Decreased serotonin2C receptor responses in male patients with schizophrenia

Psychiatry Research 226 (2015) 308–315

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Decreased serotonin2C receptor responses in male patients with schizophrenia Myung Ae Lee a,b, Karuna Jayathilake c, Min Young Sim d, Herbert Y. Meltzer c,n a

Department of Psychiatry, School of Medicine, Vanderbilt University, Nashville, TN, USA Tennessee Valley VA Healthcare System, Nashville Campus, Nashville, TN, USA c Department of Psychiatry and Behavioral Sciences, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA d Department of Psychiatry, Seoul National Hospital, Seoul, South Korea b

art ic l e i nf o

a b s t r a c t

Article history: Received 4 June 2014 Received in revised form 6 January 2015 Accepted 10 January 2015 Available online 15 January 2015

Serotonin (5-HT)2C receptors in brain affect psychosis, reward, substance abuse, anxiety, other behaviors, appetite, body temperature, and other physiological measures. They also have been implicated in antipsychotic drug efficacy and side effects. We previously reported that the hyperthermia following administration of MK-212, a predominantly 5-HT2C receptor agonist, was diminished in a small sample of patients with schizophrenia (SCH), suggesting decreased 5-HT2C receptor responsiveness. We have now studied the responses to oral MK-212 and placebo in a larger sample of unmedicated male SCH (n ¼69) and normal controls (CON) (n ¼33), and assessed the influence of comorbid substance abuse (SA) on oral body temperature, behavioral responses, etc. The placebo-adjusted oral body temperature response to MK-212 was significantly lower in SCH compared to CON and not significantly different between the SCH with or without SA. Some behavioral responses to MK-212, e.g. self-rated feelings of increased anxiety, depression and decreased calmness, or good overall feeling, were significantly lower in the SCH patients compared to CON. These results add to the evidence for diminished 5-HT2C receptor responsiveness in SCH patients compared to CON and are consistent with reported association of HTR2C polymorphisms, leading to decreased expression or function of the HTR2C in patients with SCH. & 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Serotonin 5-HT2C MK-212 Schizophrenia Temperature Anxiety

1. Introduction Serotonin (5-HT) has long been implicated in the pathophysiology and treatment of schizophrenia, beginning with the endogenous indole hallucinogen hypothesis (Rosengarten and Friedhoff, 1976), followed by numerous reports of altered 5-HT synthesis and metabolism, abnormalities in the levels of brain 5HT1A and 5-HT2A receptors, editing of 5-HT2C receptors, and coding or expression abnormalities in genes involved in the synthesis, metabolism and response to indoles and other compounds derived from tryptophan, the precursor of 5-HT (Bleich et al., 1988; Tang et al., 2014). The extensive and powerful mutual influences of 5-HT and dopamine (DA), two of the most important neurotransmitters required for reality testing, cognition, mood, motor function, and regulation of core body functions, such as energy expenditure, circadian rhythms, and endocrine function on the activity of each, led, in part, to an explicit 5-HT-DA hypothesis of schizophrenia (Meltzer et al., 1989; Meltzer and Nash,1991; van Veelen and Kahn, n Correspondence to: Feinberg School of Medicine, Northwestern University, 303 E. Chicago Avenue, #12-104, Chicago, IL 60611, USA. Tel.: þ 1 312 503-0309; fax: þ1 312 503 0348. E-mail address: [email protected] (H.Y. Meltzer).

http://dx.doi.org/10.1016/j.psychres.2015.01.007 0165-1781/& 2015 Elsevier Ireland Ltd. All rights reserved.

1999; Beaulieu, 2012; Carli and Invernizzi, 2014). Thus, it was suggested that the effect of 5-HT at various 5-HT receptors on the release and response to DA, was a crucial component of the superiority of clozapine to typical antipsychotic drugs for a variety of aspects of schizophrenia, including psychosis, without extrapyramidal side effects and eventually suicide and cognition (Meltzer et al., 1989; Meltzer and Huang, 2008). While the emphasis was initially on 5-HT2A receptors, other 5-HT receptors, including the 5-HT1A, 5-HT2C, and 5-HT7 receptors, were subsequently implicated in the treatment and pathophysiology of schizophrenia through their actions on cholinergic, noradrenergic, glutamatergic and GABAergic neurons, as well as DA neurons (Meltzer and Huang, 2008; Meltzer et al., 2012). It is of interest that selective 5-HT2A inverse agonists, e.g. pimavanserin and SR43459B, have been shown to be effective in acutely psychotic schizophrenia patients as augmentation of atypical antipsychotic drugs, or stand alone treatments (Meltzer et al., 2004, 2012a, 2012b). Many other 5-HT-related treatments for schizophrenia are in development, e.g. 5-HT2C agonists, (to be reviewed). Because of the heterogeneity of schizophrenia and the overlap of specific type of symptoms of schizophrenia with those of other neuropsychiatric disorders, including bipolar disorder, fronto-temporal dementia, and Alzheimer's disease, it is likely that only subgroups of

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patients who meet criteria for schizophrenia will manifest specific type of genetic and epigenetic 5-HT abnormalities and respond to serotonergic treatments (e.g. Iwamoto et al., 2009; Tang et al., 2014 ) We have previously reported on a subgroup of schizophrenic patients with abnormal responses to 5-HT2C receptor stimulation (Lee et al. 1992). The expression of 5-HT2C receptors has been reported to be markedly decreased in the prefrontal cortices of unmedicated (Castensson et al., 2003; Iwamoto et al., 2004) or medicated (Castensson et al., 2005) patients with SCH at the time of death, based on mRNA levels, although there is one non-replication (Dracheva et al., 2003). The serotonin (5-HT)2C receptor is one of 13 5-HT G-protein coupled receptors and is highly expressed throughout the brain, including on cell bodies or terminals of cortical, and limbic pyramidal glutamatergic neurons and γ-aminobutyric acid (GABA) interneurons (Eberle-Wang et al., 1997; Berg et al., 2008; Labonte et al., 2009; Bubar et al., 2011). 5-HT2C receptors are also present on cell bodies of ventral tegmental and nigro-striatal dopamine (DA) and acetylcholine (ACh) neurons, which is the basis for a major impact of 5-HT on reality testing, cognition, motor function, reward and substance abuse (Di Matteo et al., 2002; Bonsi et al., 2007; Berg et al., 2008). Stimulation of 5-HT2C receptors in the ventral tegmenum decreases dopamine (DA) release in the nucleus accumbens (Di Giovanni et al., 2000; Berg et al., 2008), which contributes to their efficacy in treating psychotic symptoms in patients with schizophrenia (SCH) (Rosenzweig-Lipson et al., 2012). On the other hand, stimulation of 5-HT2C receptor also decreases DA release in the cortex (Millan et al.1998), which might have an adverse effect on some types of cognition by decreasing DA-dependent signaling that is crucial for working memory (Meltzer and Huang, 2008; Huang et al., 2011). Some atypical antipsychotic drugs, e.g. clozapine, olanzapine, risperidone, and sertindole, are potent inverse agonists of 5-HT2C receptors, indicating blockade of its constitutive activity, whereas others, e.g. lurasidone, quetiapine, and aripiprazole, have weak direct effects on 5-HT2C receptors but still affect them through increasing DA release (Meltzer and Huang, 2008). Some typical antipsychotic drugs, e.g. chlorpromazine, thioridazine, spiperone, and thiothixene, are 5-HT2C neutral antagonists (Rauser et al., 2001; Kroeze et al., 2003). While 5-HT2A receptor agonism has been shown to have a major role in the action of hallucinogens, e.g. 1-(2,5-dimethoxy-4iodophenyl)-2-aminopropane (DOI) and D-lysergic acid diethylamide (LSD), 5-HT2C agonism may alsocontributess to the psychotomimetic action of DOI (Canal et al., 2010). Nevertheless, other 5-HT2C receptor agonists, because of functional selectivity (Moya et al. 2007), lack psychotomimetic properties, and also show antipsychotic drug-like action in animal models of psychosis, such as amphetamine-induced locomotor activity and pre-pulse inhibition (Marquis et al., 2007; Siuciak et al., 2007). The 5-HT2C agonist, vabicaserin, has been reported to have an antipsychotic effect in patients with SCH (Rosenzweig-Lipson et al., 2012). In addition, 6-chlor-2[1-piperazinyl]-pyrazine (MK-212), a predominantly 5-HT2C agonist, has been reported to cause anxiety when injected in the ventral hippocampus (Alves et al., 2004). Stimulation of 5-HT2C receptors in the basolateral amygdala increases anxiety (Zangrossi and Graeff, 2014). Furthermore, 5-HT2C receptors have been shown to be involved in substance abuse, most likely based upon their ability to suppress mesocorticolimbic DA neurotransmissions (Pentkowski et al., 2010; Filip et al., 2012 Cunningham et al., 2013). Preclinical studies suggest 5-HT2C agonists may be beneficial for the treatment of cocaine craving and relapse (Pentkowski et al., 2010; Filip et al., 2012; Cunningham et al., 2013). Furthermore, the cys23ser single nucleotide polymorphism (SNP) of the 5-HT2C receptor gene has been reported to affect attentional bias to cocaine cues in patients with cocaine dependence (Anastasio, et al., 2014). Parallel

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to sharing neurochemical mechanisms involved in SCH and substance abuse, the rate of comorbid substance use disorders in patients with SCH has been reported to be significantly higher than the general population (Reiger et al., 1990; Hartz et al., 2014). 5-HT2C agonists increase body temperature in man (Lee et al., 1992) and laboratory animals (Gudelsky et al., 1986; Hayashi et al., 2004). Furthermore, 5-HT2C receptors are involved in feeding behavior (Hayashi et al., 2005). 5-HT2C receptors in proopiomelanocortin neurons regulate energy and glucose homeostasis (Berglund et al., 2013). We reported that the oral body temperature increase following administration of MK-212 was markedly decreased in a substantial proportion of a sample of male and female patients with SCH (n ¼23) compared to normal controls (CON) (n¼ 22) (Lee et al., 1992), as well as in male patients with cocaine use disorder (n ¼10) compared to male CON (n ¼28) (Lee and Meltzer, 1994). Therefore it was of interest to determine the relationship between the decreased oral body temperature response in patients with SCH and comorbid substance abuse. MK-212 was shown to behave as a functionally selective 5-HT2C agonist, as indicated by its ability to suppress cortical DA efflux only in 5-HT2A knockout mice (Huang et al., 2011). MK-212 also increased cortical acetylcholine efflux via a 5-HT2C agonist mechanism (Nair and Gudelsky, 2004). 5-HT2A agonism is also the basis for stimulating corticosterone secretion in the rat by MK212 (Hemrick-Luecke and Fuller, 1996). The primary purpose of this study was to obtain additional information about the comparative effect of MK-212 on oral body temperature, endocrine and behavioral responses in male patients with SCH following MK-212. We also examined whether the responses to MK-212 were related to comorbid substance abuse.

2. Methods 2.1. Subjects Sixty nine male patients [mean age: 33.77 6.9(S.D.) years; range: 21–61 years] meeting DSM-IV criteria (APA, 1994) for SCH or schizoaffective disorder were studied. Diagnosis was established on the basis of the Schedule for Affective Disorder and Schizophrenia Lifetime (SADS-L) and Change (SADS-C) versions (Endicott and Spitzer, 1978). Thirty-nine patients had no history of substance abuse (SCH-NSA) and 30 patients had current or past history of substance abuse (SCH-SA) (mostly alcohol and/or cocaine). Thirty-three male CON [mean age: 28.6 7 7.4 years; range:19–46 years], who did not have any psychiatric illness or substance abuse in themselves, based on interview with SADS-L (Endicott and Spitzer, 1978), and their first-degree relatives were studied. Twenty male patients and 11 male CON included in the prior study, which reported the decreased oral body temperature response to MK-212 in male and female SCH (Lee et al., 1992), were included in this study, as the prior study did not examine impact of substance abuse on oral body temperature responses to MK-212. All subjects were screened for any significant medical, neurological, neuroendocrine conditions and substance abuse problems by history, physical examination and comprehensive blood and urine tests, including urine drug screen. Also, height and weight were measured to assess the effect of body mass index [BMI: weight (kg)/height (m)2] on the responses to MK-212. Demographic and clinical data on patients with SCH and CON are presented in Table 1. Patients were psychotropic drug-free (e.g. haloperidol, fluphenazine, etc.) for at least 7 days before MK-212 or placebo administration, either because of selfwithdrawal prior to admission to the study or in order to participate in the studies. No patient had received a depot neuroleptic within two cycles of their administration schedule to facilitate unmedicated status. Patients with substance abuse were required free of substance abuse at least 3 months prior to entering to the study. Patients were closely monitored with supportive care during the psychotropic drug-free period to detect early signs of deterioration, and to provide an appropriate clinical care after withdrawing patients from the study. This protocol was approved by Institutional Review Board of the University Hospital of Cleveland. Written informed consents were obtained from all patients after complete explanation of the protocol before admission to the study.

2.2. Psychopathology assessment Severity of psychopathology was evaluated at baseline by using Brief Psychiatric Rating Scale (BPRS) (Overall and Gorham, 1962) (0–6 scales), and BPRS total,

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and subscales of BPRS positive, withdrawal-retardation, and anxiety–depression were examined. 2.3. Challenge studies with MK-212 or placebo Subjects were fasted after midnight on the night before the testing day (fasted at least 8 h). On the following morning, at 8 AM, an indwelling catheter was inserted in a forearm vein. One hour later, a baseline blood sample (T0) was collected into a heparinized syringe. Immediately after, either a placebo or MK-212, 20 mg, pill was administered orally, in a randomized single-blind design. MK-212 and placebo studies were separated by at least 48 h. Additional blood samples were collected at 30-min intervals for a 3-h period. The subject was kept at bed rest during the study, except for bathroom use. Subjects were informed about how to make self-rating of behavior using visual analog scales (0–10 scale). These were completed for feeling hungry, sleepy, nauseated, dizzy, calm, anxious, irritable, depressed, good overall (a general feeling of well being), strange, and aroused. Some subjects did not comply with this so ratings were not available for all subjects.

Table 2 Baseline oral body temperature and plasma cortisol concentration, and the oral body temperature and plasma cortisol responses to MK-212 in patients with schizophrenia and normal controls.

Baseline Oral temperature (1C)a Cortisol (μg/dl)b

Schizophrenia (n ¼69)

Normal controls (n¼ 33)

36.5 7 0.4 (60) 12.2 7 3.4

36.3 7 0.3 (32) 10.17 5.1

Response to MK-212 (cm2)c Oral temperatured 0.26 7 1.70 (56) Cortisol 13.5 724.4 (63)

1.357 1.71 (30) 18.4 7 28.1

Mean 7S.D.; (n): number of subjects. a

Model: F(2,89) ¼3.77, P ¼0.03; Group: P¼ 0.008. Model: F(2,99) ¼ 3.54, P¼ 0.03; Group: P ¼0.009. c Placebo adjusted area under the curve (AUC). d Model: F(4,81) ¼ 4.09, P¼ 0.005; Group: P¼ 0.02. b

2.4. Oral body temperature measurements Oral body temperature was measured with an electronic digital thermometer (IVAC TEMP.PLUS II) at the same time as blood collections. Accuracy of the thermometer as tested in a calibrated water bath was 7 0.1 1C. The thermometer also self-checked calibration with each use. Subjects were not allowed to smoke or eat during the study period, but could drink water immediately after each temperature measurement if desired.

SCH and CON. All main effects were tested at a two-tailed α level of 0.05. All analyses were performed using SASTM (SAS Institute, Inc., Cary, NC, USA) statistical software.

2.5. Hormone assays

3.1. Age, weight, BMI, and baseline oral body temperature and plasma cortisol comparisons

Plasma was separated and kept frozen at –20 1C until assayed within 1 month of collection. The plasma cortisol concentrations were measured by double antibody radioimmunoassay (Murphy, 1967). The kit used to measure plasma cortisol was purchased from Diagnostic Products (Los Angeles, CA). The within and between assay coefficients for variation for the plasma cortisol measurements were less than 5%. 2.6. Statistical analysis The oral body temperature and plasma cortisol responses to MK-212 were determined by area under the curve (AUC), subtracting the placebo response. The univariate analysis of covariance (ANCOVA) was used to compare oral body temperature and cortisol responses to MK-212 between groups. Age, BMI and baseline oral body temperature and cortisol values were covaried as age, baseline oral body temperature and plasma cortisol were significantly different between patients with SCH and CON (Tables 1 and 2). The least significant difference test was used as a post-hoc test of significance when the overall F-Test was significant. The protocol effect of MK-212-stimulated oral body temperature and cortisol responses was evaluated by using univariate ANOVA with repeated measures. The multiple regression analysis was applied to predict psychopathology at baseline from MK-212-stimulated oral body temperature and plasma cortisol responses, as well as age, in patients with SCH. The Pearson Product Moment Correlation Coefficient was applied to examine relationships between oral body temperature and cortisol responses to MK-212. The behavioral response (visual analog measurements) was analyzed the same way as for oral body temperature and cortisol analyses. The multiple regression analysis was applied to predict behavioral responses from MK-212-stimulated oral body temperature and cortisol responses, as well as baseline behavioral scores. ANCOVA was applied with age covariation to compare weight, BMI, baseline oral body temperature and cortisol values between

Table 1 Demographic and clinical data in patients with schizophrenia and normal controls.

n

Age (years) Weight (lbs) Body Mass Index (kg/m2) Duration of Illness (years) BPRS total Positive Withdrawal-Retardation Anxiety–depression

Schizophrenia (n¼ 69)

Normal Controls (n¼33)

33.77 6.9 175.9 7 30.4 25.17 4.0 12.8 7 7.3 (68) 28.7 7 13.4 (68) 10.0 7 5.2 (68) 4.9 7 3.8 (68) 5.4 7 4.1 (67)

28.6 7 7.4 174.2 7 29.7 24.873.7

Mean 7 S.D.; (n): number of subjects; BPRS: Brief Psychiatric Rating Scale. n

t¼  3.35, d.f. ¼ 59.3, P¼ 0.001.

3. Results

Patients with SCH were significantly older than CON (t¼  3.35, d.f. ¼100, P ¼0.001) (Table 1). The averaged difference was 5.1 years. Weight and BMI were not significantly different between groups (Table 1). Baseline oral body temperature [Model: F(2,89) ¼ 3.77, P ¼0.03; Group: p¼ 0.008] and plasma cortisol concentration [Model: F(2,99) ¼ 3.54, P ¼0.03; Group: P¼ 0.009] were significantly higher in patients with SCH compared to CON (Table 2). There was no significant difference between SCH-SA and SCH-NSA in any of above variables (data not shown). 3.2. Oral body temperature and plasma cortisol responses to MK-212 in patients with schizophrenia and normal controls Oral body temperature and plasma cortisol responses to MK212 in patients with SCH and CON are given in Table 2. MK-212 significantly increased oral body temperature in CON [F(1,29) ¼ 18.62, P ¼0.0002], but did not do so in the whole SCH patient group [F(1,56)¼ 2.17, P ¼0.15], or those with and without substance abuse [SCH-NSA: F(1,30)¼2.55, P¼ 0.12; SCH-SA: F(1,24)¼ 0.05, P¼ 0.83]. The oral body temperature response to MK-212 was significantly lower in all SCH patients compared to CON [Model: F(4,81) ¼4.09, P ¼0.005; Group: P¼ 0.02] (Fig. 1). SCH-SA patients (n ¼25) had a significantly decreased oral body temperature response to MK-212 compared to CON [Model: F(5,80) ¼3.70, P¼ 0.005; Group: P ¼0.02; SCH-SA s. NC: P ¼0.006]. There was a trend in the same direction for the SCH-NSA patients (n ¼31) (SCH-NSA vs. CON: P ¼0.1) (Fig. 2). Importantly, there was no significant difference in MK-212-stimulated oral body temperature responses between SCH-NSA and SCH-SA [0.40 71.40 (S.D.) cm2 vs. 0.09 72.03 cm2, P ¼0.2]. Seven out of 30 (23.3%) CON, 11/31 (35.5%) SCH-NSA, and 12/25 (48.0%) SCH-SA had no increase in oral body temperature following MK-212 administration, which were not significantly different (data not shown). MK-212 significantly increased plasma cortisol concentrations in all SCH patients [F(1,63)¼20.52, P ¼0.0001] and CON [F(1,32) ¼ 14.11, P ¼0.0007], as well as SCH-NSA [F(1,38)¼ 8.06, P ¼0.007]and SCH-SA [F(,24)¼13.35, P ¼0.001]. The plasma cortisol response to MK-212 was not significantly different between total patients with

M.A. Lee et al. / Psychiatry Research 226 (2015) 308–315

SCH and CON [Model: F(4,91) ¼6.34, P¼ 0.0002; Group: P ¼0.55]. Similarly, there was no significant difference in cortisol responses to MK-212 in CON, SCH-NSA and SCH-SA [F(5,90) ¼5.62, P ¼0.0001; Group: P¼ 0.26].

Oral Temperature (˚C)

36.7 36.6 36.5 36.4 36.3 36.2

Time (min)

36.1 36

0

30

60

MK-212 CON

PLAC CON

MK-212 SCH

PLAC SCH

90

120

150

180

Time (min) Fig. 1. The oral body temperature response to MK-212 in patients with schizophrenia (SCH) and normal controls (CON). SCH vs CON: Model: F(4,81) ¼ 4.09, P¼ 0.005; Group: P ¼0.02.

Tempetraure response AUC (cm2)

6

4

2

0

-2

-4

-6

Normal Controls

SCH -NSA

(n =30)

(n=31)

311

3.3. Behavioral responses to MK-212 As behavioral responses to MK-212 in SCH-NSA and SCH-SA were not significantly different between groups, analyses were performed for all patients with SCH. At baseline, patients with SCH (n¼42) were more symptomatic than CON (n¼28) on a number of measures. Patients rated themselves significantly higher at baseline on the following items: feeling nauseated [Model: F(2,66)¼6.24, P¼ 0.003; Group: P ¼0.0008], dizzy[Model: F(2,67)¼5.64, P ¼0.006; Group: P¼ 0.002], anxious [Model: F(2,67) ¼9.17, P¼ 0.0003; Group: P¼ 0.0004], irritable [Model: F(2,67)¼9.69, P ¼0.0002; Group: P¼ 0.0001], depressed [Model: F(2,67) ¼9.88, P¼ 0.0002; Group: P¼ 0.0001], strange [Model: F(2,67) ¼7.18, P ¼0.002; Group: P¼ 0.0009], and aroused [Model: F(2,67)¼7.66, P ¼0.001; Group: P¼ 0.001] compared to CON (Table 3). In SCH patients w (n ¼ 36), only nausea significantly increased following MK-212 [F(1,33)¼4.68, P¼ 0.04]. In CON (n ¼25), MK212 significantly decreased self-ratings of hungry [F(1,24) ¼ 4.99, P¼ 0.04], calm [F(1,24)¼6.67, P¼ 0.02] and good overall [F(1,24) ¼ 17.52, P ¼0.0003], and increased ratings of nausea [F(1,24)¼14.76, P¼ 0.0008], dizzy [F(1,24)¼4.71, P¼ 0.04], irritable [F(1,24) ¼ 4.36, P¼ 0.05] and strange [F(1,24) ¼ 6.79, P ¼0.02]. Thus, the hunger, calming, overall good feeling responses to MK-212 were diminished in the SCH patients compared to the controls. In comparison to CON, patients with SCH showed significantly smaller changes in response to MK-212 on ratings of feeling calm [Model: F(4,56) ¼ 3.22, P¼ 0.02; Group: P ¼0.004], anxious [Model: F(4,56) ¼2.50, P¼ 0.05; Group: P ¼0.02], depressed [Model: F(4,56) ¼2.66, P¼ 0.04; Group: P ¼0.01], and feeling good overall [Model: F(4,56) ¼ 3.70, P ¼0.01; Group: P ¼0.004] (Table 3). In patients with SCH, the oral body temperature response to MK-212 showed a trend for a negative relationship with ratings of feeling calm [Model: F(2,28) ¼3.30, P ¼0.05; β¼  1.73, P ¼0.06], and a trend for a positive relationship with ratings of anxious [Model: F(2,28) ¼2.64, P ¼0.09; β ¼2.32, P¼ 0.04]. In CON, the plasma cortisol response to MK-212 showed a significant negative relationship with self-ratings of feeling hungry [Model: F(2,22) ¼ 4.66, P ¼0.02; β¼ 0.23, P ¼0.01], and significant positive relationships with dizzy [Model: F(2,22) ¼8.43, P ¼0.002; β¼ 0.23, P¼ 0.0007] and depressed [Model: F(2,22) ¼3.61, P ¼0.04; β¼0.11, P¼ 0.02].

SCH - SA* (n =25 )

Fig. 2. Comparison of the oral body temperature response to MK-212 in schizophrenic patients with and without substance abuse and normal controls. *: Model: F(5,80) ¼ 3.70, P¼ 0.005; Group: P¼ 0.02; SCH-SA vs CON: P¼ 0.006; SCH-NSA vs CON: P¼ 0.1, SCH: schizophrenia; SCH-NSA: schizophrenia without substance abuse; SCH-SA: schizophrenia with substance abuse, vertical bars are S.D.s.

3.4. Relationships between the oral body temperature and cortisol responses to MK-212, and the baseline psychopathology There was no significant correlation between BPRS total and subscale ratings, and MK-212-stimulated oral body temperature or

Table 3 Behavioral responses to MK-212 in patients with schizophrenia and normal controls. Baseline (0–10 scale)

Hungry Sleepy Nauseated Dizzy Calm Anxious Irritable Depressed Good overall Strange Aroused a

SCH vs. CON (P)

MK-212 Response (AUCa cm2)

Schizophrenia (n¼ 42)

Normal Controls (n¼ 28)

Schizophrenia (n¼ 36)

Normal Controls (n¼ 25)

5.2 7 3.0 4.17 3.2 2.17 2.4 2.5 7 3.2 4.8 7 3.0 3.3 7 2.8 3.17 2.9 3.6 7 3.1 5.5 7 2.9 2.6 7 2.9 2.2 7 2.3

3.4 7 2.6 3.3 7 2.5 0.3 7 0.6 0.4 7 0.6 6.5 7 2.6 1.0 7 1.1 0.5 7 0.8 0.6 7 1.0 6.3 7 2.6 0.5 7 0.9 0.5 7 1.0

 1.3 7 13.0 0.2 7 13.5 4.3 7 11.5  0.3 7 9.9  0.7 7 7.3  2.17 9.0  0.3 7 14.2  1.2 7 9.9  2.2 7 7.9 0.4 7 10.7  0.4 7 10.9

 6.17 13.7  1.6 7 9.8 8.7 7 11.3 4.5 7 10.3  6.6 7 12.8 2.8 7 8.7 4.6 7 10.9 1.9 7 6.8  9.17 10.8 7.0 7 13.5  1.0 7 4.6

Placebo adjusted area under the curve (AUC).

o 0.001 0.002 o 0.001 o 0.001 o 0.001 o 0.001 0.001

SCH vs. CON (P)

0.004 0.02 0.01 0.004

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cortisol responses at baseline in total patients with SCH, as well as in SCH-SA or SCH-NSA (data not shown). 3.5. Correlations between oral body temperature and cortisol responses to MK-212, and impact of the drug washout period on oral body temperature and cortisol responses to MK-212 There was no significant correlation between oral body temperature and plasma cortisol responses to MK-212 either in CON (r ¼  0.12, n ¼30, NS) or patients with SCH (r ¼0.14, n ¼52, NS). There was no significant correlation between the drug washout period and oral body temperature (r¼  0.14, n¼50, NS) and cortisol (r¼0.12, n¼ 56, NS) responses to MK-212 in patients with SCH.

4. Discussion The major findings of this study are: (1) the oral body temperature response to MK-212 was significantly decreased in patients with SCH compared to CON; (2) the oral body temperature response to MK-212 was not significantly different between SCH-NSA and SCH-SA; (3) MK-212 produced decreased feeling of hunger in CON but not SCH patients; (4) MK-212 produced significantly less dysphoric mood (e.g. less anxious or depressed) in patients with SCH compared to CON; and (5) there was no significant difference in the plasma cortisol response to MK-212 between patients with SCH and CON. The results reported here replicate and extend our previous report that the oral body temperature response to MK-212 was decreased in the SCH patient group as a whole. Kahn et al. (1992) reported decreased temperature responses to oral meta-chlorophenylpiperazine (m-CPP) (0.35 mg/kg) in patients with SCH compared to CON, but we found that m-CPP (0.5 mg/kg), did not increase oral body temperature in either SCH or CON (Maes and Meltzer, 1996). MK-212 is more selective for 5HT2C receptors than m-CPP (Kahn et al. 1992). The reported here indicate the decreased oral body temperature response to MK-212 is not related to substance abuse status. In addition, our findings of no difference in the plasma cortisol responses to MK-212 between male SCH and male CON is consistent with most, but not all (Iqbal et al., 1991) previous studies reporting no difference in the plasma cortisol responses to m-CPP in male patients with SCH compared to male CON (Kahn et al., 1992; Krystal et al., 1993; Maes and Meltzer, 1996). Thus, from a physiological point of view, the diminished response of 5-HT2C receptors may be restricted to only some functions. Further study with relate to energy metabolism is of particular interest. We found no relation between the temperature responses and body mass index (data not presented). The decreased behavioral response to MK-212 in patients with SCH is in contrast to our prior report in a small sample with MK-212 (Lee et al., 1992), and with m-CPP using 0.5 mg/kg (Maes and Meltzer, 1996), which did not show significant difference in behavioral responses between groups. The main reasons may likely to be sample size, the greater specificity of MK-212 for 5-HT2C receptors than m-CPP, and the dosage of m-CPP used. The pattern of which behavioral responses to MK-212 were less in the SCH patients compared to controls suggests there could be clinical significance to 5-HT2C receptor responses to 5-HT2C receptor stimulation. Animal studies support the conclusion that MK-212-stimulated temperature and cortisol responses are mediated by 5-HT2C and 5HT2A receptors, respectively. MK-212-stimulated hyperthermia in rats is partially blocked by ketanserin and spiperone, which are predominantly 5-HT2A receptor antagonists, and completely blocked by mianserin and pizotifen, which are mixed 5-HT2A/2C antagonists with higher affinity for 5HT2C than 5-HT2A receptors (Gudelsky et al., 1986). Hemrick-Luecke and Fuller (1996) reported that the MK-212-stimulated corticosterone

secretion in rats is mediated via 5-HT2A receptor stimulation, as it was blocked by the selective 5-HT2A inverse agonist M 100,907 (R-(þ )-α(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyletthyl)]-4-piperidinemethanol), but not by the selective 5-HT2C neutral antagonist SB 200646 [N(1-methyl-5-indolyl)–N-(3-pyridyl)urea]. These studies suggest that the MK-212-stimulated cortisol response may be mediated via 5-HT2A receptor stimulation, whereas stimulation of 5-HT2C receptors mediate the MK-212-stimulated hyperthermic response. Additional evidence for the differences in the mechanisms controlling temperature and hormonal responses is that 5-HT neurons in the dorsal raphe nucleus, preoptic area and the dorsomedial hypothalamus have been reported to be involved in body temperature regulation in rodents (DiMicco and Zaretsky, 2007; Lowry et al., 2009; Clapham, 2012), whereas rodent neuroendocrine responses to 5-HT agonists have been reported to be mediated via 5-HT receptors in the hypothalamic paraventricular nucleus (Zhang et al., 2002). Thus, the decreased oral body temperature and behavioral responses, but normal cortisol responses, to MK-212, in male patients with SCH may be related to differences in which 5-HT2 receptors mediate these responses in different brain regions (Fantegrossi et al., 2010). Variation in responses between subjects for both temperature and behavior could be related to differences in expression and editing of 5-HT2C receptors in different brain regions (see Section 1). 5-HT2C receptors exhibit constitutive activity and have multiple isoforms via RNA editing, which reduces receptor activities (Berg et al., 2008). RNA editing of the 5-HT2C receptors has been reported to be reduced (Sodhi et al., 2001) or normal (Dracheva et al., 2003) in the postmortem studies. However, no abnormalities in editing of 5-HT2C receptors in schizophrenia patients have been found in the most definitive study of this important issue (Zhu et al., 2012). In addition, differences in the complex 5-HT2C signaling mechanisms could lead to differences between subjects (see Werry et al., 2008 for review). Further research on regional differences in 5-HT2C receptor density, editing, and function, especially signaling, is needed. Significantly enhanced DA release in the striatum following an amphetamine challenge has been reported in patients with SCH (Laruelle, et al., 1996; Breier et al., 1997; Abi-Dargham et al., 2009). 5-HT2C receptor stimulation inhibits DA release in the nucleus accumbens via activation of GABA and inhibition of DA neurons in the ventral tegmental area (Bubar et al., 2011). Thus, reduced 5HT2C receptor function in patients with SCH may mediate enhanced mesoaccumbal DA release in response to amphetamine challenge. In addition, 5-HT2C receptors may be involved in striatal DA release and hypothalamic–pituitary–adrenal axis activation in response to stress. For example, the non-synonymous, putatively functional cys23ser single nucleotide polymorphism (SNP) of the 5-HT2C receptor gene has been reported to predict pain-induced striatal DA release (Mickey et al., 2012), and cortisol responses to psychological stress in humans (Brummett et al., 2012); ser23 carriers were reported to have enhanced DA (Mickey et al., 2012) and cortisol release (Brummett et al., 2012) in response to stressors, suggesting the ser form is less active than the cys. Furthermore, amphetamine-induced striatal DA release was reported to be related to cortisol responses to psychological stress (Wand et al., 2007). Thus, impaired 5-HT2C receptor function may be related to an abnormal hypothalamic–pituitary–adrenal axis responses and DA release in response to stress reported in SCH (Holtzman et al., 2013; Mizrahi et al., 2012), which may also contribute to an increase in risk for substance abuse (Piazza et al., 1990; Wise and Morales, 2010) in patients with SCH. A family based study reported that the cys23ser SNP was not associated with SCH (Murad et al. 2001). However, this does not rule out differences in 5-HT2C gene expression or editing as a possible basis for the results reported here.

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Consistent with reports of the importance of 5-HT2A and 5HT2C receptors in depression/anxiety ( Alves et al., 2004; Holmes, 2008; Zangrossi and Graeff, 2014), and diminished 5-HT2C activity in SCH (Lee et al., 1992; Kahn et al., 1992), MK-212 produced significant increases in dysphoric mood/anxiety in CON, but not in patients with SCH. In fact, patients with SCH reported nonsignificant improvement in anxiety and depression. This difference does not appear to be related to MK-212-induced physical symptoms, as there was no significant difference in self-reports of feeling sleepy, nauseous or dizzy in response to MK-212 between groups. In CON, the plasma cortisol response to MK-212 showed significant positive correlation with dysphoric mood. On the other hand, in patients with SCH, the oral body temperature response to MK-212 showed a trend for a positive relationship with anxiety, linking to 5-HT2C receptor stimulation. Thus, these results suggest that the relationship between 5-HT2C receptor stimulation and mood is disrupted in patients with SCH. A weakness of this study is that it utilized a fixed dose of MK212. To compare, we included a BMI adjustment in the analysis. The decreased oral body temperature and behavioral responses to MK-212 in patients with SCH, compared to CON, could be due to residual psychotropic drug treatment effect, pharmacokinetic difference between patients and CON, or pharmacodynamic difference. The absence of a correlation between the drug washout period, and the oral body temperature and cortisol responses to MK-212, suggest that prior treatment with psychotropic drugs was not contributory. Nash et al. (1989) have reported that MK212-stimulated corticosterone secretion and hyperthermic responses were unaffected by typical neuroleptics in rats. Although, we did not measure plasma concentration of MK-212 in this study, the plasma cortisol response to MK-212 was not significantly different between patients with SCH and CON, suggesting MK-212 concentrations were not significantly different in the patients and controls. In addition, there was no difference in body weight or BMI between groups, which might have influenced drug concentrations in brain. Thus, the decreased oral body temperature and differences in behavioral responses to MK-212 in patients with SCH compared to CON is most consistent with a pharmacodynamic difference between the two groups, particularly with regard to 5-HT2C receptor expression or function. In summary, the results of this study suggest selective reductions in 5-HT2C receptor function in patients with SCH, as assessed by the oral body temperature and behavioral responses to MK-212. Diminished 5-HT2C receptor function may be related to the increased rate of substance abuse in patients with SCH (Regier et al., 1990; Hartz et al., 2014), possibly related to some of the altered behavioral response to 5-HT2C receptor stimulation noted here. The molecular basis for this hypofunction of the 5-HT2C receptor requires further investigation. It does not appear to be due to differences in editing of the 5-HT2C receptor. Decreased expression of the 5-HT2C receptor in specific brain regions seems more likely. Additional post-mortem studies is indicated. The results reported here provide further rationale for studying 5-HT2C receptor agonists such as vabicaserin or lorcaserin in the treatment of schizophrenia.

Acknowledgments This research was supported in part by USPHS (MH 41684 and MH 41594), GCRC (MO1RR00080), the U.S. Department of Veterans Affairs, and contributions from the Weisman and Ritter Family Foundations.

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