TPH2 in personality traits of negative emotionality and in disorders of cognitive control and emotion regulation

Human variants of TPH2 have been investigated for association with personality and behavioural traits as well as with various clinical cohorts characterized by emotion dysregulation. While traits of emotionality are persistent and continuously distributed dimensions of normal human personality, pathological manifestations of cognition and emotion regulation are ubiquitous in a wide spectrum of psychiatric conditions. Variance in personality traits, including those related to failure in cognitive control and emotion regulation, such as anxiety, depression and aggression, is thought to be generated by a complex interaction of environmental factors with a number of gene products involving brain structures and circuits such as the 5-HT system. Several single nucleotide polymorphisms (SNPs) in and downstream of the transcriptional control region of TPH2 showed association with personality traits as well as cluster B and cluster C personality disorders [24]. Cluster B comprises antisocial, borderline and narcissistic personality disorders (dramatic, emotional or erratic cluster), and cluster C consists of avoidant-, dependent- and obsessive-compulsive personality disorders (anxious or fearful cluster).

Functional magnetic resonance imaging (fMRI) provides evidence that acute tryptophan depletion, which results in a transient reduction in brain 5-HT (for review, see [25] and references therein), as well as a single, potentially functional, variant in the upstream regulatory region of TPH2, bias the responsiveness of the amygdala in a face-processing task involving assessment of angry and fearful faces [26,27], indicating that allelic variation of TPH2 function may contribute to individual variability in stress responsivity and anxiety in humans. Moreover, Tph2 polymorphisms predict brain serotonin synthesis in the orbitofrontal cortex in humans estimated in vivo using positron emission tomography and α-[¹¹C]methyl-l-tryptophan trapping [28]. There is also emerging evidence from psychophysiological studies that TPH2 variation influences 5-HT synthesis in the brain and thus modulates emotional processing. Startle responses to intense noise bursts in individuals viewing pictures of negative, positive or neutral valence showed an interaction between TPH2 genotype, sex and age [29]. Two genes of the 5-HT signalling pathway, TPH2 and 5-HTT/SERT, encoding the 5-HT transporter were demonstrated to exert additive effects using event-related potentials for the early posterior negativity in a passive emotional picture perception task and fMRI in a complementary cognitive-affective task [30,31]. The additive effect in the MRI paradigm was more pronounced for visuospatial than for verbal stimuli, and more robust for negatively than for positively valenced stimuli, whereas fMRI effects were strong in the putamen, albeit also observed in the amygdala at a less stringent threshold, and in other cortical regions. These findings indicate an additive effect of two critical genes in the serotonergic regulation of neural processing of affective stimuli, and identify the putamen as a subdivision of the striatum as a critical site where interactive gene-by-gene regulation takes place. TPH2 variants were found to be associated with function of the prefrontal cortex during a response inhibition task in adult patients with ADHD, suggesting that deficient cognitive control involves a mechanism relevant to the pathophysiology of ADHD [32]. Taken together, these findings link potentially functional TPH2 variants to personality traits related to negative emotionality as well as to categorical cluster B and C personality disorders and confirm TPH2 as a susceptibility and/or modifier gene for disorders characterized by emotion dysregulation.

In line with this notion, SNP and haplotype analyses of TPH2 revealed evidence for association of TPH2 variants with depression, suicide and bipolar affective disorder, although inconsistent findings were also reported (for review, see [12]). Investigation of TPH2 expression in the brainstem of depressed patients who had committed suicide demonstrated increases in TPH2 mRNA [33,34] and protein [35–37] within the DR with evidence for specificity in distinct subdivisions. Increased TPH2 expression in depressed patients could result from both rare and frequent variants, their epistatic interaction among themselves (gene-by-gene interaction, G × G) and their interaction with early life experiences, acute stressful life events or chronic environmental adversity (gene-by-environment interaction, G × E), all of which can alter 5-HT neurotransmission and have been implicated in determining susceptibility to depression and a spectrum of co-morbid disorders, such as alcohol dependence [38].

Finally, allelic variation of TPH2 function appears to influence the risk of a variety of neuropsychiatric disorders such as ADHD and obsessive–compulsive disorder (OCD), clinical entities commonly associated with difficulties to control emotions and with a high co-morbidity of depression [39,40]. Transmission disequilibrium of potentially functional variants in the transcriptional control or in the coding region of TPH2 in ADHD [41–43], and preferential transmission of a haplotype of TPH2 in early-onset OCD [44] were reported. However, common variants in the TPH2 region did not seem to be associated with adult ADHD in a large European sample [45].

Previous SectionNext Section

3. Tph2 mutant mice

(a) Behavioural phenotypes

Various approaches have been used to experimentally alter Tph2 expression and function in mice, including the constitutive Tph2 KO reviewed here [46] (figures 1 and 2; table 1). Commonly used inbred mouse strains were found to be homozygous for the Tph2 1473G allele (e.g. BALB/c; DBA/2), resulting in 40–70% reduction in 5-HT synthesis and a 40 per cent decrease in 5-HT concentrations in frontal cortex and striatum when compared with mice homozygous for the 1473C allele (e.g. C57Bl/6; 129S1/SvJ) [47]. The strains with a less-active version of Tph2 show lower aggression and increased anxiety-like behaviour [48]. In order to clarify that these behaviours are specifically mediated by the C1473G SNP and not by other strain-specific genetic background effects, the 1473G was crossed into mouse strains that naturally express the other Tph2 allele; however, this approach yielded contradictory results [49,50]. C57Bl/6 mice homozygous for the 1473G allele as well as BALB/c mice show reduced 5-HT synthesis rates, but 5-HT tissue concentrations remain unchanged, which indicates that altered 5-HT levels and behaviour in BALB/c mice are likely to be induced by other strain-dependent factors rather than by the Tph2 G1473C SNP. On the other hand, this finding suggests that the 5-HT system is able to compensate for reduced 5-HT synthesis, which is in line with the data derived from mice carrying the human TPH2 loss-of-function R439H mutation and displaying a 50 per cent reduction in extracellular 5-HT in various brain regions [19,20].

Mice with targeted inactivation of Pet1 [17] and Lmx1b [16,51], coding for transcription factors involved in the specification of 5-HT neurons, were also generated. Both represent modification functionally upstream of the specification process rather than a selective inactivation of neuronal 5-HT synthesis. In Pet1 KO mice, 5-HT deficiency is incomplete with approximately 30 per cent of the 5-HT neurons developing and persisting in various raphe nuclei. While mice with a constitutive Lmx1b inactivation are not viable, in conditional Lmx1b KO (cKO) mice, in which the gene deletion is driven specifically in 5-HT neurons, these neurons are generated but fail to differentiate and survive. In contrast, in Tph2 ^−/− mice, raphe neurons and their projections, although devoid of Tph2 and 5-HT, are morphologically and functionally preserved [11,14].

(i) Impulsivity and aggression

Defensive aggression-like behaviour of a male resident towards male intruders is an ethologically determined response to territorial threats. Overwhelming evidence links 5-HT to impulsive and aggressive behaviour as the primary determinant of aggression control [1]. Several regions of the frontal and cingulate cortices, amygdala, septum, hypothalamus and periaqueductal grey matter are among the best documented to be involved in the neural circuitry of aggression. Serotonergic fibres extensively project to each of these regions and it is well established that both aggressiveness and increased impulsivity are associated with brain 5-HT deficiency. In the resident-intruder paradigm, Tph2 ^−/− males exhibit up to 10-fold more defensive aggressive behaviour, particularly increased impulsivity reflected by decreased latency of the first attack, number of attacks and duration of fighting than controls [46]. Chronic unpredictable stress further aggravates these traits. The impulsive and hyperaggressive behaviour of Tph2 ^−/− mice resembles the increased defensiveness reported for Pet1 KO [17] and Tph2 R439H mutants [19]. Acute treatment with 5-HT_1A and 5-HT_1B receptor agonists (or 5-HT_2A/2C antagonists) via their inhibitory action on neurotransmission (pre-synaptically or post-synaptically) was reported to reduce aggressive behaviour, and it was suggested that low 5-HT levels in the brain are associated with maladaptive forms of excessive violence rather than with natural defensiveness [3]. Because 5-HT deficiency is likely to result in impaired inhibition of engagement and sustainment of aggressive behaviour, it may explain that Tph2 ^−/− mice display exaggerated aggressive behaviour as a consequence of the failure of 5-HT-mediated inhibitory control, thus rendering these mice inept to acquire the abilities of social adjustment.

(ii) Anxiety-like behaviour and conditioned fear response

Functional variants in TPH2 are associated with anxiety-related, harm-avoidant and other personality traits of negative emotionality as well as with various clinical cohorts with neuropsychiatric disorders characterized by emotional dysregulation. Likewise, Tph2 KO mice exhibit altered anxiety-like and conditioned fear response-related behaviours in a sex-dependent mode. Although female Tph2 ^−/− mice decreased anxiety-like behaviour on the elevated plus-maze (EPM), which tests conflict-based exploration of aversive environment, genotype effects were not significant in males. Sex differences have previously been observed in mouse models with a range of genetic lesions impacting 5-HT system development and function [1,52].

Similar to Tph2 null mutant mice, Lmx1b cKO mice exhibit reduced innate anxiety-like behaviour [15], whereas for Pet1 KO mice, anxiogenic as well as anxiolytic effects were reported [17,18,53]. Selective lesion studies of ascending serotonergic pathways by neurotoxins resulted in varying degrees of anxiolytic effects dependent on experimental condition and site of injection [54–57]. As an integral part of the neural circuitry of stress responsivity, the 5-HT system has generally been viewed to exert modulatory functions. In addition, the findings derived from mouse models deficient in brain 5-HT provide evidence that 5-HT also moderates neurobiological consequences of environmental adversity, enhances appraisal of threats as well as behavioural expression of innate anxiety and fear responses, thereby permanently encoding the impact of stressful experience.

Despite reduced innate anxiety-like behaviour, Tph2 KO mice display gene dose- and sex-dependent increases in fear acquisition and memory in both cue and context trials compared with controls [46]. After chronic mild stress (CMS) experience, Tph2 ^−/− mice are insensitive to stress-induced increases in locomotor activity and resilient to stress-induced anxiety-like behaviour, but increases in fear responses are intensified. Lmx1b cKO and Pet1 KO mice also show a marked increase in freezing following fear learning [15,18]. These findings suggest that 5-HT is critical for the inhibition of exaggerated fear acquisition via neural circuits involving amygdala and connected structures such as hippocampus, medial prefrontal cortex (mPFC), hypothalamus, bed nucleus of the stria terminalis (BNST) and brainstem nuclei, including the raphe complex and locus coeruleus (LC) [58].

While anxiety and fear are assumed to be separate dimensions within a spatial continuum, it appears paradoxical only at first sight that central 5-HT deficiency leads to a dissociation of conditioned fear from innate anxiety. On the one hand, anxiety-like behaviour elicited by the EPM test corresponds to more diffuse and generalized anxiety in anticipation of potential distant danger (instinctive fear of predators) not yet identifiable and to which an escape exists by returning to the closed arm (conflict exploration versus risk). On the other hand, during the conditioning process, the animal has actually received an uncontrollable aversive stimulus and has to face instant threat from which no escape is possible. As these two experiences are distinct regarding the anxiogenic circumstances and the neural circuits involved, opposing effects of 5-HT on general anxiety and learned fear seem nevertheless plausible [59,60]. Involvement of different pathways is also indicated by differential pharmacological modulation [61]. An alternative model describing complementary effects of the central nucleus of the amygdala (CeA) and the BNST on potentiated fear (e.g. post-traumatic or panic disorder) and sustained non-associated fear (e.g. generalized anxiety disorder), respectively, was proposed, with low anxiety being associated with potentiated fear responses [62].

The amygdala is central to emotion processing and modulation of fear-related behaviour, ranging from innate anxiety to conditioned fear acquisition and retention [63]. The lateral amygdala (LA) is richly innervated by 5-HT fibres and serves as the perceptive interface, as it receives multi-modal, early sensory information from the thalamus and cortical regions [64,65] and, together with the basolateral nucleus of the amygdala (BLA), is the principal unit where fearful memory is generated and stored. Anxiety- and fear-related input is then processed towards specific downstream pathways to express appropriate behavioural responses. As an early step, the CeA is known to be the output unit for freezing behaviour in fear conditioning, while the BNST would be the effector station for sustained non-associative anxiety [62]. The LA is a cortex-like structure composed of projecting glutamatergic pyramidal cells and gamma-aminobutyric acid (GABA)ergic interneurons, which receive modulatory 5-HT projections [66] (also see §3 d). Given the behavioural profile displayed by Tph2 ^−/− mice, low innate anxiety but high fear-conditioning, it may be assumed that the basal activity or the encoding of fear-associated stimuli in amygdala are altered by 5-HT deficiency. Recording of spontaneous activity revealed hypoactivity of glutamatergic pyramidal neurons in Tph2 ^−/− mice as an electrophysiological correlate of reduced innate anxiety-like behaviour, possibly mediated via a reduced activation of the BNST (Araragi et al. manuscript in preparation).

In contrast, evoked response following cortical fibre stimulation revealed increased efficiency of the cortical-amygdaloidal pathway in Tph2 ^−/− mice, which appears to represent a neurophysiological correlate of their exaggerated fear learning and memory in fear conditioning via an over-activation of the CeA and its downstream neural circuit. Fear conditioning was previously shown to be a molecular process increasing synaptic efficacy on LA neuron dendrites as a result of input from cortical and/or thalamic fibres conveying the unconditioned (US) and conditioned stimulus (CS), commonly called long-term potentiation (LTP; reviewed in [67]). LTP in the LA appears to be a critical mechanism for storing memories of the association between the CS and US [68,69]. LTP formation may be enhanced when at least one of the involved pathways, thalamic or cortical, is more sensitive, as demonstrated in Tph2 ^−/− mice. Together with previous reports [70], the view is supported that 5-HT deficiency within the LA leads to a reduced activation of GABAergic interneurons [71], which in turn results in insufficient inhibition of glutamatergic projecting neurons and failure to delimit exaggerated fear responses. Taken together, it may be concluded that 5-HT mediates distal aversive stimuli, but is not essential for the integration of proximal or physical aversive stimuli, thus differentially regulating distinct context- and neural-pathways-dependent forms of fear or anxiety.

(iii) Depression-like behaviour

Although depression-like behaviour is challenging to model in mice, the tail suspension test (TST) and the forced swim test (FST) are widely used to validate antidepressant drugs. Tph2 ^−/− mice exhibited more behavioural despair as reflected by the onset and duration of immobility when facing a life-threatening inescapable situation, thus confirming the notion of a link between 5-HT deficiency and depression-like behaviour [46]. Contrasting results between FST and TST were reported for a different line of Tph2 ^−/− mice [23], while Pet1 KO mice did not display more behavioural despair [53], and Tph2 R439H mutant mice showed increased immobility in the TST [19]. Because extreme 5-HT deficiency also leads to a reduction in brain norepinephrine (NE; see §3 c), it is possible that the latter contributes to the observed phenotype. Remarkably, 5-HT deficiency-driven depression-like behaviour was reversed by CMS, resulting in an essentially rescued phenotype, whereas no effect of CMS was seen in controls. Although this possibility cannot be completely ruled out, it is rather unlikely that the increase in active struggle of Tph2 ^−/− mice is merely due to a CMS-induced locomotor activation, higher impulsivity or cognitive flexibility, because other behavioural paradigms failed to provide evidence for an alteration of these traits. Taken together, these findings indicate that CMS rescues behavioural consequences of emotionality in 5-HT-deficient mice, thus increasing adaptive capacity, and thus resilience, to the deleterious effects of Tph2 inactivation.

Reduction of immobility in the FST following CMS is uncommon but has been observed in previous studies using for example, parachloroamphetamine (PCA)-induced partial lesioning of 5-HT fibres followed by chronic variable stress [72]. Moreover, work in rodents by other investigators demonstrated that the experience of controllable stress may improve coping during subsequent stressful episodes by activating the mPFC, which in turn inhibits DR-mediated adverse effects of stress [73–75]. While CMS is not typically controllable stress, its ‘real life’ quality for a mouse living in a hostile, predator-infested habitat may induce similar effects in conjunction with DR malfunction owing to the lack of 5-HT synthesis [76]. Alternatively, predictable CMS appears to improve mood by increasing adult neurogenesis [77]. Multiple adaptive mechanisms along various developmental trajectories are therefore likely to be operative in Tph2 ^−/− mice to modify brain function and responses to environment adversity. From a clinical point of view, it appears rather counterintuitive that 5-HT deficiency at the same time results in anxiolytic effect and in depression-like behaviour. Nevertheless, corresponding models similar to those of the 5-HT_1A KO mouse emulate the phenotype of Tph2 ^−/− mice with increased anxiety and reduced depressive-like behaviour [78]. In depressed patients, depression is frequently accompanied by symptoms of anxiety, which are ameliorated by compounds targeting 5-HT (and NE) neurotransmission.

Anxiety disorders are frequently associated with co-morbid depression. However, ethologically relevant behaviour in mice and symptoms in depressed patients are far from being homologous. In humans, depression is complicated by conscious cognitive and emotional re-evaluation and by projections into the future, which can be anticipated as dark, hopeless and anxiety-provoking. Moreover, species-typical symptoms, such as guilt, suicidality, projection and introjection, cannot be modelled in rodents. In humans, cognitive appraisal modulates brain responses to emotional stimuli and carries the potential to counteract both genetic and environmental susceptibility factors. In this context, the regulatory role of the prefrontal cortex in controlling limbic structures is critical. Thus, a simplified, more manageable and versatile model such as the Tph2 K O mouse may help deciphering basic mechanisms and neuronal circuits involved in this dual role of 5-HT likely to be operant in humans as well. Provided that diagnostic tools allow distinction of different symptoms, the form of anxiety co-morbid with depression might be of different nature and aetiology than those of core anxiety disorders, such as generalized anxiety disorder, phobias, post-traumatic disorder and panic disorder. The dual 5-HT hypothesis further elaborated by Graeff and associates [79] describes distinct neural circuits emerging from the DR and MnR, respectively promoting and preventing anxiety and depression. While complex emotional states cannot be reduced to imbalance within a single neurotransmitter-specific circuitry, the lack of 5-HT in both DR and MnR in stress-naive Tph2 ^−/− mice is in accordance with this dual model. Taken together, these findings suggest that it is clinically relevant to understand the neural circuitry and adaptive mechanisms that mediate the amelioration of depressive symptoms by mild stressors of an enriched environment.

(b) Specification of raphe neurons lacking 5-HT synthesis

Several in vitro studies showed a morphogenic effect of 5-HT on proliferation, differentiation, migration and survival of neural cells [80–82]. During ontogeny, 5-HT appears long before maturation of raphe serotonergic neurons, suggesting a fundamental role in embryonic and brain development. Whereas in vivo studies generally underscore this notion, conditional Lmx1b KO mice, which are largely deficient in central serotonergic neurons, are viable without apparent developmental abnormalities [16,51,83]. Likewise, neuroanatomical alterations were not observed in the brain of Tph2 ^−/− mice (11,14). Conserved particularly is the expression of genes specifying a serotonergic phenotype in raphe neurons lacking 5-HT synthesis. The serotonin transporter (5-Htt/Sert) is present on the soma of raphe neurons as well as on their fibres and terminals in various projection areas, although they have lost the capacity to synthesize and release 5-HT. The 5-HT neuron-specific transcription factor Pet1, the vesicular monoamine transporter-2, as well as the somatodendritic autoreceptors 5-HT_1A are expressed by raphe cells displaying a 5-HT neuron-like morphological phenotype.

In brainstem sections of Tph2 ^−/− mice, the 5-HT-devoid neurons retain the slow, tonic pattern of firing typical of serotonergic neurons (1–2 spikes/s) demonstrating that endogenous 5-HT is not required to mediate these electrophysiological properties. Similarly, the neurons preserve the sensitivity of their pacemaker rhythm to the inhibitory effect of pharmacological 5-HT_1A receptor activation. Moreover, these neurons lack sensitivity to the inhibitory effect of Trp, confirming complete loss of Tph functionality and 5-HT-synthesis capacity. When the Tph2-dependent rate-limiting step is bypassed by supplementation with the intermediary 5-HTP, the synthesis of 5-HT is re-established as reflected by inhibition of firing activity. Because 5-HTP is selectively taken up by serotonergic neurons in the DR and converted into 5-HT by AADC [84], serotonergic-like neurons of Tph2 ^−/− mice are sensitive to endogenous 5-HT and their lack of response to Trp is not due to dysfunctional 5-HT_1A autoreceptor signalling (also see §3 e). In brainstem slices from Tph2 ^+/− mice, serotonergic neurons responded to Trp as well as the 5-HT_1A receptor agonist 8-OH-DPAT, with a decrease in firing rate similar to that observed in wild-type mice, suggesting that a gene dose-dependent reduction in 5-HT synthesis does not result in functional changes in the 5-HT system at baseline.

While genetic inactivation of the upstream transcription factors Lmx1b and Pet1 compromises the development of the majority of 5-HT neurons [16,17,51], it is concluded that intrinsic 5-HT production is neither essential for the development maintenance and survival of serotonergic neurons, nor for the molecular specification of a serotonergic-like phenotype. This further suggests that the developmental role of 5-HT in the maturation/differentiation of the 5-HT system itself has been overvalued, and the notion of an autoregulation of 5-HT system development [6,7] may need to be addressed from a new standpoint. It remains to be elucidated in detail whether subtle impairment in neurite outgrowth, axonal guidance/target finding and altered dendritic arborization occurs and whether these neurons use neuropeptides and/or other monoamines with low affinity for the 5-Htt/Sert as physiological or ‘borrowed’ neurotransmitters in establishing function and connectivity. Together, functional serotonergic-like neurons in Tph2 K O mice have lost the capacity to synthesize 5-HT from Trp but not from 5-HTP, which in vivo may originate from peripheral sources and could contribute to the remaining traces of 5-HT in the brain of Tph2 ^−/− mice.

(c) Monoamine transmitters and neurons

In Tph2 ^−/− mutants, 5-HT concentrations are markedly reduced across all brain regions and virtually absent from the serotonergic neuron-containing raphe region, with only traces detectable by HPLC, thus verifying that 5-HT synthesis within neurons depends on the activity of the Tph2 isoform [46]. While perfusion of brain with removal of the residual blood in capillaries resulted in minimal amounts of 5-HT in the rostral raphe region at the lower detection limit (less than 1.2%), it remains possible that a small number of blood components with high 5-HT content, such as platelets or mastocytes, remain trapped in capillaries of brain tissue [14]. Very low brain 5-HT levels were also detected in other Tph2 ^−/− mice [21] as well as in Tph1/Tph2 ^−/− double KO mice [23]. In addition, we previously showed that Tph1 is not upregulated in Tph2 ^−/− brain, indicating that Tph1-driven 5-HT synthesis can be ruled out in the brain [11]. However, there are several alternative explanations: (i) HPLC does not detect 5-HT but a closely related compound with the same retention time, a possibility to be resolved by mass spectrometry, (ii) the immediate 5-HT precursor (5-HTP) produced by peripheral Tph1 crosses the blood-brain barrier and could be transformed into 5-HT because AADC is ubiquitously expressed, (iii) other enzymes, such as phenylalanine hydroxylase, or as yet unknown enzymes, use tryptophan as substrate and produce 5-HT, and (iv) alternative metabolic pathways are able to produce 5-HT as end- or by-product. Of note, 5-hydroxindoleacetic acid (5-HIAA) is more reduced or even undetectable than 5-HT itself, suggesting that either the metabolic pathway of 5-HT is inhibited, with monoamine oxidase A (MAOA) activity specifically downregulated in 5-HT neurons, or the 5-HT-like traces do not represent 5-HT but another compound degraded via another pathway. Taken together, the deficiency in 5-HT is so extreme that complete dysruption of 5-HT neurotransmission in Tph2 ^−/− brain is likely despite the presence of neurons with 5-HT cell-like specification.

While dopamine concentrations are reduced only in hippocampus, 5-HT deficiency is accompanied by a consistent reduction in NE across brain regions. Furthermore, Tph2 ^−/− mice exhibited a reduced number of tyrosine hydoxylase (TH)-expressing cells in the LC, which can partly explain the lower NE content in its projection areas using unbiased stereological assessment [46]. The LC is extensively innervated by Sert-positive fibres containing 5-HT in controls and devoid of 5-HT in Tph2 ^−/− mice. It is conceivable that the absence of trophic effect of 5-HT in Tph2 ^−/− mice impacts development or survival of NE-specific neurons. Alternatively, the absence of 5-HT release may inhibit expression and activity of TH in NE neurons, presumably by indirect input from inhibitory GABAergic or excitatory glutamatergic neurons (also see §3 d), expression and activity of TH in NE neurons. Several studies reported that chronic treatment with the SSRI fluoxetine induces an increase in TH gene expression in the LC [85,86]. Conversely, 5-HT deficiency may thus downregulate TH activity in the LC, eventually reducing NE biosynthesis.

TH is also present along NE fibres projecting towards target areas and regulation terminally is likely because the DR does not seem to exert a direct inhibitory influence on the release of NE in the LC [87]. While 5-HT and NE fibres with synaptic varicosities co-localize in forebrain regions, a feedback loop, involving alpha₂-adrenergic receptors on 5-HT fibres and 5-HT₃ receptors on NE fibres, allows a reciprocal regulation of the release of both neurotransmitters by which 5-HT₃ receptors stimulate the synaptic release of NE [88]. The stimulation of the neurotransmitter release is accompanied by an activation of its synthesis, whereas the lack of stimulating effect by 5-HT on NE fibres reduces TH activity and thus NE synthesis. Overall, the findings confirm that monoaminergic systems are interdependent and subject to concomitant regulation in behaviour and psychopathology.

(d) Gamma-aminobutyric acid and interneurons

Morphogenic effects of 5-HT impact migration, differentiation and survival of GABAergic interneurons [80–82] and 5-HT influences GABAergic cell migration via 5-HT6 receptors during late embryonic stages [89], thus assisting their integration into cortical networks [90]. The BLA is fundamentally involved in the regulation of fear and anxiety [91] and is densely innervated by serotonergic fibres from the DR nucleus [92]. Within the BLA, parvalbumine (PV)/GABAergic neurons specifically express 5-HT_2A receptors [93] and tightly control glutamatergic output neurons by perisomatic inhibition [94], whereas 5-HT_2C receptors are expressed on other interneuron subtypes [95]. Furthermore, anxiogenic compounds have been shown to recruit GABAergic interneurons, including the PV-specific subpopulation in the BLA probably via serotonergic input from the DR [96]. The dorsal hippocampus was shown to be critically involved in context-dependent learning processes [97–99]. While the dorsal and ventral hippocampus are interconnected, they represent functionally separate subdivisions integrated in different neuronal networks, thus mediating diverse behaviours [100]. Furthermore, the distribution of different interneuron subtypes (including the PV-specific population) was shown to be distinct for both subregions of the hippocampus [101].

Elimination of 5-HT synthesis does not appear to affect GABA concentrations in whole-brain tissue [21], but measurement in different brain regions of Tph2 KO mice in combination with unbiased stereological assessment of GABAergic cell subpopulations in the hippocampus and amygdala revealed differential alterations [102]. While hippocampal GABA concentrations were increased in Tph2 ^−/− mice, GABA was increased in heterozygous Tph2 ^+/− mice in the amygdala compared with Tph2 ^−/− and wild-type control mice but opposite in prefrontal cortex. This was accompanied by altered cell density of GABAergic interneurons within the BLA and of PV-specific GABAergic interneurons in the CA3 region of the dorsal hippocampus.

Increased GABAergic transmission in the BLA has been associated with reduced anxiety-related behaviour [103], whereas mice deficient for GAD65 display 50 per cent reduced GABA concentrations in the amygdala and exhibit an anxiety-like phenotype [104]. In contrast, increased GABA transmission was shown in a mouse model of increased trait anxiety [105]. Tph2 ^−/− mice exhibited an altered anxiety-related phenotype with a dissociation of innate anxiety-like behaviour and conditioned fear responses but unchanged GABA concentrations. In contrast, Tph2 ^+/− mice showed an intermediate behavioural phenotype compared with Tph2 ^−/− and wild-type animals. This may be due to a counterbalancing effect of an impaired inhibition of the PFC indicated by reduced GABA concentrations in Tph2 ^+/− and of glutamate concentrations specifically increased in PFC of Tph2 ^−/− mice. Altered function of the PFC controlling other subcortical structures of the limbic system [106]—possibly as a consequence of increased activation of intercalated neurons residing at the boundary of the BLA to the central nucleus of the amygdala [107]—is likely to result in an increase in the frequency of inhibitory post-synaptic potentials in BLA output neurons. Recently, mice expressing the R439H TPH2 form were found to display increased cortical 5-HT_2A receptor expression due to diminished concentrations of 5-HT [20]. Tph2 ^+/− mice possess 20–30% reduced 5-HT concentrations in the rostral raphe but unaffected frontal cortex 5-HT concentrations. Distinct 5-HT receptor expression and activation by pyramidal cells and interneuron subtypes may lead to a disturbed control of network activity through altered gamma oscillations [108]. Therefore, elevated GABA concentrations in the Tph2 ^+/− mice may either be directly triggered by the impact of 5-HT deficiency on network activity or represent a consequence of compensatory mechanisms during development increasing expression and activation of 5-HT_1A and 5-HT2_A/C receptors. Reduced 5-HT concentrations in Tph2^+/− mice seem to be sufficient to develop normal numbers of interneurons within the BLA.

In Tph2 ^−/− mice, the overall number and density of GABAergic interneurons within the BLA were decreased [102]. Possibly as an outcome of impaired proliferation, this may represent a mechanism to cope with an imbalance of GABAergic transmission during ontogeny, and to maintain synchronous oscillatory activity, which has been shown to be important for fear learning and memory [94,109]. As PV-specific neurons were unaffected, other interneuron subpopulations might account for the decreased density of interneurons [110] and altered GABA concentrations [111]. However, because total cell numbers in the BLA remain unchanged, other populations such as glial cells or glutamatergic neurons within the BLA may be increased to account for unaffected total cell numbers. Tph2 ^−/− mice also displayed elevated concentrations of GABA in the hippocampus, with a trend towards reduced PV-specific neuron numbers in the CA3 region of the dorsal hippocampus, whereas volume, total number and density of interneurons remain unaffected. Selective activation of MnR 5-HT neurons has been reported to directly activate dorsal hippocampal interneurons [112], leading to an overall inhibition of the hippocampal formation. Dense innervations of the MnR originating beaded serotonergic axons with large spherical varicosities and fine DR axons can be found in the dorsal hippocampus [113,114]. Serotonergic fibres innervate different subpopulations of interneurons in the hippocampus acting in concert with cholinergic fibres in regulating the hippocampal processing of information [115]. GABAergic and PV-specific fast-spiking interneurons have been shown to be important for synchronous oscillatory activity of the hippocampus, which correlates with behaviour and is important for synaptic plasticity [116–118]. Therefore, reduced densities of PV-specific interneurons may represent a plausible mechanism to compensate for altered hippocampal GABA metabolism and/or disturbed synchronous oscillatory activity induced by a lack of 5-HT during development and adulthood. On the basis of the involvement of the dorsal subdivision of the hippocampus in learning and memory processes, these findings may reflect increased conditioned fear responses in Tph2 ^−/− mice and lead to a better insight into the mechanism of how early-life 5-HT deficiency impacts the development of anxiety-related disorders.

(e) Adaptive 5-HT receptor regulation

The density of 5-HT_1A and 5-HT_1B receptors and their coupling G-proteins are increased across several brain regions of male Tph2 ^−/− mice, particularly in terminal fields of the frontal cortex and septum employing quantitative autoradiography and stimulated [³⁵S]GTP-γ-S binding [46]. This finding can be explained by two mechanisms, which may be operational independently or act in concert. The opposite phenomenon was observed in mouse models characterized by robust increases in extracellular 5-HT in the brain such as monoamine oxidase A (Maoa) null mutant mice where 5-HT_1A and 5-HT_1B receptors are desensitized and downregulated [119,120] and, to a lesser extent in a brain-region-specific manner, in 5-Htt KO mice [121]. Moreover, 5-HT_1A receptors are downregulated in patients with depression and anxiety disorders as well as during SSRI treatment [122–124]. Sensitization and upregulation of 5-HT_1A and 5-HT_1B receptors in 5-HT-deficient mice may therefore be due to a direct cellular mechanism compensating for reduced 5-HT ligand availability by an increased Htr1a and Htr1b gene expression. An alternative explanation rests on evidence for a reciprocal regulation of hypothalamic-pituitary-adrenal (HPA) axis activity and 5-HT_1A receptor function. Studies in animal models demonstrated that chronic stress-induced corticosterone secretion results in a downregulation of 5-HT_1A [125,126] mediated by transcriptional repression of the Htr1a gene promoter via differential activation of intranuclear glucocorticoid and mineralocorticoid receptors [127–130]. Taken together, these data suggest that this regulatory loop also relates the low corticosterone levels to increased 5-HT_1A expression in Tph2 ^−/− mice: either low corticosterone level induces the expression of 5-Htr1a or 5-HT deficiency leads to increased expression, which is resistant to repression due to low corticosterone levels. Finally, although our electrophysiological data confirm that 5-HT_1A are functional in the raphe as autoreceptors and thus probably also in the other brain regions as heteroreceptors, it remains to be elucidated whether the 5-HT traces remaining in regions where 5-HT_1A is upregulated, such as the hippocampus, is sufficient to hyperpolarize neural cells and mediate serotonergic signalling. This possibility is however not supported by the increased aggressive behaviour observed in Tph2 ^−/− males (see §3 a).

(f) Growth, body weight and obesity across the lifespan

5-HT is implicated in the regulation of metabolic pathways influencing somatic growth, food intake and body weight. The overall life expectancy was not reduced by central 5-HT deficiency. While reduced weight gain was observed in Tph2 ^−/− females during the first 24 weeks, this growth retardation persisted in male Tph2 ^−/− mice throughout the lifespan. Hypomorphism was already observed during the early developmental period. This reduction in body weight may result from altered regulation at different levels, including reduced food intake, implicating impaired perception of energy needs and satiety, increased metabolic activity and energy expenditure or lower storage, implicating dysregulated glucose turnover, lipid and protein metabolic cycles or altered thermoregulation. Beyond the age of six months, an obesity phenotype emerges in female heterozygous Tph2 ^+/− mice and becomes more exaggerated throughout life, with accumulation of fat tissue stored in the abdominal and pericardial cavity.

Following Tph2 inactivation, growth retardation but normalization after weaning (with normal weight four months of age) was also observed by Alenina et al. [21]. In contrast, it was reported that Tph2 ^−/− mice display a reduced fat pad and size and that, at one and a half and three months, they display both reduced food intake and increased metabolism linked to altered leptin regulation [131]. These observations are unexpected in the face of reports that 5-HT or drugs increasing its release are anorexigenic via hypothalamic actions [132], reduced meal size [132], decreased body weight [133,134] and increased energy expenditure [135]. While the low body weight in Tph2 ^−/− mice contrasts data and conclusions from other investigators, the observation of age-related obesity, as reflected by excess fat storage particularly in Tph2 ^+/− females exhibiting reduced brain 5-HT, concurs. 5-HT transporter null mutant mice (5-Htt/Sert ^−/−), which display increased synaptic 5-HT but a reduced synthesis and total 5-HT brain concentrations in the face of decreased locomotor activity, also develop obesity and type 2 diabetes in adulthood, on the basis of a metabolic syndrome with insulin resistance [121]. Taken together, the gene dose- and sex-dependent divergence of body weight and fat storage in Tph2 K O mice supports the notion of a nonlinear dual effect of central 5-HT on somatic development, long-term body weight regulation and metabolic homeostasis via different pathways and endocrine systems.

Previous SectionNext Section

Поиск по сайту: