Brainstem development requires galactosylceramidase and is critical for pathogenesis in a model of Krabbe disease.

Krabbe disease (KD) is caused by a deficiency of galactosylceramidase (GALC), which induces demyelination and neurodegeneration due to accumulation of cytotoxic psychosine. Hematopoietic stem cell transplantation (HSCT) improves clinical outcomes in KD patients only if delivered pre-symptomatically. Here, we hypothesize that the restricted temporal efficacy of HSCT reflects a requirement for GALC in early brain development. Using a novel Galc floxed allele, we induce ubiquitous GALC ablation (Galc-iKO) at various postnatal timepoints and identify a critical period of vulnerability to GALC ablation between P4-6 in mice. Early Galc-iKO induction causes a worse KD phenotype, higher psychosine levels in the rodent brainstem and spinal cord, and a significantly shorter life-span of the mice. Intriguingly, GALC expression peaks during this critical developmental period in mice. Further analysis of this mouse model reveals a cell autonomous role for GALC in the development and maturation of immature T-box-brain-1 positive brainstem neurons. These data identify a perinatal developmental period, in which neuronal GALC expression influences brainstem development that is critical for KD pathogenesis.

Oxidative injuries induced by maternal low-protein diet in female brainstem.

Many studies have shown that a maternal low-protein diet increases the susceptibility of offspring to cardiovascular disease in later-life. Moreover, a lower incidence of cardiovascular disease in females than in males is understood to be largely due to the protective effect of high levels of estrogens throughout a woman's reproductive life. However, to our knowledge, the role of estradiol in moderating the later-life susceptibility of offspring of nutrient-deprived mothers to cardiovascular disease is not fully understood. The present study is aimed at investigating whether oxidative stress in the brainstem caused by a maternal low-protein diet administered during a critical period of fetal/neonatal brain development (i.e during gestation and lactation) is affected by estradiol levels. Female Wistar rat offspring were divided into four groups according to their mothers' diets and to the serum estradiol levels of the offspring at the time of testing: (1) 22 days of age/control diet: (2) 22 days of age/low-protein diet; (3) 122 days of age/control diet: (4) 122 days of age/low-protein diet. Undernutrition in the context of low serum estradiol compared to undernutrition in a higher estradiol context resulted in increased levels of oxidative stress biomarkers and a reduction in enzymatic and non-enzymatic antioxidant defenses. Total global oxy-score showed oxidative damage in 22-day-old rats whose mothers had received a low-protein diet. In the 122-day-old group, we observed a decrease in oxidative stress biomarkers, increased enzymatic antioxidant activity, and a positive oxy-score when compared to control. We conclude from these results that following a protein deficiency in the maternal diet during early development of the offspring, estrogens present at high levels at reproductive age may confer resistance to the oxidative damage in the brainstem that is very apparent in pre-pubertal rats.

Oxidative stress and Na,K-ATPase activity differential regulation in brainstem and forebrain of Wistar Audiogenic rats may lead to increased seizure susceptibility.

The Wistar Audiogenic Rat (WAR) is a well-characterized seizure-prone, inbred rodent strain that, when acutely stimulated with high-intensity sounds, develops brainstem-dependent tonic-clonic seizures that can evolve to limbic-like, myoclonic (forebrain) seizures when the acoustic stimuli are presented chronically (audiogenic kindling). In order to investigate possible mechanisms underlying WAR susceptibility to seizures, we evaluated Na,K-ATPase activity, Ca-ATPase activity, Mg-ATPase activity, lipid membrane composition and oxidative stress markers in whole forebrain and whole brainstem samples of naïve WAR, as compared to samples from control Wistar rats. We also evaluated the expression levels of α1 and α3 isoforms of Na,K-ATPase in forebrain samples. We observed increased Na,K-ATPase activity in forebrain samples and increased oxidative stress markers (lipid peroxidation, glutathione peroxidase and superoxide dismutase) in brainstem samples of WAR. The Ca-ATPase activity, Mg-ATPase activity, lipid membrane composition and expression levels of α1 and α3 isoforms of Na,K-ATPase were unaltered. In view of previous data showing that the membrane potentials from naïve WAR's neurons are less negative than that from neurons from Wistar rats, we suggest that Na,K-ATPase increased activity might be involved in a compensatory mechanism necessary to maintain WAR's brains normal activity. Additionally, ongoing oxidative stress in the brainstem could bring Na,K-ATPase activity back to normal levels, which may explain why WAR's present increased susceptibility to seizures triggered by high-intensity sound stimulation.

CD73 Controls Extracellular Adenosine Generation in the Trigeminal Nociceptive Nerves.

Purinergic signaling is involved in pain generation and modulation in the nociceptive sensory nervous system. Adenosine triphosphate (ATP) induces pain via activation of ionotropic P2X receptors while adenosine mediates analgesia via activation of metabotropic P1 receptors. These purinergic signaling are determined by ecto-nucleotidases that control ATP degradation and adenosine generation. Using enzymatic histochemistry, we detected ecto-AMPase activity in dental pulp, trigeminal ganglia (TG) neurons, and their nerve fibers. Using immunofluorescence staining, we confirmed the expression of ecto-5'-nucleotidase (CD73) in trigeminal nociceptive neurons and their axonal fibers, including the nociceptive nerve fibers projecting into the brainstem. In addition, we detected the existence of CD73 and ecto-AMPase activity in the nociceptive lamina of the trigeminal subnucleus caudalis (TSNC) in the brainstem. Furthermore, we demonstrated that incubation with specific anti-CD73 serum significantly reduced the ecto-AMPase activity in the nociceptive lamina in the brainstem. Our results indicate that CD73 might participate in nociceptive modulation by affecting extracellular adenosine generation in the trigeminal nociceptive pathway. Disruption of TG neuronal ecto-nucleotidase expression and axonal terminal localization under certain circumstances such as chronic inflammation, oxidant stress, local constriction, and injury in trigeminal nerves may contribute to the pathogenesis of orofacial neuropathic pain.

Perinatal low-protein diet alters brainstem antioxidant metabolism in adult offspring.

BACKGROUND AND OBJECTIVES: Studies in humans and animal models have established a close relationship between early environment insult and subsequent risk of development of non-communicable diseases, including the cardiovascular. Whereas experimental evidences highlight the early undernutrition and the late cardiovascular disease relation, the central mechanisms linking the two remain unknown. Owing to the oxidative balance influence in several pathologies, the aim of the present study was to evaluate the effects of maternal undernutrition (i.e. a low-protein (LP) diet) on oxidative balance in the brainstem. METHODS AND RESULTS: Male rats from mothers fed with an LP diet (8% casein) throughout the perinatal period (i.e. gestation and lactation) showed 10× higher lipid peroxidation levels than animals treated with normoprotein (17% casein) at 100 days of age. In addition, we observed the following reductions in enzymatic activities: superoxide dismutase, 16%; catalase, 30%; glutathione peroxidase, 34%; glutathione-S-transferase, 51%; glutathione reductase, 23%; glucose-6-phosphate dehydrogenase, 31%; and in non-enzymatic glutathione system, 46%. DISCUSSION: This study is the first to focus on the role of maternal LP nutrition in oxidative balance in a central nervous system structure responsible for cardiovascular control in adult rats. Our data observed changes in oxidative balance in the offspring, therefore, bring a new concept related to early undernutrition and can help in the development of a new clinical strategy to combat the effects of nutritional insult. Wherein the central oxidative imbalance is a feasible mechanism underlying the hypertension risk in adulthood triggered by maternal LP diet.

Activation of PI3K/Akt signaling in rostral ventrolateral medulla impairs brain stem cardiovascular regulation that underpins circulatory depression during mevinphos intoxication.

As the most widely used pesticides in the globe, the organophosphate compounds are understandably linked with the highest incidence of suicidal poisoning. Whereas the elicited toxicity is often associated with circulatory depression, the underlying mechanisms require further delineation. Employing the pesticide mevinphos as our experimental tool, we evaluated the hypothesis that transcriptional upregulation of nitric oxide synthase II (NOS II) by NF-κB on activation of the PI3K/Akt cascade in the rostral ventrolateral medulla (RVLM), the brain stem site that maintains blood pressure and sympathetic vasomotor tone, underpins the circulatory depressive effects of organophosphate poisons. Microinjection of mevinphos (10 nmol) bilaterally into the RVLM of anesthetized Sprague-Dawley rats induced a progressive hypotension that was accompanied sequentially by an increase (Phase I) and a decrease (Phase II) of an experimental index for the baroreflex-mediated sympathetic vasomotor tone. There were also progressive augmentations in PI3K or Akt enzyme activity and phosphorylation of p85 or Akt(Thr308) subunit in the RVLM that were causally related to an increase in NF-κB transcription activity and elevation in NOS II or peroxynitrite expression. Loss-of-function manipulations of PI3K or Akt in the RVLM significantly antagonized the reduced baroreflex-mediated sympathetic vasomotor tone and hypotension during Phase II mevinphos intoxication, and blunted the increase in NF-κB/NOS II/peroxynitrite signaling. We conclude that activation of the PI3K/Akt cascade, leading to upregulation of NF-κB/NOS II/peroxynitrite signaling in the RVLM, elicits impairment of brain stem cardiovascular regulation that underpins circulatory depression during mevinphos intoxication.

Cisplatin causes over-expression of tachykinin NK(1) receptors and increases ERK1/2- and PKA- phosphorylation during peak immediate- and delayed-phase emesis in the least shrew (Cryptotis parva) brainstem.

Scant information is available regarding the effects of cisplatin on the expression profile of tachykinin NK(1) receptors and downstream signaling during cisplatin-induced emesis. Cisplatin causes peak early- and delayed-phase emesis in the least shrew at 1-2 and 33 h post-injection. To investigate the expression profile of NK(1) receptor during both emetic phases, we cloned the cDNA corresponding to a ~700 base pairs of mRNA flanked by two stretches of nucleotides conserved among different species and demonstrated that the shrew NK(1) receptor nucleotide sequence shares ~90% sequence identity with the human NK(1) receptor. Of the 12 time-points tested, significant increases in expression levels of NK(1) receptor mRNA in the shrew brainstem occurred at 2 and 28 h post-cisplatin injection, whereas intestinal NK(1) receptor mRNA was increased at 28 h. Shrew brainstem and intestinal substance P mRNA levels also tended to increase during the two phases. Furthermore, expression levels of NK(1) receptor protein were significantly increased in the brainstem at 2, 8, and 33 h post-cisplatin. No change in brainstem 5-HT(3) receptor protein expression was observed. The temporal enhancements in NK(1) receptor protein expression were mirrored by significant increases in the phosphorylation status of the brainstem ERK1/2 at 2, 8, and 33 h post-cisplatin. Phosphorylation of PKA significantly increased at 33rd and 40th hour. Our results indicate associations between cisplatin's peak immediate- and delayed-phase vomiting frequency with increased: (1) expression levels of NK(1) receptor mRNA and its protein level, and (2) downstream NK(1) receptor-mediated phosphorylation of ERK1/2 and PKA signaling.

Redox-sensitive oxidation and phosphorylation of PTEN contribute to enhanced activation of PI3K/Akt signaling in rostral ventrolateral medulla and neurogenic hypertension in spontaneously hypertensive rats.

AIMS: The activity of phosphoinositide 3-kinase (PI3K)/serine/threonine protein kinase (Akt) is enhanced under hypertension. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of PI3K signaling, and its activity is redox-sensitive. In the rostral ventrolateral medulla (RVLM), which is responsible for the maintenance of blood pressure, oxidative stress plays a pivotal role in neurogenic hypertension. The present study evaluated the hypothesis that redox-sensitive inactivation of PTEN results in enhanced PI3K/Akt signaling in RVLM, leading to neurogenic hypertension. RESULTS: Compared to age-matched normotensive Wistar-Kyoto (WKY) rats, PTEN inactivation in the form of oxidation and phosphorylation were greater in RVLM of spontaneously hypertensive rats (SHR). PTEN inactivation was accompanied by augmented PI3K activity and PI3K/Akt signaling, as reflected by the increase in phosphorylation of Akt and mammalian target of rapamycin. Intracisternal infusion of tempol or microinjection into the bilateral RVLM of adenovirus encoding superoxide dismutase significantly antagonized the PTEN inactivation and blunted the enhanced PI3K/Akt signaling in SHR. Gene transfer of PTEN to RVLM in SHR also abrogated the enhanced Akt activation and promoted antihypertension. Silencing PTEN expression in RVLM with small-interfering RNA, on the other hand, augmented PI3K/Akt signaling and promoted long-term pressor response in normotensive WKY rats. INNOVATION: The present study demonstrated for the first time that the redox-sensitive check-and-balance process between PTEN and PI3K/Akt signaling is engaged in the pathogenesis of hypertension. CONCLUSION: We conclude that an aberrant interplay between the redox-sensitive PTEN and PI3k/Akt signaling in RVLM underpins neural mechanism of hypertension.

Differential modulation of brainstem phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2 signaling underlies WIN55,212-2 centrally mediated pressor response in conscious rats.

Our recent study demonstrated that central cannabinoid receptor 1 (CB1R) activation caused dose-related pressor response in conscious rats, and reported studies implicated the brainstem phosphatidylinositol 3-kinase (PI3K)/Akt-extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in blood pressure control. Therefore, in this study, we tested the hypothesis that the modulation of brainstem PI3K/Akt-ERK1/2 signaling plays a critical role in the central CB(1)R-mediated pressor response. In conscious freely moving rats, the pressor response elicited by intracisternal (i.c.) (R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate salt (WIN55,212-2) (15 µg) was associated with significant increases in ERK1/2 phosphorylation in the rostral ventrolateral medulla (RVLM) and the nucleus tractus solitarius (NTS). In contrast, Akt phosphorylation was significantly reduced in the same neuronal pools. Pretreatment with the selective CB1R antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) (30 µg i.c.) attenuated the neurochemical responses elicited by central CB1R activation. Furthermore, pretreatment with the ERK/mitogen-activated protein kinase kinase inhibitor 2'-amino-3'-methoxyflavone (PD98059) (5 µg i.c.) abrogated WIN55,212-2-evoked increases in blood pressure and neuronal ERK1/2 phosphorylation but not the reduction in Akt phosphorylation. On the other hand, prior PI3K inhibition with wortmannin (0.4 µg i.c.) exacerbated the WIN55,212-2 (7.5 and 15 µg i.c.) dose-related increases in blood pressure and ERK1/2 phosphorylation in the RVLM. The present neurochemical and integrative studies yield new insight into the critical role of two brainstem kinases, PI3K and ERK1/2, in the pressor response elicited by central CB1R activation in conscious rats.

[Cystathionine beta-synthase in the structural elements of the human brain and spinal cord].

Investigated by immunohistochemistry the presence and distribution of CBS in the nuclei of the spinal cord and brainstem in 8 mens of 18-44 years old, died from mechanical injury is not related to the central nervous system damage. Established that the CBS-neurons are revealed in all parts of stem and spinal cord, but their content in the studied cores, ranging from 0.9 to 17 %. Observed a definite relationship between the activity of CBS and the quantity of neurons. Large cells of motor nuclei often have a high and very high intensity of the reaction. In the sensory nuclei of a high proportion of small neurons with low activity of the enzyme.

Melittin restores proteasome function in an animal model of ALS.

Amyotrophic lateral sclerosis (ALS) is a paralyzing disorder characterized by the progressive degeneration and death of motor neurons and occurs both as a sporadic and familial disease. Mutant SOD1 (mtSOD1) in motor neurons induces vulnerability to the disease through protein misfolding, mitochondrial dysfunction, oxidative damage, cytoskeletal abnormalities, defective axonal transport- and growth factor signaling, excitotoxicity, and neuro-inflammation.Melittin is a 26 amino acid protein and is one of the components of bee venom which is used in traditional Chinese medicine to inhibit of cancer cell proliferation and is known to have anti-inflammatory and anti-arthritic effects.The purpose of the present study was to determine if melittin could suppress motor neuron loss and protein misfolding in the hSOD1G93A mouse, which is commonly used as a model for inherited ALS. Meltittin was injected at the 'ZuSanLi' (ST36) acupuncture point in the hSOD1G93A animal model. Melittin-treated animals showed a decrease in the number of microglia and in the expression level of phospho-p38 in the spinal cord and brainstem. Interestingly, melittin treatment in symptomatic ALS animals improved motor function and reduced the level of neuron death in the spinal cord when compared to the control group. Furthermore, we found increased of α-synuclein modifications, such as phosphorylation or nitration, in both the brainstem and spinal cord in hSOD1G93A mice. However, melittin treatment reduced α-synuclein misfolding and restored the proteasomal activity in the brainstem and spinal cord of symptomatic hSOD1G93A transgenic mice.Our research suggests a potential functional link between melittin and the inhibition of neuroinflammation in an ALS animal model.

Paradoxical sleep insomnia and decreased cholinergic neurons after myocardial infarction in rats.

STUDY OBJECTIVES: Acute myocardial infarction (MI) is followed, within a few hours, by neuronal loss in the central nervous system (CNS), including the limbic system, the hypothalamus, and the brainstem. Sleep before and after MI was investigated in the first experiment. In a parallel experiment, 2 weeks after MI, we quantified brainstem cholinergic neurons known to control paradoxical sleep (PS). MEASUREMENTS AND RESULTS: Data were obtained from 28 adult male Sprague-Dawley rats weighing 350-375 g and maintained under a 12-12 light-dark cycle in 2 experiments on 16 and 12 rats, respectively. The 16 animals in the first experiment were implanted with chronic electroencephalographic (EEG) and electromyographic (EMG) electrodes. A week after surgery, these animals were habituated for 2 days to the recording equipment, and baseline sleep was charted for 24 h. The next morning, MI was induced in 8 rats by occluding the left anterior descending coronary artery for 40 min. The remaining 8 rats served as sham-operated controls. Sleep was recorded again 2 weeks after MI. The number of choline acetyltransferase (ChAT)-positive neurons was counted in the second, parallel experiment on 6 MI and 6 sham rats. Compared to the sham controls, MI rats displayed longer latency to sleep onset, shorter latency to paradoxical sleep (PS), and curtailed PS duration. The number of ChAT-positive neurons in the pedunculopontine tegmentum (PPT) area of MI rats was significantly decreased compared to the sham controls, while the number of laterodorsal tegmentum (LDT) cholinergic neurons was not different. CONCLUSION: Acute MI is accompanied, within 2 weeks, by PS-specific insomnia that can be explained, at least partly, by a specific loss of cholinergic neurons in an area known to control PS.

Comparison of mRNA expression levels of selected genes in the brain stem of cattle naturally infected with classical and atypical BSE.

Since 2004 cases of atypical bovine spongiform encephalopathy (BSE) in older cattle are recorded on the basis of aberrant glycoprofiles of prion protein resistant to proteolysis (PrP(res)). The nature of those types of PrP(res) is still not fully understood but the epidemiological data indicate that their occurrence is rare. Hitherto, most BSE cases were studied on the basis of the features of pathological form of prion protein (PrP(Sc)) or lesions observed in the gray matter of the brain. Here we propose the gene expression profiling as a method to characterize and distinguish BSE types. Thus, the aim of the study was to compare the activity of some genes which are known to play a role in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Significant differences in the expression level of the selected genes in the brain stem were observed for 7 out of 11 genes tested when the results for BSE affected and healthy control animals were compared. Significant up-regulation of caspase 3, Bax and 14-3-3 protein encoding genes was apparent in the obex of all BSE affected cattle regardless of the prion type. Significant and unique to BSE H-type up-regulation was detected in prion and SOD1 genes, while BSE C-type was characterized by higher Bcl-2 and Fyn gene expression levels in respect to other BSE types and control animals. Different gene expression profiles of bovine brains infected with classical and atypical BSE indicate possible different pathogenesis or origin of the disease.

Histochemical and fluorescent analyses of mitochondrial integrity in chick auditory neurons following deafferentation.

BACKGROUND: Neurons rely exclusively on mitochondrial oxidative phosphorylation to meet cellular energy demands, and disruption of mitochondrial function often precipitates neuronal cell death. Auditory neurons in the chick brain stem (n. magnocellularis [NM]) receive glutamatergic innervation exclusively from ipsilateral eighth nerve afferents. Cochlea removal permanently disrupts afferent support and ultimately triggers apoptotic cell death in 30-50% of ipsilateral, deafferented neurons. Here, we evaluated whether disruption of mitochondrial function occurs during deafferentation-induced neuronal cell death. PURPOSE: To determine whether mitochondrial dysfunction occurs preferentially within dying NM neurons. RESEARCH DESIGN: An experimental study. All birds underwent unilateral cochlea removal. Normally innervated neurons contralateral to surgery served as within-animal controls. STUDY SAMPLE: Hatchling broiler chickens between 8 and 12 days of age served as subjects. A total of 62 birds were included in the study. INTERVENTION: Cochlea removal was performed to deafferent ipsilateral NM neurons and trigger neuronal cell death. DATA COLLECTION AND ANALYSIS: Following unilateral cochlea removal, birds were sacrificed 12, 24, 48, or 168 hours later, and brain tissue was harvested. Brainstems were sectioned through NM and evaluated histochemically for oxidative enzyme reaction product accumulation or reacted for Mitotracker Red, an indicator of mitochondrial membrane potential (m) and cytoplasmic TdT-mediated dUTP Nick-End Labeling (TUNEL), an indicator of cell death. Histochemical staining intensities for three mitochondrial enzymes, succinate dehydrogenase (SDH), cytochrome c oxidase (CO), and ATP synthase (ATPase) were measured in individual neurons and compared in ipsilateral and contralateral NM. Comparisons were made using unpaired t-tests (CO) or Kruskal Wallis one way ANOVA followed by Dunn's post hoc pairwise comparisons (ATPase, SDH). Mitotracker Red tissue was examined qualitatively for the presence of and extent of colocalization between Mitotracker Red and TUNEL label in NM. RESULTS: RESULTS showed global upregulation of all three oxidative enzymes within deafferented NM neurons compared to contralateral, unperturbed NM neurons. In addition, differential SDH and ATPase staining intensities were detected across neurons within the ipsilateral nucleus, suggesting functional differences in mitochondrial metabolism across deafferented NM. Quantitative analyses revealed that deafferented neurons with preferentially elevated SDH and ATPase activities represent the subpopulation destined to die following cochlea removal. In addition, Mitotracker Red accumulated intensely within the subset of deafferented NM neurons that also exhibited cytoplasmic TdT-mediated dUTP Nick-End Labeling (TUNEL) and subsequently died. CONCLUSIONS: Taken together, our results demonstrate that a subset of deafferented NM neurons, presumably those that die, preferentially upregulates SDH, perhaps via the tricarboxylic acid (TCA) cycle. These same neurons undergo ATPase uncoupling and an eventual loss of Deltapsi(m).

Compartment-specific regulation of plasma membrane calcium ATPase type 2 in the chick auditory brainstem.

Calcium signaling plays a role in synaptic regulation of dendritic structure, usually on the time scale of hours or days. Here we use immunocytochemistry to examine changes in expression of plasma membrane calcium ATPase type 2 (PMCA2), a high-affinity calcium efflux protein, in the chick nucleus laminaris (NL) following manipulations of synaptic inputs. Dendrites of NL neurons segregate into dorsal and ventral domains, receiving excitatory input from the ipsilateral and contralateral ears, respectively, via nucleus magnocellularis (NM). Deprivation of the contralateral projection from NM to NL leads to rapid retraction of ventral, but not the dorsal, dendrites of NL neurons. Immunocytochemistry revealed symmetric distribution of PMCA2 in two neuropil regions of normally innervated NL. Electron microscopy confirmed that PMCA2 localizes in both NM terminals and NL dendrites. As early as 30 minutes after transection of the contralateral projection from NM to NL or unilateral cochlea removal, significant decreases in PMCA2 immunoreactivity were seen in the deprived neuropil of NL compared with the other neuropil that continued to receive normal input. The rapid decrease correlated with reductions in the immunoreactivity for microtubule-associated protein 2, which affects cytoskeleton stabilization. These results suggest that PMCA2 is regulated independently in ventral and dorsal NL dendrites and/or their inputs from NM in a way that is correlated with presynaptic activity. This provides a potential mechanism by which deprivation can change calcium transport that, in turn, may be important for rapid, compartment-specific dendritic remodeling.

Brainstem adenosine A1 receptor signaling masks phosphorylated extracellular signal-regulated kinase 1/2-dependent hypotensive action of clonidine in conscious normotensive rats.

Central adenosine A(1) and A(2A) receptors mediate pressor and depressor responses, respectively. The adenosine subtype A(2A) receptor (A(2A)R)-evoked enhancement of phosphorylated extracellular signal-regulated kinase (pERK) 1/2 production in the rostral ventrolateral medulla (RVLM), a major neuroanatomical target for clonidine, contributes to clonidine-evoked hypotension, which is evident in conscious aortic barodenervated (ABD) but not in conscious sham-operated (SO) normotensive rats. We conducted pharmacological and cellular studies to test the hypothesis that the adenosine A(2A)R-mediated (pERK1/2-dependent) hypotensive action of clonidine is not expressed in SO rats because it is counterbalanced by fully functional central adenosine subtype A(1) receptor (A(1)R) signaling. We first demonstrated an inverse relationship between A(1)R expression in RVLM and clonidine-evoked hypotension in ABD and SO rats. The functional (pharmacological) relevance of the reduced expression of RVLM A(1)R in ABD rats was verified by the smaller dose-dependent pressor responses elicited by the selective A(1)R agonist N(6)-cyclopentyladenosine in ABD versus SO rats. It is important that after selective blockade of central A(1)R with 8-cyclopentyl-1,3-dipropylxanthine in conscious SO rats, clonidine lowered blood pressure and significantly increased neuronal pERK1/2 in the RVLM. In contrast, central A(1)R blockade had no influence on the hypotensive response or the increase in RVLM pERK1/2 elicited by clonidine in ABD rats. These findings support the hypothesis that central adenosine A(1)R signaling opposes the adenosine A(2A)R-mediated (pERK1/2-dependent) hypotensive response and yield insight into a cellular mechanism that explains the absence of clonidine-evoked hypotension in conscious normotensive rats.

Contribution of neuronal nitric oxide (no) synthase to N-acetylcysteine-induced increase of NO synthase activity in the brain of normotensive and hypertensive rats.

The goal of our study was to determine a contribution of nNOS to the increase of brain NO synthase activity induced by chronic N-acetylcysteine (NAC) treatment. Young 4-week-old male Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were subjected to treatment with NAC (1.5 g/kg/day) for 8 weeks. At the end of experiment total NOS activity was determined in the brainstem and cerebellum with and without specific nNOS inhibitor S-methyl-L-thiocitrulline (SMTC, 10(-6) mol/l) by measuring the formation of L-[(3)H] citrulline from L-[(3)H] arginine. Chronic NAC treatment had no effect on blood pressure (BP) of WKY, while it attenuated BP increase in young SHR. Total NOS activity was increased in the brainstem of SHR compared to WKY, but this strain difference was abolished by SMTC. Chronic NAC treatment of SHR increased total NOS activity by 32% in the brainstem and by 67% in the cerebellum. After the incubation of brainstem and cerebellum with SMTC there were no significant differences in NOS activity of NAC-treated rats compared to strain-matched controls. Taken together, nNOS seems to be responsible for the increase of total NOS activity in the brain of SHR. SMTC inhibited 86% and 70% of NAC-induced increase of total NOS activity in the brainstem and cerebellum, respectively. Thus, nNOS is responsible not only for strain differences but also for NAC-induced increase of total NOS activity in the brain.

Activation of Rho-kinase in the brainstem enhances sympathetic drive in mice with heart failure.

Rho-kinase is involved in the pathogenesis of hypertension and left ventricular remodelling after myocardial infarction (MI). In an earlier study, we had demonstrated that Rho-kinase in the brainstem contributes to hypertensive mechanisms via the sympathetic nervous system; however, it is not known whether Rho-kinase in the brainstem also contributes to sympathetic nerve activation after MI. Male Institute of Cancer Research mice (8-10 weeks old) were used for the study. Two days before coronary artery occlusion (MI group), the left ventricular function was estimated by echocardiography. Following this, Y-27632 (0.5 mM, 0.25 microL/h), a specific Rho-kinase inhibitor, or a vehicle was intracisternally infused in the mice using an osmotic mini-pump. Nine days after coronary artery occlusion, we evaluated the 24-hour urinary norepinephrine excretion (U-NE) as a marker of sympathetic nerve activity. Ten days after coronary artery occlusion, we measured organ weight and evaluated Rho-kinase activity in the brainstem by measuring the amount of phosphorylated ezrin/radixin/moesin proteins, one of the substrates of Rho-kinase. The control group underwent a sham operation. Rho-kinase activity, U-NE, and lungs and liver weight were significantly greater in the MI group compared with the control group. Left ventricular size increased and percent fractional shortening decreased in the MI group compared with the control group. Y-27632 significantly decreased Rho-kinase activity and attenuated the increase in U-NE after MI. These results demonstrate that Rho-kinase is activated in the brainstem after MI and that the activation of this pathway is involved in the resulting enhanced sympathetic drive.

Ototoxicity induced by cinnabar (a naturally occurring HgS) in mice through oxidative stress and down-regulated Na(+)/K(+)-ATPase activities.

Cinnabar, a naturally occurring mercuric sulfide (HgS), has long been used in Chinese mineral medicine for more than 2000 years; currently it is still used as a sedative for infants in Asian countries. Since methylmercury is potently ototoxic, whether cinnabar also induces hearing impairment is awaited for delineation. In this study, we attempted to explore the toxic effects of cinnabar on the auditory brainstem response (ABR) system during 2-10 weeks administration at a clinical oral dosage of 10mg/kg/day in mice. The results showed that Hg contents of the brainstem were significantly increased accompanied with gradually progressive abnormality of ABR during 4-10 weeks of cinnabar administration. The progressive increase in hearing thresholds, prolonged absolute and interwave latencies of ABR apparently exhibited a gender difference. Male mice were more sensitive to cinnabar in producing hearing impairment correlated with the biochemical alterations in plasma and brainstem, e.g. an increase of lipid peroxidation (LPO), altered Na(+)/K(+)-ATPase activities and decrease of nitric oxide (NO(x)) levels. Moreover, accumulation of Hg contents in brainstem with a greater extent was found in male mice. These findings provide important information that the clinical dosage of cinnabar (10mg/kg/day) still exhibited ototoxicity after continuously long-term exposure. The signaling pathway of oxidative stress/Na(+)-K(+)-ATPase activities/NO of brainstem (a central auditory regulatory system) probably plays an important role in the toxic mechanisms of cinnabar-induced ototoxicity. The gender difference in cinnabar-induced neurotoxic effects merits further investigation.

PACAP is expressed in sympathoexcitatory bulbospinal C1 neurons of the brain stem and increases sympathetic nerve activity in vivo.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neuropeptide present in the rat brain stem. The extent of its localization within catecholaminergic groups and bulbospinal sympathoexcitatory neurons is not established. Using immunohistochemistry and in situ hybridization, we determined the extent of any colocalization with catecholaminergic and/or bulbospinal projections from the brain stem was determined. PACAP mRNA was found in tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the C1-C3 cell groups. In the rostral ventrolateral medulla (RVLM), PACAP mRNA was found in 84% of the TH-ir neurons and 82% of bulbospinal TH-ir neurons. The functional significance of these PACAP mRNA positive bulbospinal neurons was tested by intrathecal administration of PACAP-38 in anaesthetized rats. Splanchnic sympathetic nerve activity doubled (110%) and heart rate rose significantly (19%), although blood pressure was unaffected. In addition, as previously reported, PACAP was found in the A1 cell group but not in the A5 cell group or in the locus coeruleus. The RVLM is the primary site responsible for the tonic and reflex control of blood pressure through the activity of bulbospinal presympathetic neurons, the majority of which contain TH. The results indicate 1) that pontomedullary neurons containing both TH and PACAP that project to the intermediolateral cell column originate from C1-C3 and not A5, and 2) intrathecal PACAP-38 causes a prolonged, sympathoexcitatory effect.