard agonist doses. Blocking NMDA or AMPA/kainate receptors with specific antagonists for 10 days in culture did not limit neuronal survival. Blocking metabotropic GluRs resulted in complete neuronal loss. Thus, ionotropic GluRs are not required for NM neuronal survival. However, their activity during development is affected when neurons grow in an in vitro environment that includes prevention of arrival of peripheral glutamatergic input.