Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex
Summary
Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.
Abstract
Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. Traditional in vitro electrophysiological techniques use electrical stimulation to evoke synaptic transmission. A major drawback of this method is its nonspecific nature; all axons in the region of the stimulating electrode will be activated, making it difficult to attribute an effect to a particular afferent connection. This issue can be overcome by replacing electrical stimulation with optogenetic-based stimulation. We describe a method for combining optogenetics with in vitro patch-clamp recordings. This is a powerful tool for the study of both basal synaptic transmission and synaptic plasticity of precise anatomically defined synaptic connections and is applicable to almost any pathway in the brain. Here, we describe the preparation and handling of a viral vector encoding channelrhodopsin protein for surgical injection into a pre-synaptic region of interest (medial prefrontal cortex) in the rodent brain and making of acute slices of downstream target regions (lateral entorhinal cortex). A detailed procedure for combining patch-clamp recordings with synaptic activation by light stimulation to study short- and long-term synaptic plasticity is also presented. We discuss examples of experiments that achieve pathway- and cell-specificity by combining optogenetics and Cre-dependent cell labeling. Finally, histological confirmation of the pre-synaptic region of interest is described along with biocytin labeling of the post-synaptic cell, to allow further identification of the precise location and cell type.
Introduction
Understanding the physiology of synapses and how they undergo plastic changes is fundamental for understanding how brain networks function in the healthy brain1, and how they malfunction in brain disorders. The use of acute ex vivo brain slices allows for the recording of the electrical activity of synapses from single neurons with a high signal-to-noise ratio using whole-cell patch-clamp recordings. Control of membrane potential and straightforward pharmacological manipulation allows isolation of receptor subtypes. These recordings can be made with exquisite specificity to identify the post-synaptic neuron, including laminar and sub-regional position2, cellular morphology3, presence of molecular markers4, its afferent projections5, or even if it was recently active6.
Achieving specificity of pre-synaptic inputs is, however, somewhat more challenging. The conventional method has used stimulation electrodes to excite the axons which run in a particular lamina. An example of this is in the hippocampus where local stimulation in the stratum radiatum activates synapses that project from the CA3 to the CA1 subfield7. In this instance, presynaptic specificity is achieved as CA3 input represents the sole excitatory input located within stratum radiatum which projects to CA1 pyramidal cells8. This high degree of input specificity achievable with conventional electrical presynaptic activation of CA3-CA1 axons is, however, an exception which is reflected in the intense study that this synapse has been subject to. In other brain regions, axons from multiple afferent pathways co-exist in the same lamina, for example, in layer 1 of neocortex9, thus rendering input-specific presynaptic stimulation impossible with conventional stimulating electrodes. This is problematic as different synaptic inputs may have divergent physiological properties; therefore, their co-stimulation may lead to mischaracterization of synaptic physiology.
The advent of optogenetics, the genetic encoding of photosensitive membrane proteins (opsins) such as channelrhodopsin-2 (ChR2), has allowed a vast expansion of possibilities for studying isolated synaptic projections between brain regions10,11. Here we describe a generalizable and low-cost solution to studying long-range synaptic physiology and plasticity. The optogenetic constructs are delivered in a highly specific manner using viral vectors allowing for extremely precise control of the pre-synaptic region of interest. Efferent projections will express the light-activated channel allowing for activation of these fibers in a target region. Thus, long-range, anatomically diffuse pathways that cannot be independently activated by traditional, non-specific, electrical stimulation can be studied.
We describe, as an example pathway, transduction of medial prefrontal cortex (mPFC) with adeno-associated viruses (AAVs) encoding excitatory cation-channel opsins. We then describe the preparation of acute slices from lateral entorhinal cortex (LEC), patch-clamp recordings from layer 5 LEC pyramidal neurons, and light-evoked activation of glutamatergic mPFC-LEC projections (Figure 1). We also describe the histological assessment of the injection site to confirm the location of the pre-synaptic region of interest and identification of post-synaptic cell morphology.
Protocol
Representative Results
Discussion
The protocol presented here describes a method to explore highly specific long-range synaptic projections using a combination of stereotaxic surgery to deliver AAVs encoding optogenetic constructs, and electrophysiology in acute brain slices (Figure 1). Together these techniques offer tools to characterize the physiology and plasticity of brain circuitry with high precision in long-range and anatomically diffuse pathways that were previously inaccessible using traditional, non-specific, elec…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work is supported by Wellcome grant 206401/Z/17/Z. We would like to thank Zafar Bashir for his expert mentorship and Dr. Clair Booth for technical assistance and comments on the manuscript.
Materials
| 0.2 mL tube | Fisher Scientific Ltd | 12134102 | |
| 10 µL pipette | Gilson | FD10001 | |
| 24 well plate | SARSTEDT | 83.3922 | |
| 3 way luer valve | Cole-Parmer | WZ-30600-02 | |
| 3,3′-Diaminobenzidine (DAB) substrate | Vector Laboratories | SK-4105 | |
| 40x objective | Olympus | LUMPLFLN40XW | |
| 4-aminopyridine | Hello Bio | HB1073 | |
| 4x objective | Olympus | PLN4X/0.1 | |
| AAV9-CaMKiia-hChR2(E123T/T159C)-mCherry | Addgene | 35512 | Viral titre: 3.3×1013 GC/ml |
| Achromatic lens | Edmund Optics | 49363 | Focusses visual spectrum and near-IR |
| Benchtop microcentrifuge | Benchmark Scientific | C1005* | |
| Biocytin | Sigma-Aldrich | B4261 | |
| Borosillicate glass capillary | Warner Instruments | G150F-6 | |
| Burr | Fine science tools | 19008-07 | |
| CaCl2 | Sigma-Aldrich | C5670 | |
| Camera – Qimaging Retiga Electro | Photometrics | 01-ELECTRO-M-14-C | |
| Carbachol | Tocris | 2810 | |
| Chlorhexidine surgical scrub | Vetasept | XHG008 | |
| Clippers | Andis | 22445 | AGC Super 2-Speed Detachable Blade Clipper |
| Collimation condenser lens | ThorLabs | ACL2520-A | |
| Coverslips | Fisher Scientific Ltd | 10011913 | |
| Cryostat | Leica | CM3050 S | |
| CsMeSO4 | Sigma-Aldrich | C1426 | |
| Cyanoacrylate glue | Rapid Electronics Ltd | 84-4557 | |
| Data acquisition device | National Instruments | USB-6341 BNC | |
| D-glucose | Sigma-Aldrich | G8270 | |
| Dichroic mirror 500 nm long-pass | Edmund Optics | 69899 | |
| Dichroic mirror 600 nm long-pass | Edmund Optics | 69901 | |
| Dichroic mirror cube | ThorLabs | CM1-DCH/M | |
| EGTA | Millpore | 324626 | |
| Electrode holder with side port | HEKA | 895150 | |
| Emission filter | Chroma | 59022m | |
| Excitation filter | Chroma | ET570/20x | |
| Eye gel | Dechra | Lubrithal | |
| Fine paint brush | Scientific Laboratory Supplies | BRU2052 | |
| Guillotine | World Precision Instruments | DCAP | |
| HEPES | Sigma-Aldrich | H3375 | |
| Hydrogen peroxide solution | Sigma-Aldrich | H1009 | 30% (w/w) |
| Isoflurane | Henry Schein | 988-3245 | |
| Isopentane | Sigma-Aldrich | M32631 | |
| KCl | Sigma-Aldrich | P3911 | |
| k-gluconate | Sigma-Aldrich | G4500 | |
| Kinematic fluorescence filter cube | ThorLabs | DFM1T1 | |
| LED driver | ThorLabs | LEDD1B | |
| Lidocaine ointment | Teva | 80007150 | |
| MgATP | Sigma-Aldrich | A9187 | |
| MgCl | Sigma-Aldrich | M2670 | |
| MgSO4 | Sigma-Aldrich | M7506 | |
| Micro drill | Harvard Apparatus | 75-1887 | |
| Microelectrode puller | Sutter instruments | P-87 | |
| Microinjection syringe | Hamilton | 7634-01/00 | |
| Microinjection syringe needle | Hamilton | 7803-05 | Custom specification: gauge 33, length 15mm, point style 4 – 12° |
| Microinjection syringe pump | World Precision Instruments | UMP3T-1 | |
| Mounted blue LED | ThorLabs | M470L5 | |
| Mounted green LED | ThorLabs | M565L3 | |
| Na2HPO4.7H2O | Sigma-Aldrich | S9390 | |
| NaCl | Sigma-Aldrich | S9888 | |
| NaGTP | Sigma-Aldrich | G8877 | |
| NaH2PO4 | Sigma-Aldrich | S0751 | |
| NaH2PO4.H2O | Sigma-Aldrich | S9638 | |
| NaHCO3 | Sigma-Aldrich | S5761 | |
| NIR LED | OSRAM | SFH4550 | Used for refracted IR imaging of slice, differential interference contrast (DIC) optics is another commonly used method |
| OCT medium | VWR International | RAYLLAMB/OCT | Optimal cutting temperature medium |
| Paraformaldehyde | Sigma-Aldrich | 158127 | |
| Paraformaldehyde | Sigma-Aldrich | P6148 | |
| Patch clamp amplifier | Molecular Devices | 700A | |
| Peristaltic pump | World Precision Instruments | Ministar | |
| Poly-L-lysine coated microscope slides | Fisher Scientific Ltd | 23-769-310 | |
| Recording chamber | Warner Instruments | RC-26G | |
| Scalpel blade | Swann Morton | #24 | |
| Slice anchor | Warner Instruments | SHD-26-GH/15 | |
| Stereotaxic frame | Kopf | Model 902 | |
| Stereotaxic holder for micro drill | Harvard Apparatus | 75-1874 | |
| Sucrose | Sigma-Aldrich | S0389 | |
| Surgical Microscope | Carl Zeiss | OPMI 1 FR pro | |
| Suture | Ethicon | W577H | |
| Syringe filter for intracellular recording solution | Thermo Scientific Nalgene | 171-0020 | |
| Tetrodotoxin citrate | Hello Bio | HB1035 | |
| Transfer pipettes | Fisher Scientific Ltd | 10458842 | |
| Triton X-100 | Sigma-Aldrich | X100 | |
| Upright fluorescence microscope | Leica | DM6 B | |
| VECTASHIELD Antifade Mounting Medium with DAPI | Vector Laboratories | H-1200-10 | |
| VECTASTAIN ABC-HRP kit | Vector Laboratories | PK-4000 | |
| Vibratome | Campden Instruments | 7000smz-2 | |
| WinLTP | https://www.winltp.com/ | Version 2.32 | Data acquisition software |
| Solution | |||
| aCSF | |||
| sucrose cutting solution | |||
| PFA | |||
| Intracellular? |
References
- Martin, S., Grimwood, P., Morris, R. Synaptic plasticity and memory: an evaluation of the hypothesis. Annual Review of Neuroscience. 23, 649-711 (2000).
- Poorthuis, R. B., et al. Layer-specific modulation of the prefrontal cortex by nicotinic acetylcholine receptors. Cerebral Cortex. 23 (1), 148-161 (2013).
- Scala, F., et al. Layer 4 of mouse neocortex differs in cell types and circuit organization between sensory areas. Nature Communications. 10 (1), 4174 (2019).
- Nassar, M., et al. Diversity and overlap of parvalbumin and somatostatin expressing interneurons in mouse presubiculum. Frontiers in Neural Circuits. 9, 20 (2015).
- Dembrow, N. C., Chitwood, R. A., Johnston, D. Projection-specific neuromodulation of medial prefrontal cortex neurons. Journal of Neuroscience. 30 (50), 16922-16937 (2010).
- Whitaker, L. R., et al. Bidirectional modulation of intrinsic excitability in rat prelimbic cortex neuronal ensembles and non-ensembles after operant learning. Journal of Neuroscience. 37 (36), 8845-8856 (2017).
- Skrede, K. K., Westgaard, R. H. The transverse hippocampal slice: a well-defined cortical structure maintained in vitro. Brain Research. 35 (2), 589-593 (1971).
- van Strien, N. M., Cappaert, N. L., Witter, M. P. The anatomy of memory: an interactive overview of the parahippocampal-hippocampal network. Nature Reviews Neuroscience. 10 (4), 272-282 (2009).
- Cruikshank, S. J., et al. Thalamic control of layer 1 circuits in prefrontal cortex. Journal of Neuroscience. 32 (49), 17813-17823 (2012).
- Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nature Neuroscience. 8 (9), 1263-1268 (2005).
- Nagel, G., et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proceedings of the National Academy of Sciences of the United States of America. 100 (24), 13940-13945 (2003).
- Berndt, A., et al. High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels. Proceedings of the National Academy of Sciences of the United States of America. 108 (18), 7595-7600 (2011).
- Cetin, A., Komai, S., Eliava, M., Seeburg, P. H., Osten, P. Stereotaxic gene delivery in the rodent brain. Nature Protocols. 1 (6), 3166-3173 (2006).
- Segev, A., Garcia-Oscos, F., Kourrich, S. Whole-cell patch-clamp recordings in brain slices. Journal of Visualized Experiments: JoVE. (112), (2016).
- Booker, S. A. Preparing acute brain slices from the dorsal pole of the hippocampus from adult rodents. Journal of Visualized Experiments: JoVE. (163), (2020).
- Anderson, W. W., Collingridge, G. L. Capabilities of the WinLTP data acquisition program extending beyond basic LTP experimental functions. Journal of Neuroscience Methods. 162 (1-2), 346-356 (2007).
- Basu, J., et al. Gating of hippocampal activity, plasticity, and memory by entorhinal cortex long-range inhibition. Science. 351 (6269), (2016).
- Zhang, Y. P., Oertner, T. G. Optical induction of synaptic plasticity using a light-sensitive channel. Nature Methods. 4 (2), 139-141 (2007).
- Banks, P. J., Warburton, E. C., Bashir, Z. I. Plasticity in prefrontal cortex induced by coordinated synaptic transmission arising from reuniens/rhomboid nuclei and hippocampus. Cerebral Cortex Communications. 2 (2), (2021).
- Petreanu, L., Mao, T., Sternson, S. M., Svoboda, K. The subcellular organization of neocortical excitatory connections. Nature. 457 (7233), 1142-1145 (2009).
- Swietek, B., Gupta, A., Proddutur, A., Santhakumar, V. Immunostaining of biocytin-filled and processed sections for neurochemical markers. Journal of Visualized Experiments: JoVE. (118), (2016).
- Jones, B. F., Witter, M. P. Cingulate cortex projections to the parahippocampal region and hippocampal formation in the rat. Hippocampus. 17 (10), 957-976 (2007).
- Anastasiades, P. G., Collins, D. P., Carter, A. G. Mediodorsal and ventromedial thalamus engage distinct L1 circuits in the prefrontal cortex. Neuron. 109 (2), 314-330 (2021).
- Prakash, R., et al. Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation. Nature Methods. 9 (12), 1171-1179 (2012).
- Mattis, J., et al. Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nature Methods. 9 (2), 159-172 (2011).
- Klapoetke, N. C., et al. Independent optical excitation of distinct neural populations. Nature Methods. 11 (3), 338-346 (2014).
- Hochbaum, D. R., et al. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nature Methods. 11 (8), 825-833 (2014).
- Ting, J. T., Daigle, T. L., Chen, Q., Feng, G. Acute brain slice methods for adult and aging animals: application of targeted patch clamp analysis and optogenetics. Methods in Molecular Biology. 1183, 221-242 (2014).
- Marshel, J. H., et al. Cortical layer-specific critical dynamics triggering perception. Science. 365 (6453), (2019).
- Castle, M. J., Gershenson, Z. T., Giles, A. R., Holzbaur, E. L., Wolfe, J. H. Adeno-associated virus serotypes 1, 8, and 9 share conserved mechanisms for anterograde and retrograde axonal transport. Human Gene Therapy. 25 (8), 705-720 (2014).
- Aschauer, D. F., Kreuz, S., Rumpel, S. Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One. 8 (9), 76310 (2013).
- Nathanson, J. L., Yanagawa, Y., Obata, K., Callaway, E. M. Preferential labeling of inhibitory and excitatory cortical neurons by endogenous tropism of adeno-associated virus and lentivirus vectors. Neuroscience. 161 (2), 441-450 (2009).
- Dimidschstein, J., et al. A viral strategy for targeting and manipulating interneurons across vertebrate species. Nature Neuroscience. 19 (12), 1743-1749 (2016).
- Lavin, T. K., Jin, L., Lea, N. E., Wickersham, I. R. Monosynaptic tracing success depends critically on helper virus concentrations. Frontiers in Synaptic Neuroscience. 12, 6 (2020).
- Paxinos, G., Watson, C. . The Rat Brain in Stereotaxic Coordinates. 7th edn. , (2013).
- Paxinos, G., Franklin, K. B. J. . Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates, Compact. 5th edn. , (2019).
- Nabavi, S., et al. Engineering a memory with LTD and LTP. Nature. 511 (7509), 348-352 (2014).
- Jackman, S. L., Beneduce, B. M., Drew, I. R., Regehr, W. G. Achieving high-frequency optical control of synaptic transmission. Journal of Neuroscience. 34 (22), 7704-7714 (2014).
- Xia, S. H., et al. Cortical and Thalamic Interaction with Amygdala-to-Accumbens Synapses. Journal of Neuroscience. 40 (37), 7119-7132 (2020).
- Anisimova, M., et al. Spike-timing-dependent plasticity rewards synchrony rather than causality. BioRxiv. , (2021).
- Takeuchi, T., et al. Locus coeruleus and dopaminergic consolidation of everyday memory. Nature. 537 (7620), 357-362 (2016).
