Quantitative analysis of granule cell axons and climbing fiber afferents in the turtle cerebellar cortex.

The turtle cerebellar cortex is a single flat sheet of gray matter that greatly facilitates quantitative analysis of biotylinated dextran amine labeled granule cell and olivocerebellar axons and Nissl-stained granule and Purkinje neurons. On average, ascending granule cell axons are relatively thicker than their parallel fiber branches (mean +/- SD: 0.84 +/- 0.17 vs 0.64 +/- 0.12 microm, respectively). Numerous en passant swellings, the site of presynaptic contact, were present on both ascending and parallel fiber granule cell axons. The swellings on ascending axons (1.82 +/- 0.34 microm, n = 52) were slightly larger than on parallel fibers (1.43 +/- 0.24 microm, n = 430). In addition, per unit length (100 microm) there were more swellings on ascending axons (11.2 +/- 4.2) than on parallel fibers (9.7 +/- 4.2). Each parallel fiber branch from an ascending axon is approximately 1.5 mm long. Olivocerebellar climbing fiber axons followed the highly tortuous dendrites of Purkinje cells in the inner most 15-20% of the molecular layer. Climbing fibers displayed relatively fewer en passant swellings. The spatial perimeter of climbing fiber arbors (area) increased 72% from anteriorly (1797 microm2) to posteriorly (3090 microm2) and 104% from medially (1690 microm2) to laterally (3450 microm2). Differences in the size and spacing of en passant swellings on granule cell axons suggest that ascending axons may have a functionally more significant impact on the excitability of a limited number of radially overlying Purkinje cells than the single contacts by parallel fiber with multiple orthogonally aligned Purkinje cell dendrites. The spatially restricted distribution of climbing fibers to the inner most molecular layer, the paucity of en passant swellings, and different terminal arbor areas are enigmatic. Nevertheless, these finding provide important anatomical information for future optical imaging and electrophysiological experiments.

Chronic intraventricular infusion of glial cell line-derived neurotrophic factor (GDNF) rescues some cerebellar Purkinje cells from heredodegeneration.

Cerebellar Purkinje cells degenerate in shaker mutant rats. Glia cell line-derived neurotrophic factor (GDNF) was chronically infused intraventricularly in an attempt to rescue mutant Purkinje cells from dying. Four weeks of chronic GDNF infusion delayed the degeneration of many but not all Purkinje cells. Surviving Purkinje cells formed spatially related groups interrupted by other groups of degenerated Purkinje cells. There was a positive correlation in GDNF-supported Purkinje cell survival and persistence of normal motor behaviors.

Neurodegeneration is associated to changes in serum insulin-like growth factors.

Serum levels of insulin and insulin-like growth factors and their binding proteins (IGFs and IGFBPs, respectively) are changed in human neurodegenerative diseases of very different etiology, such as Alzheimer's disease, amyotrophic lateral sclerosis, or cerebellar ataxia. However, the significance of these endocrine disturbances is not clear. We now report that in two very different inherited neurodegenerative conditions, ataxia-telangiectasia (AT) and Charcot-Marie-Tooth 1A (CMT-1A) disease, serum levels of IGFs are also altered. Both types of patients have increased serum IGF-I and IGFBP-2 levels, and decreased serum IGFBP-1 levels, while only AT patients have high serum insulin levels. Furthermore, serum IGFs are also changed in three different animal models of neurodegeneration: neurotoxin-induced motor discoordination, diabetic neuropathy, and hereditary cerebellar ataxia. In these three models, serum insulin levels are significantly decreased, serum IGF-I and IGFBP-1, -2, and -3 are decreased in diabetic and neurotoxin-injected rats, while serum IGFBP-1 is increased in hereditary ataxic rats. Altogether, these observations indicate that a great variety of neurodegenerative diseases show endocrine perturbations, resulting in changes in serum IGFs levels. These perturbations are disease-specific and are probably due to metabolic and endocrine derangements, nerve cell death, and sickness-related disturbances associated to the neurodegenerative process. Our observations strongly support the need to evaluate serum IGFs in other neurodegenerative conditions.

Olivocerebellar projections modify hereditary Purkinje cell degeneration.

Brainstem inferior olivary neurons, through their olivocerebellar efferent projections, dynamically regulate the structure and function of Purkinje neurons. To test the hypothesis that the inferior olive can epigenetically modify adult-onset hereditary Purkinje cell death, olivocerebellar projections were destroyed by 3-acetylpyridine chemoablation of the inferior olive in Shaker mutant rats. Starting around seven weeks of age, mutant Purkinje cells degenerate in a highly predictable spatial and temporal pattern. Chemoablation of the inferior olive at the onset of hereditary Purkinje cell degeneration accelerated the temporal pattern of Purkinje cell death from a natural phenotypic course of six to eight weeks to one and two weeks. When chemoablation of the inferior olive was performed three and a half weeks earlier, the onset of Purkinje cell death was accelerated by seven to 10days, but the spatial pattern and natural rate of temporal degeneration was maintained. Chemoablation of the inferior olive in normal rats did not result in any apparent death of Purkinje cells. These findings indicate that the olivocerebellar system can markedly modify hereditary Purkinje cell death. The accelerated death of Purkinje cells following chemoablation of the inferior olive can result from either the interruption of a trophic signal by climbing fiber deafferentation or parallel fiber excitotoxicity due to cortical disinhibition, but not due to olivocerebellar excitotoxicity.

X-linked transmission of the shaker mutation in rats with hereditary Purkinje cell degeneration and ataxia.

This study reports on the mode of inheritance of the shaker mutation and the development of an inbred strain of the shaker rat mutation from Sprague Dawley outbred stock onto a Wistar Furth background. Neuroanatomical and behavioral expression of the affected phenotype, through seven generations of backcross and intercross breeding, has confirmed the mode of inheritance to be X-linked. Behaviorally, affected mutants present with a wide-based ataxic gait and whole body tremor. In affected mutants calbindin immunostaining for surviving cerebellar Purkinje cells revealed widespread degeneration in the anterior lobe and in limited areas of the posterior lobe. Fast Fourier transform analysis of the tremor revealed a frequency of 3-5 Hz. As predicted by X-linked inheritance, female descendants of an affected male are carriers for the genotype and the phenotype is expressed in one-half of her male offspring. There was spatially random and limited degeneration of Purkinje cells in carrier females, but they did not display overt clinical signs of ataxia and tremor. These data provide further support for using the shaker mutant rat as an animal model for studies of mechanisms underlying human heredodegenerative diseases.

Purkinje cell transplants in Shaker mutant rats with hereditary Purkinje cell degeneration and ataxia.

Shaker mutant rats are characterized by the adult-onset degeneration of cerebellar anterior lobe Purkinje cells and temporally correlated development of ataxia and tremor. Normal E-13 Purkinje cells were transplanted into the anterior cerebellum in adult shaker mutant rats to study donor/host interactions in an animal with adult-onset heredodegeneration. Donor Purkinje cells from extraparenchymal transplant sites migrated radially into the host molecular layer and differentiated. Donor Purkinje cell dendrites expanded to fill the host molecular layer, spinous processes were apparent, and axonal projections into the host gray and white matter were observed. Donor Purkinje cells remaining in the extraparenchymal transplant sites differentiated if they were located relatively close to the host cerebellum. Donor Purkinje cells located intraparenchymally in the host white matter or granule cell layer survived, but were stunted in their development. The orthogonal movement of donor Purkinje cells away from transplant sites in the host cerebellum was spatially restricted. The findings from this study indicate that host cerebellar cortex with adult-onset heredodegeneration of Purkinje cells supports the survival and differentiation of transplanted normal embryonic Purkinje cells.

Quantitative analysis of cuneocerebellar projections in rats: differential topography in the anterior and posterior lobes.

The distribution of wheatgerm agglutinin-horseradish peroxidase-labelled mossy fibre terminals of internal and external cuneate projections to the cerebellar anterior and posterior lobes were quantitatively analysed in adult rats. Computer-based image analysis mapped the spatial distribution of labelled cuneocerebellar terminals in two-dimensional reconstructions of the unfolded cortex. Cuneocerebellar projections are mainly ipsilateral in their distribution. Cuneate projections to the anterior lobe vermis-medial paravermis terminate in well-circumscribed, irregularly-shaped patches. These terminal patches are aligned and form a longitudinally continuous, parasagittally oriented stripe in the lateral vermis-medial paravermis of lobules I-V. These terminal patches represent the topographically organized divergent projections of different parts of the internal and external cuneate nuclei. Cuneocerebellar projections to the lateral paravermis-hemisphere, particularly in the posterior part of lobule V, are organized as a transversely oriented band of terminals. Cuneocerebellar projections to the posterior lobe terminate mainly in three transversely oriented bands of terminals located at the junction between lobules. An anterior band of terminals was located in lobule VI anteriorly and was continuous with the band of terminals located in the posterolateral part of lobule V at the junction of these two lobules. Intermediate and posterior transversely oriented bands of terminals were located at the VII-VIII and VIII-IX junctions, respectively. Cuneocerebellar projections to these three bands largely appear to represent convergent projections from different parts of the cuneate nuclei. These findings are discussed in relation to similarly analysed and previously reported findings on the organization of lower thoracic-upper lumbar spinocerebellar projections and in the context of how cuneocerebellar somatosensory input may be differentially organized and processed in disparate areas of the cerebellar cortex.

Quantitative analysis of converging spinal and cuneate mossy fibre afferent projections to the rat cerebellar anterior lobe.

The convergence/divergence of mossy fibre afferent projections to the cerebellar anterior lobe from a single lumbar segment, from adjacent or widely separated lower thoracic and lumbar segments, and finally from the lower thoracic-upper lumbar spinal cord and the brainstem cuneate nuclei was quantitatively analysed in adult rats. Spinal and cuneate mossy fibre terminals were differentially labelled with biotinylated dextran amine and cholera toxin subunit B, immunohistochemically identified in the same histological sections, and their spatial distributions quantitatively plotted in computer reconstructions of the unfolded anterior lobe cortex. Afferent convergence was quantified by calculating the number of biotinylated dextran amine-labelled terminals that radially overlapped with cholera toxin-labelled terminals at points on the unfolded cortical map that represented theoretical Purkinje cells. Spino- and cuneocerebellar mossy fibre terminals are organized in patches that are oriented in parasagittally-oriented stripes or transversely oriented bands. Afferent convergence was greatest following biotinylated dextran amine and cholera toxin injections in the same or adjacent spinal lumbar segments (60 and 52%, respectively). When biotinylated dextran amine and cholera toxin were injected in a single segment differentially labelled terminals appeared randomly intermingled in common patches. There was a trend for terminals labelled from adjacent lumbar segments to be more segregated in the patches. Segmentally separated biotinylated dextran amine and cholera toxin spinal cord injections (four lumbar segments) resulted in clearly segregated (80%) biotinylated dextran amine from cholera toxin-labelled terminal patches or patches with distinct divergence of the differentially labelled terminals in the patch. Cuneocerebellar terminals labelled with biotinylated dextran amine were located in patches, stripes, and bands spatially segregated from terminal patches, stripes, and bands of cholera toxin-labelled spinal afferents except at their immediate borders where some radial overlap occurred (9-22%). These anatomical findings for a fractured somatotopy of spinal and cuneate inputs to the cerebellar anterior lobe complement neurophysiological findings for a very similar pattern of organization of cutaneous inputs to the posterior lobe, and are discussed in light of potential mechanisms for anterior lobe processing of somatosensory information.

A behavioral study of the development of hereditary cerebellar ataxia in the shaker rat mutant.

shaker Mutant rats were first identified by their abnormal motor behaviors and degeneration of cerebellar Purkinje cells and brainstem inferior olivary neurons. After 6 generations of inbreeding 77% of shaker rat mutants are mildly ataxic (identified as mild shaker mutants) and 23% are ataxic and exhibit a whole body tremor (strong shaker mutants) by 3 months of age. This study of shaker mutants from birth to 3 months of age was designed to: (1) compare the somatic and motor development of shaker mutants with age matched normal rats; (2) identify the temporal onset of motor deficits; and (3) correlate qualitative differences in Purkinje cell degeneration between 3-month-old mild and strong shaker rat mutants. Shaker mutant rats consistently weighed less than age-matched control animals. Analysis of motor-development using the hindlimb splay test demonstrated the distance between hindpaws was significantly greater in shaker mutant rats than in controls starting at 42 postnatal days (PND) of age. Hindlimb stride width was greater for shaker than control rats at 42 PNDs. However, after 42 PNDS shaker mutant average hindlimb width was narrower than controls. Forelimb stride width was consistently narrower in shaker mutants than in normal rats. Hindlimb placement was impaired in shaker rat mutants after 15 PND. Forelimb placement, cliff avoidance and surface righting were only transiently impaired in shaker mutants. Mid-air righting, performance of a geotaxic response, and climbing and jumping postural reactions were similar in shaker and normal rats. The spatial extent of Purkinje cell survival/degeneration correlated with differences in abnormal motor activity seen in 3-month-old mild and strong shaker mutants. In mild shaker rat mutants, Purkinje cells appeared to have degenerated randomly throughout the cortex. In strong shaker mutants most Purkinje cells in the anterior lobe had degenerated. In the posterior lobe Purkinje cell degeneration appeared to be numerically significant, but many surviving cells were present. Although Purkinje cell loss was not numerically quantified in this study, a strong association between the extent and type of spatial loss of Purkinje cells, and the severity of clinical signs, appears to exist.

Spatial and temporal pattern of Purkinje cell degeneration in shaker mutant rats with hereditary cerebellar ataxia.

Temporal-spatial patterns of surviving Purkinje cells were studied quantitatively in a rat mutant (shaker) with differential hereditary cerebellar ataxia and Purkinje cell degeneration. Shaker rat mutants are characterized behaviorally as mild if they are ataxic or as strong if they have ataxia and tremor. Purkinje cells degenerate in both mild and strong shaker mutants, but the temporal and spatial patterns of cell death are strikingly different. In mild shaker mutants, Purkinje cell death is temporally restricted, with 31-46% of the Purkinje cells in lobules I-IX dying by 3 months of age. Very few Purkinje cells degenerate after this age. Purkinje cell death is spatially random. In lobules I-IX, every second, third, or fourth Purkinje cell degenerates. Purkinje cells in lobule X do not degenerate. In strong shaker mutants, Purkinje cell degeneration is temporally protracted and spatially restricted. By 3 months of age, most Purkinje cells in lobules I-VIa, -b, and -d have degenerated. Numerous Purkinje cells in the paravermis of lobules VIIb-VIII have also degenerated. Surviving Purkinje cells in the vermis and lateral hemisphere of lobules VIIb-VIII are aligned in parasagittally oriented stripes or transversely oriented bands. Purkinje cells continue to degenerate in localized areas of the posterior lobe such that, by 18 months of age, surviving Purkinje cells are limited primarily to lobules VIc, VIIa, IXd, and X. Quantitative analysis indicates that none of the Purkinje cells in these lobules degenerate.

Chronic NMDA receptor blockade or muscimol inhibition of cerebellar cortical neuronal activity alters the development of spinocerebellar afferent topography.

The requirement for cerebellar cortical neuronal activity in the development of spinocerebellar afferent topography was investigated in neonatal rats. In adult rats lower thoracic-upper lumbar spinocerebellar projections are localized to sharply circumscribed patches in the granule cell layer of the cerebellar anterior lobe. In transverse sections these patches appear as sagittally oriented stripes. This pattern develops postnatally as many spinal axons which initially project between the incipient stripes are eliminated thereby sharpening the stripe boundaries. We attempted to alter cerebellar cortical neuronal activity in neonatal animals to study the effects of these changes on the development of spinocerebellar stripes. In some experiments glutaminergic excitatory synaptic transmission was chronically blocked with the N-methyl-D-aspartate (NMDA) receptor antagonist 2-aminophosphovaleric acid (APV). In other experiments postsynaptic activity was directly inhibited by the gamma-aminobutyric acid agonist muscimol. Chronic exposure to APV or to muscimol did not affect the initial development of spinocerebellar projections; many spinal axons were present in the anterior lobe and arranged in incipient stripes. Both the APV and the muscimol appeared to prevent the elimination of interstripe projections; consequently the boundaries of the stripes remained poorly defined. These findings suggest that cerebellar cortical neuronal activity is a necessary requirement for the refinement of spinal afferent topography in the anterior lobe.

Differential labeling of converging afferent pathways using biotinylated dextran amine and cholera toxin subunit B.

We report a new technique for 2-tracer anterograde labeling that permits unequivocal identification of the differentially labeled projections in the same section. One pathway is labeled with biotinylated dextran amine and is visualized as a black to dark gray diaminobenzidine (DAB)-cobalt precipitate by an avidin-biotinylated peroxidase reaction. The other pathway is labeled with cholera toxin subunit B and is visualized as a reddish-brown reaction product using DAB without cobalt as the substrate for peroxidase immunohistochemistry. To maintain serial order, sections can be processed mounted on slides without any loss of sensitivity for either tracer.

Lower thoracic upper lumbar spinocerebellar projections in rats: a complex topography revealed in computer reconstructions of the unfolded anterior lobe.

The topography of wheatgerm agglutinin-horseradish peroxidase/horseradish peroxidase-labeled mossy fiber terminals of lower thoracic-upper lumbar (T12-L3) spinal projections to the cerebellar anterior lobe was quantitatively analysed in adult rats. Computer-based image analysis mapped the orthogonal (parallel to the surface) distribution of labeled terminals in two-dimensional reconstructions of the unfoled anterior lobe cortex. The radial (perpendicular to the surface) distribution of terminals within the granule cell layer was mapped by computing whether the terminals were in either the outer- or inner-halves of this layer. The number of labeled terminals in each lobule was calculated. In the anterior lobe, lower thoracic-upper lumbar spinocerebellar projections terminate primarily in lobules II (mean 27.14%), III (mean 38.68%), and IV (mean 19.31%). Different-sized bilateral injections restricted to L1 were used to study the organization of intrasegmental spinocerebellar projections. Small injections into L1 labeled a limited number of terminals which were located either in clusters or were spatially isolated. Intermediate-sized intrasegmental injections resulted in additional clusters of labeled terminals. Many of the terminal clusters were spatially related and formed larger irregularly shaped patches. Large intrasegmental injections labeled terminal clusters and patches that were discontinuous but aligned parallel to the longitudinal (transverse) axis of lobules II-IV. Injections including segments rostral and caudal to L1 were used to study the topography of intersegmental lower thoracic-upper lumbar spinocerebellar projections. Multisegmental injections increased the number of labeled terminal clusters and patches which obscured the pattern of segmental input, but there was still a transversely oriented pattern of termination. Distinct transversely aligned terminal free areas remained apparent. Lower thoracic-upper lumbar spinocerebellar projections terminated in both the outer- and inner-halves of the granule cell layer, but overall were more numerous in the outer-half of this layer. In serially spaced sagittal sections, however, the majority of terminals alternated between the outer- and inner-halves of the granule cell layer. Outer- and inner-terminals were not spatially segregated in their orthogonal distribution. These results indicate lower thoracic-upper lumbar spinocerebellar projections have a complex three-dimensional topography in the anterior lobe. These findings are discussed in relation to previous findings for a sagittally oriented topography for lower thoracic-upper lumbar spinocerebellar projections and in the context of how cerebellar somatosensory afferent input may be organized.

The postnatal spatial and temporal development of corticospinal projections in cats.

Orthograde labeling and immunocytochemical techniques were used to study the postnatal spatial and temporal development of corticospinal projections in cats. Findings from the orthograde labeling studies indicate that there are three major phases in the spatial development of corticospinal projections: an early period (1-10 postnatal days) when cortical axons grow into the spinal gray from the white matter; an intermediate period (2-5 postnatal weeks) where corticospinal axons develop terminal arborizations in a rostral to caudal, medial to lateral and intermediate gray to dorsal and ventral horn sequence; and, a late period (6-7 postnatal weeks) during which some corticospinal projections are eliminated. The time period over which cortical axons grow into the spinal cord was determined immunocytochemically using a monoclonal antibody against a microtubule associated protein (MAP 1B) present in growing axons. The corticospinal tracts were strongly immunoreactive for MAP 1B during the first three postnatal weeks. MAP 1B immunostaining of these tracts started to decline in the fourth postnatal week and was completely absent by five weeks of age. These findings indicate that the postnatal development of corticospinal projections is spatially and temporally protracted in cats.

Absence of impulse activity in cortical neurons with transient projections to the cerebellum.

During the second postnatal week of development in cats, neurons in layer V of the primary sensorimotor cortex project transiently, by way of collaterals of pyramidal tract axons, to the cerebellum. All cerebrocerebellar collaterals are subsequently eliminated, while the collaterals in the pyramidal tract persist into the adult. To determine if the transience of the projection to the cerebellum could be due to the lack of functional activity in cerebrocerebellar projection neurons, single-unit extracellular recordings were made from neurons in the primary somatosensory cortex (S-I) in 8-14-day-old kittens. Projection neurons were identified by their antidromic activation from pyramidal tract or cerebellum. Collision experiments confirmed that some neurons had collateral projections to both structures. Recordings from both generally anesthetized as well as locally anesthetized, but awake preparations, indicated that pyramidal tract and cerebrocerebellar projection neurons never fired action potentials spontaneously or were orthodromically activated following stimulation of the medial lemniscus. Stimulation of the medial lemniscus did synaptically activate neurons in the cortex, but these were always located superficial to the antidromically activated projection neurons. These findings indicate that pyramidal tract and/or cerebrocerebellar S-I projection neurons are physiologically silent during the period of development that cortical axons are transiently present in the cerebellum, suggesting that cerebrocerebellar projections may be eliminated because of the lack of impulse activity.

Somatotopically organized transient projections from the primary somatosensory cortex to the cerebellar cortex.

The organization of transient projections from the primary somatosensory cortex (S-I) to the cerebellar cortex in neonatal kittens was examined using orthograde intraaxonal labeling techniques. Tritiated amino acid injections into face, forelimb and hindlimb areas of representation in S-I labeled mossy fiber-like terminals of cerebrocerebellar axons in different areas of the cerebellar cortex bilaterally. The hindlimb area of S-I projected to lobules I-IV in the anterior lobe and to ventral folia of the paramedian lobule (PML). Injections into forelimb areas of S-I labeled terminals in lobules IV and V and in intermediate and dorsal folia of the PML. The face area of S-I projected to the lobules V and VI, to medial folia in the ansiform and simplex lobules and to dorsal PML folia. Labeled terminals were more numerous in the cerebellar cortex contralateral to the S-I injections, except in lobules I and II and the ventral PML where the density of hindlimb input was approximately the same on both sides. These observations were supplemented by findings that small wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injections into the dorsal or ventral PML resulted in retrogradely labeled layer V pyramidal neurons in lateral (face and forelimb) and medial (hindlimb) areas of S-I respectively. The somatotopic organization of transient S-I cerebrocerebellar projections is very similar to the topography of cerebellar somatosensory afferent pathways in adult cats.

Organization of transient projections from the primary somatosensory cortex to the cerebellar nuclei in kittens.

The organization of transient projections from the primary somatosensory cortex (SI) to the cerebellar nuclei was studied in neonatal cats. Tritiated amino acids were injected into the face, forelimb, or hindlimb areas of SI in 4 to 6-day-old kittens. The animals were killed 3 to 6 days later and their brains processed for autoradiography. Labeled axons were found bilaterally in the cerebellar nuclei, but, although the distribution of label was similar on both sides, the label was always much denser on the side of the injection. Each area of SI demonstrated a characteristic pattern of projection to the cerebellar nuclei. Neurons in the hindlimb area projected to the rostral part of the anterior interpositus nucleus, the caudal part of the posterior interpositus nucleus, and the medial quadrant of the dentate nucleus. Fibers from the forelimb area were directed to the caudal part of the anterior interpositus and the rostral part of the posterior interpositus. Projections from the face area terminated principally in the caudal pole of the posterior interpositus. A small transitional area between the interpositus and fastigial nuclei was labeled with all injections. These data indicate that transient neocortical projections to the deep nuclei are organized and that the somatotopy of these projections is similar to that of other cerebellar nuclear connections.

Redirected growth of pyramidal tract axons following neonatal pyramidotomy in cats.

After the pyramidal tract at the pontomedullary junction in neonatal cats had been cut and the ipsilateral frontoparietal cortex injected with intra-axonal markers at 40 to 74 days of age, cortical axons were labeled in aberrant pathways that descended into the caudal medulla and spinal cord. Some labeled axons from the damaged pyramidal tract crossed the midline, descended with fibers in the intact pyramidal tract through the pyramidal decussation, and entered the lateral corticospinal tract. Another group of aberrant projections descended bilaterally along the ventrolateral edge of the medulla and either ended in the lateral reticular nuclei or continued into the spinal cord. Finally, some axons descended individually through the central medullary tegmentum and ended bilaterally in the spinal trigeminal, dorsal column, and lateral reticular nuclei. Although these findings suggest that pyramidal tract axons regenerate after injury, the findings from a second series of experiments refute this conclusion. In 2- to 5-day-old cats, the fluorescent dye Fast Blue was injected into the spinal cord, and 7 to 8 days later the contralateral pyramidal tract was cut. In these animals, there were never any cortical neurons retrogradely labeled with Fast Blue in the frontoparietal cortex ipsilateral to the pyramidotomy, although numerous neurons were labeled contralaterally. Control experiments confirmed that the interval between the Fast Blue injections and the pyramidotomies was long enough for retrogradely labeling cortical neurons, that the spinal cord injections did not adversely affect the retrogradely labeled cortical neurons, and following axotomy dying cortical neurons could be demonstrated directly using silver impregnation techniques. We conclude that neonatal pyramidotomy causes the death of all axotomized cortical neurons in kittens, and, therefore, the aberrant cortical projections seen caudal to the lesion must be redirected, late-developing, and undamaged cortical axons, and not regenerated axons.

Intrinsically directed pruning as a mechanism regulating the elimination of transient collateral pathways.

In neonatal cats, neurons in frontoparietal areas of the cerebral cortex have axons which branch, some collaterals project transiently to the cerebellum, whereas others project by way of the pyramidal tract to the brainstem and spinal cord and persist into the adult. If cerebrocerebellar collaterals are eliminated simply because they are exuberant, then experimentally removing the collaterals in the pyramidal tract should cause the normally ephemeral projections to the cerebellum to persist. To test this hypothesis, the pyramidal tract was cut unilaterally at the pontomedullary junction in 5-9-postnatal-day-old (PND) cats, and 35-68 days later the frontoparietal cortex ipsilateral to the pyramidotomy was injected with tritiated amino acids. From the end of the lesioned pyramidal tract, labeled axons were traced into pathways that descended aberrantly into the caudal medulla and spinal cord, but there was never any transported label in the cerebellum. In a second series of experiments, the fluorescent dye Fast blue (FB) was injected into the spinal cord (2-5 PND) prior to cutting the contralateral pyramidal tract (9-12 PND) to determine if the pyramidotomy caused the axotomized cortical neurons to die. There were no neurons labeled with FB in the frontoparietal cortex on the side of the pyramidotomy, but many retrogradely labeled neurons were present contralaterally in the cortex, suggesting that the pyramidotomy caused the death of all axotomized cortical neurons. In a final set of experiments, FB was injected into the spinal cord and the cerebellar cortex was ablated (2-3 PND) prior to cutting the pyramidal tract (9-72 PND). Cerebellar decortication results in the persistence of cerebrocerebral projections to the partially deafferented deep nuclei, therefore injections of Nuclear yellow (NY) or Diamidino yellow (DY) were made later (32-86 PND) into the cerebellar nuclei on the side of the decortication to determine if these projections persist in pyramidotomized cats. After pyramidotomies at 9 PND, there were no neurons labeled with fluorescent dyes in the ipsilateral frontoparietal cortex, indicating that the cerebrocerebellar collaterals, even under experimental conditions which normally cause them to persist, could not sustain the axotomized cortical neurons. Pyramidotomies at 24 PND or later did not cause all axotomized neurons to die since neurons labeled with FB were present in the ipsilateral cortex. These findings suggest that during development of corticosubcortical pathways there is a hierarchical.(ABSTRACT TRUNCATED AT 400 WORDS)

Exogenous cholecystokinin (CCK) reduces neonatal rat brain opioid receptor density and CCK levels.

Newborn rats were given saline or cholecystokinin8 (CCK8) (5 micrograms/kg, twice daily) i.p. for 3 weeks. On day 21, effects on brain development were assessed. CCK-like immunoreactivity was measured in 7 brain regions; a small (12-18%) but significant decrease in endogenous levels of this peptide was detected in cerebral cortex, medulla and pons of the CCK-treated rats. Morphometric measurements revealed a slight reduction in thickness of most cerebral cortical sections within the CCK-treated group. The area of a midsagittal section of the cerebellum was unchanged except for the Purkinje/granule cell layer, which was smaller in CCK-treated animals. Levels of mu-, delta- and kappa-opioid receptors were estimated by homologous displacement binding assays using selective radioligands. The CCK treatment resulted in a significant decrease in levels of mu- (11%) and delta- (13%)-sites in the cerebral cortex. Neither binding affinities nor kappa-receptor densities were altered. Other animals received the same treatment regimens for 21 days and were maintained for an additional 29 days without treatment; these rats had reductions only in cortical mu-sites (15%). Chronic intraventricular administration of CCK (0.1 microgram/h) to adult rats did not elicit a similar down-regulation of cortical mu or delta receptors, suggesting that the effects observed in neonates reflected developmental processes.