Synaptically localized GABAA receptors mediate the primary inhibition caused by propofol and tests reveal

how GABAB receptors play a role in Anesthesia A novel development

!Jordan D. RichA, Milis SunayB, Vytas Dargis-RobinsonB, and Bruce MacIverB

A Dept. of Bioengineering, University of Utah, Salt Lake City, USA B Department of Anesthesiology, Stanford School of Medicine, Palo Alto, USA !!!!

Abstract—Thanks to the recent finding of a possible binding site for propofol on GABA receptors, the possibility of developing a new intravenous anesthetic may now be a reality. This research investigates the physiological environment that propofol interacts with and touches on the relationship of GABA receptor subtypes to propofol’s inhibitory effect on the central nervous system. Thereafter, an avenue that may point toward innovation of a novel intravenous anesthetic is presented.

Index Terms—Propofol, brain, CNS, anesthesia, anesthetic, electrophysiology, neural circuit, GABA, GABAR, GABA receptor, GABAA, GABAB. (keywords)

!I. INTRODUCTION

It has been nearly 30 years since a new anesthetic has been released to the market. Propofol, the most recent anesthetic, hit the market in 1986 and has since become the most popular and prevalently used anesthetic in medicine. However, recent shortages of propofol in the United States and the underlying causes for such demonstrate a need for the introduction of a new intravenous anesthetic.

The Food and Drug Administration has approved three manufacturers for propofol, two of which are currently not in production. This is due to a combination of factors, including a costly and difficult manufacturing process that led to recalls in 2009 by both Teva Pharmaceuticals and Hospira due to contaminated product. Teva Pharmaceuticals ultimately withdrew from the market following a $500 Million judgement against it by a Nevada jury for tort claims related to the infection of patients with hepatitis C. Notwithstanding the difficulties surrounding propofol, global demand for the anesthetic is steadily rising with over $750 Million in global revenue and a projected 4% annual growth rate.

The time may be good for the development of a new intravenous anesthetic. By analyzing the physiological mechanisms of propofol induced anesthesia, indications of desirable properties in a new anesthetic may be acquired. The

recent discovery of the propofol binding site on GABAA receptors provides a basis for new development [1]. GABAA receptors localize on the cell body and dendrites of the neuron, both synaptically and extrasynaptically. Localization of the receptor is determined by the receptor’s subtype composition (See vi, vii, & viii, fig. 1) [2,3,4,5,6]. The receptor configuration further determines both the kinetics of the receptor and whether it is tonic or phasic [7,8,9]. Propofol binds to β3 subunits of synaptic, perisynaptic, and extrasynaptic tonic and phasic GABAA receptors, causing slower gating and an increase in Cl- conductance thereby inhibiting the neuron from achieving action potential [1,10,11,12,13]. Because GABA remains bound to receptors for an increased interval in the presence of propofol, extrasynaptic concentrations of GABA increase which triggers a G-protein mediated secondary signaling cascade via perisynaptic GABAB receptors that causes K+ efflux and hyperpolarizes the neuron (See iii & iv, fig. 1) [4,14,15,16].

The following study seeks to determine what effect changes in the binding probability of GABA to perisynaptic tonic GABAA receptors versus synaptic GABAA receptors have on activation of the before mentioned K+ efflux. Furosemide was employed to antagonize α6β2,3 configured perisynaptic tonic GABAA receptors, then all Cl- currents were disabled with picrotoxin [17,18]. For comparison, gabazine in a low molar concentration was employed to disable all synaptic GABAA receptors, then likewise, all Cl- currents were disabled with picrotoxin [7,18]. With Cl- currents disabled, the only notable inhibitory mechanism of the neuron is the hyperpolarizing K+ efflux induced by GABAB.!!

II. METHODS Brain excisions of isofluorane anesthetized adult sprague

dawley rats were performed in conformance with 7 U.S.C. §2131 and all regulations set forth by the National Institute of Health and the State of California with oversight of the Institutional Animal Care Committee at Stanford University. Transverse hippocampal brain slices were prepared at a

Sponsored by The Amgen Foundation

Figure 1. Diagram depicting study methodology. (i.) Transverse section of brain. (ii.) Hippocampal section of transverse brain slice depicting anatomical regions. Neural signals transmit from CA3 to CA1 via the schaffer collateral pathway (sc). Black neurons depict the orientation of pyramidal neurons; colored are interneurons. (iii.) Diagram of synaptic interaction at GABA and glutamate mediated interneural synapses [16]. Presynaptic GABAB receptors function to regulate exocytosis of neurotransmitter by decreasing Ca2+ conductance when excess GABA is present [14,16]. Postsynaptic GABAB receptors localize perisynaptic/extrasynaptic and regulate neuron membrane potential by altering K+ conductance via a G-protein mediated secondary signaling cascade [14,15,16]. (iv.) Diagram of postsynaptic GABAB receptor [16]. (v. & vi.) GABAA receptors are Cl- channels composed of 5 subunit proteins. The anesthetic propofol binds to β3 subunits of GABAA receptors increasing GABA binding affinity and causing slower gating, increased Cl- conductance, and increased GABAB activation due to increased extrasynaptic GABA [1,10]. (vii.) Seventeen known GABAA receptor subtypes: α 1-6, β 1-3, γ 1-3, δ, ε, π, ρ, and Θ [18]. GABA binding site between α and β subunits [18]. Synaptic γ2 containing receptor configurations have prevalence of ~80% phasic and ~20% tonic response [2,18]. δ configurations localize perisynaptic with tonic response [4,13,19]. (viii.) Prevalent GABAA receptor configurations [18]. **Illustrations iii, iv, and vi are the works of [16], [16], and [1] respectively.

thickness of 400nm using a vibratome (See i & ii, fig. 1). Slices were stabilized for a period of 1 hour in submersion with artificial cerebral spinal fluid (ACSF) composed (in mM) of 124 NaCl, 1.25 NaH3PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO2, and 10 glucose and bubbled with 95% O2/5% CO2 at pH 7.4. Local field potential electrophysiology was performed with a continuous perfusion of ACSF at a rate of 2-3mL/min at 22- 24 ℃. Propofol (20 µM), furosemide (20 mM), gabazine (20 µM), and picrotoxin (100 µM) were employed during their respective intervals by perfusion with the ACSF formulation. All compounds employed were reagent grade and supplied by Sigma Aldrich.

Population spike responses from the cell body layer

(stratum pyramidale) in the CA1 region of the hippocampus

were recorded by stimulating the schaffer collateral pathway with a bipolar tungsten microelectrode driven by a Grass S48 stimulator with 0.22-0.25 pulse width at 5-8 volts (See fig. 2). Glass micropipette recording electrodes filled with ACSF were employed. Signals were amplified 1000x, digitized using a National Instruments USB 6009 DAC, and stored and analyzed with Igor Pro software by Wavemetrics.

!III. RESULTS

Application of propofol caused a significant decline in population spike amplitude, signifying that less neurons were capable of achieving action potential (See A & B, fig. 3). Furosemide caused an initial partial reversal of propofol induced depression, however a continued decline in population spike amplitude followed (See A, fig. 3). In comparison, gabazine caused a consistent partial reversal of propofol induced depression (See B, fig. 3). In both cases, picrotoxin caused multiple population spikes in response to a single stimulation pulse (See C, fig. 3). By measuring the total sum of population spike amplitudes to a single stimulation pulse, it was observed that ~25% less action potentials were produced when picrotoxin was applied alongside furosemide than when picrotoxin was applied alongside gabazine (See A & B, fig. 3). !

IV. DISCUSSION The results of this study indicate two important things. First

is that synaptic GABAA receptors play a primary role in the inhibitory effect of propofol which is indicated by the observation that the population spike amplitude continued to decline after perisynaptic receptors were disabled but not when synaptic receptors were. Just as important is the finding that GABAB receptors are more attuned to changes in the probability of GABA binding to perisynaptic tonic GABAA receptors than synaptic receptors which is indicated by the depressed amplitude when perisynaptic receptors were disabled compared to when synaptic receptors were. A plausible explanation is that perisynaptic receptors function similar to a capacitor in an electrical circuit, steadying rapid changes in extrasynaptic GABA to avoid unwarranted changes in K+ efflux of the neuron [4]. This finding may similarly describe

Figure 2. Sites of stimulation and recording of hippocampal tissue.!A.

B.

C.

Figure 3. Experimental Results. (A.) Trials to determine relationship of perisynaptic tonic GABAA receptors to K+ efflux caused by perisynaptic GABAB activation. Bars (top) signify agent delivery interval. (B.) Trials to determine relationship of synaptic phasic and tonic GABAA receptors to K+ efflux. (C.) Individual population spike traces following application of select agents.

the relationship of propofol to the activation of K+ efflux via GABAB. While furosemide completely restricts the binding of GABA to the perisynaptic receptor, propofol causes GABA to remain bound to the receptor for an increased interval, thereby decreasing the likelihood of extrasynaptic GABA binding to perisynaptic GABAA receptors and increasing the likelihood of it binding to GABAB [16,17]. Accordingly, it is inferred from this study that an anesthetic designed to favor synaptic receptors and minimally bind to perisynaptic receptors would decrease the activation of K+ efflux and thereby allow for a more rapid reversal of anesthesia. It is known that a G-protein induced K+ efflux is far slower in its activation and deactivation than Cl- induced inhibition [14,15,16,18].

This model may be utilized to design a new intravenous anesthetic. It has been realized in other studies that different modalities of GABAA receptor configurations exhibit widely varying kinetics [18,20]. These variations in kinetics are notably attributed to the amino acid sequence and structure of the subunit proteins. The difference in neighboring subunit structures may affect the binding affinity of propofol to the β3 subunit. By modeling the propofol binding site and the electrical and geometrical interactions of neighboring subunits, a molecular structure that favors synaptic and disfavors perisynaptic GABAA receptor binding may be developed.

!ACKNOWLEDGMENT

All lab testing was performed at the Neuropharmacology Laboratory led by Bruce MacIver, MSc PhD at Stanford University Medical School.

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