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Electrical synapses play an important role in signaling between neurons and the synaptic connections between many neurons possess both electrical and chemical components. Although modulation of electrical synapses is frequently observed, the cellular processes that mediate such changes have not been studied as thoroughly as plasticity in chemical synapses. In the leech (Hirudo sp), the competitive AMPA receptor antagonist CNQX inhibited transmission at the rectifying electrical synapse of a mixed glutamatergic/electrical synaptic connection. This CNQX-mediated inhibition of the electrical synapse was blocked by concanavalin A (Con A) and dynamin inhibitory peptide (DIP), both of which are known to inhibit endocytosis of neurotransmitter receptors. CNQX-mediated inhibition was also blocked by pep2-SVKI (SVKI), a synthetic peptide that prevents internalization of AMPA-type glutamate receptor. AMPA itself also inhibited electrical synaptic transmission and this AMPA-mediated inhibition was partially blocked by Con A, DIP and SVKI. Low frequency stimulation induced long-term depression (LTD) in both the electrical and glutamatergic components of these synapses and this LTD was blocked by SVKI. GYKI 52466, a selective non-competitive antagonist of AMPA receptors, did not affect the electrical EPSP, although it did block the glutamatergic component of these synapses. CNQX did not affect non-rectifying electrical synapses in two different pairs of neurons. These results suggest an interaction between AMPA-type glutamate receptors and the gap junction proteins that mediate electrical synaptic transmission. This putative interaction between glutamate receptors and gap junction proteins represents a novel mechanism for regulating the strength of synaptic transmission.

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 microM. 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.

We examined the effect of the glutamatergic agonist kainate on the membrane potential, the intracellular Na+ concentration ([Na+]i), the intracellular-free Ca2+ concentration, and on the intracellular pH of Retzius neurons of the medicinal leech, Hirudo medicinalis, in order to investigate the mechanism responsible for the intracellular acidification caused by glutamatergic stimulation. The recordings were made with Na+- and pH-sensitive microelectrodes and iontophoretically injected Fura-2. Bath application of kainate evoked a marked membrane depolarization, a [Na+]i increase, and an intracellular acidification. The intracellular acidification was unaffected by reversal of the electromotive force for H+, suggesting that an influx of H+ from the interstitial space does not contribute to the acidification. While the Ca2+ channel blockers La3+ and Co2+ had no effect on the kainate-induced intracellular acidification, suggesting that a Ca2+ influx via voltage-dependent Ca2+ channels was not relevant, the acidification was reduced in Ca2+-free saline solution. In Na+-free saline solution the kainate-induced intracellular acidification was absent, suggesting the involvement of Na+ influx in generating the acidification. When injected iontophoretically Na+ induced an intracellular acidification but Li+, K+, Rb+ or Cs+ did not. Furthermore, a [Na+]i increase induced by blocking the Na+/K+ pump also led to an intracellular acidification. We conclude that the [Na+]i increase is the crucial event underlying the kainate-induced intracellular acidification. Possible mechanisms linking the [Na+]i increase to the intracellular acidification are discussed.

In patch-clamp experiments we characterized four Cl− channels (42 pS, 70 pS, 80 pS and 229 pS) underlying the large Cl− conductance of leech neuropile glial cells. They differed with respect to their gating, their rectification and their activity in the cell-attached configuration, showed the selectivity sequence I−>Br−≥Cl−>F− and were impermeable to SO42−. The four channels were blocked by NPPB, DPC, niflumic acid and DIDS and exhibited either three or four sublevel states. The outward rectifying 42 pS, 70 pS and 80 pS Cl− channels were classified as intermediate conductance Cl− channels and they could contribute to the high Cl− conductance of the glial membrane, which stabilizes the glial membrane potential. The inward rectifying 229 pS Cl− channel is very similar to vertebrate high conductance Cl− channels, which are assumed to be part of an emergency system that is activated under pathophysiological conditions. In voltage-clamp experiments we calculated that the Cl− conductance amounts to one-third of the total membrane conductance. Reduction of this Cl− conductance by Cl− channel inhibitors markedly depolarized the glial cell membrane. These prominent depolarizations depended on Na+ influx and in most cases the glial cells failed to regulate their membrane potential following wash-out of the inhibitors.

We performed patch-clamp experiments to identify distinct K + channels underlying the high K + conductance and K + uptake mechanism of the neuropile glial cell membrane on the single-channel level. In the soma membrane four different types of K + channels were characterized, which were found to be distributed in clusters. Since no other types of K + channels were observed, these appear to be the complete repertoire of K + channels expressed in the soma region of this cell type. The outward rectifying 42 pS K + channel could markedly contribute to the high K + conductance and the maintenance of the membrane potential, since it shows the highest open probability of all channels. The channel gating occurred in bursts and patch excision decreased the open probability. The outward rectifying 74 pS K + channel was rarely active in the cell-attached configuration; however, patch excision enhanced its open probability considerably. This type of channel may be involved in neuron-glial crosstalk, since it is activated by both depolarizations and increases in the intracellular Ca 2+ concentration, which are known to be induced by neurotransmitter release following the activation of neurons. The 40 pS and 83 pS K + channels showed inward rectifying properties, suggesting their involvement in the regulation of the extracellular K + content. The 40 pS K + channel could only be observed in the inside-out configuration. The 83 pS channel was activated following patch excision. At membrane potentials more negative than −60 mV, flickering events indicated voltage-dependent gating.

The present study demonstrates that stereoselective binding sites for anandamide, a naturally occurring cannabinoid substance, can be found in leech (Theromyzon tessulatum and Hirudo medicinalis) central nervous system. The anandamide binding site is monophasic and of high affinity exhibiting a Kd of approximately 32 nM with a Bmax of 550 fmol/mg protein in both animals. These sites are highly select as demonstrated by the inability of other types of signaling molecules to displace [3H]anandamide. Furthermore, this binding site is coupled to nitric oxide release. A deduced amino acid sequence (153 residues) analysis from a 480 pb amplified RT-PCR fragment cDNA exhibits a 49.3% and 47.2% sequence identity with human and rat cannabinoid receptors (CB1R), respectively. Thus, the leech cannabinoid receptor may be a G-protein coupled receptor with seven transmembrane domains as in CB1R. Moreover, this sequence exhibits highly conserved regions, particularly in the putative transmembrane domains 1 and 2. The presence of a cannabinoid receptor in these organisms indicates that this signaling system has been conserved during evolution.

Activity in non-nociceptive afferents is known to produce long-lasting decreases in nociceptive signaling, often referred to as gate control, but the cellular mechanisms mediating this form of neuroplasticity are poorly understood. In the leech, activation of non-nociceptive touch (T) mechanosensory neurons induces a heterosynaptic depression of nociceptive (N) synapses that is endocannabinoid-dependent. This heterosynaptic, endocannabinoid-dependent long-term depression (ecLTD) is observed where the T- and N-cells converge on a common postsynaptic target, in this case the motor neuron that innervates the longitudinal muscles (L-cells) that contributes to a defensive withdrawal reflex. Depression in the nociceptive synapse required both presynaptic and postsynaptic increases in intracellular Ca²⁺. Activation of the Ca²⁺-sensitive protein phosphatase calcineurin was also required, but only in the presynaptic neuron. Heterosynaptic ecLTD was unaffected by antagonists for NMDA or metabotropic glutamate receptors, but was blocked by the 5-HT₂ receptor antagonist ritanserin. Depression was also blocked by the CB1 receptor antagonist rimonabant, but this is thought to represent an effect on a TRPV-like receptor. This heterosynaptic, endocannabinoid-dependent modulation of nociceptive synapses represents a novel mechanism for regulating how injury-inducing or painful stimuli are transmitted to the rest of the central nervous system.

The peptides contained in neurons localized in the brain of the leech Theromyzon tessulatum (Hirudinae, Rhynchobdellida) and showing an immunopositive reaction with an antibody directed against angiotensin II (AII), were purified by reversed-phase HPLC. Three AII-like peptides (P1, P2 and P3) which exhibited the same retention times and chromatographic behaviors as synthetic AVII (fragment 6-8 of AII), AIV (fragment 3-8 of AII) and AII, respectively, were resolved in brain extracts. An identification of the proteins immunoreactive to an anti-AII was performed at the level of both brain extracts and in vitro brain-translated RNA products. The protein detected at the level of the brain extracts (of a molecular mass of approximately 18 kDa) is multipeptidic as it is also recognized by two other antisera, a polyclonal one directed against gamma-MSH and a monoclonal one (Tt159) raised against a leech brain epitope. It could be the pro-AII-like precursor. The protein detected at the level of in vitro brain-translated RNA products (of a molecular mass of approximately 19 kDa) could be the prepro-AII-like precursor.

Effects of serotonin (5-HT) and carbachol on Rb uptake (used as a K marker) in leech neuron and glia were studied by electron probe microanalysis (EPMA). Hirudo medicinalis ganglia were perfused 60 s in 4 mM Rb substituted normal leech Ringer's with and without 5-HT (dosage range 5-500 microM) or carbachol (range 10-1000 microM), quench frozen cryosectioned, and subjected to EPMA to determine elemental mass fractions and cell water content. Both 5-HT and carbachol altered leech neuron and glial cell elemental distribution and water content. In glial cells, a dose-dependent increase in Rb uptake was observed following 5-HT (control: 26 +/- 2 microM; 5 microM: 47 +/- 4; 50 microM: 62 +/- 4; 500 microM: 82 +/- 11 mmol/kg dry wt. +/- S.E.M.) and carbachol (10 microM: 35 +/- 3; 100 microM: 52 +/- 3; 1000 microM: 68 +/- 3 mmol/kg dry wt. +/- S.E.M.). In neurons, 5-HT and carbachol had small effects. 5-HT decreased glial and neuronal cell water content. Carbachol decreased neuronal (but not glial) water content by approximately the same amount (mean decrease 9%) regardless of dose. Both 5-HT and carbachol affected glial cell K-accumulating properties, providing evidence that certain neurotransmitters may modulate invertebrate glial cells' K clearance function.

The effects of glutaminergic agonists on neuropile glial cells and on Retzius neurones in the central nervous system (CNS) of the leech, Hirudo medicinalis were investigated using double-barrelled ion-sensitive microelectrodes. In both types of cells, bath-application of L-glutamate (Glu), kainate (Ka) and quisqualate (Qui) elicited substantial membrane depolarizations which were accompanied by increases of the intracellular Na+ activity aiNa and by concomitant decrease of the intracellular K+ activity aiK. In the glial cells, these alterations of aiNa and aiK were preceded by a transient decrease of aiNa and an increase of aiK upon administration of Ka and Qui. In both glial cells and neurones, N-methyl-D-aspartate (NMDA) did not affect Em, aiK and aiNa. As found for Ka, the neuronal as well as the glial responses to glutaminergic agonists persisted during inhibition of synaptic transmission in high Mg2+, low Ca2+ solutions. The results indicate that leech neuropile glial cells have a Ka/Qui-preferring non-NMDA glutamate receptor similar to that in the Retzius neurones.

The uptake of horseradish peroxidase (HRP), applied as an extracellular tracer, is a classical method for studying endo/exocytosis of synaptic vesicles at the ultrastructural level. It is generally not considered that HRP may affect neuronal function. Reported here is the finding that extracellularly applied HRP (0.1%) perturbs dense core vesicles in the synaptic processes of leech neurons. The strength of the effect varies with neuronal class. In sensory afferents, the number of dense core vesicles increases 5-fold, while there is only a 2-fold increase in central neurons.

In leech Retzius neurones the inhibition of the Na+/K+ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca2+]i). To elucidate the mechanism of this increase we investigated the changes in [Ca2+]i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na+/K+ pump in bathing solutions of different ionic composition. The results show that Na+/K+ pump inhibition induced a [Ca2+]i increase only if the cells depolarized sufficiently in the presence of extracellular Ca2+. Specifically, the relationship between [Ca2+]i and the membrane potential upon Na+/K+ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca2+ channels by raising the extracellular K+ concentration. It is concluded that the [Ca2+]i increase caused by inhibiting the Na+/K+ pump in leech Retzius neurones is exclusively due to Ca2+ influx through voltage-dependent Ca2+ channels.

Extracellular 4-aminopyridine (4-AP), tetraethylammonium chloride (TEA) and quinine depolarized the neuropile glial cell membrane and decreased its input resistance. As 4-AP induced the most pronounced effects, we focused on the action of 4-AP and clarified the ionic mechanisms involved. 4-AP did not only block glial K + channels, but also induced Na + and Ca 2+ influx via other than voltage-gated channels. The reversal potential of the 4-AP-induced current was −5 mV. Application of 5 mM Ni 2+ or 0.1 mM d-tubocurarine reduced the 4-AP-induced depolarization and the associated decrease in input resistance. We therefore suggest that 4-AP mediates neuronal acetylcholine release, apparently by a presynaptic mechanism. Activation of glial nicotinic acetylcholine receptors contributes to the depolarization, the decrease in input resistance, and the 4-AP-induced inward current. Furthermore, the 4-AP-induced depolarization activates additional voltage-sensitive K + and Cl − channels and 4-AP-induced Ca 2+ influx could activate Ca 2+ -sensitive K + and Cl − channels. Together these effects compensate and even exceed the 4-AP-mediated reduction in K + conductance. Therefore, the 4-AP-induced depolarization was paralleled by a decreasing input resistance.

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 ((3S,4R)-3-hydroxy-2, 2-dimethyl-4-(2-oxo-1-pyrrolidinyl)-6-phenylsulfonylchromane hemihydrate, 500 microM) 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 Ca2+ 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.

Anandamide, an endogenous cannabinoid signaling molecule, in a concentration dependent manner, initiates the release of nitric oxide (NO) from leech and mussel ganglia. SR 141716A, a cannabinoid antagonist, blocks the anandamide stimulated release of NO from these tissues. Methyl arachidonyl fluorophosphonate (MAFP), a specific anandamide amidase inhibitor, when administered to either ganglia with anandamide (10-6 M) did not increase the peak level of NO release but did significantly extend NO release from 12 to 18 min (P0.05). Lower levels of anandamide (10-8 and 10-7 M) do not stimulate the release of significant amounts of NO from these tissues. However, in the presence of MAFP (2.5 nM), the lower anandamide concentrations were able to release significant peak amounts of NO. In mussel neural tissues, the peak NO release increased from 2.2+/-1.3 nM to 8.6+/-2.1 nM. Taken together, the results indirectly demonstrate the presence of anandamide amidase in these tissues, suggesting that the enzyme may serve as an endogenous regulator of anandamide action.

The leech Theromyzon tessulatum and the marine mussel Mytilus edulis immunocytes contain a mammalian-like proenkephalin molecule. The opioid precursor was purified by gel permeation chromatography, anti-Met- and Leu-enkephalin-affinity column separation and then by reversed-phase HPLC. The amino acid sequence analysis, determined by Edman degradation, enzymatic treatments and matrix assisted laser desorption time of flight. The structure of the leech proenkephalin material demonstrates considerable amino acid sequence similarity with amphibian proenkephalin (e.g. 25.4% with Xenopus laevis) but it is smaller, 15 kDa vs. 30 kDa. In contrast, Mytilus proenkephalin is not only larger (26 kDa) but it exhibits a higher sequence identity with guinea pig proenkephalin (50%). Both of the invertebrate materials possess Met-enkephalin and Leu-enkephalin in a ratio of 3:1 for Mytilus and 1:2 in the leech. They also contain Met-enkephalin–Arg–Gly–Leu and Met-enkephalin–Arg–Phe sequences that are flanked by dibasic amino acid residues, demonstrating cleavage sites. Furthermore, using sequence comparison with bovine proenkephalin A (209–237), enkelytin (FAEPLPSEEEGESYSKEVPEMEKRYGGFM), an antibacterial peptide is found in the proenkephalin of both animals and it exhibits a 98% sequence identity with mammalian material. Finally, opioid binding experiments demonstrate the presence in leech ganglia and immunocytes of δ1 and δ2 opioid receptor subtypes as also found human and Mytilus immune cells. This report constitutes the first complete biochemical characterization of mammalian proenkephalin in invertebrates, demonstrating its origin in simpler animals.

Morphine and anandamide stimulate the release of nitric oxide (NO) in diverse tissues. The present study examines the consequences of this action on neurotransmitter release in ganglia from two invertebrates: ventral chain ganglia from the leech Hirudo medicinalis and the pedal ganglion from the mussel Mytilus edulis. In these ganglia, preloaded serotonin (5-HT) and dopamine (DA) can be released by 50 mM KCl. Anandamide, an endogenous cannabinoid substance, suppresses the potassium-stimulated release of [3H]DA (80%), but not 5-HT, in a concentration-dependent manner, from the neural tissues of both. The effect of anandamide can be antagonized by pre-exposing the neural tissues of both animals to SR 141716A, a potent cannabinoid receptor antagonist. Prior treatment of the ganglia with N-omega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, significantly diminishes the inhibitory effect of anandamide. Morphine also inhibits [3H]DA release in a naloxone- and L-NAME-sensitive manner. Anandamide and morphine act through separate mechanisms since the respective antagonists show no cross-reactivity. The NO donor, SNAP, depressed the potassium-stimulated release of preloaded [3H]DA, but not 5-HT, in the neural tissues of both animals. D-Ala2-Met5 enkephalinamide (DAMA) also inhibited the potassium-stimulated release of [3H]DA in a naloxone-sensitive process. However, the effect of DAMA was seen in the presence of L-NAME (10(-4) M), indicating that the opioid peptide inhibition of the presynaptic release of DA is not coupled to NO. We postulate that cannabinoids and their endogenous effectors play a prominent role in the regulation of catecholamine release in invertebrates via NO release as is the case for opiate alkaloids.

We determined the relationships between the intracellular free Ca2+ concentration ([Ca2+]i) and the membrane potential (Em) of six different neurones in the leech central nervous system: Retzius, 50 (Leydig), AP, AE, P, and N neurones. The [Ca2+]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 [Ca2+]i, which was abolished in Ca(2+)-free solution indicating that it was due to Ca2+ influx. The threshold membrane potential that must be reached in the different types of neurones to induce a [Ca2+]i increase ranged between -40 and -25 mV. The different threshold potentials as well as differences in the relationships between [Ca2+]i and EM were partly due to the cell-specific generation of action potentials. In Na(+)-free solution, the action potentials were suppressed and the [Ca2+]i/Em relationships were similar. The K(+)-induced [Ca2+]i increase was inhibited by the polyvalent cations Co2+, Ni2+, Mn2+, Cd2+, and La3+, 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 Ca2+ channels with similar properties.

The presence and distribution of putative nitric oxide synthase (NOS)-containing cells in whole-mount preparations of the central nervous system of the leech, Hirudo medicinalis, were studied using NADPH-diaphorase (NADPH-d) histochemistry. Specific staining occurred mainly in somata of some central neurones but NADPH-d-reactive branches and terminals were found in peripheral nerves and connectives: neuropile areas were stained weakly or unstained. Intense staining was located in many neurones on the ventral side of the segmental ganglia, including primary sensory neurones, motoneurones and interneurones, and in the anterior root ganglion. The sex ganglia contained some extra NADPH-d-positive cells. Head and tail ganglia and the dorsal side of the segmental ganglia showed less staining. Specific activity was not detected in salivary glands, crop or intestine. Controls using beta-NADPH or nitro blue tetrazolium (NBT) alone or with NBT plus alpha-NADPH, beta-NAD+, beta-NADH or beta-NADP+ did not induce specific staining. A potential NOS inhibitor, 2,6-dichlorophenol-indophenol (DPiP) at 10(-3) M, totally abolished NADPH-d-positive staining. Long-term fixation did not change the pattern of distribution of NADPH-d-positive cells. We conclude that (i) fixative-resistant NADPH-diaphorase is a characteristic marker of 12-15% of neurones in the leech CNS, and (ii) the specific distribution of the putative NOS-containing neurones suggests that NO may be a natural signal molecule in leeches.

The effects of extracellular K+ on the intracellular free Ca2+ concentration ([Ca2+]i) of neuropile glial cells and Retzius neurones in intact segmental ganglia of the leech Hirudo medicinalis were investigated by using iontophoretically injected fura-2. In both cell types, an elevation of the extracellular K+ concentration ([K+]o) caused an increase in [Ca2+]i, which was blocked by Co2+, Ni2+ and menthol, whereas nicardipine, flunarizine, omega-conotoxin GVIA and omega-agatoxin IVA were ineffective. In Ca(2+)-free solution, the K(+)-induced [Ca2+]i increase was largely suppressed in neuropile glial cells and completely abolished in Retzius neurones. The results indicate that the K(+)-induced [Ca2+]i increase was mainly due to Ca2+ influx through voltage-dependent Ca2+ channels. The Ca2+ channels of the two cell types were activated at different membrane potentials but at the same [K+]o. In both cell types, the recovery from a K(+)-induced [Ca2+]i increase was unaltered in Na(+)-free solution, indicating that active Ca2+ transport across the plasma membrane is mediated by Na(+)-independent mechanisms.

The membrane potential of neuropile glial cells and Retzius neurones in the central nervous system of the leech Hirudo medicinalis was measured using electrolyte-filled single-barreled microelectrodes. Intracellular Na+ activity (aNai) was recorded with Na(+)-sensitive double-barreled microelectrodes. Bath-application of kainate, quisqualate and L-glutamate elicited concentration-dependent membrane depolarizations in both cell types as demonstrated by dose-response curves. The competitive quinoxalinedione antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the non-NMDA glutamate receptor inhibited the membrane depolarizations in neuropile glial cells completely, but in Retzius neurones only partially. These results confirm that leech neuropile glial cells have a kainate- and quisqualate-preferring non-NMDA glutamate receptor similar to that in the Retzius neurones. The initial decrease in aNai in neuropile glial cells in kainate- or quisqualate-containing solutions and the afterhyperpolarization in these glial cells and the Retzius neurones following the removal of both glutamate antagonists, were blocked in the presence of the cardiac glycoside ouabain (10(-4) M). In saline solutions containing 42.5 mM Li+ instead of Na+ the afterhyperpolarizations were blocked in neuropile glial cells and Retzius neurones. We conclude that the initial aNai changes and the afterhyperpolarization could be due to the stimulation of the electrogenic Na+/K+ pump in the glial and neuronal membranes.

We used double-barreled pH-sensitive microelectrodes to study the mechanisms by which the intracellular pH is regulated in the connective glial cells of the medicinal leech. The experiments indicate that a Cl(-)- and HCO3(-)-dependent mechanism mediates some recovery from intracellular alkalosis even in the absence of external Na+. This suggests the presence of Na(+)-independent Cl-/HCO3- exchange in the connective glial membrane. At alkaline pHi, this exchange most likely operates in the direction of net acid loading (i.e. HCO3- efflux).

Elevation of [K+]o for 30 s from 4 to 120 mM produced a fast and reversible depolarization and transient increase in [Ca2+]i in fura-2 loaded Retzius cells of the leech. The protein phosphatase inhibitor, okadaic acid, significantly slowed the return of [Ca2+]i toward baseline without affecting the amplitude of depolarization or rate of repolarization. Furthermore, okadaic acid and another phosphatase inhibitor, calyculin A, prolonged Ba(2+)-dependent action potentials. These results suggest that the kinetics of Ca2+ influx may be regulated by the activity of phosphatases PP-1 and/or PP-2A.

Recent data suggest that IL-1-like molecules have been conserved during evolution. The signal transduction mechanism of IL-1 is not known, but kinase activation has been reported. With the aim of studying if human IL-1 has any effect on the leech nervous system, we have added this cytokine to segmental ganglia labeled with [32P]ortophosphoric acid; proteins have been separated by electrophoresis and phosphoproteins detected by autoradiography. In the present paper we show that human IL-1 and IL-6 are able to induce changes on protein phosphorylation in the leech central nervous system and that these changes are similar to those ones induced by the neurotransmitter serotonin.

A large number of oxytocin (OT)-like neurons were detected in the sex segmental ganglia (SG5, SG6) of three species of leeches belonging to different orders: Theromyzon tessulatum, Hirudo medicinalis and Erpobdella octoculata. In this latter species, an epitope close to the vertebrate OT by its C-terminal part (MSH release inhibiting factor: MIF), localized in granules of a size diameter of ca 120 nm and colocalized with FMRFamide(FMRFa)-like material was demonstrated. With reverse phase-high performance liquid chromatography, evidence was given that the two epitopes (OT and FMRFa) colocalized in the same neurons were biochemically different. A titration of OT per SG indicated that the OT-like amount was considerably higher in sex SG than in non-sex SG (ca. 5 pmol vs. ca. 0.5 pmol). Moreover, at the level of sex SG, this amount was ca. 3-fold higher in immature leeches than in mature specimens. Injections of extracts of SG of E. octoculata and of fragments of OT (Tocinoic acid or MIF) to T. tessulatum, indicated that MIF (the epitope found in the sex SG) and sex SG have the same anti-diuretic effect on the leeches injected. These results pointed to an anti-diuretic role of the leech OT-like substance.

Glial cells play a significant role in maintaining extracellular space (ECS) potassium (K) by temporarily buffering or accumulating excess ECS K and then returning that K to neurons. Yet, little is known about the relative affinity of neurons or glial cells for K when both cells are simultaneously exposed to the same ECS K, in situ. Also, the process by which glial cells return K to neurons remains unknown. Therefore, electron probe X-ray microanalysis was used to measure rubidium (Rb) uptake, as a K tracer, into leech packet neurons and glial cells, and to measure the distribution of cell water content, K, Na and Cl. When ECS Rb was increased from 4 mM to 20 mM, there was a clear preferential Rb uptake into glial cells compared to neurons. At 4 mM extracellular Rb there was only a small difference between uptake velocity of neurons and glial cells (maximum mean uptake velocity at 4 mM Rb was 1.09 for glia, and 0.41 mmol Rb/kg dry wt/s for neurons), whereas at 20 mM extracellular Rb, glial uptake velocity was dramatically greater than of neurons (max. mean Rb uptake velocity for glia was 4.3 compared to 1.47 mmol Rb/kg dry wt/s for neurons). Glial Rb uptake velocity was enhanced by low temperature (max. mean Rb uptake velocity at 20 mM ECS Rb at 6 degrees C was 6.04 for glia compared to 0.78 mmol Rb/kg dry wt/s for neurons) and by substitution of Cl with isethionate (max. mean Rb uptake velocity was 10.6 for glia compared to 1.33 mmol Rb/kg dry wt/s for neurons).(ABSTRACT TRUNCATED AT 250 WORDS)

Leech AP neurons react to axotomy by increasing excitability and resting potential of the cell body membrane. In a previous report we described single potassium channels contributing to the leak conductance in the soma membrane of AP cells. Here we compare both properties and density of single potassium leak channels in cell-free patches from normal and axotomized AP neurons. We show that properties such as single channel conductance, outward rectification, time constants of open and shut interval distributions and absence of inactivation do not significantly differ between normal and axotomized cells. On the other hand, we find that the number of channels per patch progressively increases with time after axotomy. We conclude that changes in density rather than alterations in properties of single channels can account for the increase in the resting potential, observed after axotomy.

The leech is a segmented annelid with a well characterized central nervous system. In this report, we use antibodies to map the distribution of neurons confined to selected segmental ganglia in the mud leech Haemopis marmorata . The distribution patterns of these neurons suggest 3 novel aspects of segmentation in the leech nervous system: (1) neurons are assigned to even-numbered ganglia through a mechanism which effectively counts through the leech segmental body plan by units of 2, (2) neurons are assigned to ganglia 7 and 14 through a mechanism which effectively counts in units of 7 and (3) neurons are assigned to the 2nd of 4 fused head ganglia and to the 2nd of 21 unfused midbody ganglia through a mechanism which effectively counts units from the origin of these 2 ganglia ic series. These 3 hypothetical counting mechanisms divide the central nervous system (CNS) into supersegmental units. Neurons used to define these supersegmental units have been injected with tracer and identified as interganglionic interneurons. Competitive interactions among embryonic precursors of these neurons may directly eliminate their homologs from intervening ganglia, and thus sculpture supersegmental patterns into the mature nervous system.

Phenobarbital (PNB), pentobarbital (PTB) and methohexital (MTX) decreased the maximum rate of depolarization V max and duration of divalent cation action potentials elicited in leech nociceptive neurons in Na + - free solutions containing the K + -channel blocker TEA, without significantly affecting resting membrane potential or conductance. The block of the divalent cation action potentials was reversible and dose-dependent, ED 50 for inhibition of V max being 560 μM for MTX, 800 μM for PTB and 3000 μM for PNB. This order of potency correlated well with the ratio of uncharged/charged form of the drugs at physiological pH suggesting that in leech, as in other preparations, the non-ionized form was the active one. In Na + -containing Ringer, the 3 barbiturates depolarized and decreased membrane resistance in the lateral nociceptive cells, but not the medial nociceptive cells. These results provide additional information regarding the newly described pharmacological differences among closely related neurons. These membrane actions may be related to some of the excitatory properties described for other barbiturates in invertebrate and mammalian preparations.

The distribution of 4 surface glycoproteins on axons of peripheral neurons was studied in the leech Hirudo medicinalis through monoclonal antibodies. All 4 glycoproteins have a similar molecular weight of 130 kDa. Immunohistochemical localization of these glycoproteins on tissue sections of nerves and neuropil reveals tracts of afferent axons organized as nested sets. Their distribution suggests a possible role for these molecules in mediating axon fasciculation.

Both excitatory and inhibitory motor neurons innervate longitudinal body wall muscles in the leech Hirudo medicinalis. Each inhibitory motor neuron also centrally inhibits the excitatory motor neurons that innervate its same muscle field. This central inhibition is strong, probably monosynaptic, and largely a function of graded membrane potential changes in the inhibitory motor neurons. During leech swimming, both the excitatory and inhibitory motor neurons are rhythmically active. Here, we present evidence that the inhibitory motor neurons phasically inhibit the excitatory motor neurons during swimming, thereby augmenting the amplitude of membrane potential oscillations and the burst intensity of the excitors.

Recordings of motoneuron activity during swimming, obtained from leech ventral nerve cords in situ, were compared with films of swimming leeches and with recordings of motoneuron activity from isolated nerve cords. It was found that the intersegmental phase lag in body movement is greater than the phase lag of in situ neuronal activity, which is in turn greater than the phase lag of isolated nerve cord activity. We conclude that peripheral neuronal or mechanical effects, as well as sensory feedback to the central pattern generator, contribute to the movement pattern of the intact swimming leech.

The neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) is generally believed to selectively ablate serotonergic neurons. Serotonin-containing Retzius cells (RZ) in the leech appear ablated by 5,7-DHT treatment. However, these brown, mis-shapen, non-fluorescent neurons retain their synaptic inputs, action potentials, electronic outputs, a membrane receptor, and axonal projections.

Intracellular and surface acetylcholinesterase activities were determined for individual cholinergic and non-cholinergic neurons dissected from the central nervous system of the leech. Echothiophate pretreatment was used to inhibit selectively extracellular enzyme. Cells releasing acetylcholine as a transmitter had approximately 10-fold higher levels of intracellular acetylcholinesterase activity, while all neurons had similar levels of activity associated with the cell surface. These results suggest that intracellular cholinesterase may be a useful marker for cholinergic neurons.

In the nervous system of the leech Hirudo medicinalis it has been possible to study short-term plastic changes. Depression and facilitation have been demonstrated in the fast conducting system (FCS) activity; this pathway consists of a chain of electrically linked neurons present in each ganglion. In semi-intact animals or in preparations of nerve cord and segments of body wall, both electrical stimulation of peripheral roots and tactile stimulation of the skin induced, after repetitive stimulation (0.1/s), a prolonged decrement of FCS response. Strong nociceptive stimulation applied onto the head or the body wall produced a sustained facilitation of the waned response. The same potentiation has been observed by perfusing the isolated ganglion with serotonin (5 × 10 −5 M). Such a potentiation is abolished by preincubation with methysergide, an antagonist of serotonin, and with imidazole, a cAMP-phosphodiesterase activator. Such an effect is mimicked by an analog of cAMP, db-cAMP. Simultaneous recordings of both T neurons (intracellularly) and FCS firing discharge showed that, during FCS response decrement, the T cell activity remained unchanged and no modification of conductance occurred, excluding therefore a detectable involvemnt of sensory neurons in the depression. These results suggest that short-term plastic changes of the FCS of the leech are due to a prolonged potentiation of synaptic transmission as a result of serotonin-mediated increase in cAMP.

The membrane potential of neuropile glial (NG) cells in the central nervous system of the medicinal leech and the K+ concentration in extracellular spaces (ECS) of the neuropile were measured under various experimental conditions to determine properties of a glial Na+-K+ pump. The ganglia were exposed to K+-free saline thereby loading the NG cells with intracellular Na+. Their membranes hyperpolarized transiently when the K+-free solution was replaced by a bathing medium with normal (= 4 mM) K+ concentration. The hyperpolarization increased in amplitude with time of exposure to K+ -free solution and could be abolished by ouabain or by replacing Na+ with Li+. The transient membrane hyperpolarization could not be attributed to K+ depletion in the ECS of the neuropile or to changes in membrane input conductance. In a (bathing) medium containing 5 X 10(-4) M ouabain, the K+ concentration in the ECS increased transiently, and the NG cell membrane depolarized rapidly. This short-term depolarization (duration 2-3 min) was followed by a second long-term depolarization (duration 15 min) of the NG cell membrane, which reached a steady-state 20 min after ouabain application. In a bathing medium with elevated external K+ concentrations, the amplitude of the membrane depolarization was enhanced by ouabain. This depolarizing ouabain effect was a result of K+ accumulation in the ECS. We conclude that the Na+-K+ pump does not contribute directly to the resting membrane potential of NG cells and is not directly involved in K+ homeostasis at the cellular level.

Leydig cells are electrically-coupled neurons in the segmental ganglia of the leech. This study reports that they stain specifically with high concentrations (0.05 mg/ml) of the dye neutral red, and that they contain octopamine, as demonstrated by radioenzymatic assay. Individual cell bodies were pooled and found to contain 0.37 pmol octopamine/cell body, giving an approximate intracellular concentration of 7.75 mM. Leydig cell bodies contain approximately 75% of the octopamine content of a segmental ganglia. Intracellular injection of large amounts of Lucifer yellow, coupled with long diffusions imes, revealed a previously-undescribed Leydig cell process. The probability that Leydig cells are octopaminergic neurosecretory cells is discussed.

The neurotoxin 6-hydroxydopamine (6-OHDA) is reported to selectively ablate neurons which contain catecholamines. Leeches were injected with 6-OHDA over a 4-month period in an attempt to destroy their identified dopamine neurons. Ganglionic Retzius cells (RZ), which contain serotonin, were the first to be rendered brown, misshapen, and non-fluorescent. Continued injections of 6-OHDA had similar, but less substantial, morphological and histochemical effects on the dopamine-containing neurosomata within the anterior roots. Toxin treatment reduced RZ serotonin by 75-90%, but these depleted cells retained normal electrophysiological properties. Serotonin and dopamine within ganglia, as well as dopamine within anterior roots, were reduced significantly by 6-OHDA.

We compared the molecular nature of the rat brain opiate receptor with that of the invertebrate leech, Haemopis marmorata, and the protozoan, Tetrahymena, in order to examine the issue of apparent receptor heterogeneity with respect to biochemical structure. A binding study with rat brain membrane verified that [125I]β-endorphin ([125I]βE), a broad specificity ligand, is displaced by the antagonist (-)-naloxone, but not the inactive stereoisomer (+)-naloxone; agonists considered prototypes for μ, δ, and κ opiate receptors all displayed stereospecific binding displacement. For SDS-PAGE analysis of the opiate receptor [125I]β-endorphin was covalently affixed to its recognition molecule with the cross-linking reagent DSS. Primary reaction products occur at 110, 58/55, and 29 kDa. Cross-linking products of all 3 molecular weights are effectively reversed by opiate ligands, regardless of their μ, δ, or κ specificities. Peptide mapping studies in SDS gels, using limited proteolysis, showed that the 110 kDa band can be digested into 58 and 29 kDa fragments and the 58 kDa band into a 29 kDa fragment. Additional smaller molecular weight fragments were generated from the 110, 58/55, and 29 kDa bands which shared their molecular weights. Two possible explanations for the extensive homology between the three major cross-linking products are: (1) the 110 kDa species is the opiate receptor, and the 58 and 29 kDa species are proteolytic fragments; and (2) one of the lower molecular weight species is the opiate receptor, and adjacent receptors are aggregated into the 110 kDa complex through cross-linking. An evolutionary conservation of the opiate receptor is suggested by the presence of the same 3 major cross-linking products in Tetrahymena, leech, and rat.

Summary In ion-substitution experiments supplemented by measurements of the membrane conductance, the membrane potential of neuropile glial (NG) cells in the CNS of the medicinal leech has exhibited a dependence on the external concentration of both potassium and chloride. The membrane potential was largely dependent on the external potassium concentration, as may be inferred from the change in potential as the potassium concentration of the bathing solution was changed. The external potassium concentrations had been corrected to allow for the discrepancy between intra- and extraganglionic levels found with ion-selective electrodes. A transient membrane depolarization was recorded when the chloride in the bathing medium was replaced by sulphate or glucuronate. The restoration of the normal membrane potential following the return to chloride-based saline was preceded by a transient hyperpolarization. After transfer to low-chloride solutions, the transient depolarization of the NG cell membrane was followed by a steady-state hyperpolarization. The amplitude of the steady-state hyperpolarization depended on the concentration of chloride in the bathing medium. The membrane conductance decreased in low-chloride solutions.

Intracellular recordings from T mechanosensory cells of Hirudo medicinalis showed, as previously demonstrated, that repetitive firing is followed by a long-lasting hyperpolarization. Serotonin application at two concentrations (1 μM and 50 μM) depressed this hyperpolarization by up to 2/3; the effect was dose-dependent, long-lasting and reversible. Intracellular stimulation of giant serotonergic neurons (Retzius cells, Rz) mimicked serotonin perfusion: the effect was proportional to the number of spikes fired by Retzius cells. The combined use of intracellular iontophoretic injection of horseradish peroxidase and lucifer yellow indicated the possible sites of contact between Rz and T cells. The effect of serotonin, released by Rz cells, is discussed with respect to its possible physiological significance.