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 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 investigate the hippocampal region. Accordingly, the use of animal models that resemble human cerebral disorders related to hippocampal functions, such as Alzheimer's disease, epilepsy, major depression, and stress, continues to grow.
In rodents, the hippocampus is a curved-shaped structure starting from near the medial septum towards the ventral temporal cortex. Along its longitudinal axis, the hippocampus can be divided into three different regions, each one related to specific circuitry1. The upper part constitutes the dorsal/septal hippocampus, the lower part constitutes the ventral/temporal hippocampus, and the area between them is considered the intermediate hippocampus. There is extensive literature covering the differences in cellular projections to each part, as well as reports of specific cognitive aspects processed by each5,6. Regarding its internal organization, the hippocampal regions can be separated by its functional areas. The Cornu ammonis (CA) area is subdivided in CA1, CA2, and CA3 and extends through the superior part of the hippocampus, above the dentate gyrus (DG) and the subiculum, which are the most internal hippocampal parts (Figure 1). The synapses located in these regions undergo continuous rearrangement, showing neurogenic and plastic processes throughout life3. Several studies have already shown that distinct experimental manipulations in the hippocampus result in cognitive disability7. Regarding the assessment of biochemical and molecular alterations, techniques using acute brain slices are an excellent tool to improve the knowledge regarding different aspects of the hippocampus.
Due to its precision and reproducibility, many studies that explored aspects of neurotransmission-related phenomena (enzyme activity, uptake, or release) used transverse AHS from resected hippocampus obtained by tissue chopper8,9,10,11,12. This slicing technique followed by uptake assessment is suitable for sophisticated neurochemical experiments that require the transporter activity from hippocampal tissue to be preserved. For that, the employment of a tissue chopper is preferable, since it is faster than the vibratome and provides the AHS in a proper time for experimental use with suitable accuracy.
The excitatory neurotransmission in the brain is accomplished by glutamate, the most abundant neurotransmitter, including in the hippocampus, which is dependent on glutamate signaling to a greater extent13,14. This neurotransmitter abundance is tightly controlled in the extracellular environment. Inside intracellular vesicles, however, it can reach up to 100 mM15. Once released in the synaptic cleft, glutamate is not metabolized and needs to be removed in order to avoid excitotoxicity, usually triggered as a response to an overload of glutamate14,16. The only mechanism separating toxicity from normal signaling is sodium-dependent transport through the activity of proteins located in the plasma membranes of, majorly, glial cells14,17,18,19. These transporters [GLAST (EAAT1) and GLT-1(EAAT2)] tightly regulate extracellular glutamate levels and can be modulated by a wide range of factors, such as DNA transcription, mRNA splicing and degradation, protein synthesis and targeting, amino acid transport activity, and ion channel activities20,21,22,23. Accordingly, their activity can be measured by the transport of radiolabeled substrate, as glutamate.
The use of radiolabeled substrates represents a preferable method for quantifying transporter activity since they allow tracing dynamic mechanisms such as transport across cell membranes. Besides their high sensitivity and specificity, the advantages of radiotracer experiments include their simplicity and small expense compared to competing technologies such as mass spectrometry24. Also, by using only small amounts of tracer, physiological levels of substrates are not altered, thus providing a more representative picture of the real metabolic activity scenario.
The availability of ex vivo experimental approaches is critical to support basic research on identifies novel molecular targets and drug discovery activities. Thus, considering the relevance of the glutamate uptake for glutamatergic system homeostasis and the high predominance of glutamatergic synapses in the hippocampus, this protocol demonstrates how to assess glutamate uptake activity in a fast and easy-to-reproduce method using transverse AHS from the resected hippocampus. This assay uses radiolabeled L-[3H]-Glutamate, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with specific or customized substrates, over a wide range of reaction conditions25.
Acute brain slices present many advantages and have been used to support function change under pharmacological and genetic manipulations26,27,28. Their use benefits from the following: (i) the neurochemical functionality conservation and cell-to-cell interactions; (ii) the possibility to perform numerous pharmacological and genetic manipulations to investigate pathways underlying neuronal and glial functions; (iii) precise control of the extracellular environment; and (iv) good experimental access to different hippocampal areas (such as CA1, CA3 or DG), which are kept in the same slice depending on the slicing method. Considering that distinctive slice preparation protocols might change the hippocampal regions exposed, this protocol proposes a standard technique for obtaining transverse AHS from the resected hippocampus. This simple-to-perform protocol may be used in rodent models and may allow several ex vivo approaches investigating neurochemical dynamics in different backgrounds or after in vivo manipulations29,30 (Figure 2).
Protocol
Representative Results
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<…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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
Materials
| #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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