Wentlandt, Kirsten PhD; Samoilova, Marina PhD; Carlen, Peter L. MD, FRCPC; Beheiry, Hossam El MBBCh, PhD, FRCPC
Gap junctions constitute a nonchemical form of intercellular communication rather than the well described chemical synaptic neurotransmission. They are abundant between astrocytes as well as neurons and between neurons and astrocytes. Gap junctions permit the direct exchange of current-carrying ions and small metabolites, including second messengers. A gap junction is composed of 2 hemi channels, each made of 6 connexin proteins (Cx), and formed by the docking of extracellular domains between cells, thus forming an inner pore of approximately 12.5 A (1). It has been established, by gene cloning and protein isolation, that a multigene family encodes connexin proteins consisting of at least 21 members in mammals (2). Gap junction communication is rapidly modulated by several factors, such as voltage (3), pH (4), and phosphorylation (5). There is no direct evidence that shows the linkage of gap junctions to global central nervous system (CNS) function. However, recent reports have associated neuronal gap junction communication to synchronization of large neuronal ensembles at different frequency bands (2). Such synchronization in the hippocampus leads to γ oscillations (30–80 Hz) that underlie different cognitive processes, i.e., perception, memory, and learning (6). Meanwhile, thalamic gap junctions promote synchrony leading to coherent thalamic oscillations involved in the consciousness behavior that include relaxed wakefulness, drowsiness, and sleep (2). Therefore, we can reasonably suggest that gap junction might be involved in the production of the anesthetic state.
Anesthetics have long been observed to attenuate synaptic excitation and/or enhance synaptic inhibition (7). However, anesthetics can also block direct intercellular communications via gap junctions in hepatocytes and myocytes as well as cultured astrocytes (8–11). In astrocytic primary cultures, the IV anesthetics, propofol and etomidate, at 1 μM depressed gap junction activity by 60% and 36%, respectively, and the inhaled anesthetics, halothane, enflurane, and isoflurane at 2 mM depressed gap junction activity by 80% as determined by using the scrape loading/fluorescent dye transfer technique (11). On the contrary, thiopental, diazepam and ketamine did not alter gap junction function (11). In keeping with such observations, Masaki et al. (12) recently showed that sevoflurane minimum alveolar concentration (MAC) has been reduced in rats after intracerebroventricular administration of a gap junction blocker, carbenoxolone. They also reported that sevoflurane blocked gap junction-mediated spontaneous membrane current oscillations of locus coerulus neurons in rat pontine slices (12).
The above lines of evidence indicate that there is an anesthetic effect on gap junction communications that might lead to enhancement of anesthetic potency in the whole animal. Thus, our overall hypothesis suggests that attenuation of gap junction communication by anesthetic agents can suppress specific CNS activation pathways and consequently contribute to the development of anesthesia. CNS deactivation caused by decreased gap junction communication can result in diminished ability of large neuronal ensembles to generate fast spiking activities that normally lead to different oscillatory patterns linked to cognition and consciousness. To validate such hypothesis, the first stage is to study the effects of anesthetics directly on gap junction communication using a methodology that can simulate anesthetic administration, i.e., to study the effects of anesthetics on gap junction coupling in a temporal domain of a few minutes. Therefore, the current study examined the specific hypothesis that IV and volatile anesthetics attenuate gap junction communication in cultured hippocampus slices using the recovery of fluorescence after photo bleaching (FRAP) technique. Such technique can measure changes in gap junction function in situ in a temporal resolution that is similar to anesthetic administration to humans and experimental animals. In addition, the cultured hippocampus slice is a model that has not been used to test the effects of anesthetics on gap junction function and provides the advantage of having a preserved architecture of neuronal and non-neuronal elements.
The animal care experimentation committee at the University Health Network approved all methods stated in this report.
The method for culturing brain slices has been described previously (13). Briefly, the brains of 7-day old male Wistar rats were aseptically removed after decapitation under halothane anesthesia and immersed in ice-cold dissecting medium (pH 7.15) containing 50% minimal essential medium (MEM; Gibco BRL, Gaithersburg, MD) with no bicarbonate, 50% calcium and magnesium-free balanced salt solution, 20 mM HEPES, and 7.5 mM D-glucose. Hippocampi were dissected, and coronal sections (400 μm) were obtained using a tissue chopper, then transferred to a dish containing dissecting medium at room temperature using a Pasteur pipette. The slices were then carefully separated with 2 pairs of fine forceps and transferred to sterile, porous membrane units (0.4 μm). Membrane units were placed into six-well trays. Each well contained one membrane unit and 1 mL of culture medium. The culture medium was composed of 50% MEM with Earl's salts, and L-glutamine, 25% balanced salt solution and 25% horse serum with 36 mM D-glucose, 20 mM HEPES buffer, and 50 mg/mL streptomycin-penicillin (pH adjusted to 7.2). Cultures were kept in a tissue culture incubator for 2 wk at 37°C in 5% CO2. The medium was exchanged (50% of volume) 3 times per week.
Two concentrations of each anesthetic were evaluated that were prepared in a 2% fetal calf serum/phosphate-buffered saline solution. The anesthetics were 5 and 15 μM propofol in Intralipid 10% (Zeneca, Mississauga, Ontario), 2 and 10 μM thiopental (Abbott, Saint-Etien, Quebec), and 0.64 and 2.8 mM halothane (Halocarbons Laboratories, River Edge, NJ). The concentrations of the free non-ionized form of propofol and thiopental in 2% fetal calf serum/phosphate-buffered saline solution are shown in Table 1. Control experiments were done with artificial cerebrospinal fluid (ACSF) in the electrophysiological studies and regular culture medium for FRAP procedures. Such experiments were performed with the propofol vehicle, Intralipid 10% and gap junction blocker, carbenoxolone 100 μM in ACSF or culture medium.
The method used for FRAP was similar to that described previously (15). Briefly, cultured hippocampus slices were incubated in 7 μg/mL 5,6-carboxyfluorescein diacetate in culture medium for 30 min. The cultures were then rinsed 3 times (10 min per wash) to remove excess 5,6-carboxyfluorescein. Control solution (ACSF), propofol, thiopental, halothane, or carbenoxolone was added to the culture medium to achieve final designated concentrations 15 min before imaging. Slice cultures were viewed on a BioRad MRC 600 laser-scanning confocal microscope and images were acquired and analyzed using version 7.0 COMOS software (BioRad Laboratories Canada Ltd., Mississauga, ON). First, an image was taken before photo bleaching to establish a baseline. The intensity of the laser was increased by removing neutral density filters then focused on the stratum pyramidale of the CA1 region. This area of interest was bleached to 50%–80% of the original fluorescence, then after reinsertion of the neutral density filters, imaging was resumed immediately and images were acquired continuously every 30 s. Recovery of fluorescence in the bleached area is the result of an influx of unbleached dye from neighboring cells via gap junctions. The extent of gap junction coupling between a cell and its neighbors was quantified by measuring the initial rate and percentage of fluorescence recovery. As a negative control, we used carboxyfluorescein-dextran, a fluorescent molecule similar to, but larger than, 5,6-carboxyfluorescein diacetate, was loaded with liposomes into the cultures. The large size of carboxyfluorescein-dextran, approximately 4000 Da, prevents it from passing through gap junctions and its recovery after photo bleaching was <10%. This small degree of recovery is attributed to the partially bleached cells located on the periphery of the bleached area where recovery of fluorescence is dependent upon the intracellular diffusion of the dye.
Hippocampus slice cultures were transferred to an interface-type chamber PDMI-2 (Harvard Apparatus, South Natick, MA) by cutting the membrane from the well with a scalpel blade. Experiments were conducted at 34°C and superfused with oxygenated ACSF containing (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 25 NaHCO3, 10 D-glucose, pH 7.4 when aerated with 95% O2/5% CO2 (osmolality 300 ± 5 mOsm). After the cultures had been perfused with ACSF for at least 15 min, spontaneous discharges and evoked synaptic responses were recorded extracellularly with borosilicate glass pipettes filled with NaCl 150 mM and located in the stratum pyramidale of the CA1 region. Extracellular stimulation (100-μs pulse width) was delivered orthodromically by placing a bipolar stimulating electrode (enamel-insulated nichrome wire; 125 μm diameter) in the stratum radiatum to stimulate the Schaffer collaterals. Stimulus strength was adjusted by increasing the amplitude to a minimal intensity that produced a maximal response. To evoke sustained discharges lasting seconds, a stimulus train (50 Hz for 2 s duration with maximum intensity) was delivered to the Schaffer collaterals. We refer to this type of discharge as primary after discharge (PAD) that is similar to stimulus train-induced bursting (14). Electrical signals were amplified using an Axopatch 200B amplifier with the low pass filter setting at 1–3 kHz. The recorded electrophysiologic potentials reflect the integrity of gap junction function in the cultured slices (15–20).
Spontaneous discharges were recorded on video tape (VR-10; Instrutech Corporation) and subsequently digitized and analyzed using Axotape version 2.02 software (Axon Instruments, Foster City, CA). Evoked responses were stored via a 12-bit D/A interface (Digidata 1200; Axon Instruments Inc., Foster City, CA) and analyzed using pCLAMP version 6.0.3 software (Axon Instruments, Foster City, CA).
Groups contained a maximum of two slice cultures prepared from the same litter. Statistical analyses for all experiments were performed using the Student's t-test, either paired or unpaired as required. Differences were considered significant at P < 0.05. Data are expressed as mean ± sd. The number of slices used for every treatment was calculated according to the assumption that there will be a 20% difference between control and drug effect at P < 0.05 and a statistical power of 80%.
Time-lapse imaging of FRAP was used to assess functional gap junction coupling in cultured hippocampus slices used in this study. After focal laser photo bleaching, the unbleached dye (5,6-carboxyfluorescein) can enter the cytoplasm of a bleached cell only via gap junctions from neighboring cells; hence, the degree of fluorescence recovery is directly proportional to the degree of functional gap junction coupling.
To test the accuracy of the FRAP method described in this report, a separate set of experiments was performed using the known gap junction blocker carbenoxolone 100 μM. The percent fluorescence recovery during carbenoxolone application was 11.6% ± 4.5%, thus showing a substantial degree of suppression of gap junction coupling. This indicates that the technique used is reasonably accurate and valid.
The IV anesthetic, propofol 15 μM dramatically reduced the fluorescence recovery to 46.7% ± 4.5% (n = 8), as seen in Figure 1A and B. This depression could not be attributed to propofol vehicle (intralipid 10%) as the vehicle exhibited dye-coupling levels equal to the control slices. At 8 min after photo bleaching, the percentage of fluorescence recovery was 71.2% ± 1.7% (n = 8) and 75.3% ± 3.2% (n = 8) in control slices and those treated with vehicle, respectively. Similarly, thiopental 10 μM produced significant uncoupling of gap junctions (Fig. 1A and B). Smaller concentrations of propofol 5 μM and thiopental 2 μM were also tested. It was found that during the application of such anesthetic concentrations, gap junction communication was not significantly different from control or vehicle effects (Fig. 1B). Potent uncoupling of intercellular communication was demonstrated by the application of halothane 2.8 mM. At such concentration, the FRAP was approximately 22% compared with 71.2% expressed in slices treated with control culture medium. Meanwhile, halothane 0.64 mM did not significantly suppress gap junction communication, as seen by the percent of 8 min after bleaching fluorescence when compared with control (Fig. 1A and B).
To explore the functional changes produced in hippocampus organotypic cultures during anesthetic applications, we recorded spontaneous and evoked neuronal electrophysiological activities. The evoked activities were induced by single brief-duration stimuli and tetanic stimulation of the CA1 region thus producing single evoked synaptic transients and evoked PAD, respectively. All electrophysiological recordings were performed after perfusion in ACSF for a minimum of 15 min. Control experiments were conducted in ACSF, which was later supplemented with the appropriate concentration of each anesthetic.
Spontaneous discharges (not shown) were recorded with significantly greater frequency in control cultures (0.64 ± 0.11 Hz, n = 9) when compared with those treated with propofol 15 μM and thiopental 10 μM. The frequencies recorded in propofol 15 μM and thiopental 10 μM treated slices were 0.04 ± 0.02 Hz (n = 9) and 0.04 ± 0.03 Hz (n = 9), respectively.
In control cultures, single orthodromic stimulations evoked solitary large postsynaptic potentials (5.2 ± 2.2 mV, n = 9) compared to slices exposed to propofol 15 μM (3.4 mV ± 1.7 mV, n = 9) as shown in Figure 2B and Figure 3A. Similarly, thiopental 10 μM significantly reduced such evoked postsynaptic potentials as seen in Figure 2A and Figure 4A.
Finally, cultured slices were tetanized at maximal levels for 2 s at 50 Hz to elicit a PAD. In slices perfused with ACSF, the PAD lasted for 6.0 ± 3.2 s (n = 9). The duration of these discharges was diminished in the presence of propofol 15 μM and thiopental 10 μM to 0.5 s ± 1.6 s (n = 9) and 0.2 s ± 1.1 s (n = 9), respectively (Fig. 3B and Fig. 4B).
The present investigation showed that anesthetic concentrations of propofol and thiopental attenuated gap junction coupling in the organotypic hippocampus slice. Interestingly, a halothane concentration that is equivalent to approximately 2 MAC (i.e., 0.64 mM) was unable to block gap junction communication. But a very large concentration of halothane 2.8 mM greatly suppressed gap junction coupling (16,25).
In this investigation, we cultured hippocampus slices taken from young rat brains for 2 weeks. Such slices are prone to spontaneous electrographic activity and a significant period of PAD evoked by a tetanic stimulus train. Although the mechanisms underlying the initiation, propagation, and termination of such spontaneous and evoked electrophysiological discharges are not fully understood, there is consensus that the critical requirement for these events to occur is hyper-synchronous neuronal activity (17). Meanwhile, one of the main mechanisms that promote hyper-synchrony in a neuronal network is electrical coupling via gap junctions (18–20). In the present study, IV anesthetics simultaneously decreased gap junction coupling as measured by the FRAP technique as well as the frequency of spontaneous discharges and the duration of evoked PAD. Similarly, our group previously reported that the gap junction blocker, carbenoxolone, depressed gap junction coupling, and the recorded spontaneous discharges and evoked PAD (21). Taking the above reports and our observations together, one can suggest that the anesthetic-induced depression of gap junction function observed in this report might have lead to the suppression of the spontaneous and evoked electrophysiological activities in the cultured slices. In fact, the similarity between the anesthetic and carbenoxolone effects shows that gap junction inhibition was a necessary condition for in vitro anesthesia and such inhibition might also have been sufficient to produce such an effect. Nonetheless, further investigations are necessary to elucidate the degree of involvement of gap junction function in the mechanisms of anesthetic actions.
The FRAP technique used in this report results in loading both neurons and astrocytes that exist in the cultured slices with the fluorescent probes. Consequently, the change in the kinetics of recuperation of fluorescence after photo bleaching can be explained by the direct communication through gap junctions between astrocytes, neurons and neurons and astrocytes. Hence, all types of gap junctions, i.e., astrocytic, neuronal and neruronal-astrocytic present in situ in the slices, could have been involved in the anesthetic effects. Similarly, the electrophysiological changes observed cannot be exclusively explained by the depression of gap junctions between neurons but can also result from the level of interaction between neurons and astrocytes as well as between neighboring astrocytes through gap junctions.
In the current study, we used two concentrations of each anesthetic to determine their effects on the integrity of function of gap junctions as measured by dye coupling in cultured hippocampus slices. The larger concentrations of propofol (15 μM) and thiopental (10 μM) are encountered in the brain of anesthetized animals (22–24). Both IV drugs were quite effective in suppressing dye coupling at those concentrations. Meanwhile, halothane, at approximately twice MAC (i.e., 0.64 mM) had no effect on gap junction function. A larger halothane concentration (2.8 mM) profoundly attenuated gap junction coupling. Such concentration is more than 8 times the calculated free aqueous EC50 (25). This observation is similar to others that characterized halothane as a specific gap junction blocker in many experimental models only at concentrations more than 2 mM (26). Moreover, one can conclude that anesthetic actions of volatile anesthetics may be quite different from IV anesthetics.
Because of the recent innovations in electrophysiology, tissue preparation, and cellular imaging, gap junctions and their functional significance are now being studied extensively in mammals. However, there are limitations to these techniques. For example, in this report, such complex methods are applied to a relatively small number of slices per experiment but it is still comparable to similar studies. In addition, the FRAP technique does not allow the anesthetics to be removed during the experiment to obtain recovery data. Finally, the organotypic slice cultures used are obtained from newborn brains that might have some structural and functional differences from the adult brain.
Although anesthetic-induced deactivation of the CNS is typically thought to be a result of selective depression of excitatory synapses and/or enhancement of neuronal inhibition (7,27,28), it is quite possible that such actions could be augmented by the anesthetic depression of CNS gap junction coupling. Such augmentation might be caused by the disruption in gap junction-mediated neuronal synchronization in certain neuronal ensembles in the brain (29). Hence, attenuation of gap junction function could compound the effects of anesthetic administration and their withdrawal. Further studies are necessary to address several other aspects of gap junction contributions to anesthetic actions and to answer the question of whether the anesthetic-induced alteration in gap junction function is a cause or an effect of anesthetic exposure.
1. Dermietzel R. Gap junction wiring: a “new” principle in cell-to-cell communication in the nervous system? Brain Res Rev 1998;26:176–83.
2. Sohl G, Maxeiner S, Willecke K. Expression and function of neuronal gap junctions. Nature Rev Neurosci 2005;6:191–200.
3. Moreno AP, Rook MB, Fishman GI, Spray DC. Gap junction channels: distinct voltage-sensitive and -insensitive conductance states. Biophys J 1994;67:113–9.
4. Church J, Baimbridge KG. Exposure to high-pH medium increases the incidence and extent of dye coupling between rat hippocampal CA1 pyramidal neurons in vitro
. J Neurosci 1991;11:3289–95.
5. Kwak BR. Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels. Pflugers Arch 1995;430:770–8.
6. Buhl DL, Harris KD, Hormuzdi SG, et al. Selective impairment of hippocampal gamma oscillations in connexin-36 knock-out mouse in vivo
. J Neurosci 2003;23:1013–8.
7. Richards CD. Anesthetic modulation of synaptic transmission in the mammalian CNS. Br J Anaesth 2002;89:79–90.
8. Lazrak A, Peres A, Giovannardi S, Peracchia C. Ca-mediated and independent effects of arachidonic acid on gap junctions and Ca-independent effects of oleic acid and halothane. Biophys J 1994;67:1059.
9. Burt JM, Spray DC. Volatile anesthetics block intercellular communication between neonatal rat myocardial cells. Circ Res 1989;65:829–37.
10. Mantz J, Delumeau JC, Cordier J, Petitet F. Differential effects of propofol and ketamine on cytosolic calcium concentrations of astrocytes in primary culture. Br J Anaesth 1994;72:351–3.
11. Mantz J, Cordier J, Giaume C. Effects of general anesthetics on intercellular communications mediated by gap junctions between astrocytes in primary culture. Anesthesiology 1993;78:892–901.
12. Masaki E, Kawamura M, Kato F. Attenuation of gap-junction signaling facilitated anesthetic effect of sevoflurane in the central nervous system of rats. Anesth Analg 2004;983:647–52.
13. Stoppini L, Buchs PA, Muller D. A simple method for organotypic cultures of nervous tissue. J Neurosci Meth 1991;37:173–82.
14. Stasheff SF, Bragdon AC, Wilson WA. Induction of epileptiform activity in hippocampal slices by trains of electrical stimuli. Br Res 1985;344:296–302.
15. Lee SH, Magge S, Spencer DD, Sontheimer H, Cornell-Bell AH. Human epileptic astrocytes exhibit increased gap junction coupling. Glia 1995;15:195–202.
16. Hentschke H, Schwarz C, Antkowiak B. Neocortex is the major target of sedative concentrations of volatile anaesthetics: strong depression of firing rates and increase of GABAA receptor-mediated inhibition. Eur J Neurosci 2005;21:93–102.
17. Schwartzkroin PA. Epileptogenesis in the immature central nervous system. In: Schwartzkroin PA, Wheal HV, eds. Electrophysiology of epilepsy. London: Academic Press, 1984:389–412.
18. Dudek FE, Snow RW, Taylor CP. Role of electrical interactions in synchronization of epileptiform bursts. Adv Neurol 1986;44:593–617.
19. Carlen P, Perez Velasquez JL, et al. Electric coupling in epileptogenesis. In: Spray DC, Dermietzel R, eds. Gap junctions in the nervous system. New York: Landes Co., 1996:289–99.
20. Jefferys JG. Nonsynaptic modulation of neuronal activity in the brain: electric currents and extracellular ions. Physiol Rev 1995;75:689–723.
21. Samoilova M, Li J, Pelletier MR, et al. Epileptiform activity in hippocampal slice cultures exposed chronically to bicuculline: increased gap junctional function and expression. J Neurochem 2003;86:687–99.
22. Shyr MH, Tsai TH, Tan PP, et al. Concentration and regional distribution of propofol in brain and spinal cord during propofol anesthesia in the rat. Neurosci Lett 1995;184:212–5.
23. Stabernack C, Zhang Y, Sonner JM, Laster M, Eger EI II. Thiopental produces immobility primarily by supraspinal actions in rats. Anesth Analg 2005;100:128–36.
24. Srivastava K, Hatanaka T, Katayama K, Koizumi T. Pharmacokinetic and pharmacodynamic consequences of thiopental in renal dysfunction rats: evaluation with electroencephalography. Biol Pharm Bull 1998;21:1327–33.
25. Franks NP, Lieb WR. Temperature dependence of the potency of volatile general anesthetics: implications for in vitro
experiments. Anesthesiology 1996;84:716–20.
26. Srinivas M, Rozental R, Kojima T, et al. Functional properties of channels formed by the neuronal gap junction protein connexin 36. J Neurosci 1999;19:9848–55.
27. El Beheiry H, Puil E. Anesthetic depression of excitatory synaptic transmission in neocortex. Exp Brain Res 1989;77:87–93.
28. Puil E, El Beheiry H. Anesthetic suppression of transmitter actions in neocortex. Br J Pharmacol 1990;101:61–6.
29. Perez-Velazquez JL, Carlen PL. Gap junctions, synchrony and seizures. Trends Neurosci 2000;23:68–74.
30. Cockshott ID, Douglas EJ, Plummer GF, Simons PJ. The pharmacokinetics of propofol in laboratory animals. Xenobiotica 1992;22:369–75.
31. Christensen JH, Andreason F, Jensen EB. The binding of thiopental to serum proteins determined by ultrafiltration and equilibrium dialysis. Acta Pharmacologica Toxicologica 1980;47:24–32.