Burkhanova Gulshat Foatovna

Chernova Kseniya Andreevna

Khazipov Rustem Narimanovich

Sitdikova Guzel Faritovna

Zakharov Andrey Viktorovich

Kazan Federal University Digital Repository

Inhibitory Effects of Ethanol in the Neonatal RatHippocampus In VivoKseniya Chernova1 & Gulshat Burkhanova1 & Andrey Zakharov1,2 &Roustem Khazipov1,3,4 & Guzel Sitdikova1# Springer Science+Business Media New York 2016Abstract Ethanol-induced neuroapoptosis in the developingbrain has been suggested to involve suppression of neuronalactivity. However, ethanol acts as a potent stimulant of neuro-nal activity by increasing the frequency of depolarizingGABA dependent giant depolarizing potentials in the neonatalrat hippocampal slices in vitro. Here, we show that ethanolstrongly inhibits, in a dose-dependent manner (1–6 g/kg),sharp waves and multiple unit activity in the hippocampus ofneonatal (postnatal days P4–6) rats in vivo. Thus, the effects ofethanol on the developing hippocampal network activitycardinally differ in vitro (stimulation) and in vivo (inhibition).Keywords Electroencephalography . Ethanol .Hippocampus . Sharp waves . Rat . Neonate1 IntroductionEthanol and general anesthetics induce massive neuroapoptosisin the developing brain [1, 2]. Considerable evidence obtainedin the neocortex indicates that the neuroapoptotic effects ofthese drugs involve suppression of the early activity patternsand the neuronal activity [3–5]. However, ethanol has also beenshown to increase the frequency of depolarizing GABA drivengiant depolarizing potentials and to act as a potent stimulator ofthe neuronal activity in the neonatal rat hippocampal slicesin vitro [6]. These findings go against the hypothesis that theadverse effects of ethanol in the neonatal hippocampus involvesuppression of an activity. Yet, the effects of ethanol on theearly hippocampal activity so far have not been addressedin vivo. The aim of this study was to characterize the effectsof ethanol on the electrographic activity and the neuronal firingin the hippocampus of neonatal rat pups in vivo.2 Material and MethodsThis work has been carried out in accordance with EUDirective 2010/63/EU for animal experiments and allanimal-use protocols were approved by the French NationalInstitute of Health and Medical Research (INSERM, protocolN007.08.01) and Kazan Federal University on the use of lab-oratory animals (ethical approval by the Institutional AnimalCare and Use Committee of Kazan State Medical UniversityN9-2013). Wistar rats from postnatal day (P) P4-P6 wereused. Surgery was performed under isoflurane anesthesia,and the animals were left to recover from anesthesia for morethan one hour before the recordings. Preparation of the ani-mals for the head-restrained recordings and the recordingsetups were as described previously [3]. Recordings of thesharp waves (SPWs) on the local field potential (LFP) andthe multiple unit activity (MUA) were performed from thehippocampus using linear silicone probes (16 channels,100 μm separation distance between the recording sites,Neuronexus Technologies, USA). The signals were amplifiedand filtered (×10,000; 0.1–10 kHz) using a DigitalLynx(Neuralynx, USA) amplifier, digitized at 32 kHz and savedon a PC for post-hoc analysis using custom-written functions* Andrey ZakharovAnVZaharov@kpfu.ru1 Laboratory of Neurobiology, Kazan Federal University, 18Kremlevskaya str., Kazan 420008, Russia2 Department of Physiology, Kazan State Medical University,Kazan 420012, Russia3 INMED—INSERMU901, 163 avenue de Luminy, Marseille 13273,France4 Aix-Marseille University, Marseille 13273, FranceBioNanoSci.DOI 10.1007/s12668-016-0328-4in Matlab (MathWorks, USA) as described previously [3].The Mann-Whitney and t tests were used for the group datacomparisons with a level of significance set at p < 0.05.3 Results and DiscussionThe electrical activity of the hippocampus was characterizedby SPWs which are the predominant electrographic activitypattern during the early postnatal period (Fig. 1a, b) [7]. Inkeeping with these previous studies, the SPWs synchronizedCA1 units, reversed polarity at the pyramidal cell layer, asevidenced by the current source density analysis, and occurredirregularly at a frequency of 3.7 ± 0.2 min-1 (n = 9; P4-6). Theaverage MUA in the CA1 pyramidal cell layer estimated by a1-h recording session prior to ethanol administration was3.5 ± 1.2 spikes/s.Ethanol was administered intraperitoneally at threedosage regimens: 1 g/kg + 3 g/kg (with 1-h interval),4.5 g/kg, and 6 g/kg. At the maximal dosage (6 g/kg),ethanol-induced rapid and profound inhibition of the hip-pocampal activity (Fig. 1a, c, d). Thirty minutes after ethanoladministration, SPWs and CA1 MUAwere almost completelysuppressed that was characterized by the reduction in SPWsfrequency andMUA to 7.0 ± 4.8 and 2.3 ± 1.8% of the controlvalues, respectively (n = 3, p < 0.05, t test). This was followedby partial recovery of the activity 3 h after the ethanol admin-istration (SPWs frequency recovered to 32 ± 6 % and MUA to15 ± 11 % of the control values; n = 3). Ethanol at lower doses,1 + 3 g/kg with a 1-h interval and 4.5 g/kg produced lessrapidly and less prominently, but also the long-lastingCA1pyr.c.layerEthanol 1g/kg+3g/kg0 50 100 150 20020406080100**********SPWfrequency,%time (min)+3g/kg*0Ethanol 4.5g/kg0 50 100 150 200020406080100time (min)***** * * *** * *Ethanol 6g/kgE l 1+3 g/kgthano-50 0 100 200050100150t ( )ime minCA1MUAfrequency, %E l 4.5 g/kgthano-50 0 100 200050100150t ( )ime minE l 6 g/kgthano-50 0 100 200050100150t ( )ime minb cd0 50 100 150 200020406080100time (min)* ** *** *** * ** *sink sour ec0. mV5100ms0.5mV10ssrsospa Control Ethanol(6g/kg, 30 min after i.p. injection)0.1mVLFP 300-3000HzsrsospFig. 1 Effects of ethanol on the electrical activity in newborn rathippocampus in vivo. a Example traces of CA1 hippocampal activity atdifferent depths in a P6 rat before (left) and 30min after the administrationof ethanol (right). Original and 300 Hz highpass filtered LFP—blacktraces, MUA—red bars. Sharp waves (SPWs) are marked by asterisks.Hippocampal layers stratum pyramidale, stratum oriens, and stratumradiatum are indicated as sp, so, and sr, respectively. b Example of theSPW and its current source density. c–d Time course of the CA1pyramidal cell layer MUA frequency (c) and the SPWs frequency (d)after ethanol administration at 1 g/kg + 3 g/kg, 3 g/kg, 6 g/kg (i.p.)during the 3-h recordings. Significant differences from the control values(p < 0.05) are indicated by the asterisks (Mann-Whitney test)BioNanoSci.suppression of the hippocampal activity. Thirty minutes afterethanol administration at dosage 4.5 g/kg MUA decreased to30 ± 26 % and SPWs frequency reduced to 25 ± 4 % of thecontrol values (n = 3, p < 0.05, t test). At dosage 1 g/kg, MUAshowed a tendency to decrease to 61 ± 13 % of the controlvalues but this was not significant (n = 3, p > 0.05, t test)whereas SPWs frequency reduced to 29 ± 2 % of the controlvalues (n = 3, p < 0.05, t test). These results indicate thatethanol induces rapid suppression of the hippocampal activityin the rat pups and that the effects of ethanol are dose-dependent.The inhibitory effects of ethanol were long-lasting so that by 3 hafter the injection, the electrical activity was still suppressed andwas significantly lower than in control before the ethanol injec-tion (Fig. 1c, d).Thus, ethanol at the doses inducing massive neuroapoptosisin the developing brain exerts powerful inhibitory actions onSPWs and neuronal firing in the neonatal rat hippocampus.These results differ from the stimulation of the activity de-scribed in the hippocampal slices of the neonatal rats in vitro,where ethanol was shown to increase the frequency ofdepolarizing GABA driven giant depolarizing potentials [6].Although the reasons for this sharp discrepancy in the ethanolactions in vivo and in vitro are unknown, it is plausible that itinvolves an increase in GABAergic transmission by ethanol[6], which exerts, during the neonatal period, complex excitato-ry and inhibitory network actions in the hippocampus in vitro[8, 9], but mainly inhibitory network actions in cortical circuitsof neonatal rodents in vivo [10–12]. Another mechanism couldinvolve respiratory acidosis which was shown to suppress giantdepolarizing potentials in the neonatal rat hippocampal slices[13]. However, in our previous study, ethanol at the maximaldosage of 6 g/kg did not affect oxygen saturation (SpO2), breathrate and heart rate in the neonatal rats under similar experimen-tal conditions [4]. Independently on the underlying mecha-nisms, our results support the hypothesis that ethanol-inducedhippocampal neuroapoptosis during the neonatal period in-volves suppression of neuronal activity [14, 15].4 ConclusionOur main finding is that ethanol strongly suppresses activity inthe neonatal rat hippocampus in a dose-dependent manner sim-ilarly to the ethanol actions previously described for the so-matosensory cortex [4]. These results provide mechanistic sup-port to the hypothesis that the neuroapoptotic actions of ethanoland general anesthetics involve severe suppression and particularvulnerability of the early activity patterns to these drugs.Acknowledgments This work was supported by the Program ofCompetitive Growth of Kazan Federal University, the subsidy allocatedto Kazan Federal University for the state assignment in the sphere ofscientific activities and by INSERM (LIA).References1. Ikonomidou, C., Bittigau, P., Ishimaru, M. J., Wozniak, D.F., Koch, C., Genz, K., et al. (2000). Ethanol-induced apo-ptotic neurodegeneration and fetal alcohol syndrome.Science, 287, 1056–1060.2. Ikonomidou, C., Bosch, F., Miksa, M., Bittigau, P., Vockler, J.,Dikranian, K., et al. (1999). Blockade of NMDA receptors andapoptotic neurodegeneration in the developing brain. Science,283, 70–74.3. Sitdikova, G., Zakharov, A., Janackova, S., Gerasimova, E.,Lebedeva, J., Inacio, A. R., et al. (2014). Isoflurane suppressesearly cortical activity. Annals of Clinical Translational Neurology,1, 15–26.4. Lebedeva, J., Zakharov, A., Ogievetsky, E., Minlebaeva, A.,Kurbanov, R., Gerasimova, E., Sitdikova, G., and Khazipov, R.(2015) Inhibition of Cortical Activity and Apoptosis Caused byEthanol in Neonatal Rats In Vivo. Cereb Cortex. doi:10.1093/cercor/bhv2935. Lebedeva, Y. A., Zakharova, A. V., Sitdikova, G. F., Zefirov,A. L., Khazipov, R. N. (2016). Ketamine-midazolam anes-thesia induces total inhibition of cortical activity in the brainof newborn rats. Bulletin of Experimental Biology andMedicine, 161, 15–19.6. Galindo, R., Zamudio, P. A., Valenzuela, C. F. (2005). Alcohol is apotent stimulant of immature neuronal networks: implications forfetal alcohol spectrum disorder. Journal of Neurochemistry,94, 1500–1511.7. Leinekugel, X., Khazipov, R., Cannon, R., Hirase, H., Ben Ari, Y.,Buzsaki, G. (2002). Correlated bursts of activity in the neonatalhippocampus in vivo. Science, 296, 2049–2052.8. Ben Ari, Y., Gaiarsa, J. L., Tyzio, R., Khazipov, R. (2007).GABA: a pioneer transmitter that excites immature neuronsand generates primitive oscillations. Physiological Reviews,87, 1215–1284.9. Khalilov, I., Minlebaev, M., Mukhtarov, M., Khazipov, R.(2015). Dynamic changes from depolarizing to hyperpolariz-ing GABAergic actions during giant depolarizing potentialsin the neonatal rat hippocampus. Journal of Neuroscience,35, 12635–12642.10. Minlebaev, M., Ben-Ari, Y., Khazipov, R. (2007). Network mech-anisms of spindle-burst oscillations in the neonatal rat barrel cortexin vivo. Journal of Neurophysiology, 97, 692–700.11. Kirmse, K., Kummer, M., Kovalchuk, Y.,Witte, O.W., Garaschuk, O.,Holthoff, K. (2015). GABA depolarizes immature neurons and in-hibits network activity in the neonatal neocortex in vivo. NatureCommunications, 6, 7750.12. Valeeva, G., Tressard, T., Mukhtarov, M., Baude, A., Khazipov, R.(2016). An optogenetic approach for investigation of excit-atory and inhibitory network GABA actions in mice express-ing channelrhodopsin-2 in GABAergic neurons. Journal ofNeuroscience, 36, 5961–5973.13. Ruusuvuori, E., Kirilkin, I., Pandya, N., Kaila, K. (2010).Spontaneous network events driven by depolarizing GABA actionin neonatal hippocampal slices are not attributable to deficient mi-tochondrial energy metabolism. Journal of Neuroscience, 30,15638–15642.14. Olney, John, W., (2014). Focus on apoptosis to decipher how alco-hol and many other drugs disrupt brain development. Frontiers inPediatrics, 215. Kilb, W., Kirischuk, S., Luhmann, H. J. (2011). Electrical activitypatterns and the functional maturation of the neocortex. EuropeanJournal of Neuroscience, 34, 1677–1686.BioNanoSci.

Inhibitory Effects of Ethanol In the Rat Hippocampus In Vivo

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Inhibitory Effects of Ethanol In the Rat Hippocampus In Vivo

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