mGluR7 inhibits glutamate release through a PKC-independent decrease in the activity of P/Q-type Ca2+ channels and by diminishing cAMP in hippocampal nerve terminals

The modulation of calcium channels by metabotropic glutamate receptors (mGluRs) is a key event in the fine-tuning of neurotransmitter release. Here we report that, in hippocampal nerve terminals from adult rats, the inhibition of glutamate release by the group III mGluR agonist L-2-amino-4-phosphonobutyrate (L-AP4) is largely mediated by mGluR7. In this preparation, P/Q-type Ca(2+) channels support the major component of glutamate release while the remaining release is supported by N-type Ca(2+) channels. The release associated with P/Q channels was modulated by mGluR7, either in the presence of omega-conotoxin-GVIA or after decreasing the extracellular Ca(2+) concentration [Ca(2+)](o) to abolish the contribution of N-type Ca(2+) channels. Under these conditions, L-AP4 (1 mm) reduced the evoked glutamate release by 35 +/- 2%. This inhibition was largely prevented by pertussis toxin, but it was insensitive to inhibitors of protein kinase C (bisindolylmaleimide) and protein kinase A (H-89). Furthermore, this inhibition was associated with a reduction in the Ca(2+) influx mediated by P/Q channels in the absence of any detectable change in cAMP levels. However, L-AP4 decreased the levels of cAMP in the presence of forskolin. The activation of this additional signalling pathway was very efficient in counteracting the facilitation of glutamate release induced by forskolin. Thus, mGluR7 mediates the inhibition of glutamate release at hippocampal nerve terminals primarily by inhibiting P/Q-type Ca(2+) channels, although augmenting the levels of cAMP reveals the ability of the receptor to decrease cAMP.     

Ca2+ Channels and Ca2+-Activated K+ Channels in Adult Rat Gonadotrophin-Releasing Hormone Neurones

Gonadotrophin-releasing hormone (GnRH) neurones represent the final output neurones in the neuroendocrine system for the control of reproduction. To understand the reproductive neuroendocrine system, an investigation of the intrinsic and extrinsic properties of GnRH neurones is essential. In this review, we focus on the intrinsic properties and summarise our recent findings of ion channels expressed in rat GnRH neurones. Rat GnRH neurones express all four types of high voltage-activated Ca²⁺channel (L, N, P/Q, R) and the low voltage-activated Ca²⁺ channel (T). GnRH neurones also express two types of Ca²⁺-activated K⁺ [K(Ca)] channel: the small conductance Ca²⁺-activated K⁺ (SK) channel and the large conductance Ca²⁺- and voltage-activated K⁺ (BK) channel. The activities of these Ca²⁺ and K(Ca) channels regulate cell excitability and cellular calcium homeostasis.     

Locatization of Voltaae-sensitive Ca2+ Fluxes and Neuropeptide Y Immunoreactivity to Varicosities in SH-SY5Y Human Neuroblastoma Cells Differentiated by Treatment with the Protein Kinase Inhibitor Staurosporine

The distribution of voltage-sensitive elevations of the level of Ca²⁺ in untreated SH-SY5Y cells and cells that had been induced to differentiate with staurosporine was investigated by monitoring fura-2 fluorescence in cell suspensions, and by using microfluorometry and quantitative fluorescence imaging on cell bodies and on cellular processes. Cell bodies of both types of cells displayed small Ca²⁺ elevations, which were composed of transient and sustained components. Elevations were partially sensitive to the L- and N-channel blockers nifedipine (1 μM) and ω-conotoxin GVIA (100 nM) respectively. Up to ten times higher Ca²⁺ elevations were observed in varicosities of treated cells than in cell bodies of treated and untreated cells. These elevations were insensitive to compounds known to release Ca²⁺ from intracellular stores. Elevations of Ca²⁺ were sustained, and they were insensitive to 5 pM nifedipine, 100 nM ω-agatoxin IVA and 100 nM ω-conotoxin GVIA, and partially sensitive to 2 pM ω-conotoxin GVIA, indicating predominance of non-L-type, non-N-type, non-P-type channel activity. The intracellular localization of neuropeptide Y, a marker of differentiation in these cells, was also investigated by fluorescence immunocytochemistry. Varicosities of treated cells displayed marked fluorescence when viewed in a confocal microscope. These findings show that the varicosities of staurosporine-treated cells exhibit some of the functional properties of nerve terminals. The varicosities resemble boutons en passant nerve endings and they seem to express Ca²⁺ channels different from those in the cell body.     

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.     

A Decrease in [Ca2+]c but not in cAMP Mediates L-AP4 Inhibition of Glutamate Release: PKC-mediated Suppression of this Inhibitory Pathway

We investigated the mechanism of the inhibition of glutamate release by L-2-amino-4-phosphonobutyrate (L-AP4) in cerebrocortical nerve terminals from young rats (3 weeks of age). The Ca²⁺-dependent release of glutamate was reduced by L-AP4 in a concentration-dependent manner. This inhibitory effect was prevented by pertussis toxin, insensitive to staurosporine and associated with a reduction both in the depolarization-evoked increase in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) and in forskolin-stimulated cAMP formation. However, the reduction in [Ca²⁺]_c but not in cAMP seemed to be responsible for the decrease in release, since inhibition by L-AP4 can also be observed in the absence of detectable changes in CAMP. The inhibitory modulation by L-AP4 was suppressed by the activation of protein kinase C with phorbol esters. The nerve terminals from young rats also exhibited a facilitatory pathway of glutamate release which was mediated by protein kinase C. Interestingly, stimulation of this pathway with the glutamate agonist (1 S,3R)-1-aminocyclopentane-1,3-dicarboxylate in the presence of arachidonic acid also abolished the inhibitory action of L-AP4. The dominance of the facilitatory pathway in its interaction with the L-AP4-mediated inhibitory control may provide some clues to understand the presynaptic changes during synaptic plasticity.     

Presynaptic Ca2+ channels: a functional patchwork

A key step in the release of neurotransmitter is the entry of Ca²⁺ into the presynaptic terminal via voltage-activated Ca²⁺ channels. N-type and P/Q-type Ca²⁺ channels play a predominant role but, surprisingly, their distribution across presynaptic terminals lacks any apparent order. They form a patchwork: at some terminals only N-type channels contribute to transmitter release and in others only P/Q-type channels contribute, but in many terminals both sub-types are active. The physiological implications of this non-uniform distribution are starting to emerge. Recent studies reveal that G-protein-mediated depression of N-type channels is stronger than that of P/Q-type channels, whereas voltage-dependent relief of inhibition is more pronounced for P/Q-type channels. The patchwork distribution of Ca²⁺ channel subtypes might therefore enable terminal-specific modulation of transmitter release, enhancing the power of synaptic processing.     

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.     

Different Routes of Ca2+ Influx in NMDA-Mediated Generation of Nitric Oxide and Arachidonic Acid

Nitric oxide and arachidonic acid act as inter- and intracellular messengers in the central nervous system. It is well known that the NMDA-mediated generation of nitric oxide and arachidonic acid is dependent on extracellular Ca²⁺. However, the role of voltage-dependent calcium channels (VDCCs) in this regard is poorly understood. We report here that NMDA-mediated nitric oxide production in striatal neuron cultures is blocked (80%) by the L-type VDCC antagonist nifedipine, but not by ω-conotoxin or ω-agatoxin IVA, antagonists of the N-and P-type VDCCs respectively. By contrast, none of the VDCC antagonists inhibited the NMDA-mediated release of arachidonic acid. These data indicate that permeation through different Ca²⁺ channels is responsible for the production of arachidonic acid and nitric oxide in striatal neurons.     

Axotomy-induced Changes in Ca2+ Homeostasis in Rat Sympathetic Ganglion Cells

Some of the marked biochemical and electrophysiological changes provoked by section of the axon in mature neurons suggest that the intracellular calcium concentration ([Ca²⁺]_i) may be increased. We have measured the [Ca²⁺]_i using the fluorescent indicator Indo-1 microinjected into rat superior cervical ganglion neurons. No differences in resting [Ca²⁺]_i levels were found between control neurons and cells which had been axotomized 7–10 days before. However, the rise in [Ca²⁺]_i evoked by orthodromic or antidromic stimulation and the recovery after the stimulating train were considerably slower in axotomized neurons than in control cells. We also found that the number of calbindin-D28k-immunopositive cells in the ganglion increases after axotomy, which could be related to the observed differences in calcium homeostasis.     

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.     

Adiponectin Regulate Growth Hormone Secretion Via Adiponectin Receptor Mediated Ca2+ Signalling in Rat Somatotrophs In Vitro

Obesity is associated with reduced levels of growth hormone (GH) and the disruption of pulsatile GH secretion. This results in relative GH deficiency. It is likely that a regulatory relationship between GH secretion and adipose tissue exists as the secretion of GH recovers to normal levels after a reduction in body weight. This report characterise the expression and interaction of adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) and adiponectin, respectively, in regulating the activity of GH secreting cells. Polymerase chain reaction analysis of the GH3 cell line, rat anterior pituitary gland and isolated somatotroph cells from transgenic GFP expressing mice confirmed the expression of both AdipoR1 and AdipoR2 in GH secretory cells. Because GH cells expressed both receptors, it is likely that the measured increase in GH secretion, observed in primary cultured rat pituitary cells after 30 min of incubation with full-length murine adiponectin, was mediated by a direct receptor regulated process. Adiponectin induced an increase in intracellular Ca²⁺ through both the influx of extracellular Ca²⁺ and the release of intracellular Ca²⁺ stores resulting in the secretion of GH. Furthermore, results confirm that this increase in GH secretion depended mainly on an increase in Ca²⁺ influx through L-type Ca²⁺ channels. It is concluded that adiponectin directly regulates GH secretion from somatotrophs by binding to either adiponectin receptor, and that this is mediated via a similar process observed after the stimulation of GH secretion by GH-releasing hormone.     

Presynaptic Modulation of Glutamate Release Targets Different Calcium Channels in Rat Cerebrocortical Nerve Terminals

We have studied which type/s of Ca²⁺ -channel/s support glutamate exocytosis and its modulation by presynaptic receptors in cerebrocortical nerve terminals. Depolarization of nerve terminals with 30 mM KCI induced a Ca²⁺ -dependent release of 3.64 ± 0.25 nmol/mg of protein. The addition of either 2 μM ω-conotoxin-GVIA or 200 nM ω-agatoxin-IVA reduced the KCI-evoked release by 47.7 ± 3.5% and 70.4 ± 8.9% respectively, and by 85.7 ± 4.1% when both toxins were co-applied. The activation of adenosine A₁ receptors with N ⁶-cyclohexyladenosine or the activation of rnetabotropìc glutamate receptors with L(+)-2-amino-4-phosphonobutyrate inhibited the KCI-evoked release by 41.0 ± 5.9 and 54.3 ± 10% respectively. The extent of these inhibitions was not altered by the prior addition of 2 μM ω-conotoxin-GVIA but they were significantly enhanced when ω-agatoxin-IVA was added together with the adenosine A₁ receptor agonist or the metabotropic glutamate receptor agonist, suggesting that ω-conotoxin-GVIA-sensitive and not ω-agatoxin-IVA-sensitive Ca²⁺-channels are ínvolved in the action of these inhibitory receptors. By contrast, the facilitation of glutamate release that follows the activation of the protein kinase C, either with phorbol esters or with the stimulation of phospholipase C-linked metabotropic receptors, was expressed by both ω-conotoxin-GVIA-sensitive and ω-agatoxin-sensitive Ca²⁺-channels. It is concluded that different Ca²⁺-channels support the modulation of glutamate release by presynaptic receptors.     

Glial Cells in the Mouse Hippocampus Express AMPA Receptors with an Intermediate Ca2+ Permeability

Recently, we could demonstrate that 'complex' glial cells in mouse hippocampal slices express glutamate receptor channels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate subtypes. In the present study, we further characterized this glial receptor. Since voltage-clamp control is imperfect and diffusion barriers hinder the quantitative analysis of the receptor currents in situ , the patch-clamp technique was applied to glial cells acutely isolated from the mouse hippocampal CA1 stratum radiatum subregion. A concentration-clamp technique was used which enabled very fast exchange of the extracellular solutions. Thus, it was possible to characterize the transient receptor currents with high time resolution. Application of L-glutamate, AMPA and L-homocysteate induced rapidly activating and fast desensitizing receptor currents in the suspended glial cells. In contrast, kainate induced non-desensitizing currents. The corresponding dose-response curve revealed a half-maximum of current activation at 350 μM. The current/voltage relationship of the kainate-evoked response was linear, with a reversal potential at ∼9 mV. Analysis of the reversal potential in solutions containing high concentrations of CaCl₂ confirmed earlier in situ data by demonstrating significant Ca²⁺ permeability of the glial glutamate receptor channels in the hippocampus. The kainate-induced receptor currents were markedly increased by cyclothiazide, a substance which selectively potentiates glutamate receptors of the AMPA subtype. We conclude that glial cells of the juvenile hippocampus mainly express heteromeric high-affinity AMPA receptors. Most probably, the receptor channels are assembled from the low Ca²⁺-permeable glutamate receptor-2 subunit together with Ca²⁺-permeable AMPA-preferring subunits.     

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.     

Calcium Channel Currents in Undifferentiated Human Neuroblastoma (SH-SY5Y) Cells: Actions and Possible Interactions of Dihydropyridines and ω-Conotoxin

Ca²⁺ channel currents were recorded in undifferentiated human neuroblastoma (SH-SY5Y) cells with the whole-cell patch-clamp technique, using 10 mM Ba²⁺ as charge carrier. Currents were only evoked by depolarizations to -30 mV or more positive (holding potential -80 mV), inactivated partially during 200 ms depolarizing steps, and were abolished by 150 μMCd²⁺. Currents could be enhanced by Bay K-8644 and partially inhibited by nifedipine, suggesting that they arose in part due to activation of L-type Ca²⁺ channels. Currents were also inhibited by the marine snail peptide ω-conotoxin GVIA (ω-CgTx). At a concentration of 10 nM inhibition by ω-CgTx was reversible, but at higher concentrations blockade was always irreversible. Although current inhibition by nifedipine was maximal at 1μM, supramaximal concentrations reduced the inhibitory actions of ω-CgTx in a concentration-dependent manner. Ca²⁺ channel currents evoked from a holding potential of -50 mV showed no inactivation during 200 ms depolarizations but declined in amplitude with successive depolarizing steps (0.2 Hz). Current amplitudes could be restored by returning the holding potential to -80 mV. Currents evoked from -50 mV were inhibited by nifedipine and ω-CgTx to a similar degree as those evoked from -80 mV. Our results indicate that undifferentiated SH-SY5Y cells possess L- and N-type Ca²⁺ channels which can be distinguished pharmacologically but cannot be separated by using depolarized holding potentials. Furthermore, these data suggest that nifedipine has a novel action to inhibit blockade of N-type channels by ω-CgTx.     

Switching Between Two Types of Bursting Activity of Single Ca+2-Activated K+ Channels in Dissociated Neurons

Single channel recordings of Ca²⁺-activated K⁺ currents were made from dissociated cockroach neurons by means of the gigaohm-seal patch-clamp technique. Bursts of single channel openings were composed of two distinct classes: the 'long-open burst' contained groups of long, rectangular, pulse-like openings with durations of 3.5 to 1.2 ms (depending on membrane potential), whereas the 'flickering burst' consisted of clusters of brief openings with an average duration of 0.4 ms (voltage-independent) separated by short closings with a duration of about 1.0 ms. The long-open burst and the flickering burst appeared to reflect distinct states of a single Ca²⁺-activated K⁺ channel because direct transitions between these two types of burst were often detected. We present a kinetic scheme for the gating activation pathway of a neuronal Ca²⁺-activated K⁺ channel, based on these findings.     

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.     

The patterns of spontaneous Ca2+ signals generated by ventral spinal neurons in vitro show time-dependent refinement

Embryonic spinal neurons maintained in organotypic slice culture are known to mimic certain maturation-dependent signalling changes. With such a model we investigated, in embryonic mouse spinal segments, the age-dependent spatio-temporal control of intracellular Ca²⁺ signalling generated by neuronal populations in ventral circuits and its relation with electrical activity. We used Ca²⁺ imaging to monitor areas located within the ventral spinal horn at 1 and 2 weeks of in vitro growth. Primitive patterns of spontaneous neuronal Ca²⁺ transients (detected at 1 week) were typically synchronous. Remarkably, such transients originated from widespread propagating waves that became organized into large-scale rhythmic bursts. These activities were associated with the generation of synaptically mediated inward currents under whole-cell patch-clamp. Such patterns disappeared during longer culture of spinal segments: at 2 weeks in culture, only a subset of ventral neurons displayed spontaneous, asynchronous and repetitive Ca²⁺ oscillations dissociated from background synaptic activity. We observed that the emergence of oscillations was a restricted phenomenon arising together with the transformation of ventral network electrophysiological bursting into asynchronous synaptic discharges. This change was accompanied by the appearance of discrete calbindin immunoreactivity against an unchanged background of calretinin-positive cells. It is attractive to assume that periodic oscillations of Ca²⁺ confer a summative ability to these cells to shape the plasticity of local circuits through different changes (phasic or tonic) in intracellular Ca²⁺.