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Serotonin depresses the after-hyperpolarization through the inhibition of the Na+/K+ electrogenic pump in T sensory neurones of the leech.

In T sensory neurones of the leech, a train of impulses elicited by intracellular electrical stimulation leads to an after-hyperpolarization of up to 30 mV, mainly due to the activation of the electrogenic Na+/K(+)-ATPase but partly to a Ca2(+)-activated K+ conductance. It was found that serotonin reversibly reduced the amplitude of this after-hyperpolarization. We investigated the mechanism of action of serotonin and found: (1) after inhibition of the Ca2(+)-activated K+ conductance with BaCl2 or CdCl2, serotonin was still able to reduce the after-hyperpolarization; (2) when penetration of T cells with microelectrodes leaking sodium was preceded by serotonin perfusion of the ganglia, the normal hyperpolarization due to the activation of the electrogenic pump was converted to a depolarization; (3) after long-lasting perfusion with K(+)-free saline solution (which inhibits the Na+/K+ pump), the application of CsCl caused repolarization by reactivating the electrogenic ATPase; serotonin slowed and reduced this repolarization; (4) serotonin potentiated the depolarization of T neurones caused by the inhibition of the Na+/K+ pump following cooling of ganglia and depressed the hyperpolarization after rewarming to room temperature. These data taken together suggest that serotonin directly inhibits the Na+/K+ electrogenic pump.

Action Potentials↗

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

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

Animals↗

Calcium transients in identified leech glial cells in situ evoked by high potassium concentrations and 5-hydroxytryptamine.

We have recorded the fluorescence of Fura-2-loaded, identified glial cells in the neuropile of the central nervous system of the leech Hirudo medicinalis using the ratio of emission at 350 nm excitation to that at 380 nm excitation as an indicator of intracellular Ca2+ concentration ([Ca2+]i). The neuropile glial cells were exposed by mechanically removing the overlying ganglionic capsule and the neuronal cell bodies and were then impaled using a microelectrode under visual control to inject Fura-2 ionophoretically. The resting Ca2+ level was measured using digitonin or Triton to permeabilize the cell membrane at different external concentrations of Ca2+; it was found to vary between 5 and 79 nmol l-1 and averaged 32 +/- 23 nmol l-1 (+/- S.D., N = 7). Raising the external K+ concentration from 4 to 20 mmol l-1 or adding 50 mumol l-1 5-hydroxytryptamine (5-HT) produced a rapid, reversible rise in [Ca2+]i. During prolonged exposure to high [K+] or 5-HT, [Ca2+]i remained high. Upon restoring normal external [K+] or removing 5-HT, [Ca2+]i returned to its initial resting value within 1-2 min. The responses of [Ca2+]i to high [K+] and 5-HT were greatly reduced in nominally Ca(2+)-free saline, suggesting that the [Ca2+]i transients required an influx of Ca2+ into the cells. In the presence of 5-HT, the rise in [Ca2+]i was accompanied by a decrease in the resistance and an increase in the responsiveness to K+ of the glial cell membrane, indicating the existence of a Ca(2+)-dependent K+ conductance elicited by 5-HT.

Animals↗

Evidence that acetylcholine is an inhibitory transmitter of heart interneurons in the leech.

1. In the leech, synaptic transmission between heart interneurons (HN cells) and between HN cells and heart motor neurons (HE cells) is blocked by bicuculline methiodide. 2. Gamma-aminobutyric acid, when applied focally onto the somata of HN cells or when added to the superfusate, has no effect on the membrane potential of HN cells. 3. Both acetylcholine (ACh) and the ACh agonist carbachol hyperpolarize HN cells and HE cells when applied focally onto their somata or into the neuropil or when added to the superfusate. 4. Inhibitory postsynaptic-potential-like responses elicited by focal application of carbachol onto the somata of HN cells and HE cells are blocked by bicuculline methiodide and are reversed when Cl- is injected into the cells. 5. Focal application of carbachol onto the somata of HN cells and HE cells increases membrane conductance. 6. The results indicate that HN cells use ACh as an inhibitory transmitter, that the postsynaptic receptors for ACh are blocked by bicuculline methiodide and that inhibition of HN cells and HE cells is mediated by an increased Cl- conductance.

Acetylcholine↗

Sensory and neurosecretory innervation of leech nephridia is accomplished by a single neurone containing FMRFamide.

The neural control of the excretory system of the medicinal leech Hirudo medicinalis has been characterized morphologically and chemically using light and electron microscopy, immunocytochemistry and biochemistry. Immunoreactivity against RFamide-like peptides revealed elaborate neuronal aborizations of a neurone in the nephridium, around the urinary bladder sphincter and in the central nervous system. The processes arose from the nephridial nerve cell (NNC), a previously identified receptor neurone. Using a combination of reverse-phase high pressure liquid chromatography, radioimmunoassay and subsequent Edman degradation and mass spectrometry, authentic FMRFamide has been identified as the major peptide of the NNC. Sensory and neurosecretory innervation of the nephridia is thus accomplished by a single neurone, which is thought to modulate nephridial performance.

Amino Acid Sequence↗

Switching in the activity state of an interneuron that controls coordination of the hearts in the medicinal leech (Hirudo medicinalis).

The rhythmically active heart interneuron HN(5) in the medicinal leech exhibits two distinct activity states, which have been associated with different coordination states of the two hearts. During the active state, it discharges high-frequency bursts of action potentials interrupted by rhythmic inhibitory input from other interneurons. In the inactive state, the same cell receives rhythmic inhibition but the membrane potential remains subthreshold between these volleys, producing few or no action potentials. We investigated differences in the membrane properties of the cell during the active and inactive states. The membrane potential in the active state oscillates on average between about -56 +/- 6 mV (S.D.) and -45 +/- 7 mV; the mean oscillation amplitude is 11 +/- 4 mV. In the inactive state, the membrane potential oscillates on average between -58 +/- 6 mV and -55 +/- 6 mV with a mean amplitude of 3 +/- 1 mV. The overall conductance of an HN(5) interneuron during the active state is approximately 10 nS lower than that during the inactive state, indicating that an outward current is turned off during the active state or turned on during the inactive state. This outward current is not voltage-dependent in the range -80 mV to -10 mV, as shown in voltage-clamp experiments by a linear current-voltage relationship. The reversal potential of this current is approximately -60 mV, indicating that chloride or potassium ions underlie the current. Using dynamic-clamp, we show that by adding an artificial current with a linear voltage-dependence (leak conductance) to an HN(5) interneuron (conductance 15 nS, reversal potential -60 mV), the cell can be transferred from its active to its inactive state.

Animals↗

Intracellular pH of giant salivary gland cells of the leech Haementeria ghilianii: regulation and effects on secretion.

1. Intracellular pH (pHi) and membrane potential (Em) of giant salivary gland cells of the leech, Haementeria ghilianii, were measured with double-barrelled, neutral-carrier, pH-sensitive microelectrodes. 2. Em was -51 +/- 11.2 mV and pHi was 6.98 +/- 0.1 (mean +/- S.D., N = 41) in Hepes-buffered saline (nominally HCO3(-)-free; extracellular pH, pHe = 7.4). pHi was independent of Em. 3. Amiloride (2 mmol l-1) had no effect on resting pHi or on pHi recovery from an acid load (induced by the NH4+ pre-pulse technique). Removal of external Na+ produced a progressive acidification which was blocked by amiloride, and the drug also slowed the recovery of pHi on reintroduction of Na+. The results indicate the presence of an electroneutral Na+/H+ exchanger whose access to amiloride is competitively blocked by Na+. 4. In certain smaller cells of the gland, which probably form a separate population, removal of external Na+ did not affect pHi, and recovery from an acid load was blocked by amiloride. There may, therefore, be two types of Na+/H+ exchanger, differing in reversibility and sensitivity to amiloride. 5. Recovery of pHi from NH4(+)-induced acid loading was not affected by bicarbonate-buffered saline (2% CO2; 11 mmol l-1 HCO3-) or by addition of the anion-exchange blocker SITS (10(-4) mol l-1). This suggests that there is no significant contribution of a HCO3(-)-dependent transport mechanism to pHi regulation in the gland cells. 6. Removal of external Cl- slowly reduced pHi and there was a transient increase (overshoot) in pHi when Cl- was reintroduced. These effects of Cl- are probably explained by changes in the Na+ gradient. Intracellular Na+ and Cl- activities were measured with ion-selective microelectrodes. 7. Acidification with NH4+ was difficult, probably because of the cells' poor permeability to this ion. Attempts to introduce NH4+ via the Na+ pump or Na+/Cl- transporter were not successful. The H+/K+ ionophore nigericin (1 microgram ml-1), however, produced a rapid and reversible acidification. 8. N-methylmaleimide (0.5-1 mmol l-1), which blocks proton-pumping ATPase, produced a prolonged acidification of almost 1 pH unit, well beyond the level expected for simple equilibration with pHe. The results are consistent with the presence of a vesicular proton pump, acidifying the secretory vesicles which pack the cell body. 9. NH4+ (50 mmol l-1) or trimethylamine (50 mmol l-1) increased pHi and stimulated salivary secretion, while propionate (50 mmol l-1) decreased pHi and stopped secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Amiloride↗

An endogenous peptide modulates the activity of a sensory neurone in the leech Hirudo medicinalis.

Sensory and neurosecretory innervation of each leech excretory complex, a nephridium and its bladder, is accomplished by a single neurone, the nephridial nerve cell (NNC). The NNC monitors the extracellular Cl- concentration, which ranges between 20 and 100 mmol l-1 depending on the physiological state. The NNC contains FMRFamide in its soma and sensory terminals in the nephridium. Bath or focal application of FMRFamide leads to hyperpolarization and decreases the rate of firing of the NNC, suggesting autoregulation of peptide release. Experiments under single-electrode current-clamp and voltage-clamp show that FMRFamide turns off the receptor-specific Cl- current of the NNC, indicating that FMRFamide also modulates the receptor gain.

Animals↗

Remodelling of an intact neurone in the central nervous system of the leech.

The regeneration pattern of two identified central neurones was studied in the leech Hirudo medicinalis. Anterior pagoda (AP) and mechanosensory touch-sensitive (T) neurones were stained in adult segmental ganglia, maintained in culture for 6-10 days. AP neurones, which normally project only to the contralateral nerve roots, sprouted extensively in all the available nerve paths during regeneration. Mechanosensory T cells, in the same experimental conditions, showed only a moderate growth and did not change their normal pattern of axonal projections. The observed differences in the growth pattern might account for the different electrophysiological responses to axotomy exhibited by the two types of neurone. Interruption of interganglionic connectives induced a moderate and stereotyped remodelling of the morphology of intact AP neurones, which was reminiscent of that transiently exhibited during embryonic development. This response was observed in 25% of the AP neurones we examined.

Animals↗

Intracellular Ca2+ release mediated by metabotropic glutamate receptor activation in the leech giant glial cell.

We have investigated the effects of glutamate and glutamate receptor ligands on the intracellular free Ca2+ concentration ([Ca2+]i) and the membrane potential (Em) of single, identified neuropile glial cells in the central nervous system of the leech Hirudo medicinalis. Exposed glial cells of isolated ganglia were filled iontophoretically with the Ca2+ indicator dye Fura-2. Application of glutamate (200-500 mumoll-1) caused biphasic membrane potential shifts and increases in [Ca2+]i, which were only partly reduced by either removing extracellular Ca2+ or blocking ionotropic glutamate receptors with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50-100 mumol l-1. Metabotropic glutamate receptor (mGluR) ligands had the following rank of potency in inducing a rise in [Ca2+]i: quisqualate (QQ, 200 mumol l-1) > glutamate (200 mumol l-1) > L(+)2-amino-3-phosphonopropionic acid (L-AP3, 200 mumol l-1 > trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 400 mumol l-1). The mGluR-selective antagonist (RS)-alpha-methyl-4-carboxyphenylglycine [(RS)-MCPG, 1 mmol l-1] significantly reduced glutamate-evoked increases in [Ca2+]i by 20%. Incubation of the ganglia with the endoplasmic ATPase inhibitor cyclopiazonic acid (CPA, 10 mumol l-1) caused a significant (53%) reduction of glutamate-induced [Ca2+]i transients, while incubation with lithium ions (2 mmol l-1) resulted in a 46% reduction. The effects of depleting the Ca2+ stores with CPA and of CNQX were additive. We conclude that glutamate-induced [Ca2+]i transients were mediated by activation of both Ca(2+)-permeable ionotropic non-NMDA receptors and of metabotropic glutamate receptors leading to Ca2+ release from intracellular Ca2+ stores.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

Spread of synaptic potentials through electrical synapses in Retzius neurones of the leech.

We studied the spread of excitatory postsynaptic potentials (EPSPs) through electrical synapses in Retzius neurones of the leech Haementeria officinalis. The pair of Retzius neurones in each ganglion is coupled by a non-rectifying electrical synapse. Both neurones displayed synchronous EPSPs of varying amplitudes and rise times. The kinetics of synchronous EPSPs was similar in 79 % of the EPSP pairs. In the remaining 21 %, one EPSP was smaller and slower than the other, suggesting its passive spread from the other neurone. The proportion of these events increased to 75 % in the presence of Mg(2+) in the bathing fluid. This spread of EPSPs from one neurone to another was tested by producing artificial EPSPs by current injection into the soma of one Retzius neurone. The artificial EPSPs were smaller and arrived more slowly at the soma of the coupled neurone. The coupling ratios for the EPSPs were proportional to the coupling ratio for long steady-state pulses in different neuronal pairs. Our results showed that EPSPs spread from one Retzius neurone to the other and support the idea that EPSP spread between electrically coupled neurones may contribute to the integration processes of neurones.

Animals↗

The length-tension relationship of the dorsal longitudinal muscle of a leech.

The length-tension relationship of a preparation of the dorsal body wall of the leech Haemopis sanguisuga was determined. Passive tension is low except at very long lengths of the preparation, when it rises steeply. It is due mainly to the epidermis present in the preparation. The active tension curve is very flat, with tension being reduced only at very short and very long lengths. This shape is explained in the context of the myofilament arrangement of the muscle fibres. It may be that thin filaments can form cross-bridges with different thick filaments at different lengths of the preparation.

Animals↗

Rhythmic swimming activity in neurones of the isolated nerve cord of the leech.

1. Repeating bursts of motor neurone impulses have been recorded from the nerves of completely isolated nerve cords of the medicinal leech. The salient features of this burst rhythm are similar to those obtained in the semi-intact preparation during swimming. Hence the basic swimming rhythm is generated by a central oscillator. 2. Quantitative comparisons between the impulse patterns obtained from the isolated nerve cord and those obtained from a semi-intact preparation show that the variation in both dorsal to ventral motor neurone phasing and burst duration with swim cycle period differ in these two preparations. 3. The increase of intersegmental delay with period, which is a prominent feature of swimming behaviour of the intact animal, is not seen in either the semi-intact or isolated cord preparations. 4. In the semi-intact preparation, stretching the body wall or depolarizing an inhibitory motor neurone changes the burst duration of excitatory motor neurones in the same segment. In the isolated nerve cord, these manipulations also change the period of the swim cycle in the entire cord. 5. These comparisons suggest that sensory input stabilizes the centrally generated swimming rhythm, determines the phasing of the bursts of impulses from dorsal and ventral motor neurones, and matches the intersegmental delay to the cycle period so as to maintain a constant body shape at all rates of swimming.

Action Potentials↗

Neuronal control of swimming in the medicinal leech. V. Connexions between the oscillatory interneurones and the motor neurones.

A network of intra- and intersegmental synaptic connexions has been identified in the ventral nerve cord of the leech that links the set of oscillatory interneurones of the central swim oscillator to the motor neurones commanding the swimming rhythm. Excitatory connexions lead from oscillatory interneurones to both excitatory and inhibitory motor neurones, whereas inhibitory connexions lead from oscillatory interneurones to only the inhibitory motor neurones. Connexions leading from a motor neurone back to the oscillatory interneurones were found in only one exceptional case, an inhibitory motor neurone previously known to have access to the central swim oscillator. This network of identified connexions can account reasonably well for the mechanism by which the oscillatory interneurones drive their follower motor neurones into the phasic activity pattern characteristic of the swimming movement.

Action Potentials↗

Patterns of activity and the effects of activation of the fast conducting system on the behaviour of unrestrained leeches.

1. The neural activity of the nerve cord of Hirudo medicinalis has been recorded in unrestrained animals by means of chronically implanted electrodes. 2. The Fast Conducting System (FCS) is inactive both in motionless animals and during various kinds of active behaviour (creeping, swimming, ventilation). 3. Photic and tactile stimuli applied to a motionless animal elicit a FCS discharge, which may be followed by generalized shortening. 4. Photic and tactile stimuli applied during ventilation are followed by a reversible blockade of the ongoing activity only if they are able to elicit a FCS discharge. No such effect is observed on swimming. 5. An explanation of these findings in terms of the known connexions of leech neurones is offered and a role in the control of reafferent inputs is attributed to the rectifying synapses made by FCS and T cells on the L motor neurones.

Animals↗

The roles of endogenous membrane properties and synaptic interaction in generating the heartbeat rhythm of the leech, Hirudo medicinalis.

1. Inhibitory synapses among the central neurones involved in the generation of the heartbeat rhythm of the leech were blocked by either low Cl- physiological saline or presynaptic hyperpolarizing current. 2. Low Cl- saline reversibly blocked inhibitory postsynaptic potentials (IPSPs) from the HN cells onto both other HN cells and HE cells but did not block electrical coupling among HN cells. 3. The rhythmic bursts of impulses in HE cells were abolished when IPSPs were blocked by either low Cl- saline or hyperpolarization of HN cells. 4. The rhythmic bursts of impulses in HN cells were not abolished (except in cell HN(5)) when IPSPs were blocked by low Cl- saline, but phase relations became unfixed (unless the cells were electrically coupled). 5. Both brief depolarizing and hyperpolarizing current pulses reset the rhythm of HN cells whose IPSPs were blocked by low Cl- saline. 6. The results indicate that the motor neurones to the heart (HE cells) produce rhythmic impulse bursts because their steady discharge is periodically inhibited by the HN interneurones. The pattern generated by the HN cells originates from an endogenous rhythm co-ordinated by the inhibitory interactions and electrical coupling between these cells.

Action Potentials↗

Isolation and characterization of seven lyso platelet-activating factors and two lyso phosphatidylcholines from the crude drug "suitetsu" (the leech, Hirudo nipponica).

Nine lyso glycerophospholipids were isolated in the pure state from the crude drug "Suitetsu", which is the dried body of the leech, Hirudo nipponica (Hirudidae). They were identified as 1-O-hexadecyl-(1), 1-O-octadecyl-(2), 1-O-tetradecyl-(3), 1-O-9-cis-hexadecenyl-(4), 1-O-hexadecanoyl-(5), 1-O-pentadecyl-(6), 1-O-15-methylhexadecyl-(7), 1-O-octadecanoyl-(8) and 1-O-heptadecyl-sn-glycero-3-phosphocholine (9). Two of them (5 and 8) are lysophosphatidylcholines and the other seven are lyso platelet-activating factors. One of them has an alkenyl carbon chain.

Animals↗

Six trigalactosylceramides from the leech (Hirudo nipponica).

Six neutral glycosphingolipids were isolated in the pure state from the leech, Hirudo nipponica (Annelida). In contrast to the zwitterionic monogalactosylceramides carrying a choline phosphate group so far obtained, all compounds are non-zwitterionic glycosphingolipids, trigalactosylceramides. Five compounds possess a Ga1 alpha 1-6Ga1 alpha 1-6Ga1 beta 1-Cer core, and one is unique in having a Ga1 alpha 1-6Ga1 beta 1-Cer structure. Their full structures have been determined on the bases of chemical and spectral evidence.

Animals↗