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The effects of neuropeptide urocortin 2 on the spontaneous discharge and glutamatergic neurotransmission of striatum neurons
ARTICLE IN PRESS Neuropeptides ■■ (2015) ■■–■■
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Neuropeptides j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / n p e p
The effects of neuropeptide urocortin 2 on the spontaneous discharge and glutamatergic neurotransmission of striatum neurons Chunna Liu a,*,1, Xinyu Liu b,1, Feiran Song c, Jian Li b, Xia Zhang a, Jing Yang a a b c
Department of Pharmacology, Liaoning Medical University, JinZhou 121001, China The First Affiliated Hospital of Liaoning Medical University, JinZhou 121001, China The China Medical University, ShenYang 110001, China
A R T I C L E
I N F O
Article history: Received 30 July 2014 Accepted 2 March 2015 Available online Keywords: Urocortin Neuropeptide Striatum Glutamate Spontaneous discharge Coticortropin-releasing factor
A B S T R A C T
The primary cause of the neurodegenerative process that underlies Parkinson’s disease (PD) is still unknown. Different mechanisms probably contribute to triggering neuronal death in the nigro-striatum pathway. The neuropeptide urocortin 2 (UCN2) plays an important role in the regulation of striatum (STR) neurons projection. We investigated the effects of UCN2 on spontaneous discharge and glutamatergic responses in STR for a better understanding of the pathogenesis of PD. The experiment used microiontophoresis method to observe the effects of UCN2 on STR neurons’ firing rates in vivo. Corticotrophin releasing factor receptor 2 (CRF-R2) selective inhibitor, astressin-2B (AST-2B), was administered simultaneously with UCN2 to investigate the effects of UCN2 on CRF-R2. Moreover, we further explored the effects of UCN2 on glutamatergic responses in STR neurons. We found that UCN2 could significantly inhibit the firing rate of 84% of the tested STR neurons, and its inhibitory effect followed a concentration-dependent manner. During the microiontophoresis of GLU, the excitatory firing of glutamatergic neurons could be attenuated by the addition of UCN2, but enhanced by the application of AST-2B. The results suggest that UCN2 could regulate the effects of STR neurotransmitters (GLU) via CRF-R2 and may thereby contribute to the improvement of PD. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Urocortins (UCNs) are isolated acid neuro-active peptides related to hypothalamic corticotrophin-releasing hormone (CRF) and binds with high affinity to corticotrophin-releasing hormone receptor2β, which belongs to the hypothalamic CRF family (Bale and Vale, 2004; Brar et al., 2000). Results from immunological assay have suggested that UCN mRNA was expressed in multiple parts of the brain, including the diencephalon, hypothalamic regions, the pituitary, the anterior frontal cortex, the frontal, the midbrain and the brainstem (Bagosi et al., 2014). The densest projections were also found in the intermediate part of the lateral septum, posterior division of the bed nucleus, stria terminalis, and the medial nucleus of the amygdala (Li et al., 2002). UCN had three forms-UCN1, UCN2 and UCN3. CRF receptors included two major subtypes, CRFR1 and CRF-R2. CRFR1 had a strong affinity for CRF and UCN1 peptides while UCN2 and UCN3 only showed specific binding property to CRF-R2 in rats (Nemoto et al., 2007). UCN was similar to CRF in biological
* Corresponding author. Department of Pharmacology, Liaoning Medical University, No. 40, Section 3, Songpo Road, Jinzhou City, Liaoning 121001, PR China. E-mail address: [email protected] (C. Liu). 1 These authors contributed equally to this work.
activity and, through the hypothalamus–adenohypophysis–adrenal axis, it could promote the adenohypophysis adrenal cortical hormone secretion (Riester et al., 2012). UCN not only regulated emergency response and stress in the peripheral tissues, but also participated in some functional changes in the central nervous system, including the modulation of appetite, intension, worry and anxiety, the inhibition of scald edema and the regulation of electrolyte and water balances. It has been used as an anti-epileptic drug (Cottone et al., 2007). UCN has also been reported to exert protective effects on Parkinson’s disease (PD) (Huo et al., 2012; Rivier, 2008). UCN2, a member of the corticotropin-releasing hormone family, was involved in the regulation of stress-related behaviors in rodents. It was reported (Breu et al., 2012) that male UCN2 null mice showed more passive social interactions and reduced the aggressiveness in comparison to wild-type animals. UCN2 seemed to modulate aggressive behavior in male mice. In a previous research (Hale et al., 2010), intracerebroventricular injection of UCN2 could increase c-Fos expression in serotonergic neurons in the dorsal and caudal parts of the dorsal raphe nucleus, including both subsets of serotonergic neurons that project and do not project to the ventricle/ periventricular system. Accumulative evidence showed that UCN2 was involved in the physiological activities and pathological processes in STR nucleus, which was the essential nucleus of PD pathogenesis. However, the detailed mechanism of PD remains to
http://dx.doi.org/10.1016/j.npep.2015.03.001 0143-4179/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Chunna Liu, et al., The effects of neuropeptide urocortin 2 on the spontaneous discharge and glutamatergic neurotransmission of striatum neurons, Neuropeptides (2015), doi: 10.1016/j.npep.2015.03.001
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not be elucidated. Several hypothesis supported different pathophysiological mechanisms related to oxidative stress, glutamatemediated neurotoxicity, mitochondrial energetic impairment and apoptosis (Boje, 2004). Glutamate is the predominantly fast excitatory neurotransmitter in the central nervous system and, in the presence of specific conditions, a potential neurotoxin, Glutamate mediated excitotoxicity, is a further contributor for the development in damage of the nigrostriatal that characterizes PD (Blandini, 2010). Glutamate-mediated striatal sensitization subsequently modifies the basal ganglia output in ways that favor the appearance of parkinsonian motor complications (Oh and Chase, 2002). However, it is still unclear whether UCN2 can influence basal ganglia neuronal discharge and regulate the neurotransmitters transmission, and if so, the molecular mechanism underlying its effectiveness. The present study investigates the effects of UCN2 on the STR neurons for a further comprehension on the pathogenesis of PD. At the same time, this study aims to add depth to the diagnosis and treatment of neurons having paradoxical discharge disease and seizure disorders. We used microiontophoresis to observe the effects of UCN2 and AST-2B on STR neurons’ spontaneous discharge. Moreover, the effects of UCN on Glutamatergic STR neurons (Xue et al., 2014) were also investigated in order to ascertain whether UCN do not only exist in the STR neuron, but also regulates neurotransmission and inhibits the glutamatergic exitotoxicity. 2. Materials and methods 2.1. Experimental animals Animal care and experimental protocols were treated in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and approved by the Ethics Committee of Liaoning Medical University for the Use of Experimental Animals for Research and Teaching. Eighty male Sprague-Dawley rats (weight, 180–220 g; age, 10–13 weeks) were used. 2.2. Drugs/reagents UCN2 (rats) and AST-2B were obtained from Tocris Bioscience Company, soluble to 1 mg/ml in water. Solutions were made by adding solvent directly to the vial. The vial should then be vortexed vigorously to ensure the product has completely dissolved. L-glutamic acid (GLU), MK-801 and other drugs were purchased from Sigma. Distilled-water was the vehicle in which GLU and MK-801 were dissolved as concentrated stock solutions. All drugs were diluted to their final concentrations in the bath solution immediately prior to commencing the experiments. 2.3. Methods The rats were anesthetized with intraperitoneal urethane (1g kg−1) and mounted in a stereotaxic apparatus. An incision was made in the scalp to expose the skull and a hole was drilled above the STR. For extracellular recording and microiontophoresis, seven barrel micropipettes were used, which were made with 51217 type microelectrode puller (these micropipettes were fabricated with their filling ends bent away centrifugally to avoid solutions’ contamination among the barrels with a tip diameter of around 4–8 μm). The central pipette was filled with 1% pontamine sky blue in 3.0 mol L−1 NaCl and served as the leading electrode with resistance of 5–12 MΩ (Gill et al., 2007; Liu et al., 2005). The other peripheral tubes (resistance of 20–100 MΩ) were filled with the drugs as follows: UCN2 (10−5 mol·L−1, 10−6 mol·L−1, 10−7 mol·L−1 and 10−8 mol·L−1, pH 7.5); AST-2B (CRF-2 blocker, 10−6 mol·L−1, pH 7.5); GLU (1.0 mol·L−1, pH 8.0), (±)-5-methyl-10,11-dihydro-5-H-dibenzo [a,d] cyclohepten-5,10iminehy drochloride (MK-801, 10−2 mol·L−1, pH 4.0), respectively.
According to the Paxino rat brain atlas for STR orientation, the coordinate was −0.8 mm to 1.2 mm from bregma, 2.5–3.5 mm lateral from midline and 5.0–6.0 mm below the brain surface (Liu et al., 2005). The electrode was placed into the striatum nucleus with the microelectrode pusher. The electrical signal was amplified and displayed on a storage oscilloscope and then sent to a computer system. To generate the spontaneous firing histograms, the STR neurons’ spontaneous firing rates were recorded for 2–3 min before any drug ejection was applied. The microiontophoretic apparatus was used to eject UCN2, AST-2B, GLU, and MK-801, respectively. All the drugs except GLU were ejected with cationic currents using the Neuro phore BH-2 system (Medical System Corp, Greenvale, NY, USA), and retaining currents of negative 5−10 nA were applied between ejections to prevent drug leakage. The effects of UCN2 and AST-2B on the STR neurons were observed. Subsequently, the effects of UCN2 and AST-2B on the firing rates in the presence of GLU and MK-801 were also investigated. The 0.165 mol·L−1 NaCl pipette was used as a current sink and a drug control for 10 s. In order to eliminate the possible electrical effects caused by microiontophoretic pulses, data were discarded if current ejection from this pipette elicited any changes in the spontaneous discharge. Administration must be done after the discharge induced by the previous administration did not change any more. Likewise, excessive microiontophoretic currents of more than 100 nA were never used. At the end of each experiment, the last recording site was marked by microiontophoretic ejection of pontamine sky blue through the central pipette (−20μA, 20 min). The rats were given an overdose of urethane and perfused transcardially with 0.9% normal saline followed by 4% paraformaldehyde (distilled in 0.1 mol·L−1 phosphatebuffered saline, pH 7.4) for 15–20 min. The brains were frozen at −40 °C and then cut into 30 μm slices. The recording site was verified by histological examination (Huo et al., 2012; Liu et al., 2005). 2.4. Statistical analysis The effects of drugs were determined using the following equation: Effects = (firing rate after treatment (20s) – firing rate before treatment (20s)/firing rate before treatment (20s) × 100%. Results were presented as means ± SD (standard deviation). Student’s t-test and one-way ANOVA test were used to evaluate the drug effects. 3. Results A total of 85 neurons in the STR were observed. The spontaneous firing rates of STR neurons were relatively slow and irregular, and varied from 0 to 10 Hz (6.84 ± 1.21 Hz). The waveforms were characteristics of medium spiny neurons. Burst firing were seen occasionally. 3.1. Effects of UCN2 and AST-2B on the STR neuronal activity A total of 58 STR neurons were tested in this experiment. After application of UCN2 (10−6 mol·L−1, 10−80 nA, 10 s), 84% (49/58) of the tested STR neurons showed a significant decrease in their firing rates compared to the basal level, and the neuronal average firing rates decreased from 6.98 ± 1.05 Hz to 3.93 ± 1.02 Hz (n = 58, P < 0.01). The effects of UCN2 had a latent period (n = 58, 14.56 ± 3.37s) and an obvious after-effect, which induced explosive firing rates (Fig 1a–c). Application of UCN2 microiontophoretically resulted in a decreased firing rate in an intensity-dependent manner. (Fig 2a–c). As the concentration of UCN2 (10−8 mol·L−1, 10−7 mol·L−1, 10−6 mol·L−1 and 10−5 mol·L−1, 20 nA, 10 s) increased, the neuronal firing rates were attenuated. On the other hand, 8 neurons had no responses to various levels of UCN2 and 1 neuron’s firing rates was even increased (n = 34, P < 0.01).
Please cite this article in press as: Chunna Liu, et al., The effects of neuropeptide urocortin 2 on the spontaneous discharge and glutamatergic neurotransmission of striatum neurons, Neuropeptides (2015), doi: 10.1016/j.npep.2015.03.001
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the neuronal average firing rates increased from 6.96 ± 0.93 Hz to 15.09 ± 1.28 Hz. After addition of the NMDA receptor’s blocker, MK801 (0.01 mol·L−1, 20 nA for 20 s), the spontaneous firing rates were inhibited, the neuronal firing rates decreased from 15.09 ± 1.28 Hz to 5.94 ± 1.20 Hz (n = 26, P < 0.01), and MK-801 had a significant aftereffect. The STR neuronal firing rates, which were already inhibited by MK-801, reduced the rates to 4.12 ± 0.76 Hz (n = 26, P < 0.05) by UCN2 application. In the presence of GLU for a period of 100 s, together administrated with UCN2 (20 nA, 20 s) from the 20th second to the 40th second resulted in the firing rates being significantly decreased in 73% (19/26) tested GLU neurons (compared to GLU application alone). The firing rates enhanced by GLU were decreased by UCN2 from 15.07 ± 1.97 Hz to 9.98 ± 2.04 Hz (P < 0.01). 27%
Fig. 1. (a) Effects of UCN2 (10−6 mol·L−1, 10-80 nA, 10 s) and AST-2B on striatum neuron. Values are mean ± SD for n = 58 neurons in UCN group and n = 52 neurons in AST-2B group, 80 male Sprague-Dawley rats were used. *vs Normal group, *P < 0.05, **P < 0.01; #vs UCN group, # P < 0.05, ##P < 0.01. (b) Effects of UCN2 on striatum neuron spontaneous firing rates. AST-2B was administrated during the period of UCN2. (c) The real-time recording wave of one striatum neuron.
After addition of the CRF-R2 antagonist, AST-2B (10−6 mol·L−1, 20 nA, 10 s), the spontaneous firing rates were enhanced in 78% (41/ 52) of the tested STR neurons, with the neuronal firing rates increased from 5.49 ± 1.08 Hz to 8.01 ± 1.13 Hz (n = 52, P < 0.01). The effects of AST-2B were fast without a latent period or after-effect. Moreover, in the presence of UCN2 (10−6 mol·L−1, 20 nA, 20 s), microiontophoretic application of AST-2B (20 nA, 10 s) reversed UCN2’s inhibitory effects and enhanced the firing rates in 34 out of 36 STR neurons. As demonstrated in Fig. 1b and c, the STR neuronal firing rates increased from 2.78 ± 1.47 Hz to 5.82 ± 1.12 Hz in the presence of AST-2B (n = 34, P < 0.01). 3.2. Influences of UCN2 and AST-2B on glutamatergic firing action After application of GLU (20 nA, 20 s), 26 of STR neurons showed a significant increase in their firing rates compared to the basal level,
Fig. 2. (a) The intensity-dependence of UCN2 (10 −7 mol·L −1 , 10 −6 mol·L −1 and 10−5 mol·L−1, 20 nA, 10 s) on the STR neurons. Values are mean ± SD for n = 34 neurons.*vs Normal group; #vs UCN2 10−8 mol·L−1 group; Δvs UCN2 10−7 mol·L−1 group; □vs UCN2 10−6 mol·L−1 group.*P < 0.05, **P < 0.01; #P < 0.05, ##P < 0.01; ΔP < 0.05, ΔΔP < 0.01; □P < 0.05, □□P < 0.01. (b) The intensity-dependent effects of UCN2 on STR neuron spontaneous firing rates. (c) The real-time recording wave of UCN2 effect on one striatum neuron.
Please cite this article in press as: Chunna Liu, et al., The effects of neuropeptide urocortin 2 on the spontaneous discharge and glutamatergic neurotransmission of striatum neurons, Neuropeptides (2015), doi: 10.1016/j.npep.2015.03.001
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Fig. 3. (a) Effects of UCN2 (10−6 mol·L−1, 20 nA, 20 s) and AST-2B on striatum neuron. Values are mean ± SD for n = 26 neurons. *vs Normal group; #vs GLU group. *P < 0.05, **P < 0.01; #P < 0.05, ##P < 0.01. b. Effects of UCN2 on striatum’s glutamatergic neuron spontaneous firing rates. MK-801 10−2 mol·L−1 could inhibit striatum neuron spontaneous firing rates, and the UCN2 and AST-2B were administrated during the microiontophoresis of GLU. (c) The real-time recording wave of UCN2 effect on striatum’s glutamatergic neuron.
(7/26) neurons had no response. At the 60th second, subsequent application of AST-2B (20 nA, 20 s) caused an increase in the firing rates in 58% (15/26) tested GLU neurons (compared to GLU application alone). The STR neuronal firing rates, which were already increased by GLU, increased further from 14.91 ± 2.40 Hz to 17.83 ± 2.09 Hz (n = 26, P < 0.05) by AST-2B application (Fig 3a–c). 4. Discussion The present study found that micro-electrophoresis of UCN2 could significantly reduce the discharge rate of STR neurons. This suggested that UCN2 caused inhibitory effects on STR neurons. Microelectrophoresis AST-2B alone could increase the discharge rate of the
tested STR neurons, indicating that CRF-R2 was expressed in STR neurons. It was further demonstrated that the inhibitory effects induced by UCN2 could be antagonized by CRF-R2 antagonist – AST-2B. These results further suggested that UCN2 exerted inhibitory effects on STR neurons via CRF2 receptor. Interaction was observed between UCN2 and glutamatergic neurons. It was also found that glutamatergic projected neuron fibers had an excitatory effect on STR neurons, which could be antagonized by UCN2 and synergized by AST-2B. These findings suggested that UCN2, when combined with CRF-R2 in STR neurons, can antagonize the exitotoxicity of glutamatergic neurons. The experiment further indicated that UCN2 played important regulatory effects on STR neurons and its neurotransmitters. UCN is a newly discovered neuro-endocrinal active peptide (Liu et al., 2005; Rademaker et al., 2007). As a small molecular active peptide, UCN confers protective effects via autocrine and/or paracrine pathways (Bale and Vale, 2004; Jamieson et al., 2006). Researches showed that UCN exerted anti-anxiety (Alsiö et al., 2009), anti-aging (Gill et al., 2007) and neuroprotective effects on damaged brains and relieved the damages of drug addiction (Liew et al., 2012; McGinty et al., 2010). Most researchers reported that UCN bound with CRF-R2 promoted cAMP increase, thus activating protein kinase. Brar found that mitogen activated protein kinase and phosphatidylinositol 3-kinase pathways formed important protective mechanisms of UCN (Bale and Vale, 2004; Brar et al., 2000; Rademaker et al., 2007). Physiological responses to stress coordinated by the hypothalamo– pituitary –adrenal axis were concerned with maintaining homeostasis in the presence of real or perceived challenges. Regulators of this axis were CRF and CRF related neuropeptides, including UCNs 1, 2, and 3. They mediated their actions by binding to CRF R1 and CRF R2, which were located in several stress-related brain regions (Janssen and Kozicz, 2013). Based on data obtained in animal studies, neuropeptides and their receptors might be targeted by new candidate neuropharmacons with the hope that they will become important and effective tools in the management of stress related mood disorders (Kormos and Gaszner, 2013). In a recent report (Johnson et al., 2013), rats treated with three daily UCN injections into the basolateral amygdala develop prolonged anxiety-associated behavior and vulnerability to panic-like physiological responses (i.e., tachycardia, hypertension and tachypnea) following intravenous infusions of 0.5 M sodium lactate (NaLac, an ordinarily mild interoceptive stressor). Among cultured neurons, UCN could protect hippocampal neurons, γ-aminobutyric acid (GABA) neurons and dopaminergic (DA) neurons (Brawner et al., 2008). Previous data (Skórzewska et al., 2011) suggested a role of UCN2 in the behavioral and immunocytochemical responses to stress, in which it strengthens the measures of anxiety-like responses. In addition, as a CRH-related peptide, UCN could protect the neurons of PD rat models by apomorphine, reverse the PD symptoms, and increase the level of GABA, DA and serotonin (TH) in nigra-striatum (Colosimo, 2011; Zhou et al., 2011). In a similar and consistent manner with the preservation of motor function, UCN protect the nigra from both DA depletion and loss of TH activity, indicating preservation of DA cells. UCN reversed key features of nigrostriatal damage in the hemi-parkinsonian 6-hydroxydopamine lesioned rat (Abuirmeileh et al., 2007a, 2007b). It was also suggested that UCN served to maintain sufficient functions of nigra-striatum neurons by influencing CRF receptors (Bale and Vale, 2004; Colosimo et al., 2010). These findings were of great potential significance in the treatment of PD induced by the degeneration of nigra-striatum neurons (Ziemssen et al., 2011). Our experiments suggested that in the pathological condition of PD or epilepsy, UCN2 might inhibit high frequency discharge of STR neurons and suppress GLU neuronal toxicity, thereby constraining the occurrence and development of the disease. Our data constitute the first report which demonstrated that UCN2 has inhibitory effects on STR neuron and mediates neuroprotection, thus indicating its potential therapeutic value in PD.
Please cite this article in press as: Chunna Liu, et al., The effects of neuropeptide urocortin 2 on the spontaneous discharge and glutamatergic neurotransmission of striatum neurons, Neuropeptides (2015), doi: 10.1016/j.npep.2015.03.001
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5. Conclusions The experiment showed that UCN2 could inhibit the discharge activity of STR neurons and reduce the excitation of glutamatergic neurons. UCN2 can modulate STR neurotransmitters via binding to CRF R2. Acknowledgments We thank Prof. Dongming Gao for expert technical assistance. This work was supported by the National Natural Science Foundation of China (81201037). References Abuirmeileh, A., Harkavyi, A., Lever, R., et al., 2007a. Urocortin, a CRF-like peptide, restores key indicators of damage in the substantia nigra in a neuroinflammatory model of Parkinson’s disease. J. Neuroinflammation 4, 19–21. Abuirmeileh, A., Lever, R., Kingsbury, A.E., et al., 2007b. The corticotrophin-releasing factor-like peptide urocortin reverses key deficits in two rodent models of Parkinson’s disease. Eur. J. Neurosci. 26, 417–423. Alsiö, J., Roman, E., Olszewski, P.K., et al., 2009. Inverse association of high-fat diet preference and anxiety-like behavior: a putative role for Urocortin. Genes Brain Behav. 8, 193–202. Bagosi, Z., Csabafi, K., Palotai, M., et al., 2014. The effect of urocortin I on the hypothalamic ACTH secretagogues and its impact on the hypothalamic-pituitaryadrenal axis. Neuropeptides 48, 15–20. Bale, T.L., Vale, W.W., 2004. CRF and CRF receptors: role in stress responsivity and other behaviors. Annu. Rev. Pharmacol. Toxicol. 44, 525–527. Blandini, F., 2010. An update on the potential role of excitotoxicity in the pathogenesis of Parkinson’s disease. Funct. Neurol. 25, 65–71. Boje, K.M., 2004. Nitric oxide neurotoxicity in neurodegenerative diseases. Front. Biosci. 9, 763–776. Brar, B.K., Jonassen, A.K., Stephanou, A., et al., 2000. Urocortin protects against ischemic and reperfusion injury via a MAPK-dependent pathway. J. Biol. Chem. 275, 8508–8514. Brawner, T.J., Thompson, A., et al., 2008. Urocortin expression in mouse cochlear nucleus and Scarpa’s ganglion. Laryngoscope 118, 1637–1644. Breu, J., Touma, C., Hölter, S.M., et al., 2012. Urocortin 2 modulates aspects of social behaviour in mice. Behav. Brain Res. 233, 331–336. Colosimo, C., 2011. Nonmotor presentations of multiple system atrophy. Nat. Rev. Neurol. 7, 295–298. Colosimo, C., Morgante, L., Antonini, A., et al., 2010. Non-motor symptoms in atypical and secondary Parkinsonism: the PRIAMO study. J. Neurol. 257, 5–14. Cottone, P., Sabino, V., Nagy, T.R., et al., 2007. Feeding microstructure in diet-induced obesity susceptible versus resistant rats: central effects of urocortin2. J. Physiol. 583 (Pt 2), 487–504. Gill, C.E., Konrad, P.E., Davis, T.L., et al., 2007. Deep brain stimulation for Parkinson’s disease: the Vanderbit University Medical Center experience, 1998-2004, TennMed 100, 45–47.
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Hale, M.W., Stamper, C.E., Staub, D.R., et al., 2010. Urocortin 2 increases c-Fos expression in serotonergic neurons projecting to the ventricular/periventricular system. Exp. Neurol. 224, 271–281. Huo, L.R., Liang, X.B., Li, B., et al., 2012. The cortical and striatal gene expression profile of 100 Hz electroacupuncture treatment in 6-hydroxydopamine-induced Parkinson’s disease model. Evid Based Complement Alternat. Med. 5, 1– 14. Jamieson, P.M., Li, C., Kukura, C., et al., 2006. Urocortin3 modulates the neuroendocrine stress response and is regulated in rat amygdala and hypothalamus by stress and glucocorticoids. Endocrinology 147, 4578–4588. Janssen, D., Kozicz, T., 2013. Is it really a matter of simple dualism? Corticotropinreleasing factor receptors in body and mental health. Front. Endocrinol. 2, 4–28. Johnson, P.L., Sajdyk, T.J., Fitz, S.D., et al., 2013. Angiotensin II’s role in sodium lactate-induced panic-like responses in rats with repeated urocortin 1 injections into the basolateral amygdala: amygdalar angiotensin receptors and panic. Prog. Neuropsychopharmacol Biol. Psychiatry 44, 248–256. Kormos, V., Gaszner, B., 2013. Role of neuropeptides in anxiety, stress, and depression: from animals to humans. Neuropeptides 47, 401–419. Li, C., Vaughan, J., Sawchenko, P.E., et al., 2002. Urocortin III-immunoreactive projections in rat brain: partial overlap with sites of type 2 corticotrophinreleasing factor receptor expression. J. Neurosci. 22, 991–1001. Liew, H.K., Pang, C.Y., Hsu, C.W., et al., 2012. Systemic administration of urocortin after intracerebral hemorrhage reduces neurological deficits and neuroinflammation in rats. J. Neuroinflammation 19, 9–13. Liu, C.N., Liu, X.Y., Gao, D.M., et al., 2005. Effects of SNP, GLU and GABA on the neuronal activity of striatum nucleus in rats. Pharmacol. Res. 51, 547–551. Liu, C.N., Liu, X.Y., Yang, C., et al., 2005. In vivo protective effects of urocortin on ischemia/reperfusion injury in rat heart via free radical mechanisms. Can. J. Physiol. Pharmacol. 83, 459–465. McGinty, J.F., Whitfield, T.W., Berglind, W.J., et al., 2010. Brain-derived neurotrophic factor and cocaine addiction. Brain Res. 1314, 183–193. Nemoto, T., Iwasaki-Sekino, A., Yamauchi, N., et al., 2007. Regulation of the expression and secretion of urocortin 2 in rat pituitary. J. Endocrinol. 192, 443–452. Oh, J.D., Chase, T.N., 2002. Glutamate-mediated striatal dysregulation and the pathogenesis of motor response complications in Parkinson’s disease. Amino Acids 23, 133–139. Rademaker, M.T., Charles, C.J., Richards, A.M., 2007. Urocortin administration from onset of rapid left ventricular pacing represses progression to overt heart failure. J. Am. Phys. 293, 1536–1544. Riester, A., Spyroglou, A., Neufeld-Cohen, A., et al., 2012. Urocortin-dependent effects on adrenal morphology, growth, and expression of steroidogenic enzymes in vivo. Mol. Endocrinol. 48, 159–167. Rivier, C.L., 2008. Urocortin inhibits rat Leydig cell function. Endocrinology 149, 6425–6432. Skórzewska, A., Bidzin´ski, A., Lehner, M., et al., 2011. The localization of brain sites of anxiogenic-like effects of urocortin-2. Neuropeptides 45, 83–92. Xue, X., Liu, H., Qi, L., et al., 2014. Baicalein ameliorated the upregulation of striatal glutamatergic transmission in the mice model of Parkinson’s disease. Brain Res. Bull. 103, 54–59. Zhou, B.Y., Zhao, X., Sun, H., et al., 2011. Expression of biliverdin-IX β-reductase in the nigrostriatal system in mouse and rat models of Parkinson’s disease. Chin. Pharmacol. Bull. 27, 1126–1130. Ziemssen, T., Fuchs, G., Greulich, W., 2011. Treatment of dysautonomia in extrapyramidal disorders. J. Neurol. 258, 339–345.
Please cite this article in press as: Chunna Liu, et al., The effects of neuropeptide urocortin 2 on the spontaneous discharge and glutamatergic neurotransmission of striatum neurons, Neuropeptides (2015), doi: 10.1016/j.npep.2015.03.001
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The effects of neuropeptide urocortin 2 on the spontaneous discharge and glutamatergic neurotransmission of striatum neurons Chunna Liu a,*,1, Xinyu Liu b,1, Feiran Song c, Jian Li b, Xia Zhang a, Jing Yang a a b c
Department of Pharmacology, Liaoning Medical University, JinZhou 121001, China The First Affiliated Hospital of Liaoning Medical University, JinZhou 121001, China The China Medical University, ShenYang 110001, China
A R T I C L E
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Article history: Received 30 July 2014 Accepted 2 March 2015 Available online Keywords: Urocortin Neuropeptide Striatum Glutamate Spontaneous discharge Coticortropin-releasing factor
A B S T R A C T
The primary cause of the neurodegenerative process that underlies Parkinson’s disease (PD) is still unknown. Different mechanisms probably contribute to triggering neuronal death in the nigro-striatum pathway. The neuropeptide urocortin 2 (UCN2) plays an important role in the regulation of striatum (STR) neurons projection. We investigated the effects of UCN2 on spontaneous discharge and glutamatergic responses in STR for a better understanding of the pathogenesis of PD. The experiment used microiontophoresis method to observe the effects of UCN2 on STR neurons’ firing rates in vivo. Corticotrophin releasing factor receptor 2 (CRF-R2) selective inhibitor, astressin-2B (AST-2B), was administered simultaneously with UCN2 to investigate the effects of UCN2 on CRF-R2. Moreover, we further explored the effects of UCN2 on glutamatergic responses in STR neurons. We found that UCN2 could significantly inhibit the firing rate of 84% of the tested STR neurons, and its inhibitory effect followed a concentration-dependent manner. During the microiontophoresis of GLU, the excitatory firing of glutamatergic neurons could be attenuated by the addition of UCN2, but enhanced by the application of AST-2B. The results suggest that UCN2 could regulate the effects of STR neurotransmitters (GLU) via CRF-R2 and may thereby contribute to the improvement of PD. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Urocortins (UCNs) are isolated acid neuro-active peptides related to hypothalamic corticotrophin-releasing hormone (CRF) and binds with high affinity to corticotrophin-releasing hormone receptor2β, which belongs to the hypothalamic CRF family (Bale and Vale, 2004; Brar et al., 2000). Results from immunological assay have suggested that UCN mRNA was expressed in multiple parts of the brain, including the diencephalon, hypothalamic regions, the pituitary, the anterior frontal cortex, the frontal, the midbrain and the brainstem (Bagosi et al., 2014). The densest projections were also found in the intermediate part of the lateral septum, posterior division of the bed nucleus, stria terminalis, and the medial nucleus of the amygdala (Li et al., 2002). UCN had three forms-UCN1, UCN2 and UCN3. CRF receptors included two major subtypes, CRFR1 and CRF-R2. CRFR1 had a strong affinity for CRF and UCN1 peptides while UCN2 and UCN3 only showed specific binding property to CRF-R2 in rats (Nemoto et al., 2007). UCN was similar to CRF in biological
* Corresponding author. Department of Pharmacology, Liaoning Medical University, No. 40, Section 3, Songpo Road, Jinzhou City, Liaoning 121001, PR China. E-mail address: [email protected] (C. Liu). 1 These authors contributed equally to this work.
activity and, through the hypothalamus–adenohypophysis–adrenal axis, it could promote the adenohypophysis adrenal cortical hormone secretion (Riester et al., 2012). UCN not only regulated emergency response and stress in the peripheral tissues, but also participated in some functional changes in the central nervous system, including the modulation of appetite, intension, worry and anxiety, the inhibition of scald edema and the regulation of electrolyte and water balances. It has been used as an anti-epileptic drug (Cottone et al., 2007). UCN has also been reported to exert protective effects on Parkinson’s disease (PD) (Huo et al., 2012; Rivier, 2008). UCN2, a member of the corticotropin-releasing hormone family, was involved in the regulation of stress-related behaviors in rodents. It was reported (Breu et al., 2012) that male UCN2 null mice showed more passive social interactions and reduced the aggressiveness in comparison to wild-type animals. UCN2 seemed to modulate aggressive behavior in male mice. In a previous research (Hale et al., 2010), intracerebroventricular injection of UCN2 could increase c-Fos expression in serotonergic neurons in the dorsal and caudal parts of the dorsal raphe nucleus, including both subsets of serotonergic neurons that project and do not project to the ventricle/ periventricular system. Accumulative evidence showed that UCN2 was involved in the physiological activities and pathological processes in STR nucleus, which was the essential nucleus of PD pathogenesis. However, the detailed mechanism of PD remains to
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Please cite this article in press as: Chunna Liu, et al., The effects of neuropeptide urocortin 2 on the spontaneous discharge and glutamatergic neurotransmission of striatum neurons, Neuropeptides (2015), doi: 10.1016/j.npep.2015.03.001
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not be elucidated. Several hypothesis supported different pathophysiological mechanisms related to oxidative stress, glutamatemediated neurotoxicity, mitochondrial energetic impairment and apoptosis (Boje, 2004). Glutamate is the predominantly fast excitatory neurotransmitter in the central nervous system and, in the presence of specific conditions, a potential neurotoxin, Glutamate mediated excitotoxicity, is a further contributor for the development in damage of the nigrostriatal that characterizes PD (Blandini, 2010). Glutamate-mediated striatal sensitization subsequently modifies the basal ganglia output in ways that favor the appearance of parkinsonian motor complications (Oh and Chase, 2002). However, it is still unclear whether UCN2 can influence basal ganglia neuronal discharge and regulate the neurotransmitters transmission, and if so, the molecular mechanism underlying its effectiveness. The present study investigates the effects of UCN2 on the STR neurons for a further comprehension on the pathogenesis of PD. At the same time, this study aims to add depth to the diagnosis and treatment of neurons having paradoxical discharge disease and seizure disorders. We used microiontophoresis to observe the effects of UCN2 and AST-2B on STR neurons’ spontaneous discharge. Moreover, the effects of UCN on Glutamatergic STR neurons (Xue et al., 2014) were also investigated in order to ascertain whether UCN do not only exist in the STR neuron, but also regulates neurotransmission and inhibits the glutamatergic exitotoxicity. 2. Materials and methods 2.1. Experimental animals Animal care and experimental protocols were treated in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and approved by the Ethics Committee of Liaoning Medical University for the Use of Experimental Animals for Research and Teaching. Eighty male Sprague-Dawley rats (weight, 180–220 g; age, 10–13 weeks) were used. 2.2. Drugs/reagents UCN2 (rats) and AST-2B were obtained from Tocris Bioscience Company, soluble to 1 mg/ml in water. Solutions were made by adding solvent directly to the vial. The vial should then be vortexed vigorously to ensure the product has completely dissolved. L-glutamic acid (GLU), MK-801 and other drugs were purchased from Sigma. Distilled-water was the vehicle in which GLU and MK-801 were dissolved as concentrated stock solutions. All drugs were diluted to their final concentrations in the bath solution immediately prior to commencing the experiments. 2.3. Methods The rats were anesthetized with intraperitoneal urethane (1g kg−1) and mounted in a stereotaxic apparatus. An incision was made in the scalp to expose the skull and a hole was drilled above the STR. For extracellular recording and microiontophoresis, seven barrel micropipettes were used, which were made with 51217 type microelectrode puller (these micropipettes were fabricated with their filling ends bent away centrifugally to avoid solutions’ contamination among the barrels with a tip diameter of around 4–8 μm). The central pipette was filled with 1% pontamine sky blue in 3.0 mol L−1 NaCl and served as the leading electrode with resistance of 5–12 MΩ (Gill et al., 2007; Liu et al., 2005). The other peripheral tubes (resistance of 20–100 MΩ) were filled with the drugs as follows: UCN2 (10−5 mol·L−1, 10−6 mol·L−1, 10−7 mol·L−1 and 10−8 mol·L−1, pH 7.5); AST-2B (CRF-2 blocker, 10−6 mol·L−1, pH 7.5); GLU (1.0 mol·L−1, pH 8.0), (±)-5-methyl-10,11-dihydro-5-H-dibenzo [a,d] cyclohepten-5,10iminehy drochloride (MK-801, 10−2 mol·L−1, pH 4.0), respectively.
According to the Paxino rat brain atlas for STR orientation, the coordinate was −0.8 mm to 1.2 mm from bregma, 2.5–3.5 mm lateral from midline and 5.0–6.0 mm below the brain surface (Liu et al., 2005). The electrode was placed into the striatum nucleus with the microelectrode pusher. The electrical signal was amplified and displayed on a storage oscilloscope and then sent to a computer system. To generate the spontaneous firing histograms, the STR neurons’ spontaneous firing rates were recorded for 2–3 min before any drug ejection was applied. The microiontophoretic apparatus was used to eject UCN2, AST-2B, GLU, and MK-801, respectively. All the drugs except GLU were ejected with cationic currents using the Neuro phore BH-2 system (Medical System Corp, Greenvale, NY, USA), and retaining currents of negative 5−10 nA were applied between ejections to prevent drug leakage. The effects of UCN2 and AST-2B on the STR neurons were observed. Subsequently, the effects of UCN2 and AST-2B on the firing rates in the presence of GLU and MK-801 were also investigated. The 0.165 mol·L−1 NaCl pipette was used as a current sink and a drug control for 10 s. In order to eliminate the possible electrical effects caused by microiontophoretic pulses, data were discarded if current ejection from this pipette elicited any changes in the spontaneous discharge. Administration must be done after the discharge induced by the previous administration did not change any more. Likewise, excessive microiontophoretic currents of more than 100 nA were never used. At the end of each experiment, the last recording site was marked by microiontophoretic ejection of pontamine sky blue through the central pipette (−20μA, 20 min). The rats were given an overdose of urethane and perfused transcardially with 0.9% normal saline followed by 4% paraformaldehyde (distilled in 0.1 mol·L−1 phosphatebuffered saline, pH 7.4) for 15–20 min. The brains were frozen at −40 °C and then cut into 30 μm slices. The recording site was verified by histological examination (Huo et al., 2012; Liu et al., 2005). 2.4. Statistical analysis The effects of drugs were determined using the following equation: Effects = (firing rate after treatment (20s) – firing rate before treatment (20s)/firing rate before treatment (20s) × 100%. Results were presented as means ± SD (standard deviation). Student’s t-test and one-way ANOVA test were used to evaluate the drug effects. 3. Results A total of 85 neurons in the STR were observed. The spontaneous firing rates of STR neurons were relatively slow and irregular, and varied from 0 to 10 Hz (6.84 ± 1.21 Hz). The waveforms were characteristics of medium spiny neurons. Burst firing were seen occasionally. 3.1. Effects of UCN2 and AST-2B on the STR neuronal activity A total of 58 STR neurons were tested in this experiment. After application of UCN2 (10−6 mol·L−1, 10−80 nA, 10 s), 84% (49/58) of the tested STR neurons showed a significant decrease in their firing rates compared to the basal level, and the neuronal average firing rates decreased from 6.98 ± 1.05 Hz to 3.93 ± 1.02 Hz (n = 58, P < 0.01). The effects of UCN2 had a latent period (n = 58, 14.56 ± 3.37s) and an obvious after-effect, which induced explosive firing rates (Fig 1a–c). Application of UCN2 microiontophoretically resulted in a decreased firing rate in an intensity-dependent manner. (Fig 2a–c). As the concentration of UCN2 (10−8 mol·L−1, 10−7 mol·L−1, 10−6 mol·L−1 and 10−5 mol·L−1, 20 nA, 10 s) increased, the neuronal firing rates were attenuated. On the other hand, 8 neurons had no responses to various levels of UCN2 and 1 neuron’s firing rates was even increased (n = 34, P < 0.01).
Please cite this article in press as: Chunna Liu, et al., The effects of neuropeptide urocortin 2 on the spontaneous discharge and glutamatergic neurotransmission of striatum neurons, Neuropeptides (2015), doi: 10.1016/j.npep.2015.03.001
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the neuronal average firing rates increased from 6.96 ± 0.93 Hz to 15.09 ± 1.28 Hz. After addition of the NMDA receptor’s blocker, MK801 (0.01 mol·L−1, 20 nA for 20 s), the spontaneous firing rates were inhibited, the neuronal firing rates decreased from 15.09 ± 1.28 Hz to 5.94 ± 1.20 Hz (n = 26, P < 0.01), and MK-801 had a significant aftereffect. The STR neuronal firing rates, which were already inhibited by MK-801, reduced the rates to 4.12 ± 0.76 Hz (n = 26, P < 0.05) by UCN2 application. In the presence of GLU for a period of 100 s, together administrated with UCN2 (20 nA, 20 s) from the 20th second to the 40th second resulted in the firing rates being significantly decreased in 73% (19/26) tested GLU neurons (compared to GLU application alone). The firing rates enhanced by GLU were decreased by UCN2 from 15.07 ± 1.97 Hz to 9.98 ± 2.04 Hz (P < 0.01). 27%
Fig. 1. (a) Effects of UCN2 (10−6 mol·L−1, 10-80 nA, 10 s) and AST-2B on striatum neuron. Values are mean ± SD for n = 58 neurons in UCN group and n = 52 neurons in AST-2B group, 80 male Sprague-Dawley rats were used. *vs Normal group, *P < 0.05, **P < 0.01; #vs UCN group, # P < 0.05, ##P < 0.01. (b) Effects of UCN2 on striatum neuron spontaneous firing rates. AST-2B was administrated during the period of UCN2. (c) The real-time recording wave of one striatum neuron.
After addition of the CRF-R2 antagonist, AST-2B (10−6 mol·L−1, 20 nA, 10 s), the spontaneous firing rates were enhanced in 78% (41/ 52) of the tested STR neurons, with the neuronal firing rates increased from 5.49 ± 1.08 Hz to 8.01 ± 1.13 Hz (n = 52, P < 0.01). The effects of AST-2B were fast without a latent period or after-effect. Moreover, in the presence of UCN2 (10−6 mol·L−1, 20 nA, 20 s), microiontophoretic application of AST-2B (20 nA, 10 s) reversed UCN2’s inhibitory effects and enhanced the firing rates in 34 out of 36 STR neurons. As demonstrated in Fig. 1b and c, the STR neuronal firing rates increased from 2.78 ± 1.47 Hz to 5.82 ± 1.12 Hz in the presence of AST-2B (n = 34, P < 0.01). 3.2. Influences of UCN2 and AST-2B on glutamatergic firing action After application of GLU (20 nA, 20 s), 26 of STR neurons showed a significant increase in their firing rates compared to the basal level,
Fig. 2. (a) The intensity-dependence of UCN2 (10 −7 mol·L −1 , 10 −6 mol·L −1 and 10−5 mol·L−1, 20 nA, 10 s) on the STR neurons. Values are mean ± SD for n = 34 neurons.*vs Normal group; #vs UCN2 10−8 mol·L−1 group; Δvs UCN2 10−7 mol·L−1 group; □vs UCN2 10−6 mol·L−1 group.*P < 0.05, **P < 0.01; #P < 0.05, ##P < 0.01; ΔP < 0.05, ΔΔP < 0.01; □P < 0.05, □□P < 0.01. (b) The intensity-dependent effects of UCN2 on STR neuron spontaneous firing rates. (c) The real-time recording wave of UCN2 effect on one striatum neuron.
Please cite this article in press as: Chunna Liu, et al., The effects of neuropeptide urocortin 2 on the spontaneous discharge and glutamatergic neurotransmission of striatum neurons, Neuropeptides (2015), doi: 10.1016/j.npep.2015.03.001
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Fig. 3. (a) Effects of UCN2 (10−6 mol·L−1, 20 nA, 20 s) and AST-2B on striatum neuron. Values are mean ± SD for n = 26 neurons. *vs Normal group; #vs GLU group. *P < 0.05, **P < 0.01; #P < 0.05, ##P < 0.01. b. Effects of UCN2 on striatum’s glutamatergic neuron spontaneous firing rates. MK-801 10−2 mol·L−1 could inhibit striatum neuron spontaneous firing rates, and the UCN2 and AST-2B were administrated during the microiontophoresis of GLU. (c) The real-time recording wave of UCN2 effect on striatum’s glutamatergic neuron.
(7/26) neurons had no response. At the 60th second, subsequent application of AST-2B (20 nA, 20 s) caused an increase in the firing rates in 58% (15/26) tested GLU neurons (compared to GLU application alone). The STR neuronal firing rates, which were already increased by GLU, increased further from 14.91 ± 2.40 Hz to 17.83 ± 2.09 Hz (n = 26, P < 0.05) by AST-2B application (Fig 3a–c). 4. Discussion The present study found that micro-electrophoresis of UCN2 could significantly reduce the discharge rate of STR neurons. This suggested that UCN2 caused inhibitory effects on STR neurons. Microelectrophoresis AST-2B alone could increase the discharge rate of the
tested STR neurons, indicating that CRF-R2 was expressed in STR neurons. It was further demonstrated that the inhibitory effects induced by UCN2 could be antagonized by CRF-R2 antagonist – AST-2B. These results further suggested that UCN2 exerted inhibitory effects on STR neurons via CRF2 receptor. Interaction was observed between UCN2 and glutamatergic neurons. It was also found that glutamatergic projected neuron fibers had an excitatory effect on STR neurons, which could be antagonized by UCN2 and synergized by AST-2B. These findings suggested that UCN2, when combined with CRF-R2 in STR neurons, can antagonize the exitotoxicity of glutamatergic neurons. The experiment further indicated that UCN2 played important regulatory effects on STR neurons and its neurotransmitters. UCN is a newly discovered neuro-endocrinal active peptide (Liu et al., 2005; Rademaker et al., 2007). As a small molecular active peptide, UCN confers protective effects via autocrine and/or paracrine pathways (Bale and Vale, 2004; Jamieson et al., 2006). Researches showed that UCN exerted anti-anxiety (Alsiö et al., 2009), anti-aging (Gill et al., 2007) and neuroprotective effects on damaged brains and relieved the damages of drug addiction (Liew et al., 2012; McGinty et al., 2010). Most researchers reported that UCN bound with CRF-R2 promoted cAMP increase, thus activating protein kinase. Brar found that mitogen activated protein kinase and phosphatidylinositol 3-kinase pathways formed important protective mechanisms of UCN (Bale and Vale, 2004; Brar et al., 2000; Rademaker et al., 2007). Physiological responses to stress coordinated by the hypothalamo– pituitary –adrenal axis were concerned with maintaining homeostasis in the presence of real or perceived challenges. Regulators of this axis were CRF and CRF related neuropeptides, including UCNs 1, 2, and 3. They mediated their actions by binding to CRF R1 and CRF R2, which were located in several stress-related brain regions (Janssen and Kozicz, 2013). Based on data obtained in animal studies, neuropeptides and their receptors might be targeted by new candidate neuropharmacons with the hope that they will become important and effective tools in the management of stress related mood disorders (Kormos and Gaszner, 2013). In a recent report (Johnson et al., 2013), rats treated with three daily UCN injections into the basolateral amygdala develop prolonged anxiety-associated behavior and vulnerability to panic-like physiological responses (i.e., tachycardia, hypertension and tachypnea) following intravenous infusions of 0.5 M sodium lactate (NaLac, an ordinarily mild interoceptive stressor). Among cultured neurons, UCN could protect hippocampal neurons, γ-aminobutyric acid (GABA) neurons and dopaminergic (DA) neurons (Brawner et al., 2008). Previous data (Skórzewska et al., 2011) suggested a role of UCN2 in the behavioral and immunocytochemical responses to stress, in which it strengthens the measures of anxiety-like responses. In addition, as a CRH-related peptide, UCN could protect the neurons of PD rat models by apomorphine, reverse the PD symptoms, and increase the level of GABA, DA and serotonin (TH) in nigra-striatum (Colosimo, 2011; Zhou et al., 2011). In a similar and consistent manner with the preservation of motor function, UCN protect the nigra from both DA depletion and loss of TH activity, indicating preservation of DA cells. UCN reversed key features of nigrostriatal damage in the hemi-parkinsonian 6-hydroxydopamine lesioned rat (Abuirmeileh et al., 2007a, 2007b). It was also suggested that UCN served to maintain sufficient functions of nigra-striatum neurons by influencing CRF receptors (Bale and Vale, 2004; Colosimo et al., 2010). These findings were of great potential significance in the treatment of PD induced by the degeneration of nigra-striatum neurons (Ziemssen et al., 2011). Our experiments suggested that in the pathological condition of PD or epilepsy, UCN2 might inhibit high frequency discharge of STR neurons and suppress GLU neuronal toxicity, thereby constraining the occurrence and development of the disease. Our data constitute the first report which demonstrated that UCN2 has inhibitory effects on STR neuron and mediates neuroprotection, thus indicating its potential therapeutic value in PD.
Please cite this article in press as: Chunna Liu, et al., The effects of neuropeptide urocortin 2 on the spontaneous discharge and glutamatergic neurotransmission of striatum neurons, Neuropeptides (2015), doi: 10.1016/j.npep.2015.03.001
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5. Conclusions The experiment showed that UCN2 could inhibit the discharge activity of STR neurons and reduce the excitation of glutamatergic neurons. UCN2 can modulate STR neurotransmitters via binding to CRF R2. Acknowledgments We thank Prof. Dongming Gao for expert technical assistance. This work was supported by the National Natural Science Foundation of China (81201037). References Abuirmeileh, A., Harkavyi, A., Lever, R., et al., 2007a. Urocortin, a CRF-like peptide, restores key indicators of damage in the substantia nigra in a neuroinflammatory model of Parkinson’s disease. J. Neuroinflammation 4, 19–21. Abuirmeileh, A., Lever, R., Kingsbury, A.E., et al., 2007b. The corticotrophin-releasing factor-like peptide urocortin reverses key deficits in two rodent models of Parkinson’s disease. Eur. J. Neurosci. 26, 417–423. Alsiö, J., Roman, E., Olszewski, P.K., et al., 2009. Inverse association of high-fat diet preference and anxiety-like behavior: a putative role for Urocortin. Genes Brain Behav. 8, 193–202. Bagosi, Z., Csabafi, K., Palotai, M., et al., 2014. The effect of urocortin I on the hypothalamic ACTH secretagogues and its impact on the hypothalamic-pituitaryadrenal axis. Neuropeptides 48, 15–20. Bale, T.L., Vale, W.W., 2004. CRF and CRF receptors: role in stress responsivity and other behaviors. Annu. Rev. Pharmacol. Toxicol. 44, 525–527. Blandini, F., 2010. An update on the potential role of excitotoxicity in the pathogenesis of Parkinson’s disease. Funct. Neurol. 25, 65–71. Boje, K.M., 2004. Nitric oxide neurotoxicity in neurodegenerative diseases. Front. Biosci. 9, 763–776. Brar, B.K., Jonassen, A.K., Stephanou, A., et al., 2000. Urocortin protects against ischemic and reperfusion injury via a MAPK-dependent pathway. J. Biol. Chem. 275, 8508–8514. Brawner, T.J., Thompson, A., et al., 2008. Urocortin expression in mouse cochlear nucleus and Scarpa’s ganglion. Laryngoscope 118, 1637–1644. Breu, J., Touma, C., Hölter, S.M., et al., 2012. Urocortin 2 modulates aspects of social behaviour in mice. Behav. Brain Res. 233, 331–336. Colosimo, C., 2011. Nonmotor presentations of multiple system atrophy. Nat. Rev. Neurol. 7, 295–298. Colosimo, C., Morgante, L., Antonini, A., et al., 2010. Non-motor symptoms in atypical and secondary Parkinsonism: the PRIAMO study. J. Neurol. 257, 5–14. Cottone, P., Sabino, V., Nagy, T.R., et al., 2007. Feeding microstructure in diet-induced obesity susceptible versus resistant rats: central effects of urocortin2. J. Physiol. 583 (Pt 2), 487–504. Gill, C.E., Konrad, P.E., Davis, T.L., et al., 2007. Deep brain stimulation for Parkinson’s disease: the Vanderbit University Medical Center experience, 1998-2004, TennMed 100, 45–47.
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Hale, M.W., Stamper, C.E., Staub, D.R., et al., 2010. Urocortin 2 increases c-Fos expression in serotonergic neurons projecting to the ventricular/periventricular system. Exp. Neurol. 224, 271–281. Huo, L.R., Liang, X.B., Li, B., et al., 2012. The cortical and striatal gene expression profile of 100 Hz electroacupuncture treatment in 6-hydroxydopamine-induced Parkinson’s disease model. Evid Based Complement Alternat. Med. 5, 1– 14. Jamieson, P.M., Li, C., Kukura, C., et al., 2006. Urocortin3 modulates the neuroendocrine stress response and is regulated in rat amygdala and hypothalamus by stress and glucocorticoids. Endocrinology 147, 4578–4588. Janssen, D., Kozicz, T., 2013. Is it really a matter of simple dualism? Corticotropinreleasing factor receptors in body and mental health. Front. Endocrinol. 2, 4–28. Johnson, P.L., Sajdyk, T.J., Fitz, S.D., et al., 2013. Angiotensin II’s role in sodium lactate-induced panic-like responses in rats with repeated urocortin 1 injections into the basolateral amygdala: amygdalar angiotensin receptors and panic. Prog. Neuropsychopharmacol Biol. Psychiatry 44, 248–256. Kormos, V., Gaszner, B., 2013. Role of neuropeptides in anxiety, stress, and depression: from animals to humans. Neuropeptides 47, 401–419. Li, C., Vaughan, J., Sawchenko, P.E., et al., 2002. Urocortin III-immunoreactive projections in rat brain: partial overlap with sites of type 2 corticotrophinreleasing factor receptor expression. J. Neurosci. 22, 991–1001. Liew, H.K., Pang, C.Y., Hsu, C.W., et al., 2012. Systemic administration of urocortin after intracerebral hemorrhage reduces neurological deficits and neuroinflammation in rats. J. Neuroinflammation 19, 9–13. Liu, C.N., Liu, X.Y., Gao, D.M., et al., 2005. Effects of SNP, GLU and GABA on the neuronal activity of striatum nucleus in rats. Pharmacol. Res. 51, 547–551. Liu, C.N., Liu, X.Y., Yang, C., et al., 2005. In vivo protective effects of urocortin on ischemia/reperfusion injury in rat heart via free radical mechanisms. Can. J. Physiol. Pharmacol. 83, 459–465. McGinty, J.F., Whitfield, T.W., Berglind, W.J., et al., 2010. Brain-derived neurotrophic factor and cocaine addiction. Brain Res. 1314, 183–193. Nemoto, T., Iwasaki-Sekino, A., Yamauchi, N., et al., 2007. Regulation of the expression and secretion of urocortin 2 in rat pituitary. J. Endocrinol. 192, 443–452. Oh, J.D., Chase, T.N., 2002. Glutamate-mediated striatal dysregulation and the pathogenesis of motor response complications in Parkinson’s disease. Amino Acids 23, 133–139. Rademaker, M.T., Charles, C.J., Richards, A.M., 2007. Urocortin administration from onset of rapid left ventricular pacing represses progression to overt heart failure. J. Am. Phys. 293, 1536–1544. Riester, A., Spyroglou, A., Neufeld-Cohen, A., et al., 2012. Urocortin-dependent effects on adrenal morphology, growth, and expression of steroidogenic enzymes in vivo. Mol. Endocrinol. 48, 159–167. Rivier, C.L., 2008. Urocortin inhibits rat Leydig cell function. Endocrinology 149, 6425–6432. Skórzewska, A., Bidzin´ski, A., Lehner, M., et al., 2011. The localization of brain sites of anxiogenic-like effects of urocortin-2. Neuropeptides 45, 83–92. Xue, X., Liu, H., Qi, L., et al., 2014. Baicalein ameliorated the upregulation of striatal glutamatergic transmission in the mice model of Parkinson’s disease. Brain Res. Bull. 103, 54–59. Zhou, B.Y., Zhao, X., Sun, H., et al., 2011. Expression of biliverdin-IX β-reductase in the nigrostriatal system in mouse and rat models of Parkinson’s disease. Chin. Pharmacol. Bull. 27, 1126–1130. Ziemssen, T., Fuchs, G., Greulich, W., 2011. Treatment of dysautonomia in extrapyramidal disorders. J. Neurol. 258, 339–345.
Please cite this article in press as: Chunna Liu, et al., The effects of neuropeptide urocortin 2 on the spontaneous discharge and glutamatergic neurotransmission of striatum neurons, Neuropeptides (2015), doi: 10.1016/j.npep.2015.03.001