Jonathan R. Day

GFAP mRNA increases with age in rat and human brain

Glial fibrillary acidic protein (GFAP) mRNA was examined by RNA blot hybridization in three age groups of two cohorts of male F-344 rats and in 47 human postmortem brain samples. GFAP mRNA increased in the hippocampus and striatum of 24 versus 6- to 7-month-old rats. Another astrocytic molecular marker, glutamine synthetase mRNA, did not change with age in rat brain. Rat GFAP mRNA prevalence was inversely correlated with serum testosterone but not correlated with serum corticosterone. In human hippocampus, frontal and temporal cortex, GFAP mRNA also increased in older (60-79 years) compared with middle-aged (25-59 years) individuals. In contrast, mitochondrial cytochrome oxidase subunit 1 mRNA did not change between age groups in any region. By combining the three regions for further analysis, GFAP mRNA increased with age irregardless of gender, alcoholism in the middle-aged group, or whether brains were classified as normal or neuropathologic (excluding Alzheimers disease pathology). These data indicate that increased GFAP protein or GFAP-immunoreactive astrocytes in rats and humans may result from transcriptional or post-transcriptional regulation and extend the number to three species (including mouse) showing an increase in GFAP mRNA with age. Factors that are known to regulate GFAP mRNA expression in young brains are considered as possible causes of age-related increases.

Gonadal steroids regulate the expression of glial fibrillary acidic protein in the adult male rat hippocampus

This study demonstrates that gonadal steroids (estradiol, testosterone, dihydrotestosterone) can regulate the expression of glial fibrillary acidic protein in the adult male rat brain. Previously, we showed that castration of adult male rats increased glial fibrillary acidic protein messenger RNA in the hippocampus and that this increase was additive with the increase induced by deafferenting entorhinal cortex lesions [Day et al. (1990) Molec. Endocr. 4, 1995-2002 . We extended these effects of castration and entorhinal cortex lesion to glial fibrillary acidic protein, using immunoassays. Furthermore, we found regional differences in responses to castration and inhibited by sex steroids. In contrast, hypothalamic glial fibrillary acidic protein expression was inhibited by castration. Similar regional differences were also shown for astrocyte glial fibrillary acidic protein distribution by immunocytochemistry. The regional specificity of glial fibrillary acidic protein expression after castration and sex steroid replacement is pertinent to the role of astrocytes in synaptic plasticity in unlesioned adults as well as in responses to lesions where the steroid milieu has been shown to influence sprouting.

Corticosterone differentially regulates the bilateral response of astrocyte mRNAs in the hippocampus to entorhinal cortex lesions in male rats

This study examined the effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on the levels of two astrocyte mRNAs during responses to unilateral entorhinal cortex lesions (ECL) to identify molecular mechanisms involved in glucocorticoid modulation of astrocyte activation following deafferentation. Both glial fibrillary acidic protein (GFAP) and sulfated glycoprotein-2 (SGP-2) mRNA were increased in the ipsilateral hippocampus 4 days following unilateral ECL. In unlesioned ADX rats CORT replacement decreased both messages in the hippocampus. CORT replacement suppressed the ECL-induced increase of GFAP mRNA in the contralateral, but not ipsilateral hippocampus of ADX rats. In contrast, CORT decreased SGP-2 mRNA both ipsi- and contralaterally. It is clear that several regulatory mechanisms are responsible for maintaining a physiological balance of astrocyte activity in the adult brain, and that changes in circuit integrity and the endocrine milieu can alter this balance.

Differential regulation of catalytic and non-catalytictrkB messenger RNAs in the rat hippocampus following seizures induced by systemic administration of kainate

Ribonuclease protection analysis and quantitative in situ hybridization histochemistry were used to investigate the coordination and regional expression of catalytic and non-catalytic trkB messenger RNAs in the adult rat hippocampus following systemic kainate-induced seizures. Changes in trkB expression were compared with the messenger RNA expression of its neurotrophic ligands, brain-derived neurotrophic factor and neurotrophin-3. TrkB messenger RNA expression was increased in the dentate granule cells at 1-4 h following the onset of seizures, and returned to control levels 16-24 h thereafter. In addition, seizures also induced expression of trkB messenger RNA in putative non-neuronal cells at four to seven days in the molecular layer of the dentate gyrus and the stratum lacunosum moleculare of the CA1 region. Hybridization with probes specific for the non-catalytic trkB receptor and the catalytic trkB receptor revealed that the increases at four and seven days in the molecular layers of the hippocampus reflected an up-regulation of only the non-catalytic form of the receptor. Furthermore, the neuronal increases observed 1-4 h were due to an up-regulation of both trkB TK- and trkB TK+ messenger RNAs. It was established that systemic administration of kainate increased brain-derived neurotrophic factor messenger RNA levels in the pyramidal and granule cell regions of the hippocampus 1-4 h following the onset of behaviorally manifested seizure activity. Early changes in neuronal expression of trkB TK- and trkB TK+ messenger RNA paralleled changes in brain-derived neurotrophic factor messenger RNA in the dentate granule cell and CA1 pyramidal cell layers, but not in the CA3 subregion. These data suggest that concomitant regulation of brain-derived neurotrophic factor and its cognate receptor may play a role in the selective vulnerability of hippocampal subregions to kainate-induced neuropathology. Furthermore, these data suggest a dual function for trkB receptor expression in the hippocampus following kainate-induced seizures, possibly related to both the plastic and degenerative consequences of seizure induction by kainate.

New mRNA probes for hippocampal responses to entorhinal cortex lesions in the adult male rat: A preliminary report

Three new mRNA responses were found in the hippocampus of the adult male rat after entorhinal cortex lesions (ECL) that induce synaptic reorganization. Hippocampus cDNA libraries were screened by a subtractive hybridization strategy designed to detect ECL-induced mRNAs. Partial sequencing showed clones with similarities to mouse vimentin, ferritin, and polypeptide 7B-2. A sequence similar to mouse SNAP-25 sequence was also detected. Using a mouse SNAP-25 probe, rat SNAP-25 mRNA increased in the hippocampus after ECL.

Microwave superheating enhances immunocytochemistry in the freshly frozen rat brain.

The goal of this study was to evaluate the effects of microwave irradiation on immunocytochemical staining in freshly frozen rat brain tissue using an antibody previously shown to successfully stain only formalin-fixed materials. GFAP immunoreactivity was markedly increased in sections exposed to microwave heating when compared with control tissue. Uniform staining enhancement was mainly a result of increased numbers of immunoreactive soma and large astrocytic processes. The integrity of the tissue was not affected by this treatment. Other antibodies need to be tested with this method, but our results suggest that microwave irradiation is useful for rapid antigen retrieval in freshly frozen central nervous system tissue.

Decortication and striatal mRNA : increases of mRNA for fibronectin, but not of NCAM or α-1 tubulin

Ipsilateral frontal cortex lesions damaged the corticostriatal input. Northern hybridization analysis showed increases in fibronectin (FN) mRNA, but not changes in mRNA for neural cell adhesion molecule (NCAM), neuro-filament-68 (NF-68) or alpha-tubulin (alpha-1T) 72 h post-lesion. In situ hybridization resolved a different spatial-temporal distribution. The superficial cell layer beneath the wound cavity showed transient elevations of FN mRNA that peaked at 72 h post-lesion. However, in the ipsilateral striatum, FN mRNA was maximal at later times than in the wound cavity, at 240 h post-lesion. Changes in NCAM and alpha-tubulin mRNAs in response to decortication occur only around the wound cavity but not in the deafferentated striatum. The different time courses of mRNA revealed by Northern blot analysis and in situ hybridization are most probably due to contamination of the ipsilateral striatum at dissection with superficial tissue adjacent to the wound cavity. These results suggest that cellular responses to ipsilateral decortication consist of two phases: (i) a wound healing process; and (ii) striatal responses to deafferentation.

Neurotrophins and Neurotrophin Receptors in Adult Brain Plasticity

265 Neurotrophic factors stimulate survival and growth of neurons during development and are involved in the regulation of neuronal survival, plasticity and regeneration in the adult nervous system. These growth factors offer an avenue to develop effective therapy for disorders associated with degeneration and neuron loss. Earlier studies showed that nerve growth factor (NGF) protects basal forebrain cholinergic neurons from lesion-induced degeneration and stimulates acetylcholine synthesis and release by surviving cholinergic neurons. Cell culture studies revealed that brain-derived neurotrophic factor (BDNF) promotes the developmental differentiation of both basal forebrain cholinergic and mesencephalic dopaminergic neurons. However, in adult rats, BDNF was less potent and less effective than NGF in protecting forebrain cholinergic cell bodies from axotomy-induced degeneration and, in contrast to NGF, failed to increase presynaptic cholinergic function in the hippocampus. BDNF also failed to protect dopaminergic neurons after axotomy in the adult High levels of BDNF mRNA are found in cholinergic target areas of the brain such as the hippocampus. Recent findings suggest that the expression of BDNF mRNA in the hippocampus is regulated by the cholinergic input. We compared the effects of partial and full fimbrial transections, which result in, respectively, partial and near-total cholinergic deafferentation on the expression of BDNF mRNA. Twenty one days following partial unilateral fimbrial transections there were significant decreases of BDNF mRNA expression throughout the hippocampal formation. The largest decreases were noted in pyramidal CA1 and CA3 layers and in the den-tate gyrus. The decreases amounted to 22-36% reductions compared to unlesioned control animals. BDNF mRNA levels were decreased to a greater extent (50-69%) following full unilateral fimbrial transections. Quantitative Northern blot analysis indicated that hippo-campal BDNF mRNA was decreased by 29% and 68%, 3 weeks after partial or full unilateral fimbrial transections, respectively. The extent of the reductions in BDNF mRNA levels correlated with reductions in acetylcholinesterase (ACHE) staining density and cholinergic terminal density determined by quantitative auto-radiographic analysis of [H]vesamicol binding sites. In addition, we found that chronic treatment with atropine (20 mg/kg for 14 days) decreased BDNF mRNA levels in the pyramidal CA1, CA2 and CA3 layers and in the dentate gyrus by 54%. In contrast, chronic treatment with nicotine (1.18 mg/kg for 14 days), a treatment known to desensitize nicotinic receptors, did not affect BDNF mRNA expression in the hippocampal formation. These findings provide evidence for cholinergic muscarinic regulation of BDNF mRNA expression in the adult rat hippocampal formation and …