THE USE OF CO2 AS A METHOD FOR STUDYING THEJ-REFLEXdemonstration of monoamine oxi

7.. . The inhibition of mitochon SUBHRA BASU

Department of PhysiologyUniversity College of Medical Sciences, Ring Road, New Delhi-l10016

Sammary: In order to study the J-reflex, monosynaptic reflexes were recorded from L7 or SIentral root after stimulation of the posterior biceps; and semi-tendinosus nerve (PBST) from the lower

limbin cats anaesthetisized with Pentobarbitone sodium. Intratracheal CO2 (60 ml, 100%) depressedthe monosynaptic reflexes, and the depression was comparable to the effects of right atrial phenyldiguanideinjection. Bilateral vagotomy did not abolish the response showing that the afferent pathwayofthis depression does not travel via the vagus nerve. Thus it is concluded that CO J cannot be usedID study the] -reflex.

monosynaptic reflex PDC J-reAex

INTRODUCTION

of an eiectrophoretically homog., 206: 61-70, 1970.

• Bull., 29 : 116-119, 1973.• Bioebem, J., 50 1320-326, 1952.es In MTP International Review

In 1969, Paintal redesignated the deflation receptors as the type 1 pulmonary endingsowed that the natural stimulus for these endings was pulmonary congestion. Deshpande

Devanandan (3) injected phenyl diaguanide (PDG) intra-atrially to activate the type 1rs and found reflex inhibition of monosynaptic reflexes (MSR) from hind limb muscles.This phenomenon was termed as the .T-reflex by Paintal (11). Since muscular exercise

a rise in pulmonary arterial pressure (5) which may activate the type Jvreceptors,put forward the hypothesis that f-reflex may be an important viscerosomatic reflexinate the exercise. As the simulation by natural stimulus i.e. Pulmonary congestionulate type I-receptors involves a number of technical difficulties, phenyl diguanide becameg of choice to study the characteristics of these endings (11). But it was shown that

I diguanide has got tremendous species difference to activate various lung receptors (2).a suitable stimulus to study the Jvreflex in various species had to be worked out. Dickin-Paintal had shown that CO2 activates type 1 receptors (4). It was, therefore, of

I 10 see its role in stud ying the 1 reflex.

lium salts for the detection292, 1972.

MATERIALS AND METHODS

Twelve adult cats weighing 2.4 to 5 kg were anaesthetised with intraperitoneal injectiontobarbitone sodium (40 mglkg]. The trachea was cannulated by a glass cannula withbes for injecting CO2 or air. Two polythene catheters were placed, one in the rightto inject PDG or saline and the other in the aorta to monitor the blood pressure as well

atropinise the animal (Atropine Sulphate I mg/kg). The nerve to the posterior biceps anddinosus (PBST) was dissected out carefully and cut close to the muscle. The other

of the sciatic nerve were cut. Electrical stimulation by rectangular wave pulses ofduration, and 1.5 times the threshold value was given to the PBST nerve every 2 seconds

er-silver chloride electrodes in order to stimulate all group I fibres (a and b) (10).ynaptic reflexes were recorded from the L7 or SL ventral roots after performing laminec-

from L~ to SI segments. Care was taken not to damage the dorsal root or dorsalnglia. The reflexes were displayed on the oscilloscope (Tektronix type 422) and wereaphed by Cossor camera.

'....

336 Basu October-Decemb-rInd. J. Physiol. Pha

The monosynaptic reflexes were recorded for a period of 200 to 300 seconds to showthere were any linear trend of depression (12). This was followed by a rapid injection of 2of PDG (200 ['gm) into the right atrium and the monosynaptic reflexes recorded till the recovof the pre-injection control heights were obtained. As a control, 2 ml of 0.9 % NaCI was injecinto the right atrium in place of PDG and the responses were recorded as before.

Sixty nil of 100 % CO2 was then introduced through the tracheal cannula in the end-expira-tory phase and the effects on MSR were noted. A diagrammatic sketch of the experimental designis given in Fig 1. Equivolumes of air or 100 % N~ (60 rnl) was also used to see their effectsonMSR and served as a control. As a routine, 5-10 minutes gap was given in between each seriesof experiments and the test trials repeated several times in anyone experiment. Following theseobservations bilateral vagotomy was performed, and after a gap of half to one hour, all the abovementioned series using PDG, saline, CO2 and air or N2 (100 %) were repeated. The height of each

R,VENTR1CLE LVENTRICLE

DORSAL ROOTV

tVENTRALROOT

REFLEX

Fig. 1 Represents the diagrammatic sketch of the experimental design,

.. .~

\'olume 21Nurnber 4

control reflex was measured cdered as 100%. Now on a grted as percentage of the evera120 mm of H g throughout theThe body temperature of the

•

Fig. 2 compares the effecreflexes recorded from L7 veas percentage of the average

120o •• 0 08 o• : 0

o .-. ~ .0 00 • 1)0. ...~ . .. o· 0 (70

I8C

800 •• ooe·,# •• •

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70 o•

10o

•

October-December IInd . .I. Physiol. Pharm

to 300 seconds to showy a rapid injection of 2

recorded till the recoveof0.9% NaCI was injecteded as before.

CO2 Effect on .I-Reflex 337Volume 21Number 4

control reflex was measured carefully, and the arithmatic mean of the control reflexes was consi-dered as 100%. Now on a graph paper the measured values of ~heindividual reflexes were plot-ted as percentage of the everage control values. The mean blood presure was maintained above120 mm of Hg throughout the experiment by continuous intravenous dri p of 5 % glucose in saline.The body temperature of the animals was maintained at 37°-381C.

Fig. 2 compares the effect of PDG and saline with that of CO2 and air on the monosynapticreflexesrecorded from L7 vental root. The ordinate shows the height of the reflexes presentedas percentage of the average control height taken as 100% (represented by the horizontal line).

RESULTScannulain the end-expira-of theexperimental designsed to see their effects on

ivenin between each seriesriment. Following these

f to one hour, all the abovepeated. The height of each •

o •• 0 o~ 0 o·• : 0 <fib- f)\J V • 0 Clc

o •• •• ~.o 0.. ,. 0 0

o· • 0 • • <!JI <:»0o• 0° i' Gt -1"\ ~ 00. .~ ._ 0 6> CD 0

QO~~~~o~~~2o-m~.}L~~~.~_~o~-.'.".~~o~.-.'~~o~o~o~.~~~m~~~om~----.---o 00/')0 •• I') • 0

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oe • e. 0 60" •••• 0 .~

• 0 • 0 • • •110• o~. 0 •• ~ .oo~ .0. o.~

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•190

o 0 oo~ 00., 'b <!o~~o 0

.~. • 0" 00 .c9e• .,0.0 •

o e 0 • 0• • •• o o70 o• o

• Fig. 2A: Compares the effect 0("PDG (closed circles) and saline(open circles) on MSR. Thepoint of injection is indicatedby the vertical line.

o o.c ••'..~•

• • • •o. •.. ... ••• ••

0 ••

•

o

o<0

Fig. 2B: Shows the effects of ( O2(closed circles) and air (cpencircles) injected at the pointof vertical line.

o • •

00 .-

e •••• e.• •-,.•,

•••

design.~~------~~--------~--------~~------~o 6~ J30 195 200

SECONDS

..

Oetober-Dcecmbci 1971Ind. j. Physiol. Pharrnar

The abscissa shows the time in second The filled circles (Fig 2A) show the control values andthe effect of POG on the monosynaptic reflexes. There is a variation in the control heights ran-ging from 69 % to 120 %. The vertical line demarcates the exact point of injection of POG orsaline. POG produces a depression of the monosynaptic reflexes upto 38.1 ~() (at 8th second),The depression, with gradual recovery per i ted for 54 seconds. By contra t saline did not changethe control heights (open circles) which also ranged from 73.80

0 to 125 o~ of the average controlvalue and were quite comparable to the pre-POG control height.

338 Dam

A 12C • (>

n

o

oo

• 0•

It 80

60

40

200 40

6120

0

••• Fig. 3A: Shows the cortrol MSR andthe effects of CO. (closed circles)and air open circles) on it beforevegotomy.

..

"!.- -

81,) 120SE<XJ11V5

200 240

•

.-•

•••••••

fig. 3B: Shows the respon es afterbilateral vagotomy. Thc testsubstances were injected at thevertical line .

60 • •

''t~.30

o 200

Fig 2B shows the effects of CO2 and air in the same animal. The control monosynapticreflexes before injection of CO2 (filled circles) showed variation from 66.5 to 125 %. After injec-

Volume 21Number 4

tion of CO2 (at the vertical line)with gradual recovery lasted forcircle) range from 69.8 % to 136;reflex heights.

-- ..••.. -_ .•.. - ...• -

Fig. 3 shows the comparicircles) and air (open circles) beThe reflexes are recorded fromMSR to 71.2 % (Fig. 3A): closedid not bring about any chang3B: filled circles) CO2 depressesthe depression lasts upto 38 secopen circles) .

Fig. 4 shows the actual rof CO2 and air before bilateral4 (3) and (4)]. Table I summaall the animals POG failed to

Fig. 4: Shows the os(strips I and 2

Onober-Dcu'm/)e) 1977Ind . .I. l'hysiol. Pharmac

thecontrol values andthecontrol heights ran-of injection of POG or38.1 "0 (at 8th second)

t alinedid not changeo of the average control

: Shows [he cortrol MSR andeffects of CO, (closed circles)air Open circles) on it beforetom)'.

hows the responses afterral vagotomy. The test

ances were injected at thealline.

control monosynaptic125\. After injec-

•.

CO2 Effect on J-ReAex 339

lionof CO2 (at the vertical line) the reflexes are depressed upto 37.2 % (at the 8th second) whichwithgradual recovery lasted for 32 seconds. The control values before inejction of air (opencircle)range from 69.8 % to 136% of the average. Injection of air produced no change in thesereflex heights.

Fig. 3 shows the comparison of the effects of the intratracheal injection of CO2 (filledcircles) and air (open circles) before (3A) and after bilateral vagotomy (3B) in the same animal.Thereflexes are recorded from SI ventral root. CO2 injection before vagotomy depressed theMSR to 71.2 % (Fig. 3A): closed circles which lasted for 20 seconds. Air, as seen in other series,didnot bring about any change (Fig. 3A: open circles). Following bilateral vagotomy (Fig.3B: filled circles) CO2 depresses the monosynaptic reflexes up to 44.6% (in the 14th seconds) andthedepression lasts upto 38 seconds. Air in this case also did not produce any change (Fig. 3Bopen circles).

Fig. 4 shows the actual records of monosynaptic relexes comrpising respectively the effectof CO2 and air before bilateral vagotomy [Fig. 4 (1) and (2)] and after bilateral vagotomy [Fig.4 (3) and (4)]. Table I summarises the results obtained before and after bilateral vagotomy. Inall the animals PDG failed to produce any depression of MSR after bilateral vagotomy.

Fig. 4: Shows the oscilloscopic recording of MSR and the effects of CO2 and air(strips 1 and 2) in intact animal. Strips 3 and 4 show the responses after bilateral vazotomv,

340'" Basu October-December 1977Ind. J. Physiol. Pharmac.

TABLE I: Shows the monosynaptic reflex heights in mm (Mean ±S.D.) and the range inperenthesis & the effect of CO2 and air or 100% Nz before & after bilateral vagotomy.

Before bilateral vagotomy After bilateral oagoto.ny

Cat. Control ±S.D. After CO. After air or Control After CO2 After air or»« (peak ./12 (peak depress ±S.D. (epak N2(peak

depression level) ession level) depression depressionlevel) leoel}

8 19.45±1.63 12.2 IB.O IB.19±1.B2 B.B 11.2

(14.1-22.50) (12.2-19.5) (IB.0-22.0) (15.5-22.0) (B.B-17.B) (11.2--15.5)

9 13.41±I.B4 10.0 17.5 14.31±0.09 7.0 10.4

(9.5-19.2) (10.0-16) (17.5-1B.2) (11.5-16.8) (7.0-14.B) (10.4-16.5)

10 IB.76±1.26 16.2 17.0 19.99±0.90 12.5 17.5(17.0-22.0) (16.2-21.0) (17.0-23.0) (17.4-22.5) (12.5-17.4) (17.5-20.5)

11 J9.44±1.49 14.1 17.0 19.B9±0.93 B.B 17.5

(16.5-23.5) (14.1-20.0) (17.0-23.0) (16.5-23.5) (8.B-19.0) (17.5-20.5)

12 18.9B±2.27 7.0 14.0 19.50±2.61 9.5 16.0(12.5-23.5) (7.0-20.0) (14.0-20.5) (13.8-24.0) (9.5-24.0) ( 16.0-26.0)

13 17.30±2.76 5.5 16.0 20.15±1.55 12.0 19.5

(11.0-22) (5.5-14.0) (16.0-19.0) (16.B-23.0) (12.0-22.2) (19.5-22.0)

16 18.2B±1.11 4.0 15.0 19.B6±1.64 6.0 12.0(14.B-21.0) (4.0-21.0) (15.0-23.5) (15.2-24.0) (6.0-24.0) (12.0-24.0)

17 19.68±1.34 7.0 16.0 21.B6±1.43 10.0 17.5

(15.5-22.2) 7.0-20.5) (16.0-19.0) (18.0-·25.0) (10.0-22.2) (17.5-25.0)

DISCUSSION

me 21her 4

the lung (13). The fact, howevemonosynaptic reflex, while identi

D, suggests that the activation ofonosynaptic reflexes. This effect. ished, it would indicate that brad

sion of monosynaptic reflexes (3)er vagotomy in contrast to the

e pathway for CO~ cannot be methway for J-reflex is known to beat CO2 affects CNS directly at

uestion about the use of CO2 as aresent studies are, therefore, a poimethod to study a reflex in generaill be of interest to find out the sit.S.R.

The present results show that CO2 depresses the MSR and the effects are quite comparableto the PDG depression effects. By contrast nitrogen (100 %) or air failed to produce any depres-sion. As early as 1912 Porter noted a rise in the threshold and eventuaIly the extinctionof theflexor reflex in decapitate cats, breathing 50 % CO2 in Oxygen [noted in (9) ]. Glazer (6) alsoobtained similar results in anaesthetised dogs breathing gas mixtures containing 20 % or moreCO2, In further studies King, Garrey and Bryan (8) found that in intact anaesthetised dogs thereis depression of knee jerk after breathing 2-4 % CO2, Many other workers showed that differentconcentrations of CO2 depress the monosynaptic reflexes to varying degrees (I ,9, 14). Woodburyand Karler (J 2) suggested that CO2 decreased the safety factor of transmission, and that CO2

would depress all synapses in which the safety factor is low, as it is in the monosynaptic pathwaysin the spinal cord. The result obtained in the present series though conducted under differentconditions than the earlier workers confirm the general notion that CO2 does depress the mono-synaptic reflexes. It may be argued that the volume of gases used in the present experiments,being virtually twice the tidal volume in cat, could have by itself stimulated the stretch receptors

Bradley, K., W. Schlapp and G. Scats. J. Physiol., 111 : 62P, 19jO.Dawes, C. S., J. C. Mott and J.and lungs. J. Physiol., U5 : 258-Deshpande, S.S. and M.S. DevanaJ pulmonary endings. J. Flrysiol.,Dickinson, C. J. and A. S. PaintaClin. Sci., 38 : ]\'0. 5 : P·33, 1970.Euler, U. S. von and C. Liljestrand.Acla Physiol. Scand., 12 : 301-320, 19Glazer, \V. Regulation of respiratiodioxide, sodium carbonate, sodiummuscle of the dog. Amer. J. Phys'Kalia, M. Cerebral pathways in r92P, 1969.King, C.E., W.E. Garrey and \\·.Rthe knee jerk. Amer. J. Physiol., IKirstein, L. Early effects of oxygenSupple. BO, 1951.Lloyd, D.P.C. Conduction and S)U6 : 317-326, 1943.Pai ntal, A.S. Mechanism of stimulRudomin, P. and H. Dutton. Eflof extensor moto-neurons. ). .\e\\'iddicombe, .1. G. The site of p\\'oodbury, D. M. and R. KarIer.68G-703, 196U.

This work was done underJnstitute, Delhi University, Delhi-of them. I thank Mrs. S. Chawl

October-Decem ber 1977Ind. j. Physiol. Pharmac.

cl the range intcral vagotomy.

al vagoto.ny

After CO2(epak

depressionlevel)

AIler air orNdpeakdepressionleoel )

8.8

(8.8-17.8)

7.0(7.0-14.8)

12.5(12.5-17.4)

8.8

(8.8-19.0)

9.5(9.5-24.0)

12.0

(12.0-22.2)

6.0(6.0-24.0)

10.0110.0-22.2)

11.2

(11.2--15.5)

10.4

(10.4-16.5)

17.5(17.5-20.5)

17.5

(17.5-20.5 )

16.0(16.0-2G.O)

19.5

(19.5-22.0)

12.0(12.0-24.0)

17.5(17.5-2-3.0)

ts are quite comparableto produce any depres-

lly the extinctionof the(9)]. Glazer (6) also

ntaining 20 % or moreanaesthetised dogs theres showed that differentes (1,9,14). Woodburysmission, and that CO2

onosynaptic pathwaysnducted under differentdoes depress the mono-e present experiments,d the stretch receptors

CO2 Effect on J-ReAex 341

thelung (13). The fact, however, that 60 ml of air or N2 did not being about any change inmonosynaptic reflex, while identical volume of CO2 (60 ml) brought about a significant depres-, suggests that the activation of stretch receptors or hypoxia played no part in depressing theosynaptic reflexes. This effect was J ather CO2 specific. Further as the animals were atro-

ished,it would indicate that bradycardia or hypotension could not be the possible cause of dep-ionof monosynaptic reflexes (3). Since CO2 continues to depress the monosynaptic reflexesr vagotomy in contrast to the abolition of PDG induced J-reflex (3), it would indicate thatpathway for CO~ cannot be meaningfully used to study the J-reflex in as much as the afferentwayfor J-reflex is known to be mediated via non-medullated vagal fibres (11). Also, the fact

t CO2 affects CNS directly at multi-levels and causes the depression of MSR, raised a basicestionabout the use of CO2 as a specific test sunstance to investigate a particular reflex. The

nt studies are, therefore, a pointer in this direction to show the inadequacy of using CO2 asmethodto study a reflex in general, and J-reflex in particular. Granted this limitation, it would'11 be of interest to find out the site and nature of pathway involved in CO2 indiced depression of.S.R.

ACKNOWLEDGEMENTS

This work was done under Professor A.S. Paintal and Dr. M.S. Devanandan in V.P. Chest,Institute,Delhi University, Delhi-7. The grant was given by CSIR. I am deeply indebted to allof them. I thank Mrs. S. Chawla for typing the paper.

REFERENCES

Bradley, K., W. Schlapp and G. Spaccarclli, Effect of Carbon dioxide on the spinal reAexes In decapitatedcats. J. Physiol., 111 : 62P, 1950.Dawes, G. S., J. C. Mott and J. G. \\'iddicombe. Respiratory and Cardiovascular reAexes from the heartand lungs. J. Physiol., 115 : 258-291, 1951.Deshpande, S.S. and M.S. Devanandan, Reflex inhibition of monosynaotic reAexes by stimulation of typeJ pulmonary endings. J. Fhysiol., 206 : 345-357. 1970.Dickinson, C. J. and A. S. Pain tal. Stimulation of type J pulmonary receptors in cat by carbondioxide.Clin. Sci., 38 : :'\0.5 : P-33, 1970.Euler, U. S. von and G. Liljestrand. Observations on the pulmonary arterial blood pressure in the cat.Acla Physiol, Scand., 12 : 301-320, 1946.Glazer, "V. Regulation of respiration. XXX The effects of mechanical asphyxia and administration of carbon-dioxide, sodium carbonate, sodium bicarbonate and sodium cyanide on th reflex response of the anterior tibialmuscle of the dog. Amer. J. Physiol., 88 : 562 :569, 1929.Kalia, M. Cerebral pathways in reflex muscular inhibition from type J Pulmonary receptors J. Physiol., 204 :92P, 1969.King, C.E., W.E. Garrey and \V.R. Bryan. The effect of carbondioxide, hyperventilation and anoxaemia onthe knee jerk. Amer. J. Physiol., 102 : 305-318, 1932.Kirstein, L. Early effects of oxygen lack and carbon dioxide excess on spinal reflexes. Acla Physiol, Scand, 23 :Supple. 80, 195 I.LIoyd, D.P.C_ Conduction and synaptic transmission of retlex response to stretch in spinal cat. J. Neurophysiol.,6 : 317-326, 1943.Paintal, A.S. Mechanism of stimulation of type J pulmonary rcceptors. J. Physiol., 203 : 511-523, 1969.Rudornin, P. and H. Dutton. Eflects of conditioning afferent volleys on variability of monosynaptic responsesof extensor moto-neurons. J. J\euTophyslOl., 32 : 140-157, 1969.\\'iddicombe, J. G. The site of pulmonary stretch receptors in the cat. J. Physiol., 125 : 336-3:; I, 1954.\\'oodbllry, D. :VI. and R_ Karler. The role of carbon dioxide in the nervous system. Anaesthesiology, 21686-703, 1960.