Circadian regulation of gene expression systems in the Drosophila head.

Mechanisms composing Drosophila's clock are conserved within the animal kingdom. To learn how such clocks influence behavioral and physiological rhythms, we determined the complement of circadian transcripts in adult Drosophila heads. High-density oligonucleotide arrays were used to collect data in the form of three 12-point time course experiments spanning a total of 6 days. Analyses of 24 hr Fourier components of the expression patterns revealed significant oscillations for approximately 400 transcripts. Based on secondary filters and experimental verifications, a subset of 158 genes showed particularly robust cycling and many oscillatory phases. Circadian expression was associated with genes involved in diverse biological processes, including learning and memory/synapse function, vision, olfaction, locomotion, detoxification, and areas of metabolism. Data collected from three different clock mutants (per(0), tim(01), and Clk(Jrk)), are consistent with both known and novel regulatory mechanisms controlling circadian transcription.

Defect in normal developmental increase of the brain biogenic amine concentrations in the mecp2-null mouse.

To clarify whether Mecp2 dysfunction may cause impairment of the monoaminergic and serotonergic systems, we measured the whole brain concentrations of biogenic amines and related substrates in three mecp2-null male mice and four control mice of each age at 0-42 postnatal days by HPLC methods. After 14 postnatal days, concentrations of biogenic amines were smaller in mecp2-null mice than those in control mice and at 42 postnatal days, norepinephrine, dopamine and serotonin concentrations in mecp2-null mice were significantly smaller by 25, 24 and 16%, respectively. This result suggested that the absence of Mecp2 does not impair the neurogenesis of monoaminergic and serotonergic neurons but causes succeeding impairment of those neuronal systems from 14 postnatal days.

Evidence for heritability of biogenic amine levels in the cerebrospinal fluid of rhesus monkeys.

Susceptibility to several human psychopathological disorders is under partial genetic influence, and many of these disorders have biological correlates that may form part of the basis of this vulnerability. In humans, alterations in cerebrospinal fluid (CSF) metabolite levels of the amine transmitters norepinephrine, dopamine, and serotonin have been associated with several forms of psychopathology, and altered levels of these metabolites have been found in healthy probands with a familial history of such illnesses. We report evidence for heritability of CSF levels of biogenic amine measures in rhesus monkeys, Macaca mulatta. In a pilot study of 54 monkeys with known pedigrees, significant differences among sire families were found for CSF levels of norepinephrine (p = 0.04), homovanillic acid (p = 0.02), and 5-hydroxyindoleacetic acid (p = 0.04). These data indicate that variation in bioaminergic measures is associated with pedigree, and that model systems incorporating both genetic and environmental factors can contribute to the understanding of the function of aminergic systems implicated in vulnerability to psychopathology.

Dramatic brain aminergic deficit in a genetic mouse model of phenylketonuria.

Clinical data suggest that brain catecholamines and serotonin are deficient in phenylketonuria (PKU), an inherited metabolic disorder that causes severe mental retardation and neurological disturbances. To test this hypothesis, brain tissue levels of dopamine (DA), norepinephrine (NE), 5-hydroxytryptamine (5-HT) and their metabolites were evaluated in the genetic mouse model of PKU (Pah(enu2)). Results indicated a significant reduction of 5-HT levels and metabolism in prefrontal cortex (pFC), cingulate cortex (Cg), nucleus accumbens (NAc), caudate putamen (CP), hippocampus (HIP) and amygdala (AMY). NE content and metabolism were reduced in pFC, Cg, AMY and HIP. Finally, significantly reduced DA content and metabolism was observed in pFC, NAc, CP and AMY. In pFC, NAc and CP there was also a marked reduction of DA release.

Probing conformational changes in neurotransmitter transporters: a structural context.

The Na+/Cl-dependent neurotransmitter transporters, a family of proteins responsible for the reuptake of neurotransmitters and other small molecules from the synaptic cleft, have been the focus of intensive research in recent years. The biogenic amine transporters, a subset of this larger family, are especially intriguing as they are the targets for many psychoactive compounds, including cocaine and amphetamines, as well as many antidepressants. In the absence of a high-resolution structure for any transporter in this family, research into the structure-function relationships of these transporters has relied on analysis of the effects of site-directed mutagenesis as well as of chemical modification of reactive residues. The aim of this review is to establish a structural context for the experimental study of these transporters through various computational approaches and to highlight what is known about the conformational changes associated with function in these transporters. We also present a novel numbering scheme to assist in the comparison of aligned positions between sequences of the neurotransmitter transporter family, a comparison that will be of increasing importance as additional experimental data is amassed.

Stimulant and reinforcing effects of cocaine in monoamine transporter knockout mice.

A large body of evidence supports the hypothesis that the reinforcing effects of cocaine depend on its ability to block the dopamine transporter (DAT), thereby increasing dopamine extracellular concentration within the mesocorticolimbic system. However, the fact that cocaine similarly binds to the serotonin and norepinephrine transporters (SERT and NET, respectively), raises the possibility that modulation of mesocorticolimbic dopaminergic transmission might be achieved through alternate pathways. The successful disruption of the genes coding for the DAT, the SERT and the NET offered ideal tools to determine the extent of the participation of these transporters and respective monoaminergic systems in the reinforcing effects of cocaine. Studies of cocaine-induced motor activation and maintenance of intravenous (i.v.) self-administration in DAT- and in NET-knockout (KO) mice are reviewed here, and discussed in light of new observations obtained from double monoamine transporters KO mice (i.e., DAT-KO/SERT-KO, NET-KO/SERT-KO). The reinforcing potency of cocaine is maintained in the absence of the DAT but decreased in the absence of the NET; its motivational rewarding effect is observed in the absence of the SERT, but not when both DAT and SERT are lacking. Moreover, a dichotomy between cocaine motor activating and reinforcing effects is reported. Such dichotomy is suggestive of independent mechanisms underlying the psychomotor stimulant and reinforcing effects of cocaine. Overall, these studies provide evidence that cocaine dynamically acts at multiple sites through pathways that might be exchangeable under certain circumstances.

Oligomer formation by Na+-Cl--coupled neurotransmitter transporters.

Na+-Cl--dependent neurotransmitter transporters (or neurotransmitter:Na+ symporters, NSS) share many structural and functional features, e.g. a conserved topology of 12 transmembrane spanning alpha-helices, the capacity to operate in two directions and in an electrogenic manner. Biochemical and biophysical experiments indicate that these transporters interact in oligomeric quaternary structures. Fluorescence resonance energy transfer (FRET) microscopy has provided evidence for a constitutive physical interaction of NSS at the cell surface and throughout the biosynthetic pathway. Two interfaces for protein-protein interaction have been shown to be important in NSS; these comprise a glycophorin-like motif and a leucine heptad repeat. Upon mutational modification of the latter, surface targeting is considerably impaired without concomitant loss in uptake activity. This supports a role of oligomer formation in the passage of the quality control mechanisms of the endoplasmic reticulum and/or Golgi. In contrast, oligomerisation is dispensable for substrate binding and translocation.

Polymorphisms in the dopamine, serotonin, and norepinephrine transporter genes and their relationships to monoamine metabolite concentrations in CSF of healthy volunteers.

Concentrations of monoamine metabolites (MM) in lumbar cerebrospinal fluid (CSF) have been used extensively as indirect estimates of monoamine turnover in the brain. We investigated the possible relationships between DNA polymorphisms in the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) genes and CSF concentrations of homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) in healthy volunteers (n = 66). The DAT polymorphism was not significantly associated with any of the monoamine metabolites, but a tendency for relationship with 5-HIAA was found in women. For both of the two SERT polymorphisms investigated, a functional promoter polymorphism and an intronic polymorphism without known function, significant relationships were found with CSF MHPG levels. No relationship was found between the SERT polymorphisms and CSF HVA and 5-HIAA. The NET polymorphism was associated with CSF MHPG levels but not HVA and 5-HIAA concentrations. The results suggest that SERT and NET genotypes may participate differentially in the regulation of the norepinephrine turnover rate under presumed steady-state conditions in the central nervous system. As only limited data so far indicate interactions between the serotonin and norepinephrine systems in the brain, and the NET polymorphism investigated is not known to be of functional significance, the results should be interpreted with caution until replicated.

Neurophysiology of Rett syndrome.

Neurophysiological studies on Rett syndrome (RTT) are reviewed, and pathophysiology of RTT is discussed. The electroencephalography (EEG), sensory evoked potentials (SEP), sleep-wake rhythm study and polysomnography (PSG) study showed age-dependent characteristics. PSG revealed the brainstem and midbrain monoaminergic systems are deranged from early developmental stage, that is serotonin and noradrenaline systems seem to be hypoactive and dopaminergic system is also hypoactive associated with receptor supersensitivity. These monoaminergic systems are known to influence the maturation of the higher neuronal systems at specific areas and at specific ages. Particularly the synaptogenesis of the cerebral cortex is modulated by region or layer specifically from an early stage of the development. The observations made in EEG and SEP studies also suggested specific subcortical and cortical involvements taking place during the development. The age-dependent appearance of characteristic clinical features of RTT, and the variation of the clinical severities, e.g. classical, variant, form fruste, etc., can also be explained by the specific features of these monoaminergic systems. Furthermore, analysis of the components of rapid eye movement sleep suggested the onset of RTT lies between 36 gestational weeks to 3-4 months postnatally. The discovery of the mutations of methyl-CpG-binding protein 2 (MECP2) gene as the causative gene of RTT is an epoch helping not only to understand the pathophysiology of RTT but also various neurodevelopmental disorders.

Pathophysiology of Rett syndrome from the stand point of clinical characteristics.

In this report, we reviewed the characteristics of motor development and motor symptoms of Rett Syndrome (RTT) and demarcated the early and pathognomonic motor symptom which correlates to the impairment of the higher cortical function (HCF) assessed by the ability of language. It is suggested that failure of locomotion in late infancy is the primary and pathognomonic symptom. Thus, the impairment of the neurons or neuronal systems involving locomotion is suggested as the primary lesion in the pathophysiology of RTT not only for motor dysfunction but also for the failure in the development of language and cognitive function. On the other hand the neuronal systems involving the loss of purposeful hand use and the stereotyped hand movement, the most characteristic and diagnostic symptoms of RTT appearing in early childhood, are affected later or secondarily but induce further degradation of the HCF. Hypofunction of the aminergic neurons in the brainstem and midbrain is suggested as the cause of dysfunction of these neuronal systems, for those of locomotion, the noradrenarlin (NA) and/or the serotonin (5HT) neurons and for the stereotyped hand movement the dopamine (DA) neurons. The NA and/or the 5HT neurons in the brain stem may be involved primarily and may cause dysfunction of the midbrain DA neuron directly or indirectly through affecting the pedunculopontine nuclei.

Discussant--pathophysiologies of Rett syndrome.

Studies on sleep parameters of Rett syndrome revealed hypoactivity of the noradrenaline (NA) and the serotonin (5HT) neuron in early infancy while preserving the function of the dopamine (DA) and the cholinergic neurons of the pons normally. The sleep-wake cycle remains in its development at the level of 4 months of age. Polysomnographies also showed a decrease of the function of the nigrostriatal (NS)-DA neuron in early childhood and suggested the development of receptor supersensitivity in late childhood. Neurohistochemical and neuroimaging (PET) studies revealed the hypofunction of the NS-DA neuron with receptor supersensitivity and of involvement of the cholinergic neurons to the cortical pathology, whereas no substantial pathological or histochemical abnormalities were observed in the NA and the 5HT neurons in the brainstem. The decrease of tyrosine hydroxylase without neurodegenerative changes observed in the substantia nigra of Rett syndrome had similarity to the pathology caused by excitotoxic lesion of the pedunculopontine nuclei (PPN) observed in an animal experiments. Clinically the grade of disability of locomotion was shown to correlate to the grade of the disabilities of language. These clinical manifestations were also correlated to the specific loci of the mutation in the methyl binding domain of the MECP2 gene. In rodents the axons of the brainstem 5HT neuron involved in the morphogenesis of the brain in the early developmental course disappear in neonates without apoptotic or degenerative changes in the neurons. This period corresponds to the first 1.5-2 years in humans. Thus, in Rett syndrome, the primary lesion appears in the brainstem NA and 5HT neurons which affects development of synaptogenesis of the cortex and also dysfunction of the PPN. The latter causes dysfunction of the DA neuron and the cholinergic neuron in the midbrain. The mutation of the MECP2 gene may cause early transcription of the genes which prune the axons of the aminergic neurons for the developmental morphogenesis of the central nervous system in early infancy.

Monoamine influence on neuropeptide gene expression during ontogenesis.

The present study aimed to check the hypothesis concerning the monoamine regulation of the differentiation of their target neurons during ontogenesis. For this aim, neuropeptide gene expression has been evaluated by in situ hybridization in targets for monoamines, differentiating peptidergic neurons, after monoamine depletion in rats during prenatal or early postnatal periods. In the first series of experiments, the vasopressin (VP) and oxytocin (OT) mRNA concentrations were measured in the supraoptic nucleus (SON) of rat fetuses at the 21st embryonic day (E21) following daily (E13-E20) treatment with the inhibitor of the catecholamine (CA) synthesis, alpha-methyl-m(p)-tyrosine. Similar study was performed with rats at the 11th postnatal day (P11) after daily (P2-P10) treatment with alpha-methyl-m-tyrosine and the neurotoxin, 6-hydroxydopamine. In the second series of experiments, the effect of serotonin (5-HT) depletion by the inhibitor of the 5-HT synthesis, p-chlorophenylalanine, on the vasoactive intestinal polypeptide (VIP) mRNA level in the suprachiasmatic nucleus (SCN) has been studied in fetuses and in neonates as described above. No changes were detected in the VP and OT mRNA concentration in the SON following CA depletion in fetuses, while similar treatment of neonates significantly increased both mRNA levels. On the contrary, the 5-HT depletion caused an increased VIP mRNA concentration in the SCN in fetuses but not in neonates. Thus, our data suggest a monoamine inhibitory influence on peptide gene expression in the differentiating target neurons during certain periods of ontogenesis.

Monoamine transporters: from genes to behavior.

Modulation of fast neurotransmission by monoamines is critically involved in numerous physiological functions and pathological conditions. Plasma membrane monoamine transporters provide one of the most efficient mechanisms controlling functional extracellular monoamine concentrations. These transporters for dopamine (DAT), serotonin (SERT), and norepinephrine (NET), which are expressed selectively on the corresponding neurons, are established targets of many psychostimulants, antidepressants, and neurotoxins. Recently, genetic animal models with targeted disruption of these transporters have become available. These mice have provided opportunities to investigate the functional importance of transporters in homeostatic control of monoaminergic transmission and to evaluate, in an in vivo model system, their roles in physiology and pathology. The use of these mice as test subjects has been helpful in resolving several important issues on specificity and mechanisms of action of certain pharmacological agents. In the present review, we summarize recent advances in understanding the physiology and pharmacology of monoamine transporters gained in mice with targeted genetic deletion of DAT, SERT, and NET.