The effect of water temperature on the GABAergic and reproductive systems in female and male goldfish (Carassius auratus)

This study investigated the effect of water temperature on the synthesis of the amino acid neurotransmitter gamma-aminobutyric acid (GABA). In goldfish, GABA stimulates the release of pituitary gonadotropin-II (GTH-II), which regulates gonadal function. Fish were maintained in water of 11, 18, or 24 degrees. In the female and male goldfish, GABA synthesis rates estimated following inhibition of GABA catabolism by gamma-vinyl GABA (GVG) in both the telencephalon (TEL) and the hypothalamus (HYP) were increased in fish held at 24 degrees compared to those at either 11 or 18 degrees (P < 0.05). Additionally, GABA synthesis rates in the pituitary increased in a temperature-dependent manner. Glutamate is the precursor for GABA synthesis; however, no consistent pattern was seen between glutamate and GABA synthesis rates, indicating that glutamate is not a limiting factor in GABA synthesis. Both water temperature and GVG administration increased serum GTH-II levels in female goldfish. However, in male goldfish water temperature had no significant effect on serum GTH-II levels, and GVG injection increased serum GTH-II levels only in fish maintained at 24 degrees. The effects of temperature on the levels of mRNA expression of the GABA-synthesizing enzymes glutamate decarboxylase 65 (GAD(65)) and GAD(67) were measured by semiquantitative PCR. In the TEL and HYP of female goldfish, GAD(65) was not affected, whereas temperature change from 11 to 18 degrees increased (P < 0.05) GAD(67) mRNA levels. These results demonstrate that central GABAergic systems in the goldfish are temperature sensitive.     

Regulation of GAD expression in rat pancreatic islets and brain by γ-vinyl-GABA and glucose

Summary Glutamic acid decarboxylase (GAD) is an important autoantigen in insulin-dependent diabetes mellitus (IDDM), but little is known about its regulation and function in islet cells. We investigated the effects of the GABA-transaminase inhibitor γ-vinyl-GABA (GVG) on GAD expression in rat islets and brain in vitro and in vivo. In islets incubated in high glucose culture medium there was an increase in GAD activity, GAD65 and GAD67 protein levels compared to low-glucose conditions; however, even in high glucose, GVG still significantly suppressed GAD activity and GAD67 expression. Our observations suggest that glucose and GVG act on GAD in islets through different mechanisms. Quantitative immunohistochemistry of pancreatic sections from rats treated with GVG in vivo using novel monoclonal antibodies specific for GAD65 and GAD67, showed a decrease in GAD67 expression (p < 0.005) relative to untreated rats. The effects of GVG on rat pancreatic islets were very similar to those observed in brain of rats treated with GVG in vivo. In homogenates of cerebral tissue from GVG treated rats containing both membrane-bound and soluble protein GAD67 levels were significantly decreased while GAD65 levels were not significantly changed compared to untreated rats. In contrast, in homogenates of cerebral tissues containing only soluble cytosolic protein, GVG-treatment was also significantly found to decrease GAD65 levels. Taken together, these results suggest that GVG potentially could be of use to decrease GAD expression in islet cells and consequently to deviate/inhibit the autoimmune response against the beta cells seen in IDDM. [Diabetologia (1998) 41: 530–535] Received: 19 August 1997 and in final revised form: 22 December 1997     

Role of the progesterone receptor in restrained glutamic acid decarboxylase gene expression in the hypothalamus during the preovulatory luteinizing hormone surge

Previous work by our laboratory demonstrated that activation of the progesterone receptor through exogenous administration of progesterone suppressed glutamic acid decarboxylase-67 (GAD(67)) mRNA in the hypothalamus of the estrogen-primed ovariectomized rat. Since GAD(67) is the major synthetic enzyme for the inhibitory transmitter, gamma-aminobutyric acid, the finding raised the possibility that the endogenous activation of the progesterone receptor may act to restrain GAD(67) expression during the natural preovulatory gonadotropin surge during proestrus in the rat, thereby allowing GnRH secretion and the resultant LH surge. To test this hypothesis, the progesterone receptor antagonist, RU486, was administered to regularly cycling proestrous rats and the effect on GAD(67) and GAD(65) mRNA levels in the preoptic area (POA) and medial basal hypothalamus (MBH) was examined. Serum luteinizing hormone (LH) levels were also examined in order to identify correlations between changes in POA and MBH GAD levels and production of the LH surge. GAD(67) mRNA levels in the POA were increased in the cycling rat during proestrus at 18.00 h at the peak and just preceding the termination of the LH surge. There was no change in GAD(67) mRNA levels in the MBH, and GAD(65) expression was also unchanged during proestrus in the POA and MBH. Treatment with the antiprogestin RU486 resulted in an increase in GAD(67) mRNA levels at 12.00 and 14.00 h in the POA, and in the MBH at 14.00, 16.00, and 18.00 h during proestrus, effects which preceded and correlated with the attenuated LH surge in RU486-treated rats at 18.00 h. GAD(65) mRNA levels were also elevated by RU486 at 14.00 and 16.00 h in the POA, and at 14.00 h in the MBH during proestrus. These findings suggest that the progesterone receptor plays a role in restraining GAD expression in the hypothalamus during proestrus, and that this effect may be important for the production of the GnRH and LH surge.     

Changes in hypothalamic gene expression associated with the arrest of pulsatile gonadotropin-releasing hormone release during infancy in the agonadal male rhesus monkey (Macaca mulatta)

This study examined whether changes in the levels of the messenger RNAs (mRNAs) encoding the gamma-aminobutyric acid (GABA) synthesizing enzymes, glutamate decarboxylase (GAD)65 and GAD67 and transforming growth factor-alpha (TGFalpha) in the hypothalamus are correlated with the arrest of pulsatile GnRH release during infancy in the agonadal male monkey. This experiment also provided the opportunity to examine changes in hypothalamic GnRH gene expression during this critical phase of primate development. Male rhesus monkeys were castrated at 1 week of age: four were killed 4-7 weeks after orchidectomy while pulsatile GnRH release was robust as reflected by high circulating LH levels, and four were killed at 12-15 months of age after establishing that pulsatile GnRH release had been arrested. GAD65, GAD67, TGFalpha, and GnRH mRNA levels were estimated using RNase protection assays employing homologous probes and the results were expressed relative to cyclophilin mRNA levels. GnRH peptide was measured by RIA. GAD65 and GAD67 mRNA levels in the hypothalamus of juveniles were significantly greater than those in neonatal monkeys. On the other hand, hypothalamic TGFalpha and GnRH mRNA (and peptide) levels in agonadal neonate and juvenile monkeys were indistinguishable. These results indicate that the molecular concomitants associated with bringing the hypothalamic GnRH pulse generator into check in agonadal neonatal males are not a mirror image of those previously reported at the time this neuronal network is reactivated at puberty when TGFalpha and GnRH gene expression increase and GAD65 and GAD67 mRNA levels remain unchanged. Thus, the neurobiological mechanism that reactivates pulsatile GnRH release at puberty is likely to involve more than a simple reversal of that underlying inhibition of the same network in late infancy.     

Estrogen and ovariectomy regulate mRNA and protein of glutamic acid decarboxylases and cation-chloride cotransporters in the adult rat hippocampus

17beta-Estradiol spatiotemporally regulates the gamma-aminobutyric acid (GABAergic) tone in the adult hippocampus. However, the complex estrogenic effect on the GABAergic system is still unclear. In adult central nervous system (CNS) neurons, GABA can induce both inhibitory and excitatory actions, which are predominantly controlled by the cation-chloride cotransporters NKCC1 and KCC2. We therefore studied the estrogenic regulation of two glutamate decarboxylase (GAD) isoforms, GAD65 and GAD67, as well as NKCC1 and KCC2 in the adult female rat hippocampus by immunohistochemistry and in situ hybridization. First, we focused on the duration after ovariectomy (OVX) and its effects on GAD65 protein levels. The basal number of GAD65-immunoreactive cells decreased after long-term (10 days) OVX compared to short-term (3 days) OVX. We found that, only after long-term OVX but not after short-term OVX, estradiol increased the number of GAD65-immunoreactive cells in the CA1 pyramidal cell layer. Furthermore, estradiol did not alter the GAD65-immunoreactive cell population in any other CA1 subregion. Second, we therefore focused on long-term OVX and the estrogenic regulation of GAD and cation-chloride cotransporter mRNA levels. In the pyramidal cell layer, estradiol affected GAD65, GAD67 and NKCC1 mRNA levels, but not KCC2 mRNA levels. Both GAD65 and NKCC1 mRNA levels increased within 24 h after estradiol treatment, followed by a subsequent increase in GAD67 mRNA levels. These findings suggest that basal levels of estrogen might contribute to a balance between the excitatory and inhibitory synaptic transmission onto CA1 pyramidal cells by regulating perisomatic GAD and NKCC1 expression in the adult hippocampus.     

Estriol affects prolactin and LH secretion in rats

The effects of estriol on serum prolactin (PRL) and LH levels, on the pituitary response to TRH and LHRH and on the synthesis and release of PRL from the anterior pituitary gland were investigated in female rats. The increase of serum PRL levels after estradiol administration was found to be associated with an increase of glutamic acid decarboxylase (GAD) and GABA-transaminase (GABA-T) in the hypothalamus. Thus, a study was carried out on the effects of estradiol and estriol on PRL secretion and on GAD, GABA-T and gamma-amino butyric acid (GABA) in the hypothalamus and the anterior pituitary. Under basal and TRH-stimulated conditions, estriol increased serum PRL levels, decreased basal serum LH levels, and increased the response to LHRH, in terms of LH release. Estradiol and estriol increased the synthesis and release of 3H-PRL from hemipituitary glands in incubations of pretreated animals. Both estrogens induced hyperprolactinemia, concomitantly with an increase of hypothalamic GAD and GABA-T activity. Estriol increased hypothalamic GABA concentration, but did not modify GABA concentration in the pituitary glands. Our results show that estriol, at relatively high doses, seems to be active in increasing PRL synthesis and release and in decreasing serum LH levels; it can also modify pituitary response to TRH and LHRH stimulation.     

Blockade of pro-oestrus LH surge and ovulation by GABA increase in the rat locus coeruleus

The effects of gamma-aminobutyric acid (GABA) increase at the nucleus locus coeruleus (LC) on pro-oestrus LH release and ovulation were evaluated in rats. Local microinjection of the GABA-transaminase inhibitor gamma-vinyl-GABA (GVG) produced 6 h later a marked increase in GABA in the LC. Such action caused a significant decrease of plasma LH levels and prevented the pro-oestrus LH surge. In some animals, plasma prolactin levels were also lowered, but in others its plasma concentrations were high and similar to that in controls. Ovulation did not occur in the rats treated with GVG. In additional experiments, the periventricular gray substance (PGS) close to the locus coeruleus was injected with GVG. Results obtained show a LH surge blockade and failure of ovulation in most of these rats. These findings may be interpreted on the basis of GABA action on rostral LC cells that project to the PGS. The results altogether suggest that through neurons of the locus coeruleus, GABA may exert an inhibitory role in the regulation of LH secretion.     

Localized increase of GABA levels in brain areas of the rat and inhibition of the plasma LH rise following orchidectomy

Previous studies have suggested that gamma-aminobutyric acid (GABA) exerts inhibitory actions on luteinizing hormone (LH) secretion that are likely to be mediated by modifications in noradrenergic transmission. To explore further this hypothesis we have studied the effect of increasing GABA contents in discrete areas of the brain on plasma LH levels in short-term orchidectomized rats. GABA accumulation was produced by the GABA transaminase inhibitor, gamma-vinyl-GABA (GVG). The locus coeruleus area (LC), where the noradrenaline (NA) cells projecting through the dorsal noradrenergic bundle are located, and several hypothalamic areas that are innervated by NA-containing fibers were microinjected with GVG. Most of these areas are known to be related to the neural control of LH secretion. GVG microinjected in the LC and medial preoptic area increased the GABA content and blunted significantly the acute increase of plasma LH produced by castration. Bicuculline prevented these effects. Delayed effects of GVG were observed when applied in the anterior hypothalamic area and ventromedial-arcuate nucleus area. In these latter areas, a single injection of GVG did not augment the GABA concentrations and was unable to prevent LH release, but a clear inhibitory effect took place after a second injection of GVG between 24 and 48 h after orchidectomy. Unresponsive areas to GVG treatment were the lateral preoptic area, the median eminence and the dorsal raphe. These results add support to the view that GABA inhibits LH release in rats, at discrete areas of the brain.     

Restraining action of GABA on estradiol-induced LH surge in the rat: GABA activity in brain nuclei and effects of GABA mimetics in the medial preoptic nucleus.

The relationship between GABA dynamics and LH release was studied on day 2 after subcutaneous estrogen implant in short-term ovariectomized rats. GABA accumulation, used as an index of GABA turnover, was determined in the medial preoptic nucleus (MPN), medial (MS) and lateral (LS) septal nuclei, median eminence-mediobasal hypothalamus (MBH) and locus ceruleus (LC). Measurements of GABA were performed at two different times of day (11.00 and 15.00 h), 3 h after intraperitoneal administration of gamma-vinyl-GABA (GVG), an irreversible inhibitor of GABA transaminase. Either morning or afternoon ovariectomized rats (OVX) showed a significant increase in GABA accumulation after GVG treatment in all the areas studied. Estrogen-treated OVX rats showed in the morning a lower GABA accumulation in the MPN, MBH and LC, and GABA levels remained unchanged in the LS and MS. In the afternoon, the MPN and LS showed a lower rate of GABA accumulation whereas in the MBH and LC the GABA increase was not observed. In contrast the MS showed a rate of GABA accumulation similar as in the OVX rats. Local administration in the MPN of 20 micrograms GVG, or GABA-A receptor stimulation by muscimol (50 ng), prior to the increase in plasma LH levels, prevented the occurrence of the estradiol-induced LH surge. The effect of muscimol was reversed by bicuculline (30 ng), a GABA-A receptor antagonist. Bicuculline in low doses lacked effect by itself. In conclusion, these results strongly suggest that a decreased GABAergic activity in MPN, MBH and LC precedes the estradiol-evoked LH surges in ovariectomized rats. Moreover, that in septal nuclei, a low GABAergic activity takes place well before the occurrence of plasma LH increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

A conditional tetracycline-regulated increase in Gamma amino butyric acid production near luteinizing hormone-releasing hormone nerve terminals disrupts estrous cyclicity in the rat

Gamma amino butyric acid (GABA) is the main inhibitory neurotransmitter controlling LH-releasing hormone (LHRH) secretion in the mammalian hypothalamus. Whether alterations in GABA homeostasis within discrete regions of the neuroendocrine brain known to be targets of GABA action, such as the median eminence, can disrupt the ability of the LHRH releasing system to maintain reproductive cyclicity is not known but amenable to experimental scrutiny. The present experiments were undertaken to examine this issue. Immortalized BAS-8.1 astroglial cells were genetically modified by infection with a regulatable retroviral vector to express the gene encoding the GABA synthesizing enzyme glutamic acid decarboxylase-67 (GAD-67) under the control of a tetracycline (tet) controlled gene expression system. In this system, expression of the gene of interest is repressed by tet and activated in the absence of the antibiotic. BAS-8.1 cells carrying this regulatory cassette, and cultured in the absence of tet ("GAD on"), expressed abundant levels of GAD-67 messenger RNA and GAD enzymatic activity, and released GABA when challenged with glutamate. All of these responses were inhibited within 24 h of exposure to tet ("GAD off"). Grafting "GAD on" cells into the median eminence of late juvenile female rats, near LHRH nerve terminals, did not affect the age at vaginal opening, but greatly disrupted subsequent estrous cyclicity. These animals exhibiting long periods of persistent estrus, interrupted by occasional days in proestrus and diestrus, suggesting the occurrence of irregular ovulatory episodes. Administration of the tetracycline analog doxycycline (DOXY) in the drinking water inhibited GAD-67synthesis and restored estrous cyclicity to a pattern indistinguishable from that of control rats grafted with native BAS-8.1 cells. Animals carrying "GAD on" cells showed a small increase in serum LH and estradiol levels, and a marked elevation in serum androstenedione, all of which were obliterated by turning GAD-67 synthesis off in the grafted cells. Morphometric analysis of the ovaries revealed that both groups grafted with GABA-producing cells had an increased incidence of large antral follicles (>500 micrometer) compared with animals grafted with native BAS-8.1 cells, but that within this category the incidence of steroidogenically more active follicles (i.e. larger than 600 micrometer) was greater in "GAD on" than in "GAD off" rats. These results indicate that a regionally discrete, temporally controlled increase in GABA availability to LHRH nerve terminals in the median eminence of the hypothalamus suffices to disrupt estrous cyclicity in the rat, and raise the possibility that similar local alterations in GABA homeostasis may contribute to the pathology of hypothalamic amenorrhea/oligomenorrhea in humans.     

Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase

γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, is synthesized by two glutamate decarboxylase isoforms, GAD65 and GAD67. The separate role of the two isoforms is unknown, but differences in saturation with cofactor and subcellular localization suggest that GAD65 may provide reserve pools of GABA for regulation of inhibitory neurotransmission. We have disrupted the gene encoding GAD65 and backcrossed the mutation into the C57BL/6 strain of mice. In contrast to GAD67−/− animals, which are born with developmental abnormalities and die shortly after birth, GAD65−/− mice appear normal at birth. Basal GABA levels and holo-GAD activity are normal, but the pyridoxal 5′ phosphate-inducible apo-enzyme reservoir is significantly decreased. GAD65−/− mice develop spontaneous seizures that result in increased mortality. Seizures can be precipitated by fear or mild stress. Seizure susceptibility is dramatically increased in GAD65−/− mice backcrossed into a second genetic background, the nonobese diabetic (NOD/LtJ) strain of mice enabling electroencephalogram analysis of the seizures. The generally higher basal brain GABA levels in this backcross are significantly decreased by the GAD65−/− mutation, suggesting that the relative contribution of GABA synthesized by GAD65 to total brain GABA levels is genetically determined. Seizure-associated c-fos-like immunoreactivity reveals the involvement of limbic regions of the brain. These data suggest that GABA synthesized by GAD65 is important in the dynamic regulation of neural network excitability, implicate at least one modifier locus in the NOD/LtJ strain, and present GAD65−/− animals as a model of epilepsy involving GABA-ergic pathways.     

Glutamic acid decarboxylase messenger ribonucleic acid is regulated by estradiol and progesterone in the hippocampus.

Ovarian steroids modulate learning, memory, and epileptic seizure activity, functions that are mediated in part by the hippocampus. Normal function depends on precise interactions between the inhibitory gamma-aminobutyric acid (GABA)ergic and excitatory glutamatergic neurons of the hippocampus. To determine whether estradiol and progesterone interact with GABAergic neurons, the levels of mRNA for glutamic acid decarboxylase (GAD), the rate-limiting enzyme for GABA synthesis, were measured by in situ hybridization histochemistry with 35S-labeled riboprobes complimentary to the feline GAD cDNA. The levels of mRNA for GAD were analyzed in selected region of the dorsal hippocampus and medial basal hypothalamus in ovariectomized, ovariectomized estradiol-treated, and ovariectomized estradiol- and progesterone-treated rats. In estradiol-treated rats, GAD mRNA levels increased in GABAergic neurons associated with the CA1 pyramidal cell layer, but not in the stratum oriens of CA1 or any other region of the hippocampus. Estradiol plus progesterone treatment reversed the estradiol-induced increase in GAD mRNA in CA1 and induced a small decrease in the hilus. No effect of estradiol or progesterone was observed in the dorsomedial, ventromedial, or arcuate nuclei of the hypothalamus. Estradiol or progesterone may alter cognitive performance and seizure activity by increasing or decreasing, respectively, the activity of GABAergic neurons in the hippocampus.     

Evidences for the regulation of GnRH and GTH expression by GnIH in the goldfish, Carassius auratus

Gonadotrophin-inhibitory hormone (GnIH) plays an important role in regulating of reproduction in teleosts. To clarify the mode of action of GnIH on the synthesis of gonadotropin releasing hormone (GnRH) and gonadotrophin (GtH), three GnIHR cDNAs were cloned from the goldfish brain. In situ hybridization results showed that GnIHRs were localized to the hypothalamus and pituitary. In the hypothalamus, GnIHRs were found in the NPP, NPO and NLT, whereas sGnRH neurons were reported to be located, and potentially regulated by GnIH. In the pituitary, only two GnIHRs were observed and they were localized to the PI instead of the adenohypophysis where GtH-expressing cells are localized, suggesting indirect regulation of GtH by GnIH. In vivo, intraperitoneal (i.p.) injections of synthetic goldfish GnIH-II peptide and GnIH-III peptide significantly decreased sGnRH and FSHβ mRNA levels. Only GnIH-II decreased LHβ mRNA levels significantly. In vitro, both GnIH-II and GnIH-III showed no effect on GtH synthesis, but an inhibition of GnRH-stimulated LHβ and FSHβ synthesis was observed when GnIH-III was applied to primary pituitary cells in culture. Thus, GnIH could contribute to the regulation of gonadotropin in the brain and pituitary in teleosts.     

Selective regulation of glutamic decarboxylase isoform 65, but not isoform 67, in the bed nucleus of the stria terminalis and the preoptic area of the ewe brain across the estrous cycle.

gamma-Aminobutyric acid neurons in the preoptic area (POA) of the brain may regulate GnRH neurons. The level of expression of two isoforms (65 and 67) of glutamic acid decarboxylase (GAD) in the ewe brain was determined across the estrous cycle by in situ hybridization. GAD mRNA expression (cell number and silver grains/cell) was examined in the subdivisions of the bed nucleus of stria terminalis (BnST), in the diagonal band of Broca, and the POA. The number of cells expressing GAD(65) and GAD(67) mRNA did not change across the cycle. Within the rostro-dorsal BnST, the number of silver grains/cell for GAD(65) mRNA was lower in the follicular phase than the luteal phase or at estrus. In the rostro-lateral division, expression was lower in the follicular phase. In the POA, the number of silver grains/cell for GAD(65) mRNA was lower at estrus than during the luteal phase. The number of silver grains/cell for GAD(67) mRNA did not change across the estrous cycle. GAD(65) is thought to be the active enzyme during periods of high demand of GABA and our results are consistent with the GABA neurons of BnST being most active during the luteal phase of the estrous cycle.     

Evidence for a GABAergic system in rodent and human testis: local GABA production and GABA receptors

The major neurotransmitter of the central nervous system, gamma-aminobutyric acid (GABA), exerts its actions through GABA(A), GABA(B) and GABA(C) receptors. GABA and GABA receptors are, however, also present in several non-neural tissues, including the endocrine organs pituitary, pancreas and testis. In the case of the rat testis, GABA appears to be linked to the regulation of steroid synthesis by Leydig cells via GABA(A) receptors, but neither testicular sources of GABA, nor the precise nature of testicular GABA receptors are fully known. We examined these points in rat, mouse, hamster and human testicular samples. RT-PCR followed by sequencing showed that the GABA-synthesizing enzymes glutamate decarboxylase (GAD) 65 and/or GAD67, as well as the vesicular GABA transporter vesicular inhibitory amino acid transporter (VIAAT/VGAT) are expressed. Testicular GAD in the rat was shown to be functionally active by using a GAD assay, and Western blot analysis confirmed the presence of GAD65 and GAD67. Interstitial cells, most of which are Leydig cells according to their location and morphological characteristics, showed positive immunoreaction for GAD and VIAAT/VGAT proteins. In addition, several GABA(A) receptor subunits (alpha1-3, beta1-3, gamma1-3), as well as GABA(B) receptor subunits R1 and R2, were detected by RT-PCR. Western blot analysis confirmed the results for GABA(A) receptor subunits beta2/3 in the rat, and immunohistochemistry identified interstitial Leydig cells to possess immunoreactive GABA(A) receptor subunits beta2/3 and alpha1. The presence of GABA(A) receptor subunit alpha1 mRNA in interstitial cells of the rat testis was further shown after laser microdissection followed by RT-PCR analysis. In summary, these results describe molecular details of the components of an intratesticular GABAergic system expressed in the endocrine compartment of rodent and human testes. While the physiological significance of this peripheral neuroendocrine system conserved throughout species remains to be elucidated, its mere presence in humans suggests the possibility that clinically used drugs might be able to interfere with testicular function.     

Glutamate decarboxylase 67 contributes to compensatory insulin secretion in aged pancreatic islets

Pancreatic islets play an essential role in regulating blood glucose levels. Age-dependent development of glucose intolerance and insulin resistance results in hyperglycemia, which in turn stimulates insulin synthesis and secretion from aged islets, to fulfill the increased demand for insulin. However, the mechanism underlying enhanced insulin secretion remains unknown. Glutamic acid decarboxylase 67 (GAD67) catalyzes the conversion of glutamate into γ-aminobutyric acid (GABA) and CO₂. Both glutamate and GABA can affect islet function. Here, we investigated the role of GAD67 in insulin secretion in young (3 month old) and aged (24 month old) C57BL/6J male mice. Unlike young mice, aged mice displayed glucose-intolerance and insulin-resistance. However, aged mice secreted more insulin and showed lower fed blood glucose levels than young mice. GAD67 levels in primary islets increased with aging and in response to high glucose levels. Inhibition of GAD67 activity using a potent inhibitor of GAD, 3-mercaptopropionic acid, abrogated glucose-stimulated insulin secretion from a pancreatic β-cell line and from young and aged islets. Collectively, our results suggest that blood glucose levels regulate GAD67 expression, which contributes to β-cell responses to impaired glucose homeostasis caused by advanced aging.     

Age-related changes in GAD levels in the central auditory system of the rat

Changes in the levels of gamma-aminobutyric acid (GABA) are known to occur in different parts of the brain during aging. In our study we attempted to define the effect that aging has on glutamate decarboxylase (GAD), the key enzyme in the synthesis of GABA, in the central parts of the auditory system. Age-related changes in GAD65 and GAD67 levels were investigated using immunohistochemistry and Western blotting in the inferior colliculus (IC), the auditory cortex (AC) and the visual cortex in Long-Evans rats. The results show that aging is associated with a decrease in the numbers of GAD65- and 67-immunoreactive neurons and the optical density of their somas in both the IC and AC. Western blot analysis revealed a pronounced age-related decline in the levels of GAD65 and 67 proteins in both the IC and AC. For comparison, in the visual cortex the decrease in both proteins was less pronounced than in the IC and AC. A similar pattern of age-related changes was found in Fischer 344 rats, a strain that manifests a rapid loss of hearing function with aging. The observed age-related decline in the levels of GAD65 and 67 may contribute significantly to the deterioration of hearing function that accompanies aging in mammals, including man.