Intrinsic properties of pedunculopontine cholinergic neurons

CECILIA TUBERT; GALTIERI, DANIEL; SURMEIER, DALTON JAMES

Bethesda, Washington

Congreso; 19th Annual NINDS Udall Centers Meeting; 2017

NINDS - Udall

The pedunculopontine nucleus (PPN) is a neurochemically heterogeneous structure located in the rostral brainstem. It forms part of two important regulatory systems of behavior: the reticular activating system, implicated in the regulation of the waking-sleep cycle, and the mesensephalic locomotor region, that participates in controlling the locomotion and movements (Mena-Segovia et al, 2008). They have a high degree of interconnectivity with the basal ganglia and the dopaminergic (DA) neurons of subtantia nigra pars compacta (SNc) and ventral tegmental area (VTA) (Mena-Segovia et al, 2008). PPN has at least three types of neurons: cholinergic, glutamatergic and gabaergic neurons. Cholinergic neurons in the PPN (PPN ChNs) are known to play an important functional role in movement control and in sleep. These two functions are compromised in Parkinson?s Disease (PD) (Zweig et al.,1988). Studies made in patients with PD have shown that there is a degeneration of near 40% of the cholinergic neurons of the PPN. This loss is linearly correlated with the loss of dopaminergic neurons of the SNc, and is thought to contribute to the abnormalities of gait and posture, and may be to the disturbances of the REM sleep in PD patients (Zweig et al.,1988). The determinants of the pathophysiology and loss of PPN ChNs in PD are unknown, but they could be intrinsic, extrinsic, or both, and neither of them have been deeply studied in these neurons. As a first step, we propose to characterize the intrinsic properties of PPN ChNs that might contribute to their vulnerability. In SNc DA neurons, Cav1 channels contribute to the robustness of the pacemaking, and the Ca2+ helps maintain the intracellular bioenergetics needs. It increases the generation of mitochondrial reactive oxygen and nitrogen species (ROS/RNS), which have been lined to impaired mitochondrial function, and increased vulnerability to environmental toxins and genetic mutations associated with PD (Guzman et al., 2010). We hypothesize that PPN ChNs have intrinsic determinants of vulnerability that resemble those of SNc DA neurons. To address this aim, we use a combination of slice electrophysiology and optical approaches to study PPN ChNs in mice expressing red fluorescent protein tdTomato in ChAT+ neurons. We use optical tools to quantitatively measure intracellular Ca2+ concentrations, as well as mitochondrial oxidant stress. In cell attached recordings, we found that PPN ChNs have a spontaneous firing frequency that is independent of glutamate and GABA inputs (ACSF: 6.11.0 Hz; PIC+CNQX: 6.81.1 Hz; n=7; paired t test, ns). Unlike what happens in SNc DA neurons (Guzman et al., 2010), chronic blockage of Cav1 channels in PPN ChNs with isradipine (Isr) decreases their spontaneous frequency (ACSF: 4.90.7 Hz; Isr: 2.00.4 Hz; n=13; unpaired t test, p=0.0016) and increases their irregularity (CV: ACSF: 0.30.04; Isr: 0.50.05; n=13; unpaired t test, p=0.0024). We also found that DHPG, a mGluR agonist that releases Ca2+ from de endoplasmic reticulum through second messengers, increases de firing frequency (ACSF: 4.81.3 Hz; DHPG: 7.71.2 Hz; Washout: 5.31.0 Hz; n=7; RM one way ANOVA, p=0.0003) and decreases the irregularity (CV: ACSF: 0.20.03; DHPG: 0.10.008; Washout: 0.20.02; n=7; RM one way ANOVA, p=0.0086). In a two photon setup we found that DHPH decreases the amplitude of the Ca2+ oscillations in dendrites during pacemaking (ACSF: 39.32.6 nM; DHPG: 29.32.3 nM; n=4; paired t test, p=0.0262). However, the basal Ca2+ oscillations are smaller than those in SNc DA neurons (Amplitude: PPN ChNs: 39.52.6 nM; SNc DA neurons: 200-400 nM, Guzman et al., 2017, under review). These results suggest us that PPN ChNs are different to SNc DA neurons but that it is probable that they share intrinsic mechanisms that contribute to their vulnerability in PD.