Activity-dependent release and actions of endocannabinoids in the rat hypothalamic supraoptic nucleus

Abstract

Exogenous cannabinoids have been shown to significantly alter neuroendocrine output, presaging the emergence of endogenous cannabinoids as important signalling molecules in the neuroendocrine control of homeostatic and reproductive functions, including the stress response, energy metabolism and gonadal regulation. We showed recently that magnocellular and parvocellular neuroendocrine cells of the hypothalamic paraventricular nucleus and supraoptic nucleus (SON) respond to glucocorticoids by releasing endocannabinoids as retrograde messengers to modulate the synaptic release of glutamate. Here we show directly for the first time that both of the main endocannabinoids, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), are released in an activity-dependent fashion from the soma/dendrites of SON magnocellular neurones and suppress synaptic glutamate release and postsynaptic spiking. Cannabinoid reuptake blockade increases activity-dependent endocannabinoid levels in the region of the SON, and results in the inhibition of synaptically driven spiking activity in magnocellular neurones. Together, these findings demonstrate an activity-dependent release of AEA and 2-AG that leads to the suppression of glutamate release and that is capable of shaping spiking activity in magnocellular neurones. This activity-dependent regulation of excitatory synaptic input by endocannabinoids may play a role in determining spiking patterns characteristic of magnocellular neurones under stimulated conditions.

Figure 1. Effect of cannabinoids on glutamate release

A, sample traces (1) and a running average plot of mEPSC frequency (2) from a SON magnocellular neurone showing an AEA-mediated decrease in mEPSC frequency. B, sample traces (1) and a running average plot of mEPSC frequency (2) from another magnocellular neurone showing that the CB1 receptor antagonist AM251 (1 μm) blocked the AEA-induced suppression of mEPSCs. Letter designations a and b in the running average plots correspond to traces denoted by a and b; all data points in the running average plots represent a mean ± s.e.m. of 30 s of continuous events. C, mean changes in the average mEPSC frequencies caused by the synthetic cannabinoid WIN55,212-2 (1 μm, n = 8) and the endogenous cannabinoids 2-AG (0.5–1 μm, n = 5) and AEA (0.5 μm, n = 5). The AEA effect on mEPSC frequency was blocked by AM251 (1 μm, n = 4). *P < 0.05; *P < 0.01.

Figure 2. Activity-dependent increase in endocannabinoid levels

A, high frequency stimulation (HFS) caused a significant increase in AEA (P < 0.05, n = 15) and a non-significant increase in 2-AG (n = 15) in a pairwise LC-MS-MS analysis of endocannabinoid levels in trimmed hypothalamic slices. B, the same analysis in the presence of the ionotropic glutamate receptor antagonists DNQX and AP5 (n = 6) and the sodium channel blocker TTX (n = 4) showed that blocking glutamate neurotransmission or action potentials reversed the HFS-induced increase in AEA and 2-AG.

Figure 3. Depolarization-induced retrograde endocannabinoid release

Depolarisation of a postsynaptic magnocellular neurone (arrows, 100 ms voltage steps to 0 mV at 3 Hz for 10 s) caused a transient 20% decrease in the amplitude of the EPSC evoked by electrical stimulation, or DSE, which was blocked by AM251 (1 μm). EPSCs are averages (black traces) of 3 evoked responses (grey traces) over 1 min; baseline (dashed line, 100%) represents the average of all responses over the 4-min period prior to depolarization (n = 12). *P < 0.05.

Figure 4. Blockade of cannabinoid reuptake increased endocannabinoid levels

A, continuous recording of mEPSCs (1) and running average plot of mEPSC frequency (2) from the same SON magnocellular neurone showing that the cannabinoid transporter blocker OMDM-2 (5 μm) caused a decrease in mEPSC frequency. B, continuous recording of mEPSCs (1) and running average plot of mEPSC frequency (2) from another SON neurone showing blockade of the OMDM-2-induced suppression of mEPSCs by the CB1 antagonist AM251 (1 μm). C, average changes in the mean mEPSC frequency caused by OMDM-2 (n = 5) and OMDM-2 in AM251 (n = 5). D, HFS of trimmed SON slices in the presence of the cannabinoid transporter blocker AM404 (1 μm) elicited a robust increase in the levels of both AEA and 2-AG compared to controls, measured by LC-MS-MS (n = 5). *P < 0.05; **P < 0.01.

Figure 5. Blockade of cannabinoid uptake modulates spiking activity in SON magnocellular neurones

A, bath application of the cannabinoid transporter blocker OMDM-2 (5 μm) abolished the spiking activity induced in a SON neurone by DC injection (9 pA) and high-osmolarity aCSF (340 mosmol l⁻¹). The spiking activity recovered after ∼30 min of washout of the OMDM-2. B, average changes in the mean firing frequency of SON neurones caused by the cannabinoid transporter blockers OMDM-2 (5 μm; n = 13) and AM404 (1–2 μm, n = 7). *P < 0.05.