Rabbit anti-GFP antibody (A11122, 1:1000 dilution) was from Invitrogen, mouse anti-GFP antibody (Mab3580, 1:1000 dilution), mouse anti-GAD67 antibody (Mab5406, 1:1000 dilution), and goat anti-ChAT antibody (AB144P, 1:200 dilution) were from Millipore. Mouse anti-TPH2 antibody (T0678, 1:500 dilution) was from Sigma. Rabbit anti-parvalbumin antibody (PV-28, 1:5000 dilution) was from Swant.
Generation of cell-type specific ChR2-EYFP BAC transgenic mice
The BAC transgenic mice were generated as previously described37
. Briefly, BAC clones were obtained from Children's Hospital Oakland Research Institute. ChR2-EYFP which contains the H134R mutation was engineered into the ATG exon of the specific BAC clones through homologous recombination (see Supplementary Fig. 1 and Supplementary Table 1
for detailed information). Transgenic mice were generated by the injection of modified BAC DNA constructs into fertilized oocytes, using standard pronuclear injection techniques38
. Fertilized eggs were collected from matings between C57BL/6J and CBA F1 hybrids. Genotypes were determined by PCR from mouse tail DNA samples (see Supplementary Table 2
for details). PCR-positive animals were kept as founders to establish transgenic lines by mating to C57BL/6J mice. All research involving mice has been conducted according to the Institutional Animal Care and Use Committee guidelines at Duke University. All procedures were approved by the Institutional Animal Care and Use Committee at Duke University.
Section preparation and imaging
Mouse brain section and imaging were done as previously described37
. Briefly, mice were anesthetized by the inhalation of isoflurane and were intracardially perfused with Lactated Ringers solution, followed by the fixation of 4% paraformadehyde (PFA). Mouse brains were then post-fixed in 4% PFA overnight at 4 °C. 50 μm sagittal or coronal sections were cut using a vibratome, then mounted and imaged with a ZEISS AxioImager A1 microscope using a 5 × objective or with Nikon PCM2000 confocal microscope using a 20 × objective. Rabbit or mouse anti-GFP antibody was used in co-immunostaining with anti-GAD67, anti-ChAT, anti-TPH2 or anti-parvalbumin antibody. Briefly, sections were blocked with blocking buffer (5% normal goat serum, 2% BSA, 0.2% triton X-100 in PBS) (0.1 M Tris, pH 7.6 was used for anti-GAD67 and anti-ChAT antibodies) for 1 hour at room temperature, then incubated with primary antibody overnight at 4 °C. Following incubation with the primary antibody, sections were washed with either PBS or 0.1 M Tris three times every 20 minutes, followed by incubation with Alex 488 or Cy3 conjugated secondary antibodies for 2–4 hours at room temperature, and then washed with PBS or 0.1 M Tris. Sections were transferred onto slides, dried, mounted with 0.1% paraphenylinediamine in 90% glycerol-PBS (PPD), and imaged with a Nikon PCM2000 confocal microscope.
Equipment and settings
The montage images of the mouse brain sagittal or coronal sections in Supplementary Fig. 2a, 3, 6a, 7, 12a,14a
were taken with the ZEISS AxioImager A1 microscope equipped for fluorescence with a FITC filter and an AxioCamHR camera. The software used was AxioVision Rel 4.7 with the following main settings: 5×–1× tube, X-scaling: 1.275 micrometers per pixel, Y-scaling: 1.275 micrometers per pixel, frame-pixel-distance 6.45. The exposure time was 100–200 ms. The montage images were adjusted for brightness and contrast in Photoshop and converted from RGB to CMYK mode.
The confocal images in Supplementary Fig. 2b–d, 6b–d, 12b–d and 14b,c
were taken on a Nikon PCM2000 confocal microscope. The objective lens used was 20 ×,0.75 DIC M. The main settings were: large pinhole; first dichroic slider, RGB-red, 505-green; second dichroic slider, 565 for both red and green fluorescence images; ND filter selector, 10% transmission. The software used was Simple PCI 4.06 with the following settings: capture mode, 2× integration; fast scan mode, 1× fast scan; color, PCM2000 1024 color; PMT black level: 350; PMT gain, 1,000–1,500. The confocal TIFF files were equally adjusted for brightness and contrast in Photoshop and cropped to 500 × 500 pixels. Red color was converted to magenta and the image was converted to CMYK mode.
Acute brain slice preparation and electrophysiology
Acute brain slices were prepared from predominantly mature adult (2–8 months) but in some limited cases (e.g. brainstem slices from TPH2-ChR2-EYFP
mice) from juvenile (21–30 day old) mice according to our recently reported modified adult brain slice methodology39
but with a few additional improvements. The mice were deeply anesthetized by intra-peritoneal injection of Avertin (tribromoethanol) and then trans-cardially perfused with 25–30 mL of carbogenated protective artificial cerebrospinal fluid (aCSF) of the following composition: 92 mM N-methyl-D-glucamine (NMDG), 2.5 mM KCl, 1.25 mM NaH2
, 30 mM NaHCO3
, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mM Na-ascorbate, 3 mM Na-pyruvate, 0.5 mM CaCl2
O, and 10 mM MgSO4
O. The pH of the solution was titrated to 7.3–7.4 with concentrated HCl (which provides Cl−
counter-ions for NMDG). HEPES and thiourea plus ascorbate were included as critical components to reduce edema and oxidative damage during slicing, recovery, and extended slice incubation40, 41
. Mice were then decapitated and the brains were removed into the cutting solution for an additional one minute. The brains were then rapidly embedded in 2% low melt agarose and mounted for either coronal (cortex, hippocampus, olfactory bulb, brain stem, MHb) or sagittal (cerebellum) sectioning at 300 μM thickness on a VF200 model Compresstome (Precisionary Instruments) using a zirconium ceramic injector style blade (Specialty Blades). Acute brain slices containing the habenulo-peduncular pathway were prepared using a special slicing angle of 55–60° off the horizontal axis as shown in Supplementary Fig. 10a
and as described recently 42. For midbrain slices containing the DRN the slice thickness was reduced to 200 μM to improve visualization using IR-DIC optics.
Slices were initially recovered for ≤ 20–30 minutes at room temperature (23–25 °C) in carbogenated protective cutting aCSF. In later experiments we refined the procedure by performing this initial recovery at 32–34 °C for ≤ 10–15 min rather than at room temperature, which we found provided improved visualization. The exact duration of the recovery period is critical for obtaining the optimal balance between morphological and functional preservation of the brain slices, and the timing of this recovery step exhibits clear temperature dependence, as indicated above based on extensive empirical testing. Proper implementation of this brief protective recovery step using our NMDG-based aCSF formula greatly reduces initial neuronal swelling during re-warming and enables routine preparation of healthy acute brain slices for targeted whole-cell recordings from mature adult and aging mice. After this initial recovery period the slices were transferred into a holding chamber containing room temperature carbogenated aCSF of the following composition: 119 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 12.5 mM glucose, 2 mM CaCl2.4H2O, 2 mM MgSO4.7H2O. The aCSF was supplemented with 2 mM thiourea, 5 mM Na-ascorbate, and 3 mM Na-pyruvate to improve slice health and longevity, and slices were stored for 1–5 hours prior to transfer to the recording chamber for use. The osmolarity of all solutions was measured at 300–310 mOsm and the pH was maintained at ~7.3 after equilibration under constant carbogenation. In some experiments using juvenile mice the slices were cut using standard methods (standard aCSF instead of NMDG-based aCSF for cutting and recovery). Although the slice quality was deemed to be inferior based on morphological appearance and ease of identifying cells for targeted recordings, the experimental results obtained using this standard method with juvenile mice were indistinguishable and thus were pooled.
The slices were transferred one at a time to the recording chamber of a BX51WI microscope (Olympus) equipped with infra-red DIC optics (900 nm) and epifluorescence. The slices were constantly perfused with room temperature (22–25 °C) carbogenated recording aCSF at a rate of 4 mL per min. Whole-cell patch-clamp recordings were obtained from visually identified neurons using boroscilicate glass pipettes (King Precision Glass, Inc, Glass type 8250) pulled on a horizontal pipette puller (P-87, Sutter Instruments) to a resistance of 3–4 MΩ when filled with the internal solution containing 145 mM K-Gluconate, 10 mM HEPES, 1 mM EGTA, 2 mM Mg-ATP, 0.3 mM Na2-GTP, and 2 mM MgCl2. The pH was adjusted to 7.3 with KOH and the osmolarity was adjusted to 290–300 mOsm with sucrose. This internal composition was selected to more readily distinguish spontaneous and light-evoked inhibitory synaptic events as clear outward currents at a holding potential of −50 mV or −60 mV in voltage clamp mode. The reversal potential for Cl− was experimentally determined to be −75 mV. The theoretical liquid junction potential was calculated at −11 mV and was not corrected.
Neurons expressing ChR2 were identified by visualization of membrane-targeted EYFP fluorescence around the somata of genetically labeled neuron types. A holding command of −60 mV (except where indicated) was applied to the patched cell, and once the whole-cell mode was established the cell was allowed to stabilize for 2–5 minutes. Blue laser light (473 nm) was delivered through a 200 μm diameter optic fiber (Thor Labs) positioned at the slice surface over the recorded neuron. The other end of the optic fiber was coupled to a commutator and then to the laser (Crystal Laser, 100 mW) and the laser power was controlled by an analog dial. The illuminated area was estimated as a 400 μm by 600 μm oval (0.24 mm2) and this area was used to calculate the power density following direct measurement of the power output at the optic fiber tip using a power meter (Thor Labs). Pulsing of the laser was computer- controlled using custom “optogenetics” stimulation protocols designed in the pClamp 10.2 software. Light-induced inward currents were evoked with blue laser light delivered at 1–100 Hz frequencies (0.05–5 ms pulse duration), or by continuously applying blue laser light over one second or longer. Similar protocols were applied in the current clamp mode to monitor action potential firing of the recorded neurons in response to blue light stimulation from the resting membrane potential. For tonically active neurons (often seen for striatal cholinergic neurons, cerebellar Purkinje cells, and DRN 5-HT neurons) we assessed the ability to induce action potential firing either on top of basal firing rates or after silencing basal firing with the minimal necessary hyperpolarizing current injection. For experiments using VGAT-ChR2-EYFP bran slices to assess ChR2 mediated silencing in local circuits, a layer V cortical neuron was recorded in whole-cell current clamp mode and injected continuously with 100–200 pA of depolarizing current to induce tonic firing. Blue laser light was applied to nearby ChR2-expressing cortical interneurons to assess the extent of silencing or hyperpolarization in the layer V pyramidal neuron. Cell-attached recordings were performed in some experiments to monitor action potential firing without disruption of the cytosol. Cell-attached recordings were performed in voltage clamp mode with a zero holding current. Extracellular field recordings were obtained by placement of a glass recording electrode (1–2 MΩ tip) filled with aCSF into the slice and advanced to the depth that allowed discrimination of putative single units based on the response to blue laser light stimulation (in some cases multiple units were apparent).
A Multiclamp 700B amplifier (Molecular Devices Corporation) and Digidata 1440A were used to acquire electrophysiological signals using Clampex 10.2 software (Molecular Devices). The signals were sampled at 20 kHz and low-pass filtered at 2 kHz. The series-resistance was ≤ 25 MΩ and was not compensated. Access to the recorded cells was continuously monitored, and only recordings with stable series resistance were included for analysis. All data analysis was performed in Clampfit 10.2 (Molecular Devices). Values are expressed as mean ± s.e.m. Data were tested for significance using a non-paired student t-test.
Additional acute brain slice electrophysiological recordings were conducted in the Luo laboratory using previously published procedures with only minor modification42
In vivo electrophysiology
Mice were deeply anaesthetized with isoflurane and fitted with light-weight headstages for chronic recording with multiple tetrodes in the mouse striatum43, 44
(Specialty Machining, Wayland, MA). A small opening in the skull and corresponding incision of the dura mater were made to allow entry of seven tetrodes (each made up of twisted 10 μm Ni-Cr wires) and one fiber optic cable (100 to125 μm, attached to a 1.25 mm ferrule) into the brain parenchyma. Both the tetrodes and the fiber optic cable were held individually in a series of nested polyimide tubes. The tubes, attached to microdrives, were held parallel to screws that were used to advance independently each tetrode and the fiber. After a week of post-surgical recovery, the tetrodes and the fiber optic cable were lowered, day by day, through the full dorsoventral extent of the anterior striatum (anterior-posterior + 1.5 mm, medial-lateral +1.2 mm, dorsal-ventral + 1.9 to 3.3 mm) and recordings and stimulation sites were arranged along these trajectories. The tip of the fiber optic cable was kept 0.5 mm above the tips of the tetrodes.
During neuronal recording sessions, a 16 channel preamplifier (1.7 g) connected to lightweight wires (Neuralynx) was attached to the heastage. Neuronal activity recorded on each tetrode channel was sent through the preamplifier with unity gain to two 8-channel programmable amplifiers (gain: 2,000–10,000; filter: 0.6–6 kHz) and then to a Cheetah data-acquisition system (Neuralynx). The spike waveform of each spike was digitized at 32 kHz and stored with a microsecond-precision time stamp. The fiber optic cable was coupled to a 473 nm laser (100 mW, Shanghai Dream Lasers) by connecting its ferrule to the ferrule at the end of the fiber optic cable attached to the laser. A Zirconia sleeve (Doric Lenses) was used to couple the two ferrules. The laser was controlled by a TTL circuit connecting it to a computer running Delphi software. TTL pulses were concurrently sent to the Cheetah data-acquisition system where they received a time stamp and were stored. Laser power was measured before each session through the fiber optic cable that connected the laser to the head stage fiber. The mouse was placed in a circular container (20 cm diameter) for the recording sessions and was allowed to move freely. Typically, each session consisted of 16 trials (40 s each) during which laser-on and laser-off trials were interleaved and separated by a one minute gap. The tetrodes and the fiber optic cable were moved between sessions to maximize the number of unique neurons recorded.
Unit activity containing the spikes of multiple neurons was sorted off-line into putative single units (“clusters”) according to multiple spike parameters (e.g., peak height, valley depth, peak time) on the four channels of each tetrode (DataWave Technologies). The accuracy of spike-sorting and the quality of the single units were then evaluated by (a) t-test for spike variability, (b) spike waveform overlays to confirm uniform waveforms for a given unit and different waveforms across units, and (c) autocorrelograms to detect the presence of an absolute refractory period. Based on these tests, clusters containing noise (artifacts and the activity of other units) were excluded from further analyses. All accepted units were classified as putative medium-spiny projection neurons (MSNs) fast-firing (FF) interneurons, or tonically active interneurons (TANs) based on properties of discharge patterns identified by calculating interspike intervals, autocorrelograms, and firing rates45
. MSN neurons that fired fewer than 150 spikes in a session were not included in any analysis. Histograms are displayed in bins of variable sizes time-locked to the first laser pulse for laser-on trials and the beginning of recording for laser-off trials. The order of trials was interleaved (on-off-on-off…) and the trials were separated by a 1 min gap. A paired t-test was performed to determine statistical significance of the firing rate change between laser-on and laser-off trials.