P.1.g. Basic and clinical neuroscience − Neuropharmacology S263

(range: −6.0−8.2), p = 0.08 in the occipital cortex, a region withhigh 5-HT1B receptor binding. There were no significant differ-ences between BP0 in the occipital cortex (p = 0.19) or neocortex(p = 0.36) calculated between 0−45min scan time and the fullscan time (0–120min) demonstrating excellent time stability ofthe [11C]AZ10419369 BPND. Shortening the data sets from 120 to90min did not lead to any significant differences in BP1 (p = 0.19)either, indicating that [11C]AZ10419369 BPNDs are stable whenthe duration of the PET scan is limited to 90min.

Conclusion: Based on these initial modelling on baseline data,ESRTM can be used to quantify changes in [11C]AZ10419369BPND when the pharmacological challenge is given 45−60minafter the injection of [11C]AZ10419369. This may prove especiallyuseful in the context of simultaneous PET-MR imaging whereimportant information about how vascular effects are induced byregional neurochemical or drug-induced changes can be obtained.

References

[1] Zhou, Y., Chen, M., et al., 2006, An extended simplified referencetissue model for the quantification of dynamic PET with amphetaminechallenge. NeuroImage 33, 550–563.

[2] Haahr, M.E., Fisher P.M., et al., 2013, Central 5-HT4 receptor bindingas biomarker of serotonergic tonus in humans: a [11C]SB207145 PETstudy. Molecular Psychiatry 19, 427–432.

P.1.g.101 Effects of two new neuropeptides with short

chains on cognitive functions of mice

S. Stoeva1 °, E.N. Encheva2, L.P. Tancheva3, R. Klisurov4,A. Georgieva1, T. Pajpanova5, R. Kalfin1 1Bulgarian Academyof Sciences, Institute of Neurobiology, Sofia, Bulgaria;2Medical University of Sofia, Dept. of Physiology, Sofia,Bulgaria; 3Bulgarian Academy of Neurobiology, Institute ofNeurobiology, Sofia, Bulgaria; 4Medical University of Sofia,Dept. of Pharmacology, Sofia, Bulgaria; 5Bulgarian Academy ofSciences, Institute of Molecular Biology, Sofia, Bulgaria

Introduction: Searching for biologically active peptidomimeticsas a new direction in modern pharmacology requires complex,interdisciplinary researches. On the basis of well-known activepeptides new active molecules as potential pharmacological agentswith improved pharmacokinetic properties are created. Two newlysynthesized neuropeptides (Pajpanova et al., 2013) P1 (analogueof Tyr-MIF-1) and P2 (analogue of Nociceptine) are object ofthe present study. Having in mind their molecular design − verysimilar to some neurotransmitters in the central nervous system,we decided to study their effects on the CNS.Previous data demonstrated that the new compounds are biolog-

ically active in dose 5mg/kg and have low oral and intraperitonealtoxicity. There is no prolonged toxicity or delayed effect after theirsingle administration.

Purpose of the study: To study activity of two newly syn-thesized neuropeptides on cognitive functions of white ICR malemice.

Methods: An effective dose of 5mg/kg intraperitoneally (i.p.)of new compounds was applied 20 minutes before the tests.Cognitive functions of animals were studied using the followingmethods: Step through test (for learning and memory), Rot-a-rod test (for muscular coordination) and Hole board test (forexploratory activity). Analgesic effect was evaluated by chemicalirritation test using acetic acid. The number of abdominal crampsfor 20 minutes after Acetic acid application was measured. Dose-effect analgesic activity of compound P2 was studied in doses 4, 8

and 16mg/kg b.wt. i.p. according to the same method. Interactionof the two compounds with hexobarbital (100mg/kg ip) wasmeasured as duration of sleeping time in minutes. Statistic wasperformed according ANOVA analysis.

Results: On the 48th hour after treatment with two neuropep-tides (5mg/kg ip) only peptide P1 has significant improving effecton learning and memory. P1 increased also the exploratory activityin comparison to controls. Neuromuscular coordination was notinfluence by two compounds.Compounds had the opposite effect on the nociception. P1

increased it (probably because increased local irritation), but P2significantly decreased it (by 25%). Established analgesic effect ofP2 is dose-dependent (4, 8 and 16mg/kg i.p.) but the mechanismremain unclear. We suggest that probably it is related to thechemical similarity of P2 with nociceptine and maybe is result ofinteraction with opioid receptors in CNS. In single dose the twocompounds significantly shorten duration of hexobarbital sleepingtime (P1 by 40% and P2 by 50%). The mechanism is unknownand it is possible to be a result of functional antagonism betweenneuropeptides and hexobarbital on the level of CNS and receptorsinteractions.

Conclusion: Newly synthesized neuropeptides are biologicalactive substances. P1 can improve cognitive function and curiosityin animals and P2 has dose-dependent analgesic effect. Twonew compounds P1 and P2 shorten hexobarbital sleeping timein similar degree. Future research will clarify the mechanismsunderlying of established activities.

P.1.g.102 Modulation of the conditioned emotional

response by dorsal hippocampus

TRPV1 receptor

G. Spiacci1 °, L.B.M. Resstel1 1University of Sao Paulo,Department of Pharmacology, Ribeirao Preto, Brazil

Purpose: The dorsal hippocampus (DH) is a limbic structurewhich is involved in emotional, learning and memory processes,as well as behavioral responses, such as modulation of the condi-tioned emotional response (CER) which is characterized by freez-ing behavior and autonomic changes (increases in mean arterialpressure (MAP) and heart rate (HR) [1]. The transient receptorpotential vanilloid type 1 (TRPV1) are widely expressed in brainstructures, such as the hippocampus, and this receptors appears tobe important to regulation of anxiety-related behaviors [2]. Pre-vious results from our research group has suggest that TRPV1 istonically activated during threatening condition according with theaversive situation [3], thus facilitating the expression of defensiveresponses. However, a study that pointed out the importance ofthe TRPV1 receptors present in DH on the modulation of CERin fear conditioning context has not yet been done. In the presentstudy we investigated if DH TRPV1 receptors are implicated withthe behavioral and autonomic responses associated with aversivecontext.

Methods: Male Wistar rats (220–250 g) were submitted tostereotaxic surgery to bilaterally implant guide-cannula targetingthe DH. The animals were divide in two groups, non-conditioned(not receive footshock) and conditioned context (receive foot-shock). The animals were conditioned with 6 electrical footshocks(1.5mA, 3 s), or received 3 electrical footshocks (0.85mA, 2 s).Previews results from our research group have been shown thata lower intensity footshock is necessary to observe a possible

S264 P.1.g. Basic and clinical neuroscience − Neuropharmacology

increase in CER, since methods using higher intensities promotede maximum response. Twenty-four hour after the conditioningsession a polyethylene catheter was implanted in the femoralartery for cardiovascular recordings. On test day, behavioral (freez-ing) and autonomic (MAP and HR) parameters were evaluated.Before test session, it was administered in the DH bilaterally 500nL of 6-iodonordihidrocapsaicin (6-I-CPS), a TRPV1 receptorantagonist (doses of 3 or 9 nmol) or Capsaicin, a TRPV1 agonist(doses 3 or 9 nmol) or vehicle 10min before the chamber re-exposition. The time spent in freezing and cardiovascular re-sponses were recorded for 10min during re-exposition.

Results: Our results using the high intensity protocol (1.5mA,3 s conditioning) shows that re-exposure to the conditionedcontext evoked freezing behavior (73±4% vehicle conditionedgroup) and autonomic responses, MAP and HR. The DH pre-treatment with 6-I-CPS significant reduced the expression offreezing behavior (F(2,28) = 19, P< 0.001) at the doses of 3 nmol(43±3%) and 9 nmol (55±5%), as well the autonomic response[MAP (F(14,150) = 19.91, P< 0.0001) and HR (F(14,150) = 26.80,P< 0.0001] in the conditioned group. On the other hand, resultsfrom moderate intensity protocol (0.85mA, 2 s conditioning)shows that the DH pretreatment with capsaicin increases the freez-ing behavior (F(2,16) = 3.33, P< 0.05) and the autonomic responses[MAP (F(2,16) = 4.4, P< 0.05) and HR (F(2,180)=34.2, P< 0.05)]at the dose of 9 nmol (P< 0.001) in the conditioned group.No significant differences were founded in the non-conditionedgroups.

Conclusion: According our results, the TRPV1 receptors ofDH appears to be involved of conditioned emotional responses incontext fear conditioning.

References

[1] Resstel, L.B., Joca, S.R., Correa, F.M., Guimaraes, F.S., 2008 Effects ofreversible inactivation of the dorsal hippocampus on the behavioral andcardiovascular responses to an aversive conditioned context. BehaviorPharmacology, 19(2):137−44.

[2] Marsch, R., Foeller, E., Rammes, G., Bunck, M., Kossl, M., Hols-boer, F., Zieglgansberger, W., Landgraf, R., Lutz, B., Wotjak, C.T.,2007 Reduced Anxiety, Conditioned Fear, and Hippocampal Long-Term Potentiation in Transient Receptor Potential Vanilloid Type 1Receptor-Deficient Mice. The Journal of Neuroscience, 27(4):832–839.

[3] Terzian, A.L., dos Reis, D.G., Guimaraes, F.S., Correa, F.M., Ress-tel, L.B., 2014 Medial prefrontal cortex Transient Receptor PotentialVanilloid Type 1 (TRPV1) in the expression of contextual fear condi-tioning in Wistar rats, Psychopharmacology, 231(1):149−57.

Disclosure statement: Financial Support: FAPESP (2013/07470−2).

P.1.g.103 Evaluation of the neuroprotective effects of

bioproducts from Amburana cearensis on

microglial cells

T.M. Pierdona1 °, E.V.O. Araujo1, H.H.S. Amaral1,L.B.N. Freitas2, G.S.B. Viana1, E.R. Silveira3, L.K.A.M. Leal21Federal University of Ceara, Physiology and Pharmacology,Fortaleza, Brazil; 2Federal University of Ceara, Pharmacy,Fortaleza, Brazil; 3Federal University of Ceara, Inorganic andOrganic Chemistry, Fortaleza, Brazil

Introduction: Substantial evidences have shown that activatedmicroglia is an important contributor in the progression of manyneurodegenerative disorders, such as Parkinson and Alzheimer’sdiseases. Some stimuli, such as lipopolysaccharide (LPS) areable to activate microglia with the secretion of a variety of

pro-inflammatory factors, such as citokines, reactive oxygenspecies and nitric oxide. Previous study showed the potential anti-inflammatory and/or neuroprotector of phenol compounds, such asamburoside A, and extract from Amburana cearensis A.C. Smith(Fabaceae) [1]. The objective of the present work was to study thepossible neuroprotective activity presented by dry extract (spraydryer) (DEAC), phenol fraction (PFAC) or amburoside A (AMB)obtained from A. cearensis, on rat microglial cell cultures in theabsence or presence of LPS as assessed by cytotoxicity and nitritemeasurements.

Materials and Methods: The DEAC were standardizedthrough the HPLC analysis-active principals/markers: coumarin:26.23±1.20; amburoside A: 74.54±1.45mg/g. Immortalized9L/lacZ microglial cells were maintained in Dulbecco’s ModifiedEagle Medium (DMEM) supplemented with 10% fetal bovineserum, streptomycin (10mg/ml), and penicillin (10 U/ml) at 37ºC.Cell viability was determined by the MTT assay [2]. 9L/lacZcells were seeded into 96-well plates at a density of 4×105cells/well. The cells were incubated with DEAC, AMB andPFAC (5–200mg/ml), dimethylsulfoxide (DMSO, 4%, vehicle) orTriton 0.2% (positive control). After 24 h of incubation, the MTTassay was performed. MTT solution was added to the cells, whichwere then incubated for 3 h at 37ºC under a 5% CO2 atmosphere.The absorbance was measured at 570 nm. For the assessment ofnitrite derived from nitric oxide (NO), the cells were pre-incubatedfor 60min at 37ºC with test drugs (DEAC, AMB and PFAC(5–200mg/ml), DMSO (1%) or DMEM (not treated cells group)before the exposure of LPS (125mg/ml) for 24 h. The levels ofnitrite release by cells were determined by ELISA, according tothe guidelines by the manufacturer (Roche).

Results and Discussion: The addition of DEAC, PFAC orAMB alone to the cell culture at concentrations ranging from5 to 200mg/mL did not affect cell viability even at higherconcentrations (DEAC 200: 93.78±8.12, AMB 200: 96.85±13.73,PFAC 200: 100±14.6, Triton: 22.60±10.0%) compared to thecontrol group (DMSO: 100±12.0%). The addition of test drugsbefore LPS significantly protected microglial cells against theinflammatory effect of LPS. After exposure of the cells to LPS, agreat increase (62.59%) in nitrite levels was detected compared tonot treated cells. The LPS-induced increase in nitrite levels wasreduced by the addition of DEAC, PFAC and AMB after LPS by25.7%, 37% and 33%, respectively (nitrite absorbance, not treatedcells: 0.063±0.012; control: 0.077±0.017; DEAC: 0.052±0.003,PFAC: 0.044±0.002, AMB: 0.047±0.017).

Conclusion: The DEAC, AMB and PFAC from A. cearensisdid not show any cytoxicity effect in microglial cells, protectingthese cells against LPS-induced inflammation possibly acting asan antioxidant product. The preliminary results suggest that thebioproducts from A. cearensis could be useful as therapeuticagent for the treatment of neurodegenerative diseases, althoughadditional studies are necessary.

References

[1] Leal, L.K.A.M., Nobre Junior, H.V., Cunha G.M., Moraes, M.O.,Pessoa, C., Oliveira, R.A., Silveira, E.R., Canuto, K.M., Viana, G.S.,2005. Amburoside A, a glucoside from Amburana cearensis, protectsmesencephalic cells against 6-hydroxydopamine-induced neurotoxicity.Neuroscience Letters 388, 86−90.

[2] Mosmann T., 1983. Rapid colorimetric assay for cellular growth andsurvival: application to proliferation and cytotoxicity assays. Journal ofImmunological Methods 65, 55−63.