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AN ELECTROPHYSIOLOGICAL INVESTIGATION OF MONOSIALOGANGLIOSIDE IN THE MAMMALIAN CENTRAL NERVOUS SYSTEM
by
Peter Miu
Department of Physiology McGill University, Montreal, Canada
August 1992
A thesis submitted to the Faculty ofGraduate Studies and Research in panialfulfilment ofthe requirements ofthe degree
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ofDoeror ofPhilosophy. Cl
Peter Miu. 1992
I~I
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ISBN 0-315-87969-6
Canada
Running title for the Ph. O. thesis submitted by PETER MIU:
Actions of GMl in the mammalian central nervous system.
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1 LIKE TO DEDICATE THIS THESIS TO MY WIFE, Natushka•
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•.... ta ail who eum ta science. not because they intend ta take it up ta - write or discuss or teach - bU! ta get answers ta straigh:. simple everyday problems.•
Tolstoy. Resurrection.
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Miu/Abstract/... iii
ABSTRACT This thesis focuses on the functional role of monosialoganglioside (GMl) in neuronal activity and synaptic transmission of rat hippocampal slices. The slices were placed in an interface recordinl; chamber and constantly superfused with oxygenated saline at 33 0 • Both extracellu1ar and intracellu1ar (current- and voltage-elamp) recording techniques were used to measure the field potentials and postsynaptic responses respectively. Our findings indicated that GMI induces a small inward current in CAl pyramidal neurons. depresses their high voltage activated (HVA) Ca2 + currents, and selectively facilitates the excitatory synaptic inputs while reducing the inhibitory ones. The most probable mechanism underlying the selective enhancement of excitatory inputs by GMI is an increase in glutamate release, since the amplitudes and frequency of spontaneous miniature postsynaptic responses, recorded either in the presence or absence of presynaptic ceIl firing, we;e consistently inereased. When L-glutamate was applied iontophoretically in the dendritic region, GMI transiently potentiated the postsynaptic glutamate currents, thus further indicating a GMI-induced enhancement of glutamatergic transmission. In contrast, both spontaneous and evoked inhibitory postsynaptic responses were suppressed by GMI. This effect is dependent on changes in e."{citatory inputs to inhibitory intemeurons because in the presence oftetrodotoxinandlorkynurenicacid, GMI did not alter the amplitude of the monosynaptic IPSPs or the frequency of spontaneous miniature IPSCs. Both the GMI-induced inward current and the reduction ofpostsynaptic HVA CaH currents were antagonised by kynurenic acid, suggesting that these effects might be caused by glutamate receptor activation.
By raising intraneuronal Ca2 + concentration, the
potentiated glutamate release wou1d trigger CaH -dependent CaH inactivation, and thus explain the reduction in HVA Ca2+ currents. In conclusion, most of the GMI actions observed in this project can he explained on the basis of a GM l-induced facilitation of excitatory transmission, mediated especially via enhanced glutamate release•
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Miu/Résumé/" , il'
RÉSUMÉ Les p'*"'ntes expériences, réaliS
IN RAT HIPPOCAl"lPAL SLICES . . . . . . . . . . . . . . . . . . . . . . . 1 3.2 Summary . . . . . . . . . . . .
2
3.3 Introduction . . . . . . . . . • . . . . . . . . . . . . • . . . . . . . . . . . . . . . 4
3.4 Methods
3.4.1 PreparatùJn of slices ......•.........•..•.••.... 6 3.4.2 RecorrIing techniques and arrangement
6
3.4.3 Solutions ......•........................... 9 3.5 Results
3.5.1 ExtraceIIular studies 3.5.1. i EffeclS ofexogenous GMI on eUTocellwarfieMporenti~
......•.......... 9
3.5.2 IntraceIIular stlufies 3.5.2.i EffeclS ofGMI on
passive membrane propenies . . • . . . . . . . . . . . . . 10 3.5.2.ii Ejfecrs of GMI on evoked posrsynaptic porenti~
and carrent: . • . . . • . . . . . . . . . . . . .. 11
3.5.2.iii EffeclS of GMI on isolared inhibitory inpUtS .•.•. 12
3.5.2.iv EffeclS of GMI on gluramare-evoked inward currenrs . . . . . . . . . . . .. 13
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3.5.3 Sponraneous posrsynaptic currenrs
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Miu!Table of Contents!... X
(recortIed in the absence of TTX)
3.5.3.i Spontaneous EPSCs and IPSCs recorded in ACSF
I~
3.5.3.ii Effects ofGMl on spontaneous EPSCs and IPSCs
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3.5.3.iii Effects of GMl on isolated e:r:citatory inpUts to the pyriJl7lidaI cells
15
3.5.4 Spontaneous 'miniature' PSCs (recortIed in the presence of TTX)
3.5.4.i Effects ofGMl on the miniature EPSCs in Crl3 cells . . . . . . . . . . . .. 16
3.5.4.ii Effects ofGMl on the miniature IPSCs in Crll cells
18
3.6 Discussion 3.6.1 The effects of GM1 on ex:ciJoJory synaptic transmission 3.6.l.ï Increasing presynapticfiring . . . . . . . . . . . . . . . . 18
3.6.1.ii Increasing transmitter release
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3.6.l.iii Posrsynaptic ligand-receptor interaction
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3.6.2 The effects of GM1 on inhibitory synaptic transmission
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3.7 Figures • . . • . . . . . . . . . . • . . . . . . . . . . . . . . . • . . . . . . . . . 24 3.8 References • . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . 42 CHAPTER4
4.1 MODULATION OF ffiGH VOLTAGE ACTIVATED Ca2+ CURRENTS
BY GANGLIOSIDE GMI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4.2 Snmmary •....................................... 2
4.3 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . 4 4.4 Methods
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4.4.1 PrepaTation of slices 4.4.l.ï Recording tecJmiques and arrangement
5
6
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MiuITable of Contents/...xi
7
4.4.I.ii Solutions 4.S Results
4.5.1 High voltage activared (HVA) inward
U"
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currents
4.5.I.i Effecrs ofGMI recorded wirh CsCl-jilied elecrrodes
4.5. I.ii Effecrs of Mr?" and low
or"
8 9
4.5.1. iii Effecrs of kynurenic acid on GMI-mediared responses
10
4.5.1. iv Effecrs ofbicuculline on GMI-mediared responses . . . . . . . . • . . • . . . . . . 12 4.5.1. v Effecrs ofGMI with Cs acerare-jilled eleetrodes . . . . . . . . . . . • • . . • . 12 4.6 Discussion . . . • . . . . . . . . . . . . . . . • . • • • . . . . . . • . . • . . . . 13 4.6.1 High voltage aetivared inward U" currents . . . . . . . . . . • 14 4.6.2 Effecrs of glutamate on the holding current
14
4.6.3 Involvement of second messengers
15
4.6.4 Effecrs of GMI on chloride conductances . . . . . . . • . . . . 16
4.7 Figures . . . . . . • . . • • . . . . . . . . . . . . . . . . • . . . . . . . . . . . . 18 4.8 References . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
CHAPTERS
S.l GENERAL DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 S.2 E.xogenous Versus Endogenous GMl . . . . . . . . . . • . . . . . . . . . . . 2 5.2.1 Interaction lvith adenylizte cyclase •........••..••..• 3 5.2.2 GMI degrrulatior. • • . . . • . . . . . . . . . . . . . . . • . . • . • . . 3
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S.3 The Mechanisms Underlying the Effects of Exogenous GMl 5.3.1 Phosphoinositide metabolism . . . • . . . . . . . . • . • . • • . . . • 5
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,\;jiu/Table of Contents/...xii
S.3.U Prorein kinase C S.3.l.Ü
6
cti+ /calmodulin-dependent lânase
Il
5.3.2 Indirect activalion ofglutamate receptors by GMI
6
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5.4 Consequences of GM1 Application
8
5.5 Effects of GM1 on Inhibitory Nerve Tenninals
9
5.6 Conclusions
. . . . . . . . . . . . . . . .. 10
5.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 14
CHAPI'ER6 6.1 SUMMARY AL'ID ORIGINAL CONTRIBUTIONS
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1
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Miu/list of Figures/.. .xiii
LIST OF FIGURES
Page
1.1
Parental structure of brain gangliosides
2
1.2
ChemicaI structures of sphingosine-containing lipids
4
1.3
Synethic pathways of both major and minor brain gangliosides
7
lA
Degradation pathways of brain gangliosides . . . . . . . . . • . . . . . . • . . . . 12
1.5
Working model of ganglioside metabolism . . . . . . . . . . . . . . . . . . . • . 14
2.1
A schematic diagram of a Haas-type recording chamber
3.1
GM1 enhanced the positive wave (EPSP) and population
5
spike, but did not alter the population spike-to-EPSP relation for Schaffer collateral-commissural evoked responses in the stratum pyramidale of rat hippocampal slice . . . . . . . • . . . . . . . . . . . . . . . . . . . . • • • . . . . . . . . . . . . . . 24 3.2
GM1 augmented the slope of the input-outputcurve (field EPSP to afferent volley relation) for Schaffer collateral-commissural evoked responses in the stratum radiatum . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . • . . . . . . . . . 26
3.3
GM1 selectively enhances evoked EPSP while suppressing evoked IPSPs recorded in CA1 pyramidal cells
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3.4
GM1 did not alter monosynaptica11y-evokedIPSPs, and had little effect on the voltage-sensitivity of
28
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Miullisr of Figures/...xiv monosynaptic IPSPs .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.5
GMI ttansiently potentiates inward currents elicited by glutamate applications (A)
3.6
32
In the absence of GABAA receptor-mediated IPSPs. GM 1 enhances the spontaneous TIX- and k.-ynurenate-sensitive EPSPs recorded from a CAl pyramidal cell
3.7
34
GMI increases both the frequencyand amplitude of spontaneous TIX- and kynurenate-sensitive EPSPs recorded from a CA 1 pyramidal cell . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . 36
3.8
GMI increases the frequency but not the amplitude of the spontaneous TIX-sensitive miniature EPSCs recorded from
CA3 ce11s . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . 38 3.9
GMI had no effect on the frequency cr the amplitude of the spontaneous miniature IPSCs recorded from CAl cells in the presence of TIX .. . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . 40
4.1
GMI attenuates HVA inward Ca2 + currents recorded in CAl neurons with CsCI-filled electrode . . . • . . . . . . . . . . . . . . . . . . . . . . 18
4.2
A, HVA Ca2 + current is reduced by GMI and abolished by Mn2 + and low Ca2 + perfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.3
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Low Ca2 + and kynurenic acid block GMI-induced inward shift inIH · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 2 2
4.4
Kynurenic acid (1 mM; KYN) antagonizes GMI-induced attenuation
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MiulIist of Figures/...xv
of HVA Caz+ currents
4.5
GMl-inducedattentuationofHVA Caz+ currents is more pronounced for the peak than for the 'steady state' CUITent
4.6
24
26
Histograms summarizing GMI-induced inward shift in holding current (
n of HVA Ca~+ currents by bicuculline suggest a possible interaction between GMI and GABA receptors. in particular GABAA • At present. we have insufficient information to fullyexplain this observation. in view of the fact that GM 1 does not alter spontaneous GABA release (Miu and Kmjevié, 1991a).
. In conclusion, our findings suggest that GMI modulates HVA Ca~+ currents via activation of glutamate receptor-mediated channels, and perhaps in consequence of increased [Ca~+]i results in the block3de of HVA Ca~+ currents.
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Miu/Chapter 4/... IS
4.7 Figures
Figure 4.1. GMI attenuates HVA inward Ca:'" currents, recorded in CAl neurons with CsCI-filled electrode. A) HVA inward Ca:'" currents (above) were elicited by constant depolarizing voltage commands (below). Ca:-+- current was isolated by blocking Na'" currents with TIX (1 /LM), and K'" currents with TEA (10 mM), CsCI (4 mM), and 4-AP (100 JLM). B shows (for the same cell) the current-voltage (IN) relation of the peak inward current (before, during, and after bath application of GM l, 1 /LM). Note that, GMI reversibly reduced the net Ca:-+- current over a wide voltage range, without any change in the voltage-dependence and the threshold for activation. SubstantiaJ recovery was seen after 30 min of wash. This cell was c\amped at VH -40 mV, as indicated.
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MiulChapter 4/...19
A Control
GMI
Recovery
J ..... ..... --l ..~.--------l.---.--.----
~
~
~G.~
Je-
_rL JL B V (mV)
-120-100
-sa -sa
-40 -20
1
a
20.40 , '
a -l
-2 -3
a • .0.
C4D,t..-ol
Clll Reco.ery
-4
-5 -6 -7
--=
