Evidence that G(z)-proteins couple to hypothalamic 5-HT(1A) receptors in vivo

Abstract

Using in situ hybridization and immunoblot analysis, the present studies identified G(z) mRNA and G(z)-protein in the hypothalamic paraventricular nucleus. The role of G(z)-proteins in hypothalamic 5-HT(1A) receptor signaling was examined in vivo. Activation of 5-HT(1A) receptors increases the secretion of oxytocin and ACTH, but not prolactin. Intracerebroventricular infusion (3-4 d) of G(z) antisense oligodeoxynucleotides, with different sequences and different phosphorothioate modification patterns, reduced the levels of G(z)-protein in the hypothalamic paraventricular nucleus, whereas missense oligodeoxynucleotides had no effect. Neither antisense nor missense oligodeoxynucleotide treatment altered basal plasma levels of ACTH, oxytocin, or prolactin, when compared with untreated controls. An antisense-induced decrease in hypothalamic G(z)-protein levels was paralleled by a significant decrease in the oxytocin and ACTH responses to the 5-HT(1A) agonist 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT). In contrast, the prolactin response to 8-OH-DPAT (which cannot be blocked by 5-HT(1A) antagonists) was not inhibited by G(z) antisense oligodeoxynucleotides. G(z)-proteins are the only members of the G(i)/G(o)-protein family that are not inactivated by pertussis toxin. In a control experiment, pertussis toxin treatment (1 microgram/5 microliter, i.c.v.; 48 hr before the 8-OH-DPAT challenge) did not inhibit the ACTH response, potentiated the oxytocin response, and eliminated the prolactin response to 8-OH-DPAT. Thus, pertussis toxin-sensitive G(i)/G(o)-proteins do not mediate the 5-HT(1A) receptor-mediated increase in ACTH and oxytocin secretion. Combined, these studies provide the first in vivo evidence for a key role of G(z)-proteins in coupling hypothalamic 5-HT(1A) receptors to effector mechanisms.

Fig. 1.

Diagrammatic representation of the microdissection of the hypothalamic paraventricular nucleus from coronal sections (700 μm). The landmarks used to identify the coronal section are those seen at 1.8 mm caudal to bregma according to Paxinos and Watson (1986).F, Fornix; PVN, paraventricular nucleus of the hypothalamus.

Fig. 2.

In situ hybridization of G_z mRNA in the hypothalamic paraventricular nucleus.A, High magnification (330×) of cells in the paraventricular nucleus expressing Gz mRNA; B,dark-field low level magnification of the hypothalamus.

Fig. 3.

A, Immunoblots showing the expression of G_z-proteins in the hypothalamic paraventricular nucleus. The numbers below indicate the amount of protein loaded on each lane of the gel. B,Immunoblots demonstrating the changes in G_z-proteins in the hypothalamic paraventricular nucleus after treatment with missense or G_z antisense oligodeoxynucleotides.

Fig. 4.

Effect of G_z antisense oligodeoxynucleotides on the levels of G_z-, G_i1-, and G_i2-proteins in the hypothalamic paraventricular nucleus. The data represent the mean ± SEM of seven or eight rats per group. *Significant difference from the missense and uninjected group, p < 0.05 (one-way ANOVA and Newman–Keuls' test).

Fig. 5.

Effect of completely phosphorothioate-modified 15 mer G_z antisense oligodeoxynucleotides on the oxytocin (A), ACTH (B), and prolactin (C) responses to 8-OH-DPAT (50 μg/kg, s.c.). The data represent the mean ± SEM of seven or eight rats per group. *Significant effect of G_z antisense oligodeoxynucleotides, p < 0.01 (Newman–Keuls' test).

Fig. 6.

Effect of partially phosphorothioate-modified 33 mer G_z antisense oligodeoxynucleotides on the oxytocin (A), ACTH (B), and prolactin (C) responses to 8-OH-DPAT (50 μg/kg, s.c.). The data represent the mean ± SEM of seven or eight rats per group. *Significant effect of G_z antisense oligodeoxynucleotides, p < 0.01 (Newman–Keuls' test).

Fig. 7.

Effect of pretreatment with pertussis toxin (1 μg, i.c.v.) on the oxytocin (A), ACTH (B), and prolactin (C) responses to 8-OH-DPAT (50 μg/kg, s.c.). The data represent the mean ± SEM of 8–10 rats per group. *Significant effect of pertussis toxin, p < 0.01 (Newman–Keuls' test).