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....

Protocol

All animals were generated and maintained according to the Home Office and Institutional guidelines (HO# P135148E). All rats were maintained on a 12 h light/dark cycle and given ad libitum access to food and water.

1. Transcardial perfusion of chilled ACSF

1. Prior to all experiments, place ~200 mL of sucrose-ACSF (Table 1) in the freezer at -20 °C (until semi-frozen, for slicing) and a further ~100-200 mL of filtered sucrose-ACSF on ice (for perfusion),.......

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.......

Materials

Name	Company	Catalog Number	Comments
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