Gap junction expression is required for normal chemical synapse formation.

Electrical and chemical synapses provide two distinct modes of direct communication between neurons, and the embryonic development of the two is typically not simultaneous. Instead, in both vertebrates and invertebrates, gap junction-based electrical synapses arise before chemical synaptogenesis, and the early circuits composed of gap junction-based electrical synapses resemble those produced later by chemical synapses. This developmental sequence from electrical to chemical synapses has led to the hypothesis that, in developing neuronal circuits, electrical junctions are necessary forerunners of chemical synapses. Up to now, it has been difficult to test this hypothesis directly, but we can identify individual neurons in the leech nervous system from before the time when synapses are first forming, so we could test the hypothesis. Using RNA interference, we transiently reduced gap junction expression in individual identified neurons during the 2-4 d when chemical synapses normally form. We found that the expected chemical synapses failed to form on schedule, and they were still missing months later when the nervous system was fully mature. We conclude that the formation of gap junctions between leech neurons is a necessary step in the formation of chemical synaptic junctions, confirming the predicted relation between electrical synapses and chemical synaptogenesis.

Alpha-conotoxin ImI disrupts central control of swimming in the medicinal leech.

Medicinal leeches (Hirudo spp.) swim using a metachronal, front-to-back undulation. The behavior is generated by central pattern generators (CPGs) distributed along the animal's midbody ganglia and is coordinated by both central and peripheral mechanisms. Here we report that a component of the venom of Conus imperialis, α-conotoxin ImI, known to block nicotinic acetyl-choline receptors in other species, disrupts swimming. Leeches injected with the toxin swam in circles with exaggerated dorsoventral bends and reduced forward velocity. Fictive swimming in isolated nerve cords was even more strongly disrupted, indicating that the toxin targets the CPGs and central coordination, while peripheral coordination partially rescues the behavior in intact animals.

A hormone-activated central pattern generator for courtship.

BACKGROUND: Medicinal leeches (Hirudo spp.) are simultaneous hermaphrodites. Mating occurs after a stereotyped twisting and oral exploration that result in the alignment of the male and/or female gonopores of one leech with the complementary gonopores of a partner. The neural basis of this behavior is presently unknown and currently impossible to study directly because electrophysiological recording techniques disrupt the behavior. RESULTS: Here we report that (Arg(8))-conopressin G and two other members of the oxytocin/vasopressin family of peptide hormones induce in Hirudo verbana a sequence of behaviors that closely mimic elements of spontaneous reproductive behavior. Through a series of progressively more reduced preparations, we show that one of these behaviors, a stereotyped twisting that is instrumental in aligning gonopores in preparation for copulation, is the product of a central pattern generator that consists of oscillators in ganglia M5 and M6 (the ganglia in the reproductive segments of the leech), and also in ganglion M4, which was not previously known to play a role in reproductive behavior. We find that the behavior is periodic, with a remarkably long cycle period of around five minutes, placing it among the slowest behavioral rhythms (other than diurnal and annual rhythms) yet described. CONCLUSION: These results establish the leech as a new model system for studying aspects of the neuronal basis of reproductive behavior.

From synapses to behavior: development of a sensory-motor circuit in the leech.

The development of neuronal circuits has been advanced greatly by the use of imaging techniques that reveal the activity of neurons during the period when they are constructing synapses and forming circuits. This review focuses on experiments performed in leech embryos to characterize the development of a neuronal circuit that produces a simple segmental behavior called "local bending." The experiments combined electrophysiology, anatomy, and FRET-based voltage-sensitive dyes (VSDs). The VSDs offered two major advantages in these experiments: they allowed us to record simultaneously the activity of many neurons, and unlike other imaging techniques, they revealed inhibition as well as excitation. The results indicated that connections within the circuit are formed in a predictable sequence: initially neurons in the circuit are connected by electrical synapses, forming a network that itself generates an embryonic behavior and prefigures the adult circuit; later chemical synapses, including inhibitory connections, appear, "sculpting" the circuit to generate a different, mature behavior. In this developmental process, some of the electrical connections are completely replaced by chemical synapses, others are maintained into adulthood, and still others persist and share their targets with chemical synaptic connections.

Functions of the subesophageal ganglion in the medicinal leech revealed by ablation of neuromeres in embryos.

Two general trends in the evolution of the nervous system have been toward centralization of neuronal somata and cephalization of the central nervous system (CNS). These organizational trends are apparent in the nervous system of annelid worms, including leeches. To determine if the anterior brain of the leech serves functions similar to those of the brains of more complex organisms, including vertebrates, we ablated one of the two major regions of the cephalic brain--the subesophageal ganglion (SubEG). For anatomical reasons, ablations were performed in embryos, rather than in adults. At the end of embryonic development, we observed the leeches' spontaneous behaviour and their responses to moderate touch. We observed that, although the midbody ganglia of the leech CNS display a high degree of local autonomy, the cephalic brain provides generalized excitation to the rest of the CNS, is a source of selective inhibition that modulates behaviour, integrates sensory information from the head with signals from the rest of the body, and plays an important role in organizing at least some complicated whole-body behaviours. These roles of the leech cephalic brain are common features of brain function in many organisms, and our results are consistent with the hypothesis that they arose early in evolution and have been conserved in complex nervous systems.

Embryonic electrical connections appear to pre-figure a behavioral circuit in the leech CNS.

During development, many embryos show electrical coupling among neurons that is spatially and temporally regulated. For example, in vertebrate embryos extensive dye coupling is seen during the period of circuit formation, suggesting that electrical connections could pre-figure circuits, but it has been difficult to identify which neuronal types are coupled. We have used the leech Hirudo medicinalis to follow the development of electrical connections within the circuit that produces local bending. This circuit consists of three layers of neurons: four mechanosensory neurons (P cells), 17 identified interneurons, and approximately 24 excitatory and inhibitory motor neurons. These neurons can be identified in embryos, and we followed the spatial and temporal dynamics as specific connections developed. Injecting Neurobiotin into identified cells of the circuit revealed that electrical connections were established within this circuit in a precise manner from the beginning. Connections first appeared between motor neurons; mechanosensory neurons and interneurons started to connect at least a day later. This timing correlates with the development of behaviors, so the pattern of emerging connectivity could explain the appearance first of spontaneous behaviors (driven by a electrically coupled motor network) and then of evoked behaviors (when sensory neurons and interneurons are added to the circuit).

A dye mixture (Neurobiotin and Alexa 488) reveals extensive dye-coupling among neurons in leeches; physiology confirms the connections.

Although the neuronal circuits that generate leech movements have been studied for over 30 years, the list of interneurons (INs) in these circuits remains incomplete. Previous studies showed that some motor neurons (MNs) are electrically coupled to swim-related INs, e.g., rectifying junctions connect IN 28 to MN DI-1 (dorsal inhibitor), so we searched for additional neurons in these behavioral circuits by co-injecting Neurobiotin and Alexa Fluor 488 into segmental MNs DI-1, VI-2, DE-3 and VE-4. The high molecular weight Alexa dye is confined to the injected cell, whereas the smaller Neurobiotin molecules diffuse through gap junctions to reveal electrical coupling. We found that MNs were each dye-coupled to approximately 25 neurons, about half of which are likely to be INs. We also found that (1) dye-coupling was reliably correlated with physiologically confirmed electrical connections, (2) dye-coupling is unidirectional between MNs that are linked by rectifying connections, and (3) there are novel electrical connections between excitatory and inhibitory MNs, e.g. between excitatory MN VE-4 and inhibitory MN DI-1. The INs found in this study provide a pool of novel candidate neurons for future studies of behavioral circuits, including those underlying swimming, crawling, shortening, and bending movements.

Sequential development of electrical and chemical synaptic connections generates a specific behavioral circuit in the leech.

Neuronal circuits form during embryonic life, even before synapses are completely mature. Developmental changes can be quantitative (e.g., connections become stronger and more reliable) or qualitative (e.g., synapses form, are lost, or switch from electrical to chemical or from excitatory to inhibitory). To explore how these synaptic events contribute to behavioral circuits, we have studied the formation of a circuit that produces local bending (LB) behavior in leech embryos. This circuit is composed of three layers of neurons: mechanosensory neurons, interneurons, and motor neurons. The only inhibition in this circuit is in the motor neuron layer; it allows the animal to contract on one side while relaxing the opposite side. LB develops in two stages: initially touching the body wall causes circumferential indentation (CI), an embryonic behavior in which contraction takes place around the whole perimeter of the segment touched; one or 2 d later, the same touch elicits adult-like LB. Application of bicuculline methiodide in embryos capable of LB switched the behavior back into CI, indicating that the development of GABAergic connections turns CI into LB. Using voltage-sensitive dyes and electrophysiological recordings, we found that electrical synapses were present early and produced CI. Inhibition appeared later, shaping the circuit that was already connected by electrical synapses and producing the adult behavior, LB.