Expression patterns of ciliopathy genes ARL3 and CEP120 reveal roles in multisystem development.

BACKGROUND: Joubert syndrome and related disorders (JSRD) and Jeune syndrome are multisystem ciliopathy disorders with overlapping phenotypes. There are a growing number of genetic causes for these rare syndromes, including the recently described genes ARL3 and CEP120. METHODS: We sought to explore the developmental expression patterns of ARL3 and CEP120 in humans to gain additional understanding of these genetic conditions. We used an RNA in situ detection technique called RNAscope to characterise ARL3 and CEP120 expression patterns in human embryos and foetuses in collaboration with the MRC-Wellcome Trust Human Developmental Biology Resource. RESULTS: Both ARL3 and CEP120 are expressed in early human brain development, including the cerebellum and in the developing retina and kidney, consistent with the clinical phenotypes seen with pathogenic variants in these genes. CONCLUSIONS: This study provides insights into the potential pathogenesis of JSRD by uncovering the spatial expression of two JSRD-causative genes during normal human development.

Closed-loop optogenetic control of the dynamics of neural activity in non-human primates.

Electrical neurostimulation is effective in the treatment of neurological disorders, but associated recording artefacts generally limit its applications to open-loop stimuli. Real-time and continuous closed-loop control of brain activity can, however, be achieved by pairing concurrent electrical recordings and optogenetics. Here we show that closed-loop optogenetic stimulation with excitatory opsins enables the precise manipulation of neural dynamics in brain slices from transgenic mice and in anaesthetized non-human primates. The approach generates oscillations in quiescent tissue, enhances or suppresses endogenous patterns in active tissue and modulates seizure-like bursts elicited by the convulsant 4-aminopyridine. A nonlinear model of the phase-dependent effects of optical stimulation reproduced the modulation of cycles of local-field potentials associated with seizure oscillations, as evidenced by the systematic changes in the variability and entropy of the phase-space trajectories of seizures, which correlated with changes in their duration and intensity. We also show that closed-loop optogenetic neurostimulation could be delivered using intracortical optrodes incorporating light-emitting diodes. Closed-loop optogenetic approaches may be translatable to therapeutic applications in humans.

Reduced placental size and increased apoptosis are associated with prenatal nicotine exposure in rats.

OBJECTIVE: Smoking during pregnancy has been linked to a variety of negative embryonic and neonatal consequences. Nicotine is the major constituent of tobacco smoke, which was associated with adverse impacts on histological and functional features of the placenta. This study aims to investigate the potential influence of nicotine exposure on the rat placenta and fetus. MATERIALS AND METHODS: Nicotine was administrated through the drinking water of female pregnant rats. The placental size, as well as the fetal body weight and size, were measured at E20. The mRNA expression of the Bax gene (pro-apoptotic), the Bcl-2 gene (anti-apoptotic) and the angiogenic genes VEGF, Flt-1, and HIF1 were measured in placental tissue. Furthermore, Immunohistochemistry (IHC) using Bax, caspase 9 and VEGF antibodies were performed on placental sections. RESULTS: The results of the current study showed a significant reduction in the size of the placenta along with fetal body weight in nicotine treated group compared to the control group. Apoptosis was observed to be boosted in the placentas of the nicotine-treated group. This was associated with upregulation of Bax expression combined with no change in the expression of Bcl-2 in the treated group. On the other hand, there was no difference in the expression of angiogenic factors VEGF, Flt-1, or HIF1 between tested groups. CONCLUSIONS: In utero nicotine exposure in pregnant rats showed deleterious impacts on fetus growth and weight, as well as placental size. These were accompanied by increased apoptosis within the placenta, as revealed by Bax gene upregulation.

Changing pattern of expression of parvalbumin immunoreactivity during human fetal spinal cord development.

Expression of the calcium binding protein parvalbumin (PV) by different classes of spinal neuron has been shown to be developmentally regulated in both rat and monkey. From postmortem studies of eight human cervical spinal cords ranging in age from 11 to 35 weeks postconceptional age, we report that parvalbumin immunoreactivity is similarly plastic in human lower cervical spinal cord development, with many changes occurring prenatally. At 11-14 weeks postconceptional age, there was prominent immunostaining of primary sensory afferents that could be seen coursing through the dorsal horn and extensively innervating the motoneuron pools. Motoneurons were also found to be clearly immunoreactive for choline acetyltransferase by this age. A few ventral horn neurons that were not motoneurons were also parvalbumin immunoreactive. By 24-27 weeks postconceptional age, sensory afferents were still immunoreactive, as were many other axons throughout the white matter. In addition, many ventral horn neurons were now immunoreactive as well as a few dorsal horn neurons. By 31-35 weeks postconceptional age, there was extensive immunostaining of neurons throughout the spinal cord, including a few moderately immunoreactive motoneurons. There were many immunopositive axons in all the white matter tracts except the corticospinal tracts; however, staining of sensory axons traversing the grey matter was less prominent by this age. In the rat, expression of PV by primary sensory neurons coincides with the onset of fetal limb movement. The onset of expression of PV in ventral horn neurons coincides with later developmental events after the arrival of corticospinal inputs, whereas widespread PV immunoreactivity in dorsal horn neurons marks the attainment of a mature pattern of PV expression. The extent to which expression of PV immunoreactivity can be taken to indicate landmarks in human development will be discussed.

Embryonic motoneurones grafted into the spinal cord of an adult rat can innervate a muscle.

We have previously shown that motoneurone-like cells from embryonic grafts survive and migrate into the host neuropil of adult rat spinal cord, depleted of some of its own motoneurones. We moreover demonstrated that a muscle, when connected at the site of the graft to the spinal cord of the host by its own nerve, was reinnervated by motoneurones that could be identified by retrograde labelling with HRP [11]. However, it was not clear whether these retrogradely labelled motoneurones were of graft origin. In this study we combined the use of an embryonic marker with retrograde labelling to demonstrate that grafted neurones of embryonic origin can indeed innervate a soleus muscle implant. Embryonic donor cells were labelled with bromodeoxyuridine (BrDU) by its incorporation into replicating DNA during neurogenesis. The nuclei of grafted cells were then identified in host cords by immunocytochemistry, visualising the BrDU positive nuclei with the fiuorophore Texas Red, while the fluorescent dyes Fast Blue and Diamidino Yellow were used for retrograde labelling. Examination of frozen spinal cord sections by fluorescence microscopy, at wavelengths appropriate to each fiuorophore, showed that about 12% of the neurones innervating the muscle implant also contained detectable amounts of BrDU and therefore were of graft origin.

Expression of cholinergic phenotype by embryonic ventral horn neurons transplanted into the spinal cord in the rat.

Solid grafts of E12 embryonic spinal ventral horn were transplanted into motoneuron-depleted adult lumbar spinal cord in the rat. A muscle was implanted parallel to the vertebral column with its nerve inserted into the lumbar cord at the site of transplantation so as to provide a target for innervation by the grafted neurons. Previous retrograde labelling studies have shown that modest numbers of grafted motoneuron-like cells participate in the muscle's reinnervation and these are often found outside the graft within the host spinal cord. However, Nissl stained sections show that larger numbers of neurons survive within tissue recognisable as being of graft origin. In this study we have examined the expression of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) by neurons within the graft. These enzymes are involved in cholinergic neurotransmission and are characteristic of motoneurons. Thirty-four to seventy days following transplantation the grafts contained numerous neurons with acetylcholinesterase (AChE) activity. Different patterns of AChE staining were observed which probably reflected the degree of differentiation and maturation within the graft. AChE positive neurons were found in isolation or in groups resembling developing motor pools. Most of the AChE-positive neurons appeared immature with scant cytoplasm. However, neurons could be found which appeared relatively mature with a regularly shaped nucleus, prominent nucleolus and Nissl bodies. The grafts contained few AChE-positive axons and no dense plexuses of varicose fibres around the neurons such as are found around motoneurons in the mature ventral horn. Comparisons between the size of AChE-positive neurons in the graft and the size of AChE-positive neurons in the developing ventral horn found that the size of grafted neurons to be intermediate between the sizes of spinal motoneurons at E19 and P0. Far fewer grafted neurons were found to be immunoreactive for choline acetyltransferase (ChAT) than histochemically reactive for AChE. This was consistent with our findings in the spinal cord during normal development where we found that fixation and staining procedures which labelled adult motoneurons failed to reliably demonstrate ChAT immunoreactivety in normal motoneurons prenatally, although AChE histochemical reactivity could be demonstrated as early as E16. We conclude that the grafts contain numbers of immature motoneurons which fail to proceed beyond a certain stage of development, perhaps because of a failure to form appropriate efferent and afferent connections.

Axotomy induces NADPH diaphorase activity in neonatal but not adult motoneurones.

Rat sciatic motoneurones do not normally express NADPH diaphorase activity. However, after cutting the sciatic nerve in the mid-thigh of neonates and applying the retrograde tracer fast blue, such reactivity was found colocalized with fast blue in about one-fifth of sciatic motoneurones 4 days following the lesion. This proportion of sciatic motoneurones expressing NADPH diaphorase activity remained approximately constant for 14 days after axotomy during which time extensive motoneurone death occurred. Many NADPH diaphorase-positive motoneurones appeared shrunken with degenerative changes to the nucleus. In contrast, the same lesion in adult animals failed to induce any reactivity or cause motoneurone death 4-28 days after axotomy. Induction of nitric oxide synthesising capability may play a role in the death of target-deprived immature motoneurones.

Retraction of muscle afferents from the rat ventral horn during development.

The postnatal reorganization of rat proprioreceptive muscle afferent spinal terminal fields was explored by labelling transganglionically afferents from extensor digitorum communis with cholera toxin B sub-unit at different ages. Immunocytochemistry revealed labelled afferents in all segments examined (C4-T2) as well as retrogradely labelled motoneurones (C5-T1). Dorsal horn innervation appeared similar at all ages, but there were striking changes in the ventral horn. Many afferent boutons were seen closely apposed to labelled motoneurone proximal dendrites at postnatal day 7 (P7) and P14, but in the adult such contacts were almost entirely confined to distal dendrites. Between P7 and adult, a significant decrease in bouton density was found in the area dorsomedial to the labelled motoneurones that contained labelled dendrites and antagonist motoneurones. This anatomical reorganization may explain both the increasing stretch reflex threshold and its concomitant decrease in magnitude with age, and the reduction in excitatory connections to antagonist motoneurones, previously described in developmental neurophysiological studies.

Reciprocal and Renshaw (recurrent) inhibition are functional in man at birth.

The aims were (1) to determine when in human postnatal development Group Ia reciprocal and Renshaw inhibition can be demonstrated; (2) to explore the relationship between the expression reciprocal inhibition and the disappearance of Group Ia excitatory reflexes between agonist and antagonist muscles. Studies were performed on 99 subjects, aged 1 day to 31 years, of whom 53 were neonates. A longitudinal study was also performed on 29 subjects recruited at birth and studied 3 monthly until 12 months of age. Reciprocal inhibitory and excitatory reflexes were recorded in the surface EMG of contracting biceps brachii (Bi), evoked by taps applied to the tendon of triceps brachii (Tri). Reciprocal excitatory reflexes were recorded in all but one neonate. Reciprocal inhibition was observed in 25% of neonates; evidence is provided that it was likely to have been masked by low threshold reciprocal excitation in the remaining neonates. Reciprocal inhibition was demonstrated in all subjects after 9 months of age. In four neonates there was depression of inhibition of Bi during co-contraction of Bi and Tri implying that Group Ia interneurones may be under segmental and suprasegmental control at birth. Renshaw cells, identified in human postmortem cervical spinal cord by their morphology, location and calbindin D28K immunoreactivity, were present at 11 weeks post-conceptional age (PCA) and by 35 weeks PCA had mature morphological characteristics. In four neonates reciprocal inhibitory responses in Bi disappeared when the tap to Tri evoked its own homonymous phasic stretch reflex, providing neurophysiological evidence for Renshaw inhibition of Group Ia inhibitory interneurones.

Transient expression of calcitonin gene-related peptide immunoreactivity in the ventral horn of the post-natal rat cervical spinal cord.

In addition to the well known expression of calcitonin gene-related peptide (CGRP) immunoreactivity in primary afferent fibers in the dorsal horn and in motoneurons, this study has demonstrated, in rat, transient CGRP immunoreactivity in fine caliber varicose axons throughout the ventral horn and in a group of neuron cell bodies in the medial ventral horn. This was first observed at post-natal day 7 (P7) and had disappeared by P21. Physiological studies in chick embryonic spinal cord have shown that CGRP modulates spontaneous activity during development [Carr, P.A., Wenner, P., 1998. Calcitonin gene-related peptide and effects on spontaneous activity in embryonic chick spinal cord. Dev. Brain Res. 106, 47-55]. Neural activity increases post-natally in rat where it may play a role in refinement of sensorimotor synapses. This activity may also be modulated by CGRP.

Developmental expression of parvalbumin by rat lower cervical spinal cord neurones and the effect of early lesions to the motor cortex.

Expression of calcium binding proteins (CaBPs), increasing neuronal activity and phases of synapse elimination are widely believed to be linked during development. We have employed immunocytochemistry to study the expression of the CaBP parvalbumin (PV) during the postnatal development of the lower cervical spinal cord and investigated how early lesions to the motor cortex, at the onset of corticospinal synaptogenesis, perturb the normal pattern of PV expression. This study confirms previous observations that in normal rats PV-like immunoreactivity is confined to large sensory afferents for at least 10 days postnatally (P10) and that the adult pattern of expression emerges from about P18 and involves mainly dorsal horn neurones. However, the study has also demonstrated a transient wave of expression in ventral horn neurones which reaches a maximum between P14-18 and declines thereafter. Unilateral lesions made at P7 to the forelimb motor cortex, which sends an almost completely crossed projection to the spinal cord, resulted in reduced neuronal expression of PV in the lower cervical spinal cord contralaterally at a range of ages (P14-31). The median ratio of PV positive neurones contralateral/ipsilateral to the lesion in spinal cord segments C7 and C8 was significantly lower (p < 0.01) at 56.0% (34.5-76.8 95% confidence limits, n = 14) than in sham operated controls (99.7%, range 93.7-113.6, n = 5). The lesion affected the transient wave of expression seen in ventral horn neurones during the third postnatal week as well as dorsal horn expression at older ages. We conclude that there is considerable plasticity in PV immunoreactivity during spinal cord development. PV is transiently expressed by ventral horn neurones at an age when movement control is functionally maturing. Early cortical lesions disrupt this transient phase of expression but also alter mature patterns of PV localisation. This suggests a critical role for corticospinal pathways in guiding maturation of segmental spinal cord circuitry.

Expression of nitric oxide synthase by motor neurones in the spinal cord of the mutant mouse wobbler.

The expression of nitric oxide synthase (NOS) has been studied in the spinal cord of the mutant mouse wobbler, a recessive mutation in which there is motor neurone degeneration, using nicotinamide dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. Abnormal NOS positive large neuronal profiles could be found in the ventral born of affected mutant animals but not their unaffected littermate controls. The number of abnormal profiles observed was dependent upon the age of the animal. A small number of these NOS positive large neuronal profiles were seen at the onset of the disease at 3-4 weeks of age, larger numbers were found in animals aged 5-8 weeks coincident with the main period of motor neurone death, whilst in the spinal cords of older animals aged 9-12 months, when motor neurone death is minimal, none were found. These NOS positive profiles seen in younger wobbler mouse ventral horn had a morphology and size similar to that of degenerating motor neurones seen in Nissl stained preparations. It was concluded that these NOS positive profiles were degenerating motor neurones. These observations provide further evidence that induction of nitric oxide synthase expression may play a role in motor neurone death. Though no NOS positive motor neurones were found in the spinal cords of older wobblers increased numbers of NOS positive varicose axons were observed in the ventral horn often forming tangled accumulations on the border of the grey and white matter.

The successful use of fentanyl/fluanisone ('Hypnorm') as an anaesthetic for intracranial surgery in neonatal rats.

This study reports on the successful use of fentanyl citrate and fluanisone ('Hypnorm') anaesthesia for intracranial surgery in neonatal (7-day-old) rats. Provided the anaesthetic was administered subcutaneously, the animals showed a very high survival rate in the short term (81/85, 95%) and showed no ill effects in the long term. The depth of anaesthesia was sufficient to allow the operation to be carried out without the animal reacting to any painful stimuli. However, the animals did make random movements during the period of surgical anaesthesia which were not related to any painful stimuli. Although these movements did not interfere with the surgery performed here, such movements would interfere with operations requiring greater precision, such as the localized micro-injection of neural tracers.

Elimination of muscle afferent boutons from the cuneate nucleus of the rat medulla during development.

There is developmental refinement of the proprioreceptive muscle afferent input to the rat ventral horn. This study explored the extent to which this occurs in the medulla. Muscle afferents were transganglionically labeled from the extensor digitorum communis forelimb muscle with cholera toxin B subunit. Tracer amounts and transport times were adjusted for animal size. Immunohistochemistry revealed tracer localization in the medulla and dorsal root ganglia. Labeled muscle afferent boutons were counted in the cuneate nucleus between postnatal days 7 and 42, during which time a large decrease in the density of labeled boutons was observed qualitatively. Localization of input to dorsolateral parts of the nucleus remained broadly the same at different ages, although disappearance of a marked innervation of ventromedial regions in more caudal sections was observed. Bouton counts were corrected for growth of the medulla with age, and any spread of tracer to adjacent muscles indicated by counts of labeled dorsal root ganglion neurons. There was a statistically significant, approximately 40% reduction in the number of muscle afferent boutons in the cuneate nucleus during this developmental period. Previous studies suggest that perturbations to the corticospinal input during a developmental critical period influence the eventual size of the muscle afferent input to the ventral horn. Corticocuneate fibers invade the nucleus during the same period and may influence reorganization of its muscle afferent input, making it another potential site for aberrant reflex development in cerebral palsy.

Brainstem motor nuclei respond differentially to degenerative disease in the mutant mouse wobbler.

Degenerative motoneurone diseases, whether in humans, animals, or transgenic mouse models, do not affect all types of motoneurone to the same degree. Understanding the relative differences in vulnerability of certain motor pools may be the key to developing therapies. Expression of calbindin (CB) and parvalbumin (PV) immunoreactivity, which are potentially neuroprotective calcium-binding proteins, and NADPH-diaphorase (NADPH-d) histochemical reactivity, a marker for neurodegeneration, was studied in brainstem sections from mutant wobbler mice and their normal littermates during the motoneurone degeneration phase (3-8 weeks of age). The motor trigeminal and facial nuclei reacted in a manner previously observed in spinal somatic motoneurones in the wobbler. Many motoneurones expressed moderate NADPH-d reactivity, correlated with the appearance of vacuolated motoneurones in Nissl-stained sections. This was not observed in littermate controls. Motoneurone counts from Nissl-stained sections from 14-month-old wobblers and littermates revealed significantly fewer (approximately 27%) motoneurones in the trigeminal nucleus of wobblers. In contrast, the wobbler hypoglossal nucleus contained neither vacuolated nor NADPH-d reactive motoneurones. However, expression of CB immunoreactivity by the majority of wobbler hypoglossal motoneurones was observed but not in littermate controls or in any other motor nucleus. Counts in older animals showed a smaller but still significant difference in motoneurone number between wobblers and controls (approximately 9% reduction). Finally, the wobbler abducens nucleus displayed neither vacuolated neurones, nor NADPH-d reactivity nor CB immunoreactivity. Motor nuclei innervating extraocular muscles appear to be protected in many forms of motoneurone disease in man and other species. However, there were still markedly fewer abducens motoneurones in the old wobblers compared to controls (approximately 29% reduction). Sparing of oculomotor neurones in other diseases has been attributed to their relatively high PV expression, which we also observed in the abducens nucleus of both wobblers and littermates, and to a lesser extent in the other motor nuclei too. In conclusion, our results suggest that, in the wobbler mouse, motoneurone degeneration may occur without overt signs such as cell body vacuolation and NADPH-d expression. Induced CB expression may be neuroprotective but that constitutive expression of PV may not.

Observations on the development of transplanted embryonic ventral horn neurones grafted into adult rat spinal cord and connected to skeletal muscle implants via a peripheral nerve.

Embryonic spinal cord grafts from 12-day-old rat embryos were placed into the lumbar spinal cord of adult rats depleted of sciatic motoneurones by a neonatal nerve injury. A soleus muscle was removed from the leg and implanted paravertebrally, the proximal end of its nerve connected to the graft site. Fluorescent retrograde tracers injected into the soleus implant, 37-64 days postoperatively, labelled neurones that had grown axons to the muscle. Approximately one-fifth of retrogradely labelled neurons were within the graft; however, the majority were found within the host spinal cord close to the graft. These included large neurons within the motoneurone-depleted dorsolateral ventral horn. In control experiments a muscle and nerve were implanted but no embryonic tissue grafted. Significantly fewer neurones were labelled. In some animals, one tracer was injected into the soleus muscle whilst another was applied to the cut sciatic nerve ipsilateral to the graft site. No neurones were found to project axons to both targets. In animals that received grafts prelabelled with bromodeoxyuridine (BrDU) some neurones were found to be both BrDU positive and retrogradely labelled from the soleus implant. These were most frequently within the motoneuron-depleted ventral horn ipsilateral to the graft. Thus, grafted neurones may migrate to an appropriate location within the host neuropil. Acetylcholinesterase (AChE) histochemistry showed the graft site contained immature but AChE-positive neurones. Some regions of host ventral horn contained unusually few AChE-positive nerve fibres and occasional large AChE-positive neurones, some of which were also retrogradely labelled from the implanted muscle. Studies of implanted soleus 21-90 days after transplantation showed that muscle fibres, after initial degeneration, regenerated displaying differing phenotypes, presumably under the influence of new motor innervation.

N-methyl-D-aspartate receptor blockade during development induces short-term but not long-term changes in c-Jun and parvalbumin expression in the rat cervical spinal cord.

During postnatal development, N-methyl-D-aspartate receptor (NMDA-R) expression progressively decreases in ventral and deep dorsal horns. This transient expression might play a role in activity-dependent development of segmental circuitry. NMDA-Rs were blocked unilaterally in the lower cervical spinal cord using Elvax implants that released the NMDA-R antagonist MK-801 maximally over a 2-week period from postnatal day 7 (P7) onward. At P14, the ratio of c-Jun immunoreactive motoneurons ipsilateral/contralateral to the implants was significantly increased and the ratio of parvalbumin immunoreactive neurons decreased, compared to control implants. However, at P84, MK-801-treated and control spinal cords appeared the same. Therefore, NMDA-R blockade during development only transiently altered expression of activity-dependent proteins in the spinal cord, unlike lesions to the developing motor cortex, which we have previously shown to have a permanent effect.

The effect of a peripheral nerve lesion on calbindin D28k immunoreactivity in the cervical ventral horn of developing and adult rats.

Expression of calbindin D28k (CB) immunoreactivity by putative Renshaw cells is substantially downregulated by sciatic motoneuron axotomy in the adult rat. The present study investigated the effect of median and ulnar nerve lesion at different ages on ventral horn CB immunoreactivity 7 days after the injury to see whether similar results were obtained in the cervical cord and during development. Two major differences were observed. First, axotomy induced CB immunoreactivity in some motoneurons, confirmed by retrograde labeling of the injured neurons with fast blue (FB). Observation of fluorescent phagocytic microglia revealed that some motoneuron death occurred following lesions at postnatal day 2 (P2) and P7, but not at P21 or P63. A significantly higher proportion of remaining FB labeled motoneurons expressed CB following lesion at P2 (mean 33% +/- 7.6 SD) and P7 (30.6% +/- 5.2) than at P28 (14.0% +/- 1.9). Second, CB expression by putative Renshaw cells was not significantly downregulated ipsilateral to the lesion. CB immunofluorescent putative Renshaw cells were counted in sections containing FB labeled motoneurons. No consistent differences in the numbers of Renshaw cells ipsilateral and contralateral to the lesion were found at any age. To confirm that these neurons really were Renshaw cells, the mediators of recurrent inhibition to cholinergic motoneurons, we employed double-immunofluorescence labeling with confocal microscopy. The group of CB immunopositive neurons located among the converging ventral roots in the cervical cord were closely apposed by many axon terminals immunoreactive for (i) vesicular acetylcholine transporter and (ii) cholera toxin B localized to motor axon collaterals by injection of this tracer into a distal forelimb muscle. We conclude that motoneuron axotomy need not always downregulate CB expression in associated Renshaw cells. In addition, some brachial motoneurons respond to axotomy by expressing CB.

Gephyrin-like immunoreactivity is a marker for growing axons in the central nervous system of the immature rat.

The membrane-associated protein gephyrin can form part of the glycine receptor complex at inhibitory synapses. This study provides evidence for gephyrin localisation in the developing axons of the rat brain and spinal cord, with particular reference to the corticospinal tract. Using a well-characterised monoclonal antibody (MAb 7a) gephyrin-like immunoreactivity was found expressed by growing axons, disappearing as these axons became myelinated. Immunoelectron microscopy localised the antigen to the interior of small, unmyelinated axons in the dorsal funiculus of young rats. Western blot analysis of cervical spinal cord from different post-natal ages only detected one immunoreactive band at ages when immunohistochemistry revealed gephyrin-like immunoreactivity in both the grey matter and corticospinal tract. Furthermore, the molecular weight of this band corresponded to that of gephyrin, suggesting it is present both at synapses and in axons. The known tubulin binding ability of the gephyrin may have a role in stabilisation of microtubules in growing axons.

Changes in retrogradely labelled neurones in the red nucleus and cortex after depletion of motoneurones by axotomy in neonatal rats.

Following nerve injury to the sciatic nerve in neonatal rats many motoneurones die, so that the target of the rubrospinal and corticospinal cord is altered. Retrograde labelling of neurones terminating in this region shows that, in the red nucleus as well as cerebral cortex, there is a significant reduction in labelled neurones on the side contralateral to the nerve injury. Thus neurones in the brain that give rise to descending fibres are influenced by the altered environment in the spinal cord following axotomy at birth.