TH-immunoreactive Cells Research Articles

Influence of manganese exposure via intracerebral injection on behavioristics and substantia nigra dopaminergic neurons in rats

To investigate the changes in behavioristics, substantia nigra tyrosine hydroxylase (TH) immunoreactive cells, and ultrastructure of substantia nigra neurons in rats after manganese exposure via intracerebral injection. A total of 72 healthy male Sprague-Dawley rats were randomly divided into exposure group and control group, with 36 rats in each group. The stereotactic technique was used for injection of 1 μl MnCl2·4H2O (1 mol/L) into the corpus striatum in the exposure group, and the control group was injected with the same volume of normal saline. The changes in rotational behavior, number of TH immunoreactive cells in the substantia nigra, and ultrastructure of the substantia nigra induced by apomorphine were observed at 8 hours and 1, 3, 7, 15, and 30 days after injection, and this measure was compared with manganese exposure via intraperitoneal injection. After apomorphine induction for 30 minutes, the exposure group showed a gradual increase in the number of rotations over the time of manganese exposure (F= 176.921, P<0.01) and a significantly higher number of rotations than the control group (F=482.654, P<0.01). The exposure group showed a gradual reduction in the mean A value of TH immunoreactive cells in the injured substantia nigra over the time of exposure (F=12.009, P<0.01) , and this value differed significantly between the injured substantia nigra in the exposure group and the contralateral substantia nigra in the exposure group and the injured side in the control group (F=36.131, P<0.01). At 3, 7, 15, and 30 days after exposure, the injured substantia nigra showed a significantly lower mean A value of TH immunoreactive cells than the contralateral side in the exposure group (all P<0.01). At 7, 15, and 30 days, the injured substantia nigra in the exposure group showed a significantly lower mean A value than the injured side in the control group (all P<0.01). After manganese exposure, substantia nigra neurons showed the changes including mitochondrial swelling, dilatation of the endoplasmic reticulum, and demyelination of nerve fibers in different stages, which suggested the dynamic process of dopaminergic neuron injuries. Compared with manganese exposure via intraperitoneal injection, manganese exposure via intracerebral injection can induce behavioral changes and injuries of the substantia nigra-striatum system more quickly. Manganese exposure via intracerebral injection induces behavioral changes and injuries of substantia nigra dopaminergic neurons in rats within a short time.

Neurons with different neurotransmitters in embryonic neocortical allografts in the rat sciatic nerve

Different subsets of interneurons in the Wistar rat neocortex and in neocortical transplants developing in a damaged nerve were identified by the following immunohistochemical markers: glutamate decarboxylase (GAD 67) for GABAergic nerve cells, NO-synthase (NOS) for NO-ergic neurons, choline acetyltransferase (ChAT) for cholinergic cells, and tyrosine hydroxylase for catecholaminergic structures. Twenty-eight days after surgery, individual GAD 67-ir, NO-ir, ChAT-ir, and very rarely TH-ir cells were detected in the graft. It was shown that the number of GAD 67-ir neurons per unit area in the grafts was less than in the rat neocortex P20.

IGF-1 protects dopamine neurons against oxidative stress: association with changes in phosphokinases.

Insulin-like growth factor-1 (IGF-1) is an endogenous peptide transported across the blood brain barrier that is protective in several brain injury models, including an acute animal model of Parkinson's disease (PD). Motor deficits in PD are due largely to the progressive loss of nigrostriatal dopaminergic neurons. Thus, we examined the neuroprotective potential of IGF-1 in a progressive model of dopamine deficiency in which 6-hydroxydopamine (6-OHDA) is infused into the striatum. Rats received intrastriatal IGF-1 (5 or 50µg) 6h prior to infusion of 4µg 6-OHDA into the same site and were euthanized 1 or 4weeks later. Both concentrations of IGF-1 protected tyrosine hydroxylase (TH) immunoreactive terminals in striatum at 4weeks but not at 1week, indicating that IGF-induced restoration of the dopaminergic phenotype occurred over several weeks. TH-immunoreactive cell loss was only attenuated with 50µg IGF-1. We then examined the effect of striatal IGF-1 on the Ras/ERK1/2 and PI3K/Akt pathways to ascertain whether their activation correlated with IGF-1-induced protection. Striatal and nigral levels of phospho-ERK1/2 were maximal 6h after IGF-1 infusion and, with the exception of an increase in nigral pERK2 at 48h, returned to basal levels by 7days. Phospho-Akt (Ser473) was elevated 6-24h post-IGF-1 infusion in both striatum and substantia nigra concomitant with inhibition of pro-death GSK-3β, a downstream target of Akt. These results suggest that IGF-1 can protect the nigrostriatal pathway in a progressive PD model and that this protection is preceded by activation of key pro-survival signaling cascades.

Dopaminergic-primed fetal liver mesenchymal stromal–like cells can reverse parkinsonian symptoms in 6-hydroxydopamine-lesioned mice

Background aimsCell replacement therapy is considered a promising alternative in the treatment of degenerative diseases, and in this context, mesenchymal stromal cells (MSCs) have been proposed for transplantation in Parkinson disease (PD). Thus far, the results of animal studies are found to be inconsistent and inconclusive regarding the therapeutic ability of the cells. This study investigated the efficacy of fetal liver (FL)-MSC-derived dopaminergic (DA) neuronal primed cells for correction of parkinsonian symptoms in mice. MethodsFL-MSCs were differentiated for 21 days in the presence of a combination of neurotropic factors. The extent of cellular reprogramming was analyzed by quantitative polymerase chain reaction for DA-specific neuronal gene expressions and protein expressions by immuno-cytochemistry. The functionality of the cells was determined by electrophysiology and dopamine release assays. Ten-day-primed neuron-like cells or unprimed MSCs were transplanted into the 6-hydroxydopamine (6-OHDA)-lesioned striatum using a stereotaxic device. Dopamine-secreting properties and behavioral studies were used to assess improvement of parkinsonian symptoms. ResultsThe differentiated cells expressed DA-specific genes and proteins, while exhibiting a high level of voltage-gated potassium current. Furthermore, neuronal primed cells differentiated into tyrosine hydroxylase immunoreactive and dopamine-secreting functional neuron-like cells. Symptomatic correction of PD in the recipient mice within 2 months of transplantation was also observed. DiscussionFL-MSC-derived primed neuron-like cells integrated into the striatum of PD mice, improving parkinsonian symptoms. This study demonstrates an effective cell-based therapy for PD.

PET Imaging of Serotonin Transporters With 4-[(18)F]-ADAM in a Parkinsonian Rat Model With Porcine Neural Xenografts.

Parkinson's disease (PD) is a neurodegenerative disease characterized by a loss of dopaminergic neurons in the nigrostriatal pathway. Apart from effective strategies to halt the underlying neuronal degeneration, cell replacement now offers novel prospects for PD therapy. Porcine embryonic neural tissue has been considered an alternative source to human fetal grafts in neurodegenerative disorders because its use avoids major practical and ethical issues. This study was undertaken to evaluate the effects of embryonic day 27 (E27) porcine mesencephalic tissue transplantation in a PD rat model using animal positron emission tomography (PET) coupled with 4-[(18)F]-ADAM, a serotonin transporter (SERT) imaging agent. The parkinsonian rat was induced by injecting 6-hydroxydopamine into the medial forebrain bundle (MFB) of the right nigrostriatal pathway. The apomorphine-induced rotation behavioral test and 4-[(18)F]-ADAM/animal PET scanning were carried out following 6-OHDA lesioning. At the second week following 6-OHDA lesioning, the parkinsonian rat rotates substantially on apomorphine-induced contralateral turning. In addition, the mean striatal-specific uptake ratio (SUR) of 4-[(18)F]-ADAM decreased by 44%. After transplantation, the number of drug-induced rotations decreased markedly, and the mean SUR of 4-[(18)F]-ADAM and the level of SERT immunoreactivity (SERT-ir) in striatum were partially restored. The mean SUR level was restored to 71% compared to that for the contralateral intact side, which together with the abundant survival of tyrosine hydroxylase (TH) neurons accounted for functional recovery at the fourth week postgraft. In regard to the extent of donor-derived cells, we found the neurons of the xenografts from E27 transgenic pigs harboring red fluorescent protein (RFP) localized with TH-ir cells and SERT-ir in the grafted area. Thus, transplanted E27 porcine mesencephalic tissue may restore dopaminergic and serotonergic systems in the parkinsonian rat. The 4-[(18)F]-ADAM/animal PET can be used to detect serotonergic neuron loss in PD and monitor the efficacy of therapy.

The Sensory Impact of Nicotine on Noradrenergic and Dopaminergic Neurons of the Nicotine Reward - Addiction Neurocircuitry.

The sensory experience of smoking is a key component of nicotine addiction known to result, in part, from stimulation of nicotinic acetylcholine receptors (nAChRs) at peripheral sensory nerve endings. Such stimulation of nAChRs is followed by activation of neurons at multiple sites in the mesocorticolimbic reward pathways. However, the neurochemical profiles of CNS cells that mediate the peripheral sensory impact of nicotine remain unknown. In the present study in mice, we first used c-Fos immunohistochemistry to identify CNS cells stimulated by nicotine (NIC, 40 μg/kg, IP) and by a peripherally-acting analog of nicotine, nicotine pyrrolidine methiodide (NIC-PM, 30 μg/kg, IP). Sequential double-labelling was then performed to determine whether noradrenergic and dopaminergic neurons of the nicotine reward-addiction circuitry were primary targets of NIC and NIC-PM. Double-labelling of NIC and/or NIC-PM activated c-Fos immunoreactive cells with tyrosine hydroxylase (TH) showed no apparent c-Fos expression by the dopaminergic cells of the ventral tegmental area (VTA). With the exception of sparse numbers of TH immunoreactive D11 cells, dopamine-containing neurons in other areas of the reward-addiction circuitry, namely periaqueductal gray, and dorsal raphe, were also devoid of c-Fos immunoreactivity. Noradrenergic neurons of locus coeruleus (LC), known to innervate VTA, were activated by both NIC and NIC-PM. These results demonstrate that noradrenergic neurons of LC are among the first structures that are stimulated by single acute IP injection of NIC and NIC-PM. Dopaminergic neurons of VTA and other CNS sites, did not respond to acute IP administration of NIC or NIC-PM by induction of c-Fos.

Interactions between Kisspeptin Neurons and Hypothalamic Tuberoinfundibular Dopaminergic Neurons in Aged Female Rats.

Kisspeptin neurons in the arcuate nucleus (ARC) regulate prolactin secretion, and are in physical contact with tuberoinfundibular dopaminergic (TIDA) neurons, which inhibit prolactin secretion. Prolactin levels in the blood are increased with advancing age in rats; therefore, we investigated the interactions with TIDA neurons and kisspeptin neurons in aged female rats (24 months of age), relative to those of young adult female rats (9–10 weeks of age). Plasma prolactin levels in the aged rats were significantly higher than those of young adult rats. Tyrosine hydroxylase (TH)-immunoreactive (ir) cell bodies and kisspeptin-ir nerve fibers were found in the dorsomedial ARC of both groups. The number of TH-ir cell bodies in the dorsomedial ARC did not differ significantly between groups. Additionally, no significant differences in the number of TH-ir cells in contact with kisspeptin-ir fibers was observed between groups. However, the number of kisspeptin-ir or Kiss1 mRNA-expressing cells in the ARC was significantly reduced in the aged rats compared with that of the young rats. These results suggest that the contacts between TIDA neurons and kisspeptin neurons are maintained after reproductive senescence, while production of kisspeptin in the ARC decreases significantly during aging.

Neural crest-derived resident cardiac cells contribute to the restoration of adrenergic function of transplanted heart in rodent.

We investigated whether neural crest-derived cardiac resident cells contribute to the restoration of intrinsic adrenergic function following transplantation in mice. Transplanted heart shows partial restoration of cardiac adrenergic activity with time. Both the intrinsic cardiac adrenergic system and extrinsic sympathetic re-innervation contribute to neuronal remodelling in the transplanted heart. Little is known about the origin and function of the intrinsic system in the transplanted heart. Heart from the protein 0-Cre/Floxed-Enhanced Green Fluorescent Protein double-transgenic mouse was transplanted onto the abdominal aorta of the non-obese diabetic/severe combined immunodeficient mouse to trace the fate of cardiac resident neural crest-derived cells. Sympathetic nerve fibres, which are predominantly localized to the epicardial surface of the heart, disappeared in the transplanted heart. Intramyocardial neural crest cells increased immediately, while neural crest-derived nucleated tyrosine hydroxylase (TH)-immunoreactive cells increased over 2 weeks following transplantation. The mRNA expression levels of TH, dopamine-β-hydroxylase and phenylethanolamine N-methyltransferase, and the tissue content of catecholamines in the transplanted hearts increased with time in association with an increase in the number of neural crest-derived nucleated TH-immunoreactive cells and tissue nerve growth factor levels. Iodine-123-metaiodobenzylguanidine scintigraphy showed that the uptake ability of transplanted heart for catecholamines also recovered with time. Finally, the chronotropic response to tyramine both in vivo and ex vivo reappeared 2 weeks after transplantation. Neural crest-derived adrenergic cells increased following heart transplantation. The restoration of cardiac sympathetic activities in transplanted heart is tightly coupled with an increase in the number of neural crest-derived adrenergic cells.

Tyrosine hydroxylase immunoreactivity is common in the enteric nervous system in teleosts.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines and TH immunoreactivity is indicative of cells synthesising either adrenaline/noradrenaline or dopamine. In this study, the distribution of TH immunoreactivity was examined in two distantly related teleost species, zebrafish (Danio rerio) and shorthorn sculpin (Myoxocephalus scorpius). In both species, TH-immunoreactive nerve cell bodies and varicose nerve fibres were common in the myenteric plexus of the intestine. However, no TH-immunoreactive nerve cell bodies were seen in the sculpin stomach. The TH-immunoreactive nerve cell bodies seemed to constitute a larger proportion of the total enteric population in shorthorn sculpin (50 ± 5%, n = 3067 cells) compared with zebrafish (14 ± 2%, n = 10,163 cells). In contrast, in sculpin, the TH-immunoreactive cells were smaller than the average enteric nerve cell bodies, whereas in zebrafish, the relationship was the opposite. In developing zebrafish larvae, TH-immunoreactive nerve cell bodies were common (approx. 75% of the total population) at 3days post-fertilization (dpf), but decreased in numbers between 3 and 7 dpf. In conclusion, in contrast to previous studies, TH-immunoreactive intrinsic neurons are common in the fish gut. Their role and function need to be further characterized in order to understand the potential importance of this enteric subpopulation in controlling various gut functions.

MPTP로 유도된 Parkinson's disease 동물 모델에서 열다한소탕 가감방 (MYH)의 신경 세포 보호 효과

Objectives To evaluate the neuroprotective effects of modified Yuldahanso-tang (MYH) in a Parkinson's disease mouse model. Methods 1) Four groups (each of 8 rats per group) were used in this study. 2) The neuroprotective effect of MYH was examined in a Parkinson's disease mouse model. C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg/day), intraperitoneal (i.p.) for 5 days. 3) The brains of 2 mice per group were removed and frozen at <TEX>$-20^{\circ}C$</TEX>, and the striatum-substantia nigra part was seperated. The protein volume was measured by Bradford method following Bio-Rad protein analyzing kit. Using mouse/Rat Dopamine ELISA Assay Kit. 4) The brains of 2 mice per group were separated and removed. TH-immunohistochemical was examined in the MPTP-induced Parkinson's disease mice to evaluate the neuroprotective effects of MYH on ST and SNpc. 5) Two mice out of each group were anesthetized and skulls were opened from occipital to frontal direction to take out the brains. The brains added TTC solution for 20 minutes for staining. 6) The water tank used for morris water maze test was filled with <TEX>$28^{\circ}C$</TEX> water, and a round platform of 10cm in diameter was installed for mice to step on. The study was carried out once a day within 30 seconds, keep exercising to step on the platform in the pool. 7) The brains of two mice out of each group were fixed in 10% formaldehyde solution and paraphillin substance was infiltrated. They were fragmented by microtome, and observed under an optical microscope after Hematoxylin & Eosin staining. 8) A round acrylic cylinder with its upper side open was filled with clean water and depressive mouse models were forced to swim for 15 minutes. After 24 hours the animals were put in the same equipment for 5 minutes and were forced to swim. 9) The convenient, simple, and accurate high-performance liquid chromatography (HPLC) method was established for simultaneous determination of Neurotransmitters in MPTP-MYH group. Results 1) MYH possess Dopamine cell protective effect on MPTP-induced injury in striatum and substantia nigra pars compacta. 2) MYH inhibits the loss of tyrosine hydroxylase-immunoreacitive (TH-IR) cells in the striatum and substantia nigra pars compacta on MPTP-induced injury in C57BL/6 mice. 3) MYH possesses improvement effect on MPTP-induced memory deterioration in C57BL/6 mice through the reduction of prolongated Sort of lost time by MPTP injection using the Morris water maze test. 4) MYH possesses hippocampal neuron protective effect on MPTP-induced injury in C57BL/6 mice. 5) MYH possesses improvement effect on MPTP-induced motor behaviour deficits and depression in C57BL/6 mice through the reduction of prolongated losing motion by MPTP injection using the Forced swimming test. 6) MYH increases serotonin product amount on MPTP-induced injury in C57BL/6 mice. Conclusions This experiment suggests that the neuroprotective effect of MYH is mediated by the increase in Dopamin, TH-ir cell, Hippocampus and Serotonin. Furthermore, MYH essential oil may serve as a potential preventive or therapeutic agent regarding Parkinson's disease.

Hypothalamic dopaminergic neurons in an animal model of seasonal affective disorder

Light has profound effects on mood regulation as exemplified in seasonal affective disorder (SAD) and the therapeutic benefits of light therapy. However, the underlying neural pathways through which light regulates mood are not well understood. Our previous work has developed the diurnal grass rat, Arvicanthis niloticus, as an animal model of SAD. Following housing conditions of either 12:12h dim light:dark (DLD) or 8:16h short photoperiod (SP), which mimic the lower light intensity or short day-length of winter, respectively, grass rats exhibit an increase in depression-like behavior compared to those housed in a 12:12h bright light:dark (BLD) condition. Furthermore, we have shown that the orexinergic system is involved in mediating the effects of light on mood and anxiety. To explore other potential neural substrates involved in the depressive phenotype, the present study examined hypothalamic dopaminergic (DA) and somatostatin (SST) neurons in the brains of grass rats housed in DLD, SP and BLD. Using immunostaining for tyrosine hydroxylase (TH) and SST, we found that the number of TH- and SST-ir cells in the hypothalamus was significantly lower in the DLD and SP groups compared to the BLD group. We also found that treating BLD animals with a selective orexin receptor 1 (OX1R) antagonist SB-334867 significantly reduced the number of hypothalamic TH-ir cells. The present study suggests that the hypothalamic DA neurons are sensitive to daytime light deficiency and are regulated by an orexinergic pathway. The results support the hypothesis that the orexinergic pathways mediate the effects of light on other neuronal systems that collectively contribute to light-dependent changes in the affective state.

In vitro study for exploring anti-obesity agent using the extracts from Centella spp. of Australia

Purpose: Purpose: To evaluate the autophage and neuroprotective effects of WGM, a Chinese traditional medicinal prescription in a Parkinson’s disease mouse model. Methods: Methods: Cell viability was measuredcusing a LHD-release assay and MTT assay. To find out the autophagyrelative mRNA gene expression in BV2 microglia cell line, LC3, LC3a, LC3b, Beclin-1, ATG5 were examined by RT-PCR. To find out the autophagy-relative protein expression of signal events in BV2 microglia cell line, LC3b-1, LC3b-II, Beclin1 were examined by western blotting. To find out the autophagy detection in BV2 microglia cell line. Cells were assayed for autophagy positive cells by FACS analysis. To measure the amount of dopamine in mice brain, ST-SNpc were examined by Bradford. Immunohistochemistry was examined in the MPTP-induced Parkinson’s disease mice to evaluate the neuroprotective effects of WGM on ST and SNpc. Ischemic mice brain stainedwith TTC in theMPTPinduced Parkinson’s disease to find out ischemia in mouse In order to investigate the effect of WGM on recovery of memory, we examined the memory by usingMorris watermaze test. Immunohistochemistry was examined in the MPTP-induced Parkinson’s disease mice to evaluate the neuroprotective effects of WGM on hippocampal lesion. In order to investigate the effect of WGM on recovery of behavioral deficits, we examined the motor function by using and FST. The convenient, simple, and accurate HPLC method was established for simultaneous determination of Neurotransmitters in MPTP-WGM group. Results: Results and Conclusion: WGM can enhance the induction of autophagy through key regulator beclin1 and LC3b-II. WGM inhalation in MPTP mice led to the restoration of behavioral impairment and rescued dopamine, TH-IR cell, hippocampus, serotonin. Furthermore,WGMessential oilmay serve as a potential preventive or therapeutic agent regarding Parkinson’s disease. Conclusion: Acknowledgement: This research was supported by Basic Science Research Program through the NRF funded by the Ministry of Education, Science and Technology(201401850001) Contact: Taek-Won Ahn, twahn@dju.kr

Evidence for the involvement of dopamine in stress-induced suppression of reproduction in the cichlid fish Oreochromis mossambicus.

In the present study, we examined whether stress-induced suppression of reproduction is mediated through the catecholaminergic neurotransmitter dopamine (DA) in the female cichlid fish Oreochromis mossambicus. In the first experiment, application of antibody against tyrosine hydroxylase (TH; a marker for DA) in brain sections revealed the presence of intensely stained TH immunoreactive cells in the preoptic area (POA) and nucleus preopticus (NPO) during the previtellogenic phase. These cells showed weak immunoreactivity during the vitellogenic and prespawning phases concomitant with darkly stained luteinising hormone (LH) immunoreactive content in the proximal pars distalis (PPD) of the pituitary gland and fully ripened follicles (stage V) in the ovary of control fish. However, in fish exposed to aquacultural stressors, TH secreting cells remained intensely stained in POA and NPO regions during the prespawning phase, indicating increased synthetic and secretory activity, which was reflected by a significantly higher DA content compared to controls. Increased DA activity as a result of stress was associated with a decrease in the LH immunoreactive content in the PPD and an absence of stage V follicles in the ovary. In the second experiment, administration of DA caused effects similar to those in stressed fish, whereas DA receptor antagonist domperidone (DOM) treatment significantly increased the LH content in the PPD and the number of stage V follicles in unstressed fish. On the other hand, treatment of stressed fish with DOM resulted in dark accumulations of LH immunoreactive content in the PPD accompanied by the presence of stage V follicles in the ovary. Taken together, these results suggest an additional pathway for the inhibitory effects of stress through dopaminergic neurones along the reproductive axis.

Expression of Tyrosine Hydroxylase in the Anterior Lobe of the Human Pituitary Gland: A Possible Local Autocrine/Paracrine Role in the Regulation of Pituitary Hormone Secretion

The main source of dopamine (DA) regulating anterior pituitary function is derived from the hypothalamic tuberoinfundibular (TIDA) neurons. Recent studies performed on mice have indicated the presence of a local source of DA because tyrosine hydroxylase (TH), the rate‐limiting enzyme of DA biosynthesis, has been found within the AL. Here, we provide evidence for the presence of TH in human cadaveric pituitary glands. The presence of TH is a good indicator of an intrinsic or local production of DA. Serial axial sections of pituitary glands obtained from two (70 and 87 year old) male cadavers have been histologically prepared and stained with hematoxylin‐eosin (HE) and immunohistochemistry has been performed using specific antibodies for TH. TH immunoreactivity has been found in the AL of both pituitaries. Similar to the AL of the mouse, TH positive cells have been localized bilaterally, at the border of the intermediate lobe (IL) and the AL. Incidentally, a distinct area in the left side of the AL of one of the pituitary glands has been found avoiding HE staining, which indicates a proliferation of cells forming an adenoma called “incidentaloma”. Curiously, an increased number of TH immunoreactive cells have been found in the area of the incidentaloma compared to the normal AL tissue of the same gland. The increased number of TH positive cells within the incidentaloma and the asymptomatic nature of it may suggest a compensatory elevation of local production of DA for being able to inhibit hormone secretion of the adenoma in an autocrine/paracrine manner. Therefore, a new mechanism of DAergic control of pituitary hormone secretion locating within the AL of the human pituitary gland can be hypothesized.

Sex differences and effects of prenatal exposure to excess testosterone on ventral tegmental area dopamine neurons in adult sheep.

Prenatal testosterone (T) excess in sheep results in a wide array of reproductive neuroendocrine deficits and alterations in motivated behavior. The ventral tegmental area (VTA) plays a critical role in reward and motivated behaviors and is hypothesised to be targeted by prenatal T. Here we report a sex difference in the number VTA dopamine cells in the adult sheep, with higher numbers of tyrosine hydroxylase (TH)-immunoreactive (-ir) cells in males than females. Moreover, prenatal exposure to excess T during either gestational days 30-90 or 60-90 resulted in increased numbers of VTA TH-ir cells in adult ewes compared to control females. Stereological analysis confirmed significantly greater numbers of neurons in the VTA of males and prenatal T-treated ewes, which was primarily accounted for by greater numbers of TH-ir cells. In addition, immunoreactivity for TH in the cells was denser in males and prenatal T-treated females, suggesting that sex differences and prenatal exposure to excess T affects both numbers of cells expressing TH and the protein levels within dopamine cells. Sex differences were also noted in numbers of TH-ir cells in the substantia nigra, with more cells in males than females. However, prenatal exposure to excess T did not affect numbers of TH-ir cells in the substantia nigra, suggesting that this sex difference is organised independently of prenatal actions of T. Together, these results demonstrate sex differences in the sheep VTA dopamine system which are mimicked by prenatal treatment with excess T.

Characterization of fetal antigen 1/delta-like 1 homologue expressing cells in the rat nigrostriatal system: effects of a unilateral 6-hydroxydopamine lesion.

Fetal antigen 1/delta-like 1 homologue (FA1/dlk1) belongs to the epidermal growth factor superfamily and is considered to be a non-canonical ligand for the Notch receptor. Interactions between Notch and its ligands are crucial for the development of various tissues. Moreover, FA1/dlk1 has been suggested as a potential supplementary marker of dopaminergic neurons. The present study aimed at investigating the distribution of FA1/dlk1-immunoreactive (-ir) cells in the early postnatal and adult midbrain as well as in the nigrostriatal system of 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian adult rats. FA1/dlk1-ir cells were predominantly distributed in the substantia nigra (SN) pars compacta (SNc) and in the ventral tegmental area. Interestingly, the expression of FA1/dlk1 significantly increased in tyrosine hydroxylase (TH)-ir cells during early postnatal development. Co-localization and tracing studies demonstrated that FA1/dlk1-ir cells in the SNc were nigrostriatal dopaminergic neurons, and unilateral 6-OHDA lesions resulted in loss of both FA1/dlk1-ir and TH-ir cells in the SNc. Surprisingly, increased numbers of FA1/dlk1-ir cells (by 70%) were detected in dopamine-depleted striata as compared to unlesioned controls. The higher number of FA1/dlk1-ir cells was likely not due to neurogenesis as colocalization studies for proliferation markers were negative. This suggests that FA1/dlk1 was up-regulated in intrinsic cells in response to the 6-OHDA-mediated loss of FA1/dlk1-expressing SNc dopaminergic neurons and/or due to the stab wound. Our findings hint to a significant role of FA1/dlk1 in the SNc during early postnatal development. The differential expression of FA1/dlk1 in the SNc and the striatum of dopamine-depleted rats could indicate a potential involvement of FA1/dlk1 in the cellular response to the degenerative processes.

Unaltered retinal dopamine levels in a C57BL/6 mouse model of form-deprivation myopia.

Retinal dopamine has been long implicated in the signaling pathway regulating eye growth, as evidenced by its reduced levels in myopic eyes in various species. We examined whether and how retinal dopamine levels were changed in a C57BL/6 mouse model of experimental myopia. Form-deprivation myopia (FDM) was induced in C57BL/6 mice by wearing monocular occluder for 4 weeks. Refractive errors were measured using an infrared photorefractor. Retinal dopamine/DOPAC and vitreal DOPAC levels were assessed by high-performance liquid chromatography (HPLC). Extracellular dopamine concentrations were examined by Western blot analysis of dopamine transporter (DAT) expression levels. The intactness of retinal dopaminergic system was evaluated by counting tyrosine hydroxylase (TH) immunoreactive cells, measuring the areas occupied by processes of these cells, and quantifying retinal TH expression at both protein and transcription levels. Form-deprivation myopia was successfully induced in C57BL/6 mice with the refractive status of deprived eyes being significantly different from fellow eyes. Unlike most of the previous results obtained in other myopic animal models, however, no significant changes in retinal dopamine, DOPAC, DAT, and vitreal DOPAC levels were detected in deprived eyes, either in the daytime or at night. Furthermore, neither the number of dopaminergic amacrine cells, the area size occupied by the processes of these cells, nor retinal TH expression, were altered in deprived eyes. The retinal dopamine system remains intact in C57BL/6 mice with FDM, and retinal dopamine levels are not associated with the development of FDM in this mouse strain.

Differential neuroprotective and anti-inflammatory effects of L-type voltage dependent calcium channel and ryanodine receptor antagonists in the substantia nigra and locus coeruleus.

Neuroinflammation and degeneration of catecholaminergic brainstem nuclei occur early in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Neuroinflammation increases levels of pro-inflammatory cytokines and reactive oxygen species which can alter neuronal calcium (Ca(+2)) homoeostasis via L-type voltage dependent calcium channels (L-VDCCs) and ryanodine receptors (RyRs). Alterations in Ca(+2) channel activity in the SN and LC can lead to disruption of normal pacemaking activity in these areas, contributing to behavioral deficits. Here, we utilized an in vivo model of chronic neuroinflammation: rats were infused intraventricularly with a continuous small dose (0.25 μg/h) of lipopolysaccharide (LPS) or artificial cerebrospinal fluid (aCSF) for 28 days. Rats were treated with either the L-VDCC antagonist nimodipine or the RyR antagonist dantrolene. LPS-infused rats had significant motor deficits in the accelerating rotarod task as well as abnormal behavioral agitation in the forced swim task and open field. Corresponding with these behavioral deficits, LPS-infused rats also had significant increases in microglia activation and loss of tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra pars compacta (SNpc) and locus coeruleus (LC). Treatment with nimodipine or dantrolene normalized LPS-induced abnormalities in the rotarod and forced swim, restored the number of TH-immunoreactive cells in the LC, and significantly reduced microglia activation in the SNpc. Only nimodipine significantly reduced microglia activation in the LC, and neither drug increased TH immunoreactivity in the SNpc. These findings demonstrate that the Ca(+2) dysregulation in the LC and SN brainstem nuclei is differentially altered by chronic neuroinflammation. Overall, targeting Ca + 2 dysregulation may be an important target for ameliorating neurodegeneration in the SNpc and LC.

Systemic administration of 6-OHDA to rhesus monkeys upregulates HLA-DR expression in brain microvasculature.

BackgroundWe recently developed a nonhuman primate model of cardiac dysautonomia by systemic dosing of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The aim of this study was to assess whether systemic 6-OHDA affects the central nervous system of nonhuman primates, in particular the dopaminergic nigrostriatal system.MethodsBrain sections from adult rhesus monkeys that received systemic 6-OHDA (50 mg/kg intravenously; n=5) and were necropsied 3 months later, as well as normal controls (n=5) were used in this study. Tissue was cut frozen at 40 μm on a sliding microtome, processed for immunohistochemistry, and blindly evaluated.ResultsNeither the optical density of tyrosine hydroxylase immunoreactivity (TH-ir; a dopaminergic neuronal marker) in the caudate and putamen nucleus nor the TH-ir cell number and volume in the substantia nigra showed significant differences between groups. Yet within groups, statistical analysis revealed significant individual differences in the 6-OHDA-treated group, with two animals showing a lower cell count and volume. Optical density quantification of α-synuclein-ir in the substantia nigra did not show differences between groups. As α-synuclein intracellular distribution was noted to vary between animals, it was further evaluated with a semiquantitative scale. A greater intensity and presence of α-synuclein-positive nigral cell bodies was associated with larger TH-positive nigral cell volumes. Increased human leukocyte antigen (HLA-DR; a microglial marker) expression was observed in 6-OHDA-treated animals compared with controls. HLA-DR-ir was primarily localized in endothelial cells and perivascular spaces throughout cortical and subcortical structures. Semiquantitative evaluation using a rating scale revealed higher HLA-DR-ir in blood vessels of 6-OHDA-treated animals than controls, specifically in animals with the lowest number of dopaminergic nigral neurons.ConclusionOur results demonstrate that systemic 6-OHDA administration to rhesus monkeys can affect the dopaminergic nigrostriatal system and upregulate inflammatory markers in the cerebrovasculature that persist 3 months post neurotoxin challenge. The variability of the subject response suggests differences in individual sensitivity to 6-OHDA.

Increased number of TH-immunoreactive cells in the ventral tegmental area after deep brain stimulation of the anterior nucleus of the thalamus.

Dopamine (DA) has been long implicated with the processes of memory. In long-term memory, the hippocampus and ventral tegmental area (VTA) use DA to enhance long-term potentiation, while prefrontal DA D1 receptors are involved in working memory. Deep brain stimulation (DBS) of specific brain areas have been shown to affect memory impairments in animal models. Here, we tested the hypothesis that DBS could reverse memory impairments by increasing the number of dopaminergic cells in the VTA. Rats received DBS at the level of the mammillothalamic tract, the anterior nucleus of the thalamus, and entorhinal cortex before euthanasia. These regions are part of the so-called memory circuit. Brain sections were processed for c-Fos and tyrosine hydroxylase (TH) immunocytochemistry in the VTA and the substantia nigra pars compacta (SNc). c-Fos, TH and c-Fos/TH immunoreactive cells were analyzed by means of stereology and confocal microscopy. Our results showed that DBS of the anterior nucleus of the thalamus induced substantial higher numbers of TH-immunoreactive cells in the VTA, while there were no significant differences between the experimental groups in the number of TH immunoreactive cells in the SNc, c-Fos immunoreactive cells and c-Fos/TH double-labeled cells in both the SNc and VTA. Our findings suggest a phenotypic switch, or neurotransmitter respecification, of DAergic cells specifically in the VTA which may be induced by DBS in the anterior nucleus of the thalamus.