Exposure of neuronal terminals to target-derived NGF has been hypothesized to regulate cell body responses at a distance. To test this hypothesis and, more specifically, to determine whether NGF distally regulates the synthesis of its two characterized receptors, we developed the following paradigm. Sympathetic neurons of the superior cervical ganglion (SCG) that project either to the eye or to the ear were labeled, in adult animals, with the retrograde tracers Fast Blue and Fluoro-Gold, respectively. NGF was then injected daily into the anterior chamber of one eye, exposing the terminals of the ipsilateral eye neurons to increased NGF. To control for systemic and/or localized injury effects, cytochrome C or PBS were injected into the contralateral eye of the same animals. In situ hybridization and image analysis were then used to determine neuronal levels of p75 NGF receptor, trkA, and T alpha 1 alpha-tubulin mRNAs, with the latter providing a correlative measure of neuronal sprouting. Morphological measurements revealed that exogenous, terminally-derived NGF increased the mean cross-sectional area of eye neurons by 37%. Grain counts for p75 NGF receptor mRNA increased from 2- to 6-fold in the NGF-treated neurons, and grain densities, which accounted for neuronal hypertrophy, also increased significantly. In contrast, grain counts for trkA mRNA were not significantly increased by this treatment, while T alpha 1 alpha-tubulin mRNA levels increased only 1.5- to 2-fold. No increase in grain density was detected for either of these mRNAs. The NGF-induced increased in p75 NGF receptor mRNA levels was accompanied by terminal sprouting and by an increase in the density of p75 NGF receptors on terminal neurites, as indicated by IgG-192 immunostaining of the NGF-treated iris. These data therefore suggest that, in addition to promoting local sprouting, increased target-derived NGF increases the levels of p75 NGF receptor relative to trkA on terminal neurites, by differentially regulating receptor synthesis. Such a direct regulatory feedback loop may well play an integral role in precisely modulating neuronal responses as a function of the amount of available trophic support and/or target tissue.

Animals↗

PubMed1993

Evidence that the loss of homeostatic signals induces regeneration-associated alterations in neuronal gene expression.

Axotomy of motoneurons leads to upregulation of T alpha 1 alpha-tubulin and p75 NGF receptor mRNAs. To distinguish whether these increases are due to interruption of ongoing homeostatic signals or to positive factors derived from non-neuronal cells of the injured nerve, we developed an experimental paradigm that allowed us to selectively block some facets of axonal signaling without initiating a peripheral nerve injury response. More specifically, the marginal mandibular and buccal branches of the facial nerve were locally cooled to 4-8 degrees C (a cold block) for 36-60 hr. This treatment effectively and reversibly blocked fast axonal transport, as monitored by the ability of facial motor neurons to retrogradely transport fluorogold from their terminals to their cell bodies. Light microscopy of semithin epoxy sections demonstrated that the cold block treatment did not lead to macrophage invasion or to morphological alterations in the nerve either proximal or distal to the cold block region. In situ hybridization and image analysis of retrogradely labeled facial motor neurons indicated that such a cold block induced T alpha 1 alpha-tubulin and p75 NGF receptor mRNAs to the same level as did a corresponding nerve transection. In contrast, T26 alpha-tubulin mRNA, which does not increase following axotomy, was not affected by the cold block treatment. These results suggest that neurons sense their status by a constant flow of information carried, circulated, or maintained by fast axonal transport and that the axotomy-induced increases in T alpha 1 and p75 NGF receptor mRNAs in motor neurons are, to a great extent, due to loss of such homeostatic signals.

Animals↗

PubMed1993

Induction of T alpha 1 alpha-tubulin mRNA during neuronal regeneration is a function of the amount of axon lost.

Axotomy of mature peripheral neurons leads to upregulation of T alpha 1 alpha-tubulin mRNA. To distinguish whether this increase is due to interruption of target contact or to loss of the axon itself, we developed a model system that allowed us to axotomize mature sympathetic neurons proximal or distal to their cell bodies, severing contact with target tissue in all cases. Sympathetic neurons within the same superior cervical ganglion that project via the internal and external carotid nerves to the eye and to the ear, respectively, were differentially labeled with the retrograde tracers fast blue and fluorogold, and the labeled neurons were then transected close to or far from their cell bodies. In situ hybridization analysis 5 days postaxotomy (the time of peak T alpha 1 mRNA expression) indicated that when eye and ear neurons were both axotomized close to their cell bodies. T alpha 1 mRNA increased to a similar degree in both populations. In contrast, when ear neurons were again cut close, but eye neurons of the same ganglion were transected far from their cell bodies by eye removal, peak T alpha 1 mRNA levels were two- to threefold lower in the eye neurons. Thus, the increase in T alpha 1 mRNA was much lower when only a small amount of axon was lost, even though contact with target tissue was completely interrupted in all groups. These results therefore suggest that neurons normally monitor the status of their axon via a mechanism that allows for graded responses and that the axotomy-induced increase in T alpha 1 mRNA is, to a great extent, due to loss of the nonterminal axon.

Animals↗

PubMed1992

Spatiotemporal increases in epidermal growth factor receptors following peripheral nerve injury.

Non-neuronal cells of peripheral nerve respond to axonal injury with a series of cellular changes that facilitate neuronal regeneration. To characterize the potential role of the epidermal growth factor (EGF) family of proteins in this response, we monitored the expression of EGF receptor mRNA and protein in the injured rat sciatic nerve. EGF receptor mRNA is synthesized in both primary cultured fibroblasts and Schwann cells, and Schwann cells express EGF receptor-like immunoreactivity. In situ hybridization and immunocytochemistry revealed that EGF receptor mRNA and protein are expressed in Schwann cells and fibroblasts of the sciatic nerve in vivo, and that receptor levels increase following nerve injury. Thirty-six hours postlesion, EGF receptors were expressed in gradients along the nerve both proximal and distal to the lesion, with the highest levels localized adjacent to the transection site. By 72 hr, receptor levels were maintained in a gradient in the proximal segment, but were uniformly increased throughout the portions of the distal segment that were analyzed. These changes were similar to those observed for low-affinity NGF receptor mRNA and protein, with transection causing increased expression in both Schwann cells and fibroblasts. Northern blots confirmed that primary cultured fibroblasts express low-affinity NGF receptor mRNA. To determine whether spatiotemporal gradients were a general characteristic of the nerve injury response, we monitored expression of the mRNA encoding the major myelin protein P0. Levels of P0 mRNA decreased initially in cells immediately adjacent to the transection site and, by 72 hr, were uniformly decreased throughout the distal segment. These data suggest that members of the EGF family of proteins may play a role in the peripheral nerve response to injury, and demonstrate a generalized gradient of cellular responses that commence at the transection site and progress distally in the nerve in the absence of intact axons.

Animals↗

PubMed1991

Regulation of nerve growth factor receptor gene expression by nerve growth factor in the developing peripheral nervous system.

Nerve growth factor (NGF) is a target-derived neurotrophic protein that promotes the survival and growth of developing sympathetic and sensory neurons. We have examined NGF receptor gene expression in these neurons after NGF administration. Northern blot and in situ hybridization analyses demonstrated that NGF given systemically to neonatal rats increased levels of NGF receptor mRNA in sympathetic neurons within the superior cervical ganglion. This increase was accompanied by a differential regulation of genes associated with neurotransmitter phenotype; tyrosine hydroxylase mRNA was increased, but neuropeptide Y mRNA was not. NGF receptor mRNA levels were also increased in L4-L5 dorsal root ganglia, although this mRNA was not expressed uniformly in sensory neurons of control or NGF-treated animals. Levels of T alpha 1 alpha-tubulin mRNA, a marker of neuronal growth, also increased. In contrast to developing neurons, systemic NGF did not increase NGF receptor mRNA in nonneuronal cells of the sciatic nerve. To determine if NGF regulated NGF receptor gene expression at the transcriptional level, we examined PC12 cells. NGF treatment for 6 h increased NGF receptor mRNA fourfold; this increase was inhibited by cycloheximide. Nuclear run-off transcription assays demonstrated that the increase in steady-state NGF receptor mRNA levels was mediated at the transcriptional level. In contrast, although NGF treatment increased steady-state tyrosine hydroxylase mRNA levels, this effect was not blocked by cycloheximide, and was not due to increased transcription. These data raise the possibility that transcriptional regulation of NGF receptor gene expression by target-derived NGF could be a molecular mechanism for potentiating NGF's effects on neurons during developmental periods of neuronal competition and cell death.

Adrenergic Fibers↗

PubMed1990

Increased expression of T alpha 1 alpha-tubulin mRNA during collateral and NGF-induced sprouting of sympathetic neurons.

We have examined expression of T alpha 1 alpha-tubulin mRNA in the rat superior cervical ganglion (SCG) to determine whether changes in gene expression accompany neuronal sprouting and to investigate factors that regulate growth-associated genes in intact neurons. Northern blot analysis demonstrates that levels of T alpha 1 alpha-tubulin mRNA increase in the uninjured SCG following transection of contralateral neurons that project to bilaterally innervated, but not unilaterally innervated target organs. The observed increase in uninjured neurons is associated with collateral sprouting, as measured by increased tyrosine hydroxylase immunoreactivity within the pineal gland. These data suggest that target-derived factors may regulate T alpha 1 mRNA in sprouting neurons. Consistent with this hypothesis, systemic NGF treatment of neonatal animals over a developmental interval when T alpha 1 alpha-tubulin mRNA normally decreases led to a 5- to 10-fold increase in T alpha 1 mRNA levels in developing sympathetic neurons. In addition, deafferentation of the SCG, which promotes neuronal sprouting in the ganglion, increases T alpha 1 mRNA in ganglia on the ipsilateral and contralateral sides. Together, these data demonstrate that T alpha 1 alpha-tubulin mRNA elevates as a function of neuronal sprouting, and that T alpha 1 mRNA expression in intact neurons can be regulated by extrinsic cues, including NGF and changes in connectivity.

Animals↗

PubMed1989

Immunocytochemical studies of astrocytes following injury to the cerebral cortex of the rat.

The morphological change of cerebral cortex astrocytes from protoplasmic to glial fibrillary acidic protein (GFAP)-containing cells is induced by injury. Protoplasmic astrocytes that contain no detectable amount of GFAP become filled with GFAP and their processes extend to form the glial scar around the wound. It is hypothesized that this transformation is induced by cAMP and neurotransmitters released from damaged neuronal cells. A similar mechanism may be present in other brain regions following injury or disease.

Animals↗

PubMed1989

Morphology and distribution of tanycytes in the third ventricle of the adult rat. A study using semithin methacrylate sections.

Light microscopy and semithin methacrylate sections were used to study the tanycytic projections and morphology in the floor of the third ventricle of the rat. The tanycytic cell soma was located in the ependyma. The luminal surface showed minute protrusions into the ventricular space and their basal processes projected across the width of the parenchyma of the infundibular region. During their course, tanycytic processes made contact with capillaries in the parenchyma and pial surface, suggesting that they might be involved in uptake and/or delivery mechanisms between the cerebrospinal fluid, hypothalamic cells and blood vessels.

Animals↗

PubMed1987

Adrenomedullary chromaffin cells of the rat. An ultrastructural study.

Adrenomedullary chromaffin cells of the rat were studied at the ultrastructural level. Chromaffin cells contained a large population of electron-dense-core vesicles of two types, one very electron-dense (norepinephrine) and the other moderately electron-dense (epinephrine). The vesicles showed an even distribution pattern in the cytoplasmic matrix. No physical contacts were observed between cytoplasmic and vesicle membranes to indicate exocytosis, a mechanism frequently observed in the hamster. For chromaffin cells to be used as a transplantable source of dopamine, both the cells and vesicles must survive the trauma of denervation and vascular elimination or alternatively acquire the two factors from the transplantation site.

Adrenal Medulla↗

PubMedDate not supplied

Primary Ki-1 lymphoma of the skin: expression of growth hormone receptors.

The biological significance of growth hormone (GH) in the physiology and pathophysiology of the immune system is not established. To address the site and mode of action through which GH exerts its effects on lymphocyte tumors, we applied a well-characterized monoclonal antibody directed against the hormone binding site of the receptor and were able to further characterize the tumor by immunohistochemical localization of GH receptors. Cutaneous T cell lymphomas were identified by histologic and immunomorphologic diagnosis according to the updated Kiel classification, with the application of monoclonal antibodies. Nodular tumors of the skin, identified as highly malignant Ki-1 lymphomas of large anaplastic cells, had intense GH receptor immunoreactivity. The presence of GH receptors in these proliferating tumor cells supports the hypothesis that GH is involved in paracrine-autocrine mechanisms acting locally in regulating peripheral T cell lymphoma tumor growth.

Antibodies, Monoclonal↗