Analysis of the retrograde transport of glial cell line-derived neurotrophic factor (GDNF), neurturin, and persephin suggests that in vivo signaling for the GDNF family is GFRalpha coreceptor-specific.

Neurturin (NRTN) and glial cell line-derived neurotrophic factor (GDNF) are members of a family of trophic factors with similar actions in vitro on certain neuronal classes. Retrograde transport of GDNF and NRTN was compared in peripheral sensory, sympathetic, and motor neurons to determine whether in vivo these factors are transported selectively by different neuronal populations. After sciatic nerve injections, NRTN was transported by sensory neurons of the dorsal root ganglion (DRG). Competition studies demonstrated only limited cross-competition between NRTN and GDNF, indicating selective receptor-mediated transport of these factors. By using immunohistochemistry, we identified two populations of NRTN-transporting DRG neurons: a major population of small, RET-positive, IB4-positive, non-TrkA-expressing neurons that also show the ability to transport GDNF and a minor population of calretinin-expressing neurons that fail to transport GDNF. Spinal motor neurons in the adult showed relatively less ability to transport NRTN than to transport GDNF, although NRTN prevented the cell death of neonatal motor neurons in a manner very similar to GDNF (Yan et al., 1995) and persephin (PSPN) (Milbrandt et al., 1998). Last, NRTN, like GDNF, was not transported to sympathetic neurons of the adult superior cervical ganglion (SCG) after injection into the anterior eye chamber. These data reveal a high degree of functional selectivity of GDNF family receptor-alpha (GFRalpha) coreceptor subtypes for NRTN and GDNF in vivo.

Permanent alterations of spinal cord reflexes following nerve lesion in newborn rats.

Sciatic nerve lesion in newborn rats is known to cause degeneration of a large number of axotomized motoneurones and spinal ganglion cells. Some of the surviving motoneurones exhibit abnormal firing properties and the projection pattern of central terminals of sensory neurones is altered. We report here on long-term changes in spinal cord reflexes in adult rats following neonatal nerve crush. In acutely spinalized and anaesthetized adult rats 4-6 months old in which the sciatic nerve had been crushed on one side at birth, the tibial nerve, common peroneal nerve or sural nerve were stimulated on the reinnervated and control side and reflex responses were recorded from the L5 ventral spinal roots. Ventral root responses (VRRs) to tibial and peroneal nerve stimulation on the side of the nerve lesion were significantly smaller in amplitude representing only about 15% of the mean amplitude of VRRs on the control side. The calculated central delay of the first, presumably monosynaptic component of the VRR potential was 1.6 ms on the control side while the earliest VRR wave on the side of the nerve lesion appeared after a mean central latency of 4.0 ms that seems too long to be of monosynaptic origin. These results suggest that neonatal sciatic nerve injury markedly alters the physiological properties and synaptic connectivity in spinal cord neurones and causes a marked depression of spinal cord responses to peripheral nerve stimulation.

Changes in the expression of protease-activated receptor 1 and protease nexin-1 mRNA during rat nervous system development and after nerve lesion.

HDs racI Thrombin causes profound metabolic and morphological changes in cultured neural cells via activation of the thrombin receptor, also called protease-activated receptor 1 (PAR1). PAR1 mRNA is present in the rat brain, but the role of this receptor in the nervous system remains elusive. The expression of PAR1 and the potent thrombin inhibitor protease nexin-1 (PN-1) was investigated in the developing rat brain and spinal cord and after peripheral nerve lesion. As seen by in situ hybridization, the PAR1 mRNA signal in the late embryonic and early postnatal nervous system was widespread, but generally of low intensity whereas in the adult it was more pronounced and confined to particular neuronal cells. These include the mesencephalic dopaminergic neurons, several thalamic and brainstem nuclei, the mitral cells in the olfactory bulb and the Purkinje cells in the cerebellum. In the spinal cord, PAR1 mRNA was abundant in motoneurons and a particularly high expression was detected in the preganglionic neurons of the autonomic nervous system. High PAR1 mRNA expression was also found in the dorsal root ganglia. Interestingly, strong immunoreactivity for the protease inhibitor PN-1 was present in spinal motoneuron cell bodies, although its transcript was undetectable there. In response to sciatic nerve transection, the signal intensity of PAR1 mRNA as seen by Northern analysis increased in the proximal and the distal part of the lesioned nerve and in the denervated muscle, whereas the PN-1 mRNA signal strongly increased only in the distal part of the nerve but remained unchanged in the proximal part and in the muscle. After facial nerve transection, PAR1 mRNA expression substantially decreased in facial motoneurons. No PAR1 transcript was detected in reactive astrocytes. Similar to PAR1, PN-1 mRNA which was expressed in interneurons within the facial nucleus was also decreased following facial nerve transection.

Synergistic but transient rescue effects of BDNF and GDNF on axotomized neonatal motoneurons.

Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), members of distinct families of polypeptide growth factors, have been shown to support motoneurons under various in vitro and in vivo conditions. We used a model of motoneuron cell death induced by sciatic nerve section in newborn rats and compared the efficacy of BDNF and GDNF administered alone or simultaneously in order to determine whether combinations of neurotrophic proteins can produce more potent motoneuron rescue than individual factors. The factors were administered by different methods, including (i) a single dose on to the transected nerve, (ii) continuous delivery from implanted slow-release polymer rods (BDNF) or encapsulated cells (GDNF), and (iii) repeated systemic injections (BDNF). Irrespective of the method of administration, either factor alone produced rescue effects which dramatically declined at two weeks as compared to one week post-lesion. In contrast, this decrease was significantly reduced when BDNF and GDNF were used simultaneously provided that one factor was applied on to the nerve while the other was continuously released from the rods or capsules. Other combinations in which GDNF was replaced by ciliary neurotrophic factor or axokine-1 failed to reproduce such additive activity. Two conclusions can be made from these experiments. First, when BDNF and GDNF are administered simultaneously but by distinct routes of delivery, their survival-promoting effects on the injured developing motoneurons are potentiated; second, even continuous delivery of each of these trophic factors alone cannot completely abrogate the time-dependent decline in rescue effects in this model of motoneuron cell death.

Persephin, a novel neurotrophic factor related to GDNF and neurturin.

A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.

Differential effects of neurotrophic factors on motoneuron retrograde labeling in a murine model of motoneuron disease.

It has been shown that abnormalities in axonal transport occur in several mouse models with motoneuron degeneration and also in the human disease amyotrophic lateral sclerosis. In this report, we have examined the potential of neurotrophic factors to act on axonal transport properties in a mouse mutant, progressive motor neuronopathy (pmn). This mouse mutant has been characterized as a "dying-back" motoneuronopathy, with a loss of motoneuron cell bodies and motor fibers. Retrograde transport to the spinal cord motoneurons was determined using fluorescent tracers either injected into the gastrocnemius muscle or applied directly onto the cut sciatic nerve. Because the rate of retrograde labeling was significantly reduced in the pmn, we examined the potential of neurotrophic factors to compensate for the impairment. Ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) but not glial-derived neurotrophic factor (GDNF) or nerve growth factor (NGF) were capable of significantly improving the rate of labeling. The differential effects of these factors agree with previous studies showing that molecules that promote cell survival do not necessarily compensate for axonal deficiency. Because impairment of axonal properties appears as an early event in motoneuron pathology, our results may have important clinical implications in the treatment of motoneuron diseases.

Clinical and molecular aspects of motoneurone diseases: animal models, neurotrophic factors and Bcl-2 oncoprotein.

Animal models of motor neurone disease (MND) are being increasingly used for screening molecules with clinical potential. A number of different treatments to decrease the progression of neuronal cell loss have been proposed; these include: Bcl-2 (B-cell leukaemia oncogene-2), neurotrophic factors, glutamate receptor inhibitors and Ca2+ channel antagonists. In this review Yves Sagot, Richard Vejsada and Ann C. Kato focus on the effects of neurotrophic factors and Bcl-2, both of which have been shown to prevent cell death in various experimental paradigms. Studies performed in animal models of MND have confirmed the potential of these molecules to support motoneurone survival. Some of them have been shown to act in synergy and these results are discussed in the context of molecular mechanisms leading to collaborative and synergistic activities, and also with respect to presumptive subpopulations of motoneurones, which express diverse receptors for neurotrophic factors. Finally, the current status of clinical trials for amyotrophic lateral sclerosis using neurotrophic factors will be discussed, as well as recent reports that neurotrophic factors can exert adverse effects on neuronal survival.

Cardiotrophin-1, a cytokine present in embryonic muscle, supports long-term survival of spinal motoneurons.

The muscle-derived factors required for survival of embryonic motoneurons are not clearly identified. Cardiotrophin-1 (CT-1), a cytokine related to ciliary neurotrophic factor (CNTF), is expressed at high levels in embryonic limb bud and is secreted by differentiated myotubes. In vitro, CT-1 kept 43% of purified E14 rat motoneurons alive for 2 weeks (EC50 = 20 pM). In vivo, CT-1 protected neonatal sciatic motoneurons against the effects of axotomy. CT-1 action on motoneurons was inhibited by phosphatidylinositol-specific phospholipase C (PIPLC), suggesting that CT-1 may act through a GPI-linked component. Since no binding of CT-1 to CNTFR alpha was detected, CT-1 may use a novel cytokine receptor alpha subunit. CT-1 may be important in normal motoneuron development and as a potential tool for slowing motoneuron degeneration in human diseases.

Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth.

Ciliary neurotrophic factor (CNTF) supports motor neuron survival in vitro and in mouse models of motor neuron degeneration and was considered a candidate for the muscle-derived neurotrophic activity that regulates motor neuron survival during development. However, CNTF expression is very low in the embryo, and CNTF gene mutations in mice or human do not result in notable abnormalities of the developing nervous system. We have generated and directly compared mice containing null mutations in the genes encoding CNTF or its receptor (CNTFR alpha). Unlike mice lacking CNTF, mice lacking CNTFR alpha die perinatally and display severe motor neuron deficits. Thus, CNTFR alpha is critical for the developing nervous system, most likely by serving as a receptor for a second, developmentally important, CNTF-like ligand.

Quantitative comparison of the transient rescue effects of neurotrophic factors on axotomized motoneurons in vivo.

A reproducible neuronal degeneration induced by nerve lesion in neonatal rats or mice provides a convenient in vivo assay for testing the survival-promoting activity of putative growth factors on motoneurons. The goal of this study was to compare the rescue effects of the four known neurotrophins [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4)] and two of the cytokines [ciliary neurotrophic factor (CNTF) and leukaemia inhibitory factor (LIF)] in one particular experimental model of spinal motoneuron degeneration at two different survival times. The sciatic nerve was cut in neonatal rats and the factors were applied onto the nerve stump; bovine serum albumin was used in controls. Simultaneous application of the retrograde tracer fluoro-gold made it possible to count motoneurons specifically in the sciatic pool. One week after lesion, the neurotrophins BDNF, NT-3 and NT-4, but not NGF, equally enhanced motoneuron survival compared to controls; their effects were significantly better than those of the cytokines. However, the rescue from cell death was only transitory because a great number of the motoneurons died during the second week after nerve lesion. Additional BDNF and/or CNTF supplied by repeated subcutaneous injections (1 mg/ml) over 2 weeks could not prevent this delayed motoneuron loss. These results suggest that still other factors or alternative routes of administration may be required for permanent rescue of the lesioned immature motoneurons.

BDNF-mediated rescue of axotomized motor neurones decreases with increasing dose.

Direct application of brain-derived neurotrophic factor (BDNF) to the cut end of axotomized immature motor neurones had only transient survival-promoting effects. Therefore, we have examined whether additional delivery of BDNF with repeated subcutaneous injections (1 mg/ml) could potentiate this short-term rescue of the lesioned sciatic and facial motor neurones in neonatal rats. Direct application of BDNF combined with intermittent (3-day intervals) injections slightly improved motor neurone survival. However, when BDNF was injected daily in addition to the direct application, the number of surviving lesioned motor neurones was markedly reduced. These findings, corroborated by results in embryonic spinal cord cultures, show that a dose-dependent reversal of BDNF-mediated positive effects on motor neurones occurs in vivo.

Bone fracture induces reflex muscle atrophy which is sex-dependent.

Longlasting nociceptive stimulation is known to cause atrophy of adjacent muscles. The aim of this study was to determine further the possible mechanisms of this pathological phenomenon. Unilateral fracture of the paw was performed under pentobarbital anaesthesia in several experimental groups (n = 8-11) of female and male rats. Dry muscle weights of the soleus (SOL), extensor digitorum longus (EDL), gastrocnemius (GA) and tibialis anterior (TA) were determined 7 days following the bone fracture and compared to the weight of contralateral control muscles. To demonstrate the reflex origin of this atrophy, deafferentation of the paw by dorsal root section (L4-6) was performed before or after unilateral fracture of hindlimb metatarsal bones. In female rats, the fracture resulted in a significant loss of muscle weight in all the four muscles examined. When the hindlimb was deafferented prior to the fracture, no muscle atrophy developed, and neither did deafferentation itself cause any appreciable change in muscle weight except in male rats. This supports the concept that this type of atrophy is reflex in origin. Deafferentation, when performed after the fracture, did not prevent the weight loss in extensor muscles (SOL, GA), while the flexors (EDL, TA) did not in general lose any weight. The results in male rats had a similar trend as in female rats, although the weight loss was significantly smaller. Our results showed that the mechanism of reflex muscle atrophy following metatarsal bone fracture involves a component which is dependent on afferent information from the injured paw.(ABSTRACT TRUNCATED AT 250 WORDS)

Reaching behavior in the rat: absence of forelimb peripheral input.

In order to test whether peripheral input from a moving forelimb is essential for reaching in rats, the effects of dorsal rhizotomy C5-Th2 were examined. Rats were trained to reach for a food pellet in the horizontal tube or on a tray. Reaching attempts before and after bilateral forelimb deafferentation were monitored by continual recording using magnetic induction. Deafferented animals were able to initiate and generate the motor program of reaching, but modulation of its ongoing execution was lost. Peripheral input from moving forelimb was necessary for the effective performance of grasping; the duration of the manipulative part of reaching was significantly prolonged and the success of grasping was markedly decreased. Also, the aiming of forelimb was impaired, probably by disturbing of body forelimb postural coordination. No significant changes were found in the execution of forelimb protraction. It is concluded that somesthetic feedback is not required for execution of forelimb protraction, but it is necessary for grasping.

Motor functions in rat hindlimb muscles following neonatal sciatic nerve crush.

The sciatic nerve was crushed in the right hindlimb in newborn (3-8 h old) rats. Two to four months later, electromyographic activity was recorded from both the control and reinnervated ankle extensor muscles soleus or lateral gastrocnemius and from the ankle flexor muscle tibialis anterior. Tonic postural activity was present in the extensor muscles on both sides during quiet stance. The control flexor muscles were usually silent in this situation, but the reinnervated flexors exhibited abnormal sustained activity. During locomotion, the control extensors were activated during the stance phase and their mean burst made up 61.5% of the step cycle. The control tibialis anterior muscle fired only during the swing phase, with the burst lasting 18.1% of the step cycle. In the reinnervated extensor muscles, the mean burst duration was decreased (46% of the cycle) but the basic locomotor pattern was not impaired. The reinnervated tibialis muscle, however, was activated abnormally, with one appropriate flexor burst during the swing phase and an "extensor-like" burst during the stance phase of the step. Reflex responses to stretch were weak or absent on the operated side. Histological examination showed that the reinnervated soleus and tibialis muscles were almost devoid of muscle spindles. The motor unit mean firing rates in the reinnervated soleus (22 imp/s) and lateral gastrocnemius (45 imp/s) matched those of the control muscles (25 and 42 imp/s, respectively). In contrast to the phasic, high-frequency firing (52-80 imp/s) in the control tibialis, the reinnervated tibialis motor units fired at significantly lower rates (22-56 imp/s).(ABSTRACT TRUNCATED AT 250 WORDS)

Ensemble discharge from Golgi tendon organs of cat peroneus tertius muscle.

1. The responses of individual tendon organs of the cat peroneus tertius muscle to motor-unit contractions were recorded in anesthetized cats during experiments in which all the Ib-afferent fibers from the muscle had been prepared for recording in dorsal root filaments. This was possible because the cat peroneus tertius only contains a relatively small complement of approximately 10 tendon organs. 2. Motor units of different physiological types were tested for their effects on the whole population of tendon organs in the muscle. Effects of unfused tetanic contractions were tested under both isometric and anisometric conditions. Each motor unit activated at least one tendon organ, and each tendon organ was activated by at least one motor unit. Individual slow-type units were found to act on a single or two receptors, whereas a fast-type unit could activate up to six tendon organs. 3. In one experiment, the effects of 8 motor units on 10 tendon organs were examined. One fast-twitch, fatigue resistant (FR)-type unit acted on six tendon organs, of which four were also activated by another FR unit. The contraction of each unit, on its own, elicited a range of individual responses, from weak to strong. The discharge frequencies of individual responses displayed no clear relation with the strength of contraction, nor did they accurately represent the shape of force profiles. But when all the discharges were pooled, a fairly good correspondence appeared between variations of contractile force and variations of averaged discharge frequencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Immobilization atrophy and membrane properties in rat skeletal muscle fibres.

Wet mass, resting membrane potential, frequency of miniature end-plate potentials and the concentration of [3H]ouabain-binding sites were studied after 7 days' immobilization of the rat soleus and extensor digitorum longus (EDL) muscles in the shortened or stretched position and after 3 and 7 days of remobilization. We observed that the loss of muscle mass by 37% in the rat soleus immobilized for 7 days in the shortened position is accompanied by a membrane depolarization of about 5 mV, a decrease in frequency of miniature end-plate potentials by 60% and a decrease of [3H]ouabain binding by 25%. Only minor changes were found in stretched soleus and in shortened and stretched EDL. After 3 days of remobilization of stretched soleus the muscle mass, [3H]ouabain binding and miniature end-plate potential frequency recovered to control values but the resting membrane potential continued to decrease. All changes induced by immobilization disappeared on day 7 of remobilization.

Activation of Golgi tendon organs by asynchronous contractions of motor units in cat leg muscles.

Discharges from individual tendon organs of peroneus tertius muscle activated by the isometric contractions of single motor units were recorded in anaesthetized cats. Pairs of motor units acting on the same tendon organ were stimulated asynchronously at frequencies eliciting unfused contractions. Tendon organ responses to such contractions did not display a linear relation between discharge frequency and contractile force recorded at the muscle tendon. In several instances, one of the motor units exerted a predominant influence on the response of the tendon organ, even though this unit did not produce the strongest activation of the receptor when stimulated on its own.

Unloading of tendon organ discharges by in-series motor units in cat peroneal muscles.

1. The discharges from individual Golgi tendon organs of peroneus tertius and brevis muscles were recorded in anaesthetized cats during the isometric contractions of single motor units. Upon combined contractions of several motor units, two sorts of unloading effects were observed. 2. First, the contraction of a motor unit which, by itself, was without action on a tendon organ could produce a reduction in the response of the receptor to one of its activating motor units. Unloading effects exerted by such in-parallel motor units could effectively interfere with the actions of in-series motor units on the receptor. 3. Second, the contraction of a motor unit activating a tendon organ could reduce the response of this tendon organ to the contraction of another of its activating units. This new type of unloading effect, exerted by in-series motor units, was demonstrated by the fact that the simultaneous contraction of both units elicited less discharge from the receptor than the contraction of a single unit. 4. Unfused contractions of a fast-type motor unit eliciting a response in which the tendon organ discharge was driven 1:1 at the frequency of stimulation of the motor unit, could exert unloading actions on the response of the receptor to another motor unit eliciting a higher discharge frequency. 5. In-series unloading actions were exerted not only by fast-type motor units developing large forces, but also by relatively small slow-type motor units. 6. The high incidence of in-parallel and in-series unloading effects suggests that their consequences may be functionally significant. When large numbers of motor units are being recruited in a muscle, unloading effects might result in a limitation of the Ib afferent discharges from this muscle, preventing an excessive increase of autogenetic inhibition.

Function and structure of atypical muscle spindles after neonatal nerve crush in rats.

During the early postnatal period, the differentiation and maturation of muscle spindles in the rat is still dependent on their sensory innervation. When a nerve is crushed during this period, most spindles in the denervated muscles degenerate and after reinnervation only occasional spindles of atypical structure are to be found in these muscles. We determined the basic functional properties of these atypical spindles in adult rats and attempted to correlate them with their structural characteristics. The discharge rates of 13 afferent units from the soleus or lateral gastrocnemius muscles were evaluated in response to stretch. These units were capable of a slowly adapting response to 2-4 mm stretches. Their mean discharge frequencies at any point of the ramp-and-hold stretch were, however, on an average 50% lower than normal values. The conduction velocities of afferents from the atypical spindles were in the range of 10-40 m/s. Histological examinations revealed that 90% of the atypical muscle spindles found in the soleus or lateral gastrocnemius muscles had only 1 or 2 intrafusal fibres without any nuclear accumulations as compared to four intrafusal fibres in normal muscle spindles in the rat. The proportional decrease of the discharge rate in both the dynamic and static part of the response of these atypical spindles could be due to the decreased synaptic area between the sensory terminals and the intrafusal fibres and/or to altered structural properties of the intrafusal fibres.

Observations on static and dynamic responses of muscle stretch receptors in kittens.

In 5-19-day-old kittens anesthetized with Saffan, the discharges of de-efferented triceps surae muscle receptors were recorded from afferent fibers in dorsal root filaments. The conduction velocities of the afferent fibers ranged between 7 and 30 m/s. Receptors were identified as spindles on the basis of their response to muscle stretch and, whenever possible, the pause in their discharge during muscle contraction. Spindles responding with sustained discharges to muscle extensions of less than 1 mm could be found in 5-8-day-old kittens, provided the muscles was left 'in situ', with intact skin, tendon and aponeuroses. However, such responses were rare before 10 days, and the majority of receptors essentially displayed phasic responses to muscle stretch, in agreement with previous observations. In responses to sinusoidal muscle stretching of 0.1-0.5-mm amplitude, kitten receptors were easily driven to discharge one impulse per cycle at frequencies of 10-15 Hz. But unlike adult spindles, they could not follow higher frequencies unless the amplitude of stretch was increased. The maturation of dynamic responses is slower than that of static responses in kitten spindles. In the first postnatal weeks, small changes in muscle length are poorly signaled to the central nervous system.