Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Summary

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Abstract

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

Introduction

Neuronal activity in mammals is coordinated by network oscillations, which assist information transfer within and between brain regions^1,2,3,4. Brain rhythms include oscillations ranging from very slow (< 0.8 Hz) up to ultrafast (> 200 Hz) frequencies. A large body of evidence supports involvement of network oscillations in diverse brain functions, including cognition^5,6,7,8,9,10, innate behaviors^11,12 as well as neuropsychiatric disorders such as Parkinson's disease and epilepsy^13,14,15. Selective and temporally precise methods for experimental manipulation of network oscillations are therefore essential for the development of physiologically plausible models of synchronization and for establishing causal links with behavior.

Network synchronization is mediated by diverse biological substrates and processes, ranging from molecular identity of ion channels and their kinetics to neuromodulation of excitability and network connectivity. The biological design of rhythm generators¹⁶ has been revealed for many brain rhythms, distinct aspects of which (e.g., frequency, amplitude) are often brought about by dynamics of distinct cell types and networks. For instance, inhibitory interneurons targeting the somata of principal cells are the most important players across frequency bands and brain regions^17,18, including theta^19,20, gamma^20,21, and ripple (140-200 Hz)²² oscillations. In turn, phase synchronization of distant cells is ensured by robust feed-forward signaling of pyramidal cells, which resets the firing of interneurons. A crucial parameter of oscillations, the size of the synchronized neuronal population, is closely related to the measured LFP oscillation's amplitude and, at least for fast oscillations, depends on the excitatory drive onto interneurons². In contrast, slower oscillations, like delta and theta rhythms, are generated by long-range reentrant loops, formed by cortico-thalamic^23,24 and hippocampal-medial septal projections^25,26,27, respectively. Oscillations in such circuits are brought about by interactions of signal propagation delays, excitable responses, and their frequency preference in participating cells^{28,29,30,31,32}. Inhibitory projections from GABAergic parvalbumin (PV)-positive cells of the medial septum (MS) to interneurons in the hippocampus^25,33, parahippocampal regions and entorhinal cortex²⁶ are essential for the generation of theta oscillations in the medial temporal lobe. Thus, physiological mechanisms of network oscillations and neuronal synchronization can be manipulated using optogenetics with a real-time precision.

Cell type-specific optogenetic manipulations have been applied for studies of the hippocampal and cortical oscillations in vitro^{34,35,36,37,38} and in vivo^{30,39,40,41,42,43,44,45}, including functional investigations of gamma^{5,12,36,46,47,48,49,50,51,52} and ripple oscillations^40,53,54 and sleep spindles^55,56. Recently we expressed a Cre-dependent ChR2 virus in the MS, a key region for the generation of the hippocampal theta rhythm, of PV-Cre mice. Using this preparation, features of the hippocampal theta oscillations (frequency and temporal stability) were controlled by optogenetic stimulation of inhibitory projections of the MS in the hippocampus¹¹. Furthermore, theta-frequency optogenetic stimulation of inhibitory septo-hippocampal projections evoked theta rhythm during awake immobility. The optogenetically entrained theta rhythm displayed properties of spontaneous theta oscillations in the mouse at LFP and neuronal activity levels.

Key features of this protocol include: (1) utilization of an inhibitory pathway that is physiologically critical for spontaneous theta oscillations while avoiding unspecific effects on hippocampal excitability; (2) axonal, i.e., projection-specific stimulation to minimize a direct influence on non-hippocampal MS efferents; (3) local theta-rhythmic light stimulation, ensuring a minimal direct interference with theta-rhythmic septo-hippocampal dynamics and a global bilateral entrainment of theta oscillations; (4) parametric control of theta oscillations frequency and regularity; and (5) quantification of entrainment fidelity with high temporal resolution using LFP to enable quantitative causality analysis in behaving animals. Since this preparation essentially capitalizes on a well-known role of the septo-hippocampal disinhibition in theta generation^25,30, it enables robust control over several parameters of theta oscillations in behaving mice. Studies where other less investigated pathways and cell types of the septo-hippocampal circuitry were manipulated^{38,39,47,49,50,51,52,53,54,55,56,57,58} reveal further mechanisms of the theta rhythm.

Protocol

PV-Cre knock-in male mice⁵⁹, 10-25 weeks old, were used. Mice were housed under standard conditions in the animal facility and kept on a 12 h light/dark cycle. All procedures were performed in accordance with national and international guidelines, and were approved by the local health authorities (Landesamt für Natur, Umwelt und Verbraucherschutz, Nordrhein-Westfalen).

1. Viral Injection

1. During the whole procedure, follow biological safety guidelines⁶⁰. Wear a lab coat, a surgical mask, a hairnet, and two pairs of gloves.
2. Cut tubing with a sterile scalpel or scissors to approximately 1 m length, insert it in the syringe holder block of the pump, and fix it.
   1. Fill the tubing entirely with silicon oil by using the syringe.
   2. Check whether air bubbles appear in the tubing. If air bubbles are observed, refill the tubing again with oil.
   3. Fix the plunger to the pusher block.
   4. Slowly advance the pusher towards the syringe holder block until the tip of the plunger touches the tubing.
   5. Insert the tip of the plunger into the tubing and automatically move the pusher block forward at a slow speed by selecting "Infuse" in the command window and a low infusion rate (e.g., 500 nL/min) until it reaches the syringe holder block.
3. Prepare the injection needle (34G).
   1. Remove the needle hub with a clear cut using a precision drill/grinder connected to a cutting disk. If necessary, use a needle to remove metal residues from the freshly cut surface.
   2. Thread capillary tubing (3-4 cm length) through the needle.
   3. Glue the needle to the tubing with super glue.
   4. Connect the injection needle to the end of the tubing, which connects to the microsyringe pump.
4. Attach the needle to the stereotaxic holder.
5. Withdraw air until an air bubble in the transparent tube above the needle is visible.
6. Anesthetize the mouse with 1.5-3% isoflurane in oxygen. Wait approximately 2-3 min, then confirm that the mouse is fully anesthetized by assessing its response to a toe pinch. Throughout the surgery monitor the animal's breathing and adjust isoflurane concentration if required.
7. Place the mouse in the stereotactic frame using non-traumatic ear holders.
8. Protect the eyes of the mouse with a lipid gel.
9. Inject 0.1 mL lidocaine intradermally under the head skin using a 0.01-1 mL syringe and a 26G injection cannula.
10. Shave and disinfect the mouse's head using ethanol solution. Then alternate three times 2 % chlorhexidine with ethanol to  disinfect the surgical site.
11. Perform a midline incision using fine and sharp scissors going from between the back of the ears to the level of the eyes so that the bregma and lambda are visible.
12. Clean the skull by applying approximately 50 µL of NaCl using a syringe and dry with a paper tissue and an air puff.
13. Position the mouse head by adjusting the dorso-ventral level of the nose clamp so that the bregma and lambda are on the same dorso-ventral level (± 0.3 mm).
14. Drill a hole above the MS (AP 0.98 and L 0.5 mm in reference to the bregma).
15. At this point, defrost an aliquot of the virus (should contain at least 2 µL of AAV2/1.CAGGS.flex.ChR2.tdTomato.WPRESV40) for approximately 5 min at room temperature (RT).
16. Centrifuge the aliquot at approximately 4,000 x g at RT for 1 min.
17. Pipette the 2 µL of the virus onto a piece of Parafilm; use the previously protected side.
18. Submerge the tip of the injection needle in the liquid and carefully withdraw at about 500 nL/min while observing the level of the liquid. To prevent air suctioning, stop withdrawal before the virus is entirely taken up. Adjust the withdraw rate according to the solution viscosity; a faster rate can facilitate withdrawal of a virus with higher viscosity.
19. Clean the needle with a paper tissue.
20. Check that the virus is contained in the tube and the virus and oil are separated by an air bubble. Mark the level of the virus on the tube in order to control whether virus is successfully infused during the injection.
21. Position the needle above the craniotomy and slowly insert it into the brain at the first injection point (AP 0.98, L 0.5, V -5.2, 5.5° lateral).
22. Inject 450 nL of the virus at a rate of 100-150 nL/min. Wait for 10 min. Carefully move the needle up 0.1 mm and wait another 5 min.
    1. Move the needle to the second injection point (AP 0.98, L 0.5, V -4.6) and inject another 450 nL at 100-150 nL/min.
    2. Wait again for 10 min before moving the needle up 0.1 mm. Wait another 5 min before removing the needle.
23. Suture the incision with Silk Black Braided suture using square knots. Warm the animal with a red lamp to speed up the recovery. Administer antibiotics (0.3 mL Erycinum (1:4 in sterile NaCl)) and carprofen i.p. daily 2-3 days after surgery.
24. Cut the tube above the mark that indicated the level of the virus. The tube can be used for further injections. Prepare a fresh injection needle before each injection.
    NOTE: This surgery takes about 1-1.5 h. The animal wakes up typically within 5 min after surgery. Wait a minimum time of 6 weeks for sufficient axonal expression but not longer than 5 months before performing stereotactic implantations, as expression levels begin to decrease approximately 6 months after the virus injection.

2. Preparation of Optic Fibers (Figure 1A)

1. Use multimode optic fiber (105 µm core, glass clad with silica core, 0.22 NA). Strip 125 µm cladding of the fiber core using a micro-stripper while the fiber is still attached to the fiber spool.
2. Cut the fiber to a length of approximately 2-3 cm using a diamond knife.
3. Insert the fiber in a zirconia ceramic stick ferrule (ID: 126 µm). Approximately 0.5-1 mm of the optic fiber should protrude from the convex side of the ferrule.
4. Using a needle, apply one drop of epoxy glue to both ends of the ferrule but not onto the sides of the ferrule. Alternatively, use super glue.
5. Allow the glue to dry for at least 30 min.
6. Polish the convex side of the ferrule using diamond lapping fiber polishing film (3 µm grits).
7. Test the fidelity of light transfer using an optical power meter.
   1. Set the wavelength on the power meter to the same wavelength as the laser being used.
   2. Position the patch cord with the tip facing the center of the sensor. Power on the laser and read the light output from the power meter. Record the value.
   3. Connect the optic fiber to the patch cord via a mating sleeve and position it with the tip of the fiber facing the center of the sensor. Power on the laser and read the light output from the power meter. Record the value.
   4. Calculate the transmission rate: divide the second value by the first value. If the transmission rate is below 0.5, discard the fiber, otherwise use it for implantation.
   5. Test the transmission rate for each fiber before implantation.
   6. For later experiments, adjust the light intensity output of the laser to the transmission rate of the optic fiber: set the light output from the patch cord tip to 5-15 divided by the transmission rate to achieve a final light output from the fiber tip of 5-15 mW.
      NOTE: See also ref. ⁶¹ for the preparation of optic fibers.

3. Preparation of Tungsten Wire Arrays for LFP Recordings (Figure 1B)

1. Glue several (for example 6) 100 µm silica tube guides in parallel to the sticky side of a piece of tape. Cut one piece, approximately 4-6 mm, for one wire array assembly.
2. Thread formvar-insulated 45 µm tungsten wires through the guide tubes using forceps.
3. Strip six enamel-insulated fine copper bonding wires (approximately 5 mm long) and a grounding wire (approximately 2-3 cm long) by using a scalpel to scrape away the insulation on both ends. Solder them to the nanoconnector pins.
4. Connect each bonding wire to one tungsten wire using one drop of silver conductive paint, respectively. Let dry for at least 30 min.
5. Apply a minimum amount of cement to cover the wires. Do not apply cement on the tungsten wires, which will be inserted in the brain tissue or on the upper part of the nanoconnector. Let the cement dry for at least 30 min.
6. Perform an angular cut (5-20°) of the tungsten wires using blunt stainless-steel scissors to enable reliable implantation of wires below or above the zone ventrally adjacent to the stratum pyramidale, where theta amplitude is too low for the estimation of the entrainment fidelity.
7. Deinsulate the tip (approximately 2 mm) of the ground wire by using a scalpel to scrape away the insulation. Treat it with flux and presolder.
8. Check potential cross-talks between electrodes using a digital multimeter. To do so, connect the connector pins to the multimeter, which must be set to the resistance measurement mode. Check pairwise combinations of channels; a reading on the multimeter below 5 MΩ indicates significant cross-talk.
9. Check the impedance of each wire electrode in saline using an impedance meter. Typical impedance values are below 100 kΩ.
10. To facilitate implantation, glue one optic fiber to the wire array so that the tip of the fiber is at the level of the shortest wire and the fiber tip is in close proximity, but not touching the tungsten wires. Keep the angle of the fiber as small as possible in order to prevent tissue damage during implantation.
    NOTE: See also ref. ⁶² for fabrication of tungsten wire arrays.

4. Stereotaxic Implantations

1. Perform preparations as described in steps 1.6-1.13.
2. Remove the connective tissue from the top of the skull and push down neck muscles thoroughly by approximately 2 mm to prevent muscle artifacts during the recording.
3. Clean the skull using a cotton-tip applicator and saline, and drill 4 holes (2 in the front and 2 above the cerebellum, 0.8 mm diameter) to place bone stainless-steel screws (00-96x1/16) for ground and stabilization of the implant (Figure 1D). Position the ground screw, connected to one copper wire (approximately 2-3 cm length) above the cerebellum.
4. Cover the ground-screw completely with cement to prevent muscle artifacts during the electrophysiological recordings. Build a cement ring connecting all screws (Figure 1E).
5. Perform a craniotomy above the implantation side (Hippocampus, AP -1.94, L 1.4, V 1.4 in reference to the bregma). Apply approximately 5 µL of sterile NaCl on the surface of the brain tissue.
6. Slowly lower the wire array using stereotaxis in the craniotomy. For unitary recordings, implant a silicone probe instead of a wire array⁶³; in order to prevent optoelectric light artifacts, implant the optic fiber separately in the hippocampus with the fiber tip not directly facing the probe (Figure 1C - I). For investigation of the coordination of the entrainment between hemispheres, implant an additional optic fiber in the contralateral hippocampal CA1 area.
7. Apply approximately 5 µL of warm liquid wax/paraffin oil, preheated at 70 °C, with a syringe above the implantation site to protect brain tissue.
8. Apply cement around the wire array and cover the skull with cement.
9. Apply one drop of flux to the presoldered ground/reference wire and the presoldered wire connected to the ground screw using for instance a needle, and fuse the wires using a soldering machine.
10. Cover the entire ground wire with cement.
11. Administer 0.3 mL Erycinum (1:4 in sterile NaCl) and carprofen (5mg/mL) i.p. after surgery and for at least the two days following. The mouse typically wakes up within 15 min following surgery. Warm the animal with a red lamp to speed up recovery.
12. Monitor the weight of the mouse daily for the first week following the surgery or until the weight is stable. Weight loss should not exceed 10% of the mouse weight recorded before surgery. To accelerate stabilization of weight, supply the mouse with wet food and condensed milk during the first days following the surgery.
13. To record hippocampal cellular activity during entrainment, implant a silicone probe in the hippocampus (AP -1.94, L 1.4, V 1, with subsequent lowering) as described in ref. ⁶² (Figure 1C - I). Implant the optic fiber in the hippocampus at AP -3, L 1.4, V 1.6, 39° caudal-rostral. Implant an additional optic fiber in the MS (AP +0.98, L 1, V 3.9, 15° lateral) if stimulation of cell somata is desired.

5. Optogenetic Stimulation and Electrophysiological Data Acquisition

1. Habituate the mouse to the recording setup (e.g., 15 min sessions, 1-2 sessions per day for 3 days). Examine the animal's behavior before starting with the first experiments. If the mouse is moving in the chamber, exploring the environment, sniffing, performing rearings, etc., start the first experimental session.
2. Place the mouse in a familiar chamber in the absence of other animals in the same room.
3. Attach an LED to the head-stage using adhesive tape to track the animal's position. Make sure that the LED light is captured by the camera throughout the period that the animal is exploring the recording chamber before starting the experiments. Record in the dark in order to track the LED light. Position a camera above the recording chamber.
4. Check the light output from the patch cord. Estimate the light output from the fiber tip after connection depending on transmission rate of the fiber implanted. Ensure that the light output from the tip of the fiber is between 5-15 mW, to enable reliable entrainment.
5. Connect the headstage preamplifier to the chronically implanted connector. Connect the fiberoptic patch cord to the chronically implanted hippocampal fiber for optogenetic stimulation experiments. In control light stimulation experiments, connect the optic fiber to a dummy ferrule connected to the headset.
6. Place the mouse in the recording chamber.
7. Open the software to control the stimulus generator to generate the stimulation protocol.
8. Select the channel that controls the 473 nm DPSS laser. In the first row enter 3,000 mV (1st column), time 30 ms (2nd column), value 0 mV (3rd column), time 112 ms (4th column), row repeat 840, and group repeat 1, to generate a protocol for 2 min of 7 Hz stimulation with 30 ms long pulses. Adjust the time duration in the 4th column and number of row repetitions if the stimulation at another frequency or a different duration is required. In the second row select 0 mW (1st column), time 500 h (2nd column), row repeat 1, and group repeat 1, to ensure that the laser is being switched off after the stimulation protocol is terminated.
9. Click "File > Save as" and save the file with a desired name.
10. Assure that the stimulator TTL output triggering the laser is connected to the Digital Lynx analog input board to synchronize the acquisition of electrophysiological and optogenetic data.
11. Alternatively, to parametrically regulate the variability of theta oscillation frequency, apply trains of light pulses at varying inter-pulse intervals, with periods following a Gaussian distribution. Modify the dispersion of inter-pulse intervals for different protocols, e.g., from step 3.2 to 15.1 ms². Apply these protocols to generate theta epochs with different variability of the theta frequency (Figure 6).
12. Open the software of the recording system. Click on "ACQ" to acquire and "REC" to record. Wait before initiation of the light stimulation to record the baseline behavior (e.g., 2 min to retrieve baseline speed or 30 min to extract the baseline place fields).
13. Open the software to control the stimulus generator. Click "File > Open" and select the protocol file of choice. Click "Download and Start" to initiate the light stimulation.
    NOTE: The experiment can be surveyed, and the stimulation started via remote control; this excludes the influence of the presence of the experimenter on the animal's behavior. Depending on the goal of the study, stimulation can be initiated and terminated during specific behaviors.

6. A Combined Approach for Optogenetic Entrainment and Projection-specific Inhibition of the Hippocampal Output

1. Express ChR2 in MS GABAergic cells of PV-Cre mice, as described in section 1.
2. In addition, inject a total of 2.4 µL of CamKIIα dependent halorhodopsin (eNpHR3.0, AAV2/1.CamKIIa.eNpHR3.0-EYFP.WPRE.hGH) in both dorsal hippocampal hemispheres (AP -1.7; L ± 1.05; V -2.05 and -1.4 mm; AP -1.7; L ± 1.7; V -2.05 and -1.55 mm; AP -2.3; L ± 1.5; V -2.2 and -1.3 mm; AP -2.3; L ± 2.2; V -1.65 and -2.45 mm).
3. Allow 6 weeks of expression time.
4. Implant a tungsten wire array with optic fiber in the hippocampal CA1 region, as described in section 4. Additionally, implant bilaterally optic fibers in the lateral septum (LS, AP 0.1, L 0.25, V -2.25 mm, and AP 0.5, L -0.3, V -2.7 mm).
5. Perform optogenetic theta entrainment experiments as described in steps 5.4-5.5.
   1. For simultaneous inhibition of the hippocampal output to the LS, generate a protocol stimulus generation: e.g., trigger the onset of the output channel connected to the 593 nm DPSS laser 15 s with a continuous pulse lasting in total 45 s, before triggering the pulses output to the 473 nm DPSS laser.
   2. Connect both fibers in the LS via patch cords using a multimode fiber optic coupler to a 593 nm DPSS laser.
   3. Start the recording and download and trigger the protocol to control the stimulus generation.

7. Data Processing

1. Convert the electrophysiological signals and position the tracking data with Neurophysiological Data (ND) Manager to .dat and .pos formats, respectively⁶⁴.
2. Obtain the LFP by low-pass filtering and down-sampling of the wide-band signal to 1,250 Hz using ND Manager⁶⁶.
3. In each recording, select the channel with the maximal amplitude of theta oscillations (using Neuroscope)¹⁹.
4. Detect the timestamps of the laser pulses and of the stimulation epochs with a threshold detecting algorithm (using MATLAB function findpeaks.m or similar)¹¹.
5. To import the .dat file in a multi-channel data analysis software, click "File > Import", select "binary files" as the data type, and select the .dat file. In the configuration dialog, enter the correct number of channels and a sampling rate of 1,250 Hz, click "ok", and save as .smr file. Plot power spectra by selecting "Analysis > New Result View > PowerSpectrum". In the settings, select the channel with the highest theta amplitude and 16,384 FFT size, click "New", define as "Start time" the beginning of the stimulation epoch and as "End time" the end of the stimulation epoch, and click "Process".
6. Calculate the entrainment fidelity as the ratio of the cumulative power spectral density (PSD) within the optogenetic stimulation frequency range (stimulation frequency ± 0.5 Hz), to the cumulative PSD in the theta (5-12 Hz) band with the multitaper method (NW = 3, window size 8,192) for 10 s epochs (e.g., toolkit <http://chronux.org/>).
7. Exclude recording epochs where the dominant PSD peak is ≤ 5 Hz from analysis (non- theta epochs, using MATLAB function find.m).
8. Plot the raster of the LFP power spectra for all recorded epochs according to the computed entrainment fidelity (using MATLAB functions sortrows.m and pcolor.m). Load the power spectra and entrainment fidelity by clicking "File > Open", stored variable In. Type Power = sortrows(In,1); pcolor(Power(:,2:end)).

Results

Targeting of ChR2 to GABAergic cells in the MS as described in section 1 is illustrated in Figure 2A. Optogenetic stimulation of axons of MS GABAergic cells in the dorsal hippocampus via an optic fiber which is implanted above the CA1 area entrains theta oscillations at the frequency of the stimulus in the ipsilateral (Figure 2B) as well as contralateral hemisphere (Figure 2C). Theta oscillations cou...

Discussion

Here we presented a widely accessible methodology to entrain and elicit hippocampal theta oscillations in the behaving animal. This approach can be useful for studies of theta rhythm's functions in information processing and behavior. Critical aspects of this method include: (1) choice of the opsin and targeting of ChR2 to axons of MS cells in the hippocampus, (2) robust optical and electrical features of implanted optic fiber-wire array assemblies to ensure continuous stimulation and LFP recording in behaving mice, ...

Materials

Name	Company	Catalog Number	Comments
PV-Cre mice	The Jackson Laboratory	B6;129P2-Pvalbtm1(cre)Arbr/J
Name	Company	Catalog Number	Comments
Surgery
Stereotaxis	David Kopf Instruments, Tujunga, CA, USA	Model 963	Ultra Precise Small Animal Stereotaxic Instrument
Drill bits, 0.8 mm	Bijoutil, Allschwil, Switzerland	49080HM
0.01-1 ml syringe	Braun, Melsungen, Germany	9161406V
Sterican cannulas	Braun	26 G, 0.45x25 mm BL/LB
Fine and sharp scissors	Fine Science Tools Inc., Vancouver, Canada	14060-09
Forceps	Fine Science Tools Inc.	11210-10	Dumont AA - Epoxy Coated Forceps
Blunt stainless steel scissors	Fine Science Tools Inc.	14018-14
Soldering station	Weller Tools GmbH, Besigheim, Germany	WSD 81
Erythromycin	Rotexmedica GmbH, Trittau, Germany	PZN: 10823932	1g Powder for Solution for Infusion
Name	Company	Catalog Number	Comments
Optogenetics
Hamilton pump	PHD Ultra, Harvard Apparatus, Holliston, MA, USA	model 703008	PHD Ultra Syringe Pump with push/pull mechanism
Hamilton 5 µL Syringe, 26 gauge	PHD Ultra, Harvard Apparatus	Model 75 RN SYR
Hamilton 5 µL Plunger	PHD Ultra, Harvard Apparatus	Model 75 RN SYR
Tubing	Fisher Scientific, Pittsburgh, USA	PE 20	Inner diameter 0.38 mm (.015"), Outer diameter 1.09 mm (.043")
Sterican cannulas	Braun, Melsungen, Germany	27 G, 25x0.40 mm, blunt
Precision drill/grinder	Proxxon, Wecker, Luxemburg	fbs 240/e
Cutting disks	Proxxon	NO 28812
Cre dependent channelrhodopsin	Penn Vector Core, Philadelphia, PA, USA	AV-1-18917P	Contruct name: AAV2/1.CAGGS.flex.ChR2.tdTomato, titer: 1.42x1013 vg/ml
Cam kinase dependent halorhodopsin	Penn Vector Core	AV-1-26971P	Construct name: eNpHR3.0, AAV2/1.CamKIIa.eNpHR3.0-EYFP.WPRE.hGH, titer: 2.08_1012 vg/ml
Multimode optic fiber	ThorLabs, Dachau, Germany	FG105LCA	0.22 NA, Low-OH, Ø105 µm Core, 400 - 2400 nm
Ceramic stick ferrule	Precision Fiber Products, Milpitas, CA, USA	CFLC126	Ceramic LC MM Ferrule, ID 126um
Polishing paper	Thorlabs	LF3D	6" x 6" Diamond Lapping (Polishing) Sheet
Power meter	Thorlabs	PM100D	Compact Power and Energy Meter Console, Digital 4" LCD
Multimode fiber optic coupler	Thorlabs	FCMM50-50A-FC	1x2 MM Coupler, 50:50 Split Ratio, 50 µm GI Fibers, FC/PC
Fiberoptic patch cord	Thorlabs	FG105LCA CUSTOM-MUC	custom made, 3 m long, with protective tubing, Tubing: FT030, Connector 1: FC/PC, Connector 2: 1.25mm (LC) Ceramic Ferrule
Sleeve	Precision Fiber Products, Milpitas, CA, USA	ADAL1	Ceramic Split Mating Sleeve for Ø1.25 mm (LC/PC) Ferrules
473 nm DPSS laser	Laserglow Technologies, Toronto, ON, Canada	R471005FX	LRS-0473 Series
593 nm DPSS laser	Laserglow Technologies	R591005FX	LRS-0594 Series
MC_Stimulus II	Multichannel Systems, Reutlingen, Germany	STG 4004
Impedance conditioning module	Neural microTargeting worldwide, Bowdoin, USA	ICM
Name	Company	Catalog Number	Comments
Electrophysiology
Tungsten wires	California Fine Wire Company, Grover Beach, CA, USA	CFW0010954	40 µm, 99.95%
Capillary tubing	Optronics	1068150020	ID: 100.4 µm
Omnetics nanoconnector	Omnetics Connector Corporation, Minneapolis, USA	A79038-001
Screws	Bilaney, Düsseldorf, Germany	00-96x1/16	stainless-steel
Silicone probe	NeuroNexus Technologies, Ann Arbor, MI, USA	B32
Headstage	Neuralynx, Bozeman, Montana USA	HS-8	miniature headstage unity gain preamplifiers
Silver conductive paint	Conrad electronics, Germany	530042
Liquid flux	Felder GMBH Löttechnik, Oberhausen, Germany	Lötöl ST	DIN EN 29454.1, 3.2.2.A (F-SW 11)
LED	Neuralynx	HS-LED-Red-omni-10V
Name	Company	Catalog Number	Comments
Software
MATLAB	Mathworks, Natick, MA, USA
MC_Stimulus software	Multichannel, Systems
Neurophysiological Data Manager			NDManager, http://neurosuite.sourceforge.net
Klusters			http://neurosuite.sourceforge.net, Hazan et al., 2006
Software of the recording system	Neuralynx	Cheetah	https://neuralynx.com/software/cheetah
Multi-channel data analysis software	Cambridge Electronic Design Limited, Cambridge, GB	Spike2