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Dissociated gender-specific effects of recurrent seizures on GABA signaling in CA1 pyramidal neurons: role of GABA(A) receptors - PubMed

  • ️Tue Jan 01 2008

Dissociated gender-specific effects of recurrent seizures on GABA signaling in CA1 pyramidal neurons: role of GABA(A) receptors

Aristea S Galanopoulou. J Neurosci. 2008.

Abstract

Early in development, the depolarizing GABA(A)ergic signaling is needed for normal neuronal differentiation. It is shown here that hyperpolarizing reversal potentials of GABA(A)ergic postsynaptic currents (E(GABA)) appear earlier in female than in male rat CA1 pyramidal neurons because of increased potassium chloride cotransporter 2 (KCC2) expression and decreased bumetanide-sensitive chloride transport in females. Three episodes of neonatal kainic acid-induced status epilepticus (3KA-SE), each elicited at postnatal days 4 (P4)-P6, reverse the direction of GABA(A)ergic responses in both sexes. In males, 3KA-SE trigger a premature appearance of hyperpolarizing GABA(A)ergic signaling at P9, instead of P14. This is driven by an increase in KCC2 expression and decrease in bumetanide-sensitive chloride cotransport. In 3KA-SE females, E(GABA) transiently becomes depolarizing at P8-P13 because of increase in the activity of a bumetanide-sensitive NKCC1 (sodium potassium chloride cotransporter 1)-like chloride cotransporter. However, females regain their hyperpolarizing GABA(A)ergic signaling at P14 and do not manifest spontaneous seizures in adulthood. In maternally separated stressed controls, a hyperpolarizing shift in E(GABA) was observed in both sexes, associated with decreased bumetanide-sensitive chloride cotransport, whereas KCC2 immunoreactivity was increased in males only. GABA(A) receptor blockade at the time of 3KA-SE or maternal separation reversed their effects on E(GABA). These data suggest that the direction of GABA(A)-receptor signaling may be a determining factor for the age and sex-specific effects of prolonged seizures in the hippocampus, because they relate to normal brain development and possibly epileptogenesis. These effects differ from the consequences of severe stress.

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Figures

Figure 1.
Figure 1.

Sex differences in EGABA in neonatal rat CA1 pyramidal neurons. A, A scattergram of the EGABA − Vr values in control pups is depicted. Ninety-three percent of males (blue dots) had depolarizing EGABA − Vr values until P14 and subsequently switched to hyperpolarizing. Eighty-three percent of P4–P13 females (pink dots), however, already manifested hyperpolarizing or isoelectric responses by P4. B, C, Significant differences in mean EGABA (B) and EGABA − Vr (C) between P4–P7, P8–P13, and P9–P14 male and female rat CA1 pyramidal neurons are noted. Results in B and C represent the mean ± SEM. PN, Postnatal day.

Figure 2.
Figure 2.

KA-induced seizures in neonatal rats evolve into SE but do not increase the number of Fluoro-Jade B-stained cells in the hippocampus. A, B, P7 male and female pups (n = 9) with epidural recordings were injected with kainic acid (2 mg/kg, i.p.) and monitored continuously for 6 h as well as for 4 h daily at P8–P10. The EEG derivations in A represent bipolar recordings from the right frontal and left occipital electrodes. Initially, bouts of scratching (without any change in EEG activity) interrupted by frequent periods of behavioral arrest that correlated with evolving high-amplitude EEG activity were noted. After the onset of continuous seizure activity (SE), tonic seizures (B) were also observed. Toward the end of SE, episodes of repetitive clonic jerks while the pup was lying on the side were also associated with EEG seizure patterns, as indicated by the gray bar. The ictal events became less frequent and interictal bursts of epileptiform activity were noted (asterisk). In eight of nine pups, no electrographic seizures were noted at P8–P10. C, To determine whether the three episodes of KA-SE at P4–P6 resulted in neurodegeneration in the hippocampus, Fluoro-Jade B staining was performed. There were no Fluoro-Jade B-stained cells seen in the hippocampus. The figures are representative of n = 5 rats per group. As a positive control, the hippocampal sections from an adult rat subjected to a single episode of lithium-pilocarpine SE [methods described by Galanopoulou et al. (2003b)] 48 h before being killed are shown, which demonstrate many Fluoro-Jade B-stained (green) neurons in the hilus, CA1, and CA3 pyramidal layers. Magnifications are 400×. Scale bar, 100 μm. The white arrows indicate the borders of the CA1 and CA3 pyramidal regions. PN, Postnatal day.

Figure 3.
Figure 3.

Effects of three episodes of neonatal KA-SE (KA456) and maternal separation on EGABA − Vr of male and female rat CA1 pyramidal neurons. A, Male rats; scattergram of EGABA − Vr values according to age shows a negative shift in KA456 (black triangles) versus CON (blue circles) male rats. B, Male rats; comparisons of EGABA − Vr among the three groups show that both KA456 and SS456 have hyperpolarizing EGABA − Vr. C, Female rats; scattergram of EGABA − Vr values according to age shows a transient reappearance of depolarizing EGABA − Vr in the KA456 group, between P8–13. D, Female rats; comparisons of EGABA − Vr among the three groups show more depolarizing EGABA − Vr in KA456 pups and more hyperpolarizing EGABA − Vr in SS456 pups at P8–13. Results in B and D are shown as mean ± SEM. PN, Postnatal day.

Figure 4.
Figure 4.

Increased KCC2-ir underlies the negative shift in EGABA in P10 female CON and male KA456 and SS456 groups. A, Male and female rats; representative photos (400× magnification) of CA1 pyramidal neurons from P10 CON rats using a KCC2 specific antibody, counterstained with Novared substrate (brown–red color). Increased KCC2-ir is present in female CA1 pyramidal neurons. B, Male P10 CON, KA456, and SS456 CA1 pyramidal neurons stained with KCC2-specific immunochemistry, using Novared substrate. High levels of KCC2-ir is present in KA456 rats, intermediate in SS456, and low in CON. C, Male and female rats; the results of densitometric analysis of KCC2-ir in CA1 neurons of the three treatment groups are depicted as means ± SEM. Significant sex differences are documented among CON groups. In males only, both KA456 and SS456 have increased KCC2-ir compared with CON (KA456 > SS456 > CON). PN, Postnatal day. Scale bar, 100 μm.

Figure 5.
Figure 5.

Sex- and treatment-related differences in the activity of bumetanide-sensitive NKCC1-like chloride cotransport in rat P8–14 CA1 pyramidal neurons. A, Male and female rats; representative photos (400× magnification) of the CA1 pyramidal layers of P10 CON rats, stained with NKCC1 specific antibody and counterstained with Novared substrate (brown–red color). No differences are noted in NKCC1-ir between sexes. Scale bar, 100 μm. B, Male and female rats; comparison of [EGABA]bumetanide − EGABA among CON, KA456, and SS456 groups demonstrates significant sex differences in CON and KA456 groups, but not in SS456. In males, the effect of bumetanide on EGABA is smaller in KA456 and SS456 compared with CONs. In females, the effect of bumetanide on EGABA is as follows: KA456 > CON > SS456. Results in B represent the mean ± SEM.

Figure 6.
Figure 6.

A, B, Effects of bicuculline treatment on EGABA − Vr of P9–P14 male (A) and P8–P13 female (B) rat CA1 pyramidal neurons. Bicuculline was given 10 min before kainic acid or saline injections and 6 h later at P4, P5, and P6, in both males and females. The bicuculline treatments reversed the directions of EGABA − Vr shifts in the CA1 pyramidal neurons of KA456 and SS456 groups, but their effects were not statistically significant in CON. No sex differences in EGABA − Vr were seen in BB-treated pups. Results represent the mean ± SEM. PN, Postnatal day.

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