GABA in the female brain--Oestrous cycle-related changes in GABAergic function in the periaqueductal grey matter

by T.A. Lovick
Department of Physiology, University of Birmingham B15 2TT, UK
Journal of Pharmacology, Biochemistry, and Behavior

The late luteal (premenstrual) phase of the estrous cycle coincides with a decrease in progesterone blood and brain levels. The reduction in hormone levels has been linked to an increase in anxiety symptoms in those suffering from anxiolytic disorders. Furthermore, research has suggested that the environment influences the severity of the symptoms, although the mechanism of which this occurs is unknown. The periaqueductal grey (PAG) in the midbrain is associated with the regulation of anxiolytic responses and is the center of this study.

Neuroactive progesterone metabolite allopregnanolone (ALLO) has been shown to act as a positive modulator of GABA on GABA_A receptors. Progesterone withdrawals have demonstrated an upregulation of GABA_A alpha4, beta1, and delta subunit proteins, along with changes in neuronal responsiveness to GABA within the midbrain periaqueductal grey matter. Thus, the purpose of this study was to investigate if the upregulation of the GABA_A subunits will lead to an increase in excitability in the PAG, which in turn could increase the anxiolytic symptoms seen in women suffering from PMS. The researchers believed that since the delta subunit is usually located extrasynaptically, it would be upregulated on the interneurons of the PAG circuits. The interneurons would be suppressed and the PAG output would increase. Additionally, the influence of the environment on the PAG response throughout the estrous cycle was investigated.

Animals were housed in either a “standard,” noisy environment with other animals or in an “isolated,” quiet environment with no other animals. Daily smears were taken with each group to track the estrous cycle of each rat. Two rats from each cohort were used for immunohistochemical processing. The study used immunoreactivity methods to locate PAG neurons that expressed GABA_A subunits or glutamic acid decarboxylase (GAD), a GABA synthesizing enzyme. The immunoreactive cells within the PAG were individually counted to determine the density of the cells at different points of the estrous cycle.

Extracellular recordings of individual neurons were taken while iontophoretically applying drugs within the dorsal half of the PAG. Male rats were used to study the effects of progesterone on PAG GABAergic systems by directly applying GABA, the ALLO analog ORG20599, and bicuculline (BIC), a GABA antagonist. Female rats were used to study the effects of the estrous cycle on the GABAergic tone in the PAG. BIC responses were measured at different stages of the estrous cycle. Additionally, PAG circuitry responsiveness was investigated through the use of panicogenic CCK₂ receptor agonist pentagastrin at different cycle stages.

In the male rats, GABA produced a dose-response in individual neurons within the PAG. BIC blocked all GABA response in the same cells. ALLO and ORG20599 decreased the firing rate at the same neurons in male rats. However, the firing rate decreased more slowly and was sustained over time. BIC also decreased the effect of ALLO and ORG20599, similarly to the GABA studies, and increased the firing rate by 15-29%. The results suggested that there is a GABAergic tone that is altered by progesterone metabolites.

The female rats showed similar responses to GABA and ALLO/ORG20599 injections. However, the firing rates that increased with BIC were the highest in estrous and late diestrous phases on the cycle. Thus, the excitability is increased during these phases. The females also underwent exposure to pentagastrin, the CCK₂ agonist. The firing rate of the neurons in the PAG increased the most during the estrous and late dioestrus phases of the cycle, similarly to the BIC responses.

According to the immunohistochemical studies, progesterone withdrawal in the female rats led to the increase in the GABA_A receptor subunits in neurons in the PAG. Spontaneously-cycling rats saw similar elevations in the subunits during the late dioestrus phase. GAD-positive cells were present throughout the PAG, and their levels remained constant during all phases of the cycle. However, it appeared that the neurons within elevated-expression of the subunits co-localized near the GAD neurons during the late-dioestrus phase.

Two cohorts of female animals from each environment were used to determine the influence the environment has on GABA_A receptor subunit expression. The standard-housed animals showed the expected increase in subunit expression. However, the isolated animals did not show an increase. They remained at similar levels to the proestrous phase, when subunit expression is expected to be low. All animals were cycling normally.

The study showed that progesterone, and more specifically its neuroactive metabolite ALLO, can inhibit the GABA_A neuronal firing directly through receptor-interaction. The male studies determined that the hormone can rapidly modulate the GABAergic tone within the PAG. The female studies demonstrated that the decreased levels of progesterone during the late dioestrus phase are correlated with an upregulation of subunits in the PAG. Thus, it is suggested that the cycling of hormones modulates the expression of GABA_A receptor subunits on neurons. Importantly, the delta subunit, which is located extrasynaptically, was also upregulated. Because of the increased excitability of PAG, along with the upregulated GABA_A receptor delta subunit, it is suggested that the interneurons are receiving an increased inhibition from GABA. The output neurons are receiving a reduce basal tone, which increased the “intrinsic excitability” of the PAG as a whole.

These results also explain why there is the increased sensitivity to pentagastrin during the late dioestrus phases. With a decreased GABAergic tone after the progesterone withdrawal of this phase, the CCK₂ receptors can continue to fire. The CCK₂ receptor is anxiolytic-regulating, suggesting that there is a correlation between the reduced GABA-tone from the progesterone withdrawal and increased anxiety.

The studies supported the hypothesis that the environment can influence the effects of progesterone on the GABA_A receptor subunit expression. The researchers suggest that the isolated animals underwent social-isolation stress. The influence of the stress on the animals prevented the upregulation of the subunits during late dioestrus, despite cycling normally. These results are supported by male rat studies, as well. Nevertheless, more studies need to be done to determine how the housing environment can influence the expression of the subunits.

-LivIn la Vida