Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents
Summary
The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.
Abstract
Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (>60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.
Introduction
The hippocampus is arguably the most well studied structure in the mammalian brain, due to its relatively large size and prominent laminar structure. The hippocampus has been implicated in a number of behavioral processes: spatial navigation, contextual memory, and episode formation. This is, in part, due to the relative ease of access to the dorsal portions of the hippocampus in rodents for in vivo analysis. Indeed, the major output cells are typically less than 2 mm from the pial surface.
In rodents, the hippocampus is a relatively large structure, formed of an invagination of the telencephalon extending from the dorsal septum to the ventral neocortex. It is composed of 2 major regions: the dentate gyrus and the cornu ammonis (CA); the latter of which is divided into 3 well-described sub-regions (CA1-3) that extend into the dentate gyrus hilus (formerly known as CA4), based on connectivity, cellular anatomy, and genetic properties1. This structure is maintained along the dorso-ventral extent of the hippocampus, albeit with major variations in synaptic properties2,3,4, anatomy5, genetic diversity6,7,8, and behavioral function9,10. Of the CA regions, the CA1 subfield is composed largely of glutamatergic CA1 pyramidal cells (CA1 PCs), for which 3 subtypes have been defined11, and inhibitory interneurons that make up ~10% of neurons, but are highly diverse with over 30 subtypes defined12,13,14. In addition to regional specific differences, normal aging has been shown to have dramatic effects on synaptic transmission15,16,17, anatomy18, and genetic profile19. The current gold-standard method to assess the intricacies of cellular and synaptic properties in a controlled manner is through the use of whole-cell patch-clamp recordings from acute brain slices20.
The understanding of hippocampal function is based largely on dorsal manipulation due to the ease with which it is accessed surgically or anatomically for behavioral tasks, implantation of electrodes or imaging windows, or viral plasmid expression. In many studies additionally, these procedures are performed with late-juvenile or adult rodents to prevent variability in brain structure during development. Despite this, many approaches to examine cellular and subcellular electrophysiology are performed in early- to mid-juvenile rodents, from mostly the ventro-medial portion of the hippocampus in its transverse plane21,22,23,24,25. Where the whole dorso-ventral extent has been assessed, a tissue-chopper is used to maintain the transverse extent4,26, or the experiment has been performed in young rats27 or mice28. Furthermore, cooling of tissue prior to dissection of the brain is known to preserve hippocampal structure in rats29 and neocortical neurons in mice30,31. Nevertheless, there is a paucity of detail regarding the production of brain slices from the dorsal transverse axis of the hippocampus, as generated by modified coronal slices, in mature rats.
This protocol describes an approach by which whole-cell patch-clamp recordings can be obtained from single or pairs of neurons in modified coronal slices of dorsal hippocampus from aged rats, followed by post-hoc morphological identification. Healthy brain slices are obtained following transcardial perfusion of chilled artificial cerebrospinal fluid (ACSF), facilitating measurement of electrophysiological properties from CA1 PCs and local interneurons.
Protocol
Representative Results
Discussion
Here, a protocol is described to produce high-quality brain slices from the dorsal extent of the CA1 of the hippocampus, allowing for recordings from multiple viable neurons within this region. The combinatorial approach of whole-cell recording from near-coronal slices followed by neuron visualization is critical to the confirmation of cell viability and identity.
This protocol reliably produces viable slices for 2 major reasons. Firstly, the modification to the cutting angle, as a deviation f…
Declarações
The authors have nothing to disclose.
Acknowledgements
The author wishes to thank Prof. David JA Wyllie, Dr. Emma Perkins, Laura Simoes de Oliveira, and Prof. Peter C Kind for helpful comments on the manuscript and protocol optimisation, and The Simons Initiative for the Developing Brain for providing publication costs.
Materials
| Acquisition software | Molecular Devices | pClamp 10 | |
| Adult rats | Various | n/a | Any strain of adult rat (60 days and older) |
| Amplifier | Molecular Devices | Axopatch 700B | |
| Analysis software | Freeware | Stimfit | https://github.com/neurodroid/stimfit |
| Bone Scissors | FST | 16152-12 | Littauer style |
| Capillary Glass | Harvard Apparatus | 30-0060 | Borosilicate glass pipettes with filament 1.5 mm outer diameter, 0.86 mm inner diameter. |
| Carbogen | BOC | Various | 95% O2/5% CO2 |
| CCD camera | Scientifica | SciCamPro | https://www.scientifica.uk.com/products/ |
| Chemicals/Reagents | Sigma Aldrich | Various | All laboratory reagents procured from Sigma Aldrich. |
| Cyanoacrylate (i.e. RS Pro 3) | RS Components | 918-6872 | Avoid gel based cyanoacrylate formulations |
| Digitizer | Molecular Devices | Digidata 1550B | |
| Dissection pins/needles | Various | Various | Use 16 gauge needles (above) |
| Electrophysiology system | Scientifica | SliceScope | https://www.scientifica.uk.com/products/ scientifica-slicescope |
| Fine iris scissors | FST | 14568-12 | With Tungsten-Carbide tips |
| Glass Petri dish | Fisher Scientific | 12911408 | |
| Hypodermic needles | BD Healthcare | Various | 16, 18, and 22 gauge |
| Isofluorane 100% W/W (i.e.IsoFlo) | Zoetis | 50019100 | |
| Mayo-type scissors | FST | 14110-17 | Blunt tips |
| Micromanipulators | Scientifica | Microstar | https://www.scientifica.uk.com/products/scientifica-microstar-micromanipulator |
| Paintbrush | Art store | n/a | A fine bristled paintbrush, procured from a local art shop. |
| Pasteur pipette | Fisher Scientific | 11546963 | A glass Pasteur pipette, but cut so that the blunt end is used to transfer pipette. |
| Peristaltic pump | Watson Marlow | 12466260 | Single channel peristaltic pump |
| Pipette puller | Sutter Instruments | P-97 | Other models and methods of pipette production would work equally well. |
| Plastic syringes (1, 2, 5 mL) | BD Healthcare | Various | |
| Rongeur bone tool | FST | 16021-14 | |
| Slice holding chamber | Homemade | ||
| Slice weight/harp | Harvard Apparatus | SHD-22L/15 | Alternatives would be suitable. |
| Sodium Pentobarbital (i.e. Pentoject) | Animalcare Ltd | 10347/4014 | 200 mg/mL; other formulations of pentobarbital would be suitable |
| Spatula | Bochem | 3213 | Available from Fisher Scientific |
| Syringe filters | Fisher Scientific | 10482012 | Corning brand syringe filters, 0.22 µm pore diameter. |
| Vibtratome | Leica | 1491200S001 | VT1200S model with Vibrocheck |
| Water Bath | Fisher Scientific | 15167015 | 5 Litre water bath, for example: Grant Instruments™JBA5 scientifica-scicam-pro |
Referências
- Amaral, D. G., Witter, M. P. The three-dimensional organization of the hippocampal formation: a review of anatomical data. Neurociência. 31 (3), 571-591 (1989).
- Moser, M. B., Moser, E. I. Functional differentiation in the hippocampus. Hippocampus. 8 (6), 608-619 (1998).
- Babiec, W. E., Jami, S. A., Guglietta, R., Chen, P. B., O’Dell, T. J. Differential regulation of NMDA receptor-mediated transmission by SK channels underlies dorsal-ventral differences in dynamics of schaffer collateral synaptic function. Journal of Neuroscience. 37 (7), 1950-1964 (2017).
- Papatheodoropoulos, C. Striking differences in synaptic facilitation along the dorsoventral axis of the hippocampus. Neurociência. 301, 454-470 (2015).
- O’Reilly, K. C., et al. Identification of dorsal-ventral hippocampal differentiation in neonatal rats. Brain Structure and Function. 220 (5), 2873-2893 (2015).
- Cembrowski, M. S., et al. Spatial gene-expression gradients underlie prominent heterogeneity of CA1 pyramidal neurons. Neuron. 89 (2), 351-368 (2016).
- Cembrowski, M. S., Wang, L., Sugino, K., Shields, B. C., Spruston, N. Hipposeq: a comprehensive RNA-seq database of gene expression in hippocampal principal neurons. elife. 5, 14997 (2016).
- Leonardo, E., Richardson-Jones, J., Sibille, E., Kottman, A., Hen, R. Molecular heterogeneity along the dorsal–ventral axis of the murine hippocampal CA1 field: a microarray analysis of gene expression. Neurociência. 137 (1), 177-186 (2006).
- Kheirbek, M. A., et al. Differential control of learning and anxiety along the dorsoventral axis of the dentate gyrus. Neuron. 77 (5), 955-968 (2013).
- Kjelstrup, K. B., et al. Finite scale of spatial representation in the hippocampus. Science. 321 (5885), 140-143 (2008).
- Klausberger, T., Somogyi, P. Neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations. Science. 321 (5885), 53-57 (2008).
- Booker, S. A., Vida, I. Morphological diversity and connectivity of hippocampal interneurons. Cell and Tissue Research. 373 (3), 619-641 (2018).
- Freund, T. F., Buzsaki, G. Interneurons of the hippocampus. Hippocampus. 6 (4), 347 (1996).
- Pelkey, K. A., et al. Hippocampal GABAergic inhibitory interneurons. Physiological Reviews. 97 (4), 1619 (2017).
- Barnes, C. A., Rao, G., Foster, T. C., McNaughton, B. L. Region-specific age effects on AMPA sensitivity: Electrophysiological evidence for loss of synaptic contacts in hippocampal field CA1. Hippocampus. 2 (4), 457-468 (1992).
- Potier, B., Jouvenceau, A., Epelbaum, J., Dutar, P. Age-related alterations of GABAergic input to CA1 pyramidal neurons and its control by nicotinic acetylcholine receptors in rat hippocampus. Neurociência. 142 (1), 187-201 (2006).
- Ekonomou, A., Pagonopoulou, O., Angelatou, F. Age-dependent changes in adenosine A1 receptor and uptake site binding in the mouse brain: An autoradiographic study. Journal of Neuroscience Research. 60 (2), 257-265 (2000).
- Pyapali, G. K., Turner, D. A. Increased dendritic extent in hippocampal CA1 neurons from aged F344 rats. Neurobiology of Aging. 17 (4), 601-611 (1996).
- Blalock, E. M., et al. Gene microarrays in hippocampal aging: statistical profiling identifies novel processes correlated with cognitive impairment. Journal of Neuroscience. 23 (9), 3807-3819 (2003).
- Sakmann, B., Neher, E. Patch clamp techniques for studying ionic channels in excitable membranes. Annual Review of Physiology. 46 (1), 455-472 (1984).
- Bischofberger, J., Engel, D., Li, L., Geiger, J. R., Jonas, P. Patch-clamp recording from mossy fiber terminals in hippocampal slices. Nature Protocols. 1 (4), 2075 (2006).
- Booker, S. A., Song, J., Vida, I. Whole-cell patch-clamp recordings from morphologically-and neurochemically-identified hippocampal interneurons. Journal of Visualized Experiments. (91), e51706 (2014).
- Geiger, J., et al. Patch-clamp recording in brain slices with improved slicer technology. Pflügers Archive. 443 (3), 491-501 (2002).
- Aitken, P., et al. Preparative methods for brain slices: a discussion. Journal of Neuroscience Methods. 59 (1), 139-149 (1995).
- Siwani, S., et al. OLMα2 cells bidirectionally modulate learning. Neuron. 99 (2), 404-412 (2018).
- Dong, W. Q., Schurr, A., Reid, K. H., Shields, C. B., West, C. A. The rat hippocampal slice preparation as an in vitro model of ischemia. Stroke. 19 (4), 498-502 (1988).
- Dougherty, K. A. Differential developmental refinement of the intrinsic electrophysiological properties of CA1 pyramidal neurons from the rat dorsal and ventral hippocampus. Hippocampus. 30 (3), 233-249 (2020).
- Foggetti, A., Baccini, G., Arnold, P., Schiffelholz, T., Wulff, P. Spiny and Non-spiny Parvalbumin-Positive Hippocampal Interneurons Show Different Plastic Properties. Cell Reports. 27 (13), 3725-3732 (2019).
- Newman, G. C., Qi, H., Hospod, F. E., Grundmann, K. Preservation of hippocampal brain slices with in vivo or in vitro hypothermia. Brain Research. 575 (1), 159-163 (1992).
- Ting, J. T., Daigle, T. L., Chen, Q., Feng, G. . Patch-Clamp Methods and Protocols. , 221-242 (2014).
- Ting, J. T., et al. Preparation of acute brain slices using an optimized N-methyl-D-glucamine protective recovery method. Journal of Visualized Experiments. (132), e53825 (2018).
- Hajos, N., et al. Maintaining network activity in submerged hippocampal slices: importance of oxygen supply. European Journal of Neuroscience. 29 (2), 319-327 (2009).
- Papaleonidopoulos, V., Trompoukis, G., Koutsoumpa, A., Papatheodoropoulos, C. A gradient of frequency-dependent synaptic properties along the longitudinal hippocampal axis. BMC Neuroscience. 18 (1), 79 (2017).
- Fuller, L., Dailey, M. E. . Preparation of rodent hippocampal slice cultures. (10), 4848 (2007).
- Gee, C. E., Ohmert, I., Wiegert, J. S., Oertner, T. G. Preparation of slice cultures from rodent hippocampus. Cold Spring Harbor Protocols. 2017 (2), (2017).
- Andersen, P. Brain slices – a neurobiological tool of increasing usefulness. Trends in Neurosciences. 4, 53-56 (1981).
- Moyer, J. R., Brown, T. H. . Patch-Clamp Analysis. , 135-193 (2002).
- Dougherty, K. A., et al. Differential expression of HCN subunits alters voltage-dependent gating of h-channels in CA1 pyramidal neurons from dorsal and ventral hippocampus. Journal of Neurophysiology. 109 (7), 1940-1953 (2013).
- Huang, S., Uusisaari, M. Y. Physiological temperature during brain slicing enhances the quality of acute slice preparations. Frontiers in Cellular Neuroscience. 7, 48 (2013).
- Booker, S. A., et al. Presynaptic GABAB receptors functionally uncouple somatostatin interneurons from the active hippocampal network. Elife. 9, 51156 (2020).
- Gloveli, T., et al. Orthogonal arrangement of rhythm-generating microcircuits in the hippocampus. Proceedings of the National Academy of Sciences. 102 (37), 13295-13300 (2005).
- Ordemann, G. J., Apgar, C. J., Brager, D. H. D-type potassium channels normalize action potential firing between dorsal and ventral CA1 neurons of the mouse hippocampus. Journal of Neurophysiology. 121 (3), 983-995 (2019).
- Arnold, E. C., McMurray, C., Gray, R., Johnston, D. Epilepsy-induced reduction in HCN channel expression contributes to an increased excitability in dorsal, but not ventral, hippocampal CA1 neurons. eNeuro. 6 (2), (2019).
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