Biomedical subjects

P Drapeau

Publications and source records attributed to P Drapeau.

At least 19 recordsLinked to original sources

PubMed1995

Tyrosine phosphorylation during synapse formation between identified leech neurons.

1. We have examined whether tyrosine phosphorylation is required for synapse formation between identified neurons from the central nervous system of the leech in culture. 2. Within a few hours of contact with the cell body of the serotonergic Retzius neuron (R cell), the soma of the postsynaptic pressure-sensitive neuron (P cell), but not the R cell, could be labelled intracellularly with an antibody against phosphotyrosine residues. The labelling seemed specific for P cells contacted by R cells, as it was greatly reduced in pairs of either R or P cells and in single cells. Genistein (20 microM) and lavendustin A (10 microM), selective inhibitors of tyrosine kinases, blocked the labelling of contacted P cells, whereas their ineffective analogues (genistein and lavendustin B) had no effect on labelling. 3. R cell contact also induced the loss of an extrasynaptic, depolarizing response (due to modulation of cation channels) to serotonin (5-HT) in the P cell within a few days of juxtaposing cell bodies and within an hour of contact with growth cones. Treatment of the neurons with the tyrosine kinase inhibitors (but not the ineffective analogues) prevented the loss of the depolarizing response and of single cation channel modulation by 5-HT. 4. R cells formed inhibitory, Cl(-)-dependent synapses with P cells. Synapse formation was prevented by the tyrosine kinase inhibitors but not by their ineffective analogues. These compounds had no obvious effect on neurite outgrowth or cell adhesion. We conclude that tyrosine phosphorylation is a signal during the formation of this synapse.

Animals↗

PubMed1995

Synapse formation and function: insights from identified leech neurons in culture.

Identified leech neurons in culture are providing novel insights to the signals underlying synapse formation and function. Identified neurons from the central nervous system of the leech can be removed individually and plated in culture, where they retain their characteristic physiological properties, grow neurites, and form specific synapses that are directly accessible by a variety of approaches. Synapses between cultured neurons can be chemical or electrical (either rectifying or not) or may not form, depending on the neuronal identities. Furthermore, the characteristics of these synapses depend on the regions of the cells that come into contact. The formation and physiology of synapses between the Retzius cell and its partners have been well characterized. Retzius cells form purely chemical, inhibitory synapses with pressure-sensitive (P) cells where serotonin (5-HT) is the transmitter. Retzius cells synthesize 5-HT, which is stored in vesicles that recycle after 5-HT is secreted on stimulation. The release of 5-HT is quantal, calcium-dependent, and shows activity-dependent facilitation and depression. Anterograde and retrograde signals during synapse formation modify calcium currents, responses to 5-HT, and neurite outgrowth. The nature of these synaptogenic signals is being elucidated. For example, contact specifically with Retzius cells induces a localized selection of transmitter responses in postsynaptic P cells. This effect is signaled by tyrosine phosphorylation prior to synapse formation.

Animals↗

PubMed1995

From contact to connection: early events during synaptogenesis.

When neuronal processes first come into contact, chemical synapses can form rapidly. Many neurons synthesize synaptic machinery through intrinsic programs before cell-cell interactions. During the formation of chemical synapses, contact with appropriate targets has been found to trigger intracellular signals that induce the assembly of pre-existing synaptic machinery. We propose that 'promiscuous' neurons secrete transmitter before contacting their targets, and form over-abundant synapses, which undergo additional activity-dependent refinement; 'selective' neurons, which retain their original connectivity, require concerted retrograde and anterograde signaling to ensure their correct matching.

Animals↗

PubMed1995

Signalling synapse formation between identified neurons.

We have investigated the signals between identified leech neurons during the formation of specific synapses in culture. At an inhibitory serotonergic synapse between two well-studied neurons, the postsynaptic cell has an additional (extrasynaptic) excitatory response to 5-HT which may underly a form of activity-dependent modulation. Thus, the presynaptic neuron must select which 5-HT response will be activated and which will be excluded at its synapses. The selection of these responses preceded synapse formation and was specifically induced at sites of contact with the presynaptic neuron, this not being observed for other cell pairings. Aldehyde-fixed presynaptic cells were equally effective, unless pre-treated with trypsin or wheat germ agglutinin, suggesting that contact with a specific cell-surface glycoprotein induced this physiological change in 5-HT sensitivity. The mechanism underlying the selective loss of the extrasynaptic response has been examined by single channel recording. Cation channels in the postsynaptic neuron were modulated by protein kinase C (PKC) upon binding of 5-HT to a 5-HT2 receptor. However, at sites of contact with the presynaptic neuron, the channels were no longer sensitive to PKC. Furthermore, when cation channels from uncontacted neurons were inserted or 'crammed' into contacted neurons, they were rapidly rendered insensitive to PKC, demonstrating a cytoplasmic signal for the uncoupling of channel modulation. Interestingly, the cytoplasm of contacted postsynaptic neurons showed immunoreactivity for tyrosine phosphorylation: exposure of the neurons to specific inhibitors of tyrosine kinases prevented tyrosine phosphorylation, the loss of cation channel modulation and synapse formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Voltage dependencies of the fast and slow gating modes of RIIA sodium channels.

Rat brain IIA sodium channel alpha-subunits were expressed in Xenopus oocytes, and the sodium currents were measured by intracellular voltage clamping with large agarose-tipped electrodes and by excised membrane patch-clamp recording to separate and characterize the properties of the fast and slow channel gating modes. The currents showed biexponential inactivation properties with fast and slow phases that could be isolated as distinct gating modes through differences in their inactivation properties. At holding potentials more negative than -55 mV, fast mode currents inactivated within a few milliseconds of depolarization, and could be distinguished by their rapid recovery from inactivation. Single sodium channels in the fast mode opened early after depolarization and rarely showed re-openings. At holding potentials positive to -55 mV, fast mode currents were inactivated, revealing slow mode currents which had slower activation and inactivation kinetics and showed sustained single channel activity during depolarizing pulses. The steady-state voltage dependencies of fast and slow mode activation were very similar. In contrast, slow mode inactivation occurred at potentials 27 mV more positive than fast mode inactivation. The slow mode appears to be due to destabilization of a voltage-insensitive conformation of the channel. The fast gating process dominated at high current levels, perhaps due to alpha-subunit interactions.

Animals↗

PubMed1994

Cell surface contact mediates neuronal recognition and synapse formation between two identified leech neurons.

An early event in the formation of the serotonergic synapse by the Retzius (R) onto the pressure-sensitive (P) neurons of the leech is the elimination of an extrasynaptic response to transmitter from sites of contact on the postsynaptic cell. This event during synapse formation is cell-specific in that it is elicited in vitro by contact with the presynaptic R cell but not with other neurons. In the study reported here, we investigated the nature of this interaction between R and P neurons. The loss of the extrasynaptic response of the P cell was elicited by contact with R cells fixed in a mild paraformaldehyde solution, but not by R cells treated with the proteolytic enzyme trypsin prior to fixation. As well, a variety of lectins were assayed for their ability to interfere with synapse formation. The transmitter responses of P cells plated on lectin-coated substrates were unaffected. However, exposure of the R cell to the lectin wheat germ agglutinin (WGA), but not to other lectins, prior to pairing prevented the loss of the extrasynaptic response in contacted P cells and blocked the formation of the R-P synapse in culture. We conclude that recognition by the P cell of the R cell during synapse formation may be mediated by an R cell-specific surface protein which binds wheat germ agglutinin.

Animals↗

PubMed1994

Insertion mutations of the RIIA Na+ channel reveal novel features of voltage gating and protein kinase A modulation.

A linker insertion mutagenesis strategy was developed to probe functional subdomains of the RIIA Na+ channel alpha-subunit. We describe mutations within the first two repeat domains that provide new functional information for three segments of the channel structure. 1. The insertion of two alanine residues within the short peptide segment joining helices S4 and S5 in domain II had two effects: a depolarizing shift of steady-state activation and reduced single-channel currents. These results suggest that the peptide segment following the S4 voltage sensor is involved in the activation process and is facing the ion pore. 2. An insertion immediately N-terminal to the proposed transmembrane helix S1 in domain II shifted the steady-state activation in the depolarizing direction, suggesting a functional role in channel gating. 3. Insertions in the large, cytoplasmic loop between domains I and II affect two channel functions: inactivation and protein kinase A modulation. These results demonstrate that the linker insertion approach can provide novel insights into the structure-function relationships of large, multi-domain ion channel proteins.

Amino Acid Sequence↗

PubMed1994

Segmental specificity of neuronal recognition during synapse formation between identified leech neurons.

An early event in the formation of inhibitory synapses between the serotonergic Retzius (R) neuron and the pressure-sensitive (P) neuron of the leech is the elimination of an extrasynaptic, depolarizing response to 5-HT in the P cell from sites of contact. This effect is induced specifically by contact with the R neuron but not with other identified leech neurons, including other serotonergic neurons that do not form chemical synapses with the P cell. In the reproductive (fifth and sixth) segmental ganglia, the R cells (R(5,6)) exhibit functional and morphological differences with the standard R cells (R(x)) in the other 19 segmental ganglia. In the study presented here, we tested the specificity of P cell recognition of R cells by examining whether there were differences between the R(x) and R(5,6) cells with respect to synapse formation with the P cell. R(5,6) neurons did not innervate P cells in the fifth and sixth ganglia (P(5,6)) in vivo or in vitro, nor did they form synapses with P cells from standard ganglia (P(x)) in vitro except on rare occasions, after long periods in culture. In contrast to the effects of R(5,6) neurons, R(x) neurons readily innervated both P(x) and P(5,6) cells in vitro, suggesting that the lack of an R(5,6)-P(5,6) synapse reflects differences in the presynaptic rather than the postsynaptic cell. In contrast to the R(x) cell, contact with the R(5,6) neurons had no significant effect on the responses of the P cell to 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Block of the rat brain IIA sodium channel alpha subunit by the neuroprotective drug riluzole.

The effects of riluzole, a novel neuroprotective drug with anticonvulsant and anti-ischemic properties, were studied on currents carried by cloned rat brain IIA sodium channel alpha subunits expressed in Xenopus oocytes. (i) When the oocytes were held at strongly hyperpolarized potentials to close the sodium channels and riluzole was added to the external solution, the current elicited by test depolarizing pulses was reduced within a few minutes and recovered upon washout of the riluzole. Although the currents were reduced, riluzole did not shift the peak current-voltage relationship. An inhibitory constant of 30 microM was estimated for the low affinity block of closed channels. (ii) Riluzole did not affect the time course of inactivation, and repetitive stimulation at frequencies that did not result in significant accumulation of inactivation did not affect current block. These results suggest that riluzole did not block open channels. (iii) Riluzole increased steady state inactivation by shifting its voltage dependence in the hyperpolarizing direction, by prolonging the recovery from inactivation, and by blocking more effectively at high stimulation frequencies. According to the modulated receptor theory, these results suggest that riluzole binds selectively to inactivated channels, with an inhibitory constant estimated at 0.2 microM. These results show that the riluzole binding site is on the alpha subunit of the sodium channel, and they suggest that stabilization of the inactivated state may underlie the neuroprotective properties of riluzole.

Animals↗

PubMed1993

Tyrosine kinase-dependent selection of transmitter responses induced by neuronal contact.

Transmitter receptors are localized to discrete cellular sites such that only those responses appropriate for a particular pattern of inputs are activated. How neurons select between synaptic and extrasynaptic responses during development is not understood. We have investigated how contact during synapse formation between identified leech neurons selectively suppresses the modulation of extrasynaptic channels by protein kinase C. A microelectrode with an isolated membrane patch containing channels from an uninnervated target neuron was 'crammed' into a similar cell contacted by a presynaptic partner. We report here that within a few minutes, the crammed channels were rendered insensitive to activation of protein kinase C, demonstrating the action of a cytoplasmic signal. Treatment of the neurons with selective inhibitors of tyrosine kinases, which are signalling molecules during normal and oncogenic cellular differentiation, prevented the loss of channel modulation. Thus, tyrosine kinases mediate early functional changes during specific synapse formation that are induced by neuronal contact.

Animals↗

PubMed1993

Selection of transmitter responses at sites of neurite contact during synapse formation between identified leech neurons.

1. Pressure sensitive (P) neurons of the leech Hirudo medicinalis show both an inhibitory, Cl(-)-dependent response and a depolarizing, cationic response to pipette application of serotonin (5-HT). Serotonergic Retzius (R) neurons in culture reform inhibitory, Cl(-)-dependent synapses with P neurons but fail to elicit the extrasynaptic, depolarizing response to 5-HT. We have examined the localization of the selection of 5-HT responses by testing the sensitivity of P cell growth cones and neurites to 5-HT application. 2. As measured by intracellular recording at the P cell soma, synaptic release of 5-HT from R cell processes activated only the Cl(-)-dependent response in P cell neurites. Focal application of 5-HT from a micropipette depolarized uncontacted P cell growth cones and neurites. In contrast, processes from the same P cells that were contacted by R cells were rarely depolarized by 5-HT application unless the application pipette was moved along the neurites away from the sites of contact. 3. The channels underlying the depolarizing response to 5-HT were identified in patch clamp recordings from P cell growth cones. These cation channels showed rare, brief openings in the absence of 5-HT. Application of 5-HT in the bath (outside the patch pipette) increased channel activity in uncontacted P cell growth cones but not in growth cones of the same P cells contacted by R cells. 4. We conclude that the selection of transmitter responses during synapse formation was localized to discrete sites of contact between the synaptic partners.

Action Potentials↗

PubMed1992

Cell-specific contact selects transmitter responses in an identified leech neuron.

Serotonergic Retzius (R) neurons of the leech form a Cl-dependent synapse with pressure-sensitive (P) neurons both in vivo and in vitro. However, P cells show an extrasynaptic, cationic response to application of 5-hydroxytryptamine (5-HT) which is reduced upon contact between the neurons in culture. We have examined the cellular specificity of the selection of 5-HT responses in the P cell by pairing it in culture with a variety of identified neurons. Non-synaptic sensory cells, non-serotonergic pre- and postsynaptic partners and serotonergic neurons that do not form chemical synapses with the P cell failed to alter its responses to 5-HT. The selective reduction of the extrasynaptic response to 5-HT in the P cell therefore appears to be induced specifically by contact with its only known serotonergic partner during neuronal recognition leading to synapse formation.

Animals↗

PubMed1992

Loss of extrasynaptic channel modulation by protein kinase C underlies the selection of serotonin responses in an identified leech neuron.

Pressure-sensitive (P) neurons contacted by serotonergic Retzius (R) neurons of the leech in culture selectively reduce a protein kinase C (PKC)-dependent cation response to serotonin and are innervated by the inhibitory, Cl(-)-dependent synapse seen in vivo. We have examined whether the reduction of extrasynaptic cation channel modulation is due to changes in sensitivity of the channels to second messenger. In inside-out membrane patches from single, uncontacted P cells in culture, cation channel activity was increased by rat brain PKC and cofactors. In contrast, the activity of cation channels in patches isolated from P cells paired with R cells was unaffected by PKC. These results demonstrate the loss of extrasynaptic channel modulation by PKC during synapse formation.

Animals↗

PubMed1991

Distinct receptors, second messengers and conductances underlying the dual responses to serotonin in an identified leech neurone.

1. Pressure-sensitive mechanosensory (P) neurones of the leech Hirudo medicinalis produce two responses to serotonin (5-HT): activation of a Cl- conductance and of a non-selective monovalent cation conductance. The effects of channel blockers, the receptor pharmacology and the second-messenger dependence of these responses were studied in voltage-clamped P cells in culture. Antagonists were applied by superfusion and agonists by pressure ejection. 2. Zn2+ (100 mumol l-1) and H+ (pH 6.5 and lower) reversibly reduced the Cl- conductance activated by 5-HT. The cation conductance was impermeant to calcium ions and was reduced by micromolar concentrations of the Na+ channel inhibitors amiloride and 3,4-dichlorobenzamil. 3. High concentrations of antagonists or agonists of 5-HT1 receptors and an antagonist of 5-HT3 receptors had no effect on either response of P cells to 5-HT. Micromolar concentrations of ketanserin or cyproheptadine, which selectively antagonize 5-HT2 receptors, reduced the cation but not the Cl- conductance. From these results, the receptor underlying the cation conductance appears to be of the 5-HT2 subtype, whereas the receptor activating the Cl- conductance does not fit within the mammalian classification scheme. 4. Brief (less than 500 ms) application of membrane-permeant agonists of the second messenger cyclic AMP elicited a Cl- conductance, whereas antagonists of cyclic-AMP-dependent protein kinase A reversibly suppressed the Cl- conductance elicited by 5-HT and by cyclic AMP agonists. Compounds affecting other second messenger pathways were without effect on the Cl- conductance. It therefore appears that the Cl- conductance is activated by cyclic-AMP-dependent protein kinase A. 5. Cyclic nucleotide agonists and antagonists were without effect on the cation conductance. However, brief application of phorbol esters, which activate protein kinase C, elicited an amiloride-sensitive cation current. An inhibitor of protein kinase C reduced the cation conductance activated by 5-HT and by phorbol esters. Therefore, the cation conductance appears to depend on activation of protein kinase C. 6. We conclude that 5-HT activates two types of receptor coupled to separate ionic channels via different second messenger pathways in P cells. A receptor that is distinct from the mammalian subtypes activates Cl- channels via cyclic-AMP-dependent protein kinase A. 5-HT2 receptors appear to activate cation channels by means of protein kinase C.

Animals↗

PubMed1990

Loss of channel modulation by transmitter and protein kinase C during innervation of an identified leech neuron.

When serotonergic Retzius (R) neurons of the leech contact pressure-sensitive (P) neurons in culture, P cells selectively lose a protein kinase C-dependent cationic response to serotonin and the R cell reforms the inhibitory, chloride-dependent synapse seen in vivo. In P cells not contacted by R cells, cell-attached patches contained single cation channels sensitive to serotonin and phorbol ester with characteristic properties and high incidence (present in about one-half of the patches). P cells paired with R cells had a cation channel with similar biophysical properties and incidence, but channel activity was not stimulated by serotonin and phorbol ester. These results suggest that the early clearing of the non-synaptic (excitatory) response to serotonin is due to the loss of activation by protein kinase C (and not the number) of cation channels as a prelude to inhibitory synapse formation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1989

Parallel processing and selection of the responses to serotonin during reinnervation of an identified leech neuron.

In an attempt to define the mechanism of synaptic specificity, we have been studying pairs of identified leech neurons isolated in tissue culture. The cultured neurons reform specific synapses when paired with appropriate partners in the absence of other cell types. In recent studies, we have examined in detail the reformation of a serotoninergic synapse between the Retzius cell and one of its targets, the pressure sensitive (P) cell. The P cell in vivo and its soma in vitro have two types of responses to serotonin (5-HT). From voltage clamp analysis of cultured P cells, we demonstrated the parallel activation of chloride (gCls) and monovalent cation (gCations) channels coupled to distinct receptor subtypes and gated by separate second messengers. Only gCls was activated by 5-HT released from the presynaptic Retzius cell both in vivo and in vitro. This demonstrates the remarkable specificity of the reformation of this synapse in culture since only the correct 5-HT receptor subtype is activated. An 80% reduction of gCations was observed in P cells that had failed to be innervated by Retzius cells in culture, suggesting that gCations may be lost prior to synapse formation. Retzius cells depleted of 5-HT also reduced gCations in the paired P cells and incubating single P cells in 5-HT did not reduce gCations. In addition, aldehyde-fixed Retzius cells were able to selectively reduce gCations when paired with P cells. We conclude that the loss of gCations was due to contact between the neurons. The early clearing of counter-effective receptor subtypes may be a prelude to synapse formation.

Animals↗

PubMed1989

Contact-mediated loss of the nonsynaptic response to transmitter during reinnervation of an identified leech neuron in culture.

We have examined the modification of responses to transmitter during the reformation of a specific synapse by identified leech neurons in culture. Single pressure-sensitive mechanosensory (P) neurons in culture and the soma of this cell in vivo have 2 conductances that are activated by application of 5-HT: a Cl conductance and a monovalent cation conductance (gCations); synaptic release of 5-HT by a serotoninergic Retzius cell in vivo and in culture activates only the Cl conductance and not gCations in the P cell. We have characterized the loss of gCations in P cells by manipulating the culture conditions. When 5-HT was applied from a pipette, innervated P cells and P cells paired with Retzius cells that had not formed synapses had a gCations that was markedly reduced compared with gCations in the single P cell. When 5-HT was included in the culture medium in which single P cells were grown, gCations was not reduced. When Retzius-P cell pairs were treated with reserpine, which was shown to deplete the presynaptic neuron of 5-HT, gCations in the P cell was as low as in untreated pairs. Pairing aldehyde-fixed Retzius cells with untreated P cells also resulted in the loss of gCations. The Retzius cell had both types of receptors, but synapse formation did not affect gCations in the presynaptic neuron. The results demonstrate that the loss of the nonsynaptic response of the P cell to transmitter is due to contact between the neurons rather than to the release of transmitter or trophic factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Effects of kainic acid lesions on calcium uptake and dopamine release in nerve endings isolated from rat striata.

Rats were injected intrastriatally with kainic acid and the viability of dopaminergic terminals two days following the injection was determined by comparing voltage dependent calcium uptake and dopamine release in isolated nerve endings. Evoked dopamine release remained normal following the lesion, but the initial rate of potassium stimulated calcium entry decreased by approximately 1/3. These results suggest that the excitotoxic lesion caused by acute intrastriatal injection of kainic acid results in retention of functional dopaminergic terminals.

Animals↗