Correlation of brain catecholamines with cortical acetylcholine outflow, behaviour and electrocorticogram

EUROPEAN JOURNAL OF PHARMACOLOGY 26 (1974) 63-72. NORTH-HOLLANDPUBLISHINGCOMPANY

CORRELATION OF BRAIN CATECHOLAMINES WITH CORTICAL ACETYLCHOLINE OUTFLOW, BEHAVIOUR AND ELECTROCORTICOGRAM L. BEANI, C. BIANCHI and A. CASTELLUCCI* Department of Pharmacology, University of Pisa, Italy Received 26 June 1973

Accepted 7 November 1973

L. BEANI, C. BIANCHI and A. CASTELLUCCI, Correlation of brain catecholamines with cortical acetylcholine outflow, behaviour and electrocorticogram, European J. Pharmacol. 26 (1974)63-72. The effects of changes in the brain DA/NA ratio were correlated with cortical acetylcholine outflow, behaviour and E.Co.G. in unanaesthetized, unrestrained guinea pigs. The prevailing reduction in DA content determined by a-methyl-p-tyrosine and the increase in NA levels caused by d,l-dihydroxyphenylserine, were associated with reduced acetylcholine outflow, sedation and E.Co.G. synchronization. Conversely, the predominance of DA over NA, obtained with L-dopa and Fla-63, was associated with increased acetylcholine outflow and desynchronized E.Co.G., although sedation occurred after Fla-63. The results are in agreement with the concept that DA enhances and NA restrains the functional level of the cholinergic telecenphalic structures. Acetylcholine release a-Methyl-p-tyrosine Fla-63 1. Introduction The functional interplay between cholinergic and monoaminergic structures in the brain is now considered an important step in the regulation of the final motor and behavioural output. The role played by the acetylcholine/dopamine/serotonin balance in the functioning of the extrapyramidal system is recognized (Everett, 1967; McGeer, 1970; Orzeck and Barbeau, 1970; Aquilonius and Sj~Sstrom, 1971; Costall and Olley, 1971; Ambani and Van Woert, 1972). The sleep-waking cycle appears to be regulated by complex interactions between cholinergic, noradrenergic and tryptaminergic neurones (Jouvet, 1969). Recent findings suggest that cholinergic and adrenergic structures also interact at the higher levels of central integration: the putative catecholamine releasers, amphetamine and amantadine, as well as the amine precursor L-dopa, not only stimulate locomotor activity and desynchronize the E.Co.G. but also * Present address: Istituto Malesci, Via Paisiello 8, Florence, Italy.

Behaviour D, L-Dopa

Electrocorticogram DA/NA ratio

increase the outflow of acetylcholine (ACh) from the cerebral cortex of rabbits, cats and guinea pigs (Beani et al., 1968; Pepeu and Bartolini, 1968; Beani and Bianchi, 1973). On the other hand, there is no definite evidence as to the amine responsible for stimulation of behaviour and E.Co.G. desynchronization, generally associated with cholinergic activation (Phillis, 1968; Jasper and Tessier, 1971). Some workers have stressed the role of noradrenaline (NA) (Randrup and Munkvad, 1968; Cordeau, 1970; Reiss et al., 1970; Bliss and Ailion, 1971); in contrast, others have claimed the involvement of dopamine (DA)(Van Rossum, 1964; Everett, 1970; Costa et al., 1972; Herbans et al., 1972; Schlechter and Butcher, 1972). In agreement with the latter hypothesis, we found that drugs known to increase the DA/NA ratio in the brain, concomitantly increased cortical ACh output, while opposite changes in the DA/NA ratio seemed to be associated with reduced ACh output (Beani and Bianchi, 1970). These preliminary observations required a more detailed investigation, which is now briefly reported in this paper.

64

L. Beani et al., Brain catecholamines and ACh outflow

2. Materials and methods

and Waldeck, 1958). Their values were not corrected for the recovery, which was 86.4 --- 1 (% + S.E.) for DA and 86.1 -+ 0.8 (% -+ S.E.) for NA.

2.1. Animals

Guinea pigs of both 500-600 g were used.

sexes, average weight

2.2. Cortical acetylcholine output

One epidural cup was implanted in the right or left parietal bone, 2 - 6 days before the experiment (Beani et al., 1968; Beani and Bianchi, 1970). During the experimental session, lasting 7 - 8 hr, the guinea pigs were housed in separate boxes and had free access to food and water. The epidural cups were filled with 0.7 ml of eserinized Ringer-Locke solution, renewed every 60 min. The samples were bioassayed for their ACh content on the dorsal leech muscle, as previously described (Beani et al., 1968). After having collected 2 - 3 normal samples, a given drug was injected i.p. and its effects on cortical ACh outflow and gross behaviour (estimated by direct inspection) were followed for at least 4 hr. Particular attention was paid to changes in spontaneous motility, reactivity to tactile and acoustic stimulation, and to the appearance of ataxia and stereotyped movements. 2.3. Electrocorticographic pattern

An attempt was made to correlate E.Co.G. and behaviour in some drug-treated guinea pigs. The animals had epidural electrodes made with stainless steel screws, fixed into the frontal and parietal bones. Unipolar electrocorticographic records were taken with an ink-writing electroencephalograph. 2.4. Brain catecholamine content

In another set of experiments, guinea pigs (without epidural cups) were submitted to the same drug treatment as in the ACh outflow studies. They were killed at the time when maximal changes occurred in neurotransmitter release. NA and DA of the cerebral cortex and whole brain (except cerebral cortex and cerebellum) and NA of the heart were isolated by ion exchange chromatography (Dowex 50W-X8) and estimated fluorimetrically (Ha'ggendall, 1963; Carlsson

2.5. Drugs

Freshly prepared solutions (or suspensions) of the following drugs were used: D,L-tx-methyl-p-tyrosine (a-MT); D,L-threo-dihydroxyphenylserine (D,L-dops); L-dopa; bis-(4-methyl-l-homo-piperazinylthiocarbonyl)disulflde (Fla-63), supplied by AB H/issle, G6teborg, Sweden. The dosage schedule (i.p. injection) was: D,L-dops, 200-400 mg/kg; L-dopa, 50-100 mg/kg; Fla-63, 15-25 mg/kg. This treatment was performed both in normal and a-MT-pretreated animals: a-MT was given at 18 hr, 100 mg/kg, and at 4 hr, 100-200 mg/kg, before the beginning of the experimental session.

3. Results 3.1. Drug effect on cortical acetylcholine outflow, behaviour and E. Co. G. 3.1.1. a-MT The method of semi-permanently implanted epidural cups permitted investigation of possible late effects of a-MT in cortical ACh outflow. To exclude any spontaneous decline in the neurotransmitter release from one day to another (Beani and Bianchi, 1970) only guinea pigs recently (2 days) submitted to cup implantation were used. ACh outflow was measured 2 h r before the first a-MT injection (100 mg/kg) and on the subsequent day, 2 hr after the second injection (100-200 mg/kg), i.e., at the beginning of the experimental session, before giving Ldopa, D,L-dops or Fla-63. A significant, dose-dependent decrease in ACh outflow was found after the second a-MT injection in comparison with pretreatment values (table 1). As expected, a-MT-treated guinea pigs showed reduced motor activity and closed eyes; they appeared sedated but were easily aroused by sensory stimulation; their E.Co.G. showed a clearcut synchronized pattern, promptly changed to a desynchronized pattern by mild acoustic noises (fig. 1, see p. 66).

L. Beani et al., Brain catecholamines and ACh outflow

65

Table 1 ACh release (ng/hr/cm 2 -+ S.E.) from guinea-pig cerebral cortex 2 hr before and 2 hr after c~-MT treatment, c~-MT was given i.p. 100 mg/kg at 6 pm on the first day and 100 or 200 mg/kg at 8 am on the second day. In parentheses, the percentage values with respect to that of the first collection period are given as means -+ S.E. of the percentage calculated in each experiment. Collection periods (ACh ng/hr/cm 2 ± S.E.) Experimental conditions

No. of animals

c~-MT 100 + 100 mg/kg a-MT 100 + 200 mg/kg

Before a-MT 1st

(1st day) 2nd

After a-MT 3rd

(2nd day) 4th

9

123 ± 11

122 -+ 10 (100± 4)

102 ± 9 ** (83±3)

108 ± 9 * (88±5)

6

145 ± 15

149 -+ 16 (102± 3)

102 ± 8 ** (72-+3)

109 ± 11 * ( 77± 7)

Differences statistically significant from the first period preceding drug treatment: * p < 0.05; ** p < 0.01 ; Student's t-test for paired data.

3.1.2. D,L-Dops D,L-Dops, 200 mg/kg, injected into normal animals did n o t affect A C h o u t p u t (table 2 ) a n d behaviour. The slight increase in the n e u r o t r a n s m i t t e r release at 4 - 6 hr must be regarded as a s p o n t a n e o u s event, present in u n t r e a t e d (control) guinea pigs; its possible causes have been discussed elsewhere (Beani and Bian-

chi, 1970). Conversely, D,L-dops, 400 mg/kg, significantly reduced ACh o u t p u t and caused mild sedation, associated w i t h a synchronized electrocorticographic pattern (fig. 2) (Havli~ek, 1967). In agreement w i t h our preliminary findings, the a m i n o acid effectively reduced ACh release, even at 200 mg/kg, and enhanced sedation in a-MT-pretreated guinea pigs.

Table 2 ACh release (ng/hr/cm 2 -+ S.E.) from guinea-pig cerebral cortex treated i.p. with D,L-dops 200 and 400 mg/kg. Two groups received a-MT 100 mg/kg 18 and 4 hr before D,L-dops. In parentheses, the percentage values with respect to that of the first collection period are given as means -+ S.E. of the percentage calculated in each experiment. Experimental conditions

No. of animals

Collection periods (ACh ng/hr/cm 2 ± S.E.) 1st

2nd

3rd

4th

5th

6th

117 ± 12 (109-+ 8)

123 -+ 10 (116± 9)

D,L-Dops 200mg/kg

6

109 ± 13

D,L-Dops 1111 -+ 12 ~ 122 ± 12 (103± 3) (113± 6)

118 ± 10 (111± 9)

D,L-Dops 400mg/kg

6

147±10

D,L-Dops 146-+12 ¢ 137-+14 ( 98± 1) ( 92-+ 6)

118-+11 * 121-+13" ( 80-+ 6) ( 82-+ 8)

D,L-Dops 200 mg/kg after a-MT 100 + 100 mg/kg

8

131-+ 11

D,L-Dops 400 mg/kg after a-MT 100 + 100 mg/kg

6

125+27

128± 11 ( 98-+ 4)

D,L-Dops ¢ 123-+ 16 ( 91± 8)

D,L-Dops 121+25 ~ 102+19 ( 9 7 ± 2) ( 85± 5)

117-+13 * ( 79± 8)

104-+12"* 113-+15 * 112-+ 1 0 " ( 79± 5) ( 84± 7) ( 86± 5)

82+13"* 99+15" ( 69± 6) ( 85+ 8)

100+14" ( 87+ 8)

Differences statistically significant from the first period preceding drug treatment: * p < 0.05; ** p < 0.01. Student's t-test for paired data.

L. B e a n i et al., Brain c a t e c h o l a m i n e s a n d A C h o u t f l o w

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Fig. 1. E.Co.G. record of a guinea pig before (A) and 4 hr after (B) t~-MT 200 mg/kg i.p. In each panel from the top to the

bottom: right (rf) and left (If) frontal, right (rp) and left (lp) parietal unipolar lead. In (B) note the synchronized E.Co.G. pattern. Calibration 100 t~V and 1 sec.

3.1.3. L-Dopa As previously found (Pepeu and Bartolini, 1968; Beani and Bianchi, 1970) L-dopa increased cortical ACh output within 1 hr after injection. L-dopa, 100 mg/kg, produced an effect which was of more rapid onset but otherwise no greater than that observed with 50 mg/kg. In every instance, L-dopa hardly af-

fected behaviour (the only detectable signs being mild excitation and increased responsiveness to sensory stimulations) and did not appreciably change the E.Co.G., which remained synchronized; a-MT-pretreatment did not enhance the effect of the drug but brought forward the increase in ACh outflow caused by L-dopa, 50 mg/kg, to the 1st hr. Subsequently, the

67

L. Beani et al., Brain catecholamines and A Ch outflow

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Fig. 2. E Co.G record of a guinea pig before (A) and 3 hr after (B) D,L-dops 400 mg/kg i.p In each panel from the top to the bottom: right (rf) and left (If) frontal, right (rp) and left (lp) parietal unipolar lead. In (B) note the synchronized E.Co.G. pattern with occasional spindles Calibration 100/~V and 1 see effect remained the same or slightly subsided, never reaching the large percentage increase detected in normal animals at the 3rd and 4th hr (table 3 ) 3 1 4 Fla63

Fla-63 was given to unbalance the DA/NA ratio

The drug inhibits dopamine-/3-hydroxylase at a lower dosage than diethyldithiocarbamate or disulfiram (Svensson and Waldeck, 1 9 6 9 ) Fla-63 is reported to be well tolerated by mice at 50 mg/kg and by rats at 25 mg/kg (Corrodi et al, 1 9 7 0 ) Guinea pigs, however, seem to be more sensi-

68

L. Beani et al., Brain catecholamines and A Ch outflow

Table 3 ACh release (ng/hr/cm 2 -+ S.E.) from guinea-pig cerebral cortex treated i.p. with L-dopa 50 and 100 mg/kg. Two groups received a-MT 100 mg/kg 18 and 4 hr before L-dopa. In parentheses, the percentage values with respect to that of the first collection period are given as means -+ S.E. of the percentage in each experiment. Experimental conditions

No. of animals

Collection periods (ACh ng/hr/cm 2 -+ S.E.) 1st

2nd

L-Dopa 50 mg/kg

10

95-+9

95± 9 (100 ± 1)

L-Dopa 100 mg/kg

9

70+-20

72±21 (100 ± 3)

L-Dopa50mg/kg after a-MT 100 + 100 mg/kg

7

79±

81± 9 (103 +- 4)

L-Dopal00mg/kg after ~-MT 100 + 100 mg/kg

6

71± 8

9

71± 7 (101 +- 3)

L-Dopa ~ L-Dopa ~ L-Dopa ~

L-Dopa ~

3rd

4th

5th

6th

100±10 (106 ± 3)

134-+ 9"* 163±16"* 172±17"* (145 ± 7) (173 -+ 8) (180 ± 6)

97-+24** 1 2 0 ± 3 1 " * 1 0 2 ± 2 2 " * 105+-18"* (150 ± 12) (181 ± 19) (159 ± 16) (165 -+ 17) 125±20"* 114±13" (156 ± 10) (146 +- 15)

107-+20 * (147 ± 12)

113±14" (144 ± 16)

1 2 3 ± 1 5 " * 110-+20" (170 -+ 14) (150 -+ 10)

112_+14" (142 -+ 14)

120+_18 ** (168 ± 12)

Differences statistically significant from the first period preceding drug treatment: * p < 0.05; ** p < 0.01. Student's t-test for paired data.

Table 4 ACh release (ng/hr/cm 2 ± S.E.) from guinea-pig cerebral cortex treated with Fla-63 15mg/kg. Two groups received t~-MT 100 mg/kg 18 hr before and 100 or 200 mg/kg 4 hr before Fla-63. In parentheses, the percentage values with respect to that of the first collection period are given as means ± S.E. of the percentage calculated in each experiment. Experimental conditions

No. of animals

Collection periods (ACh ng/hr/cm 2 ± S.E.) 1st

2nd

Fla-63 15 mg/kg

6

85±

5

8 5 -+ 7 (101 ± 3)

Fla-6315mg/kg after a-MT 100 + 100 mg/kg

6

64±

1

61± 3 ( 95 ± 5)

Fla-63 15 mg/kg after a-MT 100 + 200 mg/kg

8

112 +- 12

114 ± 12 (103 ± 4)

Fla-63 ~ Fla-63 ~

Fla-63 ~

3rd

4th

5th

6th

113±10" (136 +- 12)

1 2 1 -+ 9* (147 _+ 14)

147±10"* (182 +- 23)

168±14"* (205 ± 24)

73+- 8 (110 ± 12)

92± 8* (141 +- 11)

83± 7 (127 ± 9)

112+- 9** (173 ± 15)

135 ± 15 (125 ± 13)

143 ± 14 (131 ± 12)

129 ± 10 (125 ± 9)

137 -+ 11 (126 -+ 10)

Differences statistically significant from the first period preceding drug treatment: * p < 0.05; ** p < 0.01. Student's t-test for paired data.

L. Beani et al., Brain catecholamines and ACh outflow

69

Table 5 Percentage changes in brain and heart catecholamines after a-MT and tx-MT plus L-dopa, D,L-dops and Fla-63. a-MT 100 mg/kg was given i.p. 20 and 6 hr before killing. L-dopa and D,L-dops were injected 2 hr before sacrifice and Fla-63 4,hr before. The absolute values of Dopamine (DA) and Noradrenaline (NA) of control group (ng/g -+S.E.) are given in parentheses. Brain = whole brain except cerebral cortex and cerebellum. Experimental conditions

No. of animals

DA

NA

DA

NA

NA

20 9 6

(892 ± 27) 18.7-+ 2.7** 102.3 -+ 2.3

(308 ± 16) 29.9± 1.8"* 112.0± 4.5

(118 ± 11) 39.0-+ 6.3** 98.7 -+ 3.4

( 77 -+ 5) 45.3-+ 5.2** 113.4 -+ 7.9

(955 ± 42) 42.8 ± 4.0"* 143.5 +- 3.1"*

a-MT+D,L-dops 200 mg/kg

8

38.5+ 1.9"*

86.8± 5.6

33.6-+ 6.3**

76.7-+ 5.4**

a-MT + D,L-dops 400 mg/kg

8

36.2 ± 2.2"*

81.0 ± 3.3"

34.7 ± 3.2"*

90.5 ± 4.4

L-dopa 50 mg/kg

6

140.7± 7.2**

145.0± 8.2**

110.6± 6.1

a-MT + L-dopa 50 mg/kg

6

104.6 ± 5.2

ct-MT+ L-dopa 100 mg/kg

6

165.3 ± 19.3 **

Fla-63 15 mg/kg

6

95.8 ± 2.7

,~-MT+FIa-63 15 mg/kg

6

18.2± 1.1"*

Controls a-MT D,L-dops 200 mg/kg

Brain

Cerebral cortex

108.6± 9.3 88.4 ± 4.3 146.9 -+ 12.0 ** 30.8 ± 1.5'* 9.7± 0.9**

101.0 ± 19.5

Heart

93.9 ± 5.2

95.5 + 4.6 106.8 ± 8.6 125.8-+ 6.4** 104.7 ± 6.7

143.5 ± 22.7 **

155.9 ± 8.0**

89.9 ± 5.4

29.3 ± 2.5"*

97.5 ± 3.9

27.8± 2.8**

11.0± 1.3"*

164.8 -+ 20.2 **

Differences statistically significant from the control group: * p < 0.05; ** p < 0.01.

tive to the drug. Preliminary trials showed that some animals treated with 25 mg/kg i.p. died within 24 hr. At the lower dose employed, i.e. 15 mg/kg, no deaths occurred, but the animals displayed transient abdominal stretching, which developed immediately after injection and was a sign of peritoneal irritation, a progressive reduction in motility, loss of interest in food, piloerection and a higher degree of sedation than that caused by a-MT. In spite of these behavioural changes, the E.Co.G. did not differ from the desynchronized control tracing, even 4 hr after Fla-63 injection. At this time, the increase in ACh outflow, which was evident in the first hr, reached its maximum (table 4). To obtain further indirect evidence that the DA/NA unbalance was involved in the activation of the cortical cholinergic structures, catecholamine synthesis was inhibited by administering a-MT before

Fla-63; a-MT, 100 mg/kg, given twice, at 18 and 4 hr before Fla-63, appreciably curtailed the effect of Fla-63 on ACh outflow during the first 3 hr; higher doses (100 mg/kg 18 hr and 200 mg/kg 4 hr before) almost completely prevented the effect of Fla-63. It was interesting to note that the signs of Fla-63 toxicity did not seem to be enhanced by a-MT. 3.2. Changes in brain catecholamine content As shown in table 5, the a-MT-induced inhibition of amine synthesis caused the concentration of DA to fall more than that of NA, both in whole brain and in cerebral cortex (Thierry et al., 1971). D,L-dops slightly increased NA levels in normal animals and reversed NA depletion by ct-MT (Creveling et al., 1968); the latter effect, however, was not correlated to the dose. In contrast, L-dopa increased the DA content in nor-

70

L. Beani et al., Brain catecholamines and ACh outflow

mal guinea pigs and increased DA more than NA in guinea pigs treated with a-MT so that the DA/NA ratio increased. In agreement with the findings reported by Svensson and Waldeck (1969), Corrodi et al. (1970), Fla-63 did not affect DA but reduced brain NA; cardiac NA was not affected. Both amines were depleted after a-MT plus Fla-63: this treatment produced the lowest brain concentration of NA (about 10% of the control) but the DA/NA ratio was still higher than in controls.

4. Discussion The complexity of the brain has seriously thwarted any effort to clarify the kind of interaction existing between central adrenergic and cholinergic neurones. No doubt a biochemical approach to the problem could be of some help: for instance we can selectively increase or decrease the amount of amines available for physiological release by means of amine precursors and of synthesis inhibitors. On the other hand, even the most cautious choice of chemical tools does not rule out the risk of interfering with neural systems other than those selected for the investigation. For instance, it is known that competition exists between tryptophan and L-dopa at the blood-brain barrier and that displacement of brain 5-HT occurs when DA is present in excessive amounts and at non-physiological sites (Butcher et al., 1970; Ng et al., 1970; Bartholini et al., 1968; Barret and Belch, 1971; Karobath et al., 1972). Thus, the biochemical and functional picture after L-dopa administration is more complex than expected from the sole increase in brain catecholamines. Therefore, the following discussion must be regarded as a first rough attempt to check whether or not the above results fit into the hypothesis which suggests a direct relationship between the DA/NA ratio and the functional level of the telencephalic cholinergic structures. The results obtained in a-MT-pretreated guinea pigs were of particular interest, a-MT selectively inhibits catecholamine synthesis without interfering with 5-hydroxytryptamine metabolism (Spector et al., 1965) and its effects can therefore be accounted for by the reduced synaptic influence of adrenergic neurones on other neuronal pools, such as the cholinergic neurones. Because of the faster turnover of DA,

compared with NA (Udenfriend and Zaltzman-Nisemberg, 1963; Corrodi and Hanson, 1966), a-MT caused a greater depletion of DA than of NA (table 5); concomitantly sedation, E.Co.G. synchronization (Pirch and Rech, 1968) and reduction in cortical ACh outflow appeared. Although it is not known whether the DA depletion reflected exhaustion of its stores in dopaminergic terminals or indicated its preferential utilization for NA formation in noradrenergic terminals (Thierry et al., 1971), arguments in favour of the idea that NA and DA respectively depress and enhance cholinergic activity, are offered by the experiments with their immediate precursors. D,L-dops produced a biochemical, behavioural and electroencephalographic picture which was similar to that induced by a-MT. Unfortunately, the amount of drug required to obtain the effect was so high (400 mg/kg in normal animals) that non-specific influences in the physiological disposition of other amino acids, like tyrosine and tryptophan, must be considered. Another source of criticism arises from the findings of Bartholini et al. (1971) who showed that D,L-dops did not easily cross the blood-brain barrier in the rat. In agreement with the above workers, we found that the increase in brain NA content after D,L-dops, 200 mg/kg was very slight. On the other hand, the suggestion that D,L-dops had partly crossed the blood-brain barrier is supported by the findings that at 400 mg/kg, it caused sedation and E.Co.G. synchronization in normal guinea pigs and refilled NA stores in the brain and in the heart to a similar extent after ct-MT (table 5). Moreover, some kind of synergism seemed to exist between D,L-dops and a-MT: the former drug even at 200 mg/kg (ineffective in normal animals), reduced ACh outflow and enhanced sedation in a-MT-pretreated animals. The synergism between ct-MT and D,L-dops is not surprising, because of similar types of unbalance in the DA/NA ratio. A pattern of functional changes which was opposite to that induced by D,L-dops, followed L-dopa administration: mild excitation and increased ACh outflow developed during the first hour after treatment. In view of the prevailing and early increase in DA levels caused by the amino acid (Butcher et al., 1970), our results support the postulated involvement of DA in cholinergic activation. It is interesting that the effects of L-dopa were not potentiated following a-MT: at 100 mg/kg it caused a similar effect as in

L. Beani et aL, Brain catecholamines and A Ch outflow

normal animals, while at a dose of 50 mg/kg the maximum increase in cortical ACh output was lower than that found in control guinea pigs, although it was reached earlier. Accordingly, 2 hr after L-dopa, normal guinea pigs showed the only increase in brain DA, while a-MT-treated animals had almost an equal percentage refiUing of DA and NA (table 5). It is well known that the amino acid, injected during catecholamine depletion, does not cause a relevant unbalance in the ratio between the two amines, because of rapid utilization o f DA for NA synthesis (Thierry et al., 1971). Finally, there are the intriguing results obtained with Fla-63. Fla-63 caused ACh outflow to increase and the E.Co.G. pattern to remain desynchronized, it induced behavioural depression. Similar effects had been previously described using two other dopamine-/3-hydroxylase inhibitors: diethyldithiocarbamate and disulfiram (Beani and Bianchi, 1970). In our opinion, the so-called 'sedation' by Fla-63 merely represents the central aspect of complex biochemical disturbances, determined by a drug improperly used as a selective enzyme inhibitor (Moore, 1969). In any case, the effects of Fla-63 on catecholamine levels and ACh outflow fully agreed with the expectations: in fact the increase in ACh outflow was associated with a clear-cut increase in the DA/NA ratio and the attempt to prevent the unbalance through cz-MT partly succeeded in restraining the increase in ACh outflow. The above results confirm and extend our previous findings. The concomitant determination of ACh outflow and brain catecholamines support the hypothesis that every condition characterized by a relative synaptic predominance of DA over NA is accompanied by the activation of cortical cholinergic structures, whatever the absolute values o f the amines may be. It remains to be seen whether DA and NA have opposite effects on the neurosecretory processes of the cholinergic nerve terminals or on the firing rate of the cholinergic corticipetal neurones. Taking into account what we know about the adrenergic influence on the peripheral cholinergic synapses (Kosterlitz and Lees, 1972), both these hypotheses should be considered possible. However. a definite relation between ACh outflow, gross behaviour and E.Co.G. is not established. While these parameters seem to run parallel in physiological conditions (Phillis, 1968), drug-treatment can

71

disrupt their interdependence, as shown above by Fla-63 experiments and as reported previously for atropine and chlorpromazine treatment (Beani et al., 1968).

Acknowledgements The expert technical assistance of Mr. A. GiacomeUi is gratefully acknowledged. This work was supported by a grant from 'Consiglio Nazionale deUe Ricerche' Roma (no. 71.164.04-115.4872).

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