Localized suppression of cortical growth hormone-releasing hormone receptors state-specifically attenuates electroencephalographic delta waves

Abstract

Growth hormone-releasing hormone (GHRH) promotes non-rapid eye movement sleep (NREMS), in part via a well characterized hypothalamic sleep-promoting site. However, GHRH may also act in the cortex to influence sleep. Application of GHRH to the surface of the cortex changes electroencephalographic (EEG) delta power. GHRH and the GHRH receptor (GHRHR) mRNAs are detectable in the rat cortex, and the expression of cortical GHRHR is activity dependent. Here, we microinjected a GHRH antagonist or GHRHR small interfering RNA (siGHRHR) onto the somatosensory cortex surface in rats. The unilateral application of the GHRH antagonist ipsilaterally decreased EEG delta wave power during NREMS, but not wakefulness, during the initial 40 min after injection. Similarly, the injection of siGHRHR reduced cortical expression of GHRHR and suppressed NREMS EEG delta wave power during 20-24 h after injection. Using the fura-2 calcium imaging technique, cultured cortical cells responded to GHRH by increasing intracellular calcium. Approximately 18% of the GHRH-responsive cells were GABAergic as illustrated by glutamic acid decarboxylase-67 (GAD67) immunostaining. Double labeling for GAD67 and GHRHR in vitro and in vivo indicated that only a minority of cortical GHRHR-containing cells were GABAergic. Our data suggest that endogenous cortical GHRH activates local cortical cells to affect EEG delta wave power state-specifically. Results are also consistent with the hypothesis that GHRH contributes to local network state regulation.

Figure 1.

Effects of unilateral injection of GHRH antagonist on sleep. After baseline recordings on day 1, rats received 5 nmol of GHRH antagonist on one side of the Sctx and saline on the contralateral Sctx at dark onset. A, EEG delta wave power (0.5–4 Hz) during wakefulness, REMS, and NREMS after unilateral application of GHRH antagonist at dark onset. The EEG delta wave power was normalized by the baseline value recorded from the same side of the brain. The data were expressed as the normalized delta wave power of the GHRH antagonist-receiving side (■) relative to the control side (▩). The asterisk (*) indicates significant difference from control (n = 9; p < 0.05 by two-way ANOVA followed by Fisher's LSD multiple-comparison test). B, EEG power change from the baseline (BS) on the GHRH antagonist-injected side (●) and the saline-injected side (○) during 0–40 min after dark onset injections of GHRH antagonist. The asterisk (*) indicates significant differences between two sides (n = 9; p < 0.05; two-way ANOVA repeated measures followed by Fisher's LSD multiple-comparison test). Error bars indicate SEM.

Figure 2.

Effects of unilateral injection of siGHRHR on sleep. Rats received 0.1 nmol of siGHRHR on one side of the Sctx and scrambled siRNA on the contralateral Sctx at dark onset. A, EEG delta wave (0.5–4 Hz) power on the siGHRHR-receiving side (●) was significantly lower than the control side (○) during 20–24 h after injection (*p < 0.05; n = 7; two-way ANOVA repeated measures followed by Fisher's LSD multiple-comparison test). There was no significant difference between two sides on day 2 after injection. B, NREMS EEG power change from the baseline on the siGHRHR-receiving side (●) and the scrambled siRNA-receiving side (○) during 20–22 h after dark onset injections of siGHRHR. The asterisks (**) indicate significant differences between two sides (n = 7; p < 0.01; two-way ANOVA repeated measures followed by Fisher's LSD multiple-comparison test). Error bars indicate SEM.

Figure 3.

Effects of siGHRHR on GHRHR expression in vivo. Rats received microinjection of 0.1 nmol of siGHRHR on one side of the Sctx and scrambled siRNA on the contralateral Sctx at dark onset. They were killed at a time between 20 and 22 h after injection. A, Western blot of GHRHR in Sctx 20 h after siGHRHR or scrambled siRNA injection. B, C, Immunohistochemistry for GHRHR in Sctx 22 h after siGHRHR (C) compared with scrambled siRNA (B) injection. The large pyramidal neurons in layer V were dramatically reduced in GHRHR immunostaining; the arrow indicates an example of a GHRHR-immunopositive cell. Scale bar, 100 μm.

Figure 4.

GHRH-evoked activation of cultured cortical neurons. Primary cortical cells were cultured and challenged with GHRH (100 nm) on culture day 10–12 in vitro. A, Representative Ca²⁺ response to GHRH in cultured cortical neurons. Each line on graphs illustrates the Ca²⁺ level in a single neuron. GHRH was applied for the duration of the labeled bars. The bar labeled KCl was when the neurons were depolarized with a 55 mm K⁺ bath. B, Dependence of the GHRH-induced response on extracellular Ca²⁺. The bar labeled with Ca²⁺ free indicates period when Ca²⁺-free bath (Ca²⁺ was replaced by Mg²⁺) was applied.

Figure 5.

GAD67 immunostaining for GHRHR-containing cells. A, B, In primary cortical culture, GHRH-responsive cells were detected by Ca²⁺ imaging and then processed for GAD67 immunostaining. The arrows in B show the cells present in A, and one of them is GAD67 positive. Scale bar, 50 μm. C–F, Immunostaining of GHRHR and GAD67 in primary cortical cultures. C, Nuclear staining was demonstrated by Hoechst 33342 staining. D, GHRHR-immunoreactive cells. E, GAD67-immunoreactive cells. F, All three stains were merged to demonstrate triple labeling. Two cells are double labeled (arrows) by GHRHR and GAD67. Scale bar, 50 μm. G–I, Immunohistochemistry for GHRHR and GAD67 in Sctx. G, GHRHR-immunoreactive cells. H, GAD67-immunoreactive cells. I, Merged picture to show double labeling of GHRHR and GAD67. Four cells were double labeled by GHRHR and GAD67 (arrows). Scale bar, 50 μm.