Abstract
Dopaminergic (DA) midbrain neurons within the substantia nigra (SN) display an autonomous pacemaker activity that is crucial for dopamine release and voluntary movement control. Their progressive degeneration is a hallmark of Parkinson's disease. Their metabolically demanding activity-mode affects Ca2+ homeostasis, elevates metabolic stress, and renders SN DA neurons particularly vulnerable to degenerative stressors. Accordingly, their activity is regulated by complex mechanisms, notably by dopamine itself, via inhibitory D2-autoreceptors and the neuroprotective neuronal Ca2+ sensor NCS-1. Analyzing regulation of SN DA neuron activity-pattern is complicated by their high vulnerability. We studied this activity and its control by dopamine, NCS-1, and glucose with extracellular multi-electrode array (MEA) recordings from midbrain slices of juvenile and adult mice. Our tailored MEA- and spike sorting-protocols allowed high throughput and long recording times. According to individual dopamine-responses, we identified two distinct SN cell-types, in similar frequency: dopamine-inhibited and dopamine-excited neurons. Dopamine-excited neurons were either silent in the absence of dopamine, or they displayed pacemaker-activities, similar to that of dopamine-inhibited neurons. Inhibition of pacemaker-activity by dopamine is typical for SN DA neurons, and it can undergo prominent desensitization. We show for adult mice, that the number of SN DA neurons with desensitized dopamine-inhibition was increased (~60–100%) by a knockout of NCS-1, or by prevention of NCS-1 binding to D2-autoreceptors, while time-course and degrees of desensitization were not altered. The number of neurons with desensitized D2-responses was also higher (~65%) at high glucose-levels (25 mM), compared to lower glucose (2.5 mM), while again desensitization-kinetics were unaltered. However, spontaneous firing-rates were significantly higher at high glucose-levels (~20%). Moreover, transient glucose-deprivation (1 mM) induced a fast and fully-reversible pacemaker frequency reduction. To directly address and quantify glucose-sensing properties of SN DA neurons, we continuously monitored their electrical activity, while altering extracellular glucose concentrations stepwise from 0.5 mM up to 25 mM. SN DA neurons were excited by glucose, with EC50 values ranging from 0.35 to 2.3 mM. In conclusion, we identified a novel, common subtype of dopamine-excited SN neurons, and a complex, joint regulation of dopamine-inhibited neurons by dopamine and glucose, within the range of physiological brain glucose-levels.
Highlights
Dopamine and dopamine-releasing (DA) neurons are important for a variety of brain functions and processes like movement control, habit-formation, conditioning, cognition, noveltyrelated behavior, motivation, reward prediction, and glucose homeostasis (Dodson et al, 2016; Koekkoek et al, 2017; Berke, 2018; Ter Horst et al, 2018; Collins and Saunders, 2020)
multi unit activity (MUA) were discriminated from single unit activity (SUA) by a high number of events in the range before 200 ms (Figure 1C; >15% of big peak), an irregular pacemaker activity, assessed by a higher variation of the inter-spike interval (ISI, Figure 1D), and an only partial inhibition by dopamine (100 μM, bath-applied for 15 min; Figure 1E)
These criteria can be used for identifying SUA derived from Substantia nigra (SN) DA neurons, as they display in vitro in synaptic isolation a slow, very regular pacemaker-activity (∼0.5-5 Hz, coefficient of variation (CV) ISI: standard deviation (SD) (ISI): ∼5–10%), and a welldescribed full inhibition of pacemaker-activity in response to extracellular dopamine (Lacey et al, 1987; Mercuri et al, 1994; Beckstead et al, 2004; Lammel et al, 2008)
Summary
Dopamine and dopamine-releasing (DA) neurons are important for a variety of brain functions and processes like movement control, habit-formation, conditioning, cognition, noveltyrelated behavior, motivation, reward prediction, and glucose homeostasis (Dodson et al, 2016; Koekkoek et al, 2017; Berke, 2018; Ter Horst et al, 2018; Collins and Saunders, 2020). SN DA neurons display a high level of intrinsic metabolic stress already under control conditions This is due to the size and complexity of their unmyelinated axonal arbors in their striatal target regions, which is an order of magnitude greater than that of less susceptible dopamine neurons (Bolam and Pissadaki, Abbreviations: ACSF, artificial cerebrospinal fluid; AMP, adenosine monophosphate; Arc, arcuate nucleus; Cav, voltage-gated calcium channel; CFTR, cystic fibrosis transmembrane conductance regulator; CV, coefficient of variation; D2-AR, autoreceptor of the D2 type; DA, dopamine; DMN, dorsomedial nuclei; DMV, dorsal motor nucleus of the vagus; DNIP, D2/NCS-1 interaction preventing peptide; EC50, half maximal effective concentration; GE, glucose excited; GFP, green fluorescent protein; GI, glucose inhibited; GIRK, G-protein coupled inwardly rectifying K+; GK, glucokinase; GLUT, glucose transporter; HT, hypothalamus; ISI, interspike interval; K2P (TASK), two-pore domain potassium; K-ATP, ATP-sensitive K+; KO, knockout; Kv, voltage-gated potassium channel; L-Dopa, Levodopa; LH, lateral hypothalamus; MEA, multi-electrode array; MUA, multi unit activity; NCS-1, neuronal Ca2+ sensor; PBM, parabrachial medial nucleus; PBS, phosphate buffered saline; PCA, principal component analysis; PD, Parkinson’s disease; PN, post-natal; PVN, periventricular nucleus; SD, standard deviation; SE, standard error; SEM, standard error of the mean; SGLT, sodium glucose co-transporter; SN, substantia nigra; srDNIP, scrambled DNIP; SUA, single unit activity; T1R, sweet taste receptor; T2DM, diabetes mellitus type 2; TH, tyrosine hydroxylase; TRPC, transient receptor potential channel; VMH, ventromedial hypothalamus; VMN, ventromedial nucleus; VRAC, volume regulated anion channels; VTA, ventral tegmental area; WT, wildtype
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