Major role of GABAA-receptor mediated tonic inhibition in propofol suppression of supraoptic magnocellular neurons

Using slice patch clamp recording, we examined the effects of general anesthetic propofol (2,6-diisoprophlphenol) on dual modality of GABA A inhibition in supraoptic nucleus (SON) magnocellular neurosecretory cells (MNCs): conventional quantal synaptic transmission (IPSCs, I phasic ) and persistent tonic form of inhibitory current ( I tonic ). Propofol (10 μM) enhanced I tonic as shown by an inward shift in I holding (16.46 ± 2.93 pA, n = 27) and RMS increase (from 3.37 ± 0.21 pA to 4.68 ± 0.33 pA, n = 27) in SON MNCs. Propofol also prolonged the decay time of IPSCs with decreased IPSCs frequency but no significant changes in IPSCs amplitude. Overall, propofol (1–10 μM) caused much smaller increase in mean I phasic than mean I tonic at all tested concentrations. In consistent with the enhancement of GABA A currents, propofol attenuated ongoing firing activities of SON MNCs by ∼65% of control. Selective inhibition of I phasic by a GABA A antagonist, gabazine (1 μM), failed to block the propofol suppression of the firing activities, while inhibition of I tonic and I phasic by bicuculline (20 μM) efficiently blocked the propofol-induced neurodepression in SON MNCs. Taken together, our results showed that propofol facilitated I tonic with marginal increase in mean I phasic , and this could be a mechanism reducing the intrinsic SON MNCs excitability during propofol anesthesia. Keywords Propofol GABA A receptors Supraoptic nucleus Oxytocin Vasopressin Cardiovascular depression The magnocellular neurosecretory cells (MNCs) in the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON) project to the neurohypophysis [1] where they release oxytocin (OT) or vasopressin (VP) into the bloodstream, and play fundamental roles in reproduction and fluid balance homeostasis. GABA, through activation of GABA A receptors, is a major inhibitory neurotransmitter modulating neuronal excitability in the nuclei [13,20,28] . GABA A receptors, pentameric GABA-operated ion channels, produce inhibition in at least two modes in the PVN and SON [23–25] : via conventional quantal synaptic transmission (IPSCs, I phasic ) and via a persistent tonic form of inhibitory current ( I tonic ) [6,19] . The hypnotic effects of a general anesthetic, propofol (2,6-diisopropylphenol), are primarily attributed to the extended GABA A channel open times [16] and slowing desensitization [2] , both attributes of I phasic . There are also emerging evidences that I tonic , like I phasic , is facilitated by many clinically used anesthetics. I tonic is even more sensitive to anesthetics than actions on I phasic [10] and seemingly plays a substantial role in suppressing neuronal excitability [3] . Propofol is often associated with adverse cardiovascular effects, such as decreases in cardiac output [18] and arterial blood pressure [5] . The enhancement of I phasic by propofol in the PVN and SON has been known to be a possible mechanism causing cardiovascular and sympathetic depression during propofol anesthesia [12,32] . However, the functional role of I tonic during anesthesia has not been elucidated in the neurons, although I tonic plays an important role in controlling neuronal excitability in the PVN and SON [23–25] as in cerebellum [4,9] and hippocampus [31] . Here, we showed propofol facilitates tonic as well as phasic modality of GABA A inhibition in SON MNCs, and the former may play a major role in the neurodepression by the anesthetic. Male Sprague–Dawley rats (180–220 g) were housed in a 12/12-h light/dark schedule and allowed free access to food and water. All animal experimentation adheres to the policy of the Chungnam National University regarding the use and care of animals. Patch-clamp recordings from SON neurons were obtained in hypothalamic slices (300 μm) as previously described [25] . Slices containing the SON were cut using a vibrosilcer (Leica VT 100s, Leica, Bensheim, Germany) in ice-cold artificial cerebrospinal fluid (aCSF), and placed in a holding chamber containing standard oxygenated aCSF until used. The standard aCSF consists of (in mM): NaCl 126; NaHCO 3 26; KCl 5; NaH 2 PO 4 2.4; CaCl 2 M 2.4; MgCl 2 1.2; glucose 10; pH was 7.3–7.4. The medium was saturated with 95% O 2 –5% CO 2 . Electrophysiological recordings were obtained with using Axopatch200-B amplifier (Axon Instruments, Foster City, CA). Current and voltage output were filtered at 2 kHz and digitized at 10 kHz (Digidata 1322A, Axon Instruments) in conjunction with pClamp 8.2 software. Patch pipettes (borosilicate glass, 3–7 MΩ) were filled with a high Cl − containing solution (in mM): 140 KCl, 20 HEPES, 0.5 CaCl 2 , 5 EGTA, and 5 Mg 2+ ATP, pH 7.3. Spontaneous inhibitory postsynaptic currents (sIPSCs, recorded at −70 mV) were detected in the presence of the glutamate AMPA/kainate receptor antagonist, DNQX (5,7-dintroquinoxaline-2,3-dione, 10 μM), NMDA receptor antagonist AP5 ( dl -2-amino-5-phosphonopentanoic acid, 100 μM), and analyzed using MiniAnalysis (Synaptosoft). The decay phase of IPSCs was best fitted with a double-exponential function, and presented in weighted values [8] . The holding current ( I holding ) and RMS noise were measured in 50 ms epochs of traces lacking PSCs, separated by ∼800 ms. I tonic was defined as the difference in I holding before and after application of GABA A receptor blocker picrotoxin or bicuculline. RMS noise was measured in the same epochs using MiniAnalysis software. Spontaneous firing discharges were recorded in continuous mode. For firing activity, patch pipettes filled with a more physiological concentration of Cl − were used (in mM): 140 K–gluconate, 10 KCl, 10 HEPES, 0.5 CaCl 2 , 5 EGTA, and 5 Mg 2+ ATP, pH 7.3. Firing rate was calculated using MiniAnalysis, by counting the number of action potentials in 10 s bins. Mean values for each condition were then obtained. Numerical data are presented as means ± SEM. Paired Student's t -test and analysis of variance repeated measures (ANOVA-RM), followed by Tukey's post hoc tests, were used to compare the effects of a drug treatment. To determine if propofol modulate the GABA A receptor mediated tonic currents in SON neurons, we measured the holding current ( I holding ) and RMS before and during the bath application of propofol ( Fig. 1 ). Bath application of the propofol (10 μM PPF) caused a significant inward shift in I holding (from −28.67 ± 2.16 pA to −45.12 ± 3.62 pA, n = 27, P < 0.001), and RMS increase (from 3.77 ± 0.21 to 4.68 ± 0.33, n = 27, P < 0.01). These effects were blocked by the GABA A receptor blocker bicuculline (20 μM) or picrotoxin (300 μM) ( Fig. 1 A–C). Propofol (10 μM) also significantly prolonged the decay time of IPSCs with no effects on the frequency and amplitude of IPSCs ( Fig. 1 D–F). Propofol decreased the frequency of IPSCs to 2.86 ± 0.47 Hz from the control of 3.39 ± 0.64 Hz ( n = 27, P < 0.05), while the mean IPSCs amplitude were not different before and during the application of propofol (control, 203.41 ± 12.00 pA vs propofol, 195.60 ± 11.69 pA, n = 27, P > 0.3). Propofol increased the decay time constant of IPSCs to 33.82 ± 2.64 ms (range 16.30–63.81 ms) from the control of 19.09 ± 1.21 ms (range 10.45–33.78 ms) ( P < 0.01, n = 27). The vehicle consist of soybean oil has no effects on I holding and RMS as well as the main properties of IPSCs in SON neurons. Propofol (10 μM) facilitation of I tonic shown by an inward shift in I holding (22.08 ± 4.71 pA, n = 4) and RMS increase (from 3.21 ± 0.96 to 4.08 ± 0.53, n = 4) ( P < 0.01 in both cases) was also observed in putative magnocellular neurons of the PVN. The electrical properties of type I neuron [35] were confirmed in the neurons (data not shown). To evaluate the contribution of I phasic and I tonic to propofol enhancement of GABA A inhibition, we estimated and compared the propofol-facilitated charge transfer mediated by the two inhibitory modalities in SON MNCs. Despite their significant increase in IPSC decay time to ∼170% of control, due to their transient nature and rapid kinetics in addition to the frequency decrease, the overall increase of mean I phasic was negligible in the presence of propofol. The mean I phasic was calculated by multiplying the charge transfer of the averaged IPSC ( Q , the integrated area under IPSCs) by the IPSCs frequency, as previously described [22,24,25] . 10 μM PPF increased mean I phasic by 0.40 ± 0.51 pA ( n = 27), which was much smaller than the mean I tonic increase of 16.46 ± 2.93 pA ( n = 27) ( P < 0.001) ( Fig. 2 ). Although propofol (1–10 μM) increased both the decay time of IPSCs and I holding in a concentration manner, respectively, the overall increase of charge transfer was mainly mediated by I tonic at all tested concentrations ( Fig. 2 ). These results indicate that most of GABA A receptor-mediated inhibition (phasic + tonic) is carried by the tonic modality during propofol facilitation. To confirm a functional significance of propofol facilitation on phasic and tonic GABA A inhibition in SON excitability, we examined the effects of propofol on spontaneous firing activity in SON MNCs. Recorded neurons were either spontaneously active (7/21), or firing activity was evoked by small DC current injection. Two different GABA A receptor antagonists, gabazine and bicuculline, were used to differentiate between the roles of I phasic and I tonic [24,25] . Results are summarized in Fig. 3 . Propofol (10 μM) decreased the firing activity of SON neurons by 64.3 ± 4.0% ( n = 7, P < 0.01 when compared to baseline) in normal ACSF ( Fig. 3 A and B). Propofol still decreased the firing activity of SON neurons by 73.1 ± 8.7% ( n = 8, P < 0.01 when compared to baseline) in the presence of gabazine, a GABA A receptor antagonist blocks I phasic only, while it failed to depress the firing activity in the presence of bicuculline (20 μM), a GABA A receptor antagonist blocks both I phasic and I tonic . These results suggest that propofol leads to the attenuation of intrinsic neuronal excitability mainly via facilitation of tonic GABA A inhibition in SON MNCs. The main findings of these studies may be summarized as followed: (1) propofol inhibits the neuronal excitability by GABA A inhibition in magnocellular SON neurons, and (2) the inhibitory action of propofol is largely mediated by the enhancement of I tonic with minimal changes in mean I phasic in SON MNCs. Thus our data provided the significant insights into the functional role of I tonic in magnocellular neurosecretory system during anesthesia. A large bulk of information indicates that GABA, through activation of GABA A receptors, is a major neurotransmitter modulating SON neuronal excitability [13,14,20,21,28] . Furthermore, I tonic restraining a neuronal activity of SON neurons accounts for ∼80% of the total GABA A -receptor mediated current [25] . Accordingly, facilitation of I tonic may decrease SON firing discharge with decreased input resistance. Indeed, our data in the present study showed propofol decreased the ongoing firing activity in SON neurons with large and marginal potentiation of I tonic and I phasic , respectively. These results suggested that the suppression of neuronal excitability by propofol is largely mediated by I tonic in SON MNCs, as reported in hippocampal CA1 neurons [3] . The notion was further supported by our results showing that inhibition of I tonic , but not I phasic , efficiently blocked propofol-induced neurodepression in SON MNCs. Although VP and OT neurons are not identified in the present study, the potentiation effects of propofol on I tonic in 27 neurons could not be classified in 2 or more populations (data not shown). Given that I tonic is not different in VP and OT neurons in the SON [25] , the results suggest that propofol-induced neurodepression is largely mediated by I tonic in both types of SON neurons. I tonic has been observed primarily in cerebellar granule cells (CGCs) and dentate gyrus granule cells (DGGCs) [4,15,22,34,37] , where δ subunit-containing GABA A receptors mediate I tonic . More recently, the contribution of GABA A receptor γ 2 subunit in I tonic has been known in various neurons including in hippocampal CA1 neurons [27] , pyramidal neurons of neocortex [40] , and preganglionic neurons of dorsal motor nucleus of the vagus [7] . In general, δ subunit containing receptors show higher sensitivity to GABA than their classical counterpart γ 2 subunit containing receptors. Given that propofol potentiation on GABA A receptors are correlated inversely with the GABA sensitivity of each receptor [30] , our results showing the similar sensitivities of I phasic and I tonic to propofol is in the line that I tonic is mediated by γ 2 containing receptors in SON MNCs. Several distinct mechanisms potentially underlie propofol enhancement of I tonic in SON MNCs. In addition to the number and activity of presynaptic GABA release, GABA transporters (GATs) responsible for removing GABA from the extracellular space play a key role in controlling local ambient extracellular GABA concentration and I tonic . However, propofol at clinical concentrations had no effects on GABA uptake and GABA release (reverse transport) through GATs [38] . Furthermore, in consistent with previous report [12] , propofol did not enhance the frequency of GABAergic IPSCs in SON MNCs in the present study, although propofol had been found to increase GABA release in rat cortical synaptosomes [38] and DGGCs [11] . These results suggest that propofol enhanced I tonic through mechanisms other than an increase in ambient GABA concentration in SON MNCs. Both propofol potentiation of GABA action at GABA A receptors and direct propofol activation of GABA A receptors have been documented [36,39] . At ∼0.5 μM, propofol potentiates currents induced by submaximal GABA concentrations but does not directly activate GABA A receptors. At 20-fold higher concentrations, propofol directly activates receptors, causing channel opening in the absence of GABA [17,30] . Indeed, 10 μM propofol activated GABA A current in isolated SON MNCs [12] . Therefore, the enhancement of I tonic by propofol may be a result of the direct activation of GABA A receptors on the top of the facilitation of GABA A receptors already activated by low concentration of ambient GABA in our slice preparation [25] . In conclusion, our result showed propofol attenuated the intrinsic activity of SON MNCs through a postsynaptic enhancement of I tonic in conjunction with effects on synaptic transmission. Given that OT and VP peptides secretion from SON MNCs into the blood stream in the neurohypophysis are tightly locked to the electrical activity of the neurons [26,29] , the propofol enhancement of I tonic in SON neurons may suppress plasma VP level related with cardiovascular depression during propofol anesthesia. The notion is in consistent with a previous finding that propofol inhibited the somatodendritic VP release from SON slices [33] . Acknowledgment This work was supported by National Research Foundation of Korea funded by the Ministry of Education, Science and Technology ( 2010-0027606 , 2010-0015894 and R13-2007-020-01000-0 ). References [1] W. Armstrong Hypothalamic Supraoptic and Paraventricular Nuclei. The Rat Nervous System 3rd ed. 2004 Paxinos Ed. San Diego 369–388 pp. [2] D. Bai P.S. Pennefather J.F. MacDonald B.A. Orser The general anesthetic propofol slows deactivation and desensitization of GABA(A) receptors J. Neurosci. 19 1999 10635 10646 [3] M.C. Bieda M.B. 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