Ionic mechanism of 5-hydroxytryptamine induced hyperpolarization and inhibitory junctional potential in body wall muscle cells of Hirudo medicinalis.

Five-hydroxytryptamine (5-HT) causes a hyperpolarization and increased conductance of the leech body wall muscle cell membrane. If 5-HT is applied in the absence of the Cl minus ion, the response appears as a depolarization, whereas if 5-HT is applied in the absence of the K+ ion, the response is a hyperpolarization. In both cases, the conductance of the muscle cell membrane is increased. Stimulation of the peripheral nerve to the body wall muscle produces a complex junctional potential in muscle cells. Exposing the muscle to d-tubocurarine (d-TC) eliminates the excitatory component (EJP) of the complex potential. The inhibitory potential (IJP) that remains has an equilibrium potential at approximately 65 mV. Furthermore, this IJP appears as a depolarization when the nerve is stimulated in the presence of d-TC and low CL minus, whereas this is not the case if the nerve is stimulated in the presence of d-TC and low K+. The drugs BOL-148 and cyproheptadine block the IJP's in the body wall muscle. These data are interpreted as indicating that 5-HT acts on leech body wall muscle cells by increasing the conductance to the Cl minus ion and that the IJP's caused by nerve stimulation are probably the result of 5-HT release at nerve terminals. As a final point, it has been shown that the inhibition by 5-HT of the spontaneous EJP's that occur on the leech body wall muscle results from an inhibition of central neurons and not from any direct effect on the muscle cell or on peripheral synapses.     

Innervation of holothurian body wall muscle: inhibitory effects and localization of 5-HT

We investigated innervation to body wall muscles as well as distribution of 5-HT (serotonin) and its effects on longitudinal muscles of body wall (LMBW) of the sea cucumber Apostichopus japonicus. With serial sections we found neural branches and fibers extending from hyponeural part of radial nerve towards LMBW and circular muscles of body wall. With the aqueous aldehyde (Faglu) method yellow fluorescence indicating indolamines was observed in LMBW and in the mesentery connecting LMBW to the body wall. With indirect immunohistochemistry 5-HT-like immunoreactivity was observed in LMBW and in mesentery. These results strongly suggested that both LMBW and mesentery contained 5-HT. The effects of monoamine neurotransmitters were studied in LMBW. Putative neurotransmitters tested were 5-HT, adrenaline, noradrenaline, dopamine, and DOPA at the concentration of 10(-6) M. The application of 5-HT caused no contraction or relaxation, but it inhibited the contraction induced by 10(-6)-10(-5) M acetylcholine (ACh). Catecholamines were ineffective by themselves and had no effects on the contraction induced by ACh. The present histological, histochemical, and pharmacological studies strongly suggested that holothurian LMBW was innervated by inhibitory serotonergic neurons of the hyponeural nervous system.     

腺苷引起促黑素细胞膜电位超极化的离子机制

通过对青蛙垂体中叶促黑素细胞的实验已发现,腺苷激素Ａ１型腺苷受体后可引起细胞膜的超极化同时停止自发性动作电位的发放,但此现象所涉及的离子机制尚不清楚。本实验采用膜片箝技术的全细胞电流、电压记录和细胞吸附单通道电流记录方法,对此电流进行了探讨。实验结果表明：腺苷所致的细胞膜超极化是由于增加了非电压激活的钾离子通道的开放,而对超极化电压激活的内向阳离子电流（Ｉｈ）没有影响。     

5-Hydroxytryptamine-like immunoreactivity in the peripheral and central nervous systems of the leech Hirudo medicinalis

Summary Chains of segmental ganglia and various peripheral tissues from the leech (Hirudo medicinalis) were screened as whole-mount preparations for the presence of 5-hydroxytryptamine-like immunoreactivity. The gut was richly supplied with immunoreactive nerve fibres. Plexus of fibres, numerous of which were varicose, were found in the crop, with many immunopositive nerve cell bodies in the posterior region and a few in the anterior region. The intestine contained a few longitudinally oriented nerve fibres, while the rectum contained a dense network of non-varicose and varicose fibres. Fine immunopositive fibres were associated with the lateral blood vessel and reproductive organs. Many immunopositive nerve fibres ran in each of the paired connectives linking the segmental ganglia, and two fine varicose fibres were seen in Faivre's nerve. At least two immunopositive processes left each lateral segmental nerve and branched repeatedly, with many varicosities on the distal branches. The dorso-ventral and longitudinal body wall muscles both contained immunoreactive fibres, the plexus being more dense in the former muscle. The possible roles of the immunoreactive nerve fibres demonstrated in the various tissues of the leech have been discussed in relation to the known peripheral effects of serotoninergic neurone stimulation in the leech and to the actions of exogenously applied 5-hydroxytryptamine in these and other invertebrate tissues.     

Pain and tenderness in human temporal muscle induced by bradykinin and 5-hydroxytryptamine

Pain was induced in 19 healthy individuals by double-blind injections into the temporal muscle of 0.2 ml of physiological saline with or without active substances added. 5-Hydroxytryptamine (2 nmol) caused pain similar to saline, bradykinin (2 nmol) only insignificantly more pain (0.05<p<0.1), while a mixture of the two substances in half dosage (1 nmol + 1 nmol) caused pain significantly above saline (p<0.01). Variations in the response to saline did not permit a conclusion to be made on the question of induced tenderness. However, the mixture of the two substances appeared to lower the pressure-pain threshold as measured by a pressure algometer (p<0.05).     

Sensitivity changes of photoreceptor cells of Hirudo medicinalis caused by changes in extracellular calcium concentration

Extracellular recordings from the vacoule of photoreceptor cells of Hirudo medicinalis L. were performed using microelectrodes. The cells were adapted by white light flashes given at constant intervals (20 s). Response height versus relative intensity curves obtained from the same cell in physiological saline (PS) and in bathing solutions of either a) lowered calcium contents (2 ΜM/1 or less) or b) raised calcium contents (15 mM/1) were compared. The cells' adaptation state in PS was operationally defined by the ratio Q=h _A /h _S where h _A is the response height evoked by the adapting flashes, and h _S is the corresponding saturation response height. Sensitivity changes were measured by the half saturation intensity shift. Lowering extracellular calcium resulted in:

1. The response height increased and the shape of the response became more rounded and prolonged.
2. The total resistance between the vacuole and outside decreased from 8.2±1.4 MΩ (n=6) in PS to 4.6±0.4 MΩ (n=5). The resistance was independent of the cells' adaptation state.
3. A change of the cells' sensitivity occured either in direction to light adaptation or in direction to dark adaptation. It depended functionally on the ratio Q:

a) if Q was less or equal to about 0.6 the cells' sensitivity increased. b) if Q was greater than 0.6 the cells' sensitivity diminished. Raising extracellular calcium decreased the sensitivity of all cells tested independent of their adaptation states in PS. The results can be interpreted under the assumptions that 1. the sensitivity of leech photoreceptor cells is inversely proportional to the intracellular free calcium concentration and Z. intracellular calcium can interact with extracellular calcium in relatively dark adapted cells whereas in relatively light adapted cells the raise of intracellular free calcium is mainly effected by a release from intracellular stores. It is assumed that a Q value of about 0.6 separates relatively light adapted cells from relatively dark adapted cells.     

Development of dopamine-containing neurons and dopamine uptake in embryos of Hirudo medicinalis

The embryonic development of neurons which contain or take up dopamine was studied with glyoxylic acid histofluorescence in Hirudo medicinalis. Beginning at the time of the formation of the tail ganglion, one pair of dopamine-containing neurons was stained per segmental ganglion. The normal outgrowth of the cell bodies into the anterior roots was prevented in isolated and cultured chains of embryonic ganglia. Preincubation of intact embryos in dopamine led to the staining of additional neurons at certain developmental stages. These neurons presumably are the precursors of serotonin-containing cells, which have a temporary capability of taking up and storing dopamine.     

Molecular characterization and embryonic expression of innexins in the leech Hirudo medicinalis

Gap junctions are direct intercellular channels that permit the passage of ions and small signaling molecules. The temporal and spatial regulation of gap junctional communication is, thus, one mechanism by which cell interactions, and hence cell properties and cell fate, may be regulated during development. The nervous system of the leech, Hirudo medicinalis, is a particularly advantageous system in which to study developmental mechanisms involving gap junctions because interactions between identified cells may be studied in vivo in both the embryo and the adult. As in most invertebrates, gap junctions in the leech are composed of innexin proteins, which are distantly related to the vertebrate pannexins and are encoded by a multi-gene family. We have cloned ten novel leech innexins and describe the expression of these, plus two other previously reported members of this gene family, in the leech embryo between embryonic days 6 and 12, a period during which the main features of the central nervous system are established. Four innexins are expressed in neurons and two in glia, while several innexins are expressed in the excretory, circulatory, and reproductive organs. Of particular interest is Hm-inx6, whose expression appears to be restricted to the characterized S cell and two other neurons putatively identified as presynaptic to this cell. Two other innexins also show highly restricted expressions in neurons and may be developmentally regulated. Electronic Supplementary Material Supplementary material is available for this article at .     

Fluorescence marking of neuropile glial cells in the central nervous system of the leech Hirudo medicinalis

Summary Neuropile glial (NG) cells in the central nervous system of the medicinal leech, Hirudo medicinalis L., were studied by histological and intracellular electrophysiological methods. Potential profiles of single leech ganglia were mapped by advancing an electrolyte-filled microelectrode into the ganglion as far as the NG cell. A small negative potential usually appeared during or immediately after penetration of the ganglion sheath. Most of the ganglia in the chain (ganglia 1–4 and 7–21) have Retzius-cell-bodies of normal size; in these, the potential associated with the ganglion sheath was followed by a jump to a more negative potential. Superimposed action potentials were associated with entry of the electrode into a Retzius cell. When the electrode tip passed out of the cell into the center of the ganglion, another potential change was observed, namely that to the membrane potential of the anterior NG cell. This membrane potential averaged -60.2 mV and ranged from -50 to -73 mV. In ganglia 5 and 6 the Retzius-cell-bodies are particularly small, and no changes of potential associated with these cells were observed; the first potential to appear after the electrode passed through the sheath of the ganglion was the membrane potential of the NG cell. Potential profiles like those of ganglia 5 and 6 are recorded in the posterior parts of all ganglia.Potential profiles of single leech ganglia were also recorded with microelectrodes filled with the fluorescent dye Procion Yellow M4-RAN. When the presumed membrane potential of an NG cell appeared, the dye was injected into the ganglion. Subsequent histological examination with the fluorescence microscope revealed that all of the dye was contained in NG cells.Supported by a Fellowship (Heisenberg-Stipendium, Schl 169/5) and grants (Schl 169/2, 4) to W.R.S. from the Deutsche ForschungsgemeinschaftThe authors thank Gisela Geiger for excellent assistance during this work     

Axonal projections of the Retzius cells to the dorsal segmental root in the leech Hirudo medicinalis

The presence of RC's axon running in the dorsal root of the leech Hirudo medicinalis was studied by means of electrophysiological and neuroanatomical techniques. Electrical stimulation of the dorsal root gave invariably the antidromic invasion of the Retzius cell. This effect disappears after crushing the dorsal root between the stimulating electrode and the ganglion, and hence is not due to stimulus spread to the nearby posterior root. Anatomical evidence for an axon collateral running in the dorsal root was provided by catecholamine histofluorescence of the axonal branches of the RCs and by Lucifer Yellow intracellular injection into the RCs.     

Neural control of somatic muscle function in the earthworm, Allobophora longa, and in the leech, Hirudo medicinalis

Studies have been made on the electrical activity of the segmentary nerves and connectives of the abdominal nervous chain in the earthworm and leech. It was shown that the electrical activity of the isolated piece of the abdominal chain of the leech is manifested of periodic outbursts of impulsation. Presumably this central periodicity accounts for the discharge-like pattern of muscle rhythmic activity which was revealed in our earlier investigations. The electrical activity in the central nervous system of the earthworm depends on afferent influences which pass to the ganglia from the peripheral sensory nervous cells. Stimulation of the abdominal nervous chain did not result in extra discharges of muscle activity, but only affected some of the parameters of the latter.     

A comparison of receptive and non-receptive plasma membrane areas of photoreceptor cells in the leech,Hirudo medicinalis

Summary Microvillar (receptive) and external (non-receptive) portions of the plasmalemma of photoreceptor cells of Hirudo were compared electron microscopically in thin sections and freeze-fracture replicas. A morphometric approximation showed that the surface area of the microvillar membrane is about 19 times larger than that of the external membrane. The microvillar membrane most probably undergoes extensive membrane turnover. In both segments of the membrane the particles associated with the P- and the E-fracture faces are randomly distributed except at some specific sites. The particles adhere predominantly to the P-faces. The particle densities on the fracture faces of the microvillar membrane differ from those of the external membrane. The P-face particles of the external membrane appear to be larger than those of the microvillar membrane. It is suggested that the P-face particles of the microvillar membrane represent sites where the photopigment is incorporated into the membrane. The distinguishing structural features correspond to the functional differences postulated for both portions of the plasma membrane.     

Variations in the hyperpolarization potential of ethanol on muscle during reinnervation

Ethanol causes the hyperpolarization of the excitable membranes. In the Extensor Digitorum Longus (EDL) muscle of the rat the increase of resting membrane potential is 2-5% and is independent of the concentration of alcohol between 0.2 and 0.4 M, while at higher concentrations the membrane potential falls to levels equal or inferior to the normal potential. We have studied the hyperpolarization action of ethanol on the denervated muscle by crushing the sciatic nerve. Also under these conditions in which, as is known, there is a drop in the resting potential, ethanol causes hyperpolarization, however it is in general greater and it is dependent upon the concentration between 0.2 and 0.8 M.     

Photogeneration of membrane potential hyperpolarization and depolarization in non-excitable cells

We monitored femtosecond laser induced membrane potential changes in non-excitable cells using patchclamp analysis. Membrane potential hyperpolarization of HeLa cells was evoked by 780 nm, 80 fs laser pulses focused in the cellular cytoplasm at average powers of 30–60 mW. Simultaneous detection of intracellular Ca²⁺ concentration and membrane potential revealed coincident photogeneration of Ca²⁺ waves and membrane potential hyperpolarization. By using non-excitable cells, the cell dynamics are slow enough that we can calculate the membrane potential using the steady-state approximation for ion gradients and permeabilities, as formulated in the GHK equations. The calculations predict hyperpolarization that matches the experimental measurements and indicates that the cellular response to laser irradiation is biological, and occurs via laser triggered Ca²⁺ which acts on Ca²⁺ activated K⁺ channels, causing hyperpolarization. Furthermore, by irradiating the cellular plasma membrane, we observed membrane potential depolarization in combination with a drop in membrane resistance that was consistent with a transient laser-induced membrane perforation. These results entail the first quantitative analysis of location-dependent laser-induced membrane potential modification and will help to clarify cellular biological responses under exposure to high intensity ultrashort laser pulses.     

A central inhibitory action of 5-hydroxytryptamine in the leech

Previous studies indicated that 5-HT reduced the number of spontaneous excitatory junctional potentials (ejp's) that occurred in leech body wall muscle cells. The present study confirms these findings and shows that the ejp's arise from impulses in motoneuron L. This study further shows that 5-HT acts by hyperpolarizing and reducing the membrane resistance of neuron L, thus inhibiting the motoneuron and reducing the frequency of spontaneous ejp's on body wall muscle cells. These effects of 5-HT are not seen when the ganglion is bathed in a high magnesium solution, a finding that suggests that 5-HT does not act directly on the membrane of motoneuron L. This study demonstrates that 5-HT can have a central inhibitory effect on body wall muscle contractions. Previous studies provide evidence that 5-HT may act as a direct neuromuscular inhibitory transmitter and may also take part in peripheral presynaptic inhibition. Thus, if further studies confirm these suggestions, the well-known inhibitory effect of 5-HT on leech body wall muscle is a more complex process than was previously thought.     

Synaptic effects elicited in the Retzius cells of the leech Hirudo medicinalis by stimulation of the segmental roots

Electrical stimulation of the segmental roots of each ganglion of Hirudo medicinalis, elicits in both Retzius' cells inhibitory and excitatory effects. The IPSP and EPSP are chemical in nature, being dependent on the membrane potential, and suppressed by high Mg++. Selective inactivation of one RC shows that the responses of the contralateral RC are not due to electrotonic coupling between the two cells, but to synaptic actions impinging upon the membrane of both RCs. The two synaptic potentials appear to be mediated by two set of fibres with a different threshold to electrical stimulation. Their actions on the RCs appear to be polysynaptic on the basis of central latency. Simultaneous stimulation of two roots shows evidence for occlusion for IPSP and summation for EPSP, confirming the polysynaptic nature of the effects. The possible functional significance of the inhibitory and excitatory pathways, is discussed.     

Structure-activity studies of the amine receptor on the Retzius cells of the leeches Hirudo medicinalis and Haemopis sanguisuga

1. The hyperpolarising (H) responses elicited by 5-hydroxytryptamine (5-HT) or dopamine (DA) on an amine receptor on Retzius (R) cells of two leech species (Hirudo medicinalis and Haemopis sanguisuga) were increased in duration but not amplitude by imipramine, suggesting the presence of an amine uptake system.2. A structure-activity study of twenty agonists was performed on this amine receptor, and equipotent molar ratio (e.p.m.r.) values calculated. DA and other compounds lacking the β-OH group on the ethylamine side chain were very potent. Compounds possessing a β-OH could produce D, H or biphasic responses although salbutamol was inactive. Substitution on the α-carbon or terminal nitrogen of the side chain reduced potency. Compounds required an -OH group on C4 of the phenyl ring. DA agonists were preferred in a β-rotational rather than an α-rotational conformation.3. There were no major differences between the responses of the two species to agonists, except that isoprenaline hyperpolarised Hirudo R cells but was inactive on Haemopis. E.p.m.r. values varied in the two species.4. Phentolamine acted as an antagonist in Haemopis but was inactive in Hirudo. In both species atropine was an effective antagonist.5. On the basis of this study a model is proposed for a joint DA-5-HT receptor in the leech.