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. 2012 Apr;24(4):664-73.
doi: 10.1111/j.1365-2826.2011.02239.x.

Tonic Regulation of GABAergic Synaptic Activity on Vasopressin Neurones by Cannabinoids

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Free PMC article

Tonic Regulation of GABAergic Synaptic Activity on Vasopressin Neurones by Cannabinoids

L Wang et al. J Neuroendocrinol. .
Free PMC article

Abstract

Synaptic activity in magnocellular neurosecretory neurones is influenced by the retrograde (i.e. somatodendritic) release of vasopressin, oxytocin and cannabinoids (CBs). For oxytocin neurones, oxytocin exerts constitutive effects on pre-synaptic activity through its ability to release CBs post-synaptically. In the present study, we examined evoked inhibitory post-synaptic currents (eIPSCs) and spontaneous inhibitory post-synaptic currents (sIPSCs) in identified vasopressin (VP) neurones in coronal slices from virgin rats to determine: (i) the extent to which CBs may also tonically modulate VP synaptic activity; and (ii) to determine whether depolarisation-induced suppression of inhibition was present in VP neurones, and if so, whether it was mediated by VP or CBs. The CB1 antagonists AM251 (1 μm) and SR14171 (1 μm) consistently increased the frequency of sIPSCs in VP neurones without affecting their amplitude, suggesting a tonic CB presence. This effect on frequency was independent of action potential activity, and blocked by chelating intracellular calcium with 10 mm ethylene glycol tetraacetic acid (EGTA). AM251 also increased the amplitude of eIPSCs and decreased the paired-pulse ratio (PPR) in VP neurones-effects that were completely blocked with even low (1 mm EGTA) internal calcium chelation. Bouts of evoked firing of VP neurones consistently suppressed sIPSCs but had no effect on eIPSCs or the PPR. This depolarisation-induced suppression of IPSCs was reduced by AM251, and was totally blocked by 10 μm of the mixed vasopressin/oxytocin antagonist, Manning compound. We then tested the effect of vasopressin on IPSCs at the same time as blocking CB1 receptors. Vasopressin (10-100 nm) inhibited sIPSC frequency but had no effect on sIPSC or eIPSC amplitudes, or on the PPR, in the presence of AM251. Taken together, these results suggest a tonic, pre-synaptic inhibitory modulation of IPSCs in VP neurones by CBs that is largely dependent on post-synaptic calcium, and an inhibitory effect of VP on IPSCs that is independent of CB release.

Figures

Figure 1
Figure 1
CB1 receptor antagonists irreversibly increased sIPSC frequency in VP neurones without affecting amplitude. A, Effects of 1 μM AM251 on a single VP neurone. sIPSC frequency is averaged over 10 s. B, Traces of sIPSCs from the cell plotted in A. C, Effects of 1 μM SR141716 on a single VP neurone. D, Summary of effects of AM251 (left panel; (n = 14) and SR141716 (right panel; n = 5) on sIPSCs. Both antagonists significantly increased sIPSC frequency without affecting amplitude.
Figure 2
Figure 2
The CB1 antagonist AM251 (1 μM) increased the eIPSC amplitude and reduced paired pulse facilitation (PPR) in VP neurones, suggesting probability of GABA release was increased. A, Averaged traces (n = 10) from a single VP neurone showing the amplitude change (upper traces) and PPR decrease (lower traces). For both panels, traces at right are overlayed, with the traces from AM251 in gray. The PPR traces at the right are scaled to the first eIPSC from controls. B, Summary of the effects of 1 μM AM251 on PPR and eIPSC amplitude (n = 10).
Figure 3
Figure 3
Depolarization pulse protocol (DPP) reduced sIPSC frequency in VP neurones without affecting amplitude or PPR. A, DPP reduced sIPSC frequency in a single VP neurone. Each dot represents an average over 10 sec. The pair of arrowheads at time 0 indicates when DPP was applied (see text for details). Pairs of upward arrows indicate periods when PPR was tested, in a control period (left pair) and after DPP (right pair). The lower inset shows example of evoked spikes used in DPP. B, Summary of the inhibition of sIPSC frequency, but not amplitude, following DPP (n = 14). C, Example of sIPSCs in a control period (a) and following DPP (b). D, DPP failed to alter PPR in VP neurones. Averages of 10 PPR traces, with those at right overlayed (after DPP in gray) and scaled to the initial eIPSC. E, Summary of the effects of DPP on PPR and eIPSC amplitude (n = 9).
Figure 4
Figure 4
A VP receptor antagonist blocked the inhibition of sIPSCs following DPP. A, The CB1 antagonist AM251 partially blocked DPP-induced inhibition of sIPSCs in a single neurone. Dots represent averages of 10 s. Legend as in Fig. 3A. B, Summary showing continued DSI in the presence of AM251, although attenuated from control (see Fig. 3), with no effect on IPSC amplitudes or PPR (n = 12). C, Manning Compound (MC), a VP/OT antagonist, blocks DSI in a single neurone. Legend as in A. D, Summary showing MC significantly blocked DSI with no effect on IPSC amplitude or PPR (n = 8).
Figure 5
Figure 5
VP reversibly reduced sIPSCs frequency in the presence of AM251. A, VP (10 nM, application indicated by thick line) decreased sIPSC frequency in a single neurone in the continued presence of 1 μM AM251 (thin line). Dots are averages of 10 s. Double arrows indicate periods when PPR was tested. B, Summary showing VP’s inhibition of sIPSC frequency without affecting amplitude (n= 11). C, Summary showing VP’s failure to affect PPR or eIPSC amplitude (n = 9).

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