NR4A Gene Expression Is Dynamically Regulated in the Ventral Tegmental Area Dopamine Neurons and Is Related to Expression of Dopamine Neurotransmission Genes

Abstract

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.

Chronic Methamphetamine Causes Differential Expression of Immediate Early Genes in the Nucleus Accumbens and Midbrain of Rats

“…This idea is consistent with the fact that chronic D2 receptor antagonism with haloperidol was shown to increase Nr4a1 in the NAc core but not in the NAc shell, dorsal striatum or cortex [50]. Moreover, blocking of D2 receptors with the selective antagonist, ectolopride, increased Nr4a1 and Nr4a3 but not Nr4a2 mRNa levels in the ventral tegmental area (VTA) [10]. When taken together, these reports suggest region-specific effects of stimulation of dopamine receptors and the signal cascades that regulate Nr4a family gene transcription and, possibly, their target genes.…”

“…Specifically, McCoy et al [30] had reported that chronic METH caused significant decreases in Nr4a1 levels, with acute METH challenge causing normalization of Nr4a1 levels. Since the repeated METH injections were identical to those used in the previous study [30], the differences in the two studies are probably due to differential molecular events that might regulate Nr4a responses in the NAc and dorsal striatum [10,12,28,50]. This idea is consistent with the fact that chronic D2 receptor antagonism with haloperidol was shown to increase Nr4a1 in the NAc core but not in the NAc shell, dorsal striatum or cortex [50].…”

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

“…This technology is also capable of isolating small areas of tissue such as discrete nuclei or regions within the brain as shown with the isolation of the ventral tegmental area. Previous publications have demonstrated this use in brain tissue as well as to isolate pathological tissue from normal tissue 5,11 .…”

“…Based on the standard curve generated, a total of 17.3, 24.8 pg and 50.9 pg/µl was isolated from 50, 100 and 200 dopamine neurons, respectively ( Figure 10A). To measure RNA quantity with Q-PCR, a range of concentrations of whole brain RNA and RNA from the dopamine neurons were reverse transcribed and the β-actin gene was measured using Q-PCR as previously described 5 . Based on these measurements, a concentration of 3.55, 6.82 and 20.58 pg/µl was calculated from 50, 100 and 200 dopamine neurons, respectively ( Figure 10B).…”

“…The earliest approaches included direct attachment of tissue on a slide using an infrared (IR) laser and a thermoplastic film and a non-contact method using an ultraviolet (UV) cutting laser to detach a cell or tissue and collection into a tube. Subsequently, LCM has been successfully used on a variety of tissues and cell types and shown to be compatible with isolation of various molecules for downstream analysis including RNA, microRNA, DNA, and proteins, including enzymatic activity 1,[4][5][6][7] . Here LCM is demonstrated using the a LCM system which uses a cutting UV laser and a capture IR laser for cutting around cells or tissue and attaching it to an LCM cap, respectively.…”