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.

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.

Bilateral quadriceps and hamstrings muscle volume asymmetries in healthy individuals.

Determining the magnitude of quadriceps and hamstring muscle volume asymmetries in healthy individuals is a critical first step toward interpreting asymmetries as compensatory or abnormal in pathological populations. The purpose of this study was to determine the magnitude of whole and individual muscle volume asymmetries, quantified as right-left volume differences, for the quadriceps and hamstring muscles in a young and healthy population. Twenty-one healthy individuals participated: Eleven females age = 22.6 ± 2.9 years and 10 males age = 23.2 ± 3.4 years. Whole muscle group and individual muscle volume asymmetries were quantified within the context of absolute measurement error using a 95% Limits of Agreement approach. Mean muscle asymmetries ranged from -3.0 to 6.0% for all individual and whole muscle groups. Whole muscle group 95% limits of agreements represented ±11.4% and ±8.8% volume asymmetries for the hamstrings and quadriceps, respectively. Individual muscle asymmetry 95% limits of agreements ranged from ∼ ± 11-13% for the vastii muscles while the biceps femoris short-head (±33.5%), long-head (±20.9%), and the rectus femoris (±21.4%) displayed the highest relative individual asymmetries. Individual muscle asymmetries exceeded absolute measurement error in 70% of all cases, with 26% of all cases exceeding 10% asymmetry. Although whole muscle group asymmetries appear to be near the 10% assumed clinical threshold of normality, the greater magnitude of individual muscle asymmetries highlights the subject- and muscle-specific variability in volume asymmetry. Future research is warranted to determine if volume asymmetry thresholds exist that discriminate between healthy and pathological populations. Statement of Clinical Significance: Muscle volume asymmetries displayed in healthy individuals provide a reference for interpreting asymmetries in pathological populations. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:963-970, 2018.