Anthony J. Castro

The afferent connections of the inferior olivary complex in rats. An anterograde study using autoradiographic and axonal degeneration techniques

Abstract Autoradiographic and axonal degeneration techniques were employed to determine the distribution patterns of inferior olivary afferents whose origins were determined using the horseradish peroxidase method. 70 The Fink-Heimer stain for degenerating axons was used following lesions of the cerebral cortex and spinal cord, while brainstem and cerebellar afferents were mapped by tritiated leucine autoradiography. After unilateral lesions of the mid-thoracic spinal cord, degenerating axons were observed within the subnuclei a and b of the caudolateral medial accessory olive and in the caudolateral dorsal accessory olive. Degeneration after upper cervical cord lesions extended more rostrally and medially within the same olivary subdivisions. Several nuclei within the caudal brainstem projected to the inferior olivary complex. The dorsal column nuclei distributed fibers primarily contralaterally to the lateral part of the dorsal accessory olive and to the caudolateral part of the medial accessory olive; the spinal trigeminal nucleus projected contralaterally to the rostromedial dorsal accessory olive; the medial and inferior vestibular nuclei projected to the ipsilateral subnuclei b, c, and β of the medial accessory olive and to the contralateral dorsomedial cell column; the nucleus prepositus hypoglossi sent fibers to the subnuclei c and β, the dorsal cap and the ventrolateral outgrowth; the lateral reticular nucleus projected to the subnucleus a of the caudolateral medial accessory olive bilaterally; and the reticular formation distributed fibers to the dorsal accessory olive contralaterally and to the β subnucleus ipsilaterally. Study of inferior olivary complex afferents from the deep cerebellar nuclei showed a projection from the fastigial nucleus to the β subnucleus and the ventrolateral outgrowth. The dentate and interpositus nuclei demonstrated topographic connections from these nuclei to the principal olive and accessory olives, respectively. All cerebellar connections were predominantly contralateral. Analysis of mesencephalic and diencephalic areas also demonstrated several inferior olivary complex afferent systems: the caudal pretectum and the superior colliculus projected to the subnucleus c contralaterally and the dorsal lamella of the principal olive ipsilaterally; the nucleus of the optic tract sent fibers to the dorsal cap; the lateral deep mesencephalic nucleus distributed fibers to the ipsilateral dorsal accessory olive and β subnucleus; the medial terminal nucleus of the accessory optic tract projected ipsilaterally to the ventrolateral outgrowth; and several areas including the medial deep mesencephalic nucleus, periaqueductal gray, the nucleus of Darkschewitsch, the subparafascicular nucleus, the rostral red nucleus and the prerubral field all projected ipsilaterally to the principal olive, rostral medial accessory olive, ventrolateral outgrowth and, to a lesser extent, the caudal medial accessory olive, dorsal cap and β subnucleus. Lesions of the frontal cortex produced axonal degeneration primarily ipsilaterally within many olivary subdivisions, especially the medial dorsal accessory olive and the caudomedial medial accessory olive. Although some notable differences in the distribution and laterality of fibers are described, our findings generally corroborate several earlier reports which used different techniques on a variety of species. Inferior olivary afferents from functionally related areas typically demonstrated similar distribution patterns within the subdivisions of the inferior olivary complex. These patterns suggest a functional localization within the inferior olivary complex which may facilitate an understanding of afferents from areas whose functions are not clearly known.

Delayed Treatment with Monoclonal Antibody IN-1 1 Week after Stroke Results in Recovery of Function and Corticorubral Plasticity in Adult Rats

Neuronal death due to ischemic stroke results in permanent deficits in sensory, language, and motor functions. The growth-restrictive environment of the adult central nervous system (CNS) is an obstacle to functional recovery after stroke and other CNS injuries. In this regard, Nogo-A is a potent neurite growth-inhibitory protein known to restrict neuronal plasticity in adults. Previously, we have found that treatment with monoclonal antibody (mAb) IN-1 to neutralize Nogo-A immediately after stroke enhanced motor cortico-efferent plasticity and recovery of skilled forelimb function in rats. However, immediate treatment for stroke is often not clinically feasible. Thus, the present study was undertaken to determine whether cortico-efferent plasticity and functional recovery would occur if treatment with mAb IN-1 was delayed 1 week after stroke. Adult rats were trained on a forelimb-reaching task, and the middle cerebral artery was occluded to induce focal cerebral ischemia to the forelimb sensorimotor cortex. After 1 week, animals received mAb IN-1 treatment, control antibody, or no treatment, and were tested for 9 more weeks. To assess cortico-efferent plasticity, the sensorimotor cortex opposite the stroke lesion was injected with an anterograde neuroanatomical tracer. Behavioral analysis demonstrated a recovery of skilled forelimb function, and anatomical studies revealed neuroplasticity at the level of the red nucleus in animals treated with mAb IN-1, thus demonstrating the efficacy of this treatment even if administered 1 week after stroke.

Electrophysiological analysis of motor cortical plasticity after cortical lesions in newborn rats.

Intracortical microstimulation of the motor cortex in normal adult rats evoked low threshold contralateral forelimb movements and high threshold ipsilateral movements. Ablation of the opposite sensorimotor cortex in adult animals did not alter these thresholds. However, stimulation of the unablated hemisphere in adult rats that sustained unilateral sensorimotor cortical lesions as neonates elicited low threshold ipsilateral forelimb movements that were similar to contralateral movements. These low threshold ipsilateral movements may be mediated via aberrant corticofugal pathways which are known to develop following neonatal cortical lesions.

Primary cortical glial reaction versus secondary thalamic glial response in the excitotoxically injured young brain: astroglial response and metallothionein expression.

In this study we have evaluated the primary astroglial reactivity to an injection of N-methyl-D-aspartate into the right sensorimotor cortex, as well as the secondary astroglial response in the thalamic ventrobasal complex, caused by the anterograde degeneration of descending corticothalamic fibres and/or target deprivation of the developing thalamic neurons. The astroglial response was evaluated from 4 h to 30 days post-lesion, by the immunocytochemical detection of the cytoskeletal proteins glial fibrillary acidic protein and vimentin, and the antioxidant and metal binding protein metallothionein I-II. In the lesioned cortex, hypertrophied reactive astrocytes showed increased glial fibrillary acidic protein labelling that correlated with a strong expression of vimentin and metallothionein I-II. Maximal astrocytic response was seen at one week post-lesion. The glial scar that formed later on remained positive for all astroglial markers until the last survival time examined. In contrast, in the anterogradely/retrogradely affected thalamus, the induced astroglial secondary response was not as prominent as in the cortex and was characteristically transitory, being undetectable by 14 days post-lesion. Interestingly, thalamic reactive astrocytes showed increased glial fibrillary acidic protein expression but no induction of vimentin and metallothionein I-II. In conclusion, in the young brain, the pattern of astroglial reactivity is not homogeneous and is strongly dependent on the grade of tissue damage: both in response to primary neuronal death and in response to retrograde/anterograde secondary damage, reactive astrocytes show hypertrophy and increased glial fibrillary acidic protein expression. However, astroglial vimentin and metallothionein I-II expression are only observed in areas undergoing massive neuronal death, where glial scar is formed.

Microglial response to N-methyl-D-aspartate-mediated excitotoxicity in the immature rat brain.

The intracerebral injection of N‐methyl‐D‐aspartate (NMDA) has been proposed as a model for hypoxic‐ischemic insult in the immature brain. In this light, the aim of this study was to describe the time course of the microglial reaction in the areas undergoing primary degeneration at the site of intracortical NMDA injection as well as in areas undergoing secondary anterograde and/or retrograde degeneration. Fifty nanomoles of NMDA were injected in the sensorimotor cortex of 6‐day‐old rats. After survival times ranging from 10 hours to 28 days, cryostat sections were stained for routine histology and for the demonstration of microglial cells by means of tomato lectin histochemistry.

Fetal neocortical transplants grafted to the cerebral cortex of newborn rats receive afferents from the basal forebrain, locus coeruleus and midline raphe

SummaryFetal cerebral neocortex (E15–17) was grafted into the cerebral hemisphere of newborn (0–1 day old) rats. Grafts were placed into cortical aspiration lesion cavities made immediately prior to grafting. At maturity, transplant afferents were examined by injecting the retrogradely transported fluorescent dyes diamidino yellow and fast blue into the grafts. Retrogradely-labeled neurons were histologically observed within several regions of the host brain including the basal forebrain, locus coeruleus and dorsal raphe areas. The topographical distribution within these areas resembled the normal labeling patterns described in previous reports.

Transplantation of fetal cortex to the brain of newborn rats: A retrograde fluorescent analysis of callosal and thalamic projections from transplant to host

Fetal cerebral cortical tissue was transplanted into the cerebral hemisphere of 0-1-day-old rats. In some cases, the transplants were placed into or adjacent to cortical lesion cavities made immediately prior to transplantation. Injections at maturity of fast blue and diamidino yellow into the host contralateral cortex and ipsilateral thalamus demonstrated the presence of callosal and thalamic projections from transplant to host. Numerous single-labeled neurons were often arranged in cell bands or clusters. This apparent topography and the absence of double-labeling resembled normal labeling patterns.

Immunocytochemical and electron-microscopic characterization of macrophage/microglia cells and expression of class II major histocompatibility complex in the pineal gland of the rat

Interstitial cells in the pineal gland of the rat were characterized immunocytochemically using the monoclonal antibodies MRC OX-42 and ED1 for macrophages/microglia, and MRC OX-6, which recognizes major histocompatibility complex (MHC) class II antigen. A polyclonal antibody against GFAP was used to identify astrocytes. Cells immunopositive for OX-42 and/or ED1 were distributed throughout the gland; they extended processes primarily along the perivascular spaces and occasionally within the parenchyma of the gland. Ultrastructurally, these OX-42-positive cells were characterized by a nucleus with sparse heterochromatin and cytoplasmic vacuoles/lysosomes. Cells expressing MHC class II antigen had a distribution and morphology similar to OX-42-immunopositive cells, suggesting that pineal macrophages/microglia play a role as antigen-presenting cells. GFAP-positive astrocytes were concentrated at the proximal end of the pineal where the pineal stalk enters the gland. The occurrence of antigenpresenting cells in the circumventricular neuroendocrine gland has important functional implications as these cells may be mediators of neuroimmunomodulatory mechanisms, and involved in certain disease states such as autoimmune pinealitis.

Primary cortical glial reaction versus secondary thalamic glial response in the excitotoxically injured young brain: Microglial/macrophage response and major histocompatibility complex class I and II expression

The excitatory amino acid analog, N-methyl-D-aspartate, was injected intracortically into nine-day-old rats. Resulting axon-sparing lesions in the developing sensorimotor cortex, which secondarily affect thalamic neurons that become deprived of cortical targets, provide an experimental model for the study of the glial response in distantly affected areas. The microglial/macrophage response was studied using tomato lectin histochemistry and major histocompatibility complex I and II immunocytochemistry. Blood-brain barrier integrity was evaluated. In the cortical lesion site, where blood-brain barrier breakdown occurs, the rapid microglial response was restricted to the degenerating area. Microglial changes were first seen at 4 h post-injection, peaking at days 3-5. Reactive microglia changed morphology, increased tomato lectin binding and expressed major histocompatibility complex I. Additionally, some cells expressed major histocompatibility complex II. In the secondarily affected thalamus, the microglial response was not as pronounced as in the cortex, was first seen at 10 h post-injection and peaked at days 3-5. Reactive microglia showed a bushy morphology, were intensely lectin positive and expressed major histocompatibility complex I. The exceptional response of the nine-day-old brain to cortical lesions makes this model an interesting tool for studying the implications of microglial major histocompatibility factor expression in still enigmatic processes such as wound healing and plasticity.

A study of corticospinal remodelling using retrograde fluorescent tracers in rats

SummaryThe retrogradely transported fluorescent tracers Fast Blue (FB) and Diamidino Yellow (DY) were injected into the spinal cord of adult rats that sustained unilateral frontoparietal cortical lesions at birth. Analysis of the resulting cortical labeling pattern in comparison to comparably injected control aminals demonstrated an increase of retrogradely-labeled neurons within the unablated cerebral hemisphere ipsilateral to spinal cord injections. These ipsilateral labeled cells corresponded to previous descriptions, based on anterograde tracing techniques, of anomalous uncrossed corticospinal tract (CST) fibers. Additional findings indicated that the ipsilateral CST fibers are not axonal collaterals of normal, crossed CST fibers. Fluorescent tracer injections into cervical and lumbar spinal cord levels demonstrated a distribution pattern of labeled cells within the ipsilateral cortex that was similar to the topographic pattern found contralateral to spinal cord injections in normal animals. These findings support previous anatomical and electrophysiological data indicating that the neonatal cortical lesion-induced increase of ipsilateral CST fibers may be functional.