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R Robitaille

Publications and source records attributed to R Robitaille.

At least 37 records · Page 2Linked to original sources
PubMed1998

Endogenous peptidergic modulation of perisynaptic Schwann cells at the frog neuromuscular junction.

1. Although peptides are important modulators of synapses, their action on synapse-glia interactions remain unclear. The amphibian neuromuscular junction (NMJ) was used to examine the effects of substance P (SP) on perisynaptic Schwann cells (PSCs), glial cells at the frog NMJ, by monitoring changes in intracellular Ca2+. 2. SP induced Ca2+ responses that were mimicked by the neurokinin 1 receptor (NK-1) agonist septide and with a shorter delay by the SP fragment, SP(6-11). SP and SP(6-11) responses were blocked by NK-1 antagonists SR140333 and LY303870. 3. Ca2+ responses remained unchanged when extracellular Ca2+ was removed but were blocked after pertussis toxin (PTX) treatment, indicating that the receptors were linked to internal stores of Ca2+ via a PTX-sensitive G-protein. 4. The slowly hydrolysable NK-1 agonist [Sar9, Met(O2)11]-SP only induced Ca2+ responses when applied for a long period of time and not during brief, local applications, suggesting the involvement of SP hydrolysis. Acetylcholinesterase (AChE) may not be involved in SP degradation since Ca2+ responses evoked by SP were unchanged in the presence of the cholinesterase inhibitor neostigmine. 5. Ca2+ responses induced by muscarine and nerve stimulations were almost abolished when preceded by SP applications, while those induced by ATP were significantly reduced. The rundown of the nerve-evoked Ca2+ responses in PSCs was attenuated in the presence of SR140333. 6. These results indicate that endogenous SP is involved in the regulation of PSC activity and that SP is an important modulator of glial cell Ca2+ signalling and synapse-glia communication.

Acetylcholinesterase↗
PubMed1998

Modulation of synaptic efficacy and synaptic depression by glial cells at the frog neuromuscular junction.

The ability of perisynaptic glial cells to modulate transmitter release and synaptic depression was studied at the frog neuromuscular junction (nmj). Injection of GTPgammaS in perisynaptic Schwann cells (PSCs), glial cells at this synapse, induced a reduction in the amplitude of nerve-evoked synaptic responses but had no effect on the frequency, the amplitude, or the duration of the miniature endplate currents (MEPCs). Also, paired pulse facilitation was not affected. The reduction in transmitter release was mediated by pertussis toxin-(PTX) sensitive and insensitive G proteins. Blockade of G proteins in PSCs with GDPbetaS reduced synaptic depression induced by high frequency trains of stimuli, whereas activation of G proteins occluded it. Hence, the activation by endogenous neurotransmitters of G proteins in PSCs induced a profound depression in neurotransmitter release.

Animals↗
PubMed1998

Localization and characterization of nitric oxide synthase at the frog neuromuscular junction.

The frog neuromuscular junction is sensitive to nitric oxide (NO), since exogenously applied NO reduces the release of transmitter b presynaptic terminals and the size of ATP-induced Caoffresponses in perisynaptic Schwann cells. This study aimed at determining whether an NO synthase (NOS) is present at the neuromuscular junction, notably in perisynaptic Schwann cells, the glial cells at this synapse. The NADPH-diaphorase (NADPH-d) histochemical technique revealed the presence of NOS in cell bodies and presumed processes of perisynaptic Schwann cells. Incubation with NOS inhibitors, NG-nitro-L-arginine methyl ester or NG-nitro-L-arginine-acetate abolished the NADPH-d staining. Moreover, L-arginine, the precursor of NO, impeded the blockade by NOS inhibitors, establishing the NOS specificity of NADPH-d staining in frog tissue. The pattern of labelling with a polyclonal antibody against the neuronal form of NOS was similar to the NADPH-d staining, also suggesting the presence of a neuronal NOS in perisynaptic Schwann cells. Using electron microscopy the NOS immunostaining was found at the membrane and occasionally in the cytoplasm of perisynaptic Schwann cells and was not detected in the nerve terminal or muscle. There was no enzymatic or immunocytochemical labelling of NOS 6 days after denervation. It is concluded that NOS is present in frog perisynaptic Schwann cells. The presence of this endogenous NOS suggests that NO may act as a diffusible glial messenger to modulate synaptic activity and synapse formation at the neuromuscular junction.

Acetylcholinesterase↗
PubMed1997

Role of sensory-evoked NMDA plateau potentials in the initiation of locomotion.

Reticulospinal (RS) neurons constitute the main descending motor system of lampreys. This study reports on natural conditions whereby N-methyl-D-aspartate (NMDA)-mediated plateau potentials were elicited and associated with the onset of locomotion. Reticulospinal neurons responded in a linear fashion to mild skin stimulation. With stronger stimuli, large depolarizing plateaus with spiking activity were elicited and were accompanied by swimming movements. Calcium imaging revealed sustained intracellular calcium rise upon sensory stimulation. Blocking NMDA receptors on RS neurons prevented the plateau potentials as well as the associated rise in intracellular calcium. Thus, the activation of NMDA receptors mediates a switch from sensory-reception mode to a motor command mode in RS neurons.

Animals↗
PubMed1997

Muscarinic Ca2+ responses resistant to muscarinic antagonists at perisynaptic Schwann cells of the frog neuromuscular junction.

1. Acetylcholine causes a rise of intracellular Ca2+ in perisynaptic Schwann cells (PSCs) of the frog neuromuscular junction. The signalling pathway was characterized using the fluorescent Ca2+ indicator fluo-3 and fluorescence microscopy. 2. Nicotinic antagonists had no effect on Ca2+ responses evoked by ACh and no Ca2+ responses were evoked with the nicotinic agonist nicotine. The muscarinic agonists muscarine and oxotremorine-M induced Ca2+ signals in PSCs. 3. Ca2+ responses remained unchanged when extracellular Ca2+ was removed, indicating that they are due to the release of Ca2+ from internal stores. Incubation with pertussis toxin did not alter the Ca2+ signals induced by muscarine, but did block depression of transmitter release induced by adenosine and prevented Ca2+ responses in PSCs induced by adenosine. 4. The general muscarinic antagonists atropine, quinuclidinyl benzilate and N-methyl-scopolamine failed to block Ca2+ responses to muscarinic agonists. Atropine (at 20,000-fold excess concentration) also failed to reduce the proportion of cells responding to a threshold muscarine concentration sufficient to cause responses in less than 50% of cells. Only the allosteric, non-specific blocker, gallamine (1-10 microM) was effective in blocking muscarine-induced Ca2+ responses. 5. In preparations denervated 7 days prior to experiments, low concentrations of atropine reversibly and completely blocked Ca2+ responses to muscarine. 6. The lack of blockade by general muscarinic antagonists in innervated, in situ preparations suggests that muscarinic Ca2+ responses at PSCs are not mediated by any of the five known muscarinic receptors or that post-translational modification prevented antagonist binding.

Acetylcholine↗
PubMed1997

Interneuron-specific Ca2+ responses linked to metabotropic and ionotropic glutamate receptors in rat hippocampal slices.

Glutamate-mediated regulation of intracellular Ca2+ levels was examined in different populations of CA1 interneurons, using confocal microscopy and the Ca2+ indicator fluo 3-AM in rat hippocampal slices. Interneurons in basal [stratum oriens/alveus (OA)] and apical [strata radiatum and lacunosum-moleculare (R/LM)] dendritic layers responded heterogeneously to glutamate. In control medium, OA interneurons responded mostly with oscillatory Ca2+ responses, which consisted of a large Ca2+ transient and successive smaller elevations. R/LM interneurons responded mostly with biphasic responses, characterized by an initial large transient and a secondary prolonged elevation. Other interneurons in both R/LM and OA responded with transient elevations in Ca2+ levels. Ionotropic glutamate receptor antagonists (+/-)2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitro-quinoxaline-2,3-dione reduced peak Ca2+ responses in OA and R/LM cells, and blocked biphasic responses in R/LM interneurons. The metabotropic glutamate receptor antagonist (RS)-alpha-methyl-4-carboxyphenylglycine reduced peak Ca2+ responses only in OA interneurons, and prevented oscillatory responses. In low Ca2+ medium, peak responses were reduced in R/LM but not in OA interneurons, and oscillatory responses were absent. Combination of ionotropic and metabotropic receptor antagonists blocked all glutamate-evoked Ca2+ responses. Activation of different types of glutamate receptors may thus produce heterogeneous Ca2+ signals in subpopulations of CA1 interneurons. Ionotropic receptors may generate biphasic responses in interneurons in apical dendritic layers, whereas combined activation of metabotropic and ionotropic receptors may trigger oscillatory responses in interneurons of basal dendritic layers. These heterogeneous Ca2+ responses indicate that glutamate-mediated Ca2+ processes and second messenger systems differ in subpopulations of hippocampal interneurons and suggest possible postsynaptic functional specialization of interneurons.

Animals↗
PubMed1996

Localization of L-type Ca2+ channels at perisynaptic glial cells of the frog neuromuscular junction.

The presence of L-type Ca2+ channels at the frog neuromuscular junction (nmj) was studied by monitoring changes in intracellular Ca2+ evoked in presynaptic terminals and perisynaptic Schwann cells (PSCs) and by studying the distribution of Ca2+ channels using a monoclonal antibody directed against the alpha 2/delta subunit of L channels. L-type Ca2+ channel agonist and antagonist had no effect on resting level of fluorescence and nerve-evoked Ca2+ responses in presynaptic terminals. However, depolarization of PSCs induced by KCl (25 mM) produced entry of Ca2+, which was prevented by L-type Ca2+ channel blockers, in (+)R Bay K 8644 of nimodipine. Labeling of Ca2+ channels revealed an intracellular epitope with an irregular and spotty distribution along the endplate. Similar results were obtained with a fluorescent phenylalkylamine [(-)DM-BODIPY-PAA], a blocker of L-type Ca2+ channels. Ca2+ channel labeling remained in absence of nerve terminals but was absent after mechanical removal of nerve terminals and PSCs. Most Ca2+ channel spots were distributed in between bands of cholinergic receptors labeled with alpha-bungarotoxin-TRITC. Cross sections of motor endplates revealed that labeling of Ca2+ channels was found only at the level of the synaptic cleft and not all around the PSCs. We conclude that L-type Ca2+ channels are located in perisynaptic glial cells in an appropriate location to sense depolarization induced by neurotransmitters and thus may support possible roles of glial cells on synaptic function.

Aniline Compounds↗
PubMed1995

Purinergic receptors and their activation by endogenous purines at perisynaptic glial cells of the frog neuromuscular junction.

Glial cells are closely associated with synapses and are modulated by neurotransmitters released during synaptic transmission. At many synapses, ATP is released during synaptic transmission and is involved in cell-cell signaling. Since glial cells have purinoceptors, it is possible that ATP mediates synaptic neuron-glia signaling. This work aims at determining which types of purinoceptors are present on perisynaptic Schwann cells, the perisynaptic glial cells at the frog neuromuscular junction, and test their sensitivity to endogenous purines by monitoring the relative changes of intracellular Ca2+. Local application of ATP induced the release of Ca2+ from internal stores. Adenosine induced Ca2+ responses that were blocked by A1 receptor antagonists and mimicked by an A1 receptor agonist and were caused by the release of Ca2+ from internal stores via a pertussis toxin-sensitive G-protein. A2 receptor antagonists had no effect on Ca2+ responses induced by adenosine. Me-S-ATP, an ATP analog, triggered Ca2+ release from internal stores via a pertussis toxin-sensitive G-protein, consistent with the activation of P2Y receptors. L-AMP-PCP, another ATP analog, induced Ca2+ entry mainly through L-type Ca2+ channels by a pertussis toxin-insensitive mechanism, consistent with the activation of P2X receptors. Blockade of adenosine receptors did not affect glial Ca2+ responses induced by nerve evoked transmitter release. However, blockade of ATP receptors reduced the size and increased the delay of the responses. Hence, purinoceptors are present on the perisynaptic Schwann cells and are activated by endogenous ATP released during synaptic transmission.

Adenosine↗
PubMed1994

Synaptic regulation of glial protein expression in vivo.

We investigated signaling between individual nerve terminals and perisynaptic Schwann cells, the teloglial cells that cover neuromuscular junctions. When deprived of neuronal activity in vivo, either by motor nerve transection or tetrodotoxin injection, perisynaptic Schwann cells rapidly up-regulated glial fibrillary acidic protein. Addition of transcription or translation inhibitors to excised muscles prevented this increase. Stimulation of cut nerves prevented glial fibrillary acidic protein increases even when postsynaptic nicotinic receptors were blocked, but not when neurotransmitter release was blocked with omega-conotoxin GVIA. We conclude that there is a nerve terminal to glial signal, requiring presynaptic neurotransmitter release, which regulates perisynaptic Schwann cell genes. This may be a general principle since many types of glial are sensitive to transmitters applied in vitro or released in situ.

Action Potentials↗
PubMed1993

Functional colocalization of calcium and calcium-gated potassium channels in control of transmitter release.

We examined, using physiological and morphological techniques, the distribution of Ca(2+)-gated K+ (gKca) channels relative to the location of Ca2+ channels and transmitter release sites at the frog neuromuscular junction (NM). Charybdotoxin (ChTx) and iberiotoxin, blockers of gKca channels with large conductances, increase transmitter release at the frog NMJ. Intracellular Ca2+ buffers with rapid binding kinetics, dimethyl BAPTA and BAPTA, prevented the effect of ChTx, but EGTA, a Ca2+ buffer with similar affinity for Ca2+ but slower binding kinetics, did not. Dimethyl BAPTA and BAPTA, but not EGTA, caused a temporary increase in transmitter release. Labeling of gKca channels with ChTx-biotin revealed a series of bands located at the sites of Ca2+ channels, but this labeling did not occur in denervated preparations. Cross sections of NMJs revealed that gKca channels are clustered in the presynaptic membrane facing the postsynaptic membrane. We conclude that gKca channels are strategically clustered at the neurotransmitter release sites, where they can be quickly activated by Ca2+ entering the terminal.

Animals↗
PubMed1993

Calcium channels and calcium-gated potassium channels at the frog neuromuscular junction.

Two mechanisms which regulate transmitter release by regulating Ca2+ entry in the presynaptic nerve terminal were studied at the frog neuromuscular junction (nmj). First, the location of Ca2+ channels in relation to transmitter release sites and, second, the regulation of Ca2+ entry by Ca(2+)-gated potassium (gKca) channels. Ca2+ channels were disclosed using fluorescent omega-conotoxin GVIA (omega-CgTX) which blocks transmitter release and Ca2+ entry at the frog nmj. Ca2+ channels were located in bands spaced at regular intervals of 1 micron. The omega-CgTX labeling was removed following mechanical displacement of the presynaptic terminal after collagenase digestion. The bands of omega-CgTX staining matched almost perfectly the staining of cholinergic receptors with fluorescent alpha-bungarotoxin (alpha-BuTX) and therefore must be located at the active zone. The role of gKca channels in the regulation of transmitter release was assessed using charybdotoxin (ChTX) which blocks gKca channels of large and intermediate conductances. Application of ChTX (2-20 nM) induced a two-fold increase in transmitter release which was prevented when a membrane permeant Ca2+ buffer (DMBAPTA-AM) was introduced prior to the toxin application. The Ca2+ buffer by itself caused a reduction in transmitter release. Nerve-evoked Ca2+ entry in the presynaptic terminal, detected with the fluorescent indicator fluo3, was increased following gKca channel blockade by ChTX. The Ca(2+)-gated K+ channels may function to limit the duration of the presynaptic action potential and thus limit Ca2+ entry.

Animals↗
PubMed1993

A comparison of two alternatively spliced forms of a metabotropic glutamate receptor coupled to phosphoinositide turnover.

A comparison of the pharmacological and physiological properties of the metabotropic glutamate 1 alpha and 1 beta receptors (mGluR1 alpha and mGluR1 beta) expressed in baby hamster kidney (BHK 570) cells was performed. The mGluR1 beta receptor is an alternatively spliced form of mGluR1 alpha with a modified carboxy terminus. Immunoblots of membranes from the two cell lines probed with receptor-specific antipeptide antibodies showed that mGluR1 alpha migrated with an M(r) = 154,000, whereas mGluR1 beta migrated with an M(r) = 96,000. Immunofluorescence imaging of receptors expressed in BHK 570 cells revealed that the mGluR1 alpha receptor was localized to patches along the plasmalemma and on intracellular membranes surrounding the nucleus, whereas mGluR1 beta was distributed diffusely throughout the cell. Agonist activation of the mGluR1 alpha and the mGluR1 beta receptors stimulated phosphoinositide hydrolysis. At both receptors, glutamate, quisqualate, and ibotenate were full agonists, whereas trans-(+)-1-aminocyclopentane-1,3-dicarboxylate appeared to act as a partial agonist. The stimulation of phosphoinositide hydrolysis by mGluR1 alpha showed pertussis toxin-sensitive and insensitive components, whereas the mGluR1 beta response displayed only the toxin-insensitive component. The mGluR1 alpha and mGluR1 beta receptors also increased intracellular calcium levels by inducing release from intracellular stores. These results indicate that the different carboxy terminal sequences of the two receptors directly influences G protein coupling and subcellular deposition of the receptor polypeptides and suggest that the two receptors may subserve different roles in the nervous system.

Animals↗
PubMed1992

Transmitter release increases intracellular calcium in perisynaptic Schwann cells in situ.

Glial cells isolated from the nervous system are sensitive to neurotransmitters and may therefore be involved in synaptic transmission. The sensitivity of individual perisynaptic Schwann cells to activity of a single synapse was investigated, in situ, at the frog neuromuscular junction by monitoring changes in intracellular Ca2+ in the Schwann cells. Motor nerve stimulation induced an increase in intracellular Ca2+ in these Schwann cells; this increase was greatly reduced when transmitter release was blocked. Furthermore, local application of the cotransmitters acetylcholine and ATP evoked Ca2+ responses even in the absence of extracellular Ca2+. Successive trains of nerve stimuli or applications of transmitters resulted in progressively smaller Ca2+ responses. We conclude that transmitter released during synaptic activity can evoke release of intracellular Ca2+ in perisynaptic Schwann cells. This Ca2+ signal may play a role in the maintenance or modulation of a synapse. These data show that synaptic transmission involves three cellular components with both postsynaptic and glial components responding to transmitter secretion.

Animals↗
PubMed1992

Presynaptic calcium signals and transmitter release are modulated by calcium-activated potassium channels.

The regulation of synaptic transmission by Ca(2+)-activated potassium (gKca) channels was investigated at the frog neuromuscular junction (nmj). Charybdotoxin (CTX), a blocker of certain types of gKca channels, induced a twofold increase of transmitter release. Similar results were obtained with purified natural toxin, synthetic toxin, and recombinant toxin. Apamin, a blocker of a different type of gKca channel, did not alter transmitter release. CTX was ineffective after intraterminal Ca2+ buffering was increased by application of the membrane-permeant Ca2+ buffer dimethyl-BAPTA-AM. By itself, the permeant buffer first caused a slight increase in transmitter release before release was eventually decreased. This increase of release did not occur when the buffer was applied in the presence of CTX or Ba2+, another gKca channel blocker. Stimulus-evoked entry of Ca2+ in nerve terminals, detected with the fluorescent Ca2+ indicator FLUO-3, was increased after blockade of gKca channels by CTX. CTX had no effect on the amount or the time course of synaptic depression. The results are consistent with the hypothesis that CTX-sensitive gKca channels normally narrow the presynaptic action potential and thus, by indirectly regulating Ca2+ entry, can serve as powerful modulators of evoked transmitter release. In order to affect presynaptic action potentials, the gKca channels must be located close to Ca2+ channels.

Action Potentials↗
PubMed1991

Phosphatases modulate transmission and serotonin facilitation at synapses: studies with the inhibitor okadaic acid.

We examined the role of phosphatases in synaptic transmission using the permeant phosphatase inhibitor okadaic acid (OA). In the crayfish neuromuscular junction (NMJ), postsynaptic effects including increases in input resistance occurred at doses greater than 5 microM OA. At lower doses (0.5-5 microM) the effects were solely presynaptic and transmitter release increased over three-fold despite small reductions in amplitude and duration of presynaptic action potentials. Potentiating effects of serotonin on transmitter release, which depend on phosphorylation, were increased by OA. Frequency facilitation was reduced but its decay was not affected. In frog NMJs, OA increased spontaneous and evoked release two-fold through presynaptic mechanisms. An inactive analog of OA, OA tetra-acetate, had no effect on transmitter release at frog and crayfish NMJ. Therefore, phosphatases have a strong modulating influence on synaptic transmission.

Action Potentials↗
PubMed1991

Non-uniform responses to Ca2+ along the frog neuromuscular junction: effects on the probability of spontaneous and evoked transmitter release.

Spontaneous and evoked transmitter release activity was studied during selective application of Ca2+ in proximal (near the first contact of the axon on the muscle fiber) and distal regions of the frog neuromuscular junction. A new technique called "Microperfusion" was developed, which allowed us to apply a 30-microns-wide Ca2+ stream from an external pipette. The spread of this Ca2+ stream was monitored by adding Blue Dextran (40 mg/ml) to the Ca2+ solution. Microperfusion with a Ca2(+)-free Ringer containing Blue Dextran did not affect the miniature endplate potential frequency or amplitude. Changes of spontaneous transmitter release were studied either during microperfusion of Ringer containing 5 mM Ca2+ or during microperfusion of 2 mM Ca2+ simultaneously with the stimulation of the motor nerve. This second procedure also permitted study of the characteristics of evoked release. Microperfusion of Ca2+ induced a larger and more rapid increase in the miniature endplate potential frequency in proximal than in distal regions. The time required for the miniature endplate potential frequency to return to the control value after Ca2+ microperfusion was longer than the time needed to increase the frequency and this decay period was longer in the proximal region than in the distal one. Moreover, miniature endplate potentials produced in proximal regions, were typically larger and more variable than those produced in distal regions. In five experiments, the endplate potentials produced by 100-200 pulse pairs (interval of 15 ms at every 2 s) were recorded intracellularly during the microperfusion. The quantal content of the first endplate potential of the pair (EPP1) was systematically smaller in distal regions than in proximal regions. The percentage of failures and the coefficients of variation were higher in distal than in proximal regions, indicating a larger variability of quantal content. The frequency facilitation was not different between the two regions, but, however, the second stimuli of the pair usually produced a net increase of transmitter release which was greater in proximal than distal regions. Our experiments demonstrate that both the spontaneous and the evoked release are more responsive to Ca2+ application in the proximal than in the distal regions of the frog neuromuscular junction.

Animals↗
PubMed1990

Strategic location of calcium channels at transmitter release sites of frog neuromuscular synapses.

The localization of Ca2+ channels relative to the position of transmitter release sites was investigated at the frog neuromuscular junction (NMJ). Ca2+ channels were labeled with fluorescently tagged omega-conotoxin GVIA, an irreversible Ca2+ channel ligand, and observed with a confocal laser scanning microscope. The Ca2+ channel labeling almost perfectly matched that of acetylcholine receptors which were labeled with fluorescent alpha-bung-arotoxin. This indicates that groups of Ca2+ channels are localized exclusively at the active zones of the frog NMJ. Cross sections of NMJs showed that Ca2+ channels are clustered on the presynaptic membrane adjacent to the postsynaptic membrane.

Animals↗