Search

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor that influences the survival and function of several neuronal populations in the central (CNS) and peripheral nervous systems. The actions of GDNF are mediated by a multicomponent receptor complex composed of the tyrosine kinase product of c-ret and the ligand-binding protein GDNF receptor alpha (GDNFR-alpha). In the present study, we used in situ hybridization to localize cells expressing the mRNA for these GDNF receptor subunits in rat CNS. As reported previously, GDNFR-alpha and c-ret mRNA are present in the substantia nigra and ventral tegmental area, regions containing GDNF-responsive dopamine neurons. However, both mRNA were found in motor neurons of spinal cord and brainstem nuclei that innervate skeletal muscle. These areas include alpha motor neurons in the ventral horn of spinal cord and neurons in hypoglossal, facial, trigeminal, and abducens nuclei. In areas rostral to the substantia nigra, c-ret mRNA is not detected, whereas GDNFR-alpha is found in numerous brain structures, including the hippocampus, cortex, medial geniculate, and the medial habenula, the latter area expressing the highest levels of GDNFR-alpha mRNA in brain. These results provide evidence that c-ret and GDNFR-alpha mRNA are expressed in neuronal populations involved in motor function and provides further support for GDNF as a target-derived neurotrophic for these motor neurons. The observation that GDNFR-alpha mRNA is localized in several brain structures that do not contain detectable levels of c-ret mRNA indicates that either GDNFR-alpha utilizes signal transduction molecules other than c-ret in these areas or that other GDNF-like ligands that utilize GDNFR-alpha as a receptor may be present.

The levels of nerve growth factor (NGF) mRNA can be regulated in vitro and in vivo in the hippocampal formation by events associated with pharmacological activation of glutamate receptors. In the present study, the level of NGF mRNA in the hippocampal formation was examined following an intrahippocampal injection of 1 nmole fluorocitrate, which temporarily inhibits the astrocyte metabolic activity in vivo. Consistent with previous findings, fluorocitrate treatment significantly increased glutamate levels and decreased glutamine levels in the dentate gyrus as determined by in vivo microdialysis. The increased ratio of glutamate to glutamine was followed by a significant increase in NGF mRNA expression selectively in dentate gyrus granule cells. The effects of increasing glutamate levels were blocked by pretreatment with 50 nmole 2-amino-5-phosphonovalerate (AP5), a competitive antagonist that acts at the N-methyl-D-aspartate (NMDA) glutamate receptor subtype. These findings suggest that NGF mRNA expression is regulated, in part, by changes in endogenous glutamate levels, partially through enhanced excitatory neurotransmission through NMDA receptors.

One of the primary neurodegenerative events occurring in amyotrophic lateral sclerosis (ALS) is the selective loss of spinal cord alpha motor neurons. To study the potential role of apoptosis in the degeneration of these motor neurons, in situ hybridization was used to measure the expression of two apoptotic cell death genes, bcl-2 and bax, in control and ALS lumbar spinal cord sections. The strongest hybridization signal for bcl-2 mRNA in neurological and nonneurological control spinal cords was found primarily in lamina IX alpha motor neurons, while a weaker hybridization signal was found in neurons of Clarke's nucleus and the proper sensory nucleus of the dorsal horn. Surviving lamina IX motor neurons in ALS spinal cord sections also expressed bcl-2 mRNA, but at levels that were significantly and selectively decreased (4.7-fold) compared with controls. bax mRNA hybridization signal was detected in several cells throughout the gray matter in control and ALS lumbar spinal cord, but was significantly and selectively increased (2.8-fold) in ALS motor neurons. Given the proposed interactive roles of these genes in apoptosis, the present findings favor a scenario in which this mode of cell death would contribute to spinal cord motor neuron degeneration in ALS.

Total RNA from rat Schwann cells grown in culture and adult rat skeletal muscle was reverse transcribed, amplified for glial cell line-derived neurotrophic factor (GDNF) messenger RNA (mRNA) using the polymerase chain reaction (PCR), and the PCR products sequenced. Two forms of GDNF were detected in the PCR step, one of a predicted size (GDNF633) and a second smaller form missing a 78-base pair sequence (GDNF555). Sequence analysis demonstrated that GDNF633 is similar to the published sequence of GDNF differing only at three nucleotides. Southern and Northern blot analyses reveal that the two forms are probably derived from a single RNA species that is alternatively spliced. Interestingly, GDNF633 mRNA was found to be selectively upregulated in denervated rat skeletal muscle at 1-2 weeks following axotomy, providing evidence that the innervation status of the muscle may determine the expression profile of the two alternatively spliced forms. Given these findings, we suggest that GDNF may function as a target-derived trophic factor for neuronal populations innervating skeletal muscle, including sensory neurons and spinal cord motoneurons.

N-methyl-D-aspartate (NMDA) receptor activation in the hippocampal formation is thought to play an important role in learning and memory. This limbic structure contains one of the highest concentrations of NMDA binding sites in the brain. The hippocampal formation also contains high levels of nerve growth factor (NGF) mRNA and protein in the central nervous system. The expression of this neurotrophic factor may be regulated by events involving glutamatergic neurotransmission. In the present study, in situ hybridization histochemistry was used to determine the effects of NMDA receptor activation on NGF mRNA expression in the hippocampal formation. The gene encoding this neurotrophic factor was increased exclusively in the granule cells of the dentate gyrus, a hippocampal structure that receives extensive glutamatergic innervation from the entorhinal cortex. It is suggested that one consequence of glutamatergic neurotransmission in the dentate gyrus is the activation of NGF mRNA. The increased expression of this neurotrophic factor may ultimately influence the function of NGF-responsive cells innervating the hippocampal formation.

A recently cloned neurotrophic factor, termed glial cell line-derived neurotrophic factor (GDNF), has been reported to exhibit selective neurotrophic properties on ventral mesencephalon dopaminergic neurons, which degenerate in patients with Parkinson's disease. In the present study, we used reverse transcriptase followed by polymerase chain reaction (PCR) and in situ hybridization to study the expression of GDNF messenger RNA (mRNA) in the adult rat and human central nervous system (CNS). GDNF transcripts were identified using PCR in all regions of the rat CNS analyzed including striatum, hippocampus, cortex, cerebellum, and spinal cord. Interestingly, the rat hippocampal formation contained two transcripts, i.e., a larger form in addition to the amplified GDNF cDNA found in all other areas analyzed. GDNF PCR products also were observed in human striatum, hippocampus, cortex, and spinal cord, but not cerebellum, and both the striatum and hippocampal formation contained two GDNF transcripts. Finally, GDNF transcripts were detected in a rat Schwann cell line previously shown to secrete a factor that exerts a neurotrophic effect on dopaminergic neurons. In situ hybridization experiments using a cRNA probe hybridized to adult rat brain sections demonstrated no positive GDNF mRNA signal. However, intense GDNF mRNA hybridization signal was found to be associated with dorsal root ganglia in Postnatal Day 1 rats. These findings provide evidence that GDNF is detectable using PCR in a number of nervous system structures and, in some areas, GDNF is expressed in more than one form.

In the central nervous system, the highest levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNA are found in the hippocampal formation. In the present study, we report that a unilateral transection of the angular bundle, which relays cortical information via the entorhinal cortex to the hippocampal formation, increases NGF and BDNF mRNA in the ipsilateral dentate gyrus. Within 4 hours following transection, the hybridization signal for NGF and BDNF mRNA increases in stratum granulosum 3- and 5-fold, respectively, compared to control levels. This lesion-induced increase of both mRNA returns to control levels within 24 hours and is maintained for at least 5 days. The induction is not prevented by pretreatment with AP-5, CNQX, or cholinergic denervation due to transection of the fimbria-fornix. Finally, the induction of neurotrophin mRNA is preceded by an increase in c-fos mRNA. These results provide evidence that transection of the cortical input to the hippocampal formation upregulates NGF and BDNF mRNA selectively in stratum granulosum. We suggest that the increased expression of NGF and BDNF mRNA may be an early step in the synaptic rearrangement of neurotrophin responsive cholinergic afferents observed following damage to the entorhinal cortex.

In the present study, in situ hybridization was used to examine the expression of nerve growth factor (NGF) receptor (p75NGFR), trk (p140trk) and trkB (p145trkB) mRNA in spinal cord sections from patients with amyotrophic lateral sclerosis (ALS). We report that the expression of p75NGFR and p145trkB mRNA is elevated in alpha motoneurons in ALS sections. However, p140trk mRNA was not expressed in either ALS or control sections.

In the hippocampal formation, nerve growth factor (NGF) is produced in granule cells of the dentate gyrus and a few pyramidal cells of Ammon's horn. Both neuronal populations express N-methyl-D-aspartate (NMDA) receptors and receive putative glutamatergic afferents originating in the entorhinal cortex and projecting via the perforant path. We report in this study that intra-hippocampal or intraventricular injections of NMDA increase NGF mRNA levels in dentate gyrus granule cells as determined using in situ hybridization histochemistry and a solution hybridization assay. NGF mRNA induction is detected within 2 h following NMDA treatment and returns to control levels within 24 h. This NMDA effect is dose-dependent and blocked by pretreatment with 2-amino-5-phosphonopentanoic acid, a competitive NMDA antagonist. Finally, the induction of NGF mRNA is observed in the absence of detectable neurotoxicity or seizure activity. We postulate that normal physiological events associated with the activation of hippocampal NMDA receptors may regulate mRNA expression of this neurotrophic factor.

Radioactively labeled RNA probes in conjunction with in situ hybridization histochemistry have become a useful method for studying gene expression in the central nervous system. We used digoxigenin-labeled uridine triphosphate to synthesize cRNA probes for localization of nerve growth factor receptor (NGFR) mRNA in the rat basal forebrain. Detection of cells containing digoxigenin-labeled NGFR mRNA was accomplished using a digoxigenin antibody conjugated with alkaline phosphatase. NGFR mRNA-positive cells were distributed in three major cell groups in the basal forebrain: the medial septal nucleus, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis. This technique provides a rapid and sensitive method for high-resolution detection of mRNA species in the central nervous system, as well as the potential for co-localization of two different mRNA species within individual cells.

1. In situ hybridization histochemistry was used to localize nerve growth factor receptor (NGFR) mRNA in the adult rat basal forebrain. 2. In emulsion-dipped sections 35S-labeled RNA antisense probes produced a high density of silver grains over cells located in the medial septum, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis. 3. This distribution of NGFR mRNA overlaps with the distribution of NGFR protein localized using immunocytochemical techniques. 4. No hybridization signal was detected when sections were hybridized with a 35S-labeled RNA sense (control) probe. 5. We suggest that NGFRs are synthesized in these basal forebrain nuclei and transported to terminal areas where NGF is thought to be bound and internalized, an initial step in the many actions of this neurotrophic factor.