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.
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 | Vector laboratories | A2008 | |
Biocytin ≥98% (TLC) | Sigma | B4261 | |
3,5 diaminobenzidine (DAB) tablet, To prepare 5 mL | Sigma | D4293 | |
Durcupan epoxy resin A | Sigma | 44611 | |
Durcupan epoxy resin B | Sigma | 44612 | |
Durcupan epoxy resin C | Sigma | 44613 | |
Durcupan epoxy resin D | Sigma | 44614 | |
Ethanol, puriss. p.a., absolute, ≥99.8% | Sigma | 32221 | |
Gelatin | Sigma | 48723 | |
Glutaraldehyde solution, grade I, 25 % in H2O | Sigma | G5882 | |
Glycerol, ≥99% | Sigma | G5516 | |
Goat serum | Sigma | G9023 | |
Goat anti-mouse fluorescein isothiocyanate (FITC)- 14.3 mg/mL | Sigma | F2653 | |
Goat anti-rabbit Texas Red (TR)- 3.3 mg/mL | Invitrogen | T2767 | |
Hydrogen peroxide, 30% solution | Sigma | H-1009 | |
Immersion oil, viscosity 1.250 cSt (lit.) | Sigma | I0890 | |
Nickel chloride (NiCl2 . 6H2O) | Sigma | N5756 | |
Osmium tetroxide, for electron microscopy, 4% in H2O | Sigma | 75632 | |
Paraformaldehyde, reagent grade, crystalline | Sigma | P6148 | |
Picric acid, moistened with water, ≥98% | Sigma | 197378 | |
Phosphate buffer 1 M | Sigma | P3619 | |
Propylene oxide (C3H6O), 99% | Alfa Aesar | 30765 | |
Sodium tetrahydroborate | VWR | 27885.134 | |
Sucrose | Fisher scientific | S/8600/53 | |
Trizma Hydrochloride, ≥99.0% | Sigma | T5941 | |
Trizma base, ≥99.9% | Sigma | T6066 | |
Vectashield Antifade Mounting Medium, , refractive index 1.45 | Vector laboratories | H-1000 | |
Vectastain Elite ABC HRP kit | Vector laboratories | PK6100 | |
Equipment used | |||
Vibratome | Agar Scientific | ||
C4A Cupped aluminium planchettes | GA-MA & ASSOCIATES, INC. | ||
Leica DMR microscope | Leica Microsystems | ||
X-Cite 120PC Q fluorescence light source | Excelitas Technologies | ||
Leica DFC450 digital microscope camera | Leica Microsystems | ||
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 | Corel | ||
Video editing software | Adobe Premiere Pro | ||
Glass vials 14 mL | Fisher scientific |