Kathleen A. French

Development of the Leech Nervous System

Publisher Summary This chapter reveals that embryogenesis of the leech nervous system is highly determinate in the sense that during normal development the genealogical origin of each identified neuron can be traced, through a sequence of stereotyped cleavages, to the zygote. This determinacy suggests that the developmental fate of a given cell is governed by its particular line of descent. However, cellular interactions affect the fate taken on by at least some neural precursor cells, as is the case for the O/P kinship group. Although the characteristic phenotype of each identified neuron stems from a series of increasingly restrictive developmental commitments made by its ancestors, and for many neurons these developmental choices rely on cell lineage, interactions with other cells cannot be ignored as a source of significant developmental information. It is generally thought that cell fates become assigned when the developmental possibilities of pluripotent cell lines are limited by a set of sequential commitment steps that end in differentiation into the final set of phenotypes emanating from each pluripotent cell line.

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.

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.

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.

Development of spontaneous and evoked behaviors in the medicinal leech

The ontogeny of behavior in an organism must reflect developmental events in the nervous system, and it thus provides a noninvasive measure of neuronal development. This approach may be particularly fruitful in the medicinal leech because the neuronal basis of several behaviors has been characterized in adult leeches, providing a rich background against which behavioral development can be interpreted. We have investigated the order in which behaviors arise during the period of embryonic development and have determined the time at which each behavior is first expressed. Some behaviors, such as lateral ridge formation, germinal plate bending, spiral twisting, and sidewinding, were produced spontaneously by embryos. Others, such as shortening, circumferential indentation, local bending, and elongation, occurred only when they were elicited by weak mechanical stimulation. Such stimulation rarely evoked a behavioral response in young embryos (at 45% of the time required for complete embryonic development, 45% ED), but by 80% ED embryos responded to nearly 100% of the stimuli presented. In embryos older than 50% ED, the behavior most frequently evoked by stimulation of the anterior end, the posterior end, or the rear sucker was shortening. Stimulation of the midbody usually evoked behavior other than shortening, illustrating that the body was behaviorally compartmentalized, at least in part. Some behaviors observed during embryogenesis are never seen in adult leeches. For example, in response to stimulation of the midbody, young embryos produced a behavior that we have called “circumferential indentation,” whereas older embryos produced local bending, a response previously described for adults. The switch from circumferential indentation to local bending may signal the formation of new synaptic connections. J. Comp. Neurol. 402:168–180, 1998.

Mesenchyme of embryonic reproductive ducts directs process outgrowth of Retzius neurons in the medicinal leech

In the two segments of the medicinal leech (Hirudo medicinalis) that contain the male (segment 5) and the female (segment 6) reproductive ducts, the paired Retzius (Rz) neurons are distinguished by several unique properties. For example, the muscles and glands of the body wall are the primary peripheral targets of Rz neurons in standard segments [Rz(X)], whereas the muscles and glands of the reproductive ducts are the primary peripheral targets of Rz neurons in the two reproductive segments [Rz(5,6)]. In this paper, we show that organogenesis and differentiation, which generate an epithelial tube surrounded by mesenchymal cells, occur in the embryonic reproductive ducts at approximately the time when Rz processes first contact these structures. The growth cones leading one branch of the posterior axon of Rz(5,6) contact the duct mesenchymal cells. Following initiation of this contact, these posterior growth cones enlarge and send out numerous filopodia. Secondarily, growth cones leading the anterior axon of each Rz(5,6) also modify their shapes and trajectories. When embryonic reproductive ducts were transplanted into posterior (nonreproductive) segments, the branch of the posterior Rz axon near the ectopic reproductive tissue produced enlarged growth cones and extended several secondary branches into the mesenchyme of the ectopic tissue. This result suggests that the reproductive mesenchyme is attractive to, and can modify the growth of, all Rz neurons. The behavior of Rz(5,6) growth cones suggests that the reproductive mesenchyme cells provide guidance cues that control the location in which Rz axons elaborate their peripheral arborization and form synapses, and that the mesenchyme may also stimulate the production of a densely branched arbor.

Staging of middle and late embryonic development in the medicinal leech, Hirudo medicinalis.

We present a description of the last half of embryonic development in the European medicinal leech, Hirudo medicinalis, based entirely on externally visible morphological features, and establish reliably observable stages during that development. Embryogenesis, from the time fertilized eggs are deposited in an eggcase (called a cocoon) to the emergence of juveniles from the cocoon, takes approximately 4 weeks at room temperature. The stages described in this paper extend from the completion of segmentation to the appearance of the final bands of pigmentation. Developmental stages are expressed as percentages of total embryonic developmental time. This staging table was constructed for embryos kept at 20°C. In addition, the development of animals kept at 17°C or at 24°C was compared with those held at 20°C. Development proceeds more quickly at higher temperatures. Because development in embryos held at higher or lower temperatures was linearly related to the stages determined for embryos held at 20°C, the rate of development at any intermediate temperature can be predicted from the staging table at 20°C by simple multiplication. J. Comp. Neurol. 402:155–167, 1998.

Development of neuronal circuits and behaviors in the medicinal leech

We are studying the neuronal mechanisms responsible for establishing circuitry underlying the local bending response in the medicinal leech. Local bending replaces an embryonic behavior, circumferential indentation, during the time of initial chemical synaptogenesis in leech embryos. We found that the electrical connections among the motor neurons are established first, about 5% of embryonic time (almost 2 full days) before chemical connections form. The inhibitory connections from muscle inhibitors to muscle excitors are, we hypothesize, responsible for the emergence of local bending. We have also found that the central processes of the excitors--but not the inhibitors--have much longer central processes when their peripheral processes are kept from contacting their target muscles. This system should allow us to test ideas about how individual neurons find their appropriate targets to form functional neuronal circuits.

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.

Development of swimming in the medicinal leech, the gradual acquisition of a behavior

Observing the development of behavior provides an assay for the developmental state of an embryo’s nervous system. We have previously described the development of behaviors that were largely confined to one or a few segments. We now extend the work to a kinematic analysis of the development of swimming, a behavior that requires coordination of the entire body. When leech embryos first begin to swim they make little forward progress, but within several days they swim as effectively as adults. This increase in efficacy depends on changes in body shape and on improved intersegmental coordination of the swim central pattern generator. These kinematic details suggest how the swim central pattern generating circuit is assembled during embryogenesis.