Kathleen M. Guthrie

Cellular Localization of Transforming Growth Factor‐α mRNA in Rat Forebrain

Abstract: The cellular localization of transforming growth factor‐α (TGFa) mRNA in juvenile and adult rat forebrain was examined using in situ hybridization with a 35S‐labeled cRNA probe. TGFα cRNA‐labeled neuronal perikarya were distributed across many forebrain regions including the olfactory bulb, caudate‐putamen, nucleus accumbens, olfactory tubercle, ventral pallidum, amygdala, hippocam‐pal stratum granulosum and CA3 stratum pyramidale, and piriform, entorhinal, and retrosplenial cortices. TGFα cRNA‐hybridizing cells were also localized to several thalamic nuclei and to the suprachiasmatic, dorsomedial, and ventromedial nuclei of the hypothalamus. In addition, labeled cells were present in regions of white matter including the corpus callosum, anterior commissure, internal and external capsules, optic tract, and lateral olfactory tract. Thus, both neurons and glia appear to synthesize TGFα in normal brain. Hybridization densities were greater in neuronal fields at 2 weeks of age compared with the adult, suggesting a role for TGFα in the development of several forebrain systems. Our results demonstrating the prominent and widespread expression of TGFα mRNA in forebrain, combined with the extremely low abundance of epidermal growth factor mRNA in brain, support the argument that TGFα is the principal endogenous ligand for the epidermal growth factor receptor in normal brain.

Astroglial ciliary neurotrophic factor mRNA expression is increased in fields of axonal sprouting in deafferented hippocampus

Evidence that ciliary neurotrophic factor promotes axonal sprouting and regeneration in the periphery raises the possibility that this factor is involved in reactive axonal growth in the brain. In situ hybridization was used in the present study to determine whether ciliary neurotrophic factor mRNA expression is increased in association with axonal sprouting in deafferented adult rat hippocampus. In untreated rats, ciliary neurotrophic factor cRNA labeling density was high in the olfactory nerve, pia mater, and aspects of the ventricular ependyma and was relatively low within areas of white matter (fimbria, internal capsule) and select neuronal fields (hippocampal cell layers, habenula). After an entorhinal cortex lesion, hybridization was markedly increased in fields of anterograde degeneration, including most prominently the ipsilateral dentate gyrus outer molecular layer and hippocampal stratum lacunosum moleculare. Labeling in these fields was increased by 3 days postlesion, was maximal at 5 days, and returned to normal levels by 14 days. Double labeling demonstrated that, in both control and experimental tissue, ciliary neurotrophic factor mRNA was colocalized with glial fibrillary acidic protein immunoreactivity in astroglia, but it was not colocalized with markers for oligodendrocytes or microglia. These results demonstrate that astroglial ciliary neurotrophic factor expression is increased in fields of axonal and terminal degeneration and that increased expression is coincident with 1) increased insulin‐like growth factor‐1 and basic fibroblast growth factor expression and 2) the onset of reactive axonal growth. The synchronous expression of these glial factors in fields of deafferentation suggests the possibility of additive or synergistic interactions in the coordination of central axonal growth. J. Comp. Neurol. 386:137–148, 1997.

Odors increase fos in olfactory bulb neurons including dopaminergic cells

Neuronal activity may lead to long lasting changes in cell phenotype through induction of genes such as c-fos which encode transcriptional regulatory factors. Odor-activated olfactory bulb cells exhibit increases in c-fos mRNA expression. The present study examined whether odor stimulation of awake rats also leads to increases in Fos protein in these cells. The phenotype of Fos-immunoreactive cells was partially characterized using double-immunoperoxidase staining. Odor exposure increased Fos-immunoreactivity (IR) in specific sets of olfactory bulb neurons. Fos-IR was not co-localized with IR for glial fibrillary acidic protein, but was co-localized with tyrosine hydroxylase (TH)-IR in a subpopulation of dopaminergic neurons, suggesting that bulbar TH expression may be regulated in part by a Fos mechanism.

Odors regulate Arc expression in neuronal ensembles engaged in odor processing.

Synaptic activity is critical to developmental and plastic processes that produce long-term changes in neuronal connectivity and function. Genes expressed by neurons in an activity-dependent fashion are of particular interest since the proteins they encode may mediate neuronal plasticity. One such gene encodes the activity-regulated cytoskeleton-associated protein, Arc. The present study evaluated the effects of odor stimulation on Arc expression in rat olfactory bulb. Arc mRNA was rapidly increased in functionally linked cohorts of neurons topographically activated by odor stimuli. These included neurons surrounding individual glomeruli, mitral cells and transynaptically activated granule cells. Dendritic Arc immunoreactivity was also increased in odor-activated glomeruli. Our results suggest that odor regulation of Arc expression may contribute to activity-dependent structural changes associated with olfactory experience.

Induction of tyrosine hydroxylase expression in rat forebrain neurons

Olfactory nerve input is required for the normal expression of tyrosine hydroxylase (TH) by dopaminergic neurons in the glomerular region of the rodent main olfactory bulb. To determine whether the olfactory nerve exerts a similar influence on neurons in other brain regions, we performed unilateral bulbectomies in rat pups on postnatal day 5-7 and examined the brains 2-6 months later, after the regenerated olfactory nerve had penetrated the forebrain. Tissue was stained for TH, dopamine beta-hydroxylase (DBH) and olfactory marker protein immunoreactivity. We observed novel TH-immunoreactivity in neurons located in those areas of the adult forebrain which received olfactory nerve fibers, particularly the rostral extension of the subependymal layer. Many of these neurons resembled the periglomerular cells of the olfactory bulb. No cell staining for DBH was observed in these areas, suggesting the possible dopaminergic phenotype of these neurons. Our data indicate that afferent regulation of neurotransmitter expression by the olfactory nerve is not limited to the cells of the olfactory bulb.

NGF receptor increase in the olfactory bulb of the rat after early odor deprivation

In the olfactory bulb of normal rats, nerve growth factor (NGF) receptor (NGFR) immunoreactivity was largely confined to the glomerular layer. Unilateral closure of the nostril at postnatal day 2 (P2) increased NGFR immunoreactivity in the sealed bulb at both 19 and 60 days after the operation. The increase in NGFR density, measured by autoradiographic immunohistochemistry, was most dramatic 60 days postocclusion. These findings suggest that a compensatory increase in NGFRs may play a role in the maintenance of bulbar function after the early loss of sensory stimulation.

Anatomic mapping of neuronal odor responses in the developing rat olfactory bulb.

Behavioral evidence indicates that altricial mammals possess olfactory function at early developmental ages, before the olfactory bulb has matured anatomically. We monitored the early anatomic and functional development of the olfactory bulb in rat pups stimulated with odors using in situ localization of c‐fos mRNA to identify responsive postsynaptic neurons. Odor‐specific spatial patterns of neuronal activation in the glomerular layer were evident from birth, were sharply defined rather than diffuse, and remained relatively unchanged in terms of their bulbar distribution during the first 3 postnatal weeks. In neonates, focal postsynaptic responses in the glomerular layer occurred in the form of clusters of activated tufted neurons. Broad zones of activated mitral cells were located beneath these cell clusters, with scattered neurons in the underlying granule cell layer also expressing c‐fos. The cellular composition of these functional neuronal groups shifted from predominantly output neurons at the earliest ages, to increasing incorporation of interneurons as they developed postnatally. The characteristic distribution of activated neurons in the mature glomerular layer, in which the boundaries of individual glomeruli are precisely defined by cells expressing c‐fos, emerged near the end of the first week. Broad zones of cRNA hybridization in the mitral cell layer became increasingly restricted as the size of the activated granule cell population increased postnatally, correlating with the functional maturation of inhibitory circuitry. These results provide evidence that the types and distributions of neurons collectively activated by sensory input to glomeruli change as the rat olfactory bulb matures and that distinct, functional odor maps in the glomerular layer are established from birth. J. Comp. Neurol. 455:56–71, 2003.

NMDA-Receptor modulation of lateral inhibition and c-fos expression in olfactory bulb

Olfactory bulb primary output neurons, mitral/tufted cells, are glutamatergic and excite inhibitory interneurons, granule cells, by activation of both alpha-amino-3-hydroxy-5-methyl-ioxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) glutamate receptors. The data presented here demonstrate that the NMDA antagonists MK-801 and CGP39551, but not ketamine, significantly enhanced expression of c-fos mRNA by mitral cells as measured by in situ hybridization. All three antagonists significantly reduced mitral/tufted cell excitation of granule cells as measured with extracellular field potentials following antidromic stimulation of the lateral olfactory tract (LOT). In turn, the NMDA antagonists significantly reduced granule cell mediated feedback inhibition of mitral/tufted cells, as measured with field potential recordings of paired-pulse LOT stimulation, suppression of mitral/tufted cell single-unit spontaneous activity following LOT stimulation, and intracellularly recorded IPSP amplitude in mitral/tufted cells following LOT stimulation. While there was not a perfect correlation between the effects of the NMDA antagonists on c-fos mRNA expression and on inhibition, the results suggest that disinhibition of mitral/tufted cells accounts for the observed enhancement in c-fos mRNA expression induced by NMDA receptor antagonists.

Neuronal replacement in the injured olfactory bulb

The adult forebrain subventricular zone contains neural stem cells that produce neurons destined for the olfactory bulb, where interneuron populations turnover throughout life. Forebrain injuries can stimulate production of these cells, and re-direct migrating precursors from the olfactory system to areas of damage, where their region-appropriate differentiation and long-term functional integration remain a matter for debate. Paradoxically, little is known about the ability of these progenitors to replace olfactory neurons lost to injury. Their innate capacity to generate bulb neurons may give them an advantage in this regard, and using injections of N-methyl-d-aspartate to kill mature olfactory bulb neurons, combined with bromodeoxyuridine labeling to monitor the fate of adult-born cells, we investigated the potential for injury-induced neurogenesis in this system. Widespread degeneration of bulb neurons did not affect the rate of cell proliferation in the subventricular zone, or cause neuroblasts to divert from their normal migratory route. However migration was slowed by the injury, leading to the accumulation and differentiation of neuroblasts as NeuN+ cells in the rostral migratory stream within 2 weeks of their birth. Despite this, a subset of new neurons successfully invaded the damaged bulb tissue, where they expressed neuronal markers including NeuN, calretinin, GABA, and tyrosine hydroxylase, with some surviving here for as long as 6 months. To test for functional integration of cells born post-injury, we also performed smaller NMDA lesions in restricted portions of the bulb granule cell layer and observed adult-born NeuN+ cells in these areas within 5 weeks, and BrdU+ cells that expressed the immediate-early gene c-fos following odor stimulation. These data suggest that the normal neurogenic capacity of the adult subventricular zone can be adapted to replace subsets of olfactory neurons lost to injury.

Expression of the inflammatory chemokines CCL2, CCL5 and CXCL2 and the receptors CCR1–3 and CXCR2 in T lymphocytes from mammary tumor-bearing mice☆

Chemokines and their receptors have been studied in several solid tumor models as mediators of inflammation. In turn, inflammation has been implicated in the promotion and progression of tumors, and as such, chemokines have been proposed as novel molecular targets for chemotherapy. While the expression of these molecules has been described in tumor cells, endothelial cells, macrophages and neutrophils, less attention has been paid to the expression profile of these molecules by T lymphocytes in the periphery or infiltrating the tumor. Using the D1-DMBA-3 murine mammary adenocarcinoma model, we aimed to better characterize the differential expression of chemokines and/or their receptors in the host and in the tumor microenvironment, and specifically, in the T cells of tumor-bearing mice compared to normal control animals. We found that T lymphocytes from tumor-bearing mice express the pro-inflammatory chemokines, CCL2, CCL5 and CXCL2, as well as the chemokine receptors, CCR1, CCR2, CCR3 and CXCR2.