Double-labeled Cells Research Articles

Ectopic transgene expression in the retina of four transgenic mouse lines.

Retinal expression of transgenes was examined in four mouse lines. Two constructs were driven by the choline acetyltransferase (ChAT) promoter: green fluorescent protein conjugated to tau protein (tau-GFP) or cytosolic yellow fluorescent protein (YFP) generated through CRE recombinase-induced expression of Rosa26 (ChAT-CRE/Rosa26YFP). Two other constructs targeted inhibitory interneurons: GABAergic horizontal and amacrine cells identified by glutamic acid decarboxylase (GAD65-GFP) or parvalbumin (PV) cells (PV-CRE/Rosa26YFP). Animals were transcardially perfused and retinal sections prepared. Antibodies against PV, calretinin (CALR), calbindin (CALB), and tyrosine hydroxylase (TH) were used to counterstain transgene-expressing cells. In PVxRosa and ChAT-tauGFP constructs, staining appeared in vertically oriented row of processes resembling Müller cells. In the ChATxRosa construct, populations of amacrine cells and neurons in the ganglion cell layer were labeled. Some cones also exhibited GFP fluorescence. CALR, PV and TH were found in none of these cells. Occasionally, we found GFP/CALR and GFP/PV double-stained cells in the ganglion cell layer (GCL). In the GAD65-GFP construct, all layers of the neuroretina were labeled, except photoreceptors. Not all horizontal cells expressed GFP. We did not find GFP/TH double-labeled cells and GFP was rarely present in CALR- and CALB-containing cells. Many PV-positive neurons were also labeled for GFP, including small diameter amacrines. In the GCL, single labeling for GFP and PV was ascertained, as well as several CALR/PV double-stained neurons. In the GCL, cells triple labeled with GFP/CALR/CALB were sparse. In conclusion, only one of the four transgenic constructs exhibited an expression pattern consistent with endogenous retinal protein expression, while the others strongly suggested ectopic gene expression.

Impact of neonatal anoxia on adult rat hippocampal volume, neurogenesis and behavior

Neonates that suffer oxygen deprivation during birth can have long lasting cognitive deficits, such as memory and learning impairments. Hippocampus, one of the main structures that participate in memory and learning processes, is a plastic and dynamic structure that conserves during life span the property of generating new cells which can become neurons, the so-called neurogenesis. The present study investigated whether a model of rat neonatal anoxia, that causes only respiratory distress, is able to alter the hippocampal volume, the neurogenesis rate and has functional implications in adult life. MRI analysis revealed significant hippocampal volume decrease in adult rats who had experienced neonatal anoxia compared to control animals for rostral, caudal and total hippocampus. In addition, these animals also had 55.7% decrease of double-labelled cells to BrdU and NeuN, reflecting a decrease in neurogenesis rate. Finally, behavioral analysis indicated that neonatal anoxia resulted in disruption of spatial working memory, similar to human condition, accompanied by an anxiogenic effect. The observed behavioral alterations caused by oxygen deprivation at birth might represent an outcome of the decreased hippocampal neurogenesis and volume, evidenced by immunohistochemistry and MRI analysis. Therefore, based on current findings we propose this model as suitable to explore new therapeutic approaches.

Longitudinal tracking of triple labeled umbilical cord derived mesenchymal stromal cells in a mouse model of Amyotrophic Lateral Sclerosis

The translational potential of cell therapy to humans requires a deep knowledge of the interaction between transplanted cells and host tissues. In this study, we evaluate the behavior of umbilical cord mesenchymal stromal cells (UC-MSCs), labeled with fluorescent nanoparticles, transplanted in healthy or early symptomatic transgenic SOD1G93A mice (a murine model of Amyotrophic Lateral Sclerosis). The double labeling of cells with nanoparticles and Hoechst-33258 enabled their tracking for a long time in both cells and tissues. Whole-body distribution of UC-MSCs was performed by in-vivo and ex-vivo analyses 1, 7, 21days after single intravenous or intracerebroventricular administration. By intravenous administration cells were sequestered by the lungs and rapidly cleared by the liver. No difference in biodistribution was found among the two groups. On the other hand, UC-MSCs transplanted in lateral ventricles remained on the choroid plexus for the whole duration of the study even if decreasing in number. Few cells were found in the spinal cord of SOD1G93A mice exclusively. No migration in brain parenchyma was observed. These results suggest that the direct implantation in brain ventricles allows a prolonged permanence of cells close to the damaged areas and makes this method of tracking reliable for future studies of efficacy.

Nestin-Positive Ependymal Cells Are Increased in the Human Spinal Cord after Traumatic Central Nervous System Injury

Endogenous neural progenitor cell niches have been identified in adult mammalian brain and spinal cord. Few studies have examined human spinal cord tissue for a neural progenitor cell response in disease or after injury. Here, we have compared cervical spinal cord sections from 14 individuals who died as a result of nontraumatic causes (controls) with 27 who died from injury with evidence of trauma to the central nervous system. Nestin immunoreactivity was used as a marker of neural progenitor cell response. There were significant increases in the percentage of ependymal cells that were nestin positive between controls and trauma cases. When sections from lumbar and thoracic spinal cord were available, nestin positivity was seen at all three spinal levels, suggesting that nestin reactivity is not simply a localized reaction to injury. There was a positive correlation between the percentage of ependymal cells that were nestin positive and post-injury survival time but not for age, postmortem delay, or glial fibrillary acidic protein (GFAP) immunoreactivity. No double-labelled nestin and GFAP cells were identified in the ependymal, subependymal, or parenchymal regions of the spinal cord. We need to further characterize this subset of ependymal cells to determine their role after injury, whether they are a population of neural progenitor cells with the potential for proliferation, migration, and differentiation for spinal cord repair, or whether they have other roles more in line with hypothalamic tanycytes, which they closely resemble.

4,4'-Diisothiocyanostilbene-2,2'-disulfonic Acid (DIDS) Ameliorates Ischemia-Hypoxia-Induced White Matter Damage in Neonatal Rats through Inhibition of the Voltage-Gated Chloride Channel ClC-2.

Chronic cerebral hypoperfusion is believed to cause white matter lesions (WMLs), leading to cognitive impairment. Previous studies have shown that inflammation and apoptosis of oligodendrocytes (OLs) are involved in the pathogenesis of WMLs, but effective treatments have not been studied. In this study, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a chloride (Cl−) channel blocker, was injected into chronic cerebral ischemia-hypoxia rat models at different time points. Our results showed that DIDS significantly reduced the elevated mRNA levels and protein expression of chloride channel 2 (ClC-2) in neonatal rats induced by ischemia-hypoxia. Meanwhile, DIDS application significantly decreased the concentrations of reactive oxygen species (ROS); and the mRNA levels of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha TNF-α in neonatal rats with hypoxic-ischemic damage. Myelin staining was weaker in neonatal rats with hypoxic-ischemic damage compared to normal controls in corpus callosum and other white matter, which was ameliorated by DIDS. Furthermore, the elevated number of caspase-3 and neural/glial antigen 2 (NG-2) double-labeled positive cells was attenuated by DIDS after ischemia anoxic injury. Administration of DIDS soon after injury alleviated damage to OLs much more effectively in white matter. In conclusion, our study suggests that early application of DIDS after ischemia-hypoxia injury may partially protect developing OLs.

Effect of lysosomal and ubiquitin-proteasome system dysfunction on the abnormal aggregation of α-synuclein in PC12 cells.

The aim of this study was to investigate the effect of lysosomal and ubiquitin-proteasome system dysfunction on the abnormal aggregation of α-synuclein, and to analyze its role in the pathogenesis of Parkinson's disease (PD). PC12 cells subjected to nerve growth factor-induced differentiation were used as the cell model to study the dopaminergic neurons, and the lysosomal and proteasomal inhibitors trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E64) and, respectively, were used exclusively and in combination to treat the PC12 cells. The viability and metabolic state of the cells was assessed using the MTT assay; flow cytometry was used to measure the rate of cell apoptosis; and the double immunofluorescence method was applied to observe the formation of thioflavin S- and α-synuclein protein-positive aggregates and inclusion bodies in the PC12 cells. In addition, the Hoechst 33258 staining method was used to observe the apoptosis of the α-synuclein protein and thioflavin-S double-labeled cells. Following the administration of the lysosomal and proteasomal pathway inhibitors, the cell viability decreased in a concentration-dependent manner and the cell apoptosis rate increased. The proportion of PC12 cells with α-synuclein protein-positive aggregates and inclusion bodies in the E64 group was 7.94%, compared with 20.33 and 36.77% in the lactacystin and combination treatment groups, respectively. Statistical analysis indicated that the number of inclusion body-positive cells in the treatment groups was significantly higher than that in the control group (3.78%) (P<0.05). Apoptosis was evident in the double-positive cells with α-synuclein protein-positive inclusion bodies (17.29±1.54%). In conclusion, lysosomal and proteasomal dysfunction may play an important role in the pathogenesis of PD through the induction of abnormal α-synuclein protein aggregation in dopaminergic neurons.

Salivary gland homeostasis is maintained through acinar cell self-duplication.

Current dogma suggests that salivary gland homeostasis is stem cell dependent. However, the extent of stem cell contribution to salivary gland maintenance has not been determined. We investigated acinar cell replacement during homeostasis, growth, and regeneration, using an inducible CreER(T2) expressed under the control of the Mist1 gene locus. Genetic labeling, followed by a chase period, showed that acinar cell replacement is not driven by the differentiation of unlabeled stem cells. Analysis using R26(Brainbow2.1) reporter revealed continued proliferation and clonal expansion of terminally differentiated acinar cells in all major salivary glands. Induced injury also demonstrated the regenerative potential of pre-labeled acinar cells. Our results support a revised model for salivary gland homeostasis based predominantly on self-duplication of acinar cells, rather than on differentiation of stem cells. The proliferative capacity of differentiated acinar cells may prove critical in the implementation of cell-based strategies to restore the salivary glands.

Alpha-lipoic acid treatment is neurorestorative and promotes functional recovery after stroke in rats.

The antioxidant properties of alpha-lipoic acid (aLA) correlate with its ability to promote neuroproliferation. However, there have been no comprehensive studies examining the neurorestorative effects of aLA administration after the onset of ischemia. The middle cerebral artery (MCA) of adult rats was occluded for 2 hours and then reperfused. aLA (20 mg/kg) was administered in 71 animals (aLA group) through the left external jugular vein immediately after reperfusion. An equivalent volume of vehicle was administered to 71 animals (control group). Functional outcome, levels of endogenous neural precursors with neurogenesis, glial cell activation, and brain metabolism were evaluated. Immediate aLA administration after reperfusion resulted in significantly reduced mortality, infarct size, and neurological deficit score (NDS) in the test group compared to the control group. Long-term functional outcomes, measured by the rotarod test, were markedly improved by aLA treatment. There was a significant increase in the number of cells expressing nestin and GFAP in the boundary zone and infarct core regions after aLA treatment. Furthermore, significantly more BrdU/GFAP, BrdU/DCX, and BrdU/NeuN double-labeled cells were observed along the boundary zone of the aLA group on days 7, 14, and 28 days, respectively. And brain metabolism using 18F-FDG microPET imaging was markedly improved in aLA group. The effects of aLA was blocked by insulin receptor inhibitor, HNMPA (AM)3. These results indicate that immediate treatment with aLA after ischemic injury may have significant neurorestorative effects mediated at least partially via insulin receptor activation. Thus, aLA may be useful for the treatment of acute ischemic stroke.

The spino-bulbar-cerebellar pathway: organization and neurochemical properties of spinal cells that project to the lateral reticular nucleus in the rat.

In addition to classical spinocerebellar pathways, the cerebellum receives information from the spinal cord indirectly via spino-bulbar-cerebellar systems. One of the structures in this pathway is the lateral reticular nucleus (LRt). We performed series of experiments to investigate the organization and neurotransmitter content of spinoreticular tract (SRT) neurons in the lumbar spinal cord that project to the LRt. Three rats received injections of the b subunit of Cholera toxin (CTb) or Fluorogold (FG) within the left and right LRt. The majority of SRT cells (56–61%) were found within the contralateral medial intermediate gray matter where small numbers (7–10%) of double-labeled cells were also present on both sides of the cord. Six rats received unilateral spinal injections of CTb to label spinal projections to the LRt. Injections of FG were made also into the anterior lobe of the cerebellum to label LRt pre-cerebellar neurons. Terminals were found mainly ipsilateral to spinal injection sites within the central and ventrolateral regions of the LRt. Immunocytochemical analysis of SRT terminals revealed that the majority (75%) were contained vesicular glutamate transporter 2 but a minority (20%) contained the vesicular GABA transporter. The inhibitory subpopulation was found to be GABAergic, glycinergic, or contained both transmitters. Inhibitory and excitatory terminals were present within overlapping regions of the nucleus. Most CTb terminals contacting LRt pre-cerebellar neurons were excitatory (80%) whereas a minority were inhibitory and most cells (88%) received contacts from both inhibitory and excitatory terminals. This study shows that SRT axons in the LRt have the capacity to exert direct excitatory and inhibitory actions on LRt pre-cerebellar neurons. Thus spinal cord input has the capacity to facilitate or depress the activity of individual LRt cells which in turn adjust activity in the cerebellum to produce coordinated motor behaviors.

Peripheral nerve injury activates convergent nociceptive input to dorsal horn neurons from neighboring intact nerve

Previous studies demonstrated that peripheral nerve injury induced excessive nociceptive response of spinal cord dorsal horn neurons and such change has been proposed to reflect the development of neuropathic pain state. The aim of this study was to examine the spinal dorsal horn for convergence of nociceptive input to second-order neurons deafferented by peripheral nerve injury. Double immunofluorescence labeling for c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) was performed to detect convergent synaptic input to spinal dorsal horn neurons after the saphenous nerve injury. c-Fos expression and the phosphorylation of ERK were induced by noxious heat stimulation of the hindpaw and by electrical stimulation of the injured or uninjured saphenous nerve, respectively. Within the central terminal field of the saphenous nerve, the number of c-Fos protein-like immunoreactive (c-Fos-IR) cell profiles was significantly decreased at 3 days and returned to the control level by 14 days after the injury. p-ERK immunoreactive (p-ERK-IR) cell profiles were distributed in the central terminal field of the saphenous nerve, and the topographic distribution pattern and number of such p-ERK-IR cell profiles remained unchanged after the nerve injury. The time course of changes in the number of double-labeled cell profiles was similar to that of c-Fos-IR cell profiles after the injury. These results indicate that convergent primary nociceptive input through neighboring intact nerves contributes to increased responsiveness of spinal dorsal horn nociceptive neurons.

Melanin-concentrating hormone inputs to the nucleus accumbens originate from distinct hypothalamic sources and are apposed to GABAergic and cholinergic cells in the Long-Evans rat brain

Melanin-concentrating hormone [MCH] is a neuropeptide that modulates several behaviors, such as feeding and reward. Because the hedonic and rewarding features of a food also influence feeding behavior, the nucleus accumbens [Acb] has been highlighted as a key area integrating these roles. Functional data confirm that MCH acts on a subdivision of the Acb; however, considering the importance of finding anatomical and neurochemical data that correlate the previously demonstrated function of MCH, we delineated this investigation based on the following points: (1) Is there a pattern of innervation by MCH fibers regarding the subregions within the Acb? (2) Specifically, which hypothalamic nuclei synthesize MCH and innervate the Acb? (3) Finally, what are the neurochemical identities of the accumbal neurons innervated by MCH inputs? We examined the MCH immunoreactivity [MCH-ir] in the Acb in rat brains using the peroxidase technique. Additionally, after injecting retrograde neuronal tracer [Fluoro-Gold® – FG®] into subdivisions of the Acb [shell or core], we mapped single- or double-labeled cells. Moreover, using a double immunoperoxidase protocol, we investigated the MCH-ir fibers for gamma-aminobutyric acid [GABA]-ir and choline acetyltransferase [ChAT]-ir cells in the shell subdivision of the Acb [AcbSh]. We found that the MCH-ir fibers preferentially innervate the medial AcbSh, particularly the septal pole. This innervation originated from the incerto-hypothalamic area [IHy], internuclear area, lateral hypothalamic area, perifornical area, periventricular nucleus and posterior hypothalamus. Moreover, the IHy has the highest relationship between double/single retrogradely labeled cells [n=5.33±0.66/16±0.93, i.e. 33.33%] in the whole hypothalamus. Furthermore, our data suggest that MCH-ir fibers are in apposition to GABAergic and cholinergic cells in the AcbSh. Therefore, we provide anatomical support to the ongoing functional studies investigating the relation among the hypothalamus, MCH transmission into the Acb and the involvement of known neuronal phenotypes within the AcbSh.

Expression of fragile X mental retardation protein in neurons and glia of the developing and adult mouse brain

Fragile X syndrome is the most common inherited form of mental retardation and autism. It is caused by a reduction or elimination of the expression of fragile X mental retardation protein (FMRP). Because fragile X syndrome is a neurodevelopmental disorder, it is important to fully document the cell type expression in the developing CNS to provide a better understanding of the molecular function of FMRP, and the pathogenesis of the syndrome. We investigated FMRP expression in the brain using double-labeling immunocytochemistry and cell type markers for neurons (NeuN), astrocytes (S100β), microglia (Iba-1), and oligodendrocyte precursor cells (NG2). The hippocampus, striatum, cingulate cortex, retrosplenial cortex, corpus callosum and cerebellum were assessed in wild-type C57/BL6 mice at postnatal days 0, 10, 20, and adult. Our results demonstrate that FMRP is ubiquitously expressed in neurons at all times and brain regions studied, except for corpus callosum where FMRP was predominantly present in astrocytes at all ages. FMRP expression in Iba-1 and NG2-positive cells was detected at postnatal day 0 and 10 and gradually decreased to very low or undetectable levels in postnatal day 20 and adult mice. Our results reveal that in addition to continuous and extensive expression in neurons in the immature and mature brain, FMRP is also present in astrocytes, oligodendrocyte precursor cells, and microglia during the early and mid-postnatal developmental stages of brain maturation. Prominent expression of FMRP in glia during these crucial stages of brain development suggests an important contribution to normal brain function, and in its absence, to the fragile X phenotype.

The role of exogenous neural stem cells transplantation in cerebral ischemic stroke.

To observe the effects of neural stem cells (NSCs) transplantation in rats' striatum and subventricular zone (SVZ) in rat models of focal cerebral ischemia and reperfusion. Hippocampus was extracted from fetal rats with 14 days of gestation. Suspension culture was used to isolate and culture the rat's NSCs. A cerebral ischemia and reperfusion rat's model was made on the left side of the brain through occlusion of the left middle cerebral artery. Neurological signs were assessed by Zea Longa's five-grade scale, with scores 1, 2, and 3 used to determine the successful establishment of the rat's model. The NSCs were stereotaxically injected into the left striatum 24 hours after the successful rat's model was built. Rats were then randomly divided into 5 groups, namely, normal group, sham operation group, ischemia group, PBS transplantation group, and NSCs transplantation group, each of which was observed on day 3, day 7, and day 14. The ischemia-related neurological deficits were assessed by using a 7-point evaluation criterion. Forelimb injuries were evaluated in all rats using the foot-fault approach. Infarct size changes were observed through TTC staining and cell morphology and structure in the infarct region were investigated by Nissl staining. Apoptosis and apoptosis-positive cell counts were studied by Tunel assay. Expressions of double-labeling positive cells in the striatum and subventricular zone (SVZ) were observed by BrdU/NeuN and BrdU/GFAP fluorescent double-labeling method and the number of positive cells in the striatum and SVZ was counted. Results from the differently treated groups showed that right hemiplegia occurred in the ischemia group, PBS transplantation group, and NSCs transplantation group in varying degrees. Compared with the former two groups, there was least hemiplegia in the NSCs transplantation group. The TTC staining assay showed that rats in the NSCs transplantation group had smaller infarct volume than those from the PBS transplantation group. The Nissl dyeing showed that there was a large area of neuronal necrosis and apoptosis in the ischemia and PBS transplantation groups, and damage was mainly focused in the striatum. Degeneration and damage of nerve cells were significantly reduced in the NSCs transplantation group. The Tunel assay showed that the number of apoptosis-positive cells in the NSCs transplantation group was less than that in the PBS transplantation group at each time point. Double immunofluorescent labeling showed that the proliferation of endogenous neural stem cells began at the third day, reaching the peak at the 7th day, and was significantly reduced at the 14th day in the SVZ. The number of BrdU/NeuN increased significantly in the NSCs transplantation group compared to that in the PBS transplantation group (P < 0.05). The number of BrdU/GFAP decreased significantly in the NSCs transplantation group compared to that of PBS transplantation group (P < 0.05). The number of BrdU/GFAP-positive cells in the striatum was observed to be much more in the PBS transplantation group than in the NSCs transplantation group. Both neurological deficits and coordination capacity of rats with cerebral ischemia were significantly improved via transplantation of the neural stem cells. In conclusion, transplantation of neural stem cells can therefore possibly promote the differentiation of endogenous NSCs into neurons and reduce their differentiation towards glial cells. Transplantation of the neural stem cells may also change the ischemic microenvironment of striatum, possibly inhibiting the proliferation of glial cells.

Neurotensin: revealing a novel neuromodulator circuit in the nucleus accumbens-parabrachial nucleus projection of the domestic chick.

Lower brainstem projections from nucleus accumbens (Ac) subregions to the parabrachial complex (PB), the nucleus of the solitary tract and the vagal motor nuclei have been described previously in the domestic chick by our group. Such projections, particulary those from the core and rostral pole regions of Ac have not been found in mammals or pigeons. Here we report on the presence of neurotensin (NT) in the neurons projecting from different Ac subnuclei, and also from the bed nucleus of stria terminalis, to the PB in the domestic chicken. The study is based upon correlated retrograde tracing (using Fast Blue) and NT immunohistochemistry, supplemented with regional charting and quantitative analysis of double-labeled neurons. The number of retrogradely labeled cells in Ac subdivisions reflects the size of FB tracer deposit, and the degree to which it extends to the medial PB. Of all Ac subregions, the core contained the largest amount of double-labeled cells. The findings demonstrate that the anatomical pathway through which the Ac can directly modulate taste-responsive neurons of the PB employs mainly neurotensin as a neuromodulator. The observed anatomical difference between mammals and birds is either a general taxonomic feature or it reflects feeding strategies specific for the domestic chick. The results are also relevant to a better understanding of the role of NT in food intake and reward-related behaviors in birds.

Somatostatin and corticotrophin releasing hormone cell types are a major source of descending input from the forebrain to the parabrachial nucleus in mice.

The pontine parabrachial nucleus (PBN) receives substantial descending input from higher order forebrain regions that exerts inhibitory and excitatory influences on taste-evoked responses. Somatostatin (Sst) and corticotrophin releasing hormone (Crh) reporter mice were used in conjunction with injection of the retrograde tracer CTb-488 into the caudal PBN to determine the extent to which Sst and Crh cell types contribute to the descending pathways originating in the lateral hypothalamus (LH), central nucleus of the amygdala (CeA), bed nucleus of the stria terminalis (BNST), and insular cortex (IC). Five to 7 days following injections, the animals were euthanized and tissue sections prepared for confocal microscopy. Crh cell types in each forebrain site except IC project to the PBN with the greatest percentage originating in the BNST. For Sst cell types, the largest percentage of double-labeled cells was found in the CeA followed by the BNST. Few retrogradely labeled cells in the LH coexpressed Sst, whereas no double-labeled cells were observed in IC. The present results suggest that Sst and Crh cell types are a substantial component of the descending pathways from the amygdala and/or BNST to the PBN and are positioned to exert neuromodulatory effects on central taste processing.

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.

Activation of lateral hypothalamus-projecting parabrachial neurons by intraorally delivered gustatory stimuli.

The present study investigated a subpopulation of neurons in the mouse parabrachial nucleus (PbN), a gustatory and visceral relay area in the brainstem, that project to the lateral hypothalamus (LH). We made injections of the retrograde tracer Fluorogold (FG) into LH, resulting in fluorescent labeling of neurons located in different regions of the PbN. Mice were stimulated through an intraoral cannula with one of seven different taste stimuli, and PbN sections were processed for immunohistochemical detection of the immediate early gene c-Fos, which labels activated neurons. LH projection neurons were found in all PbN subnuclei, but in greater concentration in lateral subnuclei, including the dorsal lateral subnucleus (dl). Fos-like immunoreactivity (FLI) was observed in the PbN in a stimulus-dependent pattern, with the greatest differentiation between intraoral stimulation with sweet (0.5 M sucrose) and bitter (0.003 M quinine) compounds. In particular, sweet and umami-tasting stimuli evoked robust FLI in cells in the dl, whereas quinine evoked almost no FLI in cells in this subnucleus. Double-labeled cells were also found in the greatest quantity in the dl. Overall, these results support the hypothesis that the dl contains direct a projection to the LH that is activated preferentially by appetitive compounds; this projection may be mediated by taste and/or postingestive mechanisms.

Short term morphine exposure in vitro alters proliferation and differentiation of neural progenitor cells and promotes apoptosis via mu receptors.

BackgroundChronic morphine treatment inhibits neural progenitor cell (NPC) progression and negatively effects hippocampal neurogenesis. However, the effect of acute opioid treatment on cell development and its influence on NPC differentiation and proliferation in vitro is unknown. We aim to investigate the effect of a single, short term exposure of morphine on the proliferation, differentiation and apoptosis of NPCs and the mechanism involved.MethodsCell cultures from 14-day mouse embryos were exposed to different concentrations of morphine and its antagonist naloxone for 24 hours and proliferation, differentiation and apoptosis were studied. Proliferating cells were labeled with bromodeoxyuridine (BrdU) and cell fate was studied with immunocytochemistry.ResultsCells treated with morphine demonstrated decreased BrdU expression with increased morphine concentrations. Analysis of double-labeled cells showed a decrease in cells co-stained for BrdU with nestin and an increase in cells co-stained with BrdU and neuron-specific class III β-tubuline (TUJ1) in a dose dependent manner. Furthermore, a significant increase in caspase-3 activity was observed in the nestin- positive cells. Addition of naloxone to morphine-treated NPCs reversed the anti-proliferative and pro-apoptotic effects of morphine.ConclusionsShort term morphine exposure induced inhibition of NPC proliferation and increased active caspase-3 expression in a dose dependent manner. Morphine induces neuronal and glial differentiation and decreases the expression of nestin- positive cells. These effects were reversed with the addition of the opioid antagonist naloxone. Our results demonstrate the effects of short term morphine administration on the proliferation and differentiation of NPCs and imply a mu-receptor mechanism in the regulation of NPC survival.

Effects of tumor necrosis factor inhibitor on serum level of HLA-B27 and PDCD-1 in patients with ankylosing spondylitis.

The aim of this study was to evaluate the effect of tumor necrosis factor-alpha (TNF-α) inhibitor-infliximab on ankylosing spondylitis (AS) patients and detect the serum level of HLA-B27 and PDCD-1 before and after TNF inhibitor treatment. 138 patients at active stage of AS were treated with infliximab; serum was collected before and after TNF-α inhibitor treatment for analysis. Reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry, and enzyme-linked immuno sorbent assay were applied to detect the levels of HLA-B27 and PDCD-1 at different time points, which were used for statistical analysis with clinical data including two AS indicators (erythrocyte sedimentation rate--ESR and C-reactive protein--CRP). After the treatment for 6 weeks, RT-PCR showed that the gene expressions of HLA-B27 and PDCD-1 were significantly downregulated compared with baseline before infliximab treatment (P < 0.05); flow cytometry showed that the HLA-B27 and PDCD-1 double-labeled cells were significantly downregulated (P < 0.05). After 2, 6, or 10 weeks of infliximab treatment, the levels of ESR, CRP, serum HLA-B27, and PDCD-1 of the AS patients were all significantly lower than the baseline levels (P < 0.05), and the serum HLA-B27 and PDCD-1 levels were all significantly correlated with ESR (P < 0.05). Infliximab, an anti-TNF-α inhibitor, decreases significantly not only ESR and CRP, but also the serum levels of HLA-B27 and PDCD-1 in patients with AS. HLA-B27 and PDCD-1 are involved in the pathogenesis, and disease activities of AS. HLA-B27 and PDCD-1 are potentially the useful markers of AS activity and useful parameters to evaluate the effectiveness of anti-TNF-α inhibitor in treating AS.

Sex differences in the expression of estrogen receptor alpha within noradrenergic neurons in the sheep brain stem

In female sheep, high levels of estrogen exert a positive feedback action on gonadotropin releasing hormone (GnRH) secretion to stimulate a surge in luteinizing hormone (LH) secretion. Part of this action appears to be via brain stem noradrenergic neurons. By contrast, estrogen action in male sheep has a negative feedback action to inhibit GnRH and LH secretion. To investigate whether part of this sex difference is due to differences in estrogen action in the brain stem, we tested the hypothesis that the distribution of estrogen receptor α (ERα) within noradrenergic neurons in the brain stem differs between rams and ewes. To determine the distribution of ERα, we used double-label fluorescence immunohistochemistry for dopamine β-Hydroxylase, as a marker for noradrenergic and adrenergic cells, and ERα. In the ventrolateral medulla (A1 region), most ERα-immunoreactive (-ir) cells were located in the caudal part of the nucleus. Overall, there were more ERα-ir cells in rams than ewes, but the proportion of double-labeled cells was did not differ between sexes. Much greater numbers of ERα–ir cells were found in the nucleus of the solitary tract (A2 region), but <10% were double labeled and there were no sex differences. The majority of ERα-labeled cells in this nucleus was located in the more rostral areas. ERα-labeled cells were found in several rostral brain stem regions but none of these were double labeled and so were not quantified. Because there was no sex difference in the number of ERα-ir cells in the brain stem that were noradrenergic, the sex difference in the action of estrogen on gonadotropin secretion in sheep is unlikely to involve actions on brain stem noradrenergic cells.