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Differential display PCR was used to identify alternate expression of serum glucocorticoid-regulated kinase (Sgk) mRNA in diabetes-induced renal disease. Differential expression of Sgk mRNA was identified in the kidneys of normal and obese db/db mice, a model of select aspects of human diabetic nephropathy. Sgk mRNA was selectively increased in diabetic mouse kidneys. The Sgk mRNA levels remained constant in other tissues from obese db/db mice. An increase in Sgk mRNA was also observed in the human diabetic kidney. In addition, thrombin, which may play a role in the progression of renal disease, increased Sgk message in cell culture. Because the diabetes-induced increase in Sgk was only observed in the kidney, which is particularly susceptible to diabetes-induced damage, Sgk may play a role in diabetic nephropathy.

Hepatocyte growth factor (HGF) is an injury-released growth factor with diverse effects on epithelial and endothelial cells. These effects include proliferation, migration, extracellular matrix production and tubulogenesis. These activities allow HGF to function as an organizer of repair processes that bring about restoration of tubular function following renal injury. However, while HGF has been demonstrated to accelerate recovery of renal function after an acute insult, prolonged exposure to elevated levels of HGF can reduce renal function and may contribute to progressive renal disease. This review will describe the cellular activities of HGF, how they pertain to renal repair and the therapeutic application of regulating HGF activity in acute versus chronic renal disease.

Recent advances in TGF-beta signaling biology have identified smad proteins as cytoplasmic mediators of TGF-beta receptor activation. Current evidence suggests that smad proteins are transcription factors whose activity is regulated through phosphorylation by TGF-beta type I receptors. Several isoforms of the smad proteins have been identified which confer in part selectivity for different members of the TGF-beta family of receptors as well as some selectivity for downstream effects of TGF-beta activation. Therefore, smad proteins provide an attractive means for targeting select activities of TGF-beta by either inhibiting their phosphorylation or specific DNA binding modes. Because TGF-beta is a central player in the development of fibrosis, selective inhibition of smad proteins would provide an important therapeutic benefit in many acute and chronic fibrotic diseases.

Age-related increases occurred in renal thrombospondin 1 (TSP1) mRNA in F344 rats, resembling diabetes-induced TSP1 mRNA in the obese Zucker rat. TSP1 mRNA was 3.5-fold higher in 24-month-old than in 3-month-old F344 rats. TSP1 mRNA increased similarly in 5-month-old obese Zucker rats as compared with lean littermates and correlated positively with the extent of proteinuria (r = 0.71). In situ hybridization identified elevated TSP1 mRNA levels in epithelial cells of distended tubules as well as in interstitium near dilated tubules of both 24-month-old F344 rats and 5-month-old obese Zucker rats. Furthermore, thrombin increased TSP1 mRNA in mesangial and epithelial cells in culture, indicating that thrombin may contribute to elevated TSP1 expression in renal disease. Thrombin increased TSP1 mRNA within 30 min after treatment which required de novo synthesis of protein. The thrombin receptor tethered ligand peptide, SFLLRN, increased TSP1 mRNA, indicating that the thrombin-induced increase in TSP1 mRNA was due to direct thrombin receptor (PAR1) stimulation. These results show that increased TSP1 mRNA levels are a component of interstitial fibrosis seen in aged and diabetic kidneys and suggest that similar pathological changes occur in kidneys of aging and diabetic rats.

The effect of the angiotensin-converting enzyme inhibitor captopril on clusterin mRNA was examined in partially nephrectomized male rats. Urine protein excretion was measured 3, 7, and 28 days after removal of five sixths of the renal mass. Nephrectomy caused a progressive increase in clusterin mRNA levels in the remnant kidney. Maximal clusterin mRNA levels occurred 7 days after nephrectomy and declined 28 days after nephrectomy. Captopril, 250 mg/ml in drinking water, prevented the injury-induced increase in clusterin mRNA at 7 and 28 days. Captopril had no effect on clusterin in sham-operated rats. As expected, the urine protein excretion increased progressively after nephrectomy, and this was attenuated by administration of captopril in the drinking water. Therefore, clusterin is a marker of renal injury which, along with proteinuria, is modulated by angiotensin-converting enzyme inhibition.

Evidence suggests that transforming growth factor beta 1 (TGF-beta 1), a multifunctional cytokine, induces renal extracellular matrix production and glomerular hypertrophy. The aim of the present study was to investigate the effect of captopril on the expression of TGF-beta 1 mRNA in a rat model of chronic renal failure: five-sixths nephrectomy. Chronic renal disease was induced by removal of the right kidney and ligation of three blood vessels supplying the left kidney. Sham-operated animals were used as controls. RNA was extracted from the viable remnant kidney of rats 1 day and 1 and 2 weeks following five-sixths nephrectomy and from the kidneys of rats who underwent sham surgery. TGF-beta 1 mRNA was induced within 24 h of partial nephrectomy, similar to that reported for early-onset genes. Subsequently, TGF-beta 1 mRNA expression continued to increase over the next 2-4 weeks. The upregulation of TGF-beta 1 correlated with the degree of proteinuria. Both the increase in TGF-beta 1 mRNA and proteinuria were abrogated by captopril treatment. In addition, no change in expression of ALK-5 or type II TGF-beta receptors following five-sixths nephrectomy was observed. These data suggest that captopril may protect against development of glomerulosclerosis and proteinuria by reducing TGF-beta 1 expression and hence matrix production.

Hepatocyte growth factor (HGF) induces mitogenesis, chemotaxis, and tubule formation in renal epithelial cells. This study examined the effects of wortmannin and protein kinase C (PKC) inhibitors on HGF-mediated changes in metabolic activity in glomerular mesangial cells and renal epithelial carcinoma A498 cells. The extracellular acidification rate of transformed mouse glomerular mesangial cells and A498 cells was measured as an index of metabolic activity with a microphysiometer. HGF increased the acidification rate of mesangial cells and A498 cells in a concentration-dependent fashion that was inhibited completely by the tyrosine kinase inhibitor tyrophostin-23 (100 microM). The PKC inhibitors RO-32-0432 and SKF-57048 also inhibited HGF-induced acidification. The IC50 values for SKF-57048 were 59 +/- 2 and 20 +/- 10 nM in mesangial cells and A498 cells, respectively (P < 0.05). 12-O-Tetradecanoylphorbol 13-acetate (TPA), a phorbol ester that activates PKC, increased acidification in mesangial and epithelial cells similar to HGF. Wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinase (IC50 value 1-10 nM), inhibited HGF-induced acidification with an IC50 of 93 +/- 31 and 9 +/- 1 nM in mesangial and A498 cells, respectively (P < 0.05). In contrast, there was no significant difference in the IC50 value of wortmannin for epidermal growth factor (EGF)-induced acidification between mesangial and A498 cells (23 +/- 9 vs 14 +/- 1 nM, respectively). Because the IC50 value for wortmannin in inhibiting HGF but not EGF-induced acidification was an order of magnitude higher in mesangial cells than in epithelial A498 cells, a wortmannin-sensitive PI 3-kinase pathway may not be involved in HGF-mediated acidification in mesangial cells.

Clusterin is a multifunctional glycoprotein associated with development and tissue injury. Because renal function decreases with advancing age in the obese Zucker rat, clusterin mRNA expression was examined in the kidney of young adult Zucker rats and compared with age-related changes in renal clusterin mRNA expression in Fischer 344 (F344) rats. Renal clusterin mRNA levels in the obese Zucker rat were 2.5-fold higher by 3 mo of age and fourfold higher at 5 mo of age compared with the lean strain. In comparison, renal clusterin mRNA in 12-mo-old F344 rats was twofold higher than in 3-mo-old animals and was tenfold higher at 24 mo of age. Clusterin mRNA was positively correlated with urinary protein excretion and negatively correlated with creatinine clearance in Zucker rats. Clusterin was increased in select nephrons of the obese Zucker rat kidney and in 24-mo-old F344 rat kidney as assessed by in situ hybridization. Increased expression of clusterin mRNA occurred mostly in the tubular epithelium of dilated, convoluted proximal tubules. These data indicate that renal clusterin mRNA levels increase as a function of age and that age-related increases in renal clusterin and the associated tubular abnormalities are accelerated in obese Zucker rats.

Clusterin, a multifunctional protein with complement blocking activity, and fibrin, a product of thrombin's enzymatic activity, are present in the kidney during acute and chronic renal failure. The role of thrombin in regulating clusterin mRNA in the kidney is not known. The effect of thrombin on clusterin mRNA expression was examined in rat glomerular mesangial and glomerular epithelial cells, and cultured human renal proximal tubular epithelial cells by northern blot. Thrombin (10(-8) M) increased clusterin mRNA levels two- to fourfold in glomerular mesangial, glomerular epithelial, and proximal tubule epithelial cells. This was a specific effect of thrombin receptor activation because peptides corresponding to the tethered ligand of the thrombin receptor were also able to increase clusterin mRNA levels. Epidermal growth factor, insulin-like growth factor-1, and transforming growth factor-beta 1 had little or no effect on clusterin mRNA levels. The protein kinase C inhibitor RO-32-0432 (1 microM) inhibited the thrombin-induced increase in clusterin mRNA, suggesting that thrombin receptor activation may regulate renal clusterin mRNA levels through protein kinase C.

The presence of complement system proteins in amyloid plaques and the up-regulation of several complement mRNAs in neurons and glial cells in affected brain regions during Alzheimer disease (AD) provided a basis for further examination of complement protein expression in a rodent lesion model of AD. Perforant path transection in rats was used as a model for the degeneration of entorhinal cortex (EC) layer II neurons and the consequent deafferentation of the hippocampus that occurs during AD. Immunostaining for C9, a key terminal component of the complement cascade membrane attack complex (MAC), showed extracellular C9 deposition in parenchyma around the EC wound and in hippocampus as early as 1 day, and disappeared by 14 days postlesion. Apoptosis of EC layer II neurons was seen and was presumably due to severing of their axonal projections to the hippocampus by the transection lesion. However, apoptotic EC layer II neurons were not immunostained by anti-rat C9 antibody, suggesting complement was not involved in inducing apoptosis. In the deafferented hippocampus, extracellular C9 immunostaining was localized to the dentate gyrus middle molecular layer, a region of synaptic loss, dendritic degeneration, and early synaptogenesis. In addition, intracellular C9 immunostaining was seen only in select hippocampal interneurons. Dentate gyrus granule neurons and pyramidal neurons were not C9 immunostained. Clusterin (SGP-2), a soluble inhibitor of the MAC that is up-regulated in AD, was also detected in the wound area (extracellular), the dentate gyrus middle molecular layer (extracellular), and intracellularly in scattered hippocampal interneurons. The data support the hypothesis that the complement system generally participates in responses to brain injury, as well as in AD.

In the rat hippocampus and cortex, the transcription of glial fibrillary acidic protein (GFAP), an astrocyte intermediate filament protein, is inhibited by glucocorticoids. The present study examined the regulation of GFAP expression by glucocorticoids in astrocytes in vitro. Corticosterone (CORT) increased GFAP messenger RNA, protein, and transcription rates in cultured primary neonatal astrocytes, responses opposite the GFAP responses to CORT in vivo. The direction of GFAP regulation by corticosterone in vitro is reversed by coculture with neurons or by extended culture for 3 months. The switch in the direction of GFAP regulation by CORT during prolonged culture is associated with a 3-fold increased prevalence of type II glucocorticoid receptor (GR). These findings were corroborated with a promoter construct that contained 1.9 kilobases of 5'-up-stream rat GFAP DNA with a luciferase reporter. Thus, the direction of GFAP transcription to CORT is subject to the postreplicative time in culture and to interactions with neurons, in which 5'-up-stream sequences contain sufficient information to mediate the switch in the direction of the response to CORT. This in vitro model may be used to analyze how interactions of astrocytes with neurons or other cell types influence the hormonal regulation of GFAP.

This study characterizes the effect of transforming growth factor (TGF) beta 1 on clusterin expression in rat brain cells. 24 h after an acute unilateral intracerebroventricular infusion of TGF-beta 1, clusterin mRNA prevalence was increased in astrocytes that contained immunoreactive (IR) glial fibrillary acidic protein (GFAP). TGF-beta 1 selectively induced clusterin mRNA in astrocytes, as no clusterin mRNA was detected in neurons, oligodendrocytes, or microglia. TGF-beta 1 induced a bilateral increase in clusterin mRNA per astrocyte. Astrocyte hypertrophy (GFAP-IR area) was only increased on the ipsilateral side. In pure astrocyte cultures, TGF-beta 1 (200 pM) decreased clusterin mRNA levels and the rate of clusterin RNA transcription. However, in cultures of astrocytes that contained microglia and oligodendrocytes (mixed glia cultures), TGF-beta 1 caused a dose-dependent increase in astrocytic clusterin mRNA levels. The astrocytes that responded to TGF-beta 1 included two GFAP-IR subtypes, type 1 and 2. TGF-beta 1 increased clusterin protein in the conditioned medium from cultured glia, in either monotypic or mixed glial cultures. Thus, TGF-beta 1 and heterotypic cell interactions influence clusterin expression by astrocytes and may be important to the role of clusterin in multiple sclerosis, AIDS, and Alzheimer's disease.

Entorhinal cortex lesions (ECL) that damage the perforant path to the dentate gyrus of the hippocampal formation were used to model the regulation of vimentin (VIM) mRNA. ECL increased VIM mRNA in the ipsilateral hippocampus and in the ipsilateral cortex including the wound cavity within 1 day. By in situ hybridization, at 4 days post-ECL, VIM mRNA increased two-fold in the molecular layer of the dentate gyrus. VIM protein was co-localized by immunocytochemistry to astrocytes and microglia/macrophages. Transforming growth factor-beta 1 (TGF-beta 1), which was previously shown to increase in microglia/macrophages of the molecular layer after hippocampal deafferentation by ECL, was investigated as a regulator of VIM expression. Infusions of TGF-beta 1 into the lateral ventricle induced VIM mRNA with dose-dependence, e.g. infusion of 100 ng TGF-beta 1 increased VIM mRNA three-fold. The increase in VIM mRNA was localized by in situ hybridization to astrocytes and microglia in the molecular layer of the dentate gyrus. These findings further implicate TGF-beta 1 as a regulator of cytoskeletal proteins during synaptic reorganization.

Age-related increases in the expression of glial fibrillary acidic protein (GFAP) in many brain regions are observed in short- and long-lived mammals. Possible genomic mechanisms for the increase of GFAP mRNA and protein were studied in the hippocampus and cortex of male F344 rats and a longer-lived hybrid F1 (F344 x Brown Norway). No age-related changes were found in the extent of cytosine methylation at 19 CpG sites in the 5'-upstream GFAP promoter and in exon 1. With the nuclear runon assay, no change was found in the transcription rate of GFAP in the cerebral cortex or hippocampus. Thus, age-related increases in GFAP are not associated with proportionate changes in transcription rates or DNA methylation. However, the transcription of glutamine synthetase was increased by about 60%. These findings contrast with age-related loss of bulk tissue DNA methylation and decreased transcription rates of other genes reported in non-neural tissues.

Transcriptional regulation of two astrocyte genes, glial fibrillary acidic protein (GFAP) and glutamine synthase (GS), by glucocorticoids was determined by nuclear run-on assay with hippocampal tissues from adult male F344 rats. Transcriptional responses of GFAP to corticosterone were slower than those observed for GS, but were more sensitive to changes in plasma corticosterone. The transcription of GFAP did not change 2 h after the injection of 10 mg corticosterone, but was reduced by 50% at 6 and 24 h. In contrast, corticosterone increased GS transcription at 2 and 6 h. Seven days after adrenalectomy, GFAP, but not GS, transcription was increased. Corticosterone replacement (200 micrograms/ml in the drinking water) suppressed GFAP, but did not increase GS transcription in adrenalectomized rats. Therefore, GFAP transcription is more sensitive to low physiological levels of corticosterone than transcription of GS. The slower response of GFAP than GS to corticosterone suggests that glucocorticoids may have indirect effects on GFAP expression that require additional transcriptional regulators besides the glucocorticoid receptor.

Levels of glial fibrillary acidic protein (GFAP), an astrocyte-specific intermediate filament protein, are altered during development and aging, GFAP also responds dynamically to neurodegenerative lesions. Changes in GFAP expression can occur at both transcriptional and translational levels. Modulators of GFAP expression include steroids, cytokines, and growth factors. GFAP expression also shows brain region-specific responses to sex steroids and of astrocyte-neuronal interactions. The 5'-upstream sequences of rat, mouse, and human are compared for the presence of response elements that are candidates for transcriptional regulation of GFAP. We propose that the regulation of the GFAP gene has evolved a system of controls that allow integrated responses to neuroendocrine and inflammatory modulators.

Transforming growth factor-beta 1 was studied as a possible regulator of messenger RNAs in astrocytes and neurons that increase after hippocampal deafferentation by perforant path transection: tubulin alpha 1, clusterin and glial fibrillary acidic protein messenger RNA. Because transforming growth factor-beta 1 messenger RNA is increased after this lesion, we examined which messenger RNA lesion responses could be induced by transforming growth factor-beta 1 alone. Porcine transforming growth factor-beta 1 infused into the lateral ventricle elevated the messenger RNAs for tubulin alpha 1, clusterin and glial fibrillary acidic protein 24 h after infusion in the ipsilateral hippocampus. As assayed by nuclear run-on, the transcription of glial fibrillary acidic protein RNA was increased in the ipsilateral hippocampus after perforant path transection and in primary rat astrocyte cultures by transforming growth factor-beta 1. In contrast, transforming growth factor-beta 1 did not change apolipoprotein-E messenger RNA or transcription, or growth associated protein-43 messenger RNA levels. We conclude that transforming growth factor-beta 1 increases subsets of neuronal and astrocyte messenger RNAs coding for cytoskeletal proteins that are also elevated in response to experimental lesions and Alzheimer's disease. This suggests that transforming growth factor-beta 1 might be a local organizing factor of neuronal and astrocyte responses to brain injury.

TGF-beta 1 mRNA and protein were recently found to increase in animal brains after experimental lesions that cause local deafferentation or neuron death. Elevations of TGF-beta 1 mRNA after lesions are prominent in microglia but are also observed in neurons and astrocytes. Moreover, TGF-beta 1 mRNA autoinduces its own mRNA in the brain. These responses provide models for studying the increases of TGF-beta 1 protein observed in beta A/amyloid-containing extracellular plaques of Alzheimer's disease (AD) and Down's syndrome (DS) and in brain cells of AIDS victims. Involvement of TGF-beta 1 in these human brain disorders is discussed in relation to the potent effects of TGF-beta 1 on wound healing and inflammatory responses in peripheral tissues. We hypothesize that TGF-beta 1 and possibly other TGF-beta peptides have organizing roles in responses to neurodegeneration and brain injury that are similar to those observed in non-neural tissues. Work from many laboratories has shown that activities of TGF-beta peptides on brain cells include chemotaxis, modification of extracellular matrix, and regulation of cytoskeletal gene expression and of neurotrophins. Similar activities of the TGF-beta's are well established in other tissues.

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 Alzheimer's 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.

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.

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.

Prolactin (PRL) increased basal dopamine (DA) release and attenuated amphetamine (AMPH)-stimulated DA release in vitro from rat corpus striatum in a concentration-dependent manner with 10(-5) M PRL being the most effective. The effects of PRL on DA release were enhanced in the absence of extracellular calcium. PRL at 10(-5) M did not alter the DA post-superfusion content of the striatal tissue. These results indicate that the stimulatory effect of PRL on basal DA release does not require extra-cellular calcium and the inhibitory effect on AMPH-stimulated DA release is not due to depletion of DA stores.

Thios study showed responses of sulfated glycoprotein-2 (SGP-2) in the rat hippocampus after deafferenting lesion. SGP-2 is a plasma protein that also occurs in many peripheral tissues. In some circumstances, elevations of SGP-2 mRNA are associated with cell degeneration and responses to injury. This study used entorhinal cortex lesions (ECL) to partially deafferent the hippocampus by damaging the perforant path and to induce synaptic remodeling. SGP-2 mRNA is increased in hippocampal astrocytes after ECL. Western blot analysis of soluble hippocampal proteins identified 3 major forms of rat SGP-2 protein: a precursor (61 kDa) and 2 reduced subunits at 39.5 and 35 kDa. These forms increased at 4 days post ECL ipsilaterally to the lesion. By immunocytochemistry (ICC), SGP-2 showed an increased immunoreactivity on the lesioned side by 2 days post ECL that continued through 14 days post ECL. Besides immunopositive astrocytes, punctate immunochemical reaction products occurred among the degenerating fibers of the perforant path. We conclude that changes of SGP-2 protein in the hippocampus after ECL occur roughly in parallel with increases of SGP-2 mRNA. The punctate immuno-deposits could represent secreted SGP-2 and may be useful as a marker for degenerating pathways.

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.

Transforming growth factor-beta mRNA was detected with a rat TGF-beta 1 coding sequence probe as a 2.5 kb band by RNA blot hybridization of total RNA from the adult male rat hippocampus. Following electrolytic lesions of the entorhinal cortex that cause hippocampal deafferentation and synaptic remodeling, TGF-beta mRNA increases 5-fold in ipsilateral hippocampus when compared with intact controls. This increase was independent of prior adrenalectomy or corticosterone-replacement. These data demonstrate that TGF-beta gene expression increases in response to hippocampal deafferentation.

This study concerns effects of the testes on two macromolecules in the rat hippocampus that were previously not known to be responsive to this endocrine axis. Castration for 3 weeks elevated the expression of glial fibrillary acidic protein (GFAP) and sulfated glycoprotein-2 (SGP-2) in male rat hippocampus, as shown by Northern blots and immunocytochemistry. SGP-2 mRNA was colocalized with GFAP, implying increased prevalence in astrocytes after castration. During hippocampal responses to deafferentation by entorhinal cortex lesions that damage the perforant path and induce synaptic reorganization, both mRNA and protein for SGP-2 and GFAP increase. Moreover, prior castration had an additive effect with entorhinal cortex lesions in the increase in GFAP and SGP-2 mRNA. These data suggest that testicular hormones regulate hippocampal astrocyte activity in intact adult rats as well as during synaptic reorganization in response to deafferenting lesions.

The effect of Leu-enkephalin (Leu-ENK), Met-enkephalin (Met-ENK), and naloxone on potassium (K+)-evoked endogenous dopamine (DA) release from striatal tissue fragments was examined in young (2-4 month) and old (20-25 month) male rats. The K(+)-evoked DA release was significantly lower in old compared to young animals. Leu-ENK significantly reduced K(+)-evoked DA release in young animals, but neither opiate affected DA release in old animals. Naloxone, which had no effect in young animals, raised the K(+)-evoked DA response in old animals to levels seen in young control preparations. These results suggest that the age-dependent decrease in K(+)-evoked DA release may in part be due to a stronger opiate inhibitory tonus in the CS of older rats.

Young adult male rats were implanted with a push-pull cannula aimed at the dorsal and rostral areas of the caudate nucleus. Perfusate samples were collected at two-minute intervals for approximately one hour and assayed for DOPAC concentrations. Simultaneously, yawning, penile erections and grooming behavior were recorded. Yawns were induced by systemic prolactin or apomorphine injections. While mean DOPAC efflux was elevated following prolactin (PRL) and apomorphine decreased mean DOPAC efflux as expected, yawns and penile erections induced by both compounds were associated with rapid momentary decreases in DOPAC efflux in these living animals. Although yawning was associated with significant decreases in DOPAC output, not every momentary DOPAC decrease was associated with a yawn, suggesting that the 'yawning generator' most likely requires additional inputs for the expression of a yawn.

Herein we report that subcutaneous injection of low doses of ovine prolactin (oPRL) induce yawning in young adult male rats. The most effective dose of oPRL in evoking yawning was 0.25 microgram/kg body weight (5.2 yawns/60 min at 1000 hr vs 0.3 in control animals). Doses of 0.025, 0.05, 2.5, 25, and 250 micrograms/kg were less effective. Interestingly, yawning in response to oPRL changes over the course of one circadian cycle with highest frequency at 1600 hr (11 yawns/80 min vs 2 yawns/80 min in animals injected with boiled oPRL). The onset of yawning in most oPRL-treated rats began approximately 40 min after oPRL injection, whereas with apomorphine the latency to the response was about 10 min. These results indicate that oPRL in addition to other hypophysial peptides such as ACTH and MSH can stimulate yawning. It is proposed that PRL after initial activation of the nigrostriatal dopamine system secondarily induces yawning by inhibition of this system via an autoreceptor-mediated negative feedback mechanism. This may explain the long latency to the response.

The ability of superfused corpus striatal tissue fragments to release endogenous dopamine (DA) in response to potassium (K+ 30 mM) stimulation was significantly attenuated in 18- to 24-month- compared to 2- to 4-month-old male rats. These age-related effects on K+ stimulation were completely abolished with the addition of tetrodotoxin (1 microM) to the superfusion medium. Moreover, no difference in stimulated DA release was obtained between these two age groups following amphetamine stimulation (10 microM).

Herein, we evaluate the importance of the nigro-striatal dopamine system in prolactin-, apomorphine-, and physostigmine-induced yawning behavior. Bilateral 6-OH-dopamine lesions of the substantia nigra were performed on male rats (2-4 months old). The lesioned as well as control rats were injected with either physiological saline, physostigmine (200 micrograms/kg), apomorphine (50 micrograms/kg), or ovine prolactin (0.25 micrograms/kg) 72 hours after the surgical procedure. The results show that bilateral lesions of the substantia nigra did not affect physostigmine-induced yawning whereas both apomorphine- and prolactin-induced yawning were reduced by the lesion. Following the observation period the caudate nuclei were removed and analyzed for dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) content. The lesions reduced DA and DOPAC content in all treatment groups compared to the respective intact groups. Also, both DA and DOPAC concentrations were lower in the intact apomorphine and prolactin treated groups compared to intact saline controls, at times that were temporally related to the display of yawning behavior suggesting a decrease in dopamine activity following apomorphine and prolactin treatment. Interestingly, DA and DOPAC concentrations were higher in the lesioned apomorphine group compared to lesioned saline controls; however, in the lesioned prolactin group only the DA concentrations were higher when compared to lesioned saline controls. These results indicate that prolactin- and apomorphine-induced yawning require an intact nigro-striatal dopamine system and that these substances induce yawning by different mechanisms.