High Levels Of Tyrosine Hydroxylase Research Articles

Predicting outcomes with circulating adrenergic neuroblastoma mRNAs in children with relapsed and refractory neuroblastoma: A BEACON-Neuroblastoma biomarker study.

10039 Background: Children with relapsed and refractory neuroblastoma (RR-NBL) have poor outcomes. Early identification of children at greatest risk of relapse could mean timelier modifications of treatment to improve outcomes. High levels of adrenergic neuroblastoma mRNAs in blood of children with stage M neuroblastoma receiving frontline treatment predict poor outcome (Viprey PMID: 24590653). Since these markers have not been thoroughly studied in the RR-NBL population, we have prospectively evaluated the prognostic potential of the adrenergic neuroblastoma mRNAs paired-like homeobox 2B (PHOX2B ) and tyrosine hydroxylase (TH) in blood from children with RR-NBL treated in the BEACON-Neuroblastoma trial (NCT02308527). Methods: Blood samples collected at baseline from 88 children were analysed by reverse transcriptase polymerase chain reaction (RTqPCR) for PHOX2B and TH mRNAs. The prognostic power of these mRNAs was evaluated using Kaplan-Meier survival curves and Cox proportional hazards regression. Progression-free (PFS) and overall survival (OS) were calculated from the date that the blood sample was taken at screening to the date of an event; progression, disease recurrence, death or censored alive at the last clinical evaluation. Results: Of the children in this cohort, 58 (66%) had relapsed and 30 (34%) had refractory disease. Twenty-three (26%) had MYC-N amplified tumours. TH and PHOX2B mRNAs were detected in 55% and 60% of blood samples respectively; the correlation coefficient between TH and PHOX2B was 0.75. Higher levels of TH, PHOX2B mRNAs or both combined were associated with reduced PFS and OS (Table). For TH, median PFS for children with TH levels below the median was 12 months (95%CI, 4.6–13 months) versus 5.5 months (95%CI, 1.8–9.4 months) for those children with TH levels above the median. For PHOX2B, median PFS for children with PHOX2B levels below the median was 11.5 months (95%CI, 7.6–34 months), compared to 5.7 months (95%CI, 1.8–10.5 months) where levels were above the median. Conclusions: TH and PHOX2B mRNAs in blood collected at baseline identify children with refractory or relapsed neuroblastoma at greatest risk of progression or death. In the RR-NBL setting, this simple blood test could be used to stratify treatment strategies. Clinical trial information: NCT02308527. [Table: see text]

Interleukin-6 triggers toxic neuronal iron sequestration in response to pathological α-synuclein.

SUMMARYα-synuclein (α-syn) aggregation and accumulation drive neurodegeneration in Parkinson’s disease (PD). The substantia nigra of patients with PD contains excess iron, yet the underlying mechanism accounting for this iron accumulation is unclear. Here, we show that misfolded α-syn activates microglia, which release interleukin 6 (IL-6). IL-6, via its trans-signaling pathway, induces changes in the neuronal iron transcriptome that promote ferrous iron uptake and decrease cellular iron export via a pathway we term the cellular iron sequestration response, or CISR. The brains of patients with PD exhibit molecular signatures of the IL-6-mediated CISR. Genetic deletion of IL-6, or treatment with the iron chelator deferiprone, reduces pathological α-syn toxicity in a mouse model of sporadic PD. These data suggest that IL-6-induced CISR leads to toxic neuronal iron accumulation, contributing to synuclein-induced neurodegeneration.

Chronic Social Isolation Stress during Peri-Adolescence Alters Presynaptic Dopamine Terminal Dynamics via Augmentation in Accumbal Dopamine Availability.

Chronic peri-adolescent stress in humans increases risk to develop a substance use disorder during adulthood. Rats reared in social isolation during peri-adolescence (aSI; 1 rat/cage) period show greater ethanol and cocaine intake compared to group housed (aGH; 4 rats/cage) rats. In addition, aSI rats have a heightened dopamine response in the nucleus accumbens (NAc) to rewarding and aversive stimuli. Furthermore, single pulse electrical stimulation in slices containing NAc core elicits greater dopamine release in aSI rats. Here, we further investigated dopamine release kinetics and machinery following aSI. Dopamine release, across a wide range of stimulation intensities and frequencies, was significantly greater in aSI rats. Interestingly, subthreshold intensity stimulations also resulted in measurable dopamine release in accumbal slices from aSI but not aGH rats. Extracellular [Ca2+] manipulations revealed augmented calcium sensitivity of dopamine release in aSI rats. The readily releasable pools of dopamine, examined by bath application of Ro-04-1284/000, a vesicular monoamine transporter 2 (VMAT2) inhibitor, were depleted faster in aGH rats. Western blot analysis of release machinery proteins (VMAT2, Synaptogyrin-3, Syntaxin-1, and Munc13-3) showed no difference between the two groups. Tyrosine hydroxylase (TH) protein expression levels, however, were elevated in aSI rats. The greater dopamine release could potentially be explained by higher levels of TH, the rate-limiting step for dopamine synthesis. This augmented responsivity of the dopamine system and heightened dopamine availability post-aSI may lead to an increased risk of addiction vulnerability.

Overexpression of TFEB Drives a Pleiotropic Neurotrophic Effect and Prevents Parkinson’s Disease-Related Neurodegeneration

The possible implication of transcription factor EB (TFEB) as a therapeutic target in Parkinson's disease has gained momentum since it was discovered that TFEB controls lysosomal biogenesis and autophagy and that its activation might counteract lysosomal impairment and protein aggregation. However, the majority of putative direct targets of TFEB described to date is linked to a range of biological processes that are not related to the lysosomal-autophagic system. Here, we assessed the effect of overexpressing TFEB with an adeno-associated viral vector in mouse substantia nigradopaminergic neurons. We demonstrate that TFEB overexpression drives a previously unknown bona fide neurotrophic effect, giving rise to cell growth, higher tyrosine hydroxylase levels, and increased dopamine release in the striatum. TFEB overexpression induces the activation of the mitogen-activated protein kinase 1/3 (MAPK1/3) and AKT pro-survival pathways, phosphorylation of mTORC1 effectors 4E-binding protein 1 (4E-BP1) and S6 kinase B1 (S6K1), and increased protein synthesis. We show that TFEB overexpression prevents dopaminergic cell loss and counteracts atrophy and the associated protein synthesis decline in the MPTP mouse model of Parkinson's disease. Our results suggest that increasing TFEB activity might prevent neuronal death and restore neuronal function in Parkinson's disease and other neurodegenerative diseases through different mechanisms.

RT-qPCR for PHOX2B mRNA is a highly specific and sensitive method to assess neuroblastoma minimal residual disease in testicular tissue.

Neuroblastoma (NB) is the most common type of extracranial solid tumor in children with a high prevalence in toddlers. For childhood cancer survivors, preservation of reproductive potential is an important factor for quality of life. The optimization of NB minimal residual disease (MRD) detection in testicular tissue is crucial to evaluate the risk of malignant cell reintroduction. The first step in the present study was to assess the accuracy of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect tyrosine hydroxylase (TH), paired-like homeobox 2b (PHOX2B) and doublecortin (DCX) mRNA expression in frozen/thawed testicular tissues of patients with non-obstructive azoospermia (NOA) contaminated (in vitro model) with an increasing number of IMR-32 and SK-N-SH NB cells. Testicular tissues were frozen by slow or snap freezing. The second step was to determine the expression levels of these markers in testicular samples from 4 pre-pubertal males (2 with stage IV NB and 2 with non-NB malignancy). The yield of extracted RNA was similar in testicular samples frozen by slow or snap freezing. In the in vitro model, TH and DCX transcripts were detected in uncontaminated testicular tissues, whereas PHOX2B mRNA was not detected. There was a strong positive association between the number of NB cells used for contamination and PHOX2B transcript levels. For IMR-32 and SK-N-SH NB cell lines, specificity and sensitivity rates of detection were 100% for PHOX2B following in vitro contamination with 10 tumor cells. In testicular samples from pre-pubertal males with and without NB, PHOX2B mRNA expression was not observed, but high expression levels of TH and DCX mRNA were detected, which were similar to expression detected in the in vitro model. Among the markers used in blood and bone marrow for NB MRD studies, the detection of PHOX2B transcripts by RT-qPCR may provide an accurate assessment of NB cells in testicular tissues from males who require fertility preservation.

Re-Cloning the N27 Dopamine Cell Line to Improve a Cell Culture Model of Parkinson's Disease

Parkinson’s disease is characterized by the death of dopaminergic neurons in the substantia nigra. To understand the molecular mechanisms of the disease, an in vitro model is important. In the 1990s, we used the SV40 large T antigen to immortalize dopaminergic neurons derived from Embryonic Day 14 rat mesencephalon. We selected a clone for its high expression of dopaminergic neuron markers such as tyrosine hydroxylase (TH), and we named it 1RB3AN27 (N27). Because the original N27 cell line has been passaged many times, the line has become a mixture of cell types with highly variable expression of TH. In the current study, we have performed multiple rounds of clonal cultures and have identified a dopaminergic cell clone expressing high levels of TH and the dopamine transporter (DAT). We have named this new clone N27-A. Nearly 100% of N27-A cells express TH, DAT and Tuj1. Western blots have confirmed that N27-A cells have three to four times the levels of TH and DAT compared to the previous mixed population in N27. Further analysis has shown that the new clone expresses the dopamine neuron transcription factors Nurr1, En1, FoxA2 and Pitx3. The N27-A cells express the vesicular monoamine transporter (VMAT2), but do not express dopamine-beta-hydroxylase (DβH), the enzyme responsible for converting dopamine to norepinephrine. Functional analysis has shown that N27-A cells are more sensitive than N27 cells to neurotoxins taken up by the dopamine transporter such as 6-hydroxydopamine and 1-methyl-4-phenylpyridine (MPP+). The DAT inhibitor nomifensine can block MPP+ induced toxicity. The non-selective toxic effects of hydrogen peroxide were similar in both cell lines. The N27-A cells show dopamine release under basal and depolarization conditions. We conclude that the new N27-A clone of the immortalized rat dopaminergic cell line N27 should provide an improved in vitro model for Parkinson’s disease research.

TH and DCX mRNAs in peripheral blood and bone marrow predict outcome in metastatic neuroblastoma patients.

In metastatic neuroblastoma (NB) patients, accurate risk stratification and disease monitoring would reduce relapse probabilities. This study aims to evaluate the independent prognostic significance of detecting tyrosine hydroxylase (TH) and doublecortin (DCX) mRNAs by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in peripheral blood (PB) and bone marrow (BM) samples from metastatic NB patients. RT-qPCR was performed on PB and BM samples from metastatic NB patients at diagnosis, post-induction therapy and at the end of treatment for TH and DCX mRNAs detection. High levels of TH and DCX mRNAs when detected in PB and BM at diagnosis independently predicted worse outcome in a cohort of 162 metastatic NB. In the subgroup of high-risk metastatic NB, TH mRNA detected in PB remained as independent predictor of EFS and OS at diagnosis. After the induction therapy, high levels of TH mRNA in PB and DCX mRNA in BM independently predicted poor EFS and OS. Furthermore TH mRNA when detected in BM predicted worse EFS. TH mRNA in PB samples at the end of treatment is an independent predictor of worse outcome. TH and DCX mRNAs levels in PB and BM assessed by RT-qPCR should be considered in new pre-treatment risk stratification strategies to reliable estimate outcome differences in metastatic NB patients. In those high-risk metastatic NB, TH and DCX mRNA quantification could be used for the assessment of response to treatment and for early detection of progressive disease or relapses.

Intrauterine growth restriction increases the preference for palatable foods and affects sensitivity to food rewards in male and female adult rats

Clinical evidence suggests that intrauterine growth restriction (IUGR) can cause persistent changes in the preference for palatable foods. In this study, we compared food preferences, the response to food rewards, and the role of the mesolimbic dopaminergic system in feeding behavior, between IUGR and control rats. Time-mated pregnant Sprague–Dawley rats were randomly allocated to a control group (standard chow ad libitum) or a 50% food restriction (FR) group, which received 50% of the control dams׳ habitual intake. These diets were provided from gestation day 10 to the 21st day of lactation. Within 24h of birth, pups were cross-fostered and divided into four groups: Adlib/Adlib, FR/Adlib, FR/FR, Adlib/FR. Standard chow consumption was compared between all groups. Food preferences, conditioned place preference to a palatable diet, and the levels of tyrosine hydroxylase (TH) phosphorylation and D2 receptors in the nucleus accumbens were analyzed and compared between the two groups of interest: Adlib/Adlib (control) and FR/Adlib (exposed to growth restriction during the fetal period only). IUGR adult rats had a stronger preference for palatable foods, but showed less conditioned place preference to a palatable diet than controls. D2 receptors levels were lower in IUGR rats. At baseline, TH and pTH levels were higher in FR/Adlib than control males. Measurements taken after exposure to sweet foods revealed higher levels of TH and pTH in FR/Adlib than control females. These data showed that IUGR rats exhibited a preference for palatable foods, potentially due to alterations in their mesolimbic reward pathway. Additionally, the changes observed in the mesolimbic dopaminergic system of IUGR rats proved to be sex-specific.This article is part of a Special Issue entitled 1618.

Physical Training Regulates Mitochondrial Parameters and Neuroinflammatory Mechanisms in an Experimental Model of Parkinson's Disease.

This study aimed to evaluate the effects of two different protocols for physical exercise (strength and aerobic training) on mitochondrial and inflammatory parameters in the 6-OHDA experimental model of Parkinson's disease. Six experimental groups were used (n = 12 per group): untrained + vehicle (Sham), strength training + vehicle (STR), treadmill training + vehicle (TTR), untrained + 6-OHDA (U + 6-OHDA), strength training + 6-OHDA (STR + 6-OHDA), and treadmill training + 6-OHDA (TTR + 6-OHDA). The mice were subjected to strength or treadmill training for 8 weeks. PD was induced via striatal injection of 6-OHDA 24 h after the last exercise session. Mice were euthanized by cervical dislocation and the striatum and hippocampus were homogenized to determine levels of tyrosine hydroxylase (TH), nuclear factor kappa B (NF-κB) p65, and sirtuin 1 (Sirt1) by western blot; tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-17, interferon-γ (IFN-γ), and transforming growth factor β1 (TGF-β1) levels by ELISA; NO content; and complex I (CI) activity. STR + 6-OHDA mice had higher TH levels and CI activity and lower NF-κB p65 and IFN-γ levels in the striatum compared to U + 6-OHDA mice, while TTR + 6-OHDA mice had higher Sirt1 levels and CI activity in both the striatum and the hippocampus, compared to U + 6-OHDA mice. Strength training increased CI activity and TH and Sirt1 levels and reduced NO, NF-κB p65, TNF-α, IFN-γ, IL-1β, and TGF-β1 levels in 6-OHDA mice, while treadmill exercise increased CI activity and NO, TH, and Sirt1 levels and reduced NF-κB p65, TNF-α, IFN-γ, and IL-1β levels. Our results demonstrated that both treadmill training and strength training promote neuroprotection, possibly by stimulating Sirt1 activity, which may in turn regulate both mitochondrial function and neuroinflammation via deacetylation of NF-κB p65. Changes in nitric oxide levels may also be a mechanism by which 6-OHDA-induced inflammation is controlled.

Effects of developmental methylphenidate (MPH) treatment on monoamine neurochemistry of male and female rats

Attention Deficit Hyperactivity Disorder (ADHD) is estimated to affect 4–5% of the adult human population (Kessler et al., 2006; Willcutt, 2012). Often prescribed to attenuate ADHD symptoms (Nair and Moss, 2009), methylphenidate hydrochloride (MPH) can have substantial positive effects. However, there is a paucity of literature regarding its use during pregnancy. Thus, adult women with ADHD face a difficult decision when contemplating pregnancy. In this study, pregnant Sprague–Dawley rats were orally treated a total of 0 (water), 6 (low), 18 (medium), or 42 (high) mgMPH/kgbodyweight/day (divided into three doses) on gestational days 6–21 (i.e., the low dose received 2mgMPH/kgbodyweight3×/day). Offspring were orally treated with the same daily dose as their dam (divided into two doses) on postnatal days (PNDs) 1–21. One offspring/sex/litter was sacrificed at PND 22 or PND 104 (n=6–7/age/sex/treatment group) and the striatum was quickly dissected and frozen. High Performance Liquid Chromatography (HPLC) coupled to a Photo Diode Array detector (PDA) was used to analyze monoamine content in the striatum of one side while a sandwich ELISA was used to analyze tyrosine hydroxylase (TH) from the other side. Age significantly affected monoamine and metabolite content as well as turnover ratios (i.e., DA, DOPAC, HVA, DOPAC/DA, HVA/DA, 5-HT and 5-HIAA); however, there were no significant effects of sex. Adult rats of the low MPH group had higher DA levels than control adults (p<0.05). At both ages, subjects of the low MPH group had higher TH levels than controls (p<0.05), although neither effect (i.e., higher DA or TH levels) exhibited an apparent dose–response. PND 22 subjects of the high MPH treatment group had higher ratios of HVA/DA and DOPAC/DA than same-age control subjects (p<0.05). The increased TH levels of the low MPH group may be related to the increased DA levels of adult rats. While developmental MPH treatment appears to have some effects on monoamine system development, further studies are required to determine if these alterations manifest as functional changes in behavior.

Neuroblastoma mRNAs Predict Outcome in Children With Stage 4 Neuroblastoma: A European HR-NBL1/SIOPEN Study

To evaluate the hypothesis that detection of neuroblastoma mRNAs by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) in peripheral blood (PB) and bone marrow aspirates (BM) from children with stage 4 neuroblastoma are clinically useful biomarkers of risk. RTqPCR for paired-like homeobox 2b (PHOX2B), tyrosine hydroxylase (TH), and doublecortin (DCX) mRNA in PB and BM of children enrolled onto the High-Risk Neuroblastoma Trial-1 of the European Society of Pediatric Oncology Neuroblastoma Group (HR-NBL1/SIOPEN) was performed at diagnosis and after induction therapy. High levels of TH, PHOX2B, or DCX mRNA in PB or BM at diagnosis strongly predicted for worse event-free survival (EFS) and overall survival (OS) in a cohort of 290 children. After induction therapy, high levels of these mRNAs predicted worse EFS and OS in BM but not in PB. Combinations of mRNAs in BM did not add to the predictive power of any single mRNA. However, in the original (n = 182) and validation (n = 137) PB cohorts, high TH (log10TH > 0.8) or high PHOX2B (log10PHOX2B > 0.28) identify 19% of children as ultrahigh risk, with 5-year EFS and OS rates of 0%; OS rate was 25% (95% CI, 16% to 36%) and EFS rate was 38% (95% CI, 28% to 49%) in the remaining children. The magnitude of reduction in mRNA level between diagnosis and postinduction therapy in BM or PB was not of additional predictive value. High levels of TH and PHOX2B mRNA in PB at diagnosis objectively identify children with ultrahigh-risk disease who may benefit from novel treatment approaches. The level of TH, PHOX2B, and DCX mRNA in BM and/or PB at diagnosis might contribute to an algorithm to improve stratification of children for treatment.

Lentiviral Delivery of LMX1a Enhances Dopaminergic Phenotype in Differentiated Human Bone Marrow Mesenchymal Stem Cells

Human mesenchymal stem cells (MSCs) reside in the bone marrow and are known for their ability to differentiate along the mesenchymal lineage (fat, bone, and cartilage). Recent works have suggested the possibility that these cells are also capable of differentiating toward the neuroectodermal lineage. Using lentiviral gene delivery, we sought to reprogram the bone marrow-derived MSCs toward dopaminergic differentiation through delivery of LMX1a, which was reported to be a key player in dopaminergic differentiation in both developmental animal models and embryonic stem cells. Transduction of cells with fluorescent reporter genes confirmed efficiency of gene delivery. On incubation of the LMX1a transduced cells in differentiation medium, the LMX1a protein was concentrated in the cells' nuclei and specific dopaminergic developmental genes were upregulated. Moreover, the transduced cells expressed higher levels of tyrosine hydroxylase, the rate limiting enzyme in dopamine synthesis, and secreted significantly higher level of dopamine in comparison to nontransduced cells. We hereby present a novel strategy to facilitate the dopaminergic differentiation of bone marrow-derived MSCs as a possible cell source for autologous transplantation for Parkinsonian patients in the future.

Elevated Glucocorticoid Levels Are Responsible for Induction of Tyrosine Hydroxylase mRNA Expression, Phosphorylation, and Enzyme Activity in the Nucleus of the Solitary Tract during Morphine Withdrawal

Chronic opiate exposure induces neurochemical adaptations in the noradrenergic system. Enhanced responsiveness of the hypothalamo-pituitary-adrenal axis after morphine withdrawal has been associated with hyperactivity of ascending noradrenergic input from the nucleus of the solitary tract (NTS-A(2)) cell group to the hypothalamic paraventricular nucleus (PVN). This study addressed the role of morphine withdrawal-induced corticosterone (CORT) release in regulation of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis in adrenalectomized (ADX) rats supplemented with low CORT pellet (ADX plus CORT). Present results show that in sham-ADX rats, noradrenergic neurons in the NTS-A(2) became activated during morphine withdrawal, as indicated by increased TH mRNA expression. However, this induction of TH expression is not detected in ADX plus CORT rats that are unable to mount CORT secretory response to morphine withdrawal. Total TH protein levels were elevated in the NTS-A(2) from sham-operated rats during morphine dependence and withdrawal, whereas we did not find any alteration in ADX plus CORT animals. Furthermore, high levels of TH phosphorylated (activated) at Ser31 (but not at Ser40) were found in the A(2) area from sham-morphine withdrawn rats. Consistent with these effects, we observed an increase in the enzyme activity of TH in the PVN. However, induction of morphine withdrawal to ADX plus CORT animals did not alter the phosphorylation (activation) of TH in NTS-A(2) and decreased TH activity in the PVN. These results suggest the existence of a positive reverberating circle in which elevated glucocorticoids during morphine abstinence play a permissive role in morphine withdrawal-induced activation of noradrenergic pathway innervating the PVN.

Iron Deficiency Alters the Day‐Night Variation in Monoamine Levels in Mice

Monoamine metabolism in the central nervous system is altered by dietary iron deficiency, with a stronger effect seen during the active than rest span of the circadian cycle. In this report, we examined changes in intracellular and extracellular monoamine levels, synthetic enzymes, transporter and receptor densities, and responses to amphetamine‐induced dopamine (DA) efflux in iron‐deficient and iron‐sufficient mice. Extracellular striatal DA levels were 15–20% higher in all groups during the active dark phase compared to the inactive light phase, with correspondingly lower dopamine transporter (DAT) and higher tyrosine hydroxylase levels. Iron deficiency decreased DAT density by 20% and 28% in the light and dark phases, respectively, and elevated the DOPAC/DA ratio only in the dark, indicating that iron deficiency does interact with the normal diurnal cues for cyclicity. Enhanced DA efflux after amphetamine stimulation indicates no limitation on monoamine synthesis and release and is consistent with altered synaptic efficacy and perhaps recycling of DA in iron deficiency. These experimental findings provide new evidence that brain iron insufficiency does have a differential effect on the DA system at different biological times of the day and night and may be causally related to the phasic motor symptoms observed in Restless Legs Syndrome.

Oxidation of tyrosine diketopiperazine to DOPA diketopiperazine with tyrosine hydroxylase.

The diketopiperazine of DOPA was synthesized in high yield from the diketopiperazine of tyrosine using PC12 cell lysate, which expresses high levels of tyrosine hydroxylase. This represents the first use of this enzyme to prepare DOPA-containing peptides.

Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors

The aim of this study was to produce dopaminergic neurons in vitro from human embryonic stem (hES) cells following treatment of various neurotrophic factors. MB03 hES cells were induced by retinoic acid (RA) or basic fibroblast growth factor (bFGF), which were further treated with brain derived neurotrophic factor (BDNF) or transforming growth factor (TGF)-α in each induction method during neuron differentiation days. At the final differentiation stage (21 days), all treatment groups revealed very similar levels (bFGF, 76–78%; RA, 70–74%) of mature neurons (anti-NF-200) in two induction methods irrespective of the addition of BDNF or TGF-α. In addition, immunostaining and HPLC analyses revealed higher levels of tyrosine hydroxylase (20±2.3%) and dopamine (265.5±62.8 pg/ml) in the bFGF- and TGF-α-treated hES cells than in RA- or BDNF-treated hES cells. These data are one of the first reports on the generation of dopaminergic neurons of hES cells in vitro. Also, our results indicate that TGF-α may be successfully used in the bFGF induction protocol and yield higher numbers of dopaminergic neurons from hES cells.

Quantitative imaging of tyrosine hydroxylase and calmodulin in the human brain

The distributions of tyrosine hydroxylase and calmodulin in adult normal postmortem human brain were analyzed quantitatively. Consecutive coronal sections were obtained from the anterior area of the right hemisphere and were stained immunohistochemically for tyrosine hydroxylase and calmodulin. Stained sections were divided into approximately 3 million microareas at 50 μm intervals, and the immunohistochemical fluorescence intensity in each area was measured by a human brain mapping analyzer, which is a microphotometry system for analysis of the distribution of neurochemicals in a large tissue slice. Immunoreactive staining of tyrosine hydroxylase and calmodulin was observed in almost all brain regions, but its intensity varied. Relatively high levels of calmodulin were observed in brain regions with high levels of tyrosine hydroxylase, though high levels of tyrosine hydroxylase were not always observed in brain regions where high levels of calmodulin were distributed. In particular, high levels of both of tyrosine hydroxylase and calmodulin were distributed in the caudate nucleus and putamen. Previously it was shown that tyrosine hydroxylase was activated and dopamine synthesis was enhanced in the neostriatum region in mice and rats by the intracerebroventricular administration of calcium through a calmodulin-dependent system. The present results combined with these previous findings suggest that the activity of tyrosine hydroxylase in the caudate nucleus and putamen of humans may also be regulated by a calcium/calmodulin-dependent system. J. Neurosci. Res. 63:369–376, 2001. © 2001 Wiley-Liss, Inc.

Dark Agouti and Fischer 344 rats: differential behavioral responses to morphine and biochemical differences in the ventral tegmental area

We sought to identify behavioral and biochemical differences between Dark Agouti and Fischer 344 inbred rat strains to assess whether they could serve as a model of genetically determined differences in sensitivity to drugs of abuse. We compared the strains for the following traits: morphine-induced locomotor activity and sensitization; circadian variation in plasma levels of corticosterone, a hormone reported to affect sensitivity to drugs of abuse; and several biochemical parameters in the ventral tegmental area and nucleus accumbens, brain regions implicated in the locomotor activating and reinforcing actions of drugs of abuse. Fischer 344 rats exhibited greater initial locomotor responses to morphine but, unlike Dark Agouti rats, did not develop sensitization to a second morphine exposure. Fischer 344 rats displayed a marked rise in basal plasma corticosterone levels in the late light phase and early dark phase, whereas Dark Agouti rats showed no significant circadian variation in corticosterone levels. Relative to drug-naive Fischer 344 rats, drug-naive Dark Agouti rats showed higher levels of tyrosine hydroxylase and glial fibrillary acidic protein, and lower levels of neurofilament proteins, in the ventral tegmental area. In contrast, no strain differences were found in levels of tyrosine hydroxylase, specific G protein subunits or protein kinase A in the nucleus accumbens. Together, these results demonstrate that Dark Agouti rats and Fischer 344 rats exhibit differences in specific behavioral, endocrine and biochemical parameters related to sensitivity to drugs of abuse.

Immunohistochemical studies of mesolimbic dopaminergic neurons in Fischer 344 and Lewis rats

Previous work has shown that the inbred Lewis and Fischer 344 rat strains differ in several behavioral measures related to mesolimbic dopamine function. Moreover, the ventral tegmental area (VTA) of the Lewis rat has been shown to contain higher levels of tyrosine hydroxylase compared to that of the Fischer rat by blot immunolabeling procedures. To investigate structural correlates of this biochemical difference, an immunohistochemical study of VTA dopaminergic neurons in these two strains was undertaken. Results show that the density and total number of tyrosine hydroxylase-positive neurons in the VTA of the Lewis rat is about 50% of that found in the Fischer rat. In contrast, examination of the substantia nigra in the same sections revealed no differences in the density and number of tyrosine hydroxylase-positive cells between these strains. Fischer-Lewis strain differences were also evident for cholecystokinin immunoreactivity in the VTA, with much lower levels seen in the Lewis rat, consistent with the known colocalization of this neuropeptide in many VTA dopamine neurons. The finding of 50%v fewer tyrosine hydroxylase-positive neurons in the VTA of the Lewis rat, along with our earlier results showing 45% higher levels of tyrosine hydroxylase by blot immunolabeling, would suggest much higher levels of tyrosine hydroxylase per VTA neuron in this strain. However, no obvious strain difference in the cellular intensity of tyrosine hydroxylase immunoreactivity could be detected by immunohistochemistry. Finally, the density of VTA dopamine neurons was assessed in 1-week-old Fischer and Lewis rats. In contrast to the results obtained for adult animals, no difference in the number of tyrosine hydroxylase-positive neurons was apparent in these young animals, indicating that the Fischer-Lewis strain difference in VTA dopamine neurons appears later in postnatal development. These anatomical findings shed new light on the differences in the mesolimbic dopamine system between Fischer and Lewis rats that may contribute to the behavioral differences exhibited by these animals.

Effects of chronic social stress on tyrosine hydroxylase mRNA and protein levels.

Males housed in mixed sex groups quickly form dominance hierarchies; subordinates can be further subdivided into stress responsive subordinates (SRS) and non-responsive subordinates (NRS) based on corticosterone responses to a novel stressor. Tyrosine hydroxylase (TH) mRNA levels measured with in situ hybridization were elevated in locus coeruleus (LC) of NRS compared to singly or pair-housed controls; NRS also had higher TH levels than dominants. TH protein levels determined by immunoautoradiography were also higher in LC of NRS and SRS versus pair-housed controls.