Coactivation of motoneurons regulated by a network combining electrical and chemical synapses

Abstract

Electrical transmission among neurons has been considered a mechanism to synchronize neuronal activity, and rectification provides a mechanism to confine the flow of signals among the connected neurons. The question is how this type of transmission operates within complex neuronal networks. In the leech, the neurons located in position 151 of the midbody ganglion map are connected to virtually every motoneuron via rectifying electrical synapses that pass negative current to the motoneurons. These are nonspiking neurons, and here we have labeled them NS neurons. The goal of this investigation has been to assess their role in regulating motor activity and how rectifying electrical synapses contribute to the function of motor networks. The coupling between NS neurons and motoneurons was voltage sensitive: it increased as motoneurons were depolarized. In addition, excitation of motoneurons evoked hyperpolarizing synaptic responses in NS neurons, the amplitude of which depended on the membrane potential of the latter and on the motoneuron firing frequency. This hyperpolarization was mediated by chemical transmission through an interneuronal layer that spanned the nerve cord. These interactions established a feedback loop between NS and motoneurons that was regulated by the membrane potential of NS. This mechanism was responsible for the uncoupling between otherwise electrically coupled motoneurons. In this way, the NS neurons can act as "electrical neuromodulators," modifying the interaction of other neurons, depending on the activity of the system as a whole.

Fig. 1.

Scheme of the divergent connections between the NS neurons and the motoneurons. The scheme represents a circuit showing the divergent path from the bilateral pair of NS neurons (black) to the excitatory motoneurons CV, AE, cell 3, and L (gray) within a single ganglion. The NS cells are coupled to each other through nonrectifying electrical synapses and to the motoneurons through rectifying electrical synapses.CV, Circular ventral excitor; AE, annulus erector; 3, cell 3 dorsal longitudinal excitor;L, longitudinal excitor.

Fig. 2.

Simultaneous recordings from pairs of NS neurons within a ganglion. Ai, Representative simultaneous recordings of the spontaneous activity of both NS neurons in a single ganglion. Aii, Average cross-correlogram. The cross-correlograms (bin size 0.5 msec) of six different pairs of traces were averaged. The dotted lines represent the 95% confidence interval for the mean cross-correlation index.Bi, Representative simultaneous recordings of the synaptic responses of both NS neurons, evoked by a train of action potentials (15 Hz) elicited in a P cell in a single ganglion. The scheme on top represents a ganglion and the recording configuration. NSi and NSc denote NS cells ipsilateral and contralateral to the stimulated P cell, respectively. Bii, Average of three cross-correlograms (bin size 0.1 msec). The dotted lines represent the 95% confidence interval for the mean cross-correlation index.

Fig. 3.

Coupling between pairs of NS neurons as a function of the membrane potential. A, Representative recordings showing the responses of a pair of NS neurons to a current step injected into one of them (represented by the square step line on top). We define the injected NS neuron as presynaptic (top trace) and the second NS neuron as postsynaptic (bottom trace). The membrane potential at which the cells were set is indicated on the left of each trace. B, The graph displays the coupling coefficient between pairs of NS neurons as a function of the membrane potential of the presynaptic neuron. The presynaptic cell was shifted to different membrane potentials, whereas the postsynaptic NS neuron was set at −50 mV. The symbols and error bars indicate mean and SEM, respectively (n = 7). Statistical analysis used two-factor ANOVA with repeated measures.

Fig. 4.

Coupling between NS neuron and CV motoneuron as a function of the transjunctional potential. A, Representative recordings showing the responses of an NS neuron and a CV motoneuron to a hyperpolarizing square pulse (represented by thesquare step line on top) injected in the NS neuron. The membrane potential of the NS neuron was set at −50 mV, whereas the membrane potential of the CV motoneuron was set at −80 mV (Ai) or −20 mV (Aii). B, The graph shows the coupling coefficient between CV and NS neurons (●) and the input resistance of the CV motoneurons (○) as a function of the membrane potential of the CV motoneuron (top axis) and of the difference in membrane potential between the NS neurons and the CV motoneurons (bottom axis), measured at the somata (Vm_NS −Vm_CV).^§ p < 0.01 (compared with each one of the other data points of the same curve); *p < 0.01 and **p < 0.001 (compared with the value obtained at no potential difference between somata). C, The graph shows the coupling coefficient between the CV motoneurons and the NS neurons (●) and the input resistance of the NS neurons (○) as a function of the membrane potential of the NS neuron (top axis) and of the difference in membrane potential between the CV motoneurons and the NS neurons (bottom axis), measured at the somata (Vm_NS −Vm_CV). *p < 0.01 (compared with the value obtained at a membrane potential of −80 mV). The coupling coefficient was calculated as the amplitude of the response displayed by the motoneuron (postsynaptic) over that displayed by the NS neuron (presynaptic). The symbols and error bars indicate mean and SEM, respectively (n= 15, for each case). Statistical analysis used two-factor ANOVA with repeated measures.

Fig. 5.

Responses of NS neurons to motoneuron excitation.A, The two bottom traces show representative recordings of the simultaneous responses of both NS neurons within a single ganglion to the stimulation of a CV motoneuron with a square current pulse (square step line ontop). The top trace shows the recording of the CV motoneuron during the pulse injection. The small action potentials recorded in the soma reflect the passive propagation of fully developed action potentials initiated at an electrically distant site (Stuart, 1970). B, Average cross-correlogram. The cross-correlograms (bin size 0.2 msec) of 11 different pairs of traces were averaged. The broken lines represent the 95% confidence interval for the mean cross-correlation index.

Fig. 6.

Responses of NS neurons as a function of the spike frequency in the motoneuron. A, Representative recordings of an NS neuron to the activation of a CV motoneuron. Thetwo top traces correspond to a CV motoneuron, which was induced to fire at two different frequencies (indicated under thetraces) by the application of square current pulses (indicated by the square step lines ontop). The superimposed traces on thebottom correspond to the respective responses of the NS neuron. Black and gray traces indicate the corresponding recording pairings. B, Amplitude of the NS response as a function of the spike frequency of the motoneurons, stimulated with current steps of 0.5, 1, 1.5, and 2 nA. Firing frequencies were normalized to the one observed in response to the smallest current step (0.5 nA). Open symbolsrepresent five CV motoneurons; filled symbols represent four AE motoneurons. The curves are weighted fittings using the locally weighted least squared error method performed with Kaleidagraph.Broken line indicates CV data; solid lineindicates AE data.

Fig. 7.

Responses of NS neurons to the activation of motoneurons at different membrane potentials of NS. A, Representative recordings of an NS neuron, set at different membrane potentials (indicated on the left) as an AE motoneuron (top trace) was stimulated with a square step pulse. Only one of the motoneuron traces is shown displaying the activity during the stimulation period. The inset shows the fragment of the recording indicated by the dotted rectangle in an expanded temporal scale. Manipulation of the NS membrane potential, within the studied range, did not affect the spike frequency of the motoneuron. B, The graph shows the amplitude of the responses of the NS neurons to the stimulation of AE (n = 4) or CV (n = 4) motoneurons as a function of the membrane potential of the NS neuron. The stimulation protocol was like the one presented inA. The amplitude was measured only in those cases in which the hyperpolarization had its onset straight from baseline. Thesymbols and error bars indicate mean and SEM, respectively.

Fig. 8.

Responses of NS neurons to the excitation of motoneurons in a high divalents solution. A, Representative recordings showing the response of an NS neuron (bottom trace) to the stimulation of a CV motoneuron (top trace) during perfusion of the isolated ganglion with normal saline. The motoneuron trace displays the activity during the stimulus (+2 nA square current step). The insetshows the fragment of the recording indicated by the dotted rectangle in an expanded temporal scale. B, The same as in A after the ganglion has been perfused for 5 min with a solution containing 10 mmMg²⁺/10 mm Ca²⁺. It was always possible to recover the response after a 10 min washout with normal saline (n = 5). C, Superposition of the trace showed in Adisplaying the NS response in normal solution (black) and the “chemically mediated” trace(gray). The latter was obtained after subtracting the trace in A from the trace inB and represents the response mediated by the chemical transmission without the component caused by the electrical transmission.

Fig. 9.

Responses of NS neurons to the excitation of motoneurons in local and adjacent ganglia. Recordings of the responses of NS neurons to the stimulation of different motoneurons, performed in isolated three-ganglia chains (typically from midbody ganglion M7 to M9 or M10 to M12). The notation on the left indicates the identity of the recorded neurons, where the numberbetween brackets designates the ganglion number.A, Representative responses of an NS neuron to the stimulation of a local AE motoneuron [NS(8)–AE(8)] and to an AE motoneuron located in the adjacent posterior ganglion [NS(8)–AE(9)]. The two AE motoneurons were given identical stimuli (+3 nA square current step), and their responses were highly similar. The top trace shows the activity of the posterior AE [AE(9)] motoneuron during the stimulation period. The inset shows the fragment of the recording indicated by the dotted rectangle in an expanded temporal scale. B, Representative recordings showing the response of an NS neuron to the stimulation of a cell 3 in the posterior ganglion. C, Representative recordings showing the response of an NS neuron to the stimulation of a cell 1 in the posterior ganglion.

Fig. 10.

NS neurons regulate the coupling between motoneurons. Paired recordings of an NS neuron and a CV motoneuron during the stimulation of a local AE motoneuron with a square current step. Only the part of the recording corresponding to the stimulus is shown for the AE motoneuron. The inset shows the fragment of the recording indicated by the dotted rectangle in an expanded temporal scale. A, Representative recordings in normal saline as the membrane potential of the NS neuron was set at −80 mV (Ai) or at −20 mV (Aii). The membrane potential of the CV motoneuron was manipulated by injecting DC current to obtain a similar spontaneous firing rate in both conditions. B, Experiments performed in a high divalents solution (10 mmMg²⁺/10 mm Ca²⁺) as the membrane potential of the NS neuron was set at −80 mV (Bi) or −20 mV (Bii). Thegraphs show the change in frequency of the CV motoneuron (white columns) and the time integral (area) of the response of the NS neuron (striped columns) for experiments performed in normal (Aiii) and high divalents solution (Biii). The increase in frequency of the CV motoneuron during the injection of current in the AE motoneuron was measured as fp/fo, where fp is the firing frequency during the pulse and fo is the basal firing frequency, measured for 13 sec before the stimulation step. The time integral was measured during a period of 4 sec from the beginning of the pulse. The columns and error bars indicate mean and SEM, respectively (n = 3 for eachcolumn). The magnitude of both parameters is expressed using the same y-scale, with the appropriate units specified in column references. *p < 0.01 and **p < 0.001 (compared with the corresponding basal frequency); ^§ p < 0.01 (compared with the value at −80 mV). Statistical analysis used ttests

Fig. 11.

The CV and AE motoneurons are electrically coupled. Paired recordings of an AE and a CV motoneuron showing the effect of injecting a square current step in one of the neurons, as the ganglion was bathed in a solution with a high Mg²⁺/Ca²⁺ ratio.A, The AE motoneuron was stimulated with a + 3nA pulse (top panel) or a −3 nA pulse (bottom panel). B, The CV motoneuron was stimulated with a +3 nA pulse (top panel) or a −3 nA pulse (bottom panel). Qualitatively similar recordings were obtained for another five pairs of AE and CV neurons.

Fig. 12.

Scheme of the convergent connections between the NS neurons and the motoneurons. The scheme represents a circuit within a single ganglion. The NS neurons (black) are coupled to the motoneurons through rectifying connections, and the AE and CV motoneurons (dark gray) are connected between themselves through nonrectifying junctions. An interneuronal layer (light gray), shown as a single element that spans through the ganglion, along the anteroposterior axis receives excitatory input from the motoneurons and transmits hyperpolarizing signals to the NS neurons. According to this model, these synaptic sites are electrically close to the NS neuron–motoneuron junctions, and thus the hyperpolarizing synaptic potentials could pass to the motoneurons, counteracting the concomitant excitatory effect transmitted through the junctions between the motoneurons.