Fresh Brain Slices in Neuroscience Research: Physiological Methods, Advantages And Application Fields

Öz

Abstract

Neuroscience studies aiming to understand the basic properties of neurons and neuronal circuits have expanded over time to include different approaches used to examine the nervous system at different scales. These studies have gained momentum with the development of the methods used. In vivo studies and in vitro cell culture studies are frequently used in neuroscience studies. However, the limitations of in vivo studies and the out of systemic structure of in vitro culture cells are insufficient to explain neural functions. The approach known as fresh brain slices are considered to be suitable preparations for ex-perimental studies of areas out of conventional in vivo and in vitro approaches. Preserved intercellular connections and long enough vitality to allow the study of adaptive and plastic processes have led to brain slices taking an important place in neuroscience studies. Acute brain slice preparation is an ideal model for studying the details of how neurons and neuronal tissue respond to a variety of physiological conditions.
The preparation process of the brain slices differs according to the designed experimental study. In order to obtain high quality brain slices, the protocol of obtaining brain slices and the preparation of artificial cerebrospinal fluid for long-term survival are important processes. During the acquisition of electrophysiological records; to keep the cross section healthy, the preparation of the appropriate environment (pH, temperature, osmolarity), the provision of optical equipment, the provision of mechan-ical tools to stabilize the position of the microelectrodes, and the equipments of amplifying and recording electronic signals are of great importance. In this review, the use of fresh brain slices, necessary equip-ment, experimental procedures and the advantages of the technique, as well as other methods frequent-ly used in electrophysiology studies, are explained.

Keywords: Electrobiophysics, Ex-vivo recording, Field potentional, Patch-clamp

Sinirbilim Araştırmalarında Taze Beyin Kesitleri: Fizyolojik Yöntemler, Avantajları Ve Kullanım Alanları

Öz

Nöronların ve nöronal devrelerin temel özelliklerini anlamayı hedefleyen sinirbilim çalışmaları, sinir sistemini farklı ölçeklerde incelemek için kullanılan farklı yaklaşımları içerecek şekilde zamanla genişlemiştir. Kullanılan metodların geliştirilmesiyle beraber bu çalışmalar hız kazanmıştır. Canlılığın bütünüyle devam ettiği in vivo çalışmalar ve in vitro hücre kültür çalışmaları sinirbilim çalışmalarında sıklıkla kullanılmaktadır. Ancak, in vivo çalışmaların kısıtlayıcılığı ve in vitro kültür hücrelerinin sistemik yapıdan uzak oluşu sinirsel fonksiyonların açıklanmasında yetersiz kalmaktadır. Taze beyin kesitleri olarak bilinen yaklaşım klasik in vivo ve in vitro yaklaşımların erişiminden uzakta kalan alanların deneysel çalışmaları için uygun preparatlar olarak kabul edilmektedir. Hücreler arası bağlantıların korunuyor olması ve canlılığın adaptif ve plastik süreçlerin incelenmesine imkan verecek kadar uzun olması beyin kesitlerinin sinir bilim çalışmalarında önemli yer tutmasına yol açmıştır. Akut beyin kesit hazırlığı, nöronların ve nöronal dokunun çeşitli fizyolojik koşullara nasıl tepki verdiğinin ayrıntılarını incelemek için ideal bir modeldir. Beyin kesitleri çalışmalarında kesitlerin hazırlanma süreci, tasarlanan deneysel çalışmaya göre farklılık göstermektedir. Yüksek kalitede beyin kesitlerinin eldesi için, beyin kesitlerinin elde ediliş protokolü ve canlılığın uzun süre devam edebilmesi için yapay beyin omurilik sıvısının hazırlanması önemli süreçlerdir. Elektrofizyolojik kayıtların elde edilmesi esnasında; kesiti sağlıklı tutmak için uygun ortamın hazırlanması (pH, sıcaklık, osmolarite), optik donanımın sağlanması, mikroelektrotların sabit bir şekilde konumlandırmak için mekanik araçların temini ve elektronik sinyalleri yükseltme ve kaydetme araçları büyük öneme sahiptir. Bu derlemede, elektrofizyoloji çalışmalarında sıklıkla kullanılan diğer yöntemlerin yanı sıra taze beyin kesitlerinin kullanımı, gerekli ekipmanlar, deneysel prosedürler ve tekniğin sunduğu avantajlar açıklanmaktadır.

Anahtar Kelimeler

• Beyin kesitleri
• Ex-vivo kayıt
• Patch-clamp
• Elektrobiyofizik

Kaynakça

1. Referans1. Kerkut GA, and Wheal HW. (Eds.) Electrophysiology of isolated mammalian CNS preparations. Academic Press. 1981.
2. Referans2. Yamamoto C, McIlwain H. Electrical activities in thin sections from the mammalian brain maintained in chemically-defined media in vitro. J Neurochem. 1966;13(12): 1333–1343.
3. Referans3. Molleman A. Patch clamping: an introductory guide to patch clamp electrophysiology. Wiley, West Sussex, England
. 2003.
4. Referans4. Jefferys JG. Nonsynaptic modulation of neuronal activity in the brain: electric currents and extracellular ions. Physiol Rev. 1995; 75(4): 689–723.
5. Referans5. Scholfield CN. Electrical properties of neurones in the olfactory cortex slice in vitro, J. Physiol. (London). 1978;275:535-546.
6. Referans6. Voskuyl RA, and ter Keurs HEDJ. Modification of neuronal activity in olfactory cortex slices by extracellular K+, Brain Res. 1981;230:372-377.

7. Referans7. Lipton P, and Whittingham TS. The effect of hypoxia on evoked potentials in the in vitro hippocampus. J. Physiol. (London) 1979;287:427-438.
8. Referans8. Lipton P, and Heimbach CJ. The effect of extracellular potassium concentrations on protein synthesis in guinea-pig hippocampal slices, J. Neurochem. 1977;28:1347-1354.

9. Referans9. Lipton P, and Heimbach CJ. Mechanism of extracellular potassium stimulation of protein synthesis in the in vitro hippocampus, J. Neurochem. 1978;31:1299-1307.
10. Referans10. Lipton P, and Robacker K. Glycolysis and brain function: Ko stimulation of protein synthesis and K+ uptake require glycolysis. Fed Proc. 1983;42(12):2875-80.
11. Referans11. Hablitz JJ, and Lundervold A. Hippocampal excitability and changes in extracellular potassium. Exp. Neurol. 1981;71:410-420.
12. Referans12. King G1, and Somjen GG. Extracellular calcium and action potentials of soma and dendrites of hippocampal pyramidal cells. Brain Res. 1981;226:339-343.
13. Referans13. Kimelberg HK, Biddlecome R, Narumi S, and Bourke RS. ATPase and carbonic anhydrase actuities of bulk-isolated neurons, glia and synaptosome fractions from rat brain. Brain Res. 1978;141:305-323.
14. Referans14. Grisar T, and Franck G. Effect of changing potassium ion concentrations on rat cerebral slices in vitro: A study during development. J. Neurochem. 1981;36:1853-1857.
15. Referans15. Pittman QJ, Blume HW, and Renaud LP. Connections of the hypothalamic paraventricular nucleus with the neurohypophysis, median eminence, amygdala, lateral septum and midbrain periaqueductal gray: An electrophysiological study in the rat. Brain Res. 1981;215:15-28.
16. Referans16. Jefferys JGR, and Haas HL. Synchronized bursting of CAl hippocampal pyramidal cells in the absence of synaptic transmission, Nature 1982;300:448-450.
17. Referans17. Richards CD, and Sercombe R. Calcium, magnesium and the electrical activity of guinea-pig olfactory cortex in vitro. J. Physiol. (London) 1970;211:571-584.
18. Referans18. Hackett JT. Calcium dependency of excitatory chemical synaptic transmission in the frog cerebellum in vitro. Brain Res. 1976;114:35-46.

19. Referans19. Dingledine R, and Somjen G. Calcium dependence of synaptic transmission in the hippocampal slice. Brain Res. 1981;207:218-222.

20. Referans20. Schaer H. Decrease in ionized calcium by bicarbonate in physiological solutions. Pfliigers Arch. 1974;347:249-254.

21. Referans21. Dienel GA, Hertz L. Glucose and lactate metabolism during brain activation. J Neurosci Res. 2001;66:824–838.
22. Referans22. Christie JM, Jahr CE. Multivesicular release at Schaffer collateral-CA1 hippocampal synapses. J Neurosci. 2006;26:210–216.
23. Referans23. Schurr A, Payne RS, Miller JJ, Rigor BM. Study of cerebral energy metabolism using the rat hippocampal slice preparation. Methods. 1999;18:117–126.
24. Referans24. Cater HL, Chandratheva A, Benham CD, Morrison B III, Sundstrom LE. Lactate and glucose as energy substrates during, and after, oxygen deprivation in rat hippocampal acute and cultured slices. J Neurochem. 2003;37:1381–1390.
25. Referans25. Alger BE, Nicoll RA. Epileptiform burst afterhyperpolarization: Calcium-dependent potassium potential in hippocampal CA1 pyramidal cells. Science. 1980;210:1122-1124.
26. Referans26.Brown DA, Wong RKS, Prince D. Spontaneous miniature synaptic potentials in hippocampal neurons. Brain Res. 1979;177:194-199.
27. Referans27. Aydın-Abidin S, Abidin İ. 7,8-Dihydroxyflavone potentiates ongoing epileptiform activity in mice brain slice. Neuroscience Letters. 2019;703:25-31.
28. Referans28. Llinas R and Sugimori M. Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices. J. Physiol. (London) 1980a;305:171-195.
29. Referans29. Spray DC, Harris AL, and Bennett MVL. Gap junctional conductance is a simple and sensitive function of intracellular pH. Science. 1981;211:712-715.

30. Referans30. Marshall KC, and Engberg I. The effects of hydrogen ion on spinal neurons. Can. J. Physiol. Pharmacol. 1980;58:650-655.
31. Referans31. Franck G. Brain slices,The Structure and Function of Nervous Tissue, Volume VI. Structure and Physiology (G. H. Bourne, ed.), Academic Press, New York. 1972:417- 465.

32. Referans32. Xiong H, Xia J. Preparation and use of rodent hippocampal slices.Current Laboratory Methods in Neuroscience Research. 2014: 95-103.
33. Referans33. Teyler TJ. Brain slice preparation: Hippocampus, Brain Res. Bull. 1980;5:391-403.
34. Referans34. Garthwaite J, Woodhams PL, Collins MJ, and Balazs R. On the preparation of brain slices: Morphology and cyclic nucleotides, Brain Res. 1979;173:373-377.
35. Referans35. White WF, Nadler JV, and Cotman CW. A perfusion chamber for the study of CNS physiology and pharmacology in vitro, Brain Res. 1978;152:591-596.
36. Referans36. Schwartzkroin PA. Characteristics of CAl neurons recorded intracellularly in the hippocampal slice. Brain Res. 1975;85:423-435.
37. Referans37. Li CL, and McIlwain H. Maintenance of resting membrane potentials in slices of mammalian cerebral cortex and other tissues in vitro. J. Physiol. 1957;139:178-190.
38. Referans38. Haas HL, Schaerer B, and Vosmansky M. A simple perfusion chamber for the study of nervous tissue slices in vitro, J. Neurosci. Methods. 1979;1:323-325.
39. Referans39. Nicoll RA, Alger BE. A simple chamber for recording from submerged brain slices. J Neurosci Methods. 1981;4: 153–156.
40. Referans40. Stopps M, Allen N, Barrett R, Choudhury HI, Jarolimek W, Johnson M, et al. Design and application of a novel brain slice system that permits independent electrophysiological recordings from multiple slices. J Neurosci Methods. 2004;132:137–48.
41. Referans41. Dunlop J, Roncarati R, Jow B, Bothmann H, Lock T, Kowal D, et al. In vitro screening strategies for nicotinic receptor ligands. Biochem Pharmacol. 2007;74:1172–81.
42. Referans42. Graef JD, Wei H, Lippiello PM, Bencherif M, Fedorov N. Slice XVIvo: A novel electrophysiology system with the capability for 16 independent brain slice recordings. Journal of Neuroscience Methods. 2013;212: 228-233.
43. Referans43. Finkel A and Bookman R. Current Protocols in Neuroscience. 1997 by John Wiley and Sons, Inc. 6.1.1-6.1.6.
44. Referans44. Neher E, Sakmann B. Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature. 1976;260:799-802.
45. Referans45. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved Patch-Clamp Techniques for High-Resolution Current Recording from Cells and Cell-Free Membrane Patches. Pflügers Arch. 1981;391:85–100.

46. Referans46. Horn R, Marty A. Muscarinic Activation of Ionic Currents by a New Whole-Cell Recording Method. J. Gen. Physiol. 1988;92:145–159.
47. Referans47. Ashcroft, F.M. Ion Channels and Disease: Channelopathies. Boston: Academic Press, Newyork, 2000.
48. Referans48. Kass RS. The channelopathies: novel insights into molecular and genetic mechanisms of human disease. J Clin Invest. 2005;115:1986–89.
49. Referans49. Thompson RF. The search for the engram. Am. Psychol. 1976;31:209–227.

50. Referans50. Teyler TJ, Cavus I, Coussens C, DiScenna P, Grover LM, Lee YP, and Little Z. Brain Slices in Basic and Clinical Research (Schurr, A., and Rigor, B., Eds.), CRC Press, Boca Raton, FL.). 1995.
51. Referans51. Bliss TVP, and Collingridge GL. A synaptic model of memory: Long-term potentiation in the hippocampus. Nature. 1993;361:31–39.
52. 
Referans52. Martin S, Grimwood P, and Morris RGM. Synaptic plasticity and memory: An evaluation of the hypothesis. Annu. Rev. Neurosci. 2000;23:649–711.
53. Referans53. Bliss TVP, and Lomo T. Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J. Physiol. 1973;232:331–356.
54. Referans54. Collingridge GL, Kehl SJ, and McLennan H. Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus. J. Physiol. 1983;334:33–46.
55. Referans55. Lynch G, Larson J, Kelso S, Barrionuevo G, and Schottler F. Intracellular injections of EGTA block induction of hippocampal long-term potentiation. Nature. 1983;305: 719–721.
56. Referans56. Frey U, Huang YY, and Kandel ER. Effect of cAMP stimulate a late stage of LTP in hippocampal CA1 neurons. Science. 1993;260: 1661–1664.

57. Referans57. Nguyen PV, Abel T, and Kandel ER. Requirement of a critical period of transcription for induction of late phase of LTP. Science. 1994;265: 1104–1107.
58. Referans58. Abel T, Nguyen PV, Barad M, Deuel T, Kandel ER, and Bourtchouladze R. Genetic demonstration of a role for PKA in the late phase of LTP and in hippocampus-based long-term memory. Cell. 1997;88: 615–626.
59. Referans59. Giese KP, Fedorov NB, Filipkowski RK, and Silva AJ. Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning. Science. 1998;279: 870–873.

60. Referans60. Kandel ER. The molecular biology of memory storage: A dialogue between genes and synapses. Science. 2001;294: 1030–1038.
61. Referans61. Grover LM, and Teyler TJ. Two components of long-term potentiation induced by different patterns of afferent activation. Nature. 1990;347:477– 479.)
62. Referans62. Trevelyan AJ, Sussilo D, Watson BO, Yuste R. Modular propagation of epileptiform activity: evidence for an inhibitory veto in neocortex. J Neurosci. 2006;26:12447-12455.
63. Referans63. Bear J, Lothman EW. An in vitro study of focal epileptogenesis in combined hippocampal-parahippocampal slices. Epilepsy Res. 1993;14(3): 183-193.
64. Referans64. Pinto DJ, Patrick SL, Huang WC, Connors BW. Initiation, propagation and termination of epileptiform activity in rodent neocortex in vitro involve distinct mechanisms. J Neurosci. 2005;25: 8131-8140.
65. Referans65. Voskuyl RA, Albus H. Spontaneous epileptiform discharges in hippocampal slices induced by 4-aminopyridine. Brain Res. 1985;342: 54-66.
66. Referans66.Perreault P, Avoli M. Physiology and pharmacology of epileptiform activity induced by 4-aminopyridine in rat hippocampal slices. J Neurophysiol. 1991;65:771-785.
67. Referans67. Perreault P, Avoli M. 4-aminopyridine-induced epileptiform activity and a GABA-mediated long-lasting depolarization in the rat hippocampus. J Neurosci. 1992;12:104-115.
68. Referans68. Avoli M, Barbaroise M, Lucke A, Nagao T, Lopantsev V, Kohling R. Synchronous GABA-mediated potentials and epileptiform discharges in the rat limbic system in vitro. J Neurosci. 1996;16: 3912,3924.
69. Referans69. Ziburkus J, Cressman JR, Barreto E, Schiff SJ. Interneuron and pyramidal cell interplay during in vitro seizure-like events. J Neurophysiol. 2006; 95:3948-3954.
70. Referans70. Uva L, Avoli M, de Curtis M. Synchronous GABAa-receptor-dependent potentials in limbic areas of the in vitro isolated adult guinea pig brain. Eur J Neurosci. 2009;29:911-920.
71. Referans71. Ozturk H. Phoenixin-14 modulates seizure-like events in amygdala. 6. Uluslararası GAP Matematik-Mühendislik-Fen ve Sağlık Bilimleri Kongresi, Şanlıurfa, 2021:58-64.
72. Referans72. Bernard C. Hippocampal Slices: Designing and Interpreting Studies in Epilepsy Research. Models of Seizures and Epilepsy. 2006:59-72.
73. Referans73. Cronin J, Obenaus A, Houser CR, Dudek FE. Electrophysiology of dentate granule cells after kainate-induced synaptic reorganization of the mossy fibers. Brain Res. 1992;573: 305-310.
74. Referans74. Hardison JL, Okazaki MM, Nadler JV. Modest increase in extracellular potassium unmasks effect of recurrent mossy fiber growth. J Neurophysiol. 2000;84: 2380–2389.
75. Referans75. Lynch M, Sutula T. Recurrent excitatory connectivity in the dentate gyrus of kindled and kainic acid‐treated rats. J Neurophysiol. 2000;83: 693– 704.
76. Referans76. Patrylo PR, Dudek FE. Physiological unmasking of new glutamatergic pathways in the dentate gyrus of hippocampal slices from kainite-induced epileptic rats. J Neurophsiol. 1998;79(1):418-29.
77. Referans77. Patrylo PR, van den Pol AN, Spencer DD, Williamson A. NPY inhibits glutamatergic excitation in the epileptic human dentate gyrus. J Neurophysiol. 1999;82:478–483.
78. Referans78. Wuarin JP, Dudek FE. Electrographic seizures and new recurrent excitatory circuits in the dentate gyrus of hippocampal slices from kainite-treated epileptic rats. J Neurosci. 1996;16(14):4438-4448.
79. Referans79. Bernard C, Anderson A, Becker A, Poolos NP, Beck H, Johnston D. Acquired dendritic channelopathy in temporal lobe epilepsy. Science. 2004;305(5683):532-5.
80. Referans80. Brooks-Kayal AR, Shumate MD, Jin H, Rikhter TY, coulter DA. Selective changes in single cell GABA(A) receptor subunit expression and function in temporal lobe epilepsy. Nat Med. 1998;4(10):1166-72.
81. Referans81. Buhl EH, Otis TS, Mody I. Zinc-induced collapse of augmented inhibition by GABA in a temporal lobe epilepsy model. Science. 1996; 271(5247): 369-73.
82. Referans82. Cossart R, Dinocourt C, Hirsch JC, Merchan-Perez A, De Felipe J, Ben-Ari Y, et al. Dendritic but not somatic GABAergic inhibition is decreased in experimental epilepsy. Nat Neurosci. 2001. 4(1):52-62.
83. Referans83. Esclapez M, Hirsch JC, Khazipov R, Ben-Ari Y, Bernard C. Operative GABAergic inhibition in hippocampal CA1 prymidal neurons in experimental epilepsy. Proc Natl Acad Sci. 1997;94(22):12151-6.
84. Referans84. Esclapez M, Hirsch JC, Ben-Ari Y, Bernard C. Newly formed excitatory pathways provide a substrate for hyperexcitability in experimental temporal lobe epilepsy J. Comp. Neurol. 1999;408(4): 449-460.
85. Referans85. Nusser Z, Sieghart W, Somogyi P. Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells. J Neurosci. 1998;18: 1693-1703.
86. Referans86. Ratzliff AH, Howard AL, Santhakumar V, Osapay I, Soltesz I. Rapid deletion of mossy cells does not result in a hyperexcitable dentate gyrus: Implications for epileptogenesis. J Neurosci. 2004;24: 2259–2269.
87. Referans87. Scharfman HE, Goodman JH, Sollas AL. Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: Functional implications of seizure-induced neurogenesis. J Neurosci. 2000;20: 6144–6158.
88. Referans88. Su H, Sochivko D, Becker A, Chen J, Jiang Y, Yaari Y, and Beck H. Upregulation of a T‐type Ca2+ channel causes a long‐lasting modification of neuronal firing mode after status epilepticus. J Neurosci. 2002;22: 3645 – 3655 .
89. Referans89. Medvedeva YV, Lin B, Shuttleworth CW, Weiss JH. Intracellular Zn2+ accumulation contributes to synaptic failure, mitochondrial depolarization, and cell death in an acute slice oxygen-glucose deprivation model of ischemia. J. Neuroscience. 2009;29: 1105–1114.
90. Referans90. Stork CJ, Li YV. Rising zinc: a significant cause of ischemic neuronal death in the CA1 region of rat hippocampus. J. Cerebral Blood Flow & Metabolism. 2009;29: 1399–1408.
91. Referans91. Rambani K, Vukasinovic J, Glezer A, Potter SM. Culturing thick brain slices: an interstitial 3D microperfusion system for enhanced viability. J. Neuroscience Methods. 2009;180: 243–254.
92. Referans92. Taylor CP, Weber ML, Gaughan CL, Lehning EJ, LoPachin RM. Oxygen/glucose deprivation in hippocampal slices: altered intraneuronal elemental composition predicts structural and functional damage. J. Neuroscience. 1999;19: 619–629.
93. Referans93. Huchzermeyer C, Albus K, Gabriel HJ, Otahal J, Taubenberger N, et al. Gamma oscillations and spontaneous network activity in the hippocampus are highly sensitive to decreases in pO2 and concomitant changes in mitochondrial redox state. J. Neuroscience. 2008;28: 1153–1162.
94. Referans94. Hoffmann U, Pomper J, Graulich J, Zeller M, Schuchmann, et al. Changes of neuronal activity in areas CA1 and CA3 during anoxia and normoxic or hyperoxic reoxygenation in juvenile rat organotypic hippocampal slice cultures. Brain Research. 2006;1069: 207–215.
95. Referans95. Huang Y, Williams JC, Johnson SM. Brain slice on a chip: opportunities and challenges of applying microfluidic technology to intact tissues. Lab on a Chip. 2012;12: 2103–2117.
96. Referans96. Dong W-Q, Schurr A, Reid K H, Shields CB, West CA. The rat hippocampal slice preparation as an in vitro model of ıskemia. Stroke. 1988;19(4): 498-502.)
97. Referans97. Pissarek M, Reinhardt R, Reichelt C, Manaenko A, Krauss G, Illes P. Rapid assay for one-run determination of purine and pyrimidine nucleotide contents in neocortical slices and cell cultures. Brain Res.Protoc. 1999;4:314–321.
98. Referans98. Gunther A, Manaenko A, Franke H, Dickel T, Berrouschot J, Wagner A, et al. Early biochemical and histologicalchanges during hyperbaric or normobaric reoxygenation after in vitroischemia in primary corticoencephalic cell cultures of rats. Brain Res. 2002;946:130–138.
99. Referans99. Collingridge GL. The brain slice preparation: a tribute to the pioneer Henry McIlwain. J. Neurosci. Methods. 1995;59(1):5-9.