Subjects and surgery
For behavior and in vivo electrophysiology experiments we used 5 adult (30 g; 3–6 months old) male mice derived from the C57/Black6 strain and bred from the in house breeding colony. All animals were handled in accordance with institutional guidelines. For the optogenetics experiments we used adult mice (10–30 weeks old) expressing Chr2 under the thymus cell antigen 1 (Thy1) promoter (Line 18; Jackson Labs, Bowdoin, ME). All animals were handled in accordance with institutional guidelines. Mice were trained to learn a classical trace-conditioning task with a tone (7–10 kHz, 500 ms duration) as the CS and sweetened water as a reward (2000–2500 ms delay).
Mice were initially housed in a temperature-and humidity-controlled room maintained on a reversed 12 h light/dark cycle. For behavioral and in vivo physiology experiments mice were housed individually, for in vitro experiments mice were group housed. Following one week of recovery from surgery, the water consumption of the mice was limited to 1 mL per day for a week. Mice under went daily health checks, and water restriction was eased if mice fell below 75% of their body weight at the beginning of deprivation. Mice were then familiarized with the training and recording box, which was located inside a sound attenuating enclosure. Mice were trained to obtain fluid from a recessed spout in the wall of the behavioral box. Small volumes (≈0.01 ml) of water sweetened with saccharin (0.005M solution) were delivered to the spout via a computer control using custom software and electronics with a nominal time resolution of 1 kHz (to be published elsewhere). Entries to the reward port, primarily as licks, were detected using an infrared beam break positioned just in front of the spout opening.
Mice were trained to learn a classical trace conditioning task with a tone (7–10 kHz, 500 ms duration) as conditioned stimulus (CS) following the water reward with a delay of 2000–2500 ms. The intertrial intervals were chosen pseudo-randomly from a uniform distribution over the interval 20 to 40 seconds. Extinction training then involved exposing the animal to equivalent pseudo-randomly delivered repetitions of the previously conditioned CS, without solenoid activation. Each conditioning session was carried out for 40–100 trials, while the following extinction session was carried out for 80–120 trials. In a subset of behavioral sessions a distinct tone, unpaired with reward, was also delivered (e.g.
Supplementary Fig. 9
In vivo electrophysiology
Recordings were performed using 16 or 32-microwire arrays (CD Neural Technologies, Durham, NC). Electrode arrays were stereotaxically implanted under anaesthesia (isoflurane; 1.5%–2.5% in O2). The electrodes were targeted to the substantia nigra
and the ventral tegmental area of the ventral midbrain (3.0–4.5mm posterior to bregma, 0.5–2.0 mm lateral to midline and 3.5 mm below the surface of skull). Electrode arrays were mounted directly to a custom-designed microdrive and connected to the recording systems via a flexible wire coupling and connector. This configuration allowed us to advance the electrode arrays between training and recording sessions. Animals were allowed at least 1 week for recovery from surgery and initial advancement of electrodes. Approximate electrode tracks are shown in Supplemental Figure 1
. Recordings were generally initiated at multiple electrode advancement steps. We noted an enhanced probability of detecting extinction cells late in our recordings either simultaneously with dopamine units () or after dopamine units had been recorded (i.e.
at greater depth relative to the surface). However, the uncertainty present in the exact electrode position due to approximations in the drive displacement and tissue compaction made it unreasonable to report precise locations for individual recording sessions in Supplementary Figure 1
The movable electrodes were advanced in 30–60 µm increments daily to search for independent units. The voltage signals from electrodes were amplified and filtered with a sequential analog (0.1–7.6KHz bandpass) and digital filter (750–7.6KHz bandpass). Channels with detectable activity were digitized at 30 kilosamples/second, thresholded on-line, and voltage segments (30–50 samples) recorded to disk using the Cerebus Data Acquisition System (Blackrock Microsystems, Salt Lake City, UT). Spikes were re-isolated offline on the basis of wave-shape, using Plexon Offline Sorter (Plexon Inc, USA). Putative DA cells were classified according to the following criteria: 1) low firing rate (< 10 Hz), 2) relatively broad action potential (> 1.2 ms), 3) phasic CS responses with onset latencies of 40–60ms, and 4) profound (>50%) inhibition by the D2-receptor agonist quinpirole (400 µg/kg, s.c., most putative DA cells were tested, but not all).
For head-fixed recording experiments mice were implanted with a custom designed head-restraint several days prior to recording (to be described elsewhere). Mice were then habituated to the head-restraint system. Following recovery and habituation a craniotomy was made under isolflurane anesthesia as described above and electrode arrays were maintained in position by a micromanipulator (Sutter Instruments). A 200 micron core multimode fiber (ThorLabs) was affixed near the central recording wires of a 32 channel array. The entire array was slowly lowered in to the midbrain. After >1 hour of recovery recording data was obtained from alert, but quietly resting mice.
Analysis of physiology and behavioral data
Analysis was performed using custom written routines in Matlab R2011a (Mathworks, Natick, MA) and Igor Pro (Wavemetrics, Eugene, OR). Briefly, z scores were calculated as the mean subtracted PSTH divided by the standard deviation of the baseline period (2 seconds prior to the stimulus). Responses were calculated from more than 40 trials of acquisition and extinction. A phasic response was specified to occur in the first 150 ms after stimulus onset with a width defined as the first point 2 s.d. above baseline prior to and after the peak response. Mean responses were quantified as the integral of the response (z score, or rate) within the peak window, trialwise responses were integrals or spike counts within the same window. Significant differences between extinction and acquisition were defined by comparing equivalent numbers of trials during stable behavior. Units in which both the ranksum and Kruskal-Wallis test were significant (p<0.05) were labeled significant. P values reported for all pairwise comparisons of means are taken from two-tailed t-tests. Significant correlations were assessed using a t transformation of the data and evaluating the Pearson correlation.
To confirm the position of recording sites, mice were killed by anesthetic overdose (isoflurane, >3%), perfused with phosphate-buffered saline (PBS) then paraformaldehyde (4% w/vol. in PBS). Brains were post-fixed for 24 hours and then rinsed in saline. Whole brains were then sectioned (50–100 µm thickness) using a vibrating microtome (VT–1200; Leica Microsystems, Germany). Electrode tracks were mapped onto standard atlas sections by visual inspection using counter-staining or autofluorescence for registration.
In vitro electrophysiology
Briefly, mice were deeply anaesthetized under isoflurane, decapitated and the brains were dissected out into ice-cold modified artificial cerebral spinal fluid (aCSF) (in mM: 52.5 NaCl, 100 Sucrose, 26 NaHCO3, 25 Glucose, 2.5 KCl, 1.25 NaH2PO4, 1 CaCl2, 5 MgCl2 and in uM: 100 Kynurenic Acid) that had been saturated with 95%O2/5%CO2. 300 µM thick coronal slices were cut (Leica VT1200S; Leica Microsystems, Germany), transferred to a holding chamber and incubated at 35°C for 30 minutes in modified aCSF (in mM: 119 NaCl, 25 NaHCO3, 28 Glucose, 2.5 KCl, 1.25 NaH2PO4, 1.4 CaCl2, 1 MgCl2, 3 Na Pyruvate and in uM: 400 Ascorbate and 100 Kynurenic Acid, saturated with 95%O2/5%CO2) and then stored at room temperature.
For recordings, slices were transferred to a recordings chamber perfused with modified aCSF (in mM: 119 NaCl, 25 NaHCO3, 18 Glucose, 2.5 KCl, 1.25 NaH2PO4, 1.4 CaCl2, 1 MgCl2, 3 Na Pyruvate and in µM: 400 Ascorbate and saturated with 95%O2/5%CO2) maintained at 32–34°C, at a flow rate of 2–3mL per minute. Patch pipettes (resistance 5–8 MΩ) were pulled on a laser micropipette puller (Model P-2000, Sutter Instrument Co., Sunnyvale, CA) and filled with one of the following intracellular solutions: Current-clamp recordings of spike activity used a KGluconate based intracellular solution (in mM: 137.5 KGluconate, 2.5 KCl, 10 HEPES, 4 NaCl, 0.3 GTP, 4 ATP, 10 phosphocreatine, pH 7.5). Voltage-clamp recordings for IPSC measurements used a CeMeSO4 based intracellular solution (in mM: 114 CeMeSO4, 4 NaCl, 10 HEPES, 5 QX314, 0.3 GTP, 4 ATP, 10 phosphocreatine, pH 7.5). Alexa Fluor 488 or Alexa Fluor 568 was commonly added to intracellular solution to aid cell visualization and post-hoc reconstruction. In some experiments the following were added as indicated in the text: 10µM CNQX or 5µM NBQX, 50µM D-AP5, 10µM GABAzine were diluted from stock in the aCSF. All drugs were obtained from Tocris Biosciences, Inc. Intracellular recordings were made using a MultiClamp700B amplifier (Molecular Devices, Sunnyvale, CA) interfaced to a computer using a analog to digital converter (PCI-6259; National Instruments, Austin, TX) controlled by custom written scripts (available at dudmanlab.org) in Igor Pro (Wavemetrics, Eugene, OR). Photostimulation was carried out using a dual scan head raster scanning confocal microscope and control software developed by Prairie Systems, (Middleton, WI) and incorporated into a BX51 upright microscope (Olympus America, Inc., Center Valley, PA).
Viral overexpression of ChR2
An adeno-associated virus (kindly provided by the Sternson laboratory at Janelia Farm Research Campus) with a cre-dependent ChR2 transgene was injected into the SN of mice in which cre-recombinase was expressed under the control of the glutamatic acid decarboxylase 2 gene in a fashion similar to that previously described47
. Briefly, under deep anaesthesia a small craniotomy was made over the SN (−3 mm AP, 1 mm ML, −4.2 mm DV). A glass pipette was used to pressure inject small volumes of virus (20–50 nL per injection site). Animals were allowed to recover for at least 2 weeks following infection and before in vitro
brain slices were prepared as described above.
Optical stimulation and imaging
The optics were designed to minimize the spread of the laser in the x,y dimensions of the focal plane while accentuating the focus in z by underfilling the back aperature of the objective. Stimulation intensity was controlled by pulse duration (0.2 –1 ms). Stimulation typically consisted of 9×9 maps of stimulation sites with independent stimuli being delivered in a pseudo-random (non-neighbor) sequence at an interstimulus interval of >=150 ms). Stimulation strength was modulated by gating the laser at maximal power (473 nm; AixiZ or 488 nm; BlueSky Research) with varying durations using timing signals from an external pulse controller, PrairieView software, and the internal power modulation circuitry of the laser or an external Pockels cell (Conoptics, Danbury, CT) with indistinguishable results.
Wide-field activation of ChR2 was accomplished using blue LED (470 nm; ThorLabs, Newton, NJ) transmitted through the fluorescence light path of the BX51 microscope. LED intensity and timing were controlled through a TTL-triggered variable current source (ThorLabs, Newton, NJ).
The simple model described here was inspired by the canonical theta neuron model from Gutkin and Ermentrout48
. The DAm
was implemented in Matlab R2011a (Mathworks, Natick, MA) with minor modification from previous models. We modified the model to simulate a neuron with an intrinsic bias towards tonic activity that could be perturbed by input stimuli. The phase of the oscillator was solved using numerical integration of a differential equation for phase:
- dθ/dt = b(1-cosθ) + K(1+cosθ)
A ‘spike’ was determined as the phase reset at θ=pi. The intrinsic bias b
was introduced to drive a tonically active oscillator independent of stimuli. A large parameter space of the model was examined (Supplementary Fig. 8
) by altering the magnitude of b
(minimum: 0.0005 a.u., maximum: 0.02 a.u.), the amplitude (±0.005 a.u., ±0.5 a.u.) and decay time constant (10 ms, 200 ms) of the exponentially decaying of Istim
. For each parameter combination 100 iterations were run. PSTHs were calculated with 1 ms resolution and smoothed with a gaussian kernel (σ = 10 ms). The full-width half maximum of inhibition and pause duration were calculated as in analysis of in vitro