In situ characterization of a rectifying electrical junction.

Electrical synapses play significant roles in neural processing in invertebrate and vertebrate nervous systems. The view of electrical synapses as plain bidirectional intercellular channels represents a partial picture because rectifying electrical synapses expand the complexity in the communication capabilities of neurons. Rectification derives, mostly, from the sensitivity of electrical junctions to the transjunctional potential (V(j)) across the coupled cells. We analyzed the characteristics of this sensitivity and their effect on neuronal signaling, studying rectifying junctions present in the leech nervous system. The NS neurons, a pair of premotor nonspiking neurons present in each midbody ganglion, are electrically coupled to virtually every excitatory motor neuron. Studied at rest, only hyperpolarizing signals can be transmitted from NS to the motoneurons, and only depolarizing signals are conducted in the opposite direction. Our results show that small changes in the NS membrane potential (V(m)) exerted an effective control of the firing frequency of the CV motoneurons (excitor of circular muscles). This effect revealed the existence of a threshold V(j) across which the electrical synapse shifts from a nonconducting to a conducting state. The junction can operate as a relatively symmetrical bidirectional bridge provided that the transmitted signals do not cross this threshold transjunctional potential.

Network interactions among sensory neurons in the leech.

Interactions among mechanosensory neurons, sensitive to touch, pressure and nociceptive stimuli in the leech nervous system were studied in isolated ganglia and in body-wall preparations. Pairs of touch-pressure, touch-nociceptive and pressure-nociceptive neurons were tested by suprathreshold stimulation of one neuron while recording the response of the other, in both directions. Pressure and nociceptive stimulation evoked depolarizing and hyperpolarizing responses in touch cells, mediated by interneurons. The relative expression of these responses depended on the stimulus duration. One or two pressure cell spikes produced, predominantly, a depolarization of the touch cells, and increasing number of spikes evoked a hyperpolarization. Nociceptive cells produced primarily the hyperpolarization of touch cells at any stimulus duration. When touch cells were induced to fire by injection of positive current into the soma, stimulation of pressure cells inhibited touch cell activity. However, when touch cells were induced to fire by peripheral stimulation, pressure cell activation failed to inhibit touch cell firing. The results suggest that excitation of pressure and nociceptive cells would not limit the responses of touch cells to peripheral stimuli, but would inhibit the firing of touch cells evoked by their central connectivity network.

Convergence of mechanosensory inputs onto neuromodulatory serotonergic neurons in the leech.

By the frequency-dependent release of serotonin, Retzius neurons in the leech modulate diverse behavioral responses of the animal. However, little is known about how their firing pattern is produced. Here we have analyzed the effects of mechanical stimulation of the skin and intracellular stimulation of mechanosensory neurons on the electrical activity of Retzius neurons. We recorded the electrical activity of neurons in ganglia attached to their corresponding skin segment by segmental nerve roots, or in isolated ganglia. Mechanosensory stimulation of the skin induced excitatory synaptic potentials (EPSPs) and action potentials in both Retzius neurons in a ganglion. The frequency and duration of responses depended on the strength and duration of the skin stimulation. Retzius cells responded after T and P cells, but before N cells, and their sustained responses correlated with the activity of P cells. Trains of five impulses at 10 Hz in every individual T, P, or N cell in isolated ganglia produced EPSPs and action potentials in Retzius neurons. Responses to T cell stimulation appeared after the first impulse. In contrast, the responses to P or N cell stimulation appeared after two or more presynaptic impulses and facilitated afterward. The polysynaptic nature of all the synaptic inputs was shown by blocking them with a high calcium/magnesium external solution. The rise time distribution of EPSPs produced by the different mechanosensory neurons suggested that several interneurons participate in this pathway. Our results suggest that sensory stimulation provides a mechanism for regulating serotonin-mediated modulation in the leech.

Processing of sensory signals by a non-spiking neuron in the leech.

The non-spiking neurons 151 are present as bilateral pairs in each midbody ganglion of the leech nervous system and they are electrically coupled to several motorneurons. Intracellular recordings were used to investigate how these neurons process input from the mechanosensory P neurons in isolated ganglia. Induction of spike trains (15 Hz) in single P cells evoked responses that combined depolarizing and hyperpolarizing phases in cells 151. The phasic depolarizations, transmitted through spiking interneurons, reversed at around -20 mV. The hyperpolarization had two components, both reversing at around -65 mV, and which were inhibited by strychnine (10 micromol l(-1)). The faster component was transmitted through spiking interneurons and the slower component through a direct P-151 interaction. Short trains (<400 ms) of P cell spikes (15 Hz) evoked the phasic depolarizations superimposed on the hyperpolarization, while long spike trains (>500 ms) produced a succession of depolarizations that masked the hyperpolarizing phase. The amplitude and duration of the hyperpolarization reached their maximum at the initial spikes in a train, while the depolarizations persisted throughout the duration of the stimulus train. Both phases of the response were relatively unaffected by the spike frequency (5-25 Hz). The non-spiking neurons 151 processed the sensory signals in the temporal rather than in the amplitude domain.

Differential channeling of sensory stimuli onto a motor neuron in the leech.

We studied a specific sensory-motor pathway in the isolated leech ganglia. Pressure-sensitive mechanosensory neurons were stimulated with trains of action potentials at 5-20 Hz while recording the responses of the annulus erector motorneurons that control annuli erection. The response of the annulus erector neurons was a succession of excitatory postsynaptic potentials followed by inhibitory postsynaptic potentials. The excitatory postsynaptic potentials had a brief time-course while the inhibitory postsynaptic potentials had a prolonged time-course that enabled their temporal summation. Thus, the net effect of pressure-sensitive neuron stimulation on the annulus erector neurons was inhibitory. Both phases of the response were mediated by chemical transmission; the excitatory postsynaptic potentials were transmitted via a monosynaptic pathway, and the inhibitory postsynaptic potentials via a polysynaptic one. The pattern of expression of this dual response depended on the field of innervation of the sensory neuron and it was under the influence of cell 151, a non-spiking interneuron, that could regulate the expression of the hyperpolarization. The interaction between pressure-sensitive neurons and annulus erector neuron reveals how sensory specificity, connectivity pattern and regulatory elements interplay in a specific sensory-motor network.

Long-lasting depolarization of leech neurons mediated by receptors with a nicotinic binding site

The serotonergic Retzius neurons of the leech midbody ganglia respond in a complex manner to pressure pulses of acetylcholine (ACh) applied onto their soma with a fast depolarization followed by a slower hyperpolarization and an additional delayed long-lasting depolarization. The delayed depolarization is the subject of the present study. The delayed depolarization could be elicited by long (>1 s) ACh pressure pulses or by short pulses (10 ms) of carbachol, nicotine and DMPP, but not by muscarinic agonists. It was inhibited by bath application of nicotine (10-100 micromol l-1), strychnine (100 micromol l-1) and atropine (10-100 micromol l-1). Nicotinic antagonists that blocked the fast depolarization and the slow hyperpolarization (100 micromol l-1 mecamylamine and d-tubocurarine) did not affect the delayed depolarization induced by carbachol. Partial replacement of the extracellular Na+ by glucamine caused a decrease in the amplitude of the response and a shift of its reversal potential to more negative values. Carbachol pulses applied to Retzius neurons of the ganglia innervating the reproductive segments elicited delayed depolarizations of much smaller amplitude than the ones recorded in Retzius neurons from standard segments. The delayed depolarization could be elicited by the application of short agonist pulses onto different loci over the surface of the ganglion, at a distance from the soma. Isolated cultured Retzius neurons did not exhibit the delayed depolarization although they readily expressed the earlier phases of the complex cholinergic response. Carbachol pulses applied to the soma of other neurons in the leech ganglion produced a variety of specific responses.The results suggest that the delayed depolarization was produced by the activation of a cationic conductance mediated by receptors with a pharmacological profile similar to that of the 9 nicotinic receptors and was not a byproduct of the early phases of the cholinergic response. The response seemed to be initiated in the extensive neuropilar processes of the Retzius cell, enabling a persistent excitatory signal.

Basal acetylcholine release in leech ganglia depolarizes neurons through receptors with a nicotinic binding site

The response of Retzius neurons, the main neuronal source of serotonin in the leech nervous system, to cholinergic agonists has been extensively investigated. In this study, we analyzed the effects of inhibiting the acetylcholinesterase (AChE) activity in the leech midbody ganglion on the electrophysiological activity of the Retzius neurons. Bath application of neostigmine and physostigmine (0.1-100 &mgr;mol l-1) produced, after a delay, a strong depolarization of the Retzius neurons with a dose-dependent amplitude and latency. The amplitude of this depolarization increased as the extracellular level of Ca2+ increased and decreased as the extracellular level of Ca2+ decreased. The response to neostigmine and physostigmine was inhibited by curare (100 &mgr;mol l-1), nicotine (10 &mgr;mol l-1), atropine (100 &mgr;mol l-1) and strychnine (100 &mgr;mol l-1), but was not affected by mecamylamine (100 &mgr;mol l-1) or hexamethonium (100 &mgr;mol l-1). Superfusion with solutions containing 100 &mgr;mol l-1 strychnine or atropine produced a progressive hyperpolarization of the Retzius neurons, while superfusion with 100 &mgr;mol l-1 curare did not. The hyperpolarization induced by atropine was inhibited in the presence of curare. Other neurons in the ganglion showed distinctive responses to the AChE inhibitors that were coincident with their responses to cholinergic agonists. The results suggest the existence of a basal level of acetylcholine (ACh) release in the leech ganglion that is powerfully counteracted by endogenous AChE activity. Under control conditions, this basal release appears to be sufficient to generate an ACh tonus that regulates the membrane potential of Retzius neurons. Since these neurons can support a sustained firing rate, which is dependent on the membrane potential, the results presented in this report suggest that the basal ACh tonus regulates the output of these neuromodulatory serotonergic neurons.

Long-lasting depolarization of leech neurons mediated by receptors with a nicotinic binding site.

The serotonergic Retzius neurons of the leech midbody ganglia respond in a complex manner to pressure pulses of acetylcholine (ACh) applied onto their soma with a fast depolarization followed by a slower hyperpolarization and an additional delayed long-lasting depolarization. The delayed depolarization is the subject of the present study. The delayed depolarization could be elicited by long (> 1 s) ACh pressure pulses or by short pulses (10 ms) of carbachol, nicotine and DMPP, but not by muscarinic agonists. It was inhibited by bath application of nicotine (10-100 mumol l-1), strychnine (100 mumol l-1) and atropine (10-100 mumol l-1). Nicotinic antagonists that blocked the fast depolarization and the slow hyperpolarization (100 mumol l-1 mecamylamine and d-tubocurarine) did not affect the delayed depolarization induced by carbachol. Partial replacement of the extracellular Na+ by glucamine caused a decrease in the amplitude of the response and a shift of its reversal potential to more negative values. Carbachol pulses applied to Retzius neurons of the ganglia innervating the reproductive segments elicited delayed depolarizations of much smaller amplitude than the ones recorded in Retzius neurons from standard segments. The delayed depolarization could be elicited by the application of short agonist pulses onto different loci over the surface of the ganglion, at a distance from the soma. Isolated cultured Retzius neurons did not exhibit the delayed depolarization although they readily expressed the earlier phases of the complex cholinergic response. Carbachol pulses applied to the soma of other neurons in the leech ganglion produced a variety of specific responses. The results suggest that the delayed depolarization was produced by the activation of a cationic conductance mediated by receptors with a pharmacological profile similar to that of the alpha 9 nicotinic receptors and was not a byproduct of the early phases of the cholinergic response. The response seemed to be initiated in the extensive neuropilar processes of the Retzius cell, enabling a persistent excitatory signal.

Basal acetylcholine release in leech ganglia depolarizes neurons through receptors with a nicotinic binding site.

The response of Retzius neurons, the main neuronal source of serotonin in the leech nervous system, to cholinergic agonists has been extensively investigated. In this study, we analyzed the effects of inhibiting the acetylcholinesterase (AChE) activity in the leech midbody ganglion on the electrophysiological activity of the Retzius neurons. Bath application of neostigmine and physostigmine (0.1-100 mumol l-1) produced, after a delay, a strong depolarization of the Retzius neurons with a dose-dependent amplitude and latency. The amplitude of this depolarization increased as the extracellular level of Ca2+ increased and decreased as the extracellular level of Ca2+ decreased. The response to neostigmine and physostigmine was inhibited by curare (100 mumol l-1), nicotine (10 mumol l-1), atropine (100 mumol l-1) and strychnine (100 mumol l-1), but was not affected by mecamylamine (100 mumol l-1) or hexamethonium (100 mumol l-1). Superfusion with solutions containing 100 mumol l-1 strychnine or atropine produced a progressive hyperpolarization of the Retzius neurons, while superfusion with 100 mumol l-1 curare did not. The hyperpolarization induced by atropine was inhibited in the presence of curare. Other neurons in the ganglion showed distinctive responses to the AChE inhibitors that were coincident with their responses to cholinergic agonists. The results suggest the existence of a basal level of acetylcholine (ACh) release in the leech ganglion that is powerfully counteracted by endogenous AChE activity. Under control conditions, this basal release appears to be sufficient to generate an ACh tonus that regulates the membrane potential of Retzius neurons. Since these neurons can support a sustained firing rate, which is dependent on the membrane potential, the results presented in this report suggest that the basal ACh tonus regulates the output of these neuromodulatory serotonergic neurons.

Target tissues affect cellular properties of cocultured leech Retzius neurons.

The development of many neurons, including the Retzius (Rz) neurons of the medicinal leech, is shaped in part by interactions with other cells in the environment. To explore the nature of the interaction between growing Rz processes and potential target tissues, adult Rz neurons were cultured directly in contact with some of the tissues that normally serve as their targets in vivo. The morphology of the regenerated processes of these neurons varied depending upon the identity of the target tissues, but other cellular properties remained unchanged. In particular, although during normal development contact with peripheral targets determines the sign of Rz neurons' response to acetylcholine (ACh) applied to the soma, these cultured neurons maintained their original response to ACh even after as long as 2 weeks in culture on novel targets. Hence, some features of cultured adult Rz neurons varied depending upon the conditions, whereas other features remained fixed.

Widespread mechanosensory activation of the serotonergic system of the medicinal leech.

The serotonergic system of the medicinal leech comprises a small number of iterated, identified neurons, of which the Retzius (Rz) neurons are major components. Activity in pressure mechanosensory (P) cells sufficient to elicit locomotory and defensive behaviors also excites Rz neurons. We characterized the interactions between P and Rz neurons within the ganglion and at different distances along the nerve cord. 2. Within a ganglion 1) P cells excited both Rz neurons, electrically close to the site of electrical coupling between the Rz neurons; 2) each of the four P cells had similar effects on the Rz neurons; and 3) homologous contralateral P cells shared interneuronal pathways. These data show that P cells provide nearly identical bilateral information onto Rz neurons. 3. Along the nerve cord 1) every P cell excited Rz neurons in ganglia anterior and posterior to the site of stimulation; 2) the signal was carried the entire length of the nerve cord along interneuronal pathways with similar overall (but regionally different) conduction velocities in the two directions; 3) the amplitude of the Rz responses was smaller as the distance to the activated P cell increased; 4) the rate of change of the amplitude along the cord was larger when the signal traveled from front-to-back than in the opposite direction. 4. These data shows that mechanosensory input from any segment could excite Rz neurons along the cord, in proportion to the intensity of the stimulus.

Segment-specific modulation of the electrophysiological activity of leech Retzius neurons by acetylcholine.

1. The acetylcholine responses of Retzius neurons were electrophysiologically and pharmacologically characterized in situ and in culture. Single-electrode voltage-clamp was used to record currents from leech Retzius neurons from standard segments [Rz(X)] and from reproductive segments [Rz(5,6)]. 2. A 1 s pressure pulse of acetylcholine (ACh) produced a fast inward current followed by a slower outward current in Rz(X) neurons, whereas it produced only an outward current in Rz(5,6) neurons. These segment-specific responses were maintained when the two types of Retzius neurons were isolated in culture for up to 12 days. 3. The inward current of Rz(X) reversed at around -25 mV and was partially carried by Na+. This cationic current desensitized rapidly. The outward current of Rz(X) and Rz(5,6) neurons reversed at around -65 mV and was carried by Cl-. This anionic current desensitized very slowly upon prolonged applications of ACh. 4. The expression of the ACh-induced outward current in Rz(X) was season-dependent and was recorded in a larger proportion of Rz(X) neurons during the summer than during the winter. The expression of the ACh-induced outward current in Rz(5,6) did not show any seasonal pattern. 5. The fast inward current of Rz(X) was also elicited by nicotine; it was blocked by d-tubocurarine, hexamethonium and mecamylamine, but was not affected by alpha-bungarotoxin. The outward current of Rz(X) and Rz(5,6) was also elicited by nicotine and by 4-[N-(3-chlorophenyl)carbamoxyloxy]2-butynyltrimethylammonium chloride (a muscarinic agonist); it was blocked by d-tubocurarine and by alpha-bungarotoxin, but it was not affected by hexamethonium or mecamylamine. 6. The results show that the serotonergic Retzius neurons of the leech could be tonically inhibited by ACh. In addition, the Retzius neurons from standard segments could also be phasically excited by ACh. The receptors responsible for the excitation fit into the classification of neuronal nicotinic receptors, whereas the receptors mediating the inhibition are closer in type to the muscular nicotinic receptor.

Developmental regulation of segment-specific cholinergic receptors on Retzius neurons in the medicinal leech.

Retzius (Rz) neurons in the midbody ganglia of medicinal leeches responded to ACh, applied to their somata, in a manner that depended upon the neuron's segmental location: Rz neurons in ganglia from midbody segments 5 and 6 [Rz(5,6)] hyperpolarized, whereas Rz neurons from all other segments [Rz(X)] depolarized. Midbody segments 5 and 6 are notable because they contain the male and female reproductive organs. Both types of Rz neurons responded to ACh in a complex way, but the initial phase of each response appeared to be nicotinic because nicotinic agonists evoked the responses and nicotinic antagonists blocked them. The reversal potentials of the responses and the effects of changing the internal and external Cl- concentration indicated that the hyperpolarizing response of Rz(5,6) neurons depended upon Cl- whereas the depolarizing response of Rz(X) neurons did not. The segmentally characteristic responses of Rz neurons arose during embryonic development. Removing the reproductive ducts [the peripheral targets of Rz(5,6)] early in embryogenesis caused the Rz(5,6) neurons to depolarize in response to ACh rather than to hyperpolarize. This result indicates that development of the characteristic response of Rz neurons to ACh is strongly influenced by interactions between the neurons and their appropriate target tissues.

Effect of omega-conotoxin GVIA on neurotransmitter release at the mouse neuromuscular junction.

The effect of omega-conotoxin GVIA (omega-CgTx) was studied on spontaneous, K(+)-induced and electrically evoked neurotransmitter release at the neuromuscular junction of mouse diaphragm. omega-CgTx decreased the frequency and amplitude of basal and K(+)-induced miniature end plate potentials. This effect was abolished by raising the extracellular Ca2+ concentration. omega-CgTx had no effect on the quantal content of the electrically evoked release in this preparation.

Ca2+ role on the effect of phorbol esters on the spontaneous quantal release of neurotransmitter at the mouse neuromuscular junction.

The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the release process was studied in presence of different extracellular Ca2+ concentrations, in the mouse phrenic-diaphragm preparation. Hemidiaphragms were incubated for 2 h at room temperature in the presence or absence of TPA. TPA increased the basal frequency of miniature end plate potentials (mepp's) in a dose-dependent manner, resulting in a maximal increase of 280% at a concentration of 0.5 microM. An inverse relationship between extracellular Ca2+ concentration and TPA effect was observed: at high extracellular concentrations of Ca2+ the action of TPA decreased significatively, while at low Ca2+ concentrations the effect of TPA was remarkably augmented. The highest effect of TPA was obtained when tested in a calcium-free medium. TPA also increased mepp frequency stimulated by 10 mM K+. As at basal conditions, the effect of TPA was higher at lower concentrations of extracellular calcium. The results suggest that the effect of stimulation of PKC on neurotransmitter release at the mice neuromuscular junction is not exerted at the level of calcium influx to the nerve terminal. Moreover the action of calcium and TPA seems to be superimposed. The effect of K+ on neurotransmitter release could be explained not only by depolarization of the nerve terminal but by increasing the pool of activable PKC.

Electrical properties of normal, denervated and organ-cultured slow fibres of toad cruralis muscles.

Two types of fibres were characterized in the cruralis muscle of the toad using electrophysiological techniques: the slow and the fast fibres. Five to ten slow fibres were easily identified on the inner face of this muscle. The cruralis slow fibres developed the ability to produce action potentials 40 days after the sciatic nerve was transected at the hip level, while the slow fibres of the pyriformis muscle showed a latent period of 17 days after the same surgical treatment. However, when in addition to this procedure the nerve was transected at the point where it enters the muscle but without damaging the slow fibres, the latency was about 20 days. The slow fibres of the cruralis muscle maintained in organ culture developed the ability to produce action potentials in 24 days. During the winter the slow fibres of in vivo denervated cruralis and pyriformis muscles did not develop the ability to produce regenerative responses. More-over organ-cultured cruralis muscles taken from winter toads showed this same inability. These results further support the idea that the excitability of slow fibres is under the control of a neural factor rather than of activity. The seasonal dependence points to the fact that the metabolic state of the muscle is of crucial importance in determining the development of excitability of slow fibres.