Acetylcholinesterase inhibitor pretreatment alters stress-induced expression of acetylcholinesterase transcripts in the mouse brain.

The role of the rare soluble splice variant of acetylcholinesterase (AChE-R) in anxiety behavior was assayed using the elevated plus maze (EPM). The effects of pretreatment with restraint stress and the acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP) were tested, as these treatments are known to enhance the expression of AChE-R in several brain regions. Mice from the CD-1 outbred and C57BL/6 inbred strains were randomly assigned to seven treatment groups: homecage control, elevated plus maze without pretreatment, 3 days restraint stress or 3 days pretreatment with saline or one of three doses of DFP, for a total of 14 groups. All mice, except homecage controls, were tested twice on the elevated plus maze. AChE-R transcript expression was increased following elevated plus maze stress in hippocampus and amygdala, but not in the prefrontal cortex of CD-1 and not in C57BL/6 mice. Saline-injected C57BL/6 mice had reduced expression of AChE-R transcripts compared to untreated C57 BL/6 mice. DFP pretreatment reversed the stress-induced changes, increased AChE-R transcripts in CD-1 mice. AChE-R expression in the striatum and amygdala were positively correlated with anxiety in the EPM.

Critical role of the embryonic mid-hindbrain organizer in the behavioral response to amphetamine and methylphenidate.

The embryonic mid-hindbrain organizer, which is composed of a transient cell population in the brainstem, controls the development of dopaminergic and serotonergic neurons. Different genes determining the position and activity of this embryonic structure have been implicated in dopamine- and serotonin-associated disorders. Mouse mutants with a caudally shifted mid-hindbrain organizer, are hyperactive, show increased numbers of dopaminergic neurons and a reduction in serotonergic cells. In the present study we used these mutants to gain insights into the genetic and developmental mechanisms underlying motor activity and the response to psychostimulants. To this end, we studied the motor activity of these animals after exposure to methylphenidate and amphetamine and characterized their dopaminergic and serotonergic innervation. Saline-treated mutants showed increased locomotion, more stereotypic behavior and a decrease in rearing compared to wild-type mice. This baseline level of activity was similar to behaviors observed in wild-type animals treated with high doses of psychostimulants. In mutants methylphenidate (5 or 30 mg/kg) or amphetamine (2 or 4 mg/kg) did not further increase activity or even caused a decrease of locomotor activity, in contrast to wild-type mice. Fluoxetine (5 or 10 mg/kg) reduced hyperactivity of mutants to levels observed in wild-types. Transmitter measurements, dopamine and serotonin transporter binding assays and autoradiography, indicated a subtle increase in striatal dopaminergic innervation and a marked general decrease of serotonergic innervation in mutants. Taken together, our data suggest that mice with an aberrantly positioned mid-hindbrain organizer show altered sensitivity to psychostimulants and that an increase of serotonergic neurotransmission reverses their hyperactivity. We conclude that the mid-hindbrain organizer, by orchestrating the formation of dopaminergic and serotonergic neurons, is an essential developmental parameter of locomotor activity and psychostimulant response.

The anxiolytic effect of chronic inositol depends on the baseline level of anxiety.

Inositol, a precursor for membrane phosphoinositides involved in signal transduction, has been found to be clinically effective in a number of psychiatric disorders and to reverse behavioural effects of lithium. To gain insight into the mechanism of action of inositol, it is critical to establish its efficacy in animal models. Following the initial report by Cohen et al. (1997b) that inositol was anxiolytic in the elevated plus maze model of anxiety, the effect of chronic intraperitoneal and chronic dietary inositol administration in rats was tested in four experiments. There was a significant increase in closed arm and total arm entries following chronic injection of inositol, but no effect of inositol when it was given chronically in rat chow. Because the first 2 experiments suggested that the mode of drug administration affected the control levels of anxiety (open arm entries and time in open arms) in control groups, the effect of chronic dietary inositol was tested in rats that were exposed to a mild and a more severe form of stress. Chronic saline injections elevated anxiety in the plus maze, which was only marginally affected by chronic dietary inositol. Following 3 weeks administration of 5% dietary inositol rats were pre-exposed to a cat. There was a clear increase in number of entries into open arms, suggesting an anxiolytic effect of inositol.

Interactions of lithium and drugs that affect signal transduction on behaviour in rats.

The therapeutic mechanism of the action of lithium in the treatment of bipolar affective disorder is not known, in spite of a burgeoning number of biochemical studies linking lithium to signal transduction processes. This article reviews a decade of studies examining the behavioural manifestations of manipulating inositol, cyclic adenosine monophosphate (cAMP) and G proteins in rats. Inositol, forskolin, dibutyryl cAMP and pertussis toxin all interacted with lithium when rearing behavior was measured. Lithium potentiated the increase in locomotion induced by injections of cholera toxin into the nucleus accumbens, consistent with the hypothesis that it inactivates inhibitory G proteins. More specific interactions were found between lithium and inositol following cholinergic and serotonergic stimulation. Inositol, but not forskolin, attenuated lithium-pilocarpine seizures and the enhancement of the serotonin syndrome; however, inositol had no effect on lithium-induced attenuation of wet dog shakes following an injection of 5-hydroxytryptophan. Behavioural evidence supports biochemical findings suggesting that lithium's interactions with the phoshphatidyl inositol and cyclic AMP signal transduction systems may be relevant to its therapeutic effects in bipolar disorder. Further research on more specific behaviours may elucidate the relevant pharmacological mechanisms underlying the therapeutic effect of lithium.

Chronic dietary inositol enhances locomotor activity and brain inositol levels in rats.

myo-Inositol has been found to be clinically effective in depression, obsessive compulsive disorder (OCD) and panic disorder when given chronically per os. The present study examined the effects of chronic dietary inositol in rats on locomotor behaviour. Regional brain levels of inositol were analyzed by gas chromatography. Chronic dietary inositol significantly enhanced locomotion and rearing in rats and elevated inositol levels by 36% in the cortex and 27% in hippocampus. No differences in inositol levels were found in the striatum or cerebellum. The stimulatory effects of inositol may be related to its effects as an atypical antidepressant in depressed patients.

Behavioral reversal of lithium effects by four inositol isomers correlates perfectly with biochemical effects on the PI cycle: depletion by chronic lithium of brain inositol is specific to hypothalamus, and inositol levels may be abnormal in postmortem brain from bipolar patients.

The inositol depletion hypothesis of lithium (Li) action has been criticized, because depletion of inositol after chronic Li treatment has not been reproducible, effects of inositol to reverse Li-induced behaviors occurred also with epi-inositol, a unnatural isomer, and because inositol is ubiquitous in brain and hard to relate to the pathogenesis of affective disorder. Therefore, we review our studies showing that lithium depletion of brain inositol occurs chronically in the hypothalamus, a region not previously examined; that behavioral effects of four different inositol isomers including epi-inositol correlate perfectly with their biochemical effects; and that inositol in postmortem human brain is reduced by 25% in frontal cortex of bipolars and suicides as compared with controls. Because inositol in postmortem brain is reduced and not increased in bipolar patients, the relationship between inositol, lithium, and affective disorder is complex.

Augmentation of lithium's behavioral effect by inositol uptake inhibitors.

Lithium inhibits the enzyme inositol monophosphatase and thus obstructs the enzymatic degradation of inositol triphosphate (IP3) to inositol in the phosphate-phosphoinositide (PIP) cycle. This inhibition may result in reduced availability of the second messengers IP3 and DAG that are derivates of the PIP cycle, and this action is currently a leading hypothesis regarding lithium's therapeutic and prophylactic effect in affective disorders. Inositol is also available to the cell by uptake from the intercellular matrix, and therefore it is possible that compounds that block the uptake may have lithium-like effects. To test this hypothesis, the present study evaluates the effects of two inositol uptake inhibitors, the carbohydrate L-fucose and the cyclodepsipeptide nordidemnin, in a behavioral model of pilocarpine-induced seizures known to be enhanced by lithium. We tested the possibility that L-fucose produces lithium-like effects, or that L-fucose or nordidemnin augment lithium's behavioral effects. Results indicate that acute ICV treatment with L-fucose did not by itself have a lithium-like effect in the behavioral model, but significantly augmented lithium's effect when combined with lithium treatment. Nordidemnin treatment showed similar effects. The results suggest that when inositol monophosphatase is inhibited by lithium, further restriction of cellular inositol availability may result in an augmentation of lithium's behavioral effects. It is possible that such manipulations may be applicable in the treatment of patients with affective disorders, especially patients who are poor responders to lithium monotherapy.

Lithium, but not carbamazepine, potentiates hyperactivity induced by intra-accumbens cholera toxin.

Elevated G protein abundance and/or function has been implicated in the pathophysiology and pharmacotherapy of bipolar affective disorder. To test the interactions between chronic lithium and carbamazepine on behavioral changes induced by cholera toxin (CTX), which catalyzes ADP-ribosylation and constitutively activates G alphas/olf, rats were given chronic dietary lithium, carbamazepine (CBZ), or regular food (REG) and injected bilaterally in the nucleus accumbens (nACC) with CTX (400 ng/ml/side) or vehicle. Locomotor activity was tested daily for 2 weeks after the injection. CTX increased locomotor activity, but a significant interaction between drug treatment and CTX reflected a two- to threefold increase of CTX-induced hyperactivity in the lithium-treated group. In contrast, on day 1, the CBZ-CTX group was significantly more active than the the LI-CTX and REG-CTX groups, both of which had suppressed locomotor activity. There was a significant reduction in CTX-catalyzed ADP ribosylation of G alphas (52 kDa and 45 kDa) in the nucleus accumbens in all three CTX-treated groups. The potentiation of the behavioral effect of CTX by lithium supports the hypothesis that lithium interacts with G proteins; however, the mechanism of interaction appears to be more complex than direct attenuation of G alphas function, as previously suggested.

Lithium-induced inositol depletion in rat brain after chronic treatment is restricted to the hypothalamus.

The influence of acute and chronic lithium administration on inositol content was examined in five different regions of the rat brain: caudate, cerebellum, cortex, hippocampus and hypothalamus. After acute administration of lithium at doses of 3, 6 or 10 mEq kg-1, no significant reductions of inositol were found in any brain region. Also no significant changes were observed in cortex, caudate, hippocampus and cerebellum after chronic treatment with lithium-containing diet, which led to brain concentrations of lithium in the therapeutic range. However, a moderate but significant reduction of inositol was under these conditions observed in the hypothalamus. At basal conditions, ie in control rats not treated with lithium, the inositol content in various brain areas was different, the hypothalamus containing the highest inositol concentration (4.4 mmol kg-1 wet weight) and the cortex the lowest (2.3 mmol kg-1 wet weight). It is concluded that chronic lithium treatment at therapeutically relevant brain concentrations does not evoke major changes in the inositol content of the brain but induces a moderate decrease which is restricted to the hypothalamus. The results are discussed with respect to the potential function of the hypothalamus in affective disorders.

Inositol has behavioral effects with adaptation after chronic administration.

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. We hereby report effects of inositol on 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.

Reduced [3H]cyclic AMP binding in postmortem brain from subjects with bipolar affective disorder.

Findings of increased Gs alpha levels and forskolin-stimulated adenylyl cyclase activity in selective cerebral cortical postmortem brain regions in bipolar affective disorder (BD) implicate increased cyclic AMP (cAMP)-mediated signaling in this illness. Accumulating evidence suggests that intracellular levels of cAMP modulate the abundance and disposition of the regulatory subunits of cAMP-dependent protein kinase (cAMP-dPK). Thus, in the present study, we tested further whether hyperfunctional Gs alpha-linked cAMP signaling occurs in BD by determining [3H]cAMP binding, a measure of the levels of regulatory subunits of cAMP-dPK, in cytosolic and membrane fractions from discrete brain regions of postmortem BD brain. Specific [3H]cAMP (5 nM) binding was determined in autopsied brain obtained from 10 patients with DSM-III-R diagnoses of BD compared with age- and postmortem delay-matched controls. [3H]cAMP binding was significantly reduced across all brain regions in cytosolic fractions of BD frontal (-22%), temporal (-23%), occipital (-22%) and parietal (-15%) cortex, cerebellum (-36%), and thalamus (-13%) compared with controls, but there were no differences in [3H]cAMP binding in the membrane fractions from these same regions. These results suggest that changes occur in the cAMP-dPK regulatory subunits in BD brain, possibly resulting from increased cAMP signaling. The possibility that antemortem lithium and/or other mood stabilizer treatment may contribute to the above changes, however, cannot be ruled out.

Oral maternal inositol supplementation does not increase rat conceptus inositol levels.

Lithium (Li) is an effective drug for prophylaxis and treatment of major affective disorders. It is teratogenic both to animals and human beings. Depletion of inositol is associated both with lithium side effects and teratogenesis. There is no direct evidence showing that in humans Li-teratogenesis is associated with the phosphoinositol (PI) cycle. It is conceivable that the teratogenic effect of Li in humans is also associated with inositol depletion and therefore is amenable to inositol supplementation. To test the hypothesis that oral inositol may cross the placental barrier and may be useful as a protective supplement to lithium therapy during pregnancy, we studied the effect of 2.5% inositol in drinking water on embryonic inositol levels in rats. There was no effect on fetal inositol concentration. However, weight of embryos of mothers receiving inositol was significantly higher. These data do not support the concept that inositol supplementation may be useful in preventing human Li-induced teratogenesis.

The effect of acute and chronic lithium on forskolin-induced reduction of rat activity.

Forskolin is a diterpene derivative that activates adenylate cyclase and raises cAMP levels in the cell. Both i.p. and i.c.v. forskolin cause behavioral hypoactivity. Lithium has been reported for many years to block cAMP accumulation, but the behavioral relevance of this biochemical effect is not clear. We studied the effect of acute and chronic lithium on icv forskolin-induced hypoactivity. Acute lithium had no effect, but chronic lithium significantly blocked forskolin-induced hypoactivity. The effect of chronic lithium occurred with both forskolin in DMSO and with a water-soluble forskolin derivative. These results suggest that this behavioral model can be used to investigate whether new inhibitors of adenylate cyclase possess lithium-like effects.

Differential uptake of myo-inositol in vivo into rat brain areas.

Oral inositol has been reported to have antidepressant and antipanic properties in humans. Inositol enters the brain poorly and high doses are required. Natural uptake processes and specific transporters are involved. We here report that intraperitoneally administered inositol is taken up differently by various brain areas and that brain areas have different baseline inositol levels. These effects could be important in understanding the differential effects of lithium-induced lowering of inositol and of behavioral effects of exogenous inositol.

How does lithium work on manic depression? Clinical and psychological correlates of the inositol theory.

How lithium works in manic-depressive illness is unknown. Recently, however, a powerful hypothesis has been gaining momentum. Distinguished by its testability and clinical implications, the inositol depletion hypothesis of lithium action is relevant to treatment of lithium side effects, to the development of new compounds with the clinical profile of lithium, and to new experimental treatment of depression.

Lack of effect of ECS on rat brain inositol monophosphatase activity and inositol levels and of i.c.v. inositol on ictal and post-ictal length.

Lithium (Li) has often been compared to ECT in therapeutic spectrum and mechanism. Inhibition of inositol monophosphatase and reduction of brain inositol are major mechanisms of Li action. Many Li effects in animals and humans are reversible by inositol. We therefore studied interactions of ECS and inositol. ECS in rats did not reduce brain inositol monophosphatase activity or brain inositol levels. Intracerebroventricular injection of 10 mg inositol, a dose that reverses Li effects, had no effect on seizure length or post-ictal length.

Lithium attenuates hypokinesia induced by immobilization stress in rats.

1. Hypokinesia following immobilization stress in rats is attenuated by anti-depressant drugs used in the treatment of unipolar depression. Lithium has anti-depressant effects both clinically and in other animal models of depression, but the mechanism of its anti-depressant effect has not been elucidated. 2. To determine if lithium reverses immobilization-induced hypokinesia, the effects of lithium and immobilization stress were tested in a fully factorial 2 x 2 design. 3. Half the rats were fed chronic dietary lithium, while the other half ate regular chow. Half of each group were exposed to one hour immobilization, while the other half remained in their home cages until the test. Activity was measured for 20 min in an automated activity meter. 4. Stress significantly reduced activity, but a significant interaction between stress and lithium was found, indicating that lithium attenuated the effect of stress. 5. Lithium-induced attenuation of immobilization stress may serve as an animal model for the anti-depressant effects of lithium.

Double-blind, controlled trial of inositol treatment of depression.

OBJECTIVE: CSF levels of inositol have been reported to be lower than normal in depressed subjects. The authors administered inositol to depressed patients in a double-blind, controlled trial. METHOD: Under double-blind conditions, 12 g/day of inositol (N = 13) or placebo (N = 15) was administered to depressed patients for 4 weeks. RESULTS: The overall improvement in scores on the Hamilton Depression Rating Scale was significantly greater for inositol than for placebo at week 4. No changes were noted in hematology or in kidney or liver function. CONCLUSIONS: This may be the first use of the precursor strategy for a second messenger rather than a neurotransmitter in treating depression. Although inositol had a significant antidepressant effect in this study, replication is crucial.

Myo-inositol attenuates the enhancement of the serotonin syndrome by lithium.

Lithium elicits opposite effects on two behavioural syndromes in rats: enhancement of the 5-HT1A-linked serotonin syndrome and attenuation of the 5-HT2-linked wet dog shakes. The ability of intracerebroventricular (ICV) myo-inositol or forskolin to reverse the enhancement of the serotonin syndrome by lithium was tested in rats that were fed chronic dietary lithium or control diet and injected with the serotonin agonist 5-MeODMT (5-methoxy-N, N-dimethyltryptamine). Lithium enhanced the total serotonin syndrome score and particularly flat posture and tremor. Inositol, but not forskolin, mitigated the effects of lithium. Inositol was also injected in the lateral ventricle of rats pretreated with chronic dietary lithium or regular rat chow for 3 weeks and injected with carbidopa and L-5-hydroxytryptophan (5-HTP). Lithium attenuated wet dog shakes, but inositol had no significant effect on lithium-treated or control rats. These findings suggest that the enhancement of the serotonin syndrome by lithium may be related to lithium-induced inositol depletion.