neurochemical effects of nicotine on glutamate and gaba mechanisms in the rat brain

Acta Ph,ystol Scand 1991, 141, 241-250 ADONIS 000 167729 100036R

Neurochemical effects of nicotine on glutamate and GABA mechanisms in the rat brain

M. P E R E Z DE L A M O R A " , J. M E N D E Z - F R A N C O " , R. S A L C E D A " , J. A. A G U I R R E and K. F U X E " Department of Neurosciences, Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico, Mexico DF, Mexico and Department of Histology and Neurobiology, Karolinska Institute, Stockholm, Sweden

P ~ R F . ~ DE LA MORA, M., M~NDEZ-FRANCO, J., SALCEDA, R., AGUIRRE, J. A. & FUXE, K. 1990. Neurochemical effects of nicotine on glutamate and GABA mechanisms in the rat brain. Acta Physiol Scand 141, 241-250. Received 13 February 1990, accepted 29 September 1990. ISSN 0001-6772. Department of Neurosciences, Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico, Mexico DF, Mexico and Department of Histology and Neurobiology, Karolinska Institute, Stockholm, Sweden.

The effects of nicotine on y-aminobutyric acid (GABA) and glutamate mechanisms were studied in several rat brain regions both in vivo and in vitro. In vivo acute intermittent injections of nicotine decrease GABA utilization in the hypothalamus and glutamate levels within the nucleus caudatus and the subcortical limbic forebrain (mainly tuberculum olfactorium and nucleus accumbens). Glutamic acid decarboxylase activity was slightly increased in several regions, when the rats were treated with a single convulsant dose of nicotine and killed at the moment of the convulsions but it was not affected by a single injection nor by intermittent acute administration of non-convulsant doses of nicotine. In vitro nicotine elicited release of ~-[~H]glutamate from synaptosomal preparations obtained from the frontoparietal cortex, nucleus caudatus and hypothalamus. The effect was dose-dependent and it was not blocked by mecamylamine. It was also Ca2+ independent. The possibilities are discussed that the decreased GABA utilization in the hypothalamus may be related to certain neuroendocrine actions of nicotine and that the nicotine-induced glutamate release might be involved in some of the physiological and toxicological effects of nicotine.

Key words ; nicotine, GABA, GABA utilization, glutamate, glutamate release, brain, rat.

Nicotine, particularly in the form of tobacco, has for many years been one of the most consumed drugs and is known to have a wide variety of actions both at the peripheral and at the central nervous system of mammals. Nicotine is inter ulia able to affect cognitive, neuroendocrine and motor functions (see book by Nordberg et al. 1989). Furthermore, trophic actions of nicotine have recently been recognized (Janson et al. 1988) and many studies agree with the notion

Correspondence : Prof. Kjell Fuxe, Department of Histology and Neurobiology, Karolinska Institute, Box 60400, S-104 01 Stockholm, Sweden.

that nicotine is responsible for the dependence, tolerance and withdrawal symptoms observed in chronic smokers (Benowitz 1988). Although it is commonly admitted that nicotine effects should be mediated by a number of different neuro- transmitter systems in the brain most of the biochemical studies so far have been focused on catecholamine and acetylcholine neurons (Fuxe et al. 1987, 1989, Beani et al. 1989) and very little is known on the interaction of nicotine with the aminoacidergic neuronal systems. T h e aim of this work was to analyze whether or not y- aminobutyric acid (GABA) and glutamate neurons could be involved in the central effects of nicotine.

24 1

242 M . Pe'rec de la Mora et al.

M A T E R I A L A N D METHODS

215 male Wistar rats (160-190 g ) maintained with water and food ad libitum were used for all the experiments. Nicotine (I-nicotine hydrogen ( +)- tartrate) dissolved in saline was injected intra- peritonially at the doses (free base) indicated under Results. Nicotine was either administered as a single dose or as 4 intermittent injections given at 30 min intervals. The rats were killed by decapitation at 2-5 min following the single injection of nicotine or 30 min after the last injection in the intermittent administration experiments. In all the experiments in which GABA levels were measured and nicotine was given intermittently, 125 mg kg-' i.p. mercapto- propionic acid was injected 2.5 min before killing the animals in order to prevent the post-mortem increase of GABA levels (Van der Heyden & Korf 1978).

A special knife made up of two parallell blades (3.0 mm apart) was used to obtain tissue slices from which the desired regions were either dissected or punched out. The subcortical limbic forebrain (mainly tuberculum olfactorium +nucleus accumbens), nucleus caudatus and frontoparietal cortex were taken out from a slice, with the posterior blade of the knife positioned 1.0 mm rostral to the rostral border of the chiasma opticum. The hippocampal formation and the substantia nigra were dissected out from a similar slice but with the rostral knife blade located at the level of the mammillary bodies. The hypothalamus was manually dissected from the remaining tissue. The dissected regions were wrapped in aluminium foil, weighed and unless otherwise stated stored in liquid nitrogen until analysed.

GABA and gluturnate deterrninalaons

Protein-free extracts were prepared by homogenizing the tissue in 1.5 ml 807; (vol vo1-l) ethanol and centrifuging the homogenates for 10 min at 3.000 rpm in the cold. The supernatants were decanted and evaporated to dryness under an infrared lamp. The extracts were reconstituted in 300-500 pl water and from them an aliquot was taken for measuring either GABA or glutamate. Both amino acids were quanti- tated enzymatically with either L,-glutamate dehydrogenase or GABA-transaminase + succinic semi- aldehyde dehydrogenase (Gabase, Perez de la Mora e l ul. 1975, 1989). The reduced pyridine nucleotides formed in the corresponding reactions were measured after their transformation to highly fluorescent derivatives.

Glutarnic acid decarboxylase actawty determinataun

T o measure glutamic acid decarboxylase (GAD) activity the dissected regions were homogenized in 500 p l of a 50 mM K-phosphate buffer pH 7.0, con-

taining 0.5 mhi pyridoxal phosphate (PLP) and 10 mM aminoethylisothiouronium (AET). The homogenates were supplemented with a small volume of a concentrated Triton X-100 solution to give a 0.50,6 concentration and were left standing in ice for at least 10 min before the assay of the enzyme activity. When GAD activity was measured either in the presence or absence of PLP the homogenates were prepared without this coenzyme and divided into portions. To one portion a small aliquot of a concentrated PLP solution was added to give a 0.5 mM concentration and the same volume of water was added to the other. GAD activity was measured by triplicate in a way similar to that described by Albers & Brady (1959) with some modifications. T o a glass micro-tube (0.3 x 25 mm) containing 8 pl of 87.5 n M ~-[l-"c]- glutamate (Sp activity 37.8 pCi/mmol), 0.5 mM PLP and 50 mMK-phosphate buffer pH 7.0 were added 62 yl homogenate. The micro-tube was joined to another micro-tube containing 50 yl Hyamine hydroxide through a 12 cm latex tube, and both micro-tubes were incubated for 20 min in a water bath at 37 "C. At the end of the incubation period, the reaction was stopped by the injection of 75pl 3.8 MII,SO,. In order to allow for a complete evolution and absorption of ["CC]CO, in Hyamine the measuring device in a nearly extended position was incubated for an additional 60 min at 40 "C in an electric oven. Finally, the I Iyamine micro-tube was dropped into a vial containing 10 ml of scintillation cocktail (4.0 g 2,5-diphenyloxazole (PPO) and 0.1 g (2(4-niethyl-5-phenyloxazolyl) benzene (DMPOPOP) per 1 1 toluene) and its content pumped out with the aid of a glass rod. The radioactivity was determined in a Tri-Carb Packard Scintillation counter with SO'+" efficiency.

['Hklutamate release

The release of ~-["II]glutamate was studied in a synaptosomal preparation obtained from non-frozen tissue by the procedure of Loscher et al. (1985). Electron-microscopic evaluation of the preparations obtained showed that synaptosomes accounted for at least 75 yo of all the structural elements present in the fraction. Membrane fragments and damaged synaptosomes were the major contaminants. In order to study the release of glutamate synaptosomes (300-400 p g protein) were transfered to flasks containing 1.5 ml of oxygenated Krebs-Ringer medium (1 18 mM NaCI, 1.2 mM KH,PO,, 4.7 mM KCI, 1.17 mM MgSO,, 2.5 mM CaCI,, 25 mM NaHCO, and 5.6 mM glucose, pH 7.4) and preincubated at 37" for 5 min L- ['Hlglutamate (2 pCi) was added and the incubation continued for 10 min at allow for the uptake. At the end of the incubation the medium was filtered through a Millipore filter (0.6 mm) and the filter containing the trapped synaptosomes was carefully sliced. Pieces

Nicotine, glutamate and GABA 243

FRONTOPARIETAL LIMBIC HYPOTHALAMUS CORTEX

T

AREAS

r * i

NUC. CAUDATUS

I - * l

r * i

HIPPOCAMPAL FORMATION

Fig. 1. Effects of acute intermittent injections of nicotine on regional brain glutamate levels. Rats were injected (i.p.) every 30 min with either saline or nicotine (1.0 or 3.0 mg kg-' x 4). Thirty min after the last injection the animals were killed. Absolute glutamate levels were 8.3 $0.2, 12.0+0.5, 11.4 k 0.4, 7.7 k 0.5 and 7.5 0.24 pmol g-' wet tissue for the frontoparietal cortex, the limbic areas (subcortical ; mainly nucleus accumbens and tuberculum olfactorium), the nucleus caudatus, the hypothalamus and the hippocampal formation, respectively. The statistical analysis of the results was carried out by the test Treatments versus control (non-parametrical procedures). "P < 0.05. Twelve rats were used for the frontoparietal cortex, the limbic areas and the nucleus caudatus groups and 6 for the rest of the groups. Open bars; control, obliquely stripped bar:

. . 1.0 mg kg-l x 4; and horizontally stripped bar . . .

3.0 mg kg-l x 4.

of the filter were transfered to glass superfusion chambers of 0.25 ml volume. Four chambers were arranged in parallel1 using a 4-channel polystaltic pump. The synaptosomes were superfused with a warm and oxygenated medium at a flow rate of 1.5 ml min '. After 8 min, the medium was changed for one containing the stimulating substances and the superfusion was continued for a further 6rnin. The fractions were collected every minute and at the end of the experiments the pieces of the filter were transferred to scintillation vials containing 5 ml Instagel and counted for radioactivity in a Packard scintillation Spectrometer. The efflux of radioactive glutamate was expressed as the efflux rate constant (ERC), which shows the percent of the total radioactivity incor- porated, which is released per minute (Lopez-Colom6 et al. 1976).

Materials

~-[3,4,-'H]gIutamic acid (Sp Act 42.3 Ci mmol-') and ~-['~C]glutamic acid were obtained from DuPont, New England Nuclear. Hyamine hydroxide was from

Amersham Laboratories. PLP, AET, PPO, DMPOPOP were purchased from Sigma Chemical Co, St Louis, MO, USA. t-glutamic acid dehydrogenase is a product of Boehringer-Mannheim, Mann- heim, Germany. I-nicotine hydrogen( + )tartrate was obtained from BDH Chemicals Ltd., Poole England and gabaculine from Fluka Chemie AG, Switzerland. All other chemicals were of the puriest grade available.

R E S U L T S

Efects of nazcotine on GARA a.nd glutamate levels

As shown in Fig. 1, nicotine when administered intermittently to rats induced a significant 20 decrease in glutamate levels in the subcortical limbic areas and in the nucleus caudatus, while no changes were found in the cortex and the hypothalamus. Under the same conditions and in agreement with Fung & Reed (1988) nicotine was unable to modify GABA levels in the

244 M . Pirez de la Mora et al.

5 150-

u 2 8

125-

w z 2 a

100- #

Table 1. Effects of nicotine on glutamate decarboxylasc activity in various brain areas

Dose Treatment mg kg-'

Nuc caudatus

Saline 0 Nicotine 3 Nicotine 5

9.4 k 0.3 9.7 k 0.5 9.4 k0 .09

Saline 0 Nicotine 3 Nicotine 5

17.0k0.6 18.5k0.5 18.0k0.4

1,imbic Frontoparietal areas cortex

~~ ~ ~ ~- __

no PLP

13.4k0.6 11.2 f 0.6 11.4kO.9 12 .8k0 .5 12 .9k0 .5 11.9$0.8

with PLP ~- ~

Hypothalamus - ~~ ~

- - ~-

45.0+ 1.1 1 49.6k 1.7 * 50.4+ 1.1 J

Hippocampus -~~

-

19.02 1.0 20.9 k 0.6 21.2 k 0 . 3

Rats were injected i.p. with nicotine at the dose indicated (free base) and were killed at the moment they show a tonic-clonic convulsion (1.3-1.7 min). Figures are pmoles g-' h-' SEM, n = 6. Statistical analysis was made by the test Treatments versus control (non-parametrical procedures). * P < 0.05. GAD was measured either with or without PLP in the incubation mixture as described in Materials and Methods.

75 ! I I I

SALINE GABACULINE GABACULINE +

NICOTINE Fig. 2. Effects of acute intermittent injections of nicotine on the gabaculine-induced GABA accumulation in the hypothalamus. The rats were injected (i.p.) at 0 time either with saline, gabaculine (75 mg kg-') or gabaculine (75 mg kg-')+nicotine (2 mg kg-I). Additionally nicotine (2 mg kg-') was administered again to the gabaculine + nicotine-treated group at 30, 60 and 90 min. All the rats were killed at 120 min. For statistical procedures see Fig. 1.

nucleus caudatus and the subcortical limbic Negative results (data not shown) were found areas as well as in the frontoparietal cortex and on both glutamate and GABA levels when a the hypothalamus (data not shown). single nicotine dose of 1.0 or 3.0 mg kg-' was

Nico t ine , g lu tamate and GABA 245

250

200

150

100

50

( 5 )

0 20 100 200 0 20 100

NICOTINE (JIM) 200

Fig. 3. Effects of nicotine on the spontaneous release of ~-[~H]glutamate from (a) frontoparietal and (b) caudate synaptosomes. The nicotine-induced release of ~-[~H]glutamate evoked by various concentrations of nicotine was studied as indicated under Material and Methods. The effects of ~-[~H]glutamate were calculated as efflux rate constants (ERC), which are defined as the percent of total tissue radioactivity, which is released. In the figure the peak of the nicotine stimulated ERC is given in percent of the ERC value of the previous non-stimulated fraction (means & SEM). Number of experiments in parenthesis. The results were analysed by the Jonckheere-Terpstra test for ordered alternatives and the null hypothesis was rejected at a level of P < 0.01 for both regions.

administered and the rats were killed either at 2.5 min after the injection or when an obvious increase in muscle tone was first noticed (2-3 min).

Effects of nicotine on G A D activity

In agreement with the lack of effects of nicotine on GABA levels the administration of the drug under the same treatment schedules was in- effective in modifying GAD-activity in the same brain areas (data not shown). Since a decrease in GAD-activity due to low PLP levels has been responsible for the production of a number of drug-induced convulsions (Tapia et al. 1969) we determined the effects of convulsant doses of nicotine (3.&5.0 mg/kg) on G A D activity in the presence and in the absence of PLP. As can be seen in Table 1, a slight general 1&15 yo increase in G A D activity was found at the moment of convulsions in most regions studied. Thus, when GAD-activity was measured in the presence of its coenzyme GAD activity was found to be increased in the subcortical limbic areas, fron-

9

toparietal cortex and hypothalamus. Further- more, in the absence of PLP GAD-activity was also higher in the hypothalamus and hippocampus.

Effects o f nicotine on gabaculine-induced GABA accumulation

As shown in Fig. 2, the intermittent injection of nicotine reduced the GABA accumulation in the hypothalamus after blocking degradation of GABA with gabaculine (Rando & Bangerter 1977). This effect was specific, since the gabaculine induced increase of GABA levels (pmoles g-') ( P < 0.001) in the nucleus caudatus (1.4f0.05 to 3.5f0.22, meansfSEM, n = 9), the frontoparietal cortex (1.1 f 0.01 to 4.1 f 0.2, n = 9), the hippocampal formation (1.4 f 0.08 to 3.9 f 0.3, n = 4) and the substantia nigra (6.2k0.3 to 9.5f0.2, n = 5 ) was not significantly altered by this nicotine treatment (103f8 ; 112f10; l O O + S ; 104+7 percent, respectively).

ACT 141

246 M . Pe'rex de la Mora et al.

3 0 1 m

0 X

s 20- ;s 8 rA z

!i 2 W

x 10-

I I I i 6 7 8 9 10 1 1 1 2

MINUTES

0

Fig. 4. The CaZT independency of the nicotine- induced release of ~-['H]glutamate from nucleus caudatus synaptosomes is shown. For details, see legend in Fig. 5 and Materials and Methods. The results are expressed as means & SEM of 4 different experiments. The Ca2+ free medium contained, 5.0mM Mg SO, and 1 . 0 m ~ EGTA. .----.. Normal medium; x----X Ca2+ free medium. The arrow shows the time at which nicotine (100 ,UM) was introduced.

Effect of nicotine on ~ - [ ~ H l g l u t a m a t e release

As shown in Figure 3, nicotine elicited a dose- related significant release of ~- [~H]glu tamate in the frontoparietal cortex and the nucleus caudatus. A similar but somewhat attenuated effect was found in the hypothalamus, while only a trend for ~- [~H]glu tamate release was present in the hippocampal formation (data not shown). T h e effect of nicotine was Caz+ independent (Fig. 4). When synaptosomes from the frontoparietal cortex and the nucleus caudatus were preincubated (10 min) and superfused in the presence of 10 mM mecamylamine, the release of ~- [~H]glu tamate expressed in percent of the prestimulated mean value was non-significantly decreased in the frontoparietal cortex from 214 f 10 (yo; means SEM; nicotine 100 mM alone group ; 3 experiments) to 166 f 33 (nicotine 100 mM + mecamylamine 10 mM group ; 4 experiments). ~- [~H]glu tamate release was induced from synaptosomes by 15 mM KCl, and nicotine at a 100 mM concentration was found to enhance

this release in the frontoparietal cortex but not in the hypothalamus, the nucleus caudatus and the hippocampal formation (data not shown) (Fig. 5). I n order to rule out the possibility that intermediates derived from the metabolism of ~- [~H]glu tamate could account for the nicotine- induced release of radioactivity the effect of nicotine was studied on ~- [~H]glu tamate release from cortical synaptosomes after the purification of the superfusion fractions by ion-exchange chromatography in Dowex 50 X-8. Although the radioactivity released by nicotine from the synaptosomes is reduced a significant effect of nicotine is still present after the purification of the fractions (Fig. 6). Furthermore, when the peak fraction was concentrated and chromato- graphed by thin layer chromatography in silica gel plates developed in ethanol : water (70 : 30) 90% of the radioactivity released migrated as L-glutamate (data not shown).

D I S C U S S I O N

I n the present study we have evaluated the effect of nicotine on the activity of two aminoacidergic systems possessing either major excitatory (glutamatergic) or inhibitory GABAergic effects on the central nervous system. We have found that in comparison with the wide spectrum of actions on the catecholaminergic systems (see Fuxe et al. 1987, Nordberg et al. 1989) nicotine has a limited influence on the aminoacidergic systems (see also Fung & Reed 1988). Thus, nicotine in vivo was unable to modify a number of GABAergic parameters such as GABA levels and in most areas studied the glutamate-induced GABA accumulation. There was only a decrease of this last parameter in the hypothalamus. Since GABA accumulation after GABA degradation blockade seems to occur in nerve terminals and is nerve-impulse-dependent (PCrez de la Mora et al. 1977), the decrease in the gabaculine- induced GABA accumulation reported in this work after the intermittent injection of nicotine may indicate a decrease of GABA utilization in the hypothalamus.

T h e increases in G A D activity found in some brain areas were rather modest (1&15 yo) and relatively unspecific and can be interpreted as compensatory changes produced by an en- hancement in brain excitability due to an increase in glutamate release (see below). Thus, they were only observed, when the animals were


80

60 m

z X

t 2 z 40 s v)

w t U X

20 3 U LL Lu

0 + \ . , . , 1 . 1

6 0 1 0 12

1 b '

1 1 . I 1 6 8 1 0 12

MINUTES

Fig. 5. Effects of nicotine on the K+-stimulated release of ~-[~H]glutamate from (a) frontoparietal and (b) hypothalamic synaptosomes. See Fig. 3 and Materials and Methods for details. The arrows show the time at which nicotine (100 /AM) and potassium (15 mM) or potassium (15 mM) alone was introduced. The results are means SEM of at least 3 different experiments. In the statistical analysis the peak values were obtained for each curve and the results (KCI alone us KCI + nicotine) compared with a double-tailed Student's t-test. "P < 0.05. .----. 15 mM KCI, X----X 15 mM KCI+ 100 ,UM nicotine.

killed at the time of the convulsions and no changes were observed following either the intermittent or the single acute treatment with non-convulsant doses of nicotine. GAD activity was slightly increased when

measured in the absence of PLP in the hippocampus but such an increase in enzyme activity disappeared when GAD activity was measured in the presence of PLP. T h e reason for this difference is unclear. However, in the rat brain two forms of GAD exist which differ in their dependence of PLP. Furthermore, the relative amounts of the dependent and independent forms seem to differ from region to region (Denner & W u 1985). Thus, a slight increase of PLP as seen following the administration of a variety of psychoactive drugs, may increase GAD activity only when measured in the absence of saturating PLP concentrations in those regions, in which the PLP-dependent form of the enzyme predominates. T h e modest increase in GAD activity together with the predominance of the PLP-dependent GAD in the hippocampus suggests that this might be a possible explanation.

Nicotine was also found to interact in vivo with glutamatergic neurons, since a decrease in glutamate levels was found in nucleus caudatus and in the subcortical limbic forebrain (tuberculum olfactorium and nucleus accumbens). T h e possibility that nicotine diminished glutamate levels in these regions by an effect on the general metabolism seems unlikely in view of the fact that this effect it is not observed in other regions.

fn vitro it was found that nicotine, as in the case with catecholaminergic and cholinergic neurons (Beani et al. 1989, Wonnacott et al. 1989) increases the release of ~-[~H]glutamate. Thus, we have found that nicotine in high concentrations evokes a dose-related release of ~- [~H]glu tamate from synaptosomes of the frontoparietal cortex, the nucleus caudatus and the hypothalamus. T h e effect of nicotine was Ca2+ independent and mecamylamine was unable to block it. Furthermore, nicotine selectively enhanced the K+-stimulated release of glutamate from cortical synaptosomes.

T h e possibility that the radioactivity released by nicotine from synaptosomes was accounted

9-2

248

80

rn 60

K

2

o 40

P

s z u < x 2 20

W

0

M . Pei-en de la Mora et al.

-u , I I 1 1

8 9 10 1 1 12

MINUTES

Fig. 6. The effects of nicotine on the spontaneous release of ~-[~H]glutamate from frontoparietal cortex synaptosomes with or without purification of the fractions obtained in the superfusion. The release of ~-[~H]glutamate was studied as described under Materials and Methods. An aliquot of each fraction was analysed for radioactivity and the rest was acidified to pH 1.0 and passed through a 4 x 80 mm Dowex 50 X-8 column (H-form). The column was washed with water to eliminate organic acids and the amino acids were eluted with 5 m12 M NH,OH. The eluted amino acid fractions were concentrated to about 1.0 ml under an infrared lamp and its radioactivity estimated. The arrow shows the time, at which nicotine (100 /AM)

was introduced. The results obtained before (X----X) and after (@----a) ion exchange chromatography of the fractions are indicated. A similar effect was observed with or without purification of the fractions.

for by radioactive Krebs-cycle intermediates was ruled out in control experiments in which the fractions obtained during the superfusion were purified by ion-exchange chromatography and the peak fraction was evaluated by thin layer chromatography. T h e results of such experiments showed that ~- [~H]glu tamate is actually released by nicotine.

T h e nicotine-induced release of ~ - [ ~ H ] g l u t a - mate found in this paper is in line with the effects of this drug on the release of amino acid neurotransmitters. Thus, it has been found that in addition to stimulating the release of catechol-

amines and acetylcholine (Ach) nicotine enhances in vzvo the outflow of ~- [~H]aspar ta te from cerebral cortex and increases in vitro the electrically induced release of ~-[~H]aspartate and the spontaneous release of GABA from cortical and striatal slices (Levi et al. 1980, Beani et al. 1989) as well as from hippocampal synaptosomes (Wonnacott et al. 1989). However, in contrast to the low concentrations of nicotine required to stimulate the release of catechol- amines (see Wonnacott et al. 1989) and Ach (see Beani et al. 1989) the spontaneous release of glutamate reported in this paper although dose- dependent is elicited at concentrations well above 50 ,UM. These results explain the lack of effect of nicotine on the spontaneous release of D-

[3H]aspartate reported by Beani et al. (1989) in striatal slices, since in that study nicotine was tested in the range of 6 . 2 - 6 2 , ~ ~ . T h e Ca2+ independency and the mecamylamine insensi- tivity of the nicotine induced glutamate release is not surprising, since a similar situation is found when high concentrations of nicotine are used to stimulate dopamine (DA) release (West- fall et al. 1989). T h e mechanism by which nicotine releases glutamate might therefore, be similar to that operating for the release of DA at high nicotine concentrations. Nicotine might e.g. release glutamate by a displacement mechanism or by the activation of an atypical receptor, as suggested by Westfall et al. (1989), which could lead to the release of glutamate through a non-exocytotic process similar to that suggested for the Ca2+ independent release of GABA (Cunningham & Neal 1981). T h e high synaptosomal glutamate concentration (Ryall 1964, Mangan & Whittaker 1966) and the fact that glutamate release is only partially Ca2+ dependent (Nadler et al. 1977) lend support to this possibility. T h e decreased glutamate levels found in this work could be explained by the stimulatory effect of nicotine on glutamate release. T h e finding that nicotine increases the outflow of ~- [~H]aspar ta te in vzvo from the parietal cortex (Beani et al. 1989) is in line with this interpretation.

T h e findings reported in this paper could have important physiological and toxicological impli- cations. Thus, from a neuroendocrine point of view the decrease in GABA turnover found in the hypothalamus may be involved in the nicotine-induced decrease in prolactin secretion (Fuxe et al. 1987, 1989). Thus, this reduction is


mediated via an increase in DA turnover within the median eminence and these DA nerve terminals may be controlled by an inhibitory GABAergic system (see Fuxe et al. 1979). From a toxicological point of view the increased release of glutamate in the central cortex and possibly in the nucleus caudatus are of interest since it may have a role in nicotine-induced convulsions. I t seems possible that at least some of the toxic neurological effects characteristic of nicotine poisoning can be attenuated by the use of glutamate receptor antagonists.

In conclusion, the results of this work indicate that nicotine decreases GABA turnover in the hypothalamus and releases glutamate from several brain regions.

This work has been supported by a Grant from the Swedish Tobacco Monopoly and by funds from Consejo Nacional de Ciencia y Tecnologia (Conacyt) Mexico (Clave: 890757 (SA-7). J. A. Aguirre was supported by Ministerio de Educacion y Ciencia (Spain).

R E F E R E N C E S ALBERS, T. & BRADY, R.O. 1959. The distribution of

glutamic decarboxylase in the nervous system of the rhesus monkey. 3 Biol Chem 234, 926-928.

BEANI, L., BIANCHI, C., FERRARO, L., NILSSON, L., NORDBERG, A,, ROMANELLI, L., SPALLUTO, P., SUNDWALL, A. & TANGANELLI, S. 1989. Effect of nicotine on the release of acetylcholine and amino acids in the brain. In: Nicotinic receptors in the CNS. Their role in synaptic transmission (ed. A. Nordberg, K. Fuxe, B. Holmstedt & A. Sundwall), pp. 149-155. Progress in Brain Res Vol 79, Elsevier, Amsterdam.

BENOWITZ, N.L. 1988. Pharmacological aspects of cigarette smoking and nicotine addiction. New Eng 3 Med 319, 1318-1330.

CUNNINGHAM, J. & NEAL, M.J. 1981. On the mechanism by which veratridine causes a Ca2+- independent release of GABA from brain slices. Br 3 Phurmucol73, 655-667.

DENNER, L.A. & WU, J.-Y. 1985. Two forms of rat brain glutamic acid decarboxylase differ in their dependence of free pyridoxal phosphate. 3 Neuro- chem 44, 957-965.

FUNG, Y.K. & REED, J.A. 1989. Effect of nicotine on central GABAergic system. Gen Pharmacol 19,

FUXE, K., ANDERSSON, K., OGREN, S.O., PEREZ DE LA

MORA, M., SCHWARCZ, R., HOKFELT, T., ENEROTH, P., GUSTAFSSON, J.-A. & SKETT, P. 1979. GABA

533-536.

neurons and their interaction with monoamine neurons. An anatomical, pharmacological and func- tional analysis. In : GABA-neurotransmitters. Pharmacochemical, biochemical and pharmacological aspects (eds. P. Krogsgaard-Larsen, J. Scheel- Kriiger, C.H. Kofod), pp. 74-94. Munksgaard, Copenhagen.

FUXE, K., ANDERSON, K., ENEROTH, P., HARFSTRAND, A., NORDBERG, A. & AGNATI, L.F., 1987. Effects of nicotine and exposure to cigarette smoke on discrete dopamine and noradrenaline nerve terminal systems of the telencephalon and diencephalon of the rat: Relationship to reward mechanisms and neuroendocrine functions and distribution of nicotinic binding sites in brain. In: Tobacco, Smoking and Nicotine (eds. W.R. Martin, G.R. van Loon, E.T. Iwamoto & L. Davis), pp. 255-262. Plenum Press, New York.

FUXE, K., ANDERSON, K., ENEROTH, P., JANSSON, A,, VON EULER, G., TINNER, B., BJELKE, B. & AGNATI, L.F. 1989. Neurochemical mechanisms underlying the neuroendocrine actions of nicotine : Focus on the plasticity of central cholinergic nicotinic receptors. In: Nicotinic Receptors in the C N S . Their Role in Synaptic Transmission (eds. A. Nordberg, K. Fuxe, B. Holmstedt & A. Sundwall), pp. 197- 207. Progress in Brain Res Vol. 79, Elsevier, Amsterdam.

JANSON, A.M., FUXE, K., AGNATI, L.F., KITAYAMA, I., HARFSTRAND, A,, ANDERSON, K. & GOLDSTEIN, M. 1988. Chronic nicotine treatment counteracts the disappearance of tyrosine-hydroxylase immuno- reactive nerve cell bodies, dendrites and terminals in the mesostriatal dopamine system of the male rat after partial hemitransection. Brain Res 455,

LEVI, G., GALLO, V. & RAITERI, M. 1980. A re- evaluation of veratridine as a tool for studying the depolarization-induced release of neurotransmitters from the nerve endings. Neurochem Res 5,281-295.

LOPEZ-COLOME, A.M., ERLIJ, D. & PASANTES- MORALES, H. 1976. Different effects of calcium flux blocking agents on light and potassium stimulated release of taurine from retina. Bruin Res 113,

LOSCHER, W., BOHME, G., MULLER, F. & PAGLIUSI, S. 1985. Improved method for isolating synaptosomes from 11 regions of one brain : Electron microscopic and biochemical characterization and use in the study of drug effects on nerve terminal g-aminobutyric acid in vivo. 3 Neurochem 45, 879-889.

MANGAN, J.L. & WHITTAKER, V.P. 1966. The distribution of free amino acids in subcellular fractions of guinea pig brain. Biochem 3 98, 128-137.

NADLER, J.V., WHITE, W.F., VACA, K.W., REDBURN, D.A. & COTMAN, C.W. 1977. Characterization of putative amino acid transmitter release from slices of rat dentate gyrus. 3 Neurochem 22, 717-724.

332-345.

527-534.

250 M . Pe'rez de la Mora et al.

NORDBERG, A., FUXE, K., HOLMSTEDT, B. & SUND- WALL, A. 1989. Nicotinic receptors in the CNS. Their role in synaptic transmission. Progress in Brazn Res Vol. 79, Elsevier, Amsterdam.

PEREZ DE LA MORA, M., FUXE, K., HOKFELT, T. & 1-JUNGDAHL, A. 1975. Effect of apomorphine on the GABA turnover in the DA cell group rich area of the mesencephalon. Evidence for the involvement of an inhibitory GABAergic feedback control of the ascending DN neurons. Neurosci Let t 1,

PEREZ DE LA M ~ R A , M., FUXE, K., HOKFELT, T. & LJUNGDAHL, A. 1977. Evidence for a nervous impulse flow-dependent GABA accumulation in the substantia nigra after treatment with g- glutamyl-hydrazide. Neurosci Let t 5, 75-82.

P ~ R E Z DE LA MORA, M., M~NDEZ-FRANCO, J., SALCEDA, R. & RIESGO-ESCOVAR, R. 1989. A glutamate dehydrogenase-based method for the assay of L-

glutamic acid : Formation of pyridine nucleotide fluorescent derivatives. Anal Biochem 180,248-252.

RANDO, R.R. & BANGERTER, F.W. 1977. The in vivo inhibition of GABA transaminase by gabaculine. Btochem Biophys Res Comm 76, 127G1281.

RYALL, R.W. 1964. The subcellular distribution of acetylcholine, substance P, 5-hydroxytryptamine,

109-111.

g-aminobutyric acid and glutamic acid in brain homogenates. 3 Neurochem 11, 131-145.

TAPIA, R., PEREZ DE LA MORA, M. & MASSIEU, G. 1969. Correlative changes of pyridoxal kinase, pyridoxal-5'-phosphate and glutamate decarboxylase in brain during drug-induced convulsions. Ann N Y Acad Sc i 166, 257-266.

VAN DER HEYDEN, J.A.M. & KORF, J. 1978. Regional levels of GABA in the brain : Rapid semiautomated assay and prevention of postmortem increase by 3- mercapto-propionic acid. 3. Neurochem 31,197-203.

WESTFALL, T.C., MEREU, G., VICKERY, L., PERRY, H., NAES, L. & YOOM, K.-W.P. 1989. Regulation by nicotine of midbrain dopamine neurons. In: Nic- otinic Receptors in the CNS. Their Role in Synaptic Transmission (eds. A. Nordberg, K. Fuxe, B. Holmstedt & A. Sundwall), pp. 173-185. Progress in Brain Res Vol. 79, Elsevier, Amsterdam.

WONNACOTT, S., IRONS, J., RAPIER, C., THORNE, B. & LUNT, G.G. 1989. Presynaptic modulation of transmitter release by nicotinic receptors. In: Nicotinic Receptors in the CNS. Their Role in Synaptic Transmission (eds. A. Nordberg, K. Fuxe, B. Holmstedt & A. Sundwall), pp. 157-163. Pro- gress in Brain Res Vol. 79, Elsevier, Amsterdam.

neurochemical effects of nicotine on glutamate and gaba mechanisms in the rat brain

Documents