Electrical transmission among neurons in the buccal ganglion of a mollusc, Navanax inermis

The opisthobranch mollusc, Navanax, is carnivorous and cannibalistic. Prey are swallowed whole by way of a sudden expansion of the pharynx. The buccal ganglion which controls this sucking action was isolated and bathed in seawater. Attention was focused upon 10 identifiable cells visible on the ganglion''s rostral side. Two cells were observed simultaneously, and each was penetrated with two glass microelectrodes, one for polarizing the membrane and the other for recording membrane potential variations. The coupling coefficients for direct current flow and action potentials of several identified cells were tabulated. Attenuation was essentially independent of the direction of current flow, but depended upon the relative size of the directly and indirectly polarized cells. The attenuation of subthreshold sinusoidally varying voltages increased with frequency above about 1 Hz. The coupling coefficient for spikes was lower than for DC due to greater high frequency attenuation. There is considerable similarity in the spontaneous PSP''s of all cells, which is not due to the electrical coupling but to input from a common source. The 10 cells were not chemically interconnected but some were electrically connected to interneurons which fed back chemically mediated PSP''s. The feedback can be negative or positive depending upon the membrane potential of the postsynaptic cell. We conclude that electrical coupling among the 10 cells plays a minor role in sudden pharyngeal contractions but that the dual electrical-chemical coupling with interneurons may be important in this respect.     

Initiation of swimming activity by trigger neurons in the leech subesophageal ganglion

The aim of this study was to identify neurons in the subesophageal ganglion of the medicinal leech which initiate swimming activity and to determine their output connections. We found two bilaterally symmetrical pairs of interneurons, Tr1 and Tr2, located in the first division of the subesophageal ganglion which initiate swimming activity in the isolated nervous system when depolarized with brief (1-3 s) current pulses. Tr1 and Tr2 are considered trigger neurons because elicited swimming episodes outlast the stimulus duration, and because the length of elicited swim episodes is nearly independent of the intensity with which Tr1 and Tr2 are stimulated. Tr1 and Tr2 have similar morphologies. The neurites of both cells cross contralaterally in the subesophageal ganglion, project posteriorly, and exit the subesophageal ganglion in the contralateral connective. The axons of Tr1 and Tr2 extend as far posterior as segmental ganglion 18 of the ventral nerve cord. Tr1 provides direct excitatory drive to three groups of segmental neurons which are capable of initiating swimming: swim-initiating interneurons (cells 204 and 205), serotonin-containing interneurons (cells 61 and 21), and the serotonergic Retzius cells. In addition, all Retzius cells in the subesophageal ganglion are excited directly by Tr1. These three groups of neurons are excited even if Tr1 stimulation is subthreshold for swim initiation. In contrast to Tr1, Tr2 stimulation evokes transient inhibition in swim-initiating and serotonin-containing interneurons, and has little immediate effect on Retzius cells. In addition, Tr2 indirectly inhibits several oscillator neurons, including cells 208, 33, and 60. When Tr1 is stimulated during a swimming episode the swim period decreases for several cycles, while stimulation of Tr2 during swimming episodes reliably resets the ongoing swimming rhythm. Our findings indicate that Tr1 and Tr2 are trigger neurons which initiate swimming activity by different pathways. These neurons also have functional interactions with the swim oscillator network since either Tr1 or Tr2 stimulation during swimming can modulate the ongoing swimming rhythm.     

Initiation of swimming activity by trigger neurons in the leech subesophageal ganglion

In papers I and II of this series, we described two pairs of interneurons, Tr1 and Tr2, in the leech subesophageal ganglion which can trigger swimming activity in the isolated central nervous system (CNS). In this paper, we describe sensory inputs to these trigger neurons from previously identified mechanosensory neurons. We found that: Weak mechanical stimulation (stroking) of a body wall flap attached to a segmental ganglion in an otherwise isolated CNS excites the contralateral Tr1 slightly. Strong mechanical stimulation (pinching) of a mid-body wall flap evokes a burst of impulses in the contralateral Tr1. For both means of stimulation the effects on the ipsilateral Tr1 and on the Tr2 cell pair were much weaker. Stroking a body wall flap attached to the head ganglion (supra- and subesophageal ganglia) in an otherwise isolated CNS excites both Tr1s and both Tr2s, although the effect is weaker for the Tr2s. Pinching strongly excites both trigger neurons bilaterally. Pressure and nociceptive mechanosensory neurons (P and N cells) in the subesophageal ganglion and the first segmental ganglion appear to make direct excitatory synapses with the contralateral Tr1 and Tr2. Mechanosensory interactions with the ipsilateral trigger neurons appear to be indirect. Functional inactivation of Tr1 by hyperpolarization does not prevent swim initiation either by weak mechanical stimulation of a body-wall flap or by intracellular stimulation of P cells.2+ We conclude that the trigger neurons, Tr1 and Tr2, provide an excitatory pathway by which mechanosensory stimulation can initiate leech swimming activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Frequency-dependent coupling between rhythmically active neurons in the leech.

The heart excitor (HE) cells, a set of rhythmically active motor neurons, drive the heartbeat of the medicinal leech. Their activity is gated by inhibitory input from a network of interneurons, but that influence may be modified locally by electrotonic coupling between the HE cells. In this paper I analyze that electrotonic coupling by applying direct current and alternating current signals, and compare the results with predictions based on linear cable theory. The electrotonic junction itself appears to be conventional, but because of the membrane properties of the HE cells, the coupling strength depends upon both the frequency and polarity of the signal and the phase of heartbeat cycle when the signal is applied.     

Data transmission between neurons

Summary By modulating sinusoidally the impulse frequency of the inhibitory input to the slowly adapting stretch receptor of Crustacea, the impulse frequency of the sensory neuron becomes sinusoidally modulated. The systems involved are linear for amplitudes of modulation up to 70% of the steady state frequency. The transfer function is given and some of its implications are discussed.     

Chronic pregabalin inhibits synaptic transmission between rat dorsal root ganglion and dorsal horn neurons in culture

In this study, we have examined the properties of synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons, placed in co-culture. We also examined the effect of the anti-hyperalgesic gabapentinoid drug pregabalin (PGB) at this pharmacologically relevant synapse. The main method used was electrophysiological recording of excitatory post synaptic currents (EPSCs) in DH neurons. Synaptic transmission between DRG and DH neurons was stimulated by capsaicin, which activates transient receptor potential vanilloid-1 (TRPV1) receptors on small diameter DRG neurons. Capsaicin (1 μM) application increased the frequency of EPSCs recorded in DH neurons in DRG-DH co-cultures, by about 3-fold, but had no effect on other measured properties of the EPSCs. There was also no effect of capsaicin in the absence of co-cultured DRGs. Application of PGB (100 μM) for 40-48 h caused a reduction in the capsaicin-induced increase in EPSC frequency by 57%. In contrast, brief preincubation of PGB had no significant effect on the capsaicin-induced increase in EPSC frequency. In conclusion, this study shows that PGB applied for 40-48 h, but not acute application inhibits excitatory synaptic transmission at DRG-DH synapses, in response to nociceptive stimulation, most likely by a presynaptic effect on neurotransmitter release from DRG presynaptic terminals.     

Chronic pregabalin inhibits synaptic transmission between rat dorsal root ganglion and dorsal horn neurons in culture

In this study, we have examined the properties of synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons, placed in co-culture. We also examined the effect of the anti-hyperalgesic gabapentinoid drug pregabalin (PGB) at this pharmacologically relevant synapse. The main method used was electrophysiological recording of excitatory post synaptic currents (EPSCs) in DH neurons. Synaptic transmission between DRG and DH neurons was stimulated by capsaicin, which activates transient receptor potential vanilloid-1 (TRPV1) receptors on small diameter DRG neurons. Capsaicin (1 μM) application increased the frequency of EPSCs recorded in DH neurons in DRG-DH co-cultures, by about 3-fold, but had no effect on other measured properties of the EPSCs. There was also no effect of capsaicin in the absence of co-cultured DRGs. Application of PGB (100 μM) for 40–48 h caused a reduction in the capsaicin-induced increase in EPSC frequency by 57%. In contrast, brief preincubation of PGB had no significant effect on the capsaicin-induced increase in EPSC frequency. In conclusion, this study shows that PGB applied for 40–48 h, but not acute application inhibits excitatory synaptic transmission at DRG-DH synapses, in response to nociceptive stimulation, most likely by a presynaptic effect on neurotransmitter release from DRG presynaptic terminals.     

Statistical independence and neural computation in the leech ganglion

 In this report, the input/output relations in an isolated ganglion of the leech Hirudo medicinalis were studied by simultaneously using six or eight suction pipettes and two intracellular electrodes. Sensory input was mimicked by eliciting action potentials in mechanosensory neurons with intracellular electrodes. The integrated neural output was measured by recording extracellular voltage signals with pipettes sucking the roots and the connectives. A single evoked action potential activated electrical activity in at least a dozen different neurons, some of which were identified. This electrical activity was characterized by a high degree of temporal and spatial variability. The action potentials of coactivated neurons, i.e. activated by the same mechanosensory neuron, did not show any significant pairwise correlation. Indeed, the analysis of evoked action potentials indicates clear statistical independence among coactivated neurons, presumably originating from the independence of synaptic transmission at distinct synapses. This statistical independence may be used to increase reliability when neuronal activity is averaged or pooled. It is suggested that statistical independence among coactivated neurons may be a usual property of distributed processing of neuronal networks and a basic feature of neural computation. Received: 20 September 1999 / Accepted in revised form: 3 March 2000     

Temporal correlation between neuronal tail ganglion activity and locomotion in the leech,Hirudo medicinalis

Intracellular and extracellular recordings were performed in the posterior ventral nerve cord of restrained crawling preparations of the medicinal leech,Hirudo medicinalis. Short-latency neuronal activities in the tail ganglion nerves correlated with different phases of crawling behavior. Eight neurons with characteristic activation patterns during crawling were identified morphologically and physiologically in the tail ganglia of 23 preparations. The axons of four of these neurons projected through posterior tail brain nerves; four ascending interneurons had projections in the connectives or in Faivre's nerve. These interneurons are suitable candidates for carrying information between the front end and the tail end of the animal to coordinate the behavioral components during a crawling step.     

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.     

Spatial-specific action of serotonin within the leech midbody ganglion

Serotonin is a conspicuous neuromodulator in the nervous system of many vertebrates and invertebrates. In previous experiments performed in the leech nervous system, we compared the effect of the amine released from endogenous sources [using selective serotonin reuptake inhibitors (SSRIs), e.g. fluoxetine] with that of bath-applied serotonin. The results suggested that the amine does not reach all its targets in a uniform way, but produces the activation of an interneuronal pathway that generated specific synaptic responses on different neurons. Taking into account that the release of the amine is often regulated at the presynaptic level, we have investigated whether autoreceptor antagonists mimic the SSRIs effect. We found that methiothepin (100 microM) produced similar effects than fluoxetine. To further test the hypothesis that endogenous serotonin produce its effect by acting locally at specific sites, we analyzed the effect of iontophoretic applications of serotonin. We found a site in the neuropil of the leech ganglia where serotonin application mimicked the effect of the SSRIs and the 5-HT antagonist. The results further support the view that the effect of serotonin exhibits a spatial specificity that can be relevant to understand its modulatory actions.     

Subsynaptic modulation of excitatory chemical transmission produced by metacerebral neurons in the mollusk buccal ganglion

The effects of stimulating neuron LMcl (which secretes serotonin from the axonal endings) on the response of buccal cells LBc2 and LBc3 to stimulation of synaptic (predominantly cholinergic) inputs from iron peripheral neurons were investigated inHelix pomatia. Stimulation of neuron LMc1 was found to modulate such response LBc2 and LBc3 producing an increase in the former and decrease in the latter. Results of pharmacological action show the modulation of excitatory chemical transmission produced by LMc1 in the buccal ganglion to be subsynaptic.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 4, pp. 539–546, July–August, 1989.     

Anatomical analysis of contacts between identified neurons that control heartbeat in the leech Hirudo medicinalis

Summary The rhythmic constriction of the heart tubes in the leech Hirudo medicinalis is controlled by an identified set of motor neurons (HE cells) and interneurons (HN cells) (reviewed by Calabrese and Peterson 1983). Electrophysiological recordings have indicated particular synaptic relationships among HE and HN cells. In the present study, the synaptic framework mediating the interactions among HE cells and HN cells was examined anatomically. Using light and electron microscopy of physiologically identified, HRP-injected cells, we have examined the zones of interaction and types of contacts between specific cells. HE cells, which have very fine, threadlike processes, interact with their contralateral homologues throughout most of the middle third of the ganglionic neuropil. When HE-cell neuntes come together, the apposed plasma membranes are rigidly parallel, separated by an intercellular gap of 6 nm, for up to 6 m. These specializations must form the structural basis for the strong electrical coupling observed (Peterson 1983) between HE-cell pairs. HE cells also emit from the main neurite a series of extremely fine processes that extend dorsally. These appear in the light microscope to contact processes of the ipsilateral HN cell of the same ganglion, and are also in a position to make contact with the axons of more anterior HN cells. The intraganglionic processes of HN cells, which are studded with large varicosities, ramify in part of the region of neuropil occupied by HE-cell processes, as well as more posteriorly. Contacts between HE and HN cells, which are known to be mostly inhibitory synaptic contacts, are seen in the electron microscope to be formed between medium-diameter HN processes, which are filled with clear round synaptic vesicles, and multiple fine tendrils of the HE cell that surround the HN process. Certain HN cells form reciprocal inhibitory synapses with their contralateral homologues. These contacts occur near the midline, sometimes in the major mass of neuropil and sometimes embedded in the extracellular material that ensheathes the neuropil. The contacts are between medium-and small-diameter profiles that are both filled with synaptic vesicles. Our findings indicate that various classes of physiological interactions among HE and HN cells are mediated by anatomically distinct types of contacts and, at least in some cases, are segregated from each other on the neuritic trees of the cells.     

Effects of transition metal ions on spontaneous electrical activity and chemical synaptic transmission of neurons in the medicinal leech

Leech neurons exposed to salines containing inorganic Ca²⁺-channel blockers generate rhythmic bursts of impulses. According to an earlier model, these blockers unmask persistent Na⁺ currents that generate plateau-like depolarizations, each triggering a burst of impulses. The resulting increase in intracellular Na⁺ activates an outward Na⁺/K⁺ pump current that contributes to burst termination. We tested this model by examining systematically the effects of six transition metal ions (Co²⁺, Ni²⁺, Mn²⁺, Cd²⁺, La³⁺, and Zn²⁺) on the electrical activity of neurons in isolated leech ganglia. Each ion induced bursting activity, but the amplitude, form, and persistence of bursting differed with the ion used and its concentration relative to Ca²⁺. All ions tested suppressed chemical synaptic transmission between identified motor neurons, consistent with block of voltage-dependent Ca²⁺ currents in these cells. In addition, a strong correlation between suppression of synaptic transmission and burst amplitudes was obtained. Finally, burst duration was increased and the rate of repolarization decreased in reduced K⁺ saline, as expected for pump-dependent repolarization. These results provide further support for the hypothesis that a novel form of oscillatory electrical activity driven by persistent Na⁺ currents and the Na⁺/K⁺ pump occurs in leech ganglia exposed to Ca²⁺-channel blockers. Accepted: 15 May 1997     

Nicotinic synapses formed between chick ciliary ganglion neurons in culture resemble those present on the neurons in vivo

We studied nicotinic synapses between chick ciliary ganglion neurons in culture to learn more about factors influencing their formation and receptor subtype dependence. After 4--8 days in culture, nearly all neurons displayed spontaneous excitatory postsynaptic currents (sEPSCs), which occurred at about 1 Hz. Neurons treated with tetrodotoxin displayed miniature EPSCs (mEPSCs), but these occurred at low frequency (0.1 Hz), indicating that most sEPSCs are actually impulse driven. The sEPSCs could be classified by decay kinetics as fast, slow, or biexponential and, reminiscent of the situation in vivo, were mediated by two major nicotinic acetylcholine receptor (AChR) subtypes. Fast sEPSCs were blocked by alpha-bungarotoxin (alpha Bgt), indicating dependence on alpha Bgt-AChRs, most of which are alpha 7 subunit homopentamers. Slow sEPSCs were unaffected by alpha Bgt, and were blocked instead by the alpha 3/beta 2-selective alpha-conotoxin-MII (alpha CTx-MII), indicating dependence on alpha 3*-AChRs, which lack alpha 7 and contain alpha 3 subunits. Biexponential sEPSCs were mediated by both alpha Bgt- and alpha 3*-AChRs because they had fast and slow components qualitatively similar to those comprising simple events, and these were reduced by alpha Bgt and blocked by alpha CTx-MII, respectively. Fluorescence labeling experiments revealed both alpha Bgt- and alpha 3*-AChR clusters on neuron somata and neurites. Colabeling with antisynaptic vesicle protein antibody suggested that some alpha 3*-AChR clusters, and a few alpha Bgt-AChR clusters are associated with synaptic sites, as is the case in vivo. These findings demonstrate the utility of ciliary ganglion neuron cultures for studying the regulation of nicotinic synapses, and suggest that mixed AChR subtype synapses characteristic of the neurons in vivo can form in the absence of normal inputs or targets.     

Electrical connection between two bursting neurons inHelix pomatia

In some preparations of the CNS ofHelix pomatia, two neurons with bursting activity may be present in the right parietal ganglion, where usually there is only one bursting neuron RPal. If electrical activity of these neurons is recorded simultaneously, fluctuations of membrane potential are almost completely synchronized. Artificial depolarization and hyperpolarization of the membrane of one neuron caused depolarization or hyperpolarization of the other neuron. During long-term recording of the activity of both neurons synchronous modulation of their bursting activity was observed. Modulating factor (a peptide fraction obtained from the water-soluble part of snail brain homogenate) led to potentiation of the bursting activity of both neurons. It is concluded from the results of these experiments that two bursting RPal neurons, connected electrically with one another, may exist in the snail nervous system. In cases when the parameters of pacemaker activity of these two neurons are closely similar, electrical connection guarantees synchronization of their bursting activity and ensures a common frequency of changes in their membrane potential.