Biomedical subjects

P Drapeau

Publications and source records attributed to P Drapeau.

At least 19 recordsLinked to original sources

PubMed1998

Neurite growth patterns leading to functional synapses in an identified embryonic neuron.

We explored the relationship between neurite outgrowth and the onset of synaptic activity in the central neuropil of the leech embryo in vivo. To follow changes in early morphology and the onset of synaptic activity in the same identified neuron, we obtained whole-cell patch-clamp recordings and fluorescent dye fills from dorsal pressure-sensitive (P) cells, the first neurons that could be reliably identified in the early embryo. We followed the development of the P cell from the first extension of neurites to the elaboration of an adult-like arbor. After the growth of primary neurites, we observed a profuse outgrowth of transient neurites within the neuropil. Retraction of the transient neurites left the primary branches studded with spurs. After a dormant period, stable secondary branches grew apparently from the spurs and became tipped with terminals. At this time, neurites of the Retzius (R) cell, a known presynaptic partner in the adult, were observed to apparently contact the terminals. Although voltage-dependent currents were seen in the P cell at the earliest stage, spontaneous synaptic activity was only observed when terminals had formed. Spontaneous release was observed before evoked release could be detected from the R cell. Our results suggest that transient neurites are formed during an exploratory phase of development, whereas the more precisely timed outgrowth of stable neurites from the spurs signals functional differentiation during synaptogenesis. Because spurs have also been observed in neurons of the mammalian brain, they may constitute a primordial synaptic organizer.

Animals↗

PubMed1998

Ionotropic and metabotropic activation of a neuronal chloride channel by serotonin and dopamine in the leech Hirudo medicinalis.

1. Cl- channels on the pressure-sensitive (P) neuron in the leech are directly activated by synaptic release of serotonin (5-HT) and are indirectly stimulated by the cAMP second messenger pathway, suggesting an unusual dual regulation of the channels. We have investigated the mode of action of 5-HT and dopamine (DA) on a Cl- channel in adult P cells in culture by recording from cell-attached patches. 2. 5-HT increased Cl- channel activity only when included in the recording pipette and not when applied in the bath. 3. Pipette or, more effectively, bath application of DA led to an increase in Cl- channel activity. This effect was blocked by the potent and specific dopaminergic (DA1) receptor blocker, SCH-23390. 4. The stimulation by DA, but not by 5-HT, was also blocked by the cAMP-dependent protein kinase A (PKA) inhibitor Rp-cAMP and was mimicked by the membrane-permeant cAMP analogue dibutyryl cAMP (db-cAMP). 5. Our results show that 5-HT directly gates a Cl- channel that is also activated by DA via the cAMP pathway. This study demonstrates that a ligand-gated channel can be independently operated by another transmitter acting via a second messenger pathway.

Animals↗

PubMed1998

Time course of the development of motor behaviors in the zebrafish embryo.

The development and properties of locomotor behaviors in zebrafish embryos raised at 28.5 degrees C were examined. When freed from the chorion, embryonic zebrafish showed three sequential stereotyped behaviors: a transient period of alternating, coiling contractions followed by touch-evoked rapid coils, then finally, organized swimming. The three different behaviors were characterized by video microscopy. Spontaneous, alternating contractions of the trunk appeared suddenly at 17 h postfertilization (hpf), with a frequency of 0.57 Hz, peaked at 19 hpf at 0.96 Hz, and gradually decreased to <0.1 Hz by 27 hpf. Starting at 21 hpf, touching either the head or the tail of the embryos resulted in vigorous coils. The coils accelerated with development, reaching a maximum speed of contraction before 48 hpf, which is near the time of hatching. After 27 hpf, touching the embryos, particularly on the tail, could induce partial coils (instead of full coils). At this time, embryos started to swim in response to a touch, preferentially to the tail. The swim cycle frequency gradually increased with age from 7 Hz at 27 hpf to 28 Hz at 36 hpf. Lesions of the central nervous system rostral to the hindbrain had no effect on the three behaviors. Lesioning the hindbrain eliminated swimming and touch responses, but not the spontaneous contractions. Our observations suggest that the spontaneous contractions result from activation of a primitive spinal circuit, while touch and swimming require additional hindbrain inputs to elicit mature locomotor behaviors.

Aging↗

PubMed1997

Requirement for tyrosine phosphatase during serotonergic neuromodulation by protein kinase C.

Tyrosine kinases and phosphatases are abundant in the nervous system, where they signal cellular differentiation, mediate the responses to growth factors, and direct neurite outgrowth during development. Tyrosine phosphorylation can also alter ion channel activity, but its physiological significance remains unclear. In an identified leech mechanosensory neuron, the ubiquitous neuromodulator serotonin increases the activity of a cation channel by activating protein kinase C (PKC), resulting in membrane depolarization and modulation of the receptive field properties. We observed that the effects on isolated neurons and channels were blocked by inhibiting tyrosine phosphatases. Serotonergic stimulation of PKC thus activates a tyrosine phosphatase activity associated with the channels, which reverses their constitutive inhibition by tyrosine phosphorylation, representing a novel form of neuromodulation.

Action Potentials↗

PubMed1997

Channel modulation by tyrosine phosphorylation in an identified leech neuron.

1. We have examined the effects of tyrosine phosphorylation on a spontaneously active cation channel that also participates in the modulation of pressure-sensitive (P) neurons in the leech. Cation channel activity in cell-attached or isolated, inside-out membrane patches from P cells in culture was monitored before and after treatments that altered the level of tyrosine phosphorylation. 2. In cell-attached recordings from intact P cells, bath application of genistein, an inhibitor of tyrosine kinases, resulted in a 6.6 +/- 2.6-fold increase in channel activity with no change in the mean open time or amplitude. Daidzein, an inactive form of genistein, was without effect. Addition of pervanadate, a membrane-permeant inhibitor of tyrosine phosphatases, had no effect on its own and blocked the effect of subsequent addition of genistein. 3. In inside-out P cell membrane patch recordings, exposure to a catalytically active fragment of a tyrosine phosphatase resulted in a 10.3 +/- 3.6-fold increase in channel activity with no change in the mean open time or amplitude. Orthovanadate had no effect on channel activity and, when added with the phosphatase, prevented the increase in activity. 4. Our results demonstrate that the basal activity of cation channels is increased by tyrosine dephosphorylation, suggesting a constitutive modulation of channel activity under resting conditions.

Animals↗

PubMed1996

Modulation of conduction block in leech mechanosensory neurons.

Conduction block is a mechanism of activity-dependent neuronal plasticity, but little is known about its possible neuromodulation. Extensive activity in leech touch (T), pressure (P), and nociceptive (N) mechanosensory neurons results in conduction block of their minor receptive fields. We have examined whether the duration of conduction block could be modulated by the serotonergic Retzius neurons or by application of serotonin (5-HT). Activation of one Retzius cell reduced the duration of conduction block in T and P cell posterior fields, but their anterior fields and N cell fields were unaffected. Perfusion with 5-HT had stronger effects, reducing the duration of conduction block in T, P, and lateral N cells in the posterior fields and either reducing or more often enhancing the expression of conduction block in anterior fields. The effects of 5-HT on posterior fields were blocked by the nonspecific 5-HT antagonist methysergide and were partly suppressed by the 5-HT2 antagonist ketanserin. To determine the site of 5-HT action, the central ganglion or peripheral skin was perfused independently. T and to a greater extent P cells showed a preferential sensitivity to application of 5-HT onto the central ganglion. Interestingly, medial N cells exhibited a progressive decrease in the duration of conduction block during repeated trials ("wind-up") that was unaffected by 5-HT. We conclude that secretion of 5-HT by the Retzius cells has a central modulatory effect on the duration of conduction block in T, P, and lateral N cells.

Animals↗

PubMed1996

Modulation and selection of neurotransmitter responses during synapse formation between identified leech neurons.

1. Serotonin (5-HT) modulates two different responses in the pressure sensitive neurons (P) of the leech: an inhibitory, Cl- dependent synaptic response and a depolarizing extrasynaptic response. 2. Serotonergic Retzius cells (R) in vivo and in culture elicit inhibitory Cl- dependent responses in P neurons. Moreover, at discrete sites of contact between R and P cells, the excitatory response to 5-HT is gradually lost prior to synapse formation. This phenomenon is specifically mediated by R cells. 3. The extrasynaptic response is mediated by cation channels sensitive to protein kinase C (PKC). Cation channels are present at the sites of contact but they become insensitive to PKC. Moreover, cation channels from single P cells are no longer modulated by PKC if they are inserted (by cramming the patch pipette) into the cytoplasm of a P cell in contact with an R cell. 4. Blockers of tyrosine kinases prevent the uncoupling of cation channel modulation and inhibit synapse formation between the R and the P neurons. 5. We suggest that cell contact induces an intracellular, tyrosine kinase-dependent signal as part of the mechanism of neuronal recognition leading to synapse formation.

Animals↗

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

Generating a phage display antibody library against an identified neuron.

The generation of monoclonal antibodies by conventional hybridoma technology is limited by the diversity of the clones and the difficulties of screening the antibodies and of their large-scale production from isolated clones. As an alternative approach, we have investigated the suitability of phage display libraries for the production of recombinant antibodies against an identified neuron. Mice were immunized with isolated leech Retzius (R) neurons. The spleen poly A+ RNA was isolated and first-strand cDNA was prepared. The variable regions of light- and heavy-chain IgG molecules were amplified by the polymerase chain reaction (PCR) and separate libraries of each were constructed and combined in the pComb8 vector to yield a combinatorial library of approximately 10(7) transformants. Single R neurons that were plated in culture bound approximately 100 phages on a first screen and several thousand when the first batch was re-screened. Of these, 96 individual phage colonies were isolated and used for immunocytochemistry with leech CNS ganglia: 41 exhibited general staining, 20 showed no detectable staining and 30 stained selective subsets of neurons including (but not specific for) the R neuron. The phage display library approach thus simplifies the screening of large libraries with small numbers of (and even single) cells. However, the combinatorial antibodies with binding activity must still be tested individually by immunocytochemistry, which is further limited by their apparently low affinity.

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↗