Cholinergic and endocannabinoid neuromodulatory effects overlap on neurons of the pedunculopontine nucleus of mice

Abstract

The pedunculopontine nucleus (PPN) is a part of the reticular activating system (RAS) and one of the main sources of the cholinergic fibers in the midbrain, while it is also subject to cholinergic modulation. This nucleus is thought to have an important role in REM sleep and wakefulness. Several neuromodulatory mechanisms were described in the PPN, but overlaps of the endocannabinoid and cholinergic effects have not been demonstrated yet.We showed that PPN neurons respond to carbachol in a heterogeneous way: they were depolarized and increase firing rate, hyperpolarized and decrease firing frequency, or lack response. The effect of carbachol was similar to our previous observations with type 1 cannabinoid (CB1) receptor agonists; therefore, we investigated whether different neuromodulatory effects elicit the same action on a certain neuron. A marked but not full overlap was revealed: all neurons depolarized by carbachol were depolarized by the CB1 receptor agonist ACEA, and all neurons lacking response to carbachol lacked response to ACEA, as well. However, neurons hyperpolarized by carbachol were depolarized, hyperpolarized or not affected by the ACEA .Summarizing our data, we found that certain neurons of the PPN respond to muscarinic and cannabinoid stimulations in a similar but not identical way: the same cells were depolarized or not affected by both drugs, whereas neurons hyperpolarized by carbachol responded to ACEA in a heterogeneous way.3

Astrocyte-Dependent Slow Inward Currents (SICs) Participate in Neuromodulatory Mechanisms in the Pedunculopontine Nucleus (PPN)

“…Also surprisingly, we found that changes in SIC frequency and area by neuromodulatory actions depend on previous SIC activity and not on the neuromodulatory agent itself: if original SIC activity was high, neuromodulatory actions decreased it, while in case of low SIC activity, neuromodulatory agents had a stimulatory action on them. Explaining these results along with the previously obtained data (Kőszeghy et al, 2015 ; Kovács et al, 2015 , 2017 ), we hypothesize that neuromodulatory actions increase ambient glutamate level, which stimulate appearance of SICs by activating NMDA receptors, if the majority of NMDA receptors are available for activation. However, if NMDA receptors are already activated by a previously increased ambient glutamate level, further glutamate increase results NMDA receptor desensitization/inactivation.…”

“…Cholinergic stimulation resulted depolarization or hyperpolarization on PPN neurons, leaving a distinct neuronal population unaffected (Ye et al, 2010 ). Cannabinoid agonists exerted similar actions on the PPN, showing marked overlap with the cholinergic actions (Kőszeghy et al, 2015 ; Kovács et al, 2015 , 2017 ). In overlap with carbachol-initiated hyperpolarization, serotonin also elicited tonic hyperpolarization on the same PPN neurons (Leonard and Llinás, 1994 ).…”

“…Our experimental protocols were approved by the Committee of Animal Research of the University of Debrecen (5/2015/DEMÁB). The homozygous floxed-stop- tdTomato (B6;129S6-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze/ J; Jax mice accession number: 007905), -channelrhodopsin-2 (ChR2) (B6;129S-Gt(ROSA)26Sor tm32(CAG-COP4*H134R/EYFP)Hze/ J; Jax number: 012569), glutamate decarboxylase type 2 (GAD2)-cre (Gad2 tm2(cre)Zjh/ J; Jax number: 010802), choline acetyltransferase (ChAT)-cre (B6;129S6-Chat tm2(cre)Lowl/ J; Jax number: 006410) and glial fibrillary acidic protein (GFAP)-cre (B6.Cg-Tg(Gfap-cre)73.12Mvs/J; Jax number: 012886) strains with C57BL6/J background originated from Jackson Laboratories (Bar Harbor, ME, USA) and were crossed in the animal facility of the University of Debrecen, Department of Physiology, following the recommendations of Jackson Laboratories (Kőszeghy et al, 2015 ; Kovács et al, 2015 , 2017 ). Eight to 16 days old offsprings heterozygous both for the cre driver and the floxed-stop genes, expressing tdTomato fluorescent protein in a GFAP-, ChAT- or GAD2- dependent way ( n = 18, 17 and 10, respectively), as well as animals expressing ChR2 in a GFAP-dependent way, from both sexes ( n = 17), were used for the slice electrophysiology experiments.…”

“…Similarly, endocannabinoids also depolarized or hyperpolarized PPN neurons by eliciting tonic currents. We previously demonstrated that the tonic currents on PPN neurons develop via activation of astrocytes (Kőszeghy et al, 2015 ; Kovács et al, 2015 , 2017 ). Astrocytes, in turn, release glutamate, which depolarize or hyperpolarize neurons in a tonic way via activation of different sets of metabotropic glutamate receptors.…”

The M-current contributes to high threshold membrane potential oscillations in a cell type-specific way in the pedunculopontine nucleus of mice

“…There are more overlaps of neuromodulatory actions in the PPN. Orexin and ghrelin (Kim et al, 2009 ), or endocannabinoid and cholinergic actions depolarize the same neuronal population (Kovács et al, 2015 ). The similar regulation of the M-current might have a role in overlaps of the neuromodulatory effects on PPN neurons.…”