Localization and functional roles of corticotropin-releasing factor receptor type 2 in the cerebellum.

The corticotropin-releasing factor (CRF) type 2 receptor has three splice variants alpha, beta, and gamma. In the rodent brain only CRFR2alpha is present. In the cerebellum, CRF-R2alpha has two different isoforms: a full-length form (fl) and truncated (tr). Both forms CRF-R2 have a unique cellular distribution. During postnatal cerebellar development, the expression patterns of tr and fl isoforms are changing. This suggests that, CRF and the related peptide urocortin (UCN) could play distinct roles in the immature and adult cerebellum, acting via different receptors subtypes. This review focuses on differences in the distribution of each isoform of CRF-R2 in view of their relationship to CRF and UCN release sites and their possible functional implications. Moreover, it includes novel findings of molecular pathways activating CRF-R2 isoforms through which CRF and UCN excert their specific actions.

Neural compensation after early lesions: a clinical view of animal experiments.

The widely held view, known as Kennard's principle, that lesions in the human central nervous system in the perinatal period are followed by better compensation than equivalent lesions sustained at a later age is under challenge from results of animal experiments. Experiment in rats have demonstrated, contrastingly, that lesions at early stages generally lead to more behavioral impairments than lesions of a similar size and location at adult stages. It also has been demonstrated that early lesions often lead to extensive neural reorganizations. Certain experiments even indicated that the extent of these reorganizations (e.g., after lesions in the motor cortex or the cerebellum) is inversely related to the degree of behavioral compensation. The question arises, why there is a discrepancy between the results in the human, suggesting enhanced functional recovery after lesions at perinatal ages, and those from animal experiments? One explanation is that lesions in animal experiments often are much larger than those occurring in the human. Secondly, the timing of the insult relative to the brain development seems to be critical. For each neural system a specific relation seems to exist between the timing of the insult, the degree of the neuronal reorganization and the behavioral consequences. Results from experimental research have suggested that the recruitment of stem cells in the brain after early lesions in certain regions might play a role in the repair of lesioned areas and also, that stem cells might play a role in the rescue of compromised cells. Ultimately such therapies might help to alleviate the consequences of perinatal brain lesions in the human.

The glutamate receptor delta 2 in relation to cerebellar development and plasticity.

Understanding what are the mechanisms that strengthen, stabilize and restrict synaptic innervation is a relevant topic in glutamate receptor delta 2 (GluRdelta2)-related research. It also involves targeting and selection of afferent input during formation of the neuronal circuitry in the cerebellar cortex and its functioning. This review will focus on the role of GluRdelta2, one of the main players in this field. Special emphasis will be placed on the processes that regulate the rapid translocation from climbing fibres to parallel fibres of GluRdelta2 and the role of GluRdelta2 in the reduction of supernumerary climbing fibre contacts on a single Purkinje cell. Furthermore, GluRdelta2 knockout mice show ataxia and impaired motor coordination, suggesting that the presence of GluRdelta2 plays an important role in controlling cerebellar functioning.

Functional recovery after transection of the sciatic nerve at an early age: a pilot study in rats.

It is often clinically noted that peripheral nerve transections in infants and very young children seem to show better functional recovery after nerve repair compared with adult patients. There can be several reasons for this (e.g. outgrowing axons might have maintained their potential to locate their genuine target muscle, the distance from the lesion site to the target organ is shorter, or there is enhanced compensational capacity of central brain areas at early stages of development). We decided to study the effects on walking performance of sciatic nerve transection and repair in nine rats at the youngest age at which appreciable numbers of motoneurons survive transaction (10th day of life). The follow-up period was 52 weeks. Walking movements were recorded on video and performance was qualitatively assessed. The stance factor and the sciatic function index were calculated and abnormalities such as automutilation of the toes or flexion contractures were noted. Postmortem inspection aimed to examine the continuity of the regenerated nerve and the presence of neuromas. Nine days after the transection and nerve repair, walking performance in all rats was close to the level in rats that had not undergone the procedure. Exorotation of the hind leg on the operated side was observed in only two rats. However, 20 days after lesioning the walking performance started to deteriorate, and walking patterns in all but one rat showed severe disability until the end of the observations. Only one out of the nine animals walked relatively undisturbed, with a sciatic function index approaching normal values but with an abnormal stance factor. We conclude that a sciatic nerve transection in rats aged 10 days leads to long-term impairments in walking patterns.

The dynamic developmental localization of the full-length corticotropin-releasing factor receptor type 2 in rat cerebellum.

Corticotropin releasing factor receptor 2 (CRF-R2) is strongly expressed in the cerebellum and plays an important role in the development of the cerebellar circuitry, particularly in the development of the dendritic trees and afferent input to Purkinje cells. However, the mechanisms responsible for the distribution and stabilization of CRF-R2 in the cerebellum are not well understood. Here, we provide the first detailed analysis of the cellular localization of the full-length form of CRF-R2 in rat cerebellum during early postnatal development. We document unique and developmentally regulated subcellular distributions of CRF-R2 in cerebellar cell types, e.g. granule cells after postnatal day 15. The presence of one or both receptor isoforms in the same cell may provide a molecular basis for distinct developmental processes. The full-length form of CRF-R2 may be involved in the regulation of the first stage of dendritic growth and at later stages in the controlling of the structural arrangement of immature cerebellar circuits and in the autoregulatory pathway of the cerebellum.

CRF and urocortin differentially modulate GluRdelta2 expression and distribution in parallel fiber-Purkinje cell synapses.

Corticotropin-releasing factor (CRF) and urocortin (UCN) are closely related multifunctional regulators, governing, among other processes, Purkinje cell development. Here, we investigate the effects of CRF and UCN on Purkinje cells in organotypic slices. We show that both peptides upregulate delta2 ionotropic glutamate receptor gene expression, and increase the abundance of the receptor in the postsynaptic density. However, only UCN treatment results in increased delta2 protein level per Purkinje cell, implying the existence of posttranscriptional regulation of GluRdelta2 mRNA. CRF, in contrast, reduces the number of delta2-positive dendritic shafts per cell, implying that the increase of GluRdelta2 in remaining synapses may be mainly due to its retargeting. We further observed different patterns of GluRdelta2 distribution in the zone of postsynaptic density upon CRF and UCN treatment. CRF treatment results in a clustered distribution of GluRdelta2 along the postsynaptic density, whereas UCN treatment provides a linear distribution.

Postural control in man: the phylogenetic perspective.

Erect posture in man is a recent affordance from an evolutionary perspective. About eight million years ago, the stock from which modern humans derived split off from the ape family, and from around sixty-thousand years ago, modern man developed. Upright gait and manipulations while standing pose intricate cybernetic problems for postural control. The trunk, having an older evolutionary history than the extremities, is innervated by medially descending motor systems and extremity muscles by the more recent, laterally descending systems. Movements obviously require concerted actions from both systems. Research in rats has demonstrated the interdependencies between postural control and the development of fluent walking. Only 15 days after birth, adult-like fluent locomotion emerges and is critically dependent upon postural development. Vesttibular deprivation induces a retardation in postural development and, consequently, a retarded development of adult-like locomotion. The cerebellum obviously has an important role in mutual adjustments in postural control and extremity movements, or, in coupling the phylogenetic older and newer structures. In the human, the cerebellum develops partly after birth and therefore is vulnerable to adverse perinatal influences. Such vulnerability seems to justify focusing our scientific research efforts onto the development of this structure.

Long-term reinnervation effects after sciatic nerve lesions in adult rats.

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 on the control side. These increases, compared to data at 21 weeks, are interpreted as an aging effect.

Early hypergravity exposure effects calbindin-D28k and inositol-3-phosphate expression in Purkinje cells.

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 (2g). 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 1g. 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 1g at P5 or P10 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.

Rehabilitation strategy using enhanced housing environment during neural regeneration.

The influence of an enriched environment on the recovery of nerve function was studied after a sciatic nerve lesion and repair. A sciatic nerve gap of 15 mm was bridged in 12 rats using autologous nerve grafts. The rats were housed either in an enriched environment or in standard cages. In the enhanced housing environment, the rats were forced to move by dissociating food and water sources, including wire for foot gripping instead of flat plastic floors, and wooden play toys. Locomotor behavior was recorded on tape with a digital videorecorder and behavioral data were compared with those of a group of six unoperated rats. The video-recordings were analyzed for the stance factor (SF) as well as several other aspects of the rat's walking pattern. Walking was evaluated between 10 and 21 weeks after the operation. Differences in walking behavior between rats raised in an enriched environment and rats raised in standard cages could not be demonstrated. Differences in walking behavior between male and female rats were not found either. But data differed significantly at all ages with rats of the control group. Automutilation of parts of the denervated foot revealed a significant difference in both experimental groups, occurring less often in the enriched environment group.

The postnatal developmental expression pattern of urocortin in the rat olivocerebellar system.

Urocortin belongs to the family of corticotropin-releasing factor (CRF)-like peptides, which play an important role in sensorimotor coordination. CRF induces locomotor activity, and urocortin has an inhibitory effect. Here, we document the regional and subcellular localization of urocortin in the developing rat cerebellum to compare it with CRF. During the first postnatal week, urocortin immunoreactivity (UCN-ir), within the white matter and cerebellar cortex, was strongest in vermal lobules I, II, IX, and X, closely followed by lobules IV, V, and VIII; lobules VI and VII showed the weakest labeling. Cortical immunoreactivity was in the form of puncta that encircled Purkinje cell somata. By postnatal day (PD) 12, UCN-ir had increased appreciably in all lobules. In Purkinje cells, labeling was spread throughout their somata and proximal dendrites. By PD 15, labeling in lobules I-IV appeared to wane, yet still prevailed in the central and posterior lobules. This anterior-to-posterior gradient persisted through to adulthood. The study shows that urocortin and CRF have similar regional distribution profiles during development, suggesting synergistic roles within the vestibulocerebellum. The onset of the adult distributional pattern of urocortin at the stage when rats are capable of fluent walking patterns further strengthens the correlation between CRF-like peptides and postural control. An important difference between urocortin and CRF is the localization of urocortin, and not CRF, within Purkinje cells, implying that urocortin probably has an additional role in modulating the signals emanating from the cerebellar cortex to the deep cerebellar nuclei.

Development of sensory motor reflexes in 2 G exposed rats.

During gestation and early postnatal development, the animal's size and weight rapidly increase. Within that period, gravity affects sensory and motor development. We studied age-dependent modifications of several types of motor reflexes in 5 groups of rats conceived, born and reared in hypergravity (HG; 2 g). These rats were transferred to normal gravity (NG; 1 g) at various postnatal days, and their behavioral reflexes were compared with a control group which was constantly kept under NG. HG induced a retarded development of vestibular dependent reflexes. Other types of motor behavior were not delayed.

Development of the locomotor system in 2 G exposed rats.

Several studies have shown the detrimental effects of microgravity exposure on the locomotor development in young rats. The opposite situation, i.e. hypergravity, which strongly stimulates several sensory systems and in particular the vestibular system, has unknown effects on the development of locomotion. This study reports 1) the temporal course of walking development of rats which were conceived and born in 2 g, and subsequently transferred to 1 g at different postnatal ages, and 2) the correlated modifications of soleus and tibialis anterior muscles.

Clumsiness and disturbed cerebellar development: insights from animal experiments.

Cerebellar functioning has been implied in the fine adjustments of muscle tone, in the coordination and the feed-forward control of movements and posture, as well as in the establishment and performance of motor skills. The cerebellar cortex in mammals develops late in neuro-ontogeny and an extrapolation from experimental results indicates that in the human the proliferation of the granule cells and the development of circuitry in the cerebellar cortex starts only in the last trimester of pregnancy and lasts until beyond the first birthday. This late development makes the cerebellar development particularly vulnerable to situations like an insufficient supply of nutrients, which may follow placental dysfunction, or to side effects of pharmacological treatments like the administration of corticosteroids in the postnatal period. We studied whether such situations might also lead to motor impairments. In rats, the effects of undernutrition during the brain growth spurt were investigated as well as those of corticosteroids administered in a period that is analogous to the 7th to 8th month of pregnancy in the human. Both these interferences affect cerebellar development and our results in rats indicate that they also lead to retardations in the emergence of certain reflexes, as well as to longer lasting motor impairments during locomotion. Extrapolation of these results strongly suggests that a disturbed cerebellar development should be considered as an important etiological factor in clumsiness in human children.

Toe out angle: a functional index for the evaluation of sciatic nerve recovery in the rat model.

In experimental peripheral nerve studies, the rat sciatic nerve model is widely used to examine functional outcome following nerve injury and repair. A variety of evaluation methods exist in the literature, but an adequate selection continues to be a critical point for the researcher. Rats with sciatic nerve injury typically ambulate with an external rotation of the foot. A new functional assessment instrument, the toe out angle (TOA) is quantified using computerized gait analysis. We compared Sciatic Functional Index (SFI) with TOA parameter after peripheral nerve transection and entubulation repair. We found a good correlation between SFI and TOA measurements in terms of predicting functional recovery. Moreover, the TOA provides information on the biomechanical consequences of the external rotation of the foot in the stance phase of walking.

Reinnervation of muscles after transection of the sciatic nerve in adult rats.

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.