Tyrosine Hydroxylase-immunoreactive Neurons Research Articles

Cotransplantation of Olfactory Ensheathing Cells and Schwann Cells Combined with Treadmill Training Promotes Functional Recovery in Rats with Contused Spinal Cords

The present study investigated the ability of cotransplantation of Schwann cells (SCs) and olfactory ensheathing cells (OECs) combined with treadmill training in facilitating neuronal plasticity and promoting hindlimb function recovery of subacute moderate thoracic (T10) spinal cord contusion in rats. Two weeks postinjury, SCs were injected directly into the lesion, while OECs were injected into the adjacent tissues. The treadmill training with the rats began postinjury on day 7, with each session lasting 20 ± 10 min per day, 5 days per week, for 10 weeks. At the 11th week postinjury, OECs were found migrating longitudinally and laterally from the injection site to the injury site through the gray and white matter, while some traveled along the central canal or pia. The SCs remained densely packed and concentrated at the transplant site. The transplanted SCs supported ingrowth of numerous, densely populated neurofilament-positive (NF(+)), MBP(+) axons. The OECs promoted elongation of moderate NF(+), GAP-43(+) axons and a few MBP(+) axons in parallel with OEC processes. The GFAP immunoreactivity in the spared tissue surrounding the graft of SCs and OECs at the lesion site was less intense than that in the DMEM group. Treadmill training had no effect on GFAP immunoreactivity. Treadmill training increased the number of TH-immunoreactive neurons in the gray matter of L2 spinal cord. Moreover, cotransplantation of OECs and SCs significantly increased the BBB score during 5-8 weeks postinjury alongside treadmill training between 5 and 11 weeks. Cotransplantation of OECs and SCs combined with treadmill training resulted in the highest BBB score at 4 and 11 weeks. The study details the differential mechanisms of neuronal plasticity: (1) axon growth and remyelination induced by cotransplantation of OECs and SCs and (2) neuron plasticity below the lesion enhanced by treadmill training. The synergistic effects of the combined strategy enhance functional recovery. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

Allopregnanolone Reinstates Tyrosine Hydroxylase Immunoreactive Neurons and Motor Performance in an MPTP-Lesioned Mouse Model of Parkinson's Disease

Restorative/protective therapies to restore dopamine neurons in the substantia nigra pars compacta (SNpc) are greatly needed to effectively change the debilitating course of Parkinson's disease. In this study, we tested the therapeutic potential of a neurogenic neurosteroid, allopregnanolone, in the restoration of the components of the nigrostriatal pathway in MPTP-lesioned mice by measuring striatal dopamine levels, total and tyrosine hydroxylase immunoreactive neuron numbers and BrdU-positive cells in the SNpc. An acute treatment (once/week for two weeks) with allopregnanolone restored the number of tyrosine hydroxylase-positive and total cell numbers in the SNpc of MPTP-lesioned mice, even though this did not increase striatal dopamine. It was also noted that MPTP treated mice to which allopregnanolone was administered had an increase in BrdU-positive cells in the SNpc. The effects of allopregnanolone in MPTP-lesioned mice were more apparent in mice that underwent behavioral tests. Interestingly, mice treated with allopregnanolone after MPTP lesion were able to perform at levels similar to that of non-lesioned control mice in a rotarod test. These data demonstrate that allopregnanolone promotes the restoration of tyrosine hydroxylase immunoreactive neurons and total cells in the nigrostriatal tract, improves the motor performance in MPTP-treated mice, and may serve as a therapeutic strategy for Parkinson's disease.

Transplantation of neural stem cells co-transfected with Nurr1 and Brn4 for treatment of Parkinsonian rats

Neural stem cells (NSCs) tranplantation has great potential for the treatment of neurodegenerative disease such as Parkinson's disease (PD). However, the usage of NSCs is limited because the differentiation of NSCs into specific dopaminergic neurons has proven difficult. We have recently demonstrated that transgenic expression of Nurr1 could induce the differentiation of NSCs into tyrosine hydroxylase (TH) immunoreactive dopaminergic neurons, and forced co-expression of Nurr1 with Brn4 caused a dramatic increase in morphological and phenotypical maturity of these neurons. In this study, we investigated the effect of transplanted NSCs in PD model rats. The results showed that overexpression of Nurr1 promoted NSCs to differentiate into dopaminergic neurons in vivo, increased the level of dopamine (DA) neurotransmitter in the striatum, resulting in behavioral improvement of PD rats. Importantly, co-expression of Nurr1 and Brn4 in NSCs significantly increased the maturity and viability of dopaminergic neurons, further raised the DA amount in the striatum and reversed the behavioral deficit of the PD rats. Our findings provide a new potential and strategy for the use of NSCs in cell replacement therapy for PD.

Preproglucagon (PPG) neurons innervate neurochemicallyidentified autonomic neurons in the mouse brainstem

Preproglucagon (PPG) neurons produce glucagon-like peptide-1 (GLP-1) and occur primarily in the nucleus tractus solitarius (NTS). GLP-1 affects a variety of central autonomic circuits, including those controlling the cardiovascular system, thermogenesis, and most notably energy balance. Our immunohistochemical studies in transgenic mice expressing YFP under the control of the PPG promoter showed that PPG neurons project widely to central autonomic regions, including brainstem nuclei. Functional studies have highlighted the importance of hindbrain receptors for the anorexic effects of GLP-1.In this study, we assessed YFP innervation of neurochemically identified brainstem neurons in transgenic YFP–PPG mice. Immunoreactivity for YFP plus choline acetyltransferase (ChAT), tyrosine hydroxylase (TH) and/or serotonin (5-HT) was visualised with two- or three-colour immunoperoxidase labelling using black (YFP), brown and blue-grey reaction products.In the dorsal motor nucleus of the vagus (DMV), terminals from fine YFP-immunoreactive axons closely apposed a small proportion of ChAT-positive and rare TH-positive/ChAT-positive motor neurons, mostly ventral to AP. YFP-immunoreactive innervation was virtually absent from the compact and loose formations of the nucleus ambiguus. In the NTS, some TH-immunoreactive neurons were closely apposed by YFP-containing axons. In the A1/C1 column in the ventrolateral medulla, close appositions on TH-positive neurons were more common, particularly in the caudal portion of the column. A single YFP-immunoreactive axon usually provided 1–3 close appositions on individual ChAT- or TH-positive neurons. Serotonin-immunoreactive neurons were most heavily innervated, with the majority of raphé pallidus, raphé obscurus and parapyramidal neurons receiving several close appositions from large varicosities of YFP-immunoreactive axons.These results indicate that GLP-1 neurons innervate various populations of brainstem autonomic neurons. These include vagal efferent neurons and catecholamine neurons in areas linked with cardiovascular control. Our data also indicate a synaptic connection between GLP-1 neurons and 5-HT neurons, some of which might contribute to the regulation of appetite.

Abstract 635: Knockdown Of Tyrosine Hydroxylase In The Nucleus Of The Solitary Tract (NTS) Reduces Persistent Increase In Blood Pressure During Chronic Intermittent Hypoxia (CIH)

A2 neurons in caudal NTS express tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, and respond to stressors such as hypoxia. We tested the hypothesis that TH knockdown in NTS reduces cardiovascular responses to CIH, an animal model that mimics the arterial hypoxemia observed during sleep apnea in humans. Adult male Sprague-Dawley rats were implanted with radiotelemetry transmitters and adeno-associated viral constructs (GeneDetect, Inc) with a GFP reporter having either short hair-pin RNA for TH (shRNA) or scrambled virus (scRNA) were injected into caudal NTS. A group of no injection, no CIH (control) rats were also used. Baseline mean arterial pressure (MAP) and heart rate (HR) were collected from all 3 groups for at least 4 days after which the virus injected rats were exposed to 7 days CIH (alternating 3 min periods of 10% O2 and 21% O2 from 8am to 4pm; from 4pm to 8am the rats were exposed to 21% O2). In shRNA injected rats (n=9) 7 day exposure to CIH increased MAP and HR during the day by 6.6±1.8 mmHg and 49.9±7.7 bpm and during the night by 0.3±1.8 mmHg and -4.5±1.3 bpm compared to baseline. In scRNA injected rats (n=10) 7 day exposure to CIH increased MAP and HR during the day by 7.7±1.2 mmHg and 65.3±7.9 bpm and during the night by 4.8±1.5 mmHg and 0.0±4.9 bpm compared to baseline. In control rats (n=8) MAP and HR were 1.5±1.0 mmHg and -8.4±3.8 bpm during the day and 1.8±0.9 mmHg and -10.8±2.9 bpm during the night compared to baseline. CIH induced significant changes in MAP and HR during the day in both the scRNA (p=.006 MAP; p<.001 HR) and shRNA (p=.007 MAP; p<.001 HR) groups. Compared to control rats, during the night CIH induced significant changes in MAP in the scRNA rats (p=0.02) but not in the shRNA group and no significant changes in HR in either group. The number of TH-immunoreactive neurons was reduced by 20% in sections with GFP fluorescence; shRNA 28±1 cells/section compared to scRNA 35±1 cells/section (p=.005). CIH induces an increase in MAP that persists beyond the period of exposure to CIH. Knockdown of TH in the NTS reduces this persistent increase in MAP induced by exposure to CIH. This indicates that NTS A2 neurons play a role in the cardiovascular responses to CIH and suggests they may play a similar role in the pathology of sleep apnea in humans.

A novel strategy for intrastriatal dopaminergic cell transplantation: Sequential “nest” grafting influences survival and behavioral recovery in a rat model of Parkinson's disease

Neural transplantation in experimental parkinsonism (PD) is limited by poor survival of grafted embryonic dopaminergic (DA) cells. In this proof-of-principle study we hypothesized that a first regular initial graft may create a “dopaminergic” environment similar to the perinatal substantia nigra and consequently stimulate a subsequent graft. Therefore, we grafted ventral mesencephalic neurons sequentially at different time intervals into the same target localization. Rats with a unilateral lesion of the dopamine neurons produced by injections of 6-hydroxydopamine (6-OHDA) received E14 ventral mesencephalon derived grafts into the DA-depleted striatum. In the control group we grafted all 6 deposits on the first day (d0). The other 4 groups received four graft deposits distributed over 2 implantation tracts followed by a second engraftment injected into the same site 3, 6, 14 and 21 days later. Quantitative assessment of the survival of tyrosine hydroxylase-immunoreactive neurons and graft volume revealed best results for those DA grafts implanted 6 days after the first one. In the present study, a model of short-interval sequential transplantation into the same target-site, so called “nest” grafts were established in the 6-OHDA rat model of PD which might become a useful tool to further elucidate the close neurotrophic and neurotopic interactions between the immediate graft vicinity and the cell suspension graft. In addition, we could show that the optimal milieu was established around the sixth day after the initial transplantation. This may also help to further optimize current transplantation strategies to restore the DA system in patients with PD.

L-DOPA-induced increase in TH-immunoreactive striatal neurons in parkinsonian mice: Insights into regulation and function

Tyrosine hydroxylase (TH)-immunoreactive (ir) neurons have been found in the striatum after dopamine depletion; however, little is known about the mechanism underlying their appearance or their functional significance. We previously showed an increase in striatal TH-ir neurons after l-DOPA treatment in mice with unilateral 6-OHDA lesions in the striatum. In the present study, we further examined the time-course and persistence of the effects of chronic l-DOPA treatment on the appearance and regulation of TH-ir neurons as well as their possible function. We found that the l-DOPA-induced increase in striatal TH-ir neurons is dose-dependent and persists for days after l-DOPA withdrawal, decreasing significantly 10days after l-DOPA treatment ends. Using hemiparkinsonian D1 receptor knock-out (D1R−/−) and D2 receptor knock-out (D2R−/−) mice, we found that the D1R, but not the D2R, is required for the l-DOPA-induced appearance of TH-ir neurons in the dopamine-depleted striatum. Interestingly, our experiments in aphakia mice, which lack Pitx3 expression in the brain, indicate that the l-DOPA-dependent increase in the number of TH-ir neurons is independent of Pitx3, a transcription factor necessary for the development of mesencephalic dopaminergic neurons. To explore the possible function of l-DOPA-induced TH-ir neurons in the striatum, we examined dopamine overflow and forelimb use in l-DOPA-treated parkinsonian mice. These studies revealed a tight spatio-temporal correlation between the presence of striatal TH-ir neurons, the recovery of electrically stimulated dopamine overflow in the lesioned striatum, and the recovery of contralateral forelimb use with chronic l-DOPA treatment. Our results suggest that the presence of TH-ir neurons in the striatum may underlie the long-duration response to l-DOPA following withdrawal. Promotion of these neurons in the early stages of Parkinson's disease, when dopamine denervation is incomplete, may be beneficial for maintaining motor function.

Human Albumin Prevents 6-Hydroxydopamine-Induced Loss of Tyrosine Hydroxylase in In Vitro and In Vivo

Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis.

Tenuigenin Protects Dopaminergic Neurons from Inflammation‐Mediated Damage Induced by the Lipopolysaccharide

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive death of dopaminergic neurons in the substantia nigra pars compacta (SNpc). To study if tenuigenin (TEN), the main active component of Polygala tenuifolia, can protect dopaminergic neurons from inflammation-mediated damage in vivo. We observed the effects of TEN on lipopolysaccharide (LPS) induced PD model by behavioral analysis, high-performance liquid chromatography, immunohistochemistry and enzyme-linked immunoadsorbent assay, etc. We showed that a single intranigral dose of LPSs (10 μg) induced microglial activation, reduced the survival ratio of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the SNpc and reduced dopamine (DA) content in the striatum. Treatment with 300 mg/kg TEN once per day over 14 weeks improved the survival rate of TH-ir neurons in the SNpc to 75%, on the non-injected side. Treatment with 200 or 300 mg/kg TEN once per day over 14 weeks significantly improved DA levels in the striatum to 73% and 81% on the non-injected side, respectively. The excessive production of cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, was abolished by TEN administration. Our results suggest that TEN may play a role in protecting dopaminergic neurons against inflammatory challenge.

Distribution of neurons containing catecholamines in brain of hermit crab Pagurus middendorffii and of king crab Paralithodes camtschaticus (Anomura, Decapoda)

Distribution of catecholamine-containing neurons in the brain of two species of crustaceans—hermit crab Pagurus middendorffii and the king crab Paralithodes camtschaticus was studied by immunocytochemical methods using an antibody against tyrosine hydroxylase (TH). In both species of the studied crustaceans, morphologically different types of TH-immunoreactive neurons are detected in cell clusters in brain protocerebrum, deutocerebrum, and tritocerebrum. Similarities and differences in the topography, numbers, and morphology of TH-positive neurons in different brain structures in the hermit crab and king crab are discussed.

GIRK2 expression in dopamine neurons of the substantia nigra and ventral tegmental area

G-protein-regulated inward-rectifier potassium channel 2 (GIRK2) is reported to be expressed only within certain dopamine neurons of the substantia nigra (SN), although very limited data are available in humans. We examined the localization of GIRK2 in the SN and adjacent ventral tegmental area (VTA) of humans and mice by using either neuromelanin pigment or immunolabeling with tyrosine hydroxylase (TH) or calbindin. GIRK2 immunoreactivity was found in nearly every human pigmented neuron or mouse TH-immunoreactive neuron in both the SN and VTA, although considerable variability in the intensity of GIRK2 staining was observed. The relative intensity of GIRK2 immunoreactivity in TH-immunoreactive neurons was determined; in both species nearly all SN TH-immunoreactive neurons had strong GIRK2 immunoreactivity compared with only 50-60% of VTA neurons. Most paranigral VTA neurons also contained calbindin immunoreactivity, and approximately 25% of these and nearby VTA neurons also had strong GIRK2 immunoreactivity. These data show that high amounts of GIRK2 protein are found in most SN neurons as well as in a proportion of nearby VTA neurons. The single previous human study may have been compromised by the fixation method used and the postmortem delay of their controls, whereas other studies suggesting that GIRK2 is located only in limited neuronal groups within the SN have erroneously included VTA regions as part of the SN. In particular, the dorsal layer of dopamine neurons directly underneath the red nucleus is considered a VTA region in humans but is commonly considered the dorsal tier of the SN in laboratory species.

Curcumin protects nigral dopaminergic neurons by iron-chelation in the 6-hydroxydopamine rat model of Parkinson’s disease

Curcumin is a plant polyphenolic compound and a major component of spice turmeric (Curcuma longa). It has been reported to possess free radical-scavenging, iron-chelating, and anti-inflammatory properties in different tissues. Our previous study showed that curcumin protects MES23.5 dopaminergic cells from 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro. The present study aimed to explore this neuroprotective effect in the 6-OHDA-lesioned rat model of Parkinson's disease in vivo. Rats were given intragastric curcumin for 24 days. 6-OHDA lesioning was conducted on day 4 of curcumin treatment. Dopamine content was assessed by high-performance liquid chromatography with electrochemical detection, tyrosine hydroxylase (TH)-containing neurons by immunohistochemistry, and iron-containing cells by Perls' iron staining. The dopamine content in the striatum and the number of TH-immunoreactive neurons decreased after 6-OHDA treatment. Curcumin pretreatment reversed these changes. Further studies demonstrated that 6-OHDA treatment increased the number of iron-staining cells, which was dramatically decreased by curcumin pretreatment. The protective effects of curcumin against 6-OHDA may be attributable to the iron-chelating activity of curcumin to suppress the iron-induced degeneration of nigral dopaminergic neurons.

Effects of cannabinoids Δ(9)-tetrahydrocannabinol, Δ(9)-tetrahydrocannabinolic acid and cannabidiol in MPP+ affected murine mesencephalic cultures

Cannabinoids derived from Cannabis sativa demonstrate neuroprotective properties in various cellular and animal models. Mitochondrial impairment and consecutive oxidative stress appear to be major molecular mechanisms of neurodegeneration. Therefore we studied some major cannabinoids, i.e. delta-9-tetrahydrocannabinolic acid (THCA), delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in mice mesencephalic cultures for their protective capacities against 1-methyl-4-phenyl pyridinium (MPP+) toxicity. MPP+ is an established model compound in the research of parkinsonism that acts as a complex I inhibitor of the mitochondrial respiratory chain, resulting in excessive radical formation and cell degeneration. MPP+ (10μM) was administered for 48h at the 9th DIV with or without concomitant cannabinoid treatment at concentrations ranging from 0.01 to 10μM.All cannabinoids exhibited in vitro antioxidative action ranging from 669±11.1 (THC), 16±3.2 (THCA) to 356±29.5 (CBD) μg Trolox (a vitamin E derivative)/mg substance in the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) assay. Cannabinoids were without effect on the morphology of dopaminergic cells stained by tyrosine hydroxylase (TH) immunoreaction. THC caused a dose-dependent increase of cell count up to 17.3% at 10μM, whereas CBD only had an effect at highest concentrations (decrease of cell count by 10.1–20% at concentrations of 0.01–10μM). It influenced the viability of the TH immunoreactive neurons significantly, whereas THCA exerts no influence on dopaminergic cell count.Exposure of cultures to 10μM of MPP+ for 48h significantly decreased the number of TH immunoreactive neurons by 44.7%, and shrunken cell bodies and reduced neurite lengths could be observed. Concomitant treatment of cultures with cannabinoids rescued dopaminergic cells. Compared to MPP+ treated cultures, THC counteracted toxic effects in a dose-dependent manner. THCA and CBD treatment at a concentration of 10μM lead to significantly increased cell counts to 123% and 117%, respectively. Even though no significant preservation or recovery of neurite outgrowth to control values could be observed, our data show that cannabinoids THC and THCA protect dopaminergic neurons against MPP+ induced cell death.

Adeno‐Associated Virus Labeling of Catecholaminergic neurons in Nucleus of Solitary Tract (NTS) of Rats

A2 catecholaminergic neurons located within caudal and sub‐postremal NTS play a critical role in modulating responses to physiological stress and project to multiple brain stem and forebrain hypothalamic regions. A2 neurons by definition express tyrosine hydroxylase, the rate‐limiting enzyme in catecholamine synthesis. In vitro analyses of A2 neurons in the rat have been limited as identification of A2 neurons requires labeling of neuron and post‐mortem immunohistochemistry. As a result, one does not know the phenotype of the neuron at the time of study. To overcome this challenge we have used commercially available adeno ‐ associated virus (AAV) vector mediated delivery of green fluorescent protein (GFP) labeled TH promoter (AAV‐GFP‐TH), which will incorporate into the TH genome and express GFP with the TH expression. To verify that GFP expression was restricted to catecholaminergic neurons, conventional immunohistochemistry for TH was performed on sections from AAV‐GFP‐TH injected animals. We found that, 78.4 ± 3.07% TH immunoreactive neurons to be GFP labeled and 95 ± 2.40% (n=3 sections) of the GFP expressing neurons of caudal NTS to be TH‐immunoreactive, making our technique a reliable and accurate way to label A2 neurons prior to in vitro study. Brain slices from these rats can be used for direct visualization of TH‐immunoreactive neurons without any need for further processing.

Physical (In)activity dependent changes in the morphology of RVLM neurons

The rostral ventrolateral medulla (RVLM) contains neurons critical for cardiovascular regulation. Functional plasticity in RVLM neurons may contribute to increased sympathoexcitation in disease states. Here, we aimed to define morphological differences in RVLM cardiovascular neurons in sedentary vs physically active rats, hypothesizing that increased sympathoexcitation in sedentary rats would correlate with more dendritic branching. Physically active rats were provided with in‐cage running wheels for 14 weeks and ran 230 ± 81 km (n=4). We used immunohistochemistry for tyrosine hydroxylase (TH) in 150 μm brainstem sections and tracing software to reconstruct TH‐immunoreactive neurons at the caudal pole of the facial nucleus. Comparison of dendrites in 3 sedentary vs 4 active rats showed an average length of 1111±54 μm vs. 686±86 μm (p<0.05) and 9.7±0.7 branch nodes vs. 7.0±1.1 (p=0.058). A Sholl analysis showed 82±4.2 vs. 52.8±6.2 total intersections (p<0.05). These data suggest that greater dendritic branching in RVLM cardiovascular neurons contributes to increased sympathoexcitation and incidence of cardiovascular disease in sedentary individuals. (F30‐HL105003; NHMRC 480414; R01‐HL096787).

Orexin A in rat rostral ventrolateral medulla is pressor, sympatho‐excitatory, increases barosensitivity and attenuates the somato‐sympathetic reflex

The rostral ventrolateral medulla (RVLM) maintains sympathetic nerve activity (SNA), and integrates adaptive reflexes. Orexin A-immunoreactive neurones in the lateral hypothalamus project to the RVLM. Microinjection of orexin A into RVLM increases blood pressure and heart rate. However, the expression of orexin receptors, and effects of orexin A in the RVLM on splanchnic SNA (sSNA), respiration and adaptive reflexes are unknown. The effect of orexin A on baseline cardio-respiratory variables as well as the somato-sympathetic, baroreceptor and chemoreceptor reflexes in RVLM were investigated in urethane-anaesthetized, vagotomized and artificially ventilated male Sprague-Dawley rats (n= 50). orexin A and its receptors were detected with fluorescence immunohistochemistry. Tyrosine hydroxylase-immunoreactive neurones in the RVLM were frequently co-localized with orexin 1 (OX(1) ) and orexin 2 (OX(2) ) receptors and closely apposed to orexin A-immunoreactive terminals. Orexin A injected into the RVLM was pressor and sympatho-excitatory. Peak effects were observed at 50 pmol with increased mean arterial pressure (42 mmHg) and SNA (45%). Responses to orexin A (50 pmol) were attenuated by the OX(1) receptor antagonist, SB334867, and reproduced by the OX(2) receptor agonist, [Ala(11) , D-Leu(15) ]orexin B. Orexin A attenuated the somato-sympathetic reflex but increased baroreflex sensitivity. Orexin A increased or reduced sympatho-excitation following hypoxia or hypercapnia respectively. Although central cardio-respiratory control mechanisms at rest do not rely on orexin, responses to adaptive stimuli are dramatically affected by the functional state of orexin receptors.

Susceptibility to a parkinsonian toxin varies during primate development

Symptoms of Parkinson's disease typically emerge later in life when loss of nigrostriatal dopamine neuron function exceeds the threshold of compensatory mechanisms in the basal ganglia. Although nigrostriatal dopamine neurons are lost during aging, in Parkinson's disease other detrimental factors must play a role to produce greater than normal loss of these neurons. Early development has been hypothesized to be a potentially vulnerable period when environmental or genetic abnormalities may compromise central dopamine neurons. This study uses a specific parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to probe the relative vulnerability of nigrostriatal dopamine neurons at different stages of primate development. Measures of dopamine, homovanillic acid, 1-methyl-pyridinium concentrations and tyrosine hydroxylase immunoreactive neurons indicated that at mid-gestation dopamine neurons are relatively vulnerable to MPTP, whereas later in development or in the young primate these neurons are resistant to the neurotoxin. These studies highlight a potentially greater risk to the fetus of exposure during mid-gestation to environmental agents that cause oxidative stress. In addition, the data suggest that uncoupling protein-2 may be a target for retarding the progressive loss of nigrostriatal dopamine neurons that occurs in Parkinson's disease and aging.

Curcumin Protects Nigrostriatal Dopaminergic Neurons and Reduces Glial Activation in 6-Hydroxydopamine Hemiparkinsonian Mice Model

This study investigated the effects of curcumin on nigrostriatal dopaminergic (DA) neurons and glial response in 6-hydroxydopamine (6-OHDA) hemiparkinsonian mice. Following unilateral intrastriatal 6-OHDA injection, mice were daily injected with curcumin for seven days, beginning on the day of lesion. Seven days after 6-OHDA lesioning, sections from the striatum and the substantia nigra pars compacta (SNpc) were collected and immunohistochemically stained for DA neurons and reactive glia. Curcumin decreased 6-OHDA-induced loss of nigral tyrosine hydroxylase-immunoreactive (TH-IR) neurons and striatal TH-IR fibers. The neuroprotection was coincided with a significant attenuation of microglial and astroglial reaction in the SNpc and the striatum. These results suggest that the neuroprotective effects of curcumin in 6-OHDA-lesioned mice may be mediated through its anti-inflammatory properties or direct protection on nigral DA neurons, thereby reducing neuronal injury-induced glial activation.

Blockade of mGluR5 Reverses Abnormal Firing of Subthalamic Nucleus Neurons in 6-Hydroxydopamine Partially Lesioned Rats

Activation of metabotropic glutamate receptor 5 (mGluRs) in the subthalamic nucleus (STN) results in burst-firing activity of STN neurons, which is similar to that observed in Parkinson's disease (PD). We examined the effects of chronic and systemic treatment with 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective mGluR5 antagonist, in firing activity of STN neurons in partially lesioned rats by 6-hydroxydopamine (6-OHDA). In 6-OHDA-lesioned rats treated with vehicle, injection of 6-OHDA (4 microg) into the medial forebrain bundle produced a partial lesion causing 36% loss of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the substantia nigra pars compacta (SNpc). The 6-OHDA lesion in vehicle-treated rats showed an increasing firing rate and a more irregular firing pattern of STN neurons. Whereas chronic, systemic treatment of MPEP (3 mg/kg/day, 14 days) produced neuroprotecive effects on the TH-ir neurons and normalized the hyperactive firing activity of STN neurons in 6-OHDA partially lesioned rats. These data demonstrate that partial lesion of the nigrostriatal pathway increases firing activity of STN neurons in the rat, and chronic, systemic MPEP treatment has the neuroprotective effect and reverses the abnormal firing activity of STN neurons, suggesting that MPEP has an important implication for the treatment of PD.

Neurorescue Effect of Rosmarinic Acid on 6-Hydroxydopamine-Lesioned Nigral Dopamine Neurons in Rat Model of Parkinson's Disease

Rosmarinic acid (RA) is a naturally occurring polyphenolic compound. It has been reported that RA possessed antioxidant and anti-inflammatory properties. Our previous study showed that RA could protect MES23.5 dopaminergic cells against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro. The purpose of this study was to explore the neuroreparative (neurorescue) effect of RA on 6-OHDA-lesioned rat model of Parkinson's disease (PD) in vivo. In this study, the rats were given RA orally after intrastriatal 6-OHDA lesion. Results showed that the dopamine content in the striatum decreased and the numbers of tyrosine hydroxylase-immunoreactive neurons reduced after 6-OHDA treatment. RA treatment after 6-OHDA administration could restore these changes. Further studies demonstrated that 6-OHDA treatment increased the iron-staining positive cells, which were markedly decreased by RA treatment. Moreover, RA suppressed the increased ratio of Bax/Bcl-2 at gene level induced by 6-OHDA. This indicates that the neurorescue effects of RA against 6-ODHA-induced degeneration of the nigrostriatal dopaminergic system were achieved by decreasing nigral iron levels and regulating the ratio of Bcl-2/Bax gene expression.