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Preferential effects of caffeine on limbic and cortical dopamine systems
BIOL
761
PSYCHIATRY
1988;23:761-768
Preferential Effects of Caffeine on Limbic and Cortical Dopamine Systems G. R. Stoner, L. R. Skirboll, Sidney Werkman, and D. W. Hommer
In this study,
of midbrain pamine
we investigated dopamine
neurons
of caffeine
The action
and other antagonist
consistent
with
involving
It appears
socortical project
release,
that,
projecting
in the rat, dopamine
during
also antagonized
caffeine
neurons,
qffect
with the adenosine
at adenosine
of caffeine. of dopamine
amphetamine
inhibits
has no effect
(A9 group).
lines of evidence
the dopaminergic
administration but
of do-
numerous
the effects
to that observed
diazepam
rates
but had no significant
by pretreatment
depression
on the activit?
the firing
zona compacta
antagonists
also antagonized
similar
nigra
conJrming
qf cafleine-induced
the benzodiazepine
group),
blocked
act as competitive
a mechanism
administration
depressed
in the substantia
(L-PIA),
haloperidol
dopamine
Finally.
signi$cantly
Al0 was completely
in
xanthines
dopamine
.feine.
neurons
L-phenyl-isopropyl-adenosine
caffeine
of acute caffeine
Caffeine
in the ventral tegmental area (Al0
rates of dopamine
on thejring
agonist
the effects
neurons.
receptors.
The
This ,finding neuron
is
activit?
administration. effects
mesolimbic
on dopamine
that
of cafand
neurons
methat
to the striatum.
Introduction Caffeine is the most widely used psychotropic substance in the world. Its use by hospitalized psychiatric patients is common (Winstead 1975). Various lines of evidence suggest that some of the behavioral effects of caffeine may be mediated through brain catecholamine systems. In this regard, administration of caffeine has been shown to increase plasma catecholamines in humans (Robertson et al. 1978). In vivo and in vitro neurochemical studies have demonstrated that caffeine and other methylxanthines effect both central norepinephrine (Waldeck 1971, Karasawa et al. 1976) as well as central dopamine (DA) turnover (Waldeck 197 1; Carrodi et al. 1972; Govoni et al. 1984). Behaviorally, caffeine and other methylxanthines potentiate the rotational behavior induced by DA agonists in rats with unilateral lesions of the nigrostriatal pathway (Fredholm et al. 1983). Methylxanthines also increase the locomotor stimulation induced by DA infused directly into the nucleus accumbens of the rat (Anden and Jackson 1975). In light of the effects of caffeine on brain catecholamines and the proposed catecho-
From
the Electrophysiology
Addre\\
reprmt
Columbian Received
a? 19X8 Society
requests Way.
Unit. to Dr.
Seattle.
June 8. 1987: revised
of Bwlogical
Psychiatry
Clmical D.
WA
W
Neuroscience Hammer.
Branch.
GRECC
NIMH.
(1828).
Bethesda,
Veterans
MD.
Administration
Medical
Center.
1660
South
98108.
August
28,
1987.
ooo6-3223/Xx/$03
so
762
BIOL PSYCHIATRY 1988;23:761-768
G.R. Stoner et al.
laminergic involvement in the pathophysiology of schizophrenia (Stevens 1973), it is interesting to note that there are several case reports of caffeine inducing or worsening psychosis (McManamy and Schube 1936; Mikkelsen 1978; Shoul et al. 1984). In a controlled study of caffeine’s effects on hospitalized psychotic patients, it was found that during a period of decaffeinated coffee use, there was a significant decrease in the patients’ symptoms. When the patients returned to using coffee containing caffeine, their psychotic symptoms worsened (DeFreiter and Schwartz 1979). In order to determine how caffeine might be exerting its putative, psychosis-worsening action, we examined the effect of caffeine on the firing rates of two populations of midbrain dopaminergic neurons. Midbrain DA neurons in the rodent have generally been divided into two populations: the ventral tegmental area (AlO) (VTA); and the substantia nigra zona compacta (A9) (SNZC). A10 sends projections primarily to the frontal cortex and limbic structures, whereas A9 projects primarily to the striatum. These systems have been well defined both biochemically (Bunney and Aghajanian 1976; Tassin et al. 1976) and anatomically (Fuxe et al. 1974; Hofelt et al. 1974). Furthermore, as methylxanthines act as competitive antagonists at adenosine receptors (Schwabe and Trost 1980; Williams and Risley 1980; Daly et al. 1981; Snyder et al. 1981a,b; Murphy and Snyder, 1982), we reasoned that an adenosine agonist would block any action of caffeine that is mediated through the adenosine receptor. Therefore, we examined the ability of pretreatment with the adenosine agonist L-phenyl-isopropyladenosine (L-PIA) to block the effects of caffeine on midbrain DA neurons. In addition, as caffeine is known to release brain catecholamines, we examined the ability of haloperidol, a DA antagonist, to block the effects of caffeine. Finally, because the benzodiazepine diazepam has been shown to block stress-induced changes in DA turnover in frontal cortex (Lavielle et al. 1978), we pretreated animals with diazepam to determine if it would antagonize caffeine-induced changes in firing rates of midbrain DA cells.
Methods Male Sprague-Dawley rats weighing between 200 and 250 g were used. Animals were anesthetized with chloral hydrate (400 mg/kg ip), and anesthesia was maintained during the course of the experiment, with additional intravenous chloral hydrate as needed. Rats were mounted in a stereotaxic apparatus, the scalp and periosteum were reflected, and a 3-mm burr hole was drilled over either the SNZC or VTA. Cells recorded from the SNZC were from a region defined in the atlas of Paxinos and Watson (1982) as 1.5-2.5 mm lateral to midline and 2.7-3.5 mm anterior to the interaural line. VTA cells were recorded from a region O-l.0 mm lateral to the midline and 2.7-3.5 mm anterior to the interaural line. Single barrel micropipettes (WPI lB150F) filled with 2 M NaCl saturated with 2.0% pontamine sky blue dye and with an impedance of 408 Mohm (measured at 60 Hz) were lowered into the brain using a hydraulic microdrive (Narishige, MO-8, Tokyo, Japan). Electrode potentials were passed through a high-impedance amplifier (WPI 750, New Haven, CT) and monitored on an oscilloscopeand audiomonitor. The firing of single units was counted over IO-set epochs using a window discriminator and a rate meter, and the rates were displayed on a chart recorder and thermal printer. Core body temperature was monitored with a thermister rectal probe and was maintained at 3637°C with a heating pad. Extracellular potentials were identified as belonging to DA neurons on the basis of spike duration (>2 msec), shape (initial notched segment followed by a triphasic potential), and firing rate (<9 spikes/set) (Bunney et al. 1973). All drugs were administered via the lateral tail vein. Caffeine was administered at 2-
Limbic and Cortical Dopamine:
Caffeine Effect
BIOL PSYCHIATRY 1988;23.761-768
763
min intervals in cumulative doses up to 40 mg/kg. Baseline firing rate was calculated from the 60-set interval following the injection of drug. The study was divided into two parts. First, we compared the effects of caffeine on DA neurons in the VTA with its effects on DA neurons in the SNZC (n = 13 in each group). Then we examined the ability of various drug pretreatments to affect caffeine-induced slowing of VTA DA neurons. The drug pretreatments were: saline, L-PIA (0.03 mg/kg), haloperidol (0.5 mg/kg), and diazepam (0.5 mg/kg) (n = 13 in each group). The pretreatments were administered iv a minimum of 2 min and a maximum of 10 min before administration of the first dose of caffeine. This variation in interval between drug pretreatment and caffeine administration was the result of the transient fluctuations in firing rate sometimes observed after drug pretreatment (particularly with L-PIA) and our desire to have a stable baseline prior to caffeine administration. Only one cell was tested per animal. Following the experiment, recording sites were confirmed by an iontophoretic ejection of pontamine sky blue dye through the recording electrode. This marked the recording site. The animal was then killed by overdose of chloral hydrate. The brain was removed and placed in 4% paraformaldehyde. Frozen serial sections were cut at 20-pm intervals through the brain on a cryostat. Data were used only from animals with recording sites within VTA or SNZC.
Results There was no significant difference between the basal firing rate of DA neurons in the VTA and SNZC. Cells in the SNZC fired at 4.2 + 0.5 spikeslsec, and cells in the VTA fired at 4.0 ? 0.6 spikeslsec (NS, group r-test, t = 0.3, df = 24). Caffeine administration produced a highly significant decrease in the activity of DA neurons in the VTA (twoway ANOVA with repeated measures on the dose, Fc1.24J = 16, p < 0.03). In contrast to the inhibitory effect of caffeine in the VTA, caffeine had no effect on the firing of DA neurons in the SNZC (two-way ANOVA with repeated measures, F,1.74) = 0.9, NS) (Figures 1 and 2). In the second part of the study, we examined the effects of pretreatment with haloperidol, L-PIA, diazepam, or saline on the ability of caffeine to decrease the activity of VTA neurons. None of these pretreatments had a significant effect on activity of VTA neurons (ANOVA, Fc3.4X) = 1.31, NS). Haloperidol, however, did produce a slight increase in firing that was not statistically significant. Pretreatment with haloperidol. LPIA, or diazepam blocked the ability of caffeine (40 mg/kg cumulative dose) to slow the firing of VTA neurons (ANOVA, Fc3,4Xj= 5.12, p < 0.004). Duncan’s multiple range test showed that only the saline pretreatment group had a significant change in firing rate following caffeine administration @ < 0.01) (Figure 3).
Discussion Intravenous administration of caffeine depresses the firing rates of dopaminergic cells in VTA. but has no significant effect on dopaminergic cells in SNZC. This selective inhibition was antagonized by pretreatment with the adenosine agonist L-PIA, the dopamine antagonist haloperidol, and the benzodiazepine diazepam. Our finding that caffeine selectively affects the mesolimbic-mesocortical as compared to the nigrostriatal dopaminergic system suggests that this agent has a preferential action on neural pathways implicated in emotional responsivity (Thierry et al. 1976) and the pathophysiology of schizophrenia (Hofelt et al. 1974).
764
BIOL PSYCHIATRY 1988;23:761-768
G.R. Stoner et al.
I
5 min
I
Figure 1. the firing tegmental effect on inhibition
’
5 min
’
These chart recorder tracings show the effects of intravenously administered caffeine on rates of dopamine neurons in the substantia nigra zona compacta (SNZC) and the ventral arrea (VTA). The triangles indicate the dose of caffeine in mg/kg.‘(A) Caffeine had no the activity of this SNZC neuron. (B) In contrast, caffeine administration led to an of the firing of this VTA neuron.
For many years, the actions of caffeine and other xanthines were attributed to their ability to inhibit phosphodiesterases and subsequent elevation of cyclic adenosine monophosphate (AMP) concentration (Sutherland and Rall 1958; Butcher and Sutherland 1962). Later, it was reported that methylxanthines antagonize the actions of adenosine (Sattin and Rall 1970) and that this antagonism occurs at doses that have no effect on phosphodiesterase activity (Fredholm 1980). Our finding that L-PIA antagonized the inhibitory effect of caffeine on VTA cells is in agreement with an adenosine receptor-mediated action of caffeine. Pretreatment with the DA antagonist haloperidol also antagonized the effect of caffeine. Our results with caffeine are similar to those reported by Bunney and Aghajanian (1973) for amphetamine. These authors found that haloperidol antagonizes amphetamineinduced inhibition of dopaminergic neurons in both VTA and SNZC and proposed that the amphetamine-induced decrease in the firing rate of dopaminergic neurons resulted from an increase in DA release in DA terminal regions. Similar to amphetamine, caffeine also promotes the release of brain catecholamines (Berkowitz et al. 1970) and increases action potential-dependent dopamine release in synaptosomes prepared from rat corpus striatum (Chou et al. 1982). As caffeine and amphetamine both release DA and inhibit DA neuron activity in a haloperidol-reversible manner, it is reasonable to suggest that if caffeine does increase psychosis, it may act through a similar mechanism to amphetamine.
Limbic and Cortical Dopamine:
Caffeine
Effect
BIOL PSYCHIATRY iY88;23:7hi-76X
765
Our findings demonstrate, however, that unlike amphetamine, caffeine acts preferentially on AIO. Thus, it is possible that administration of a high dose of caffeine may provide a more specific model of the pathophysiology of schizophrenia than does amphetamine. Finally, in light of data that benzodiazepines block stress-induced increases in DA turnover in cortica1 areas (Lavielle et al. 1979), we asked if pretreatment with diazepam might antagonize the effect of caffeine on VTA. We found that diazepam completely antagonized the effect of caffeine. The preferential action of caffeine on the VTA as compared to the SNZC DA systems could reflect differences in DA-mediated feedback mechanisms. However, with regard to the DA autoreceptor-mediated feedback, the evidence for differential sensitivity in SNZC and VTA is controversial. Anden et al. f 1982, 1983) report that the selective DA receptor-autoreceptor agonist B-HT 920 reduces the DA synthesis rate constant equally in neurons projecting to striatum, olfactory tubercle, and nucleus accumbens. In contrast. Bannon et al. (1981) report that mesocortical DA cells lack synthesis-modulating autoreceptors. In addition, Chido et al. (1984) report that VTA dopamine neurons show a complete absence of autoreeceptors, whereas White et al. (1984) report a reduced autoreceptor sensitivity in the VTA. With regard to differential neuronal feedback pathways, detailed neuroanatomical studies have been described for both systems (Nauta and Mehler 1966; Nauta et al. 1978). As more extensive feedback pathways have been documented for the nigrostriatal system as compared to the VTA, where less dense innervation has been described, it is unlikely that these feedback systems account for caffeine’s preferential
Figure 2. Caffeine administration produces a dose-dependent slowing of VTA dopamine neurons, but has no effect on SNZC dopamine neurons (n = 13 in each group).
766
BIOL PSYCHIATRY 1988:23:76l-768
SALINE
HALQPEWDOL
DIAZLPAM
L-PM
Figure 3. Pretreatment with haloperidol, diazepam, or the adcnosine agonist, L-PlA blocks the ability of caffeine (40 mg/kg, cumulative dose) to inhibit the firing of WA dopam& neurons (ANOVA Ff3.48 = 5.12, p < ~.~4~. The asterisk indicates that onfy saline pretreatment was followed by a signiticant caffeine administration.
decrease
in firing rate (p < 0.01, Duncan’s multiple range test) after
action in the VTA. The preferential action of caffeine in VTA more likely reflects differences in receptor populations on VTA versus SNZC neurons. However. the precise mechanism by which caffeine selectively affects VTA remains to be dete~~i~~ed. In conclusion, we have demonstrated that caffeine selectively inhibits DA ceils in the VTA. In addition to providing a possible site of action for the effects of caffeine on vigilance and attention, these results also may explain the clinical observation that caffeine can exacerbate schizophrenic symptoms. Adenosine agonists might have some use in the reduction of schizophrenic symptoms and. unlike other antipsychotic agents. they may not produce basal gangiia dysfunction (i.e.. Parkinson side effects). Fu~hermore~ as diazepam blocks both caffeine-induced and stress-induced changes in mesoiimbic and mesocortical dopaminergic function, benzodiazepines may also have some efficacy in the reduction of stress-induced exacerbations of psychosis.
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321:lW104.
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27:666-670.
K, Thornstiom U (1982): Selective stimulation of riqxamine by B-HI’920 and B-HT933, respectively. Naunvra-Schtniedeher[:
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BIOL PSYCHIATRY 1988:23:761-768
Caffeine Effect
767
Anden N-E, Isson H, ROS E, Thornstrom U (1983): Effects of B-HT920 and B-HT933 on dopamine and noradrenaline autoreceptors in the rat brain. Acta Pharm~~l TOX~CO~ 52:5 l-56. Bacopoulos NG, BUS~OS G, Redmond DE, Baulu J, Roth RH (1978): Regional sensitivity of primate brain dopaminergi~ neurons and halo~ridoi; alterations folIowing chronic treatment. Bruin Res 157:396-401.
Bannon MJ, Michaud RL, Roth RH (198 I): Mesocortical dopamine neurons: Lack of autoreceptors modulating dopamine synthesis. Molec Phnrmacol 19:270-275. Berkowitz BA, Tarver JH, Spector S (1970): Release of norepinephrine system by theophylline and caffeine. Eur J Pharmacol 10:64-7 I
in the central nervous
Bruns RF, Daly JW. Snyder SH (1980): Adenosine receptors in brain membranes: Binding of N6-~y~Iohexyl ~~H]adenosine and 1,3-diethyl-8-[3H] phenylxanthine. Proc Nat1 Arad Sci USA 77:554-555 1. Bunney ES, Aghajanian GK (1976): Dopamine and norepinephrine innervated cells in the rat prefrontal cortex. Pharmacological differentiation using micro-iontophoretic techniques. Life Sci 10:1782-1792. Bunney BS, Aghajanian GK ( 1978): d-Amphetamine-induced depression of central dopamine neurons: Evidence for mediation by both autoreceptors and a striato-nigrai feedback pathway. Nauny-Schmiedebeq
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304125%261.
Bunncy B.S. Walters JR, Roth RH. Aghajanian GK (1973): Dopaminergic antipsychotic drugs and amphetamine on single cell activity. J Pharmacol
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in biological materials. J Eirtl
Chido L, Bannon M. Grace A, Roth R, Bunney BS (1984): Evidence for the absence of impulseregulating somatodendritic and synthesis-modulating nerve terminals autoreceptors on subpopulations of mesocortical dopamine neurons. .Neuroscience 12: I -I 6. Chou DT, Cuzzone H, Springstead J, Hirsch K (1982): Effects of caffeine and amphetamine-SOa on dopamine uptake and release mechanisms in rat corpus striatum. Fed Proc 41:462Y. Corrodi H, Fuxe K. Jonsson G ( 1972):Effects of caffeine on central monoamine neurons. J Phorm Pharmocol
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Daly JW, Burns RF, Snyder SH (1981): Adenosine receptors in the central nervous Relationship to the central actions of methylxanthines. Life Sri 28:2083-2097.
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1:129- 132.
Fredholm BB, Herrera-Marschitt M, Jonzon B, Lindstrom K, Ungerstedt U (1983): On the mcchanism by which methylxanthines enhance apomorphine induced rotation in the rat. Phurmnt*oi Biochem Behcw 19535-54 I .
Fuxe K. Hofelt T, Johansson 0. Jonsson G, Lidbrink P, Ljungdahl A ( 1974): The origin of d~)pamin~ nerve terminals
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Govoni S. Petkov VV, Montefusco 0. Missale C, Battaini F. Spano SF, Trabucchi M (1984): Differential effects of caffeine on hydroxyphcnylacetic acid concentrations in various rat brain dopuminergic structures. J Pharm Phrrrmrcc~ol 36:458~60. Greden JF. Procter A, Victor B ( 198 I}: Caffeine associated with greater use of other psychotropic agents. Corny ~.s~~tt~oit~ 22:565-57 I,
fMdt
T. Ljungdah~ A. Fuxe K, Johansson 0 f 1974): Dopamine nerve terminals in the rat limbic cortex: Aspects of the dopamine hypothesis of schizophrenia. S&~zr~~ 184: 177-179.
Karasawa T. Furukawa K. Yoshida K, Shimizu M (t976): Effect of theophyhinc metabolism in the rat brain. i%r J Pttwmncy~i 37:97-104.
on monoamrnc
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Lavielle S, Tassin JP, Thierry AM, Bfanc G, Het-ve D, Ba~h~kmy C, Glowinski J t 197X): blockade by benzodiazepine of the selective high increase in do~rn~ne turnover induced h\ \trc”‘;< in mesocortical dopaminergic neurons of the rat. Brc~in Res 16858%594. Lidbrink P, Farnebo LO ( 1973): Uptake and release of NA in rat cerebraf cortex in VI\C: No ctfect of bcnzodiazepincs and barbituates. NertropharmtrcoloRv 12:84-X7. McManamy MC, Schube PC (1936): Caffeine intoxication: Report of’ a case the symptoms which amounted to a psychosis. N En,@ J Med 215:616-620. Mikkefsen
EJ ( 1978): Caffeine and schizophrenia.
Murphy KM, Snyder SH (1982): Heterogeneity
J C’lin P.sychirrrp
of adenosine
or
39732-735
A I receptor binding m bram nssue
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Nauta WJH. Mehler WR ( 1966): Projections
of the lentiform nucfeus in the monkey. ~intin Re.5
15-42.
Nauta WJH. Smith GP, Faulf RLM, Domesick VP ( 1978): Efferent c~~nne~tions and niprat afferents of the nucleus accumbens septi: In the rat. ~~~~r~~.s~,je~~*e 3:385401. Paxinos G. Watson c‘ f f 982): Tlrr Ret Brurn in Stereotnxrt~ C~owdinntc~. Sydney:
Acader~r~
Press.
Robertson D. Frofick JC, Can RK, Watson JJ, Hollitiefd JW. Shand DG, Gates JA (19’78~:Effects of caffeine on pfasma renin activity, catecholamines and blood pressure. N En~l .I MP~ 298: i 8 I IXS. Sattin A, Ralf TW ( 1970): The effect of adenosine and adeninc nucfeotides on the adenosrne S’-phosphate content of guinea pig cerebral cortex slices. MCI/ Phurmacol 6: 13-23
3’.
Scatton B ( 1977): Differential regional development of tolerance to incrcasc in dopaminc tumovscr upon repeated neurofeptic administration. Eur J Phormacni 46:363-369. Schwabe VC. Trost 7‘ ( 1980): Characterization of adenosine receptors in rat brain by I - I i3HjNhphenyf~soipiopy-adenosine. N~ui?~~f ~~hmiedeber~ Arch ~h~r~luc~~i 3 f 3: 179-I X7. Shouf PW, Farrell MK. Mafoney MJ (f984): Caffeine anorexia nervosa. I Pediutr 105:493-495.
toxicity as a cause of acute psychosis
Snyder SH, Buns RF, Daly JW, lnnis RR (fY81a): Muftiple nemotransmitters brain: Amines. adenosine, and chokcystokin. Fed Prow 40: 142-146.
receptors
in
in the
Snyder SH, Katims JJ, Annau Z. Bruns RF, Daly JW (198 I b): Adenosine receptors and bchaviorai actions of methylxanthines. Pror Ncrtl Acad Sci lJSA 78:3060-3264. Stevens J ( 1973): An anatomy of schizophrenia.
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of cyclic adenim rebonufeotide
Tassin JP, Cheramy A, Blanc G. ‘f’hierry AM, Gowinski J (1976): ‘Topographical distrtbution oi dopaminergic innervation and of dopaminergic receptors in the rat striatum. I. Micr~st~mat~on of [jH]DA uptake and DA content in microdiscs. Brain Res 141:291--301. Thierry AM. Tassin JP, Blanc G, Giowinsk~ J (1976): Selective dopaminergic system by stress. Nature flortdl 26?:242-244. Wafdeck B ( I97 I ): Some effects of’ caffeine and aminophylfine in the brain. J Phwm Pharmacof 23:X24-830.
activation
of the m~~4~co~ical
on the turnover of catechofamines
Williams M, Risley E ( 1980): Biochemical characterization of putative central purinergrc receptors by using 2-chforo[‘H]adenosine. a stable analog of adenosine. Proc Nat1 Acnd Sci USA 77:6892-6896. Winstead DK (1976): Coffee consumption 1450.
among psychiatric inpatients. Am J Pswhiufry
White F. Wang R (1984): AI0 dopamine neuron: Role of autoreceptors of sensitivity to dopamine agonists. Lije Sci 34:f 161-i 170.
in determining
i33: l44’uring rate
761
PSYCHIATRY
1988;23:761-768
Preferential Effects of Caffeine on Limbic and Cortical Dopamine Systems G. R. Stoner, L. R. Skirboll, Sidney Werkman, and D. W. Hommer
In this study,
of midbrain pamine
we investigated dopamine
neurons
of caffeine
The action
and other antagonist
consistent
with
involving
It appears
socortical project
release,
that,
projecting
in the rat, dopamine
during
also antagonized
caffeine
neurons,
qffect
with the adenosine
at adenosine
of caffeine. of dopamine
amphetamine
inhibits
has no effect
(A9 group).
lines of evidence
the dopaminergic
administration but
of do-
numerous
the effects
to that observed
diazepam
rates
but had no significant
by pretreatment
depression
on the activit?
the firing
zona compacta
antagonists
also antagonized
similar
nigra
conJrming
qf cafleine-induced
the benzodiazepine
group),
blocked
act as competitive
a mechanism
administration
depressed
in the substantia
(L-PIA),
haloperidol
dopamine
Finally.
signi$cantly
Al0 was completely
in
xanthines
dopamine
.feine.
neurons
L-phenyl-isopropyl-adenosine
caffeine
of acute caffeine
Caffeine
in the ventral tegmental area (Al0
rates of dopamine
on thejring
agonist
the effects
neurons.
receptors.
The
This ,finding neuron
is
activit?
administration. effects
mesolimbic
on dopamine
that
of cafand
neurons
methat
to the striatum.
Introduction Caffeine is the most widely used psychotropic substance in the world. Its use by hospitalized psychiatric patients is common (Winstead 1975). Various lines of evidence suggest that some of the behavioral effects of caffeine may be mediated through brain catecholamine systems. In this regard, administration of caffeine has been shown to increase plasma catecholamines in humans (Robertson et al. 1978). In vivo and in vitro neurochemical studies have demonstrated that caffeine and other methylxanthines effect both central norepinephrine (Waldeck 1971, Karasawa et al. 1976) as well as central dopamine (DA) turnover (Waldeck 197 1; Carrodi et al. 1972; Govoni et al. 1984). Behaviorally, caffeine and other methylxanthines potentiate the rotational behavior induced by DA agonists in rats with unilateral lesions of the nigrostriatal pathway (Fredholm et al. 1983). Methylxanthines also increase the locomotor stimulation induced by DA infused directly into the nucleus accumbens of the rat (Anden and Jackson 1975). In light of the effects of caffeine on brain catecholamines and the proposed catecho-
From
the Electrophysiology
Addre\\
reprmt
Columbian Received
a? 19X8 Society
requests Way.
Unit. to Dr.
Seattle.
June 8. 1987: revised
of Bwlogical
Psychiatry
Clmical D.
WA
W
Neuroscience Hammer.
Branch.
GRECC
NIMH.
(1828).
Bethesda,
Veterans
MD.
Administration
Medical
Center.
1660
South
98108.
August
28,
1987.
ooo6-3223/Xx/$03
so
762
BIOL PSYCHIATRY 1988;23:761-768
G.R. Stoner et al.
laminergic involvement in the pathophysiology of schizophrenia (Stevens 1973), it is interesting to note that there are several case reports of caffeine inducing or worsening psychosis (McManamy and Schube 1936; Mikkelsen 1978; Shoul et al. 1984). In a controlled study of caffeine’s effects on hospitalized psychotic patients, it was found that during a period of decaffeinated coffee use, there was a significant decrease in the patients’ symptoms. When the patients returned to using coffee containing caffeine, their psychotic symptoms worsened (DeFreiter and Schwartz 1979). In order to determine how caffeine might be exerting its putative, psychosis-worsening action, we examined the effect of caffeine on the firing rates of two populations of midbrain dopaminergic neurons. Midbrain DA neurons in the rodent have generally been divided into two populations: the ventral tegmental area (AlO) (VTA); and the substantia nigra zona compacta (A9) (SNZC). A10 sends projections primarily to the frontal cortex and limbic structures, whereas A9 projects primarily to the striatum. These systems have been well defined both biochemically (Bunney and Aghajanian 1976; Tassin et al. 1976) and anatomically (Fuxe et al. 1974; Hofelt et al. 1974). Furthermore, as methylxanthines act as competitive antagonists at adenosine receptors (Schwabe and Trost 1980; Williams and Risley 1980; Daly et al. 1981; Snyder et al. 1981a,b; Murphy and Snyder, 1982), we reasoned that an adenosine agonist would block any action of caffeine that is mediated through the adenosine receptor. Therefore, we examined the ability of pretreatment with the adenosine agonist L-phenyl-isopropyladenosine (L-PIA) to block the effects of caffeine on midbrain DA neurons. In addition, as caffeine is known to release brain catecholamines, we examined the ability of haloperidol, a DA antagonist, to block the effects of caffeine. Finally, because the benzodiazepine diazepam has been shown to block stress-induced changes in DA turnover in frontal cortex (Lavielle et al. 1978), we pretreated animals with diazepam to determine if it would antagonize caffeine-induced changes in firing rates of midbrain DA cells.
Methods Male Sprague-Dawley rats weighing between 200 and 250 g were used. Animals were anesthetized with chloral hydrate (400 mg/kg ip), and anesthesia was maintained during the course of the experiment, with additional intravenous chloral hydrate as needed. Rats were mounted in a stereotaxic apparatus, the scalp and periosteum were reflected, and a 3-mm burr hole was drilled over either the SNZC or VTA. Cells recorded from the SNZC were from a region defined in the atlas of Paxinos and Watson (1982) as 1.5-2.5 mm lateral to midline and 2.7-3.5 mm anterior to the interaural line. VTA cells were recorded from a region O-l.0 mm lateral to the midline and 2.7-3.5 mm anterior to the interaural line. Single barrel micropipettes (WPI lB150F) filled with 2 M NaCl saturated with 2.0% pontamine sky blue dye and with an impedance of 408 Mohm (measured at 60 Hz) were lowered into the brain using a hydraulic microdrive (Narishige, MO-8, Tokyo, Japan). Electrode potentials were passed through a high-impedance amplifier (WPI 750, New Haven, CT) and monitored on an oscilloscopeand audiomonitor. The firing of single units was counted over IO-set epochs using a window discriminator and a rate meter, and the rates were displayed on a chart recorder and thermal printer. Core body temperature was monitored with a thermister rectal probe and was maintained at 3637°C with a heating pad. Extracellular potentials were identified as belonging to DA neurons on the basis of spike duration (>2 msec), shape (initial notched segment followed by a triphasic potential), and firing rate (<9 spikes/set) (Bunney et al. 1973). All drugs were administered via the lateral tail vein. Caffeine was administered at 2-
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min intervals in cumulative doses up to 40 mg/kg. Baseline firing rate was calculated from the 60-set interval following the injection of drug. The study was divided into two parts. First, we compared the effects of caffeine on DA neurons in the VTA with its effects on DA neurons in the SNZC (n = 13 in each group). Then we examined the ability of various drug pretreatments to affect caffeine-induced slowing of VTA DA neurons. The drug pretreatments were: saline, L-PIA (0.03 mg/kg), haloperidol (0.5 mg/kg), and diazepam (0.5 mg/kg) (n = 13 in each group). The pretreatments were administered iv a minimum of 2 min and a maximum of 10 min before administration of the first dose of caffeine. This variation in interval between drug pretreatment and caffeine administration was the result of the transient fluctuations in firing rate sometimes observed after drug pretreatment (particularly with L-PIA) and our desire to have a stable baseline prior to caffeine administration. Only one cell was tested per animal. Following the experiment, recording sites were confirmed by an iontophoretic ejection of pontamine sky blue dye through the recording electrode. This marked the recording site. The animal was then killed by overdose of chloral hydrate. The brain was removed and placed in 4% paraformaldehyde. Frozen serial sections were cut at 20-pm intervals through the brain on a cryostat. Data were used only from animals with recording sites within VTA or SNZC.
Results There was no significant difference between the basal firing rate of DA neurons in the VTA and SNZC. Cells in the SNZC fired at 4.2 + 0.5 spikeslsec, and cells in the VTA fired at 4.0 ? 0.6 spikeslsec (NS, group r-test, t = 0.3, df = 24). Caffeine administration produced a highly significant decrease in the activity of DA neurons in the VTA (twoway ANOVA with repeated measures on the dose, Fc1.24J = 16, p < 0.03). In contrast to the inhibitory effect of caffeine in the VTA, caffeine had no effect on the firing of DA neurons in the SNZC (two-way ANOVA with repeated measures, F,1.74) = 0.9, NS) (Figures 1 and 2). In the second part of the study, we examined the effects of pretreatment with haloperidol, L-PIA, diazepam, or saline on the ability of caffeine to decrease the activity of VTA neurons. None of these pretreatments had a significant effect on activity of VTA neurons (ANOVA, Fc3.4X) = 1.31, NS). Haloperidol, however, did produce a slight increase in firing that was not statistically significant. Pretreatment with haloperidol. LPIA, or diazepam blocked the ability of caffeine (40 mg/kg cumulative dose) to slow the firing of VTA neurons (ANOVA, Fc3,4Xj= 5.12, p < 0.004). Duncan’s multiple range test showed that only the saline pretreatment group had a significant change in firing rate following caffeine administration @ < 0.01) (Figure 3).
Discussion Intravenous administration of caffeine depresses the firing rates of dopaminergic cells in VTA. but has no significant effect on dopaminergic cells in SNZC. This selective inhibition was antagonized by pretreatment with the adenosine agonist L-PIA, the dopamine antagonist haloperidol, and the benzodiazepine diazepam. Our finding that caffeine selectively affects the mesolimbic-mesocortical as compared to the nigrostriatal dopaminergic system suggests that this agent has a preferential action on neural pathways implicated in emotional responsivity (Thierry et al. 1976) and the pathophysiology of schizophrenia (Hofelt et al. 1974).
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I
5 min
I
Figure 1. the firing tegmental effect on inhibition
’
5 min
’
These chart recorder tracings show the effects of intravenously administered caffeine on rates of dopamine neurons in the substantia nigra zona compacta (SNZC) and the ventral arrea (VTA). The triangles indicate the dose of caffeine in mg/kg.‘(A) Caffeine had no the activity of this SNZC neuron. (B) In contrast, caffeine administration led to an of the firing of this VTA neuron.
For many years, the actions of caffeine and other xanthines were attributed to their ability to inhibit phosphodiesterases and subsequent elevation of cyclic adenosine monophosphate (AMP) concentration (Sutherland and Rall 1958; Butcher and Sutherland 1962). Later, it was reported that methylxanthines antagonize the actions of adenosine (Sattin and Rall 1970) and that this antagonism occurs at doses that have no effect on phosphodiesterase activity (Fredholm 1980). Our finding that L-PIA antagonized the inhibitory effect of caffeine on VTA cells is in agreement with an adenosine receptor-mediated action of caffeine. Pretreatment with the DA antagonist haloperidol also antagonized the effect of caffeine. Our results with caffeine are similar to those reported by Bunney and Aghajanian (1973) for amphetamine. These authors found that haloperidol antagonizes amphetamineinduced inhibition of dopaminergic neurons in both VTA and SNZC and proposed that the amphetamine-induced decrease in the firing rate of dopaminergic neurons resulted from an increase in DA release in DA terminal regions. Similar to amphetamine, caffeine also promotes the release of brain catecholamines (Berkowitz et al. 1970) and increases action potential-dependent dopamine release in synaptosomes prepared from rat corpus striatum (Chou et al. 1982). As caffeine and amphetamine both release DA and inhibit DA neuron activity in a haloperidol-reversible manner, it is reasonable to suggest that if caffeine does increase psychosis, it may act through a similar mechanism to amphetamine.
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Our findings demonstrate, however, that unlike amphetamine, caffeine acts preferentially on AIO. Thus, it is possible that administration of a high dose of caffeine may provide a more specific model of the pathophysiology of schizophrenia than does amphetamine. Finally, in light of data that benzodiazepines block stress-induced increases in DA turnover in cortica1 areas (Lavielle et al. 1979), we asked if pretreatment with diazepam might antagonize the effect of caffeine on VTA. We found that diazepam completely antagonized the effect of caffeine. The preferential action of caffeine on the VTA as compared to the SNZC DA systems could reflect differences in DA-mediated feedback mechanisms. However, with regard to the DA autoreceptor-mediated feedback, the evidence for differential sensitivity in SNZC and VTA is controversial. Anden et al. f 1982, 1983) report that the selective DA receptor-autoreceptor agonist B-HT 920 reduces the DA synthesis rate constant equally in neurons projecting to striatum, olfactory tubercle, and nucleus accumbens. In contrast. Bannon et al. (1981) report that mesocortical DA cells lack synthesis-modulating autoreceptors. In addition, Chido et al. (1984) report that VTA dopamine neurons show a complete absence of autoreeceptors, whereas White et al. (1984) report a reduced autoreceptor sensitivity in the VTA. With regard to differential neuronal feedback pathways, detailed neuroanatomical studies have been described for both systems (Nauta and Mehler 1966; Nauta et al. 1978). As more extensive feedback pathways have been documented for the nigrostriatal system as compared to the VTA, where less dense innervation has been described, it is unlikely that these feedback systems account for caffeine’s preferential
Figure 2. Caffeine administration produces a dose-dependent slowing of VTA dopamine neurons, but has no effect on SNZC dopamine neurons (n = 13 in each group).
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SALINE
HALQPEWDOL
DIAZLPAM
L-PM
Figure 3. Pretreatment with haloperidol, diazepam, or the adcnosine agonist, L-PlA blocks the ability of caffeine (40 mg/kg, cumulative dose) to inhibit the firing of WA dopam& neurons (ANOVA Ff3.48 = 5.12, p < ~.~4~. The asterisk indicates that onfy saline pretreatment was followed by a signiticant caffeine administration.
decrease
in firing rate (p < 0.01, Duncan’s multiple range test) after
action in the VTA. The preferential action of caffeine in VTA more likely reflects differences in receptor populations on VTA versus SNZC neurons. However. the precise mechanism by which caffeine selectively affects VTA remains to be dete~~i~~ed. In conclusion, we have demonstrated that caffeine selectively inhibits DA ceils in the VTA. In addition to providing a possible site of action for the effects of caffeine on vigilance and attention, these results also may explain the clinical observation that caffeine can exacerbate schizophrenic symptoms. Adenosine agonists might have some use in the reduction of schizophrenic symptoms and. unlike other antipsychotic agents. they may not produce basal gangiia dysfunction (i.e.. Parkinson side effects). Fu~hermore~ as diazepam blocks both caffeine-induced and stress-induced changes in mesoiimbic and mesocortical dopaminergic function, benzodiazepines may also have some efficacy in the reduction of stress-induced exacerbations of psychosis.
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