1.Holm DM, et al. An improved flow microfluoremeter for rapid measurement of cell fluorescence.Exp. Cell . Res. , 1973;80:105

1.Arnaout MA.Structure andfunction of the leukocyte adhesion molecule CD11/CD18.Blood,1991;75(5):1037

1.Aird F,etal.Corticotropin-releasing factor mRNA in rat thymus and spleen.Proc Natl AcadSci USA,1993;90:7104

核心提示:Introduction The mitochondrial respiratory chain produces energy which is stored as an electrochemical graIntroduction

2.Steinkamp JA, et al.Multiparameter analysis and sorting of mammalian cells . Exp . Cell Res. , 1974;84:15~32

2.Autran B,et al. T cell receptorγ/δ+lymphocyte subsets during HIV infection. Clin Exp Innunol,1989;75:206

2.Amano Y,et al.Inhibition byglucocorticoids of the formation of interliukinlα,interleukinlβ and interleukin-6:mediation by decreasedmRNa stability.Mol Pharmacol,1993;43:176

3.Steen HB, et al. Differentialof light-scattering detection in arc—lamp –illumination flow cytometry.Cytometry, 1985;6(3):273~275

3.Barber EK,et al.Tthe CD4 andCD8 antigens are coupled to a protein tyrosine kinase(p56lck)thatphosphorylates the CD3complex.PNAS,1989;86:3277

3.Araujo al.Trophic effectsof interleukin-4,7 and 8 on hippocampla cultures:potential involvement ofglia-derived factor.Brain Res,1993;600:49


4.Loken MR, et al. Flow cytometryas an analyticaal preparetive tool in immunology. J. Immunol. Methods., 1982;50(3):85~112

4.Bevilacqua MP,et al.Endothelialleucocyte adhesion molceule 1,An inducible receptor for neutrophils related tocomplement regulatory proteins and lectins.Science,1989;243:1160

4.Ban EM,et al.Identification ofsuppressin-producing cells in the rat pituitary.Endocrinology,1993;133:241

5.Smart YC, et al. Flowcytometric ceumeration of absolute lymphocyte number in peripheral blood usingtwo parameters of light scatter. Cy-tometry, 1985;6(2):172~174

5.Carel JM,et al.Dual antigenrecognition by B cells,Immunol Today,1993;14(1):8

5.Berkenbosch F,etal.CRF-producing neurons in the rat activated by interleukin-1.Science,1987;238:524

JC-1 staining

6.Dvaies EG, et al. Lymphocytesubpopulations in primary immunodeficience disorders. Arch . Dis. Child. , 1983;58:346~351

6.Eiden LE,et al.HIV interactionswith CD4:continum of conformations and consequences. ImmunolToday,1992;13(6):291

6.Bernadini R,et al.Rathypothalamic CRH secretion in vitro is stimulated by IL-1 in aneicosanoid-dependent manner.Life Sci,1990;47:1601

2.1 Materials JC-1 fluorescent probe, plastic tubes for FACS analysis, complete medium, i.e. RPMI added with 10% fetal calf serum, PBS.

7.Campbell A, et al. Lymphocytesubpopulations in the blood of newborn infants. Clin. Exp . Immunol. ,1974;18:469~482

7.Figdor CG,et al.On the mode ofaction of LFA-1. Immunol Today,1990;11(8):277

7.Berczi I.Pituitary function andImmunity.CRC Press Inc.Boca Raton Florida.1986

2.2. Methodology 1. Harvest cells from experimental samples, bring total volume up to 1 mL of fresh complete medium.

8.Shackney SE. The use of flowcytometry in the diagnosis and biological characterization of the nonHodgkin’slymphomas. Ann . NY. Acad. Sci., 1986;486:171~177

8.Hirsch R,et al.Effects of invivo administration of anti-CD3 monoclonal antibody on T cell function in mice.j Immunol,1989;142(3):737

8.Buzzetti R,et al.A criticalassessment of the interactions between the immune system and thehypothalamus-pituitary-adrenalaxis.j Endocrinol,1989;120:183

  1. Stain cell suspension with 2.5 mg/mL JC-1. Shake cell suspension until the dye is well dissolved, giving a uniform red-violet color. To do this, it is also possible to vortex vigorously the suspension immediately after the addition of the probe.

  2. Keep the samples in a dark place at room temperature for 15-20 minutes. The duration of the staining depends upon the cell type, but in our hands all the cells used responded quite well to the treatment. Wash twice centrifuging at 500 g for 5 min with a double volume of PBS.

  3. Resuspend in 0.3 mL of PBS, then analyze immediatly with the flow cytometer, typically equipped with a 488 nm argon laser. Set the value of photomultiplier detecting the signal in FL1 at about 390 V, and FL2 PMT at 320 V; FL1-FL2 compensation should be around 4.0%, while FL2-FL1 compensation around 10.6%. This is however the classical setting of the instrument we use in our laboratory, and it has to be taken into account that, as each instrument has a different sensitivity, a different setting can be necessary to obtain an optimal signal. Concerning instruments, the staining has been tested on several different apparatus such as an Excel, from Coulter , an Elite in Paris, some FACSCAN, a FACSTAR Plus and a FACSCalibur, from Becton Dickinson , a Biorad Brite and a Partec , and they work perfectly as well. Obviously, compensations have to be set in a different way.

  1. Foucar K, et al. Flowcytometry in lymphoma. Am. J. Surg. Pathol., 1986;10(8):584~585

9.Hogg N.The leukocyte integrins.Immunol Today,1989;10(4):111

9.Celada A,et al.Repression ofmajor histocompatibility complex I-a expression by glucocorticoids:theglucocorticoid receptor inhibits the DNa binding of the X box DNA bindingprotein.J Exp Med ,1993;177:691


10.Hoffonan RA, et al.Immunofluorescent analysis of blood cells by flow cytometry. Int. J.Immuno—Pharmacol, 1981;3(3):249~254

10.Holoshitz J,et al.Isolation ofCD4 CD8mycobacteria-reactive T lymphocyte clones from rheumatoid arthritissynovial fluid. Nature,1989;339:226

10.Chao CC,et al.Activatedmicroglia mediate nediate neuronal cell injury via a nitric oxide mechanism.JImmunol,1992;149:2736

3.1 Background information The technique of JC-1 staining has been developed with the intent to detect DY in intact, viable cells. For this purpose JC-1 acts as a marker of mitochondrial activity, since the formation of J-aggregates, which give red emission, is reversible. Cells with high DY are those forming J-aggregates, thus showing high red fluorescence. On the other hand, cells with low DY are those in which JC-1 maintains monomeric form, thus showing only green fluorescence. Normally green fluorescence of depolarized cells is a little bit higher than that of polarized ones simply because of the presence of a higher amount of JC-1 monomers.

11.Fahey JL, et al. Quantitativechanges in T—helpper or T—suppressor /cytotoxic lymphocyte subsets that distinguishacquired immune defi-ciency syndrome from other immunesubset disorders. Am. J. Med., 1984;76:95~100

11.Kavanough AF,et al.Role ofCD18 in adhesion and transendotlial migration of T cell. JImmunol,1991;146(12):4149

11.Christ M,et al.Theinositol-1,4,5-trisphosphate system is involved in repid effects of aldosteronein human mononuclear leukocytes.J cLIN eNDOCRINOl mETAB,193;77:1452

  1. Eichner RD, et al.LAV/HTLV-III plays a dominant role in the etiology of AIDS. AIDS RES., 1984;1(4):237~241

  2. Smith BR, et al. Circulationmonoclonal B lymphocytes in non—Hodgkin’s lymphoma. J. Med., 1984;311:1476~81

  3. Committee on Human LeukocyteDifferentiation Antigens, IUIS-WHo Nomenclature Subcommittee. DifferentiationHuman. Leukocyte Antigens:a proposednomenclature. Immunol. Today, 1984;5:158~159

12.Kevin H,et al.Murinehematopoietic cell surface antigen expression. Immunol Today,1988;9(11):344

12.Cirino G,etal.Anti-inflammatory action of an N-terminal peptide from humanlipocortin-1,Brit J Pharmacol,1990;104:573

15.Cleary ML, et al.Immunoglobulin gene rearrangements as a diagnositic criterion of B celllymphomas. Proc. Natl. Acad. Sci. USA, 1984;81:593~597

13.Knapp W,et al.Towards a betterdefinition of human leucocyte surface molecules. Immunol Today,1989;10(8)253

13.DuBois CM,et al.Hematopoieticgrowth factors and hlucocorticoids synergize to mimic the effects of IL-1 ongranulocyte differentiation and IL-1 receptor induction on bone marrow cells invi.Exp Hematol,1993;21:303

  1. Kroemer G., Zamzani N., Susin S.A. Mitochondrial control of apoptosis. Immunol. Today, 18: 44-51, 1997.

  2. Susin S.A., Zamzami N., Castedo M., Daugas E., Wang H.G., Geley S., Fassy F., Reed J.C., Kroemer G. The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis. J. Exp. Med., 186: 25-37, 1997.

  3. Kroemer G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nature Med., 3: 614-620, 1997.

  4. Cossarizza A., Kalashnikova G., Grassilli E., Chiappelli F., Salvioli S., Capri M., Barbieri D., Troiano L., Monti D., Franceschi C. Mitochondrial modifications during rat thymocyte apoptosis: a study at the single cell level. Exp. Cell Res., 214: 323-330, 1994.

  5. Richter C., Schweizer M., Cossarizza A., Franceschi C. Control of apoptosis by the cellular ATP level. FEBS Lett., 378: 107-110, 1996.

  6. Gorman A.M., Samali A., McGowan A.J., Cotter T.G. Use fo flow cytometry techniques in studying mechanisms of apoptosis in leukemic cells. Cytometry, 29: 97-105, 1997.

  7. Yang J., Liu X., Bhalla K., Kim C.N., Ibrado A.M., Cai J., Peng T.I., Jones D.P., Wang X. Prevention of apoptosis by bcl-2: release of cytochrome c from mitochondria blocked. Science, 275: 1129-1132, 1997.

  8. De Maria R., Lenti L., Malisan F., d'Agostino F., Tomassini B., Zeuner A., Rippo M.R., Testi R. Requirement for GD3 ganglioside in CD95- and ceramide-induced apoptosis. Science, 277: 1652-1654, 1997.

  9. Maftah A., Petit J.M., Ratinaud M.H.A.J., R. 10-N nonyl-acridine orange: a fluorescent probe which stains mitochondria independently of their energetic state. Biochem. Biophys. Res. Commun., 164: 185-190, 1989.

  10. Lopez-Mediavilla C., Orfao A., Gonzales M., Medina J.M. Identification by flow cytometry of two distinct rhodamine-123-stained mitochondrial populations in rat liver. FEBS Lett., 254: 115-120, 1989.

  11. Terasaki M., Song J., Wong J.R., Weiss M.J., Chen B.L. Localization of endoplasmic reticulum in living and glutaraldehyde-fixed cells with fluorescent dyes. Cell, 38: 101-108, 1984.

  12. Salvioli S., Ardizzoni A., Franceschi C., Cossarizza A. JC-1, but not DiOC6 or rhodamine 123, is a reliable fluorescent probe to assess DY in intact cells. Implications for studies on mitochondrial functionality during apoptosis. FEBS Lett., 411: 77-82, 1997.

  13. Cossarizza A., Baccarani Contri M., Kalashnikova G., Franceschi C. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide . Biochem. Biophys. Res. Commun., 197: 40-45, 1993.

  14. Cossarizza A., Salvioli S., Franceschini M.G., Kalashnikova G., Barbieri D., Monti D., Grassilli E., Tropea F., Troiano L., Franceschi C. Mitochondria and apoptosis: a cytofluorimetric approach. Fund. Clin. Immunol., 3: 67-68, 1995.

  15. Cossarizza A., Ceccarelli D., Masini A. Functional heterogeneity of isolated mitochondrial population revealed by cytofluorimetric analysis at the single organelle level. Exp. Cell Res., 222: 84-94, 1996.

  16. Hada H., Honda C., Tanemura H. Spectroscopic study on the J-aggregate of cyanine dyes. I. Spectral changes of UV bands concerned with J-aggregate formation. Photogr. Sci. Eng., 21: 83-91, 1977.

  17. Reers M., Smith T.W., Chen L.B. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry, 30: 4480-4486, 1991.

  18. Smiley S.T., Reers M., Mottola-Hartshorn C., Lin M., Chen A., Smith T.W., Steele G.D., Chen L.B. Intracellular heterogeneity in mitochondrial membrane potential revealed by a J-aggregate-forming lipophilic cation JC-1. Proc. Natl. Acad. Sci. USA, 88: 3671-3675, 1991.

  19. Johnson L.V., Walsh M.L., Bockus B.J., Chen L.B. Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. J. Cell Biol., 88: 526-535, 1981.

  20. Goldstein S., Korczack L.B. Status of mitochondria in living human fibroblasts during growth and senescence in vitro: use of the laser dye rhodamine 123. J. Cell Biol., 91: 392-398, 1981.

  21. Darzynkiewicz Z., Staiano-Coico L., Melamed M.R. Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation. Proc. Natl. Acad. Sci. USA, 78: 2383-2387, 1981.

  22. Petit P.X., Lecoeur H., Zorn E., Dauguet C., Mignotte B., Gougeon M.-L. Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J. Cell Biol., 130: 157-167, 1995.

  23. Jenssen H.-L., Redmann K., Mix E. Flow cytometric estimation of transmembrane potential of macrophages - A comparison with microelectrode measurements. Cytometry, 7: 339-346, 1986.

  24. Petit P.X., O'Connor D., Grunwald D., Brown S.C. Analysis of the membrane potential of rat- and mouse-liver mitochondria by flow cytometry and possible applications. Eur. J. Biochem., 194: 389-397, 1990.

  1. Weinberg DS, et al.Cytofluorometric detection of B cell clonalexcess:a new approachto the diagnosis of B cell lymphoma. Blood, 1984;63:1080~1087

  2. Gray JW, et al. Cell cycleanalysis using flow cytometry. Int. J. Radian . Biol., 49(2):237~255

14.Littman DR,et al.The isolationand sequence of the gene encoding T8, A molecule defining functional classes ofT lymphocytes. Cell,198540(1):237

14.Effros RB,et al.Influence ofage and caloric restriction on macrophage IL-6 and TNF production.Lymphok CytokRes,1991;10:347

Appendix 1: Stock solution:

18.Marti GE, et al. Normal humanblood density gradient lymphocyte subset analysis. An interlaboratory flowcytometric comparison of 85 nor-mal adults.Am.J. Jematol., 1985;20(1):41~52

15.Marx J.Biologists turn on to“off-enzymes”.Science,1991;251:744

15.Flecher-Chiappini SE,etal.Glucocorticoid-prolactin interactions in Nb2lymphomacells:antiproliferative versus anticytolytic effects.Proc Soc Exp BiolMed,1993;202:345

JC-1 is dissolved in N,N’-dimethylformamide at the concentration of 2.5 mg/ml.

  1. Levy EM, et al. Defective Tcell differentiation in acquired immunedefecience syndrome (AIDS).J. Clin.Immunol., 1986;77(6):1756~1761

  2. Schjnitzer B, et al. Americanadult T cell leukemja/lymphoma:aflowcytometric and morphological study. Ann . NY. Acad. Sci. 1986;486:256~267

  3. Van dilla MA, et al. (eds )flow Cytometry:Instrument and Data Analysis. London:Academic Press, 1985

  4. Melamed MR, et al, (eds) flowCytometry and sorting . 2nd ed . New York:John Wiley Sons, 1990

  5. Iwao Nishiya et al, (eds)flow cytometry and Image Analysis for clinical applications, Excerpta Medica.Netherlands, 1991

16.Modlin RL,et al.Lymphocytesbearing antigen-specific T-cell receptors accumulate in human infection diseaselesions .Nature,1989;339:544

16.Freidin M and KesslerJA.Cytokine regulation of substance Pexpression in sympathetic neurons. ProcNatl Acad Sci UCA,1991;88:3200

It is stored at -20°C. Light sensitive.


17.O'Brien RL,et al.Stimulationof a major subset of lymphocytes expressing T cell receptor γδby an antigenderived from mycobacterium tuberculosis. Cell,1989;57:667

17.Garcia I,et al.β-Endorphininhibits IL-2 release and expression of IL-2receptors in Con A-stimulatedsplenic lymphocytes.Lymphok Cytok Res,1992;11:339

Appendix 2: Reagents

18.Patel MD,et al.MultipleKinases and signal transduction.JBC,1987;262(12):5831

18.Guilian D and LachmanL.Interleukin 1 stimulation of astroglial proliferation after graininjury.Science,1984;228:497

JC-1 Molecular Probes, Eugene, OR, USA catalog No.: T-3168

19.Pinto A,et al.New moleculesburst at the leukocyte surface A comprehensive review based on the 5thinternational workshop on leukocyte differentiation antigens Boston,USA,3-7November,1993.Leukemia,1994;8(3):347

19.Haines KA,etal.Chemoattraction of neurtrophils by substance P and transforming growthfactor β1 is inadequately explained by current models of lipid remodelling.JImmunol,1993;151:1491

Note: colture medium, saline solutions and washing buffers are depending on the cell type which is used for the experimental procedure . For blood white cells, RPMI 1640 with 10% heat inactivated foetal calf serum, 100 IU/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine is normally used as complete colture medium.

20.Rivas A,et al.CD4 moleculesare associated with the antigen receptor complex on activated but not resting Tcells. j Immunol,1988;140(9):2912

20.Hart RP,et al.Substance P geneexpression is regulated by interleukin-1 in cultured sympathetic ganglia.JNeurosci Res ,1991;29:282

Appendix 3: Equipment

21.Rudd CE.CD4 CD8 and theTCR-CD3 complex:a novel class of protein tyrosine kinase receptor. ImmunolToday,1990;11(11):400

21.Heagy W,et al.Opioid receptoragonists and Ca2+modulation by interleukin-1 in cultured sympatheticganglia.J Neurosci Res,1991;29:282

Flow Cabinet TC60 Gelaire
Flow Cytometer FACScan Becton Dickinson
Incubator CO2-AUTO-ZERO Heraeus
Centrifuge Minifuge RF Heraeus
Pipetman P20, P200, P1000 Gilson
Vortex Vibrofix VF1 Electronic Janke & Kunkel-Ika Labortechnik

22.Sanders ME,et al.Human naiveand memory t cells:reinterpretation of helper-inducer and suppressor-inducersubsets. Immunol Today,1988;9(7):195

22.Hiramoto R,etal.Identification of specific pathways of communication between the CNS and NKcell system.Life Sci,1993;53:527

Appendix 4: Glossary

23.Schlossman SF,et al.Leucocytetyping V:white cell differentiation antigens. Oxford University Press, Oxford1994 in press

23.Jonakati GM.Neural immuneinterations in sympathetic ganglia.TINS,1993;16:419

Mitochondrial membrane potential is generated by mitochondrial electron transport chain, which drives a proton flow from matrix through inner mitochondrial membrane to cytoplasm, thus creating an electrochemical gradient. This gradient is in turn responsible for the formation of ATP molecules by F0-F1 ATP synthase. For this reason Dy is an important parameter for mitochondrial functionality and an indirect evidence of energy status of the cell.

24.Sukhatme VP,et al.Gene for thehuman T cell differentiation antigen Leu-2/T8 is closely linked to the lightchain locus on chronosome 2. J Exp. Med,1985;161(2):429

24.Katsuura G,et al.Interleukin-1beta increases prostaglanken E2 in rat astrocyte cultrues:modulatoryeffect of neuropeptides.Endocrinology,1989;124:3125

25.Williames AF.A year in thelife of the immunoglobulin superfamily. Immunol Today ,1987;8(10):298

25.Kim KS,et al.Modulation ofbloodbrain barrier permeability by tumor necrosis facto and antibody to tumornecrosis factor in the rat.Lymphok Cytok rES,1992;11:293


26.kIMAALL ES,et al. SubstanceP,neurokinin A,and neurokinin Binduce generation of IL-1 like activity inP388D1 cells.j Immunol,1988;141:356427.

27.Koike K,et al.Tumor necrosisfactor-alpha increases release of arachidonate and prolactin from rat anteriorpituitary cells.Endocrinology,1991;128:2791

28.Kraiem A,et al.Effects ofgamma-interferon on Dr antigen expression,growth,3.5.3'-triidothyroninesecretion ,iodide uptake and cyclic adenosine monophosphate accumulation incultured human thyroid cells.j Clin Endocrin Metab,1990;71:817

29.Kroemer G,et al.Physiology andpathology of an immunoendocrine feedback loop.Immunol Today,1988;9:163

30.Lee SC,etal.Macrophage-colony-stimulating factor in human fetal astrocytes andmicroglia.Differential regulation by cytokines and lipopolysaccharide,andmodulation of class II MHC on microglia.j Immunol,1993;150:594

31.Louis J-C,et al. CNTFprotection of oligodendrocytes against natural and tumor necrosisfactor-induced death.Science,1993;259:689

32.Low KG,et al.Differentialregulation of proenkephalin expression in astrocytes bycytokines.Endocrinology,1992;131:1908

33.Lyson K,et al.Involvement ofarachidonic acid cascade in IL-6 stimulated CRF release invitro.Neuroendocrinol,1992;55:708

34.McGeer PL,et al.Reactivemicroglia are positive for HLA-Dr in the substantia nigra of Parkinson's andAlzheimer's disease brains.Neurology,1988;38:1285

35.Milenkovic L,et al.Cachectinalters anterior pituitary hormone release by a direct action in vitro.Proc Natl Acad Sci USA,1989;86:2418

36.Millington G and BuckinghamJC,Thymic peptides and neuroendocrine immunecommunication.JEndocrinol.1992;133:163

37.Muller H,et al.Interfaeronalpha 2-induced stimulation of ACTH and cortisol secretion inman.Neuroendocrinol,1991;54:499

38.Naiboh Y,et al.Interleukin-6stimulates the secretion of corticotropin in conscious freely movingrats.BBRC,1988;155:1459

39.Nistico G.Communications amongceutral nervous system,neuroendocrine and immune systems:interleukin 2.ProgrNeurodiol,1993;10:463

40.Nohava K,et al.Neurons andneuroblastoma as source of macrophage colony stimulating factor.Eur J Immunol.

41.Patterson PH and NawaH.Neuronal differentiation factors/cytokines and synaptic piasticity.Neuron10(suppl)1993;10(suppl):123

42.Perry VH,et al.Macrophages andinflammation in the central nervous system.TINS,1993;16:268

43.Rabin BS,et al.Bidirectionalinteraction between the central nervous system and the immune system.CriticalRev Immunol,1989;9:279.

44.Rogister B,et al.Transforminggrowth factor beta as a neuroglial signal during peripheral nervous systemresponse to injury.J.Neurosci Res,1993;34:32

45.Selmaj KW,et al.Proliferationof astrocytes in vitro in response to cytokines.A primary role for tumornecrosis factor.J Immunol,1990;144:129

46.Shieh JH,et al.Cytokines and dexamethasone modulation ofIL-1receptors on human neutrophils in vitro.J Immunol.1993;150:3515

47.Spangelo BL,et al.Induction ofinterlukin-6 release by interleukin-1 in rat anterior pituitary cells invitro:Evidence for an eicosanoid dependent mehanism.Endocrinology,1991;129:2886

48.Spinedi E,et al.Cytokinestimulates the CRH but not the vasopressin neuronal system:evidence for amedian eminence site of interleukin-6action.Neuroendocrinol.1992;56:46

49.Tsagarakis S,etal.Interleukin-1 directly stimulates the release of corticotropin releasingfactor from rat hypothalamus,Neuroendocrinol,1989;49:98

50.Tsitsiloni OE,et al.Expressionof α-thymosins in human tissues in normal and abnormal growth.Proc Natl AcadSci USA.1993;90:9504

51.Vankelecom H,et al.Evidencethat folliculostillate cells mediate the inhibitory effect of interferon-γ onhormone secretion in rat anterior pituitary cell cultures.Endocrinology,1992;130:3537

52.Yamashita H,et al.Effects ofintekleukin 1β on membrane potentials of hypothalamic meurons in rats invitro.Amm NY Acad Sci,1993;689:689