Use of amfonelic acod to discriminate between classical and atypical neuroleptics and neurotensin: an in vivo voltammetric study

86

Bram Research, 544 (1991) 86-93 © 1991 Elsevier Soence Pubhshers B V (Biomedical Division) 0006-8993/91/$03.50 ADONIS 0006899391164211

BRES 16421

Use of amfonelic acid to discriminate between classical and atypical neuroleptics and neurotensin: an in vivo voltammetric study R. R i v e s t 1, E B . J o l i c o e u r ~ a n d C . A . M a r s d e n 2 1Department of Psychmtry, Medtcal School, Untversay of Sherbrooke, Que (Canada) and 2Departmentof Phystology and Pharmacology, Medical School, Queens's Medical Centre, Umverstty of Nottingham, Nottmgham (UK) (Accepted 6 October 1990)

Key words Neurotensm; Neurolepttc, Strmtum; Voltammetry m VlVO,Dopamlne metabolism; DOPAC

Previous ex VlVOstudies have shown that the non-amphetamine stimulant amfonehc acid potentiates the increase in DOPAC induced by classical but not by atypical neuroleptics In the present study, we have demonstrated that this neurochemlcal model can be used to discriminate typical from atypical neuroleptics in vivo using differential pulse voitammetry with carbon fibre electrodes. The study also compared the effect of intracerebroventricular (i c v.) administration of neurotensin, on extraceilular strlatal DOPAC following amfonelic acid, with the effects of both classical and atypical neuroleptlcs. Sahne or amfonelic acid (2 5 mg/kg s c ) were administered; followed 5 mln later by the classical neuroleptics haioperidol, perphenazine, or the atypical neuroleptlcs ciozaplne, thiondazine, or by neurotensin After drug administration extraceilular striatal DOPAC was recorded every 5 min for 90 mln Amfonehc acid did not alter basal striatal DOPAC but potentiated the increase in DOPAC induced by halopendoi (1.0 and 0 05 mg/kg s c.) and perphenazme (10 mg/kg s c.). Both clozapine (30 mg/kg i.p.) and thiorldazine (20 mg/kg s.c.) increased extracellular DOPAC, but pretreatment with amfonelic acid prevented the increase in DOPAC produced by both drugs Neurotensin (10/~g, 1 c v.), in a similar manner to the atypmal neuroleptlcs, increased extracellular DOPAC in the striatum and the effect was prevented by amfonehc acid. The present study demonstrates that pretreatment with amfonelic acid is a valuable tool to dlscrlmmate between classical and atypical neuroleptlcs in vivo The results also indicate that neurotensln in the presence of amfonelic acid has a profile similar to the atypical neuroleptics INTRODUCTION The efficacy of most neuroleptics (e.g. haloperidol) to attenuate the symptoms of schizophrenia has been correlated with their D 2 d o p a m i n e r e c e p t o r antagomst properties in the nucleus accumbens and the striatum ~2" 53. A m a j o r p r o b l e m associated with these drugs is the induction of extrapyramidal side-effects and tardive dyskinesias believed to result from blockade of d o p a m i n e receptors in the striatum 39. H o w e v e r , some neuroleptics (such as clozapine and thioridazine) are distinguished by having a lower incidence of extrapyramidal side effects TM 24 and thus have been n a m e d 'atypical' neuroleptics. The study of the effects of neurotensin (NT) in the central nervous system has shown that it has a close interaction with central dopaminergic systems and therefore a potential implication in dopaminergic related n e u r o p a t h o l o g y 16"28"33"47"51. Because many of the effects induced by central administration of N T are also produced by neuroleptics, it has been p r o p o s e d that N T possesses antidopaminergic activity and thus neurolepticlike p r o p e r t i e s 4s. In agreement with this hypothesis, both

neuroleptics and N T increase the synthesis, metabolism and release of dopaminergic mesolimbic or nigrostriatal terminals 5'7'26"32"46'65 and reduce l o c o m o t o r hyperactivity mduced by direct administration of d o p a m i n e or dopamine agonists into the nucleus accumbens 9'29'32. However, while some simtlarities between the actions of NT and neuroleptics are obvious, there are also m a j o r differences. Thus N T does not bind to the d o p a m i n e receptors in limb~c and striatal tissue 46. H o w e v e r , the peptide reduces the affinity of the d o p a m i n e agonist [3H]N-propylnorapomorphine at d o p a m i n e D 2 receptors 3 and changes the high affinity binding state of D 1 agonists to a low affinity state 44. These results suggest that N T may act as a m o d u l a t o r at d o p a m i n e receptors. Using positron emission t o m o g r a p h y in schizophrenic patients, it has been r e p o r t e d that clinical doses of classical neuroleptics result in a high percentage of D 2 d o p a m i n e receptor occupancy while both D t and D 2 d o p a m i n e receptors were occupied after t r e a t m e n t with clozapine TM. The atypical neuroleptics also have relatively high affinity for muscarimc 42 and serotonin (5-HT2) receptors 41 but there is no evidence that N T shares these properties.

Correspondence. R Rivest, Department of Physiology and Pharmacology, Medical School, Queen's Medical Centre, University of Nottingham, Nottingham NG7-2UH, U K

87 W i t h the need to characterise the n e u r o p h a r m a c o l o g ical p r o p e r t i e s of neuroleptics in o r d e r to develop new effective drugs, various tests have been p r o p o s e d to discriminate b e t w e e n classical and atypical neuroleptics. First, it was suggested that, in contrast to classical neuroleptics, the atypical antipsychotics are virtually devoid of cataleptogenic activity in rats when used at low but effective doses63; a p r o p e r t y shared with N T 27.

tics (clozapine, thioridazine) in vivo by monitoring extracellular D O P A C in the striatum using differential pulse voltammetry with carbon fibre micro-electrodes22. Moreover, we have compared the effects of the selected neuroleptics with central administration of N T to investigate if the in vivo neurochemical effects of N T resemble those of atypical neuroleptics more than those of the classical drugs.

H o w e v e r , thioridazine which is generally considered an atypical neuroleptic has cataleptogenic activity34. A n o t h e r test uses a p o m o r p h i n e o r a m p h e t a m i n e to induce s t e r e o t y p y and p r o p o s e s that classical neuroleptics antagonise s t e r e o t y p y induced by both drugs while atypical neuroleptics are w e a k or inactive 9'36. Similarly to atypical

MATERIALS AND METHODS

neuroleptlcs N T attenuates l o c o m o t o r hyperactivity but not s t e r e o t y p y induced by a p o m o r p h i n e or amp h e t a m i n e 15'27. M o r e recent studies have s e p a r a t e d the different forms of s t e r e o t y p y and found that atypical neuroleptics have a m o r e specific effect on behaviours such as licking, sniffing, yawning and penile erection induced by a p o m o r p h i n e or a m p h e t a m i n e 14,5°'58. N T also reduces certain aspects of s t e r e o t y p y such as yawning and penile erection 3° but, contrary to the atypical neuroleptics, has no effect on sniffing and licking behaviours. H o w e v e r , in general the neuropharmacological effects of N T m o r e closely r e s e m b l e those of atypical neuroleptics. Until now, most comparisons between the effects of N T and neuroleptics have e m p l o y e d behavioural studies. H o w e v e r , a neurochemical m o d e l for discrimination b e t w e e n classical and atypical neuroleptics has been p r o p o s e d where the d o p a m i n e m e t a b o l i t e ( D O P A C ) is m e a s u r e d in the striatum ex vivo after administration of neuroleptics to rats p r e t r e a t e d with amfonelic acid 2°'4°'6°. In this neurochemical model, amfonelic acid potentiates the increase in D O P A C induced by classical neuroleptics but blocks the effect of atypical neuroleptics 2°'4°'6°. A m f o n e l i c acid is an indirect dopaminergic stimulant which p r o d u c e s behavioural hyperactivity c o m p a r a b l e to amphetamine 56, but the mechanism of action is different from amphetamine as it does not increase the basal extracellular level of dopamine or D O P A C in the striatum 61. Two mechanisms of action have been proposed for arnfonelic acid. First, specific inhibition of re-uptake of dopamine 2'56 , second, facilitation of the transfer of neuronal dopamine from the vesicular pool to an impulse releasable compartment 6'3L43. It is probably the transfer of dopamine to the impulse-releasable pool that results in amfonelic acid potentiating the effects of electrical stimulation of the medial forebrain bundle 17 and classical neuroleptics z°'4°'6°'6~ on dopamine release and metabolism. In the present study, we e x a m i n e d w h e t h e r amfonelic acid can be used to discriminate between two classical (haloperidol, p e r p h e n a z i n e ) and two atypical neurolep-

Animals Male Wlstar rats weighing between 250 and 280 g were used. They were housed, 5 per cage, in a room (21 °C) with a 12 h light/dark cycle Food and water were avatlable ad libitum.

Voltammemc procedures Working electrodes made from one pyrolytic 12 #m carbon fibre, reference and auxihary electrodes were prepared as previously describedsS. To obtain sensitivity and adequate separation between ascorblc acid, catechol and indoles, the working electrodes were electrically pretreated as described22. First, working, reference and auxthary electrodes were ~mmersed m a phosphate-buffered saline solution (pH 7.4, 0 1 M) and connected to a polarograph (Princeton Apphed Research 174A). A triangular waveform current (0 to 3.4 V, 70 Hz) was applied for 20 s, then a continuous potential of +1.5 V, -0.9 V for 5 s each. This was followed by another continuous potential varying from 1.1 V to 1.5 V, depen&ng on the sensitivity of the electrode Sensmvity and selectixaty of the electrodes were examined by perfomung one voltammetric recording m a phosphatebuffered soluUon containing ascorhic acid (5 × 10-3 M), DOPAC (1 x 10-4 M) and 5-HIAA (5 x 10-5 M). Electrodes showing poor separation, low sensitivaty or high charging current were discarded. Differential pulse voltammetnc parameters for the in vitro recording were as follows: potential range' -0 2 to 0.350 V, increase scan rate: 5 mV/s, pulse amplitude 5 mA, pulse frequency 2 5/s.

Surgery and experimental procedure Rats were anaesthetlsed w~th chloral hydrate (400 mg/kg i.p.) and maintained dunng the experiment with additional injections (70 mg/kg/h). Ammals were mounted on a stereotaxic frame with the upper incisor bar set at 3.3 mm below the mteraural line. One hole (approx. 2 mm o.d.) was made in the cranium above the striatum. The dura was carefully broken and removed using a hypodermic needle. For the admimstration of NT, a guide cannula (23 ga) was stereotaxically implanted 2 mm above the left lateral ventricle and fixed using dental cement (Espe Durelon). The coordinates used were A/P: -0.2; L: 1.4; V: 4.2, with reference to bregma49. The working electrode was stereotaxically implanted m the medio dorsal part of the striatum (A/P: 0 7, L: 3.0, V: 4.0) with reference to Bregman49. Auxiliary and reference electrodes were positioned on the surface of the brain. Electrodes were connected to the polarograph and the DOPAC oxidation peak was recorded every 5 min during a 60-min stabdisation penod before the administration of drugs or NT. To detect DOPAC excluswely, the initial potential applied to the working electrode was set to -50 mV which corresponded with the end of the ascorbic acid peak and the scan was stopped at 200 mV corresponding to the beginning of the 5-HIAA + uric acid peak. Sahne (1 ml/kg s.c ) or amfonelic acid (2.5 mg/kg s.c.) were administered followed 5 mm later by saline (s.c.) or haloperidol (0 05 and 1 0 mg/kg s.c ), perphenazine (10 mg/kg s.c.), clozapine (30 mg/kg Lp.), thioridazine (20 mg/kg s.c.), or NT (10/~g/10/~1, LC.V.) NT was rejected over 60 s. Extracellular DOPAC was recorded every 5 rain for the following 90 rain.

Drugs Amfonelic acid (Biochem Res.) was dissolved in saline-NaOH 0.05 M and the pH was adjusted to approx, pH 8-9 with saline-HC1

88

00i

,

O

I " ~ "/~. j . . / ~ "

Bregma 0.70 mm

Fig. 1. Representative micrograph (left side) showing the location of a voltammetric electrode m the striatum. The arrow shows the tip of the electrode On the right side is the appropriate diagram from Paxmos and Watson (1982) showing (0) the position of the electrode. Note the small brain damage produced by the voltammetric electrode.

i

150- ~

400

@

@

@

300 •

@

|

|

i

@

•

@

•

i

!

|

!

@

•

!

i

0 I0 20 30 40 50 60 70 80 90 Ttme t,-,,,)

Fig. 3. Effect of amfonehc acid on the halopendol-induced increase in extracellular stnatal DOPAC of anaestheslzed rats. Sahne (1 mg/kg s c ) or amfonelic aod (2 5 mg/kg s.c.) were administered followed 5 mm later by saline, or halopendol (0.05 mg/kg s.c.) DOPAC peak height was monitored every 10 min during 90 min The extracellular DOPAC basal level was estimated from the means of the last 4 signals before administration and represents 100% Groups are' amfonehc acid + saline (-), sahne + haloperldol (11), amfonelic acid + haloperidol (El) Each value is the mean of 6 recordings + S E.M Significant differences, *P < 0.05 are for comparisons between sahne-haloperidol and amfonelic acid-halopendol (Dunnett's test)

1 M Ciozapme (Sandoz) and Perphenazme (Sigma) were dissolved in saline-HC1 0 3 M and the pH adjusted to 4-5 with sallne-NAOH 1 M Haloperldol (Janssen) was obtained m injectable ampoule Thiondazine (Sandoz) and NT (gift from Dr. S St-Pierre) were dissolved m sahne.

Data

O[JOlllllli' Time(.In) Fig 2. Effect of amfonehc acid on the halopendol-mduced increase m extracellular stnatal DOPAC of anaestheslzed rats Saline (1 mg/kg s c.) or amfonehc acid (2 5 mg/kg s c ) were administered followed 5 mm later by saline, or halopendol (1 mg/kg s c.) DOPAC peak height was momtored every 10 min during 90 mm The extracellular DOPAC basal level was estimated from the means of the last 4 signals before administration and represents 100% Groups are sahne + saline (-); saline + halopendol (as); amfonehc aod + saline (A), amfonehc acid + halopendol (D) Each value is the mean of 6 recordings + S E.M Sigmficant differences, *P < 0.05 are for comparisons between sallne-haloperidol and amfonehc actd-halopendol (Dunnett's test)

The DOPAC peak was recorded every 5 min but for clanty the data are presented at 10-rain intervals. Peak heights were measured and converted to a percentage of the basal level calculated from the means of the last 4 peaks before drug admimstraUon. Results were analysed by two-way ANOVA's for repeated measurements. Significant differences between control injection and mdwidual doses of the peptide for each time were assessed by means of Dunnett's test (*P < 0 05)

Histology The placement of electrodes m the stnatum were checked histologically Brains were removed, frozen and sectioned wih a microtome, slices mounted on glass slides and stained with cresyl violet for examination (Fig. 1). For the verification of the ICV injection, 2/zl of mk was rejected using the same procedure as for NT The brains were removed, sliced transversally at the rejection site and the dispersion of mk m the ventricle was directly checked, RESULTS

Interaction between amfonehc acid and classical neurolepttcs T h e D O P A C p e a k r e c o r d e d in t h e s t r i a t u m d e c r e a s e d

89

!"

150

125

•

•

"

"

"

"

"

0 0

10

20

30

40

50

60

70

80

90 0

0

!

|

!

10

20

30

,

40

!

|

!

!

|

50

60

70

80

90

(rain)

Time

Fig 4 Effect of amfonelic acid on the perphenazine-induced increase in extracellular stnatal DOPAC of anaesthesized rats, Saline (ml/kg s c.) or amfonehc acid (2.5 mg/kg s.c ) were administered followed 5 rain later by saline or perphenazine (10 mg/kg s c.). DOPAC peak height was monitored every 10 mm during 90 mln. The extracellular DOPAC basal level was estimated from the means of the last 4 signals before administration and represents 100%. Groups are' amfonehc acid + saline (-); saline + perphenazine (m), amfonehc acid + perphenazme (I-1) Each value is the mean of 6 record, ngs+ S E.M. Significant differences, *P < 0.05 are for comparisons between saline-perphenazme and amfonehc acld-perphenazine (Dunnett's test) 175'

Time (..,I.) Fig. 6. Effect of amfonelic acid on the thloridazine-induced increase m extracellular striatal DOPAC of anaesthesized rats. Saline (ml/kg s.c.) or amfonelic acid (2.5 mg/kg s.c.) were administered followed 5 mm later by saline or thiorldazine (20 mg/kg s.c.). DOPAC peak height was momtored every 10 mm during 90 rain. The extracellular DOPAC basal level was estimated from the means of the last 4 signals before admin,stration and represents 100%. Groups are: amfonelic acid + saline (-); saline + thioridazme (m); amfonelic acid + thiondazine (I-1). Each value is the mean of 6 recordings + S.E.M Significant differences, *P < 0.05 are for comparisons between sahne-thioridazine and amfonelic acid-thioridazine (Dunnett's test).

slightly (maximal decrease 8%) during a period of 90 min after saline administration. Amfonelic acid (2.5 mg/kg s.c.) did not alter the basal level of D O P A C (Fig. 2). 125' I

i 100

I

I

I

75

0

,

0

10

|

20

|

|

30

40

|

50

|

60

|

70

!

80

|

90

Time (,-,,,] Fig. 5. Effect of amfonelic aod on the clozapme induced increase in extracellular striatal DOPAC of anaestheslzed rats. Saline (ml/kg s c.) or amfonehc acid (2 5 mg/kg s.c.) were administered followed 5 min later by sahne or clozapme (30 mg/kg i.p.) DOPAC peak height was momtored every 10 min dunng 90 rain The extracellular DOPAC basal level was estimated from the means of the last 4 signals before administration and represents 100% Groups are: amfonehc aod + saline (-); saline + clozapme (m); amfonehc acid + clozapine (F1). Each value Is the mean of 6 recordings + S.E M. Significant differences, *P < 0.05 are for comparisons between saline-clozapine and amfonehc acld-clozapme (Dunnett's test)

Haloperidol (0.05 and 1.0 mg/kg s.c.) increased the extracellular D O P A C peak by 44% + 5.4 and 85% + 13.9 resp. 90 min after administration (Figs. 2 and 3). W h e n amfonelic acid was injected 5 min before haloperidol, the increase in D O P A C was significantly greater compared with haloperidol (0.05 and 1.0 mg/kg s.c.) alone (78% _+ 5.2 and 227% + 23.4 of the basal level after 90 min resp. (Fig. 2 and 3)). The administration of perphenazine (10 mg/kg s.c.) also increased the height of the D O P A C peak (87% + 6.8 after 90 m i n (Fig. 4)) and pre-administration of amfonelic acid also significantly potentiated in the height of the peak at 70, 80, and 90 rain following administration of p e r p h e n a z i n e with an increase of 153% + 13.2 after 90 min.

Interaction between amfonelic acid and atypical neuroleptics or N T Clozapine (30 mg/kg i.p.) produced a gradual increase in extracellular D O P A C in the striatum (51% + 5.2 after 90 min, Fig. 5). However, p r e t r e a t m e n t with amfonelic

90

I7't 0

0

p r o d u c e d a smaller but significant mcrease in extracellular D O P A C ; maximal increase of 25% + 5.4 during the 90 min recording (Fig. 6). P r e t r e a t m e n t with amfonelic acid again p r e v e n t e d the increase in D O P A C p r o d u c e d by the neuroleptics (Fig. 6). Finally, NT (10/~g i.c.v.) significantly increased extracellular D O P A C m the striatum (maximal increase of 27% over the 90-min recordlng period) and this effect was also antagonised by p r e t r e a t m e n t with amfonelic acid (Fig. 7). A summary of the effects of amfonelic acid on the increase in striatal extracellular D O P A C m d u c e d by typical, and atypical neuroleptics and N T 90 min after their administration is shown in Fig. 8. !

|

|

|

I0

20

30

40

!

50

,

60

~

70

,

80

!

90

Time 0,,h,) Fig 7. Effect of amfonehc acid on the NT-induced increase in extraceUular striatal DOPAC of anaesthesized rats Sahne (mi/kg s c.) or amfonelic acid (2.5 mg/kg s c.) were admimstered followed 5 min later by saline or NT (10/zg/10/~i 1.c v.). DOPAC peak height was monitored every 10 mm during 90 mm. Extracellular DOPAC basal level was estimated from the means of the last 4 signals before administration and represents 100%. Groups are: amfonelic aod + saline (-); saline + NT ( I ) , amfonehc acid + NT ([3). Each value is the mean of 6 recordings + S.E.M. Significant differences, *P < 0.05 are for comparisons between saline-NT and amfonehc acid-NT (Dunnett's test). acid totally p r e v e n t e d the increase in the height of the D O P A C p e a k (Fig. 5). Thioridazine (20 mg/kg s.c.)

4ool

Typical

~

ST

J 200

100 ¸ llalop

~

Perp

flmg/lqp IO.0t)ml/l~ I I 0 m a / l ~

Clmt

Thior

(~Oml/blll ~0ml/Iqa

NT (t0~lllcv)

Tr~tmemta

Fig 8. Effect of amfonehc acid on the increase of striatal extracellular DOPAC induced by typical, atypical neuroleptlcs and NT of anaesthesized rats. Saline (ml/kg s.c.) or amfonehc acid (2.5 mg/kg s c.) were admimstered followed 5 min later by neuroleptlcs or NT. Groups are: saline + neuroleptlcs or NT (hatched column), and amfonellc acid + neuroleptics or NT (filled column) DOPAC peak height was momtored every 10 mm during 90 mln and the data obtained at 90 min after administration are present. The extracellular DOPAC basal level was estimated from the means of the last 4 s~gnals before administration and represents 100%

DISCUSSION The use of amfonelic acid to discriminate between classical and atypical neuroleptics by m e a s u r e m e n t of striatal tissue D O P A C has been previously described 2°" 40.60 In the present study, using differential pulse v o l t a m m e t r y with carbon fibre electrodes to m o n i t o r extracellular striatal D O P A C , we d e m o n s t r a t e that this m o d e l can discriminate between both types of neuroleptics in vivo. M o r e o v e r , we present evidence using the amfonelic acid test that i.c.v, administration of N T produces a neurochemical profile similar to the atypical neuroleptlcs. The m e a s u r e m e n t of electroacttve c o m p o u n d s in the brain using differential pulse v o l t a m m e t r y with electrically p r e t r e a t e d carbon fibre electrodes allows the selective recording of 3 separated peaks in the striatum which have been identified as ascorbic acid, catechols and indoles together with uric acid 11. T h e identification of the voltammetric p e a k at 80 m V as D O P A C is based on two main observations First, pharmacological investigations using the same electrode type have shown that monoamine oxidase inhibitors decrease the p e a k indicating that the signal r e c o r d e d is a m e t a b o l i t e rather than an amine 22"38. Second, the extracellular concentration of the amines are below the limit of detection of the electrodes whde D O P A C is present at high extracellular concentrations in the striatum and therefore the signal r e c o r d e d in the present study is most p r o b a b l y due to the oxidation of extracellular D O P A C 22'38. Amfonelic a o d had no effect on extracellular basal striatal D O P A C in anaesthesized rats. This result confirms previous work indicating that amfonelic acid treatment does not alter either tissue levels of d o p a m i n e or D O P A C 2'6'40"56'6° o r their extracellular levels in freely moving rats using intracerebral dialysis61. In view of the potent behavioural effects of amfonelic acid 1'6 it is surprising that the drug produces no changes in d o p a m m e metabolism but this m a y relate to its inhibition of

91 dopamine reuptake 6~ and a recent study has shown that dopamine reuptake inhibitors produce little effect on extracellular dopamine and DOPAC 25. In agreement with previous in vitro2°'4°'56'6° and in v i v o 61 studies, amfonelic acid markedly potentiated the increase in striatal DOPAC induced by a classical dopamine antagonist neuroleptics 12'53 probably by increasing the availability of dopamine for release within the nerve endings 2°'4°'56'6°'61. A similar enhancement of dopamine release is seen when amfonelic acid is given prior to electrical stimulation of the medial forebrain bundle 17. Acute administration of the atypical neuroleptics clozapine and thioridazine also increased striatal dopamine metabolism in agreement with previous e x v i v o 59 and in vivo studies 37'4s. It has been proposed that atypical neuroleptics act specifically on the mesolimbic dopaminergic system to attenuate psychotic symptoms 13, an effect resulting in increased neuronal firing in the ventral tegmental area 23'62 and dopamine release in the nucleus accumbens 35. However, this specificity of action has been a subject of controversys'37'48'57 and the present study further demonstrates that acute administration of either clozapine or thioridazine does not produce a selective increase in mesolimbic dopamine metabolism. Nevertheless, a different effect on dopamine metabolism in the striatum between typical and atypical neuroleptics was observed when the animals were pre-treated with amfonelic acid. Thus in agreement with previous studies 4°'6°, amfonelic acid did not potentiate but rather totally inhibited the increase m extracellular striatal DOPAC induced by atypical neuroleptics. These results indicate an interaction between amfonelic acid and atypical neuroleptics in the striatum and show that while both classes of neuroleptics increase dopamine metabolism, the mechanisms involved may differ. Preliminary data from this laboratory suggest that in the nucleus accumbens amfonelic acid also potentiates the increase in DOPAC induced by typical neuroleptics but blocks the effect of an atypical neuroleptic. The exact reason why amfonelic acid can discriminate between classical and atypical neuroleptics remains to be determined. A major difference between the two classes of drugs is their selectivity and affinity for dopamine, acetylcholine and serotonin receptors. Both haloperidol and perphenazine are potent D2 dopamine antagonists. The blockade of dopamine receptors results in an increase in neuronal activity and this effect is potentiated by amfonelic acid. In comparison with these two drugs, clozapme and thioridazine have much lower affinity on dopamine receptors. It has been reported that clozapine acts more selectively on D 1 receptors at low doses (5 and 10 mg/kg) while affecting both D 1 and D 2 receptors at

higher doses 1°. From the present data it is not possible to determine if selectivity for a dopamine receptor subtype is important because the dose of clozapine (30 mg/kg) was too high to have a selective effect on D1 receptors. It therefore would be interesting to study the interaction between amfonelic acid and specific D 1 and D 2 antagonists. Apart from the possible participation of D~ and D2 receptors m the effect of the two atypical neuroleptics, it is possible that other neurotransmitters may be involved since existing atypical neuroleptics possess relatively good affinity for cholinergic and serotonergic receptors41' 42 Therefore, an alternative explanation is that the increase in dopamine metabolism induced by the atypical neuroleptics involves the indirect effects of other neurotransmitters. The action of" amfonelic acid on these systems is not known. It is unlikely that the potentiation of the dopamine metabolism by amfonelic acid depends on the amplitude of the increase in DOPAC because haloperidol (1 mg/kg) and perphenazine (10 mg/kg) produced similar increases in DOPAC but the potentiation by amfonelic acid was greater with haloperidol. Moreover, a smaller dose of haloperidol (0.05 mg/kg) increased the extracellular striatal DOPAC to a similar extent to clozapine (30 mg/kg) but pretreatment with amfonelic acid potentiated the effect of haloperidol but blocked that of clozapine. Intracerebroventricular administration of NT also increased striatal dopamine metabolism in agreement with earlier reports 32'46. Furthermore, like atypical neuroleptics, the effects of NT were antagonised by amfonelic acid. It is interesting that NT can be distinguished from typical neuroleptics in the present study because unlike this class of neuroleptics NT does not bind to dopamine receptors 46. The mechanism by which NT increases dopamine metabolism in the striatum is unclear but may involve an interaction between cholinergic and NT systems in this region 19. Therefore, as with atypical neuroleptics, the increase of dopamine metabolism after i.c.v, administration of NT could involve the indirect action of neurotransmitters other than the dopamine. The present in vivo neurochemical study supports the view that NT resembles the atypical rather than typical neuroleptics 28. Further studies are required to establish the mechanisms involved and the clinical potential of NT of stable analogues as neuroleptics. In conclusion, the present study demonstrates that the coadministration of amfonelic acid with classical neuroleptics potentiates the increase in extracellular striatai DOPAC in the striatum of the anaesthetized rat while antagonising the effects of atypical neuroleptics on extracellular DOPAC indicating that amfonelic acid can be used to discriminate in vivo between the two types of neuroleptics. In this model, NT appears to interact with

92 amfonelic acid in a similar way to atypical neuroleptics, suggesting a similar neurochemical mechanism may be involved in the effects of NT and atypical neuroleptics on striatal dopaminergic systems. It is suggested that, in contrast to typical neuroleptics which directly increase dopamine metabolism, the effects of both atypical neuREFERENCES 1 Aceto, M.D., Harris, L S., Lesher, G Y , Pearl, J and Brown Jr., T . G , Pharmacologic studies with 7-benzyi-l-ethyl-l,4-dihydro-4-oxo-l,8-naphthyridine-3-carboxyhc acid, J Pharmacol Exp Ther, 158 (1967) 286-293 2 Aceto, M.D., Botton, I., Levitt, M., Martin, R , Bentley, H.C and Speight, P T., Pharmacological properties and mechanism of action of amfonelic aod, Eur. J. Pharmacol., 10 (1970) 344-354. 3 Agnatl, L.F, Fuxe, K., Benfenati, E and Basttistinl, N , Neurotensln in vitro markedly reduces the affinity in subcortical hmbic 3H-N-propylnorapomorphine binding site, Acta Physiol Scand., 119 (1983) 459-461. 4 Angrade, R. and Aghalanian, G K., Neurotensln selectively activates dopamlnerg~c neurons of the substantia nigra, Soc Neuroscl. Abstr., 7 (1981) 553 5 Blaha, C.D., Phillips, A . G , Flblger, H.C and Lane, R F , Effects of neurotensin on dopamme release in the nucleus accumbens' comparisons with atypical antipsychotic drug action, Ann. N Y. Acad. Sct., 537 (1988) 478-480 6 Braestrup, C., Biochemical differentiation of amphetamine vs methylphenidate and nomifensine in rats, J Pharm Pharmacol, 29 (1977) 463-470 7 Carlsson, A and Llndqvlst, M , Effect of chlorpromazme or haloperidol on formaaon of 3-methoxytyramme and normetanephnne in mouse brain, Acta Pharmacol., 20 (1963) 140-144 8 ChIodo, L.A and Bunney, B S , Typical and atypical neuroleptics, differentml effects of chronic administration on the actwlty of A9 and A10 midbrain dopammergic neurons, J Neurosct, 3 (1983) 1607-1619 9 Costali, B. and Nayior, R . J , A comparison of the abdmes of typical neuroleptlc agents and of thioridazine, clozapine, sulpiride and metoclopram~de to antagonise the hyperactwity induced by dopamme applied intracerebrally to areas of the extrapyramidal and mesohmbic systems, Eur. J. PharmacoL, 40 (1976) 9-19 10 Coward, C.M., Imperato, A., Urwler, S and White, T . G , Biochemical and behavloural properties of clozapme, Psychopharmacology, 99 (1989) $6-S12 11 Crespi, E , Sharp, T , Maidment, N T and Marsden, C . A , Dffferentml pulse voltammetry: s~multaneous m vivo measurement of ascorblc aod, catechols and 5-hydroxymdoles m the rat stnatum, Brain Research, 322 (1984) 135-138 12 Creese, I., Burt, D.R. and Snyder, S , Dopamme receptor binding predicts chnical and pharmacological potenoes of antischizophremc drugs, Science, 192 (1976) 4811-4813 13 Crow, TJ., Deakin, J F W and Longden, A., The nucleus accumhens - - a possible site of antipsychotlc acUon of neuroleptic drugs 9, Psychol Med, 6 (1977) 213-221 14 Dubuc, I., Protals, P , Colboc, O and Constentm, J., Antagomsm of the apomorphine-lnduced yawning by 'atypical' neuroleptlcs, Neuropharmacology, 21 (1982) 1203-1210. 15 Ervm, G.N , Nemeroff, C B. and Prange J r , A.J., Neurotensm blocks certain amphetamine-reduced behaviors, Nature, 291 (1981) 73-76. 16 Ervm, G N and Nemeroff, C B , Interactions of neurotensm with dopamme-containmg neurons m the central nervous system, Prog. Neuro-Psychopharmacol. Btol Psychiatry, 12 (1988) $53-$69. 17 Ewing, A G , Bigelow, J C and Wightman, R M , Direct m wvo momtormg of dopamme released from two strlatal corn-

roleptics and NT may involve indirect action antagonised by amfonelic acid. Acknowledgements This work was supported by the Weilcome Trust, the MRC and by a Scholarship to R R by the FCAR of Quebec partments in the rat, Science, 221 (1983) 169-171. 18 Farde, L., Wiesel, F A . , Nordstrom, A L and Sedvall, G., D1and D2-dopamine receptor occupancy during treatment with conventional and atypical neuroleptics, Psychopharmacology, 99 (1989) $28-$31 19 Frey, P, Lls, M and Coward, D . M , Neurotensm concentration m rat striatum and nucleus accumbens, further studies of their regulation, Neurochem lnt, 12 (1988) 33-38. 20 Fuller, R.W. and Snoddy, H.D , Flumezapine and zotepine: 5-hydroxytryptamlne antagonism not involved m the lack of synergism of these anttpsychoac drugs with amfonehc aod in rats, J Pharm Pharmacol., 37 (1985) 755-756. 21 Gerlach, J. and Simmelsgard, H , Tardwe dyskmesia during and following treatment with haloperldol, blperiden, thiondazine and clozapme, Psychopharmacology, 59 (1978) 105-112. 22 Gonon, F , Buda, M. and Pujoi, J.F, Treated carbon fiber electrode for measunng catechols and ascorbic acid. In C.A Marsden ( E d ) , Measurement of Neurotransmitter Release, John Wiley and Sons, Chichester, 1984, pp 153-171. 23 Hand, T H , Hu, X.T and Wang, R . Y , Differential effects of acute clozapine and halopendol on the activity of ventral tegmental (A10) and nigrostnatal (A9) dopamlne neurons, Brain Research, 415 (1987) 257-269. 24 Herman, E and Pleasure, H., Clinical evaluation of thlorldazine and chlorpromazine in chronic schizophrenia, Dis Nerv Systm, 24 (1963) 54-59 25 Hurd, Y L. and Ungerstedt, U., In vivo neurochem~cal profile of dopamine uptake lnhibltors and releases m rat caudateputamen, Eur J Pharmacol, 166 (1989) 251-260 26 Imperato, A and DI Chiara, G., Dopamine release and metabolism in awake rats after systemic neuroleptics as studied by transstriatal dialysis, J Neurosct, (1985) 297-306. 27 Johcoeur, EB., De Mlchelle, G , Barbeau, A. and St-Pwrre, S., NeurotensIn affects hyperactivity but not stereotypy reduced by pre and post synaptic dopamlnergic stimulation, Neurosct Btobehav. Rev, 7 (1983) 285-390 28 Johcoeur, EB., Rioux, F and St-Pierre, S , Neurotensln. In Handbook of Neurochemlstry, Vol 8, 1985, pp. 93-113 29 Johcoeur, C B , Rivest, R , St-Pierre, S , Gagne, M.A and Dumais, M , The effects of neurotensln and [D-Tyrll]-NT on the hyperactivity induced by intra-accumbens administration of a potent dopamlne receptor agonlst, Neuropepudes, 6 (1985) 143-156 30 Johcoeur, E B , Gagne, M.A , Rwest, R , Drumheller, A. and St-Pierre, S , Atypical neuroleptic-hke effects of neurotensin, in press 31 Juorlo, A V , The effects of amfonehc acid and some other central stimulants on mouse strlatal tyramme, dopamlne and homovanllhc acid, Br J Pharmacol, 77 (1982) 511-515 32 Kahvas, P W , Nemeroff, C B and Prangc J r , A J , Neurotensm mlcroinlectlon into the nucleus accumbens antagomzes dopamme-mduced increase m locomotion and rearing, Neuroscience, 11 (1984) 919-930 33 Kltabgl, P , Neurotensln modulates dopamme neurotransmissIon at several levels along brain dopamlnergic pathways, Neurochem lnt, 14 (1989) 11-19. 34 Krelskott, H , Behavioral pharmacology of antlpsychotlcs In F Hoffmelster and G Stille (Eds), Handbook of Experimental Pharmacology, Vol 55, Sprmger-Verlag, Berlin, 1980, pp 59-89 35 Lane, R F and Blaha, C D , Acute thlondazme stimulates

93 mesolimbic but not nigrostriatal dopamme release: demonstration by m vivo electrochemistry, Brain Research, 408 (1987) 317-320 36 Ljunberg, T. and Ungerstedt, U., Classification of neuroleptlc drugs according to their ability to inhibit apomorphlne-lnduced locomotion and gnawing: evidence for two different mechanisms of acuon, Psychopharmacology, 56 (1978) 239-247. 37 Maidment, N.T. and Marsden, C.A. Acute administration of clozapme, thiondazine and metoclopramide increases extracellular DOPAC and decreases extracellular 5-HIAA, measured in the nucleus accumbens and striatum of the rat using m vivo voltammetry, Neuropharmacology, 26 (1987) 187-193. 38 Marsden, C.A., Joseph, M.H., Kruk, Z.L., Maidment, N.T., O'Neill, R.D., Schenk, J.P. and Stamford, J.A., In wvo voltammetry - - Present electrodes and methods, Neuroscience, 25 (1988) 389-400. 39 Marsden, C.D., Tarsy, D. and Baldessarini, R.J., Spontaneous and drug-induced movement disorders in psychotic patients. In D.E Benson and D. Blumer (Eds.), Psychiatry Aspects of Neurological Dzsease, Grune and Stratton, New York, 1975, 219 PP. 40 Mcmdlen, B.A., ComparaUve effects of classical and atypical antipsychotlc drugs in combination with a non-amphetamine stimulant on rat brain dopamme metabohsm, J. Pharm Pharmacol., 33 (1981) 544-546. 41 Meltzer, H.Y., Matsubara, S. and Lee, J.C, Classdication of typical and atypical drugs on the basis of dopamme D-l, D-2 and serotonin2 pk, values, J. Pharmacol. Exp. Ther, 251 (1989) 238-246 42 Miller, R.J. and Hdey, C.R., Antl-muscarinic properties of neuroleptics and drugs-induced parkinsomsm, Nature, 248 (1974) 596-597 43 Miller, H.H. and Shore, P.A, Effects of amphetamine and amfonelic acid on the disposltmn of striatal newly synthestzed dopamine, Eur J Pharmacol, 78 (1982) 33-44. 44 Miyoshi, R., Kito, S., Ishiba, H. and Nakashlma, M., Modulation of dopamine D-1 receptor bindings by neurotensin in the rat stnatum, Neurochem. lnt, 15 (1989) 493-496. 45 Nemeroff, C.B., Neurotensm: perchance an endogenous neuroleptic, Bml. Psychiatry, 15 (1980) 283-302 46 Nemeroff, C.B., Luttlnger, D , Hernandez, D.E., Mailman, R.B., Mason, G.A., Davis, D., Wlderlov, E., Frye, G.D., Kilts, C.A, Beaumont, K., Breese, G.R. and Prange Jr., A.J, Interaction of neurotensin and brain dopaminergic systems biochemical and behavmral studies, J. Pharmacol Exp. Ther., 225 (1983) 337-345. 47 Nemeroff, C.B., The lnteractmn of neurotensm with dopaminerglc pathways m the central nervous system basic neurobmlogy and lmphcat~ons for the pathogenesis and treatment of schizophrenia, Psychoneuroendocrmology, 11 (1986) 15-37. 48 O'Connors, W.T., Drew, K L. and Ungerstedt, U., Differences m dopamme release and metabolism m rat strlatal subregions following acute clozapme using m wvo mlcrodialysis, Neurosct Lea., 98 (1989) 211-216. 49 Paxmos, G and Watson, C., The Rat Brain in Stereotaxtc Coordinates, Academic Press, New York, 1982 50 Prota~s, P, Dubuc, I. and Constentm, J , Pharmacological

51 52

53 54

55

56 57

58 59

60

61

62 63 64 65

characteristics of dopamine receptors involved in the dual effect of dopamme agomsts on yawning behaviour in rats, Eur. J. Pharmacol., 94 (1983) 271-282 Quirlon, R., Interaction between neurotensin and dopamme in the brain: an ovemew, Pepudes, 4 (1983) 609-615 Schotte, A. and Leysen, J.E., Autoradiographic evidence for the locahzation of high affinity neurotensin binding sites on dopaminergic nerve terminals in the nigrostriatal and mesolimblc pathways in rat brain, J Chem. Neuroanat., 2 (1989) 253-257 Seeman, P., Lee, T., Chau-Wong, M. and Wong, K., Antipsychotlc drug doses and neuroleptic/dopamine receptors, Nature, 261 (1976) 717-719. Seutm, V., Massotte, L. and Dressem A., Eiectrophysiological effects of neurotensm on dopaminerg~c neurones of the ventral tegmental areas of the rat m vitro, Neuropharmacology, 28 (1989) 949-954. Sharp, T., Maidment, N.T, Brazell, M.P, Zetterstrom, T., Ungerstedt, U., Bennett, G W. and Marsden, C.A., Changes m monoamlne metabolites measured by s~multaneous in wvo differential pulse voltammetry and intracerebral dialysts, Neurosctence, 12 (1984) 1213-1221. Shore, EA., Actions of amfonelic aod and other non-amphetamine stimulants on the dopamine neuron, J. Pharm. Pharmacol, 28 (1976) 855-857. Stamford, J.A., Kruk, Z.L. and Milar, J., Actions of dopamine antagonists on sumulated striatal and limbic dopamine release: an m vlvo voitammetnc study, Br J. Pharmacol., 94 (1988) 924-932. Tschanz, J.T. and Rebec, G.V, Atypical antipsychotlc drugs block selective components of amphetamine-reduced stereotypy, Pharmacol. Biochem, Behav., 31 (1988) 519-522. Waldemeier, EC. and Mmtre, L , On the relevance of preferential increases of mesohmbic versus striatal dopamine turnover for the predictmn of ant~psychot~cactivity of psychotropic drugs, J Neurochem., 27 (1976) 589-597. Waldmeier, P C., Huber, H , Hemnch, M. and Stoecklin, K., Discrimination of neurolept~csby means of their interactmn with amfonelic acid: an attempt to characterize the test, Bmchem. Pharmacol., 34 (1985) 39-44. Westerink, B.H.C., Damsma, G., De Vnes, J.B. and Koning, H , Dopamine re-uptake mhibitors show inconsistent effects on the m vtvo release of dopamine as measured by intracerebral dialysis in the rat, Eur. J. Pharmacol, 135 (1987) 123-128. White, F J. and Wang, R Y., Differential effects of classical and atypical antlpsychotic drugs on A9 and A10 dopamme cells, Sctence, 221 (1983) 1054-1057. Worms, P., Broekkamp, C L E and Lloyd, K.G., Behavioral effects of neuroleptics. In R.H. Roth (Ed.), Neuroleptics, Raven, New York, p. 93 Young, W.S and Kuhar, M.J., Neurotensin receptor Iocahzation by bght microscope autoradiography m rat brain, Brain Research, 206 (1981) 273-285. Zetterstrom, T., Sharp, T. and Ungerstedt, U., Effect of neuroleptic drugs on striatal dopamme release and metabolism in the awake rat studied by lntracerebral dialysis, Eur J. Pharmacol., 106 (1984) 27-37