Transgenesis enables mapping of segmental ganglia in the leech Helobdella austinensis.

The analysis of how neural circuits function in individuals and change during evolution is simplified by the existence of neurons identified as homologous within and across species. Invertebrates, including leeches, have been used for these purposes in part because their nervous systems comprise a high proportion of identified neurons, but technical limitations make it challenging to assess the full extent to which assumptions of stereotypy hold true. Here, we introduce Minos plasmid-mediated transgenesis as a tool for introducing transgenes into the embryos of the leech Helobdella austinensis (Spiralia; Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae). We identified an enhancer driving pan-neuronal expression of markers, including histone2B:mCherry, which allowed us to enumerate neurons in segmental ganglia. Unexpectedly, we found that the segmental ganglia of adult transgenic H. austinensis contain fewer and more variable numbers of neurons than in previously examined leech species.

Phase-Specific Motor Efference during a Rhythmic Motor Pattern.

Neuronal circuits that control motor behaviors orchestrate multiple tasks, including the inhibition of self-generated sensory signals. In the hermaphroditic leech, T and P mechanosensory neurons respond to light touch and pressure on the skin, respectively. We show that the low threshold T cells were also sensitive to topological changes of the animal surface, caused by contraction of the muscles that erect the skin annuli. P cells were unresponsive to this movement. Annuli erection is part of the contraction phase of crawling, a leech locomotive behavior. In isolated ganglia, T cells showed phase-dependent IPSPs during dopamine-induced fictive crawling, whereas P cells were unaffected. The timing and magnitude of the T-IPSPs were highly correlated with the activity of the motoneurons excited during the contraction phase. Together, the results suggest that the central network responsible for crawling sends a reafferent signal onto the T cells, concomitant with the signal to the motoneurons. This reafference is specifically targeted at the sensory neurons that are affected by the movements; and it is behaviorally relevant as excitation of T cells affected the rhythmic motor pattern, probably acting upon the rhythmogenic circuit. Corollary discharge is a highly conserved function of motor systems throughout evolution, and we provide clear evidence of the specificity of its targets and timing and of the benefit of counteracting self-generated sensory input.SIGNIFICANCE STATEMENT Neuronal circuits that control motor behaviors orchestrate multiple tasks, including inhibition of sensory signals originated by the animal movement, a phenomenon known as corollary discharge. Leeches crawl on solid surfaces through a sequence of elongation and contraction movements. During the contraction, the skin topology changes, affecting a subpopulation of mechanosensory receptors, T (touch) neurons, but not P (pressure) sensory neurons. In the isolated nervous system, T neurons were inhibited during the contraction but not during the elongation phase, whereas P cells were unaffected throughout crawling. Excitation of T cells during the contraction phase temporarily disrupted the rhythmic pattern. Thus, corollary discharge was target (T vs P) and phase (contraction vs elongation) specific, and prevented self-generated signals to perturb motor behaviors.

A tale of two leeches: Toward the understanding of the evolution and development of behavioral neural circuits.

In the animal kingdom, behavioral traits encompass a broad spectrum of biological phenotypes that have critical roles in adaptive evolution, but an EvoDevo approach has not been broadly used to study behavior evolution. Here, we propose that, by integrating two leech model systems, each of which has already attained some success in its respective field, it is possible to take on behavioral traits with an EvoDevo approach. We first identify the developmental changes that may theoretically lead to behavioral evolution and explain why an EvoDevo study of behavior is challenging. Next, we discuss the pros and cons of the two leech model species, Hirudo, a classic model for invertebrate neurobiology, and Helobdella, an emerging model for clitellate developmental biology, as models for behavioral EvoDevo research. Given the limitations of each leech system, neither is particularly strong for behavioral EvoDevo. However, the two leech systems are complementary in their technical accessibilities, and they do exhibit some behavioral similarities and differences. By studying them in parallel and together with additional leech species such as Haementeria, it is possible to explore the different levels of behavioral development and evolution.

Feedback Signal from Motoneurons Influences a Rhythmic Pattern Generator.

Motoneurons are not mere output units of neuronal circuits that control motor behavior but participate in pattern generation. Research on the circuit that controls the crawling motor behavior in leeches indicated that motoneurons participate as modulators of this rhythmic motor pattern. Crawling results from successive bouts of elongation and contraction of the whole leech body. In the isolated segmental ganglia, dopamine can induce a rhythmic antiphasic activity of the motoneurons that control contraction (DE-3 motoneurons) and elongation (CV motoneurons). The study was performed in isolated ganglia where manipulation of the activity of specific motoneurons was performed in the course of fictive crawling (crawling). In this study, the membrane potential of CV was manipulated while crawling was monitored through the rhythmic activity of DE-3. Matching behavioral observations that show that elongation dominates the rhythmic pattern, the electrophysiological activity of CV motoneurons dominates the cycle. Brief excitation of CV motoneurons during crawling episodes resets the rhythmic activity of DE-3, indicating that CV feeds back to the rhythmic pattern generator. CV hyperpolarization accelerated the rhythm to an extent that depended on the magnitude of the cycle period, suggesting that CV exerted a positive feedback on the unit(s) of the pattern generator that controls the elongation phase. A simple computational model was implemented to test the consequences of such feedback. The simulations indicate that the duty cycle of CV depended on the strength of the positive feedback between CV and the pattern generator circuit.SIGNIFICANCE STATEMENT Rhythmic movements of animals are controlled by neuronal networks that have been conceived as hierarchical structures. At the basis of this hierarchy, we find the motoneurons, few neurons at the top control global aspects of the behavior (e.g., onset, duration); and within these two ends, specific neuronal circuits control the actual rhythmic pattern of movements. We have investigated whether motoneurons are limited to function as output units. Analysis of the network that controls crawling behavior in the leech has clearly indicated that motoneurons, in addition to controlling muscle activity, send signals to the pattern generator. Physiological and modeling studies on the role of specific motoneurons suggest that these feedback signals modulate the phase relationship of the rhythmic activity.

Graded boosting of synaptic signals by low-threshold voltage-activated calcium conductance.

Low-threshold voltage-activated calcium conductances (LT-VACCs) play a substantial role in shaping the electrophysiological attributes of neurites. We have investigated how these conductances affect synaptic integration in a premotor nonspiking (NS) neuron of the leech nervous system. These cells exhibit an extensive neuritic tree, do not fire Na(+)-dependent spikes, but express an LT-VACC that was sensitive to 250 µM Ni(2+) and 100 µM NNC 55-0396 (NNC). NS neurons responded to excitation of mechanosensory pressure neurons with depolarizing responses for which amplitude was a linear function of the presynaptic firing frequency. NNC decreased these synaptic responses and abolished the concomitant widespread Ca(2+) signals. Coherent with the interpretation that the LT-VACC amplified signals at the postsynaptic level, this conductance also amplified the responses of NS neurons to direct injection of sinusoidal current. Synaptic amplification thus is achieved via a positive feedback in which depolarizing signals activate an LT-VACC that, in turn, boosts these signals. The wide distribution of LT-VACC could support the active propagation of depolarizing signals, turning the complex NS neuritic tree into a relatively compact electrical compartment.

Wide propagation of graded signals in nonspiking neurons.

Signal processing in neuritic trees is ruled by the concerted action of passive and active membrane properties that, together, determine the degree of electrical compartmentalization of these trees. We analyzed how active properties modulate spatial propagation of graded signals in a pair of nonspiking (NS) neurons of the leech. NS neurons present a very extensive neuritic tree that mediates the interaction with all the excitatory motoneurons in leech ganglia. NS cells express voltage-activated Ca(2+) conductances (VACCs) that, under certain experimental conditions, evoke low-threshold spikes. We studied the distribution of calcium transients in NS neurons loaded with fluorescent calcium probes in response to low-threshold spikes, electrical depolarizing pulses, and synaptic inputs. The three types of stimuli evoked calcium transients of similar characteristics in the four main branches of the neuron. The magnitude of the calcium transients evoked by electrical pulses was a graded function of the change in NS membrane potential and depended on the baseline potential level. The underlying VACCs were partially inactivated at rest and strongly inactivated at -20 mV. Stimulation of mechanosensory pressure cells evoked calcium transients in NS neurons whose amplitude was a linear function of the amplitude of the postsynaptic response. The results evidenced that VACCs aid an efficient propagation of graded signals, turning the vast neuritic tree of NS cells into an electrically compact structure.

Motor neural networks in the leech.

Leeches display robust motor patterns and exhibit a relatively simple nervous system where neurons are unambiguously identified. This brief article focuses on Hirudo verbana and summarizes how research in this organism has contributed to insights in the field of motor control, where networks have been studied from population down to individual neuron perspectives.

Effect of a nonspiking neuron on motor patterns of the leech.

Premotor and motoneurons could play regulatory roles in motor control. We have investigated the role of a premotor nonspiking (NS) neuron of the leech nervous system in two locomotive patterns: swimming and crawling. The NS neuron is coupled through rectifying electrical junctions to all the excitatory motoneurons examined. In addition, activation of motoneurons evokes chemically mediated inhibitory responses in NS. During swimming and crawling, the NS membrane potential (Vm(NS)) oscillated phase locked to the motor output. Hyperpolarization or depolarization of NS had no effect on swimming, but hyperpolarization of NS slowed down the crawling activity and decreased the motoneuron firing frequency. Depolarization of NS increased the motoneuron activity, and, at stages where the crawling pattern was fading, depolarization of NS reinstated it. Future work should determine if NS is actually a member of the central pattern generator or a regulatory element.

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 Mg²âº 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 Mg²âº. In response to 5-s pulses, DE-3 exhibited a fast initial adaptation, a slow recovery and a very slow late adaptation. High Mg²âº abolished the initial high frequency. The frequency-voltage relationship for the rest of the response was highly similar in normal and in high Mg²âº saline. MN-L exhibited a minor initial adaptation and then fired steadily. High Mg²âº 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.

Intersegmental Interactions Give Rise to a Global Network.

Animal motor behaviors require the coordination of different body segments. Thus the activity of the networks that control each segment, which are distributed along the nerve cord, should be adequately matched in time. This temporal organization may depend on signals originated in the brain, the periphery or other segments. Here we evaluate the role of intersegmental interactions. Because of the relatively regular anatomy of leeches, the study of intersegmental coordination in these animals restricts the analysis to interactions among iterated units. We focused on crawling, a rhythmic locomotive behavior through which leeches move on solid ground. The motor pattern was studied ex vivo, in isolated ganglia and chains of three ganglia, and in vivo. Fictive crawling ex vivo (crawling) displayed rhythmic characteristics similar to those observed in vivo. Within the three-ganglion chains the motor output presented an anterior-posterior order, revealing the existence of a coordination mechanism that occurred in the absence of brain or peripheral signals. An experimental perturbation that reversibly abolished the motor pattern in isolated ganglia produced only a marginal effect on the motor activity recorded in three-ganglion chains. Therefore, the segmental central pattern generators present in each ganglion of the chain lost the autonomy observed in isolated ganglia, and constituted a global network that reduced the degrees of freedom of the system. However, the intersegmental phase lag in the three-ganglion chains was markedly longer than in vivo. This work suggests that intersegmental interactions operate as a backbone of correlated motor activity, but additional signals are required to enhance and speed coordination in the animal.

Premotor nonspiking neurons regulate coupling among motoneurons that innervate overlapping muscle fiber population.

Recent work indicated that co-activity of different motoneurons (MNs) in the leech can be regulated through a network that is centered on a pair of nonspiking (NS) neurons. Here, we investigate whether this effect generalizes to different types of MNs that display differential co-activity patterns in different motor behaviors: the dorsal longitudinal excitors DE-3 and the dorsal and ventral excitors MN-L. The data indicates that both motoneurons are coupled to the NS neurons through rectifying junctions that are activated when the motoneuron membrane potential becomes more negative than that of the NS, and that they exert an inhibitory synaptic potential on NS via a polysynaptic pathway. In addition, DE-3 and MN-L are linked by junctions that allow mutual excitation but the transmission of excitatory signals from MN-L to DE-3 depended on NS membrane potential. The results support the view that NS neurons can play a central role in orchestrating the co-activity of MNs during motor behaviors.

Functional contributions of electrical synapses in sensory and motor networks.

Intercellular interactions in the nervous system are mediated by two types of dedicated structural arrangements: electrical and chemical synapses. Several characteristics distinguish these two mechanisms of communication, such as speed, reliability and the fact that electrical synapses are, potentially, bidirectional. Given these properties, electrical synapses can subserve, in addition to synchrony, three main interrelated network functions: signal amplification, noise reduction and/or coincidence detection. Specific network motifs in sensory and motor systems of invertebrates and vertebrates illustrate how signal transmission through electrical junctions contributes to a complex processing of information.

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

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.

Gap junctions: their importance for the dynamics of neural circuits.

Electrical coupling through gap junctions constitutes a mode of signal transmission between neurons (electrical synaptic transmission). Originally discovered in invertebrates and in lower vertebrates, electrical synapses have recently been reported in immature and adult mammalian nervous systems. This has renewed the interest in understanding the role of electrical synapses in neural circuit function and signal processing. The present review focuses on the role of gap junctions in shaping the dynamics of neural networks by forming electrical synapses between neurons. Electrical synapses have been shown to be important elements in coincidence detection mechanisms and they can produce complex input-output functions when arranged in combination with chemical synapses. We postulate that these synapses may also be important in redefining neuronal compartments, associating anatomically distinct cellular structures into functional units. The original view of electrical synapses as static connecting elements in neural circuits has been revised and a considerable amount of evidence suggests that electrical synapses substantially affect the dynamics of neural circuits.

Recurrent inhibition in motor systems, a comparative analysis.

The review proposes a comparison between recurrent inhibition in motor systems of vertebrates and the leech nervous system, where a detailed cellular and functional analysis has been accomplished. A comparative study shows that recurrent inhibition is a conserved property in motor systems of phylogenetically distant species. Recurrent inhibition has been extensively characterized in the spinal cord of mammals, where Renshaw cells receive excitatory synaptic inputs from motoneurons (MNs) and, in turn, exert an inhibitory effect on the MNs. In the leech, a recurrent inhibitory circuit has been described, centered around a pair of nonspiking (NS) neurons. NS are linked to every excitatory MN through rectifying electrical junctions. And, in addition, the MNs are linked to the NS neurons through hyperpolarizing chemical synapses. Functional analysis of this leech circuit showed that heteronymous MNs in the leech are electrically coupled and this coupling is modulated by the membrane potential of NS neurons. Like Renshaw cells, the membrane potential of NS neurons oscillates in phase with rhythmic motor patterns. Functional analysis performed in the leech shows that NS influences the activity of MNs in the course of crawling suggesting that the recurrent inhibitory circuit modulates the motor performance.

Calcium spikes in a leech nonspiking neuron.

The NS neurons are nonspiking cells, present as pairs in each midbody ganglion of the leech nervous system, which display a very extensive arborization. They were shown to regulate the coactivation of motoneurons. Here we have investigated the electrophysiological properties of these neurons under the hypothesis that transmission along the extensive neurites requires the aid of voltage-dependent conductances. The results indicate that NS neurons respond to electrical stimulation with a spike-like event, which was not an all-or-none but rather a graded phenomenon that depended on the intensity and duration of the electrical stimulus. The spike-like response was activated at a membrane potential of approximately -50 mV; its amplitude was a logarithmic function of the extracellular Ca2+ concentration and was unaffected by a broad range of changes in the extracellular Na+ concentration; intracellular application of tetraethylammonium (TEA) caused a large increase in its amplitude and duration. These data indicate that NS neurons bear voltage-dependent low-threshold Ca2+ and TEA-sensitive K+ conductances that could contribute to shaping synaptic signals, or transmission along the extensive neuritic tree.

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.

Selective serotonin reuptake inhibitors induce spontaneous interneuronal activity in the leech nervous system.

Serotonin [5-hydroxytryptamine (5-HT)] is a conspicuous neuromodulator of sensory-motor networks that affects a variety of neurons at different levels of the network hierarchy. Because of its many possible targets, it has been difficult to obtain a comprehensive picture of how 5-HT achieves its final modulatory output on any given network. Our hypothesis is that the profile of 5-HT actions is dictated by its pattern of release from endogenous sites. We tested this hypothesis in the leech nervous system by means of a selective serotonin reuptake blocker (SSRI), fluoxetine. Fluoxetine evoked barrages of synaptic potentials in identified sensory, motor, and interneurons. This effect was mimicked by the tricyclic antidepressants imipramine and clomipramine, and by the SSRI citalopram, with relative efficacies that matched their known relative selectivities for the 5-HT transporter. The synaptic responses evoked by fluoxetine in different neurons were temporally correlated, suggesting that they had a common origin. The profile of the synaptic responses matched that expected from the activation of the mechanosensory pressure cells, known to act by polysynaptic pathways. The results suggest that endogenous 5-HT acted on cord spanning interneurons. On the other hand, bath-applied 5-HT evoked an effect different from that of the SSRI. Taken together, the results evidenced that the pattern of action of the monoamine is dictated by the spatial distribution of the 5-HT release sites.

Modulation of mechanosensory responses by motoneurons that regulate skin surface topology in the leech.

Central regulation of somatosensory signals has been extensively studied, but little is known about their regulation in the periphery. Given the widespread exposure of the skin sensory terminals to the environment, it is of interest to explore how somatosensory sensitivity is affected by changes in properties of the skin. In the leech, the annuli that subdivide the skin can be erected under the control of the annulus erector (AE) motoneurons. To analyze whether this surface change influences mechanosensory sensitivity, we studied the responses of low threshold mechanosensory T cells to mechanical stimulation of the skin as AE motoneurons were activated. In segments of the body wall connected to the corresponding ganglion and submerged in an aqueous environment, T cells responded to localized bubbling on the skin and to water flow parallel to its surface. Excitation of AE motoneurons diminished these responses in a way that depended on the motoneuron firing frequency. Video recordings established that the range of AE firing frequencies that produced effective annulus erection coincided with that influencing T cell responses. In isolated ganglia, AE firing had no effect on T cell excitability, suggesting that annulus erection diminished T cell responsiveness to mechanical input. Counteracting this effect, mechanosensory inputs inhibited AE motoneurons. However, because depolarization of AE cells caused a decrease in their input resistance, the more active the motoneuron, the less sensitive it became to inhibitory signals. Thus when brought to fire, AE motoneurons would stay "committed" to a high activity level, and this would limit sensory responsiveness to incoming mechanical signals.