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. 2009 Jan 23;158(2):623-34.
doi: 10.1016/j.neuroscience.2008.09.061. Epub 2008 Nov 1.

Resting intracellular calcium concentration, depolarizing Gamma-Aminobutyric Acid and possible role of local estradiol synthesis in the developing male and female hippocampus

J L Nuñez  1 , M M McCarthy
Affiliations

Resting intracellular calcium concentration, depolarizing Gamma-Aminobutyric Acid and possible role of local estradiol synthesis in the developing male and female hippocampus

J L Nuñez et al. Neuroscience. .

Abstract

The maturation of the hippocampus is impacted by a multitude of factors, including the regulation of intracellular calcium levels. Depolarizing actions of Gamma-Aminobutyric Acid (GABA) can profoundly alter intracellular calcium in immature hippocampal neurons via influx through voltage-gated calcium channels. We here report fundamental sex differences in properties of depolarizing GABA responses and in resting intracellular calcium in neonatal cultured hippocampal neurons. The effects of the estrogen receptor antagonist, ICI 182,780, and the estradiol-synthesis inhibitor, formestane, indicate the sex differences in depolarizing GABA responses are at least in part due to de novo estradiol synthesis by female neurons, whereas a sex difference in resting calcium is independent of steroids. We postulate that local estradiol synthesis in cultured female hippocampal neurons affects the kinetics of either the GABA(A) receptor or voltage sensitive calcium channels. These data highlight the fact that immature hippocampal neurons exhibit fundamentally different physiological properties in males versus females. Elucidating how and where immature male and female neurons differ is essential for a complete understanding of normal rodent brain development.

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Figures

Figure 1
Figure 1. Effect of sex and age on resting intracellular calcium concentration in developing hippocampal neurons
(A) Mean baseline (resting) intracellular calcium concentration ([Ca2+]i) on day in vitro 2 and 7 in male and female hippocampal neurons. On day in vitro 2 (DIV2), females had a significantly elevated baseline [Ca2+]i as compared to males (Tukey’s, #p<0.05). Data represent mean ± SEM [Ca2+]i, with data obtained from 5–6 culture runs, with two to three fields of view per culture run for a total n of 10–12. # indicates significant difference from DIV2 female cultures. (B) Representative pseudocolored image of baseline [Ca2+]i male hippocampal neurons on DIV 2. (C) Representative pseudocolored image of baseline [Ca2+]i female hippocampal neurons on DIV 2. Scale bar = 20μm. (D) Effect of numerous regulators of intracellular calcium. Treatment with the ionotropic glutamate receptor antagonists AP5, MK-801, NBQX and NAS (100μM of each antagonist), voltage sensitive calcium channel antagonist, diltiazem (10μM), extracellular calcium chelator EGTA (500μM) in the presence of calcium free physiological saline solution, intracellular calcium chelator BAPTA-AM (1μM), and intracellular calcium re-uptake inhibitor CPA (1μM) were assessed for their impact on resting intracellular calcium levels in male and female cultured hippocampal neurons on DIV 2. The baseline [Ca2+]i in both male and female hippocampal neurons was significantly reduced by the intracellular calcium chelator BAPTA-AM and CPA (Tukey’s, *p<0.05). However, addition of these agents did not eliminate the sex difference in baseline [Ca2+]i. Blockade of extracellular sources of calcium (observed following calcium free PSS + EGTA treatment) likewise significantly reduced baseline [Ca2+]i (Tukey’s, *p<0.05, significant difference from vehicle treated cultures within the same sex), and eliminated the sex difference in baseline intracellular calcium concentration by reducing females baseline [Ca2+]i to the level of males. These data are interpreted as indicating the sex difference in resting [Ca2+]i is due to increased influx from external sources in females. Data represent the mean ± SEM [Ca2+]i value, from a total of five culture runs, with one to two fields of view per culture run, providing an overall n of 5 to 10 per group.
Figure 2
Figure 2. Effect of sex on peak magnitude of muscimol-induced calcium transients in developing hippocampal neurons
(A) There was a significant sex difference in the maximal increase in [Ca2+]i following muscimol (10μM) application in hippocampal neurons on day in vitro 2 in cells classified as “high” responders (greater than 75% increase over baseline; ANOVA; #p<0.01. significant difference from females), but not in cells classified as “low” responders (less than 75% but greater than 10% increase over baseline). Data represent mean ± SEM peak [Ca2+]i value, with data obtained across 3 culture runs, with two fields of view per culture run, providing an overall n of 6 per group. (B) Histogram of calcium responses from individual male and female hippocampal neurons. Visual inspection of the distribution pattern illustrates the distinction between “high” and “low” responding neurons. Data were obtained from 55 responding male and female hippocampal neurons. (C) Effect of antagonizing the muscimol-induced increase in [Ca2+]i in “high” responding hippocampal neurons by blockade of ionotropic glutamate receptors using the NMDA antagonists AP5 and MK-801, the AMPA antagonists NBQX and NAS (100μM of each antagonist), voltage sensitive calcium channel blocker diltiazem (10μM), extracellular calcium entry using a combination of the calcium chelator EGTA (500μM) in the presence of calcium free physiological saline solution, intracellular calcium release using the calcium chelator BAPTA-AM (1μM), intracellular calcium re-uptake using the inhibitor CPA (1μM) and sodium channels using tetrodotoxin (1μM). There was a significant main effect of antagonist treatment on response to muscimol application in male and female hippocampal neurons on DIV 2 (ANOVA: p<0.03). Antagonists of both voltage sensitive calcium channels and extracellular calcium entry significantly eliminated the muscimol-induced increase in [Ca2+]i in both male and female hippocampal neurons (Tukey’s, *p<0.01, significant difference from vehicle treated cultures of the same sex), indicating the source of muscimol-induced calcium is the same for both sexes. Data represent mean ± SEM peak [Ca2+]i value, obtained across 3 to 4 culture runs, with two fields of view per culture run for an n of 4 to 8 per group.
Figure 3
Figure 3
Representative calcium responses from individual (A) male and (B) female hippocampal neurons. Responses were obtained from one individual field of view, documenting alterations in [Ca2+]i following transient muscimol (10μM) administration.
Figure 4
Figure 4. Time to and duration of elevated intracellular calcium post muscimol application are affected by inhibiting estradiol synthesis or antagonizing the estrogen receptor
(A) Female hippocampal neurons on DIV 2 had significantly faster rise time to peak intracellular calcium concentration subsequent to muscimol administration compared to all other groups (Tukey’s, #p<0.01, significant difference from male vehicle treated cultures), which was significantly reduced to the level of male hippocampal neurons by treatment with both the estrogen receptor antagonist ICI 182,780 (8, 24 and 48 hour pretreatment) and the estradiol synthesis inhibitor formestane (48 hour pretreatment) (Tukey’s, *p<0.01, significant difference from vehicle treated cultures of the same sex). Data represent mean ± SEM time to peak [Ca2+]i, with data obtained across three culture runs, with two fields of view per culture run for an n of 6 per group. Muscimol was applied for 50 seconds, with the time to the greatest increase [Ca2+]i during the 50 second administration window analyzed. (B) Female (vehicle and 8 hour ICI 182,780 pretreatment) hippocampal neurons on DIV 2 had significantly faster decay time than all other groups (Tukey’s, #p<0.01, significant difference from male vehicle treated cultures), which was significantly reduced to the level of male hippocampal neurons by treatment with both ICI 182,780 (24 and 48 hour pretreatment) and formestane (48 hour pretreatment) (Tukey’s, *p<0.01, significant difference from vehicle treated cultures of the same sex.). Data represent mean ± SEM duration of [Ca2+]i response, with data obtained across three culture runs, with one to two fields of view per culture run for an n of 4 to 6 per group. The decay time was the time following muscimol application (i.e. the start of PSS administration) required to re-attain one-half baseline intracellular calcium concentration.
Figure 5
Figure 5. Estrogen receptor activation and estradiol synthesis affect excitatory GABA responses in the developing female hippocampus
(A) Female hippocampal neurons on DIV 2 show enhanced muscimol-induced calcium response following pretreatment with 17β-estradiol relative to all other groups (Tukey’s, *p<0.01, significant difference from vehicle treated cultures). Pretreatment with the selective estrogen receptor a agonist PPT likewise increased the magnitude of muscimol-induced calcium responses (Tukey’s, #p<0.05), with no effect of the selective estrogen receptor β agonist DPN. Data represent mean ± SEM peak [Ca2+]i value, data obtained across 3 culture runs, with one to two fields of view per culture run, providing an overall n of 4 to 6 per group. (B) Hippocampal cells from DIV 2 female cultures show significantly attenuated levels of the phosphorylated form of the chloride co-transporter, pNKCC1, following pretreatment with the estrogen receptor antagonist, ICI 182,780, relative to all other groups (Tukey’s, *p<0.05, significant difference from vehicle treated cultures). Pretreatment with 17β-estradiol produced a marginal effect (Tukey’s, p < 0.07). Data are expressed as grayscale integrative area density/GAPDH (means ± SEM, n = 6 per group across two culture runs).
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