Estrogen and Ovariectomy Regulate mRNA and Protein of Glutamic Acid Decarboxylases and Cation-Chloride Cotransporters in the Adult Rat Hippocampus

Abstract

17β-Estradiol spatiotemporally regulates the γ-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.

Mass spectrometric assay and physiological–pharmacological activity of androgenic neurosteroids

“…While estradiol have been shown to be proconvulsant in several studies (Buterbaugh, 1989;Reddy, 2004c), there are also studies that support an inhibiting effect of estrogens on cortical excitability (Weiland, 1992;Nakamura et al, 2004), suggesting that the effects of estrogens on seizures are contradictory. The action of estrogens on seizures depends on factors such as treatment duration, dosage, hormonal status and seizure model.…”

Seizure susceptibility in intact and ovariectomized female rats treated with the convulsant pilocarpine

“…One mechanism would be the decrease in GABAergic inhibition caused by a loss of estradiol and progesterone, which would lead to enhanced seizure propagation given that GABAergic inhibition serves as a brake on many, if not all, relevant glutamatergic pathways. There are several studies that have shown that both estradiol and progesterone maintain GABAergic function: (1) estradiol increases synthesis of the enzyme responsible for GABA biosynthesis, glutamic acid decarboxylase (GAD), in the hippocampus (Weiland, 1992), (2) estradiol maintains many processes and proteins that are important to GABAergic function, such as K+/Cl− cotransporters (Nakamura et al, 2005), and (3) progesterone, via allopregnanolone, normally enhances activity at GABA A receptors (Kokate et al, 1994). In a study of seizures initiated by cyclosporin-A (Tominaga et al, 2001), estradiol replacement to ovariectomized rats reversed the effects observed after ovariectomy, consistent with the hypothesis that estradiol may play a critical role in maintaining GABAergic inhibitory tone.…”

“…The most likely explanation is that acute reduction in reproductive steroids on diestrus 2 is quite distinct in its effects, relative to chronic reduction in the ovariectomized rat. The importance of the chronic loss of estradiol is supported by studies showing that GAD levels decline progressively as the time after ovariectomy increases beyond several days (Nakamura et al, 2005). Thus, the ovariectomized rat is likely to have less GAD than the diestrus 2 rat, with a greater reduction in GABAergic function as a result.…”

“…Valente et al tested their animals just 4 days after ovariectomy, whereas our ovariectomized rats were tested at least 15 days after ovariectomy. Our animals may have had a considerably lower level of GAD than the animals used by Valente et al (2002), given the data from Nakamura et al (2005), and this would be likely to facilitate status.…”

“…Ovariectomy does not immediately terminate responses to the hormones present at the time of surgery (Clark et al, 1981); there are potentially lasting effects of the surgery itself; and, moreover, it may be many days after ovariectomy before sex steroid concentrations fall to stable low levels. In addition, as mentioned above, the time after ovariectomy could also influence seizure susceptibility because GABA levels decline during the week after ovariectomy (Nakamura et al, 2005), and the brain and periphery adjust in other ways to the dramatic loss of steroids. Stress is also known to alter the expression in the brain of a number of factors involved in the regulation of neuronal activity (Avishai-Eliner et al, 2002;Cavus and Duman, 2003).…”