of new inositol analogs that could expand psychoactive drug development possibilities via second messenger manipulation. Keywords: Inositol, PI cycle, anxiety ...
J Neural Transm (1997) 104:299-305
_Journal of_ Neural Transmission 9 Springer-Verlag 1997 Printed in Austria
Inositol has behavioral effects with adaptation after chronic administration H. Cohen, M. Kotler, Z. Kaplan, M. A. Matar, O. Kofman, and R. H. Belmaker Ministry of Health Mental Health Center, Faculty of Health Sciences, Ben Gurion University of the Negev, Beersheva, Israel Accepted October 25, 1996
Summary. Inositol is a simple dietary polyol that serves as a precursor in important second messenger systems. Inositol in pharmacological doses has been reported recently to be therapeutic in depression, panic disorder and obsessive compulsive disorder. W e hereby report effects of inositol in the elevated plus maze model of anxiety. These results should allow development of new inositol analogs that could expand psychoactive drug development possibilities via second messenger manipulation. Keywords: Inositol, PI cycle, anxiety, animal model. Introduction Inositol is a simple polyol precursor of phosphatidylinositol (PI). PI is a key juncture of intracellular second messenger generation in response to several important brain neurotransmitters (Baraban et al., 1989). The enzyme which generates PI from inositol (and cytidine monophosphate phosphatidic acid (CMP-PA)) has been reported to be unsaturated at physiological inositol concentrations (Ghalayini et al., 1985). Lithium (Li) reduces brain inositol by inhibiting inositol-monophosphatase, the enzyme required for regeneration of inositol from inositol-triphosphate (IP3) degradation products or from glucose. The role of this Li-induced reduction of inositol in lithium's mechanism of action is controversial (Jope et al., 1994). Some groups have found that Li-induced reduction of inositol does not affect PI turnover, whereas others have reported effects of inositol to reverse lithium's effects on behavior (Kofman et al., 1993a). The dose of inositol necessary to reverse lithium effects in animals, in vitro or in vivo, is high (Again et al., 1994; Kofman et al., 1993). Effects of inositol on human behavior were first studied by Levine et al. (1995a) who found antidepressant effects of 12gm daily inositol over four weeks in a double-blind controlled trial. Benjamin etal. (1995) reported anti-
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panic effects in a d o u b l e blind c o n t r o l l e d trial with 1 2 g m of inositol administ e r e d o v e r f o u r weeks. Fux et al. (1996) f o u n d that 18 g m inositol a d m i n i s t e r e d daily o v e r six w e e k s significantly r e d u c e d s y m p t o m s of obsessive-compulsive disorder. N o t h e r a p e u t i c effects of inositol a d m i n i s t r a t i o n w e r e f o u n d in s c h i z o p h r e n i a or a t t e n t i o n deficit h y p e r a c t i v i t y d i s o r d e r ( A D H D ) (Levine et al., 1994, 1995b; B e l m a k e r et al., 1995). Inositol has b e e n n o t e d as a f o l k - r e m e d y for anxiety a n d d e p r e s s i o n in the British p h a r m a c o p o e i a ( R e y n o l d s et al., 1993). Inositol has also b e e n r e p o r t e d to be t h e r a p e u t i c in r e s p i r a t o r y distress s y n d r o m e ( R D S ) of infants at doses of 1 2 0 - 1 6 0 m g / k g daily ( H a l l m a n et al., 1986, 1992), w h e r e it was s p e c u l a t e d that it a u g m e n t s synthesis of p h o s p h a t i d y l c h o l i n e ( H a l l m a n et al., 1984). Inositol s u p p l e m e n t a t i o n to p r e v e n t or r e v e r s e diabetic n e u r o p a t h y is c o n t r o v e r s i a l ( A d e n d r u p et al., 1989; G r e e n e et al., 1981); t h e m e c h a n i s m is speculatively via its effects o n p r o t e i n kinase C ( P K C ) ( K i m et al., 1991). P r e l i m i n a r y d a t a of effects of o n e dose of acute inositol o n rat b e h a v i o r w e r e published ( C o h e n et al., 1996). W e n o w verify a n d e x t e n d these results with a d o s e - r e s p o n s e curve i n t r a p e r i t o n e a l (i.p) a n d to i n t r a c e r e b r o v e n tricular (icv) and c h r o n i c effects. Material and methods Adult male Sprague-Dawley rats weighing 200-250gm were habituated to the housing conditions for at least ten days. During that time the rats were handled once daily. Handling consisted of picking the rats up with a gloved hand. The animals were housed 4 per cage in an animal room with stable temperature and reversed 12 hour light/dark cycle with ad libitum food and water. All testing was performed during the dark phase using a dim light. All inositol given was myo-inositol (Sigma) except when noted to be L-chiroinositol, the biological inactive stereoisomer. Behavioral testing was conducted in an elevated plus-maze made of Perspex (Stout et al., 1994; File, 1993). This apparatus consists of two open arms and two enclosed arms, with an open roof. The apparatus is configured such that the two open arms are opposite one other. The maze is elevated to 0.5 meter. Rats were placed individually in the central platform, facing different arms on different days in randomized order, for 6 min sessions recorded on video camera. Ratings were made by reviewing the video recordings by observers blind to the original treatment conditions. The parameters rated were: (1) time spent in the open versus closed arms; (2) the number of entries into each arm; (3) time spent in the central platform. Experiment I - Acute ip dose response curve
One hundred eighty four adult Sprague-Dawley rats were used in these experiments. Inositol (10% solution in 0.9% saline) was injected intraperitoneally at doses from 2.5 gm/ kg to 0.03 gm/kg. Glucose plus mannitol in a ratio of 1 : 3 was used as control. Behavior was measured six hours after ip inositol as in Agam et al. (1994). Each dose was run on a separate day vs control and a positive control of CCK-4 (50 ~g/kg) ip was run simultaneously at each dose. There were about ten rats in each group on each experimental day. For data analysis the control data were pooled (Fig. 1). Experiment H - Chronic intraperitoneal (ip) inositol
Thirty adult male rats were randomly divided into three groups receiving a daily ip injection of: (1) inositol 1.25 gm/kg over 14 days; (2) glucose-mannitol 1.25 gm/kg (at ratio
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Fig. 1. The effect of decreasing doses of acute ip inositol on time spent in the open and closed arms. Time spent in the open arms, one way ANOVA: F = 8.923, p = 0.000. Post Hoc LSD Tests: ,1p = 0.0035; ,2p = 0.005; ,3p = 0.00008; ,4p = 0 . 0 0 0 0 ; * s p = 0.002. Time spent in the closed arms, one way ANOVA: F = 10.51, p = 0.000. Post Hoc LSD Tests: #6p = 0.003; #7p = 0.0008; #sp = 0.0000; #gp = 0.0000; #10p = 0.002
1:3) over 14 days; (3) inositol 1.25gm/kg over 13 days + glucose-mannitol on the 14th day. After 14 days, behavior of the different groups was tested in the elevated plus maze. Rats were tested two hours after the last injection. The dose of chronic inositol was based on preliminary data (Kofman et al., 1993). The glucose-mannitol control was chosen to control for inositol's caloric value (1/3 of glucose) and osmotic effects.
Experiment III - Intracerebroventricular (icy) inositol Thirty adult male rats were implanted with guide cannulae unilaterally in the lateral ventricle using standard stereotaxic procedures under pentobarbital anesthesia (Kofman et al., 1993b). Coordinates for the cannula placement were 0.8ram posterior to bregma, 1.4mm lateral to midline, and 5.0ram below skull surface. At least 1 week after recovery from surgery the rats were randomly divided into three groups receiving an icv injection of: (1) myo-inositol 2.5mg in 40~tl artificial cerebrospinal fluid (CSF); (2) myo-inositol 5.0 mg in 40 ~tl artificial CSF; (3) L-chiro-inositol 5.0rag in 40 ~tl artificial CSF. Two hours following the icv injection the rats were tested in the elevated plus maze. The time point was chosen based on previous data (Bersudsky et al., 1993).
Statistical analyses Statistical analyses were performed using the SPSS software program. One way Analysis of Variance (ANOVA) was performed for between-group differences on time spent in the open versus closed arms, the number of entries into each arm, the ratio between those parameters and time spent in the central platform. Post Hoc Least Significant Difference (LSD) (p < 0.05) tests indicated that there was a significant difference between groups.
Results A c u t e ip i n o s i t o l (Fig. 1) d e c r e a s e d t i m e s p e n t in t h e o p e n a r m s o f a n e l e v a t e d plus m a z e a n d i n c r e a s e d t i m e s p e n t in t h e c l o s e d a r m s , c o m p a r e d w i t h a
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Time spent in the open arms (rain) 1 Time spent in the closed arms (min) 2 Number of entries to the open arms 3 Number of entries to the closed arms 4 Time spent in the central platform 5
myo-inositol 14 days (N = 10)
Control 14 days (N = 10)
I
II
myo-inositol 13 days + control 1 day ( N = 10) III
Post-hoc LSD tests
1.3 +_ 0.3
0.4 _ 0.2
1.0 • 0.12
I r
4.55 _+ 0.3
5.6 + 0.2
5.0 + 0.13
I r
3.0 + 0.6
1.5 +_ 0.6
4.9 + 0.6
13.3 _+ 1.53
7.0 _+ 1.63
9.1 • 0.8
I r II r I r I r I r IIr
0.16 • 0.035
0.01 • 0.007
0.09 • 0.03
III III III III
Results are expressed as mean + SEM. One way ANOVA: 1F = 4.24, P = 0.025; 2F = 5.63, P = 0.009; 3F = 7.73, P = 0.0002; 4F = 5.47, P = 0.01; 5F = 8.38, P = 0.0015
m i x t u r e of 1/3 glucose and 2/3 m a n n i t o l to control for b o t h osmotic and f o o d value effects of inositol. T h e g l u c o s e - m a n n i t o l m i x t u r e alone h a d no effects vs saline (results n o t shown). T h e inositol effects w e r e similar to a "positive c o n t r o l " of C C K - 4 50 ~g/kg, a k n o w n anxiogenic t r e a t m e n t , that was r u n in e a c h e x p e r i m e n t (Crawley, 1994). R e v i e w of video recordings of rat b e h a v i o r by an e x p e r i e n c e d b e h a v i o r a l p h a r m a c o l o g i s t (O.K.) did not r e v e a l o t h e r b e h a v i o r s t h a t could explain t h e results via a m e c h a n i s m o t h e r t h a n anxiety. T h e effect on t i m e in o p e n and closed arms is significant at doses as low as 0.2 gm/kg. T h e r e w e r e n o significant effects on the p a r a m e t e r s of entries to t h e o p e n or closed arms or time spent in the central platform. Daily ip inositol (Table 1) for 14 days c a u s e d a reversal o f the b e h a v i o r c a u s e d by a single dose o f ip inositol. T i m e in closed arms was significantly r e d u c e d and t i m e in o p e n arms significantly i n c r e a s e d (see Table 1). N u m b e r s of entries into o p e n arms was i n c r e a s e d n o n significantly but n u m b e r of entries (as o p p o s e d to time spent) into the closed arms was significantly i n c r e a s e d (see T a b l e 1), suggesting p e r h a p s a stimulant as well as anxiolytic effect. A third g r o u p of animals was t r e a t e d with myo-inositol for 13 days and g l u c o s e - m a n n i t o l on the 14th day, to e v a l u a t e w i t h d r a w a l f r o m inositol effects. T r e n d s to r e d u c e d t i m e in closed arms and i n c r e a s e d t i m e in o p e n arms w e r e n o t significant, as was the t r e n d t o w a r d i n c r e a s e d n u m b e r of entries into closed arms. H o w e v e r , n u m b e r of entries into o p e n arms was still significantly increased. T i m e spent in the central p l a t f o r m was also significantly increased, a l t h o u g h this was of small m a g n i t u d e and u n c l e a r m e a n i n g . Icy inositol (Table 2) was studied to d e t e r m i n e the central n a t u r e of m y o inositol effects and stereospecificity using as c o n t r o l chiro-inositol, a biologically inactive i s o m e r which is n o t available in quantities n e c e s s a r y for ip use.
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Inositol has behavioral effects with adaptation Table 2. ICV inositol
myo-inositol 2.5 mg in 40 gl artificial CSF (N = 10) II
Chiro-inositol 5 mg in 40 ~tl artificial CSF (N = 10) III
Post-hoc LSD tests
1.3 _+ 0.2
0.48 _+ 0.16
0.37 -+ 0.14
4.6 + 0.2
5.4 + 0.2
5.6 -+ 0.15
4.4 _+ 0.8
2.1 _+ 0.7
2.6 _+ 1.0
I r I r I r I r NS
10.4 + 0.9
8.9 _+ 1.3
6.5 + 1.2
NS
0.08 _+ 0.05
0.03 --_ 0.02
NS
myo-inositol 5 mg in 40 gl artificial CSF (N = 10) I Time spent in the open arms (min) 1 Time spent in the closed arms (min)2 Number of entries to the open arms3 Number of entries to the closed arms4 Time spent in the central platform5
0.1 + 0.04
II III I1 III
Results are expressed as mean _+ SEM. One way ANOVA: ~F = 8.07, P = 0.0017; 2F = 8.81, P = 0.001; 3NS; 4F = NS; 5F = NS
Icv myo-inositol at doses of 5 m g but not 2.5mg had acute anxiolytic effects similar to the effects of chronic ip inositol. Discussion
The present results suggest that inositol has central, stereospecific effects on behavior that adapt with chronic treatment. The acute central effect is opposite to the acute peripheral effect, suggesting that different brain areas m a y be exposed to inositol acutely administered icv than ip. Differences in accessibility of brain areas after ip inositol have b e e n d o c u m e n t e d (Patishi et al., 1996). A d a p t a t i o n of behavioral effects after chronic t r e a t m e n t is characteristic of m a n y psychoactive compounds, and m a y be due to receptor down-regulation or other processes. In humans, clomipramine has antipanic efficiency after chronic t r e a t m e n t but may cause anxiety acutely (Ramos et al., 1993). Given inositol's role as a second messenger precursor, possible mechanisms of adaptation include PKC down-regulation (Manji et al., 1996) or G protein down regulation (Warsh et al., 1996). It is important to note that the acute effect of ip inositol does not disappear with time but changes direction. Like m a n y antidepressants in humans which show increased "activity" effects in some animal models of depression, inositol is also therapeutic in h u m a n panic disorder and would be expected to show "sedative" effects u n d e r certain o t h e r conditions. It is also possible that acute ip inositol is anxiogenic because of peripheral effects, similar to intravenous CCK-4, which m a y have gastrointestinal effects (Rodriguez et al., 1994). As this effect of inositol tolerates out, the central anxiolytic effect b e c o m e apparent ip, as it is apparent icy even acutely.
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Present efforts at drug d e v e l o p m e n t based on second m e s s e n g e r m a n i p u lation are increasingly promising. T h e lowest active dose of inositol in the p r e s e n t e x p e r i m e n t (0.2gm/kg) agrees well with doses effective in h u m a n panic disorder, 1 2 g m p e r day ( B e n j a m i n et al., 1995). Inositol administration m a y indirectly regulate p h o s p h o l i p a s e C activity (Batty et al., 1995) and can reverse n e u r o t r a n s m i t t e r i n d u c e d desensitization ( R a h m a n et al., 1993).
Acknowledgments L-chiro-Inositol was the generous gift of Prof. W. Sherman and Prof. L. Anderson. Supported by a Germany-Israel Foundation Grant (O.K. and R.H.B.), and an Institute for Psychobiology in Israel Grant (Z.K. and H.C.).
References Arendrup K, Gregersen G, Hawley J, Hawthorne JN (1989) High-dose dietary myoinositol supplementation does not alter the ischemia phenomenon in human diabetics. Acta Neurol Scand 80:99-102 Agam G, Shapiro J, Bersudsky Y, Kofman O, Belmaker RH (1994) High-dose peripheral inositol raises brain inositol levels and reverses behavioral effects of inositol depletion by lithium. Pharmacol Biochem Behav 49:341-343 Baraban JM, Worley PF, Snyder SH (1989) Second messenger systems and psychoactive drug actioni focus on the phosphoinositol system and lithium. Am J Psychiatry 146: 1251-1260 Batty HI, Downes CP (1995) The mechanism of muscarinic receptor-stimulated phosphatidylinositol resynthesis in 1321N1 astrocytoma cells and its inhibition by Li +. J Neurochem 65(5): 2279-2289 Belmaker RH, Bersudsky Y, Benjamin J, Agam G, Levine J, Kofman O (1995) Manipulation of inositol-linked second messenger systems as a therapeutic strategy in psychiatry. In: Gessa G, Fratta W, Pani L, Serra G (eds) Depression and mania. From neurobiology to treatment. Raven Press, New York Benjamin J, Levine J, Fux M, Aviv A, Levy D, Belmaker RH (1995) Inositol treatment for panic disorder: a double-blind placebo-controlled crossover trial. Am J Psychiatry 152:1081-1086 Bersudsky Y, Vinnitsky I, Grisaru N, Kofman O, Belmaker RH (1993) Dose-response and time-curve of inositol prevention of Li-pilocarpine seizures. Eur Neuropsychopharmacol 2 [Special issue]: 428-429 Cohen H, Bar-Haim N, Kotler M (1996) Acute inositol induces anxiety in rats. Biol Psychiatry 40:426-427 Crawley JN (1994) Interactions between cholecystokinin and other neurotransmitter systems. In: Bradwejn J (ed) Cholecystokinin and anxiety. From neuron to behavior. Biomedical Publishers, CRC Press File SE (1993) The interplay of learning and anxiety in the elevated plus maze. Behav Brain Res 58(1-2): 199-202 Fux M, Levine J, Aviv A, Belmaker RH (1966) Inositol treatment of obsessivecompulsive disorder. Am J Psychiatry 153:1219-1221 Ghalayini A, Eichberg J (1985) Purification of phosphatidylinositol synthetase from rat brain by CDP-diacylglycerol affinity chromatography and properties of the purified enzyme. J Neurochem 44:175-182 Greene DA, Brown MJ, Braunstein SN, Schwartz SS, Asbury AA, Winegrad AI (1981) Comparison of clinical course and sequential electrophysiological tests in diabetics with symptomatic polyneuropathy and its implications for clinical trials. Diabetes 30: 139-147
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Hallman M (1984) Effect of extracellular myo-inositol on surfactant phospholipid synthesis in the fetal rabbit lung. Biochim Biophys Acta 795:67-78 Hallman M, Jarvenpaa AL, Pohjavuori (1986) Respiratory distress syndrome and inositol supplementation in preterm infants. Arch Dis Childhood 61:1076-1083 Halhnan M, Bry K, Hoppu K, Lapp M, Pohjavuori M (1992) Inositol supplementation in premature infants with respiratory distress syndrome. N Engl J Med 326:1233-1239 Jope RS, Williams MB (1994) Lithium and brain signal transduction systems. Biochem Pharmacol 47:429-434 Kim J, Kyriazi H, Greene DA (1991) Normalization of NA+-K+-ATPase activity in isolated membrane fraction from sciatic nerves of streptozocin-induced diabetic rats by dietary myo-inositol supplementation in vivo or protein kinase C agonists in vitro. Diabetes 40:558-567 Kofman O, Sherman WR, Katz V, Belmaker RH (1993) Restoration of brain myoinositol levels in rats increases latency to lithium-pilocarpine seizures. Psychopharmacology 110:229-234 Kofman O, Belmaker RH (1993a) Biochemical, behavioral and clinical studies of the role of inositol in lithium treatment and depression. Biol Psychiatry 34:839-852 Kofman O, Bersudsky Y, Vinnitsky I, Alpert C, Belmaker RH (1993b) The effect of peripheral inositol injection on rat motor activity models of depression. Israel J Med Sci 29:580-586 Levine J, Goldberger I, Rapaport A, Schwarts M, Schilds C, Elizur A, Belmaker RH, Shapiro J, Again G (1994) CSF inositol levels in schizophrenia are unchanged and inositol is not therapeutic in anergic schizophrenia. Eur Neuropsychopharmacol 4: 487-490 Levine J, Barak Y, Gonsalves M, Szor H, Elizur A, Kofman O, Belmaker RH (1995a) A double-blind controlled trial of inositol treatment of depression. Am J Psychiatry 152: 792-794 Levine J, Ring A, Barak Y, Elizur A, Belmaker RH (1995b) Inositol may worsen attention deficit disorder with hyperactivity. Hum Psychopharmacol 10:481-484 Manji HK, Bersudsky B, Chen G, Belmaker RH, Potter WZ (1996) Modulation of protein kinase C isozymes and substrates by lithium: the role of myo-inositol. Neuropsychopharmacology 15:370-381 Patishi Y, Lubrich B, Berge M, Kofman O, Van Calker D, Belmaker RH (1996) Differential uptake of myo-inositol in vivo into rat brain areas. Eur Neuropsychopharmacol 6:73-75 Rahman S, Neuman RS (1993) Myo-inositol reduces serotonin (5-HTz) receptor induced homologous and heterologous desensitization. Brain Res 631:349-351 Ramos RT, Gentil V, Gorenstein C (1993) Clomipramine and initial worsening in panic disorder: beyond the "jitteriness syndrome". J Psychopharmacol 7(3): 265-269 Reynolds JEF (1993) Martindale: the extra pharmacopoeia, 30th ed. The Pharmaceutical Press, London Rodriguez SA, Martin MT, Fernandez E, Gonalons E (1994) Cecocolonic motility in the chicken. Effects of cholecystokinin. Life Sci 55(22): 1743-1755 Stout JC, Weiss JM (1994) An animal model for measuring behavioral responses to anxiogenic and anxiolytic manipulation. Pharmacol Biochem Behav 47(3): 459-465 Warsh JJ, Li PP (1996) Second messenger systems and mood disorders. Curr Opin Psychiatry 9:23-29 Authors' address: Prof. R. H. Belmaker, M.D., Beer-Sheva Mental Health Center, Ben-Gurion University of the Negev, P.O. Box 4600, Beer-Sheva, Israel Received July 30, 1996
