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46 P.P. Kale, V. Addepalli / Int. J. D
compensatory increase at E18. To resolve whether this delayn neurogenesis is due to defect in differentiation from progeni-ors, double labeling with the mitotic marker BrdU and cell cycle
arker Ki67 was performed at E16, and cell cycle quitting fractionas analyzed by flow cytometry and 5 folds decrease was seennder hypothyroidism. It could be either from diminished mitoticool of ventricular zone (VZ) resident apical progenitors or subven-ricular zone (SVZ) resident intermediate progenitor cells (IPCs).
apping of mitotic figures by p-Histone3 immunostaining revealedsignificant reduction in the mitotic progenitors in intermediate
one and increase in the VZ progenitors under hypothyroidism,ndicating loss of proliferating IPCs. This was further confirmed
ith the significant decrease in IPCs specific marker Tbr2 underypothyroidism. The loss of indirect neurogenesis by IPCs waslso evident in the reduction of telencephalic thickness underaternal hypothyroidism. Cultured neuronal progenitors showed
xpression TH transporter MCT8, De-iodinase-II and TH receptorR�1 indicating that IPCs can metabolize maternal TH and explainsheir regulation by altered TH status during development. Taken,ogether we describe direct regulation of neurogenesis by maternalH as seen by diminished pool of IPCs during development underypothyroidism.
yclic AMP pathway independent induction of neurite exten-ion and their networking in the motor neuron cell line NSC34
jeet Kumar, Himanshu K. Mishra, Priyanka Dwivedi, Jamuna R.ubramaniam ∗
Department of Biological Sciences and Bioengineering, Indian Institutef Technology, Kanpur 208016, India
-mail address: [email protected] (J.R. Subramaniam).Matrix stromal cells (HUMS) derived from the human umbili-
al cord secrete several neurotrophic factors. The HUMS cells andheir secreted factors are shown to provide some amount of neu-oprotection in the neurodegenerative disease models of mice. Buthe exact mechanism of protection is not well understood. Here, weeport that the HUMS cells secrete six neurotrophic factors, namely,T-3, NGF, BDNF, VEGF, IGF-1 and GDNF(NFs). These NFs present
n the conditioned medium of the HUMS cells induce differentia-ion, neurite extension and neural networking of a motor neuronell line, NSC34. These motor neurons express the tyrosine kinaseeceptors for the above trophic factors (except for BDNF), which arerucial for neurite extension. The tyrosine kinase inhibitor, K252a,rastically reduces CM induced neurite extension. Further, all theTFs need to be neutralized simultaneously with their antibodies
o abrogate neurite extension, proving the flexibility and prudentackup mechanism of the system. Intriguingly, none of the phe-omenon – differentiation, neurite extension or neural networkingequired cAMP second messenger system coupling as evidenced byAMP pathway activator or inhibitor treatment of the NSC34 cellsith or without CM.
solation and characterization of centrosomes from cellsxpressing modulated levels of Stil
. Chatterjee ∗, S. Mani
Centre for Neuroscience, Indian Institute of Science, Bangalore 560012,ndia
Introduction: Primary microcephaly is an autosomal recessiveongenital disorder which results from hypoplasia of the cerebral
uroscience 30 (2012) 640–671
cortex, characterized by a greater than three standard deviationsbelow the age and sex related mean for head circumference. All theproteins linked to this disorder (MCPH1, CDK5RAP2, ASPM, CENPJand STIL) localize to the centrosome, suggesting a role for the cen-trosome in neurogenesis during brain development.
STIL is a mitotic check point gene which controls the transitionfrom G2 to M phase in the cell cycle. It is expressed in all dividingcells and its expression decreases upon differentiation.
An understanding of the structural components of the centro-some and their functions thereof, has been elusive and isolatedcentrosomes are potentially useful for chemical and structuralstudies of the organelle.
Methodology: ESD3 cells were transfected with overexpressionand downregulation vectors for Stil. Centrosome isolation was donefollowing Moudjou and Bornens (1998). It was isolated on a sucrosegradient, which serves to purify them on the basis of sedimentationvelocity. Coomassie Blue staining was done to check the profileof the isolated proteins and the fractions were characterized bystaining with acetylated tubulin antibodies (centriole marker) andgamma tubulin antibodies (PCM marker).
Results: Coomassie Blue staining showed that proteins rangingfrom 10 kDa to 250 kDa had been isolated onto the sucrose cushion.Bands of 48 kDa (gamma tubulin) and 55 kDa (acetylated tubulin)were seen in a few fractions, indicating the successful isolation ofcentrosomes from the cells.
Discussion: Proteomic characterization of centrosomes will helpin greater understanding of its structural components, functionalrelevance and its potential role in cell signalling. A comparisonof centrosomal components, between normal, high and low Stilexpression conditions, will help in delineating the effects of thisgene on centrosomal function and provide clues as to the possiblerole of centrosomes in neurogenesis itself.
Orexinergic neurons modulate REMS by influencing locuscoeruleus neurons
R.C. Choudhary, H.K. Srivastava ∗, B.N. Mallick
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
Neural mechanism of rapid eye movement sleep (REMS) regu-lation is being actively investigated. Locus coeruleus (LC) REM-OFFneuronal activity reduces upon sleep onset and eventually becomesquiescent through REMS. Activation of orexinergic-neurons inthe perifornical area (Pef) contribute to wakefulness and theirloss is associated with narcolepsy, a REMS-disorder. Althoughorexinergic-neurons innervate virtually the entire brain, nora-drenalin (NA)-ergic neurons in the LC, in particular, receive heaviestprojections. Independent studies have shown that stimulation oforexinergic-neurons and local microinjection of orexin into LCreduced REMS. Thus, although it was suggestive that stimulationof orexinergic-neurons mediated their action possibly by activat-ing LC-neurons, it was unknown if orexinergic-neurons directlyaffected the LC-neurons to modulate REMS, which was investigatedin this study.
Male wistar rats (250–275 g) were stereotaxic surgicallyprepared with implanted electrodes for electrophysiological sleep-wake-REMS recording and with bilateral guide cannulae into LC aswell as Pef for local microinjections. After recovery from surgery,sleep-waking-REMS were recorded in freely moving normallybehaving chronically prepared rats simultaneously with or withoutmicroinjection(s) of chemicals in either LC or Pef or both.
Glutamate stimulation of orexinergic-neurons significantlyincreased waking and decreased sleep; REMS-duration/h wasdecreased but not REMS-frequency/h. The effects however, were
P.P. Kale, V. Addepalli / Int. J. D
revented if orexinergic receptor1 antagonist was simultaneouslyicroinjected into the LC along with glutamate microinjection into
he Pef. Control studies were performed by saline injection.The findings confirm that the effect of activation of Pef-
rexinergic neurons is routed through the LC. Since REMS-durationnd not its frequency was affected it is likely that orexin is involvedn maintenance of REMS. The findings in vivo help understandinghe neural regulation of REMS in normal and altered states.
Acknowledgements: Funding from CSIR and DBT.
oradrenaline deficiency in brain may facilitate progression oflzheimer’s disease
. Singh, S.S. Komath, B.N. Mallick ∗
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
-mail address: [email protected] (B.N. Mallick).Introduction: REM sleep (REMS) is a unique but essential
hysiological process; however, why it evolved and its detailedechanism of action is unknown. Although elevated noradrenaline
NA) level is undesirable for health, some NA is necessary for normalhysiological processes including priming the system for REMS.rain NA level is modulated by quantity of REMS. Alzheimer’sisease (AD) is an age associated neurodegenerative disease charac-erized by loss of memory and altered cognitive behaviours whichre also affected by REMSD. In AD the locus coeruleus (LC) neurons,he major source of NA in the brain, are degenerated and cortical NAevels decrease; such altered NA level may have bearing on AD. Aey event in AD pathogenesis is the conversion of the amyloid betaeptide from its soluble monomeric to its aggregated morphology
n the brain. Hence, we investigated the effect of NA on aggregationroperties of amyloid beta.
Methods: In vitro, amyloid beta (1–40) fibril was measured byhioflavin T (ThT) fluorescence assay, circular dichorism (CD) spec-roscopy and transmission electron microscopy (TEM).
Results: ThT assay showed that fibrillation of amyloid beta1–40) was inhibited by NA in a dose dependent manner. Timeourse of changes in the far-UV CD spectra of the A�40 in thebsence or presence of NA showed that in presence of NA amyloideta (1–40) did not form beta-sheet structure; and TEM confirmedhat NA inhibited amyloid beta (1–40) fibrillation.
Discussion: The results of this study demonstrated that NAnhibits amyloid beta aggregation in a concentration dependent
anner. Pending confirmation, we propose that increased NA, aspon REMSD for example may be a compensatory mechanism to
nhibit Amyloid beta aggregation to slow down the process of AD,hich however, may be facilitated by prolonged REMSD.
Acknowledgement: AIRF-JNU and Funding from, DBT, DST andGC-resource networking.
oradrenalin protects ROS generation in C6-glial cells by Fe++-helation
. Singh, B.N. Mallick ∗
School of Life Sciences, Jawaharlal Nehru University, New Delhi10067, India
-mail address: [email protected] (B.N. Mallick).
Recently we have proposed that rapid eye movement sleepREMS) serves house keeping function of the brain (Mallick andingh, Sleep Med. Rev., 2011); the action is likely to be mediated by
uroscience 30 (2012) 640–671 647
many ways. REMS modulates noradrenalin (NA) level in the brainand REMS loss is extremely damaging. Glia possesses neurotrophicproperties and their loss may lead to severe malfunctioning of thebrain. Due to availability of very little antioxidants, brain is vul-nerable to oxidative damage. NA is among very few antioxidants,possessing neuro-protective property, available in the brain. Lossof NA-ergic neurons and decreased NA-level are reported in vari-ous neurodegenerative diseases. Therefore, we investigated if NAprotects brain by reducing reactive oxygen species (ROS).
C6-cells (glia), cultured on cover-slips, were treated withH2O2 in presence and absence of NA for 2 h. Treated and controlcells were loaded with ROS-indicator dichlorofluorescein acetate(DCFDA) and fluorescence estimated by fluorescence microscopy.10 �M NA significantly reduced ROS level in normal as wellas H2O2-induced ROS generation in C6-cells. Further, ferrozinechelation assay was performed to evaluate whether this protectionwas due to chelation of Fe++, which participates in Fenton-reactionfor ROS generation. We found that NA significantly chelated Fe++,maximum when NA was 10-fold to Fe++ concentration; detailedmechanism will be presented.
Findings of this study suggest that increased NA chelates Fe++
preventing them to initiate Fenton-reaction and possibly attemptsto protect the brain where antioxidants are very little. Further studyis necessary to establish if similar mechanism exists for neurons andif it works in vivo as well. Pending confirmation the findings suggestthat increased NA in the brain during REMS-deprivation may thusbe an attempt to reduce oxidative stress to maintain homeostasis.
Acknowledgement: Funding from CSIR, UGC, ICMR, JC Bose Fel-lowship and DST-FIST.
Benzo[a]pyrene-induced learning impairment may be pro-tected by noradrenaline: possibly by modulating Ca2+-influx
Manorama Patri, Abhishek Singh, Birendra N. Mallick ∗
School of Life Sciences, Jawaharlal Nehru University, New Delhi110067, India
E-mail address: [email protected] (B.N. Mallick).Introduction: Benzo[a]pyrene (B[a]P), a carcinogen, enters into
our body through contaminated food, water, cigarette-smoke,polluted air and impairs learning and memory. Although its mech-anism of action is unknown, it is reported to elevate noradrenalin(NA) level. Learning and memory is affected after rapid eye move-ment sleep deprivation when NA level is also increased in the brain.NA directly or indirectly modulates various brain functions includ-ing neuronal growth, development, learning and memory. Hence,we studied in vivo and in vitro if NA could modulate B[a]P-inducedeffects related to learning and memory.
Methodology: Single dose of 0.1 �M B[a]P in 10 �l of DMSO wasintracisternally injected into postnatal-day-5 rats and after 4–5weeks their learning and memory was assessed by Morris watermaze; DMSO treated and naïve rats were used as controls. SinceNMDA-receptors (NR1 and NR2B) are involved in learning andmemory, they were estimated by immunostaining. The effect ofB[a]P on NR1/NR2B and intracellular-Ca2+ in presence/absence ofNA (0.1, 1, 10 �M) in C6 and Neuro2a was estimated.
Results: Intracisternal administration of B[a]P in vivo signif-icantly (p < 0.05) reduced spatial learning, but not long-termmemory (probe test). The density of both receptor subunits, NR1and NR2B significantly (p < 0.01) increased in the hippocampus.Although B[a]P significantly increased NR1 in both the cell-lines,NR2B significantly (p < 0.05) increased in C6, but significantly(p < 0.01) decreased in Neuro2a even after NA-treatment. NA in vitro