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 properties^2,3,4, anatomy⁵, genetic diversity^6,7,8, and behavioral function^9,10. 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 defined^12,13,14. In addition to regional specific differences, normal aging has been shown to have dramatic effects on synaptic transmission^15,16,17, 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 plane^{21,22,23,24,25}. Where the whole dorso-ventral extent has been assessed, a tissue-chopper is used to maintain the transverse extent^4,26, 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 mice^30,31. 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

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), bubbling with carbogen (95% O₂/5% CO₂).
2. Collect an adult rat from its home cage and place it for ~30 minutes in a holding cage in the procedure room to acclimatize to noise and light levels.
3. Prepare the dissection tools (Figure 1A), the perfusion area and the injectable anesthetic (approx. 1 mL of 200 mg/mL sodium pentobarbital for a final concentration of 100 mg/kg).
4. Prepare an appropriate anesthesia chamber by placing a small swab of tissue paper or cotton wool inside. Introduce 1-2 mL of volatile isoflurane anesthetic to the absorbent material in the chamber.
5. Place the rat in an anesthesia chamber to sedate. Monitor breathing until the breathing rate drops to ~1 shallow breath per second.
6. At this point, start bubbling semi-frozen sucrose-ACSF with carbogen on ice for use during slicing.
7. Weigh the rat and note its weight.
8. Terminally anesthetize the rat by injecting the prepared sodium pentobarbital into the intraperitoneal cavity. The dose of sodium pentobarbital should be 100 mg/kg, calculated from the previously taken weight and stock concentration of drug. Place the rat in a holding chamber and allow 0.5-5 minutes for the onset of terminal anesthesia.
9. Confirm cessation of reflexes: test both corneal blink (touch the pupil) and hind-paw pinch (lift the leg and pinch the hind-paw) reflexes using a blunt probe (i.e., rounded forceps). Once reflexes have ceased, pin the rat to the polystyrene or cork surgical board using hypodermic needles.
10. Open the chest cavity and place the cannula at the base of the left ventricle of the heart. Puncture the right atrium and immediately start perfusion with the ice-cold (0-1 °C) sucrose-ACSF (using a peristaltic pump at 50 mL/minute).
11. Once full exchange of fluids and cooling of the body has occurred (<5 minutes), remove the cannula and pins, and then decapitate using a guillotine.
12. Carefully and rapidly remove the skull using bone scissors and Rongeur bone tools (Figure 1A,1B).
    1. Start by making 2x bilateral cuts through the foramen magnum using the bone scissors and remove the skull to the lambda suture with the Rongeurs. Cut carefully along the midline suture with the bone scissors to just behind the eyes.
    2. Make 2x bilateral cuts through the skull, perpendicular to the midline. Using the Rongeurs, open the skull along the midline. Take extra care to remove pia mater using fine scissors or a hooked needle.
13. Scoop the brain out of the skull using a blunt spatula, severing the cranial and optic nerves with side to side compression. Place the brain into carbogenated, semi frozen (0– 1 °C) sucrose-ACSF for 1-2 minutes prior to slicing.

2. Preparation of brain slices from dorsal hippocampus

1. Remove the perfused brain from the semi-frozen (0–1 °C) sucrose-ACSF and place in a glass Petri dish lined with filter paper. Place the brain onto its ventral surface.
2. Using a scalpel (No. 22 blade), remove the posterior portion of the brain at ~10° from vertical to create a flat surface to glue the brain to the stage (Figure 1C).
3. Apply a small amount of cyanoacrylate glue to the stage of the vibratome. Spread the glue to make a thin film approximately 50% larger than the cross-sectional area of the cut surface of the brain.
4. Lift the brain out of the glass dish onto a spatula, cut side down, using a paintbrush to guide the tissue. Blot the brain with a piece of tissue to remove excess ACSF and slide the brain, cut surface down, onto the center of the glue. Using a Pasteur pipette, add 1-2 mL of ice-cold sucrose-ACSF over the brain to remove glue away from brain block.
5. Place the brain into the slicing chamber and flood with semi-frozen sucrose ACSF. Use a spoon or spatula to keep excess ice away from brain block, and then carbogenate (Figure 1D).
6. Move the blade of the vibratome into position: ~1 mm from the dorsal surface of the brain and vertically ~1 mm anterior to bregma. Ensure that the blade is fully submerged, and remove bubbles using a paintbrush.
7. Start slicing the brain. To trim down to the dorsal hippocampus, use a speed of 0.1-0.2 mm/s, with a horizontal blade movement of 1-1.5 mm and a reciprocal oscillatory rate of ~90 Hz. When slicing the dorsal hippocampus, reduce the speed to 0.05-0.1 mm/s.
8. Collect slices of dorsal hippocampus (nominally 3-4 full slices or 6-8 hemisected slices) per brain. If longitudinal slices of ventro-medial hippocampus are required, continue slicing. Once the dorsal hippocampus has been sliced, there is no need cut extra tissue beyond approximately the position of the 3^rd ventricle. Stop the vibratome, separate the slice with a bent hypodermic needle, and collect in the base of the slicing chamber.
   NOTE: Slices can be 250-500 µm thick, depending on experimental requirements. For recordings from the dorsal hippocampus, typically use 400 µm thick slices to preserve as much of the local network, whilst allowing the suitable microscopy conditions.
9. Trim the slices to contain only the hippocampus and overlying cortex under a dissecting microscope. Transfer slices to the pre-warmed 35 °C chamber with the anterior surface facing up.
10. Determine the storage chamber based on the experiment to be performed. To obtain high quality whole-cell patch-clamp or extracellular field recordings close to the slice surface in submerged recording chambers, store in submerged chambers. Alternatively, store in a liquid/gas interface chamber for recordings of oscillatory network activity or interface extracellular field recordings.
11. For submerged storage conditions, allow slices to recover at 35 ºC for 30 minutes from the time of the last slice entering the storage chamber. This allows for reactivation of metabolic processes and re-sealing of cut neurites. After 30 minutes, transfer the storage chamber to room temperature.

3. Recording the dorsal hippocampal neurons

1. Fabricate recording patch pipettes from capillary glass. This protocol uses 1.5 mm outer diameter, 0.86 mm inner diameter borosilicate glass with filament, which yields a tip resistance of 3-5 MΩ when filled with intracellular solution (Table 1). Keep intracellular solutions chilled on ice to prevent degradation of energetic components and filtered prior to use (syringe filter, pore size: 0.2 μm).
2. Carbogenate recording ACSF and pre-warm in a water bath (35-40 ºC). Deliver the carbogenated and pre-warmed ACSF to the recording chamber via perfusion tubing assisted by a peristaltic pump. Start perfusion several minutes prior to transferring a slice into the chamber.
3. Stop the perfusion and transfer a brain slice to the recording chamber with the anterior surface facing up. Hold the slice in place with a platinum ring with single fibers of silk attached to form a “harp” shape. Position the slices so that stratum pyramidale of CA1 runs perpendicular to the axis of the first recording pipette.
4. Restart the flow of carbogenated and pre-warmed (35-40 ºC) recording ACSF (Table 1) at an optimal rate of 6-8 mL∙min^-1.
   NOTE: High flow rates (6-8 mL∙min^-1) are optimal for maintaining network activity in slices³². Lower flow rates (i.e., 2-3 mL∙min^-1) can be used to maintain slice stability for imaging experiments or where biologically relevant network activity is not required.
5. Assess slice quality using infrared differential inference contrast (IR-DIC) optics with 40x objective magnification (visualized with a CCD camera). Assume good slice quality if a large number of ovoid-shaped, moderately contrasted CA1 PCs can be seen in str. pyramidale at depths of 20-30 μm below a smooth and lightly dimpled surface (Figure 2A). Poor quality slices contain large numbers of highly contrasted, shrunken or swollen cells, with an uneven slice surface.
6. Fill patch pipettes with an intracellular solution (e.g., based on an intracellular [Cl^-] of 24 mM) to allow comparison with other published data.
7. Perform whole-cell patch-clamp recordings as previously described²². Exclude cells from analysis if the membrane potential (V_M) on break-through is more depolarized than -50 mV, the series resistance is >30 MΩ; or the series resistance changes by >20% over the course of the recording. Under these recording conditions, series resistance is typically in the range of 8 – 25 MΩ and stable for up to 1 hour.
8. To examine CA1 intrinsic excitability from the dorsal hippocampus, test intrinsic physiological properties with whole-cell recordings with the following protocols in current-clamp configuration:
   1. From the resting membrane potential with no bias current applied, apply small (-10 pA, 500 ms) current steps repeated 30 times.
   2. From -70 mV with bias current applied, apply hyper to depolarizing current steps of 500 ms duration (-100 to +400 pA, 25 pA steps) with 3 repetitions of a family of traces.
   3. Apply a sinusoidal wave of 100 pA peak-to-peak amplitude, with variable frequency from 0.1 to 20 Hz. Repeat 3 times.
   4. Apply a 5x 2 nA, 2 ms stimuli to drive action potentials at 20, 40, 60, 80, 100 Hz. 10 sweeps per frequency.
   5. From a -70 mV voltage-clamp, apply 5 minutes of spontaneous excitatory postsynaptic currents (EPSC) for recording.
9. To reseal cells for histological analysis following successful recording, produce outside-out patches by slowly retracting the patch pipette. When an increase in series resistance is observed from experimental levels to >1 GΩ as measured by a -5 mV test pulse, raise the holding potential to -40 mV and retract the pipette fully.
10. Perform additional recordings in the same slice to satisfy the required statistical power of the experimental design.
11. Remove brain slices containing recorded neurons from the recording chamber, place in 24-well plate, replace the ACSF with 4% paraformaldehyde (in 0.1 M phosphate buffer) and leave overnight.
12. The next day, replace the PFA with 0.1 M phosphate buffer and store until histological processing. Visualize cells with fluorescent-conjugated streptavidin as previously described²².

النتائج

The protocol described above allows for the preparation of viable slices from the septal pole of the dorsal hippocampus in mature rats. A key factor in this protocol is the perfusion of chilled sucrose-ACSF, prior to slice preparation, resulting in healthy CA1 PCs proximal to the slice surface. The quality of the slice produced is assessed visually under IR-DIC optics, and healthy cells identified as having large, ovoid-shaped cell bodies are located throughout the full extent of stratum pyramidale, from the com...

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