Bidirectional regulation of stress responses by galanin in mice: involvement of galanin receptor subtype 1

Abstract

The neuropeptide galanin has been shown to play a role in psychiatric disorders as well as in other biological processes including regulation of pain threshold through interactions with three G-protein coupled receptors, galanin receptor subtypes 1-3 (GalR1-3). While most of the pharmacological studies on galanin in stress-related disorders have been done with rats, the continuous development of genetically engineered mice involving galanin or its receptor subtype(s) validates the importance of mouse pharmacological studies. The present study on mice examined the homeostatic, endocrinological and neuroanatomical effects of the galanin, injected intracerebroventricularly (i.c.v.), in regulation of stress responses after restraint stress. Furthermore, the roles of GalR1 on these effects were studied using GalR1 knockout (KO) mice. The core body temperature and the locomotor activity were monitored with radio telemetry devices. Galanin (i.c.v.) decreased locomotor activity and exerted a bidirectional effect on the restraint stress-induced hyperthermia; a high dose of galanin significantly attenuated the stress-induced hyperthermic response, while a low dose of galanin moderately enhanced this response. The bidirectional effect of galanin was correlated with changes in stress hormone levels (adrenocorticotropic hormone and corticosterone). To neuroanatomically localize the effects of galanin on stress response, cFos immunoreactivity was assessed in galanin receptor rich areas; paraventricular nucleus (PVN) of the hypothalamus and the locus coeruleus (LC), respectively. A high dose of galanin significantly induced cFos activity in the LC but not in the PVN. In GalR1KO mice, a high dose of galanin failed to induce any of the above effects, suggesting the pivotal role of GalR1 in decreased locomotor activity and stress-resistant effects caused by galanin i.c.v. injection studied here.

Figure 1

The effects of galanin injection (icv) on the core body temperature and the locomotor activity. Mice were injected vehicle or galanin (3 or 30 nmol, icv) and kept in individual home cages for 60 min while being measured the core body temperature (a) and the locomotor activity (b) with the implanted radio telemetry devices. The injection-related stress increased body temperature in vehicle- and galanin-treated mice and the injection of galanin decreased locomotor activity (* p < 0.05, ** p < 0.01; vs. Vehicle). The data represents means ± SEM (n ≥ 6).

Figure 2

The effects of galanin injection (icv) on the restraint stress-induced hyperthermia test (a) and on its associated plasma corticosterone (b) and ACTH (c) levels. Mice were injected vehicle or galanin (3 or 30 nmol, icv), kept in individual home cages for 60 min and then given the restraint stress for 40min, followed by the blood sample collection (13:00 - 15:00). Both vehicle- and galanin-injected mice showed the increased core body temperature in response to restraint stress. A low dose of galanin (3 nmol, icv) showed the significantly increased core body temperature while a high dose of galanin (30 nmol, icv) showed the markedly decreased core body temperature, compared to the vehicle injection (a; * p < 0.05, ** p < 0.01; vs. Vehicle). These changes were correlated with corticosterone and ACTH levels (b, c; * p < 0.05, ** p < 0.01; vs. Vehicle, # p < 0.05, ## p < 0.01; vs. Galanin 3 nmol). The data represents means ± SEM (n ≥ 7).

Figure 3

The effects of galanin injection (icv) on cFos immunoreactivity in the LC (a) and the PVN (b) of restraint-stressed mice. Mice were injected vehicle or galanin (3 or 30 nmol, icv), kept in individual home cages for 60 min and then given the restraint stress for 40 min, followed by the perfusion of brain samples which were later immunostained for cFos. In the LC, galanin 30 nmol injection significantly increased cFos immunoreactivity (* p < 0.05 vs. Vehicle). There were no significant differences of cFos activation in the PVN between vehicle- and galanin-injected groups. The data represents means ± SEM (n ≥ 6). The levels of cFos activation are shown relative to each vehicle-treated group that is set as 100 %.

Figure 4

The basal plasma corticosterone (a) and ACTH (b) levels in WT mice and in GalR1KO mice, and the effects of galanin injection (icv) on the core body temperature (c) and the locomotor activity (d) in GalR1KO mice. Mice treatments are as described in Figure 1. There were no significant differences in basal corticosterone and ACTH levels between WT and GalR1KO mice. In GalR1KO mice, there were no significant differences in core body temperature and in locomotor activity between vehicle- and galanin-injected groups. The data represents means ± SEM (n ≥ 6). n.s.; not significant

Figure 5

The effects of galanin injection (icv) on the restraint stress-induced hyperthermia test in GalR1KO mice (a) and its comparison between WT and GalR1KO mice at 40 min after the restraint stress exposure (b). Plasma corticosterone (c) and ACTH (d) levels were measured with or without restraint stress in WT and GalR1KO mice. Mice treatments are as described in Figure 2. There was no effect of 30 nmol galanin on restraint stress-induced hyperthermia in GalR1KO mice and the lower core body temperature observed in WT mice at 40 min after galanin 30 nmol injection, compared to vehicle injection, was not observed in GalR1KO mice. These results were well correlated with corticosterone and ACTH levels. The data represents means ± SEM (n ≥ 6). ** p < 0.01

Figure 6

The effects of galanin injection (icv) on cFos immunoreactivity in the LC of restraint-stressed WT and GalR1KO mice. The injection of 30 nmol galanin increased cFos immunoreactivity in WT mice (* p < 0.05, vs. Vehicle), however this effect of galanin was not observed in GalR1KO mice. The data represents means ± SEM (n ≥ 5).