Plasma and urine pharmacokinetics of niacin and its metabolites from an extended-release niacin formulation.

OBJECTIVE: To characterize plasma and urine pharmacokinetics of niacin and its metabolites after oral administration of 2,000 mg of extended-release (ER) niacin in healthy male volunteers. METHODS: Niacin ER was administered to 12 healthy male subjects following a low-fat snack. Plasma was collected for 12 h post dose and was analyzed for niacin, nicotinuric acid (NUA), nicotinamide (NAM) and nicotinamide-N-oxide (NNO). Urine was collected for 96 h post dose and analyzed for niacin and its metabolites, NUA, NAM, NNO, N-methylnicotinamide (MNA) and N-methyl-2-pyridone-5-carboxamide (2PY). RESULTS: Mean niacin Cmax and AUC(0-t) values were 9.3 microg/ml and 26.2 microg x h/ml and were the highest of all analytes measured. Peak niacin and NUA levels occurred at 4.6 h (median) while tmax for NAM and NNO were 8.6 and 11.1 h, respectively. The mean plasma terminal half-life for niacin (0.9 h) and NUA (1.3 h) was shorter as compared to NAM (4.3 h). Urine recovery of niacin and metabolites accounted for 69.5% of the administered dose; only 3.2% was excreted as niacin. The highest recovery was for 2PY (37.9%), followed by MNA (16.0%) and NUA (11.6%). Mean half-lives for 2PY and MNA calculated in urine were 12.6 and 12.8 h, respectively. CONCLUSIONS: Niacin was extensively metabolized following oral administration, and about 70% of the administered dose is recovered in urine in 96 h as niacin, NUA, MNA, NNO, NAM and 2PY. The plasma levels of the parent niacin were higher than its metabolites though only about 3% of the unchanged drug is recovered in urine.

Definition of a DNA repair domain in the genomic region containing the human p53 gene.

The human p53 gene is repaired in UV (254 nm)-irradiated xeroderma pigmentosum group C (XP-C) cells as part of a large genomic region that is about twice the size of the gene. Surrounding genomic regions are not repaired. Through DNA cloning and measurements of DNA repair, we mapped the location of the repair domain, including the terminal regions, relative to the topological features of the gene. The domain includes only the DNA strand that is transcribed and extends in both 3' and 5' directions beyond the promoter and transcription termination sites. No transcriptional activity other than that associated with the p53 gene was detected. The results suggest that nontranscribed regions adjacent to the p53 transcribed regions are efficiently repaired in XP-C cells. This means that factors associated with transcription other than RNA polymerase II and the associated transcription repair coupling factor must also play a role in the selective repair process in XP-C cells. We also found that a DNA fragment that contains the p53 promoters is nearly twice as sensitive to cyclobutane pyrimidine dimer induction by UV irradiation than are the surrounding fragments, which have the expected sensitivity.

Transient cerebrocerebellar projections in kittens: postnatal development and topography.

Orthograde and retrograde labeling techniques were used to study the ontogenesis of transient cerebrocerebellar projections in kittens. Tritiated amino-acid or horseradish peroxidase injections were made into the coronal gyrus of the primary somatosensory cortex of kittens 1-70 postnatal days old. Orthogradely labeled axons were observed bilaterally in the superior and inferior cerebellar peduncles in kittens between 6 and 49 postnatal days of age. Most cerebrocerebellar axons labeled on the ipsilateral side arise from the pyramidal tract as it courses through the pontine nuclei. These axons descend through the pontine tegmentum as a diffusely organized corticotegmental tract and enter the ipsilateral superior cerebellar peduncle. Fewer cerebrocerebellar axons leave the pyramidal tract caudal to the pontine nuclei and project into the contralateral superior cerebellar peduncle. Cerebrocerebellar projections through the superior cerebellar peduncles terminate primarily in the cerebellar nuclei, where they are localized in the interpositus nuclei and in immediately adjacent areas of the dentate and fastigial nuclei. More caudally, labeled axons exit from the pyramidal tract and take a superficial route around the ventrolateral brainstem into the inferior cerebellar peduncles bilaterally. These projections are more numerous contralaterally and are directed primarily to the internal granule cell layer of the posterolateral folia of the anterior lobe, the posteromedial simplex lobule, and the dorsal paramedian lobule. Horseradish peroxidase injections were made into the cerebellar posterior lobe and deep nuclei and the results from these cases showed that the cerebrocerebellar pathway originates from pyramidal neurons in layer V primarily in the coronal, the precoronal, and the anterior and posterior sigmoid gyri on both sides. In these gyri, many of the HRP-positive neurons were found in clusters of two to five neurons, aligned in anterior-posterior strips. The results from all experiments provide evidence about the ontogeny of cerebrocerebellar projections. Projections through the superior cerebellar peduncles generally develop at 6-8 postnatal days of age, whereas projections through the inferior cerebellar peduncles first are seen at 8-10 days postnatally. Cerebrocerebellar projections reach their maximum development in the second postnatal week but sharply decrease in density during the third postnatal week. No cerebrocerebellar projections were observed after the seventh postnatal week of development. Possible functional implications for this transient projection are discussed.

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.

Organizational features of the cat and monkey cerebellar nucleocortical projection.

The organization of the cerebellar nucleocortical projection in the cat and the monkey has been studied using orthograde and retrograde neuroanatomical tracing techniques. Injections of tritiated leucine in the cat cerebellar nuclei orthogradely labeled nucleocortical fibers throughout their course to the cerebellar cortex. Their branch points in the corpus medullare, in the folial white matter, and in the granular layer were evident from the dense, continuous distribution of silver grains overlying these labeled axons. The results from the cat showed that the cerebellar nucleocortical projection is organized principally into three rostrocaudally oriented longitudinal cortical zones. Fastigial nucleocortical fibers were directed principally to the medial 1.5-2.5 mm of the ipsilateral vermis, with a lighter projection to the lateral vermis ipsilaterally and to the medial area of the vermis contralaterally. The interposed nuclei projected mainly to the paravermis-medial hemispheric zone of the cerebellar cortex. Nucleocortical fibers from the posterior interposed nucleus projected principally to the paramedian lobule, to the medial hemispheric area of Crus I and the lobus simplex, and to the flocculus and paraflocculus. Nucleocortical projections from the anterior interposed nucleus coursed to the anterior lobe paravermis and to the ventral folia of the paramedian lobule. A lighter projection from the interposed nuceli was found to the lateral edge of the vermis and into intermediate areas of the hemisphere. Dentatocortical fibers were directed into the lateral folia of Crus I and Crus II of the lateral hemispheric zone, with a ligher projection to intermediate areas of the hemisphere of the posterior lobe and along the lateral edge of the anterior lobe hemisphere. Along the periphery of each cortical zone, the nucleocortical projection from adjacent deep nuclei overlapped slightly. The retrograde transport of horseradish peroxidase (HRP) from injection sites in the lateral hemisphere, in the medial hemisphere--paravermis, and in the vermis labeled neurons localized mainly within the dentate, interposed, and fastigial nuclei, respectively. Retrograde labeling experiments carried out in monkeys indicated that the organization of the nucleocortical projection in this species is different than that of the cat. In the primate, the nucleocortical projection to the lateral hemisphere, to the medial hemisphere--paravermis, and to the vermis appeared to arise principally from the dentate nucleus. There was a secondary input to the paravermis and vermis arising from the interposed and fastigial nuclei, respectively. This evidence suggests that the cerebellar nucleocortical system undergoes a significant phylogenetic change in its organization between the cat and primate. These organization differences are discussed in light of possible functional implications.

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.

The postnatal development of corticotrigeminal projections in the cat.

The postnatal development of corticotrigeminal projections was studied in kittens following 3H-amino acid injections into the face area of the primary somatosensory cortex. Corticofugal axons grow into the brainstem and form the pyramidal tract prenatally. Corticotrigeminal projections begin to develop at the end of the first postnatal week. The earliest corticotrigeminal axons grow out of the pyramidal tract caudally and project into laminae III-V of the spinal trigeminal (Vs) nucleus caudalis. During the second postnatal week, corticotrigeminal axons grow out of the pyramidal tract in a caudal to rostral sequence and project up to the ventromedial borders of Vs-interpolaris, Vs-oralis, and to the principal trigeminal nucleus. Corticotrigeminal axons pause at the periphery of these nuclei for 1-2 days before penetrating the trigeminal neuropil and forming terminal arborizations in a centripetal direction. Coincident with the development of cortical projections to the principal trigeminal nucleus, some of the labeled axons which were in lamina III of Vs-caudalis project into lamina I and terminate. This sequence of development of corticotrigeminal projections closely parallels, albeit at a later time, the sequence of formation of the trigeminal nuclei, suggesting that the temporal sequence of cytogenesis of trigeminal neurons may be a factor which regulates their order of innervation by afferents. Corticotrigeminal projections develop bilaterally and, during the second postnatal week, are relatively equal in density in the ipsilateral and contralateral nuclei. Many of the ipsilateral corticotrigeminal projections are lost, however, after the second postnatal week, so that by the fourth postnatal week, corticotrigeminal projections are mainly contralateral and adultlike in their distribution. It remains to be determined whether the transience of ipsilateral corticotrigeminal projections is due to selective elimination of axon collaterals or to neuronal death.

The anatomical organization of the cerebello-olivary projection in the cat.

The cerebello-olivary pathway in the cat has been examined using orthograde and retrograde neuroanatomical tracing techniques. The orthograde transport of 3H-leucine from injection sites in the deep cerebellar nuclei labeled dentate and interpositus projections to the rostral two-thirds of the contralateral inferior olivary complex. These projections are topographically organized, with the dentate nucleus projecting to the principal olivary nucleus and the posterior and anterior interpositus nuclei projecting to the medial and dorsal accessory olives respectively. Fibers from the ventral half of the dentate nucleus terminate in the lateral bend and ventral lamina of the principal olive, whereas the medial and lateral parts of the dorsal half of the nucleus project to the medial and lateral regions of the dorsal lamina respectively. It is apparent that the more caudal parts of the interpositus nuclei project to areas of the medial and dorsal accessory olives near the caudal end of the principal olivary nucleus, whereas neurons in the more rostral parts of the interpositus nuclei project to the more rostral areas of the accessory olivary nuclei. A connection between the fastigial ncleus and the inferior olive could not be demonstrated. The retrograde transport of horseradish peroxidase (HRP) from injections sites in the inferior olive labeled cells throughout the contralateral dentate and interpositus nuclei. The labeled cells were especially numerous in the ventral parts of the dentate and posterior interpositus nlclei. These HRP-positive neurons were consistently small (10--15 mu) ovoid or spindle-shaped cells, with relatively large nuclei and light-staining Nissl substance. This evidence strongly suggests that the cerebello-olivary pathway originates from a population of small neurons in the dentate and interpositus nuclei and projects to specific, topographically defined areas in the contralateral inferior olive.

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.

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.

Anatomical and physiological evidence for a cerebellar nucleo-cortical projection in the cat.

Combined neuroanatomical and electrophysiological experiments were performed to test the hypothesis that axon collaterals of neurons in the cerebellar nuclei project to the cerebellar cortex in cats. The anatomical studies demonstrated that (a) following the injection of tritiated leucine into the deep cerebellar nuclei, labeled fibers could be traced into the granular layer of the cerebellar cortex, and (b) following the injection of horseradish peroxidase into the cerebellar cortex, retrogradely labeled horseradish peroxidase-positive neurons were identified in the deep nuclei. The electrophysiological experiments confirmed the anatomical findings. Neurons in the dentate and interposed nuclei, identified by their antidromic activation from the brachium conjunctivum, could also be activated antidromically from the cerebellar surface. Collision experiments demonstrated that projections from the deep cerebellar nuclei to the cerebellar cortex are in part collaterals of efferent neurons projecting through the brachium conjunctivum. Care was taken to ensure that all recordings were obtained from the region of cell somata in order to minimize the likelihood of recording from neuronal elements passing through the cerebellar nuclei. These combined neuroanatomical and electrophysiological studies provide strong evidence supporting the existence of a collateral system from cerebellar output neurons to the cerebellar cortex. The existence of this collateral system emphasizes that the cerebellar cortex and cerebellar nuclei may comprise a functional unit in which these collaterals may serve as a substrate for feedback control of the cerebellar cortex by the cerebellar output.

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.

Heat-sensitivity and the immune response of a methylcholanthrene-induced tumor.

Our results indicate that this methylcholanthrene (MCA)-induced tumor is immunogenic and that its heat-sensitivity is linked to the immune response of the host. Stimulation or reduction of the latter caused a corresponding change in the former. A therapeutic heat-dose failed to protect a majority of the treated mice against a second tumor challenge.

Celecoxib does not significantly alter the pharmacokinetics or hypoprothrombinemic effect of warfarin in healthy subjects.

The objective of this study was to determine the effects of celecoxib, an anti-inflammatory/analgesic agent that primarily inhibits COX-2 and not COX-1 at therapeutic doses, on the steady-state pharmacokinetic profile and hypoprothrombinemic effect of racemic warfarin in healthy volunteers. Twenty-four healthy adult volunteers on maintenance doses of racemic warfarin (2-5 mg daily), stabilized to prothrombin times (PT) 1.2 to 1.7 times pretreatment PT values for 3 consecutive days, were randomized to receive concomitant celecoxib (200 mg bid) or placebo for 7 days in an open-label, multiple-dose, randomized, placebo-controlled, parallel-group study of warfarin pharmacokinetics and PT. Steady-state exposure of S- and R-warfarin (area under the curve [AUC]) and maximum plasma concentration (Cmax) in subjects receiving celecoxib were within 2% to 8% of the warfarin AUC and Cmax in subjects receiving placebo during the concomitant treatment period. In addition, PT values were not significantly different in subjects receiving warfarin and celecoxib concomitantly compared with subjects receiving warfarin and placebo. In conclusion, concomitant administration of celecoxib has no significant effect on PT or steady-state pharmacokinetics of S- or R-warfarin in healthy volunteers.

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.

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.

The organization of cerebellar afferent projections to the paramedian lobule in neonatal cats.

This study sought to determine whether cerebellar afferent pathways, that are topographically organized in adult cats, are similarly ordered during the postnatal development and maturation of the cortex, or whether the projections are first distributed randomly in the cortex before becoming organized. Injections of wheat germ agglutinin-horseradish peroxidase were made into dorsal (dPML) or ventral (vPML) divisions of the paramedian lobule (PML) in neonatal (0- to 21-days-old) and adult cats and the ensuing distributions of retrogradely labeled neurons in the lateral reticular nuclei, the inferior olive and the pontine nuclei were compared. Magnocellular and parvicellular neurons in the dorsomedial and dorsolateral parts of the ipsilateral lateral reticular nuclei project respectively to dPML and vPML in all neonatal and adult cats. Olivocerebellar projections were entirely crossed, in most cases, with neurons projecting to the dPML more rostral and medial in the dorsal and medial accessory nuclei and in the principal olive than neurons which project to the vPML. A parasagittal zonal organization of olivocerebellar projections was present in newborn cats. Neurons were labeled in the ipsilateral inferior olive following dPML injections in 1- to 4-day-old kittens, but not in older kittens or in adult cats. Pontocerebellar projections were bilateral with a contralateral predominance. In adult and neonatal cats, labeled neurons were clustered together and formed rostral-caudal oriented columns dorsomedial and ventromedial to the pyramidal tract after injections in the contralateral dPML and vPML and bilaterally in the dorsolateral pons after dPML injections. These results show that lateral reticulo-, olivo- and pontocerebellar projections to the PML which are topographically organized in adult cats are organized similarly in newborn cats. Studies in prenatal cats are required in order to determine whether these cerebellar afferents are ever randomly distributed in the cerebellar anlage or whether these projections are ordered as they grow into the cerebellum.

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)

The corticotrigeminal projection in the cat. A study of the organization of cortical projections to the spinal trigeminal nucleus.

The projection from the cerebral cortex to the spinal trigeminal nucleus has been studied light microscopically in adult cats. Both orthograde degeneration and orthograde intra-axonal labeling techniques have been applied. Our results indicate that the projection from the coronal gyrus (face area of primary somatosensory cortex) to the spinal trigeminal complex is somatotopically organized. In subnucleus caudalis this somatotopy is organized dorsoventrally and appears to match the somatotopic distribution of the divisional trigeminal afferents. Hence cortical fibers originating from the posterior coronal gyrus (upper representation) project ventrolaterally into caudalis where division I trigeminal afferents terminate. Likewise cortical fibers from the anterior coronal gyrus (jaw and tongue representation) terminate dorsomedially in caudalis to overlap with division III trigeminal afferents. In contrast, the distribution of corticofugal afferents to the rostral spinal trigeminal subnuclei (pars interpolaris and oralis) is organized mediolaterally. Therefore in these subnuclei the cortical projection does not appear to overlap the dorsoventral lamination of the divisional trigeminal afferents. In addition, our results suggest that the cortical projection to subnucleus caudalis includes fibers which terminate in the marginal zone (lamina I) and its extensions into the spinal trigeminal tract (the interstitial cells of Cajal). We have been unable to document a projection from the proreate gyrus to the spinal trigeminal complex.