1Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, 2Department of Biomedical Engineering, School of Engineering, University of California, Irvine
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Ikrar, T., Olivas, N. D., Shi, Y., Xu, X. Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation. J. Vis. Exp. (56), e3109, doi:10.3791/3109 (2011).
Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.
1. Brain slice preparation
2. Equipment startup
The overall system implementation is illustrated in Figure 1.
3. Setting up slice perfusion
4. Whole cell patch-clamping recording
5. Laser scanning photostimulation
6. Photostimulation data analysis
Our newly developed methodology and software implementation11 is applied to the detection and measurement of photostimulation-evoked synaptic events in the raw data maps. As exemplified in Figure 2, color-coded maps are constructed to illustrate the pattern of synaptic input to the recorded neuron.
7. Representative Results:
Example results of electrophysiological recording and photostimulation mapping are shown in Figure 2. Targeted recordings of specific inhibitory cell types are made possible by using transgenic mice expressing GFP in known inhibitory cell types. Given the diversity of inhibitory interneurons, analyses of GFP expression, intrinsic electrophysiology and morphological characteristics (Figure 2A) are combined to arrive at the final cell type classification. Figure 2B-C illustrate that laser scanning photostimulation allows for extensive mapping of local cortical circuit connections to single inhibitory neurons. The raw data map is shown in Figure 2C. The direct uncaging responses are excluded from data analysis (Figure 2C). The quantitative input maps are color-coded, shown in Figure 2D-F. The example fast-spiking inhibitory interneurons received strong excitatory synaptic input (EPSCs) from layer 4 and deeper layers. By relating the cell's synaptic input organization to defined cortical pathways, we are able to infer its possible role (e.g., feedback and feedforward inhibition) in cortical information processing.
Figure 1. General system instrumentation for laser scanning photostimulation. Our overall system consists of photostimulation control, video imaging, and electrophysiological recording systems. We adopted the design of the LSPS system described previously7,17. A laser unit is used to generate 355 nm UV laser for glutamate uncaging. The laser beam is directed through the optical path of our system. Short durations of laser flashes (e.g., 1- 3 ms) are controlled by using an electro-optical modulator (i.e., pockels cell) and a mechanical shutter. Laser beam power is modulated by a neutral density gradient wheel and monitored by diverting a small fraction of the laser beam with a glass coverslip to a photodiode. The laser scanning system includes an X-Y pair of scan mirrors, the scan lens, the tube lens, and the objective lens. The mirrors deliver the laser beam through a scan lens; then the beam enters the microscope via a dichroic mirror and is focused by a custom-made UV-transmitting tube lens. The beam underfills the back aperture of the microscope objective to provide a more columnar (as opposed to conical) illuminating beam, keeping the mapping as two-dimensional as possible by reducing the axial resolution. Under the 4x objective, the laser beam forms uncaging spots, each approximating a Gaussian profile with a width of 153 μm laterally at the focal plane (see the insert). Various laser stimulation positions can be achieved through galvanometers-driven XY scanning mirrors, as the mirrors and the back aperture of the objective are in conjugate planes, translating mirror positions into different scanning locations at the objective lens focal plane. During uncaging, a variable number of patterned sites that covers the whole field are stimulated sequentially in a nonraster, nonrandom sequence to avoid revisiting the vicinity of recently stimulated sites. Please refer to Xu et al. (2010) for more detailed information.
Figure 2. Mapping local excitatory circuit connections to a GFP-expressing fast-spiking interneuron in layer 4 of mouse primary sensory cortex. A1 and A2 show the DIC and GFP fluorescent images of the targeted cell under the 60x objective in the living brain slice, while A3 shows the cell morphology revealed by biocytin staining in the post-recording, fixed slice. A4 shows the firing patterns of the recorded GFP cell, characteristic of a fast-spiking inhibitory interneuron, in response to intrasomatic current injections of different strengths. B show the slice image superimposed with the 16 x16 photostimulation sites (*). The cell location is indicated by the small red circle. C shows an array of photostimulation-evoked response traces from the stimulation locations shown in B, while the cell was held at -70 mV to detect inward excitatory currents. Different forms of photostimulation responses are illustrated by the traces at locations of 1 and 2, which are expanded and separately shown in the insert. The trace 1 is an example of the direct responses (indicated by the red arrowheads) to glutamate uncaging on the cell body and promximal dendrites. Other traces in 2 are typical examples of excitatory synaptic input responses (blue). Direct responses and synaptic inputs can be distinguished by their amplitudes and response latencies. D, E and F are the color-coded maps (16x16 sites) of average EPSC amplitude, the EPSC numbers, and the first detected EPSC latency per site, respectively, for the raw data map shown in C. The average input amplitude from each stimulation site is the mean amplitude of EPSCs in the analysis window, with the baseline spontaneous response subtracted from the photostimulation response of the same site. The number of EPSCs and the arrival time or latency of the first detected EPSC per site are also measured and plotted. Please refer to (Shi et al., 2010) for more details.
Photostimulation-based mapping techniques have been effectively applied for analyzing cortical circuits. Laser scanning photostimulation combined with whole cell recording allows high resolution mapping of laminar distributions of presynaptic input sources to single neurons, because the simultaneous recording from a postsynaptic neuron with photostimulation of clusters of presynaptic neurons at many different locations provides quantitative measures of spatial distribution of excitatory or inhibitory inputs. This technique aided by using transgenic mice expressing GFP in known inhibitory cell types has greatly facilitated our current work toward elucidating local inhibitory cortical networks. In the example study, we used caged glutamate to activate neurons in a spatially restrict manner upon laser uncaging. It should be noted that glutamate uncaging can be replaced with photoactivation via channelrhodopsin or other genetically encoded photosensitive molecules in specific cell types12,13. Thus the photostimulation technique is possible to target not only specific cortical regions but also specific subset of neurons within their participating circuits14-16. In addition, we recently developed a new photostimulation-based mapping technique9 that incorporates the spatial precision of activation that can be achieved by laser-scanning photostimulation with rapid and high-temporal resolution assessment of evoked network activity that can be achieved by voltage-sensitive dye imaging. Unlike combination of whole cell recordings with photostimulation for mapping local circuit inputs to individually recorded neurons, this innovation can be used to map cortical circuit output and functional connections at the level of neuronal populations.
No conflicts of interest declared.
We thank Tran Huynh, Andrew San Antonio, Jerry Lin for their technical assistance. This work was funded by the National Institutes of Health grants DA023700 and DA023700-04S1 to X.X.
|transgenic mouse lines||Jackson lab or other sources||Please refer to Xu and Callaway (2009)|
|GFP goggles||BLS Ltd., Hungary|
|MNI caged glutamate (4-methoxy-7-nitroindolinyl-caged l-glutamate)||Tocris Bioscience, Ellisville, MO||Cat No. 1490|
|electrode puller||Sutter Instrument, Novato, CA||P-97|
|glass tubes for making electrodes||BF150-86-10|
|Multiclamp 700B amplifier||Molecular Devices, Sunnyvale, CA||Multiclamp 700B|
|digital CCD camera||Q-imaging, Austin, TX||Retiga 2000|
|Research microscope||Olympus, Tokyo, Japan||BW51X|
|UV laser unit||DPSS Lasers, Santa Clara, CA||model 3501|
|Other equipment for Laser scanning phostimulation||Please refer to Xu et al. (2010)|