Multi-Electrode Array Analysis Identifies Complex Dopamine Responses and Glucose Sensing Properties of Substantia Nigra Neurons in Mouse Brain Slices

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.

Targeted Ablation of Primary Cilia in Differentiated Dopaminergic Neurons Reduces Striatal Dopamine and Responsiveness to Metabolic Stress

“…An inhibition of spontaneous activity in response to dopamine (either sustained or with prominent desensitization) has been described as typical for SN DA neurons, and it is mediated by dopamine autoreceptors of the D2 type [ 42 ]. However, we and others have described another type of SN neuron that is excited by dopamine, in similar abundance [ 5 , 43 , 44 ]. These dopamine-excited cells were either silent in the absence of dopamine, or they were active, with a similar pacemaker frequency as the dopamine-inhibited SN DA neurons ( Figure 3 A, Table S4 , and [ 5 ]).…”

“…Vital brain slices from adult mice were prepared, MEA recordings were performed, and MEA data were analyzed essentially as previously described [ 5 ]. Briefly, after brain removal, coronal midbrain slices (200 μm) were prepared using a vibratome (Leica VT1200S, amplitude 1.4 mm, speed 0.6 mm/s) with ceramic blades (Campden Instruments Ltd.) in ACSF at ~36 °C.…”

“…To define the basal firing rate, the mean firing rate of a stable 10 min control period at the beginning of each recording was calculated. As a measure of pacemaker regularity, the mean interspike interval (ISI = 1/frequency) and its standard deviation (SD) were determined as previously described [ 5 ]. For analysis of firing rates in dopamine, firing rates in the last minute in dopamine (i.e., minute 15) were normalized to the mean firing rate during the 10 min control period.…”

“…In particular, when PC are disrupted, intrinsic activities of pancreatic beta-cells are dysregulated because of abnormal glucose sensing [ 4 ]. Similarly, substantia nigra (SN) dopaminergic (DA) neurons also appear to be sensitive to changes in the extracellular glucose concentration, increasing their activity in high glucose [ 5 ]. In addition, previous data based on systemic glucose injections in rats also show an inhibitory effect on the firing of SN DA neurons [ 6 ].…”

A simple parametric representation of the Hodgkin-Huxley model

“…Besides, it is worth mentioning other more complex related problems such as multi-channel Spike Sorting and the function identification of neurons in brain circuits. These questions that are tackled in [47][48][49].…”