Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Summary

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Abstract

Glutamate removal from the extracellular space by high-affinity Na⁺-dependent transporters is essential to ensure that the brain's intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats' experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 µm thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[³H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Introduction

The hippocampus, a brain structure buried deep in the medial temporal lobe of each hemisphere, where high cognitive functions lie, is one of the most studied entities of the central nervous system (CNS). The function of the hippocampus is strongly related to declarative and spatial memory. This structure also plays a part in emotional behavior and in the regulation of hypothalamic functions^1,2,3,4. Ever since it was confirmed, important mechanisms of memory formation and storage take place in this region and the field began to deeply investi....

Protocol

All procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the local Ethics Committee (project approval # 33732/CEUA-UFRGS). All efforts were made to minimize discomfort and the number of animals used in the experiments.

1. Preparing Hank's Balanced Salt Solution (HBSS)

1. In a 1 L beaker, add approximately 0.5 L of sterile or double-distilled water and begin stirring vigorously on a magnetic stirrer. Add the following HBSS components (in mM): 137 NaCl, 0.63 Na₂HPO₄, 4.17 NaHCO₃, 5.36 KCl, 0.44 KH₂PO₄, 0.41....

Results

Glutamate uptake is one of the most important mechanisms controlling neurotransmission in the brain. The hippocampus, specifically, is a critical place in glutamate signaling, being an important hub connecting memory, cognition, and emotions in the brain. Following the protocol, adult male Wistar rats were used to generate representative results. Animals were anesthetized using isoflurane 3% until unconscious. After dissecting the brain, hippocampi were removed and placed in the chopper table perpendicularly to the blade.......

Discussion

The presented protocol shows an easy-to-perform glutamate uptake assessment using hippocampal slices. The results demonstrate that AHS regularly takes up around 60 fmol of radiolabeled L-[³H]-Glutamate, that the thickness of the slice (protein amount) did not influence the L-[³H]-Glutamate uptake (data not shown), and that the dorsal, intermediate, and ventral parts of the hippocampus exhibited similar performances when obtained from naïve adult male Wistar rats (Figure 3A<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#11 scalpel blade	Swann-Morton	525
100 mm glass petri dish	Common suppliers
110 mm diameter Whatman Filter	Sigma Aldrich	WHA1001110
42.5 mm diameter Whatman Filter	Sigma Aldrich	WHA1001042
24-well cell culture plate	Falcon	353047
Becker	Common suppliers
Blades for the tissue chopper	Wilkinson	3241
Bone rongeur	Erwin Guth	9,00,005
CaCl2	Sigma Aldrich	C4901
D-[2,3-3H]-Aspartic acid	PerkinElmer	NET581001MC	11.3 Ci/mmol (37 MBq)
D-Glucose	Sigma Aldrich	G8270
N-Methyl-D- Glucamine	Sigma Aldrich	M2004
HEPES	Sigma Aldrich	H3375
Hidex 300 SL	Hidex Oy.	Super Low Level #425-020
Iris scissors	Erwin Guth	8,00,040
Isoflurane	Cristalia (São Paulo, Brazil)	4,10,525	1 mL/mL
KCl	Sigma Aldrich	P3911
KH2PO4	Sigma Aldrich	P0662
L-[3,4-3H]-Glutamic Acid	PerkinElmer	NET490005MC	49.7 Ci/mmol (185 MBq)
MgCl2	Sigma Aldrich	M8266
MgSO4	Sigma Aldrich	M7506
Na2HPO4	Sigma Aldrich	S9763
NaCl	Sigma Aldrich	S9888
NaHCO3	Sigma Aldrich	S5761
Plastic Pasteur pipette	Common suppliers
Scintillation liquid	PerkinElmer	1200.437 for 1 x 5 Liter	Optiphase HiSafe 3
Small surgical scissors	Erwin Guth	8,00,040
Small tweezers	Erwin Guth	6,00,131
Spare chopping discs for the chopper	Common suppliers
Standard scissors	Erwin Guth	8,00,010
Thin brushes (size 0 or 2)	Common suppliers
Thin double-ended spatula	Erwin Guth	470.260E
Tissue Chopper	Ted Pella, Inc.	10180