Search PubMed⌕ Search

Biomedical subjects

R D McKay

Publications and source records attributed to R D McKay.

At least 55 records · Page 3Linked to original sources
PubMed1995

Re-expression of the intermediate filament nestin in reactive astrocytes.

The intermediate filament nestin is highly expressed in multipotential stem cells of the developing central nervous system (CNS). During neuro- and gliogenesis, nestin is replaced by cell type-specific intermediate filaments, e.g. neurofilaments and glial fibrillary acidic protein (GFAP). In this study, we demonstrate that nestin expression is re-induced in reactive astrocytes in the lesioned adult brain. Following ischaemic and mechanical lesioning, a strong and sustained expression of nestin was noted in GFAP-positive cells surrounding the lesion site. Lesion experiments in transgenic mice carrying the lacZ gene under control of regulatory sequences from the nestin gene suggested that the upregulation of nestin in reactive astrocytes is mediated via the same sequences that control nestin expression during CNS development. These observations and recent data on the co-expression of glial and neuronal marker antigens in reactive astrocytes point to a close relationship between proliferating astrocytes and neuroepithelial precursor cells.

Animals↗
PubMed1995

Host-guided migration allows targeted introduction of neurons into the embryonic brain.

The stereotyped positions occupied by individual classes of neurons are a fundamental characteristic of CNS cytoarchitecture. To study the regulation of neuronal positioning, we injected genetically labeled neural precursors derived from dorsal and ventral mouse forebrain into the telencephalic vesicles of embryonic rats. Cells from both areas were found to participate in the generation of telencephalic, diencephalic, and mesencephalic brain regions. Donor-derived neurons populated the host brain in distinct patterns and acquired phenotypic features appropriate for their final location. These observations indicate that neuronal migration and differentiation are predominantly regulated by non-cell-autonomous signals. Exploiting this phenomenon, intrauterine transplantation allows generation of controlled chimerism in the mammalian brain.

Animals↗
PubMed1995

Functions of basic fibroblast growth factor and neurotrophins in the differentiation of hippocampal neurons.

Restrictions in neuronal fate occur during the transition from a multipotential to a postmitotic cell. This and later steps in neuronal differentiation are determined by extracellular signals. We report that basic fibroblast growth factor is mitogenic for stem cells and is a differentiation factor for calbindin-expressing hippocampal neurons. The neurotrophin NT-3 is a differentiation factor for the same neurons but does not affect proliferation. NT-3 and brain-derived neurotrophic factor promote the maturation of neurons derived from stem cells that have been grown in vitro. These results define functions for basic fibroblast growth factor and neurotrophins in the differentiation processes that direct a multipotential stem cell to a specific neuronal fate.

Animals↗
PubMed1995

Cerebellar precursors transplanted to the neonatal dentate gyrus express features characteristic of hippocampal neurons.

During the development of the CNS, a salient issue is whether neuronal phenotype is defined by the lineage or by the environment of precursor cells. Transplants permit these two possibilities to be tested, as cell fate can be examined in a new location. Dissociated cerebellar cells from newborn rats treated with tritiated thymidine or from NSE-lacZ transgenic mice were grafted into the dentate gyrus of the developing hippocampus. Implanted cells integrated into the granule cell layer, which contains the cell bodies of host granule neurons. Immunohistochemistry showed that grafted cells in the granule cell layer, like the host hippocampal granule neurons, were calbindin positive and upregulated FOS in a seizure paradigm. Electron microscopic analysis also showed that cells grafted to the dentate gyrus share features with host dentate neurons. These assays indicate that transplanted cerebellar cells acquired morphological and antigenic features characteristic of hippocampal neurons. These results show that metencephalic precursors are capable of differentiating in response to signals in the telencephalon, suggesting that the environment controls the regional fate of neuronal precursor cells during neurogenesis.

Animals↗
PubMed1995

Dopaminergic microtransplants into the substantia nigra of neonatal rats with bilateral 6-OHDA lesions. I. Evidence for anatomical reconstruction of the nigrostriatal pathway.

Reconstruction of the nigrostriatal pathway by long axon growth derived from dopamine-rich ventral mesencephalic (VM) transplants grafted into the substantia nigra may enhance their functional integration as compared to VM grafts implanted ectopically into the striatum. Here we report on a novel approach by which fetal VM grafts are implanted unilaterally into the substantia nigra (SN) of 6-hydroxydopamine (6-OHDA)-lesioned neonatal pups at postnatal day 3 (P3) using a microtransplantation technique. The results demonstrate that homotopically placed dopaminergic neurons survive and integrate well into the previously 6-OHDA-lesioned neonatal SN region. Moreover, the tyrosine hydroxylase (TH)-positive neurons extended axons rostrally along the white matter tract of the internal capsule closely following the course of the original nigrostriatal pathway. The graft reestablished a TH-positive axon terminal network in the ipsilateral caudate-putamen, with the highest density in the medial and central parts. Retrograde labeling with Fluoro-Gold from the host striatum demonstrated that most of the transplant neurons giving rise to the graft-derived fiber outgrowth were TH-positive, but revealed also a small proportion of projecting neurons which were TH-negative. Amphetamine-induced striatal Fos expression was normalized in the caudate-putamen ipsilateral to the intranigral VM grafts, showing hyperexpression in some areas of the striatum, and the apomorphine-induced Fos expression seen in the 6-OHDA-lesioned animals was completely reversed on the grafted side. These findings indicate that the graft-derived dopaminergic reinnervation of the striatum is functional. The microtransplantation strategy may provide new avenues for the exploration of morphological and functional integration of fetal dopamine neurons in the nigrostriatal system and give new insights into the mechanisms controlling long-distance axon growth in the brain.

Amphetamine↗
PubMed1995

CNS-derived neural progenitor cells for gene transfer of nerve growth factor to the adult rat brain: complete rescue of axotomized cholinergic neurons after transplantation into the septum.

A CNS-derived conditionally immortalized temperature-sensitive neural progenitor (CINP) cell line was used to generate NGF-secreting cells suitable for intracerebral transplantation. The cells were transduced by repeated retroviral infection, using a vector containing the mouse NGF cDNA under the control of the LTR promoter. Subcloning at the permissive temperature (33 degrees C) identified a highly NGF-secreting clone (NGF-CINP), which contained multiple copies of the transgene and released NGF at a rate of 2 ng/hr/10(5) cells in vitro, both at 33 and 37 degrees C, which was approximately 1 order of magnitude higher than what was possible to achieve in the heterogeneously infected cell cultures. After transplantation to the brain, the NGF-CINPs differentiated into cells with a predominant glia-like morphology and migrated for a distance of 1-1.5 mm from the implantation site into the surrounding host tissue, without any signs of overgrowth and tumor formation. Grafts of NGF-CINP cells implanted into the septum of adult rats with complete fimbria-fornix lesion blocked over 90% of the cholinergic cell loss in the medial septum and grafts placed in the intact striatum induced accumulation of low-affinity NGF receptor positive fibers around the implantation site. Expression of the NGF transgene in vivo was demonstrated by RT-PCR at 2 weeks after grafting. It is concluded that the immortalized neural progenitors have a number of advantageous properties that make them highly useful experimental tools for gene transfer to the adult CNS.

Animals↗
PubMed1995

Dopaminergic regulation of transcription factor expression in organotypic cultures of developing striatum.

Dopamine is a major neurotransmitter in neural systems innervating the striatum, and dopamine receptors are expressed during early pattern formation in the developing striatum. To test for the functional responsiveness of developing striatal neurons to dopaminergic stimulation, we established an organotypic slice culture of newborn rat striatum. We analyzed the effects of dopamine receptor agonists and of adenylate cyclase and protein kinase activation on striatal neurons by measuring the induction of Fos-like and Fra-like proteins in the cultured striatum. Fos-like and Fra-like proteins were induced in striatal neurons by activation of D1-like dopamine receptors but not by activation of D2-like receptors. The induction of Fos-like protein was mainly in striosomes and a medial compartment next to the ventricular zone, whereas Fra-like protein was induced in the striatal matrix as well. cAMP analogs and forskolin induced widespread expression of both Fos-like and Fra-like proteins. Our findings thus suggest that neurons of developing striosome and matrix compartments not only have different functional coupling of D1-like receptors to adenylate cyclase, but also have distinct maturational programs for dopaminergic regulation of individual transcription factors. Finally, despite evidence that protein kinase was involved in the induction of Fos-like protein, experiments with kinase inhibitors suggested that the induction of Fos-like protein had unusual pharmacological characteristics and raised the possibility that a novel protein kinase A-like molecule may have been involved in the induction. The cultured striatal slice preparation should provide a valuable tool for analyzing the molecular determinants of striatal development and function.

Amino Acid Sequence↗
PubMed1994

Morphological specification of trigeminal neurites depends on target fields.

Primary sensory neurons bridge the sensory periphery to the central nervous system (CNS) via their two axonal processes. The morphological patterning of the terminals of each process in its respective target is unique. Whether the differences between peripheral and central axons result from an intrinsic developmental program of the ganglion cell body, or from target-derived signals is not known. To explore this issue, we have used an explant coculture system in which embryonic (E15) trigeminal ganglion explants were placed between a vibrissa pad and a brainstem explant, but the explants were rotated 180 degrees relative to their normal orientation. In other experiments, individual ganglia were placed between two vibrissa pad explants or between two slices taken through the brainstem. The cultures were fixed after several days and ganglion cell processes were labeled with the lipophilic tracer DiI. Results of the ganglion rotation experiments suggest that trigeminal axons which would be directed centrally in vivo can regenerate into peripheral targets, and peripheral axons can grow into CNS tissue. Similarly, in cocultures with two peripheral or two central targets, both processes of trigeminal ganglion cells can simultaneously invade vibrissa pad explants or project into brainstem slices. Moreover, in all cocultures the differentiation of each set of processes is specific to the target innervated by it. These results show that the axons of embryonic sensory neurons are not selective in their choice of targets, and that their morphological patterning is dictated by target-derived signals.

Animals↗
PubMed1994

5-Hydroxytryptamine type 2A receptors regulate cyclic AMP accumulation in a neuronal cell line by protein kinase C-dependent and calcium/calmodulin-dependent mechanisms.

The effects of 5-hydroxytryptamine (5-HT)2A receptor activation on cAMP formation were studied in a cell line derived from embryonic rat cortex (A1A1). 5-HT (EC50 = 0.87 microM) amplified the amount of cAMP formed in response to 5'-N-ethylcarboxamidoadenosine (an adenosine A2 receptor agonist), cholera toxin, and forskolin after 15 min of coincubation in the presence of the phosphodiesterase inhibitor rolipram. This effect of 5-HT was blocked by 10 nM ketanserin as well as by 10 nM spiperone, indicating a response mediated by the 5-HT2A receptor subtype. Similarly, cAMP accumulation was enhanced by coincubation with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore A23187. After exposure to PMA for 24 hr (PKC-depleted cells), 5-HT and A23187 still enhanced cAMP formed in response to forskolin and 5'-N-ethylcarboxamidoadenosine, whereas the amplifying effects of PMA were abolished. Analysis by Western blots and PKC activity measurements revealed that, of three PKC isoforms detected in A1A1 cells (alpha, delta, and epsilon), only the calcium-independent isoform PKC-epsilon remained in membrane fractions after long term PMA treatment. In PKC-depleted cells, 5-HT-mediated amplification was greatly reduced after treatment with the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl)-ester or the calmodulin antagonists calmidazolium and N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride. In addition, 5-HT-mediated amplification of cAMP accumulation was reduced by the PKC inhibitor staurosporine in normal cells but was unaffected in PKC-depleted cells. In conclusion, these data suggest that 5-HT2A receptor activation can amplify cAMP formation in A1A1 cells by two distinct pathways coupled to the hydrolysis of inositol phosphates, i.e., PKC and calcium/calmodulin.

Adenosine↗
PubMed1993

Target-derived influences on axon growth modes in cultures of trigeminal neurons.

Cellular and molecular signals involved in axon elongation versus collateral and arbor formation may be intrinsic to developing neurons, or they may derive from targets. To identify signals regulating axon growth modes, we have developed a culture system in which trigeminal ganglion cells are challenged by various target tissues. Embryonic day 15 (E15) rat trigeminal ganglion explants were placed between peripheral (vibrissa pad) and central nervous system targets. Normally, bipolar trigeminal ganglion cells extend one process to the vibrissa pad and another to the brainstem trigeminal complex. Under coculture conditions, the peripheral processes invade the vibrissa pad explants and form a characteristic circumfollicular pattern. Central processes of E15 ganglion cells invade many, but not all, central nervous system tissues. In isochronic (E15) central nervous system explants such as brainstem, olfactory bulb, or neocortex, these central processes elongate and form a "tract" but have virtually no arbors. However, in more mature targets (e.g., a section from postnatal brainstem or neocortex), they form arbors instead of a tract. We conclude from these observations that whether trigeminal axons elongate to form a tract, or whether they begin to arborize, is dictated by the target tissue and not by an intrinsic developmental program of the ganglion cell body. The explant coculture system is an excellent model for analysis of the molecular basis of neuron-target interactions.

Animals↗
PubMed1993

Hybrid cell extinction and re-expression of Oct-3 function correlates with differentiation potential.

The Oct-3 gene is expressed in highly undifferentiated cells and is implicated in mammalian early embryogenesis. We have generated a series of hybrid cells between pluripotent embryonal carcinoma cells (Oct-3+) and fibroblasts (Oct-3-), and have studied the regulation and function of Oct-3. Upon fusion, the hybrid cells differentiated to nestin+/Brn-2+ cells resembling neuroepithelial stem cells. Expression of Oct-3 was extinguished at the transcriptional level in all the hybrid cells examined. The Oct-3 modulating activity required for the Oct-3-mediated enhancer activation was also extinguished. When the Oct-3 transactivating function was introduced into the hybrid cells, they transformed into morphologically distinct nestin-/Brn-2- cells ('revertants'). When the 'revertant' cells subsequently lost Oct-3 expression, they differentiated back to nestin+/Brn-2+ cells. The close correlation between the phenotypic changes and the gain/loss of Oct-3 function indicates that Oct-3 can induce dedifferentiation of the neural cells.

Animals↗
PubMed1993

A hypothermic miniaturized stereotaxic instrument for surgery in newborn rats.

We describe a simple, scaled-down instrument which enables accurate, reproducible stereotaxic placements into specific sites in the brain of the newborn rat. The instrument is specially designed for the administration of long-term hypothermia, yet permits the use of alternative methods of anesthesia. The design of the head-stabilizing mechanism allows head positioning to be finely adjusted to achieve precise horizontal and vertical zero planes. This adaptability also allows the device to accommodate a large range of animal sizes and levels of maturity. Furthermore, the apparatus can be fitted onto a conventional adult stereotaxic frame or used by itself in combination with a free-standing manipulator. As a model preparation, we describe a procedure for stereotaxic surgery in the post-natal day (P1) rat. The versatility of the instrument has permitted successful stereotaxic surgery in adolescent as well as neonatal rats, newborn and adult mice, and newborn hamsters.

Animals↗
PubMed1993

Monoclonal antibodies to a rat nestin fusion protein recognize a 220-kDa polypeptide in subsets of fetal and adult human central nervous system neurons and in primitive neuroectodermal tumor cells.

Nestin is the major intermediate filament protein of embryonic central nervous system (CNS) progenitor cells. To identify proteins involved in early stages of lineage commitment in the developing human CNS we generated monoclonal antibodies to a TrpE-rat nestin fusion protein. This resulted in a monoclonal antibody (designated NST11) that did not recognize authentic human nestin, but did recognize a novel neuron-specific human polypeptide expressed in a subset of embryonic and adult CNS neurons as well as in medulloblastomas. NST11 immunoreactivity was abundant in developing spinal cord motor neurons, but was extinguished in these neurons by 17 weeks gestation. NST11 also labeled Purkinje cells at 17 weeks gestation, but Purkinje cells continued to express the NST11 antigen throughout gestation as well as in the adult cerebellum, and NST11 immunoreactivity was more abundant in Purkinje cells than in any other human CNS neurons. No NST11 immunoreactivity was detected in cells of the adult human peripheral nervous system or in a variety of adult non-neural human tissues. Further, NST11 almost exclusively stained cerebellar medulloblastomas. In Western blots of immature and mature human cerebral and cerebellar extracts, NST11 did not bind human nestin, but did detect an immunoband with a molecular weight of 220 kd. A similar immunoband was detected in medulloblastoma-derived cell lines with a neuron-like phenotype. These findings suggest that the NST11 monoclonal antibody recognizes a novel protein expressed by a subpopulation of immature and mature human CNS neurons, medulloblastomas, and medulloblastoma-derived cell lines.

Adult↗
PubMed1992

Cellular targets and trophic functions of neurotrophin-3 in the developing rat hippocampus.

The expression of the neurotrophins and trk receptors in the hippocampus has directed attention toward their roles in the development and maintenance of this region. We have examined the effects of the neurotrophins NT-3, BDNF, and NGF in cultures of developing rat hippocampal cells by two criteria: rapid induction of c-fos and neurotrophic responses. The selective induction of c-fos mRNA suggests the presence of functional receptors for NT-3 and BDNF, but not NGF, in embryonic hippocampal cultures. The NT-3-responsive cells were localized in pyramidal neurons of areas CA1 through CA3 and dentate granular and hilar cells of postnatal organotypic slices, as detected by c-Fos immunocytochemistry. In addition to immediate early responses, NT-3 caused a 10-fold increase in the number of cells expressing the neuronal antigen calbindin-D28k. This increase was dose dependent, with maximal stimulation at 10 ng/ml. In contrast, BDNF elicited small but significant calbindin responses. These results indicate biological responses to NT-3 in the CNS and suggest roles for for this neurotrophin during hippocampal neurogenesis.

Animals↗
PubMed1992

Changes in neurotrophin responsiveness during the development of cerebellar granule neurons.

Neurotrophins and their receptors are widespread in the developing and mature CNS. Identifying the differentiation state of neurotrophin-responsive cells provides a basis for understanding the developmental functions of these factors. Studies using dissociated and organotypic cultures of rat cerebellum demonstrated that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) affect developing granule cells at distinct stages in differentiation. While early granule neurons in the external germinal layer responded to BDNF, more mature granule cells responded to NT-3. BDNF, but not NT-3, enhanced survival of granule cells in cultures of embryonic cerebella. Thus, BDNF and NT-3 have distinct sequential functions that are likely to be critical in the development of the cerebellum. BDNF may promote the initial commitment, while NT-3 may direct the subsequent maturation of granule cells.

Analysis of Variance↗
PubMed1992

Establishment and characterization of a human primitive neuroectodermal tumor cell line from the cerebral hemisphere.

The primitive neuroectodermal tumors (PNET) comprise a class of malignant nervous system neoplasms that afflict children. These tumors consist of cells that are morphologically identical to the primitive neuroepithelial cells normally seen in early stages of neural embryogenesis, supporting the notion that PNET result from a disturbance in the process of normal neuronal or glial differentiation. In the central nervous system, PNET occur most commonly in the cerebellum (medulloblastomas), but only occasionally in the cerebral hemispheres. We report here the establishment and characterization of a new human cell line (PFSK) derived from a PNET from the cerebral hemisphere of a child. The growth characteristics of PFSK cells were typical of an immortalized, transformed cell line. Cytogenetic and molecular genetic studies showed that three different sublines were present. In one of these sublines, sequences from chromosome 17 had been lost during establishment in culture. Immunocytochemical studies showed that PFSK cells expressed nestin, an intermediate filament protein normally expressed by neuroepithelial stem cells during neurulation. The PFSK cells did not express antigens typically found in terminally differentiated neurons or glia, indicating that this tumor cell line might represent neuroepithelial stem cells prior to commitment to a neuronal or glial lineage.

Brain Neoplasms↗