Published: November 20th, 2018
The protocol outlined here describes the immunofluorescence analysis, biocytin recovery and high-quality reconstructions of hippocampal CA2 interneurons following the intracellular electrophysiological recordings in vitro, allowing neuronal characterization and ultimately fine neuronal anatomy to be studied.
How cortical network activity processes information is of importance to a large number of basic and clinical scientific questions. The protocol described here identifies the basic building blocks of this circuitry. The in-depth studies of cortical regions will ultimately provide other scientists with the circuit components needed for an understanding of how the brain acquires, processes and stores information and what goes wrong in disease, while the electrophysiological and morphological data are widely used by computational neuroscientists in the construction of model networks that explore information processing. The protocol outlined here describes how biocytin-filled cells recorded in the CA2 region of the hippocampus are recovered and then reconstructed in 3D. Additionally, the protocol describes the demonstration of calcium binding protein or peptide content in recorded interneurons.
The cortex and hippocampus are structures of such complexity that the classification of neuronal subtypes1,2,3,4, maps of the connections between them5,6,7,8,9,10,11 and how this circuitry supports cognitive functions12,13,14,15 are still under intense study and the subject of continuing debate. For example, to understand the details and complexities of the circuitry and to coordinate data obtained from many different studies, it is extremely helpful to be able to define and describe the components, but it remains a matter for debate how many different classes of neurons exist, or even whether it is possible to define all neurons as belonging to a specific class. Computational tools that can build and test circuits with varying degrees of complexity are being developed16,17,18, but central to these endeavors is the need for detailed studies of the cell types and of the properties of the connections between them. A large amount of information about the local circuitry of the neocortex of adult rats has already been collected using the protocol described here10,19,20,21,22. Although the "wiring diagram" is far from complete, some clear patterns or rules have emerged. Moreover, although some details vary, these rules are common to two mammalian species (rat and cat) and across several neocortical regions, allowing the development of basic building blocks that are likely to be equally applicable to human neocortex. The technique described here is used to extend our understanding of the functional map of cortical circuitry by identifying the presynaptic and postsynaptic neurons involved in connections in the regions that have not been studied in detail before, using a protocol that allows excellent tissue preservation and remarkable staining recovery in adult brain tissue. Data on the local circuitry in CA2 and neuronal properties in this subfield have been collected with this method by combining intracellular electrophysiological recordings (paired recordings with sharp microelectrodes) with biocytin filling, immunofluorescence, histological procedures and highly-detailed neuronal reconstructions, allowing direct comparison with the neighboring CA regions23,24,25.
The technique described in this article has been developed over the years to obtain detailed neuronal anatomy allowing the correct classification of the cells and high quality and accurate reconstructions of both their dendritic and axonal arbors, data that can be correlated with electrophysiological data collected from paired recordings using sharp electrodes. The histological protocol has been optimized to preserve the ultrastructure of the neurons and obtain excellent recovery of both dendritic (including spines) and axonal arbors. For example, the principle of the double fixation technique by firstly immersing in a fixative solution and secondly post-fixing in osmium tetroxide gives a good contrast for light microscopy26. A small amount of glutaraldehyde and picric acid solution are added to the fixative solution to enhance antibody penetration and to preserve the cells' ultrastructure as suggested in a previous study27. The permeabilization of the brain slices using the freeze-thaw method combined with cryo-protection with sucrose, rather than a traditional detergent, also provides optimal preservation of the tissues for detailed morphological analysis of the recorded cells. In addition, the visualization particularly of very fine structures is improved by reducing background staining, with the incubations with hydrogen peroxide (H2O2) and sodium borohydride (NaBH4). Adding nickel chloride (NiCl2) to the horseradish peroxidase (HRP) reaction to obtain a black pigment reaction product also increases the contrast.
The following protocol describes the procedures used to ascertain excellent tissue preservation and highly detailed neuronal 3D reconstructions following intracellular recordings in vitro. The description of the slice preparation, intracellular paired recordings using sharp electrodes and subsequent histological procedures used in our laboratory have been previously reported28. Although the protocol applies to the cells filled intracellularly with biocytin in 450–500 μm thick slices, the same protocol may be used following whole-cell recordings. However, the use of thinner slices will result in less complete reconstructions of the cells.
All procedures used throughout this study were carried out according to the British Home Office regulations with regard to the Animal Scientific Procedures Act 1986.
1. Determination of Calcium Binding Protein or Protein Content of Interneurons and Biocytin Visualization Following Electrophysiological Recordings and Biocytin Filling
Note: At the end of the electrophysiological recordings, slices that contain biocytin-filled cells are fixed overnight prior to histological procedures. The fixative solution will be replaced with a single change of 0.1 M phosphate buffer the next morning, if the rest of the procedure is carried out on another day, in order to prevent tissue damage. The fixation solution (4% paraformaldehyde, 0.2% saturated picric acid solution, 0.025% glutaraldehyde solution in 0.1 M phosphate buffer (PB)) must be made fresh on the day of the recordings for best results.
2. 3D Neuronal Reconstructions
NOTE: Neurolucida software is used. Instructions provided below only apply to a specific neurone reconstruction system (Table of Materials). The sections obtained from the cutting are matched prior to the reconstructions using a microscope.
Neurons in hippocampal CA2 were filled with biocytin following electrophysiological recordings (Figures 1Ac, Ad and 1Bc, Bd). The slices were fixed overnight following the recordings and the neurochemistry and morphological characterization of neurons were revealed following the protocol described here.
The calcium binding protein or protein content of filled interneurons was determined by incubating the slices first with primary antibodies and then with fluorescently labelled secondary antibodies. The firing characteristics of the interneurons during the recordings will dictate the choice of primary antibodies used. Avidin-AMCA was used to visualize the biocytin-filled interneuron and anti-mouse fluorescein isothiocyanate (FITC) and goat anti-rabbit Texas Red (TR) were used to characterize the interneuronal neurochemistry (Figure 1Bb).
Following the fluorescence visualization, an HRP protocol was used to reveal the biocytin (Figure 1Ab). The fine detailed anatomy of CA2 interneurons was then drawn in 3D using a neuron reconstruction software (Figure 2 and Figure 3a). A video of a 3D reconstruction of a basket cell recorded and filled in CA2 is displayed in Video. Neuronal reconstructions were considered as complete if both the dendritic and axonal arbors were confined within the depth of the 450-500 μm slice. Poor axonal arbor staining was assessed by the presence of truncated branches with open endings at the top or the bottom of the slice or by a staining that was limited to the axon initial segment and very proximal branches. Figure 4 represents the examples of a good and a poor HRP staining following biocytin visualization.
Morphometric analysis of 3D reconstructions (Figure 3) can be carried out to demonstrate branch complexity, soma surface area and the surface area and volume of the dendritic and axonal arbors.
Figure 1: Neuronal reconstructions of two types of basket cells recorded and filled in the hippocampal CA2 region and correlated electrophysiological data obtained following intracellular recordings in vitro. This figure has been modified from previous studies23,24. SO Stratum Oriens, SP Stratum Pyramidale, SR Stratum Radiatum, SLM Stratum Lacunosum Moleculare. (A) Aa: Reconstruction of a CA2 basket cell with restricted dendritic and axonal arbor using a drawing tube (1000X). The dendrites are in black, and the axon is in red. Ab: Image of the biocytin-filled basket cell following the avidin-HRP protocol described here. Ac: Representative trace of voltage responses to hyperpolarizing and depolarizing current injection of a CA2 basket cell with restricted dendritic and axonal arbor. Ad: Example of a CA2 pyramid to narrow arbor basket cell connections recorded using sharp electrodes. Composite excitatory post-synaptic potential (EPSP) averages show brief train depression apparent during responses to trains of three spikes. (B) Ba: 2D reconstruction of a CA2 basket cell with wide dendritic and axonal arbors using a drawing tube (1000X). The dendritic tree of this basket cell (in black) extended radially through all layers of the CA2 region and horizontally in SO and SP of the CA2 and CA3 regions. One horizontal dendrite also reached the CA1 region. The axon (in red) extended to the CA3 and CA1 regions. Bb: The biocytin-filled (AMCA staining) basket cell was PV-immunopositive (FITC staining) and CB-immunonegative (Texas-Red staining). Bc: Representative trace of voltage responses to hyperpolarizing and depolarizing current injection of a CA2 basket cell with wide dendritic and axonal arbor. Bd: Composite EPSP averages show brief train facilitation apparent during responses to trains of three spikes. Examples of other types of interneurons recorded in CA2 can be found in previous studies25,30. Please click here to view a larger version of this figure.
Figure 2: 3D cell body reconstruction. (A) 3D tracing of the cell body. View of the different contours traced at different z positions whilst focusing through the cell body. (B) 3D view of the different contours. (C) 3D view of the cell body at a different angle. Please click here to view a larger version of this figure.
Figure 3: Morphometry analyses of 3D neuronal reconstructions. (A) 3D reconstruction of a CA2 narrow arbor basket cell. Each dendritic branch is represented by a color (green, blue, red and pink) and axon is in light blue. (B) Dendrogram of the basket cell representing the number of dendritic branches and length of each segment contained within spheres concentric with the soma and at 100 μm intervals from the soma. The colors on the dendrogram correspond to those of the dendrites in A. (C) Example of morphometric analysis performed on CA2 pyramidal cells (adapted from a previous study23). The number of dendritic branches was plotted against the distance from the soma of CA2 pyramidal cells. Please click here to view a larger version of this figure.
Figure 4: Examples of good (A) and poor (B) HRP staining. (A) Biocytin recovery revealed a very well filled interneuron in CA2. The cell body (CB) is darkly stained and has clear outlines. The dendrites are beaded and displayed some spines (represented by red stars). The axonal arbor (A) is dense and presents small boutons. (B) Example of a poor dendritic and axonal staining of 2 pyramidal cells in CA2. The staining of the CB is faint with no clear outline. Poor staining is often associated with shorter electrophysiological recordings resulting in the presence of very few biocytin-filled branches. Please click here to view a larger version of this figure.
Video 1: 3D neuronal reconstruction of a CA2 basket cell with restricted dendritic arbor (also referred to as CA2 narrow arbor basket cell) with its soma in stratum pyramidale, dendrites spanning all layers and axon in CA2 stratum pyramidale and adjacent stratum oriens and radiatum. Very few branches reached the proximal CA3 stratum oriens and CA3 stratum pyramidale. This cell was filled with biocytin following electrophysiological recordings and sections were processed with avidin-HRP following the protocol described here. Due to slicing, only the axon within the depth of the slice was recovered, though the dendrites are intact. Dendrites are in dark pink and axon in white. Layer and region boundaries have been added at the beginning of the video. 3D reconstruction by Georgia Economides- 3D video by Svenja Falk. The video was recorded with a neuron reconstruction software as stated in Step 2.17 and edited with a video editing software. Link to the video:30. Please click here to download this video.
|4% paraformaldehyde, 0.2% saturated picric acid solution, 0.025% glutaraldehyde solution in 0.1 M phosphate buffer (PB)
|0.1M Phosphate Buffer pH 7.6
|Add 100 mL of stock 1 M Phosphate Buffer to 900 mL of distilled water
|Phosphate buffered saline (PBS) pH 7.4/7.5
|Add 10 mL of 0.1M phosphate buffer, 0.2 g of KCl and 8.76 g of NaCl to 990 mL of distilled water
|TRIS buffer pH 7.5
|Dissolve 5.72 g of Tris Hydrochloride and 1.66 g of Tris Base in 50 mL of distilled water. Then make up to 1 L with distilled water.
|Buffered glutaraldehyde and paraformaldehyde fixative solution
|4% paraformaldehyde, 0.2% saturated picric acid solution, 0.025% glutaraldehyde solution in 0.1 M Phosphate buffer.
|Solution to be made at least 30 min before use from the ABC kit. Add 1 drop of solution A and 1 drop of solution B to 2.5 mL of PBS.
|Durcupan epoxy resin:
|To make 20 pots: 20 g of component A, 20 g of component B, 0.6 g of component C and 0.4 g of component D-
|Protect the balance from spills by covering the plate with a circle of filter paper. Carefully weigh the reagents into a tripour beaker in the proportions stated above. Mix thoroughly by vigorously stirring using two wooden sticks for at least 5 min. The mixture should become a uniform density dark brown colour. Place the beaker into the oven at ~50 °C for a maximum of 10 min to remove as many air bubbles as possible. NOTE: The resin will start to cure if you leave the beaker in the oven longer than 10 min. Decant the resin out into plastic pots or 5 mL syringes, date them and store in the -20 °C freezer ready for use.
Table 1: Table of solutions.
Electrophysiological recordings in vitro (Figure 1 Ac,d and Bc,d) combined with histochemical and immunohistochemical procedures enable the detailed morphology, calcium binding protein content and identity of adult cortical interneurons recorded to be revealed. In the CA2 region, this technique allowed the study of the local circuitry for the first time and revealed subclasses of interneurons that had not been previously described in CA1 or CA3: wide dendritic and axonal arbor basket cells (Figure 1B), bistratified cells and SP-SR interneurons.
The protocol described here has been optimized to preserve the ultrastructure of the neurons and obtain excellent recovery of both dendritic (including spines) and axonal arbors. Critical steps includes the use of the double fixation technique to enhance contrast for light microscopy26 and the addition of glutaraldehyde and picric acid solution to the fixative solution to enhance antibody penetration and preserve the neuronal ultrastructure27. Gentle freeze-thaw permeabilization gives better preservation of fine structure, while osmication and resin embedding reduce z-plane shrinkage28. In addition, the visualization of very fine structures (fine axons with small boutons for example) is improved by incubating the sections with H2O2 and NaBH4 to reduce background staining. Contrast can also be increased with the addition of NiCl2 to the HRP reaction.
The histological procedure detailed here offers excellent results in terms of reproducibility and reliability. However, the duration of the electrophysiological recordings will determine the quality of the biocytin/fluorescence staining, with shorter recordings usually associated with poor axonal staining. The choice of recording protocols (intracellular recordings using sharp electrodes vs. whole-cell patch clamping) may also influence biocytin retention and preservation of fine anatomy.
While the difficulties encountered in preserving fine structure during histological processing described here and the time taken to reconstruct at 100X magnification (1-4weeks depending on the complexity of the axon) are appreciated, this method gives an accurate representation of dendritic and axonal diameters. The use of less demanding protocols to reveal biocytin-labelling is understandable, however, these often preclude clear visualization of fine axonal branches. Detergents, to promote entry of Avidin-HRP to reveal the biocytin and antibodies, are often necessary in thick sections, but can disrupt fine structure. Neuroscientists search constantly for semiautomatic methods of reconstruction, but, for now and for axons especially, biocytin-HRP with manual reconstruction remains the gold standard31.
Highly detailed neuronal reconstructions, especially accurate drawings of axonal boutons and nodes, the presence or absence of myelin and more generally the drawing of complete axonal arbor, with the representation of accurate axon-diameter changes along its length, provide further information for accurate identification of a distinct type of interneurone. Although many interneurons may not fit exactly into a specific class, the technique described above provides correlated data on neuronal electrophysiological properties, the short-term plasticity associated with a specific type of connection and detailed neuronal reconstructions, allowing the wiring diagram, in the CA2 region, for example, to be studied in detail.
Fine, detailed structure is often simplified in computational models. While understandable, this results in the loss of the information that could prove critical in the future. Analysis of detailed 3D reconstructions with parallel synaptic data will allow the addition of further criteria for interneuronal classification. Data can be deposited in public repositories and used by modellers to explore the outcome of sporadic changes in axon diameter and myelination on action potential propagation computationally.
The authors have nothing to disclose.
This research has received funding from Novartis Pharma (Basel) and the Medical Research Council awarded to Prof Alex Thomson, the Biotechnology and Biological Sciences Research Council (BBSRC- BB/G008639/1), the Physiological Society, the European Union's Horizon 2020 Framework Programme for Research and Innovation under the Specific Grant Agreement No. 720270 (Human Brain Project SGA1) and under the Specific Grant Agreement No. 785907 (Human Brain Project SGA2). We are extremely grateful to Prof Alex Thomson for setting up the protocol in other cortical regions, securing funding and for her continuous support for this project. Contributions made by lab-members who helped optimising the recovery protocol and reconstructed CA2 neurones are gratefully acknowledged: J. Deuchars, H. Pawelzik, D. I. Hughes, A. P. Bannister, K. Eastlake, H. Trigg, N. A. Botcher.
|Avidin-7-amino-4-methylcoumarin-3-acetic acid (Avidin-AMCA) antibody- 20.8 mg/mL
|Biocytin ≥98% (TLC)
|3,5 diaminobenzidine (DAB) tablet, To prepare 5 mL
|Durcupan epoxy resin A
|Durcupan epoxy resin B
|Durcupan epoxy resin C
|Durcupan epoxy resin D
|Ethanol, puriss. p.a., absolute, ≥99.8%
|Glutaraldehyde solution, grade I, 25 % in H2O
|Goat anti-mouse fluorescein isothiocyanate (FITC)- 14.3 mg/mL
|Goat anti-rabbit Texas Red (TR)- 3.3 mg/mL
|Hydrogen peroxide, 30% solution
|Immersion oil, viscosity 1.250 cSt (lit.)
|Nickel chloride (NiCl2 . 6H2O)
|Osmium tetroxide, for electron microscopy, 4% in H2O
|Paraformaldehyde, reagent grade, crystalline
|Picric acid, moistened with water, ≥98%
|Phosphate buffer 1 M
|Propylene oxide (C3H6O), 99%
|Trizma Hydrochloride, ≥99.0%
|Trizma base, ≥99.9%
|Vectashield Antifade Mounting Medium, , refractive index 1.45
|Vectastain Elite ABC HRP kit
|C4A Cupped aluminium planchettes
|GA-MA & ASSOCIATES, INC.
|Leica DMR microscope
|X-Cite 120PC Q fluorescence light source
|Leica DFC450 digital microscope camera
|Rapidograph technical drawing pen 0.18mm
|London graphics centre
|0.18mm rapidograph nib
|London graphics centre
|Rapidograph technical drawing pen 0.25mm
|London graphics centre
|0.25mm rapidograph nib
|London graphics centre
|Neurolucida system including PC workstation, stage, camera and joystic for XYZ stage control
|Microbrighfield (MBF) Bioscience
|Neurolucida software version 2017
|Microbrighfield (MBF) Bioscience
|CorelDRAW graphics Suite X5
|Video editing software
|Adobe Premiere Pro
|Glass vials 14 mL
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