RELEASE OF ACETYLCHOLINE FROM THE BRAIN IN VIVO: SOME ... · technique Fig. 2. Possibility of...

ACTA NEUROBIOL. EXP. 1980, 40: 687-707

RELEASE OF ACETYLCHOLINE FROM THE BRAIN IN VIVO: SOME COMMENTS ON ESTIMATION METHODS AND THEIR

APPLICATION

Andrzej WIERASZKO

Department of Biochemistry of Nervous System and Muscle Nencki Institute of Experimental Biology

Warsaw, Poland

Abstract. Techniques (push-pull cannula, cup, brain ventricles perfusion) allowing estimation of the amount of the ACh released in vivo from the brain are described. The main attention is paid to biochemi- cal, physiological and morphological factors influencing the amount of ACh released and available for estimation. Conditions of experiments for each of these techniques are described in details. The amount of ACh released in different physiological states of several animal specles is compared. Typical applications of all three methods are glven.

INTRODUCTION

The aim of this review is to describe some of the methods used to determine the amount of neurotransmitters, specifioally acetylcholine (ACh), released in the brain of anesthetized or freely moving animals. Pepeu in his recent review (71) has described some pharmacological aspects of this subj,ect. Th,is review will therefore concentrate mainly on the factors influencing the accuracy of these techniques and their applications.

FACTORS INFLUENCING THE RELEASE OF ACETYLCHOLIXE

General rules for the determination of the amount of ACh released in vivo

It is generally accepted that the major mod,e of neuron communica- tion in the brain is through the release of neurotransmitters. These che-

10 - Acta Neurobiol. Exp. 3 i 8 0

mica1 compounds are released from presynaptic nerve terminals following excitation, diffuse across the synaptic cleft and interact with postsynaptic receptors, thus producing either a depolarization or hy- perpola~nzation of the postsynaptic membrane. Inactivation of the neurotransmitter may take place by: a) enzymatic hydrolysis of the neurotransmitter (e.g.ACh); b) reuptake by the cell from which it has been released (e.g. noradrenaline); c) uptake by glial cells (as is postulated e.g. for glutamic acid) or by all three simulbaneously.

Inhibition of hydrolysing enzymes or blocking of uptake elevates the amount of neurotransmitter released into the synaptic cleft. As a result of diffusion the concentration of neurobransmitfter also 'increses in the extracellular space in the ventricles, and on the surface of the brain.

Depending on the area of the brain the following techniques may be adopted to measure the amount of released neurotransmitter: a) push-pull calnnula technique for suboartical structures; b) cup technique for the surface of the brain.

One of the most intensively investigated neurotransmitters is acetylcholine. It is released by cholinergic nerve terminals in constant multimolecular amounts: quanta (31, 45, 52). After depolarization of the postsynaptic membrane, ACh is hydrolysed by aoetylcholinesterase (AChE) to choline and acetic acid. Some of the ACh, which is not hydrolysed may diffuse from the synaptic cleft into the extracellular space (53) as it is shown in Fig. 1. The final concentnation of ACh in the extracellular space depends on the balance between released and hydmlysed neurotransmitter.

Application of the above mentioned techniques for estimation of the amount of released ACh is shown in Fig. 2.

As suggested by Katz and Miledi (53), a high concentration of ACh in the synaptic cleft following presynaptic stimulation of pevipheral neTves partially inhibits AChE activity. Thus more ACh would appear in the extracellular space but the amount of choline available for uptake and ACh synthesis would decline. In this way the concentration of ACh in the synaptic cleft could regulate funther synthesis and release of ACh. The amount of ACh diffusing from the synaptic cleft depends on sevenal factors: a) the suriace, area, and volume of the synaptic cleft; b) the temperature of the brain (it is especially important to wanm the animal or the exposed tissue during acute experiments); c) presynaptic and postsynaptic membrane potentials; d) the concentration of ions in the synaptic cleft; e) AChE activity; f ) the density of receptor sites on the postsynaptic membrane to which ACh may attached (53). If one wants to estimate the amount of ACh rele-

Fig. 1. Scheme illustrating diffusion of ACh in the extracellular space: A, active AChE; B, inhibited AChE; 1, ACh released into extracellular space; 2, synaptic vesicles containing ACh; 3, synaptic cleft; 4, postsynaptic density. Note the difference in ACh concentration (degree of stippling) in the extracellular space

between A and B.

} cup techique

the nerve release

i difiusic

ACh Fn the extracellular

i dif f ie ic

ACh in the ventriclee I psh-pull

~n canula technique

Fig. 2. Possibility of applying push-pull cannula and cup techniques for estimation of ACh released in vivo.

ased in vivo it is necessary to inhibit the activity of AChE. This is usually done by adding physostigmine or diisopropylfluorophosphate to perfusing media. ACh, whioh is thlen not hydrolysed upon release can diffuse through the extracellular space to reach the tip of the push-pull cannula or the cup on the surface of the cortex.

The role of the extracellular space in the estimation of ACh released in vivo

As has been mentioned, diffusion of ACh through the extracellular space is t~he crucial point for determination of neurotransmitter release. If the push-pull cannula is placed in the brain ventricles (this technique 1s often used and will be described below) ACh additionally must diffuse through the ependyma and into the cerebrospinal fluid (CSF), where traces of neurotransmitter synthetized mainly non enzymatically have been found recently (3, 38). The extracellular space in the brain may be genenally defined as a compartment between the membranes of neurons and glia cells. Measurement of its size may be done chemi- cally (75) or by measuring electrioal resistance (33) cr using electron microscopy (33). For chemical estimation inulin is often used. This compound is not taken up by neurons and glia and thus remains in the extracellular space. The size of the extracellular space can be expressed in several different ways: mllg dry weight or percent of wet weight - (22). It must be mentioned that there is still a large discrepancy in measurements of the size of the extracellular space from 0 to 40°/o of wet weight (cf. 22). Extracellular space estimated e.g. for the hippocampus using inulin has been determined as 8O/o of the wet weight and with electron microscopy only as 1.5O/o of the wet weight (49). Chemical compounds of low molecular weight can diffuse through the extracellular space, as the distance between the membranes of neurons and glia is some few hundred angstroms (75). It has also been reported that the extracellular space may chanlge during anesthesia (76).

Influence of K+ and Ca++ on ACh release

Stimulation dependent release of ACh by potassium ions has b e ~ n found (6, 13, 41). Synaptic membranes are more permeable to K+ than Na+. Due to this property surface membrane potentials can be recor- ded, the size of which depends upon differences between intra- and extracellular concentration of patasslum ions (8). Extracellular concentration of potassium ions rises following stimulation (47, 48) and may reach level of 12 mM. Further increment of the concentration

of potassium (e.g. spreading depression-47) may evoke increasing membrane depolarization followed by swelling (51). Potassium ions react in1 some way with membranes, changing their structure and facilita- t ~ n g the release of neurotransmitters which may be long lasting. Increase of extracellular potassium concentration is followed by calcium-dependent increase in postsynaptic minliature end plate potentials (MEPP) in the frog rectus abdominis (15, 68). The pwsence of potassium ions facilitates the inte~action of Ca+f with the mechanisms underlying nleurotransmitter release (30). These conclusions are fruther supported by data obtained from rat brain synaptosomes (1, 9, 36, 37). Increasing K+ ion concentration stimulates the uptake of calcium ions by synaptosomes. Simultaneously, inlcreases in release of ACh and noradrenaline have been observed. Other mechanisms regulating ACh release have also been postulated (94). During nerve cell excitation, the increased contentration of Ca++ inside the cell temporarily inhibits the activity of the ion pump Na, K-ATPase, thus stimulating ACh release (94). However, it is not clear if ATPase (ATP phosphohydro- lase) inhibition plays a direct or indirect role in this stimulation- dependent ACh release. Bath sodium and potassium ions are located in nerve endings in a compantment sensitive to osmotic shock. On the other hand Ca++ ions are mainly bound by mitochondria in this compartment In a ATP-dependent manner (93). The various localization of these ions suggests different mechanisms for the regulation of thzlr concentration in the extracellular space. S9, the concentration of Na+ and K+ ions would be mainly regulated by osmotic strength and activity of the Na+-K+ pump. In the regulation of the concentration of Ca++ ions additionally mitochondria would participate. This is in agre- ement with the theory, according to which movement of syn~p t i c vesicles towards the presynaptic membrane could be due to contraction of actin-like protein (59, 74). As for these processes calcium ions are crucial, their concentration inside the nerve cell may regulate ACh release. Influence of anoxia on the synthesis and release of ACh (55) and the flattening of the EEG (5, 16) as a result of disappearance of nleuronal activity may be at least partially explained by the role of mitochondria in the ACh release process.

Thus it is clear tkat local changes in the concentration of K+ and Ca++ ions may regulate release of ACh and possibly of other neurotransmitter in different brain regions. The next question to be answe- red is: how fast this local increase in K+ concentration by nerve cell activity may spread out and influence surrounding areas of the brain1. There are only few data on this problem. In the cat cerebral cortex potassium ions diffuse with a constant speed of 1.03 f 0.16mm*/h (34).

As diffusion takes place through the extwcellular space (11) its size and shape may have implication for neurotransmission (62).

Axonal transport of ACh

Axmal tnamport of ACh estimated by Haggendal and his m-war- kers (42) in $he peI;ipheral nervous system is about 5 m m h which is too slow to maintain an adequate ACh level in nerve endings (80). On the other hand, as turnover of ACh in nerve endings of the 'CNS is very high and newly synthetized ACh is preferentially released (26), one can conclude that inhibitors of axonal transport would not significantly influence ACh release.

The uptake of ACh

As has been previously mentioned, inactivation of neurotransmitters takes place mainly by reuptake. Acetylcholine is mainly hydmlysed by AChE after being released (85). Reuptake of t hk neumtransmitter also exists and has to be taken into oonsideration in release experiments. Cerebral slices take up ACh from the medium, following AChE inhibition, in an energy-dependent way (73). Concentration of ACh in slices was six times higher compared to the surrounding medium. The uptake of both ACh and ch l ine is inhibited by hemicholinium (27, 73, 82). This point is important as hemicholinium is often used in experiments in vitro as a drug specifically inhibiting ch l ine uptake only.

ACh uptake varies according to the cell type, brain region (73), and concentration of this neurut~ansmittw in the extracellular space (66, 82). The latter is especially important as AChE is inhibited during push-pull cannula and cup estimation of ACh. Finally, high concentration of K+ ions in vitro inhibits ACh uptake (38). This again emphasi- ses the importance of potassium ions in the regulation of the ACh concentration in the extracellular space.

The influence of choline concentration on ACh release

Choline is one of the substrates for biosynthesis of ACh. It can not be synthetized in the brain and is supplied mainly by the liver via the blocd stream (1). Injection of choline or its precursors (20, 43, 44, 70), or addition of these compounds to the diet (21) increase the amount of ACh synthetized in the rat brain. The observed increase depends on the particular brain region which suggests a correlaticun between the brain region and the efficiency af the choline uptake system (15). The nerve endings of the centmil nervous system possess two uptake systems for cboline: with high and low affinity (60, 89). The high affini-

ty system is able to take up choline even if it is p m e n t in the extracellular space or synaptic cleft in very low contentnation. On the other hand, the low affinity system can work only in a high choline concentration. It is very probable, that the low affinity system exists in all types of nerve endings. On the other hand, the high affinity, sodium- dependent system is supposed to act only in nerve endings from which' ACh is released (96). It is especially interesting that a correlation between the amount of released ACh and cholinle uptake has been found (16, 83, 84).

More details about the regulation of ACh biosynthesis can be found in an earlier review (89).

Recent evidence (14) indicates that the ooncentration of Kf ions influences the tonus of 'smooth muscle in blood vessels in the brain. This might suggest a correlation between blood flow (regulated by K+) and the amount of cholinle delivered to the cells.

THE RULES AND CONDITIONS OF WORKING OF PUSH-PULL CANNULA

The push-pull oannula was first used in 1958 (35), but it was ap- plied for the first time to the brain by Gaddum in 1961, (39). Figure 3 illustrates the push-pull cannula and perfusing system. Two thin tubes, either side by side, or one inside the other are introduced into the brain (67). The latter arrangement is used more often due to smaller brain damage. Recently (65, 81) a guide tube chronically implanted into the brain has been described and the push-pull cannula is introduced through this tube before each experiment. Outlets of both push-pull cannulla tubes are separated outside of skull. Artificial cerebro-spinal fluid is pumped in with constant speed through the internal tube and pumped out through the external tube. A constant speed of perfusion is very important. Any decrease in the outflow produces an increase in the pressure in the brain tissue and may cause damage of the nerve cells surrounding the tip of the push-pull cannula. ACh can be measured in the outflow and consequently its release by brain tissue estimated per unit of time.

Little has been published about the optimal working conditions of the push-pull cannula. In 1975 Yaksh (95) described experiments in which the rat hypathalamus was injected with radioactive urea. Urea does not excite nerve cells, is not taken up or released by them, and mainly diffuses through the extracellular space. Following implantation of a push-pull cannula, urea was washed out with artificial cerebrospinal fluid. The largest outflow of radioactivity was found when the speed of perfusion was 80 yllmin. Further increase in the s p e d

of perfusion evoked a drop in the amount of elueted urea. The best results may be obtained (86), when the internal tube protrudes from the external one for a distance of 0.75 mm (see parameter "h" on Fig. 3). Perfusion can not be interrupted and even a one minute break evokes a long-lasting increase in the amount of the urea in the eluate.

Fig. 3. Schematic illustration 01 the push-pull cannula arrangement (according to 77, modified, not to scale); A, push-pull cannula implanted into brain tissue; 1, inside tube; 2, outside tube; 3, dental- cement fixing the push-pull cannula with the screw to the bones of the skull; 4, skull: 5, brain tissue; B, the coupled syringe system; C, t ip of the push-pull cannula (not to scale); h, the distance

by which the inside tube protrudes from the outside one.

Damage of the brain tissue produced by implantation of the push- pull cannula and perfusion may both be irqportant problems. As the push-pull cannula destroys some cells during implantation, its diameter should be as small as possible (about l mm). The damage due to perfusionl is least if artificial cerebrospinal fluid is isotonic with natu- ral CSF. Both hypo- and hypertonic solutions evoked large changes in brain tissue (95). Tubes used for perfusion should not be made from polyvinyl chloride (PVC), as this material contains some substances toxic to brain tissue ( lo) , The contact area between the perfusion fluid and brain tissue was estimated after examination with an isotonic dye solution to be 1.3-1.5 mm3 ( 6 , 23, 64, 92). In these conditions, dege- nemting cells were same 2 mm from the tip of the push-pull cannula (65).

The time from the implantation of the push-pull cannula to the beginning of the experiments also seems to be important. Introduction of the push-pull cannula (1.3 mm diameter) into the lateral ventricles of the rabbit brain evoked increase in CSF pressure 1 h after surgery (32). Five to six hours later, the pressure was about five times higher, as compared to normal conditions. It decreased 45 h later but even after 75 h the pressure was still twice as high as in controls. Varia- tions in CSF pressure can be followed by other alterations in1 the tissue surrounding the push-pull cannula e.g. swelling of the brain, damage of the blood vessels and the changes evoked by local hypoxia (32). Fortunately, damage to the blood brain barrier appeared to be only partial with rapid recovery (32). A push-pull cannula system may be used to measure the amount of released neurotransmitters, as well as for introducing drugs to the brain (79).

Examples of the application of the push-pull cannula are described in part VI of this review.

VENTRICULAR PERFUSION

Ventricular perfusion is based on the principles of the push-pull canlnula technique. Two tubes (not connected with each other) are introduced in two different places in the brain ventricles. Artificial cerebro-spinal fluid is pumped in by one of these tubes and pumped out by the other. The neurotransmitter released from nerve cells following stimulation spreads out through the extracellular space and reaches the brain ventricles from which it may be washed out during perfusion. Figu~?e 4 shows two arrangements of ventricular perfusion.

As the ventricle walls are composed of different brain regions it is not possible to establish exactly from which brain structure release takes place. This disadvantage may be partially overcome by the addition of a dye to the perfusing fluid (66), which marks the perfusion area. As it has been mentioned before, released neurotransmitter has to diffuse to the ventricles through the ependyma. Changes in per- meability evoked by experimental conditions may influence the composition of cerebrospinal fluid. It is clear from this short description that results obtained with this method are not precisely defined. Per- fusion of ventricles bas been used to investigate, among others: (i) ACh disappearance from brain ventricles (57), (ii) metabolism and transport of serotonin (69) and noradrenaline (4), (iii) changes in the concentration of electrolytes (12, 28) and in CAMP (54) in the cerebrospinal fluid. More details concerning tbis method can be found in the review by Myers (66).

Fig. 4. Scheme illustrating the measurement of the amount of neurotransmitter in brain ventricles (according to 67 modified); A, side view; B, front view; 1, inflow tube; 2, bones of the skull; 3, outflow tube; 4, lateral brain ventricle; 5, I11 brain ventricle; 6, IV brain ventricle. Arrows indicate the direction of per-

fusion. Dotted parts of the ventricles illustrate perfused area.

DESIGN AND APPLICATION OF THE CUP TECHNIQUE; ESTIMATION OF ACH

The cup technique was first described by McIntosh and Oborin in 1953 (58). The idea of this method is to measure the amount of neurotransmitter released into a small cup placed on the surface of the cortex (see Fig. 5). The area of cup depends on the species of animal and ranges from 0.3 cmz in the rat (2) to about 3 ern2 in the sheep (63). The cup is filled with artificial cerebro-spinal fluid. Acetylcholine released from the nerve cells diffuses after AChE inhibition in all di- rections th~ough the extracellular space and reaches, among other places, the cup on the cortioal surface.

The cup technique can be used for acute and chronic experiments, just like the push-pull cannulla. In acute experiments b t h cranium and underlying dura should be removed because only rabbit's dura is fully permeable for ACh (77). However, an inbact dura is essential to maintain brain integrity in chronic experiments. The cup is slightly pressed against the brain to avoid leakage of fluid. It is useful to pack the rim of the cup and surface of the cortex with parafin or silica gel,

if fluid essapes (18). The level of fluid in the cup should be stable during the experiment, but sometimes it may rise. This usually means that cerebno-spinal fluid is filling the cup due to pressure inside the brain. To overcome this, a syringe may be inserted in the large brain ventricle (63). The fluid in the cup should be mixed comtantly with oxygen (63) or carbondioxide mixbure (77) to avaid inhibition of ACh diffusion by high concentxition of this neurotransmitter close to the cortex surface. It is not necessary to mix the fluid in the cup, if the fluid is changed frequently.

Fig. 5. Diagram of the cup technique: (not to scale): 1, inflow tube; 2, outflow tube; 3, the cup; 4, dental cement fixing cup with the bones of the skull (5); 6,

dura; 7, brabn tissue. For detail explanation see text.

It is very useful to estimate, simultaneously with ACh release, diffusion of a neutral substance e.g. urea (23). Lack of changes in urea release and changes in ACh release following nerve cell stimulation suggests that the latter is specific. On the other hand, interpretation of the data is difficult as chmges in the amount of urea released after stimulation have also been reported (9 1).

It is important to know the volume of bnain tissule from which ACh may diffuse. It has been found with radioactive choline (18) that using a cup of 1 cm2 crossection (during a 10 min collection period) most of the ACh diffusing from the surflace is limited by the rim of the cup and to a depth of 2 mm. The amount of ACh which diffuses from the depth greater than 2 mm sharply decreases. Similar results have been

found by Lancaster (56) during estimation of ACh diffusion through cat cortex slices. Moreover, to obtain any diffusion of ACh in his experiments it was necessary to inhibit AChE by 99O/o.

EXAMPLES OF APPLICATION OF T H E PUSH-PULL CANNULA AND T H E C U P TECHNIQUE

Push-pull cannula

Although the push-pull cannula technique is not generally used in1 neurochemical studies, this technique has provided some interesting results. Collier and Mitchell (23, 25) have found an increase in the release of ACh from the visual cortex of the rabbit following light stimulation. Treatment of the mbbit with a small stress increases the release of noradrenaline and serotonin from the, olfactory bulb (19). Dependence of the amount of serotonin release on different phases of sleep strongly suggests participation of this n'eunotransmitter in sleep regulation. This technique has also been used to describe which parts of the hypothalamus of the monkey (64) and cat (18) release the largest amount of ACh. Recently this method was introduced for estimation of glutamic acid release from hippocampal slices (90).

Myers experiments deserve special attention (67). Two push-pull cannulae were introduced to identical brain structures of different animals. The outlet of the first push-pull cannula placed in the brain of one animal was connected with the inlet of the push-pull cannula of the brain of the second animal. The first animal was trained to exhibit a particular behavior. Different substances, among them probably neurotransmitters released during these tests from the brain of the first animal were washed out by the push-pull cannula and transporlted to the brain of the second animal. Similar behavior was than observed in the second animal.

From the results obtained with the push-pull technique presented in Table I, it can be seen that variability of the data is very high. The amount of ACh released (nglml of perfusing fluid) ranges from 0.1 (64), to 200 (see 25). This variability can not be explained by differences between animals or brain structures. On the other hand, one can suggest that the period of time between1 surgery and the start of the experiments is of importance. S h o ~ t t ime (hours) is related to higher ACh release, whereas longer periods of time (days) correlate with lower ACh release (compare 24 and the rest of Table I). The influence of anesthesia on ACh release is also seen.

The amount of acetylcholine released in different brain areas of various mammals estimated with push-pull cannula technique.8 From introducting of push-pull cannula to the start of the experiment; b In artificial cerebro-spinal fluid used for perfusion; The data are recalculated.

I AChE I

Bra~n ~nhbltor / Stare ACh

I area perfus~on surgery

I

eserlne con- of the released References centrat~on I animal I ng/ml/mlnc

g/mlb I

Cup technique

I Vlsual 1 33 3h 1 lo-' Consc~ous 200 25

Rabb~t I cortex

-- 33 1 7h 25 -- -- - - - --

Monkey I Hypotha- (.%facaca lamus 30-50 5-7 days lo-4 Conw~our 0 . 1 4 5

-- -- 64

,~atiaita) ' Nucleus 17 7 days ! 0 Consc~ous I 0 5 - 29 I caudatus 1 I

1 ~~~l~~~ 120 1 ==I 0.8-5.2 61

caudatus anestheb~a - - - -

Cat 1 100 3-6 days I 0 Consc~ous, 16.5 I 40

1 1 Slow 12.5 1

With the cup technique (58) correlation between electrical activity of the brain and the amount of ACh released has been found. Sti- mulation of the l'ateral geniculat,e body (part of the visual pathway in the brain), evoked an increase in ACh released from the visual cortex. Similar results were obtained following light stimulati~on of the retina (23). Frequency dependent increase of ACh release from the surface of the cortex has been found during electrical stimql'ation of the reti- cular formation (87). Using a slightly modified cup, Beani and Bianchi were abl,e to determine changes in ACh release from the rabbit cortex up to 2 wk followinlg surgery (18). Later the cup gets displaced from the skull due to osteolytic processes. Beani and co-work,ers have found a correlation between changes in EEG evoked by pentobarbital and am- phetamine and changes in, animal behavior and ACh release. According to these authors the dura has no influence on ACh re1eas.e.

Szerb and his co-workers (88) found, using a pr,etrigeminal preparation of the cat (97), thfat there is a strong negative correlation between

1

1 wave 1 I

sleep - --

1 I P a r a d o x l c a l 2 ~ sleep

cortical ACh output and ACh content. It means that cholinergic nerve terminals in the aat cortex are unable to maintain a stable level of ACh during its increased release. Recently (17) an influence of phosphatidylserine injected int~avenously on ACh output from the rat bmin cortex has been reported.

Time aft- surgery lhwrsl

1 2 3 L S 6 7 8 9 1 0

Behavior ol the animal

Fig. 6. Release of ACh from the cortex of freely moving rabbit during different states of arousal estimated with the push-pull cannula (according to 25 modified); A, anesthetized animal (implantation of push-pull cannula); B, conscious animal (exploration of the cage, drinking, eating); C, quiet animal; rare movements; D, active animal, E, quiet animal, F, anesthetized with Nembutal (30 mglkg i.v.);

G, death after lethal dose of Nembutal.

An attempt to cmrelate the amount of Aah released from the cortex and the type of behavior of the anirln1a.l is shlown in Fig. 6. I t is, however, only possible to suggest that higher activity and state of arousal of the animal is a m p a n i e d by an increase in ACh release. On the other hand, a correlation between ACh released by different parts of the rabbit cortex and different behavimal tests performed by these animals has been demonstrated (77, 78). Some results obtained with the cup technique are illustrated in Table 11.

The measurements of ACh release were mainly taken from visual and temporal cortices. In most expenirnents the dura had been removed but it is not clear if it really affects A0.h release, as no special studies were performed. It is difficult to compare results obtained in various laboratories mainly due to the different experimental conditions. The period of time between the surgery and the start of the

The amount of acetylcholine released on the surface of some areas of the brain cortex of various mammals estimated with cup technique. a the data are recalculated; Eserine (about 10-4g/mI) in the cup was used in all experiments except (50, 88).

Animal

P'

Brain cortex

Cat

ACh released ng/min/cm2

Dura - Removed I Intact

p- -

Rabbit

References Time after surgery State of the animal

Auditory 0.5h Visual 1 O.5h

Somatosensory temporal

Not specified

Suprasylvian gyms

I t 9 7 - . ? - 9 1 - 3 ) - - -

No information 0.5h

1-2h 2-3days

-- - -- . -

Visual l h No information

2h No information

0.5h Auditory , Not specified 30h

Rat Temporal -

l h

I Somatosensory No information Sheep / Temporal I NO information

Nembutal Allobarbitone + urethane

Allobarb. + urethane Slow wave sleep Paradoxical sleep Conscious

Allobarb. + urethane Allobarb. + urethane Pretrigeminal prepar. Allobarb. + urethane Pretrigeminal prepar. Unai~esthetized

Allobarb. + urethane Allobarb. + urethane Conscious Allobarb. + urethane Allobarb. + urethane Allobarb. + urethane Conscious -- Conscious

experiment was usually a few hours but some authors often neglected to define it. It may be suggested that chronic experiments are more reliable than acute ones for estimation of ACh release. The different anesthetics and AChE inhibitors which have been used make it im- possible to use any of these results as a reference and they may only be of use as relative data.

CONCLUDING REMARKS

The techniques presented for estimating ACh release in vivo have both benefits and drawbacks. Each must be used with proper perspec- tive in accordance with the type of information they can yield. The method of ventricular perfusion is rather an introductory technique. If changes in the level of a compound appear following brain stimulation it may be suggested that this compound plays a rol? in the CNS. The push-pull cannula technique allows precise localization of the part of the brain respo'nsible for the observed changes. Both t~chn i - ques may be used not only for perfusi'on, but also for introducinlg drugs into brain tissue.

On the other hand, the cup technique is used fc'r release experiments on th'e surface of the bnain. Compared to the push-pull cannula, this techniqu'e is less oonyenient as: (i) it is not easy to use in chronic experiments, ( i i ) the temperature of eyposed cortex in acute expxi- ments should be maintained at the same level during the experiment which is not easy, (iii) introducing a push-pull cannula into the brain probably involves less stress than cup impllantation.

If one compares Tables I and I1 it is obvious that the cup technique has been used more often. This technique is seemingly easier to perform. It may be concluded, that the potential possibilities of the push-pull cannula, for which the theoretical basis is now known, are still w,aiting to be used.

I t is a pleasure to thank Prof. dr. Stella Niemierko for her continuous interest and helpful advice during the preparation of this review and Boiena Dqbrowska for her technical assistance i n the preparation of this manuscript. This investiga- tion was supported by Project 10.4.1.01 of t h e Polish Academy of Sciences.

REFERENCES

1. ANSEL, G. B. and SPANNER, S. 1971. Studies on the origin of choline in the brain of the rat. Biochem. J. 122: 741-750.

2. AQUILIONIUS, S. M., LUNHOLM, B. and WINBLADH, B. 1972. Effects of some anticholinergic drugs on cortical acetylcholine release and motor activity in rats. Eur. J. Pharmacol. 20: 226230.

3. AQUILIONIUS, S. M. and ECKERNAS, S. A. 1976. Choline acetyltransferase in human cerebrospinal fluid: non-enzymatically and enzymatically cata- lyzed acetylcholine synthesis. J. Neurochem. 27: 317-318.

4. ASGHAR. K. and WAY, E. L. 1970. Active removal of mprphine from the cerebral ventricles. J. Pharm. Exp. Ther. 175: 75-83.

5. ASTRUP, J. and NORBERG, K. 1976. Potassium activity in cerebral cortex in rats during progressive sedere hypoglycemia. Brain Res. 103: 418-423.

6. BAKER, L. A. 1976. Modulation of synaptosomal high affinity choline Gans- port. Life Sci. 18: 725-732.

7. BEANI, L., BIANCHI, C., SANTINOCENTO, L. and MARCHETTI, P. 1968. The cerebral acetylcholine release in conscious rabbits with semiperma- mently implanted epidural cups. Int. J. Neuropharmacol. 7: 469-481.

8. BLAUNSTEIN, M. P. and GOLDING, J. M., 1975. Membrane potentials in pinched-off presynaptic nerve terminals monitored with a fluorescent probe: evidence that synaptosomes have potassium diffusion potentials. J. Physiol. 247: 589-615.

9. BLAUNSTEIN, M. P. 1975. Effects of potassium, veratridine and scorpion venom on calcium accumulation and transmitter release by nerve terminals in vitro. J. Physiol. 247: 617-655.

10. BOWERY, N. G. and LEWIS, G. P. 1968. Pharmacological activity in polyvinyl chloride (PVC) tubing. Br. J. Pharmacol. 34: 207.

11. BRANCHO, H. and ORKAND, R. K. 1972. Neuron-glia interaction dependence on temperature. Brain Res. 36: 416-419.

12. BRADBURY, M. W. B. and KLEEMAN, C. K. 1969. The effect of chronic osmotic disturbance on the concentration of cations in cerebrospinal lfluid. J. Physiol. 204: 181-183.

13. BROWNING, E. T. and SCHULMAN, M. P. 1968. [14C] acetylcholine synthesis by cortex slices of ra t brain. J. Neurochem. 15: 1392-1405.

14. CAMERON, I. R., CARONNA, J., MILLER, R. and LINTON, R. A. F. 1976. The action of +K a t the cerebral vessels. In E. Betz (ed.), Ionic action on vascular smooth muscle with special regard to brain vessels. Springer- Verlag, Berlin, p. 92-96.

15. CARROLL, P. T. and BUTERBAUCH, G. G. 1975. Regional differences in high affinity choline transport velocity in guinea-pig brain. J. Neuro- chem. 24: 229-232.

16. CARROLL, P. T. and GOLDBERG, A. M. 1975. Relative importance of choline transport to spontaneous and potassium depolarized release of ACh. J. Neurochem. 25: 523-527.

17. CASAMENTI, F., MANTOVANI, P., AMADUCCI, L. and PEPEU, G. 1979. Effect of phosphatidylserine on acetylcholine output from the cerebral cortex of the rat. J. Neurochem. 32: 529-533.

18. CHAKRIN, L. W., MARCHBANKS, .R. M., MITCHELL, J. F. and WHIT- TAKER, V. P. 1972. The origin of the acetylcholine released from the surface of the cortex. J. Neurochem. 19: 2727-2736.

19. CHASE, H. C. and KOPIN, J. J. 1968. Stimulus induced release of substances from olfactory bulb using the push-pull cannula Nature (Lond.) 217: 466- 467.

20. COHEN, E. L. and WURTMAN, R. J. 1975. Brain acetylcholine: increase after systemic choline administration. Life Sci. 16: 1095-1102.

11 - Acta Neurobiol. Exp. 3/80

21. COHEN, E. L. and WURTMAN, R. J. 1976. Brain acetylcholine: control by dietary choline. Science 191: 561-562.

22. COHEN, S. R. 1972. The estimation of intracellular space of brain tissue in vitro. Res. Meth. Neurochem. 1: 179-219.

23. COLLIER, B. and MITCHELL, J. F. 1966. The central release of acetylcholine during stimulation of the visual pathway. J. Physiol. 184: 239-254.

24. COLLIER, B. and MURRAY-BROWN, N. 1968. Validity of a method measuring transmitter release from the central nervous system. Nature (Lond.) 218: 484-485.

25. COLLIER, B. and MITCHELL, J. F. 1967. The central release of acetylcholine during consciousness and after brain lesions. J. Physiol. 188: 83-98.

26. COLLIER, B. 1969. The preferential release of newly synthesized transmitter by a sympathetic ganglion. J . Physiol. 205: 341-352.

27. CREESE, R. and TAYLOR, D. B. 1966. Labelled carbamylcholine in brain slices of the rat. J. Physiol. 183: 68 P.

28. CSERR, H. 1965. Potassium exchange between cerebrospinal fluid, plasma, and brain. Am. J. Physiol. 209: 1219-1226.

29. DELGADO, J. M. R. and RUBINSTEIN, L. 1964. Intracerebral release of neurohumors in unanesthetized monkeys. Arch. Int. Pharmacodyn. 150: 530-546.

30. DOWNIE, E. E. 1970. New model for transmitter release a t the presynaptic membrane. J. Theor. Biol. 28: 297-300.

31. ECCLES, J. C. 1964. The physiology of synapses. New York, Academic Press. 32. EDVINSSON, L., NIELSEN, K. C., OWMAN, Ch. and WEST, K. A. 1971.

Alterations in intracranial pressure, blood brain barrier, and brain edema after sub-chronic implantation of a cannula into the brain of conscious animals. Acta Physiol. Scand. 82: 527-531.

33. FENSTENMACHER, J. D., CHON-LUH LI. and LEVIN, V. A. 1970. Extra- cellular space of the cerebral cortex of normothermic and hypothermic cats. Exp. Neurol. 27: 101-114.

34. FISCHER, R. S., PEDLEY, T. A. and PRINCE, D. A. 1976. Kinetics of potassium movement in normal cortex. Brain Res. 101: 223-237.

35. FOX, R. H. and HILTON, S. M. 1958. Bradykinin formation in human skin as a factor in heat vasodilatation. J. Physiol. 142: 219-232.

36. FREEMAN, J . J., CHOI, R. L. and JENDEN, D. J. 1975. Plasma choline: its turnover and exchange with brain choline. J. Neurochem. 24: 729-734.

37. FREEMAN, J. J. and JENDEN, D. J. 1976. The source of choline for acetylcholine synthesis in brain. Life Sci. 19: 949-962.

38. FUTAMACHI, J. K., MUTANI, R. and PRINCE, D. A. 1974. Potassium activity in rabbit cortex. Brain Res. 75: 5-25.

39. GADDUM, J. H. 1961. Push-pull cannulae. J. Physiol. 155: 1-2 P. 40. GADEA-CIRIA, M., STADLER, H., LLOYD, H. G. and BARTHOLINI, G.

1973. Acetylcholine release within the cat striatum during the sleep- wakefulness cycle. Nature 243: 518-519.

41. GREWAAL, D. S. and QUASTEL, J. H. 1973. Control of synthesis and release of radioactive acetylcholine in brain slices from the rat. Biochem. J. 132: 1-14.

42. HAGGENDAL, C. J., DAHLSTROM, A. B. and SAUNDERS, N. R. 1973. Axonal transport and acetylcholine in rat preganglionic neurones. Brain Res. 58: 494-499.

43. HAUBRICH, D. R., WANG, P. F. L. and WEDEKING, P. W. 1974. Role of choline in biosynthesis of acetylcholine. Fed. Proc. 33: 477 (Abstr.).

44. HAUBRICH, D. R., WANG, P. F. L., CLODY, D. E. and WEDEKING, P. W. 1976. Increase i n r a t brain acetylcholine induced by choline or deanol. Life Sci. 17: 975-980.

45. HEBB, C. 1972. Biosynthesis of acetylcholine in nervous tissue. Physiol. Rev. 52: 918-957.

46. HEMSWORTH, B. A. and MITCHELL, J . F. 1969. Characteristics of acetylcholine release mechanism i n the auditory cortex Br. J. Pharmacol. 36: 161-170.

47. HERTZ, L. 1977. Drug-induced alterations of ion distribution a t the cellular level of the central nervous system. Pharm. Rev. 35-65.

48. HERTZ, L. 1978. An intense potassium uptake into astrocytes, its futher enhancement by high concentration of potassium and its possible invol- vement in potassium homeostasis a t the cellular level. Brain Res. 145: 202-208.

49. IZQUIERDO, I. 1975. The hippocampus and learning. Prog. Neurobiol. (Oxf.) 5: 37-75.

50. JASPER, H. H. and TESSIER, J. 1971. Acetylcholine liberation from cerebral cortex during paradoxical (REM) sleep. Science 172: 601-602.

51. KAMINO, K. INOUYE, K. and INOUYE, A. 1973. Potassium ion induced swelling of nerve-endings particles by high-scattering measurement. BBA 330: 39-52.

52. KATZ, B. 1971. Quanta1 mechanism of neuronal transmitter release. Science 173: 123-126.

53. KATZ, B. and MILEDI, R. 1973. The binding of acetylcholine to receptors and its removal f rom the synaptic cleft. J. Physiol. 231: 549-574.

54. KORF, J., BOER, P. H. and FEKKES, D. 1976. Release of cerebral cyclic AMP into push-pull perfusates in freely moving rats. Brain Res. 113: 551-561.

55. KSIgZAK, H., KOMENDER, B. and GROMEK, A. 1973. The effect of barbitu- rate anesthesia on acetylcholine content and intracellular localization of acetylcholinesterase i n the central nervous system. Acta Physiol. Pol. 24: 455-463.

56. LANCASTER, R. 1971. Measurement of the ra te of acetylcholine diffusion through a brain slice and its significance in studies of the cellular distribution of acetylcholinesterase. J. Neurochem. 18: 2329-2334.

57. LEVINGER, I. M. and EDERY, H. 1971. Removal of acetylcholine during perfusion of liquor-space and its influence on outflow volume. Experientia 27: 291-293.

58. MACINTOSH, F. C. and OBORIN, P. E. 1953. Release of acetylcholine from intact cerebral cortex. XIX Int. Physiol. Congr., p. 580-581. Abstr.

59. MAHEDRAN, C. and NICKLAS, W. J., BERL, S. 1974. Evidence for calcium- sensitive component i n brain actomyosin-like protein (neurostenin). J. Neurochem. 23: 497-501.

60. MARCHBANKS, R. M. 1968. The uptake of I4C -choline into synaptosomes in vitro. Biochem. J. 110: 533-541.

61. MCLENNAN, H. 1964. The release of acetylcholine and of 3-hydroxytryptamine from the caudate nucleus. J . Physiol. 174: 152-161.

62. MCLENNAN, H. 1957. The diffusion of potassium, sodium, sucrose and inulin i n the extracellular spaces of mammalian tissues. BBA 24: 1-8.

63. MITCHELL, J. F. 1963. The spontaneous and evoked release of acetylcholine f rom the cerebral cortex. J. Physiol. 165: 98-116.

64. MYERS, R. D. and BELESLIN, D. B. 1970. The spontaneous release of 5-hydroxytryptamine and acetylcholine within the diencephalon of the unanesthetized Rhesus monkey. Exp. Brain Res. 11: 539-552.

65. MYERS, R. D. 1970. An improved push-pull cannula system for perfusing a n isolated region of the brain. Psychol. Behav. 5: 243-246.

66. MYERS, R. D. 1972. Methods in perfusing different structures of the brain. In R. D. Myers (ed.), Methods i n psychobiology Academic Press, New York, Vol. 2, p. 169-211.

67. MYERS, R. D. 1974. Handbook of drug and chemical stimulation of the brain. In R. D. Myers (ed.), Behavioral, pharmacological and physiological aspects. Van Nostrand and Reinhold Co., New York, p. 55-59.

68. OKADA, K. 1973. Effect of calcium and magnesium on spontaneous transmitter release accelerated by raised potassium. Brain Res. 53: 237-242.

69. PALAIC, D., PAGE, J. H. and KHAIVALLAH, P. A. 1967. Uptake and metabolism of [ W ] - serotonin in rat brain J. Neurochem. 14: 63-69.

70. PEDATA, F., WILRASZKO, A. and PEPEU, G. 1977. Erfect of choline, phosp- horylcholine and dimethylaminoethanol on brain acetylcholine level i n the rat. Pharmacol. Res. Commun. 2: 755-761.

71. PEPEU, G. 1977. The release of acetylcholine from the brain: a n approach to 'the study of the central cholinergic mechanisms. Prog. Neurobiol. (Oxf.) 2: 257-288.

72. PHILLIS, J. W. 1968. Acetylcholine release from the cerebral cortex: its role in cortical arousal. Brain Res. 7: 378-389.

73. POLAK, R. L. 1969. The influence of drugs on the uptake of acetylcholine by slices of ra t cerebral cortex. Br. J. Pharmacol. 36: 144-152.

74. PUSZKIN, S. and KOCHWA, P. 1974. Regulation of neurotransmitter release by a complex of actin with relaxing protein isolated from ra t brain synaptosomes. J. Biol. Chem. 249: 7711-7714.

75. RALL, D. P. and FENSTENMACHER, J. D. 1971. Volume of cerebral extracellular fluids i n ion homeostasis of the brain. I11 B. K. Siesjo and S. C. Sorensen (ed.), The regulation of hydrogen and potassium ion concentration i n cerebral intra- and extracellular fluids. p. 29-34.

76. RAMPTON, D. S. and RAMSAY, D. J. 1974. The effects of pentobarbitone anesthesia on the volume and composition of the extracellular fluid of dogs. J. Physiol. 237: 521-533.

77. RASMUSSON, D. and SZERB, J. 1975. Cortical acetylcholine release during operant behaviour i n rabbits. Life Sci. 16: 683-690.

78. RASMUSSON, D. and SZERB, J. C. 1976. Acetylcholine release from visual and sensimotor cortices of conditioned rabbits: the effects of sensory cuing and patterns responding. Brain Res. 104: 243-259.

79. REDGRAVE, R. and HORREL, R. J. 1976. Potentiation of central reward by localized perfusion of acetylcholine and hydroxytryptamine. Nature 262: 305-307.

80. SAUNDERS, N. R. 1975. Axonal transport of acetylcholine. In P. G. Waser (ed.), Cholinergic mechanisms. Raven Press, New York, p. 175-185.

81. SCHERBER, A,, STAIB, A. H. and OELSZNER, W. 1973. Die anwendung der

Acc

push-pull methode zur erfassung der acetylcholinfreisetzung im der ratte. Acta Biol. Med. Ger. 30: 397-405.

SCHUBERTH, J. and SUNDWALL, A. 1967. Effects of some drugs on the uptake of acetylcholine in cortex slices of mouse brain. J. Neurcchem. 14: 807-812.

SIMON, J. R. 'and KUHAR, M. J. 1975. Impulse flow regulation of high affinity choline uptake i n brain cholinergic nerve terminals. Nature 255: 162-163.

SIMON, J. R., ATWEH, S. and KUHAR, M. J. 1976. Sodium-dependent high- affinity choline uptake: a regulatory step in the synthesis of acetylcholine. J. Neurochem. 26: 909-922.

STORM-MATHISEN, J. 1977. Localization of transmitter candidates in the brain: the hippocampal formation as a model. Prog. Neurobiol. (Oxf.) 8: 119-181.

SZERB, J. C. 1967. Model experiments with Gaddum's pusn-pull cannula. Can. J. Physiol. Pharmacol. 45: 613-620.

SZERB, J. C. 1967. Cortical acetylcholine release and clectroencephalographic arousal. J. Physiol. 192: 329-343.

SZERB, J. C., MALIK, H. and HUNTER, E. G. 1970. Relationship between acetylcholine content and release i n the cat's cerebral cortex. Can. J. Physiol. Pharmacol. 48: 780-790.

WIERASZKO, A. 1975. Regulation of the biosynthesis of acetylcholine (in polish). Postepy Biochem. 21: 57-73.

WIERASZKO, A. and LYNCH, G. 1979. Stimulation-dependent release of possible transmitter substances from hippocampal slices studied with localized perfusion. Brain Res. 160: 372-376.

WINSON, J. and GERLACH, J. L. 1971. Stressor induced release of substances from the ra t amygdala detected by the push-pull cannula. Nature. New Biol. 230: 251-253. I

VEALE, W. L. 1972. A stereotaxic method for the push-pull perfusion of discrete regions of brain tissue of the unanesthetized rabbit. Brain Res. 42: 479-481.

VICKERS, G. R. and DOWDALL, M. J. 1976. Calcium uptake in preterminal central synapses: importance of mitochondria. Exp. Brain Res. 25: 429-445.

VIZI, E. S. 1975. Release mechanisms of acetylcholine and the role of Na+- K+-activated ATPase. In P. G. Waser (ed.), Cholinergic mechanisms. p. 199-211.

YAKSH, T. L. 1974. Factors influencing performance of the push-pull cannula in the brain. J. appl. Physiol. 37: 428-434.

YAMAMURA, H. J. and SNYDER, S. H. 1972. Choline: high-affinity uptake by rat brain synaptosomes. Science 178: 626-628.

ZERNICKI, B. 1974. Isolated cerebrum of the pretrigeminal cat. Arch. Ital. Biol. 112: 350-371.

zep ted 10 January 1980

Andrzej WIERASZKO, Nericki Institute of Experimental Biology, Pasteura 3 , 02-093 Warbaw, Poland.

RELEASE OF ACETYLCHOLINE FROM THE BRAIN IN VIVO: SOME ... · technique Fig. 2. Possibility of...

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