3.1 Electroporation Enables Uptake of Various Dyes in Multiple Different Circuits
Slices containing either the cortex, the hippocampus, the main olfactory bulb, or the accessory olfactory bulb from mice were used to test the local electroporation method. Dextran-conjugated dyes were initially used to test the protocol in each brain area. Immediately after completion of the protocol, dye was present throughout the tissue in a larger area surrounding the electrode location, which eventually dissipated leaving only the area of tissue which had taken up the dye. The number of loaded cells varied greatly with the number of pulses given and with stimulation intensities. We eventually established two protocols, one with a greater number of pulses with higher amplitudes for loading large areas and numerous cells and another with much fewer pulses with smaller amplitudes for more focal targeting and labeling of a single or a few cells (see methods).
As a result, we were able to electroporate both large areas of the cortex, hippocampus and main olfactory bulb (), and also target loading to a fairly small and well-defined area using the low intensity protocol. As an example, we found that we could electroporate a single glomerulus (25 μm) as seen via a GFP-transgenic mouse line (Del Punta, Puche et al., 2002
) in which sensory neurons expressing the V2r1b receptor type also express tau-GFP allowing us to observe axonal terminations in individual glomeruli in the accessory olfactory bulb (). In some instances, many cells deep to the stimulation electrode were labeled following electroporation. For example, in the CA3 region of the hippocampus, a confocal stack acquired starting just below the site of electroporation showed dense labeling of many cells (). Anterograde and retrograde projections were clearly visible in all circuits. Indeed we were able to visualize processes from loaded cells up to several hundred microns away from the site of electroporation () as well as visualize the distinct morphology of individually loaded cells and their processes (). In all these examples, dye loading was complete within 25 – 30 minutes following the beginning of the electroporation.
Electroporation of Various Circuits
3.2 Electroporation Can Provide for Discrete Labeling of Specific Neuronal Circuits
We used acute coronal brain slices to test whether this technique was capable of targeted electroporation of specific processing units, for instance the mitral cell to glomerular layer connections within the olfactory bulb. Three consecutive glomeruli were identified under DIC and targeted for electroporation using three separable hydrazide dyes (Alexa Fluor 488, 594, and 647). Each glomerulus was electroporated separately with a different electrode using the high intensity stimulation protocol (see methods). High intensity protocols typically labeled 3 - 10 mitral cells per glomerulus whereas low intensity protocols typically labeled only 1 - 2 (see and ).
Completeness of Electroporation Protocol
Targeted Whole-Cell Patch Clamp via Electroporation
We found that each glomerulus was individually labeled with surrounding periglomerular and external tufted cells () as well as connected mitral cells for each glomerulus (). We found little to no overlap between electroporated glomeruli at the level of the glomerular layer for each labeled glomerulus () consistent with the known anatomy of this system (Schoppa and Urban, 2003
). The resultant mitral cell loading labeled unique populations with each population connecting exclusively to the corresponding electroporated glomerulus (). As a result, this technique allowed us to fluorescently identify individual processing units in the olfactory bulb.
Specificity of Electroporated Circuits
Following electroporation, we also performed immunohistochemistry on sections containing labeled cells. After staining for the potassium channel Kv. 1.2, we found that we could examine the expression of local potassium channels in specific populations of neurons by observing the overlap between labeled neurons via electroporation () with immunohistochemistry (). Indeed, we observed overlap between labeled cells and Kv. 1.2 staining as seen in , where two electroporated external tufted cells also stained positive for Kv. 1.2 (arrows). This illustrates that this technique provides a useful tool for examining the overlap of the expression of individual proteins within local neuronal circuits of interest.
3.3 Rapid Uptake and Diffusion of Dye After Electroporation
One of the major limitations of previous dye loading techniques such as the use of lipophilic dyes, including DiI, is the relatively slow uptake of dye and subsequent loading of somata and processes. For imaging approaches to be useful in slice electrophysiology experiments, both rapid uptake and diffusion of excess dye are essential for overall slice quality, the quality of whole-cell recordings, and calcium imaging of cellular activity. To assess the time required for dye uptake we acquired images at a series of time points following electroporation.
Immediately after the electroporation protocol we observed bright fluorescence through the tissue in the region in which the electrode had been placed. In a few minutes following electroporation, the fluorescence became less uniform presumably as the dye in the extracellular space diffused away, leaving behind labeled processes. Detectable levels of dye were seen in the somata of cells ≈200 μm away immediately after the completion of the electroporation protocol (data not shown). Sufficient levels for targeted whole-cell patch recordings were seen at 5 minutes post-hoc ().
Rapid Uptake and Diffusion of Dye
We quantified this observation by calculating the ratio of pixel intensities across the cell body over the pixel intensities at a fixed point in the surrounding tissue. This ratio increased dramatically at each time point () as the dye diffused towards the cell body, and the dye in the surround tissue was cleared. We found nearly complete diffusion of dye by 20 minutes after electroporation (). All experiments including calcium imaging and targeted whole-cell patch clamp recording were performed 15-25 minutes post electroporation.
For usefulness of this technique purely for anatomical purposes, we also wondered what fraction of the cells from the population projecting to a target region were labeled after a single electroporation protocol. As before, the glomerular system provided a perfect setting to answer this question by electroporating the same glomerulus twice with two differently colored dyes. To do this, we used a theta glass electrode, filling one chamber with Alexa 594 hydrazide, and the other chamber with Alexa 488 hydrazide (). We then electroporated a random glomerulus using the high intensity protocol with the red chamber only and observed the number of labeled periglomerular, external tufted, and mitral cells ().
Without moving the electrode, we then electroporated the same glomerulus with the same protocol using the green chamber to then observe the labeled cells from the second electroporation epoch (). If each electroporation protocol was 100% effective in labeling all cells projecting to a given glomerulus, we would expect all yellow cells. In fact, when we counted the number of labeled cells in each channel, we saw that, on average, 93% percent of labeled cells (29 Red, 32 Green in Glomerulus 1, 39 Red, 41 Green in Glomerulus 2), were labeled during the first electroporating epoch while the second electroporation epoch only labeled a combined 5 additional cells in both glomeruli (). This suggests that, at least for the high intensity protocol, each electroporation epoch is effective in labeling the vast majority of cells projecting into a target region.
3.4 Electroporation Enables Targeted Whole-Cell Patch Clamp While Preserving Cell Viability
Many slice electrophysiology experiments require the ability to target and record from cells based on their connectivity. This includes single cells or pairs of cells that innervate a particular layer or which send their dendrites to a particular layer. One example of such a system is the mouse olfactory system where the primary output neurons of the olfactory bulb, mitral cells, target a single primary dendrite typically to one glomerulus. Individual glomeruli are innervated by olfactory sensory neurons expressing a single receptor gene (Mombaerts, Wang et al., 1996
; Ressler, Sullivan et al., 1994
) such that all mitral cells innervating a given glomerulus receive homotypic input. Thus targeting cells based on the glomerular termination of their dendrites allow analysis of neurons that receive the same inputs and should have comparable receptive fields.
To determine if this technique would enable us to not only label multiple mitral cells innervating a given glomerulus, but to also use this fluorescence to target and record from those cells, we first electroporated random glomeruli in the main olfactory bulb using the low intensity stimulation protocol. We observed loading of (in most cases) surrounding periglomerular cells, external tufted cells, and typically 1 -2 mitral cells (, ). We used fluorescence to target labeled mitral cells/tufted cells for whole-cell patch clamp to record and fill these cells with a second fluorescent dye (, ). For patched mitral cells, we injected both square current pulses and filtered white noise current pulses (generated by convolving white noise with a 3 msec alpha function), to probe the responses of electroporated cells to various inputs ().
Electrophysiological Recordings from Electroporated Cells
To assess the effect of electroporation on the intrinsic properties of these mitral cells, we recorded the changes in membrane potentials in current clamp to 25 pA hyperpolarizing currents for 500 ms (). Input resistances were calculated when the membrane potential reached steady state (). Th fitting the membrane potential trace between the initial potential and the steady state potential using a mean-square error function (). We found no significant differences in the input resistance (control R=19±7 MΩ, electroporated R=37±34 MΩ, P=0.29 ANOVA) or membrane time constants (control T=14±4.5 ms, electroporated T=17±9.8 ms, P=0.56 ANOVA) between mitral cells recorded under control conditions (N=5) and those recorded after they had been electroporated (N=4). In addition, these values are consistent with previously reported values in vitro
(Margrie, Sakmann et al., 2001
3.5 Electroporation of Calcium Indicators Enables Calcium Imaging of Local Neuronal Circuits
We found that both high and low intensity protocols worked well with multiple dextran conjugated dyes and hydrazide dyes (see ) and allowed for multi-dye loading within a given circuit. However, these fluorescent dyes only allow for morphological reconstruction and targeted whole-cell patch clamp recording of labeled neurons. To determine if our technique would also allow for the examination of physiological activity of multiple neurons within a circuit, we proceeded to label populations of neurons with a calcium indicator dye that serves as a proxy for neuronal activity via calcium imaging (Kerr, Greenberg et al., 2005
; Ohki, Chung et al., 2005
; Tank, Sugimori et al., 1988
We choose dextran-conjugated Oregon Green Bapta as a calcium indicator and electroporated within the mitral cell layer in the accessory olfactory bulb. We found intense labeling of both mitral cells and granule cells within the bulb as a result (). In order to evoke activity within these neurons and to test whether the electroporated calcium indicator retained its calcium sensitivity, we placed a theta glass stimulation electrode nearby labeled mitral and granule cells (). Stimulation evoked calcium transients in nearby cells that had been loaded with Dextran-OGB () indicating that electroporation of calcium-sensitive dyes could be used to study the physiological activity of targeted populations of neurons.
Calcium Activity in Local Neuronal Circuit