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1.  Differential Effects of Pharmacologic and Genetic Modulation of NMDA Receptor Activity on HIV/gp120-Induced Neuronal Damage in an In Vivo Mouse Model 
HIV-associated neurocognitive disorder (HAND) consists of motor and cognitive dysfunction in a relatively large percentage of patients with AIDS. Prior work has suggested that at least part of the neuronal and synaptic damage observed in HAND may occur due to excessive stimulation of NMDA-type glutamate receptors (NMDARs). Here, we compared pharmacological and genetic manipulation of NMDAR activity using an improved derivative of the NMDAR antagonist memantine, termed NitroMemantine, and the modulatory NMDAR subunit GluN3A in the HIV/gp120 transgenic (tg) mouse model of HAND. Interestingly, we found that while both NitroMemantine and GluN3A have been shown to inhibit NMDAR activity, NitroMemantine protected synapses in gp120 tg mice, but overexpression of GluN3A augmented the damage. Given recent findings in the field, one explanation for this apparently paradoxical result is the location of the NMDARs primarily affected, with NitroMemantine inhibiting predominantly extrasynaptic pathologically-activated NMDARs, but GluN3A disrupting normal NMDAR-mediated neuroprotective activity via inhibition of synaptic NMDARs.
PMCID: PMC4821006  PMID: 26374431
HIV-associated neurocognitive disorder (HAND); AIDS; NMDA-type glutamate receptors; GluN3A; gp120; memantine; NitroMemantine
2.  Attenuation of Cystitis and Pain Sensation in Mice Lacking Fatty Acid Amide Hydrolase 
Endocannabinoids, such as N-arachidonylethanolamine (AEA, also called anandamide), exert potent analgesic and anti-inflammatory effects. Fatty acid amide hydrolase (FAAH) is primarily responsible for degradation of AEA, and deletion of FAAH increases AEA content in various tissues. Since FAAH has been shown to be present in the bladder of various species, we compared bladder function, severity of experimental cystitis, and cystitis-associated referred hyperalgesia in male wild type (WT) and FAAH knock-out (KO) mice. Basal concentrations of AEA were greater, and the severity of cyclophosphamide (CYP)-induced cystitis was reduced in bladders from FAAH KO compared to WT mice. Cystitis-associated increased peripheral sensitivity to mechanical stimuli and enhanced bladder activity (as reflected by increased voiding frequency) were attenuated in FAAH KO compared to WT mice. Further, abundances of mRNA for several pro-inflammatory compounds were increased in bladder mucosa after CYP treatment of WT mice, and this increase was inhibited in FAAH KO mice. These data indicate that endogenous substrates of FAAH, including the cannabinoid AEA, play an inhibitory role in bladder inflammation and subsequent changes in pain perception. Therefore, FAAH could be a therapeutic target to treat clinical symptoms of painful inflammatory bladder diseases.
PMCID: PMC4355044  PMID: 25374388
Fatty acid amide hydrolase; N-arachidonylethanolamine; Cystitis; Pain; Mice
3.  Neural cell adhesion molecule NrCAM is expressed in the mammalian inner ear and modulates spiral ganglion neurite outgrowth in an in vitro alternate choice assay 
Neuron-glial-related cell adhesion molecule (NrCAM) is a neuronal cell adhesion molecule involved in neuron-neuron and neuron-glial adhesion as well as directional signaling during axonal cone growth. NrCAM has been shown to be involved in several cellular processes in the central and peripheral nervous systems, including neurite outgrowth, axonal pathfinding and myelination, fasciculation of nerve fibers, and cell migration. This includes sensory systems such as the eye and olfactory system. However, there are no reports on the expression/function of NrCAM in the auditory system. The aim of the present study was to elucidate the occurrence of NrCAM in the mammalian cochlea and its role in innervation of the auditory end organ. Our work indicates that NrCAM is highly expressed in the developing mammalian cochlea (position consistent with innervation). Moreover, we found that NrCAM, presented in stripe micropatterns, provide directional cues to neonatal rat inner ear spiral ganglion neurites in vitro. Our results are consistent with a role for NrCAM in the pathfinding of spiral ganglion dendrites toward their hair cell targets in the sensory epithelium.
PMCID: PMC4355056  PMID: 25407819
growth cone guidance; neurite outgrowth; inner ear; NrCAM; spiral ganglion
4.  Role of pGlu-Serpinin, a Novel Chromogranin A-Derived Peptide in Inhibition of Cell Death 
Chromogranin A (CgA) is a member of the granin family of molecules found in secretory granules of endocrine and neuro-endocrine cells. Here, we have identified a new 23-mer CgA-derived peptide secreted from pituitary AtT-20 cells, which we named pyroGlu-serpinin (pGlu-serpinin). LC–MS studies of peptides in conditioned medium of AtT-20 cells indicate that pGlu-serpinin is derived from initial processing of mouse CgA at paired basic residues, Arg461–Arg462 and Arg433–Arg434, to yield a previously described 26 amino acid peptide, serpinin. Three amino acids are then cleaved from the N terminus of serpinin, yielding a peptide with an N-terminal glutamine, which is then subsequently pyroglutaminated. Immunocytochemistry showed co-localization of pGlu-serpinin with adrenocorticotropic hormone in secretory granules of AtT-20 cells, and it was released in an activity-dependent manner. Functional studies demonstrated that pGlu-serpinin was able to prevent radical oxygen species (hydrogen peroxide)-induced cell death of AtT-20 cells and cultured rat cerebral cortical neurons at a concentration of 1 and 10 nM, respectively. These data indicate that pGlu-serpinin has anti-apoptotic effects that may be important in neuroprotection of central nervous system neurons and pituitary cells. Furthermore, pGlu-serpinin added to the media of AtT-20 cells up-regulated the transcription of the serine protease inhibitor, protease nexin-1 (PN-1) mRNA. pGlu-serpinin’s ability to increase levels of PN-1, a potent inhibitor of plasmin released during inflammatory processes causing cell death, may play a role in protecting cells under adverse pathophysiological conditions.
PMCID: PMC4792120  PMID: 21537909
Serpinin; Chromogranin A; Neuroprotection; Protease nexin-1; Pyroglutamination
5.  Microvascular lesions by estrogen-induced ID3: its implications in cerebral & cardiorenal vascular disease 
Severe symptoms of cerebral and cardiorenal vascular diseases can be triggered when cerebral, coronary, or glomerular arterioles grow inappropriately as a result of abnormal cell proliferation. The risk factor(s) and molecular mechanisms responsible for microvascular lesion formation are largely unknown. Although controversial, both animal and epidemiological studies have shown that estrogen increases the risk of stroke which may be due to microvascular lesions. Since microvascular diseases are characterized by excessive vessel growth, it is plausible that estrogen-induced neovascularization contributes to the growth of microvascular lesions. We present evidence for how ID3 overexpression in endothelial cells contributes to the development of an estrogen-induced neovascular phenotype with an additional focus on Pyk2 kinase. Our data showed that ID3 overexpression increased neovascularization, cell migration, and spheroid growth of human cerebral microvascular endothelial cells, hCMEC/D3. ID3 overexpressing cells showed significant estrogen-induced G2/M phase transition. Estrogen treatment increased both ID3 phosphorylation and total protein that was inhibited by tamoxifen; and Pyk2 mediated estrogen-induced ID3 mRNA expression. These findings suggest that Pyk2 signals ID3 expression and ID3 is necessary for estrogen-induced neovascularization in hCMEC/D3 cells. A better understanding of how microvascular lesions depend on ID3 may open new avenues for prevention and treatment of neurological diseases.
PMCID: PMC4320014  PMID: 25129100
6.  Sleep fragmentation has differential effects on obese and lean mice 
Chronic sleep fragmentation (SF), common in patients with sleep apnea, correlates with the development of obesity. We hypothesized that SF differentially affects neurobehavior in lean wildtype (WT) and obese pan-leptin receptor knockout (POKO) mice fed the same normal diet. First we established an SF paradigm by interrupting sleep every 2 min during the inactive light span. The maneuver was effective in decreasing sleep duration and bout length, and in increasing sleep state transition and waking, without significant rebound sleep in the dark span. Changes of sleep architecture were evident in the light span and consistent across days 1–10 of SF. There was reduced NREM, shortened sleep latency, and increased state transitions. During the light span of the first day of SF, there also was reduction of REM and increased delta power of slow wave sleep. Potential effects of SF on thermal pain threshold, locomotor activity, and anxiety were then tested. POKO mice had a lower circadian amplitude of pain latency than WT mice in the hot plate test, and both groups had lowest tolerance at 4 pm (ZT 10) and longest latency at 4 am (ZT 22). SF increased the pain threshold in WT but not POKO mice when tested at 8 am (ZT 2). Both the POKO mutation and SF resulted in reduced physical activity and increased anxiety, but there was no additive effect of these two factors. Overall, SF and the POKO mutation differentially regulate mouse behavior. The results suggest that obesity can blunt neurobehavioral responses to SF.
PMCID: PMC4320048  PMID: 25152064
sleep fragmentation; sleep architecture; obesity; pain; anxiety
8.  Regulation Of Rod Photoreceptor Differentiation By STAT3 Is Controlled By A Tyrosine Phosphatase 
Signal pathways that reduce the levels of tyrosine phosphorylated STAT3 (pSTAT3) allow late retinal progenitors to exit the cell cycle and enter a terminal differentiation pathway into rod photoreceptors. In the mouse retina we previously identified PKC-β1 and -γ isoforms as essential components of a key signal pathway and IGF-1 as a major extrinsic factor regulating rod formation. In this manuscript we demonstrate that PKC decreases phosphotyrosine but not phosphoserine on STAT3 in neonatal mouse retinas. Neither IGF-1 nor PMA induced a significant change in the levels of STAT3, or in the levels of the key proteins regulating STAT3 degradation, SOCS3 and PIAS3. Treatment of neonatal mouse retinal explants with sodium orthovanadate inhibited the PKC-mediated reduction in pSTAT3, indicating a role for a phosphatase. Addition of the PTEN inhibitor bpV(phen) to explant cultures treated with IGF-1 or PMA had no effect on the reduction in pSTAT3 levels, but the effect of both IGF-1 and PMA was blocked by a concentration of the inhibitor NSC87877 that is selective for the phosphatases Shp1 and Shp2. Inhibition of Shp1/2 phosphatases was also sufficient to abolish the IGF1-mediated induction of rod photoreceptor differentiation in the retina explants cultures. We conclude that one or both of these phosphatases are key components regulating the formation of rod photoreceptors in mouse retina.
PMCID: PMC4293205  PMID: 25108518
rod photoreceptor; IGF1; STAT3; PTEN; Shp1; Shp2
9.  Molecular neuroimaging of post-injury plasticity 
Nerve injury induces long-term changes in neuronal activity in the primary somatosensory cortex (S1), which has often been implicated as the origin of sensory dysfunction. However, the cellular mechanisms underlying this phenomenon remain unclear. C-fos is an immediate early gene, which has been shown to play an instrumental role in plasticity. By developing a new platform to image real-time changes in gene expression in vivo, we investigated whether injury modulates the levels of c-fos in layer V of S1, since previous studies have suggested that these neurons are particularly susceptible to injury.
The yellow fluorescent protein, ZsYellow1, under the regulation of the c-fos promoter, was expressed throughout the rat brain. A fiber-based confocal microscope that enabled deep brain imaging was utilized and local field potential were collected simultaneously. In the weeks following limb denervation in adult rats (n=10), sensory stimulation of the intact limb induced significant increases in c-fos gene expression in cells located in S1, both contralateral (affected, 27.6±3 cells) and ipsilateral (8.6±3 cells) to the injury, compared to controls (n=10, 13.4±3 and 1.0±1, respectively, p-value <0.05). Thus, we demonstrated that injury activates cellular mechanisms that are involved in reshaping neuronal connections and this may translate to neurorehabilitative potential.
PMCID: PMC4257867  PMID: 24909382
Immediate early genes; plasticity; injury; somatosensory cortex; optical imaging; gene expression
10.  Regulation of bed nucleus of the stria terminalis PACAP expression by stress and corticosterone 
Single nucleotide polymorphisms (SNP) in the genes for pituitary adenylyl cyclase-activating peptide (PACAP) and the PAC1 receptor have been associated with stress-related psychiatric disorders. Although from recent work we have argued that stress-induced PACAP expression in the bed nucleus of the stria terminalis (BNST) may mediate stress-related psychopathology, it is unclear whether stress-induced increases in BNST PACAP expression require acute or repeated stressor exposure, and whether increased BNST PACAP expression is related to stress-induced increases in circulating glucocorticoids. In the current work, we have used quantitative real-time polymerase chain reaction (qPCR) to assess transcript expression in brain punches from rats after stressor exposure paradigms or corticosterone injection. BNST PACAP and PAC1 receptor transcript expression was increased only after 7 days of repeated stressor exposure; no changes in transcript levels were observed 2 or 24 hours after a single restraint session. Moreover, repeated corticosterone treatment for 7 days was not sufficient to reliably increase BNST PACAP transcript levels, suggesting that stress-induced elevations in corticosterone may not be the primary drivers of BNST PACAP expression. These results may help clarify the mechanisms and temporal processes that underlie BNST PACAP induction for intervention in stress-related anxiety disorders.
PMCID: PMC4162870  PMID: 24614974
pituitary adenylate cyclase-activating peptide (PACAP); bed nucleus of the stria terminalis (BNST); stress; corticosterone; glucocorticoids
11.  PACAP27 is protective against Tat-induced neurotoxicity 
Human immunodeficiency virus type-1 (HIV) infection of the central nervous system promotes neuronal injury and apoptosis that culminate in HIV-associated neurocognitive disorders. Viral proteins, such as transactivator of transcription (Tat), have emerged as leading candidates to explain HIV-mediated neurotoxicity, though the mechanism remains unclear. To determine the effects of Tat, rat cortical neurons were exposed to nanomolar concentrations of Tat for various time points. Within a few hours, Tat induced the production of reactive oxygen species (ROS), and other indices of mitochondrial destabilization. In addition, we observed a significant induction of DNA double strand breaks (DSBs) by Tat. We next investigated the neuroprotective activity of the pituitary adenylate cyclase-activating polypeptide 27 (PACAP27) against these cardinal features of Tat-induced neurodegeneration. PACAP27 (100 nM) inhibited all Tat-mediated toxic effects including DNA DSBs. Importantly, PACAP27 prevented the induction of neuronal loss induced by Tat. The neuroprotective effect of PACAP27 is correlated with its ability to release the anti-apoptotic chemokine CCL5. Our data support a mechanism of Tat neurotoxicity in which Tat induces mitochondrial destabilization, thus increasing the release of ROS, which causes DNA DSBs leading to cell death. PACAP27, through CCL5, mitigates the effects of Tat-induced neuronal dysfunction, suggesting that PACAP27 could be a new strategy for an adjunct therapy against HIV-associated neurocognitive disorders.
PMCID: PMC4185276  PMID: 24696163
DNA damage; oxidative stress; mitochondria; CCL5; HIV; gp120
12.  Calcium influx, but not intracellular calcium release, supports PACAP-mediated ERK activation in HEK PAC1 receptor cells 
In HEK cells expressing GFP-tagged PAC1Hop1 receptors, PACAP augments ERK phosphorylation through two parallel pathways; one through PACAP/PAC1 receptor internalization/endosome MEK/ERK signaling, the other through PLC/DAG/PKC activation. We examined whether elevation of intracellular calcium ([Ca2+]i) was required for either of the PACAP/PAC1 receptor-mediated ERK activation mechanisms. The PACAP (25 nM)-induced elevation of [Ca2+]i was greater with cells maintained in Ca2+-containing than in Ca2+-deficient solution, suggesting that both calcium release from internal stores and calcium influx contributed to the rise in [Ca2+]i. A thapsigargin-induced increase in [Ca2+]i also was greater with calcium in the external solution. OAG, the cell permeable analogue of DAG, increased [Ca2+]i, but only in Ca2+-containing solution. Decreasing external calcium or depleting internal calcium stores did not block PACAP-induced PAC1 receptor internalization. Omission of calcium from the external solution, but not thapsigargin pretreatment, significantly blunted PACAP-stimulated ERK phosphorylation. The PKC inhibitor BimI decreased PACAP-mediated ERK activation in both Ca2+-containing or Ca2+-deficient solutions. In contrast, following Pitstop 2 pretreatment to block endocytic mechanisms, PACAP activated ERK only when calcium was present in the external solution. We conclude that the endosome signaling pathway is largely calcium-independent whereas calcium influx appears necessary for the PLC/DAG/PKC component of PACAP-induced ERK activation.
PMCID: PMC4194181  PMID: 24723666
Pituitary adenylate cyclase activating polypeptide; PAC1 receptor internalization; ERK phosphorylation; thapsigargin; IP3-mediated calcium release
13.  Pituitary adenylate cyclase activating polypeptide (PACAP) dilates cerebellar arteries through activation of large-conductance Ca2+-activated (BK) and ATP-sensitive (KATP) K+ channels 
Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent vasodilator of numerous vascular beds, including cerebral arteries. Although PACAP-induced cerebral artery dilation is suggested to be cyclic AMP (cAMP)-dependent, the downstream intracellular signaling pathways are still not fully understood. In this study, we examined the role of smooth muscle K+ channels and hypothesized that PACAP-mediated increases in cAMP levels and protein kinase A (PKA) activity result in the coordinate activation of ATP-sensitive K+ (KATP) and large-conductance Ca2+-activated K+ (BK) channels for cerebral artery dilation. Using patch-clamp electrophysiology, we observed that PACAP enhanced whole-cell KATP channel activity and transient BK channel currents in freshly isolated rat cerebellar artery myocytes. The increased frequency of transient BK currents following PACAP treatment is indicative of increased intracellular Ca2+ release events termed Ca2+ sparks. Consistent with the electrophysiology data, the PACAP-induced vasodilations of cannulated cerebellar artery preparations were attenuated by approximately 50% in the presence of glibenclamide (a KATP channel blocker) or paxilline (a BK channel blocker). Further, in the presence of both blockers, PACAP failed to cause vasodilation. In conclusion, our results indicate that PACAP causes cerebellar artery dilation through two mechanisms: 1) KATP channel activation and 2) enhanced BK channel activity, likely through increased Ca2+ spark frequency.
PMCID: PMC4201911  PMID: 24744252
14.  Role of c-Jun N-terminal Kinase (JNK) activation in micturition reflexes in cyclophosphamide (CYP)-induced cystitis in female rats 
c-Jun N-terminal Kinase (JNK) is member of the Mitogen-Activated Protein Kinase (MAPK) family, activated through phosphorylation following cytokine exposure and stress. In this study, phosphorylation of JNK was examined in the urinary bladder with CYP-induced cystitis and the effects of SP600125, a selective inhibitor of phosphorylation of JNK, on urinary bladder function were assessed using conscious, open outlet, cystometry with continuous instillation of intravesical saline. We induced bladder inflammation in adult female Wistar rats by injecting CYP intraperitoneally to produce acute (150 mg/kg; 4 hr), intermediate (150 mg/kg; 48 hr) and chronic (75 mg/kg; every third day for 10 days) treatments. Western blotting of urinary bladder demonstrated a significant (p ≤ 0.01) increase (i.e., phosphorylation) in JNK activation with 4 hr and 48 hr CYP-induced cystitis. Immunohistochemistry and image analyses demonstrated a significant (p ≤ 0.01) increase in JNK activation in the urothelium with 4 hr and 48 hr CYP-induced cystitis. Blockade of JNK phosphorylation significantly (p ≤ 0.01) increased bladder capacity and intercontraction void intervals in CYP-treated rats (4 hr and 48 hr). Furthermore, blockade of JNK phosphorylation reduced (p ≤ 0.01) neuropeptide (substance P, calcitonin gene-related peptide) expression in the urinary bladder with CYP-induced cystitis (4 hr and 48 hr). In contrast, blockade of JNK phosphorylation was without effect on bladder function or neuropeptide expression in urinary bladder in control (no inflammation) rats. Blockade of JNK phosphorylation may represent a novel target for improving urinary bladder function with CYP-induced cystitis.
PMCID: PMC4209338  PMID: 24763745
micturition; western blot; cystometry; phosphorylation; IHC; neuropeptides
15.  Shared Pathways Among Autism Candidate Genes Determined by Co-expression Network Analysis of the Developing Human Brain Transcriptome 
Journal of Molecular Neuroscience  2015;57(4):580-594.
Autism spectrum disorder (ASD) is a neurodevelopmental syndrome known to have a significant but complex genetic etiology. Hundreds of diverse genes have been implicated in ASD; yet understanding how many genes, each with disparate function, can all be linked to a single clinical phenotype remains unclear. We hypothesized that understanding functional relationships between autism candidate genes during normal human brain development may provide convergent mechanistic insight into the genetic heterogeneity of ASD. We analyzed the co-expression relationships of 455 genes previously implicated in autism using the BrainSpan human transcriptome database, across 16 anatomical brain regions spanning prenatal life through adulthood. We discovered modules of ASD candidate genes with biologically relevant temporal co-expression dynamics, which were enriched for functional ontologies related to synaptogenesis, apoptosis, and GABA-ergic neurons. Furthermore, we also constructed co-expression networks from the entire transcriptome and found that ASD candidate genes were enriched in modules related to mitochondrial function, protein translation, and ubiquitination. Hub genes central to these ASD-enriched modules were further identified, and their functions supported these ontological findings. Overall, our multi-dimensional co-expression analysis of ASD candidate genes in the normal developing human brain suggests the heterogeneous set of ASD candidates share transcriptional networks related to synapse formation and elimination, protein turnover, and mitochondrial function.
Electronic supplementary material
The online version of this article (doi:10.1007/s12031-015-0641-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4644211  PMID: 26399424
Autism spectrum disorder; Gene co-expression network; Synaptogenesis; Mitochondrion; Apoptosis
16.  Effects of ceftriaxone on chronic ethanol consumption: a potential role for xCT and GLT1 modulation of glutamate levels in male P rats 
Alterations in glutamatergic neurotransmission have been suggested to affect many aspects of neuroplasticity associated with alcohol/drug addiction. We have previously shown that ceftriaxone, a β-lactam antibiotic known to upregulate glutamate transporter 1 (GLT1), reduced ethanol intake after five weeks of free-choice ethanol drinking paradigm in male alcohol-preferring (P) rats. Evidence suggests that differential effects involving alterations of glutamatergic neurotransmission occur after long term ethanol consumption. In this study, we tested whether the efficacy of administration of ceftriaxone persists after 14 weeks of free access to 15% and 30 % ethanol in male P rats. After 14 weeks of ethanol consumption, male P rats were administered ceftriaxone (100 mg/kg, i.p.) or saline vehicle for five days. We found that ceftriaxone treatment resulted in a significant reduction in ethanol intake starting from Day 2 (48 hours after the first i.p. injections of ceftriaxone) through Day 14, 10 days after final injection. Western blot analysis of brain samples from animals euthanized 24 h after treatment with the last dose of ceftriaxone revealed a significant upregulation of cystine/glutamate exchanger (xCT) and GLT1 levels in prefrontal cortex, nucleus accumbens and amygdala as compared to saline vehicle-treated group. These findings demonstrated the effectiveness of ceftriaxone in attenuating ethanol intake in a chronic consumption paradigm. These might be due in part through upregulation of both xCT and GLT1 levels in brain reward regions. Thus, the drug has a potential therapeutic action for the treatment of alcohol dependence.
PMCID: PMC4127185  PMID: 24535561
glutamate; alcohol-preferring rats; chronic ethanol exposure; xCT; GLT1
17.  Study of the spatial correlation between neuronal activity and BOLD fMRI responses evoked by sensory and channelrhodopsin-2 stimulation in the rat somatosensory cortex 
In this work we combined optogenetics tools with high-resolution blood oxygenation level dependent functional MRI (BOLD fMRI), electrophysiology, and optical imaging of cerebral blood flow (CBF), to study the spatial correlation between the hemodynamic responses and neuronal activity. We first investigated the spatial and temporal characteristics of BOLD fMRI and the underlying neuronal responses evoked by sensory stimulations at different frequencies. The results demonstrated that under dexmedetomidine anesthesia, BOLD fMRI and neuronal activity in the rat primary somatosensory cortex (S1) have different frequency - dependency and distinct laminar activation profiles. We the found that localized activation of channelrhodopsin-2 (ChR2) expressed in neurons throughout the cortex induced neuronal responses that were confined to the light stimulation S1 region (<500 μm) with distinct laminar activation profile. However, the spatial extent of the hemodynamic responses measured by CBF and BOLD fMRI induced by both ChR2 and sensory stimulation were greater than 3 mm. These results suggest that due to the complex neurovascular coupling it is challenging to determine specific characteristics of the underlying neuronal activity exclusively from the BOLD fMRI signals.
PMCID: PMC4104155  PMID: 24443233
18.  lynx1 supports neuronal health in mouse dorsal striatum during aging: an ultrastructural investigation 
Nicotinic acetylcholine receptors have been shown to participate in neuroprotection in the aging brain. Lynx protein modulators dampen the activity of the cholinergic system through direct interaction with nicotinic receptors. Although lynx1 null mutant mice exhibit augmented learning and plasticity, they also exhibit macroscopic vacuolation in the dorsal striatum as they age, detectable at the optical microscope level. Despite the relevance of the lynx1 gene to brain function, little is known about the cellular ultrastructure of these age-related changes. In this study, we assessed degeneration in the dorsal striatum in 1-, 3-, 7-, and 13-month-old mice, using optical and transmission electron microscopy. We observed a loss of nerve fibers, a breakdown in nerve fiber bundles, and loss of neuronal nuclei in the 13-month-old lynx1 null striatum. At higher magnification, these nerve fibers displayed intracellular vacuoles and disordered myelin sheaths. Few or none of these morphological alterations were present in younger lynx1 null mutant mice, or in heterozygous lynx1 null mutant mice at any age. These data indicate that neuronal health can be maintained by titrating lynx1 dosage, and that the lynx1 gene may participate in a trade-off between neuroprotection and augmented learning.
PMCID: PMC4265479  PMID: 25027556
nicotinic acetylcholine receptors; cholinergic system; prototoxins; neurotoxins; neurodegeneration; plasticity; learning and memory
19.  Novel Insights Into M3 Muscarinic Acetylcholine Receptor Physiology and Structure 
Recent studies with M3 muscarinic acetylcholine receptor (M3R) mutant mice suggest that drugs selectively targeting this receptor subtype may prove useful for the treatment of various pathophysiological conditions. Moreover, the use of M3R-based designer G protein-coupled receptors (GPCRs) has provided novel insights into how Gq-coupled GPCRs can modulate whole-body glucose homeostasis by acting on specific peripheral cell types. More recently, we succeeded in using X-ray crystallography to determine the structure of the M3R bound to the bronchodilating drug tiotropium, a muscarinic antagonist (inverse agonist). This new structural information should facilitate the development of orthosteric or allosteric M3R-selective drugs that are predicted to have considerable therapeutic potential.
PMCID: PMC3966988  PMID: 24068573
Muscarinic acetylcholine receptor; G protein-coupled receptor; X-ray structure; Mutant mouse models; Muscarinic receptor physiology
20.  Preclinical Studies on Neurobehavioral and Neuromuscular Effects of Cocaine Hydrolase Gene Therapy in Mice 
Cocaine hydrolase gene transfer of mutated human butyrylcholinesterase (BChE) is evolving as a promising therapy for cocaine addiction. BChE levels after gene transfer can be 1,500-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. Because mutated BChE is approximately 70 % as efficient in hydro-lyzing acetylcholine as wild-type enzyme, it is important to examine the impact on cholinergic function. Here, we focused on memory and cognition (Stone T-maze), basic neuromuscular function (treadmill endurance and grip strength), and coordination (Rotarod). BALB/c mice were given adeno-associated virus vector or helper-dependent adenoviral vector encoding mouse or human BChE optimized for cocaine. Age-matched controls received saline or luciferase vector. Despite high doses (up to 1013 particles per mouse) and high transgene expression (1,000-fold above baseline), no deleterious effects of vector treatment were seen in neurobehavioral functions. The vector-treated mice performed as saline-treated and lucif-erase controls in maze studies and strength tests, and their Rotarod and treadmill performance decreased less with age. Thus, neither the viral vectors nor the large excess of BChE caused observable toxic effects on the motor and cognitive systems investigated. This outcome justifies further steps toward an eventual clinical trial of vector-based gene transfer for cocaine abuse.
PMCID: PMC3983183  PMID: 24085526
Cocaine; Addiction; Gene therapy; Butyrylcholinesterase; Adeno-associated viral vector; Helper-dependent adenoviral vector
21.  Cocaine-and Amphetamine-Regulated Transcript Peptide (CARTp): Distribution and Function in Rat Urinary Bladder 
We investigated the distribution of CARTp(55-102) in rat lower urinary tract and evaluated its effect on urinary bladder function in vitro. Immunohistochemistry and a vertical isolated tissue bath system were used. Neurons, clusters of non-neuronal endocrine cells, and nerve fibers stained positive for CARTp(55-102) in young adult rat urinary bladder. In addition, a dense plexus of CARTp-immunoreactive (IR) nerve fibers was detected in ureters and small blood vessels in the bladder. The CARTp-expressing neuronal elements were nitric oxide synthase (NOS)- and tyrosine hydroxylase (TH)-IR, whereas all non-neuronal CARTp-IR elements stained positively only for TH (100%). In isolated bladder strips, CARTp significantly increased the amplitude of electric field stimulation (EFS)-induced detrusor contractions at stimulation frequencies ≤ 12.5 Hz (p ≤ 0.001) as well as amplitude and frequency of spontaneous phasic urinary bladder smooth muscle (UBSM) contractions (p ≤ 0.05). The responses to CARTp stimulation were dose-dependent and increased in the presence of the urothelium. To determine if the CARTp-increase in nerve mediated contractions may involve an action of CARTp on specific neural pathways, we blocked cholinergic, purinergic and adrenergic pathways and determined CARTp actions on EFS-medicated contractions. CARTp enhancement of EFS-mediated contractions does not involve alteration in purinergic, adrenergic or cholinergic pathways. The study demonstrates that CARTp(55-102) is highly expressed in rat urinary bladder. CARTp increased the amplitude of EFS-induced detrusor contractions as well as the amplitude and frequency of spontaneous phasic urinary bladder smooth muscle contractions. We conclude that CARTp may alter the release of compounds from the urothelium that leads to an enhancement of UBSM contractility/excitability.
PMCID: PMC4454392  PMID: 24740629
neuropeptides; immunohistochemistry; intramural ganglia; NOS; TH
22.  Mouse Striatal Dopamine Nerve Terminals Express α4α5β2 and Two Stoichiometric Forms of α4β2*-Nicotinic Acetylcholine Receptors 
Wild-type and α5 null mutant mice were used to identify nicotinic cholinergic receptors (nAChRs) that mediate α-conotoxin MII (α-CtxMII)-resistant dopamine (DA) release from striatal synaptosomes. Concentration–effect curves for ACh-stimulated release (20 s) were monophasic when wild-type synaptosomes were assayed but biphasic with synaptosomes from the α5 null mutant. Deleting the α5 gene also resulted in decreased maximal ACh-stimulated α-CtxMII-resistant DA release. When a shorter perfusion time (5 s) was used, biphasic curves were detected in both wild-type and α5 null mutants, indicative of high- and low-sensitivity (HS and LS) activity. In addition, DHβE-sensitive (HS) and DHβE-resistant (LS) components were found in both genotypes. These results indicate that α-CtxMII-resistant DA release is mediated by α4α5β2, (α4)2(β2)3 (HS), and (α4)3(β2)2 (LS) nAChRs.
PMCID: PMC4386732  PMID: 19693710
Neuronal nicotinic acetylcholine receptors; Synaptosomes; subunit null mutation; α-Conotoxin MII
23.  The Influence of Low Doses of Zearalenone on Distribution of Selected Active Substances in Nerve Fibers Within the Circular Muscle Layer of Porcine Ileum 
Journal of Molecular Neuroscience  2015;56(4):878-886.
The aim of this study was to investigate, whether low doses (25 % of no observable adverse effect levels values) of zearalenone (ZEN) can affect the expression of active substances in nerve fibers in the muscular layer of porcine ileum. The study was performed on ten immature pigs divided into two groups: experimental group (n = 5), where zearalenone (10 μg/kg body weight) was given for 42 days, and control animals (n = 5), where placebo was administered. Fragments of ileum of all animals were processed for single-labelling immunofluorescence technique using the antibodies against vasoactive intestinal peptide, neuronal form of nitric oxide synthase, cocaine and amphetamine regulatory peptide, galanin, pituitary adenylate cyclase-activating peptide-27 and substance P. The number of nerve fibers immunoreactive to particular substances was evaluated by the counting of nerves per observation field (0.1 mm2). Low doses of zearalenone caused the clear changes in the expression of substances studied. The number of nerve fibers immunoreactive to the majority of substances increased in experimental animals. The exception was only galanin, the expression of which was less after administration of zearalenone. The obtained results for the first time show that even low doses of zearalenone can affect the nerve fibers in the digestive tract.
PMCID: PMC4529468  PMID: 25772391
Zearalenone; Mycotoxins; Nerve fibers; Gastrointestinal tract; Immunohistochemistry; Pig
24.  Small-Molecule Anticonvulsant Agents with Potent in vitro Neuroprotection and Favorable Drug-like Properties 
Severe seizure activity is associated with reoccurring cycles of excitotoxicity and oxidative stress that result in progressive neuronal damage and death. Intervention with these pathological processes is a compelling disease-modifying strategy for the treatment of seizure disorders. We have optimized a series of small molecules for neuroprotective and anticonvulsant activity as well as altered their physical properties to address potential metabolic liabilities, to improve CNS penetration and to prolong the duration of action in vivo. Utilizing phenotypic screening of hippocampal cultures with nutrient medium depleted of antioxidants as a disease model, cell death and decreased neuronal viability produced by acute treatment with glutamate or hydrogen peroxide were prevented. Modifications to our previously reported proof of concept compounds have resulted in a lead which has full neuroprotective action at < 1 nM and antiseizure activity across six animal models, including the kindled rat, and displays excellent pharmacokinetics including high exposure to the brain. These modifications have also eliminated the requirement for a chiral molecule, removing the possibility of racemization and making large scale synthesis more easily accessible. These studies strengthen our earlier findings which indicate that potent, multifunctional neuroprotective anticonvulsants are feasible within a single molecular entity which also possesses favorable CNS-active drug properties in vitro and in vivo.
PMCID: PMC3945118  PMID: 24277343
neuroprotection; glutamate toxicity; oxidative stress; hippocampal cultures; epilepsy; anticonvulsant; pharmacokinetics
25.  Vasoactive Intestinal Peptide in Early Spondyloarthritis: Low Serum Levels as a Potential Biomarker for Disease Severity 
Journal of Molecular Neuroscience  2015;56(3):577-584.
Spondyloarthritis (SpA) is a family of inflammatory diseases sharing clinical, genetic, and radiological features. While crucial for tailoring early interventions, validated prognostic biomarkers are scarce in SpA. We analyze the correlation between serum levels of vasoactive intestinal peptide (VIP) and disease activity/severity in patients with early chronic inflammatory back pain. The study population comprised 54 patients enrolled in our early chronic inflammatory back pain register. We collected demographic information, clinical data, laboratory data, and imaging findings. VIP levels were measured by enzyme immunoassay in serum samples from 162 visits. The association between independent variables and VIP levels was analyzed using longitudinal multivariate analysis nested by patient and visit. No significant differences were observed in VIP levels between these two groups. Lower levels of VIP were significantly associated with a higher Bath Ankylosing Spondylitis Disease Activity Index (BASFI) score, presence of bone edema in magnetic resonance imaging (MRI) scan, and lower hemoglobin levels. Coexistence of cutaneous psoriasis was independently associated with lower VIP levels, and similar trend was observed for enthesitis. We conclude that SpA patients with low serum VIP levels had worse 2-year disease outcome, suggesting that serum VIP levels could be a valid prognostic biomarker.
PMCID: PMC4477066  PMID: 25711477
Neuroimmunomodulation; Spondyloarthritis; Vasoactive intestinal peptide; Prognosis biomarker; Bath Ankylosing Spondylitis Disease Activity Index

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