Emerging Role of Kinin B1 Receptor in Persistent Neuroinflammation and Neuropsychiatric Symptoms in Mice Following Recovery from SARS-CoV-2 Infection.

Evidence suggests that patients with long COVID can experience neuropsychiatric, neurologic, and cognitive symptoms. However, these clinical data are mostly associational studies complicated by confounding variables, thus the mechanisms responsible for persistent symptoms are unknown. Here we establish an animal model of long-lasting effects on the brain by eliciting mild disease in K18-hACE2 mice. Male and female K18-hACE2 mice were infected with 4 × 103 TCID50 of SARS-CoV-2 and, following recovery from acute infection, were tested in the open field, zero maze, and Y maze, starting 30 days post infection. Following recovery from SARS-CoV-2 infection, K18-hACE2 mice showed the characteristic lung fibrosis associated with SARS-CoV-2 infection, which correlates with increased expression of the pro-inflammatory kinin B1 receptor (B1R). These mice also had elevated expression of B1R and inflammatory markers in the brain and exhibited behavioral alterations such as elevated anxiety and attenuated exploratory behavior. Our data demonstrate that K18-hACE2 mice exhibit persistent effects of SARS-CoV-2 infection on brain tissue, revealing the potential for using this model of high sensitivity to SARS-CoV-2 to investigate mechanisms contributing to long COVID symptoms in at-risk populations. These results further suggest that elevated B1R expression may drive the long-lasting inflammatory response associated with SARS-CoV-2 infection.

Lipid metabolism in dopaminergic neurons influences light entrainment.

Dietary lipids, particularly omega-3 polyunsaturated fatty acids, are speculated to impact behaviors linked to the dopaminergic system, such as movement and control of circadian rhythms. However, the ability to draw a direct link between dopaminergic omega-3 fatty acid metabolism and behavioral outcomes has been limited to the use of diet-based approaches, which are confounded by systemic effects. Here, neuronal lipid metabolism was targeted in a diet-independent manner by manipulation of long-chain acyl-CoA synthetase 6 (ACSL6) expression. ACSL6 performs the initial reaction for cellular fatty acid metabolism and prefers the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA). The loss of Acsl6 in mice (Acsl6-/- ) depletes neuronal membranes of DHA content and results in phenotypes linked to dopaminergic control, such as hyperlocomotion, impaired short-term spatial memory, and imbalances in dopamine neurochemistry. To investigate the role of dopaminergic ACSL6 on these outcomes, a dopaminergic neuron-specific ACSL6 knockout mouse was generated (Acsl6DA-/- ). Acsl6DA-/- mice demonstrated hyperlocomotion and imbalances in striatal dopamine neurochemistry. Circadian rhythms of both the Acsl6-/- and the Acsl6DA-/- mice were similar to control mice under basal conditions. However, upon light entrainment, a mimetic of jet lag, both the complete knockout of ACSL6 and the dopaminergic-neuron-specific loss of ACSL6 resulted in a longer recovery to entrainment compared to control mice. In conclusion, these data demonstrate that ACSL6 in dopaminergic neurons alters dopamine metabolism and regulation of light entrainment suggesting that DHA metabolism mediated by ACSL6 plays a role in dopamine neuron biology.

COVID-19 Infection Enhances Susceptibility to Oxidative Stress-Induced Parkinsonism.

BACKGROUND: Viral induction of neurological syndromes has been a concern since parkinsonian-like features were observed in patients diagnosed with encephalitis lethargica subsequent to the 1918 influenza pandemic. Given the similarities in the systemic responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection with those observed after pandemic influenza, there is a question whether a similar syndrome of postencephalic parkinsonism could follow coronavirus disease 2019 infection. OBJECTIVE: The goal of this study was to determine whether prior infection with SARS-CoV-2 increased sensitivity to a mitochondrial toxin known to induce parkinsonism. METHODS: K18-hACE2 mice were infected with SARS-CoV-2 to induce mild-to-moderate disease. After 38 days of recovery, mice were administered a non-lesion-inducing dose of the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and euthanized 7 days later. Subsequent neuroinflammation and substantia nigra pars compacta (SNpc) dopaminergic (DA) neuron loss were determined and compared with SARS-CoV-2 or MPTP alone. RESULTS: K18-hACE2 mice infected with SARS-CoV-2 or MPTP showed no SNpc DA neuron loss after MPTP. In mice infected and recovered from SARS-CoV-2 infection, MPTP induced a 23% or 19% greater loss of SNpc DA neurons than SARS-CoV-2 or MPTP, respectively (P < 0.05). Examination of microglial activation showed a significant increase in the number of activated microglia in both the SNpc and striatum of the SARS-CoV-2 + MPTP group compared with SARS-CoV-2 or MPTP alone. CONCLUSIONS: Our observations have important implications for long-term public health, given the number of people who have survived SARS-CoV-2 infection, as well as for future public policy regarding infection mitigation. However, it will be critical to determine whether other agents known to increase risk for PD also have synergistic effects with SARS-CoV-2 and are abrogated by vaccination. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Hypothalamic kinin B1 receptor mediates orexin system hyperactivity in neurogenic hypertension.

Brain orexin system hyperactivity contributes to neurogenic hypertension. We previously reported upregulated neuronal kinin B1 receptor (B1R) expression in hypertension. However, the role of central B1R activation on the orexin system in neurogenic hypertension has not been examined. We hypothesized that kinin B1R contributes to hypertension via upregulation of brain orexin-arginine vasopressin signaling. We utilized deoxycorticosterone acetate (DOCA)-salt hypertension model in wild-type (WT) and B1R knockout (B1RKO) mice. In WT mice, DOCA-salt-treatment increased gene and protein expression of orexin A, orexin receptor 1, and orexin receptor 2 in the hypothalamic paraventricular nucleus and these effects were attenuated in B1RKO mice. Furthermore, DOCA-salt- treatment increased plasma arginine vasopressin levels in WT mice, but not in B1RKO mice. Cultured primary hypothalamic neurons expressed orexin A and orexin receptor 1. B1R specific agonist (LDABK) stimulation of primary neurons increased B1R protein expression, which was abrogated by B1R selective antagonist R715 but not by the dual orexin receptor antagonist, ACT 462206, suggesting that B1R is upstream of the orexin system. These data provide novel evidence that B1R blockade blunts orexin hyperactivity and constitutes a potential therapeutic target for the treatment of salt-sensitive hypertension.

Acyl-CoA synthetase 6 is required for brain docosahexaenoic acid retention and neuroprotection during aging.

The omega-3 fatty acid docosahexaenoic acid (DHA) inversely relates to neurological impairments with aging; however, limited nondietary models manipulating brain DHA have hindered a direct linkage. We discovered that loss of long-chain acyl-CoA synthetase 6 in mice (Acsl6-/-) depletes brain membrane phospholipid DHA levels, independent of diet. Here, Acsl6-/- brains contained lower DHA compared with controls across the life span. The loss of DHA- and increased arachidonate-enriched phospholipids were visualized by MALDI imaging predominantly in neuron-rich regions where single-molecule RNA in situ hybridization localized Acsl6 to neurons. ACSL6 is also astrocytic; however, we found that astrocyte-specific ACSL6 depletion did not alter membrane DHA because astrocytes express a non-DHA-preferring ACSL6 variant. Across the life span, Acsl6-/- mice exhibited hyperlocomotion, impairments in working spatial memory, and increased cholesterol biosynthesis genes. Aging caused Acsl6-/- brains to decrease the expression of membrane, bioenergetic, ribosomal, and synaptic genes and increase the expression of immune response genes. With age, the Acsl6-/- cerebellum became inflamed and gliotic. Together, our findings suggest that ACSL6 promotes membrane DHA enrichment in neurons, but not in astrocytes, and is important for neuronal DHA levels across the life span. The loss of ACSL6 impacts motor function, memory, and age-related neuroinflammation, reflecting the importance of neuronal ACSL6-mediated lipid metabolism across the life span.

Nurr1 deficiency shortens free running period, enhances photoentrainment to phase advance, and disrupts circadian cycling of the dopamine neuron phenotype.

Neurological and neuropsychiatric disorders, including addiction, schizophrenia, and Parkinson's disease (PD), involve dysfunction in midbrain dopamine (DA) neurotransmission with severity of disease symptoms and progression associated with disrupted circadian rhythms. The nuclear transcription factor Nurr1, essential for DA neuron (DAN) development, survival, and maintenance, is also known to interact with circadian rhythm regulating clock proteins. In the Nurr1-null heterozygous (+/-) mice, a Nurr1 deficient model which reproduces some of the alterations in DA function found in schizophrenia and PD, we measured, using wheel-running activity, the free running period (tau) and photoperiod entrainment. Because Nurr1 has a role in regulating the DA phenotype, we also measured the circadian fluctuations in the number of DANs using tyrosine hydroxylase (TH) immunofluorescence. In Nurr1 +/- mice, tau was significantly shorter and entrainment to a 6 h earlier shift in the dark cycle was accelerated. The Nurr1 wild-type (+/+) mice cycled DAN numbers across time, with a significantly greater number (∼2-fold increase) of DANs at zeitgeber time (ZT) 0 than ZT12. The +/- mice, however, did not cycle the DA phenotype, as no differences in DAN numbers were observed between ZT0 and ZT12. Additionally, the +/- mice had significantly fewer DANs at ZT0 but not at ZT12 as compared to +/+ mice. Based these data, circadian rhythms and fluctuations in the DA phenotype requires normal Nurr1 function. A better understanding is needed of the mechanisms regulating the DA phenotype and subsequent neurotransmission across the circadian cycle and how this is altered in circadian rhythm and DA neurotransmission-associated disorders.

Quantitative Immunohistochemistry to Measure Regional Expression of Nurr1 in the Brain and the Effect of the Nurr1 Heterozygous Genotype.

The transcription factor Nurr1 is a member of the steroid hormone nuclear receptor superfamily. Ablation of Nurr1 expression arrests mesencephalic dopamine neuron differentiation while attenuation of Nurr1 in the subiculum and hippocampus impairs learning and memory. Additionally, reduced Nurr1 expression has been reported in patients with Parkinson's disease and Alzheimer's disease. In order to better understand the overall function of Nurr1 in the brain, quantitative immunohistochemistry was used to measure cellular Nurr1 protein expression, across Nurr1 immunoreactive neuronal populations. Additionally, neuronal Nurr1 expression levels were compared between different brain regions in wild-type mice (+/+) and Nurr1 heterozygous mice (+/-). Regional Nurr1 protein was also investigated at various time points after a seizure induced by pentylenetetrazol (PTZ). Nurr1 protein is expressed in various regions throughout the brain, however, a wide range of Nurr1 expression levels were observed among various neuronal populations. Neurons in the parietal and temporal cortex (secondary somatosensory, insular, auditory, and temporal association cortex) had the highest relative Nurr1 expression (100%) followed closely by the claustrum/dorsal endopiriform cortex (85%) and then subiculum (76%). Lower Nurr1 protein levels were found in neurons in the substantia nigra pars compacta and ventral tegmental area (39%) followed by CA1 (25%) and CA3 (19%) of the hippocampus. Additionally, in the parietal and temporal cortex, two distinct populations of high and medium Nurr1 expressing neurons were observed. Comparisons between +/- and +/+ mice revealed Nurr1 protein was reduced in +/- mice by 27% in the parietal/temporal cortex, 49% in the claustrum/dorsal endopiriform cortex, 25% in the subiculum, 33% in substantia nigra pars compacta, 22% in ventral tegmental area, and 21% in CA1 region of the hippocampus. Based on these data, regional mechanisms appear to exist which can compensate for a loss of a Nurr1 allele. Following a single PTZ-induced seizure, Nurr1 protein in the dentate gyrus peaked around 2 h and returned to baseline by 8 h. Since altered Nurr1 expression has been implicated in neurologic disorders and Nurr1 agonists have showed protective effects, understanding regional protein expression of Nurr1, therefore, is necessary to understand how changes in Nurr1 expression can alter brain function.

Region Specific Effects of Aging and the Nurr1-Null Heterozygous Genotype on Dopamine Neurotransmission.

The transcription factor Nurr1 is essential for dopamine neuron differentiation and is important in maintaining dopamine synthesis and neurotransmission in the adult. Reduced Nurr1 function, due to the Nurr1-null heterozygous genotype (+/-), impacts dopamine neuron function in a region specific manner resulting in a decrease in dopamine synthesis in the dorsal and ventral striatum and a decrease in tissue dopamine levels in the ventral striatum. Additionally, maintenance of tissue dopamine levels in the dorsal striatum and survival of nigrostriatal dopamine neurons with aging (>15 months) or after various toxicant treatments are impaired. To further investigate the effects of aging and the Nurr1-null heterozygous genotype, we measured regional tissue dopamine levels, dopamine neuron numbers, body weight, open field activity and rota-rod performance in young (3-5 months) and aged (15-17 months) wild-type +/+ and +/- mice. Behavioral tests revealed no significant differences in rota-rod performance or basal open field activity as a result of aging or genotype. The +/- mice did show a significant increase in open field activity after 3 min of restraint stress. No differences in tissue dopamine levels were found in the dorsal striatum. However, there were significant reductions in tissue dopamine levels in the ventral striatum, which was separated into the nucleus accumbens core and shell, in the aged +/- mice. These data indicate that the mesoaccumbens system is more susceptible to the combination of aging and the +/- genotype than the nigrostriatal system. Additionally, the effects of aging and the +/- genotype may be dependent on genetic background or housing conditions. As Nurr1 mutations have been implicated in a number of diseases associated with dopamine neurotransmission, further data is needed to understand why and how Nurr1 can have differential functions across different dopamine neuron populations in aging.

Evaluation of a modified infraorbital approach for a maxillary nerve block for rhinoscopy with nasal biopsy of dogs.

OBJECTIVE To determine whether a maxillary nerve block via a modified infraorbital approach, applied before rhinoscopy and nasal biopsy of dogs, would decrease procedural nociception, minimize cardiorespiratory anesthetic effects, and improve recovery quality. ANIMALS 8 healthy adult hound-type dogs PROCEDURES In a crossover study, dogs received 0.5% bupivacaine (0.1 mL/kg) or an equivalent volume of saline (0.9% NaCl) solution as a maxillary nerve block via a modified infraorbital approach. A 5-cm, 20-gauge over-the-needle catheter was placed retrograde within each infraorbital canal, and bupivacaine or saline solution was administered into each pterygopalatine region. Rhinoscopy and nasal biopsy were performed. Variables monitored included heart rate, systolic arterial blood pressure (SAP), mean arterial blood pressure (MAP), diastolic arterial blood pressure (DAP), plasma cortisol and norepinephrine concentrations, purposeful movement, and pain scores. After a 14-day washout period, the other treatment was administered on the contralateral side, and rhinoscopy and nasal biopsy were repeated. RESULTS SAP, MAP, and DAP were significantly higher for the saline solution treatment than for the bupivacaine treatment, irrespective of the time point. Plasma cortisol concentrations after saline solution treatment were significantly higher 5 minutes after nasal biopsy than at biopsy. Heart rate, norepinephrine concentration, purposeful movement, and pain score were not significantly different between treatments. CONCLUSIONS AND CLINICAL RELEVANCE Maxillary nerve block via a modified infraorbital approach prior to rhinoscopy and nasal biopsy reduced procedural nociception as determined on the basis of blood pressures and plasma cortisol concentrations during anesthesia. These findings warrant further evaluation in dogs with nasal disease.

Decreased anxiety in juvenile rats following exposure to low levels of chlorpyrifos during development.

Exposure to chlorpyrifos (CPF) during the late preweanling period in rats inhibits the endocannabinoid metabolizing enzymes fatty acid hydrolase (FAAH) and monoacylglycerol lipase (MAGL), resulting in accumulation of their respective substrates anandamide (AEA) and 2-arachidonylglycerol (2-AG). This occurs at 1.0mg/kg, but at a lower dosage (0.5mg/kg) only FAAH and AEA are affected with no measurable inhibition of either cholinesterase (ChE) or MAGL. The endocannabinoid system plays a vital role in nervous system development and may be an important developmental target for CPF. The endocannabinoid system plays an important role in the regulation of anxiety and, at higher dosages, developmental exposure to CPF alters anxiety-like behavior. However, it is not clear whether exposure to low dosages of CPF that do not inhibit ChE will cause any persistent effects on anxiety-like behavior. To determine if this occurs, 10-day old rat pups were exposed daily for 7 days to either corn oil or 0.5, 0.75, or 1.0mg/kg CPF by oral gavage. At 12h following the last CPF administration, 1.0mg/kg resulted in significant inhibition of FAAH, MAGL, and ChE, whereas 0.5 and 0.75mg/kg resulted in significant inhibition of only FAAH. AEA levels were significantly elevated in all three treatment groups as were palmitoylethanolamide and oleoylethanolamide, which are also substrates for FAAH. 2-AG levels were significantly elevated by 0.75 and 1.0mg/kg but not 0.5mg/kg. On day 25, the latency to emerge from a dark container into a highly illuminated novel open field was measured as an indicator of anxiety. All three CPF treatment groups spent significantly less time in the dark container prior to emerging as compared to the control group, suggesting a decreased level of anxiety. This demonstrates that repeated preweanling exposure to dosages of CPF that do not inhibit brain ChE can induce a decline in the level of anxiety that is detectable during the early postweanling period.

Chronic Toxoplasma gondii in Nurr1-null heterozygous mice exacerbates elevated open field activity.

Latent infection with Toxoplasma gondii is common in humans (approximately 30% of the global population) and is a significant risk factor for schizophrenia. Since prevalence of T. gondii infection is far greater than prevalence of schizophrenia (0.5-1%), genetic risk factors are likely also necessary to contribute to schizophrenia. To test this concept in an animal model, Nurr1-null heterozygous (+/-) mice and wild-type (+/+) mice were evaluate using an emergence test, activity in an open field and with a novel object, response to bobcat urine and prepulse inhibition of the acoustic startle response (PPI) prior to and 6 weeks after infection with T. gondii. In the emergence test, T. gondii infection significantly decreased the amount of time spent in the cylinder. Toxoplasma gondii infection significantly elevated open field activity in both +/+ and +/- mice but this increase was significantly exacerbated in +/- mice. T. gondii infection reduced PPI in male +/- mice but this was not statistically significant. Aversion to bobcat urine was abolished by T. gondii infection in +/+ mice. In female +/- mice, aversion to bobcat urine remained after T. gondii infection while the male +/- mice showed no aversion to bobcat urine. Antibody titers of infected mice were a critical variable associated with changes in open field activity, such that an inverted U shaped relationship existed between antibody titers and the percent change in open field activity with a significant increase in activity at low and medium antibody titers but no effect at high antibody titers. These data demonstrate that the Nurr1 +/- genotype predisposes mice to T. gondii-induced alterations in behaviors that involve dopamine neurotransmission and are associated with symptoms of schizophrenia. We propose that these alterations in murine behavior were due to further exacerbation of the altered dopamine neurotransmission in Nurr1 +/- mice.

Laser capture microdissection--a demonstration of the isolation of individual dopamine neurons and the entire ventral tegmental area.

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

NR4A gene expression is dynamically regulated in the ventral tegmental area dopamine neurons and is related to expression of dopamine neurotransmission genes.

The NR4A transcription factors NR4A1, NR4A2, and NR4A3 (also known as Nur77, Nurr1, and Nor1, respectively) share similar DNA-binding properties and have been implicated in regulation of dopamine neurotransmission genes. Our current hypothesis is that NR4A gene expression is regulated by dopamine neuron activity and that induction of NR4A genes will increase expression of dopamine neurotransmission genes. Eticlopride and γ-butyrolactone (GBL) were used in wild-type (+/+) and Nurr1-null heterozygous (+/-) mice to determine the mechanism(s) regulating Nur77 and Nurr1 expression. Laser capture microdissection and real-time PCR was used to measure Nurr1 and Nur77 mRNA levels in the ventral tegmental area (VTA). Nur77 expression was significantly elevated 1 h after both GBL (twofold) and eticlopride (fourfold). In contrast, GBL significantly decreased Nurr1 expression in both genotypes, while eticlopride significantly increased Nurr1 expression only in the +/+ mice. In a separate group of mice, haloperidol injection significantly elevated Nur77 and Nor1, but not Nurr1 mRNA in the VTA within 1 h and significantly increased tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNA expression by 4 h. These data demonstrate that the NR4A genes are dynamically regulated in dopamine neurons with maintenance of Nurr1 expression requiring dopamine neuron activity while both attenuation of dopamine autoreceptors activation and dopamine neuronal activity combining to induce Nur77 expression. Additionally, these data suggest that induction of NR4A genes could regulate TH and DAT expression and ultimately regulate dopamine neurotransmission.

Immunomodulator expression in trophoblasts from the feline immunodeficiency virus (FIV)-infected cat.

BACKGROUND: FIV infection frequently compromises pregnancy under experimental conditions and is accompanied by aberrant expression of some placental cytokines. Trophoblasts produce numerous immunomodulators that play a role in placental development and pregnancy maintenance. We hypothesized that FIV infection may cause dysregulation of trophoblast immunomodulator expression, and aberrant expression of these molecules may potentiate inflammation and compromise pregnancy. The purpose of this project was to evaluate the expression of representative pro-(TNF-α, IFN-γ, IL-1ß, IL-2, IL-6, IL-12p35, IL-12p40, IL-18, and GM-CSF) and anti-inflammatory cytokines (IL-4, IL-5, and IL-10); CD134, a secondary co-stimulatory molecule expressed on activated T cells (FIV primary receptor); the chemokine receptor CXCR4 (FIV co-receptor); SDF-1α, the chemokine ligand to CXCR4; and FIV gag in trophoblasts from early-and late-term pregnancy. METHODS: We used an anti-cytokeratin antibody in immunohistochemistry to identify trophoblasts selectively, collected these cells using laser capture microdissection, and extracted total RNA from the captured cell populations. Real time, reverse transcription-PCR was used to quantify gene expression. RESULTS: We detected IL-4, IL-5, IL-6, IL-1ß, IL-12p35, IL-12p40, and CXCR4 in trophoblasts from early-and late-term pregnancy. Expression of cytokines increased from early to late pregnancy in normal tissues. A clear, pro-inflammatory microenvironment was not evident in trophoblasts from FIV-infected queens at either stage of pregnancy. Reproductive failure was accompanied by down-regulation of both pro-and anti-inflammatory cytokines. CD134 was not detected in trophoblasts, and FIV gag was detected in only one of ten trophoblast specimens collected from FIV-infected queens. CONCLUSION: Feline trophoblasts express an array of pro-and anti-inflammatory immunomodulators whose expression increases from early to late pregnancy in normal tissues. Non-viable pregnancies were associated with decreased expression of immunomodulators which regulate trophoblast invasion in other species. The detection of FIV RNA in trophoblasts was rare, suggesting that the high rate of reproductive failure in FIV-infected queens was not a direct result of viral replication in trophoblasts. The influence of placental immune cells on trophoblast function and pregnancy maintenance in the FIV-infected cat requires additional study.

Repeated developmental exposure to chlorpyrifos and methyl parathion causes persistent alterations in nicotinic acetylcholine subunit mRNA expression with chlorpyrifos altering dopamine metabolite levels.

Organophosphates (OPs), commonly used as insecticides, inhibit acetylcholinesterase, the enzyme responsible for the inactivation of synaptic acetylcholine, which results in elevated acetylcholine neurotransmission. Nigrostriatal dopamine neurons receive substantial cholinergic innervation and express a number of nicotinic acetylcholine receptor subunits. Since epidemiological data have implicated pesticides in the incidence of Parkinson's disease, the current experiment investigated how repeated, developmental exposure to the OPs chlorpyrifos (CPS) or methyl parathion (MPT) affects striatal dopamine levels and dopamine neuron gene expression. Newborn rats were treated daily via oral gavage with corn oil vehicle, CPS, or MPT from postnatal days (PND) 1-21. Rats were sacrificed at PND 22 and 50. Levels of dopamine and its metabolites 3,4 dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured in the striatum and mRNA expression was measured in the substantia nigra. At 22 days of age, CPS and MPT treatment had no effect on dopamine, DOPAC or HVA levels. At 50 days of age, CPS significantly elevated DOPAC levels and elevated dopamine turnover (DOPAC/dopamine) but did not affect dopamine or HVA levels. MPT had no significant effects on any of these parameters. Interestingly, both CPS and MPT treatments caused a significant alteration in the ratio of alpha7 to alpha6 nicotinic acetylcholine receptor (nAChR) subunit expression in the substantia nigra with a non-significant elevation in alpha6 and a reduction in alpha7 at 22 days. At 50 days of age, a significant elevation in alpha6 nAChR subunit was observed in the MPT treated rats. No differences in dopamine neuron transcription factors (Nurr1 or Lmx1b) or neurotransmission genes were observed. These data demonstrate that repeated exposure to OPs during postnatal maturation can have a significant effect on dopamine neurochemistry, primarily by modifying dopamine metabolism, which can persist for up to 1 month (CPS) and alter acetylcholine subunit expression (CPS and MPT).

Alterations in amphetamine-stimulated dopamine overflow due to the Nurr1-null heterozygous genotype and postweaning isolation.

The nuclear receptor Nurr1 functions to regulate dopamine neurotransmission, as Nurr1-null heterozygous (+/-) mice have alterations in dopamine function and, when raised in isolation immediately after weaning, have disruptions in sensorimotor gaiting, a behavior altered in schizophrenia and modulated by dopamine neurotransmission. The goal of this study was to determine nigrostriatal and mesoaccumbens dopamine neurotransmission using microdialysis in +/- and wild-type (+/+) mice raised in groups or isolation. In the striatum, isolation significantly reduced amphetamine-stimulated dopamine overflow and levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). The +/- genotype alone caused a small, nonsignificant reduction in basal dopamine levels but a significant reduction in basal DOPAC levels. In the nucleus accumbens shell, the +/- genotype elevated basal dopamine levels. Isolation had genotype specific effects, causing an elevation in amphetamine-stimulated dopamine overflow in +/- mice but a reduction in +/+ mice, resulting in a large difference in stimulated dopamine overflow when comparing the +/+ and +/- isolated mice. These data indicate that a deletion of a single allele of Nurr1, which produces only subtle changes alone, when coupled with a developmental stressor, can dramatically alter mesoaccumbens dopamine neurotransmission. These observations demonstrate how the combination of genetic predisposition and an environmental insult during development can cause dysfunction of dopamine neurotransmission and could contribute to diseases such as schizophrenia or attention deficit hyperactivity disorder.

Regulation of GTP cyclohydrolase I expression by orphan receptor Nurr1 in cell culture and in vivo.

Nurr1 is an orphan nuclear transcription factor essential for the terminal differentiation of dopamine (DA) neurons in the ventral midbrain (VM). To identify the Nurr1-target genes, we carried out microarray and quantitative real-time PCR analyses of Nurr1 null and wild-type mice in VM at embryonic day (E) 12.5 and shortly after birth (P0). In addition to the absence of mRNAs of DA synthesizing enzymes, the guanosine 5'-triphosphate (GTP) cyclohydrolase I (GTPCH) was also substantially reduced in the VM of Nurr1-null mice. GTPCH is the first enzyme in the synthesis pathway of tetrahydrobiopterin (BH4), an essential cofactor for tyrosine hydroxylase in DA synthesis. In the mouse, Nurr1 and GTPCH mRNA were first detected at E10.5, and GTPCH transcription paralleled that of Nurr1. Small interfering RNA targeted against Nurr1 decreases GTPCH expression in MC3T3-E1 osteoblasts in cell culture. Cotransfection of Nurr1 and the GTPCH-luciferase (luc) reporter increased the luc activity by about threefold in N2A cells. Additional analysis using 5'-deletions and mutants revealed that Nurr1 activates GTPCH transcription indirectly through the proximal promoter region, in the absence of the nerve growth factor-induced clone B (NGFI-B) responsive element-like sites, similarly, as recently reported for DA transporter regulation by Nurr1.

Reduced tyrosine hydroxylase and GTP cyclohydrolase mRNA expression, tyrosine hydroxylase activity, and associated neurochemical alterations in Nurr1-null heterozygous mice.

The nuclear receptor Nurr1 is essential for the development of midbrain dopamine neurons and appears to be an important regulator of dopamine levels as adult Nurr1-null heterozygous (+/-) mice have reduced mesolimbic/mesocortical dopamine levels. The mechanism(s) through which reduced Nurr1 expression affects dopamine levels has not been determined. Quantitative real-time PCR revealed a significant reduction in tyrosine hydroxylase (TH) and GTP cyclohydrolase (GTPCH) mRNA in ventral midbrain of +/- mice as compared to wild-type mice (+/+). The effect on TH expression was only observed at birth, while reduced GTP cyclohydrolase was also observed in the adult ventral tegemental area. No differences in dopamine transporter, vesicular monoamine transporter, dopamine D2 receptor or aromatic amino acid decarboxylase were observed. Since TH and GTPCH are both involved in dopamine synthesis, regulation of in vivo TH activity was measured in these mice. In vivo TH activity was reduced in nucleus accumbens and striatum of the +/- mice (24.7% and 15.7% reduction, respectively). In the striatum, gamma-butyrolactone exacerbated differences on +/- striatal TH activity (29.8% reduction) while haloperidol equalized TH activity between the +/+ and +/-. TH activity in the nucleus accumbens was significantly reduced in all conditions measured. Furthermore, dopamine levels in the striatum of +/- mice were significantly reduced after inhibition of dopamine synthesis or after haloperidol treatment but not under basal conditions while dopamine levels in the nucleus accumbens were reduced under basal conditions. Based on these data the +/- genotype results in changes in gene expression and impairs dopamine synthesis which can affect the maintenance of dopamine levels, although with differential effects between mesolimbic/mesocortical and nigrostriatal dopamine neurons. Together, these data suggest that Nurr1 may function to modify TH and GTPCH expression and dopamine synthesis.

Erythropoietin receptor signalling is required for normal brain development.

Erythropoietin, known for its role in erythroid differentiation, has been shown to be neuroprotective during brain ischaemia in adult animal models. Although high levels of erythropoietin receptor are produced in embryonic brain, the role of erythropoietin during brain development is uncertain. We now provide evidence that erythropoietin acts to stimulate neural progenitor cells and to prevent apoptosis in the embryonic brain. Mice lacking the erythropoietin receptor exhibit severe anaemia and defective cardiac development, and die at embryonic day 13.5 (E13.5). By E12.5, in addition to apoptosis in foetal liver, endocardium and myocardium, the erythropoietin receptor null mouse shows extensive apoptosis in foetal brain. Lack of erythropoietin receptor affects brain development as early as E10.5, resulting in a reduction in the number of neural progenitor cells and increased apoptosis. Corresponding in vitro cultures of cortical cells from Epor(-/-) mice also exhibited decreases in neuron generation compared with normal controls and increased sensitivity to low oxygen tension with no surviving neurons in Epor(-/-) cortical cultures after 24 hour exposure to hypoxia. The viability of primary Epor(+/+) rodent embryonic cortical neurons was further increased by erythropoietin stimulation. Exposure of these cultures to hypoxia induced erythropoietin expression and a tenfold increase in erythropoietin receptor expression, increased cell survival and decreased apoptosis. Cultures of neuronal progenitor cells also exhibited a proliferative response to erythropoietin stimulation. These data demonstrate that the neuroprotective activity of erythropoietin is observed as early as E10.5 in the developing brain, and that induction of erythropoietin and its receptor by hypoxia may contribute to selective cell survival in the brain.