Tyrosine hydroxylase conditional KO mice reveal peripheral tissue-dependent differences in dopamine biosynthetic pathways.

Dopamine (DA) exerts well-known functions in the brain as a neurotransmitter. In addition, it plays important physiological roles in peripheral organs, but it is largely unknown how and where peripheral DA is synthesized and regulated. Catecholamines in peripheral tissues are either produced within the tissue itself and/or derived from sympathetic neurons, which release neurotransmitters for uptake by peripheral tissues. To evaluate DA-producing ability of each peripheral tissue, we generated conditional KO mice (cKO mice) in which the tyrosine hydroxylase (TH) gene is ablated in the sympathoadrenal system, thus eliminating sympathetic neurons as a DA source. We then examined the alterations in the noradrenaline (NA), DA, and 3,4-dihydroxyphenylalanine (DOPA) contents in peripheral organs and performed immunohistochemical analyses of TH-expressing cells. In the heart and pancreas of cKO mice, both the TH protein and NA levels were significantly decreased, and the DA contents were decreased in parallel with NA contents, indicating that the DA supply originated from sympathetic neurons. We found TH-immunoreactive cells in the stomach and lung, where the TH protein showed a decreasing trend, but the DA levels were not decreased in cKO mice. Moreover, we found a significant correlation between the DA content in the kidney and the plasma DOPA concentration, suggesting that the kidney takes up DOPA from blood to make DA. The aforementioned data unravel differences in the DA biosynthetic pathway among tissues and support the role of sympathetic neurons as a DA supplier.

Descending Dopaminergic Inputs to Reticulospinal Neurons Promote Locomotor Movements.

Meso-diencephalic dopaminergic neurons are known to modulate locomotor behaviors through their ascending projections to the basal ganglia, which in turn project to the mesencephalic locomotor region, known to control locomotion in vertebrates. In addition to their ascending projections, dopaminergic neurons were found to increase locomotor movements through direct descending projections to the mesencephalic locomotor region and spinal cord. Intriguingly, fibers expressing tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine synthesis, were also observed around reticulospinal neurons of lampreys. We now examined the origin and the role of this innervation. Using immunofluorescence and tracing experiments, we found that fibers positive for dopamine innervate reticulospinal neurons in the four reticular nuclei of lampreys. We identified the dopaminergic source using tracer injections in reticular nuclei, which retrogradely labeled dopaminergic neurons in a caudal diencephalic nucleus (posterior tuberculum [PT]). Using voltammetry in brain preparations isolated in vitro, we found that PT stimulation evoked dopamine release in all four reticular nuclei, but not in the spinal cord. In semi-intact preparations where the brain is accessible and the body moves, PT stimulation evoked swimming, and injection of a D1 receptor antagonist within the middle rhombencephalic reticular nucleus was sufficient to decrease reticulospinal activity and PT-evoked swimming. Our study reveals that dopaminergic neurons have access to command neurons that integrate sensory and descending inputs to activate spinal locomotor neurons. As such, our findings strengthen the idea that dopamine can modulate locomotor behavior both via ascending projections to the basal ganglia and through descending projections to brainstem motor circuits.SIGNIFICANCE STATEMENT Meso-diencephalic dopaminergic neurons play a key role in modulating locomotion by releasing dopamine in the basal ganglia, spinal networks, and the mesencephalic locomotor region, a brainstem region that controls locomotion in a graded fashion. Here, we report in lampreys that dopaminergic neurons release dopamine in the four reticular nuclei where reticulospinal neurons are located. Reticulospinal neurons integrate sensory and descending suprareticular inputs to control spinal interneurons and motoneurons. By directly modulating the activity of reticulospinal neurons, meso-diencephalic dopaminergic neurons control the very last instructions sent by the brain to spinal locomotor circuits. Our study reports on a new direct descending dopaminergic projection to reticulospinal neurons that modulates locomotor behavior.

NAD+ cellular redox and SIRT1 regulate the diurnal rhythms of tyrosine hydroxylase and conditioned cocaine reward.

The diurnal regulation of dopamine is important for normal physiology and diseases such as addiction. Here we find a novel role for the CLOCK protein to antagonize CREB-mediated transcriptional activity at the tyrosine hydroxylase (TH) promoter, which is mediated by the interaction with the metabolic sensing protein, Sirtuin 1 (SIRT1). Additionally, we demonstrate that the transcriptional activity of TH is modulated by the cellular redox state, and daily rhythms of redox balance in the ventral tegmental area (VTA), along with TH transcription, are highly disrupted following chronic cocaine administration. Furthermore, CLOCK and SIRT1 are important for regulating cocaine reward and dopaminergic (DAergic) activity, with interesting differences depending on whether DAergic activity is in a heightened state and if there is a functional CLOCK protein. Taken together, we find that rhythms in cellular metabolism and circadian proteins work together to regulate dopamine synthesis and the reward value for drugs of abuse.

Prefrontal D1 dopamine signaling is required for temporal control.

Temporal control, or how organisms guide movements in time to achieve behavioral goals, depends on dopamine signaling. The medial prefrontal cortex controls many goal-directed behaviors and receives dopaminergic input primarily from the midbrain ventral tegmental area. However, this system has never been linked with temporal control. Here, we test the hypothesis that dopaminergic projections from the ventral tegmental area to the prefrontal cortex influence temporal control. Rodents were trained to perform a fixed-interval timing task with an interval of 20 s. We report several results: first, that decreasing dopaminergic neurotransmission using virally mediated RNA interference of tyrosine hydroxylase impaired temporal control, and second that pharmacological disruption of prefrontal D1 dopamine receptors, but not D2 dopamine receptors, impaired temporal control. We then used optogenetics to specifically and selectively manipulate prefrontal neurons expressing D1 dopamine receptors during fixed-interval timing performance. Selective inhibition of D1-expressing prefrontal neurons impaired fixed-interval timing, whereas stimulation made animals more efficient during task performance. These data provide evidence that ventral tegmental dopaminergic projections to the prefrontal cortex influence temporal control via D1 receptors. The results identify a critical circuit for temporal control of behavior that could serve as a target for the treatment of dopaminergic diseases.

Tlx3 controls cholinergic transmitter and Peptide phenotypes in a subset of prenatal sympathetic neurons.

The embryonic sympathetic nervous system consists of predominantly noradrenergic neurons and a very small population of cholinergic neurons. Postnatal development further allows target-dependent switch of a subset of noradrenergic neurons into cholinergic phenotype. How embryonic cholinergic neurons are specified at the prenatal stages remains largely unknown. In this study, we found that the expression of transcription factor Tlx3 was progressively restricted to a small population of embryonic sympathetic neurons in mice. Immunostaining for vesicular acetylcholine transporter (VAChT) showed that Tlx3 was highly expressed in cholinergic neurons at the late embryonic stage E18.5. Deletion of Tlx3 resulted in the loss of Vacht expression at E18.5 but not E12.5. By contrast, Tlx3 was required for expression of the cholinergic peptide vasoactive intestinal polypeptide (VIP), and somatostatin (SOM) at both E12.5 and E18.5. Furthermore, we found that, at E18.5 these putative cholinergic neurons expressed glial cell line-derived neurotrophic factor family coreceptor Ret but not tyrosine hydroxylase (Ret(+)/TH(-)). Deletion of Tlx3 also resulted in disappearance of high-level Ret expression. Last, unlike Tlx3, Ret was required for the expression of VIP and SOM at E18.5 but not E12.5. Together, these results indicate that transcription factor Tlx3 is required for the acquisition of cholinergic phenotype at the late embryonic stage as well as the expression and maintenance of cholinergic peptides VIP and SOM throughout prenatal development of mouse sympathetic neurons.

Neurobiological consequences of acute footshock stress: effects on tyrosine hydroxylase phosphorylation and activation in the rat brain and adrenal medulla.

Stress activates selected neuronal systems in the brain and this leads to activation of a range of effector systems. Our aim was to investigate some of the relationships between these systems under basal conditions and over a 40-min period in response to footshock stress. Specifically, we investigated catecholaminergic neurons in the locus coeruleus (LC), ventral tegmental area and medial prefrontal cortex (mPFC) in the brain, by measuring tyrosine hydroxylase (TH) protein, TH phosphorylation and TH activation. We also measured the effector responses by measuring plasma adrenocorticotrophic hormone, corticosterone, glucose and body temperature as well as activation of adrenal medulla protein kinases, TH protein, TH phosphorylation and TH activation. The LC, ventral tegmental area and adrenal medulla all had higher basal levels of Ser19 phosphorylation and lower basal levels of Ser31 phosphorylation than the mPFC, presumably because of their cell body versus nerve terminal location, while the adrenal medulla had the highest basal levels of Ser40 phosphorylation. Ser31 phosphorylation was increased in the LC at 20 and 40 min and in the mPFC at 40 min; TH activity was increased at 40 min in both tissues. There were significant increases in body temperature between 10 and 40 min, as well as increases in plasma adrenocorticotropic hormone at 20 min and corticosterone and glucose at 20 and 40 min. The adrenal medulla extracellular signal-regulated kinase 2 was increased between 10 and 40 min and Ser31 phosphorylation was increased at 20 min and 40 min. Protein kinase A and Ser40 phosphorylation were increased only at 40 min. TH activity was increased between 20 and 40 min. TH protein and Ser19 phosphorylation levels were not altered in any of the brain regions or adrenal medulla over the first 40 min. These findings indicate that acute footshock stress leads to activation of TH in the LC, pre-synaptic terminals in the mPFC and adrenal medullary chromaffin cells, as well as changes in activity of the hypothalamic-pituitary-adrenal axis.

Loss of locus coeruleus noradrenergic neurons alters the inflammatory response to LPS in substantia nigra but does not affect nigral cell loss.

In Parkinson's disease (PD), destruction of noradrenergic neurons in the locus coeruleus (LC) may precede damage to nigral cells and subsequently exaggerate dopaminergic cell loss. We examine if destruction of the locus coeruleus with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) alters dopaminergic cell loss in substantia nigra (SN) initiated by lipopolysaccharide (LPS) in the rat through an effect on glial cell activation. In rats, a single intraperitoneal dose of DSP-4 administered 8 days previously, caused a marked loss of tyrosine hydroxylase positive neurons in LC but no change in dopaminergic cell number in SN. Unilateral nigral LPS administration resulted in marked dopaminergic cell death with reactive microgliosis associated with enhanced p47 phox in OX-6 and OX-42 positive microglia. There was proliferation of inducible nitric oxide synthase (iNOS)-positive cells, formation of 3-nitrotyrosine (3-NT) and proliferation of astrocytes that expressed glial cell line-derived neurotrophic factor (GDNF). Following combined DSP-4 treatment and subsequent administration of LPS, unexpectedly, no further loss of tyrosine hydroxylase (TH)-immunoreactivity (-ir) occurred in the SN compared to the effects of LPS alone. However, there was a marked alteration in the morphology of microglial cell and a reduction of 3-NT- and iNOS-ir was evident. Expression of p47 phox was downregulated in microglia but up-regulated in TH-ir neurons. No further change in GFAP-ir was observed compared to that produced by DSP-4 alone or LPS alone, but the expression of GDNF was markedly reduced. This study suggests that in contrast to previous reports, prior LC damage does not influence subsequent nigral dopaminergic cell degeneration induced by LPS. Rather it appears to attenuate the microglial response thought to contribute to disease progression in PD.

Cloning and expression analysis of tyrosine hydroxylase and changes in catecholamine levels in brain during ontogeny and after sex steroid analogues exposure in the catfish, Clarias batrachus.

Tyrosine hydroxylase (Th) is the rate-limiting enzyme for catecholamine (CA) biosynthesis and is considered to be a marker for CA-ergic neurons, which regulate the levels of gonadotropin-releasing hormone in brain and gonadotropins in the pituitary. In the present study, we cloned full-length cDNA of Th from the catfish brain and evaluated its expression pattern in the male and female brain during early development and after sex-steroid analogues treatment using quantitative real-time PCR. We measured the CA levels to compare our results on Th. Cloned Th from catfish brain is 1.591 kb, which encodes a putative protein of 458 amino acid residues and showed high homology with other teleosts. The tissue distribution of Th revealed ubiquitous expression in all the tissues analyzed with maximum expression in male and female brain. Copy number analysis showed two-fold more transcript abundance in the female brain when compared with the male brain. A differential expression pattern of Th was observed in which the mRNA levels were significantly higher in females compared with males, during early brain development. CAs, l-3,4-dihydroxyphenylalanine, dopamine, and norepinephrine levels measured using high-performance liquid chromatography with electrochemical detection in the developing male and female brain confirmed the prominence of the CA-ergic system in the female brain. Sex-steroid analogue treatment using methyltestosterone and ethinylestradiol confirmed our findings of the differential expression of Th related to CA levels.

Inputs underlying the ON-OFF light responses of type 2 wide-field amacrine cells in TH::GFP mice.

In the mammalian retina, two types of catecholaminergic amacrine cells have been described. Although dopaminergic type 1 cells are well characterized, the physiology of type 2 cells is, so far, unknown. To target type 2 cells specifically, we used a transgenic mouse line that expresses green fluorescent protein under the control of the tyrosine hydroxylase promoter. Type 2 cells are GABAergic and have an extensive dendritic arbor, which stratifies in the middle of the inner plexiform layer. Our data suggest that type 2 cells comprise two subpopulations with identical physiological properties: one has its somata located in the inner nuclear layer and the other in the ganglion cell layer. Immunostaining with bipolar cell markers suggested that type 2 cells receive excitatory inputs from type 3 OFF and type 5 ON bipolar cells. Consistently, patch-clamp recordings showed that type 2 cells are ON-OFF amacrine cells. Blocking excitatory inputs revealed that different rod and cone pathways are active under scotopic and mesopic light conditions. Blockade of inhibitory inputs led to membrane potential oscillations in type 2 cells, suggesting that GABAergic and glycinergic amacrine cells strongly influence type 2 cell signaling. Among the glycinergic amacrine cells, we identified the VGluT3-immunoreactive amacrine cell as a likely candidate. Collectively, light responses of type 2 cells were remarkably uniform over a wide range of light intensities. These properties point toward a general function of type 2 cells that is maintained under scotopic and mesopic conditions.

Vitamin D and Parkinson's disease.

Parkinson's disease (PD) is the second most common form of neurodegeneration among the elderly population. PD is clinically characterized by tremors, rigidity, slowness of movement, and postural imbalance. Interestingly, a significant association has been demonstrated between PD and low levels of vitamin D in the serum, and vitamin D supplement appears to have a beneficial clinical effect on PD. Genetic studies have provided the opportunity to determine which proteins link vitamin D to PD pathology, e.g., Nurr1 gene, toll-like receptor, gene related to lipid disorders, vascular endothelial factor, tyrosine hydroxylase, and angiogenin. Vitamin D also exerts its effects on cancer through nongenomic factors, e.g., bacillus Calmette-Guerin vaccination, interleukin-10, Wntß-catenin signaling pathways, mitogen-activated protein kinase pathways, and the reduced form of the nicotinamide adenine dinucleotide phosphate. In conclusion, vitamin D might have a beneficial role in PD. Calcitriol is best used for PD because it is the active form of the vitamin D(3) metabolite and modulates inflammatory cytokine expression. Further investigation with calcitriol in PD is needed.

Engineering of L-tyrosine oxidation in Escherichia coli and microbial production of hydroxytyrosol.

The hydroxylation of tyrosine is an important reaction in the biosynthesis of many natural products. The use of bacteria for this reaction has not been very successful due to either the over-oxidation to ortho-quinone when using tyrosinases from bacteria or plants, or the lack of the native cofactor, tetrahydrobiopterin (BH4), needed for the activity of tyrosine hydroxylases (TH). Here, we demonstrate that an Escherichia coli cofactor, tetrahydromonapterin (MH4), can be used as an alternative cofactor for TH in presence of the BH4 regeneration pathway, and tyrosine hydroxylation is performed without over-oxidation. We used this platform for biosynthesis of one of the most powerful antioxidants, hydroxytyrosol. An endogenous aromatic aldehyde oxidase was identified and knocked out to prevent formation of the side product, and this resulted in nearly exclusive production of hydroxytyrosol in engineered E. coli. Finally, hydroxytyrosol production from a simple sugar as a sole carbon source was demonstrated.

The retina is a target for GnRH-3 system in the European sea bass, Dicentrarchus labrax.

The European sea bass expresses three GnRH (Gonadotrophin Releasing Hormone) forms that exert pleiotropic actions via several classes of receptors. The GnRH-1 form is responsible for the endogenous regulation of gonadotrophin release by the pituitary gland but the role of GnRH-2 and GnRH-3 remains unclear in fish. In a previous study performed in sea bass, we have provided evidence of direct links between the GnRH-2 cells and the pineal organ and demonstrated a functional role for GnRH-2 in the modulation of the secretory activity of this photoreceptive organ. In this study, we have investigated the possible relationship between the GnRH-3 system and the retina in the same species. Thus, using a biotinylated dextran-amine tract-tracing method, we reveal the presence of retinopetal cells in the terminal nerve of sea bass, a region that also contains GnRH-3-immunopositive cells. Moreover, GnRH-3-immunoreactive fibers were observed at the boundary between the inner nuclear and the inner plexiform layers, and also within the ganglion cell layer. These results strongly suggest that the GnRH-3 neurons located in the terminal nerve area represent the source of GnRH-3 innervation in the retina of this species. In order to clarify whether the retina is a target for GnRH, the expression pattern of GnRH receptors (dlGnRHR) was also analyzed by RT-PCR and in situ hybridization. RT-PCR revealed the retinal expression of dlGnRHR-II-2b, -1a, -1b and -1c, while in situ hybridization only showed positive signals for the receptors dlGnRHR-II-2b and -1a. Finally, double-immunohistochemistry showed that GnRH-3 projections reaching the sea bass retina end in close proximity to tyrosine hydroxylase (dopaminergic) cells, which also expressed the dlGnRHR-II-2b receptor subtype. Taken together, these results suggest an important role for GnRH-3 in the modulation of dopaminergic cell activities and retinal functions in sea bass.

Dopamine-dependent neurotoxicity of alpha-synuclein: a mechanism for selective neurodegeneration in Parkinson disease.

The mechanism by which dopaminergic neurons are selectively lost in Parkinson disease (PD) is unknown. Here we show that accumulation of alpha-synuclein in cultured human dopaminergic neurons results in apoptosis that requires endogenous dopamine production and is mediated by reactive oxygen species. In contrast, alpha-synuclein is not toxic in non-dopaminergic human cortical neurons, but rather exhibits neuroprotective activity. Dopamine-dependent neurotoxicity is mediated by 54 83-kD soluble protein complexes that contain alpha-synuclein and 14-3-3 protein, which are elevated selectively in the substantia nigra in PD. Thus, accumulation of soluble alpha-synuclein protein complexes can render endogenous dopamine toxic, suggesting a potential mechanism for the selectivity of neuronal loss in PD.

Bee venom reduces neuroinflammation in the MPTP-induced model of Parkinson's disease.

AIM: This study was designed to investigate the anti-inflammatory effects of bee venom (BV) in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson's disease (PD). METHOD: MPTP was administered by intraperitoneal (IP) injection at 2-hr intervals over an 8-hr period. Mice were then subjected to BV subcutaneous injection and sacrificed on days 1 and 3 following the final MPTP injection. The loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) was assessed by tyrosine hydroxylase (TH) immunohistochemistry. Microglial activation was measured by immunohistochemistry for macrophage antigen complex-1 (MAC-1) and inducible nitric oxide synthase (iNOS). The staining intensities of MAC-1 and iNOS were quantified with respect to optical density. RESULT: In animals treated with MPTP, the survival percentages of TH+ cells in the SNpc were 32% on day 1 and 46% on day 3 compared with normal mice. In BV-treated mice, the survival percentages of TH+ cells improved to 70% on day 1 and 78% on day 3 compared with normal mice. BV treatment also resulted in reduced expression of the inflammation markers MAC-1 and iNOS in the SNpc. CONCLUSION: These data suggest that BV injection may have a neuroprotective effect that attenuates the activation of the microglial response, which has implications for the treatment of PD.

Advanced research on dopamine signaling to develop drugs for the treatment of mental disorders: regulation of dopaminergic neural transmission by tyrosine hydroxylase protein at nerve terminals.

5R-L-Erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) is an essential cofactor for tyrosine hydroxylase (TH). Recently, a type of dopa-responsive dystonia (DRD) (DYT5, Segawa's disease) was revealed to be caused by dominant mutations of the gene encoding GTP cyclohydrolase I (GCHI), which is the rate-limiting enzyme of BH(4) biosynthesis. In order to probe the role of BH(4) in vivo, we established BH(4)-depleted mice by disrupting the 6-pyruvoyltetrahydropterin synthase (PTS) gene (Pts(-/-)) and rescued them by introducing human PTS cDNA under the control of the human dopamine ß-hydroxylase (DBH) promoter (Pts(-/-)-DPS). The Pts(-/-)-DPS mice developed hyperphenylalaninemia. Interestingly, tyrosine hydroxylase protein was dramatically reduced in the dopaminergic nerve terminals of these mice, and they developed abnormal posture and motor disturbance. We propose that the biochemical and pathologic changes of Pts(-/-)-DPS mice are caused by mechanisms common to human DRD, and understanding these mechanisms could give us insight into other movement disorders.

Dose optimization for long-term rAAV-mediated RNA interference in the nigrostriatal projection neurons.

Short-hairpin RNA (shRNA)-mediated gene knockdown is a powerful tool for targeted gene silencing and an emerging novel therapeutic strategy. Recent publications, however, reported unexpected toxicity after utilizing viral-mediated shRNA knockdown in vivo. Thus, it is currently unclear whether shRNA-mediated knockdown strategy can be used as a safe and efficient tool for gene silencing. In this study, we have generated rAAV vectors expressing shRNAs targeting the rat tyrosine hydroxylase (TH) mRNA (shTH) for testing the efficacy of in vivo TH knockdown in the nigral dopaminergic neurons. At high titers, not only the shTH vectors but also the scrambled and green fluorescence protein (GFP)-only controls caused cell death. In a dose-response study, we identified a dose window leading to >60% decrease in TH(+) neurons without any change in vesicular monoamine transporter-2 (VMAT2) expression. Moreover, using the safe and efficient dose, we showed that dopamine (DA) synthesis rate was significantly reduced and this lead to emergence of motor deficits in the shTH-expressing rats. Interestingly, these animals showed very robust and long-lasting recovery after a single systemic L-3,4-dihydroxyphenylalanine (L-DOPA) administration beyond what can be achieved in 6-hydroxydopamine (6-OHDA)-lesioned rats. Our results have implications for both mechanistic and therapeutic studies utilizing long-term shRNA-mediated gene silencing in the nigrostriatal projection system.

Annual and periovulatory changes in tyrosine hydroxylase activity in the ovary of the catfish Heteropneustes fossilis.

In the present study, tyrosine hydroxylase (TH, the rate-limiting enzyme in catecholamine synthesis) activity was demonstrated in the ovary of the catfish to elucidate the possible physiological role of catecholamines in the gonad. The ovary is innervated by seven pairs of nerves, originating from the paired sympathetic chain lying dorsal to the posterior kidney. Ovarian TH activity showed a significant annual variation (P<0.001, one-way ANOVA), correlating with gonado-somatic index. Activity was low from December to February (resting phase), increased from March to July (recrudescent phase) and then decreased in post-spawning phase (August-November). The annual pattern was similar to that of the brain. An intraperitoneal injection of 100 IU hCG/fish induced significant periovulatory changes in TH activity with the peak rise at 16 h, and the activity decreased after egg-stripping (P<0.001, one-way ANOVA). Brain TH activity showed similar periovulatory changes. The results suggest that catecholamine synthesis is increased during both ovarian recrudescence and spawning of the annual reproductive cycle, implying a functional role in ovarian growth, maturation and ovulation.

Differences in behavior between surface and cave Astyanax mexicanus may be mediated by changes in catecholamine signaling.

Astyanax mexicanus is a teleost fish that is in the process of allopatric speciation. Ancestral Astyanax are found in surface rivers and derived blind forms are found in cave systems. Adaptation to life in nutrient poor caves without predation includes the evolution of enhanced food seeking behaviors and loss of defensive responses. These behavioral adaptations may be mediated by changes in catecholaminergic control systems in the brain. We examined the distribution of tyrosine hydroxylase, a conserved precursor for the synthesis of the catecholamines dopamine and noradrenaline, in the brains of surface and cave Astyanax using immunohistochemistry. We found differences in tyrosine hydroxylase staining in regions that are associated with nonvisual sensory perception, motor control, endocrine release, and attention. These differences included significant increases in the diameters of tyrosine hydroxylase immunoreactive soma in cave Astyanax in the olfactory bulb, basal telencephalon, preoptic nuclei, ventral thalamus, posterior tuberculum, and locus coeruleus. These increases in modulation by dopamine and noradrenaline likely indicate changes in behavioral control that underlie adaptations to the cave environment.

Cross-talk between protein kinase A and mitogen-activated protein kinases signalling in the adaptive changes observed during morphine withdrawal in the heart.

Our previous studies have shown that morphine withdrawal induced an increase in the expression of protein kinase (PK) A and mitogen-activated extracellular kinase (MAPK) pathways in the heart during morphine withdrawal. The purpose of the present study was to evaluate the interaction between PKA and extracellular signal-regulated kinase (ERK) signaling pathways mediating the cardiac adaptive changes observed after naloxone administration to morphine-dependent rats. Dependence on morphine was induced by a 7-day subcutaneous implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (2 mg/kg). ERK1/2 and tyrosine hydroxylase (TH) phosphorylation was determined by quantitative blot immunolabeling using phosphorylation state-specific antibodies. Naloxone-induced morphine withdrawal activates ERK1/2 and phosphorylates TH at Ser31 in the right and left ventricle, with an increase in the mean arterial blood pressure and heart rate. When N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a PKA inhibitor, was infused, concomitantly with morphine, it diminished the expression of ERK1/2. In contrast, the infusion of calphostin C (a PKC inhibitor) did not modify the morphine withdrawal-induced activation of ERK1/2. The ability of morphine withdrawal to activate ERK that phosphorylates TH at Ser31 was reduced by HA-1004. The present findings demonstrate that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation (phosphorylation) of TH.

Effects of ionizing radiation on locomotory behavior and mechanosensation in Caenorhabditis elegans.

Locomotory behavior (motility) and mechanosensation are of vital importance in animals. We examined the effects of ionizing radiation (IR) on locomotory behavior and mechanosensation using a model organism, the nematode Caenorhabditis elegans. Bacterial mechanosensation in C. elegans induces the dopamine-mediated slowing of locomotion in the presence of bacteria (food), known as the basal slowing response. We previously reported an IR-induced reduction of locomotory rate in the absence of food. In the present study, we observed a similar IR-induced reduction of locomotory rate in the cat-2 mutant, which is defective in bacterial mechanosensation. The dose response pattern of the locomotory rate in the presence of food was relatively flat in wild-type animals, but not in cat-2 mutants. This suggests that the dopamine system, which is related to bacterial mechanosensation in C. elegans, might have a dominant effect on locomotory rate in the presence of food, which masks the effects of other stimuli. Moreover, we found that the behavioral responses of hydrogen peroxide-exposed wild-type animals are similar to those of IR-exposed animals. Our findings suggest that the IR-induced reduction of locomotory rate in the absence of food is mediated by a different pathway from that for bacterial mechanosensation, at least partially through IR-produced hydrogen peroxide.