Department of Pharmacology, Kurnool Medical College,. Kumoo1518 ... This investigation was supported by a research grant from the Indian Council of Medical.
Jap. J. Physiol., 23, 241-250, 1973
RELEASE OF ACETYLCHOLINE FROM PERFUSED CEREBRAL VENTRICLES IN UNANAESTHETIZED DOGS DURING WAKING AND SLEEP* P. S. R. K. HARANATHand H. VENKATAKRISHNA-BHATT Department of Pharmacology, Kurnool Medical College, Kumoo1518 002 (Andhra Pradesh), India
Summary The cerebral ventricles were perfused with sterile artificial cerebrospinal fluid in unanaesthetized dogs from a cannula in the lateral ventricle to a cannula either in the upper cervical subarachnoid space or in the aqueduct of Sylvius. The perfusate was assayed for acetylcholine on rat blood pressure or -frog rectus muscle. Even when no eserine was added to the perfusion fluid the output of acetylcholine in 475 samples taken during wakeful state was between 0.01 to 5 ng/min in 354 (74.5 %) and above 5 ng in 53 (11.2 %). No acetylcholine was detected in 68 samples (14.3 %). In 129 samples obtained during sleep (excluding periods of REM sleep) no acetylcholine was detected in 75 (58 %) samples and an output of less than 1 ng/min was observed in the rest. Acetylcholine appeared in the effluent of samples taken during sleep when rapid eye movements occurred or before waking.
The release of acetylcholine from the brain in conscious state was so far studied only with the cerebral cortex. CELESIAand JASPER (1966) studied the release of acetylcholine from the somatosensory cerebral cortex in conscious cats which had been operated on under ether and local anaesthesia and then maintained immobile with gallamine triethiodide. Nylon cups were placed over the cortex and were filled with perfusion fluid containing 75/kg/ml neostigmine. They reported an acetylcholine output of 2-4ng/min em" cortex. JASPER and TESSIER (1971) in similar studies investigated the rate of liberation of acetylcholine from the surface of prostigmine-treated cerebral cortex in unanaesthetized cats during states of slow wave sleep, paradoxical or activated sleep and wakefulness. They reported the rate of liberation of free acetylcholine as 1.2 ng/min em" of cortical surface during slow wave sleep, 2.2 ng/min during paradoxical sleep and 2.1 ng/min during wakeful state. COLLIERand MITCHELL(1967) studied Received for publication August 15, 1972 * This investigation was supported by a research grant from the Indian Council of Medical Research, New Delhi-ll0 016. 241
242
P. S. R. K. HARANATH and H. VENKATAKRISHNA-BHATT
the release of acetylcholine from the visual cortex in conscious rabbits using indwelling 'push pull cannulae.' On perfusion with Ringer-Locke's solution containing eserine 100 f-lg/ml,they observed a high output of acetylcholine closely related to the state of activity of the animal. BEANIet al. (1968) studied the acetylcholine output from the cortex in conscious rabbits with semipermanent epidural cups filled with Ringer's solution containing 100 f-lg/ml eserine. The output of acetylcholine was about 1 ng/min em" cortex. In the present study the release of acetylcholine from the perfused cerebral ventricle was studied in unanaesthetized dogs. In these experiments acetylcholine could be detected in the perfusate even when no cholinesterase inhibitor was added to the perfusion fluid. It was further found that the release of acetylcholine was nil or diminished during sleep and increased sometimes during rapid eye movement (REM) sleep or just before the animal woke up. MATERIALS AND METHODS
Fifty-eight dogs of either sex weighing 5.8 to 14kg were used. In dogs under pentobarbitone anaesthesia (30 mg/kg), under aseptic conditions, a Collison cannula was placed in the left lateral ventricle using the method described by FELDBERG and SHERWOOD (1953) for cats. Cervical subarachnoid cannulation. With the animal still anaesthetized, a polyethylene tube of suitable length was introduced into the subarachnoid space of the cervical region. For this purpose the laminae of two upper lumbar vertebrae were removed and the spinal cord with its meningeal coverings was exposed. A small niche was made into the subarachnoid space to admit a polyethylene tube (1.0 mm outer diameter and 0.5 mm inner diameter). The tube was pushed up into the cervical subarachnoid space as far as it could go. Its correct placement was checked by measuring the length of the tube introduced and by ensuring a free flow of fluid. The tube was stoppered and fixed to the . skin margin. The operated area was covered with a protective metal or perspex shield to prevent the removal of the cannula by the dog. The introduction of this subarachnoid cannula in a few preliminary experiments either through the cisterna magna or at the cervical region after removing the 5th and 6th vertebral arches did not yield satisfactory results. The animals were given intramuscular injection of streptomycin and penicillin during the post-operative period. Aqueductal cannulation. A polyvinyl tube (outer diameter 2 mm and inner diameter 1 mm) Silastic" (Dow and Corning) was placed into the aqueduct under aseptic conditions using the method described by BHATIACHARYA and FELDBERG (1958) for cats. The tube was tied in position with anchoring stitches to the atlanto-occipital membrane and to cervical muscles. There were residual nervous effects for the next 2 or 3 days in varying degrees in the dogs during the postCerebral ventricular cannulation.
.•...
ACETYLCHOLINE RELEASE FROM CEREBRAL VENTRICLES
243
operative period in the form of ataxia and paresis of the limbs, a symptom complex generally seen in animals in which the cerebellum was handled through the open cisterna magna. The placement of the aqueductal cannula was checked at the end of the investigation by perfusing with methylene blue (0.1 %) from the lateral ventricle and on post-mortem examination it was confirmed that the fourth ventricle was not stained. Perfusion of cerebral ventricles. The animal moved about normally on the next day of operation. The cerebral ventricles were perfused daily for the next four days following operation. The ventricular cannula served as inlet for the perfusion fluid and the outflow was collected from the cervical subarachnoid cannula or the aqueductal cannula. Before starting the perfusion, the stilette of the outflow cannula was removed to allow a free flow of cerebrospinal fluid. Subsequently, sterile artificial cerebrospinal fluid without any eserine was perfused at 0.15 ml/min with a continuous slow injector. The outflow was usually a little more or less than the input and varied sometimes with the position of dog. The artificial cerebrospinal fluid had the following composition (g/liter): NaCI 8.1, KCl 0.25, CaCl 0.14, MgC1 0.11, NaHC0 1.76, NaH P0 0.07, urea 0.13 and glucose 0.61 (MERLIS,1940). Hydrochloric acid (0.1 N) 0.1 ml/ml of perfusate was added to the samples when the efHuent was collected for 30 min periods so as to prevent the destruction of acetylcholine before the sample was assayed. No acid was added if the samples were collected for 5-10 min periods and assayed immediately on rat blood pressure within 5 min after collection. A minute to minute record of the behaviour of the animal was maintained throughout the period of perfusion. The position of the animal (standing or lying), the degree of alertness and response and the state of wakefulness or sleep were noted. The animal was considered asleep if it was in a sleeping posture with all the muscles relaxed, with eyes closed beyond 3 min and with the respirations slow, regular and deep. Since the animals scarcely slept during the daytime, the collections were made at night after 10 p.m. in experiments studying the release of acetylcholine during sleep. Inulin studies. Inulin 5 mg % in 0.85 % NaCI was perfused in the same way as artificial cerebrospinal fluid at 0.15 ml/min as described above and the effluent collected every 10 min was chemically estimated for inulin according to VARLEY (1969). The inulin content in the successive 10 min samples expressed as percentage of the inulin input' during the same period is represented in Fig. 1. These experiments were performed to know how much of the acetylcholine released in the cerebral ventricles can be washed out under the present perfusion system. It took about 20-30 min for a steady state of perfusion to be achieved with a constant perfusion of inulin from the lateral ventricle. The outflow concentration of inulin was over 80 % of its input. The results with inulin suggest that the amount of acetylcholine recovered in the effluent would represent more than 80 % of the actual amount released provided it is not destroyed 2
2
3
2
4
244
P. S. R. K. HARANATH
and H. VENKATAKRISHNA-BHATT
100
~ "'"' c:
2c: 50 0 o
.~ ::; c:
0'
60' Time
Fig. 1. Inulin content of effluent (10 min samples) from perfused cerebral ventricles expressed as percentage of its input. Inulin 5 mg% in saline was perfused at 0.15 ml/rnin.
on its way. Assay. The samples were assayed for acetylcholine-like activity on the frog rectus preparation in the first seven experiments and on rat blood pressure in subsequent experiments. Assay on rat blood pressure was preferred in the later experiments as it was found to be more sensitive to acetylcholine. The method described by MAcINTOSHand PERRY(1950) was used for assay of acetylcholine on frog rectus muscle. The assay of acetylcholine on rat blood pressure was carried out according to the method of STRAUGHAN (1958) modified by VENKATAKRISHNA-BHATT and HARANATH (1970). To show that the substance being assayed by both these methods was acetylcholine or a very similar choline ester, the following procedures were used. The samples were tested before and after curarisation on frog rectus preparation and before and after atropinisation on the rat blood pressure preparation. The occurrence of acetylcholine-like response on two different preparations (one stimulant and other depressant) and also their abolition after application of specific acetylcholine antagonists suggested that the substance could be acetylcholine or a similar choline ester. In three experiments the assay of samples was repeated both on frog rectus preparation and rat blood pressure and similar values were obtained. Thereafter the assay was done only on rat blood pressure. When a part of the sample was made alkaline (PH 12.0), boiled, again acidified to pH 5 and assayed, it was shown to have lost its acetylcholine-like activity confirming that the substance being assayed was acetylcholine or a similar choline ester. Standard acetylcholine solutions were made with acidified artificial cerebrospinal fluid used for perfusion.
ACETYLCHOLINE
RELEASE FROM CEREBRAL VENTRICLES
245
RESULTS
A total of 628 samples of uneserinized perfusate obtained from 58 dogs was assayed for acetylcholine. Most of these samples were collected for 30 min. But in experiments in which release of acetylcholine during sleep was studied, samples were collected for shorter periods of 5 to 10 min. Table 1 gives break Table 1. Acetylcholine output into the effiuent on perfusion of cerebral ventricles. Frequency of occurrence of different rates of output. Parenthesized figures represent number of samples in rapid eye movement sleep. Total number of samples Awake Asleep
475 153(24)
Number of samples containing acetylcholine expressed as output ngjrnin Nil 68 80(5)
0.01-0.1 86 17(0)
0.1-1
1-5
5-10
10-20
>20
187 48(11)
81 7(7)
16 1(1)
20 0
17 0
up of acetylcholine content in the samples both while the animals were awake and asleep and also when the animals showed rapid eye movements during sleep (in parenthesis). . Release of acetylcholine during wakeful state
Acetylcholine was present in varying amounts in most of the 475 samples which include 34 samples of aqueductal effluent obtained during wakefulness. The output of acetylcholine was usually between 0 and 1 rig/min. It can be seen from Table 1that an output up to 5 ng/min was frequent and values above 5 ng/min were observed only in a few instances. The high output of over 20 ng/min was observed only in three dogs. Out of these 17 samples of subarachnoid effluent 11 were from a single dog with a maximum output of 117.7 rig/min. The remaining 6 samples were from 2 dogs with maximum output of 30 and 50 ng/min in each. In only 14.3 % of the samples no acetylcholine-like activity could be detected. The output of acetylcholine varied from dog to dog and even in the same dog from day to day. This can be seen from Table 2, which gives acetylcholine output in one dog in 4 successive days where the perfusion was carried on for periods of over 3 hr each day. There were some fluctuations in the output of acetylcholine even during the same day. Release of acetylcholine during sleep Subarachnoid effluent. The cerebral ventricles were perfused from lateral
ventricle to cervical subarachnoid space in 22 dogs who slept for varying periods during the period of observation. The output of acetylcholine in the effluent during sleep was less than that during wakeful state. Out of the 153 samples obtained during sleep, 24 were during rapid eye movement sleep. In the rest
246'
P. S. R. K. HARANATH
·
and H. VENKATAKRISHNA-BHATf
Table 2. Acetylcholine output during wakeful state in the effluent in successive half hour samples on perfusion of cerebral ventricles on 4 consecutive days after operation. Perfusion with uneserinized artificial cerebrospinal fluid. Day of collection after operation
Acetylcholine output expressed as ngjmin in successive half hour samples 1 1.00 2.43 0.38 0.73
1st 2nd 3rd 4th
2
3
4
5
6
1. 73 1.85 1.06 0.40
1.00 2.10 1.50 0.37
nil 3.67 1.36 0.80
0.80 4.00 0.28 0.67
nil 0.13 0.27 0.80
of the 129 samples, 75 (58 %) did not show any acetylcholine and even in the balance of 54 samples the output was less than 1 ng/min (Table 1). In six experiments, samples were collected at intervals of 30 min and the dogs were sometimes both asleep and awake during the same 30 min period. The output of acetylcholine in such samples where the animal was asleep for parts of the period was low in many instances though not always. Hence in the subsequent 16 experiments, the cerebral ventricles were perfused at nights when the animals slept more frequently and for longer intervals. Further the samples were collected frequently every 5 or 10 min and were immediately assayed for acetylcholine within 5 min of their collection. Figure 2 shows one such experiment. It can be seen that the 4
~
.s 3 E no
.e
2
--
;:J 0
.c U ~On
CEREBRAL
1.0
~
-S 0.5 s: u
« 60'
120' Time
Fig. 3. Acetylcholine output ng/min (bottom histogram), volume of each 5 min sample (top histogram) in wakeful state and sleep during perfusion of cerebral ventricles. Sleep periods indicated by black bars and rapid eye movement (REM) by inverted triangles T.
more. Out of 24 samples obtained during rapid eye movement sleep in all the experiments 19 samples showed acetylcholine output ranging from 0.1 to 7.8 ngj min and 5 samples did not show any acetylcholine (Table 1). Aqueductal effluent. In the previous experiments, the area under perfusion was large and included the cerebral ventricles as well as the subarachnoid space. Hence in five experiments, to study the release of acetylcholine in the cerebral ventricles alone, the perfusion was from the lateral ventricle but the outflow was collected from an aqueductal cannula. The dead space in the collection system was much reduced in this method of perfusion. There were residual nervous effects as a result of operative handling of elevation of the cerebellum and other structures in dogs in the form of paresis of the limbs on the next day. In two of these animals who were able to raise the head, to get up and to respond to calls properly, the cerebral ventricles were perfused from lateral ventricle to aqueduct. Figure 4 gives the output of acetylcholine in the aqueductal effluent in one of these
.
