Cigarette Smoking and Tryptophan Hydroxylase 2 mRNA in the Dorsal Raphe Nucleus in Suicides.

Cigarette smoking is associated with suicide and mood disorders and stimulates serotonin release. Tryptophan hydroxylase (TPH2) synthesizes serotonin and is over-expressed in suicides. We determined whether smoking is associated with TPH2 mRNA in suicides and controls. TPH2 mRNA was measured postmortem in the dorsal raphe nucleus (DRN) of controls (N = 26, 17 nonsmokers and nine smokers) and suicides (N = 23, 5 nonsmokers and 18 smokers). Psychiatric history was obtained by psychological autopsy. TPH2 mRNA was greater in suicide nonsmokers than suicide smokers, control smokers and control nonsmokers (p = 0.006). There was more TPH2 mRNA throughout the DRN. Smoking interferes with the TPH2 mRNA increase observed in suicide nonsmokers. The absence of altered TPH2 expression in non-suicide smokers suggests no pharmacological effect of smoking.

Alcoholics have more tryptophan hydroxylase 2 mRNA and protein in the dorsal and median raphe nuclei.

BACKGROUND: Chronic alcohol use depletes brain serotonin (5-hydroxytryptamine [5-HT]), yet we previously found more tryptophan hydroxylase 2 (TPH2), the rate-limiting biosynthetic enzyme for 5-HT, in the dorsal raphe nucleus (DRN) of alcoholics. We sought to determine whether the increase in amount of TPH2 enzyme is associated with more TPH2 mRNA gene expression in the DRN of a new cohort of alcoholics and controls. METHODS: TPH2 mRNA and protein were measured by in situ hybridization and immunoautoradiography, respectively, in the DRN and median raphe nucleus (MRN) of age- and sex-matched pairs (n = 16) of alcoholics and nonpsychiatric controls. Alcohol use disorder diagnosis and medical, psychiatric, and family histories were obtained by psychological autopsy. Age and sex were covariates in the analyses. RESULTS: TPH2 mRNA in alcoholics was greater in the DRN and MRN compared to controls (DRN: controls: 3.6 ± 1.6, alcoholics: 4.8 ± 1.8 nCi/mg of tissue, F = 4.106, p = 0.02; MRN: controls: 2.6 ± 1.2, alcoholics: 3.5 ± 1.1 nCi/mg of tissue, F = 3.96, p = 0.024). The difference in TPH2 mRNA was present in all DRN subnuclei (dorsal [DRd]: 135%, interfascicular [DRif]: 139%, ventral [DRv]: 135%, ventrolateral [DRvl]: 136% of control p < 0.05) except the caudal subnucleus. Alcoholics also had more TPH2 protein in the DRN and MRN than controls (DRN: controls: 265 ± 47, alcoholics: 318 ± 47 µCi/g, F = 8.72, p = 0.001; MRN: controls: 250 ± 33, alcoholics: 345 ± 39 µCi/g, F = 7.78, p = 0.001). There is a positive correlation between TPH2 protein and mRNA expression in the DRN (r = 0.815, p < 0.001), suggesting that the higher amount of TPH2 protein is due to an increase in TPH2 gene expression. CONCLUSIONS: These findings suggest that greater TPH2 gene expression is the basis for more TPH2 protein in the DRN and MRN in alcoholics.

Elevated serotonin and 5-HIAA in the brainstem and lower serotonin turnover in the prefrontal cortex of suicides.

Using high pressure liquid chromatography, we find more brainstem 5-HT and 5-HIAA in suicides compared with nonpsychiatric, sudden death controls throughout the rostrocaudal extent of the brainstem DRN and MRN. This suggests that 5-HT synthesis in suicides is greater within all DRN subnuclei and the MRN compared with controls.

Neuronal tryptophan hydroxylase expression in BALB/cJ and C57Bl/6J mice.

BALB/c is an inbred stress-sensitive mouse strain exhibiting low brain serotonin (5-HT) content and a 5-HT biosynthetic enzyme tryptophan hydroxylase (Tph2) variant reported to have lower catalytic activity compared with other inbred base strains. To evaluate other mechanisms that may explain low 5-HT, we compared BALB/cJ mice and a control inbred strain C57Bl/6J mice, for expression of Tph2 mRNA, TPH2 protein and regional levels of 5-HT and its metabolite 5-hydroxyindoleacetic acid. Tph2 mRNA and TPH2 protein in brainstem dorsal raphe nuclei was assayed by in situ hybridization and immunocytochemistry respectively. 5-HT and 5-hydroxyindoleacetic acid were determined by HPLC. BALB/cJ mice had 20% less Tph2 mRNA and 28% fewer TPH2 immunolabeled neurons than C57Bl/6J mice (t = -2.59, p = 0.02). The largest difference in Tph2 transcript expression was in rostral dorsal raphe nuclei (t = 2.731, p = 0.008). 5-HT was 15% lower in the midbrain and 18% lower in the cerebral cortex of BALB/cJ compared with C57Bl/6J mice (p < 0.05). The behavioral differences in BALB/cJ mice relative to the C57Bl/6J strain may be due in part, to fewer 5-HT neurons and lower Tph2 gene expression resulting in less 5-HT neurotransmission. Future studies quantifying expression per neuron are needed to determine whether less expression is explained by fewer neurons or also less expression per neuron, or both.

Partial nucleotide sequences and expression patterns of frog (Rana pipiens) ephrin-A2 and ephrin-A5 mRNA.

We have generated 362 bp and 547 bp partial sequences for Rana pipiens ephrin-A2 and ephrin-A5 mRNA, respectively. Translation homologies for the comparable segments of cDNA of chicken, mouse and human are 90.8, 86.9 and 84.4% for the ephrin-A2 sequence and 85.7, 85.0 and 85.0% for the ephrin-A5 sequence. Digoxigenin-labeled riboprobes were prepared and applied by means of in situ hybridization to whole-mounts of the brains of mature adults and expression patterns in tadpoles were also explored. The RNA probes revealed similar posterior (high) to anterior (low) expression gradients in the adult tectum, demonstrating that both ephrin-As are expressed in the adult Ranid frog tectum. Only the ephrin-A2 probe was tested on tadpole brain, yielding an appropriately graded expression pattern similar to the adult.

Fiber order of the normal and regenerated optic tract of the frog (Rana pipiens).

In the normal frog, axons from the peripheral retina arising at the temporal pole course superficially in the middle stream of the diencephalic optic tract. Axons from the nasal pole course in two streams running in the opposite margins of the tract, dorsonasal axons ventrally, ventronasal axons dorsally. Axons from the dorsal and ventral poles of the retina occupy the intervals between the aforementioned middle and marginal streams. Axons from more central regions of the retina tend to occupy deeper levels of the optic tract. The regenerated optic tract does not regain its normal organization, e.g., axons of peripheral nasal origin are spread out widely over the entire width of the tract. However, axons from the temporal pole of the retina do return approximately to their original location in the middle stream. The concentration of temporal axons in the middle stream of the optic tract after regeneration may now be understood in terms of the expression pattern of the ephrin-A class of receptor tyrosine kinase ligands in the cellular matrix of the optic tract. The ephrin-As, which have a repellent effect on growing temporal retinal axons, are concentrated in and along the margins of the diencephalic optic tract and essentially absent from its middle stream. It is proposed here that peripheral temporal axons may be forced into this middle region by their avoidance of the higher levels of ephrin-A expression in the tract margins. In contrast, the growth pattern of regenerating peripheral nasal axons would not be affected by the ephrin-A gradient in the optic tract.

Persistence of graded EphA/Ephrin-A expression in the adult frog visual system.

Many studies have demonstrated the involvement of the EphA family of receptor tyrosine kinases and their ligands, ephrin-A2 and -A5, in the development of the temporonasal axis of the retinotectal/collicular map, but the role of these molecules in optic nerve regeneration has not been well studied. Noting that the characteristic gradients of the EphA/ephrin-A family that are expressed topographically in the retina and tectum of embryonic chicks and mice tend to disappear after birth, we took as our starting point an analysis of EphA and ephrin-A expression in leopard frogs (Rana pipiens and utricularia), species capable of regenerating the retinotectal map as adults. For the EphA family to be involved in the regeneration, one would expect these topographic gradients to persist in the adult or, if downregulated after metamorphosis, to be reexpressed after optic nerve injury. Using EphA3 receptor and ephrin-A5 ligand alkaline phosphatase in situ affinity probes (RAP and LAP, respectively) in whole-mount applications, we report that reciprocally complementary gradients of RAP and LAP binding persist in the optic tract and optic tectum of postmetamorphic frogs, including mature adults. EphA expression in temporal retinal axons in the optic tract was significantly reduced after nerve section but returned during regeneration. However, ephrin-A expression in the tectal parenchyma was not significantly elevated by either eye removal, with degeneration of optic axons, or during regeneration of the retinotectal projection. Thus, the present study has demonstrated a persisting expression of EphA/ephrin-A family members in the retinal axons and tectal parenchyma that may help guide regenerating fibers, but we can offer no evidence for an upregulation of ephrin-A expression in conjunction with optic nerve injury.