Differential effect of cannabinoid agonists and endocannabinoids on histamine release from distinct regions of the rat brain

Abstract

Cannabinoids exert complex actions on neurotransmitter systems involved in cognition, locomotion, appetite, but no information was available so far on the interactions between the endocannabinoid system and histaminergic neurons that command several, similar behavioural states and memory. In this study, we investigated the effect of cannabimimetic compounds on histamine release using the microdialysis technique in the brain of freely moving rats. We found that systemic administration of the cannabinoid receptors 1 (CB1-r) agonist arachidonyl-2'chloroethylamide/N-(2chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA; 3 mg/kg) increased histamine release from the posterior hypothalamus, where the histaminergic tuberomamillary nuclei (TMN) are located. Local infusions of ACEA (150 nm) or R(+)-methanandamide (mAEA; 1 microm), another CB1-r agonist, in the TMN augmented histamine release from the TMN, as well as from two histaminergic projection areas, the nucleus basalis magnocellularis and the dorsal striatum. When the endocannabinoid uptake inhibitor AM404 was infused into the TMN, however, increased histamine release was observed only in the TMN. The cannabinoid-induced effects on histamine release were blocked by co-administrations with the CB1-r antagonist AM251. Using double-immunofluorescence labelling and confocal laser-scanning microscopy, CB1-r immunostaining was found in the hypothalamus, but was not localized onto histaminergic cells. The modulatory effect of cannabimimetic compounds on histamine release apparently did not involve inhibition of gamma-aminobutyric acid (GABA)ergic neurotransmission, which provides the main inhibitory input to the histaminergic neurons in the hypothalamus, as local infusions of ACEA did not modify GABA release from the TMN. These profound effects of cannabinoids on histaminergic neurotransmission may partially underlie some of the behavioural changes observed following exposure to cannabinoid-based drugs.

Fig. 1

Schematic diagram and photomicrographs showing the position of the microdialysis probes. Rats were implanted with one probe in the tuberomamillary nuclei (TMN) to deliver drugs locally and measure neurotransmitter release, and another probe in either the nucleus basalis magnocellularis (NBM) (A) or the dorsal striatum (B) to measure histamine release. A single probe was implanted in the TMN, when measuring γ-aminobutyric acid (GABA), or when drugs were administered i.p. (C). (D–F) Representative histological structures showing the actual site of probe placement.

Fig. 2

Time course of histamine release from the tuberomamillary nuclei (TMN) of freely moving rats after systemic administration of the CB1-r agonist, arachidonyl-2′chloroethylamide/N-(2chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA). Histamine release was measured in fractions collected every 15 min. ACEA was administered i.p., whereas control animals received 1 mL/kg of vehicle containing 60% ethanol. Control values of spontaneous histamine release were calculated for each experiment by averaging the mean of four initially collected 15 min samples. Histamine release was expressed as a percentage of spontaneous release. The arrow indicates the time of i.p. injections. Represented are means ± SEM of eight–four experiments. *P < 0.05 vs. last sample before drug treatment (ANOVA and Fischer’s test).

Fig. 3

Influence of cannabinoid administration into the tuberomamillary nuclei (TMN) on histamine release from the TMN and nucleus basalis magnocellularis (NBM) of freely moving rats. Histamine was measured in 15-min fractions and expressed as a percentage of spontaneous release, calculated as described in Fig. 2. (A) Arachidonyl-2′chloroethylamide/N-(2chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA) was infused into the TMN and histamine release was measured from the TMN (lower panel) and the NBM (upper panel). (B) R(+)-Methanandamide (mAEA) was infused in the TMN and histamine release was measured in the TMN (lower panel) and NBM (upper panel). Bars indicate the period of drug application. Shown are means ± SEM of four–five experiments. **P < 0.01; *P < 0.05 vs. last sample before drug treatment (ANOVA and Fisher’s test).

Fig. 4

Effect of cannabinoid infusion into the tuberomamillary nuclei (TMN) on spontaneous histamine release from the TMN and dorsal striatum of freely moving rats. Histamine was measured in 15-min fractions and expressed as a percentage of spontaneous release, calculated as described in Fig. 2. (A) Arachidonyl-2′chloroethylamide/N-(2chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA) was infused into the TMN and histamine release was measured from the TMN (lower panel) and the dorsal striatum (upper panel). (B) R(+)-Methanandamide (mAEA) was infused in the TMN and histamine release was measured in the TMN (lower panel) and dorsal striatum (upper panel). Bars indicate the period of drug application. Shown are means ± SEM of four–five experiments. **P < 0.01; *P < 0.05 vs. last sample before drug treatment (ANOVA and Fisher’s test).

Fig. 5

Endocannabinoids modulate histamine release in the tuberomamillary nuclei (TMN) of freely moving rats. Histamine was measured in 15-min fractions and expressed as a percentage of spontaneous release, calculated as described in Fig. 2. (A) AM404 was infused into the TMN and histamine release was measured from the TMN (lower panel) and nucleus basalis magnocellularis (NBM; upper panel). (B) AM251 was infused into the TMN and histamine release was measured from the TMN and (lower panel) and NBM (upper panel). Bars indicate the period of drug application. Shown are means ± SEM of five experiments (A and B). ** P < 0.01; *P < 0.05 vs. last sample before drug treatment (ANOVA and Fisher’s test).

Fig. 6

Cannabinoid-induced increase of histamine release involves activation of CB1-r in the tuberomamillary nuclei (TMN) of freely moving rats. Histamine was measured in 15-min fractions and expressed as a percentage of spontaneous release, calculated as described in Fig. 2. (A and B) The selective CB1-r antagonist AM251 was infused into the TMN 15 min before adding R(+)-methanandamide (mAEA) and maintained in the perfusing medium during administration of the agonist. (C) AM251 was infused in the TMN 15 min before adding AM404 and maintained during administration of the endocannabinoid uptake inhibitor. Bars indicate the period of drug application. Each point represents the means ± SEM of six (A and B) and three experiments (C).

Fig. 7

CB1 receptors are not detectable on TMN histaminergic neurons. (A and B) The photomicrographs show HDC-positive cells (green) in the E2–E3 subdivision of the TMN and the sparse immunoreactivity for CB1-r (red). (C and D) In the E4 subdivision, the density of immunoreactive fibres is higher than in E2–E3, although no co-localization of anti-HDC and anti-CB1-r immunoreactivity was observed. Note the denser CB1-r immunostaining surrounding what appear to be clusters of HDC-immunonegative cells (arrowhead). Insets show optical scan volumes of the histaminergic cell framed in (D). (E) Anti-CB1-r antibodies densely stained the neuropil in the CA3 region of the hippocampus. Pyramidal cells are immunonegative but surrounded by immunoreactive fibres. HDC positive projections are also visible. (F) The photomicrograph shows HDC positive cells in the TMN. Preadsorption of the anti-CB1-r antibodies with glutathione-S-transferase-conjugated fusion protein resulted in no anti-CB1-r immunostaining. PY, pyramidal cell layer; Scale bars: 120 μm (A and E); 20 μm (B and F); 30 μm (C and D).

Fig. 8

Comparison of the effects of bicuculline, and of bicuculline in the presence of selective CB1-r ligands. Bicuculline (A) or bicuculline plus arachidonyl-2′chloroethylamide/N-(2chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA) (B) were infused in the tuberomamillary nuclei (TMN), and histamine release was measured in the TMN (lower panels) and nucleus basalis magnocellularis (NBM; upper panels). Histamine was measured in 15-min fractions and expressed as a percentage of spontaneous release, calculated as described in Fig. 2. Bars indicate the period of drug application. Each point represents the means ± SEM of six–seven experiments (ANOVA and Fisher’s test). (C) 15-min changes of histamine release in the presence of ACEA, bicuculline and ACEA + bicuculline. Percentage histamine release was calculated by averaging histamine content in the samples collected during pharmacological stimulations. **P < 0.01, *P < 0.05 (ANOVA and Fisher’s test). (D) AM251 (200 nM) was infused in the TMN 15 min before adding bicuculline (10 μM) and maintained in the perfusion medium throughout the experiment. Histamine was measured in 15-min fractions and expressed as a percentage of spontaneous release, calculated as described in Fig. 2. Bars indicate the period of drug application. Each point represents the means ± SEM of six experiments. **P < 0.01, *P < 0.05 (ANOVA and Fisher’s test).