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Lithium effects on noradrenergic-linked adenylate cyclase activity in intact rat brain: an in vivo microdialysis study
Brain Research, 538 (1991) 333-336
333
Elsevier BRES 24484
Lithium effects on noradrenergic-linked adenylate cyclase activity in intact rat brain: an in vivo microdialysis study Monica I. Masana, Jose A. Bitran, John K. Hsiao, Ivan N. Mefford and William Z. Potter Section of Clinical Pharmacology, Clinical Neuroscience Branch, National Institute of Mental Health, Bethesda, MD 20892 (U.S.A.)
(Accepted 2 October 1990) Key words: Lithium; Cyclic adenosine monophosphate; Beta-receptor; In vivo microdialysis; Norepinephrine
The effects of chronic lithium treatment on adenylate cyclase activity in intact rat brain were examined using in vivo microdialysis. Basal extracellular cyclic adenosine monophosphate (AMP) increased in a dose-dependent manner after norepinephrine was added to the perfusate. Chronic lithium treatment increased basal brain extracellular fluid cyclic AMP levels, while decreasing the magnitude of the cyclic AMP response to stimulation with 100/~M norepinephrine.
Elucidating lithium's neurochemical mechanism of action has been a major goal of psychopharmacological research. Considerable data have accumulated demonstrating that lithium treatment alters signal transduction mechanisms. For example, lithium attenuates agonist and depolarization-induced phosphoinositide (PI) turnover 8 and recently, phorbol ester-induced protein kinase C translocation has been reported to be inhibited by lithium treatment zt. A particularly enduring line of research, however, has focused on lithium's effects on adenylate cyclase (AC). Dousa and Hechter were the first to demonstrate that lithium, in vitro, inhibited norepinephrine (NE) stimulated formation of 3",5"-cyclic adenosine monophosphate (cAMP) in rat brain homogenate 2. Since that time, lithium treatment has been shown to attenuate fl-adrenoceptor stimulated AC activity in membrane, slice and synaptosomal preparations from rat brain in vitro as well as ex vivo (lithium treatment of an intact animal with subsequent in vitro assay of AC activity) 3. Similarly, the post-receptor stimulation of AC by forskolin has been shown to be inhibited by lithium 1, as is the stimulation of AC via the stimulatory guanine nucleotide binding protein (Gs) 1'13. Previous studies of brain AC activity have been limited to examining lithium's effects in vitro or ex vivo. It has not been possible, as of yet, to assess signal transduction in vivo, in intact animals. Tissue integrity may be an important issue since, in previous studies, the particular preparation used affected the results obtained. For instance, the chronic administration of desipramine 16 or
chronic electroconvulsive shock 15 induces a significant decrease in forskolin-stimulated cyclic AMP production in rat cortical slices, whereas in a cortical membrane preparation from similarly treated animals, forskolinstimulated AC activity was significantly increased. Previous studies have demonstrated that cAMP in extracellular fluid correlated highly with intracellular content after receptor activation of AC in rat brain slices 5"9. Recently, Egawa and co-workers used in vivo microdialysis to sample cAMP from brain extracellular fluid and showed a dose-dependent NE induced increase in cAMP efflux 5. They suggested that microdialysis might be a useful method for assessing /3adrenoceptor linked AC activity in vivo 5'2°. In the present study, we use in vivo microdialysis to demonstrate that in intact animals, chronic lithium treatment increases basal AC activity, while significantly attenuating the cAMP response to NE stimulation in rat prefrontal cortex. Male Sprague-Dawley rats (Taconic Farm) weighing 350-450 g were used. Rats had free access to water and food. The control group was fed ordinary rat chow while animals treated chronically with lithium were fed Li2CO 3 supplemented chow (0.165% by weight) for 8 weeks. Serum lithium levels were determined by flame photometry. Microdialysis probes were fabricated as previously described 7, using a semipermeable polymeric membrane (polyacrylonitrile, 3 mm long, 0.3 mm diameter, 2.83 mm 2 in surface area, 40,000 MW cutoff, Hospal Medical) attached to probe bodies of concentric design. Recoveries
Correspondence: M.I. Masana, Section on Clinical Pharmacology, Clinical Neuroscience Branch, National Institute of Mental Health, Building
10, Room 2D46, Bethesda, MD 20892, U.S.A.
334 in vitro in an unstirred solution at a flow rate of 1/A/min were (mean + S.E.M.) 20.0 _+ 1.6% for cAMP (n = 27) (determined for each probe after each experiment). Rats were anesthetized with chloral hydrate (480 mg/kg i.p.) and probes were implanted using a stereotaxic apparatus into the prefrontal cortex at antero-posterior 1.7 mm from bregma, lateral 1.0 mm, vertical 4.5 mm ~7. The rats were maintained under anesthesia throughout the experiment. The probes were perfused with artificial CSF (concentrations in mM): CaCI 2 1.2, MgCI 2 1.2, KCI 3.0, NaCI 120, sodium phosphate 3.0 (pH adjusted to 7.4), containing 30 /~M of the phosphodiesterase inhibitor, rolipram (donated by Boehringer Ingelheim, Germany), at a flow rate of 1 ktl/min. Samples were collected every 30 min for 2 h and then every 15 min thereafter. After 2.5 h, NE was added to the perfusate for one 15-rain sample, then 4 more 15-min samples were collected. Sample volumes were checked by weighing, after which the samples were placed on ice until c A M P was assayed. Dialysate c A M P concentrations were determined with a commercially available radioimmunoassay kit (125IcAMP, Amersham). Detection limit was 0.5 fmol/tube and basal levels were at least 3 times the detection limit. Basal c A M P was taken as the average c A M P concentration in dialysate for the 1 h prior to NE stimulation. The average of the c A M P levels during the period of NE stimulation and the next 15-rain period was taken as the effect of NE. When propranolol was tested, 1 p M propranolol was added to the perfusate 30 min before NE stimulation and continued throughout the rest of the experiment. Statistical analysis was performed using Student's t-test or analysis of variance of repeated measures ( A N O V A R ) with c A M P levels (basal and NE-stimulated) as a repeated factor and a between group treatment factor (either dose, 1 0 - 6 t o 10 3 M NE, absence
or presence of propranolol, or control versus lithium, depending on the A N O V A R ) , after log transformation of the data to linearize variance. A significant effect of a treatment on the c A M P response to NE stimulation would be evident as a significant interaction between the repeated factor and the treatment factor (e.g. greater increases in c A M P response to NE with greater doses of NE). Concentrations of c A M P were elevated initially, decreasing to a steady basal level after about 1.5 h (see Fig. 1 for a representative experiment). Addition of rolipram, a phosphodiesterase inhibitor, was necessary in order to detect basal c A M P levels. Infusion through the probe of 100 # M NE increased c A M P levels in the 15-rain period of the NE infusion followed by a further increase during the next 15-rain period (Fig. 1.). The c A M P responses to different concentrations of NE are shown in Fig. 2. There was a dose-response relationship up to 1 mM NE (F3. w = 37.717, P < 0,001). Addition of 1 /~M propranolol largely blocked the c A M P response to 100/~M NE (mean _+ S.E.M.: basal 0.45 _+ 0.33, NE 0.67 _+ 0.46 fmol/min, Fl, 4 = 19.524, P = 0.0115, compared to the effect of 100 /~M NE in the absence of propranolol). This result was consistent with a fl-adrenoceptor-mediated response. Chronic treatment of animals with lithium, produced a serum lithium concentration of 0.85 + 0.05 mM. Lithium-treated animals had significantly higher basal c A M P levels as shown in Table I. Eleven of these animals (5 lithium-treated and 6 controls) were tested for NE stimulation and the results are shown in Fig. 3. The absolute magnitude ( A ) of the c A M P response to NE was less in the lithium-treated than in the control animals (F1, 9 = 14.907, P = 0.0038) (Fig. 3A). This was
1.6
o
Basal
¢
I
"
'
1.4
1.2"
1.2
10-
08-
0
E
0.0
O. < u
0.6
04-
0.4 02-
0.2
00 30
60
90
120
TIME (rnin)
150
180
210
240
NE
Fig. 1. cAMP levels in dialysate at various times after implantation of a mierodialysis probe in prefrontal cortex. The horizontal bar indicates when 100 ~M NE was added to perfusate. Data from one representative experiment are shown.
10-7
10 .6
10 "s
NOREPINEPHRINE
I0 "4
10 °3
10 .2
(tool/l)
Fig. 2. Effect of different NE doses, on cAMP release. Open symbols: basal levels, closed symbols: NE-stimulated levels. Results are expressed as fmol/min and each point represents the average of at least 3 experiments (mean + S.E.M).
335 accounted for by an increase in basal levels after lithium treatment, since cAMP levels after stimulation with 100 /~M NE were similar in both groups. Lithium's effects were evident as well when the response to NE was expressed as a percentage of basal levels (mean + S.E.M.: control: 270.6 + 23.4%, lithium 157.9 + 12.4%, P < 0.01, Student's t-test) (Fig. 3B). NE stimulation produced a dose-dependent increase in cAMP content in dialysate that was blocked by the fl-blocker propranolol. These in vivo microdialysis results, in conjunction with previous findings demonstrating close correlations between intracellular and extracellular cAMP content in tissue slices 5'9, suggest that sampling of extracellular cAMP by in vivo microdialysis can index AC activity. The increase in cAMP following NE stimulation found in this study was comparable to that of previous investigators 5, although our basal levels obtained in the presence of rolipram were higher than those in which a phosphodiesterase inhibitor was not added (11.7 fmol/30 min in the present experiment compared with 2.6 fmol/30 min in Egawa and co-workers' experimentS). Inhibition of phosphodiesterase activity might also account for the continued cAMP spillover we observed in the second 15-min period following NE infusion (Fig. 1). Use of microdialysis to assess AC activity may represent a more physiological measurement than methods using isolated membranes or brain slices. While implantation of the probe produces acute damage in its immediate vicinity, neuronal projections to this area should largely be preserved, and the brain as a whole will remain intact. Tissue integrity may be an important factor in determining lithium's effects on basal AC activity. For instance, studies using crude synaptosomal fractions or membranes 6,18 have not found alterations in basal AC activity after lithium treatment, while lithium
TABLE I Effect o f chronic lithium treatment on basal c A M P in the dialysate
Lithium-treated animals were fed during at least 4 weeks with a Li2CO3 supplemented chow (serum lithium concentration: 0.85 + 0.05 mM). Basal c A M P (fmol/min )
Control Lithium
14 11
0.47 + 0.06 1.21 + 0.21'
has been shown to raise basal cAMP accumulation in tissue slices 3 and intact glial cells in culture 19. As with these latter studies, basal cAMP levels in dialysate increased significantly after chronic lithium treatment. The mechanism causing increased basal cAMP is unclear. It is possible (albeit unlikely, given the lack of effect in membrane preparations) that lithium increases basal adenylate cyclase activity. Another possibility is that lithium increases extrusion (or decreases clearance) of cAMP into the extracellular fluid. A final possibility is that lithium increases presynaptic release of NE (as has been shown in rat cortex synaptosomes4), leading to increased basal fl-adrenoceptor mediated cAMP formation. The degree of stimulation of AC activity by NE is decreased after lithium, however. While the cAMP concentrations in dialysate after stimulation with 100/~M NE were roughly the same in lithium-treated and control animals, the magnitude of change from baseline (whether expressed as absolute amount of change or as percent change) was significantly less after lithium treatment. This effect of chronic lithium treatment is, for the most part, consistent with earlier studies, both in vitro and ex vivo, demonstrating blunted NE-stimulated AC activity
300 •
Basal
t
~NE
11 A
n
*P < 0.005 in comparisonwith control animals.
1.6 1.4
Treatment
T
1.2
/¢
~200 r,
0.8 0.6 0.4 0.2 0
-
Control
-
i_ Lithium
7r, X <~ 100
Control
Lithium
Fig. 3. Effects of chronic lithium treatment on cAMP response to stimulation with 100 gM NE. A: data expressed as fmol/min. *ANOVAR: FI. 9 = 14.907, P = 0.0038, for the interaction between NE stimulation and lithium treatment. B: data expressed as percentage of the corresponding basal level. Column heights represent the mean + S.E.M. of at least 5 experiments. *P < 0.01 versus control.
336 after lithium t r e a t m e n t 2'3,14. W h i l e absolute c A M P con-
h o w e v e r . R e c e n t studies h a v e d e m o n s t r a t e d that addition
c e n t r a t i o n s after N E stimulation w e r e not d e c r e a s e d after
of M g 2+ does not r e v e r s e the effects of c h r o n i c lithium on
lithium in the p r e s e n t study (unlike p r e v i o u s studies), this
f l - a d r e n e r g i c - l i n k e d A C 11 while G T P d o e s 12. This sug-
was p r o b a b l y due to the increase in basal levels after
gests that c h r o n i c lithium t r e a t m e n t has an additional
lithium t r e a t m e n t . F u r t h e r m o r e , since 100 # M N E is on
inhibitory effect on A C , p e r h a p s
the linear part of the c A M P d o s e - r e s p o n s e curve (Fig.
n u c l e o t i d e p r o t e i n - d e p e n d e n t step.
involving a guanine
2), it m a y be r e a s o n a b l e to c o n c l u d e that the smaller
T h e p r e s e n t study is the first that we are a w a r e of to
r e s p o n s e after lithium t r e a t m e n t r e p r e s e n t s a d e c r e a s e in
d e m o n s t r a t e the effects of c h r o n i c lithium t r e a t m e n t on
f l - a d r e n o c e p t o r s t i m u l a t e d A C activity. T h e r e h a v e b e e n a n u m b e r of investigations into the
a signal t r a n s d u c t i o n system in a living rat brain, and serves as partial v a l i d a t i o n of e a r l i e r investigations that
m e c h a n i s m by which lithium affects brain A C activity.
used less a n a t o m i c a l l y intact p r e p a r a t i o n s . T h e p r e s e n t
A c u t e a d d i t i o n of lithium i n t e r f e r e s with A C by c o m p e t -
findings also s u p p o r t the use of microdialysis as a m e t h o d
ing with M g 2÷ for a r e g u l a t o r y site on the e n z y m e 1°'13.
for assessing signal t r a n s d u c t i o n in vivo. Investigations
The
are u n d e r way to c h a r a c t e r i z e l i t h i u m ' s effects on o t h e r
mechanism
by which
chronic lithium
treatment
a t t e n u a t e s A C r e s p o n s e s a p p e a r s to be m o r e c o m p l e x ,
c o m p o n e n t s of the r e c e p t o r - l i n k e d A C system.
1 Andersen, P.H. and Geisler, A., Lithium inhibition of forskolinstimulated adenylate cyclase, Neuropsychobiology, 12 (1984) 1-3. 2 Dousa, T. and Hechter, O., Lithium and brain adenylate cyclase, Lancet, i (1970) 834-835. 3 Ebstein, R.P., Hermoni, M. and Belmaker, R.H., The effect of lithium on noradrenaline-induced cyclic AMP accumulation in rat brain: inhibition after chronic treatment and absence of supersensitivity, J. Pharmacol. Exp. Ther., 213 (1980) 161-167. 4 Ebstein, R.P., Lerer, B., Shlaufman, M., Belmaker, R.H., The effect of repeated electroconvulsive shock treatment and chronic lithium feeding on the release of norepinephrine from rat cortical vesicular preparations, Cell Mol. Neurobiol., 3 (1983) 191-201. 5 Egawa, M., Hoebel, B.G. and Stone, E.A., Use of microdialysis to measure brain noradrenergic receptor function in vivo, Brain Research, 458 (1988) 303-308. 6 Geisler, A. and Klysner, R., Combined effect of lithium and flupenthixol on striatal adenylate cyclase (letter), Lancet, i (1977) 430. 7 Hsiao, J.K., Ball, B.A., Morrison, P.E, Mefford, I.N. and Bungay, P.M., Effects of different semipermeable membranes on in vitro and in vivo performance of microdialysis probes, J. Neurochem., 54 (1990) 1449-1452. 8 Kendall, D.A. and Nahorski, S.R., Acute and chronic lithium treatments influence agonist and depolarization-stimulated inositol phospholipid hydrolysis in rat cerebral cortex, J. Pharmacol. Exp. Ther., 241 (1987) 1023-1027. 9 Lazareno, S., Marriott, D.B. and Nahorski, S.R., Differential effects of selective and non-selective neuroleptics on intracellular and extracellular cyclic AMP accumulation in rat striatal slices, Brain Research, 361 (1985) 91-98. 10 Mork, A. and Geisler, A., Mode of action of lithium on the catalytic unit of adenylate cyclase from rat brain, Pharmacol. Toxicol., 60 (1987) 241-248. 11 Mork, A. and Geisler, A., The effects of lithium in vitro and ex vivo on adenylate cyclase in brain are exerted by distinct
mechanisms, Neuropharmacology, 28 (1989) 307-311. 12 Mork, A. and Geisler, A., Effects of GTP on hormonestimulated adenylate cyclase activity in cerebral cortex, striatum, and hippocampus from rats treated chronically with lithium, Biol. Psychiatry, 26 (1989) 279-288. 13 Newman, M.E. and Belmaker, R.H., Effects of lithium in vitro and ex vivo on components of the adenylate cyclase system in membranes from the cerebral cortex of the rat, Neuropharmacology, 26 (1987) 211-217. 14 Newman, M.E., Lichtenberg, P. and Belmaker, R.H., Effects of lithium in vitro on noradrenaline-induced cyclic AMP accumulation in rat cortical slices after reserpine-induced supersensitivity, Neuropharmacology, 24 (1985) 353-355. 15 Newman, M.E., Salomon, H. and Lerer, B., Electroconvulsive shock and cyclic AMP signal transduction: effects distal to the receptor, J. Neurochem., 46 (1986) 1667-1669. 16 Newman, M.E., Lipot, M. and Lerer, B., Differential effects of chronic administration of desipramine on the cyclic AMP response in cortical slices and membranes in the rat, Neuropharmacology, 26 (1987) 1127-1130. 17 Paxinos, G. and Watson, C., The rat brain in stereotaxic coordinates, Academic Press, Australia, 1986. 18 Reches, A., Ebstein, R.P. and Belmaker, R.H., The differential effect of lithium on noradrenaline- and dopamine-sensitive accumulation of cyclic AMP in guinea pig brain, Psychopharmacology, 58 (1978) 213-216. 19 Schimmer, B.P., Effects of catecholamines and monovalent cations on adenylate cyclase activity in cultured glial tumor cells, Biochem. Biophys. Acta, 252 (1971) 567-573. 20 Stone, E.A., Egawa, M. and Colbjornsen, C.M., Catecholamine-induced desensitization of brain beta adrenoceptors in vivo and reversal by corticosterone, Life Sci., 44 (1989) 209-213. 21 Wang, H.Y. and Friedman, E., Lithium inhibition of protein kinase C activation-induced serotonin release, Psychopharrnacol. (Berlin), 99 (1989) 213-218.
333
Elsevier BRES 24484
Lithium effects on noradrenergic-linked adenylate cyclase activity in intact rat brain: an in vivo microdialysis study Monica I. Masana, Jose A. Bitran, John K. Hsiao, Ivan N. Mefford and William Z. Potter Section of Clinical Pharmacology, Clinical Neuroscience Branch, National Institute of Mental Health, Bethesda, MD 20892 (U.S.A.)
(Accepted 2 October 1990) Key words: Lithium; Cyclic adenosine monophosphate; Beta-receptor; In vivo microdialysis; Norepinephrine
The effects of chronic lithium treatment on adenylate cyclase activity in intact rat brain were examined using in vivo microdialysis. Basal extracellular cyclic adenosine monophosphate (AMP) increased in a dose-dependent manner after norepinephrine was added to the perfusate. Chronic lithium treatment increased basal brain extracellular fluid cyclic AMP levels, while decreasing the magnitude of the cyclic AMP response to stimulation with 100/~M norepinephrine.
Elucidating lithium's neurochemical mechanism of action has been a major goal of psychopharmacological research. Considerable data have accumulated demonstrating that lithium treatment alters signal transduction mechanisms. For example, lithium attenuates agonist and depolarization-induced phosphoinositide (PI) turnover 8 and recently, phorbol ester-induced protein kinase C translocation has been reported to be inhibited by lithium treatment zt. A particularly enduring line of research, however, has focused on lithium's effects on adenylate cyclase (AC). Dousa and Hechter were the first to demonstrate that lithium, in vitro, inhibited norepinephrine (NE) stimulated formation of 3",5"-cyclic adenosine monophosphate (cAMP) in rat brain homogenate 2. Since that time, lithium treatment has been shown to attenuate fl-adrenoceptor stimulated AC activity in membrane, slice and synaptosomal preparations from rat brain in vitro as well as ex vivo (lithium treatment of an intact animal with subsequent in vitro assay of AC activity) 3. Similarly, the post-receptor stimulation of AC by forskolin has been shown to be inhibited by lithium 1, as is the stimulation of AC via the stimulatory guanine nucleotide binding protein (Gs) 1'13. Previous studies of brain AC activity have been limited to examining lithium's effects in vitro or ex vivo. It has not been possible, as of yet, to assess signal transduction in vivo, in intact animals. Tissue integrity may be an important issue since, in previous studies, the particular preparation used affected the results obtained. For instance, the chronic administration of desipramine 16 or
chronic electroconvulsive shock 15 induces a significant decrease in forskolin-stimulated cyclic AMP production in rat cortical slices, whereas in a cortical membrane preparation from similarly treated animals, forskolinstimulated AC activity was significantly increased. Previous studies have demonstrated that cAMP in extracellular fluid correlated highly with intracellular content after receptor activation of AC in rat brain slices 5"9. Recently, Egawa and co-workers used in vivo microdialysis to sample cAMP from brain extracellular fluid and showed a dose-dependent NE induced increase in cAMP efflux 5. They suggested that microdialysis might be a useful method for assessing /3adrenoceptor linked AC activity in vivo 5'2°. In the present study, we use in vivo microdialysis to demonstrate that in intact animals, chronic lithium treatment increases basal AC activity, while significantly attenuating the cAMP response to NE stimulation in rat prefrontal cortex. Male Sprague-Dawley rats (Taconic Farm) weighing 350-450 g were used. Rats had free access to water and food. The control group was fed ordinary rat chow while animals treated chronically with lithium were fed Li2CO 3 supplemented chow (0.165% by weight) for 8 weeks. Serum lithium levels were determined by flame photometry. Microdialysis probes were fabricated as previously described 7, using a semipermeable polymeric membrane (polyacrylonitrile, 3 mm long, 0.3 mm diameter, 2.83 mm 2 in surface area, 40,000 MW cutoff, Hospal Medical) attached to probe bodies of concentric design. Recoveries
Correspondence: M.I. Masana, Section on Clinical Pharmacology, Clinical Neuroscience Branch, National Institute of Mental Health, Building
10, Room 2D46, Bethesda, MD 20892, U.S.A.
334 in vitro in an unstirred solution at a flow rate of 1/A/min were (mean + S.E.M.) 20.0 _+ 1.6% for cAMP (n = 27) (determined for each probe after each experiment). Rats were anesthetized with chloral hydrate (480 mg/kg i.p.) and probes were implanted using a stereotaxic apparatus into the prefrontal cortex at antero-posterior 1.7 mm from bregma, lateral 1.0 mm, vertical 4.5 mm ~7. The rats were maintained under anesthesia throughout the experiment. The probes were perfused with artificial CSF (concentrations in mM): CaCI 2 1.2, MgCI 2 1.2, KCI 3.0, NaCI 120, sodium phosphate 3.0 (pH adjusted to 7.4), containing 30 /~M of the phosphodiesterase inhibitor, rolipram (donated by Boehringer Ingelheim, Germany), at a flow rate of 1 ktl/min. Samples were collected every 30 min for 2 h and then every 15 min thereafter. After 2.5 h, NE was added to the perfusate for one 15-rain sample, then 4 more 15-min samples were collected. Sample volumes were checked by weighing, after which the samples were placed on ice until c A M P was assayed. Dialysate c A M P concentrations were determined with a commercially available radioimmunoassay kit (125IcAMP, Amersham). Detection limit was 0.5 fmol/tube and basal levels were at least 3 times the detection limit. Basal c A M P was taken as the average c A M P concentration in dialysate for the 1 h prior to NE stimulation. The average of the c A M P levels during the period of NE stimulation and the next 15-rain period was taken as the effect of NE. When propranolol was tested, 1 p M propranolol was added to the perfusate 30 min before NE stimulation and continued throughout the rest of the experiment. Statistical analysis was performed using Student's t-test or analysis of variance of repeated measures ( A N O V A R ) with c A M P levels (basal and NE-stimulated) as a repeated factor and a between group treatment factor (either dose, 1 0 - 6 t o 10 3 M NE, absence
or presence of propranolol, or control versus lithium, depending on the A N O V A R ) , after log transformation of the data to linearize variance. A significant effect of a treatment on the c A M P response to NE stimulation would be evident as a significant interaction between the repeated factor and the treatment factor (e.g. greater increases in c A M P response to NE with greater doses of NE). Concentrations of c A M P were elevated initially, decreasing to a steady basal level after about 1.5 h (see Fig. 1 for a representative experiment). Addition of rolipram, a phosphodiesterase inhibitor, was necessary in order to detect basal c A M P levels. Infusion through the probe of 100 # M NE increased c A M P levels in the 15-rain period of the NE infusion followed by a further increase during the next 15-rain period (Fig. 1.). The c A M P responses to different concentrations of NE are shown in Fig. 2. There was a dose-response relationship up to 1 mM NE (F3. w = 37.717, P < 0,001). Addition of 1 /~M propranolol largely blocked the c A M P response to 100/~M NE (mean _+ S.E.M.: basal 0.45 _+ 0.33, NE 0.67 _+ 0.46 fmol/min, Fl, 4 = 19.524, P = 0.0115, compared to the effect of 100 /~M NE in the absence of propranolol). This result was consistent with a fl-adrenoceptor-mediated response. Chronic treatment of animals with lithium, produced a serum lithium concentration of 0.85 + 0.05 mM. Lithium-treated animals had significantly higher basal c A M P levels as shown in Table I. Eleven of these animals (5 lithium-treated and 6 controls) were tested for NE stimulation and the results are shown in Fig. 3. The absolute magnitude ( A ) of the c A M P response to NE was less in the lithium-treated than in the control animals (F1, 9 = 14.907, P = 0.0038) (Fig. 3A). This was
1.6
o
Basal
¢
I
"
'
1.4
1.2"
1.2
10-
08-
0
E
0.0
O. < u
0.6
04-
0.4 02-
0.2
00 30
60
90
120
TIME (rnin)
150
180
210
240
NE
Fig. 1. cAMP levels in dialysate at various times after implantation of a mierodialysis probe in prefrontal cortex. The horizontal bar indicates when 100 ~M NE was added to perfusate. Data from one representative experiment are shown.
10-7
10 .6
10 "s
NOREPINEPHRINE
I0 "4
10 °3
10 .2
(tool/l)
Fig. 2. Effect of different NE doses, on cAMP release. Open symbols: basal levels, closed symbols: NE-stimulated levels. Results are expressed as fmol/min and each point represents the average of at least 3 experiments (mean + S.E.M).
335 accounted for by an increase in basal levels after lithium treatment, since cAMP levels after stimulation with 100 /~M NE were similar in both groups. Lithium's effects were evident as well when the response to NE was expressed as a percentage of basal levels (mean + S.E.M.: control: 270.6 + 23.4%, lithium 157.9 + 12.4%, P < 0.01, Student's t-test) (Fig. 3B). NE stimulation produced a dose-dependent increase in cAMP content in dialysate that was blocked by the fl-blocker propranolol. These in vivo microdialysis results, in conjunction with previous findings demonstrating close correlations between intracellular and extracellular cAMP content in tissue slices 5'9, suggest that sampling of extracellular cAMP by in vivo microdialysis can index AC activity. The increase in cAMP following NE stimulation found in this study was comparable to that of previous investigators 5, although our basal levels obtained in the presence of rolipram were higher than those in which a phosphodiesterase inhibitor was not added (11.7 fmol/30 min in the present experiment compared with 2.6 fmol/30 min in Egawa and co-workers' experimentS). Inhibition of phosphodiesterase activity might also account for the continued cAMP spillover we observed in the second 15-min period following NE infusion (Fig. 1). Use of microdialysis to assess AC activity may represent a more physiological measurement than methods using isolated membranes or brain slices. While implantation of the probe produces acute damage in its immediate vicinity, neuronal projections to this area should largely be preserved, and the brain as a whole will remain intact. Tissue integrity may be an important factor in determining lithium's effects on basal AC activity. For instance, studies using crude synaptosomal fractions or membranes 6,18 have not found alterations in basal AC activity after lithium treatment, while lithium
TABLE I Effect o f chronic lithium treatment on basal c A M P in the dialysate
Lithium-treated animals were fed during at least 4 weeks with a Li2CO3 supplemented chow (serum lithium concentration: 0.85 + 0.05 mM). Basal c A M P (fmol/min )
Control Lithium
14 11
0.47 + 0.06 1.21 + 0.21'
has been shown to raise basal cAMP accumulation in tissue slices 3 and intact glial cells in culture 19. As with these latter studies, basal cAMP levels in dialysate increased significantly after chronic lithium treatment. The mechanism causing increased basal cAMP is unclear. It is possible (albeit unlikely, given the lack of effect in membrane preparations) that lithium increases basal adenylate cyclase activity. Another possibility is that lithium increases extrusion (or decreases clearance) of cAMP into the extracellular fluid. A final possibility is that lithium increases presynaptic release of NE (as has been shown in rat cortex synaptosomes4), leading to increased basal fl-adrenoceptor mediated cAMP formation. The degree of stimulation of AC activity by NE is decreased after lithium, however. While the cAMP concentrations in dialysate after stimulation with 100/~M NE were roughly the same in lithium-treated and control animals, the magnitude of change from baseline (whether expressed as absolute amount of change or as percent change) was significantly less after lithium treatment. This effect of chronic lithium treatment is, for the most part, consistent with earlier studies, both in vitro and ex vivo, demonstrating blunted NE-stimulated AC activity
300 •
Basal
t
~NE
11 A
n
*P < 0.005 in comparisonwith control animals.
1.6 1.4
Treatment
T
1.2
/¢
~200 r,
0.8 0.6 0.4 0.2 0
-
Control
-
i_ Lithium
7r, X <~ 100
Control
Lithium
Fig. 3. Effects of chronic lithium treatment on cAMP response to stimulation with 100 gM NE. A: data expressed as fmol/min. *ANOVAR: FI. 9 = 14.907, P = 0.0038, for the interaction between NE stimulation and lithium treatment. B: data expressed as percentage of the corresponding basal level. Column heights represent the mean + S.E.M. of at least 5 experiments. *P < 0.01 versus control.
336 after lithium t r e a t m e n t 2'3,14. W h i l e absolute c A M P con-
h o w e v e r . R e c e n t studies h a v e d e m o n s t r a t e d that addition
c e n t r a t i o n s after N E stimulation w e r e not d e c r e a s e d after
of M g 2+ does not r e v e r s e the effects of c h r o n i c lithium on
lithium in the p r e s e n t study (unlike p r e v i o u s studies), this
f l - a d r e n e r g i c - l i n k e d A C 11 while G T P d o e s 12. This sug-
was p r o b a b l y due to the increase in basal levels after
gests that c h r o n i c lithium t r e a t m e n t has an additional
lithium t r e a t m e n t . F u r t h e r m o r e , since 100 # M N E is on
inhibitory effect on A C , p e r h a p s
the linear part of the c A M P d o s e - r e s p o n s e curve (Fig.
n u c l e o t i d e p r o t e i n - d e p e n d e n t step.
involving a guanine
2), it m a y be r e a s o n a b l e to c o n c l u d e that the smaller
T h e p r e s e n t study is the first that we are a w a r e of to
r e s p o n s e after lithium t r e a t m e n t r e p r e s e n t s a d e c r e a s e in
d e m o n s t r a t e the effects of c h r o n i c lithium t r e a t m e n t on
f l - a d r e n o c e p t o r s t i m u l a t e d A C activity. T h e r e h a v e b e e n a n u m b e r of investigations into the
a signal t r a n s d u c t i o n system in a living rat brain, and serves as partial v a l i d a t i o n of e a r l i e r investigations that
m e c h a n i s m by which lithium affects brain A C activity.
used less a n a t o m i c a l l y intact p r e p a r a t i o n s . T h e p r e s e n t
A c u t e a d d i t i o n of lithium i n t e r f e r e s with A C by c o m p e t -
findings also s u p p o r t the use of microdialysis as a m e t h o d
ing with M g 2÷ for a r e g u l a t o r y site on the e n z y m e 1°'13.
for assessing signal t r a n s d u c t i o n in vivo. Investigations
The
are u n d e r way to c h a r a c t e r i z e l i t h i u m ' s effects on o t h e r
mechanism
by which
chronic lithium
treatment
a t t e n u a t e s A C r e s p o n s e s a p p e a r s to be m o r e c o m p l e x ,
c o m p o n e n t s of the r e c e p t o r - l i n k e d A C system.
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