Table Of ContentEffect of histamine on the electric activities of cerebellar Purkinje cell
Lin Tang
Department of Physics, Nanjing University, Nanjing 210093, China
(January, 1999)
The effect of histamine (HA) on the electric activities of Purkinje cell (PC) is studied on the
cerebellum slice. We find that: (1) HA’s main effect on PC is excitative (72.9%); there are also a
smallamountofPCshowinginhibitive(10.2%)orno(16.9%)responsetoHA.(2)Differentfromthe
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conventional opinion, HA’s excitative effect on PC is mutually conducted by H1 and H2 receptors;
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the antagonist for H1 receptor could weaken HA’s excitative effect on PC, while the antagonist for
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H2receptorcould weakenorevenblocktheexcitativeeffectofHAonPC.(3)PC’s reaction toHA
1
isrelatedtoitsintrinsicdischargefrequency;thereexistsafrequencyatwhichPCishighlysensitive
n toHA,andwellabovethisfrequencyPCbecomesstableagainstHA.Theseresultsindicatethatthe
a
histaminergicafferentfibrecanadjustPC’selectricactivitiesbyreleasingHA,andtherebyinfluence
J
theglobal function of thecerebellar cortex; and that just liketheγ region of cerebrum,cerebellum
1
may also havesome sort of characteristic frequency.
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PACS number: 87.10.+e
]
h
p Key words: cerebellar cortex; Purkinjecell; histamine; receptor; cerebellum slice
-
o
I. INTRODUCTION GR,basketcellBA,star-likecellST,andGolgicellGO).
i
b Hence we see that the cerebellar afferent fibres and the
s. Cerebellum deserves more extensive studies than was intermediateneurons,withPCactingasthecore,consti-
c conventionally realized. Historically, cerebellum was tute the basic neural circular that is responsible for the
si thought of as mainly a motor control organ, while re- sensoryfunctionofthecerebellarcortex,andthecerebel-
y cent researches reveal that it has many other functions, larcortextogetherwiththedeepnucleiundertakevarious
h andithasmoreintimateconnectionstootherpartsofthe functions of the cerebellum.
p
brain [1]. Histamine (HA), a neurotransmitter or neuro- InourlaboratorytherehasbeenstudiesofHA’seffects
[
modulator in the brain, plays an important role in the oncertainneuronsofthecerebellum,suchasthegranular
1 functions and interactions of various parts of the brain, cells [3,4], while HA’s effect on the neurons in the cere-
v andalsointhestudiesofthesefunctionsandinteractions. bellar cortex is not yet intensively studied. Considering
4 For instance, the neuroanatamic researches revealed the that PC is the only efferent neuronof the cerebellar cor-
0
existence of the hypothalamus-cerebellum histaminergic tex,wearegoingtoinvestigateHA’seffectontheelectric
0
2 path [2,3], and shows that hypothalamus has great in- activities of PC, based on our accumulated experiences
0 fluence on the cerebellar activities and hence plays an in studying the influences of aminergic materials such as
9 important role in coordinatingthe functions of the body norepinephrine (NA) and serotonin (5-HT) on the spon-
9 andviscera. Besides,HAhasthepossibleroleofcontrol- taneousandinduceddischargeactivitiesofthecerebellar
s/ ling the cerebellarcirculation,andHA receptorsare also PC; so as to learn more about the role of aminergic af-
c found in the neurons of the cerebellar cortex [2,3]. ferent system in the process of information treatment in
si Cerebellum is made up of the outer dark matter (cor- the cerebellar cortex.
y tex), inner white matter, and three pairs of deep nuclei
h
lying in the heart of the white matter. These nuclei are
p thefastigialnucleusFN,interposednucleusIN,andden- II. EXPERIMENTAL MATERIAL AND METHOD
:
v tatenucleusDN.Theafferentfibrestothecerebellumare
Xi mainly from the vestibule, spinal cord, and the cerebral We use for our experiments 19 mature SD rats (200-
cortex. Theyformsynapticconnectionswiththeneurons 250g). Anaesthetizearatbyinjectingbetchloramineshy-
r
a inthe cerebellardeepnucleiandcortex. Thesynapsesof drochloride (4mg/100g) into the abdominal cavity, take
Purkinjecell(PC)maketheefferentfibresofthecerebel- out the cerebellum right after cutting the head, wash
larcortex,theyaremainlyprojectedintothedeepnuclei, the cerebellum with frozen artificial cerebrospinal fluid
◦
then the neurons there stick out fibres which make the (ACSF,4 C),stickitontotheoperatingtableofamicro-
cerebellar output; a small amount of PC synapses are tome(atwhichthecerebellumissoakedinfrozenACSF),
directly projected into the vestibular nucleus. andcuta400µmthickofarrow-likeslicefromthevermis.
The cerebellar cortex can the divided into three lay- Theprocessofmakingthesliceshouldbedonewithin20
ers: the (out-most) molecular layer, PC layer, and the minutes. Then move the so obtained slice into a record-
granular layer. It contains three kinds of afferent fibres ingtrough,andbeginthe experimentafter15minutesof
(musciform fibre MF, crawl fibre CF, and monoaminer- hatching. The recordingtrough is continuously irrigated
gic fibre), and five kinds of neurons (PC, granular cell (3ml/min)byACSF(33±0.2◦C),andisaeratedwiththe
1
mixedairof95%O2+5%CO2. Theconcentrationsofvar- 59 cells with relatively stable spontaneous discharge for
ious elements in ACSF are (mmol/l): NaCl 124, KCl 5, ourstudy. Theirspontaneousdischargefrequenciesrange
KH2PO4 1.2, MgSO4 1.3, CaCl2 2.4, NaHCO3 26, glu- from4.48to78.32Hz. Wefindthatofthe59cells,83.1%
cose 10. Put a tiny glass electrode (filled in with colored (49/59) are affected by HA, the other 16.9% (10/59)
conducting fluid) at the PC layer of the X leaflet of the show no response. And of the 49 responsive ones, 87.8%
cerebellar cortex to make out-cell records of PC’s dis- (43/49) are excited, the other 12.2% (6/49) are inhib-
chargeactivity. ThisisbecausetheXleafletreceivedthe ited. (See Fig. 1) Therefore HA’s effect on PC is mainly
least mechanical wound in making the slice, and hence excitative. From Figs. 1B, 1C, we can see that there is
the cerebellum slice should be so put in the recording anevidentincreaseofthevariationofthecell’sdischarge
trough that the X leaflet be well hatched by the ACSF frequencyastheirrigatingconcentrationofHAincreases.
and the mixed air.
We base on the following criteria to single outPC dis- Excited
charge signals: (1) position of the electrode: PCs in the Inhibited
cerebellarcortexareof lineartype, namely,the cellbod- No reaction
ies concentrate at one end and the dendrites stick to the
other end, thereby forma PC layerinthe cerebellar cor- A
tex. We put the recording electrode at the out side of
the PC layer and near to the molecular layer, therefore
there is very little chance to catch a discharge signal of
the granular cell; (2) discharge wave shape: the PC ac-
tionpotentialhaslargemagnitude(0.700±0.143µV)and
thick contour [5], moreover, the effective recording dis-
tance of PC discharge is 50µm, much longer than that
of the granular cell (20-30µm) [4]; (3) cell numbers: in
the cerebellar cortex there are much less GO, ST, and
BA than PC, and hence the dischargesignals fromthese
cells may be ignored.
Because the nerve cells in the brain have the charac- B
ter of continuously producing impulses, and often at the
frequency of tens of times per second, the physiological
status of a neuron is usually symbolized by its discharge
frequency. Undertheenvironmentalstimulation,thedis-
chargefrequenciesofcentralneuronswillvariate,andthe
intrinsic potentials will also deviate. Hence we magnify
the PC discharge signal, choose a relatively small time
constant for the recording system, so as to differentiate
theinputsignalandtransformtheslowlyincreasingwave
intoasharphighpeakwave,thenuseapulsediscrimina-
tortoconvertthepeaksignalintoTTLpulseandinputit
into the computer, and then draw the post-stimulus his-
togram. ThuswecandescribePC’sreactiontochemicals
bythepost-stimulushistogramofitsdischargefrequency. C
Allthedrugsherearecontemporarilycompoundedus-
ing ACSF, and are used to irrigate the cerebellum slice
separately. When doing the experiments of studying the
receptormechanismofHA’seffectonPC,thesliceiscon-
tinuouslyirrigatedbythereceptorantagonistforover10
minutes before by HA.
FIG. 1. HA’s effect on PC; (A) shows the percentage of
III. RESULTS PC’s various reactions to HA; (B) and (C) show HA’s ex-
citative and inhibitive effects on PC, respectively; numbers
abovethe barrepresent HA’sirrigating concentration.
A. The effect of HA on PC’s electric activities
Another important observation is that whether or
Because intermittent discharge is often due to the ill not a PC shows response to HA may have some con-
state of PC or indistinguishability of cell discharge sig- nection with its spontaneous discharge frequency. (cf.
nal from other signals, we choose in 29 cerebellum slices Fig. 2) The spontaneous discharge frequencies of the 59
2
tested cells are 4.48-78.32 Hz, while that of the 49 cells on PC. Since HA’s main effect on PC is excitative, we
which show response to HA is 21.34±12.69Hz (M±SD), makestudy for the excitativeeffect. For this purpose we
this is significantly lower than the average frequency observe the influences of Triprolidine (hyper-specific an-
(37.00±7.73 Hz) of the 10 cells unaffected by HA. How- tagonist for H1 receptor) and Ranitidine (hyper-specific
ever, whether a cell is excited or inhibited by HA seems antagonist for H2 receptor) on PC’s excitative reaction
to have nothing to do with its intrinsic frequency. The toHA,andfindthatlow-concentrationTriprolidine(0.5-
average frequencies of the 43 excited cells and of the 6 1.0µmol/l) could weaken HA’s excitative effect on PC,
inhibited cells are 21.53±13.20 and 20.00±8.80 Hz, re- whilelow-concentrationRanitidine(0.9-1.0µmol/l)could
spectively, which are not much different (P>0.5, t test). weaken or even block HA’s excitative effect on PC. (cf.
This differs fromhow HA’s effect on the cerebellar gran- Fig. 3).
ular cells is related to their intrinsic frequencies [3]. We H3 receptor is a presynaptic self-receptor, it adjusts
also find that among the 49 responsive cells, those who the presynaptic release of HA [2]. In this research we
have higher intrinsic discharge frequencies are more sen- havenotinvestigatedits roleinHA’s excitative effecton
sitive to HA (there are 8 responsive cells with intrinsic PC.
frequencies higher than 30 Hz, and 7 of them show re-
sponsetoHAatthelowestHAconcentrationoflessthan
30µmol/l).
18
A
16
14
12
mber of cells180 EInxhciibtaitteedd A
Nu No reaction
6
4 B
2
0
0~10 10~20 20~30 30~40 40~
100
90
80
C
70
% of responsive cells 456000 RNeos rpeoancstiivoen B FIG. 3. Influence of receptor antagonist on HA’s excita-
30 tive effect on PC; (A) shows the normal excitative effect
20 of HA (50µmol/l) on PC; (B) shows that after irrigating
PC with ACSF containing 0.5µmol/l of Triprolidine for 15
10
minutes, HA’s excitative effect on PC is significantly weak-
0 ened;(C)showsthatafterirrigatingPCwithACSFcontain-
0~10 10~20 20~30 30~40 40~
ing 0.9µmol/l of Ranitidine, HA’s excitative effect on PC is
Frequency (Hz)
blocked.
FIG.2. PC’s discharge frequency and reaction to HA
IV. DISCUSSIONS
B. The receptor conducting mechanism of HA’s
excitative effect on PC
A. Data-taking system
Itisknownthattherearethreesub-typesofHArecep-
torsinbrain: H1,H2,andH3,thereforeitisnecessaryto In this experiment we used the data-taking system:
study the receptor conducting mechanism of HA’s effect oscillometer-pulse discriminator-computer, and drawn
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the post-stimulus histogram, then evaluate PC’s reac- those with higher frequency are highly sensitive to HA.
tion to chemicals by the post-stimulus histogram of We therefore speculate that there exists a critical fre-
the discharge frequency. The sampling bin width is 3, quency,acellwithintrinsicfrequencyneartothiscritical
point=900, interval=1000, one data represents the dis- point will significantly change its states by only a small
charge times of a cell in one second. Since a cell dis- amount of HA, while as its frequency goes well above
charges at very high frequency and its electric signals this point, it becomes stable. In connection with the in-
transmitatveryhighspeed,itgivesveryrichinformation fluenceofNAand5-HAonthespontaneousandinduced
in one second. We know that the information carried by electric activities of cerebellar PC [6], we go further to
aneuronisrepresentedbyitsfrequencydistributionover speculate that histaminergic and other aminergic affer-
the action potential, therefore if we can obtain the fre- ent systems might, through their coordinations and/or
quencyspectrum,andmakethecombinedanalysesofthe antagonism, adjust the synaptic transmission efficiency
frequencyandpowerspectra,wewillgetgreateramount ofMF-PF(parallelfibre)-PCandCF-PC,oradjustPC’s
information. Besides,what we take in this experiment is sensitivity to signalsfrom MF andCF, and thereby take
out-cell record of a cell’s discharge activities, and PC is part in the the global process of the sense movement of
in connection with various other cells in the cerebellum the cerebellar neuron net. That many kinds of neural
slice,the measurementinsuchcasemay be disturbed by active materials mutually act on the neuron is a com-
the environment, and the excitative or inhibitive reac- mon pattern of signal transmission of the nerve system;
tion of PC may actually be a combined result of various suchmutualactionmayhappenatthe presynapse,post-
influences including those from the environment. These synapse, or even postreceptor level. HA’s effect on the
are to be improved in future studies. cerebellar PC is to adjust the excitativity level of the
neuronand the neuron’s sensitivity to input information
from outside of the cerebellum, and adjust the sleep or
awake state of the cortex. The aminergic afferent fibres
B. HA’s effect on PC
have synaptic or non-synaptic chemical transmission ef-
fect on the cerebellar cortex, their non-synaptic trans-
Histamine is an important neurotransmitter or neuro- mission may not encode the fast phase-like information,
modulator, and acts as an inter-cell messenger. HA can but hyperfinely adjusts the membrane potential and ba-
hardly penetrate the blood-brain barrier, hence must be sic discharge level or the target neuron.
produced by the histaminergic neurons. It can be re-
leased through depolarization and by means of calcium
dependence. The hypothalamus-cerebellum histaminer- C. The receptor conducting mechanism of HA’s
gic fibres in the brain are casted by the hypothalamus excitative effect on PC
tuberous mastoidnucleus(HA pericaryon)into the cere-
bellar cortex and the cerebellar deep nuclei (HA nerve Of the three sub-types of HA receptors in the brain
end) [2,3]. Cerebellum-hypothalamus histaminergic sys- (H1, H2, and H3), H3 receptor is a presynaptic self-
tem can bring about many functions via the inter-cell receptor. The acting mechanism of H1 receptor is
messenger HA. The hypothalamus-cerebellum histamin- through some sub-type of G protein to activate PLC,
ergic projecting fibres end at the cerebellar cortex as hydrolize4,5-PIP2tobe1,4,5-IP3andDG,thesetwosec-
spreadingmulti-layerfibres,andtheir nerveends mainly ondarymessengersgofurthertotriggervariousbiological
spreadingly adjust the functions of the peripheral neu- effects, such as stimulating endoplasmic reticulum to re-
ronsinthevarixform;these aremuchlikethe serotonin- leaseCa+andthencetriggertheionchannels[2]. TheH2
ergic and norepinephrinergic afferent fibres of the cere- receptor brings effect mainly via G protein-AC-cAMP:
bellum [2,4]. HAcombineswiththeH2receptorofthecellmembrane,
Purkinje cell is the only kind of efferent neuron of the the H2 receptor is then deformed and triggers G protein
cerebellar cortex, it is a deformation of the multi-pole to give off its β and γ sub-radicals, and on the α sub-
cell, and has very strong dentrite. Our research shows radical GDP is replaced by GTP, then AC is activated
that PC’s discharge activity is significantly affected by andcatalyzesATPtotransformtocAMP,cAMPactsas
HA. From our observationsin Sec. IIIA, andassociating a secondary messenger and triggers a series of reactions,
how the reaction of cerebellar granular cells to HA is re- suchasactivatingPKAandthentriggeringionchannels,
latedto their intrinsic dischargefrequencies [3], we spec- ordirectly triggeringion channels. Becausemany highly
ulate that the cerebellar afferent fibres might be mainly efficient enzymes take part in these reactions, small sig-
responsiblefor adjusting the basic dischargelevelsofthe nals are magnified step by step.
cerebellarneurons: asthedischargefrequencyofatarget ItisusuallythoughtthatH1andH2receptorsconduct
celllies ina certainregionit losessensitivity to HA; and HA’s excitative and inhibitive effects on neurons respec-
as the discharge frequency lies out of this region the cell tively [2]. However, this is in contradiction with our re-
reacts to HA, and thereby its frequency tends to that portinSec. IIIB that low-concentrationH1 receptoran-
region. Moreover, the high-frequency region is a low- tagonistcouldweakenHA’sexcitativeeffectonPC,while
sensitivity region to HA, while for the responsive cells, low-concentration H2 receptor antagonist could weaken
4
or even block HA’s excitative effect on PC. This makes in the experiment. It is straightforward to check that
ustoconjecturethatbothH1andH2receptorsinvolvein u0 has a minimum value at an intermediate f0, just as
HA’sexcitativeeffectonPC,andH2receptoristhemain the experimental results show. Eq. (4) is much like the
conductor. There has been report that HA’s excitative relation in the Hopfield model. But further study of the
effectonthegranularcellsofthecerebellarcortexandon connectiontotheHopfieldmodelwouldrequiremorede-
the cells of the vestibular inner-side nucleus is mutually tailed knowledge about the relation between f and u,
conducted by H1 and H2 receptors [2]. Histochemistry which is not possible in the present experiment, for PC
researches also revealthat in the cerebellar cortex of rat cannotlivethroughalongenoughtime to allowtests for
thedensityofH2receptorishigherthanthatofH1recep- various HA concentrations; hence the simulation here is
tor. All these partly support our conjecture. This may merely a rough one.
suggest that the H2-related signal transmission chain in
cell differs from one type of neuron to another, and such
difference determines whether the H2 receptor conducts
E. Characteristic frequency in the cerebellum?
excitative or inhibitive effect; but the particular mecha-
nism of signal transmission is still to be studied.
Eqs. (1,4) show a pattern very similar the character-
istic frequency of the cerebrum (the γ region of around
D. Mathematical simulation
40 Hz): there exist a particular frequency at which the
target cell is highly sensitive to HA, and well above this
To systematically study the biophysical or biochemi- frequency the cell loses sensitivity to HA. For PC this
cal mechanisms of the reactions of cells, it is often help- insensitive region is 25.00-46.24 Hz, with the average
ful to make mathematical simulations of the reactions. 37.00±7.72,whichisclosetotheγ region. Thesesuggest
Here we give a simulation for the relation between PC’s thatcerebellummightalsoexhibitsomesortofcharacter-
(excitative) reaction to HA (measured by its maximum istic frequency to external stimulations. Further studies
variation of discharge frequency under the action of HA of such characteristic frequency would necessarily take
atacertainirrigatingconcentration)anditsintrinsicdis- into account of the interactions of various cell, such as
charge frequency f0. To be concrete, we choose a fixed in the HR model [7]; these we hope to accomplish in the
frequency variation A, and denote by u0 the irrigating future.
concentration of HA at which the maximum change of a
cell’s frequency would be A, then study the relation be-
tween u0 and f0. For this purpose we first look at how
V. ACKNOWLEDGEMENTS
the maximum frequency f of a cell under the action of
HA is related to HA’s irrigating concentration u.
As we noted in Sec. IIIA, PC’s reaction to HA has
The author owes special thanks to Prof. Jiang-Jun
something to do with its intrinsic discharge frequency:
Wang and Prof. Bing Zhu for valuable instructions, to
high-frequency cells are insensitive to HA, while for the
LeTianandJieMaforcollaborationsinthisexperiment,
cellsaffectedbyHA,thehigher-frequencyonesarehighly
and to Feng Dong, Qin Xi, and Guo-Ning Hu for kind
sensitive to HA, and at low intrinsic frequencies f in-
help and suggestions.
creases steadily with u. We therefore assume that f is
an S-like function of u:
f(u)=fc/(1+exp(−(u−uc))). (1)
As u=0, f(0) is just the intrinsic frequency f0:
f0 =f(0)=fc/(1+exp(uc)). (2)
[1] See,eg.,J.H.Gao,L.M.Parsons,J.M.Bower,J.Xiong,J.
According to our above explanations, we write Li,P.T.Fox,Science272,545(1996); Thompson,Science
238, 1729 (1987); and references therein.
f(u0)=f0+A=fc/(1+exp(−(u0−uc))). (3) [2] J.C. Schwartz et al, Physio. Rev.71, 1 (1991).
[3] W.C.Li,J.J.Wangetal,ScienceBulletin41,2269(1996)
From Eqs. (2,3) we can derive the u0-f0 relation:
(in Chinese).
u0 =ln(fc/f0−1)−ln(fc/(f0+A)−1). (4) [4] W.C. Li, J.J. Wang et al, to bepublished.
[5] C.M. Huang,H.Mu and C.F. Hsiao, Brain Res. 619, 313
Herethephysiologicalmeaningoffcisthemaximumdis- (1993).
charge frequency of the cell, and A is an arbitrarily cho- [6] J.J. Wang, et al, J. Physio. 48, 581 (1996) (in Chinese).
sen minimum frequency variation that can be observed [7] R.R.Llinas, Science 242, 1654 (1988).
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