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Spasticity is a common and disabling symptom observed in patients with central nervous system diseases, including amyotrophic lateral sclerosis, a disease affecting both upper and lower motor neurons. In amyotrophic lateral sclerosis, spasticity is traditionally thought to be the result of degeneration of the upper motor neurons in the cerebral cortex, although degeneration of other neuronal types, in particular serotonergic neurons, might also represent a cause of spasticity. We performed a pathology study in seven patients with amyotrophic lateral sclerosis and six control subjects and observed that central serotonergic neurons suffer from a degenerative process with prominent neuritic degeneration, and sometimes loss of cell bodies in patients with amyotrophic lateral sclerosis. Moreover, distal serotonergic projections to spinal cord motor neurons and hippocampus systematically degenerated in patients with amyotrophic lateral sclerosis. In SOD1 (G86R) mice, a transgenic model of amyotrophic lateral sclerosis, serotonin levels were decreased in brainstem and spinal cord before onset of motor symptoms. Furthermore, there was noticeable atrophy of serotonin neuronal cell bodies along with neuritic degeneration at disease onset. We hypothesized that degeneration of serotonergic neurons could underlie spasticity in amyotrophic lateral sclerosis and investigated this hypothesis in vivo using tail muscle spastic-like contractions in response to mechanical stimulation as a measure of spasticity. In SOD1 (G86R) mice, tail muscle spastic-like contractions were observed at end-stage. Importantly, they were abolished by 5-hydroxytryptamine-2b/c receptors inverse agonists. In line with this, 5-hydroxytryptamine-2b receptor expression was strongly increased at disease onset. In all, we show that serotonergic neurons degenerate during amyotrophic lateral sclerosis, and that this might underlie spasticity in mice. Further research is needed to determine whether inverse agonists of 5-hydroxytryptamine-2b/c receptors could be of interest in treating spasticity in patients with amyotrophic lateral sclerosis.

Aggression, which comprises multi-faceted traits ranging from negative emotionality to antisocial behaviour, is influenced by an interaction of biological, psychological and social variables. Failure in social adjustment, aggressiveness and violence represent the most detrimental long-term outcome of neurodevelopmental disorders. With the exception of brain-specific tryptophan hydroxylase-2 ( Tph2 ), which generates serotonin (5-HT) in raphe neurons, the contribution of gene variation to aggression-related behaviour in genetically modified mouse models has been previously appraised (Lesch 2005 Novartis Found Symp . 268 , 111–140; Lesch & Merschdorf 2000 Behav. Sci. Law 18 , 581–604). Genetic inactivation of Tph2 function in mice led to the identification of phenotypic changes, ranging from growth retardation and late-onset obesity, to enhanced conditioned fear response, increased aggression and depression-like behaviour. This spectrum of consequences, which are amplified by stress-related epigenetic interactions, are attributable to deficient brain 5-HT synthesis during development and adulthood. Human data relating altered TPH2 function to personality traits of negative emotionality and neurodevelopmental disorders characterized by deficits in cognitive control and emotion regulation are based on genetic association and are therefore not as robust as the experimental mouse results. Mouse models in conjunction with approaches focusing on TPH2 variants in humans provide unexpected views of 5-HT's role in brain development and in disorders related to negative emotionality, aggression and antisocial behaviour.

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) is a popular party drug known to cause selective serotonergic damage. Here we examined the long-term recovery and aging of serotonergic fibers and levels of brain-derived neurotrophic factor (BDNF) after intermittent MDMA administration (15 mg kg(-1) i.p. every 7th day for 4 weeks, MDMA ×4) and a single-dose treatment (15 mg kg(-1) i.p., MDMA ×1) in adolescent/young adult male Dark Agouti rats. After MDMA treatment, tryptophan hydroxylase-immunoreactive fiber density decreased and then recovered in all brain regions. Recovery was more pronounced in the MDMA ×4 group compared with the MDMA ×1 group, but similar long-term BDNF responses were found after both treatments. Twenty-two months after treatment, there were fewer clusters of aberrant serotonergic fibers in the parietal cortex in the MDMA ×4 group compared with the MDMA ×1 group. There was no difference in the density of microglial cells or astrocytes in treated groups versus the control 22 months after the treatments. These results indicate that recovery of serotonergic fibers is faster after intermittent MDMA treatment than after single-dose administration, and differences in BDNF levels per se are unlikely to account for this difference. Moreover, it seems that intermittent MDMA treatment attenuates the morphological signs of aging in serotonergic fibers. In addition, neither intermittent nor single-dose MDMA exposition of young animals induces accelerated aging processes or neurodegeneration in senescence, as indicated by the unaltered densities of microglial cells and astrocytes in the treated groups compared with the control.

Several lines of evidence implicate a dysregulation of tryptophan hydroxylase (TPH)-dependent serotonin (5-HT) synthesis in emotional behaviour and stress, and point to its relevance for the etiology and pathogenesis of various neuropsychiatric disorders. We therefore studied different animal models featuring reduced Tph2 expression to investigate the consequences of impaired brain 5-HT synthesis on neuronal development. Specifically, brain-specific conditional and time-specific inducible Tph2 knockout (KO) models were generated and investigated for altered serotonergic neuron-specific gene expression. Raphe neurons of a brain-specific constitutive Tph2 KO were completely devoid of Tph2-positive neurons and, consequently, 5-HT in the brain, and also displayed no compensatory up-regulation of Tph1 expression. In contrast, an inducible Tph2 KO mouse facilitates the generation of a brain-specific 5-HT-reduction model selectively during adult life. This resulted in a highly reduced number of Tph2-positive cells and thus 5-HT in the brain. Intriguingly, expression studies detected no alteration in the expression of genes relevant to the 5-HT system in the brain-specific Tph2 KO and the 5-HT-reduction models. These findings confirm the specificity of Tph2 in brain 5-HT synthesis across the lifespan, yet also suggest that neither developmental nor adult 5-HT synthesis is required for the expression of genes specific for serotonergic signalling. The formation of the serotonergic system thus seems to be a preserved expressional pattern due to intrinsic cellular programs which occurs also in the absence of its key molecule, namely 5-HT.

As soon as in the 1960's, the role of serotonin (5-Hydroxytryptamin, 5-HT) in psychiatric disorders was realized, which was further substantiated by several lines of evidence amounting to a huge body of knowledge. The indolamine 5-HT belongs to the class of monoamine transmitters and can be found in the serotonergic neurons of the raphe nuclei in the brain stem. In the periphery, it is mainly present in the gastrointestinal system and the pineal gland. 5-HT is implicated in a variety of cognitive, emotional and vegetative behaviors, as well as in the regulation of circadian rhythms. Apart from its role as a neurotransmitter, it has an important function in prenatal development, where its expression pattern is tightly regulated, and in adult neurogenesis. The numerous effects of 5-HT are mediated by specific pre- and postsynaptic receptors, whose localization and functions are further described here. The serotonin transporter (SERT), which accomplishes the re-uptake of 5-HT into the neuron following its release in the synaptic cleft, not only has an important role in the termination of serotonergic neurotransmission but is also an important drug target for antidepressant compounds. In this part of the review, the neurobiological underpinnings of 5-HT synthesis, metabolism, and neurotransmission as well as the corresponding physiological consequences are summarized, while in the second part, an overview on clinical findings is provided and critically discussed.

Several lines of evidence implicate a dysregulation of the serotonin (5-HT) system in emotional behavior and stress, and point to its relevance for the etiology and pathogenesis of various neuropsychiatric disorders. This is evidenced by behavioral pharmacology as well as genetic studies, yet the impact of genetic variation within the 5-HT system on human disorders remains controversial. The generation of tissue-specific and inducible knockout mice lacking genes belonging to the 5-HT system further established the importance of the 5-HT system for neuronal development and the regulation of emotions. This part of the review provides a summary and critical discussion of genetic, neurobiological and pharmacological studies along with recent clinical research. Together, these data underscore the complex effects of 5-HT on human behavior and psychiatric disorders. Epigenetic mechanisms add to the complexity of the 5-HT system and will be increasingly studied in the coming years. Thus, the serotonergic system still remains in the centre of current hypotheses regarding the pathogenesis of disorders with the shared feature of emotional dysregulation.

Although therapeutic interventions in attention-deficit/hyperactivity disorder (ADHD) still focus on the dopaminergic system, recent studies indicate a serotonergic dysfunction in this disease as well. In that respect, several variants of the tryptophan hydroxylase gene (TPH2), which codes for the rate-limiting enzyme in the biosynthesis of serotonin (5-HT), have been associated with ADHD. The rs4570625 G-allele polymorphisms of the TPH2 gene have already been related to altered reactivity of the brain during perception tasks with emotional stimuli in healthy adults. Here we investigated the influence of the ADHD related risk alleles for rs4570625 and for rs11178997 on prefrontal brain function during cognitive response control in large samples of adult ADHD patients (n=124) and healthy controls (n=84). Response control was elicited with a Go-NoGo task (continuous performance test; CPT) performed during recording of an ongoing EEG. From the resulting event-related potentials in the Go- and NoGo conditions of the CPT, the NoGo-anteriorization (NGA) has been calculated as a valid neurophysiological parameter for prefrontal brain function. In the current study, ADHD risk alleles of both polymorphisms were found to be associated with a reduction in the NGA in both healthy controls and ADHD patients. These findings are in line with the notion that genetic variations associated with altered serotonergic neurotransmission are also associated with the function of the prefrontal cortex during response inhibition. This mechanism might also be relevant in the pathophysiology of ADHD.

The involvement in neural plasticity and the mediation of effects of repeated stress exposure and long-term antidepressant treatment on hippocampal neurogenesis supports a critical role of brain-derived neurotrophic factor (BDNF) in the pathophysiology of affective and other stress-related disorders. A previously reported valine to methionine substitution at amino-acid position 66 (BDNF Val66Met) seems to account for memory disturbance and hippocampal dysfunction. In the present study, we evaluated the impact of the BDNF Val66Met polymorphism on individual differences in personality traits in a sample of healthy volunteers in relation to other common gene variants thought to be involved in the pathophysiology of affective disorders, such as the serotonin transporter promoter polymorphism (5-HTTLPR) and a variable number of tandem repeat polymorphism of the dopamine transporter gene (DAT VNTR). Personality traits were assessed using the NEO personality inventory (NEO-PI-R) and Tridimensional Personality Questionnaire (TPQ). There was a significant DAT VNTR-dependent association between NEO-PI-R Neuroticism and the BDNF Val66Met polymorphism. Among individuals with at least one copy of the DAT 9-repeat allele, carriers of the BDNF Met allele exhibited significantly lower Neuroticism scores than noncarriers. This interaction was also observed for TPQ Harm Avoidance, a personality dimension related to Neuroticism. Our results support the notion that allelic variation at the BDNF locusFin interaction with other gene variantsFinfluences anxietyand depression-related personality traits. Neuropsychopharmacology (2007) 32, 2552–2560; doi:10.1038/sj.npp.1301383; published online 28 March 2007

Rationale While brain serotonin (5-HT) function is implicated in gene-by-environment interaction (GxE) impacting the vulnerability-resilience continuum in neuropsychiatric disorders, it remains elusive how the interplay of altered 5-HT synthesis and environmental stressors is linked to failure in emotion regulation. Objective Here, we investigated the effect of constitutively impaired 5-HT synthesis on behavioral and neuroendocrine responses to unpredictable chronic mild stress (CMS) using a mouse model of brain 5-HT deficiency resulting from targeted inactivation of the tryptophan hydroxylase-2 (Tph2) gene. Results Locomotor activity and anxiety- and depression-like behavior as well as conditioned fear responses were differentially affected by Tph2 genotype, sex, and CMS. Tph2 null mutants (Tph2−/−) displayed increased general metabolism, marginally reduced anxiety- and depression-like behavior but strikingly increased conditioned fear responses. Behavioral modifications were associated with sex-specific hypothalamic-pituitary-adrenocortical (HPA) system alterations as indicated by plasma corticosterone and fecal corticosterone metabolite concentrations. Tph2−/− males displayed increased impulsivity and high aggressiveness. Tph2−/− females displayed greater emotional reactivity to aversive conditions as reflected by changes in behaviors at baseline including increased freezing and decreased locomotion in novel environments. However, both Tph2−/− male and female mice were resilient to CMS-induced hyperlocomotion, while CMS intensified conditioned fear responses in a GxE-dependent manner. Conclusions Our results indicate that 5-HT mediates behavioral responses to environmental adversity by facilitating the encoding of stress effects leading to increased vulnerability for negative emotionality. Electronic supplementary material The online version of this article (doi:10.1007/s00213-015-3879-0) contains supplementary material, which is available to authorized users.

Panic disorder is a common psychiatric disorder characterized by recurrent anxiety attacks and anticipatory anxiety. Due to the severity of the symptoms of the panic attacks and the frequent additional occurrence of agoraphobia, panic disorder is an often debilitating disease. Elevation of central serotonin levels by drugs such as clomipramine represents one of the most effective treatment options for panic disorder. This points to an important role of dysregulation of the serotonergic system in the genetic etiology of panic disorder. The novel brain-specific 5-HT synthesizing enzyme, tryptophan hydroxylase-2 (TPH2), which represents the rate-limiting enzyme of 5-HT production in the brain, may therefore be of particular importance in panic disorder. We focused on the putative transcriptional control region of TPH2 and identified two novel common single nucleotide polymorphisms (SNPs) of TPH2 in and close to this region. Moreover, a recently described loss-of-function mutation of TPH2 which results in an 80% reduction of serotonin production, was assessed. In an analysis of the putative transcriptional control region SNPs in a sample of panic disorder patients and controls no association of the disorder with the TPH2 SNPs or haplotypes was found. Moreover, the loss-of-function R441H mutation of TPH2 was not present in the panic disorder patients. The results of this first study of TPH2 in panic disorder argue against an importance of allelic variation of TPH2 in the pathogenesis of panic disorder with or without agoraphobia.

The tryptophan hydroxylase-2 gene (TPH2) codes for the enzyme of serotonin (5-HT) synthesis in the brain and variation of TPH2 has been implicated in disorders of emotion regulation. Here, we used functional magnetic resonance imaging (fMRI) to demonstrate that a potentially functional variant of TPH2 modulates amygdala responsiveness to emotional stimuli of both negative and positive valence.

Response to psychopharmacologic drugs is genetically complex, results from an interplay of multiple genomic variations with environmental influences, and depends on the structure or functional expression of gene products, which are direct drug targets or indirectly modify the development and synaptic plasticity of neural networks critically involved in their effects. During brain development, the serotonin (5HT) system, which is commonly targeted by antidepressant, anxiolytic, and antipsychotic drugs, controls neuronal specification, differentiation, and phenotype maintenance. While formation and integration of these neural networks is dependent on the action of multiple proteins, converging lines of evidence indicate that genetically controlled variability in the expression of the 5HT transporter (5HTT) is critical to the development and plasticity of distinct neurocircuits. The most promising finding to date indicate an association between the response time as well as overall response to serotonin reuptake inhibitors (SSRIs) and a common polymorphism (5HTTLPR) within the transcriptional control region of the 5HTT gene (SLC6A4) in patients with depressive disorders. The formation and maturation of serotonergic and associated systems, in turn, are influencing the efficacy of serotonergic compounds in a variety of psychiatric conditions. Based on the notion that complex gene x gene and gene x environment interactions in the regulation of brain plasticity are presumed to contribute to individual differences in psychopharmacologic drug response, the concept of developmental psychopharmacogenetics is emerging.

While multiple lines of evidence implicate the 5-HT1A receptor in the pathophysiology of anxiety and depression as well as in the mechanism of action of anxiolytics/antidepressants, its relevance to the therapeutic effectiveness of these drugs has been a matter of considerable debate (for review see Griebel, 1995; Hensler, 2003; Hjorth et al., 2000; Lesch et al., 2003). In the current issue of the International Journal of Neuropsychopharmacology, however, both Serretti et al. (2004) and Lemonde et al. (2004) make a strong argument for contribution of a functional 5-HT1A receptor gene variant in the pharmacogenetics of antidepressant treatment with prototypic tricyclics and selective serotonin reuptake inhibitors (SSRIs). Furthermore, the third study in this series by Huang and associates (2004) reveals an association of allelic variation of 5-HT1A receptor expression in a wide spectrum of psychopathology including schizophrenia, substance abuse, and panic disorder. Not unexpectedly, the failure to detect a consistent effect of this gene variation on 5-HT1A receptor functionality in the mature brain as indicated by both receptor binding in post-mortem brain and in-vivo receptor responsivity further supports a critical role of the 5-HT1A receptor in engineering neurodevelopmental processes which may have the potential to set the stage for the brain's permissiveness for psychopathology in later life. The availability of an increasing number of functional gene variants within the serotonergic pathway together with integration of emerging concepts of developmental genetics of complex traits will provide the groundwork for the molecular dissection of syndromal dimensions and treatment response.

Association between the low-activity variant of a polymorphism in the transcriptional control region of the serotonin transporter (5-HTTLPR) and neuroticism or harm avoidance was found in several but not all studies. The authors analyzed the influence of 5-HTTLPR variants on personality disorders.Patients with personality disorders (N=320) and healthy volunteers (N=281) were studied with the Revised NEO Personality Inventory and the Tridimensional Personality Questionnaire. All were genotyped for 5-HTTLPR variants.No differences in 5-HTTLPR genotype distribution were detected between patients with cluster B and C personality disorders and comparison subjects. In contrast, among patients with a cluster C diagnosis, carriers of the low-activity short allele of the 5-HTTLPR exhibited higher neuroticism scores than noncarriers.These findings support the notion that there is no general association between the 5-HTTLPR and anxiety-related traits and that differential gene effects and/or gene-by-environment interactions are likely operative in distinct clinical subpopulations.

The 5-HT1A receptor plays a critical role in the pathophysiology of anxiety and depression as well as in the mode of action of anxiolytic and antidepressant drugs. Human 5-HT1A gene transcription is modulated by a common C-1016G single nucleotide polymorphism (SNP) in its upstream regulatory region. In the present study, we evaluated the role of the HTR1A-1019 polymorphism in the modulation of individual differences in personality traits by an association study of a sample of healthy volunteers. Personality traits were assessed with two different methods, NEO personality inventory (NEO-PI-R) and Tridimensional Personality Questionnaire (TPQ). There was a significant effect of the HTR1A-1019 polymorphism on NEO Neuroticism with carriers of the G allele showing higher scores than individuals homozygous for the C variant. The effect was primarily due to associations with the Neuroticism facets Anxiety and Depression. Carriers of the G allele also exhibited higher TPQ Harm Avoidance scores. Our findings indicate a role of allelic variation in 5-HT1A receptor expression in the development and modulation of anxiety- and depression-related personality traits.

While tryptophan hydroxylase-2 (Tph2) null mutant (Tph2(-/-)) mice are completely deficient in brain serotonin (5-HT) synthesis, the formation of serotonergic neurons and pathfinding of their projections are not impaired. However, 5-HT deficiency, during development and in the adult, might affect morphological and functional parameters of other neural systems. To assess the influence of 5-HT deficiency on γ-amino butyric acid (GABA) systems, we carried out measurements of GABA concentrations in limbic brain regions of adult male wildtype (wt), heterozygous (Tph2(+/-)) and Tph2(-/-) mice. In addition, unbiased stereological estimation of GABAergic interneuron numbers and density was performed in subregions of amygdala and hippocampus. Amygdala and prefrontal cortex displayed significantly increased and decreased GABA concentrations, respectively, exclusively in Tph2(+/-) mice while no changes were detected between Tph2(-/-) and wt mice. In contrast, in the hippocampus, increased GABA concentrations were found in Tph2(-/-) mice. While total cell density in the anterior basolateral amygdala did not differ between genotypes, the number and density of the GABAergic interneurons were significantly decreased in Tph2(-/-) mice, with the group of parvalbumin (PV)-immunoreactive (ir) interneurons contributing somewhat less to the decrease than that of non-PV-ir GABAergic interneurons. Major morphological changes were also absent in the dorsal hippocampus, and only a trend toward reduced density of PV-ir cells was observed in the CA3 region of Tph2(-/-) mice. Our findings are the first to document that life-long reduction or complete lack of brain 5-HT transmission causes differential changes of GABA systems in limbic regions which are key players in emotional learning and memory processes. The changes likely reflect a combination of developmental alterations and functional adaptations of emotion circuits to balance the lack of 5-HT, and may underlie altered emotional behavior in 5-HT-deficient mice. Taken together, our findings provide further insight into the mechanisms how life-long 5-HT deficiency impacts the pathogenesis of anxiety- and fear-related disorders.

Brain serotonin (5-HT) is implicated in a wide range of functions from basic physiological mechanisms to complex behaviors, including neuropsychiatric conditions, as well as in developmental processes. Increasing evidence links 5-HT signaling alterations during development to emotional dysregulation and psychopathology in adult age. To further analyze the importance of brain 5-HT in somatic and brain development and function, and more specifically differentiation and specification of the serotonergic system itself, we generated a mouse model with brain-specific 5-HT deficiency resulting from a genetically driven constitutive inactivation of neuronal tryptophan hydroxylase-2 (Tph2). Tph2 inactivation (Tph2-/-) resulted in brain 5-HT deficiency leading to growth retardation and persistent leanness, whereas a sex- and age-dependent increase in body weight was observed in Tph2+/- mice. The conserved expression pattern of the 5-HT neuron-specific markers (except Tph2 and 5-HT) demonstrates that brain 5-HT synthesis is not a prerequisite for the proliferation, differentiation and survival of raphe neurons subjected to the developmental program of serotonergic specification. Furthermore, although these neurons are unable to synthesize 5-HT from the precursor tryptophan, they still display electrophysiological properties characteristic of 5-HT neurons. Moreover, 5-HT deficiency induces an up-regulation of 5-HT\(_{1A}\) and 5-HT\(_{1B}\) receptors across brain regions as well as a reduction of norepinephrine concentrations accompanied by a reduced number of noradrenergic neurons. Together, our results characterize developmental, neurochemical, neurobiological and electrophysiological consequences of brain-specific 5-HT deficiency, reveal a dual dose-dependent role of 5-HT in body weight regulation and show that differentiation of serotonergic neuron phenotype is independent from endogenous 5-HT synthesis.

E-pub, udgivelsesdato: 2010-May-18 A wide range of physiological processes and neuronal functioning is modulated by the serotonergic system. Serotonin (5-HT) plays an important role during early brain development. Moreover, dysfunction of the 5-HT system is implicated in psychiatric disorders, especially in affective disorders. Little is known, however, about the transcriptional mechanisms leading to a functional 5-HT system in humans. The Fifth Ewing Variant (FEV), an E-twenty-six (ETS) transcription factor, is assumed to be involved in the transcription of gene(s) in the serotonergic pathway and to play a role in early brain development. To investigate its specificity, we performed an expression analysis of FEV in different human brain regions utilizing quantitative real-time polymerase chain reaction. Our results demonstrate that FEV is not exclusively expressed in serotonergic neurons, but, on the contrary, also in several non-serotonergic brain regions such as locus coeruleus, caudate nucleus and putamen. In the latter two regions, FEV expression levels actually were higher when compared with the pons and the medulla oblongata, which contain the raphe nuclei. Additionally, we examined whether genetic variance in the FEV gene contributes to the susceptibility towards affective disorders. Direct re-sequencing, however, did not provide evidence for FEV mutations in patients, and neither were non-coding single nucleotide polymorphisms associated with disease. FEV therefore might not account for the genetic risk towards depression or bipolar disorder. Furthermore, the specificity of FEV for the serotonergic system should be reconsidered.

3,4-Methylenedioxymethamphetamine (MDMA, “ecstasy”) is a widely used recreational drug known to cause selective long-term serotonergic damage. The aim of this study was to characterize the ultrastructure of serotonergic pericarya and proximal neurites in the dorsal raphe nucleus as well as the ultrastructure of serotonergic axons in the frontal cortex of adolescent Dark Agouti rats 3 days after treatment with 15 mg/kg i.p. MDMA. Light microscopic immunohistochemistry and pre-embedding immunoelectron microscopy with a novel tryptophan hydroxylase-2 (Tph2) specific antibody, as a marker of serotonergic structures. Light microscopic analysis showed reduced serotonergic axon density and aberrant swollen varicosities in the frontal cortex of MDMA-treated animals. According to the electron microscopic analysis, Tph2 exhibited diffuse cytoplasmic immunolocalization in dorsal raphe neuronal cell bodies. The ultrastructural-morphometric analysis of these cell bodies did not indicate pathological changes or significant alteration in the cross-sectional areal density of any examined organelles. Proximal serotonergic neurites in the dorsal raphe exhibited no ultrastructural alteration. However, in the frontal cortex among intact fibers, numerous serotonergic axons with destructed microtubules were found. Most of their mitochondria were intact, albeit some injured axons also contained degenerating mitochondria; moreover, a few of them comprised confluent membrane whorls only. Our treatment protocol does not lead to ultrastructural alteration in the serotonergic dorsal raphe cell bodies and in their proximal neurites but causes impairment in cortical serotonergic axons. In these, the main ultrastructural alteration is the destruction of microtubules although a smaller portion of these axons probably undergo an irreversible damage.

Dysregulation of tryptophan hydroxylase (TPH)-dependent serotonin (5-HT) synthesis, has been implicated in various neuropsychiatric disorders, although the differential expression pattern of the two isoforms is controversial. Here, we report a comprehensive spatio-temporal isoform-specific analysis of TPH1 and TPH2 expression during pre- and postnatal development of mouse brain and in adult human brain. TPH2 expression was consistently detected in the raphe nuclei, as well as in fibers in the deep pineal gland and in small intestine. Although TPH1 expression was found in these peripheral tissues, no significant TPH1 expression was detected in the brain, neither during murine development, nor in mouse and human adult brain. In support of TPH2 specificity in brain 5-HT synthesis, raphe neurons of Tph2 knockout mice were completely devoid of 5-HT, with no compensatory activation of Tph1 expression. In conclusion, our findings indicate that brain 5-HT synthesis across the lifespan is exclusively maintained by TPH2.

The relative contribution of the two tryptophan hydroxylase (TPH) isoforms, TPH1 and TPH2, to brain serotonergic system function is controversial. To investigate the respective role of TPH2 in neuron serotonin (5-HT) synthesis and the role of 5-HT in brain development, mice with a targeted disruption of Tph2 were generated. The preliminary results indicate that in Tph2 knockout mice raphe neurons are completely devoid of 5-HT, whereas no obvious alteration in morphology and fiber distribution are observed. The findings confirm the exclusive specificity of Tph2 in brain 5-HT synthesis and suggest that Tph2-synthesized 5-HT is not required for serotonergic neuron formation.

The gaseous messenger nitric oxide (NO) has been implicated in a wide range of behaviors, including aggression, anxiety, depression, and cognitive functioning. To further elucidate the physiological role of NO and its down-stream mechanisms, we conducted behavioral and expressional phenotyping of mice lacking the neuronal isoform of nitric oxide synthase (NOS-I), the major source of NO in the central nervous system. No differences were observed in activity-related parameters; in contrast to the a priori hypothesis, derived from pharmacological treatments, depression-related tests (Forced Swim Test, Learned Helplessness) also yielded no significantly different results. A subtle anxiolytic phenotype however was present, with knockdown mice displaying a higher open arm time as compared to their respective wildtypes, yet all other investigated anxiety-related parameters were unchanged. The most prominent feature however was gender-independent cognitive impairment in spatial learning and memory, as assessed by the Water Maze test and an automatized holeboard paradigm. No significant dysregulation of monoamine transporters was evidenced by qRT PCR. To further examine the underlying molecular mechanisms, the transcriptome of knockdown animals was thus examined in the hippocampus, striatum and cerebellum by microarray analysis. A set of > 120 differentially expressed genes was identified, whereat the hippocampus and the striatum showed similar expressional profiles as compared to the cerebellum in hierarchical clustering.

Variation in the tryptophan hydroxylase-2 gene (TPH2) coding for the rate-limiting enzyme of serotonin (5-HT) synthesis in the brain modulates responses of limbic circuits to emotional stimuli and has been linked to a spectrum of clinical populations characterized by emotional dysregulation. Here, we tested a set of common single nucleotide polymorphisms (SNPs) in and downstream of the transcriptional control region of TPH2 for association with personality traits and with risk for personality disorders in two cohorts comprising of 336 healthy individuals and 420 patients with personality disorders. Personality dimensions were assessed by the Tridimensional Personality Questionnaire (TPQ) and the revised NEO Personality Inventory (NEO-PI-R). Personality disorders were diagnosed with the Structured Clinical Interview of DSM-IV and were allocated to clusters A, B, and C. Individual SNP and haplotype analyses revealed significant differences in genotype frequencies between controls and cluster B as well as cluster C patients, respectively. In both patient groups, we observed overrepresentation of T allele carriers of a functional polymorphism in the upstream regulatory region of TPH2 (SNP G-703T, rs4570625) which was previously shown to bias responsiveness of the amygdala, a structure critically involved in emotionality. Furthermore, significant effects of TPH2 variants on anxiety-related traits defined primarily by the TPQ Harm Avoidance were found in healthy individuals. The results link potentially functional TPH2 variants to personality traits related to emotional instability as well as to cluster B and cluster C personality disorders. These findings implicate alterations of 5-HT synthesis in emotion regulation and confirm TPH2 as a susceptibility and/or modifier gene of affective spectrum disorders.

Dysfunction of the central serotonergic system has been implicated in the pathophysiology of obsessive-compulsive disorder (OCD). The genetic contribution to the development of OCD is particularly high in early-onset OCD. The aim of this study was to investigate the effect of polymorphic variants in the gene of the novel brain-specific tryptophan hydroxylase-2 (TPH2), the rate-limiting enzyme of serotonin (5-HT) synthesis in the brain, in OCD with disease onset in childhood and adolescence. We analysed two common single nucleotide polymorphisms (SNPs) of TPH2 in the putative transcriptional control region and in intron 2 of the TPH2 gene in a unique family-based sample of OCD patients with onset of the disease in childhood and adolescence comprising 71 complete, independent trios. The transmission disequilibrium test was used to determine transmission of alleles and haplotypes from parents to offspring. In this first study of TPH2 in OCD, analysis of the SNPs, rs4570625 and rs4565946, revealed a significant preferential transmission of haplotype G-C to children and adolescents with OCD. Moreover, a trend towards preferential transmission of the C allele of SNP rs4565946 to the patients was found. The genotype relative-risk estimate for homozygous C allele carriers of SNP rs4565946 was 2.58 (95% CI 0.98-6.82). In conclusion, the results link TPH2 variations to the pathogenesis of early-onset OCD and further support the aetiological relevance of 5-HT signalling in OCD. Copyright © 2005 CINP.

Genetic variants of the monoamine oxidase A (MAOA) have been associated with aggression-, anxiety-, and addiction-related behavior in several nonclinical and clinical populations. Here, we investigated the influence of allelic variation of MAOA activity on aggressionrelated personality traits and disease risk in patients with personality disorders. Personality disorders were diagnosed with the Structured Clinical Interview of DSM-IV and were allocated to cluster A, B, and C. Personality features were assessed by the revised NEO Personality Inventory and the Tridimensional Personality Questionnaire. The genotype of the MAOA gene-linked polymorphic region (MAOA-LPR) was determined in 566 patients with personality disorders and in 281 healthy controls. MAOA genotype was significantly associated with cluster B personality disorders (w 2 ¼ 7.77, p ¼ 0.005, df ¼ 1) but not with cluster C personality disorders. In total, 26.0% of cluster B patients were hemi- or homozygous for the low-activity variant of the MAOA genotype, compared to 16.4% in the control group. Associations between MAOA variants and personality domains related to impulsivity and aggressiveness were inconsistent. Our findings further support the notion that allelic variation of MAOA activity contributes modestly to the balance of hyper- (impulsiveaggressive) and hyporeactive (anxious-depressive) traits. Neuropsychopharmacology (2005) 30, 1711–1718. doi:10.1038/sj.npp.1300737; published online 4 May 2005

The neurobiology of anxiety is complex, reflecting the cumulative physiological effects of multiple genes. These genes are interactive with each other and with the environment in which they are expressed. Variation in genes coding for proteins that control serotonin (5-HT) system development and plasticity, establish 5-HT neuron identity, and modulate 5-HT receptor-mediated signal transduction and cellular pathways have been implicated in the genetics of anxiety and related disorders. Here, we selected anxiety and avoidance as paradigmatic traits and behavior and cover both traditional studies with inbred murine strains and selected lines which have been modified by gene knockout technologies. The design of a mouse model partially or completely lacking a gene of interest during all stages of development (constitutive knockout) or in a spatio-temporal context (conditional knockout) is among the prime strategies directed at elucidating the role of genetic factors in fear and anxiety. In many cases, knockout mice have been able to confirm what has already been anticipated based on pharmacological studies. In other instances, knockout studies have changed views of the relevance of 5-HT homeostasis in brain development and plasticity as well as processes underlying emotional behavior. In this review, we discuss the pertinent literature regarding phenotypic changes in mice bearing inactivation mutations of 5-HT receptors, 5-HT transporter, monoamine oxidase A and other components of the serotonergic pathway. Finally, we attempt to identify future directions of genetic manipulation in animal models to advance our understanding of brain dysregulation characteristic of anxiety disorders.

Panic disorder is a common anxiety disorder which frequently co-occurs with agoraphobia. A functional promoter polymorphism in the serotonin receptor 1A (5-HT1A) gene has been found to be associated with major depression as well as anxiety- and depression-related personality traits. We investigated a possible association between this 5-HT1A gene promoter polymorphism and panic disorder by genotyping the −1019C>G single nucleotide polymorphism in 134 panic-disorder patients with and without agoraphobia and matched 134 controls. In our sample no significant evidence of allelic association in the combined panic-disorder group was found. However, our results show a significant association with the G allele in patients with panic disorder with agoraphobia ( p =0.03, n =101). In conclusion, our findings do not support a major contribution of this polymorphism to the pathogenesis of panic disorder, but provide evidence for a possible role in the subgroup with agoraphobia.

Context Human personality is characterized by substantial heritability but few functional gene variants have been identified. Although rodent data suggest that the neuronal isoform of nitric oxide synthase (NOS-I) modifies diverse behaviors including aggression, this has not been translated to human studies. Objectives To investigate the functionality of an NOS1 promoter repeat length variation ( NOS1 Ex1f variable number tandem repeat [VNTR]) and to test whether it is associated with phenotypes relevant to impulsivity. Design Molecular biological studies assessed the cellular consequences of NOS1 Ex1f VNTR; association studies were conducted to investigate the impact of this genetic variant on impulsivity; imaging genetics was applied to determine whether the polymorphism is functional on a neurobiological level. Setting Three psychiatric university clinics in Germany. Participants More than 3200 subjects were included in the association study: 1954 controls, 403 patients with personality disorder, 383 patients with adult attention-deficit/hyperactivity disorder (ADHD), 151 with familial ADHD, 189 suicide attempters, and 182 criminal offenders. Main Outcome Measures For the association studies, the major outcome criteria were phenotypes relevant to impulsivity, namely, the dimensional phenotype conscientiousness and the categorical phenotypes adult ADHD, aggression, and cluster B personality disorder. Results A novel functional promoter polymorphism in NOS1 was associated with traits related to impulsivity, including hyperactive and aggressive behaviors. Specifically, the short repeat variant was more frequent in adult ADHD, cluster B personality disorder, and autoaggressive and heteroaggressive behavior. This short variant came along with decreased transcriptional activity of the NOS1 exon 1f promoter and alterations in the neuronal transcriptome including RGS4 and GRIN1 . On a systems level, it was associated with hypoactivation of the anterior cingulate cortex, which is involved in the processing of emotion and reward in behavioral control. Conclusion These findings implicate deficits in neuronal signaling via nitric oxide in moderation of prefrontal circuits underlying impulsivity-related behavior in humans.

Panic disorder is an anxiety disorder with an estimated heritability of 48%. Associations findings have been obtained with candidate genes from both serotonergic and noradrenergic pathways including regulatory and coding variants of the serotonin receptor 1A gene, the monoamine oxidase A gene, the catechol-O-methyltransferase gene and the norepinephrine transporter gene.In the present study, an analysis of interactions between the functional serotonin receptor 1A polymorphism, the norepinephrine transporter variants and the other respective polymorphisms of the above-mentioned genes is reported. The analysis is based on genotype results from 115 cases and 115 age and sex-matched controls.A nominally significant (P=0.04) interaction between the serotonin receptor 1A and the catechol-O-methyltransferase polymorphisms was observed. Stratified analysis revealed that the odds ratio of each polymorphism was highest in the presence of the low-risk genotype(s) of the other polymorphism and low in the presence of the high-risk genotype(s) of the other polymorphism.This is the first possible interaction of genetic variations in panic disorder that has been observed. As the sample size was small and no adjustment for multiple testing was made, the assessment of the interacting risk alleles needs replication in a larger sample with higher power.