Activity of Neurochemically Heterogeneous Dopaminergic Neurons in the Substantia Nigra during Spontaneous and Driven Changes in Brain State

Electrophysiological recordings.

Experimental procedures were performed on adult male Sprague Dawley rats (Charles River) and were conducted in accordance with the Animals (Scientific Procedures) Act, 1986 (United Kingdom), and the European Communities Council Directive (86/609/EEC).

Electrophysiological recordings were made in 42 rats (240–370 g). Anesthesia was induced with isoflurane (Isoflo, Schering-Plough) and maintained with urethane (1.3 g/kg, i.p.; ethyl carbamate, Sigma), and supplemental doses of ketamine (30 mg/kg, i.p.; Ketaset, Willows Francis) and xylazine (3 mg/kg, i.p.; Rompun, Bayer), as described previously (Magill et al., 2001). All wound margins were infiltrated with the local anesthetic bupivacaine (0.75% w/v; Astra) and corneal dehydration was prevented with application of Hypromellose eye drops (Norton Pharmaceuticals). Animals were then placed in a stereotaxic frame. Body temperature was maintained at 37 ± 0.5°C using a homeothermic heating device (Harvard Apparatus).

Anesthesia levels were assessed by examination of the electrocorticogram (ECoG, see below), and by testing reflexes to a cutaneous pinch or gentle corneal stimulation. The electrocardiogram (ECG) and respiration rate were also monitored constantly to ensure appropriate anesthetic levels (see below). Saline solution (0.9% w/v NaCl) was applied to all areas of exposed cortex to prevent dehydration.

The ECoG was recorded via a 1 mm diameter steel screw juxtaposed to the dura mater above the right frontal cortex (AP: +2.7 mm, ML: 2.0 mm in relation to bregma (Paxinos and Watson, 1986)), and was referenced against another screw implanted in the skull above the cerebellum. Raw ECoG was bandpass filtered (0.3–1500 Hz, −3 dB limits) and amplified (2000×, DPA-2FS filter/amplifier; Scientifica) before acquisition. The ECG was differentially recorded via two silver wires inserted into the skin of the left forelimb and hindlimb. Raw ECG was bandpass filtered (10–100 Hz) and amplified (5000×, DPA-2FS; Scientifica) before acquisition. A discrete craniotomy was performed above the right and/or left SN, and the dura mater removed for insertion of recording electrodes. Extracellular recordings of action potentials of SN neurons were made using 10–25 MΩ glass electrodes (tip diameter ∼1.5 μm), which contained saline solution (0.5 m NaCl) and Neurobiotin (1.5% w/v, Vector Laboratories). Electrode signals were amplified (10×) through the active bridge circuitry of an Axoprobe-1A amplifier (Molecular Devices Corp.), bifurcated, then differentially filtered to extract local field potentials (LFPs) and unit activity. The LFPs were recorded after further amplification (100×; DPA-2FS; Scientifica) and “wideband” filtering (between 0.3 and 5000 Hz; DPA-2FS; Scientifica). Single units were recorded after alternating current coupling, further amplification (100×; DPA-2FS; Scientifica), and standard bandpass filtering (between 300 and 5000 Hz; DPA-2FS; Scientifica). A Humbug (Quest Scientific) was used in place of a traditional “notch” filter to eliminate mains noise at 50 Hz (Brown et al., 2002). Spikes were often several millivolts in amplitude and always exhibited an initial positive deflection. Neuronal firing was recorded during slow-wave activity (SWA), which accompanies deep anesthesia and is similar to activity observed during natural sleep, and during episodes of cortical activation, which contain patterns of activity that are more analogous to those observed during the awake, behaving state (Steriade, 2000). Transition from SWA to the activated brain state is exemplified by obliteration of the cortical slow oscillation (∼1 Hz), as well as δ (1–4 Hz) and spindle (7–14 Hz) oscillations (Steriade, 2000).

After recordings of spontaneous activity, the responses of individual neurons and the ECoG to two standard types of aversive somatosensory stimuli (a pinch or an electrical stimulus applied to the hindpaw contralateral to the recording site) were characterized. Pinches of 15 s duration were delivered using pneumatically driven forceps that consistently delivered a standard pressure of 183 g/mm² (Pollard, 2000; Ungless et al., 2004), which has previously been shown to exceed the threshold of pain in behaving rats (Cahusac et al., 1990). Electrical stimuli (single current pulses of 5 mA intensity and 2 ms duration) were delivered at 0.5 Hz (Coizet et al., 2006) for 100 trials. The timings of stimuli delivery were precisely controlled by an external pulse generator (Master-8; A.M.P.I.). The animals did not exhibit a marked change in ECG or respiration rate, nor a hindpaw withdrawal reflex, in response to the pinch or the electrical stimuli, thus indicating anesthesia was adequate throughout recordings.

Juxtacellular labeling of single neurons.

To locate the recorded neurons and enable an analysis of their morphological and neurochemical properties, they were then labeled with Neurobiotin (Vector Laboratories) by the juxtacellular method (Pinault, 1996; Magill et al., 2000). Briefly, the electrode was advanced slowly toward the neuron while a microiontophoretic current was applied (1–10 nA positive current, 200 ms duration, 50% duty cycle). The optimal position of the electrode was identified when the firing pattern of the neuron was robustly modulated by the current injection. It was necessary to modulate the neuronal firing by the microiontophoretic current for at least 2 min, but preferably longer, to obtain reliable labeling. The Neurobiotin was then left to transport along neuronal processes for up to 12 h. After the recording and labeling sessions, the animals were given a lethal dose of ketamine (150 mg/kg) and perfused via the ascending aorta with 100 ml of 0.01 m PBS at pH 7.4, followed by 300 ml of 0.1% w/v glutaraldehyde and 4% w/v paraformaldehyde in 0.1 m phosphate buffer, pH 7.4, and then by 100 ml of PBS. Brains were then left in PBS or fixative solution at 4°C until they were sectioned 24–72 h later.

Histochemistry.

The fixed brains were sectioned at 60 μm in the parasagittal plane on a vibrating blade microtome (VT1000S; Leica Microsystems,). Selected tissue sections were then incubated (4°C, 2 nights) in PBS containing primary antibodies raised against tyrosine hydroxylase (TH, mouse anti-TH; 1:1000, Sigma) and calbindin (rabbit anti-CB; 1:5000, Swant, or goat anti-CB; 1:500, Santa Cruz Biotechnology). Triton X-100 (0.3% v/v) and 1% w/v bovine serum albumin (Sigma) were included in the incubation mixture. Fluorescent conjugates were used to visualize Neurobiotin (streptavidin-CY3; 1:1000, 4°C, overnight; Zymed), TH immunoreactivity (AlexaFluor 488-conjugated donkey anti-mouse; 1:1000, 4°C, overnight; Invitrogen) and CB immunoreactivity (CY5-conjugated donkey anti-rabbit or CY5-conjugated donkey anti-goat; 1:500, 4°C, overnight; Jackson Immunoresearch Laboratories). Sections were then mounted on slides for viewing with a conventional epifluorescence microscope (Leica Microsystems), or a laser-scanning confocal microscope (Zeiss).

After analysis in the fluorescence microscope, standard histochemical techniques were used to visualize the Neurobiotin-filled neurons with a permanent peroxidase reaction product for light microscopy. Unless otherwise stated, histochemical incubations were performed at room temperature. Sections were washed in PBS and incubated overnight (4°C) in avidin-biotin peroxidase complex (ABC Elite; 1:100; Vector) in PBS (with or without 0.3% v/v Triton X-100) and 1% w/v bovine serum albumin. After washing, the sections were incubated in hydrogen peroxide (0.002% w/v; Sigma) and diaminobenzidine tetrahydrochloride (DAB; 0.025% w/v; Sigma) in the presence of nickel ammonium sulfate (0.5% w/v; Sigma) dissolved in Tris buffer (0.05 m, pH 8.0) for 5–30 min. Neurobiotin-filled neurons were labeled intensely with an insoluble, black/blue precipitate. Sections containing the cell body were then incubated in rabbit anti-mouse IgG (1:200, 1 h; Dako), washed with PBS, and incubated in mouse peroxidase anti-peroxidase (PAP; 1:100, 2 h; Dako). After this, the DAB procedure was repeated (omitting the nickel ammonium sulfate, and DAB prepared in Tris buffer pH 7.6), so that the TH-immunoreactive (dopaminergic) neurons of the substantia nigra were labeled with a brown precipitate.

Electrophysiological data analysis.

All biopotentials were digitized on-line using a Power 1401 Analog-Digital converter (Cambridge Electronic Design) and a PC running Spike2 acquisition and analysis software (version 5.15; Cambridge Electronic Design). Unit activity, LFPs and the ECoG were sampled at 16.7 kHz, 16.7 kHz and 5.6 kHz, respectively. Data from the recording sessions were inspected visually and epochs of stationary cortical activity were identified. A portion of the coincident spike train composed of 150 spikes was then isolated and used for our standard statistical analyses. Measurements of the extracellular action potential waveform were taken from these data epochs, as were spontaneous firing rate (Hz) and the coefficient of variation of spiking (CV, a standard measure of regularity). Autocorrelograms of unit activity (50 ms bins) and ECoG power spectra (frequency resolution of 0.17 Hz) were constructed using standard procedures in Spike2. To test whether dopaminergic neurons engaged in bursting activity, the classical criteria outlined by Grace and Bunney were used (Grace and Bunney, 1984a). Briefly, bursts were defined as beginning when 2 action potentials occurred within 80 ms of each other, and ending when an action potential did not occur for 160 ms. Previous studies have additionally categorized the spontaneous firing patterns of dopaminergic neurons into 3 groups: “pacemaker”, “random” or “bursty” (Tepper et al., 1995; Paladini and Tepper, 1999). When possible, we tested our neurons according to this additional classification scheme. To achieve this, autocorrelograms (5 ms bins, plotted over 2 s) were constructed from data epochs containing ≥1000 spikes. Cells that exhibited 3 or more equally spaced peaks in the autocorrelogram were classified as pacemakers, cells that exhibited a distinct initial peak followed by a return to steady state were classified as bursty, and all others were classified as random (Tepper et al., 1995; Paladini and Tepper, 1999).

The slow (∼1 Hz) oscillation that dominates SWA, as recorded in ECoGs or basal ganglia LFPs, does not closely approximate a sinusoidal waveform, and contains active and inactive components of varied shape and duration. This makes detecting the peaks (or troughs) of this oscillation difficult, and also limits the utility of several analyses that are commonly used for defining the relationships between unit activity and ECoG/LFP oscillations (e.g., spike-triggered waveforms or circular statistics). Thus, we used an alternative method of quantifying the temporal relationship between the cortical slow oscillation and unit activity (Mallet et al., 2008). Single-unit activity was converted so that the “time stamp” of each spike was represented by a single digital event (Spike2). Raw ECoGs were initially down-sampled to 1.79 kHz off-line, high-pass filtered at 0.3 Hz to remove any slow drift in DC potential, and then bandpass filtered at 0.4–1.6 Hz to isolate the cortical slow oscillation (MATLAB). The zero-voltage crossings of this filtered ECoG signal were then used to define the start and end points of active (and inactive) components. However, active and inactive components were only considered as such after thresholding for voltage (amplitude difference between components of ≥0.3 mV) and power (ratio of component power, which takes into account the signal energy over time, was >0.15). Thus, only robust slow oscillations were analyzed. Moreover, active components were only accepted if they were preceded or followed by an inactive component and vice versa. After defining active and inactive components of ECoGs, coincident spikes were automatically assigned to one of 14 bins (7 bins each for active and inactive components: MATLAB). Spike counts per bin across all accepted oscillation components were then normalized (by converting to firing rate) to take into account the variable durations of active and inactive components, and then displayed in an “activity histogram” (Fig. 1. For statistical definition of relationships between unit activity and the cortical slow oscillation, we used Pearson's χ² test (Excel, Microsoft) to assess the goodness of fit of the observed firing, as indicated in the activity histogram, to the expected firing. The null hypothesis, which dictated the expected firing, was that firing during the active component was the same as that during the inactive component, i.e., that unit activity was not modulated in time with the slow oscillation. Significance for the χ² test was set at p < 0.05.

To quantify the responses of dopaminergic neurons to the pinch stimulus, a baseline of spontaneous unit activity recorded for 30 s immediately preceding pinch onset was first established. This baseline was compared with the activity both during, and 15 s immediately after, the pinch. The mean firing rate and SD of activity during the baseline period were calculated. Firing rate was plotted against time (1 s bins), and the number of bins above and below 2 SDs from the baseline mean rate were calculated. A neuron was defined as significantly inhibited or excited by a pinch stimulus if 2 consecutive histogram bins within the 15 s stimulus period lay outside 2 SDs from the baseline mean rate. A neuron was defined as exhibiting a rebound excitation if 2 consecutive histogram bins lay above 2 SDs from the mean up to 5 s after stimulus offset. To quantify the responses of dopaminergic neurons to the electrical stimulus, artifacts were first removed from the spike train (Spike2, custom script), and then peri-stimulus time interval histograms (PSTHs) of unit activity were constructed from 0.5 s before to 1 s after the stimulus delivery (20 ms bins) (Coizet et al., 2006). A neuron was defined as exhibiting a significant response to the electrical stimulus if 2 consecutive PSTH bins lay ouside 2 SDs from the baseline mean rate (measured as the 500 ms of activity before the stimulus). To assess the influence of the electrical stimulus on cortical activity, peristimulus evoked potentials (as recorded in the ECoG) were calculated.

Statistical analysis.

The single-sample Kolmogorov–Smirnov test was used to judge whether data sets were normally distributed (p ≤ 0.05 to reject). Because some data sets were not normally distributed, we used nonparametric statistical testing throughout (SigmaStat, Systat Software Inc.). The Mann–Whitney U test was used for comparisons of unpaired data, whereas the Wilcoxon signed rank test was used to compare paired data sets. Significance for all statistical tests was set at p < 0.05 unless noted otherwise.