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Biochemistry

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

Published: October 2nd, 2021

DOI:

10.3791/62292

1Graduate Program in Biochemistry, Universidade Federal do Rio Grande do Sul, 2Department of Biochemistry, Universidade Federal do Rio Grande do Sul, 3Department of Biological Sciences, Universidade Federal de Ouro Preto, 4Programa de Pós-Graduação em Ciências Biológicas, Universidade Federal de Ouro Preto

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.

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.

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 functions1,2,3,4. Ever since it was confirmed, important mechanisms of memory formation and storage take place in this region and the field began to deeply investi....

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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.......

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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.......

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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<.......

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Authors receive financial support from the Brazilian National Institute of Science and Technology in Excitotoxicity and Neuroprotection [465671/2014-4], CNPq [438500/2018-0], and [152189/2020-3], FAPERGS/CAPES/DOCFIX [18/2551-0000504-5], CAPES [88881.141186/ 2017-01], CNPq [460172/2014-0], PRONEX, FAPERGS/CNPq [16/ 2551-0000475-7], FAPERGS/MS/CNPq/SESRS-PPSUS [30786.434.24734.23112017]

UFOP - MODALIDADE: "EDITAL PROPP 19/2020 AUXÍLIO À PUBLICAÇÃO DE ARTIGOS CIENTÍFICOS - 2020", PROCESSO N.: 23109.000929/2020-88

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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

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