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In this study the effects of hypergravity were analyzed on cerebellar Purkinje cells during early development in rats. The cerebellum is a key structure in the control and the adaptation of posture and anti-gravity activities. This holds particularly when external conditions are modified. Three groups of rats were conceived, born and reared in hypergravity (2 g). At postnatal day 5 (P5), P10 or P15, they were exposed to normal gravity and at P40, the cerebella were investigated on the expression of calbindin-D28k and inositol-3-phosphate (IP3) in Purkinje cells. Control animals were bred in the same conditions but at I g. Immunoreactivity of Purkinje cells was studied in lobules III and IX of the vermis. Lobule IX of the vermis is one of the targets of primary otolithic vestibular projections, and lobule III served as a control, being much less related with vestibular inputs. The results show that hypergravity induces a decrease in calbindin and IP3 labeling in 20% of Purkinje cells of lobule IX without any change in lobule III. Animals transferred from 2g to 1 g at P5 or PI 0 showed the most pronounced effects and much less at P15. This study demonstrates that early development of the cerebellum is highly sensitive to changes in gravity. Ages until P10 are critical for the development of vestibulo-cerebellar connections, and in particularly the calcium signaling in Purkinje cells. (c) 2005 Published by Elsevier Ireland Ltd.

Transection of the sciatic nerve in adult rats induces drastic changes in hindleg muscles. Earlier, we demonstrated that the reinnervated soleus (SOL) muscle, 21 weeks after a transection mainly contains type II fibers. This is in striking contrast to normal muscle, which consists to 80% of type I muscle fibers. Also we observed 13.9% of the fibers to be polyneurally innervated. The problem of the present study is whether these changes are reversible after longer survival periods. Therefore, the SOL was studied 60 weeks after transection and reconstruction by an autologous nerve graft. In six rats, we studied muscle fiber distributions by monoclonal antibodies, and innervation patterns by cholinesterase staining and AgNO3 impregnation.Still at 60 weeks, only 20% of the muscle fibers are of type I and this is similar to results at 21 weeks, indicating that no recovery to the normal has been reached by that age. Furthermore, 20% of the endplates in the reinnervated SOL were polyneurally. innervated, but we also observed this in 10% of the endplates an the control side. These increases, compared to data at 21 weeks, are interpreted as an aging effect. © 2005 Elsevier GmbH. All rights reserved.

Disruption of peripheral nerves due to trauma is a frequently Occurring clinical problem. Gaps in the nerve are bridged by guiding the regenerating nerves along autologous grafts or artificial guides. This review gives an overview oil the different methods of nerve repair techniques. Conventional suturing techniques are discussed Lis well as the use of e.g. biological, synthetic, non-degradable or degradable nerve guides. Functional assessment showed that repair of a gap with a bio-degradable guide is superior to that with autologous grafts. But still, long lasting changes were observed in the Sciatic Function Index (SFI), abnormal walking patterns, disturbed Electro Myo Graphic (EMG) patterns, next to shifts in the histochemical properties of the muscles and longlasting abnormalities in neuromuscular contacts. These phenomena are explained by an at-random reinnervation. When transecting the nerve at young ages, this did not lead to enhanced recovery. Rearing rats operated at adult age in an enriched environment, also had no beneficial effect. Future research should aim at developing longer guides, possibly lined with Schwann cells, or additives, improving specific reinnervation of the former target areas. (C) 2003 Elsevier Ltd. All rights reserved.

The precise outgrowth and arborization of dendrites is crucial for their function as integrators of signals relayed from axons and, hence, the functioning of the brain. Proper dendritic differentiation is particularly resonant for Purkinje cells as the intrinsic activity of this cell-type is governed by functionally distinct regions of its dendritic tree. Activity-dependent mechanisms, driven by electrical signaling and trophic factors, account for the most active period of dendritogenesis. An as yet unexplored trophic modulator of Purkinje cell dendritic development is corticotropin-releasing factor (CRF) and family member, urocortin, both of which are localized in climbing fibers. Here, we use rat organotypic cerebellar slice cultures to investigate the roles of CRF and urocortin on Purkinje cell dendritic development. Intermittent exposure (12 h per day for 10 days in vitro) of CRF and urocortin induced significantly more dendritic outgrowth (45% and 70%, respectively) and elongation (25% and 15%, respectively) compared with untreated cells. Conversely, constant exposure to CRF and urocortin significantly inhibited dendritic outgrowth. The trophic effects of CRF and urocortin are mediated by the protein kinase A and mitogen-activating protein kinase pathways. The study shows unequivocally that CRF and urocortin are potent regulators of dendritic development. However, their stimulatory or inhibitory effects are dependent upon the degree of expression of these peptides. Furthermore, the effects of CRF and urocortin on neuronal differentiation and re-modeling may provide a cellular basis for pathologies such as major depression, which show perturbations in the expression of these stress peptides.

Corticotropin-releasing factor (CRF)-like proteins act via two G-protein-coupled receptors (CRF-R1 and CRF-R2) playing important neuromodulatory roles in stress responses and synaptic plasticity. The cerebellar expression of corticotropin-releasing factor-like ligands has been well documented, but their receptor localization has not. This is the first combination of a light microscopic and ultrastructural study to localize corticotropin-releasing factor receptors immunohistologically in the developing rat cerebellum. Both CRF-R1 and CRF-R2 were expressed in climbing fibres from early stages (post-natal day 3) to the adult, but CRF-R2 immmunoreactivity was only prominent throughout the molecular layer in the posterior cerebellar lobules. CRF-R1 immunoreactivity was concentrated in apical regions of Purkinje cell somata and later in primary dendrites exhibiting a diffuse cytoplasmic appearance. In Purkinje cells, CRF-R1 immunoreactivity was never membrane bound post-synaptically in dendritic spines while CRF-R2 immunoreactivity was found on plasmic membranes of Purkinje cells from post-natal day 15 onwards. We conclude that the localization of these receptors in cerebellar afferents implies their pre-synaptic control of the release of corticotropin-releasing factor-like ligands, impacting on the sensory information being transmitted from afferents. Furthermore, the fact that CRF-R2 is membrane bound at synapses, while CRF-R1 is not, suggests that ligands couple to CRF-R2 via synaptic transmission and to CRF-R1 via volume transmission. Finally, the distinct expression profiles of receptors along structural domains of Purkinje cells suggest that the role for these receptors is to modulate afferent inputs.

Summary The functional recovery after peripheral nerve lesions is generally poor. We studied whether changes in muscles after reinnervation might explain such disappointing results. The functional recovery after peripheral nerve lesions is generally poor. Changes in muscle morphology and neuromuscular innervation might partly explain this lack of compensation. In order to test this hypothesis, we studied muscular differentiation in the soleus, gastrocnemius and tibialis anterior muscles at 7, 15 and 21 weeks after a sciatic nerve lesion in adult rats. In the gastrocnemius and tibialis muscles the percentages of type II muscles fibres were decreased at 7 and 15 weeks but at 21 weeks they again approached normal values. The soleus muscle, however, was permanently decreased in size and this muscle, in contrast to the normal soleus muscle, contained mainly type II fibres. The morphology of the endplates showed distinct stages of degeneration and reinnervation. Two weeks after denervation, in rats in which reinnervation was prevented, all 3 muscles contained considerable numbers of morphologically abnormal endplates and, after 7 weeks, no endplates were detected. During reinnervation, endplates showing signs of acetylcholinesterase activity were observed in all 3 muscles from 7 weeks. At later ages a shift towards morphologically normal endplates occurred, but complete recovery was not observed. Endplates in all 3 muscles were polyneurally innervated at 7 weeks. Although these percentages decreased over age, polyneural innervation was still present at 21 weeks. We conclude that the changes in the distribution of fibre types, abnormal endplate morphology and polyneural innervation may in part explain the poor functional recovery after peripheral nerve lesions.

Polyneural innervation was studied in the psoas muscle in developing rats from P4 till P25 and at adult age, with the combined silver-acetylcholinesterase technique. Nerve endings were counted, and endplates were measured. These data were compared with such data in the human. The end of polyneural innervation in the rat (around P20) and in the human (around 12 weeks postterm age) in both cases coincides with a transformation in motor behavior and postural control. The rat's psoas muscle at early stages is less heavily innervated than this muscle in the human. Up to three axons per motor end-plate were counted at P4, but in the human up to five axons at 25 weeks of post menstrual age. This difference might be related to the lower percentage of type I muscle fibers in the rat. (C) 1998 John Wiley & Sons, Inc.

The ultrastructural characteristics of identified dendrite bundles in the rat spinal cord were analyzed following retrograde tracing from the soleus muscles. Dendrite bundles are arranged in networks that an heavily interconnected by means of gap junctions. The bundles are formed by at least 10 crossing dendrites travelling in different focal planes. In between dendrites, elongated gap junctional complexes are frequently found. Dendrite lamellar bodies, recently described to occur in relation with gap junctions in the central nervous system were not observed in the present study. (C) 1998 Elsevier Science B.V.

The anatomical distribution and the content of dendrite bundles were studied in the spinal cord of adult rats. We injected retrogradely transported Cholera Toxin subunit B into muscles in order to label motoneurons and their dendrites. Twenty-one muscles in the trunk and in extremities were selected which are involved in a variety of motor tasks. Reconstructions were made from consecutive sections and the organization of dendrites was studied. In addition, the distributions of fiber types in the muscles were estimated on the basis of ATPase and succinic dehydrogenase-stained sections. Dendrite bundles are confined to pools of muscles which have postural tasks. Two different types can be distinguished. In the first type, longitudinal dendrite bundles extend over the length of the pool and also transversal bundles occur. Such pools innervate the axial musculature, and the soleus and flexor carpi ulnaris muscles in the extremities. These muscles contain relatively high percentages of slow twitch type I muscle fibers. In the second type, the occurrence of bundles is confined to a few zones within the pool. Such pools innervate muscles which are involved in postural tasks but in addition they also subserve other movement tasks. Distinct regions within these muscles are characterized by high percentages of type I muscle fibers.

Cerebellar hemispherectomy before the 10th day in rats leads to extensive neuronal remodelling. In the present study the problem was studied whether such early lesions also have effects on the maturation of the soleus and the extensor digitorum longus muscles in the hindleg as well as on the formation of dendrite bundles from motoneurons innervating the soleus muscle. Results indicate consistent left-to-right differences in the numbers of muscle fibres but no differences in muscle differentiation. Dendritic bundles of soleus motoneurons, at the side ipsilateral to the cerebellar lesion are absent or less conspicuous in comparison to the contralateral side or to those bundles in normal rats. Cerebellar lesioning at the 30th day does not affect dendritic bundles.

After cerebellar hemispherectomy before the 10th day of life aberrant cerebello-rubral projections develop from the nuclei in the remaining hemisphere, which terminate on the ipsilateral side. These aberrant fibres stem from separate parent cells in the lateral and interposed nuclei. The problem addressed by the present study was whether the number of neurones in these nuclei is increased after early cerebellar hemispherectomy. Results demonstrate that the number of neurones was increased by 50-60%. The volumes of the nuclei were also increased (14-31%) but much less so, which indicates a relative decrease in neuropil per neuron.

Functional recovery after transection of the sciatic nerve in adult rats is poor, probably because of abnormalities in reinnervation. Denervation and reinnervation patterns were studied morphologically in the lateral gastrocnemius (LGC), tibialis anterior (TA), and soleus (SOL) muscles for 21 weeks after nerve transection (motor endplates by acetylcholinesterase staining; nerves by silver impregnation). Motor endplates in the TA showed improving morphology with age, and, at 21 weeks, three-quarters of these were normal. Poorest recovery was observed in the SOL, as, at 21 weeks, only one-third of the motor endplates had a normal morphology. Polyneuronal innervation initially was more pronounced in the SOL, but, at 21 weeks, 10% of the motor endplates in all three muscles were still polyneuronally innervated. Our results indicate important differences in the reinnervation of these three hindleg muscles, and, even at 5 months, abnormalities were still present. These factors may in part explain the abnormal locomotion in rats as well as the limited recovery of function observed clinically in humans after nerve transection. (C) 2002 Wiley Periodicals, Inc.

Reinnervation after peripheral nerve transections generally leads to poor functional recovery. In order to study whether changes in muscles might be a contributing factor in this phenomenon we studied muscle morphology and fibre type distributions after sciatic nerve transection in the rat hind limb. Proximally, before the bifurcation in the tibial and common peroneal nerve, a 12 mm segment of the sciatic nerve was resected, reversed and re-implanted as an autologous nerve graft. After survival periods of 7, 15 and 21 weeks the lateral gastrocnemius, tibialis anterior and soleus muscles were dissected, stained with mATP-ase, and fibre type distributions were studied. In addition, numbers of muscle fibres were counted, and cross sectional areas were calculated. After 7 weeks, cross sectional areas were decreased in all muscles. In the gastrocnemius and tibialis anterior muscles the fibre number remained unaltered but the hypotrophy had been reversed at later ages. The number of muscle fibres in the soleus muscle remained decreased over the entire period of observation. The percentages of type II fibres in the gastrocnemius and tibialis anterior muscles were decreased at 7 and 15 weeks but these again approached normal values at 21 weeks. The type I fibres, however, remained arranged in groups. In the soleus muscle a large increase in the percentage of type II muscle fibres was observed and this remained until 21 weeks. We conclude that a non-selective reinnervation and later readjustments by regression of polyneural innervation may in part explain the changes in distributions of various fibre types.

The effects of lesions in the sciatic nerve were studied in adult rats, In the left hindleg, a segment 12 mm long was resected from the proximal part of the nerve, before the bifurcation into the peroneal and tibial nerves, This segment in a reversed orientation was used as a nerve graft. EMG patterns in the tibialis anterior and the gastrocnemius muscles at both sides were recorded during locomotion in six rats after recovery periods varying from 15 to 21 weeks. The specificity of axonal outgrowth was studied in nine rats by retrogradely labeling the motoneurons with unconjugated Cholera Toxin subunit B (CTB) after injections into the gastrocnemius, the soleus, and the tibialis anterior muscles at both sides. EMG patterns at the operated side were irregular and we often observed coactivation of the gastrocnemius and tibialis anterior muscle. Moreover, burst activity was badly adjusted to the phases of the stepcycle, Retrogradely labeling indicated that the pools of motoneurons innervating the respective muscles at the left side had increased in volume. Neuronal diameters were slightly decreased but a considerable decrease was observed in dendritic branching and dendrite bundles in the pools of the SOL and in the GC were absent. No consistent trends in neuronal numbers at the affected side in comparison to the right side were detected. We conclude that axons, sprouting from the proximal stump of the sciatic nerve, innervate the muscles aselectively and that the motoneurons of origin maintain their original activation pattern. (C) 2000 academic Press.

Motoneuronal pools of muscles that subserve postural tasks contain dendrite bundles. We investigated in the rat the development of these bundles in the pools of the long back muscles and related this to postural development. Motoneurons and their dendrites were retrogradely labeled by injecting unconjugated cholera toxin subunit B (CTB) into the muscles of 54 normal rats from birth until adulthood and into 18 rats that were vestibularly deprived from the 5th postnatal day (P5). Dendrite bundles coursing in a transverse direction already occurred at P1. From P4, the first longitudinal bundles could be observed, but the major spurt in development occurred between P6 and P9, when conspicuous bundles developed coursing in rostro-caudal and tranverse directions. This is the age when rats become able to stand freely and walk a few steps. Around P20, the dendrite bundles attained their adult characteristics. Vestibular deprivation by plugging both semicircular horizontal canals did not lead to a retarded development of dendrite bundles nor to a changed morphology. This finding is remarkable, as behavioral analysis showed a delay in postural development by about 3 days. We hypothesize that dendrite bundles in the pools of the long back muscles function to synchronize the motoneurons in different spinal cord segments.

The aim of this study was to compare functional nerve recovery after reconstruction with a biodegradable p(DLLA-epsilon-CL) nerve guide filled with modified denatured muscle tissue (MDMT), or an autologous nerve graft. We evaluated nerve recovery using walking track analysis (measurement of the sciatic function index [SFI]) and electrostimulation tests. Functional nerve recovery after reconstruction with a biodegradable p(DLLA-epsilon-CL) nerve guide filled with MDMT was faster when compared with nerve reconstruction using an autologous nerve graft. We conclude that in case of a short nerve gap in the rat, reconstruction can best be carried out using a p(DLLA-epsilon-CL) biodegradable nerve guide filled with MDMT.

Stimulation of the inferior alveolar nerve (IAN) elicits the so-called jaw-opening reflex (JOR) which is associated with excitation of the jaw-opening muscles and inhibition of the jaw-elevator muscles. High threshold afferents with their cell bodies located in the trigeminal ganglion (tg) are responsible for the activation of the jaw-opening muscles, whereas low threshold afferents with their cell bodies in the mesencephalic trigeminal nucleus (Me5) are involved in the inhibition of the jaw-elevator muscles (Dessem, Iyadurai & Taylor, 1988). The central neuronal routing of the JOR is not fully understood yet. High threshold short latency JOR activity is mediated through a disynaptic pathway involving interneurones in the trigeminal brainstem nuclear complex (TBNC). However, the location of interneurones involved in high-threshold long latency JOR activity is still disputed. This study indicates that the supratrigeminal area (Su5), adjacent to the Me5, contains elements of a neuronal pathway involved in reflex activation of the jaw-opening muscles.

The double labelling technique has been applied to study the quantitative relations between aberrant and normal cerebello-rubral projections in rats hemicerebellectomized on the 2nd, 5th, 10th, 20th or 30th day after birth, respectively. In the group of rats lesioned on the 2nd day about 30% of cells in the lateral nucleus and 20% in the interpositus nuclei are labelled retrogradely by the fluorescent tracer injected in the aberrant ipsilateral projection area. These values decreased in the groups of rats operated on the 5th or 10th day and reached values of about 1–2% in rats lesioned on the 20th or 30th day. These values have been also found in control rats. Both in experimental rats and in control rats less than 0.5% of cells are double labelled from both projection areas. This implies that the aberrant cerebello-rubral fibres stem from separate parent cells and are not collaterals from normally projecting fibres.

Early animal experiments suggested that brain lesions at an early stage of brain maturation have less severe effects on later behavior than similar lesions in adult animals. From this so-called Kennard principle the notion has been derived that the capacity for functional compensation after brain trauma is greater for the immature than for the mature brain. However, more recent investigations — often involving a meticulous analysis of short and long term behavioral sequelae — rather provides evidence against the existence of any simple relation between early lesions and increased compensational capacity.

During the early stages of mammalian ontogeny muscle fibres are innervated by more than one axon. This polyneural innervation is replaced by mononeural innervation in the course of development. The regression of polyneural innervation in the psoas muscle in the human is the topic of the present study. Innervation patterns were studied in fetuses from 15 1 2 weeks of post menstrual age (PMA) and in babies until 80 weeks PMA (40 weeks after term age) and compared to data from two adults. Motor endplates were stained by a combined acetylcholinesterase stain. Innervation patterns and motor endplate morphology were studied and the sizes of endplates were measured. As a main result of our study polyneural innervation of the psoas muscle remains at a level of about 2 endings per endplate (range 1–5 terminals) until 18–25 weeks PMA and decreases thereafter. From 52 weeks PMA (12 weeks post term) onwards, muscle fibres are predominantly mononeurally innervated. During development the morphology of the terminal patterns of the nerve endings becomes more complex and the size of endplates increases, implying that the adult pattern of muscle innervation is reached at the age at which a major functional transformation in the neurobehavioural repertoire occurs (i.e. the end of the second and the beginning of the third month).