Magnesium-resistant excitatory synaptic potentials in the leech Retzius cell.

Postsynaptic potentials (PSPs) recorded from leech Retzius cells in response to stimulation of interganglionic connective could not be reversed by soma depolarization or abolished by 40 mM Mg ion, nor could input resistance changes be detected during them. Alteration of external Cl and K over a tenfold range provided no clear evidence that the PSPs involved a conductance change to either ion. The method of extrapolation yielded an apparent PSP equilibrium potential of about -20 mV. The steep portion of the relationship between Retzius cell action potential amplitude and membrane potential extrapolated to an apparent reversal potential of -13 mV. It is likely that the connective-to-Retzius cell PSPs were principally electrical events. Their apparent reversal potentials could have been in the range associated with chemical synapses because they traversed an electrical synapse with a variable coupling resistance, or because the polarizing currents, passing "backwards" across electrical synapses, changed the amplitude of the presynaptic action potentials.     

The dynamic electrical behaviour of the electrotonic junction between Retzius cells in the leech

Linear frequency domain analysis of subthreshold current flow and cell to cell action potential timing studies have been used to examine the dynamic electrical behaviour of the electrotonic junction between Retzius cells in the leech. Both types of electrical transmission may be completely explained if the junction is modelled by a single resistive element. The possible role of axonal cable properties in the apparent junctional characteristics were considered, but the form of the predicted frequency response functions for such cables make it unlikely that they are involved in the observed electrical behaviour.     

Persistent inward currents in cultured Retzius cells of the medicinal leech

Current-clamp studies of cultured leech Retzius cells revealed inward rectification in the form of slow voltage sags in response to membrane hyperpolarization. Sag responses were eliminated in Na⁺-free saline and blocked by Cs⁺, but not Ba²⁺. Voltage clamp experiments revealed a Cs⁺-sensitive inward current activated by hyperpolarization negative to −70 mV. Cs⁺ decreased the frequency of spontaneous impulses in Retzius cells of intact ganglia. Plateau potentials were evoked in Retzius cells following block of Ca²⁺ influx with Ni²⁺ and suppression of K⁺ currents with internal tetraethylammonium. Plateau potentials continued to be expressed with Li⁺ as the charge carrier, but were eliminated when Na⁺ was replaced with N-methyl-d-glucamine. A persistent Na⁺ current with similar pharmacology that activated positive to −40 mV and reached its peak amplitude near −5 mV was identified in voltage-clamp experiments. Inactivation of the persistent Na⁺ current was slow and incomplete. The current was revealed by slow voltage ramps and persisted for the duration of 5-s voltage steps. Persistent Na⁺ current may underlie Na⁺-dependent bursting recorded in neurons of intact ganglia exposed to Ca²⁺-channel blockers. Accepted: 22 September 1998     

Sensitivity of the resting sodium conductivity of leech Retzius nerve cells to tetrodotoxin

Simultaneously, the effect of sodium-free medium and tetrodotoxin (3 X 10(-8) M/ml) were investigated on some passive electrophysiological properties of leech Retzius nerve cells. Complete replacement of Na+ with Tris or addition of tetrodotoxin to the leech Ringer was followed by an increase of input resistance in contrast to the cell-to-cell interaction which was not affected by such a procedure. At the same time tetrodotoxin was not able to block repetitive spike activity. The data imply the existence of two types of Na+ channel in leech Retzius nerve cells.     

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.     

Pharmacological control of the pattern of activity in leech Retzius neurones

1. Changes in the activity pattern of leech Retzius (R) cells were investigated using intracellular recording. 2. The presence of an after-hyperpolarisation (AHP) is closely related to activity pattern; regular firing being associated with an AHP, bursting with its absence. 3. Increasing external calcium (Cao), cyclic AMP levels or activity of kinase A enhanced the AHP. 4. Bursting was induced by low Cao, EGTA, barium, cobalt or injection of phorbol ester. 5. Reduction of Cao to zero caused long paroxysmal depolarising shifts of potential which could be reversed to bursting by cobalt, IBMX or injection of kinase A catalytic subunit. 6. The possible roles of a calcium-activated potassium channel and protein phosphorylation in regulating the activity of the cell are discussed.     

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.     

Calcium influx into dendrites of the leech Retzius neuron evoked by 5-hydroxytryptamine

5-Hydroxytryptamine (5-HT) is a ubiquitous neurotransmitter and neuromodulator that affects neural circuits and behaviours in vertebrates and invertebrates. In the present study, we have investigated 5-HT-induced Ca(2+) transients in subcellular compartments of Retzius neurons in the leech central nervous system using confocal laser scanning microscopy, and studied the effect of 5-HT on the electrical coupling between the Retzius neurons. Bath application of 5-HT (50mM) induced a Ca(2+) transient in axon, dendrites and cell body of the Retzius neuron. This Ca(2+) transient was significantly faster and larger in dendrites than in axon and cell body, and was half-maximal at a 5-HT concentration of 5-12mM. The Ca(2+) transient was suppressed in the absence of extracellular Ca(2+) and by methysergide (100mM), a non-specific antagonist of metabotropic 5-HT receptors, and was strongly reduced by bath application of the Ca(2+) channel blocker Co(2+) (2mM). Injection of the non-hydrolysable GTP analogue GTPgammaS increased and prolonged the dendritic 5-HT-induced Ca(2+) transient. The non-selective protein kinase inhibitor H7 (100mM) and the adenylate cyclase inhibitor SQ22536 (500 mM) did not affect the Ca(2+) transient, and the membrane-permeable cAMP analogue dibutyryl-cAMP (500 mM) did not mimic the effect of 5-HT application. 5-HT reduced the apparent electrical coupling between the two Retzius neurons, whereas suppression of the Ca(2+) influx by removal of external Ca(2+) improved the transmission of action potentials at the electrical synapses which are located between the dendrites of the adjacent Retzius neurons. The results indicate that 5-HT induces a Ca(2+) influx through calcium channels located primarily in the dendrites, and presumably activated by a G protein-coupled 5-HT receptor. The dendritic Ca(2+) increase appears to modulate the excitability of, and the synchronization between, the two Retzius neurons.     

The activity of leech motoneurons during motor patterns is regulated by intrinsic properties and synaptic inputs

The activity of motoneurons during motor patterns depends on their intrinsic properties and on synaptic inputs. This study analyzed the properties of two leech motoneurons: the excitors of dorsal longitudinal muscles (DE-3) and of dorsal and ventral longitudinal muscles (MN-L) in basal conditions (normal and high Mg²⁺ saline) and during crawling. The voltage–current relationships in DE-3 and MN-L were similar. The curves exhibited the largest slope around resting potential, showed marked inward and outward rectification, and were not affected by high Mg²⁺. In response to 5-s pulses, DE-3 exhibited a fast initial adaptation, a slow recovery and a very slow late adaptation. High Mg²⁺ abolished the initial high frequency. The frequency–voltage relationship for the rest of the response was highly similar in normal and in high Mg²⁺ saline. MN-L exhibited a minor initial adaptation and then fired steadily. High Mg²⁺ diminished the frequency–voltage relationship. During crawling DE-3 and MN-L fired in phase and their frequency–voltage curves overlapped with the lower end of the curves obtained in basal conditions. The results suggest that the activity of these motoneurons during crawling was regulated, to a large extent, by synaptic inputs.     

Action potential prolongation: an effect of physostigmine (eserine) upon Retzius cells in the leech C.N.S

Physostigmine (PHY; eserine) prolongs the action potentials in the Retzius cells within leech ganglia to about 800 ms. The effect was reversible and occurred at concentrations of 1-10 mM which are several orders of magnitude greater than those required to inhibit cholinesterase. The prolonged action potentials showed an early, spike-like depolarization followed by a plateau. The initial depolarization exhibited a strong dependence on external Na+ while the amplitude of the plateau had somewhat less Na+ dependence: 52 and 24 mV/decade, respectively. The duration of the plateau was increased by elevating Na+ and decreased by elevating Ca2+. Increasing the action potential frequency, by intracellular stimulation, decreased both the duration and amplitude of the plateau. Neostigmine, di-isopropylphosphofluoridate, and acetylcholine did not prolong RZ action potentials. Thus, the membrane effects of physostigmine appear to be independent of any inhibition of cholinesterase or accumulation of acetylcholine.     

Perioral synaptic connections and their possible role in the feeding behavior of Hydra

Electron microscopic observations of serially sectioned perioral neurons revealed a complex synaptic organization in which reciprocal synapses were observed for the first time in Hydra. Sensory cells had reciprocal synapses with each other and with ganglion cells, which in turn had reciprocal synapses with each other. A two-way chemical synapse with vesicles on both sides of the paramembranous densities was observed between ganglion cells; none was found between sensory cells. Ganglion cell axons participated in serial axo-axo-epitheliomuscular synapses. Two-cell pathways formed by direct sensory cell-nematocyte or neuromuscular synapses and three-cell pathways forming indirect sensory cell-ganglion cell-nematocyte or neuromuscular synaptic interconnections were found. It is possible that either simple direct changes in or direct effects on threshold stimuli could trigger both nematocyst discharge and/or muscular contraction and effect more complex intermediate pathways modulating feeding behavior. Each large epitheliomuscular cell enveloped from one to four sensory cells in the perioral region. The concentration of sensory cells around the mouth and their complex synaptic connections with each other and with ganglion and effector cells support our hypothesis for neural control of feeding behavior in Hydra.     

Calcium-induced calcium release contributes to somatic secretion of serotonin in leech Retzius neurons

We analyzed the contribution of calcium (Ca2+)-induced Ca2+ release to somatic secretion in serotonergic Retzius neurons of the leech. Somatic secretion was studied by the incorporation of fluorescent dye FM1-43 upon electrical stimulation with trains of 10 impulses and by electron microscopy. Quantification of secretion with FM1-43 was made in cultured neurons to improve optical resolution. Stimulation in the presence of FM1-43 produced a frequency-dependent number of fluorescent spots. While a 1-Hz train produced 19.5+/-5.0 spots/soma, a 10-Hz train produced 146.7+/-20.2 spots/soma. Incubation with caffeine (10 mM) to induce Ca2+ release from intracellular stores without electrical stimulation and external Ca2+, produced 168+/-21.7 spots/soma. This staining was reduced by 49% if neurons were preincubated with the Ca2+- ATPase inhibitor thapsigargin (200 nM). Moreover, in neurons stimulated at 10 Hz in the presence of ryanodine (100 microM) to block Ca2+-induced Ca2+ release, FM1-43 staining was reduced by 42%. In electron micrographs of neurons at rest or stimulated at 1 Hz in the ganglion, endoplasmic reticulum lay between clusters of dense core vesicles and the plasma membrane. In contrast, in neurons stimulated at 20 Hz, the vesicle clusters were apposed to the plasma membrane and flanked by the endoplasmic reticulum. These results suggest that Ca2+-induced Ca2+ release produces vesicle mobilization and fusion in the soma of Retzius neurons, and supports the idea that neuronal somatic secretion shares common mechanisms with secretion by excitable endocrine cells.     

The ionic mechanisms associated with the excitatory response of kainate, L-glutamate, quisqualate, ibotenate, AMPA and methyltetrahydrofolate on leech Retzius cells

Intracellular recordings were made from Retzius cells from segmental ganglia of the leech, Hirudo medicinalis. The ionic mechanisms of the following compounds were examined: L-glutamate, ibotenate, quisqualate, AMPA, kainate, methyltetrahydrofolate and carbachol. All these compounds depolarise and excite Retzius cells. In sodium-free Ringer, the responses to L-glutamate, kainate, ibotenate and AMPA were greatly reduced, the response to quisqualate was reduced, the response to methyltetrahydrofolate was normal while the response to carbachol was abolished. In sodium-free high calcium Ringer the responses to L-glutamate, ibotenate and carbachol were absent, the responses to quisqualate and AMPA greatly reduced, the responses to methyltetrahydrofolate and kainate were normal. The methyltetrahydrofolate and kainate responses in sodium-free high calcium Ringer were greatly reduced on addition of cobalt. All the responses are associated with an increase in conductance, the increase being the largest in the case of kainate. It is concluded that the response to L-glutamate, ibotenate and carbachol are dependent on sodium, the responses to quisqualate and AMPA are mainly sodium dependent, possibly with a small calcium component. The kainate response in normal Ringer is largely sodium dependent but in sodium-free Ringer calcium can completely substitute for sodium. The methyltetrahydrofolate response appears to be sodium independent but at least partly calcium dependent. These studies provide further evidence that L-glutamate and ibotenate act on a common receptor on leech Retzius cells while kainate acts on a separate receptor which can activate a calcium ionophore. It is probable that methyltetrahydrofolate acts on a different ionophore system to kainate. N-Methyl-D-aspartate has no agonist activity on any of these receptors.