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In Living Brain Slices (Neocortex/Whole-Cell Recording/Intrinsic Circuits) EDWARD M Proc. Natl. Acad. Sci. USA Vol. 90, pp. 7661-7665, August 1993 Neurobiology Photostimulation using caged glutamate reveals functional circuitry in living brain slices (neocortex/whole-cell recording/intrinsic circuits) EDWARD M. CALLAWAY* AND LAWRENCE C. KATZt Department of Neurobiology, Box 3209, Duke University Medical Center, Durham, NC 27710 Communicated by Dale Purves, May 6, 1993 ABSTRACT An approach for high-spatial-resolution map- stimulated axons of passage along with cell bodies did not ping of functional circuitry in living mammalian brain slices limit its utility. However, mapping circuitry in the dense has been developed. The locations of neurons making func- neuropil of the mammalian central nervous system necessi- tional synaptic connections to a single neuron are revealed by tates a method that does not stimulate axons of passage. In photostimulation of highly restricted areas of the slice (50-100 addition, the method ofFarber and Grinvald (7) was based on ,um in diameter) while maintaining a whole-cell recording of the light-induced formation of small transient pores in the the neuron ofinterest. Photostimulation is achieved by bathing neuronal membrane and, thus, could only be used to activate brain slices in a molecularly caged form ofthe neurotransmitter a particular neuron a few times before it was killed by glutamate [L-glutamic acid a-(4,5-dimethoxy-2-nitrobenzyl) phototoxic damage. Furthermore, the performance of their ester], which is then converted to the active form by briefpulses photostimulation probes varied between cell types and was (<1 ms in duration) ofultraviolet irradiation. Direct activation occasionally species-specific. Here we report development ofreceptors on recorded neurons in rat hippocampus and ferret of a photostimulation method, based on the use of caged visual cortex demonstrates that photostimulation is reliable and glutamate (8), designed to achieve high-resolution mapping of reproducible and can be repeated at the same site at least 30 functional microcircuitry in living mammalian brain slices. times without obvious decrement in neuronal responsiveness. This approach is based on the fact that virtually all neurons Photostimulation of presynaptic neurons at sites distant to the in the mammalian central nervous system can be activated by recorded neuron evoked synaptic responses in hippocampal the excitatory amino acid neurotransmitter glutamate. By and cortical cells at distances of up to several millmeters from combining photostimulation with whole-cell patch-clamp re- the recorded neuron. Stimulation of 25-100 distinct presyn- cordings, the regions within a brain slice that contain neurons aptic sites while recording from a single postsynaptic neuron making functional connections to a single neuron can be was easily achieved. Caged glutamate-based photostimulation mapped with high resolution and the strengths of the con- eliminates artifacts and limitations inherent in conventional nections can be measured. stimulation methods, including stimulation ofaxons ofpassage, desensitization, and poor temporal resolution of "puffer" pipettes, and current artifacts of iontophoretic application. MATERIALS AND METHODS This approach allows detailed physiological investigation and Overview. Brain slices were prepared by conventional manipulation of the complex intrinsic circuitry of the mam- methods and submerged in artificial cerebral spinal fluid malian brain. (ACSF) containing 1 mM "caged glutamate" [L-glutamic acid a-(4,5-dimethoxy-2-nitrobenzyl) ester]. The synthesis of this Understanding the detailed organization of neuronal net- compound and some of its properties, along with several works is important for elucidating computations responsible other amino acid neurotransmitters, were described by for brain function. The limitations of presently available Wilcox et al. (8). Whole-cell recordings were obtained from methods for revealing such circuits are especially apparent in neurons in selected regions of brain slices and maintained the study of detailed local circuits, as in the mammalian while stimulating small groups of neurons at selected neigh- cerebral cortex, in which very closely spaced neurons often boring locations within the slice. Stimulation was achieved by make up distinctly different components of the network (1). converting caged glutamate to active glutamate by using an Conventional physiological and anatomical methods, such as intense 800-,sec-duration flash of ultraviolet irradiation fo- current source density analysis (2, 3), single-unit recordings, cused through the epifluorescence optical pathway of an cross-correlation analyses, intracellular recording, and dye inverted microscope. The inverted microscope was mounted filling (4-6), allow only limited insight into the connectivity on an x-y stage, allowing it to be moved freely beneath the of neuronal assemblies. To overcome these limitations, Far- fixed stage containing the brain slice without disrupting the ber and Grinvald (7) developed an approach based on light- whole-cell recording. Repeated stimulation at closely spaced based neuronal stimulation ("photostimulation") for dissect- locations resulted in high-resolution functional mapping of ing neuronal networks. They designed photostimulation mol- the local circuitry impinging on the recorded neuron. A ecules (analogs of voltage-sensitive dyes) that allowed detailed description of the technique is provided below. stained neuronal elements to be activated by small spots of Slice Preparation and Whole-Cell Recording. Brain slices laser light and were able to sequentially stimulate numerous were prepared from the hippocampus, somatosensory cor- neurons in invertebrate ganglionic preparations to identify tex, or visual cortex of rats aged from 2 weeks postnatal to those that were presynaptic to an intracellularly impaled neuron. Since single neurons could easily be targeted for Abbreviations: CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; stimulation in the isolated ganglia, the fact that their method AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionate; TTX, tetrodotoxin; EPSC, excitatory postsynaptic current. *Present address: Department of Physiology and Neuroscience The publication costs of this article were defrayed in part by page charge Program, University ofColorado School ofMedicine, Campus Box payment. This article must therefore be hereby marked "advertisement" B138, 4200 East Ninth Avenue, Denver, CO 80262. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 7661 Downloaded by guest on October 1, 2021 7662 Neurobiology: Callaway and Katz Proc. Natl. Acad. Sci. USA 90 (1993) adult, from the visual cortex of ferrets aged 4-5 weeks laser alignment spot on each image and then superimposed to postnatal, or from the visual cortex of adult cats. Slices (400 create a single map. ,um thick) were cut using a specialized slicer and maintained at 33-34°C in an interface holding chamber as described (1). RESULTS At least 1 h after cutting, individual slices were moved to a recording chamber on the fixed stage of an inverted micro- Direct Neuronal Stimulation. For this photostimulation scope and submerged in ACSF containing 1 mM L-glutamic method to be useful for mapping circuitry, uncaged glutamate acid a-(4,5-dimethoxy-2-nitrobenzyl) ester [caged glutamate must be capable of stimulating neurons sufficiently to pro- (8), obtained from Molecular Probes] at room temperature. In duce an action potential. We used whole-cell recordings in a some control experiments, the ACSF contained no caged voltage-clamp mode (holding potential, -66 mV) to monitor glutamate. The slice was held against the coverslip on the currents evoked by light flashes positioned directly under the bottom of the chamber with small pieces of silver wire placed recorded neurons. Large inward currents were invariably over its ends. About 5 ml of ACSF was continously reper- measured and were large enough to overcome the voltage fused through the recording chamber with a peristaltic pump clamp, resulting in regenerative action potentials (Fig. 1). (at -0.5 ml/min) and reoxygenated with 95% 02/5% CO2 by Inward currents were evoked in all 30 cells tested including bubbling in a 5-ml syringe used as a secondary reservoir. 5 in rat hippocampus, 4 in rat cerebral cortex, 13 in ferret Whole-cell recordings were obtained in selected regions of visual cortex, and 8 in cat visual cortex. No decrement in brain slices as described by Blanton et al. (9). The electrodes responsiveness was observed with repeated stimulation of used had resistances of 5-10 Mfl and were filled with the neurons, which were in some cases activated by as many as following solution: 130 mM cesium gluconate/10 mM 30 flashes. Responses were only observed when the objective CsCl2/10 mM Hepes/li mM EGTA/1 mM CaCl2/1 mM was focused into the slice itself. Focusing the objective even MgCl2/2 mM ATP/0.3 mM GTP, pH 7.4. After whole-cell slightly below the slice greatly attenuated the responses. In recordings were obtained, neurons were voltage-clamped at several cases we noted that relatively small changes in focus -66 mV and the whole-cell currents evoked by "photostim- (10-50 ,um) produced significant changes in the magnitude of ulation" were recorded using an Axopatch 1-D amplifier response, implying that the technique can provide at least (Axon Instruments, Burlingame, CA) and saved using some z-axis resolution, especially when high-numerical- PCLAMP software (Axon Instruments).
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