Daniel L. Tolbert

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 Alzheimers 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.

Quantitative and qualitative characteristics of dentate and fastigial afferents identified by electron microscopic autoradiography in the cat thalamus.

Dentato- and fastigiothalamic afferents were identified in the VM and medial VA and VL using electron microscopic (EM)-autoradiography. Synaptic vesicles in labeled dentate and fastigial boutons differed significantly in both their size and shape, which allows these two types of terminals to be distinguished in the normal neuropil. Differences in the mode of termination of cerebellar afferents upon the neurons in the thalamic nuclei studied are also discussed.

Excessive running induces cartilage degeneration in knee joints and alters gait of rats.

The goal of this study was to develop an aggressive running regimen for modeling osteoarthritis (OA) in rats. Twelve Wistar rats were randomly placed into either a running group or a non‐running group to serve as the control. The running rats used a motorized treadmill to run either 30 km in 3 weeks or 55 km in 6 weeks. Each week, the prints of hind paws were obtained when rats were made to walk through a tunnel. The resulting prints were digitalized for analyses of stride length and step angle. The histology of the knees was examined at 3 and 6 weeks and the OA pathology in the knees was quantified by Mankins score. Osteoarthritic pathology developed in the knees of the running rats, including decreased proteoglycan content, uneven type II collagen distribution in the cartilage matrix, increased MMP‐13 expression, expanded calcified cartilage zone, and clefts and defects in articular cartilage. The pathology worsened from running for 3 to 6 weeks. Gait analysis revealed an inverse correlation between paw angle and the grades of OA pathology. In conclusion, excessive running induces joint degeneration and a unique gait pattern in rats.

GDNF and IGF-I trophic factors delay hereditary Purkinje cell degeneration and the progression of gait ataxia

Neurotrophic factors GDNF and/or IGF-I were chronically infused into shaker mutant rats to rescue cerebellar Purkinje neurons from adult-onset heredodegeneration. The natural expression of the shaker mutation is characterized by spatially restricted degeneration of Purkinje cells that occurs earlier and faster in an anterior vermal compartment and slightly later and more slowly in a posterior vermal compartment. Gait ataxia and whole body tremor develop concomitant with the degeneration of Purkinje neurons. The number and spatial distribution of surviving Purkinje neurons, identified by cell-specific calbindin immunoreactivity, were quantitatively analyzed in mid-sagittal sections and correlated with quantitative movement analysis of hindlimb gait patterns. Compared to the number of surviving Purkinje cells in age-matched, non-infused, or saline-infused control mutants, 4 weeks of infusion of GDNF or IGF-I rescued many anterior compartment Purkinje cells from early degeneration. However, 2 and 4 weeks after cessation of GDNF or IGF-I infusion, respectively, the number and spatial distribution of surviving Purkinje cells was comparable to that observed in age-matched controls. Eight weeks of infusion of trophic factors did not support the continued survival of most anterior compartment Purkinje cells and was partially, and probably only transiently, neuroprotective for some posterior compartment Purkinje cells. When GDNF and IGF-I were infused together for 4 weeks the number of surviving Purkinje cells was additively greater than with either factor alone. Behaviorally, 4 weeks of infusion of trophic factors delayed the development of gait ataxia. Infused GDNF appeared to preserve hip stability, whereas IGF-I stabilized step length. Tremor was attenuated with 8 weeks of infusion of GDNF or IGF-I. GDNF-infused animals showed low power tremor frequencies, whereas IGF-I infusion resulted in a single large power peak with decreased numbers of low-amplitude frequencies. Collectively these findings indicate that exogenous trophic factors can delay the onset of hereditary Purkinje cell degeneration and gait ataxia. Quite surprisingly, GDNF and IGF-I appeared to act on disparate populations of mutant Purkinje cells, whose differential survival affected different aspects of locomotion.

The transience of cerebrocerebellar projections is due to selective elimination of axon collaterals and not neuronal death.

Fluorescent dyes were used to determine firstly if the transience of cerebrocerebellar projections in neonatal kittens is due to the selective elimination of axon collaterals or to neuronal death; and secondly, if the cerebrocerebellar projection neurons lived, did any maintain a projection to the brainstem or spinal cord. Injections of Fast Blue were made into the cerebellar cortex and deep nuclei in 7-9 postnatal days old kittens, the age in which cortical axons grow into the cerebellum. Later, at 31-71 postnatal days of age, when the transient cerebrocerebellar projections have disappeared, injections of Nuclear Yellow were made into the brainstem or the spinal cord. In the frontoparietal cortex, numerous neurons were labeled with Fast Blue suggesting that the disappearance of cerebrocerebellar projections is due primarily to the selective elimination of axon collaterals and not neuronal death. Moreover, many of the cortical neurons labeled with Fast Blue also were labeled with Nuclear Yellow which shows that many of the cortical neurons with transient collateral projections to the cerebellum in the neonate maintain a projection to brainstem or spinal targets in older animals.

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.

EM-Autoradiography of cerebellar nucleocortical terminals in the cat

SummaryThe findings from the present EM-autoradiography experiments identify the cerebellar nucleocortical projection as a mossy fiber-type pathway. In these studies orthogradely labelled axons and terminals were observed in the folial white matter and granule cell layer, with only background levels of silver grains over the Purkinje cell and molecular layers. Most nucleocortical axons synapsed within cerebellar glomeruli either at en passant contacts along dilated segments of unmyelinated axons or at multilobulated (terminal) expansions of the axons. Typically these synaptic junctions were of the asymmetrical type containing clear round vesicles. Usually, each labelled mossy fiber rosette synapsed on a large number of small terminal dendrites of granule cells; however, occasionally a series of 4–8 asymmetric contacts were noted on a single large dendrite. Labelled nucleocortical terminals were occasionally present in the nonglomerular neuropil between granule cell bodies where they contacted large dendrites of Golgi cells.

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 collateral origin of a transient cerebrocerebellar pathway in kittens. A study using fluorescent double-labeling techniques

A transient pathway from the sensorimotor neocortex to the cerebellum recently has been described in the kitten which appears late in the first postnatal week, develops maximally at days 9 and 10 and then gradually disappears. The transience of this pathway has been investigated in the present study using the retrograde transport of fluorescent dyes. In one set of experiments Fast Blue was injected into the caudal medulla of neonatal kittens while Nuclear Yellow was injected into the opposite cerebellum 3-10 days later. Neurons labeled with Fast Blue were found bilaterally throughout lamina V of the frontoparietal cortex. Neurons labeled with Nuclear Yellow also were found in lamina V. However, their distribution was predominantly ipsilateral to the cerebellar injection and generally was limited to the anterior and posterior sigmoid gyri, and the coronal gyrus. Most importantly, many neurons were labeled with both dyes, indicating that at least part of the transient cerebrocerebellar projection is derived from collateral branches of corticobulbar axons. A second set of experiments was done to determine if the transience of the cerebrocerebellar pathway is due to a retraction of collaterals of corticofugal axons or due to the death of the cortical neurons themselves. In these experiments True Blue was injected into the cerebella of kittens 8-10 days after birth and allowed to survive for 40-75 days. No neurons were labeled with True Blue in the frontoparietal cortex, although numerous neurons were labeled in the precerebellar nuclei of the brainstem. In a control set of experiments, True Blue was injected into the caudal medulla in animals at 7 postnatal days.(ABSTRACT TRUNCATED AT 250 WORDS)

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.