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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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 or months later.

Abstract

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 µm 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.

Introduction

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 Ramón y Cajal's seminal histological studies1. The advent of single-cell recording tech....

Protocol

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.......

Representative Results

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 2, the morphology of a biocytin-filled neuron in a thick section (300 µm) was revealed using streptavidin visualized in re.......

Discussion

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, .......

Acknowledgements

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

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Materials

NameCompanyCatalog NumberComments
NaCl Sigma S7653Immunostaining
KCL Fluka60129Immunostaining
Na2HPO4 SigmaS7907Immunostaining
KH2PO4 Sigma229806Immunostaining
Triton X-100SigmaT8787Immunostaining
Guinea pig anti CB1 SigmaAf530-1Immunostaining
Mouse anti CCKCURE, UCLAcourtesy of G. OhningImmunostaining
Rabbit anti ParvalbuminSwantPV27Immunostaining
Streptavidin, Alexa Fluor conjugateMolecular ProbesS11227Immunostaining
Normal goat serumSigmaG9023Immunostaining
Vectashield Vector LabsH-1000Immunostaining
Secondary AntibodiesInvitogen Molecular probesAlexa Fluor conjugated dyesImmunostaining
Labnet orbit low speed shakerBioexpressS-2030-LSImmunostaining
ForcepsDumont11231-30Immunostaining
Slide foldersEMS71520Immunostaining
Vibratome VT 1200 SLeica14048142066Electrophysiology
Multiclamp 700B amplifierMolecular devicesMulticlamp 700BElectrophysiology
pCLAMP 10 SoftwareMolecular devicespCLAMP 10 Electrophysiology
DigitizerMolecular DevicesDigidata 1440 digitizerElectrophysiology
Filter tipsNalgene171-0020Electrophysiology
SonicatorFisher Scientific15-335-100Electrophysiology
MicroloadersEppendorf930001007Electrophysiology
BiocytinSigmaB4261Electrophysiology

References

  1. Garcia-Lopez, P., Garcia-Marin, V., Freire, M. The histological slides and drawings of cajal. Front Neuroanat. 4, 9 (2010).
  2. Klausberger, T., Somogyi, P. Neuronal diversity and temporal dynamics: the unity of hippocampal c....

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