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1.  [No title available] 
PMCID: PMC3939038  PMID: 24334024
2.  Contaminating levels of zinc found in commonly-used labware and buffers affect glycine receptor currents 
Brain research bulletin  2013;100:10.1016/j.brainresbull.2013.10.012.
Zinc is an allosteric modulator of glycine receptor function, enhancing the effects of glycine at nM to low μM concentrations, and inhibiting its effects at higher concentrations. Because of zinc’s high potency at the glycine receptor, there exists a possibility that effects attributed solely to exogenously-applied glycine in fact contain an undetected contribution of zinc acting as an allosteric modulator. We found that glycine solutions made up in standard buffers and using deionized distilled water produced effects that could be decreased by the zinc chelator tricine. This phenomenon was observed in three different vials tested and persisted even if vials were extensively washed, suggesting the zinc was probably present in the buffer constituents. In addition, polystyrene, but not glass, pipets bore a contaminant that enhanced glycine receptor function and that could also be antagonized by tricine. Our findings suggest that without checking for this effect using a chelator such as tricine, one cannot assume that responses elicited by glycine applied alone are not necessarily also partially due to some level of allosteric modulation by zinc.
PMCID: PMC3885322  PMID: 24177173
Zinc; Tricine; Allosteric modulation; Glycine receptor; electrophysiology; Xenopus oocytes
3.  Prenatal Dexamethasone, as Used in Preterm Labor, Worsens the Impact of Postnatal Chlorpyrifos Exposure on Serotonergic Pathways 
Brain research bulletin  2013;100:44-54.
This study explores how glucocorticoids sensitize the developing brain to the organophosphate pesticide, chlorpyrifos. Pregnant rats received a standard therapeutic dose (0.2 mg/kg) of dexamethasone on gestational days 17–19; pups were given subtoxic doses of chlorpyrifos on postnatal days 1–4, (1 mg/kg, <10% cholinesterase inhibition). We evaluated serotonin (5HT) synaptic function from postnatal day 30 to day 150, assessing the expression of 5HT receptors and the 5HT transporter, along with 5HT turnover (index of presynaptic impulse activity) in brain regions encompassing all the 5HT projections and cell bodies. These parameters are known targets for neurodevelopmental effects of dexamethasone and chlorpyrifos individually. In males, chlorpyrifos evoked overall elevations in the expression of 5HT synaptic proteins, with a progressive increase from adolescence to adulthood; this effect was attenuated by prenatal dexamethasone treatment. The chlorpyrifos-induced upregulation was preceded by deficits in 5HT turnover, indicating that the receptor upregulation was an adaptive response to deficient presynaptic activity. Turnover deficiencies were magnified by dexamethasone pretreatment, worsening the functional impairment caused by chlorpyrifos. In females, chlorpyrifos-induced receptor changes reflected relative sparing of adverse effects compared to males. Nevertheless, prenatal dexamethasone still worsened the 5HT turnover deficits and reduced 5HT receptor expression in females, demonstrating the same adverse interaction. Glucocorticoids are used in 10% of U.S. pregnancies, and are also elevated in maternal stress; accordingly, our results indicate that this group represents a large subpopulation that may have heightened vulnerability to developmental neurotoxicants such as the organophosphates.
PMCID: PMC3891922  PMID: 24280657
Chlorpyrifos; Dexamethasone; Glucocorticoids; Organophosphate pesticides; Preterm delivery; Serotonin
4.  Knockout of vascular early response gene worsens chronic stroke outcomes in neonatal mice 
Brain research bulletin  2013;98:111-121.
Vascular early response gene (Verge) is a novel immediate early gene that is highly expressed during developmental angiogenesis and after ischemic insults in adult brain. However, the role of Verge after neonatal injury is not known. In the present study, we investigated the hypothesis that Verge contributes to vascular remodeling and tissue repair after neonatal ischemic injury. The Rice–Vanucci model (RVM) was employed to induce neonatal stroke in both Verge knockout (KO) and wild-type (WT) postnatal day 10 (P10) mice. Histological and behavioral outcomes at acute (24 h), subacute (7 days) and chronic (30 days) phases were evaluated. Angiogenesis, neurogenesis, and glial scar formation were also examined in the ischemic brain. No significant differences in outcomes were found between WT and Verge mice at 24 h or 7 days after stroke. However genetic deletion of Verge led to pronounced cystic cavitation, decreased angiogenensis and glial scar formation in the ischemic hemisphere compared to WT mice at 30 days. Verge KO mice also had significantly worse functional outcomes at 30 days which was accompanied by decreased neurogenesis and angiogenesis in the ischemic hemisphere. Our study suggests that Verge plays an important role in the induction of neurogenesis and angiogenesis after ischemia, contributes to improved tissue repair, and enhances chronic functional recovery.
PMCID: PMC4275796  PMID: 23973431
Angiogenesis; Ischemic stroke; Neonate; Neurogenesis; Vascular early response gene
5.  In vivo SPECT and ex vivo autoradiographic brain imaging of the novel selective CB1 receptor antagonist radioligand [125I]SD7015 in CB1 knock-out and wildtype mouse 
Brain research bulletin  2013;91:46-51.
We aimed to evaluate the novel high-affinity and relatively lipophilic CB1 receptor (CB1R) antagonist radioligand [125I]SD7015 for SPECT imaging of CB1Rs in vivo using the multiplexed multipinhole dedicated small animal SPECT/CT system, NanoSPECT/CTPLUS (Mediso, Budapest, Hungary), in knock-out CB1 receptor knock-out (CB1R-/-) and wildtype mice. In order to exclude possible differences in cerebral blood flow between the two types of animals, HMPAO SPECT scans were performed, whereas in order to confirm the brain uptake differences of the radioligand between knock-out mice and wildtype mice, in vivo scans were complemented with ex vivo autoradiographic measurements using the brains of the same animals. With SPECT/CT imaging, we measured the brain uptake of radioactivity, using %SUV (% standardised uptake values) in CB1R-/- mice (n = 3) and C57BL6 wildtype mice (n = 7) under urethane anaesthesia after injecting [125I]SD7015 intravenously or intraperitoneally. The Brookhaven Laboratory mouse MRI atlas was fused to the SPECT/CT images by using a combination of rigid and non-rigid algorithms in the Mediso Fusion™ (Mediso, Budapest, Hungary) and VivoQuant (inviCRO, Boston, MA, USA) softwares. Phosphor imager plate autoradiography (ARG) was performed on 4 μm-thin cryostat sections of the excised brains. %SUV was 8.6 ± 3.6 (average ± SD) in CB1R-/- mice and 22.1 ± 12.4 in wildtype mice between 2 and 4 h after injection (p < 0.05). ARG of identically taken sections from wildtype mouse brain showed moderate radioactivity uptake when compared with the in vivo images, with a clear difference between grey matter and white matter, whereas ARG in CB1R(-/-) mice showed practically no radioactivity uptake. [125I]SD7015 enters the mouse brain in sufficient amount to enable SPECT imaging. Brain radioactivity distribution largely coincides with that of the known CB1R expression pattern in rodent brain. We conclude that [125I]SD7015 should be a useful SPECT radioligand for studying brain CB1R in mouse and rat disease models.
PMCID: PMC4182908  PMID: 23318272
Endocannabinoid CB1 receptor (CB1R); Single photon emission computed tomography (SPECT); Molecular imaging biomarker; [125I]SD7015; Knock-out CB1R-/- mouse; Multiplexed multipinhole dedicated small animal SPECT/CT system
6.  Editorial 
PMCID: PMC4180212  PMID: 24513428
7.  The decrease of dopamine D2/D3 receptor densities in the putamen and nucleus caudatus goes parallel with maintained levels of CB1 cannabinoid receptors in Parkinson’s disease: A preliminary autoradiographic study with the selective dopamine D2/D3 antagonist [3H]raclopride and the novel CB1 inverse agonist [125I]SD7015 
Brain research bulletin  2012;87(6):504-510.
Cannabinoid type-1 receptors (CB1Rs) modulate synaptic neurotransmission by participating in retrograde signaling in the adult brain. Increasing evidence suggests that cannabinoids through CB1Rs play an important role in the regulation of motor activities in the striatum. In the present study, we used human brain samples to examine the relationship between CB1R and dopamine receptor density in case of Parkinson’s disease (PD).
Post mortem putamen, nucleus caudatus and medial frontal gyrus samples obtained from PD patients were used for CB1R and dopamine D2/D3 receptor autoradiography. [125I]SD7015, a novel selective CB1R inverse agonist, developed by a number of the present co-authors, and [3H]raclopride, a dopamine D2/D3 antagonist, were used as radioligands. Our results demonstrate unchanged CB1R density in the putamen and nucleus caudatus of deceased PD patients, treated with levodopa (l-DOPA). At the same time dopamine D2/D3 receptors displayed significantly decreased density levels in case of PD putamen (control: 47.97 ± 10.00 fmol/g, PD: 3.73 ± 0.07 fmol/g (mean ± SEM), p < 0.05) and nucleus caudatus (control: 30.26 ± 2.48 fmol/g, PD: 12.84 ± 5.49 fmol/g, p < 0.0005) samples. In contrast to the putamen and the nucleus caudatus, in the medial frontal gyrus neither receptor densities were affected.
Our data suggest the presence of an unaltered CB1R population even in late stages of levodopa treated PD. This further supports the presence of an intact CB1R population which, in line with the conclusion of earlier publications, may be utilized as a pharmacological target in the treatment of PD. Furthermore we found discrepancy between a maintained CB1R population and a decreased dopamine D2/D3 receptor population in PD striatum. The precise explanation of this conundrum requires further studies with simultaneous examination of the central cannabinoid and dopaminergic systems in PD using higher sample size.
PMCID: PMC4180092  PMID: 22421165
Parkinson’s disease; Endocannabinoid CB1 receptor; Dopamine D2/D3 receptor; Molecular imaging biomarker; Human brain autoradiography; Striatum
8.  Glutamate transporter type 3 mediates isoflurane preconditioning-induced acute phase of neuroprotection in mice 
Brain research bulletin  2013;98:23-29.
A pre-exposure to isoflurane reduces ischemic brain injury in rodents (isoflurane preconditioning). This neuroprotection has acute and delayed phases. Our previous in vitro studies suggest that the acute phase may involve excitatory amino acid transporters (EAAT). We determine whether this protection involves EAAT3, the major neuronal EAAT. Adult male EAAT3 knockout mice and their wild-type littermates were exposed or were not exposed to 1.5% isoflurane for 30 min. Sixty minutes later, they were subjected to a 90- or 60-min middle cerebral arterial occlusion (MCAO). Their neurological outcomes were evaluated 24 h after the MCAO. In another experiment, cerebral cortex was harvested for Western blotting at 30 min after animals were exposed to 1.5% isoflurane for 30 min. Here, we showed that isoflurane reduced brain infarct volumes and improved neurological functions of wild-type mice after a 90-min MCAO. However, isoflurane pre-exposure did not change the neurological outcome of EAAT3 knockout mice no matter whether the MCAO was for 90 min or 60 min. Isoflurane increased phospho-Akt, a survival-promoting protein, in the wild-type mice but not in the EAAT3 knockout mice. The isoflurane-induced neuroprotection in the wild-type mice was abolished by LY294004, an Akt activation inhibitor. LY294004 alone did not affect the neurological outcome of the wild-type or EAAT3 knockout mice after focal brain ischemia. These results suggest that the isoflurane preconditioning-induced acute phase of neuroprotection involves EAAT3. The downstream event includes Akt activation.
PMCID: PMC3805665  PMID: 23827345
Akt; glutamate transporter; isoflurane; neuroprotection; preconditioning
9.  Biochemical, behavioral, physiologic, and neurodevelopmental changes in mice deficient in monoamine oxidase A or B 
Brain research bulletin  2001;56(5):453-462.
The availability of mutant mice that lack either MAO A or MAO B has created unique profiles in the central and peripheral availability of serotonin, norepinephrine, dopamine, and phenylethylamine. This paper summarizes some of the current known phenotypic findings in MAO A knock-out mice and contrast these with those of MAO B knock-out mice. Differences are discussed in relation to the biochemical, behavioral, and physiologic changes investigated to date, as well as the role played by redundancy mechanisms, adaptational responses, and alterations in neurodevelopment.
PMCID: PMC4109811  PMID: 11750790
Monoamine oxidase; Serotonin; Norepinephrine; Phenylethylamine; Dopamine
10.  Putative genes mediating the effects of orexins in the posterior paraventricular thalamus on neuroendocrine and behavioral adaptations to repeated stress 
Brain research bulletin  2012;89(0):203-210.
Exposure to repeated stress is often associated with psychopathology. However, our understanding of the underlying neural circuitry that regulates responses to repeated stress is limited. The posterior paraventricular thalamus (pPVT) is a brain region responsible for transmission of multimodal sensory information to limbic structures that regulate responses to both acute and repeated stress. Orexin-containing cells originating in the hypothalamus heavily innervate the pPVT. Our previous work has shown that activation of orexin1 receptors in the pPVT during repeated swim stress is important for facilitation of the hypothalamic-pituitary-adrenal (HPA) axis response to subsequent novel restraint. However, the genes responsible for these orexin-mediated adaptations to repeated stress are not known. Using a custom PCR array we examined the expression of 186 specific mRNAs in the pPVT of animals exposed to repeated swim stress (4 days of 15 min swim/day) with or without direct pPVT microinfusion of the orexin1 receptor antagonist SB334867 prior to each daily swim stress. Tissue was collected the next morning under basal non stressed conditions. Repeated stress and/or orexin receptor blockade significantly altered expression of only 9 specific genes including growth factors (Vegfa, Bax and Mt3), G-protein coupled receptors (Adora2a, Grm2 and Crhr1), immune-related genes (Ptgs2 and Cx3cr1) and an epigenetic-related gene (Hdac5). These genes represent potential targets for further characterization of orexin-mediated adaptations to repeated stress in the pPVT.
PMCID: PMC4068329  PMID: 22982687
Hypocretin; Hypothalamic pituitary adrenal; PCR array; Crhr1; Vegfa; Bax; Mt3; Ptgs2; Ador2a; Grm2; Cx3cr1; Hdac5
11.  Diabetic plasticity of non-adrenergic non-cholinergic and P2X-mediated rat bladder contractions 
Brain research bulletin  2013;95:40-45.
We investigated the plasticity effects of diabetes mellitus and diuresis on the non-adrenergic non-cholinergic (NANC) and purinergic (P2X-type) contractile responses in longitudinal rat bladder strips. Female Sprague-Dawley rats received streptozotocin to induce diabetes, or sucrose in water to induce diuresis as a control condition for polyuria. Experiments were carried out at four weeks after treatments, using bladders from not-treated rats as control. Urinary bladder strips were electrically stimulated throughout the experiments to generate neurally evoked contractions (NEC). In all cases, P2X-mediated purinergic contractions were evaluated at the beginning and end of the stimulations with α,β-Methylene Adenosine Triphosphate (α,βMeATP). The NANC responses were assessed by using two independent protocols. First, cholinergic receptors were activated with carbachol (CCh), followed by inhibition of the muscarinic component with atropine. In the second protocol, the application order for CCh and atropine was reversed. The NANC response, unmasked with the application of atropine, and the P2X purinergic contractions were analyzed. NANC contractions in diabetic bladder strips are more resistant to the desensitizing effects caused by activation of cholinergic receptors. In early stages of experimental diabetes, NANC responses in diabetic strips are less sensitive to functional inhibition mediated by the cholinergic activation. However, P2X-mediated purinergic contractions are more sensitive to desensitization in diabetic or diuretic bladders. For instance preventing muscarinic receptor activation with atropine does not counteract the desensitization of purinergic contractions in either diabetic or diuretic strips. We suggest that diabetes may induce a plasticity of the NANC and P2X-mediated bladder contractile responses. The first one may be associated with diabetic neuropathic damage to bladder nerves, while impaired P2X purinergic contractions might be associated with detrusor hypertrophy observed in diabetic and diuretic strips.
PMCID: PMC3681287  PMID: 23562604
Diabetic bladder dysfunction; purinergic receptor; cholinergic receptor; non-adrenergic non-cholinergic response; neurally evoked contraction; detrusor muscle
12.  Enhance, delete, incept: Manipulating hippocampus-dependent memories☆ 
Brain Research Bulletin  2014;105(100):2-7.
•Newly developed methods allow manipulation of hippocampus-dependent memories.•Cued reactivation during sleep and transcranial stimulation can enhance memories.•Pharmacological agents can delete memories.•Optogenetics and DREADDs can be used to incept memories.•Electrophysiology and fMRI have been used to investigate the underlying mechanisms.
Here we provide a brief overview of recent research on memory manipulation. We focus primarily on memories for which the hippocampus is thought to be required due to its central importance in the study of memory. The repertoire of methods employed is expanding and includes optogenetics, transcranial stimulation, deep brain stimulation, cued reactivation during sleep and the use of pharmacological agents. In addition, the possible mechanisms underlying these memory changes have been investigated using techniques such as single unit recording and functional magnetic resonance imaging (fMRI).
This article is part of a Special Issue entitled ‘Memory enhancement’.
PMCID: PMC4058530  PMID: 24397964
Memory consolidation; Sleep; Hippocampus; Fear conditioning; Extinction; Reconsolidation
13.  Construction of pathways to promote axon growth within the adult central nervous system 
Brain research bulletin  2010;84(0):300-305.
Inducing significant axon growth or regeneration after spinal cord injury has been difficult, primarily due to the poor growth supportive environment and low intrinsic growth ability of neurons within the CNS. Neurotrophins alone have been shown to readily induce regeneration of sensory axons after dorsal root lesions, however if neurotrophin gradients are expressed within the spinal cord these axons fail to terminate within appropriate target regions. Under such conditions, addition of a “stop” signal reduces growth into deeper dorsal laminae to support more specific targeting. Such neurotrophin gradients alone lose their effectiveness when lesions are within the spinal cord, requiring a combined treatment regime. Construction of pathways using combined treatments support good regeneration when they increase the intrinsic growth properties of neurons, provide a bridge across the lesion site, and supply a growth supportive substrate to induce axon growth out of the bridge and back into the host. Neurotrophin gradients distal to the bridge greatly enhance axon outgrowth. In disorders where neuronal circuits are lost, construction of preformed growth supportive pathways sustain long distance axon growth from a neuronal transplant to distal target locations.
PMCID: PMC4038358  PMID: 20554000
Axon regeneration; Spinal cord injury; Neurotrophin; Gradient; Guidance molecules
14.  Distribution of secretagogin-containing neurons in the basal forebrain of mice, with special reference to the cholinergic corticopetal system 
Brain research bulletin  2013;94:1-8.
Cholinergic and GABAergic corticopetal neurons in the basal forebrain play important roles in cortical activation, sensory processing, and attention. Cholinergic neurons are intermingled with peptidergic, and various calcium binding protein-containing cells, however, the functional role of these neurons is not well understood. In this study we examined the expression pattern of secretagogin (Scgn), a newly described calcium-binding protein, in neurons of the basal forebrain. We also assessed some of the corticopetal projections of Scgn neurons and their co-localization with choline acetyltransferase (ChAT), neuropeptide-Y, and other calcium-binding proteins (i.e., calbindin, calretinin, and parvalbumin). Scgn is expressed in cell bodies of the medial and lateral septum, vertical and horizontal diagonal band nuclei, and of the extension of the amygdala but it is almost absent in the ventral pallidum. Scgn is co-localized with ChAT in neurons of the bed nucleus of the stria terminalis, extension of the amygdala, and interstitial nucleus of the posterior limb of the anterior commissure. Scgn was co-localized with calretinin in the accumbens nucleus, medial division of the bed nucleus of stria terminalis, the extension of the amygdala, and interstitial nucleus of the posterior limb of the anterior commissure. We have not found co-expression of Scgn with parvalbumin, calbindin, or neuropeptide-Y. Retrograde tracing studies using Fluoro Gold in combination with Scgn-specific immunohistochemistry revealed that Scgn neurons situated in the nucleus of the horizontal limb of the diagonal band project to retrosplenial and cingulate cortical areas.
PMCID: PMC3628291  PMID: 23376788
basal forebrain; calcium-binding proteins; choline acetyltransferase; co-localization; cortical projection
15.  Effects of Social Housing on Hippocampal Dendrites and Behavior in Ovariectomized Rats 
Brain research bulletin  2012;92:69-75.
Social stress is both species and gender specific. For female rats, individual housing and social instability housing conditions are associated with behavioral indicators of stress and depression. The present study directly compared the effects of six weeks of individual housing, social instability and mixed sex, semi-crowded housing in a visible burrow system (VBS) on ovariectomized female rats. Paired, stable housing was used as the control. Behavioral tests were conducted two, four and six weeks into the housing manipulations and included sucrose consumption, social interest, and activity in the open field. Following a series of four behavioral tests, animals were sacrificed and brains were processed for Golgi impregnation. Basal dendrites of CA3 hippocampal neurons were measured. Results indicate that the individual housing and social instability groups were comparable to the control group for all measures. In contrast, the rats housed in the VBS exhibited reduced activity in open field testing, and alterations in social interest. Dendritic lengths were also reduced in those animals living in the VBS in comparison to the animals housed in pairs. To our knowledge, this is the first report of behavioral and neural effects of VBS housing on female rats. Further research is necessary to determine what facets of the VBS housing are responsible for the behavioral and neural changes.
PMCID: PMC3594123  PMID: 23194666
depression; social stress; hippocampal dendrites; open field behavior; anxiety
16.  Nucleus accumbens lesions modulate the effects of Methylphenidate 
Brain research bulletin  2010;82(0):293-301.
The psychostimulant methylphenidate (MPD, Ritalin) is the prescribed drug of choice for treatment of ADHD. In recent years, the diagnosis rate of ADHD has increased dramatically, as have the number of MPD prescriptions. Repeated exposure to psychostimulants produces behavioral sensitization in rats, an experimental indicator of a drug’s potential liability. In studies on cocaine and amphetamine, this effect has been reported to involve the nucleus accumbens (NAc), one of the nuclei belonging to the motive circuit. The aim of this study was to investigate the role of the NAc on the expression of behavioral sensitization as a response to MPD exposure. In the present study, 20 male Sprague-Dawley rats were divided randomly into three groups: an intact control group, a sham operated group, and a NAc bilateral electrical lesion group. Locomotor activity was assessed for the first two hours following 2.5 mg/kg MPD injection, using open field monitoring systems. Recordings were made during six days of continuous MPD administration, and then upon re-challenge with the same dose following three days of washout. Acute MPD exposure elicited an increase in locomotor activity in all three groups. However, the NAc lesion group exhibited significantly increased locomotor activity in comparison to sham and control groups. Chronic MPD did not elicit sensitization in the NAc lesion group, while both sham and control groups did exhibit behavioral sensitization to repetitive MPD administration. These findings suggest that the NAc plays a significant role in eliciting locomotor activity as an acute effect of MPD, and in the expression of sensitization due to chronic MPD exposure.
PMCID: PMC3935495  PMID: 20470871
Nucleus accumbens; Ritalin; psychostimulant; behavioral sensitization
Brain research bulletin  2012;91:1-7.
The abrupt discontinuation of prolonged benzodiazepine treatment elicits a withdrawal syndrome with increased anxiety as a major symptom. The neural mechanisms underlying benzodiazepine physical dependence are still insufficiently understood. Flumazenil, the non-selective antagonist of the benzodiazepine binding site of GABAA receptors was capable of preventing and reversing the increased anxiety during benzodiazepine withdrawal in animals and humans in some, but not all studies. On the other hand, a number of data suggest that GABAA receptors containing α1 subunits are critically involved in processes developing during prolonged use of benzodiazepines, such are tolerance to sedative effects, liability to physical dependence and addiction. Hence, we investigated in the elevated plus maze the level of anxiety 24 h following 21 days of diazepam treatment and the influence of flumazenil or a preferential α1-subunit selective antagonist βCCt on diazepam withdrawal syndrome in rats. Abrupt cessation of protracted once-daily intraperitoneal administration of 2 mg/kg diazepam induced a withdrawal syndrome, measured by increased anxiety-like behavior in the elevated plus maze 24 h after treatment cessation. Acute challenge with either flumazenil (10 mg/kg) or βCCt (1.25, 5 and 20 mg/kg) alleviated the diazepam withdrawal-induced anxiety. Moreover, both antagonists induced an anxiolytic-like response close, though not identical, to that seen with acute administration of diazepam. These findings imply that the mechanism by which antagonism at GABAA receptors may reverse the withdrawal-induced anxiety involves the α1 subunit and prompt further studies aimed at linking the changes in behavior with possible adaptive changes in subunit expression and function of GABAA receptors.
PMCID: PMC3578024  PMID: 23149168
elevated plus maze; βCCt; antagonism; benzodiazepines; physical dependence
18.  Effect of repeated exposure to MDMA on the function of the 5-HT transporter as assessed by synaptosomal 5-HT uptake 
Brain research bulletin  2013;91:52-57.
Recent studies have demonstrated that a preconditioning regimen (i.e., repeated low doses) of MDMA provides protection against the reductions in tissue concentrations of 5-HT and 5-HT transporter (SERT) density and/or expression produced by a subsequent binge regimen of MDMA. In the present study, the effects of preconditioning and binge treatment regimens of MDMA on SERT function were assessed by synaptosomal 5-HT uptake. Synaptosomal 5-HT uptake was reduced by 72% 7 days following the binge regimen (10 mg/kg, ip every 2 hr for a total of 4 injections). In rats exposed to the preconditioning regimen of MDMA (daily treatment with 10 mg/kg for 4 days), the reduction in synaptosomal 5-HT uptake induced by a subsequent binge regimen was significantly less. Treatment with the preconditioning regimen alone resulted in a transient 46% reduction in 5-HT uptake that was evident 1 day, but not 7 days, following the last injection of MDMA. Furthermore, the preconditioning regimen of MDMA did not alter tissue concentrations of 5-HT, whereas the binge regimen of MDMA resulted in a long-term reduction of 40% of tissue 5-HT concentrations. The distribution of SERT immunoreactivity (ir) in membrane and endosomal fractions of the hippocampus also was evaluated following the preconditioning regimen of MDMA. There was no significant difference in the relative distribution of SERTir between these two compartments in control and preconditioned rats. The results demonstrate that SERT function is transiently reduced in response to a preconditioning regimen of MDMA, while long-term reductions in SERT function occur in response to a binge regimen of MDMA. Moreover, a preconditioning regimen of MDMA provides protection against the long-term reductions in SERT function evoked by a subsequent binge regimen of the drug. It is tempting to speculate that the neuroprotective effect of MDMA preconditioning results from a transient down-regulation in SERT function.
PMCID: PMC3578032  PMID: 23318273
MDMA; serotonin; serotonin transporter
19.  Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia 
Brain research bulletin  2003;60(0):423-433.
The molecular and cellular origin of the primary neurons of the inner ear, the vestibular and spiral neurons, is reviewed including how they connect to the specific sensory epithelia and what the molecular nature of their survival is. Primary neurons of the ear depend on a single basic Helix-Loop-Helix (bHLH) protein for their formation, neurogenin 1 (ngn1). An immediate downstream gene is the bHLH gene neuronal differentiation (NeuroD). Targeted null mutations of ngn1 results in absence of primary neuron formation; targeted null mutation of NeuroD results in loss of almost all spiral and many vestibular neurons. NeuroD and a later expressed gene, Brn3a, play a role in pathfinding to and within sensory epithelia. The molecular nature of this pathfinding property is unknown. Reduction of hair cells in ngn1 null mutations suggests a clonal relationship with primary neurons. This relationship may play some role in specifying the identity of hair cells and the primary neurons that connect with them. Primary neuron neurites growth to sensory epithelia is initially independent of trophic factors released from developing sensory epithelia, but becomes rapidly dependent on those factors. Null mutations of specific neurotrophic factors lose distinct primary neuron populations which undergo rapid embryonic cell death.
PMCID: PMC3904733  PMID: 12787865
Inner ear; Primary neurons; Development; Molecular origin; Primary neuron survival; Pathfinding
20.  Near-infrared laser illumination transforms the fluorescence absorbing X-Gal reaction product BCI into a transparent, yet brightly fluorescent substance 
Brain research bulletin  2005;70(1):33-43.
The β-galactosidase protein generated by the bacterial LacZ gene is widely used to map gene expression patterns. The ease of its use is only rivaled by green fluorescent protein, which can be used in combination with various other procedures such as immunocytochemistry, flow cytometry, or tract tracing. The β-galactosidase enzymatic reaction provides potentially a more sensitive assay of gene expression than green fluorescent protein. However, the virtual impermeability and tendency to absorb light over a wide range limit the use of the most frequently used β-galactosidase substrate, X-Gal, in combination with other fluorescent labeling procedures. Here, we provide details on a simple photoactivation procedure that transforms the light-absorbing X-Gal product, 5-bromo-4-chloro-3-indolyl (BCI) precipitate, into an intensely fluorescent product excited by 488 and 633 nm light. Photoactivation is achieved through exposure to 730 nm near-infrared light emitted from a femtosecond titanium-doped Sapphire laser. Photoactivation of BCI occurs in tissue sections suspended in buffered saline, glycerol, or even embedded in epoxy resin. A protocol for the use of BCI photoactivation is here provided. Importantly, the BCI photoactivated product is photoswitchable, displaying bistable photochromism. This permits the use of the fluorescent product in a variety of co-localization studies in conjunction with other imaging modalities. As with other bistable and photoswitchable products, the BCI reaction product shows concentration quenching at high density and can be degraded by continuous exposure to intense 730 nm illumination. Therefore, care must be taken in developing imaging strategies. Our findings have implications for the use of X-Gal in gene and protein detection and provide a novel substrate for high density digital information storage.
PMCID: PMC3904734  PMID: 16750480
β-Galactosidase; X-Gal reaction; Confocal microscopy; Gene expression; Two photon excitation; Fluorescence
21.  Development of vestibular afferent projections into the hindbrain and their central targets 
Brain research bulletin  2003;60(0):497-510.
In contrast to most other sensory systems, hardly anything is known about the neuroanatomical development of central projections of primary vestibular neurons and how their second order target neurons develop. Recent data suggest that afferent projections may develop not unlike other sensory systems, forming first the overall projection by molecular means followed by an as yet unspecified phase of activity mediated refinement. The latter aspect has not been tested critically and most molecules that guide the initial projection are unknown.
The molecular and topological origin of the vestibular and cochlear nucleus neurons is also only partially understood. Auditory and vestibular nuclei form from several rhombomeres and a given rhombomere can contribute to two or more auditory or vestibular nuclei. Rhombomere compartments develop as functional subdivisions from a single column that extends from the hindbrain to the spinal cord. Suggestions are provided for the molecular origin of these columns but data on specific mutants testing these proposals are not yet available. Overall, the functional significance of both overlapping and segregated projections are not yet fully experimentally explored in mammals. Such lack of details of the adult organization compromises future developmental analysis.
PMCID: PMC3901526  PMID: 12787869
Vestibular; Hindbrain; Sensory system
22.  Use of a modified prepulse inhibition paradigm to assess complex auditory discrimination in rodents 
Brain research bulletin  2007;76(0):1-7.
Prepulse inhibition (PPI; also termed startle reduction or reflex modification, see [15]) provides an efficient and accurate method to assess both simple and complex acoustic discrimination in rodents [18]. Assessment of acoustic processing using PPI is less time consuming than operant conditioning paradigms, allows for the testing of many subjects simultaneously, and largely eliminates confounds due to motivation and attention [6]. Moreover, PPI procedures allow for data acquisition from the first day of testing, and can be used on rats as young as P14–15 [14, 31, 43]. For these and additional reasons, the PPI paradigm has more recently been adapted to the assessment of complex acoustic discrimination (tone sequences and FM sweeps), and applied to the study of normally developing as well as neuropathologically affected rodent populations.
The purpose of the current review is to provide a background on the PPI paradigm, and to summarize what has been learned more recently using modified versions of PPI with rodent models.
PMCID: PMC3888703  PMID: 18395604
23.  Probing the role of the sodium/calcium exchanger in pentylenetetrazole-induced generalized seizures in rats 
Brain research bulletin  2012;90:52-57.
The Na+/Ca2+ exchanger (NCX) is thought to play an important role in the pathogenesis of pentylenetetrazole (PTZ)-induced tonic flexion in mice. Here, I investigated the expression of PTZ-induced generalized clonic and tonic-clonic seizures in rats, using two potent NCX reverse mode inhibitors, KB-R7943 and SN-6 for NCX subtypes 3 (NCX3) and 1 (NCX1), respectively. Pretreatment with KB-R7943 (3, 10, 30 mg/kg; p.o.) significantly reduced the expression of PTZ-induced generalized seizures with clonic and tonic-clonic components in 12–62% and 25–62% of the treated animals, respectively. In the remaining animals that exhibited seizures, KB-R7943 (3 mg/kg; p.o.) pretreatment significantly delayed the onset of the first seizure episode and reduced the seizure severity. Following pretreatment with SN-6 (0.3, 1, 3, 10, 30 mg/kg; p.o.), clonic and tonic-clonic PTZ-induced generalized seizures were reduced in 25–50% and 38–63% of treated animals, respectively. SN-6 (0.3, 1, 3, mg/kg; p.o.) also significantly reduced PTZinduced seizure severity scores, but did not alter seizure latencies. KB-R7943 (3, 30 mg/kg; p.o.) or SN-6 (3, 30 mg/kg; p.o.) administration potentiated the sub-anticonvulsant dose of diazepam (2.5 mg/kg; i.p.) that suppresses clonic and tonic-clonic PTZ-induced seizures. These findings suggested that Ca2+ influx via the NCX in reverse mode contributes to a neuronal hyperexcitability that leads to clonic and tonic-clonic generalized seizures and that the NCX1 and NCX3 isoforms may serve as novel molecular targets for seizure suppression.
PMCID: PMC3527654  PMID: 22995283
KB-7943; SN-6; anticonvulsant; hyperexcitability; clonic seizures; tonic-clonic seizures
24.  Ascorbic Acid Efficiently Enhances Neuronal Synthesis of Norepinephrine from Dopamine 
Brain research bulletin  2012;90:35-42.
Ascorbic acid enhances synthesis of norepinephrine from dopamine in adrenal chromaffin cells by serving as a co-factor for chromaffin granule dopamine β-hydroxylase (DβH). However, there is controversy regarding in situ kinetics of the ascorbate effect in chromaffin cells, as well as whether they apply to neuronal cells. In this study we evaluated the stimulation of norepinephrine synthesis from dopamine in cultured SH-SY5Y neuroblastoma cells. These cells contained neither ascorbate nor norepinephrine in culture, but when provided with dopamine, they generated intracellular norepinephrine at rates that were stimulated several fold by intracellular ascorbate. Ascorbate-induced increases in norepinephrine synthesis in dopamine-treated cells were linear over 60 minutes, despite saturation of intracellular ascorbate. Norepinephrine accumulation after 60 minutes of incubation with 100 μM dopamine was half-maximal at intracellular ascorbate concentrations of 0.2 – 0.5 mM, which fits well with the literature Km for ascorbate of DβH using dopamine as a substrate. Moreover, these ascorbate concentrations were generated by initial extracellular ascorbate concentrations of less than 25 μM due to concentrative accumulation by the ascorbate transporter. Treatment with 100 μM dopamine acutely increased cellular superoxide generation, which was prevented by ascorbate loading, but associated with a decrease in intracellular ascorbate when the latter was present at concentrations under 1 mM. These results show that ascorbate promptly enhances norepinephrine synthesis from dopamine by neuronal cells, that it does so at physiologic intracellular concentrations in accord with the kinetics of DβH, and that it both protects cells from superoxide and by providing electrons to DβH.
PMCID: PMC3527656  PMID: 23022576
dopamine; norepinephrine; dopamine β-hydroxylase; oxidative stress; SH-SY5Y neuroblastoma cells
25.  Norepinephrine inhibition in juvenile male zebra finches modulates adult song quality 
Brain research bulletin  2012;90:132-136.
During development, male zebra finches learn a song that they eventually use in courtship and defense of nest sites. Norepinephrine (NE) is important for learning and memory in vertebrates, and this neuromodulator and its receptors are present throughout the brain regions that control song learning and production. The present study used the neurotoxin N-(2-Chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP4) to reduce brain levels of NE in juvenile males. This manipulation inhibited the development of quality songs, with some birds producing syllables that were unusually long and/or contained frequencies that were predominantly higher than normal. These results suggest that NE is important for the acquisition of typical song.
PMCID: PMC3527669  PMID: 23160069
Songbird; development; DSP4; catecholamine

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