CSF dopamine is elevated in first-episode psychosis and associates to symptom severity and cognitive performance

Background: The hypothesis of dopamine dysfunction in psychosis has evolved since the mid-twentieth century. However, clinical support from biochemical analysis of the transmitter in patients is still missing. The present study assessed dopamine and related metabolites in the cerebrospinal fluid (CSF) of first-episode psychosis (FEP) subjects. Methods: Forty first-episode psychosis subjects and twenty healthy age-matched volunteers were recruited via the Karolinska Schizophrenia Project, a multidisciplinary research consortium that investigates the pathophysiology of schizophrenia. Psychopathology, disease severity, and cognitive performance were rated as well as cerebro- spinal fluid concentrations of dopamine and related metabolites were measured using a sensitive high-pressure liquid chromatography assay. Results: CSF dopamine was reliably detected in 50 % of healthy controls and in 65 % of first-episode psychosis subjects and significantly higher in first-episode psychosis subjects compared to age-matched healthy controls. No difference in CSF dopamine levels was observed between drug-naive subjects and subjects with short exposure to antipsychotics. The dopamine concentrations were positively associated with illness severity and deficits in executive functioning. Conclusions: Dopamine dysfunction has long been considered a cornerstone of the pathophysiology of schizo- phrenia, although biochemical support for elevated brain dopamine levels has been lacking. The results of the present study, showing that FEP subjects have increased CSF dopamine levels that correlate to disease symptoms, should fill the knowledge gap in this regard.


Introduction
The dopamine hypothesis of schizophrenia was formulated in the 1960s (Carlsson and Lindqvist, 1963;Carlsson et al., 2001;Snyder, 1973) and is still one of the most compelling models of schizophrenia pathophysiology. Primary evidence supporting this theory arise from the observation that antipsychotics block dopamine D 2 receptors (Creese et al., 1976;Farde et al., 1988;Kapur et al., 2000;Seeman and Lee, 1975;Seeman et al., 1976), whereas psychotomimetic drugs, like amphetamine, augment dopaminergic neurotransmission (Angrist et al., 1974;Laruelle et al., 1996;Lieberman et al., 1987). However, over the years, this theory has been refined to include a nuance that involves dopaminergic hyperactivity in mesolimbic dopamine pathways, concomitant with a dopaminergic hypofunction in the mesocortical pathway (Davis et al., 1991), thought to account for the positive (Laruelle et al., 1996) and the negative symptoms (including cognitive deficits) of the disorder (Slifstein et al., 2015), respectively. Over the last decade positron emission tomography (PET) studies have provided strong evidence of abnormal dopamine signaling in schizophrenia. Thus, subjects with schizophrenia show elevated dopamine synthesis and release capacity also in the ventral and dorsal striatum (Howes et al., 2012;McCutcheon et al., 2018McCutcheon et al., , 2019, concominent with a slightly increased striatal D 2 -dopamine receptor density (Howes et al., 2012;Frankle et al., 2018;McCutcheon et al., 2020). In addition, subjects with first-episode psychosis (FEP) show lower dopamine D 2 receptor binding in the thalamus, suggesting aberrant dopaminergic functioning in subregions relaying to the prefrontal cortex (Plaven-Sigray et al., 2022).
Taken together, there is compelling evidence implicating altered dopamine neurotransmission in schizophrenia. However, robust biochemical evidence from cerebrospinal fluid (CSF) studies of involvement of dopamine in psychosis is still lacking. Relatively few studies have been conducted to analyze dopamine or its main metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the CSF from individuals with schizophrenia. These studies were all performed decades ago utilizing analyzing techniques with lower sensitivity than today's methods of monoamine detection and have not allowed for a consensus. Further, the overall picture is blurred by the simultaneous hypo-and hyperdopaminergic conditions proposed in the disease and the use of antipsychotics that directly impact dopaminergic neurotransmission.
We here analyze dopamine and its main metabolites DOPAC and HVA in the CSF, utilizing a sensitive top-of-the-art analytical assay, in well-characterized groups of FEP subjects and age-matched healthy controls.

Subject population
All FEP and healthy control subjects (>18 years) were recruited between March 2011 and January 2014 as part of the Karolinska Schizophrenia Project (KaSP), a multidisciplinary research consortium that investigates the pathophysiology of schizophrenia. The human study was granted ethical approval by the Regional Ethics Committee in Stockholm (2010/879-31-1) and conformed to the tenets of the Declaration of Helsinki with written informed consent obtained from each participant.
The Positive and Negative Syndrome Scale (PANSS), the Clinical Global Impression and the Global Assessment of Functioning (GAF) were applied to assess functional impact and symptom severity. Alcohol consumption was assessed by the Alcohol Use Disorders Identification Test (AUDIT) questionnaire and the Drug Use Disorders Identification Test (DUDIT) was used to screen for drug-related problems.
The exclusion criteria were severe somatic or neurological disease, substance abuse, autism spectrum disorder, and >1 month of antipsychotic drug treatment. The absence of major brain abnormalities was confirmed using magnetic resonance imaging. Although subjects were examined during the initial observation period which typically occurs before the initiation of antipsychotic treatment, 16 of the 40 FEP subjects (40 %) were treated with antipsychotic drugs at the time of CSF sampling as decided by the treating physician. Twelve out of 40 subjects were naive to all medications. For additional information on pharmacological treatment at the time of CSF sampling, see Table 1.
Twenty healthy controls (11 males and 9 females) were recruited from the same catchment area by advertisement and underwent similar clinical evaluation as the FEP subjects. To confirm absence of psychiatric illness, the healthy controls were screened using the Mini International Neuropsychiatric interview. The exclusion criteria were first-degree relatives with psychotic illness or bipolar disorder, former or current use of illegal drugs, severe neurologic and/or somatic disease. All healthy controls were free from medication and any form of substance abuse, including alcohol evaluated with AUDIT and DUDIT.

CSF sampling
CSF sampling was performed approximately two weeks after admission (14.88 ± 1.78 days). A lumbar puncture was performed in the right decubitus position during daytime (from 07.45 to 13.15) after a night's sleep. Most individuals (n = 36, 23 FEP subjects and 13 healthy controls) underwent the lumbar puncture between 07.45 and 10.00 and the remaining (n = 24, 17 FEP subjects and 7 healthy controls) between 10.00 and 13.15. No significant correlations between the point of time for lumbar puncture and CSF levels of dopamine (all: r = − 0.20, p = 0.24; FEP subjects: r = − 0.18, p = 0.37; healthy controls: r = − 0.23, p = 0.52) were observed.
Eighteen mL CSF was withdrawn from the L 4-5 interspace using a disposable atraumatic needle (22G Sprotte, Geisingen, Germany), collected in a plastic test tube, protected from light. Following separation of cells and supernatant through centrifugation (Sigma 5810R, Eppendorf, Hamburg, Germany at 3500 r.p.m. (1438g) for 10 min), CSF supernatant was divided into 10 aliquots and frozen at − 80 • C within 1 h of sampling.
To avoid inflammatory conditions or infections at the time of CSF sampling a fresh CSF aliquot was analyzed for cell numbers, albumin, immunoglobulin G content and the presence of immunoglobulin G and immunoglobulin M antibodies to Borrelia, as well as with immune electrophoresis.

Analysis of CSF dopamine, DOPAC and HVA
On the analysis day, the samples were placed in the laboratory − 20 • C refrigerator and defrosted individually not earlier than 5 min before the injection. The samples were analyzed randomly by an operator not informed of the sample populations. CSF was analyzed for dopamine, DOPAC, and HVA with a reversed-phase liquid chromatography (HPLC) system including a pump (Bischoff Chromatography, Leonberg, Germany), an Agilent Eclipse XDB-C18 column (4.6 × 150 mm, Agilent Technologies, Inc., CA, USA), and a highsensitivity analytical cell (ESA 5011; ESA Inc., Chelmsford, MA, USA) controlled by a potentiostat (Coulochem III; ESA Inc.) with an applied potential of − 350 mV for detection. The mobile phase consisted of 55 mM sodium acetate buffer (pH 4.1, 10 % methanol) with 1.16 mM octanesulfonic acid and 0.01 mM EDTA disodium Na2EDTA. The flow rate for the mobile phase was 0.7 mL/min. The signals from the detector were analyzed by Datalys Azur Software (Grenoble, France). The retention time was approximately as follows; Dopamine (7 min), DOPAC (4 min), and HVA (10 min). Depending on variations in baseline noise, the lowest levels of dopamine detection were within a range of 0.02-0.1 nM. All values below 0.02 nM were regarded as non-detectable. Dopamine was detected in the CSF of 36 (10 controls and 26 FEP) subjects.

Cognitive assessments
Participants were evaluated with the Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) Consensus Cognitive Battery (Nuechterlein et al., 2008), including the following cognitive domains and tests: attention/vigilance (Continuous Performance Test-Identical Pairs); speed of processing (Trail Making Test: Part A (TMT-A), Brief Assessment of Cognition in Schizophrenia: Symbol Coding (BACS-SC), Category Fluency: Animal Naming (Fluency)); visual learning (Brief Visuospatial Memory Test-Revised); verbal learning (Hopkins Verbal Learning Test-Revised (HVLT-R)); working memory (Wechsler Memory Scale-3rd Edition (WMS-III): Spatial Span (SS), Letter-Number Span (LNS)); reasoning and problem solving (Neuropsychological Assessment Battery: Mazes) and social cognition (Mayer-Salovey-Caruso Emotional Intelligence Test: Managing Emotions).

Statistics
The normality of the data was determined using Shapiro-Wilk test. Analysis of potential confounders was performed using binary logistic regression or χ 2 -test. One healthy control was considered an outlier regarding CSF dopamine level (Grubb's test, CSF dopamine: 2.39 nM) and therefore removed from further analysis. CSF levels of dopamine, DOPAC and HVA were compared between FEP subjects and healthy controls using unpaired t-test with Welch's correction (unequal variance; two-tailed tests of significance). The same statistical test was performed to determine the effects of various medications on CSF dopamine. All reported correlation coefficients are Pearson's r. A Bonferroni correction was applied to control for multiple testing with regard to the correlation analyses between performance in different cognitive tests and CSF dopamine, DOPAC and HVA levels, giving a significant threshold of 0.005 (0.05/10). All analyses were performed using Prism version 8.0 (GraphPad Software, La Jolla, CA, USA), SPSS Statistics version 26.0 (IBM, Armonk, NY, USA), or R statistics (R Development Core Team, Vienna, Austria). Statistical significance was considered when P < 0.05.

Demographic and clinical characteristics of the study population
As shown in Table 2, healthy controls (n = 20) and FEP subjects (n = 40) did not differ in terms of age, BMI, or gender at the study enrollment. However, 27 % (n = 11) of the FEP subjects used tobacco (smoking or snuff) while all healthy controls were tobacco-free.

CSF dopamine in FEP subjects versus control subjects
Dopamine was reliably detected in 50 % of the healthy controls and 65 % of the FEP subjects. Dopamine were significantly elevated in FEP subjects as compared to the healthy controls ( Fig. 1; 0.88 ± 0.15 nM, n = 26 vs. 0.43 ± 0.09 nM, n = 9; p = 0.018). The demographics of FEP subjects with detectable CSF dopamine did not significantly differ from those without detectable dopamine. No significant correlations between CSF dopamine and age and BMI, were observed either in FEP subjects or healthy controls (Supplementary Table S1). CSF DA levels did not differ between FEP subjects using tobacco and those who did not (p = 0.97). CSF dopamine levels did not differ between treated and drug-naive FEP subjects with regard to treatment with antipsychotic drugs (p = 0.38), benzodiazepines (p = 0.92), zopiclone (p = 0.78), antihistamines (p = 0.21), or various combinations of these drugs (p = 0.89) (see Table 3).
Finally, all FEP subjects were reassessed 1.5 years after the first evaluation. CSF dopamine levels at inclusion did not differ between FEP subjects who met the DSM-IV criteria for schizophrenia and subjects who did not (0.98 ± 0.21 nM, n = 17 vs. 0.68 ± 0.21 nM, n = 9; p = 0.33) at the follow-up evaluation (see Supplementary Fig. 1).

CSF DOPAC and HVA in FEP subjects versus healthy controls
The results from CSF DOPAC and CSF HVA analysis in FEP subjects and healthy controls as are shown in Fig. 2. DOPAC and HVA was reliably detected in the CSF of all FEP subjects and healthy controls. The CSF levels of DOPAC (13.61 ± 3.90 nM in FEP subjects, n = 40 vs. 13.88 ± 6.19 nM in healthy controls, n = 19, p = 0.97) and HVA (274.4 ± 16.79 in FEP subjects, n = 40 vs. 247.3 ± 21.63 in healthy controls, n = 19, p = 0.33) did not differ significantly between FEP subjects and healthy controls ( Fig. 2A and B). Notably, a negative correlation was found between CSF dopamine and CSF HVA in healthy controls (r = − 0.84, p = 0.0043) (Supplementary Table 2). No other correlations between CSF dopamine and HVA or DOPAC were observed.

Correlations between CSF dopamine or dopamine metabolites with symptoms or functioning in FEP subjects
Correlations between CSF dopamine and metabolites with PANSS, GAF and CGI scores are presented in Table 4. A positive correlation was observed between CSF dopamine and CGI (r = 0.56, p = 0.003), indicating that subjects with severe illness have higher levels of CSF dopamine. With regard to the metabolites, levels of CSF DOPAC correlated negatively with disease severity as measured by CGI (r = − 0.33, p = 0.037). HVA correlated negatively with symptom severity as measured by negative (r = − 0.34, p = 0.032) or total (r = − 0.31. p = 0.049) psychopathology subscale of PANSS, respectively.

Correlations between CSF dopamine or dopamine metabolites with cognitive performance in FEP subjects and healthy controls
Correlations between CSF dopamine and metabolites with cognitive performance in FEP subjects and healthy controls are presented in Table 4 and Supplementary Table 3, respectively. Higher levels of CSF dopamine correlated with slower speed of processing as measured by the TMT A (r = 0.60, p = 0.002) and verbal fluency (r = − 0.58, p = 0.003) tests in FEP subjects. Both correlations remained significant following Bonferroni correction (threshold of p < 0.005). In the healthy control group, higher levels of CSF dopamine correlated with better social cognition performance on the MSCEIT (r = 0.88, p = 0.002) and poorer working memory performance on the WMS-III (r = − 0.78, p = 0.013). Following Bonferroni correction, only correlation with the MSCEIT test remained significant (threshold of p < 0.005).

Discussion
Ever since the formation of the dopamine hypothesis of schizophrenia in the mid-1960s, direct support from the analysis of dopamine in the CSF from subjects with schizophrenia has been lacking. The results of the present study, utilizing a well-characterized cohort of FEP subjects and healthy controls as well as a sensitive analytical assay to detect the low concentrations of CSF dopamine and its metabolites, should fill the knowledge gap in this regard. The observed group difference and correlations of CSF dopamine concentration with disease severity, cognitive performance, and symptomatology strongly supports an essential role of dopamine in schizophrenia.
The present results, showing that CSF dopamine levels are elevated in FEP subjects, are indirectly supported by the higher number of FEP subjects with detectable CSF dopamine levels compared to that of the healthy controls. The elevation of CSF dopamine was independent of age, gender, BMI, or tobacco use. Similarly, we found no differences in CSF dopamine levels between FEP subjects with antipsychotic treatment and those that were drug naïve. The present results contrast the very few previously reported analyses showing no difference in CSF dopamine between controls and subjects with schizophrenia Gattaz et al., 1983;McCutcheon et al., 2020). However, most previous DOPAC, nM HVA, nM  studies were performed 30-40 years ago, and the discrepancy between those and present results may be primarily related to the advanced sensitivity and specificity of up-to-date analyzing techniques. The presently indicated increased dopaminergic neurotransmission in FEP subjects appears to be related explicitly to dopamine since no differences in DOPAC or HVA between FEP subjects and healthy controls were observed. This discrepancy is obscure since DOPAC and HVA are the primary metabolites of dopamine, but our results are in line with several previous studies showing no differences in CSF metabolites between subjects with schizophrenia and healthy controls (Berger et al., 1980;Miller et al., 1996;Nyback et al., 1983). However, the relatively low number of individuals in the present study may hinder the appearance of an accurate picture in this regard. In this context, though, it is worth mentioning that a recent study from our group showed that bipolar disorder subjects with history of psychosis displayed significantly higher CSF HVA concentrations than healthy controls or subjects with bipolar disorder without a history of psychosis (Sellgren et al., 2016).
Considering the literature on dopamine synthesis capacity and psychotic symptoms, we expected a positive correlation between CSF dopamine and PANSS, however, this hypothesis was not supported by the results. Instead, CSF dopamine concentration was found to correlate with CGI so higher CSF dopamine levels were associated with enhanced illness severity. In addition, higher CSF dopamine concentrations were associated with slower speed of processing. In healthy controls, higher CSF dopamine concentrations were associated with a poor working memory but improved social cognition performance. Despite the lack of correlation to PANSS, the association of CSF dopamine concentrations with the severity of disease and cognitive parameters in FEP subjects further strengthen the validity of our results.
We observed no difference in CSF levels of dopamine, HVA, and DOPAC between subjects medicated with antipsychotics and without such treatment. Our findings reasonably well follow results from animal experiments showing that chronic treatment with dopamine D 2 -receptor antagonists produces a moderate decrease in HVA (see Gasnier et al., 2021), while, in other studies, no significant effects of chronic olanzapine treatment on HVA or DOPAC were observed (Nicolaou, 1980;Jordan et al., 2004;Arjona et al., 2004). Clinical studies are sparse (see McCutcheon et al., 2020) but show that levels of dopamine are increased in schizophrenia subjects receiving antipsychotic treatment (Gattaz et al., 1983; and that reductions in dopamine or its metabolites occur following the withdrawal of treatment Bagdy et al., 1985). Of interest, antipsychotic treatment did not alter dopamine synthesis capacity in FEP subjects (Jauhar et al., 2019). In the present study, though, the relatively low number of FEP subjects treated with antipsychotics may generate insufficient statistical power to definitively evaluate if CSF dopamine is influenced by antipsychotic treatment. Thus, the possible influence of antipsychotic medication cannot be entirely excluded.
The pathophysiology of psychosis has been a focus of psychiatry research for decades. Although a dysfunctional dopamine system in schizophrenia is suggested to involve both a hyper-and a hypofunction (c.f. Introduction), the present finding of heightened CSF dopamine levels in FEP subjects likely reflects facilitated dopamine neurotransmission in the brain. However, one must bear in mind that the FEP subjects were investigated during their first acute psychosis. Thus, such subjects may show more prominent positive symptoms than those with chronic schizophrenia. In fact, in our follow-up analysis, we observed that while negative symptoms remain within the same magnitude after one and a half years, their positive symptoms significantly decline (data not shown). Thus, transitioning to a state of chronic schizophrenia, where negative and positive symptoms may be more stabilized, a simultaneous dopaminergic hypo-and hyperactivity may counterbalance CSF dopamine levels.
Growing evidence suggests that dopaminergic dysfunction in schizophrenia is triggered by disruption of upstream afferent circuits controlling midbrain dopamine neurons. It is well known that dopamine neuron activity is under tonic inhibitory control by GABAergic afferents that operate through GABA-B in particular (Erhardt et al., 1999Grace, 2010). Thus, an attenuated GABAergic tone, e.g. induced by psychotomimetic N-methyl-D-aspartate (NMDA)-receptor antagonist like phencyclidine (French et al., 1991;French et al., 1993;Schwieler et al., 2006;Zhang et al., 1992) or the endogenous NMDA-receptor antagonist kynurenic acid (Erhardt et al., 2001;, lead to activation of midbrain dopamine neurons. Such mechanisms may tentatively underlie the presently observed elevation of CSF dopamine. In line with this view, we previously observed reduced levels of CSF GABA in essentially the same cohort as in the present study, (Orhan et al., 2018).
In conclusion, utilizing a well-characterized cohort of subjects with FEP and healthy controls, and state-of-the-art HPLC analysis, we provide direct evidence, for the first time, for increased levels of CSF dopamine in FEP subjects. This observation is supported by its correlation with disease severity, cognitive impairments, and symptomatology.

Role of funding source
No funding sources had any role in the study design, in the collection, analysis, and interpretation of data, in the writing of the report, or in the decision to submit the paper for publication.

Declaration of competing interest
All authors declare they have no competing interest.