Neuromuscular development in the absence of programmed cell death: phenotypic alteration of motoneurons and muscle.

The widespread, massive loss of developing neurons in the central and peripheral nervous system of birds and mammals is generally considered to be an evolutionary adaptation. However, until recently, models for testing both the immediate and long-term consequences of preventing this normal cell loss have not been available. We have taken advantage of several methods for preventing neuronal death in vivo to ask whether rescued neurons [e.g., motoneurons (MNs)] differentiate normally and become functionally incorporated into the nervous system. Although many aspects of MN differentiation occurred normally after the prevention of cell death (including the expression of several motoneuron-specific markers, axon projections into the ventral root and peripheral nerves, ultrastructure, dendritic arborization, and afferent axosomatic synapses), other features of the neuromuscular system (MNs and muscle) were abnormal. The cell bodies and axons of MNs were smaller than normal, many MN axons failed to become myelinated or to form functional synaptic contacts with target muscles, and a subpopulation of rescued cells were transformed from alpha- to gamma-like MNs. Additionally, after the rescue of MNs in myogenin glial cell line-derived neurotrophic factor (MyoGDNF) transgenic mice, myofiber differentiation of extrafusal skeletal muscle was transformed and muscle physiology and motor behaviors were abnormal. In contrast, extrafusal myofiber phenotype, muscle physiology, and (except for muscle strength tests) motor behaviors were all normal after the rescue of MNs by genetic deletion of the proapoptotic gene Bax. However, there was an increase in intrafusal muscle fibers (spindles) in Bax knock-out versus both wild-type and MyoGDNF mice. Together, these data indicate that after the prevention of MN death, the neuromuscular system becomes transformed in novel ways to compensate for the presence of the thousands of excess cells.

Complete dissociation of motor neuron death from motor dysfunction by Bax deletion in a mouse model of ALS.

The death of cranial and spinal motoneurons (MNs) is believed to be an essential component of the pathogenesis of amyotrophic lateral sclerosis (ALS). We tested this hypothesis by crossing Bax-deficient mice with mice expressing mutant superoxide dismutase 1 (SOD1), a transgenic model of familial ALS. Although Bax deletion failed to prevent neuromuscular denervation and mitochondrial vacuolization, MNs were completely rescued from mutant SOD1-mediated death. However, Bax deficiency extended lifespan and delayed the onset of motor dysfunction of SOD1 mutants, suggesting that Bax acts via a mechanism distinct from cell death activation. Consistent with this idea, Bax elimination delayed the onset of neuromuscular denervation, which began long before the activation of cell death proteins in SOD1 mutants. Additionally, we show that denervation preceded accumulation of mutant SOD1 within MNs and astrogliosis in the spinal cord, which are also both delayed in Bax-deficient SOD1 mutants. Interestingly, MNs exhibited mitochondrial abnormalities at the innervated neuromuscular junction at the onset of neuromuscular denervation. Additionally, both MN presynaptic terminals and terminal Schwann cells expressed high levels of mutant SOD1 before MNs withdrew their axons. Together, these data support the idea that clinical symptoms in the SOD1 G93A model of ALS result specifically from damage to the distal motor axon and not from activation of the death pathway, and cast doubt on the utility of anti-apoptotic therapies to combat ALS. Furthermore, they suggest a novel, cell death-independent role for Bax in facilitating mutant SOD1-mediated motor denervation.

Adaptive roles of programmed cell death during nervous system development.

The programmed cell death (PCD) of developing cells is considered an essential adaptive process that evolved to serve diverse roles. We review the putative adaptive functions of PCD in the animal kingdom with a major focus on PCD in the developing nervous system. Considerable evidence is consistent with the role of PCD in events ranging from neurulation and synaptogenesis to the elimination of adult-generated CNS cells. The remarkable recent progress in our understanding of the genetic regulation of PCD has made it possible to perturb (inhibit) PCD and determine the possible repercussions for nervous system development and function. Although still in their infancy, these studies have so far revealed few striking behavioral or functional phenotypes.

Role of programmed cell death in normal neuronal development and function.

The consequences of eliminating the process of programmed cell death during the development of the nervous system is examined by reviewing studies in the genetic model organisms Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus, where mutations of cell death genes have eliminated or reduced programmed cell death in the nervous system. In many cases, genetic elimination of cell death leads to embryonic mortality or gross anatomical malformations; however, there are cases where animals develop normally but with excess neurons and glia in the nervous system. Undead cells either differentiate and function as working neurons, in some instances being of smaller size, or fail to differentiate and lack normal connections with their targets. Changes in motor control and sensory processing are generally not observed, except for during the most complex of behaviors. Examination of organisms where death genes have been genetically eliminated reveals that programmed cell death may play an important role in sculpting gross brain structure during early development of the neural tube. In contrast, the consequences of preventing neuronal cell death at later developmental stages (e.g. during vertebrate synapse formation) are just beginning to be understood.

Sacral dorsal horn neurone activity during micturition in the cat.

The excitability of two groups of neurones located in different parts of the sacral spinal cord were examined during micturition in decerebrate adult cats. One group of cells, characterized by their activation by pudendal cutaneous afferents, was located in the dorsal commissure of the first and second sacral spinal segments. The second group, located in the dorsal horn of the first sacral spinal segment, was excited by group II muscle and cutaneous afferents. Micturition was evoked by distension of the urinary bladder or by electrical stimulation of the pontine micturition centre. Tonic firing was induced in the neurones by ejection of DL-homocysteic acid from the recording extracellular micropipette. The instantaneous firing frequency of 11/17 sacral dorsal grey commissure neurones was decreased from 7 to 100 % during micturition, and on average was about half of the prevoid firing frequency. It is hypothesized that these sacral neurones are interposed in polysynaptic excitatory pathways from sacral perineal afferents to sphincter motoneurones and that they are subject to direct postsynaptic inhibition during micturition. One other cell showed no change in firing during micturition, two displayed complex patterns of modulation, while 3/17 of the dorsal grey commissure neurones increased their firing rate 30 to 1000 % during micturition. It is hypothesized that the excited neurones may be part of the inhibitory pathways mediating postsynaptic inhibition of sphincter motoneurones or sacral primary afferent depolarization during micturition. Alternatively, they may be part of the excitatory urethral-bladder reflex circuitry. A small (5-15 %) but significant decrease in firing was observed in 4/5 of the group II rostral sacral neurones examined; the firing of a fifth neurone was unchanged. The depression of group II neurones may serve to suppress unwanted hindlimb reflexes that could disrupt micturition.

Membrane properties related to the firing behavior of zebrafish motoneurons.

The physiological and pharmacological properties of the motoneuron membrane and action potential were investigated in larval zebrafish using whole cell patch current-clamp recording techniques. Action potentials were eliminated in tetrodotoxin, repolarized by tetraethylammonium (TEA) and 3,4-diaminopyridine (3,4-AP)-sensitive potassium conductances, and had a cobalt-sensitive, high-threshold calcium component. Depolarizing current injection evoked a brief (approximately 10-30 ms) burst of action potentials that was terminated by strong, outwardly rectifying voltage-activated potassium and calcium-dependent conductances. In the presence of intracellular cesium ions, a prolonged plateau potential often followed brief depolarizations. During larval development (hatching to free-swimming), the resting membrane conductance increased in a population of motoneurons, which tended to reduce the apparent outward rectification of the membrane. The conductances contributing to action potential burst termination are hypothesized to play a role in patterning the synaptically driven motoneuron output in these rapidly swimming fish.

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.

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.

Activation of embryonic red and white muscle fibers during fictive swimming in the developing zebrafish.

Sub-threshold, motoneuron-evoked synaptic activity was observed in zebrafish embryonic red (ER) and white (EW) muscle fibers paralyzed with a dose of D-tubocurarine insufficient to abolish synaptic activity to determine whether muscle activation was coordinated to produce the undulating body movements required for locomotion. Paired whole-cell recordings revealed a synaptic drive that alternated between ipsilateral and contralateral myotomes and exhibited a rostral-caudal delay in timing appropriate for swimming. Both ER and EW muscle were activated during fictive swimming. However, at the fastest fictive swimming rates, ER fibers were de-recruited, whereas they could be active in isolation of EW fibers at the slowest fictive swimming rates. Prior to hatching, fictive swimming was preceded by a lower frequency, more robust and rhythmic synaptic drive resembling the "coiling" behavior of fish embryos. The motor activity observed in paralyzed zebrafish closely resembled the swimming and coiling behaviors observed in these developing fishes. At the early developmental stages examined in this study, myotomal muscle recruitment and coordination were similar to that observed in adult fishes during swimming. Our results indicate that the patterned activation of myotomal muscle is set from the onset of development.