Published: October 2nd, 2021
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....
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)
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.......
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<.......
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....
|#11 scalpel blade
|100 mm glass petri dish
|110 mm diameter Whatman Filter
|42.5 mm diameter Whatman Filter
|24-well cell culture plate
|Blades for the tissue chopper
|Hidex 300 SL
|Super Low Level #425-020
|Cristalia (São Paulo, Brazil)
|Plastic Pasteur pipette
|1200.437 for 1 x 5 Liter
|Optiphase HiSafe 3
|Small surgical scissors
|Spare chopping discs for the chopper
|Thin brushes (size 0 or 2)
|Thin double-ended spatula
|Ted Pella, Inc.
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