J. Physiol. (1979), 294, pp. 239-253 With 8 text-figures Printed in Great Britain
239
EFFECT OF INTRAVENTRICULAR INJECTION OF TRANSMITTER SUBSTANCES AND TEMPERATURE ON AUTONOMIC FUNCTIONS OF CONSCIOUS SHEEP
BY A. M. BEAL* AND J. BLIGHt From the Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge, CB2 4AT
(Received 22 January 1979) SUMMARY
1. Changes in cardiovascular and renal functions following injection of noradrenaline, 5-hydroxytryptamine and carbachol into one lateral cerebral ventricle were investigated in conscious sheep at ambient air temperatures of 0 and 40 'C. The dose rates used were known to produce predictable changes in thermoregulation in sheep. 2. The changes in body temperature, respiratory frequency and shivering caused by the intracerebroventricular (i.c.v.) injections of noradrenaline, 5-hydroxytryptamine and carbachol were as reported previously. 3. The cardiovascular and renal responses of the sheep at both high and low ambient temperatures were consistent for each transmitter indicating that these activities had no major dependence on the type of thermoregulatory response. 4. i.c.v. injection of noradrenaline increased heart rate and decreased pulse pressure but caused no change in mean arterial blood pressure (B.P.). Urine flow rate, sodium clearance, potassium clearance and osmolal clearance were decreased whereas solute-free water reabsorption was unaltered. 5. ic.v. injection of 5-hydroxytryptamine caused no significant alterations in either cardiovascular or renal function. 6. i.c.v. injection of carbachol increased systolic and diastolic B.P., heart rate and haematocrit. Sodium, potassium and osmolal clearances were increased after administration of carbachol. 7. The changes in renal function after noradrenaline and carbachol injection could be explained by the concurrent changes in cardiovascular function. INTRODUCTION
Injection of the monoamines, noradrenaline and 5-hydroxytryptamine and of acetylcholine or cholinomimetic substances into the cerebral ventricles or anterior hypothalamus of various species has been found to cause marked changes in thermoregulation. In sheep, the consistent but ambient-temperature dependent effects of * Present address: School of Zoology, University of New South Wales, P.O. Box 1, Kensington, N.S.W., Australia 2033. t Present address: Institute of Arctic Biology, University of Alaska, Fairbanks, Alaska 99701.
0022-3751/79/4890-0976 $01.50 © 1979 The Physiological Society.
A. M. BEAL AND J. BLIGH intracerebroventricular (i.c.v.) injections of noradrenaline, 5-hydroxytryptamine and carbachol have been interpreted as indicative of specific synaptic roles of these substances in the central processes of thermoregulation (Bligh, Cottle & Maskrey, 1971). There is, however, no reason to assume that these or related substances which occur naturally in the preoptic/anterior hypothalamus and which may be synaptic transmitters, act only on thermoregulation. Other autonomic functions may also be modified by i.c.v. injections of these putative transmitters. In anaesthetized rats, rabbits, cats and dogs injection of noradrenaline into the lateral cerebral ventricle causes changes in cardiovascular function (Baum & Shropshire, 1973; Toda, Matsuda & Shimamoto, 1969; Smookler, Severs, Kinnard & Buckley, 1966; McCubbin, Kaneko & Page, 1960; Bhargava, Mishra & Tangri, 1972). Little is known, however, about the cardiovascular responses of conscious animals to the i.c.y injection of noradrenaline, 5-hydroxytryptamine or carbachol, and whether or not any alterations in cardiovascular function caused by such i.c.v. injections relate to concurrent changes in vascular peripheral resistance associated with 240
thermoregulation. Here we report on an investigation of changes which occur in other autonomically controlled systems when noradrenaline, 5-hydroxytryptamine and carbachol are injected into the lateral cerebral ventricle at dose rates which cause thermoregulatory effects in unshorn sheep. The systems observed were (1) the cardiovascular system which has long been known to be regulated by the autonomic nervous system and (2) the kidney, the autonomic innervation of which can influence renal blood flow, renin release and renal function by mechanisms that are not clearly understood. The effects of these i.c.v. injections were investigated at ambient temperatures of 40 and 0 TC, which are outside the range of thermoneutrality, to ensure that any observed effect of the drugs on cardiovascular and renal functions were not directly related to thermoregulatory activity. A preliminary report of this work has been presented to the Physiological Society (Beal & Bligh, 1976). METHODS
Experimental procedures Four unshorn Soay ewes weighing 20*0-25*5 kg were used. Each sheep had the right common carotid artery exteriorized in a loop of skin and had a permanent cannulation of one lateral cerebral ventricle (Barton, Bligh & Sharman, 1969). Before each experiment a vinyl cannula (1.4 mm i.d., 2*0 mm o.d.; Portex Limited) was inserted under local anaesthesia into one jugular vein using the technique of Seldinger (1953). The exteriorized carotid artery was cannulated with a disposable plastic cannula (Braunula, size 1; Armour Pharmaceutical Company Limited) directed towards the heart to facilitate blood pressure recording. After a catheter (Casper pattern; Rusch) had been inserted into the urinary bladder the sheep were harnessed and restrained singly in a standing position in climatic chambers maintained at either 40 or 0 IC. Rectal temperature and respiratory movements were monitored by techniques previously described by Bligh et al. (1971). The arterial cannula was connected by a thick-walled polyethylene tube filled with heparinized saline (500 i.u./ml.) to a pressure transducer outside the climatic chamber. Urine reaching the end of the bladder catheter was pumped rapidly to a collection site outside the chamber thereby reducing the number of times the room was entered to obtain samples. The sheep were allowed to become accustomed to the experimental conditions for 60 min after which an I.c.v. injection of 04 ml. sterile isotonic saline was given. Sixty minutes later the sheep received a second i.c.v. injection consisting either of 0 4 ml. saline (control treatment)
AUTONOMIC FUNCTION AFTER I.C.V. INJECTION
241
or of one of the three test substances dissolved in 0-2 ml. saline followed by a further 0-2 ml. saline to ensure all the drug was flushed into the ventricular space. The test substances used in this study were L-noradrenaline bitartrate (Sigma Chemical Company) at 20 n-mole/kg, 5-hydroxytryptamine creatinine sulphate (Sigma Chemical Company) at 40 n-mole/kg and carbachol (British Drug Houses) at 3 n-mole/kg. Urine collections were made serially over 15 min periods from the beginning of the acclimatization period until 120 min after the second i.c.v. injection. Jugular blood samples were taken at each mid-period in plastic syringes heparinized with 1 drop of heparin (5000 i.u./ml.). The sheep were subjected to this experimental procedure at intervals of not less than 7 days and the second i.c.v. injection of either saline or saline plus drug was given randomly at both 0 and 40 'C.
Analytical procedures (1) Blood samples were centrifuged in glass tubes at 2400 g for 10 min to provide plasma for analysis. (2) Microhaematocrit determinations were made in triplicate within 15 min of sampling on blood spun at 12,000 g for 10 min in a microhaematocrit centrifuge (Hawksley). (3) Sodium and potassium concentrations in plasma were estimated simultaneously and in duplicate by emission flame photometry in an oxygen-propane flame (Autotechnicon) using mixed standards in appropriate ranges to correct for mutual interference. (4) Osmolality was estimated in duplicate by freezing-point depression using a Fiske osmometer. (5) Arterial blood pressure was recorded for the last 30 sec of each 5 min throughout every experiment using a pressure transducer (Bell and Howell Limited, Type 4-327-L221) coupled to an amplifier and recorder (Devices Instruments Limited, Type M4). Systolic blood pressure was estimated from the recorder chart as the mean of the sum of all systolic pressure values recorded during the three 30 sec records of each 15 min urine collection. Diastolic blood pressure was estimated from the recorder chart in a similar manner to the systolic pressure. Mean arterial blood pressure was calculated from the systolic and diastolic pressures using the approximation. BP-Diastolic BP Mean arterial BP = Diastolic 3 (6) Heart rate was calculated as the mean rate/min over the three 30 sec periods of pressure recording in each 15 min urine collection.
BP+Systolic
Statistical procedures Analysis of covariance was applied to the data for each corresponding sample during the final 120 min of observation. The two covariates used in the analysis of each variable were the values of that variable corresponding to the urine collection period immediately preceding each of the two i.c.v. injections (i.e. periods 2 and 6 on the Figures). When the analyses of covariance produced significant variance ratios the differences between individual treatments were found using Tukey's w procedure (i.e. honestly significant difference (h8d) procedure) as described by Steel & Torrie (1960). The results of the hed analyses are presented as levels of significance in Table 1. RESULTS
Repeated i.c.v. injection of saline (control) At an environmental temperature of 40 'C (three animals) the rectal temperature of the sheep was reasonably constant throughout the 4 hr of observation. The animals panted with a mean respiratory frequency of 230 + 24-9 (S.E. of mean) breaths/min over the period of experiment. When the ambient temperature was 0 'C (three animals) the animals shivered but were able to maintain their rectal temperature unaltered and their mean respiratory rate was 17 + 2-2 breaths/min for the experimental period.
242 A. M. BEAL AND J. BLIGH The patterns of change in cardiovascular and renal function were similar for experiments at either ambient temperature. Therefore the data for the three control experiments at 40 0C and the three control experiments at 0 0C have been combined and the mean results are presented in Figs. 1 and 2. At both temperatures, systolic, diastolic and mean arterial B.P. and heart rate fell slightly over the period of observation whereas the haematocrit was unaltered Saline I
130
_
X 110 o
0
Saline
+hF
90
_
750
-
0
50
90
4070
-
3 ~
I
~
~
~~~~~~~~ I
40~
351m .. o m 0 I 1. Ssoidatlcadmaareiabloprsuehrtaean Fig.~~~~~. ee
30
i
emoci
i -30 0 60
120
~~~~180
Fig. 1. Systolic, diastolic and mean arterial blood pressures, heart rate and haematocrit before and after injection of 0n4ml. isotonic saline into the lateral cerebral ventricle of sheep (n = 6; means+ S.E. of mean).
(Fig. 1). Neither of the two ixc.v. injections of isotonic saline had any appreciable effect on this trend. Urine flow, sodium excretion and clearance, potassium excretion and clearance, osmolal clearance and solute-free water reabsorption declined during the initial 60 min regardless of environmental temperature and this trend continued after the first i.c.v. injection of saline. The second saline injection was followed by small and non-synchronous increases in urine flow, sodium excretion, potassium excretion and osmolal clearance during the subsequent 120 min of some experiments
(Fig, 2).
AUTONOMIC FUNCTION AFTER
I.C.V. INJECTION
243
i.c.v. injection of drugs During the first 2 hr of the experiments in which noradrenaline, 5-hydroxytryptamine or carbachol were injected the protocol was identical with the control experiments and the results obtained were similar to those for the corresponding time intervals of the control experiment. As the changes in cardiovascular and Saline
Saline
20 OC~
a,
1-0
X-.
0Q.5
C.-
c.E 40 _
=
0
.20
I_
C
o
3-0
E
2-0[__
0o 1-0 _I_
Q
-30
0
60 120 Time (min)
180
Fig. 2. Urine flow, renal sodium clearance, renal potassium clearance, renal osmolal
clearance and solute-free water reabsorption before and after injection of 0 4 ml. isotonic saline into the lateral cerebral ventricle of sheep (n = 6; means + S.E. of mean).
renal functions which followed I.c.v. injection of each of the above compounds were consistent for any one of the three substances injected, irrespective of whether the environmental temperature was 40 or 0 0C, the data for cardiovascular and renal parameters has been presented as the combined results for both temperatures in Figs. 3-8.
A. M. BEAL AND J. BLIGH
244
Noradrenaline (20 n-mole/kg) At an ambient temperature of 40 TC (four animals) injection of noradrenaline was followed by a fall in respiratory rate from 238 + 22-0 to a minimum of 94+ 18-3 breaths/min within 30 min and the pre-injection frequency of respiration was not observed again until 105 min after injection. Rectal temperature increased from Saline r
NA
130 _
E
_
E ,
110_
a)_ O
90
0
.70
.0
50 E
120 _ _
e,100 _ I
80
60
be 35 _
_
0
,,30 25
-30
0
60 120 Time (min)
180
Fig. 3. Systolic, diastolic and mean arterial blood pressures, heart rate and haematocrit before and after injection of noradrenaline (NA) at 20 n-mole/kg into the lateral cerebral ventricle of sheep (n = 8; means + s.E. of mean).
39-4 + 0-11 to 40-3 + 0-05 0C by 105 min post-injection and decreased thereafter. At an ambient temperature of 0 0C (four animals) respiratory frequency and intensity of shivering were unaltered by noradrenaline injection and no consistent changes in rectal temperature were observed. At both environmental temperatures systolic B.P. usually continued to fall for 30-60 min immediately following noradrenaline injection and then rose slightly. Diastolic B.P. increased slightly after noradrenaline injection in most experiments
AUTONOMIC FUNCTION AFTER I.C.V. INJECTION 245 so that mean B.P. was stable at this time (Fig. 3). Although the levels of systolic and diastolic B.P. were not significantly different from those of the control experiment the combined effect of these changes in B.P. resulted in the pulse pressure being significantly lower than that of the control experiment at the same time periods (Table 1). Heart rate rose following noradrenaline administration to reach statistical -
.C
Saline
E
1-0
NA l
I_
CC
60 '-
C
60
E 2
0
180
Fig. 4. Urine flow, renal sodium clearance, renal potassium clearance, renal osmolal clearance and solute-free water reabsorption before and after injection of noradrenaline (NA) at 20 n-mole/kg into the lateral cerebral ventricle of sheep (n = 8; means + 5.E. Of mean).
significance after 45 min (Fig. 3; Table 1) whereas the haematocrit showed a nonsignificant fall in value (Fig. 3). The rates of urine flow, sodium excretion and clearance, potassium excretion and clearance and osmolal clearance fell during the 45 min after noradrenaline injection to minimum values which were significantly lower than those following injection of saline alone (Fig. 4; Table 1). Solute-free water reabsorption was not altered significantly.
246
A. M. BEAL AND J. BLIGH
5-Hydroxytryptamine (40 n-mole/kg) At 40 0C (two animals), injection of 5-hydroxytryptamine was followed by an increase in respiratory frequency from 201 + 16-3 to a maximum of 254 + 12-0 breaths/ min within 30 min and respiration returned to pre-injection rates by 60 min after injection. Rectal temperature declined from 39'4 + 0 30 to 39 1 + 0'57 0C during the first 60 min post-injection and rose again thereafter. At 0 TC (three animals), 5TABLE 1. Summary of the final h8d analyses of cardiovascular and renal function for 120 min immediately following injection of isotonic saline (control), noradrenaline (NA), 5-hydroxytryptamine (5-HT) and carbachol (Carb) into the lateral cerebral ventricle of sheep. Every variable on the ordinate of Figs. 1-8 was tested and omission of a comparison from this Table means that no significant differences were found. Differences are indicated as levels of significance (* = P < 0-05; ** = P < 001; *** = P < 0*001; n.s. = not significant). P is the probability of erroneous rejection of the null hypothesis (i.e. type 1 error)
Variable (d.f. = 23) Systolic blood pressure Diastolic blood pressure Mean blood pressure Pulse pressure Heart rate
Comparison control 1 v8. Carb *
Haematocrit Urine flow Sodium clearance
Potassium clearance Osmolal clearance
Periods following i.c.v. drug injection
2
3
4
*
*
*
5 ***
6 ***
7 ***
8 ***
Carb
**
***
***
**
*
*
**
**
Carb
**
*
*
**
**
**
**
***
NA NA Carb Carb NA NA Carb NA Carb NA Carb
* n.s. ** n.s. n.s. n.s. n.s. n.s. n.s. n.s. *
* * *** ** ** * * ** ** * **
* n.s. ** * ** ** ** ** * ** **
n.s. * ** * * *** ** * ** ** **
n.s. * n.s. n.s. * *** * n.s. * * *
n.s. * n.s. n.s.
n.s. n.s. n.s.
n.s. n.s.
n.s. ** n.s.
n.s. * * n.s.
n.s. n.s. ** n.s. n.s. n.s. n.s. n.s.
n.s.
n.S. n.s. ** n.s. n.s. n.s. n.s. n.s.
hydroxytryptamine caused no consistent changes in respiratory frequency but shivering was stopped or reduced and rectal temperature fell from 39'7 + 0 39 to a minimum of 38*9 + 0-51 TC during the 105 min following injection. At both temperatures, systolic B.P. tended to be lowest immediately after 5hydroxytryptamine injection and both systolic and diastolic B.P. as well as heart rate increased toward the end of the experiment (Fig. 5). Except for the rate of urine flow which was low during the last 45 min of observation (Fig. 6) all other changes in renal and cardiovascular parameters were not statistically significant.
Carbachol (3 n-mole/kg) At 40 TC (four animals), injection of carbachol was followed by a reduction in respiratory rate from 233 + 26-3 to a minimum of 109 + 21-2 breaths/min during the first 15 min post-injection after which respiration increased progressively to reach pre-injection rate at approximately 75 min post-injection. Rectal temperature
AUTONOMIC FUNCTION AFTER I.C.V. INJECTION 247 rose from 39-3 + 0-2 to 40-1 + 0-29 0C during the 60 min following carbachol injection and declined thereafter. At 0 'C (four animals) the respiratory rate before carbachol injection was 19 + 3-3 breaths/min rising to a maximum of 33 + 5-2 breaths/min at 30 min post-injection and then falling slowly toward pre-carbachol rates until the end of 120 min period of observation. Mean respiration rate was correlated with Saline
CI
5-HT 4
130
E E : 110
90 0 0
70
50
90
L
o
70
50 40
6
1
o
a
o35
EK_ 30
-30
60
0
120
180
Time (min)
Fig. 5. Systolic, diastolic and mean arterial blood pressures, heart rate and haematocrit before and after injection of 5-hydroxytryptamine (5-HT) at 40 n-mole/kg into the lateral cerebral ventricle of sheep (n
=
5; means ± S.E. of mean).
mean heart rate throughout this treatment (r.4 = 0-80; P < 0-001). The intensity of shivering increased after carbachol injection and the rectal temperature of the sheep rose from 39-0 + 0-13 'C to a maintained maximum of 39-7 + 0-18 'C after 45 min and commenced to fall after 75 min. At both ambient temperatures i.c.v. injection of carbachol was followed by similar marked increases in systolic, diastolic and mean arterial B.]?., heart rate and haematocrit to levels significantly above those for the corresponding times in the control experiment (Fig. 7; Table 1). Mean urine flow doubled to reach maximum in 30 min
A. M. BEAL AND J. BLIGH 248 in parallel with the increases in B.P. and heart rate. Sodium excretion and clearance, potassium excretion and clearance, osmolal clearance and solute-free water reabsorption increased to maxima within 60 min and declined thereafter (Fig. 8). Statistically the rates of sodium excretion and clearance, potassium excretion and clearance, and osmolal clearance following carbachol were significantly higher than those for the corresponding times after the second saline injection of the control experiment (Table 1). Saline
2-0
5-HT
-
3 .
10
X "
20
=M
E
1.0~~~~~~~~~~8 z ~~3
0
0
120
Fig. 3 6. Urn ~ fornlsduceane, reaoasu
18
larne
ea
soa
clearance and solute-free water reabsorption before and after injection of 5-hydroxytryptamine (5-HT) at 40 n-mole/kg into the lateral cerebral ventricle of sheep (n = 5; means ± 5.E. of mean).
DISCUSSION
The injection of noradrenaline, 5-hydroxytryptamine and carbachol into one lateral cerebral ventricle of the sheep caused consistent changes in body temperature, respiratory frequency and shivering which were dependent on whether the animals
AUTONOMIC FUNCTION AFTER I.C.V. INJECTION 249 were subjected to high or low ambient air temperature. At an ambient temperature of 40 0C both noradrenaline and carbachol caused reduced respiratory frequency and increased body temperature whereas 5-hydroxytryptamine injection was followed by a rise in respiratory frequency and a slight fall in body temperature. When the sheep were subjected to an ambient temperature of 0 0C, injection 150
Saline
Carb
130
0,E ° E 110
239
EFFECT OF INTRAVENTRICULAR INJECTION OF TRANSMITTER SUBSTANCES AND TEMPERATURE ON AUTONOMIC FUNCTIONS OF CONSCIOUS SHEEP
BY A. M. BEAL* AND J. BLIGHt From the Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge, CB2 4AT
(Received 22 January 1979) SUMMARY
1. Changes in cardiovascular and renal functions following injection of noradrenaline, 5-hydroxytryptamine and carbachol into one lateral cerebral ventricle were investigated in conscious sheep at ambient air temperatures of 0 and 40 'C. The dose rates used were known to produce predictable changes in thermoregulation in sheep. 2. The changes in body temperature, respiratory frequency and shivering caused by the intracerebroventricular (i.c.v.) injections of noradrenaline, 5-hydroxytryptamine and carbachol were as reported previously. 3. The cardiovascular and renal responses of the sheep at both high and low ambient temperatures were consistent for each transmitter indicating that these activities had no major dependence on the type of thermoregulatory response. 4. i.c.v. injection of noradrenaline increased heart rate and decreased pulse pressure but caused no change in mean arterial blood pressure (B.P.). Urine flow rate, sodium clearance, potassium clearance and osmolal clearance were decreased whereas solute-free water reabsorption was unaltered. 5. ic.v. injection of 5-hydroxytryptamine caused no significant alterations in either cardiovascular or renal function. 6. i.c.v. injection of carbachol increased systolic and diastolic B.P., heart rate and haematocrit. Sodium, potassium and osmolal clearances were increased after administration of carbachol. 7. The changes in renal function after noradrenaline and carbachol injection could be explained by the concurrent changes in cardiovascular function. INTRODUCTION
Injection of the monoamines, noradrenaline and 5-hydroxytryptamine and of acetylcholine or cholinomimetic substances into the cerebral ventricles or anterior hypothalamus of various species has been found to cause marked changes in thermoregulation. In sheep, the consistent but ambient-temperature dependent effects of * Present address: School of Zoology, University of New South Wales, P.O. Box 1, Kensington, N.S.W., Australia 2033. t Present address: Institute of Arctic Biology, University of Alaska, Fairbanks, Alaska 99701.
0022-3751/79/4890-0976 $01.50 © 1979 The Physiological Society.
A. M. BEAL AND J. BLIGH intracerebroventricular (i.c.v.) injections of noradrenaline, 5-hydroxytryptamine and carbachol have been interpreted as indicative of specific synaptic roles of these substances in the central processes of thermoregulation (Bligh, Cottle & Maskrey, 1971). There is, however, no reason to assume that these or related substances which occur naturally in the preoptic/anterior hypothalamus and which may be synaptic transmitters, act only on thermoregulation. Other autonomic functions may also be modified by i.c.v. injections of these putative transmitters. In anaesthetized rats, rabbits, cats and dogs injection of noradrenaline into the lateral cerebral ventricle causes changes in cardiovascular function (Baum & Shropshire, 1973; Toda, Matsuda & Shimamoto, 1969; Smookler, Severs, Kinnard & Buckley, 1966; McCubbin, Kaneko & Page, 1960; Bhargava, Mishra & Tangri, 1972). Little is known, however, about the cardiovascular responses of conscious animals to the i.c.y injection of noradrenaline, 5-hydroxytryptamine or carbachol, and whether or not any alterations in cardiovascular function caused by such i.c.v. injections relate to concurrent changes in vascular peripheral resistance associated with 240
thermoregulation. Here we report on an investigation of changes which occur in other autonomically controlled systems when noradrenaline, 5-hydroxytryptamine and carbachol are injected into the lateral cerebral ventricle at dose rates which cause thermoregulatory effects in unshorn sheep. The systems observed were (1) the cardiovascular system which has long been known to be regulated by the autonomic nervous system and (2) the kidney, the autonomic innervation of which can influence renal blood flow, renin release and renal function by mechanisms that are not clearly understood. The effects of these i.c.v. injections were investigated at ambient temperatures of 40 and 0 TC, which are outside the range of thermoneutrality, to ensure that any observed effect of the drugs on cardiovascular and renal functions were not directly related to thermoregulatory activity. A preliminary report of this work has been presented to the Physiological Society (Beal & Bligh, 1976). METHODS
Experimental procedures Four unshorn Soay ewes weighing 20*0-25*5 kg were used. Each sheep had the right common carotid artery exteriorized in a loop of skin and had a permanent cannulation of one lateral cerebral ventricle (Barton, Bligh & Sharman, 1969). Before each experiment a vinyl cannula (1.4 mm i.d., 2*0 mm o.d.; Portex Limited) was inserted under local anaesthesia into one jugular vein using the technique of Seldinger (1953). The exteriorized carotid artery was cannulated with a disposable plastic cannula (Braunula, size 1; Armour Pharmaceutical Company Limited) directed towards the heart to facilitate blood pressure recording. After a catheter (Casper pattern; Rusch) had been inserted into the urinary bladder the sheep were harnessed and restrained singly in a standing position in climatic chambers maintained at either 40 or 0 IC. Rectal temperature and respiratory movements were monitored by techniques previously described by Bligh et al. (1971). The arterial cannula was connected by a thick-walled polyethylene tube filled with heparinized saline (500 i.u./ml.) to a pressure transducer outside the climatic chamber. Urine reaching the end of the bladder catheter was pumped rapidly to a collection site outside the chamber thereby reducing the number of times the room was entered to obtain samples. The sheep were allowed to become accustomed to the experimental conditions for 60 min after which an I.c.v. injection of 04 ml. sterile isotonic saline was given. Sixty minutes later the sheep received a second i.c.v. injection consisting either of 0 4 ml. saline (control treatment)
AUTONOMIC FUNCTION AFTER I.C.V. INJECTION
241
or of one of the three test substances dissolved in 0-2 ml. saline followed by a further 0-2 ml. saline to ensure all the drug was flushed into the ventricular space. The test substances used in this study were L-noradrenaline bitartrate (Sigma Chemical Company) at 20 n-mole/kg, 5-hydroxytryptamine creatinine sulphate (Sigma Chemical Company) at 40 n-mole/kg and carbachol (British Drug Houses) at 3 n-mole/kg. Urine collections were made serially over 15 min periods from the beginning of the acclimatization period until 120 min after the second i.c.v. injection. Jugular blood samples were taken at each mid-period in plastic syringes heparinized with 1 drop of heparin (5000 i.u./ml.). The sheep were subjected to this experimental procedure at intervals of not less than 7 days and the second i.c.v. injection of either saline or saline plus drug was given randomly at both 0 and 40 'C.
Analytical procedures (1) Blood samples were centrifuged in glass tubes at 2400 g for 10 min to provide plasma for analysis. (2) Microhaematocrit determinations were made in triplicate within 15 min of sampling on blood spun at 12,000 g for 10 min in a microhaematocrit centrifuge (Hawksley). (3) Sodium and potassium concentrations in plasma were estimated simultaneously and in duplicate by emission flame photometry in an oxygen-propane flame (Autotechnicon) using mixed standards in appropriate ranges to correct for mutual interference. (4) Osmolality was estimated in duplicate by freezing-point depression using a Fiske osmometer. (5) Arterial blood pressure was recorded for the last 30 sec of each 5 min throughout every experiment using a pressure transducer (Bell and Howell Limited, Type 4-327-L221) coupled to an amplifier and recorder (Devices Instruments Limited, Type M4). Systolic blood pressure was estimated from the recorder chart as the mean of the sum of all systolic pressure values recorded during the three 30 sec records of each 15 min urine collection. Diastolic blood pressure was estimated from the recorder chart in a similar manner to the systolic pressure. Mean arterial blood pressure was calculated from the systolic and diastolic pressures using the approximation. BP-Diastolic BP Mean arterial BP = Diastolic 3 (6) Heart rate was calculated as the mean rate/min over the three 30 sec periods of pressure recording in each 15 min urine collection.
BP+Systolic
Statistical procedures Analysis of covariance was applied to the data for each corresponding sample during the final 120 min of observation. The two covariates used in the analysis of each variable were the values of that variable corresponding to the urine collection period immediately preceding each of the two i.c.v. injections (i.e. periods 2 and 6 on the Figures). When the analyses of covariance produced significant variance ratios the differences between individual treatments were found using Tukey's w procedure (i.e. honestly significant difference (h8d) procedure) as described by Steel & Torrie (1960). The results of the hed analyses are presented as levels of significance in Table 1. RESULTS
Repeated i.c.v. injection of saline (control) At an environmental temperature of 40 'C (three animals) the rectal temperature of the sheep was reasonably constant throughout the 4 hr of observation. The animals panted with a mean respiratory frequency of 230 + 24-9 (S.E. of mean) breaths/min over the period of experiment. When the ambient temperature was 0 'C (three animals) the animals shivered but were able to maintain their rectal temperature unaltered and their mean respiratory rate was 17 + 2-2 breaths/min for the experimental period.
242 A. M. BEAL AND J. BLIGH The patterns of change in cardiovascular and renal function were similar for experiments at either ambient temperature. Therefore the data for the three control experiments at 40 0C and the three control experiments at 0 0C have been combined and the mean results are presented in Figs. 1 and 2. At both temperatures, systolic, diastolic and mean arterial B.P. and heart rate fell slightly over the period of observation whereas the haematocrit was unaltered Saline I
130
_
X 110 o
0
Saline
+hF
90
_
750
-
0
50
90
4070
-
3 ~
I
~
~
~~~~~~~~ I
40~
351m .. o m 0 I 1. Ssoidatlcadmaareiabloprsuehrtaean Fig.~~~~~. ee
30
i
emoci
i -30 0 60
120
~~~~180
Fig. 1. Systolic, diastolic and mean arterial blood pressures, heart rate and haematocrit before and after injection of 0n4ml. isotonic saline into the lateral cerebral ventricle of sheep (n = 6; means+ S.E. of mean).
(Fig. 1). Neither of the two ixc.v. injections of isotonic saline had any appreciable effect on this trend. Urine flow, sodium excretion and clearance, potassium excretion and clearance, osmolal clearance and solute-free water reabsorption declined during the initial 60 min regardless of environmental temperature and this trend continued after the first i.c.v. injection of saline. The second saline injection was followed by small and non-synchronous increases in urine flow, sodium excretion, potassium excretion and osmolal clearance during the subsequent 120 min of some experiments
(Fig, 2).
AUTONOMIC FUNCTION AFTER
I.C.V. INJECTION
243
i.c.v. injection of drugs During the first 2 hr of the experiments in which noradrenaline, 5-hydroxytryptamine or carbachol were injected the protocol was identical with the control experiments and the results obtained were similar to those for the corresponding time intervals of the control experiment. As the changes in cardiovascular and Saline
Saline
20 OC~
a,
1-0
X-.
0Q.5
C.-
c.E 40 _
=
0
.20
I_
C
o
3-0
E
2-0[__
0o 1-0 _I_
Q
-30
0
60 120 Time (min)
180
Fig. 2. Urine flow, renal sodium clearance, renal potassium clearance, renal osmolal
clearance and solute-free water reabsorption before and after injection of 0 4 ml. isotonic saline into the lateral cerebral ventricle of sheep (n = 6; means + S.E. of mean).
renal functions which followed I.c.v. injection of each of the above compounds were consistent for any one of the three substances injected, irrespective of whether the environmental temperature was 40 or 0 0C, the data for cardiovascular and renal parameters has been presented as the combined results for both temperatures in Figs. 3-8.
A. M. BEAL AND J. BLIGH
244
Noradrenaline (20 n-mole/kg) At an ambient temperature of 40 TC (four animals) injection of noradrenaline was followed by a fall in respiratory rate from 238 + 22-0 to a minimum of 94+ 18-3 breaths/min within 30 min and the pre-injection frequency of respiration was not observed again until 105 min after injection. Rectal temperature increased from Saline r
NA
130 _
E
_
E ,
110_
a)_ O
90
0
.70
.0
50 E
120 _ _
e,100 _ I
80
60
be 35 _
_
0
,,30 25
-30
0
60 120 Time (min)
180
Fig. 3. Systolic, diastolic and mean arterial blood pressures, heart rate and haematocrit before and after injection of noradrenaline (NA) at 20 n-mole/kg into the lateral cerebral ventricle of sheep (n = 8; means + s.E. of mean).
39-4 + 0-11 to 40-3 + 0-05 0C by 105 min post-injection and decreased thereafter. At an ambient temperature of 0 0C (four animals) respiratory frequency and intensity of shivering were unaltered by noradrenaline injection and no consistent changes in rectal temperature were observed. At both environmental temperatures systolic B.P. usually continued to fall for 30-60 min immediately following noradrenaline injection and then rose slightly. Diastolic B.P. increased slightly after noradrenaline injection in most experiments
AUTONOMIC FUNCTION AFTER I.C.V. INJECTION 245 so that mean B.P. was stable at this time (Fig. 3). Although the levels of systolic and diastolic B.P. were not significantly different from those of the control experiment the combined effect of these changes in B.P. resulted in the pulse pressure being significantly lower than that of the control experiment at the same time periods (Table 1). Heart rate rose following noradrenaline administration to reach statistical -
.C
Saline
E
1-0
NA l
I_
CC
60 '-
C
60
E 2
0
180
Fig. 4. Urine flow, renal sodium clearance, renal potassium clearance, renal osmolal clearance and solute-free water reabsorption before and after injection of noradrenaline (NA) at 20 n-mole/kg into the lateral cerebral ventricle of sheep (n = 8; means + 5.E. Of mean).
significance after 45 min (Fig. 3; Table 1) whereas the haematocrit showed a nonsignificant fall in value (Fig. 3). The rates of urine flow, sodium excretion and clearance, potassium excretion and clearance and osmolal clearance fell during the 45 min after noradrenaline injection to minimum values which were significantly lower than those following injection of saline alone (Fig. 4; Table 1). Solute-free water reabsorption was not altered significantly.
246
A. M. BEAL AND J. BLIGH
5-Hydroxytryptamine (40 n-mole/kg) At 40 0C (two animals), injection of 5-hydroxytryptamine was followed by an increase in respiratory frequency from 201 + 16-3 to a maximum of 254 + 12-0 breaths/ min within 30 min and respiration returned to pre-injection rates by 60 min after injection. Rectal temperature declined from 39'4 + 0 30 to 39 1 + 0'57 0C during the first 60 min post-injection and rose again thereafter. At 0 TC (three animals), 5TABLE 1. Summary of the final h8d analyses of cardiovascular and renal function for 120 min immediately following injection of isotonic saline (control), noradrenaline (NA), 5-hydroxytryptamine (5-HT) and carbachol (Carb) into the lateral cerebral ventricle of sheep. Every variable on the ordinate of Figs. 1-8 was tested and omission of a comparison from this Table means that no significant differences were found. Differences are indicated as levels of significance (* = P < 0-05; ** = P < 001; *** = P < 0*001; n.s. = not significant). P is the probability of erroneous rejection of the null hypothesis (i.e. type 1 error)
Variable (d.f. = 23) Systolic blood pressure Diastolic blood pressure Mean blood pressure Pulse pressure Heart rate
Comparison control 1 v8. Carb *
Haematocrit Urine flow Sodium clearance
Potassium clearance Osmolal clearance
Periods following i.c.v. drug injection
2
3
4
*
*
*
5 ***
6 ***
7 ***
8 ***
Carb
**
***
***
**
*
*
**
**
Carb
**
*
*
**
**
**
**
***
NA NA Carb Carb NA NA Carb NA Carb NA Carb
* n.s. ** n.s. n.s. n.s. n.s. n.s. n.s. n.s. *
* * *** ** ** * * ** ** * **
* n.s. ** * ** ** ** ** * ** **
n.s. * ** * * *** ** * ** ** **
n.s. * n.s. n.s. * *** * n.s. * * *
n.s. * n.s. n.s.
n.s. n.s. n.s.
n.s. n.s.
n.s. ** n.s.
n.s. * * n.s.
n.s. n.s. ** n.s. n.s. n.s. n.s. n.s.
n.s.
n.S. n.s. ** n.s. n.s. n.s. n.s. n.s.
hydroxytryptamine caused no consistent changes in respiratory frequency but shivering was stopped or reduced and rectal temperature fell from 39'7 + 0 39 to a minimum of 38*9 + 0-51 TC during the 105 min following injection. At both temperatures, systolic B.P. tended to be lowest immediately after 5hydroxytryptamine injection and both systolic and diastolic B.P. as well as heart rate increased toward the end of the experiment (Fig. 5). Except for the rate of urine flow which was low during the last 45 min of observation (Fig. 6) all other changes in renal and cardiovascular parameters were not statistically significant.
Carbachol (3 n-mole/kg) At 40 TC (four animals), injection of carbachol was followed by a reduction in respiratory rate from 233 + 26-3 to a minimum of 109 + 21-2 breaths/min during the first 15 min post-injection after which respiration increased progressively to reach pre-injection rate at approximately 75 min post-injection. Rectal temperature
AUTONOMIC FUNCTION AFTER I.C.V. INJECTION 247 rose from 39-3 + 0-2 to 40-1 + 0-29 0C during the 60 min following carbachol injection and declined thereafter. At 0 'C (four animals) the respiratory rate before carbachol injection was 19 + 3-3 breaths/min rising to a maximum of 33 + 5-2 breaths/min at 30 min post-injection and then falling slowly toward pre-carbachol rates until the end of 120 min period of observation. Mean respiration rate was correlated with Saline
CI
5-HT 4
130
E E : 110
90 0 0
70
50
90
L
o
70
50 40
6
1
o
a
o35
EK_ 30
-30
60
0
120
180
Time (min)
Fig. 5. Systolic, diastolic and mean arterial blood pressures, heart rate and haematocrit before and after injection of 5-hydroxytryptamine (5-HT) at 40 n-mole/kg into the lateral cerebral ventricle of sheep (n
=
5; means ± S.E. of mean).
mean heart rate throughout this treatment (r.4 = 0-80; P < 0-001). The intensity of shivering increased after carbachol injection and the rectal temperature of the sheep rose from 39-0 + 0-13 'C to a maintained maximum of 39-7 + 0-18 'C after 45 min and commenced to fall after 75 min. At both ambient temperatures i.c.v. injection of carbachol was followed by similar marked increases in systolic, diastolic and mean arterial B.]?., heart rate and haematocrit to levels significantly above those for the corresponding times in the control experiment (Fig. 7; Table 1). Mean urine flow doubled to reach maximum in 30 min
A. M. BEAL AND J. BLIGH 248 in parallel with the increases in B.P. and heart rate. Sodium excretion and clearance, potassium excretion and clearance, osmolal clearance and solute-free water reabsorption increased to maxima within 60 min and declined thereafter (Fig. 8). Statistically the rates of sodium excretion and clearance, potassium excretion and clearance, and osmolal clearance following carbachol were significantly higher than those for the corresponding times after the second saline injection of the control experiment (Table 1). Saline
2-0
5-HT
-
3 .
10
X "
20
=M
E
1.0~~~~~~~~~~8 z ~~3
0
0
120
Fig. 3 6. Urn ~ fornlsduceane, reaoasu
18
larne
ea
soa
clearance and solute-free water reabsorption before and after injection of 5-hydroxytryptamine (5-HT) at 40 n-mole/kg into the lateral cerebral ventricle of sheep (n = 5; means ± 5.E. of mean).
DISCUSSION
The injection of noradrenaline, 5-hydroxytryptamine and carbachol into one lateral cerebral ventricle of the sheep caused consistent changes in body temperature, respiratory frequency and shivering which were dependent on whether the animals
AUTONOMIC FUNCTION AFTER I.C.V. INJECTION 249 were subjected to high or low ambient air temperature. At an ambient temperature of 40 0C both noradrenaline and carbachol caused reduced respiratory frequency and increased body temperature whereas 5-hydroxytryptamine injection was followed by a rise in respiratory frequency and a slight fall in body temperature. When the sheep were subjected to an ambient temperature of 0 0C, injection 150
Saline
Carb
130
0,E ° E 110
