Identified Leech Neurons Research Articles

Functional properties and cell type specific distribution ofIh channels in leech neurons

The hyperpolarisation-activated cation current (I(h)) has been described in many vertebrate and invertebrate species and cell types. In neurons, I(h) is involved in rhythmogenesis, membrane potential stabilisation and many other functions. In this work, we investigate the distribution and functional properties of I(h) in identified leech neurons of intact segmental ganglia. We found I(h) in the mechanosensory touch (T), pressure (P) and noxious (N) neurons, as well as in Retzius neurons. The current displayed its largest amplitude in P neurons and we investigated its biophysical and pharmacological properties in these cells. I(h) was half-maximally activated at -65 mV and fully activated at -100 mV. The current mutually depended on both Na(+) and K(+) with a permeability ratio p(Na)/p(K) of ∼0.21. The reversal potential was approximately -35 mV. The time course of activation could be approximated by a single time constant of ∼370 ms at -60 mV, but required two time constants at -80 mV of ∼80 and ∼560 ms. The current was half-maximally blocked by 0.3 mmol l(-1) Cs(+) but was insensitive to the bradycardic agent ZD7288. The physiological function of this channel could be a subtle alteration of the firing behaviour of mechanosensory neurons as well as a stabilisation of the resting membrane potential.

Regulation of the mechanosensitive cation channels by ATP and cAMP in leech neurons

Single-channel recordings were used to study the modulation of stretch-activated channels (SACs) by intracellular adenosine nucleotides in identified leech neurons. These channels exhibited two activity modes, spike-like (SL) and multiconductance (MC), displaying different polymodal activation. In the absence of mechanical stimulation, internal perfusion of excised patches with ATP induced robust and reversible activation of the MC but not of the SL mode. The ATP effect on channel activity was dose-dependent within a range of 1 μM–1 mM and was induced at different values of intracellular pH and Ca 2+. The non-hydrolyzable ATP analog AMP-PNP, ATP without Mg 2+ or ADP also effectively enhanced MC activity. Adenosine mimicked the effect of its nucleotides. At negative membrane potentials, both ATP and adenosine activated the channel. Moreover, ATP but not adenosine induced a flickering block. Addition of cAMP during maximal ATP activation completely and reversibly inhibited the channel, with activation and deactivation times of minutes. However, cAMP alone only induced a weak and rapid channel activation, without inhibitory effects. The expression of these channels in the growth cones of leech neurons, their permeability to Ca 2+ and their sensitivity to intracellular cAMP are consistent with a role in the Ca 2+ oscillations associated with cell growth.

Extracellular matrix glycoproteins inhibit neurite outgrowth of different types of identified leech neurons in culture

We explored the contribution of inhibitory peanut-binding extracellular matrix glycoproteins to the regeneration of characteristic outgrowth patterns by different types of identified neurons. Adult leech neurons were isolated one by one and plated in culture on a substrate that consisted of the capsules that encase the CNS ganglia. On the inside surface of this substrate, a combination of growth-promoting and -inhibiting extracellular matrix glycoproteins regulates the regeneration of distinctive outgrowth patterns by different neuron types. The role of inhibitory glycoproteins that bind to peanut lectin was studied by perturbation experiments in which peanut lectin was added to the culture medium. The effects of peanut lectin on the outgrowth patterns depended on the specific cell type that was tested. Anterior pagoda neurons, which on capsules produce a bipolar outgrowth pattern, in the presence of the lectin multiplied the number of primary neurites and the total neurite length and also lost their bipolarity. Annulus erector motoneurons, which on capsules grow poorly, in the presence of peanut lectin sprouted 70% more neurites and duplicated their total neurite length. By contrast, Retzius neurons which grow profusely on ganglion capsules, in the presence of peanut lectin increased the number of primary neurites without increasing their total neurite length or branch points. When neurons were plated on plastic, peanut lectin added to the culture medium did not affect the growth of neurons, thus showing that the effects of peanut lectin were induced by blocking the binding of neurons to inhibitory glycoproteins on the capsules. These results show that regeneration of different neuron types has different regulation by inhibitory extracellular matrix molecules.

Ca2+ influx into identified leech neurons induced by 5-hydroxytryptamine.

The role of 5-hydroxytryptamine (5-HT, serotonin) in the control of leech behavior is well established and has been analyzed extensively on the cellular level; however, hitherto little is known about the effect of 5-HT on the cytosolic free calcium concentration ([Ca(2+)](i)) in leech neurons. As [Ca(2+)](i) plays a pivotal role in numerous cellular processes, we investigated the effect of 5-HT on [Ca(2+)](i) (measured by Fura-2) in identified leech neurons under different experimental conditions, such as changed extracellular ion composition and blockade of excitatory synaptic transmission. In pressure (P), lateral nociceptive (N1), and Leydig neurons, 5-HT induced a [Ca(2+)](i) increase which was predominantly due to Ca(2+) influx since it was abolished in Ca(2+)-free solution. The 5-HT-induced Ca(2+) influx occurred only if the cells depolarized sufficiently, indicating that it was mediated by voltage-dependent Ca(2+) channels. In P and N1 neurons, the membrane depolarization was due to Na(+) influx through cation channels coupled to 5-HT receptors, whereby the dose-dependency suggests an involvement in excitatory synaptic transmission. In Leydig neurons, 5-HT receptor-coupled cation channels seem to be absent. In these cells, the membrane depolarization activating the voltage-dependent Ca(2+) channels was evoked by 5-HT-triggered excitatory glutamatergic input. In Retzius, anterior pagoda (AP), annulus erector (AE), and median nociceptive (N2) neurons, 5-HT had no effect on [Ca(2+)](i).

L-type Ca 2+ channel antagonists block voltage-dependent Ca 2+ channels in identified leech neurons

We investigated the effect of L-type Ca 2+ channel antagonists on the Ca 2+ influx through voltage-gated Ca 2+ channels in leech Retzius, Leydig, AP, AE, P, and N neurons. The efficacy of the antagonists was quantified by monitoring their effect on the increase in the intracellular free Ca 2+ concentration ([Ca 2+] i; measured by Fura-2) that was induced by depolarizing the cell membrane by raising the extracellular K + concentration. This K +-induced [Ca 2+] i increase was blocked by the phenylalkylamines verapamil, gallopamil, and devapamil, the benzothiazepine diltiazem, as well as by the 1,4-dihydropyridine nifedipine. The blocking effect of the three phenylalkylamines was similar, being most pronounced in P and N neurons and smaller in Leydig, Retzius, AP, and AE neurons. Contrastingly, diltiazem and nifedipine were similarly effective in the neurons investigated, whereby their efficacy was like that of the phenylalkylamines in Retzius, Leydig, AP, and AE neurons. Depending on cell type and blocking agent, the concentrations necessary to suppress the K +-induced [Ca 2+] i increase by 50% were estimated to vary between 5 and 190 μM. At high concentrations, the phenylalkylamines and diltiazem by themselves caused a marked [Ca 2+] i increase in Leydig, P, and N neurons, which is probably due to activation of the caffeine-sensitive ion channels present in the plasma membrane of these cells. Together with previous observations, the results indicate a distant relationship of the voltage-gated Ca 2+ channels present in many if not all leech neurons to vertebrate L-type Ca 2+ channels.

L-type Ca2+ channel antagonists block voltage-dependent Ca2+ channels in identified leech neurons

We investigated the effect of L-type Ca2+ channel antagonists on the Ca2+ influx through voltage-gated Ca2+ channels in leech Retzius, Leydig, AP, AE, P, and N neurons. The efficacy of the antagonists was quantified by monitoring their effect on the increase in the intracellular free Ca2+ concentration ([Ca2+]i; measured by Fura-2) that was induced by depolarizing the cell membrane by raising the extracellular K+ concentration. This K+-induced [Ca2+]i increase was blocked by the phenylalkylamines verapamil, gallopamil, and devapamil, the benzothiazepine diltiazem, as well as by the 1,4-dihydropyridine nifedipine. The blocking effect of the three phenylalkylamines was similar, being most pronounced in P and N neurons and smaller in Leydig, Retzius, AP, and AE neurons. Contrastingly, diltiazem and nifedipine were similarly effective in the neurons investigated, whereby their efficacy was like that of the phenylalkylamines in Retzius, Leydig, AP, and AE neurons. Depending on cell type and blocking agent, the concentrations necessary to suppress the K+-induced [Ca2+]i increase by 50% were estimated to vary between 5 and 190 μM. At high concentrations, the phenylalkylamines and diltiazem by themselves caused a marked [Ca2+]i increase in Leydig, P, and N neurons, which is probably due to activation of the caffeine-sensitive ion channels present in the plasma membrane of these cells. Together with previous observations, the results indicate a distant relationship of the voltage-gated Ca2+ channels present in many if not all leech neurons to vertebrate L-type Ca2+ channels.

Extracellular matrix glycoproteins inhibit neurite production by cultured neurons.

We have analyzed the role of extracellular matrix glycoproteins in the formation of a bipolar outgrowth pattern of identified leech neurons in culture. Adult anterior pagoda (AP) neurons cultured on the inner surface of the ganglion capsules that surround central nervous system, generate two processes oriented in opposite directions. This pattern differs from those produced by these neurons cultured on other substrates, and is similar to the pattern of developing AP neurons at embryonic day 10. We used different lectins to identify subsets of glycoproteins in the extracellular matrix (ECM) of the capsules and to study their contribution to the formation of the bipolar outgrowth pattern. ECM glycoproteins binding to peanut agglutinin (PNA) or Galanthus nivalis aglutinin (GNA) lectins were detected in ganglion capsules and in ganglion extracts that had been separated by electrophoresis and blotted to nitrocellulose membranes. Four protein bands bound to PNA lectin and six other bands, including laminin subunits, bound to GNA lectin. Other lectins failed to recognize any of the proteins. For AP neurons cultured on capsules, addition of PNA lectin to the culture medium produced a dose-dependent increase in the number of primary neurites without affecting their shape, length or number of branch points. However, PNA lectin used as substrate did not affect sprouting of AP neurons. Our results suggest that PNA-binding extracellular matrix glycoproteins regulate the formation of the bipolar pattern of AP neurons by inhibiting the formation of neurites.

ATP-inhibited K + channels and membrane potential of identified leech neurons

The effect of the ATP-inhibited K + channel on the membrane potential of leech Retzius neurons was analyzed using electrolyte-filled single-barrelled microelectrodes. The membrane potential was independent of the external nutrient supply during a period of 11 h, probably because the internal energy reserves were sufficient. The K + channel activator HOE 234 ((3 S,4 R)-3-hydroxy-2,2-dimethyl-4-(2-oxo-1-pyrrolidinyl)-6-phenylsulfonylchromane hemihydrate, 500 μM) induced a membrane hyperpolarization. In the presence of HOE 234, action potentials occurred with a reduced after-hyperpolarization and were discharged in bursts, possibly because of an inhibition of Ca 2+ channels. The blocker of ATP-inhibited K + channels tolbutamide did not significantly alter the membrane potential. In the absence of tolbutamide, the metabolic inhibitors iodoacetate, azide and cyanide (10 mM) evoked membrane hyperpolarizations, but in the presence of 1 mM tolbutamide their hyperpolarizing actions were reduced or abolished while membrane depolarizations were intensified. We conclude that ATP-inhibited K + channels in the soma membrane of leech Retzius neurons provide coupling of cellular metabolism to electrical activity and ionic fluxes.

Outgrowth patterns and directed growth of identified neurons induced by native substrates in culture.

In this study, we have tested how various identified leech neurons in culture grow on surfaces that they normally contact in situ. Neurons were cultured either on ganglion capsules from which neurons had been removed or on skin. On these substrates, outgrowth patterns were characteristic for each cell type. Retzius cells plated on capsules extended bundles of thick, fasciculated processes with few branching points and in the opposite direction a tangle of fine neurites. Anterior pagoda (AP) neurons plated on capsules extended two single processes in opposite directions but failed to grow on skin. Sensory P and N neurons on capsules extended multiple processes. On skin, P neurons extended only two long branches in opposite directions over the superficial body wall. N neurons on skin extended multiple processes. Varicosities were common in the processes of P and N neurons on capsules or skin. The branching patterns described here bore closer resemblance to those in the developing or adult nervous system than to those on Concanavalin A or laminin-enriched extract. Pairs of Retzius or AP neurons plated at a distance on the same capsule extended neurites from one neuron toward the other and formed contacts. Such directed growth failed in hybrid pairs of Retzius and AP neurons or in pairs plated on laminin-enriched extract or Concanavalin A. Our results suggest that multiple growth-promoting molecules anchored to the extracellular matrix may cooperate in regulating the branching pattern of neurons, fasciculation, and direction of growth.

Different determinants on growth and synapse formation in cultured neurons.

The influence of substrate and target on growth and synapse formation was investigated in identified leech neurons in culture. Nociceptive neurons and annulus erector motoneurons were cultured on ganglion capsules, a substrate they encounter in the leech. Within a few hours, single nociceptive cells sprouted profusely, whereas annulus erector cells failed to grow. When annulus erector neurons were plated near a nociceptive cell on the same capsule both neurons grew, made contact and formed an electrical synapse, different from the chemical synapse they form in the ganglion, but identical to that formed when plated on Concanavalin A. These results suggest that substrate and target have complementary effects regulating growth, but fail to define the type of synapse formed.

Voltage-dependent Ca 2+ influx into identified leech neurones

We determined the relationships between the intracellular free Ca 2+ concentration ([Ca 2+] i) and the membrane potential ( E m) of six different neurones in the leech central nervous system: Retzius, 50 (Leydig), AP, AE, P, and N neurones. The [Ca 2+] i was monitored by using iontophoretically injected fura-2. The membrane depolarization evoked by raising the extracellular K + concentration ([K +] o) up to 89 mM caused a persistent increase in [Ca 2+] i, which was abolished in Ca 2+-free solution indicating that it was due to Ca 2+ influx. The threshold membrane potential that must be reached in the different types of neurones to induce a [Ca 2+] i increase ranged between −40 and −25 mV. The different threshold potentials as well as differences in the relationships between [Ca 2+] i and E m were partly due to the cell-specific generation of action potentials. In Na +-free solution, the action potentials were suppressed and the [Ca 2+] i/ E m relationships were similar. The K +-induced [Ca 2+] i increase was inhibited by the polyvalent cations Co 2+, Ni 2+, Mn 2+, Cd 2+, and La 3+, as well as by the cyclic alcohol menthol. Neither the polyvalent cations nor menthol had a significant effect on the K +-induced membrane depolarization. Our results suggest that different leech neurones possess voltage-dependent Ca 2+ channels with similar properties.

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.

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.

Distribution and functional properties of glutamate receptors in the leech central nervous system

1. The effect of kainate and other glutamatergic agonists on the membrane potential (Em), the intracellular Na+ activity (aNai), and the intracellular free Ca2+ concentration ([Ca2+]i) of identified leech neurons and neuropile glial cells was measured with conventional and ion-sensitive microelectrodes, as well as with the use of the iontophoretically injected fluorescent indicators sodium-binding benzofuran isophthalate and Fura-2. 2. In Retzius neurons, AE, L, 8, and 101 motoneurons, and in the unclassified 50 neurons (Leydig cells) and AP neurons, as well as in neuropile glial cells, bath application of 100 microM kainate evoked a marked membrane depolarization and an increase in aNai and [Ca2+]i. The kainate-induced aNai increase persisted in solutions with high Mg2+ concentration in which synaptic transmission is blocked. 3. A membrane depolarization as well as an increase in aNai and [Ca2+]i was also evoked by L-glutamate, quisqualate, and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). The agonist-induced [Ca2+]i increase was inhibited by 6,7-dinitroquinoxaline-2,3-dione (DNQX). 4. In Ca(2+)-free solution, the kainate-induced [Ca2+]i increase was abolished in the neurons and in neuropile glial cells, whereas membrane depolarization and aNai increase were unchanged. In Na(+)-free solution, kainate had no effect on Em, aNai, or [Ca2+]i in the neurons. 5. In the mechanosensory T, P, and N neurons, kainate induced considerably smaller membrane depolarizations than in the other neurons or in neuropile glial cells, and it had no significant effect on aNai or [Ca2+]i. 6. It is concluded that in leech segmental ganglia the majority of the neurons and the neuropile glial cells, but probably not the mechanosensory neurons, possess glutamate receptors of the AMPA-kainate type. In the neurons, the [Ca2+]i increase caused by glutamatergic agonists is due to Ca2+ influx through voltage-dependent Ca2+ channels that are activated by the agonist-induced membrane depolarization.

Segmental diversification of an identified leech neuron correlates with the segmental domain in which it expresses Lox2, a member of the Hox gene family.

The cellular colocalization of LOX2 protein and small cardioactive peptide (SCP)-like immunoreactivity was studied in the nerve cord of the glossiphoniid leech Helobdella triserialis. Of the six neurons that express SCP in the midbody segments 7 to 17, only one, the MPS neuron, expresses LOX2 protein. The medial paired SCP (MPS) neurons are segmentally repeated and can be divided into three contiguous segmental domains according to cell body size and the timing and level of SCP expression. MPS neurons located in the anterior and middle segmental domains express LOX2 protein. In the middle domain, large MPS neurons begin to accumulate SCP shortly after the end of embryonic development, whereas in the anterior domain the MPS neurons are smaller and begin to express SCP at a later stage. In the posterior domain the MPS neurons exhibit a third phenotype -- they have large cell bodies, express low levels of SCP starting from the midjuvenile stage, and do not show detectable LOX2 expression. Lineage tracer injections showed that the MPS neurons arise from a stereotyped cell lineage and are descended from the O teloblast stem cell. In midbody ganglia 2 to 6 and 18 to 21, there are lineally homologous neurons that do not express either LOX2 protein or SCP. Thus, the boundaries of LOX2 expression coincide precisely with two of the segmental boundaries of MPS differentiation, suggesting that expression of LOX2 at the level of this single identified neuron governs some, but not all, aspects of the neuron's segmental diversification.

Membrane Responses Evoked By Organic Buffers In Identified Leech Neurones

Using single-electrode current-clamp and two-electrode voltage-clamp, different identified neurones of the leech (Hirudo medicinalis L.) were shown to hyperpolarize, or to exhibit outward currents (usually >2 nA), during bath application of the organic buffers Mops, Pipes and Mes (10 mmol l-1). Tris and Hepes had little or no effect on the membrane properties of neurones when they were added to a bathing saline buffered by CO2-HCO3- at a constant pH of 7.4. Outward currents evoked by buffers obtained from two suppliers, Roth and Sigma, were not significantly different, except in the case of Pipes. Steady-state currents evoked by Pipes supplied by Roth were eightfold larger than steady-state currents evoked by Pipes from Sigma. A transient current peak always present in responses evoked by Pipes from Roth was never present in responses evoked by Pipes from Sigma. Outward currents evoked by buffers were due to a conductance increase and appeared to be carried by Cl-. In low-Cl- saline, hyperpolarizations evoked by Mops in Retzius cells were reduced. When Cl- was injected into heart motor neurones, the hyperpolarizations evoked by Mops reversed. Tetraethylammonium (TEA+) injected into heart interneurones did not block Mops-evoked hyperpolarizations. Mops-evoked outward currents in Retzius cells were partially blocked by d-tubocurarine and bicuculline methiodide, and the latter also partially blocked Mops-evoked hyperpolarizations in HE cells. Since d-tubocurarine partially blocked acetylcholine-evoked Cl- currents in Retzius cells and bicuculline methiodide partially blocked carbachol-evoked hyperpolarizations in HE cells, Mops appears to act on the cholinergic receptors of these neurones by mediating a Cl- conductance.

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.

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.

Segmental specialization of calcium-activated potassium conductances in an identified leech neuron

1. Retzius (R) neurons of the fifth and sixth segmental ganglia of the leech, called R(5,6) neurons are specialized to innervate the adjacent reproductive organs and are morphologically and functionally distinct from R neurons of standard ganglia [R(x) cells]. In this study the electrical properties of the R(x) and R(5,6) neurons were compared under current-clamp and voltage-clamp conditions. 2. The action-potential waveforms of R(x) and R(5,6) cells were similar except for the presence in the R(5,6) cells of a long afterhyperpolarization (AHP) following action potentials arising from the resting membrane potential but not from more depolarized potentials. Its role may thus be to inhibit firing of the R(5,6) neurons at rest or in response to weak depolarizing stimuli. 3. In the presence of the Ca2+ channel blocker Cd2+, the long AHP of the R(5,6) was abolished, and the action potentials of all R cells were identical. 4. Under voltage clamp, current kinetics and densities were similar between R(x) and R(5,6) cells for Ca2+ currents, delayed and inward rectifier K+ currents, and a rapid Ca(2+)-activated K+ current (IKc) that is common to the two cell types. The R(5,6) cells, however, expressed a second Ca(2+)-activated K+ current that was not observed in the R(x) cells. This current, called IKAHP, activated and inactivated more slowly than IKC, with a time course similar to that of the AHP observed under physiological conditions. 5. Neither IKC nor IKAHP was blocked by high concentrations of charybdotoxin or apamin, which block vertebrate Ca(2+)-activated K+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

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)