Research centers on minority institutions (RCMI): II.

The goal of the Research Centers in Minority Institutions (RCMI) is to develop biomedical and behavioral research at institutions with 50% minority enrollment (African Americans, Hispanics, Native Hawaiians, Pacific Islanders, Native Americans and Alaska Natives) who have been underrepresented in the biomedical sciences. The program has made available resources vital to scientific development and progress. While these resources have included, equipment, personnel supplies, Core laboratories etc, important effective approaches to research also have been emerging.

Activation and endocytic internalization of melanocortin 3 receptor in neuronal cells.

Melanocortins play a central role in autonomic modulation of metabolism by acting through a family of highly homologous G protein-coupled receptors. Studies with gene knockout mice have implicated neural melanocortin receptors, MC3R and MC4R, in the etiology of obesity, insulin resistance, and salt-sensitive hypertension. In an attempt to better understand the mechanisms of function of these receptors, we expressed MC3R and MC4R in neuronal cells and demonstrated their co-localization to several membrane regions. We now show that in cultured neuronal cells, MC3R localizes to lipid rafts and undergoes endocytic internalization upon activation by gamma-MSH through a protein kinase-sensitive pathway. The appearance of the internalized receptor in lysosomes suggests that it is subsequently degraded. The expression of protein kinase A regulatory subunits and of c-Jun and c-Fos was analyzed by either immunoblotting or real-time PCR. No discernable changes were observed in the expression levels of these protein kinase A and protein kinase C responsive genes. Immunohistochemical studies showed a robust expression of MC3R protein in brain nuclei with relevance to cardiovascular function and fluid homeostasis further supporting the notion that the physiological effects of melanocortins on the cardiovascular system arise from effects on the central nervous system.

The claustrum: a historical review of its anatomy, physiology, cytochemistry and functional significance.

The claustrum (Cl) is a subcortical structure located in the basolateral telencephalon of the mammalian brain. It has been a subject of inquiry since the mid-nineteenth century. The Cl can be identified in a number of species, and appears as a phylogenetically related nucleus in Insectivores, Prosimians and Marsupials. Ontogenetic investigations have been the subject of much debate over the years. There are three hypotheses for claustral development. To date, the "hybrid theory" has garnered the most support. Pathological conditions specifically associated with the Cl, while few in number, are of interest from a functional perspective. Several cases of claustral agenesis have been reported. The implications of these clinical reports are discussed. Claustral neuroanatomy at the light-microscopic and electron-microscopic level is reviewed. The morphology of the claustral neuron consists of several types, which roughly corresponds to the neuron's location within distinct claustral subdivisions. The interconnectivity of the Cl with the cerebral cortex is rather complex and reflective of complex functional interrelationships. Several researchers have investigated the angioarchitecture of the Cl. It appears that vessels permeating the insula also vascularize the Cl. Literature investigating the neurotransmitters and overall chemical neuroanatomy of the Cl is extensive. These studies clearly demonstrate that the Cl is richly innervated with a wide and diverse array of neurotransmitters and neuromodulators. Lesion, stimulation and recording experiments demonstrate that the functional and physiologic capacity of the Cl is quite robust. A recurring theme of claustral function appears to be its involvement in sensorimotor integration. This may be expected of the Cl, given the degree ofheterotopic, heterosensory convergence and its interconnectivity with the key subcortical nuclei and sensory cortical areas. The Cl remains a poorly understood and under investigated nucleus. Therefore, a review of the world literature through 1986 prior to the advent of the "molecular revolution" is presented. This diverse and extensive body of knowledge is reviewed in the areas ofphylogeny, ontogeny, pathology, angioarchitecture, cytochemistry, anatomy and physiology. Theories of possible claustral function are also noted. It is hoped that this work will stimulate research scientists to further investigate the functional interrelationships of the Cl as well as to aim with far greater precision and accuracy towards a deeper understanding of its raison d'etre. The recent efforts in neurosciences by Sir Francis Crick and Christof Koch implicating the Cl in visual consciousness, is an important step in understanding just what its functions could encompass. Efforts in molecular neurosciences will be indispensable for a mechanistic understanding of these functions. Currently research efforts are underway from many perspectives. In considering the past scientific literature on the Cl, it is interesting to regard that this once obscure brain structure, may serve as a model system for the study of one of the most interesting and complex brain functions-consciousness.

Coexistence of Alzheimer disease neuropathology with herpes simplex encephalitis.

Several unusual features were observed during routine histopathological confirmation of a clinical diagnosis of Alzheimer's disease (AD) in an 85-year-old, right-handed, married male. The patient presented with a 12-year history of slowly progressive cognitive impairment, which increased in severity just prior to death. Detailed postmortem examination of the frontal lobes revealed a significant number of neuritic plaques and neurofibrillary tangles. Multifocal spongiform encephalopathic changes, mononuclear perivascular infiltrates, subcortical demyelination and gliosis were also found. Of particular interest were well-defined neuronal and astrocytic intranuclear inclusion bodies (Cowdry type I and I), suggestive of viral disease. Electron microscopy, immunohistochemical and immunohistofluorescent studies confirmed a Herpes simplex type I encephalitis (HSV-I). These histological results and the clinical history of progression suggest that reactivation of a latent viral infection may have contributed to the rapid progression of dementia prior to death. The present analysis underscores the fact that multiple etiologic factors may act simultaneously to produce dementia. While one such process may be identified or diagnosed (in the present case AD), it is necessary to be open to the possibility that another mechanism may come into play during the time course of that illness. A differential diagnosis may be difficult when the symptoms of the two disease processes are very similar. Such may be the case if there is reactivation of a previously undiagnosed herpes virus infection. With the development of PCR and in situ hybridization diagnosis will be simplified and more definitive.

The horseradish peroxidase technique for cell lineage studies.

The identification of cell lineage for a given cell type of a particular tissue is an important step in understanding how this process contributes to histogenesis. The importance in understanding cell lineage has relevance for both theoretical and practical reasons. For example, delineating and identifying histogenic principals is required to advance stem cell research and tissue regeneration. To document cell lineage in a given experimental preparation, a number of techniques have been developed. This typically requires the injection of a tracer compound into a founder cell. As this cell produces progeny, the tracer is passed on to the daughter cells. By detecting the tracer in the progeny or daughter cells the investigator can determine which cells originated from the cell that was originally injected with the tracer. By using such an approach it is possible to trace the developmental path from the first cells of the embryo to the specialized cells making the tissue of the adult. A number of tracer compounds have been used with good results in lineage tracing. One of these tracer compounds is horseradish peroxidase (HRP). Several variations of the technique are available depending on what species are studied or what histological requirements are made by the study. A basic technique that can be adapted to individual needs is presented. Included in this protocol on lineage tracing are the procedures for injection, fixation, and the microscope evaluation of labelled cells in the Helobdella triseralis embryo. A brief discussion of the technique will note its advantages and disadvantages. Examples of labelled cell preparations are given to illustrate the technique.

Expression and distribution of protein kinase C isozymes in brain tissue of spontaneous hypertensive rats.

Hypertension activates many endocrine, neuroendocrine and metabolic responses. How hypertension alters these functions remains unknown. Consequently the pathophysiology of hypertension related illnesses are incompletely understood. Protein kinase C (PKC) isoforms play an important role in cellular signal transduction and altered PKC activity has been reported in spontaneous hypertensive rats (SHR). In order to understand the role that PKC plays in hypertension, we hypothesized that PKC activity is significantly expressed in synaptosomal preparations from the brains of SHRs. In addition, the neuroanatomical distribution of this expression was mapped and compared to control animals. The brains were further studied for signs of neuropathology. Total PKC activity was significantly increased in synaptosomal samples isolated from the forebrain, midbrain, and hindbrain of SHR rats. Westem blot analysis identified PKC-alpha, -beta, -gamma, -delta, -epsilon and -zeta in all brain regions. Immunohistochemical analyses indicated that PKC-gamma was localized in cell bodies and processes in many autonomic cardiovascular control regions. These results suggest that PKC may be an important modulator of autonomic blood pressure control.

Adhesion of Candida albicans to brain tissue of Macaca mulata in an ex vivo assay.

An ex vivo adhesion assay was used to examine adhesion of Candida albicans yeast cells to brain tissue of the primate Macaca mulata. Tissues from frontal lobes and striatum (caudate, putamen, and portions of the globus pallidus) were used in the assay. Yeast cells adhered to gray matter at about six times the level of adhesion to white matter. The fungus was able to bind to different cell types within the cortex, basal ganglia, and white matter. Binding to neurons, small neurons or glia, endothelial cells, and neuropil was observed.

Polysaccharide storage myopathy in canine phosphofructokinase deficiency (type VII glycogen storage disease).

A severe, progressive myopathy developed in an 11-year-old, phosphofructokinase (PFK)-deficient, male, English Springer Spaniel dog. Results from a routine neurological examination were normal. Examination of histologic sections of skeletal muscle revealed large accumulations of material in some myofibers. These deposits were pale, basophilic, somewhat flocculent, and slightly granular with hematoxylin and eosin stain. Most fascicles examined in sections of limb and trunk muscles were affected to some degree, with up to 10% of muscle fibers being involved. Deposits stained strongly with periodic acid-Schiff and were resistant to digestion by alpha amylase but were removed by incubation with gamma amylase. Deposits were faintly positive with Gomori's methenamine silver technique and alcian blue (pH 2.5) and were brown-gray with Lugol's iodine solution but were negative with other stains. Based on staining characteristics, the deposits seemed to consist primarily of an amylopectin-like polysaccharide(s). Alcian blue staining (pH 2.5) was removed by treatment with neuraminidase but not with hyaluronidase, indicating that some sialic acid residues were also present. Electron microscopically, the deposits were composed of short granular filaments, small granules and amorphous material. They were not membrane bound. The morphologic appearance and staining characteristics of the deposits were remarkably similar to deposits previously described in human PFK-deficient myopathy. As expected, total PFK activities were markedly reduced when assayed in skeletal muscles of this dog. In contrast with other PFK-deficient dogs, muscle glycogen in this animal was not increased above that of normal dogs.

Astrocytic responses to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in cat and mouse brain.

Glial fibrillary acidic protein immunohistochemistry was used as a selective marker for regional reactive gliosis in the striatum and ventral mesencephalon in cats and mice exposed to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Thirty mice (C-57 black strain) were injected with 30 mg/kg intraperitoneally (IP) MPTP.HCl for seven days. Five adult cats were injected with 10 mg/kg IP MPTP.HCl for seven days. Animals were killed five to seven days after the last MPTP injection. Reactive gliosis was observed throughout the mouse striatum but not in the substantia nigra. In contrast, reactive gliosis was topographically represented in the cat caudate nucleus with a dorsal-ventral and medial-lateral gradient evident. Gliosis was also observed in the putamen and the substantia nigra, pars compacta. Tyrosine hydroxylase immunocytochemistry revealed a loss of dopamine in the mouse striatum but no loss of substantia nigra neurons. Nigral neurons were destroyed in the cat. These results suggest that MPTP may destroy nigrostriatal dopamine cell bodies and terminals in the cat while destruction in the mouse is at least initially confined to striatal terminals.

Localization of cytomegalovirus proteins and genome during fulminant central nervous system infection in an AIDS patient.

Approximately one-half of autopsied acquired immune deficiency syndrome (AIDS) patients demonstrate probable human cytomegalovirus (CMV) infection of the central nervous system (CNS). Because CMV in brain tissue or cerebrospinal fluid is difficult to culture, we used antisera, and radioactive probes to diagnose CMV infection in the brain of an autopsied AIDS patient, who died of a fulminant CNS and systemic infection with CMV, suggesting a complete seeding of the ependymal regions possibly followed by a uniform ventriculofugal spread of the virus deep into the parenchyma. Cytomegalic cells were observed in optic nerve, retina, ependymal and subependymal regions of the brain and in the motor (but not sensory) root-CNS junctions. Immunocytochemistry demonstrated viral antigen predominantly in cytomegalic cells, which also stained positively for glial fibrillary acidic protein, S-100, or neuron-specific enolase, but not a common leukocyte antigen. Virions were visible in these cells examined by electron microscopy. No viral replication was observed in pineocytes, pituicytes or the choroid plexus. Morphologically normal cells that were CMV antigen-negative proved to be infected after in situ hybridization with well-defined human CMV DNA fragments. Hence, morphologically normal glia and neurons show restricted replication of CMV, indicating that such cells may be latently infected.

The detection of serotonin: an additional approach in diagnosing amebiasis.

The diagnosis of amebiasis is often difficult and time consuming. Entamoeba histolytica has been shown to contain serotonin. This fact might be exploited in the clinical setting. Assaying for serotonin may provide valuable diagnostic information.

Stereotaxic atlas of the trigeminal ganglion in rat, cat, and monkey.

Stereotaxic maps of the trigeminal ganglion of rat, cat and monkey are presented. Each ganglion drawing is a composite from means of coordinates from several ganglions sampled. Depth coordinates are supplied in relation to both the dorsal surface of each ganglion and the cranial bone directly underlying the ganglion.

Trigeminal influences on entopeduncular units.

Because considerable work has implicated the basal ganglia in oral-ingestive behavior, an assessment was made of the effects of trigeminal stimuli upon entopeduncular single units. Units wre recorded extracellularly in awake, paralyzed and locally anesthetized cats. The effects of two types of sensory input were tested. Afferents from periodontal mechano-receptors involved in reflex jaw opening were stimulated via electrodes in the inferior dental nerve. Afferents from stretch receptors involved in reflex jaw closure were stimulated via electrodes in the trigeminal mesencephalic nucleus. A significant proportion of cells responded to both types of stimulation. The data were discussed in the context of a basal ganglionic role in oropharyngeal motor processes and a more general role in movement per se.