Pierre Drapeau

Time course of the development of motor behaviors in the zebrafish embryo

The development and properties of locomotor behaviors in zebrafish embryos raised at 28.5 degrees C were examined. When freed from the chorion, embryonic zebrafish showed three sequential stereotyped behaviors: a transient period of alternating, coiling contractions followed by touch-evoked rapid coils, then finally, organized swimming. The three different behaviors were characterized by video microscopy. Spontaneous, alternating contractions of the trunk appeared suddenly at 17 h postfertilization (hpf), with a frequency of 0.57 Hz, peaked at 19 hpf at 0.96 Hz, and gradually decreased to <0.1 Hz by 27 hpf. Starting at 21 hpf, touching either the head or the tail of the embryos resulted in vigorous coils. The coils accelerated with development, reaching a maximum speed of contraction before 48 hpf, which is near the time of hatching. After 27 hpf, touching the embryos, particularly on the tail, could induce partial coils (instead of full coils). At this time, embryos started to swim in response to a touch, preferentially to the tail. The swim cycle frequency gradually increased with age from 7 Hz at 27 hpf to 28 Hz at 36 hpf. Lesions of the central nervous system rostral to the hindbrain had no effect on the three behaviors. Lesioning the hindbrain eliminated swimming and touch responses, but not the spontaneous contractions. Our observations suggest that the spontaneous contractions result from activation of a primitive spinal circuit, while touch and swimming require additional hindbrain inputs to elicit mature locomotor behaviors.

Development of the locomotor network in zebrafish

The zebrafish is a leading model for studies of vertebrate development and genetics. Its embryonic motor behaviors are easy to assess (e.g. for mutagenic screens), the embryos develop rapidly (hatching as larvae at 2 days) and are transparent, permitting calcium imaging and patch clamp recording in vivo. We review primarily the recent advances in understanding the cellular basis for the development of motor activities in the developing zebrafish. The motor activities are generated largely in the spinal cord and hindbrain. In the embryo these segmented structures possess a relatively small number of repeating sets of identifiable neurons. Many types of neurons as well as the two types of muscle cells have been classified based on their morphologies. Some of the molecular signals for cellular differentiation have been identified recently and mutations affecting cell development have been isolated. Embryonic motor behaviors appear in sequence and consist of an early period of transient spontaneous coiling contractions, followed by the emergence of twitching responses to touch, and later by the ability to swim. Coiling contractions are generated by an electrically coupled network of a subset of spinal neurons whereas a chemical (glutamatergic and glycinergic) synaptic drive underlies touch responses and swimming. Swimming becomes sustained in larvae once the neuromodulatory serotonergic system develops. These results indicate many similarities between developing zebrafish and other vertebrates in the properties of the synaptic drive underlying locomotion. Therefore, the zebrafish is a useful preparation for gaining new insights into the development of the neural control of vertebrate locomotion. As the types of neurons, transmitters, receptors and channels used in the locomotor network are being defined, this opens the possibility of combining cellular neurophysiology with forward and reverse molecular genetics to understand the principles of locomotor network assembly and function.

Synchronization of an Embryonic Network of Identified Spinal Interneurons Solely by Electrical Coupling

There is a need to understand the mechanisms of neural synchronization during development because correlated rhythmic activity is thought to be critical for the establishment of proper connectivity. The relative importance of chemical and electrical synapses for synchronization of electrical activity during development is unclear. We examined the activity patterns of identified spinal neurons at the onset of motor activity in zebrafish embryos. Rhythmic activity appeared early and persisted upon blocking chemical neurotransmission but was abolished by inhibitors of gap junctions. Paired recordings revealed that active spinal neurons were electrically coupled and formed a simple network of motoneurons and a subset of interneurons. Thus, the earliest spinal central pattern generator consists of synchronously active, electrically coupled neurons.

Mutations in the KIAA0196 Gene at the SPG8 Locus Cause Hereditary Spastic Paraplegia

Hereditary spastic paraplegia (HSP) is a progressive upper-motor neurodegenerative disease. The eighth HSP locus, SPG8, is on chromosome 8p24.13. The three families previously linked to the SPG8 locus present with relatively severe, pure spastic paraplegia. We have identified three mutations in the KIAA0196 gene in six families that map to the SPG8 locus. One mutation, V626F, segregated in three large North American families with European ancestry and in one British family. An L619F mutation was found in a Brazilian family. The third mutation, N471D, was identified in a smaller family of European origin and lies in a spectrin domain. None of these mutations were identified in 500 control individuals. Both the L619 and V626 residues are strictly conserved across species and likely have a notable effect on the structure of the protein product strumpellin. Rescue studies with human mRNA injected in zebrafish treated with morpholino oligonucleotides to knock down the endogenous protein showed that mutations at these two residues impaired the normal function of the KIAA0196 gene. However, the function of the 1,159-aa strumpellin protein is relatively unknown. The identification and characterization of the KIAA0196 gene will enable further insight into the pathogenesis of HSP.

In vivo recording from identifiable neurons of the locomotor network in the developing zebrafish

The zebrafish is a popular model for developmental studies due to its accessibility by cellular, molecular and genetic approaches. As a complement to these other methods, we have devised an exposed hindbrain/spinal cord preparation in the curarized zebrafish embryo and larva that permits intracellular labeling and patch clamp recording from individually identified sensory neurons, motoneurons and interneurons in vivo. Regular bursts of synaptic potentials and action potentials were observed under whole-cell current clamp in embryonic motoneurons and in some identified interneurons. Larval neurons showed prolonged depolarizations with synaptically driven bursts of action potentials. Frequent spontaneous synaptic potentials were observed and synaptic currents were effectively space clamped. It is thus feasible to study in vivo the properties of identifiable neurons of the developing locomotor network in the zebrafish, including their synaptic activity, firing patterns and interconnections.

Steps during the development of the zebrafish locomotor network.

This review summarizes recent data from our lab concerning the development of motor activities in the developing zebrafish. The zebrafish is a leading model for studies of vertebrate development because one can obtain a large number of transparent, externally and rapidly developing embryos with motor behaviors that are easy to assess (e.g. for mutagenic screens). The emergence of embryonic motility was studied behaviorally and at the cellular level. The embryonic behaviors appear sequentially and include an early, transient period of spontaneous, alternating tail coilings, followed by responses to touch, and swimming. Patch clamp recording in vivo revealed that an electrically coupled network of a subset of spinal neurons generates spontaneous tail coiling, whereas a chemical (glutamatergic and glycinergic) synaptic drive underlies touch responses and swimming and requires input from the hindbrain. Swimming becomes sustained in larvae once serotonergic neuromodulatory effects are integrated. We end with a brief overview of the genetic tools available for the study of the molecular determinants implicated in locomotor network development in the zebrafish. Combining genetic, behavioral and cellular experimental approaches will advance our understanding of the general principles of locomotor network assembly and function.

Serotoninergic Modulation of Chloride Homeostasis during Maturation of the Locomotor Network in Zebrafish

During development, neural networks progress through important functional changes such as the generation of spontaneous activity, the expression of a depolarizing chloride gradient, and the appearance of neuromodulation. Little is known about how these processes are integrated to yield mature behaviors. We showed previously that, during the maturation of the locomotor network of the zebrafish, endogenous serotonin (5HT) increased motor activity by reducing intervals of inactivity, without affecting the active swim periods that are the target of 5HT in other and more mature preparations. Because membrane properties were constant during the rest intervals, we examined here whether 5HT modulates chloride homeostasis. We compared the effects of blocking (inward) chloride cotransport with bumetanide to the effects of 5HT and its antagonists, both behaviorally by video imaging and cellularly by whole-cell and gramicidin-perforated patch recordings. Bumetanide mimicked the effects of 5HT antagonists, by prolonging rest intervals without affecting the properties of swim episodes (duration; frequency; extent of depolarization) either behaviorally or during fictive swimming. Furthermore, bumetanide and 5HT antagonists suppressed the amplitude of depolarizing responses evoked by ionophoresis of glycine onto spinal neurons in the presence of tetrodotoxin and transiently suppressed the amplitude of responses to glycine measured after fictive swimming. The effects of bumetanide contrasted with and occluded the effects of 5HT. We suggest that, during development, endogenous 5HT modulates chloride homeostasis during the quiescent intervals and thereby offsets the long periods of quiescence commonly observed in developing networks to allow expression of sustained and behaviorally relevant activity.

Ionotropic and metabotropic activation of a neuronal chloride channel by serotonin and dopamine in the leech Hirudo medicinalis

1 Cl− channels on the pressure‐sensitive (P) neuron in the leech are directly activated by synaptic release of serotonin (5‐HT) and are indirectly stimulated by the cAMP second messenger pathway, suggesting an unusual dual regulation of the channels. We have investigated the mode of action of 5‐HT and dopamine (DA) on a Cl− channel in adult P cells in culture by recording from cell‐attached patches. 2 5‐HT increased Cl− channel activity only when included in the recording pipette and not when applied in the bath. 3 Pipette or, more effectively, bath application of DA led to an increase in Cl− channel activity. This effect was blocked by the potent and specific dopaminergic (DA1) receptor blocker, SCH‐23390. 4 The stimulation by DA, but not by 5‐HT, was also blocked by the cAMP‐dependent protein kinase A (PKA) inhibitor Rp‐cAMP and was mimicked by the membrane‐permeant cAMP analogue dibutyryl cAMP (db‐cAMP). 5 Our results show that 5‐HT directly gates a Cl− channel that is also activated by DA via the cAMP pathway. This study demonstrates that a ligand‐gated channel can be independently operated by another transmitter acting via a second messenger pathway.

Loss of channel modulation by transmitter and protein kinase c during innervation of an identified leech neuron

When serotonergic Retzius (R) neurons of the leech contact pressure-sensitive (P) neurons in culture, P cells selectively lose a protein kinase C-dependent cationic response to serotonin and the R cell reforms the inhibitory, chloride-dependent synapse seen in vivo. In P cells not contacted by R cells, cell-attached patches contained single cation channels sensitive to serotonin and phorbol ester with characteristic properties and high incidence (present in about one-half of the patches). P cells paired with R cells had a cation channel with similar biophysical properties and incidence, but channel activity was not stimulated by serotonin and phorbol ester. These results suggest that the early clearing of the non-synaptic (excitatory) response to serotonin is due to the loss of activation by protein kinase C (and not the number) of cation channels as a prelude to inhibitory synapse formation.

Tyrosine phosphorylation during synapse formation between identified leech neurons.

1. We have examined whether tyrosine phosphorylation is required for synapse formation between identified neurons from the central nervous system of the leech in culture. 2. Within a few hours of contact with the cell body of the serotonergic Retzius neuron (R cell), the soma of the postsynaptic pressure‐sensitive neuron (P cell), but not the R cell, could be labelled intracellularly with an antibody against phosphotyrosine residues. The labelling seemed specific for P cells contacted by R cells, as it was greatly reduced in pairs of either R or P cells and in single cells. Genistein (20 microM) and lavendustin A (10 microM), selective inhibitors of tyrosine kinases, blocked the labelling of contacted P cells, whereas their ineffective analogues (genistein and lavendustin B) had no effect on labelling. 3. R cell contact also induced the loss of an extrasynaptic, depolarizing response (due to modulation of cation channels) to serotonin (5‐HT) in the P cell within a few days of juxtaposing cell bodies and within an hour of contact with growth cones. Treatment of the neurons with the tyrosine kinase inhibitors (but not the ineffective analogues) prevented the loss of the depolarizing response and of single cation channel modulation by 5‐HT. 4. R cells formed inhibitory, Cl(‐)‐dependent synapses with P cells. Synapse formation was prevented by the tyrosine kinase inhibitors but not by their ineffective analogues. These compounds had no obvious effect on neurite outgrowth or cell adhesion. We conclude that tyrosine phosphorylation is a signal during the formation of this synapse.