Photoperiodic effects on monoamine signaling and gene expression throughout development in the serotonin and dopamine systems.

Photoperiod or the duration of daylight has been implicated as a risk factor in the development of mood disorders. The dopamine and serotonin systems are impacted by photoperiod and are consistently associated with affective disorders. Hence, we evaluated, at multiple stages of postnatal development, the expression of key dopaminergic (TH) and serotonergic (Tph2, SERT, and Pet-1) genes, and midbrain monoamine content in mice raised under control Equinox (LD 12:12), Short winter-like (LD 8:16), or Long summer-like (LD 16:8) photoperiods. Focusing in early adulthood, we evaluated the midbrain levels of these serotonergic genes, and also assayed these gene levels in the dorsal raphe nucleus (DRN) with RNAScope. Mice that developed under Short photoperiods demonstrated elevated midbrain TH expression levels, specifically during perinatal development compared to mice raised under Long photoperiods, and significantly decreased serotonin and dopamine content throughout the course of development. In adulthood, Long photoperiod mice demonstrated decreased midbrain Tph2 and SERT expression levels and reduced Tph2 levels in the DRN compared Short photoperiod mice. Thus, evaluating gene × environment interactions in the dopaminergic and serotonergic systems during multiple stages of development may lead to novel insights into the underlying mechanisms in the development of affective disorders.

Is dopamine transporter-mediated dopaminergic signaling in the retina a noninvasive biomarker for attention-deficit/ hyperactivity disorder? A study in a novel dopamine transporter variant Val559 transgenic mouse model.

BACKGROUND: Dopamine (DA) is a critical neuromodulator in the retina. Disruption of retinal DA synthesis and signaling significantly attenuates light-adapted, electroretinogram (ERG) responses, as well as contrast sensitivity and acuity. As these measures can be detected noninvasively, they may provide opportunities to detect disease processes linked to perturbed DA signaling. Recently, we identified a rare, functional DA transporter (DAT, SLC6A3) coding substitution, Ala559Val, in subjects with attention-deficit/hyperactivity disorder (ADHD), demonstrating that DAT Val559 imparts anomalous DA efflux (ADE) with attendant physiological, pharmacological, and behavioral phenotypes. To understand the broader impact of ADE on ADHD, noninvasive measures sensitive to DAT reversal are needed. METHODS: Here, we explored this question through ERG-based analysis of retinal light responses, as well as HPLC measurements of retinal DA in DAT Val559 mice. RESULTS: Male mice homozygous (HOM) for the DAT Val559 variant demonstrated increased, light-adapted ERG b-wave amplitudes compared to wild type (WT) and heterozygous (HET) mice, whereas dark-adapted responses were indistinguishable across genotypes. The elevated amplitude of the photopic light responses in HOM mice could be mimicked in WT mice by applying D1 and D4 DA receptor agonists and suppressed in HOM mice by introducing D4 antagonist, supporting elevated retinal DA signaling arising from ADE. Following the challenge with amphetamine, WT exhibited an increase in light-adapted response amplitudes, while HOM did not. Total retinal DA content was similar across genotypes. Interestingly, female DAT Val559 HOM animals revealed no significant difference in photopic ERG responses when compared with WT and HET littermates. CONCLUSIONS: These data reveal that noninvasive, in vivo evaluation of retinal responses to light can reveal physiological signatures of ADE, suggesting a possible approach to the segregation of neurobehavioral disorders based on the DAT-dependent control of DA signaling.

Photoperiod programs dorsal raphe serotonergic neurons and affective behaviors.

The serotonergic raphe nuclei of the midbrain are principal centers from which serotonin neurons project to innervate cortical and sub-cortical structures. The dorsal raphe nuclei receive light input from the circadian visual system and indirect input from the biological clock nuclei. Dysregulation of serotonin neurotransmission is implicated in neurobehavioral disorders, such as depression and anxiety, and alterations in the serotonergic phenotype of raphe neurons have dramatic effects on affective behaviors in rodents. Here, we demonstrate that day length (photoperiod) during development induces enduring changes in mouse dorsal raphe serotonin neurons­programming their firing rate, responsiveness to noradrenergic stimulation, intrinsic electrical properties, serotonin and norepinephrine content in the midbrain, and depression/anxiety-related behavior in a melatonin receptor 1 (MT1)-dependent manner. Our results establish mechanisms by which seasonal photoperiods may dramatically and persistently alter the function of serotonin neurons.

Circadian perinatal photoperiod has enduring effects on retinal dopamine and visual function.

Visual system development depends on neural activity, driven by intrinsic and light-sensitive mechanisms. Here, we examined the effects on retinal function due to exposure to summer- and winter-like circadian light cycles during development and adulthood. Retinal light responses, visual behaviors, dopamine content, retinal morphology, and gene expression were assessed in mice reared in seasonal photoperiods consisting of light/dark cycles of 8:16, 16:8, and 12:12 h, respectively. Mice exposed to short, winter-like, light cycles showed enduring deficits in photopic retinal light responses and visual contrast sensitivity, but only transient changes were observed for scotopic measures. Dopamine levels were significantly lower in short photoperiod mice, and dopaminergic agonist treatment rescued the photopic light response deficits. Tyrosine hydroxylase and Early Growth Response factor-1 mRNA expression were reduced in short photoperiod retinas. Therefore, seasonal light cycles experienced during retinal development and maturation have lasting influence on retinal and visual function, likely through developmental programming of retinal dopamine.

Circadian rhythm of contrast sensitivity is regulated by a dopamine-neuronal PAS-domain protein 2-adenylyl cyclase 1 signaling pathway in retinal ganglion cells.

Spatial variation in light intensity, called spatial contrast, comprises much of the visual information perceived by mammals, and the relative ability to detect contrast is referred to as contrast sensitivity (Purves et al., 2012). Recently, retinal dopamine D4 receptors (D4Rs) have been implicated in modulating contrast sensitivity (Jackson et al., 2012); however, the cellular and molecular mechanisms have not been elucidated. Our study demonstrates a circadian rhythm of contrast sensitivity that peaks during the daytime, and that its regulation involves interactions of D4Rs, the clock gene Npas2, and the clock-controlled gene adenylyl cyclase 1 (Adcy1) in a subset of retinal ganglion cells (RGCs). Targeted disruption of the gene encoding D4Rs reduces the amplitude of the contrast sensitivity rhythm by reducing daytime sensitivity and abolishes the rhythmic expression of Npas2 and Adcy1 mRNA in the ganglion cell layer (GCL) of the retina. Npas2(-/-) and Adcy1(-/-) mice show strikingly similar reductions in the contrast sensitivity rhythm to that in mice lacking D4Rs. Moreover, Adcy1 transcript rhythms were abolished in the GCL of Npas2(-/-) mice. Luciferase reporter assays demonstrated that the Adcy1 promoter is selectively activated by neuronal PAS-domain protein 2 (NPAS2)/BMAL1. Our results indicate that the contrast sensitivity rhythm is modulated by D4Rs via a signaling pathway that involves NPAS2-mediated circadian regulation of Adcy1. Hence, we have identified a circadian clock mechanism in a subset of RGCs that modulates an important aspect of retinal physiology and visual processing.

Retinal dopamine mediates multiple dimensions of light-adapted vision.

Dopamine is a key neuromodulator in the retina and brain that supports motor, cognitive, and visual function. Here, we developed a mouse model on a C57 background in which expression of the rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase, is specifically disrupted in the retina. This model enabled assessment of the overall role of retinal dopamine in vision using electrophysiological (electroretinogram), psychophysical (optokinetic tracking), and pharmacological techniques. Significant disruptions were observed in high-resolution, light-adapted vision caused by specific deficits in light responses, contrast sensitivity, acuity, and circadian rhythms in this retinal dopamine-depleted mouse model. These global effects of retinal dopamine on vision are driven by the differential actions of dopamine D1 and D4 receptors on specific retinal functions and appear to be due to the ongoing bioavailability of dopamine rather than developmental effects. Together, our data indicate that dopamine is necessary for the circadian nature of light-adapted vision as well as optimal contrast detection and acuity.

Dopamine D4 receptor activation controls circadian timing of the adenylyl cyclase 1/cyclic AMP signaling system in mouse retina.

In the mammalian retina, dopamine binding to the dopamine D4 receptor (D4R) affects a light-sensitive pool of cyclic AMP by negatively coupling to the type 1 adenylyl cyclase (AC1). AC1 is the primary enzyme controlling cyclic AMP production in dark-adapted photoreceptors. A previous study demonstrated that expression of the gene encoding AC1, Adcy1, is downregulated in mice lacking Drd4, the gene encoding the D4R. The present investigation provides evidence that D4R activation entrains the circadian rhythm of Adcy1 mRNA expression. Diurnal and circadian rhythms of Drd4 and Adcy1 mRNA levels were observed in wild-type mouse retina. Also, rhythms in the Ca²âº-stimulated AC activity and cyclic AMP levels were observed. However, these rhythmic activities were damped or undetectable in mice lacking the D4R. Pharmacologically activating the D4R 4 h before its normal stimulation at light onset in the morning advances the phase of the Adcy1 mRNA expression pattern. These data demonstrate that stimulating the D4R is essential in maintaining the normal rhythmic production of AC1 from transcript to enzyme activity. Thus, dopamine/D4R signaling is a novel zeitgeber that entrains the rhythm of Adcy1 expression and, consequently, modulates the rhythmic synthesis of cyclic AMP in mouse retina.

Nonmotor symptoms of Parkinson's disease revealed in an animal model with reduced monoamine storage capacity.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is characterized by the loss of dopamine neurons in the substantia nigra pars compacta, culminating in severe motor symptoms, including resting tremor, rigidity, bradykinesia, and postural instability. In addition to motor deficits, there are a variety of nonmotor symptoms associated with PD. These symptoms generally precede the onset of motor symptoms, sometimes by years, and include anosmia, problems with gastrointestinal motility, sleep disturbances, sympathetic denervation, anxiety, and depression. Previously, we have shown that mice with a 95% genetic reduction in vesicular monoamine transporter expression (VMAT2-deficient, VMAT2 LO) display progressive loss of striatal dopamine, L-DOPA-responsive motor deficits, alpha-synuclein accumulation, and nigral dopaminergic cell loss. We hypothesized that since these animals exhibit deficits in other monoamine systems (norepinephrine and serotonin), which are known to regulate some of these behaviors, the VMAT2-deficient mice may display some of the nonmotor symptoms associated with PD. Here we report that the VMAT2-deficient mice demonstrate progressive deficits in olfactory discrimination, delayed gastric emptying, altered sleep latency, anxiety-like behavior, and age-dependent depressive behavior. These results suggest that the VMAT2-deficient mice may be a useful model of the nonmotor symptoms of PD. Furthermore, monoamine dysfunction may contribute to many of the nonmotor symptoms of PD, and interventions aimed at restoring monoamine function may be beneficial in treating the disease.

Essential roles of dopamine D4 receptors and the type 1 adenylyl cyclase in photic control of cyclic AMP in photoreceptor cells.

Light and dopamine regulate many physiological functions in the vertebrate retina. Light exposure decreases cyclic AMP formation in photoreceptor cells. Dopamine D(4) receptor (D(4)R) activation promotes light adaptation and suppresses the light-sensitive pool of cyclic AMP in photoreceptor cells. The key signaling pathways involved in regulating cyclic AMP in photoreceptor cells have not been identified. In the present study, we show that the light- and D(4)R-signaling pathways converge on the type 1 Ca(2+)/calmodulin-stimulated adenylyl cyclase (AC1) to regulate cyclic AMP synthesis in photoreceptor cells. In addition, we present evidence that D(4)R activation tonically regulates the expression of AC1 in photoreceptors. In retinas of mice with targeted deletion of the gene (Adcy1) encoding AC1, cyclic AMP levels and Ca(2+)/calmodulin-stimulated adenylyl cyclase activity are markedly reduced, and cyclic AMP accumulation is unaffected by either light or D(4)R activation. Similarly, in mice with disruption of the gene (Drd4) encoding D(4)R, cyclic AMP levels in the dark-adapted retina are significantly lower compared to wild-type retina and are unresponsive to light. These changes in Drd4-/- mice were accompanied by significantly lower Adcy1 mRNA levels in photoreceptor cells and lower Ca(2+)/calmodulin-stimulated adenylyl cyclase activity in retinal membranes compared with wild-type controls. Reduced levels of Adcy1 mRNA were also observed in retinas of wild-type mice treated chronically with a D(4)R antagonist, L-745870. Thus, activation of D(4)R is required for normal expression of AC1 and for the regulation of its catalytic activity by light. These observations illustrate a novel mechanism for cross-talk between dopamine and photic signaling pathways regulating cyclic AMP in photoreceptor cells.

Temporal coupling of cyclic AMP and Ca/calmodulin-stimulated adenylyl cyclase to the circadian clock in chick retinal photoreceptor cells.

cAMP signaling pathways play crucial roles in photoreceptor cells and other retinal cell types. Previous studies demonstrated a circadian rhythm of cAMP level in chick photoreceptor cell cultures that drives the rhythm of activity of the melatonin synthesizing enzyme arylalkylamine N-acetyltransferase and the rhythm of affinity of the cyclic nucleotide-gated channel for cGMP. Here, we report that the photoreceptor circadian clock generates a rhythm in Ca(2+)/calmodulin-stimulated adenylyl cyclase activity, which accounts for the temporal changes in the cAMP levels in the photoreceptors. The circadian rhythm of cAMP in photoreceptor cell cultures is abolished by treatment with the l-type Ca(2+) channel antagonist nitrendipine, while the Ca(2+) channel agonist, Bay K 8644, increased cAMP levels with continued circadian rhythmicity in constant darkness. These results indicate that the circadian rhythm of cAMP is dependent, in part, on Ca(2+) influx. Photoreceptor cell cultures exhibit a circadian rhythm in Ca(2+)/calmodulin-stimulated adenylyl cyclase enzyme activity with high levels at night and low levels during the day, correlating with the temporal changes of cAMP in these cells. Transcripts encoding two of the Ca(2+)/calmodulin-stimulated adenylyl cyclases, type 1 and type 8 (Adcy1 and Adcy8), displayed significant daily rhythms of mRNA expression under a light-dark cycle, but only the Adcy1 transcript rhythm persisted in constant darkness. Similar rhythms of Adcy1 mRNA level and Ca(2+)/calmodulin-stimulated adenylyl cyclase activity were observed in retinas of 2-week-old chickens. These results indicate that a circadian clock controls the expression of Adcy1 mRNA and Ca(2+)/calmodulin-stimulated adenylyl cyclase activity; and calcium influx into these cells gates the circadian rhythm of cAMP, a key component in the regulation of photoreceptor function.

Int7G24A variant of transforming growth factor-beta receptor type I is associated with invasive breast cancer.

PURPOSE: The transforming growth factor-beta (TGF-beta) signaling pathway has been frequently implicated in breast cancer. An intronic variant (Int7G24A) of TGF-beta receptor type I (TGFBR1) is associated with kidney and bladder cancers in our recent study. We hypothesize that this germline variant may be involved in development and progression of breast cancer. EXPERIMENTAL DESIGN: Case-control studies were designed from archived paraffin-embedded tissue specimens from the same geographic area with a homogenous ethnic population. We analyzed 223 patients (25 with preinvasive tumors and 198 with invasive and metastatic breast cancers) and 153 noncancer controls. The Int7G24A was identified by PCR-RFLP. Another germline deletion (TGFBR1*6A) and somatic mutations in the TGFBR1 were also analyzed by PCR and single-strand conformational polymorphism. RESULTS: The Int7G24A allele was evident in 32% of patients with preinvasive neoplasms and 48% of patients with invasive breast cancers compared with 26% controls (P = 0.00008). In addition, 11 (5.6%) homozygous Int7G24A carriers were found in patients with invasive breast cancers, whereas only 3 (2%) homozygous carriers were found in the control group. The TGFBR1*6A allele was not significantly associated with breast cancer patients and only one somatic mutation was found in 71 breast cancers. CONCLUSION: These data suggest that the germline Int7G24A variant may represent a risk factor for invasive breast cancer and a marker for breast cancer progression. A separate study with a larger sample size is warranted to validate the association of the Int7G24A with human breast cancer.