The authors would like to acknowledge the support from NIH/NINDS R01 NS069861 and NJCBIR CBIR14IRG024 to VS.

1. Biocytin Filling during Electrophysiology
NOTE: The readers can refer to alternative sources for basic patch-clamp recording techniques and instrumentation19-22, which are not elaborated upon here. The steps detailed here assume that the equipment and procedures for patch-clamp recordings are already established, and the description will be restricted to details related to biocytin-filling and post-hoc immunostaining. All experiments outlined in this manuscript were performed on ra...

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V., Zaitsev, A. V., Gonzalez-Burgos, G., Lewis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+heterogeneity+of+fast-spiking+interneurons:+chandelier+versus+basket+cells.">Electrophysiological heterogeneity of fast-spiking interneurons: chandelier versus basket cells.</a> PLoS One. 8 (8), e70553 (2013).</li><li>Gupta, A., Elgammal, F. S., Proddutur, A., Shah, S., Santhakumar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decrease+in+tonic+inhibition+contributes+to+increase+in+dentate+semilunar+granule+cell+excitability+after+brain+injury.">Decrease in tonic inhibition contributes to increase in dentate semilunar granule cell excitability after brain injury.</a> J Neurosci. 32 (7), 2523-2537 (2012).</li><li>Fish, K. N., Hoftman, G. D., Sheikh, W., Kitchens, M., Lewis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin-containing+chandelier+and+basket+cell+boutons+have+distinctive+modes+of+maturation+in+monkey+prefrontal+cortex.">Parvalbumin-containing chandelier and basket cell boutons have distinctive modes of maturation in monkey prefrontal cortex.</a> J Neurosci. 33 (19), 8352-8358 (2013).</li><li>Yu, J., Swietek, B., Proddutur, A., Santhakumar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dentate+cannabinoid-sensitive+interneurons+undergo+unique+and+selective+strengthening+of+mutual+synaptic+inhibition+in+experimental+epilepsy.">Dentate cannabinoid-sensitive interneurons undergo unique and selective strengthening of mutual synaptic inhibition in experimental epilepsy.</a> Neurobiol Dis. 89, 23-35 (2016).</li><li>Yu, J., Swietek, B., Proddutur, A., Santhakumar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dentate+total+molecular+layer+interneurons+mediate+cannabinoid-sensitive+inhibition.">Dentate total molecular layer interneurons mediate cannabinoid-sensitive inhibition.</a> Hippocampus. 25 (8), 884-889 (2015).</li><li>Varga, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+fission+of+parvalbumin+interneuron+classes+during+fast+network+events.">Functional fission of parvalbumin interneuron classes during fast network events.</a> Elife. 3, (2014).</li><li>Kawashima, T., Okuno, H., Bito, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+era+for+functional+labeling+of+neurons:+activity-dependent+promoters+have+come+of+age.">A new era for functional labeling of neurons: activity-dependent promoters have come of age.</a> Neural Circuits Revealed. , 109 (2015).</li><li>Krook-Magnuson, E., Luu, L., Lee, S. H., Varga, C., Soltesz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ivy+and+neurogliaform+interneurons+are+a+major+target+of+mu-opioid+receptor+modulation.">Ivy and neurogliaform interneurons are a major target of mu-opioid receptor modulation.</a> J Neurosci. 31 (42), 14861-14870 (2011).</li><li>Szabadics, J., Soltesz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+specificity+of+mossy+fiber+innervation+of+GABAergic+cells+in+the+hippocampus.">Functional specificity of mossy fiber innervation of GABAergic cells in the hippocampus.</a> J Neurosci. 29 (13), 4239-4251 (2009).</li><li>Iball, J., Ali, A. 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Critical Steps within the Protocol 
Filling the patched cell with biocytin is the most crucial step to ensure the full recovery of the morphology. For full recovery of the cell, it is essential to select an optimal slice orientation to minimize the severing of processes during slicing. This orientation may differ based on the circuit and cell type under examination. Next, it is essential to allow adequate time for the biocytin to diffuse to the dendrites and axons. Some dyes, ...

The brain is known for diversity in the structural and functional characteristics of its individual neuronal elements. Understanding the roles of distinct neuronal types in brain function and pathology requires characterization and unambiguous identification of neurons. Structurally, the morphological features defined by somato-dendritic location determine the potential inputs that a given neuron receives, while the pattern of axonal arborization identifies potential postsynaptic targets. The structural diversity of neurons has been appreciated since the days of Ram&#243;n y Cajal&#39;s seminal histological studies1. The advent of single-cell recording techniques revealed that structurally distinct neurons also show differences in firing patterns and synaptic characteristics. The diversity in structure and physiology is particularly evident in GABAergic inhibitory neurons2,3. In addition, it has become increasingly apparent that structurally similar neurons can express different neurochemical markers and show corresponding functional differences4. Similarly, neurons with the same neurochemical markers can have distinct structures and functions5-10. Thus, in practice, the analysis of the functional characteristics of neurons and their role in the network entails defining both the morphological and neurochemical identities. Even with the advent of reporter mouse lines targeting specific neurochemical markers, it is often necessary to determine morphology and subtype identity based on immunohistology11.
The standard method used to characterize cells recorded in acute brain slices is to fill them with biocytin or neurobiotin during the recording, fix the sections in paraformaldehyde (PFA) following the recordings, and use immunohistochemistry to reveal the morphology and neurochemistry. Since the thickness of sections for slice physiology are typically 300 &#181;m or more, and because most antibodies fail to penetrate all the way through that depth, the slices need to be re-sectioned to 60 &#181;m or less to allow for simultaneous immunostaining for biocytin and neurochemical markers12-14. Unfortunately, resectioning is laborious; risks loss of tissue during sectioning; and can lead to differential tissue shrinkage, complicating morphological reconstructions. Additionally, prior knowledge of morphology could help narrow down the candidate markers that are likely to be expressed by the cells. We have modified the standard biocytin immunohistology protocols to allow serial processing of sections first for the recovery of morphology and then for the identification of potential neurochemical markers.
Immunohistochemistry is the study of antigen distribution in tissues or cells and can be visualized using an enzyme, fluorescent labels, radioactive elements, or gold colloid particles15. The procedure involves using primary antibodies to specifically tag and amplify one or more specific antigens, followed by the use of fluorescent secondary antibodies targeting the primary antibody for visualization. Due to the need to distinguish the fluorescence spectra of each secondary antibody without overlap, only a limited number of antigens can be examined simultaneously. Thus, prior knowledge of morphology could be useful in selecting the candidate neurochemical markers for cell classification. Conceptually, the rationale behind serial processing of already-stained sections is based on the premise that immunolabeling for one protein or peptide should not interfere with antigenicity and subsequent immunolabeling for a structurally independent peptide16. This lack of interference is due to the binding of the antibodies to a specific protein epitope on an antigen and therefore allows for the simultaneous staining of multiple antigens in the same tissue. The number of antigens revealed by immunostaining is limited by the need for non-overlapping spectra of the fluorescent secondary antibodies and by the need to target individual antigens with antibodies raised in different species so as to eliminate cross-reactivity17,18. While this is the reasoning behind serial rather than simultaneous labeling with two distinct antibodies that may interact, to our knowledge, immunostaining for a second antigen has not been reported after the completion of immunolabeling for one or more antigens on mounted sections. Here, we describe a method for serial immunostaining of previously stained and mounted sections. While we detail this process for a serial immunolabeling procedure for the recovery of morphology followed by staining for protein/peptide markers in thick sections, the same procedures can be used in standard, thin histological sections as well. In addition, we describe a practical approach to fill recorded neurons with biocytin and the process to dislodge the electrode from the cell upon completion of recordings to optimize the filling of the axonal and dendritic arbors of neurons, as presented in our recent work6,8.
The most crucial advantage of the procedure described here is that the morphology of the recorded cell can be fully recovered and imaged before attempting to resection or immunostain the slices. Although issues with the penetration of certain antibodies may render it necessary to resection slices for secondary immunostaining, the procedures detailed here would eliminate the need to reconstruct complex neurons from multiple sections and would avoid issues due to tissue loss and differential shrinkage, which can compromise reconstruction following resectioning. An added advantage is that the process will reduce cost, time, effort, and expensive antibodies by limiting immunostaining and re-sectioning to slices in which biocytin-filled neurons are recovered. The most practical aspect is the additional immunostaining that can be performed on sections stained months before using the aforementioned technique. In particular, the recovery of morphology would considerably reduce the potential that physiological data from the cells is discarded due to an inability to obtain a basic morphological characterization of the cell type.

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		<tr height="20">
			<td height="20" style="height:20px;width:335px;">NaCl&#160;</td>
			<td style="width:211px;">Sigma</td>
			<td style="width:203px;">&#160;S7653</td>
			<td style="width:151px;">Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCL&#160;</td>
			<td>Fluka</td>
			<td>60129</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Na2HPO4&#160;</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">KH2PO4&#160;</td>
			<td>Sigma</td>
			<td>229806</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Guinea pig anti CB1&#160;</td>
			<td>Sigma</td>
			<td>Af530-1</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CCK</td>
			<td>CURE, UCLA</td>
			<td>courtesy of G. Ohning</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti Parvalbumin</td>
			<td>Swant</td>
			<td>PV27</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptavidin, Alexa Fluor conjugate</td>
			<td>Molecular Probes</td>
			<td>S11227</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal goat serum</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vectashield&#160;</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Secondary Antibodies</td>
			<td>Invitogen Molecular probes</td>
			<td>Alexa Fluor conjugated dyes</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Labnet orbit low speed shaker</td>
			<td>Bioexpress</td>
			<td>S-2030-LS</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Dumont</td>
			<td>11231-30</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Slide folders</td>
			<td>EMS</td>
			<td>71520</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vibratome VT 1200 S</td>
			<td>Leica</td>
			<td>14048142066</td>
			<td>Electrophysiology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multiclamp 700B amplifier</td>
			<td>Molecular devices</td>
			<td>Multiclamp 700B</td>
			<td>Electrophysiology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pCLAMP 10 Software</td>
			<td>Molecular devices</td>
			<td>pCLAMP 10&#160;</td>
			<td>Electrophysiology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digitizer</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440 digitizer</td>
			<td>Electrophysiology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter tips</td>
			<td>Nalgene</td>
			<td>171-0020</td>
			<td>Electrophysiology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonicator</td>
			<td>Fisher Scientific</td>
			<td>15-335-100</td>
			<td>Electrophysiology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microloaders</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td>Electrophysiology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biocytin</td>
			<td>Sigma</td>
			<td>B4261</td>
			<td>Electrophysiology</td>
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immunostaining of biocytin-filled and processed sections for neurochemical markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

Upon successful completion, the sections retain the biocytin fill and the immunolabeling performed in step 2 and can be imaged using confocal or epifluorescence microscopy. In addition, the processed sections will also show immunostaining for the antigen labeled during the subsequent processing in step 3. In the section illustrated in Figure&#160;2, the morphology of a biocytin-filled neuron in a thick section (300 &#181;m) was revealed using streptavidin visualized in re...

Watch this Scientific Journal Video about Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers at JoVE.com

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

The goal of this protocol is to present an efficient method for identifying neurons patched during electrophysiological recordings using biocytin and immunostaining for recovery of morphology prior to re-staining with specific antibodies to determine neurochemical identity. So this method describes an important step needed to study the diversity in neuronal structure and function. This is essential to understand their role in brain networks.The main advantage of using a sequential processing techniques, is that it eliminates the data loss because of inability to recover morphology. So this helps also guide the neurochemical markers to be tested in these cells. Although this method is described for morphological recovery of cells in thick sections, it can also be applied to thin sections and on previously stained and mounted samples.Generally neutrals new to this method may have difficultly in resealing the soma while detaching from the cell, and may fail to allow adequate time for successful fills. Visual demonstration of this method is critical as specific techniques are required for effective biocytin filling of cells in order to recover the complete morphology. To begin this procedure, in voltage clamp configuration apply small brief voltage steps using the membrane test function in bath mode while approaching the cell.Patch onto the cell by applying suction. Then, hold the cell at negative 70 milivolts. Note the development of a gigaohm seal.Compensate for capacitive transients and then apply controlled suction with a syringe or by mouth to achieve whole cell configuration. Estimate whole cell capacitance and series resistance, and stop the membrane test pulses. Series resistance of less than 10 megaohm is optimal.This will ensure the establishment of whole cell recording conditions to allow for biocytin filling. Upon completion of the physiological recordings, re-establish the patch clamp configuration by slowly moving the recording pipette in small steps, alternating upward and outward in voltage clamp mode. The most critical step in accomplishing morphological recovery of cells, is to ensure the re-sealing of the neuronal soma while detaching the recording pipette from the cell.Using the membrane test function, visualize the loss of capacitive transients and the collapse of current responses to a straight line, which indicates the re-sealing of the cell and the establishment of an outside out patch at the pipette tip. In the meantime, holding the cell at a depolarized potential will facilitate the resealing process. After detaching the pipette from the cell, retain the slice in the recording chamber for three to five minutes to ensure the dye transporting to the distal dendritic and axonal processes.Then, transfer the slice to a 24-well plate containing 4%PFA for fixation. 24 to 48 hours after PFA fixation, transfer all the fixed slices to zero point one molar PBS for storage prior to immunostaining. In this procedure, wash the tissues three times for 10 minutes each in zero point one molar PBS.Next, block the tissues with 10 percent blocking serum for one hour at room temperature. Subsequently, incubate the sections in primary antibodies CB1R at room temperature overnight to identify cannabinoid receptor type 1 expression. Then, wash the brain sections three times for 10 minutes each in 0.1 molar PBS.Streptavidin and secondary antibody to first primary antibody are both added to the well plate and kept overnight at four degrees celsius. Subsequently, incubate the sections in streptavidin red dye conjugate at four degrees celsius overnight in the dark to reveal the biocytin. After that, wash the brain sections three times for 10 minutes each in 0.1 molar PBS.To protect the florescence and to facilitate restaining in the future, mount the sections at an equally spaced mounting medium. Enseal the edges of the cover slips with clear nail polish. In this step, strip the nail polish off of the glass slide with a cotton swab of acetone.Remove the cover slip carefully using fine tip forceps and put a few droplets of 0.1 molar PBS on the sections. It is critical not to damage the process tissue while unmounting the tissue from the aqueous mounting medium. Carefully remove the sections from the slide using a fine paintbrush and wash them in 0.1 molar PBS for 24 to 48 hours on a shaker, and kept away from light in four degrees Celsius.To ensure complete removal of the mounting medium, wash the sections one to two times on the following day in 0.1 molar PBS for 10 minutes each time. Then, incubate the sections with a second primary antibody cholecystokinin for one day. Wash the brain sections three times for 10 minutes each in 0.1 molar PBS.Subsequently, stain the sections with a fluorophore conjugated secondary antibody goat anti-mouse. Making sure that the excitation emission spectra of the secondary antibody do not overlap with streptavidin and prior immunofluorescent stains. Afterward, mount the sections in aqueous mounting medium.Enseal the edges of the cover slip with clear nail polish. Following successful biocytin fill and the first immunostaining process, the neuronal structure can be visualized and imaged as shown in the confocal image obtained at 60x magnification. The section was also immunostained for CB1R along with biocytin, and the CB1R immunostaining in the biocytin expressing neuronal soma is indicated by the arrowhead.The same section was processed for CCK immuno-reactivity after one week, and the image shows successful recovery of CCK immuno labeling in several neurons indicated by the asterisk. Note that the biocytin labeled cell indicated by the arrowhead did not express CCK. A merged image of panels A, B, and C show that all three labels are visualized robustly, and the overlay between biocytin and CB1R is evident.The image shows that the second immunostaining process does not compromise the signal from the prior staining process. Morphology of the biocytin filled cell was obtained from the confocal images prior to this CCK immunostaining. Crucially, in addition to the soma and dendrite, axons can be visualized, and co-labeling of CB1R and biocytin labeled axonal processes is observed.While attempting this procedure, it's important to target healthy cells located below the slice surface to avoid neurons with severed processes. And that could compromise the recover of the morphology. Once mastered, morphological recovery of biocytin filled cells can be reliably obtained during the patch clamp recording.Successful implementation of serial immunostaining technique will improve efficiency of data recovery and facilitate neuronal characterization during standard patch clamp recordings. After watching this video, you should have a good understanding on how to effectively use biocytin filled cells to recover neuronal morphology and to determine neurochemical markers.

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Research

JoVE Journal

Neuroscience

22.7K Views.  Rutgers New Jersey Medical School. The goal of this protocol is to present an efficient method for identifying neurons patched during electrophysiological recordings using biocytin and immunostaining for recovery of morphology prior to re-staining with specific antibodies to determine neurochemical identity. So this method describes an important step needed to study the diversity in neuronal structure and function. This is essential to understand their role in brain networks.The main advantage of using a sequential proc...