Transcriptional Regulation, Signaling Pathways, and Subcellular Localization of Corticotropin-Releasing Factor Receptors in the Central Nervous System.

Corticotropin-releasing factor (CRF) receptors CRF-R1 and CRF-R2 are differentially distributed in body tissues, and although they respond differentially to stimuli due to their association with different signaling pathways, both receptors have a fundamental role in the response and adaptation to stressful stimuli. Here, we summarize the reported data on different forms of CRF-R1 and CRF-R2 regulation as well as on their subcellular localization. Although the presence of R1 has been described at pre- and postsynaptic sites, R2 is mainly associated with postsynaptic densities. Different studies have provided valuable information on how these receptors regulate responses at a central level, elucidating different and sometimes synergistic roles in response to stress, but despite their high sequence identity, both receptors have been described to be differentially regulated both by their ligands and by transcriptional factors. To date, and from the point of view of their promoter sequences, it has not yet been reported how the different consensus sites identified in silico could be modulating the transcriptional regulation and expression of the receptors under different conditions, which strongly limits the full understanding of their differential functions, providing a wide field to increase and expand the study of the regulation and role of CRF receptors in the CRF system. SIGNIFICANCE STATEMENT: A large number of physiological functions related to the organization of the stress response in different body tissues are associated with the corticotropin-releasing factor system. This system also plays a relevant role in depression and anxiety disorders, as well as being a direct connection between stress and addiction. A better understanding of how the receptors of this system are regulated would help to expand the understanding of how these receptors respond differently to both drugs and stressful stimuli.

Type 1 Corticotropin-Releasing Factor Receptor Differentially Modulates Neurotransmitter Levels in the Nucleus Accumbens of Juvenile versus Adult Rats.

Adversity is particularly pernicious in early life, increasing the likelihood of developing psychiatric disorders in adulthood. Juvenile and adult rats exposed to social isolation show differences in anxiety-like behaviors and significant changes in dopamine (DA) neurotransmission in the nucleus accumbens (NAc). Brain response to stress is partly mediated by the corticotropin-releasing factor (CRF) system, composed of CRF and its two main receptors, CRF-R1 and CRF-R2. In the NAc shell of adult rats, CRF induces anxiety-like behavior and changes local DA balance. However, the role of CRF receptors in the control of neurotransmission in the NAc is not fully understood, nor is it known whether there are differences between life stages. Our previous data showed that infusion of a CRF-R1 antagonist into the NAc of juvenile rats increased DA levels in response to a depolarizing stimulus and decreased basal glutamate levels. To extend this analysis, we now evaluated the effect of a CRF-R1 antagonist infusion in the NAc of adult rats. Here, we describe that the opposite occurred in the NAc of adult compared to juvenile rats. Infusion of a CRF-R1 antagonist decreased DA and increased glutamate levels in response to a depolarizing stimulus. Furthermore, basal levels of DA, glutamate, and γ-Aminobutyric acid (GABA) were similar in juvenile animals compared to adults. CRF-R1 protein levels and localization were not different in juvenile compared to adult rats. Interestingly, we observed differences in the signaling pathways of CRF-R1 in the NAc of juveniles compared to adult rats. We propose that the function of CRF-R1 receptors is differentially modulated in the NAc according to life stage.

Social isolation of adolescent male rats increases anxiety and K+ -induced dopamine release in the nucleus accumbens: Role of CRF-R1.

Early life adversity can disrupt development leading to emotional and cognitive disorders. This study investigated the effects of social isolation after weaning on anxiety, body weight and locomotion, and on extracellular dopamine (DA) and glutamate (GLU) in the nucleus accumbens (NAc) and their modulation by corticotropin releasing factor receptor 1. On the day of weaning, male rats were housed singly or in groups for 10 consecutive days. Anxiety-like behaviors were assessed by an elevated plus maze (EPM) and an open field test (OF). Neurotransmitter levels were measured by in vivo microdialysis. Single-housed rats spent less time, and entered more, into the closed arms of an EPM than group-housed rats. They also spent less time in the center of an OF, weighed more and showed greater locomotion. In the NAc, no differences in CRF, or in basal extracellular DA or GLU between groups, were observed. A depolarizing stimulus increased DA release in both groups but to higher levels in isolated rats, whereas GLU increased only in single-housed rats. Blocking CRF-R1 receptors with CP-154,526 decreased DA release in single-housed but not in group-housed rats. The corticotropin releasing factor receptor type 1 receptor antagonist also decreased GLU in group-housed animals. These results show that isolating adolescent rats increases anxiety, body weight and ambulation, as well as the sensitivity of dopaminergic neurons to a depolarizing stimulus. This study provides further evidence of the detrimental effects of social isolation during early development and indicates that dysregulation of the CRF system in the NAc may contribute to the pathologies observed.

Discovery of a macromolecular complex mediating the hunger suppressive actions of cocaine: Structural and functional properties.

Cocaine not only increases brain dopamine levels but also activates the sigma1 receptor (σ1 R) that in turn regulates orexigenic receptor function. Identification of interactions involving dopamine D1 (D1 R), ghrelin (GHS-R1a ), and σ1 receptors have been addressed by biophysical techniques and a complementation approach using interfering peptides. The effect of cocaine on receptor functionality was assayed by measuring second messenger, cAMP and Ca2+ , levels. The effect of acute or chronic cocaine administration on receptor complex expression was assayed by in situ proximity ligation assay. In silico procedures were used for molecular model building. σ1 R KO mice were used for confirming involvement of this receptor. Upon identification of protomer interaction and receptor functionality, a unique structural model for the macromolecular complex formed by σ1 R, D1 R, and GHS-R1a is proposed. The functionality of the complex, able to couple to both Gs and Gq proteins, is affected by cocaine binding to the σ1 R, as confirmed using samples from σ1 R-/- mice. The expression of the macromolecular complex was differentially affected upon acute and chronic cocaine administration to rats. The constructed 3D model is consistent with biochemical, biophysical, and available structural data. The σ1 R, D1 R, and GHS-R1a complex constitutes a functional unit that is altered upon cocaine binding to the σ1 R. Remarkably, the heteromer can simultaneously couple to two G proteins, thus allowing dopamine to signal via Ca2+ and ghrelin via cAMP. The anorexic action of cocaine is mediated by such complex whose expression is higher after acute than after chronic administration regimens.

Cross-talk between dopamine D1 and corticotropin releasing factor type 2 receptors leads to occlusion of their ERK1/2 signaling.

One manner in which G protein-coupled receptors potentiate, increase, and change their functionality is through the formation of heteromers in a specific cellular context. Previously, we have shown that dopamine D1 receptor (D1R) and the corticotropin releasing factor receptor type-2α (CRF2α) heteromerize in HEK293T cells, enabling D1R to mobilize intracellular calcium in response to D1R agonists. In this study, we further investigated the pharmacological properties of the CRF2α-D1R heteromer and the consequences of the heteromerization in their signaling and subcellular localization when both receptors are co-expressed in HEK293T cells. Using immunoprecipitation assays, we observed that the addition of 10 µM dopamine in the incubation medium significantly decreased the amount of CRF2α on the cell surface of cells expressing both receptors. The presence of agonists of both receptors increased the interaction between CRF2α and D1R as assessed by co-immunoprecipitation. However, the presence of agonists of both receptors resulted in a lesser efficient activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase. Using a synaptosomal preparation of rat prefrontal cortex devoid of post-synaptic elements, we found that CRF2α and D1R co-localize in synaptic terminals of the rat medial prefrontal cortex and that the simultaneous activation of both receptors also occluded phosphorylation of extracellular signal-regulated kinase. These results strengthen the idea that the heteromer CRF2a-D1R is an entity functionally different from each receptor that composes it and suggests that its formation is enhanced by CRF and dopamine co-transmission, as occurs in stress and addiction.

Inhibitory Control of Basolateral Amygdalar Transmission to the Prefrontal Cortex by Local Corticotrophin Type 2 Receptor.

BACKGROUND: Basolateral amygdalar projections to the prefrontal cortex play a key role in modulating behavioral responses to stress stimuli. Among the different neuromodulators known to impact basolateral amygdalar-prefrontal cortex transmission, the corticotrophin releasing factor (CRF) is of particular interest because of its role in modulating anxiety and stress-associated behaviors. While CRF type 1 receptor (CRFR1) has been involved in prefrontal cortex functioning, the participation of CRF type 2 receptor (CRFR2) in basolateral amygdalar-prefrontal cortex synaptic transmission remains unclear. METHODS: Immunofluorescence anatomical studies using rat prefrontal cortex synaptosomes devoid of postsynaptic elements were performed in rats with intra basolateral amygdalar injection of biotinylated dextran amine. In vivo microdialysis and local field potential recordings were used to measure glutamate extracellular levels and changes in long-term potentiation in prefrontal cortex induced by basolateral amygdalar stimulation in the absence or presence of CRF receptor antagonists. RESULTS: We found evidence for the presynaptic expression of CRFR2 protein and mRNA in prefrontal cortex synaptic terminals originated from basolateral amygdalar. By means of microdialysis and electrophysiological recordings in combination with an intra-prefrontal cortex infusion of the CRFR2 antagonist antisauvagine-30, we were able to determine that CRFR2 is functionally positioned to limit the strength of basolateral amygdalar transmission to the prefrontal cortex through presynaptic inhibition of glutamate release. CONCLUSIONS: Our study shows for the first time to our knowledge that CRFR2 is expressed in basolateral amygdalar afferents projecting to the prefrontal cortex and exerts an inhibitory control of prefrontal cortex responses to basolateral amygdalar inputs. Thus, changes in CRFR2 signaling are likely to disrupt the functional connectivity of the basolateral amygdalar-prefrontal cortex pathway and associated behavioral responses.

CRF binding protein facilitates the presence of CRF type 2α receptor on the cell surface.

Corticotropin releasing factor binding protein (CRF-BP) was originally recognized as CRF sequestering protein. However, its differential subcellular localization in different brain nuclei suggests that CRF-BP may have additional functions. There is evidence that CRF-BP potentiates CRF and urocortin 1 actions through CRF type 2 receptors (CRF2R). CRF2R is a G protein-coupled receptor (GPCR) that is found mainly intracellularly as most GPCRs. The access of GPCRs to the cell surface is tightly regulated by escort proteins. We hypothesized that CRF-BP binds to CRF2R, exerting an escort protein role. We analyzed the colocalization of CRF-BP and CRF2R in cultured rat mesencephalic neurons, and the localization and interaction of heterologous expressed CRF-BP and CRF2αR in yeast, human embryonic kidney 293, and rat pheochromocytoma 12 cells. Our results showed that CRF-BP and CRF2R naturally colocalize in the neurites of cultured mesencephalic neurons. Heterologous expression of each protein showed that CRF-BP was localized mainly in secretory granules and CRF2αR in the endoplasmic reticulum. In contrast, CRF-BP and CRF2αR colocalized when both proteins are coexpressed. Here we show that CRF-BP physically interacts with the CRF2αR but not the CRF2ßR isoform, increasing CRF2αR on the cell surface. Thus, CRF-BP emerges as a GPCR escort protein increasing the understanding of GPCR trafficking.

Mechanisms of Kappa Opioid Receptor Potentiation of Dopamine D2 Receptor Function in Quinpirole-Induced Locomotor Sensitization in Rats.

Background: Increased locomotor activity in response to the same stimulus is an index of behavioral sensitization observed in preclinical models of drug addiction and compulsive behaviors. Repeated administration of quinpirole, a D2/D3 dopamine agonist, induces locomotor sensitization. This effect is potentiated and accelerated by co-administration of U69593, a kappa opioid receptor agonist. The mechanism underlying kappa opioid receptor potentiation of quinpirole-induced locomotor sensitization remains to be elucidated. Methods: Immunofluorescence anatomical studies were undertaken in mice brain slices and rat presynaptic synaptosomes to reveal kappa opioid receptor and D2R pre- and postsynaptic colocalization in the nucleus accumbens. Tonic and phasic dopamine release in the nucleus accumbens of rats repeatedly treated with U69593 and quinpirole was assessed by microdialysis and fast scan cyclic voltammetry. Results: Anatomical data show that kappa opioid receptor and D2R colocalize postsynaptically in medium spiny neurons of the nucleus accumbens and the highest presynaptic colocalization occurs on the same dopamine terminals. Significantly reduced dopamine levels were observed in quinpirole, and U69593-quinpirole treated rats, explaining sensitization of D2R. Presynaptic inhibition induced by kappa opioid receptor and D2R of electrically evoked dopamine release was faster in U69593-quinpirole compared with quinpirole-repeatedly treated rats. Conclusions: Pre- and postsynaptic colocalization of kappa opioid receptor and D2R supports a role for kappa opioid receptor potentiating both the D2R inhibitory autoreceptor function and the inhibitory action of D2R on efferent medium spiny neurons. Kappa opioid receptor co-activation accelerates D2R sensitization by contributing to decrease dopamine release in the nucleus accumbens.

Aminochrome induces dopaminergic neuronal dysfunction: a new animal model for Parkinson's disease.

L-Dopa continues to be the gold drug in Parkinson's disease (PD) treatment from 1967. The failure to translate successful results from preclinical to clinical studies can be explained by the use of preclinical models which do not reflect what happens in the disease since these induce a rapid and extensive degeneration; for example, MPTP induces a severe Parkinsonism in only 3 days in humans contrasting with the slow degeneration and progression of PD. This study presents a new anatomy and develops preclinical model based on aminochrome which induces a slow and progressive dysfunction of dopaminergic neurons. The unilateral injection of aminochrome into rat striatum resulted in (1) contralateral rotation when the animals are stimulated with apomorphine; (2) absence of significant loss of tyrosine hydroxylase-positive neuronal elements both in substantia nigra and striatum; (3) cell shrinkage; (4) significant reduction of dopamine release; (5) significant increase in GABA release; (6) significant decrease in the number of monoaminergic presynaptic vesicles; (7) significant increase of dopamine concentration inside of monoaminergic vesicles; (8) significant increase of damaged mitochondria; (9) significant decrease of ATP level in the striatum (10) significant decrease in basal and maximal mitochondrial respiration. These results suggest that aminochrome induces dysfunction of dopaminergic neurons where the contralateral behavior can be explained by aminochrome-induced ATP decrease required both for anterograde transport of synaptic vesicles and dopamine release. Aminochrome could be implemented as a new model neurotoxin to study Parkinson's disease.

Corticotropin-Releasing Factor Receptors and Their Interacting Proteins: Functional Consequences.

The corticotropin-releasing factor (CRF) system, which is involved in stress, addiction, and anxiety disorders such as depression, acts through G-protein-coupled receptors (GPCRs) known as type-1 and type-2 CRF receptors. The purpose of this review is to highlight recent advances in the interactions of CRF receptors with other GPCRs and non-GPCR proteins and their associated functional consequences. A better understanding of these interactions may generate new pharmacological alternatives for the treatment of addiction and stress-related disorders.

Corticotropin-releasing factor type-2 receptor and corticotropin-releasing factor-binding protein coexist in rat ventral tegmental area nerve terminals originated in the lateral hypothalamic area.

There is significant functional evidence showing that corticotropin-releasing factor type-2 receptor (CRF2R) and corticotropin-releasing factor-binding protein (CRF-BP) regulate glutamatergic synapses onto ventral tegmental area (VTA) dopaminergic neurons. It has been shown that CRF requires CRF-BP to potentiate N-methyl-D-aspartate receptors in dopaminergic neurons through CRF2R, and that increases glutamate release in cocaine-treated rats through the activation of CRF2R only by agonists with high affinity to CRF-BP. Furthermore, this CRF-mediated increase in VTA glutamate is responsible for stress-induced relapse to cocaine-seeking behaviour. However, there is a lack of anatomical evidence to explain the mechanisms of CRF actions in VTA. Thus, it was studied whether CRF2R and CRF-BP are expressed in VTA nerve terminals, using a synaptosomal preparation devoid of postsynaptic elements. The current results show that both proteins are co-expressed in glutamatergic and γ-aminobutyric acid (GABA)ergic VTA synaptosomes. A main glutamatergic input to the VTA that has been associated to addictive behaviour is originated in the lateral hypothalamic area (LHA). Thus, this study was focused in the LHA-VTA input using orexin as a marker of this input. The results show that CRF2R and CRF-BP mRNA and protein are expressed in the LHA, and that both proteins are present in orexin-positive VTA synaptosomes. The results showing that CRF2R and CRF-BP are expressed in the LHA-VTA input give anatomical support to suggest that this input plays a role in stress-induced relapse to cocaine-seeking behaviour.

A comprehensive survey of non-canonical splice sites in the human transcriptome.

We uncovered the diversity of non-canonical splice sites at the human transcriptome using deep transcriptome profiling. We mapped a total of 3.7 billion human RNA-seq reads and developed a set of stringent filters to avoid false non-canonical splice site detections. We identified 184 splice sites with non-canonical dinucleotides and U2/U12-like consensus sequences. We selected 10 of the herein identified U2/U12-like non-canonical splice site events and successfully validated 9 of them via reverse transcriptase-polymerase chain reaction and Sanger sequencing. Analyses of the 184 U2/U12-like non-canonical splice sites indicate that 51% of them are not annotated in GENCODE. In addition, 28% of them are conserved in mouse and 76% are involved in alternative splicing events, some of them with tissue-specific alternative splicing patterns. Interestingly, our analysis identified some U2/U12-like non-canonical splice sites that are converted into canonical splice sites by RNA A-to-I editing. Moreover, the U2/U12-like non-canonical splice sites have a differential distribution of splicing regulatory sequences, which may contribute to their recognition and regulation. Our analysis provides a high-confidence group of U2/U12-like non-canonical splice sites, which exhibit distinctive features among the total human splice sites.

Reduced dopamine and glutamate neurotransmission in the nucleus accumbens of quinpirole-sensitized rats hints at inhibitory D2 autoreceptor function.

Dopamine from the ventral tegmental area and glutamate from several brain nuclei converge in the nucleus accumbens (NAc) to drive motivated behaviors. Repeated activation of D2 receptors with quinpirole (QNP) induces locomotor sensitization and compulsive behaviors, but the mechanisms are unknown. In this study, in vivo microdialysis and fast scan cyclic voltammetry in adult anesthetized rats were used to investigate the effect of repeated QNP on dopamine and glutamate neurotransmission within the NAc. Following eight injections of QNP, a significant decrease in phasic and tonic dopamine release was observed in rats that displayed locomotor sensitization. Either a systemic injection or the infusion of QNP into the NAc decreased dopamine release, and the extent of this effect was similar in QNP-sensitized and control rats, indicating that inhibitory D2 autoreceptor function is maintained despite repeated activation of D2 receptors and decreased dopamine extracellular levels. Basal extracellular levels of glutamate in the NAc were also significantly lower in QNP-treated rats than in controls. Moreover, the increase in NAc glutamate release induced by direct stimulation of medial prefrontal cortex was significantly lower in QNP-sensitized rats. Together, these results indicate that repeated activation of D2 receptors disconnects NAc from medial prefrontal cortex and ventral tegmental area. Repeated administration of the dopamine D2 receptor agonist quinpirole (QNP) induces locomotor sensitization. We found that the NAc of QNP-sensitized rats has reduced glutamate levels coming from prefrontal cortex together with a decreased phasic and tonic dopamine neurotransmission but a conserved presynaptic D2 receptor function. We suggest that locomotor sensitization is because of increased affinity state of D2 post-synaptic receptors.

Exposure to repeated immobilization stress inhibits cocaine-induced increase in dopamine extracellular levels in the rat ventral tegmental area.

A higher vulnerability to drug abuse has been observed in human studies of individuals exposed to chronic or persistent stress, as well as in animal models of drug abuse. Here, we explored the effect of repeated immobilization stress on cocaine-induced increase in dopamine extracellular levels in VTA and its regulation by corticotropin-releasing factor (CRF) and GABA systems. Cocaine (10mg/Kg i.p.) induced an increase of VTA DA extracellular levels in control rats. However, this effect was not observed in repeated stress rats. Considering the evidence relating stress with CRF, we decided to perfuse CRF and CP-154526 (selective antagonist of CRF1 receptor) in the VTA of control and repeated stress rats, respectively. We observed that perfusion of 20µM CRF inhibited the increase of VTA DA extracellular levels induced by cocaine in control rats. Interestingly, we observed that in the presence of 10µM CP-154526, cocaine induced a significant increase of VTA DA extracellular levels in repeated stress rats. Regarding the role of VTA GABA neurotransmission, cocaine administration induced a significant increase in VTA GABA extracellular levels only in repeated stress rats. Consistently, cocaine was able to increase VTA DA extracellular levels in repeated stress rats when 100µM bicuculline, an antagonist of GABAA receptor, was perfused intra VTA. Thus, both CRF and GABA systems are involved in the lack of response to cocaine in the VTA of repeated stress rats. It is tempting to suggest that the loss of response in VTA dopaminergic neurons to cocaine, after repeated stress, is due to an interaction between CRF and GABA systems.

Withdrawal from chronic amphetamine reduces dopamine transmission in the rat lateral septum.

The lateral septum (LS) is a brain nucleus implicated in the addictive process. This study investigated whether withdrawal from chronic amphetamine (AMPH) induces alterations in dopamine (DA) transmission within the LS. Male Sprague-Dawley rats were injected with AMPH (2.5 mg/kg i.p.) or saline during 14 days and thereafter subjected to 24 hr or 14 days of withdrawal. After these withdrawal periods, we measured DA extracellular levels by in vivo microdialysis, DA tissue levels, and tyrosine hydroxylase (TH) and vesicular monoamine transporter-2 (VMAT2) expression in the LS. Our results showed a significant decrease in K(+) -induced release of DA in the LS of AMPH-treated rats, 14 days after withdrawal compared with saline-treated rats. There were no significant differences in DA tissue content and TH expression. Interestingly, there was a decrease of LS VMAT2 expression in AMPH-treated rats, 14 days after withdrawal compared with saline-treated rats. This is the first neurochemical evidence showing that withdrawal from repeated AMPH administration decreases K(+) -induced DA release in the rat LS. Our results suggest that this decrease in DA releasability could be due to a decrease in DA vesicular uptake. The possibility that these neurochemical changes are associated with AMPH abstinence syndrome should be further explored.

Corticotropin-releasing factor system in the lateral septum: Implications in the pathophysiology of obesity.

Obesity is a pandemic associated with lifestyles changes. These include excess intake of obesogenic foods and decreased physical activity. Brain areas, like the lateral hypothalamus (LH), ventral tegmental area (VTA), and nucleus accumbens (NAcc) have been linked in both homeostatic and hedonic control of feeding in experimental models of diet-induced obesity. Interestingly, these control systems are regulated by the lateral septum (LS), a relay of γ-aminobutyric (GABA) acid neurons (GABAergic neurons) that inhibit the LH and GABAergic interneurons of the VTA. Furthermore, the LS has a diverse receptor population for neurotransmitters and neuropeptides such as dopamine, glutamate, GABA and corticotropin-releasing factor (CRF), among others. Particularly, CRF a key player in the stress response, has been related to the development of overweight and obesity. Moreover, evidence shows that LS neurons neurophysiologically regulate reward and stress, although there is little evidence of LS taking part in homeostatic and hedonic feeding. In this review, we discuss the evidence that supports the role of LS and CRF on feeding, and how alterations in this system contribute to weight gain obesity.

Reactive oxygen species modulate locomotor activity and dopamine extracellular levels induced by amphetamine in rats.

The increase of dopamine (DA) in the reward system is related to the reinforcing effects of drugs of abuse and hyper locomotion induced by psychostimulants. The increase of DA induced by drugs of abuse generates high amounts of ROS by monoamines metabolization. It has been showed that ROS could modulate psychomotor response and reinforcing effects induced by drugs of abuse as cocaine and methamphetamine (METH). The aim of this study is to evaluate the relation of ROS and amphetamine (AMPH). Here, we show that pretreatment of the ROS scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) attenuates the induction of locomotion and oxidative stress generated in nucleus accumbens (Nac) by acute AMPH administration. Interestingly, TEMPOL also attenuates the increase of DA induced by AMPH in Nac. Finally, TEMPOL reduces DAT phosphorylation when AMPH is co-infused in Nac synaptosomes. Taking together, our results suggest that ROS modulate AMPH effects in rats.

Nurr1 regulates RET expression in dopamine neurons of adult rat midbrain.

Genesis of midbrain dopamine (DA) neurons depends on Nurr1, a nuclear receptor expressed during development and adulthood in these neurons. Nurr1 is required for the expression of genes of dopaminergic phenotype such as tyrosine hydroxylase and DA transporter. The expression of the tyrosine kinase receptor RET also depends on Nurr1 during development. However, it is unknown whether RET expression is regulated by Nurr1 during adulthood, and the mechanism by which Nurr1 regulates RET expression. Using an adeno-associated vector-delivered anti-Nurr1 ribozyme, we knocked-down Nurr1 expression unilaterally in the substantia nigra (SN) of adult rats. Animals injected with the ribozyme displayed a 57.3% decrease in Nurr1 mRNA in the SN accompanied by decreased DA extracellular levels in the striatum. RET mRNA in the injected SN and RET protein in the ipsilateral striatum decreased 76.9% and 47%, respectively. Tyrosine hydroxylase and DA transporter mRNA did not change in Nurr1 knocked-down SN. Nurr1 induced the transcription of the human RET promoter in cell type and concentration-dependent manner. Nurr1 induction of RET promoter is independent of NBRE elements. These results show that the expression of RET in rat adult SN is regulated by Nurr1 and suggest that RET is a transcriptional target of this nuclear receptor.

Activation of GABA-B receptors induced by systemic amphetamine abolishes dopamine release in the rat lateral septum.

The lateral septum is a brain nucleus involved in various mental disorders such as anxiety and drug addiction. In the present study, we investigated whether systemic amphetamine, known to release dopamine (DA) in nucleus accumbens, will also release DA in lateral septum. Our results show that systemic amphetamine administration (2 mg/kg i.p.) induced a significant increase in DA extracellular levels in nucleus accumbens but not in lateral septum. Interestingly, intralateral septum perfusion of amphetamine through the microdialysis probe induced a significant increase in DA extracellular levels. To test if GABAergic neurotransmission in lateral septum was responsible for inhibiting the release of DA when amphetamine was administered systemically, we perfused a GABA-B selective antagonist (CGP-52432) intra lateral septum. Systemic amphetamine administration induced a significant increase in lateral septum DA release when CGP-52432 was concomitantly superfused. Our results indicate that the systemic administration of amphetamine induces an increase in lateral septum GABA release and the consequent activation of GABA-B receptors counteracting the direct effect of amphetamine on lateral septum DA release.

Repeated treatment with the κ-opioid agonist U-69593 increases K+-stimulated dopamine release in the rat medial prefrontal cortex.

Acute activation of κ-opioid receptors (KOR) decreases dopamine (DA) extracellular levels in both the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAc). Also, the acute activation of KOR prevents alterations in behavior and neurochemistry occurring after repeated use of psychostimulants. Opposing to the acute effects, repeated administration of the KOR agonist, U-6593, potentiates both high-potassium and amphetamine induced DA release in the NAc, suggesting that repeated activation of KOR sensitizes mesolimbic dopaminergic neurotransmission. This study investigated the effect of repeated treatment with U-69593 on basal and stimulated DA and serotonin (5HT) extracellular levels in the rat mPFC. Rats were injected once daily with U-69593 (0.16-0.32 mg/kg) or vehicle for 4 days. One day after the last injection, microdialysis experiments assessing DA and 5HT extracellular levels in mPFC were conduced. The repeated treatment with U-69593 significantly augmented potassium-stimulated DA extracellular levels, without affecting potassium-stimulated 5HT extracellular levels, suggesting an increase in DA releasability.