The present findings provide strong evidence to indicate that the neurosteroid environment in the hippocampus has bi-directional effects on seizure susceptibility in EtOH naïve mice and during EtOH withdrawal. Intra-hippocampal ALLO was anticonvulsant, whereas FIN was proconvulsant, in EtOH naïve mice. During EtOH withdrawal, animals were tolerant to the anticonvulsant effect of ALLO infusion and more sensitive to the pro-convulsant effects of inhibiting 5α-reductase. The present results indicate that GABAA
receptor sensitivity in the hippocampus is a dynamic process and that alterations in local endogenous neurosteroid levels can have behavioral consequences. Consistent with the idea that manipulation of local ALLO concentration can produce physiologically relevant effects, Belelli and Herd (2003)
demonstrated that the use of inhibitors to block the oxidation of ALLO (thereby increasing ALLO levels) unmasked a GABAergic inhibitory tone in hippocampal dentate gyrus neurons. The present findings represent one of the first demonstrations that bi-directional manipulation of hippocampal ALLO levels produces opposite behavioral consequences that are consistent with alterations in GABAergic inhibition.
The anticonvulsant effect of bilateral infusion of ALLO into the CA1 region of the hippocampus was similar to that seen with systemic injection. The potent anticonvulsant effect of intro-hippocampal ALLO against forebrain (predominantly limbic) convulsant endpoints was documented by the 157% and 107% increases in the PTZ threshold dose for onset to MC Twitch and FF Clonus, respectively. This result is consistent with the involvement of the hippocampus in the limbic seizure circuit (Gale, 1988
), and with the contribution of GABAA
receptors in the hippocampus to the anticonvulsant effect of ALLO (Rhodes and Frye, 2005a
). For comparative purposes, systemic administration of a 5 mg/kg dose of ALLO increased the threshold dose of PTZ for onset to FF Clonus by 2-fold in air-exposed WSP mice (Finn et al., 2006
). Taken in conjunction with the present findings, activation of GABAA
receptors in the hippocampus following ALLO infusion is sufficient to produce an anticonvulsant effect against limbic convulsion endpoints that is comparable to that seen following systemic injection of ALLO.
Use of FIN to decrease endogenous ALLO levels had a proconvulsant effect in EtOH naïve mice, as evidenced by the 18% and 17 % decrease in the PTZ threshold dose for onset to MC Twitch and FF clonus respectively. This effect of FIN was selective for limbic convulsion endpoints, as RB Clonus and THE endpoints were not significantly affected. Recent work in progesterone-primed female rats also found a potent proconvulsant effect of intra-hippocampal FIN (10 μg/side) against PTZ-induced convulsions (Rhodes and Frye, 2005a
). Notably, the increased seizure susceptibility following FIN infusion in the study by Rhodes and Frye (2005a)
corresponded to a significant decrease in hippocampal ALLO levels. Taken in conjunction with the present finings, hippocampal ALLO levels can impact PTZ sensitivity.
The change in seizure susceptibility in naïve mice following infusion of FIN was less pronounced than that seen with ALLO. One explanation for this difference could be the manner in which the local neurosteroid environment was manipulated. That is, infusion of FIN would decrease basal endogenous ALLO concentrations whereas infusion of ALLO would increase ALLO levels to supra-physiological concentrations. Although ALLO levels were not measured in the current studies, previous reports indicate that microinjection of a 0.1 μg ALLO dose increased ALLO concentrations 3-fold over basal levels (Frye and Rhodes, 2006
) and that infusions of FIN (10 μg) into the ventral tegmental area or hippocampus reduced ALLO levels by approximately 60%–80% respectively (Frye and Vongher, 2001
; Rhodes and Frye, 2005a
Another consideration with regard to the FIN data is that inhibition of 5α-reductase could alter the concentrations of other 5α-reduced steroids, some of which are GABAergic (e.g., the deoxycorticosterone metabolite tetrahydrodeoxycorticosterone), or could shift local steroid metabolism to favor the production of steroids with proconvulsant effects such as corticosterone or estrogen Additionally, FIN administration may produce 3α
-HP) by inducing the reduction of progesterone by 3α
-hydroxysteroid dehydrogenase (Purdy et al., 1990
-HP is a potent agonist at the GABAA
receptor, but is not found in high concentrations in rodents (Purdy et al., 1990
). While all of these steroids must be taken into consideration, ALLO is the most potent GABAA
receptor agonist and is found in physiological ranges in the blood and brain.
One concern with the interpretation of microinjection studies is whether a drug’s effect is due to a specific action at the site of the microinjection or to drug diffusion (primarily, to sites distal to the injection target). The results of our anatomical control studies strongly suggested that the effect of ALLO following microinjection into the CA1 was not due to diffusion up the injector track, but to a localized neurosteroid action at the injection target. Thus, these anatomical and diffusion controls provide evidence that the effects of ALLO and FIN were likely confined to the CA1 of the hippocampus.
EtOH withdrawal significantly increased sensitivity to PTZ in WSP-mice, consistent with an overall proconvulsant effect of EtOH withdrawal. However, the change in PTZ sensitivity was only true for the later two convulsion endpoints, RB clonus and THE. This finding is consistent with previous data -from our lab, whereby seizure prone genotypes (e.g. WSP and DBA/2J) do not exhibit an increased sensitivity to all PTZ convulsion endpoints during EtOH withdrawal (Finn and Crabbe, 1999
; Finn et al., 2006
; Finn et al., 2000
). It is likely that the main effect of EtOH withdrawal for all 4 seizure endpoints was due in part, to the concomitant decrease in sensitivity to ALLO’s anticonvulsant effect during withdrawal.
Notably, EtOH withdrawal was accompanied by decreased sensitivity to the anticonvulsant effect of intra-hippocampal ALLO administration in mice and increased sensitivity to the proconvulsant effects of intra-hippocampal FIN. Our reasoning for using two different measures of sensitivity was based on the following. With regard to ALLO, we had previously shown that the anticonvulsant effect of ALLO against withdrawal-related HICS was very transient (Finn et al., 1995
). We subsequently determined that the assessment of ALLO sensitivity during EtOH withdrawal was much more quantitative when the examination was limited to a single time point and that timed tail infusion of PTZ was a highly sensitive measure of sensitivity at a single time point of withdrawal (Finn et al., 2006
). Further, we wanted to directly compare the change in sensitivity to ALLO during EtOH withdrawal following systemic versus intra-hippocampal administration, since we had recently found that functional sensitivity of GABAA
receptors to ALLO was significantly reduced during EtOH withdrawal (Finn et al., 2006
). Specifically, the potency of ALLO to potentiate GABA (10 M) stimulated chloride flux was significantly reduced (rightward shift in the dose-response curve and significant increase in EC50
) concomitant with a reduction in efficacy of ALLO in the 60–600 nM concentration range during EtOH withdrawal. Consistent with this decrease in the functional sensitivity of GABAA
receptors to ALLO during EtOH withdrawal, our data indicate that the anticonvulsant effect of intra-hippocampal ALLO was significantly reduced during EtOH withdrawal. These results provide further evidence for the critical involvement of the hippocampus in mediating tolerance to the anticonvulsant effect of ALLO during EtOH withdrawal.
We chose a different tactic to assess sensitivity to FIN during EtOH withdrawal. While FIN produced a slight but significant proconvulsant effect in naïve WSP mice when PTZ threshold dose was the dependent measure, we reasoned that quantifying the effect of FIN on EtOH withdrawal severity would be best achieved by an examination of the withdrawal time course (which had never been done). We also were asking a slightly different question, namely, does suppression of endogenous GABAergic neurosteroids during the development of physical dependence alter the expression of EtOH withdrawal? Interestingly, four daily infusions of FIN produced a greater proconvulsant effect during EtOH withdrawal, suggesting that decreasing endogenous ALLO levels during the development of physical dependence produced a greater decrease in GABAergic inhibition and concomitant increase in withdrawal severity. This result contrasts with recent findings in our lab showing that FIN systemically administered during the development of physical dependence decreased withdrawal severity (Finn et al., 2004b
; Gorin et al., 2005
). However, systemically administered FIN significantly decreased BEC during EtOH exposure and upon the initiation of withdrawal, suggesting that the decrease in withdrawal severity in the earlier work was due to an indirect effect on EtOH pharmacokinetics. Notably, intra-hippocampal FIN did not alter BEC in the current study, suggesting that the increase in withdrawal severity was due to the manipulation of GABAergic neurosteroid tone in the hippocampus.
Decreased sensitivity to ALLO, which we have characterized as tolerance to the anticonvulsant effect of ALLO, was only observed in one other seizure prone genotype during EtOH withdrawal, the DBA/2J inbred mouse strain (Finn et al., 2000
). In contrast, rats, C57BL/6J and WSR mice exhibit increased sensitivity to the anticonvulsant effect of ALLO and alphaxalone during EtOH withdrawal (Beckley et al., 2008
; Cagetti et al., 2004
; Devaud et al., 1996
; Finn et al., 2000
). Collectively, data to date suggest that the plasticity of GABAA
receptors during EtOH withdrawal may differ between seizure prone and resistant genotypes, particularly with regard to ALLO sensitivity.
Evidence indicates that neurosteroid modulation of GABAA
receptors can undergo dynamic change. For example, one mechanism implicated in the timed release of oxytocin from hypothalamic neurons involves fluctuations in ALLO levels and a concomitant decrease in sensitivity of GABAA
receptors to ALLO (Brussaard et al., 1997
; Koksma et al., 2003
). This decreased GABAergic inhibition, due in part to a switch in GABAA
receptor subunit expression as well as to the activity of constitutive phosphatases and kinases (which would alter the phosphorylation state of GABAA
receptors), allows for the timed release of oxytocin. Chronic intermittent EtOH exposure has been shown to alter the responsivity of synaptic and extrasynaptic GABAA
receptors (Liang et al., 2004
), which also exhibit differential sensitivity to GABAergic neurosteroids (Belelli and Lambert, 2005
). Chronic intermittent EtOH exposure also increased the localization of α
4 subunits within GABAergic synapses, which would decrease the sensitivity to neurosteroids at synaptic GABAergic receptors (Liang et al., 2006
). A number of mechanisms have been suggested to contribute to the changes in GABAA
receptor sensitivity to different modulators during EtOH withdrawal, such as alterations in subunit expression and assembly of GABAA
receptors, post-translational modifications, alterations in receptor trafficking or changes in the subcellular or synaptic localization of receptors (Kumar et al., 2004
). However, the specific mechanism(s) underlying the tolerance to ALLO during EtOH withdrawal in WSP mice remains to be determined.
In conclusion, the present findings are the first demonstration that bi-directional manipulation of hippocampal ALLO levels produces opposite behavioral consequences that are consistent with alterations in GABAergic inhibition. These results have important implications for understanding neurosteroid plasticity as it pertains to disorders such as EtOH withdrawal.