# Interesting article linking the GABA-Serotonin Hypothesis



## David Kozin (Jan 11, 2005)

I am reprinting this article without permission, and I hope that individuals will be able to comment on it. However, it is quite interesting....

It will take an individual with a strong background to read the end section of the work.
The image below may not display, but is a good look at the interaction of Serotonin receiving cells with GABA outputs and the effect on pyramidal cells. There are some key phrases in this article that are interesting.










Background

This study tested the hypothesis that deficits in γ-aminobutyric acid type A (GABAA) receptor function might create a vulnerability to the psychotogenic and perceptual altering effects of serotonergic (5-HT2A/2C) receptor stimulation. The interactive effects of iomazenil, an antagonist and partial inverse agonist of the benzodiazepine site of the GABAA receptor complex, and m-chlorophenylpiperazine (m-CPP), a partial agonist of 5-HT2A/2C receptors, were studied in 23 healthy male subjects.
Methods

Subjects underwent 4 days of testing, during which they received intravenous infusions of iomazenil/placebo followed by m-CPP/placebo in a double-blind, randomized crossover design. Behavioral, cognitive, and hormonal data were collected before drug infusions and periodically for 200 min after.
Results

Iomazenil and m-CPP interacted in a synergistic manner to produce mild psychotic symptoms and perceptual disturbances without impairing cognition. Iomazenil and m-CPP increased anxiety in an additive fashion. Iomazenil and m-CPP interacted in a synergistic manner to increase serum cortisol.
Conclusions

Gamma-aminobutyric acid-ergic deficits might increase the vulnerability to the psychotomimetic and perceptual altering effects of serotonergic agents. These data suggest that interactions between GABAA and 5-HT systems might contribute to the pathophysiology of psychosis and dissociative-like perceptual states.

Biological Psychiatry
Vol: 59 Issue: 2, January 15, 2006
pp: 128-137

PII: S0006-3223(05)00771-7
DOI: 10.1016/j.biopsych.2005.06.020
Copyright ? 2005 Society of Biological Psychiatry All rights reserved.
Original article
γ-Aminobutyric Acid?Serotonin Interactions in Healthy Men: Implications for Network Models of Psychosis and Dissociation

Deepak Cyril D?Souza a, b, , Email Address, Roberto B. Gil a, b, d, Edward Zuzarte a, b, e, Lisa M. MacDougall a, b, Lia Donahue a, b, John S. Ebersole f, Nashaat N. Boutros a, b, Tom Cooper g, John Seibyl b, c, John H. Krystal a, b
a Schizophrenia Biological Research Center, West Haven Veterans Affairs Medical Center, West Haven, Connecticut
b Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut
c Department of Nuclear Medicine, Yale University School of Medicine, New Haven, Connecticut
d Schizophrenia Research Unit, Columbia University, New York, New York
e Sheppard Pratt Health System, Timonium, Maryland
f Department of Neurology, Chicago University School of Medicine, Chicago, Illinois
g Nathan Kline Institute, Orangeburg, New York

Received 25 January 2005; revised 29 April 2005; accepted 17 June 2005
Background

This study tested the hypothesis that deficits in γ-aminobutyric acid type A (GABAA) receptor function might create a vulnerability to the psychotogenic and perceptual altering effects of serotonergic (5-HT2A/2C) receptor stimulation. The interactive effects of iomazenil, an antagonist and partial inverse agonist of the benzodiazepine site of the GABAA receptor complex, and m-chlorophenylpiperazine (m-CPP), a partial agonist of 5-HT2A/2C receptors, were studied in 23 healthy male subjects.
Methods

Subjects underwent 4 days of testing, during which they received intravenous infusions of iomazenil/placebo followed by m-CPP/placebo in a double-blind, randomized crossover design. Behavioral, cognitive, and hormonal data were collected before drug infusions and periodically for 200 min after.
Results

Iomazenil and m-CPP interacted in a synergistic manner to produce mild psychotic symptoms and perceptual disturbances without impairing cognition. Iomazenil and m-CPP increased anxiety in an additive fashion. Iomazenil and m-CPP interacted in a synergistic manner to increase serum cortisol.
Conclusions

Gamma-aminobutyric acid-ergic deficits might increase the vulnerability to the psychotomimetic and perceptual altering effects of serotonergic agents. These data suggest that interactions between GABAA and 5-HT systems might contribute to the pathophysiology of psychosis and dissociative-like perceptual states.

Key Words: Iomazenil; GABA; m -CPP; serotonin; psychosis; perceptions; anxiety; schizophrenia.

Article Outline

Methods and Materials

Participants

Drugs

Measures

Schedule of Testing

Statistical Analyses

Results

Behavioral Measures

BPRS Four-Key Positive Symptoms Subscale

Perceptual Alterations

Anxiety

Cognitive Measures

Plasma m -CPP Levels

Neurochemical Measures

Serum Cortisol

Serum Prolactin

Follow-Up Safety Data

Discussion

Psychosis

Dissociation

Model Circuitry

Anxiety

Hormones

Limitations

References

One-(m-chlorophenyl)piperazine (m -CPP) has been used to probe serotonin (5-HT) receptor function in humans. One-(m-chlorophenyl)piperazine binds to several 5-HT receptor subtypes, with highest affinity (Kd = 21 nmol/L) for 5-HT2C, followed by affinity (Kd = 200?400 nmol/L) for 5-HT2A, 5-HT1A, 5-HT1B, and 5-HT3 receptor subtypes (Hamik and Peroutka 1989; Hoyer and Neijt 1988 [ Hamik and Peroutka 1989, Hoyer and Neijt 1988]). One-(m-chlorophenyl)piperazine has partial agonist activity at 5-HT2C, 5-HT1A, 5-HT1B, and 5-HT1D receptor subtypes, showing intrinsic activity only 40%?60% that of the endogenous ligand (Conn and Sanders-Bush 1987; Kahn and Wetzler 1991; Porter et al 1999; Thomas et al 1996 [ Conn and Sanders-Bush 1987, Kahn and Wetzler 1991, Porter et al 1999, Thomas et al 1996]); m -CPP was previously classified as an antagonist at 5-HT2A receptors, although recent studies suggest that it is a potent partial agonist at 5-HT2A receptors with maximal effects comparable to (+) lysergic acid diethylamide (Grotewiel et al 1994; Willins and Meltzer 1997 [ Grotewiel et al 1994, Willins and Meltzer 1997]). It is also an antagonist at 5-HT2B and 5-HT3 receptors (Conn and Sanders-Bush 1987; Fiorella et al 1995; Thomas et al 1996 [ Conn and Sanders-Bush 1987, Fiorella et al 1995, Thomas et al 1996]) and an inhibitor of 5-HT reuptake ( Baumann et al 1995). Several lines of evidence suggest that the effects of m- CPP appear to be predominantly mediated by a combination of partial agonist activity at 5-HT2C and 5-HT2A receptors (Grotewiel et al 1994; Kennett and Curzon 1991; Simansky and Schechter 1988; Willins and Meltzer 1997 [ Grotewiel et al 1994, Kennett and Curzon 1991, Simansky and Schechter 1988, Willins and Meltzer 1997]).

In healthy subjects, m -CPP has anxiogenic and euphoric effects (Charney et al 1987; Zuardi 1990 [ Charney et al 1987, Zuardi 1990]). Its anxiogenic effects appear to be mediated by 5-HT2C receptor activation ( Abi-Dargham et al 1997, for review). In patients with panic disorder and post-traumatic stress disorder (PTSD), the anxiogenic effects of m -CPP are increased, and panic attacks are frequently elicited (Germine et al 1994; Kahn et al 1988b, 1988c; Southwick et al 1997; Wetzler et al 1996 [ Germine et al 1994, Kahn et al 1988b, Kahn et al 1988c, Southwick et al 1997, Wetzler et al 1996]). One-(m-chlorophenyl)piperazine elicits prominent dissociative-like perceptual states including depersonalization, derealization, and flashbacks to previous traumatic experiences in patients with PTSD ( Southwick et al 1997); m- CPP has also been reported to induce transient depersonalization and derealization in patients with obsessive-compulsive disorder, social phobia, and borderline personality disorder ( Simeon et al 1995).

Studies with m -CPP in schizophrenia patients have produced mixed results; a relatively small subgroup of schizophrenic patients experience a worsening of psychosis (Iqbal et al 1991; Krystal et al 1993 [ Iqbal et al 1991, Krystal et al 1993]), whereas others show no change (Breier et al 1993; Maes and Meltzer 1996 [ Breier et al 1993, Maes and Meltzer 1996]) or improvements in psychosis ( Kahn et al 1992). Relative to control subjects, unmedicated schizophrenic patients showed a selective vulnerability to the psychotogenic, but not anxiogenic or endocrine, effects of m -CPP (Abi-Saab et al 2002; Krystal et al 1993 [ Abi-Saab et al 2002, Krystal et al 1993]). We observed varying degrees of worsening of psychosis with m -CPP administration in schizophrenic patients, and speculated that this variable vulnerability to worsening of psychosis with m -CPP administration (Abi-Saab et al 2002; Krystal et al 1993 [ Abi-Saab et al 2002, Krystal et al 1993]) might be a result of varying levels of γ-aminobutyric acid (GABA) receptor dysfunction reported in schizophrenia ( Benes and Berretta 2001).

This study tested the hypothesis that a GABAergic deficit produced by iomazenil might predispose healthy subjects to exhibit the increase in anxiety, psychosis, and perceptual alterations associated with m- CPP administration observed in schizophrenia and PTSD, as discussed previously. Iomazenil (Ro 16-0154), the iodine analog of the benzodiazepine (BZ) receptor competitive antagonist flumazenil, has high affinity and selectivity for BZ receptors (Kd = .5 nmol/L) ( Johnson et al 1990). Iomazenil is a BZ receptor competitive antagonist with inverse agonist effects (Beer et al 1990; Schubiger and Hasler 1989 [ Beer et al 1990, Schubiger and Hasler 1989]) (Hoffman La-Roche data on file). Unlike the competitive antagonist flumazenil, which antagonizes the effects of BZ agonists but lacks intrinsic pharmacological effects ( Hunkeler et al 1981), inverse agonists have intrinsic pharmacological effects opposite to those of BZs ( Tallman and Gallager 1985). BZ receptor inverse agonists interfere with the function of the BZ receptor in coupling the GABA type A (GABAA) receptor and chloride channel (Polc 1995; Sieghart 1992 [ Polc 1995, Sieghart 1992]). Anxiogenic effects of BZ inverse agonists are hallmarks of their reduction in GABAA receptor function (Bradberry et al 1991; Dorow et al 1983; Gentil et al 1989; Murphy et al 1996; Sarter et al 2001 [ Bradberry et al 1991, Dorow et al 1983, Gentil et al 1989, Murphy et al 1996, Sarter et al 2001]), and higher doses are epileptogenic. Iomazenil has anxiogenic effects and, at higher doses, proconvulsant effects in humans that are consistent with inverse agonist activity at BZ receptors (Randall, personal communication, 1995).

The hypothesized relationship between GABA and 5-HT systems that is the basis of this report is informed by a series of preclinical studies of the limbic cortex (Gellman and Aghajanian 1994; Marek and Aghajanian 1994; Sheldon and Aghajanian 1991 [ Gellman and Aghajanian 1994, Marek and Aghajanian 1994, Sheldon and Aghajanian 1991]) and the medial prefrontal cortex (Aghajanian and Marek 1997, 1999; Araneda and Andrade 1991; Marek and Aghajanian 1999; Marek and Gewirtz 1999; Zhou and Hablitz 1999 [ Aghajanian and Marek 1997, Aghajanian and Marek 1999, Araneda and Andrade 1991, Marek and Aghajanian 1999, Marek and Gewirtz 1999, Zhou and Hablitz 1999]). Activation of 5-HT2A receptors results in two different effects on pyramidal cells. First, activation of postsynaptic 5-HT2A receptors directly depolarizes most cells. Second, activation of cortical 5-HT2A receptors induces glutamate release from thalamocortical afferents onto the apical dendrites of layer V pyramidal cells and increases the excitatory influence on layer V pyramidal cells. These pyramidal cells also have GABAergic interneurons which synapse on them and provide an inhibitory tone. Serotonin activates GABAergic interneurons in the piriform cortex (Gellman and Aghajanian 1994; Sheldon and Aghajanian 1991 [ Gellman and Aghajanian 1994, Sheldon and Aghajanian 1991]), the hippocampus ( Piguet and Galvan 1994), and the neocortex ( Aghajanian and Marek 1997), resulting in local inhibition of pyramidal neurons. Iomazenil, by interfering with GABA?s ability to activate BZ-GABAA sites located on pyramidal neurons, would reduce inhibitory tone ( Figure 1). In the absence of inhibitory tone, activation of 5-HT2A receptors located on pyramidal neurons, such as would occur with m -CPP, could result in unopposed excitation of pyramidal neurons ( Figure 1). The latter would lead to a loss of associative functions, disruption of normal gating mechanisms and, eventually, psychotic symptoms; however, m -CPP and iomazenil are not regionally selective and, hence, their interactive effects could result from effects in brain regions other than the areas hypothesized.

Figure 1: Schema of of γ-aminobutyric acid(GABA)?Serotonin interactions in the cortex. BZ = Benzodiazepine receptors; 5-HT2a = serotonin 2a receptors; m -CPP = m -chlorphenylpiperazine.

Methods and Materials
Participants

The study was conducted at the Neurobiological Studies Unit (VA [Department of Veterans Affairs] Connecticut Healthcare System, West Haven, Connecticut) with the approval of the Institutional Review Board at VA Connecticut. Healthy male volunteers (n = 23) were recruited from the community by public advertisements and were paid for their participation. Female subjects were excluded from this study, because the teratogenic potential of iomazenil had not been studied. Subjects were informed about the potential for psychosis, anxiety, panic, blurred vision, dry mouth, decreased concentration, confusion, dizziness, drowsiness, and nausea. Subjects were paid $100 per test day for a total of $400. After obtaining informed consent, subjects underwent a structured psychiatric interview for DSM-IIIR ( Spitzer et al 1990) conducted by a trained rater and were carefully screened for any DSM-IV Axis I or Axis II lifetime psychiatric or substance abuse disorder (excluding nicotine) and family history of major Axis I disorder. The history provided by subjects was confirmed by a telephone interview conducted with an individual (spouse or family member) identified by the subject before screening. A history was obtained to exclude individuals with psychiatric illness, medical or neurologic conditions, substance abuse disorders, and known psychiatric illness in first-degree relatives. Finally, subjects underwent a general physical and neurologic examination, EKG, and laboratory tests (serum electrolytes, liver function tests, complete blood count with differential and urine toxicology). About 20% of volunteers failed to meet the inclusion/exclusion criteria. Given the proconvulsant potential of iomazenil, a baseline electroencephalogram (EEG) was conducted and read by a qualified neurologist (JSE or NB).
Drugs

The m -CPP (Bristol-Myers, Wallingford, Connecticut), 10 ? .1 mg, was dissolved in 10 mL normal saline solution. Under sterile conditions, the solution was passed through a .22 μm polymer filter. An aliquot was subjected to sterility and pyrogenicity testing, whereas the remainder was stored at −20?C to be used later for testing. A dose of .1 mg/kg m -CPP was administered intravenously over 20 min as per previous studies (Charney et al 1987; Krystal et al 1993 [ Charney et al 1987, Krystal et al 1993]). Iomazenil for intravenous (IV) injection was prepared by dissolving iomazenil powder (Hoffman La-Roche, Basel, Switzerland) in sterile ethanol (5% volume) and mixing the concentrate with sterile normal saline to prepare a .05-mg/mL concentration. Before use, product concentration was verified by high-performance liquid chromatography and tested for sterility and pyrogenicity. Iomazenil was administered intravenously at a dose of 3.7 μg/kg over 10 min. On the basis of displacement studies in nonhuman primates (Abi-Dargham et al 1994; Laruelle et al 1993 [ Abi-Dargham et al 1994, Laruelle et al 1993]), this dose of iomazenil (3.7 μg/kg IV) is estimated to occupy about 25% of BZ binding sites in the brain ( Innis et al 1991). The production of anxiety, a hallmark of the reduction of GABAA receptor function by BZ inverse agonists, at this dose in human studies at our center (Randall, personal communication, 1995) suggests that iomazenil is functionally relevant at the dose studied.
Measures

On the basis of studies of m -CPP effects in schizophrenic patients (Charney et al 1987; Krystal et al 1993 [ Charney et al 1987, Krystal et al 1993]) and ketamine effects in healthy subjects ( Krystal et al 1994a), the principal outcome measure was the four-key positive symptom subscale of the Brief Psychiatric Rating Scale (BPRS), which has items for hallucinatory behavior, conceptual disorganization, unusual thought content, and suspiciousness (Overall and Gorham 1962; Woerner et al 1988 [ Overall and Gorham 1962, Woerner et al 1988]). The four-key positive symptoms subscale of the BPRS was selected as an index of the positive symptoms of schizophrenia on the basis of previous reports indicating their validity and utility (Krystal et al 1994a; Overall and Gorham 1962 [ Krystal et al 1994a, Overall and Gorham 1962]) and their inclusion within the empirically derived thought disorder factor of the BPRS( Vieweg and Hedlund 1984). In our previous study with m -CPP, schizophrenic patients had peak increases of at least 3 points (4.3 ? 1.2) on the four-key subscale (BPRS), whereas healthy subjects did not (.05 ? .3) ( Krystal et al 1993). Furthermore, a ≥ 3-point change in the BPRS four-key positive symptoms subscale is within the range of improvement observed in clinical trials of schizophrenia patients ( Kane et al 1988). On the basis of these data, clinically significant psychotic symptoms were defined as a ≥ 3-point increase in the BPRS four-key positive symptom subscale score. Perceptual alterations were measured with the Clinician Administered Dissociative Symptoms Scale (CADSS) ( Bremner et al 1998), a scale consisting of 19 subjective items and 8 objective items (0 = not at all, 4 = extremely) that is sensitive to the perceptual altering effects of other drugs (e.g., delta-9-tetrahydrocannabinol and ketamine) (D?Souza et al 2004; Krystal et al 1994a [ D?Souza et al 2004, Krystal et al 1994a]). In validating the CADSS as an instrument to assess dissociative states in PTSD patients, we showed that perceptual distortions at an elemental level (i.e., changes or distortions in body perception, time perception, environmental stimuli, etc.) were common features of PTSD. Anxiety was measured with the clinician-rated anxiety item of the BPRS and a self-rated visual analog scale (Patient Rated Analog Scale: PRAS). The latter was scored in millimeters, from the left-hand side of a 100-mm line to a perpendicular mark at a point corresponding to the apparent magnitude of the feeling state reported and exhibited by the subject (range: 0, ?not at all,? to 100, ?most ever?) ( Charney et al 1987). The same research assistant rated all 3 days of a subject, and the same staff rated both schizophrenia patients and healthy control subjects. Interrater reliability sessions were conducted every 1?2 months. Intraclass correlation (ICC) for the BPRS and CADSS were consistently greater than .85. General measures of cognition included tasks of immediate and delayed recall, orientation and attention (serial sevens) adapted from the Mini-Mental State Examination (MMSE) ( Folstein et al 1975), and abstract reasoning (proverbs and similarities).

Plasma m -CPP levels obtained at the +35 and 80 time points were assayed to exclude the possibility that the behavioral interactions of m -CPP and iomazenil were due to a pharmacokinetic interaction between the two drugs. Plasma m -CPP levels were analyzed at the Nathan Kline Institute, Orangeburg, New York by Tom Cooper with a published liquid chromatographic procedure ( Suckow et al 1990) with ultraviolet (UV) detection. After the addition of 75 mg of internal standard (1-[0-tolyl]piperazine), m -CPP is extracted from 1-mL plasma, made alkaline with .6 mol/L carbonate buffer, with 6 mL of methyl-tert -butyl ether. After mixing and centrifuging, the organic extract is back-extracted with .25 mL of acidic phosphate buffer, mixed and centrifuged. Chromatography was carried out with a trimethylsilyl bonded silica column with a mobile phase consisting of 82% phosphate buffer, 18% acetonitrile, adjusted to pH similar to 3. At a flow rate of 1.8 mL/min, m -CPP and the internal standard were separated and detected at a UV wavelength of 214 nm in <10 min. Interassay coefficient of variation of m -CPP did not exceed 6.8% (range 50 to 3 ng/mL) (n = 10, for each concentration). Day-to-day variation of m -CPP quality controls at 100, 25, and 5 ng/dL did not exceed 2.3%, 4.9%, and 9.5 %, respectively. The minimum quantifiable limit for m -CPP was set at 3 ng/mL.

One-(m-chlorophenyl)piperazine has been demonstrated to increase plasma cortisol and prolactin (Kahn et al 1988a; Krystal et al 1993 [ Kahn et al 1988a, Krystal et al 1993]). Hormonal assays were conducted to provide a behavioral-independent measure of the interactive effects of m -CPP and iomazenil. All hormonal analyses were run in duplicate pairs. Radioimmunoassay kits were used to determine plasma levels of prolactin (Serono Diagnostics, Norwell, Massachusetts; intra-assay [intraclass correlation coefficient] and interassay coefficients of variation were 3% and 7%, respectively) and cortisol (Baxter Travenol Diagnostics, Incstar Corporation, Stillwater, Minnesota; intra-assay [intraclass correlation coefficient] and interassay coefficients of variation were 3% and 5%, respectively).
Schedule of Testing

Subjects completed a 4-day test paradigm involving the administration of placebo or active iomazenil, followed by placebo or active m -CPP, in a randomized, counterbalanced order under double-blind conditions. Each test day was separated by 72 hours to minimize any carryover effects. Subjects were asked to refrain from using alcohol, street drugs, psychotropic medications, or caffeinated beverages for 2 weeks before testing through the entire study. On each test day, subjects were instructed to fast after breakfast and report to the test facility around 3 pm. A urine toxicology was conducted to rule out recent illicit drug use. The first 5 subjects underwent testing with continuous electroencephalography to monitor for any seizure activity. Because there was no evidence of seizure activity on EEG, the remainder of subjects (n = 18) did not undergo EEG monitoring.

After obtaining IV access at −90 min and obtaining baseline assessments at −90 min and −30 min, subjects were administered active iomazenil or placebo (saline) intravenously over a 10-min period at −15 min, after which, behavioral ratings were conducted. Five minutes after the iomazenil infusion, subjects were administered active m -CPP or placebo (saline) intravenously over 20 min. The timing of m -CPP administration was scheduled to coincide with the peak effect of iomazenil. Behavioral ratings were conducted at the −30, −5, +35, +65, +80, +110, +140, and +200 min time points; vital signs were recorded at the −90, −30, −5, +35, +65, +80, +110, +140, and +200 min time points; and neuropsychological tests were conducted at the +45 min time point. Blood was sampled for hormones at the −30, −5, +35, +65, +80, +110, +140, and +200 time points and, for m -CPP levels, at the −15, +35, and +80 time points. The timing of behavioral assessments fit the time course of the behavioral response to m -CPP in previous studies (Abi-Saab et al 2002; Krystal et al 1993, 1994b, 1996, 1997 [ Abi-Saab et al 2002, Krystal et al 1993, Krystal et al 1994b, Krystal et al 1996, Krystal et al 1997]). The timing of behavioral assessments also fits with BZ receptor occupancy by iomazenil (Abi-Dargham et al 1994; Laruelle et al 1993 [ Abi-Dargham et al 1994, Laruelle et al 1993]).
Statistical Analyses

Measures of anxiety, psychosis, and perceptual alterations were analyzed, consistent with the stated study hypothesis. Plasma m -CPP levels were also analyzed to exclude a pharmacokinetic interaction. Each response variable was analyzed with a univariate repeated measures analysis of variance (ANOVA) with subject factors of iomazenil (active versus placebo), m -CPP (active versus placebo), and time points. The data were first checked for skewness and, if necessary, transformed with a square root transformation before analysis. If the data showed significant lack of sphericity, Huynh-Feldt adjustments were made to the degrees of freedom. All main effects and interactions in the repeated measures model were tested at α = .05 level. When time interactions with either drug alone or in combination were significant, post hoc multiple comparisons were performed with Dunnett?s test comparing the mean across time for each condition to the active iomazenil?active m-CPP conditions (3 comparisons). A maximum of three post hoc tests were run for each omnibus test with an overall (experiment-wise) α level of .05. Except when noted, standard errors of the mean were used to indicate variance. Data were analyzed with the Statistical Package for the Social Sciences (SPSS, Chicago, Illinois).
Results

Of 23 subjects studied, 1 dropped out after completing three test sessions and 2 dropped out after completing one test session. The two subjects who dropped out after completing only one test session completed the active iomazenil?active m -CPP condition. These two subjects dropped out after withdrawing consent, whereas the third subject could not complete the study because of time constraints. The duration between test sessions was 6.65 days (? 5.51 days). Subjects were 38.7 ? 9.9 years old (range 21?52 years) and weighed 196.1 ? 37.6 lbs. (range 135?270 lbs) (all values presented are mean ? SD). To improve clarity of the presentation, when both the treatment condition (dose) effect and the treatment condition (dose) by time interaction were significant, only the latter are reported in the text.
Behavioral Measures
BPRS Four-Key Positive Symptoms Subscale

Iomazenil alone (iomazenil ? time interaction: [F (7,133) = 7.959, p <.001]), m -CPP alone (m -CPP ? time: [F (7,133) = 9.635, p < .001]), and the combination of iomazenil and m -CPP (iomazenil ? m -CPP ? time [F (7,133) = 6.027, p < .003]) induced transient positive psychotic symptoms of schizophrenia, measured by the BPRS four-key subscale ( Figures 2 A & 2B).

Figure 2: (A) Effects of iomazenil and m-CPP on the Brief Psychiatric Rating Scale (BPRS) four-key positive symptom subscale. T-shaped bars indicate SEMs. Iomazenil ? time [F (7,133) = 7.9, p < .001], m -chlorphenylpiperazine (m -CPP) ? time [F (7,133) = 9.6, p < .001], iomazenil ? m -CPP ? time [F (7,133) = 6, p < .003]. (B) Scatterplot of the dose-related peak change in BPRS four-key positive symptom score. T-shaped bars represent the mean and SEM of peak change at each dose. Clinically significant psychosis induced by the combination of iomazenil and m-CPP (n = 23). Whereas 7 of 20 subjects (35%) met the threshold of a ≥3-point increase in BPRS four-key positive symptom score on the iomazenil?m -CPP test day, none of the subjects did on the other three test days.

The increases in positive symptom scores induced by the combination of iomazenil and m -CPP were greater than those produced by either drug alone, as determined by post hoc contrasts (Dunnett?s) (active iomazenil?active m -CPP vs. placebo iomazenil?active m -CPP, p < .0001, and active iomazenil?active m -CPP vs. active iomazenil?placebo m -CPP, p < .0023). Thus, the interaction iomazenil and m -CPP were synergisitic (i.e., the effects of the combination of iomazenil and m -CPP was significantly greater than the addition of the two individual drug effects).

The mean increases in BPRS four-key positive symptom scores induced by iomazenil alone and m -CPP alone, although statistically significant, were small and of questionable clinical significance. The combination of iomazenil and m -CPP was unique in inducing transient, but clinically significant psychosis; not all subjects had increases in positive symptom scores, as shown in the scatter plot ( Figure 2 B). Whereas 7 of 20 subjects (35%) met the threshold of a ≥3-point increase in BPRS four-key positive symptom score (described earlier) on the iomazenil?m -CPP test day, none of the subjects did on the other three test days.

The typical positive psychotic symptoms elicited by the combination of iomazenil and m -CPP were suspiciousness and unusual thought content. For example, one subject reported that ?staff were talking about me behind my back.? Another subject reported feeling that he ?could not trust the staff, as they were not telling me when the infusion was over.? One-(m-chlorophenyl)piperazine and the combination of iomazenil and m -CPP increased scores on the suspiciousness and unusual thought items of the BPRS?consistent with these reports?but had no effect on the conceptual disorganization and hallucinatory behavior items. Rather than hallucinations, subjects reported perceptual alterations (discussed later). The increases in positive symptoms scores were modest, transient, and resolved both spontaneously and completely.
Perceptual Alterations

Iomazenil alone (iomazenil ? time [F (3,51) = 2.87, p < .044]), m -CPP alone (m -CPP ? time [F (3,51) = 9.87, p < .0001]), and the combination of iomazenil and m -CPP (iomazenil ? m -CPP ? time [F (7,133) = 4.56, p < .006]) increased perceptual alteration scores, measured by the subjective items subscale of the CADSS, in a statistically significant manner ( Figure 3). The increases in subject-rated perceptual alteration scores induced by the combination of iomazenil and m -CPP were greater than those produced by either drug alone, as determined by post hoc contrasts (Dunnett?s) (active iomazenil?active m -CPP vs. placebo iomazenil?active m-CPP, p < .008, and active iomazenil?active m -CPP vs. active iomazenil?placebo m -CPP, p < .002).

Figure 3: Perceptual altering effects of iomazenil and m -chlorphenylpiperazine (m -CPP) on the subject-rated Clinician Administered Dissociative Symptoms Scale (CADSS). T-shaped bars indicate SEMs. Iomazenil ? time [F (3,57) = 2.88, p < .044], m -CPP ? time [F (3,57) = 9.87, p < .015], iomazenil ? m -CPP ? time [F (3,57) = 4.56, p < .006].

The perceptual alterations reported by subjects included ?I feel like I?m floating?like in a dream,? or ?my clothes are moving,? or ?objects look much larger?I can hear the IV dripping?I feel like I?m locked in a sauna?time is moving very slowly.? These perceptual alterations were not scored as hallucinations, because when challenged, subjects were able to test reality. The perceptual alterations induced by the combination of iomazenil and m -CPP resembled the perceptual alterations induced by the dissociative anesthetic ketamine but were relatively muted in intensity.

Iomazenil alone (iomazenil ? time: [F (3,36) = 3.4, p < .07]) and the combination of iomazenil and m -CPP (iomazenil ? m -CPP ? time [F (3,36) = 4.88, p < .01]) increased perceptual alteration scores measured by the objective items subscale of the CADSS in a statistically significant manner; m -CPP alone did not increase clinician-rated perceptual alteration scores (m -CPP ? time [F (3,36) = 2, p = .14]). Post hoc contrasts (Dunnett?s) between the increases in clinician-rated perceptual alteration scores induced by the combination of iomazenil and m -CPP and those produced by either drug alone did not meet the threshold of adjusted statistical significance (active iomazenil?active m -CPP vs. placebo iomazenil?active m -CPP, p = .5, and active iomazenil?active m -CPP vs. active iomazenil?placebo m -CPP, p < .02).
Anxiety

Iomazenil (iomazenil ? time [F (7,133) = 4.3, p < .002]) and m -CPP (m -CPP ? time [F (7,133) = 8.5, p < .001]), but not the combination of iomazenil and m -CPP (iomazenil ? m -CPP ? time [F (7,133) = 1.2, p = .33]), produced statistically significant increases in clinician-rated BPRS anxiety scores ( Figure 4). Post hoc contrasts (Dunnett?s) revealed that, whereas the increase in anxiety produced by the combination of iomazenil?m -CPP was statistically greater than the increase in anxiety produced by iomazenil alone [F (7,133) = 4.2, p < .0002] and m -CPP [F (7,133) = 3, p < .01] alone, it did not exceed the sum of the increases in anxiety produced by each drug alone. These data suggest that the anxiogenic effects of iomazenil and m -CPP are additive.

Figure 4: Anxiogenic effects of iomazenil and m -chlorphenylpiperazine (m -CPP) on the Brief Psychiatric Rating Scale (BPRS) and subject-rated Visual Analog Scale (VAS). T-shaped bars indicate SEMs. BPRS anxiety : iomazenil ? time [F (7,133) = 4.3, p < .002], m -CPP ? time [F (7,133)= 8.5, p < .001], iomazenil ? m -CPP ? time [F (7,133) = 1.2, p < .33] (ns); Visual Analog Scale anxiety : iomazenil ? time [F (7,133) = 3.7, p < .001], m -CPP ? time [F (7,133) = 6.3, p < .001], iomazenil ? m -CPP ? time [F (7,133) = 1.24, p < .19] (ns).

Similarly, iomazenil alone (iomazenil ? time [F (7,133) = 3.7, p < .001]) and m -CPP alone (m -CPP ? time [F (7,133) = 6.3, p < .001]), but not the combination of iomazenil and m -CPP (iomazenil ? m -CPP ? time [F (7,133) = 1.24, p < .19]), produced statistically significant increases in self-reported anxiety scores measured by the Visual Analog Scale. Post hoc contrasts (Dunnett?s) revealed that, whereas the increase in anxiety produced by the combination of iomazenil?m -CPP was statistically greater than the increase in anxiety produced by iomazenil alone [F (7,133) = 6.3, p < .0002] and m -CPP [F (7,133) = 3.4, p < .004] alone, it did not exceed the sum of the increases in anxiety produced by each drug alone.

To estimate the contribution of anxiety to the psychotic effects of iomazenil?m -CPP, a one-way analysis of covariance (ANCOVA) was conducted to detect differences between drug conditions (placebo?placebo vs. iomazenil?m -CPP) on four-key positive symptoms (BPRS). The ANCOVA revealed significant [F (1,24) = 7.6, p < .009] differences in BPRS four-key scores between the placebo?placebo and iomazenil?m -CPP, despite covarying for anxiety scores.
Cognitive Measures

There were no significant effects of any of the four conditions on any of the simple cognitive measures (orientation and arithmetic) used in the study.
Plasma m -CPP Levels

There were no significant differences in plasma m -CPP levels on the placebo?m -CPP condition versus the iomazenil?m -CPP condition at the +35 (34.83 ? 9.64 vs. 55.41 ? 54.41; [F (1,22) = 1.68, p < .2]) or +80 (25.25 ? 21.09 vs. 32.83 ? 32.61; [F (1,22) = .457, p < .5]) time points in an interim analysis involving the first twelve subjects. Because there were no significant pharmacokinetic interactions in the first 12 subjects (50% of the sample), m -CPP assays were not conducted on the remaining subjects.

Additional analyses were conducted to determine whether differences in the behavioral response to m -CPP across conditions were related to differences in m -CPP levels between the m -CPP alone and iomazenil?m -CPP conditions. An initial univariate ANOVA revealed no interactions between drug condition (m -CPP alone vs. iomazenil?m -CPP) and m -CPP blood levels [F = .08, p < .77] on four-key positive symptoms, the primary outcome measure. A subsequent univariate ANCOVA was conducted on four-key positive symptom scores with m -CPP levels at the +35 time point as the covariate. In this analysis, differences in BPRS four-key scores between the m -CPP alone and iomazenil?m -CPP conditions remained statistically significant [F (1,24) = 8.48, p < .008], despite covarying for blood m -CPP levels. These data suggest that differences in positive symptom scores between the iomazenil?m -CPP and placebo?m -CPP conditions cannot be explained simply on the basis of differences in plasma m-CPP levels.
Neurochemical Measures
Serum Cortisol

Iomazenil alone, m -CPP alone, and the combination of iomazenil and m -CPP alone increased serum cortisol levels ( Figure 5). Repeated measures ANOVAs revealed statistically significant effects of iomazenil (iomazenil ? time: [F (7,126) = 13.2, p < .0001]) and m -CPP (m -CPP ? time: [F (7,126) = 32.9, p < .0001]), and the combination of iomazenil and m -CPP (iomazenil ? m -CPP ? time: [F (7,126) = 3, p < .005]) on serum cortisol levels.

Figure 5: Effects of iomazenil and m -CPP on serum cortisol and prolactin. T-shaped bars indicate SEMs. Cortisol : iomazenil ? time [F (7,126) = 13.2, p < .001], m -CPP ? time [F (7,126) = 32.9, p < .001], iomazenil ? m -CPP ? time [F (7,126) = 3.1, p < .035]; Prolactin : iomazenil ? time [F (7,112) = 3.2, p < .048], m -CPP ? time [F (7,112) = 12.8, p < .001], iomazenil ? m -CPP ? time [F (7,112) = 2, p < .144].

Serum Prolactin

Iomazenil alone (iomazenil ? time: [F (7,112) = 3.2, p < .048]), m -CPP alone (m -CPP ? time: [F (7,112) = 12.8, p < .001]), but not the combination of iomazenil and m -CPP (iomazenil ? m -CPP ? time: [F (7,112) = 2, p < .144]), increased serum prolactin levels in a statistically significant manner.
Follow-Up Safety Data

By the end of the testing session, none of these healthy subjects had any lingering signs or symptoms of psychosis or anxiety. Furthermore, none of the subjects required extended observation, psychopharmacological interventions, or hospitalization. Follow-up evaluation with the total and four-key BPRS scores did not reveal any lingering effects or changes detected by the BPRS in any of the subjects studied, including those who dropped out before completing all four-test sessions.
Discussion

The principal finding of this study is that psychosis and perceptual alterations, which are not seen in healthy subjects administered m -CPP or iomazenil, emerge when m -CPP is administered after iomazenil. The lack of differences in m -CPP blood levels across conditions and the selective nature of the interactive effects suggests a pharmacodynamic rather than a pharmacokinetic interaction.

The preservation of orientation and several simple cognitive functions suggest that the symptomatic changes induced by the combination of iomazenil and m -CPP reflect a more focused change in consciousness that might have greater relevance to psychiatric disorders (e.g., psychosis, where orientation is generally preserved).
Psychosis

Our data are consistent with previous studies in which neither m -CPP alone ( Krystal et al 1993) nor iomazenil (Randall, personal communication, 1995) alone produced clinically significant psychosis in healthy subjects or in subjects with other psychiatric disorders other than schizophrenia. Whereas the psychotic symptoms induced by the iomazenil?m -CPP combination were not characteristic of a specific disorder, the fact that m -CPP induces psychosis only in schizophrenia ties the current findings to schizophrenia. Furthermore, additional analyses suggest that it is unlikely that anxiety drives the psychotic symptoms induced by the iomazenil?m -CPP combination.
Dissociation

The combination of iomazenil and m -CPP induced dissociative-like perceptual alterations that have been shown to be common features of PTSD (Bremner et al 1998, 1992 [ Bremner et al 1998, Bremner et al 1992]); PTSD patients have been observed to experience dissociative-like perceptual effects induced by m -CPP ( Rainey et al. 1987; Southwick et al. 1997), but not iomazenil (Randall, personal communication, 1995). The capacity of iomazenil to create a propensity for m -CPP?induced dissociation-like perceptual effects in healthy subjects renders plausible the hypothesis that deficits in GABA systems in PTSD ( Bremner et al 2000) contribute to the propensity for m -CPP?induced dissociative-like perceptual effects that are distinctive of this disorder ( Southwick et al 1997).

Other BZ receptor inverse agonists, FG7142 and RO 15-3505, have been reported to induce anxiety and psychotic-like symptoms in healthy volunteers (Dorow et al 1983; Gentil et al 1989; Sarter et al 2001 [ Dorow et al 1983, Gentil et al 1989, Sarter et al 2001]). The fact that iomazenil, a weaker partial inverse agonist, induced psychosis but did so only when combined with m -CPP highlights a potential role of GABA?5-HT interactions in the pathophysiology of psychosis and dissociative states.
Model Circuitry

Whereas the precise basis of the psychotogenic and perceptual altering of the combination of iomazenil and m -CPP is not known with certainty, the model circuitry described earlier ( Figure 1) might provide a framework to understand the findings of this study. In brief, m -CPP?induced excitation of cortical pyramidal cells, in the backdrop of an iomazenil-induced GABAergic deficit, could disrupt the equilibrium between excitatory and inhibitory transmission within corticolimbic networks, leading to psychosis, perceptual alterations, and other dissociative phenomenon. We suggest, however, somewhat distinct circuitries underlying the psychotogenic and dissociative effects of the combination of iomazenil and m -CPP effects. In studies at our center, m -CPP induced psychosis and not dissociation in schizophrenia patients (Abi-Saab et al 2002; Krystal et al 1993 [ Abi-Saab et al 2002, Krystal et al 1993]) while inducing dissociation, but not psychosis, in PTSD patients ( Southwick et al 1997). This separation suggests that the psychotogenic and dissociative effects of m -CPP emerge in distinct neurobiological contexts and, perhaps, distinct circuits. We hypothesize that iomazenil creates a vulnerability to both conditions, because it produces a non-selective GABA deficit that encompasses the circuitry related to the psychotogenic and dissociative symptoms of m -CPP.

Several groups have described abnormalities in GABA systems in schizophrenia and bipolar disorder ( Benes and Berretta 2001; Benes et al 1998; Lewis 2000; Pierri et al 1999; Woo et al 1998). Furthermore, some, although not all, in vivo brain imaging (Single Photon Emission Computed Tomography) studies (Abi-Dargham et al 1999; Ball et al 1998; Busatto et al 1997; Schroder et al 1997; Verhoeff et al 1999 [ Abi-Dargham et al 1999, Ball et al 1998, Busatto et al 1997, Schroder et al 1997, Verhoeff et al 1999]) suggest reduced BZ receptor binding in schizophrenia. It has been speculated that the loss of the normal inhibitory tone set by GABAergic interneurons in schizophrenia might create a vulnerability to excitatory inputs on pyramidal cells, resulting in psychosis. A similar mechanism might underlie the psychotomimetic, perceptual altering and cognitive effects of the NMDA (N-methyl-D-aspartate) receptor antagonist, ketamine (reviewed in Krystal et al 2003).

The results of this study suggest that the dissociative-like perceptual effects of m -CPP in PTSD patients might be related to inhibitory (GABAergic) deficits in networks modulating anxiety and sensory processing, a finding consistent with other basic and clinical data (Chambers et al 1999; Krystal et al 1995, 1999 [ Chambers et al 1999, Krystal et al 1995, Krystal et al 1999]).
Anxiety

When given in combination, iomazenil and m -CPP, both known anxiogenic agents, produced anxiogenic effects that did not exceed the sum of their independent anxiogenic effects. The selective blockade of m -CPP?induced anxiety by 5-HT2A/2C receptor antagonists, but not by 5-HT1A or 5-HT3 receptor antagonists (Kahn et al 1990; Meltzer and Maes 1995; Seibyl et al 1991; Silverstone and Cowen 1994 [ Kahn et al 1990, Meltzer and Maes 1995, Seibyl et al 1991, Silverstone and Cowen 1994]) or GABAA /BZ receptor agonists (Kennett et al 1989; Wallis and Lal 1998 [ Kennett et al 1989, Wallis and Lal 1998]), suggests that its anxiogenic effects are mediated by 5-HT2 receptors. Furthermore, the BZ receptor inverse agonist, RO 15-4513, fails to substitute for m -CPP in drug discrimination studies ( Gatch et al 2002), and only GABAA /BZ receptor agonists consistently block the anxiogenic effects of GABAA receptor antagonist such as pentylenetetrazole ( Williams et al 1989). Taken collectively, these data suggest the anxiogenic effects of each drug might not overlap.
Hormones

Both iomazenil and m- CPP induced stress (anxiety), which is known to be associated with increased cortisol levels. The cortisol response to m- CPP administration has been linked to its anxiogenic effects (Kahn et al 1988b; Takamatsu et al 2003 [ Kahn et al 1988b, Takamatsu et al 2003]). The 5-HT2 receptor antagonist ritanserin has been shown to attenuate the cortisol response to m- CPP (Abi-Saab et al 2002; Seibyl et al 1991 [ Abi-Saab et al 2002, Seibyl et al 1991]). In contrast, BZ inverse agonists have been reported to increase cortisol levels ( De Boer et al 1991), and BZ receptor agonists have been reported to attenuate the cortisol response to m- CPP ( Sevy et al 1994). Thus, it is not clear whether the cortisol findings in this study reflect the increased stress associated with the combination of iomazenil and m- CPP (i.e., a non-specific effect) or a specific interplay between GABAA receptors and 5-HT receptors modulating cortisol response.
Limitations

One-(m-chlorophenyl)piperazine has a broad spectrum of effects on 5-HT function, making it difficult to attribute its effects to any one receptor subtype. Studies with more selective 5-HT receptor agonists and antagonists might clarify the role of 5-HT receptor subtypes in psychotic states. The psychotic effects of m -CPP are most likely mediated by 5-HT2C receptors because: 1) pretreatment with the 5-HT2 receptor antagonist ritanserin attenuates psychosis induced by m -CPP in unmedicated schizophrenic patients ( Abi-Saab et al 2002), and clozapine, but not haloperidol, treatment blocks the propsychotic effects of m -CPP in schizophrenic patients (Krystal et al, unpublished data); and 2) m -CPP is both more potent and more efficacious at 5-HT2C than at 5-HT2A receptors ( Conn and Sanders-Bush 1987).

Iomazenil levels were not measured, and therefore, the potential impact of differential iomazenil levels across test conditions cannot be excluded. Finally, this psychopharmacological approach does not provide a direct assessment of GABA?5-HT system interactions.

In conclusion, the results of this study emphasize the importance of GABA?5-HT interactions and the role of these interactions in the pathophysiology of psychosis, dissociative states, and anxiety. γ-aminobutyric acid deficits might predispose to the production or exacerbation of psychosis and perceptual alterations in the context of serotonergic activation, and this mechanism might apply to several disorders where GABA deficits have been described. Furthermore, the findings of this study lend support to the notion that dysfunction within a network involving several neurotransmitters might underlie the pathophysiology of psychosis and dissociative-like perceptual states.

We acknowledge support from the 1) Department of Veterans Affairs (Schizophrenia Biological Research Center, Alcohol Research Center, National Center for PTSD, and Merit Review Program (JK), 2) National Institute of Alcohol Abuse and Alcoholism (KO2 AA 00261-04 to JK, R03 AA11413-02 To DCD), 3) National Institute of Mental Health (RO1 MH61019-02 to DCD) (P50 MH44866-15 to JK), 4) National Institute of Drug Abuse (1 DA12382-01 to DCD), 5) Stanley Foundation (DCD), 6) Donaghue Foundation (DCD), and 7) NARSAD (National Alliance for Research on Schizophrenia and Depression) Young Investigator Award (EZ); JS discloses equity interest in Molecular Neuroimaging.

We wish to acknowledge the critical contributions to this research program made by the research staff of the Biological Studies Unit, West Haven VA Medical Center, including Elizabeth O?Donell, R.N.; Angelina Genovese, R.N.; Gina Mcmanus, R.N.; Robert Sturwold, RPh. We would also like to thank Mr. Jason Fletcher for his assistance with statistical analyses.
Address reprint requests to: Deepak Cyril D?Souza, M.D., VA Connecticut Healthcare System 116A, Psychiatry Service, 950 Campbell Avenue, West Haven, CT 06516
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## comfortably numb (Mar 6, 2006)

Holy shat that was a long read but an interesting one. I admit that i scipped over some of it due to my short attention span and the fact that im now hungry.

Ive heard of mcpp before i believe it's making the rounds in some places as a adulterant in ecstasy tabs. Ive read some trip reports of it and it causes nasty side effects in some people. It's also a metabolite of trazodone but i dont know if it's responsible for any of trazodone's anti-depressant effects.

This article might point us in the right direction of why some people get dp/dr from serotonic drugs. Mcpp does seem to produce bad reactions in alot of people from what ive read though. Maybe they should do a test with a more agreeable 5-ht2a agonist.


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