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This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.
Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.
Defining connectivity between brain regions is necessary to understand neural circuits. Classical anatomical tracing methods allow establishing interregional connectivity, and lesion studies help to understand the hierarchical organization of information flow. For example, brain circuits for spatial orientation and head direction signaling involve the directional flow of information from the thalamus to the presubiculum. This has been demonstrated by lesion studies of antero-dorsal thalamic nuclei (ADN) that degrade the head direction signal in the downstream dorsal presubiculum, as well as the parahippocampal grid cell signal1,2.
The functional connectivity between brain areas is more difficult to establish at a cellular and subcellular level. In the hippocampus, a highly organized anatomy allows to investigate pathway-specific synaptic connections using electrical simulation in the slice preparation. Stimulation electrodes placed in stratum radiatum of CA1 can be used to specifically stimulate Schaffer collateral input from CA33. Stimulating electrodes placed in stratum lacunosum moleculare of CA1 will activate the perforant path input to CA14,5. Electrical stimulation activates neurotransmitter release from axon terminals; however, it activates neurons with somata near the stimulation site as well as axons of passage. It is therefore of limited use for studying afferents from defined brain regions when fibers of different regions of origin intermingle in the target structure, as is typically the case in the neocortex.
Neurons may also be stimulated with light. Optical methods include the photoactivation of caged glutamate, which can be combined with one- or two-photon laser scanning. Multiple closely spaced sites may be stimulated sequentially, with no mechanical damage to the tissue6. This has been successfully used to map synaptic receptors as well as activate individual neurons7. While glutamate uncaging can be used for local circuit analysis, it does not allow for specific activation of long-range inputs.
A method of choice for the investigation of long-range connectivity in neuronal circuits is the use of virus-mediated channelrhodopsin expression. Using in vivo stereotaxic injections as described here, the expression of light-gated ion channels can be targeted and spatially restricted to a desired brain region. In this way, channelrhodopsins are effective for mapping excitatory or inhibitory connectivity from one region to its target. Transfected axons terminals may be stimulated with light in a brain slice preparation, and patch-clamp recordings as a read-out allow examination of the functions and strengths of specific circuit components in the brain8. The optogenetic approach combined with stereotaxic injection of a virus offers unprecedented specificity and genetic control9. Stimulating with light additionally allows for both high temporal and spatial precision10,11.
The presubiculum is a six-layered cortical structure at the transition of the hippocampus and the para-hippocampal formation12,13. It receives important synaptic input from the ADN11 but also from several other cortical and subcortical regions14. Thus, the selective stimulation of thalamic axons terminals within a presubicular slice is not possible with electrical stimulation nor glutamate uncaging. Described in this protocol are methods to determine functional connectivity between brain regions (ADN and presubiculum) using precise stereotaxic injections of viral vectors expressing light-gated channels. Also described is the photostimulation of axons terminals of projecting neurons in their target region, coupled with whole-cell patch-clamp recordings of post-synaptic neurons in the brain slice preparation.
All procedures were performed in accordance with the European Community Council Directive (2010/63/EU) and approved by the ethics committee of Paris Descartes University. The experimenter must obtain authorization for the procedure to comply with local regulations.
1. Planning of the experiment
2. Stereotaxic surgery
3. Solutions for acute slice recordings and fixation
4. Preparation of brain slices
5. Whole-cell patch-clamp recording
6. Biocytin revelation
The procedure presented here was used to express a blue light-sensitive channelrhodopsin (Chronos) fused to GFP in the antero-dorsal nucleus of the thalamus (ADN), by stereotaxic injection of anterograde adeno-associated virus. The stereotaxic coordinates were determined according to a mouse brain atlas and tested by injecting 200 nL of fluorescent tracer fluoro-ruby. The animal was sacrificed 10 min after the injection, and the brain was extracted and fixated overnight. Coronal brain sections were prepared to examine th...
In vivo viral injection to express light-sensitive opsins in a defined brain area is a choice method for the optogenetic analysis of long-range functional connectivity10,11,17,18. Stereotaxic injections offer the possibility to precisely target a specific area of the brain. The coexpression of an opsin with a fluorescent reporter conveniently allows evaluation of the successful expression and c...
The authors declare no competing financial interests.
We thank Bertrand Mathon, Mérie Nassar, Li-Wen Huang, and Jean Simonnet for their help in the development of previous versions of the stereotaxic injection protocol and Marin Manuel and Patrice Jegouzo for technical help. This work was supported by the French Ministry for Education and Research (L. R., L. S.), Centre National des Etudes Spatiales (M. B.), and Agence Nationale de la Recherche Grant ANR-18-CE92-0051-01 (D. F.).
Name | Company | Catalog Number | Comments |
0.5 mm bur | Harvard Apparatus | 724962 | |
10 µL Hamilton syringe | Hamilton | 1701 RN - 7653-01 | |
10X PBS solution | Thermofisher Scientific | AM9624 | text |
36% PFA | Sigma-Aldrich | F8775 | |
470 nm LED | Cairn Research | P1105/470/LED DC/59022m | use with matched excitation filter 470/40x and emission filter for GFP |
AAV5.Syn.Chronos-GFP.WPRE.bGH | Penn Vector Core | AV-5-PV3446 | lot V6026R, qTiter GC/ml 4.912e12, ddTiter GC/ml 2.456e13 |
All chemicals | Sigma | ||
Bath temperature controler | Luigs & Neumann | SM7 | Set at 34°C |
beveled metal needle | Hamilton | 7803-05 | 33 gauge, 13mm, point style 4-20° |
Big scissors | Dahle Allround | 50038 | |
Biocytin | Sigma | B4261 | final 1-3 mg/ml |
Borosilicate Capillaries | Havard Apparatus | GC150-10 | 1.5 mm outer, 0.86 inner diameter |
Brown Flaming electrode puller | Sutter Instruments | P-87 | |
BupH Phosphate Buffered Saline pack | Thermofisher Scientific | 28372 | |
butterfly needle for perfusion | Braun | Venofix A | 24G |
CCD Camera | Andor | DL-604M | |
Confocal Microscope | Zeiss | LSM710 | 20X |
curved forceps | FST | 11011-17 | |
CY5 configuration (confocal) | Helium-Neon 633nm (5,0 mW) laser; Mirror: MBS 488/561/633 | ||
CY5 configuration (epifluo) | Nikon/Chroma | Fluorescent light (Intensilight); Excitation filter: BP645/30; Dichroic mirror: 89100 BS ; Emission filter: BP705/72 | |
DAPI | Sigma | D9542 | |
DAPI configuration (epifluo) | Nikon/Chroma | Fluorescent light (Intensilight); Cube: Semrock Set DAPI-5060C-000-ZERO (Excitation: BP 377/50; Mirror: BS 409; Emission: BP 447/60) | |
Digidata 1440A | Axon Instruments | ||
Digital handheld optical meter | ThorLabs | PM100D | Parametered on 475 nm |
Double egde stainless steel razor blades | Electron Microscopy Sciences | 72000 | Use half of the blade in the slicer |
Dual Fluorescent Protein Flashlight | Nightsea | DFP-1 | excitation, 440-460 nm; emission filter on glasses, 500 nm longpass. |
EGTA | Sigma | E4368 | final 0,2 mM |
Epifluorescence Microscope | Nikon | Eclipse TE-2000E | 10 or 20X |
Filter paper | Whatman | ||
Fluoro-Ruby 10% | Millipore | AG335 | disolve 10 mg in 100 µl of distilled water ; inject 150 to 300 nl |
GFP configuration (epifluo) | Nikon/Chroma | Fluorescent light (Intensilight); Cube: Filter Set Nikon B-2E/C FITC (Excitation: BP 465-495; Mirror: BS 505; Emission: BP 515-555) | |
Heatingplate | Physitemp | HP4M | |
Heparin choay 5000 U.I./ml | Sanofi | 5 ml vial | |
HEPES | Sigma | H3375 | final 10 mM |
High speed rotary micromotor kit | Foredom | K.1070 | maximum drill speed 38,000 rpm |
Internal solution compounds : | |||
Isolated Pulse Stimulator | A-M Systems | 2100 | |
KCl | Sigma | P4504 | final 1,2 mM |
Ketamine 1000 | Virbac | ||
Ketofen 10% | Merial | 100 mg/ml : dilute 1 µl in 1ml total (0,1%) | |
Laocaine (lidocaine) | MSD | 16,22 mg/ml : dilute 1 ml in 4 ml total (around 4%) | |
LED hi power spot for surgery | Photonic (via Phymep) | 10044 | |
LED Power Supply | Cairn Research | OptoLED Light Source | |
Manipulators | Luigs & Neumann | SM-7 | |
Mg-ATP 2H20 | Sigma | A9187 | final 4 mM |
MgCl2 | Sigma | 63069 | final 2 mM |
Micro temperature controler | Physitemp | MTC-1 | |
Milk powder | Carnation | ||
MultiClamp 700B | Axon Instruments | ||
Na Phosphocreatine | Sigma | P7936 | final 10 mM |
Na3-GTP 2H20 | Sigma | G9002 | final 0.4 mM |
needle holder/hemostat | FST | 13005-14 | |
pClamp acquisition software | Axon Instruments | ||
Peristaltic pump | Gilson | Minipuls 3 | 14-16 on the display for 2-3 ml/min |
Potassium gluconate (K-gluconate) | Sigma | G4500 | Final 135 mM |
ProLong Gold antifade mounting medium | Thermofisher Scientific | P36390 | |
Rompun 2% (xylazine) | Bayer | ||
small scissors | FST | 14060-09 | |
Sodium chloride 0.9% | Virbac | dilute 8.5 mL in 10 ml total | |
Stereomicroscope VISISCOPE SZT | VWR | 630-1584 | |
Stereotaxic frame with digital display | Kopf | Model 940 | Small animal stereotaxic instrument |
Streptavidin-Cy3 conjugate | Life technologies | 434315 | |
Streptavidin-Cy5 conjugate | Thermofisher Scientific | S32357 | |
Superglue3 Loctite | Dutscher | 999227 | 1g tube |
Suture filament Ethilon II 4-0 polyamid | Ethicon | F3210 | |
Syringe pump | kdScientific | Legato 130 - 788130 | Use Infuse and Withdraw modes |
Tissue slicer | Leica | VT1200S | speed 0.07, amplitude 1. |
tubing | Gilson | F117942, F117946 | Yellow/Black, Purple/Black |
upright microscope | Olympus | BX51W1 | |
Versi-dry bench absorbant paper | Nalgene |
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