Synaptic Mechanisms of Thiopental-induced Alterations in Synchronized Cortical Activity

Two separate experimental protocols were used in the current study. EEG-like activity was pharmacologically evoked in neocortical regions, while patch clamp and evoked field excitatory postsynaptic potential (EPSP) recordings were performed in hippocampal cortex. Hippocampal rather than neocortical neurons were patched because of difficulties associated with eliciting synchronous monosynaptic evoked inhibitory postsynaptic currents (IPSCs) in neocortex, and because of difficulties associated with patching onto sparsely distributed neocortical somata. It was shown that thiopental produced similar effects on hippocampal and neocortical EEG activity, as shown in the preceding in vivo article. [10]Previous studies have shown that theta EEG activity in hippocampal slices [12–14,16]shares a similar frequency (3.5–7.5 Hz.), amplitude (50–250 micro Volt), and pharmacologic profile with the neocortical xi-like oscillations observed in the current study.

Experiments were performed on slices isolated from juvenile male Sprague-Dawley rats (weighing 80–120 g). Experimental protocols were approved by the Institutional Animal Care Committee at Stanford University and adhered to published guidelines of the National Institutes of Health, Society for Neuroscience, and American Physiological Society. Rats were anesthetized with diethyl ether and their hearts were stopped with a blow to the back of the thorax. Brains were removed to cold (1–2 degrees Celsius) oxygenated artificial cerebrospinal fluid (ACSF, see Materials). Brains were sectioned in the coronal plane into 450-micro meter thick slices using a vibratome (Vibraslice, Boston, MA). Slices containing both neocortical and hippocampal areas were then hemisected and placed on filter papers at the interface of a humidified carbogen (95% O²/5% CO sub 2) gas phase and ACSF liquid phase. Slices were allowed at least 1 h to recover from the slicing procedure before submersion in ACSF in a recording chamber. The ACSF was saturated with carbogen gas and perfused at a rate of 2.5 ml/min, at room temperature (21–24 degrees C). Rapid and accurate solution changes were made using a ValveBank8 computerized perfusion system (AutoMate Scientific, Oakland, CA). Thiopental concentrations were measured using high-performance liquid chromatography. [17].

In vivo theta EEG activity has been shown to be associated with activation of both ascending cholinergic and GA-BAergic inputs. [18]Cholinergic inputs are thought to depolarize pyramidal neurons, whereas GABAergic inputs have been shown to selectively innervate inhibitory neocortical interneurons, [19,20]suggesting that activation of these GABAergic afferents results in neocortical disinhibition. These endogenous inputs were mimicked in neocortical slice micro-EEG experiments by using ACSF containing the cholinergic agonist carbachol (100 micro Meter), and the GABA^Aantagonist bicuculline (10 micro Meter). Gamma-Aminobutyric acid (GABA) was not used because in addition to blocking inhibitory interneuron activity, it would directly inhibit pyramidal neurons and interfere with cholinergic excitation of these cells.

Pharmacologically evoked EEG-like oscillations were recorded with low resistance (< 2 M Omega) glass microelectrodes filled with ACSF and placed in layer 2 or 3 of the neocortex for most experiments (Figure 1). These micro-EEG signals were amplified X10,000–50,000 (model 210A; Brown-Lee Precision, San Jose, CA), filtered 1–30 Hz band-pass, 60 Hz notch (Cyber Amp 380, Axon Instrument, Foster City, CA), digitized (256 or 2,048 Hz; DataWave Technologies, Longmont, CO) and stored on computer disk for further analysis. Micro-EEG spectral analysis was accomplished using fast Fourier transforms on 2.5-s long epochs of data using DataWave software.

Before drug application, each neocortical slice displayed a 20-min baseline consisting of trains of spontaneous theta-like micro-EEG activity in ACSF containing carbachol (100 micro Meter) and bicuculline (10 micro Meter). All pharmacologic agents were applied in ACSF containing carbachol and bicuculline, and drug application was continued until steady-state effects were achieved (i.e., burst suppression or isoelectric activity). Drug washout began within 15 min of achieving a steady-state effect.

Whole cell recording microelectrodes (4–8 M Omega) contained an internal solution comprising (in mM): K-gluconate 100; ethylene glycol-bis(beta-amino-ethyl ether) N,N,N',N'-tetra acetic acid 10; MgCl sub 2 5, N-[Z-hydroxy-ethyl]piperazine-N'-[Z-ethane sulfonic acid](HEPES) 40; adenosine triphosphate 0.3; and guanosine triphosphate 0.3, pH = 7.2 and osmolarity = 280–290 mOsm. Monosynaptic IPSCs were evoked in stratum radiatum with a bipolar-stimulating electrode (5 V, 250 micro second, 0.033 Hz), in the presence of 6-cyano-7-nitroquinoxaline-2,3-dion and (plus/minus)-2-amino-5-phosphonovaleric acid (see Materials) to block excitatory glutamate-mediated transmission. Stimulating electrodes were placed for optimal GABA^A, slow IPSC activation. [21]Cells were voltage clamped at -60 mV (Axoclamp 2A, Axon Instrument), which is typical for resting membrane potentials in hippocampal pyramidal cells. [22]Signals were amplified (X500), filtered (DC--10 kHz band-pass; Axon Instrument), digitized (10 kHz; DataWave Technologies) and stored on computer disk for further analysis.

Field EPSPs were evoked (3–8 V, 250 micro second, 0.1 Hz) with a bipolar tungsten stimulating electrode placed in stratum radiatum of area CA1. EPSPs were recorded with low resistance (< 2 M Omega) glass electrodes filled with ACSF also placed in stratum radiatum. [23]Signals were amplified (X5,000), filtered (DC to 10 kHz) and digitized.*.

Rats were obtained from Simonsen Laboratories (Gilroy, CA). Thiopental, carbamylcholine chloride (carbachol), and atropine sulfate were obtained from Sigma (St. Louis, MO). (-)-Bicuculline methiodide, muscimol HBr, (plus/minus)-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitro-quinoxaline-2,3-dione were supplied by Research Biochemicals International (Natick, MA). All solutions were made with spectrophotometric grade water (Omnisolve) supplied by EM Science (Gibbstown, NJ). The ACSF had the following ionic composition (in mM): Na sup + 151.25; K sup + 3.5; Ca sup ++ 2.0; Mg sup ++ 2.0; Cl sup - 130.5; HCO³sup - 26; SO⁴sup - 2.0; H²PO⁴sup - 1.25; and glucose 10. Chemicals for the ACSF were reagent grade or better and obtained from J.T. Baker (Philadelphia, PA).

Spontaneous trains of neocortical theta-like micro-EEG activity, lasting up to 9 s and ranging in amplitude from 20 to 450 micro Volt, appeared in vitro during bath application of carbachol and bicuculline (Figure 1). These theta trains occurred 0.5–2 times per minute and, like cholinergically driven type II theta in vivo, [18]were blocked by the muscarinic receptor antagonist atropine (0.5 micro Meter). This theta-1ike micro-EEG activity was not observed throughout the entire neocortex, rather it was confined to a bilateral strip of medial occipital cortex that runs rostrocaudally and included four anatomically defined areas: 29d, 18b, 17, and 18a, [24]also known as the occipital association areas: Oc2MM, Oc2MM/Oc2ML, Oc1M/Oc1B, and Oc2L, respectively. [25]Previous in vivo studies also have demonstrated the occurrence of theta EEG activity within these anatomically defined cortical regions. [26,27]The neurons and synchronizing circuits generating these theta frequency oscillations must be intrinsic to neocortex because isolated mini-slices of Oc2MM cortex produced theta-like activity, and paired electrode differential recordings revealed a phase reversal in this cortical region, indicating the presence of a local neuronal generator. The narrow bandwidth and sinusoidal nature of in vitro neocortical micro-EEG activity (Figure 1) may be caused by removal of other ascending modulatory influences such as catecholamine or indolamine afferents.

Thiopental concentrations in brain slices were matched to calculated in vivo thiopental levels that produced progressively deeper stages of anesthesia. [9,10]In brain slices, fast Fourier transform analysis revealed that 20 micro Meter thiopental produced a threefold decrease in micro-EEG frequency, from theta (7.3+/-0.9 Hz, mean+/- SD; n = 19) to delta (2.5+/-0.5 Hz, n = 11). This decrease was comparable to a threefold slowing of EEG frequencies observed in vivo Figure 2. Slice micro-EEG amplitudes increased approximately 375% during transitions from theta to delta frequency oscillations, also similar to results obtained in vivo. [10]Higher concentrations of thiopental (50 micro Meter; n = 11) produced burst suppression micro-EEG patterns, characterized by large amplitude (200–500 micro Volt) burst discharges separated by brief periods of isoelectric activity (Figure 2(A and C)). Burst activity in slices was either monophasic or biphasic with a sharp negativity followed by a low amplitude positive overshoot. In vitro bursts were typically separated by 0.5–3 s, whereas in vivo bursts were more varied (separated by 0.1–5 s). Increasing thiopental levels to 100 micro Meter produced isoelectric activity, which reversed to theta-like activity after barbiturate washout (5 of 5 brain slices). Thus, a similar progression in EEG profiles was observed in vivo and in vitro over a clinically relevant concentration range.

Thiopental effects on inhibitory currents were examined using whole cell patch clamp recordings. In the presence of 20 micro Meter thiopental, monosynaptic evoked IPSC half width (t¹/2) increased approximately threefold from 135+/-22 ms (n = 8) to 400+/-40 ms (n = 5), whereas IPSC amplitudes remained relatively unchanged (Figure 3(A and B)). This threefold increase in IPSC t¹/2 was associated with the threefold slowing in micro-EEG frequencies observed during theta to delta transitions also produced by 20 micro Meter thiopental (Figure 2).

To compare thiopental effects on IPSCs with effects on micro-EEG slowing, theta and delta rhythms were approximated as sine waves (Figure 3(C)). Theta and delta sine wave periodicities of 137 and 397 ms were calculated from experimental mean theta (7.3 Hz) and delta (2.5 Hz) oscillation frequencies, respectively. Modeled EEG waves were superimposed on IPSCs recorded in the presence or absence of 20 micro Meter thiopental (Figure 3(C)). Control and thiopental-prolonged IPSC amplitudes were compared at times corresponding to the periodicity of either theta or delta frequency oscillations, and revealed that both IPSCs had decayed approximately 38% from their peak values over these time periods (Figure 3C). The inhibitory current amplitude (approximately 140 pA) associated with these time points may represent a critical degree of inhibition such that EEG generating neurons discharge and then remain inhibited until recurrent IPSCs decay below this critical level (i.e., after 137 ms in control conditions vs. 397 ms in the presence of 20 micro Meter thiopental). Thus, micro-EEG oscillation frequency appeared to depend on IPSC decay times. The thiopental-induced increase in variability of IPSC t¹/2 (Figure 3(B)) was also apparent in the increased standard deviation for delta micro-EEG activity (Figure 3(C)), as expected if IPSC prolongation resulted in the observed decrease in micro-EEG frequencies.

Whole cell voltage clamp recordings revealed a tonic activation of inhibitory currents (approximately 100 pA;Figure 3(A)) at thiopental concentrations (50 micro Meter) that elicited sustained burst suppression micro-EEG activity. To test whether tonic GABA^A-mediated hyperpolarization contributed to burst suppression activity, micro-EEG effects of the GABA^Aagonist muscimol were studied. Muscimol (1 micro Meter) produced a direct transition from theta to burst suppression activity without a slowing to delta frequencies (5 of 5 slices;Figure 4). The time course of this effect was rapid with bursts typically occurring 3–5 min after muscimol application, and recovery to theta-like activity occurring 2–4 min after drug removal. Muscimol-induced burst suppression activity was similar to burst suppression activity produced by 50 micro Meter thiopental in that high amplitude (greater or equal to 200 micro Volt) monophasic or biphasic bursts occurred with an interburst interval of 0.5–3.0 s.

Thiopental effects on glutamate-mediated transmission were investigated using evoked field EPSPs in area CA1 of the rat hippocampus. These EPSPs are glutamate mediated, [28]and can be completely blocked by concurrent application of the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dion and (plus/minus)-2-amino-5-phosphonovaleric acid. [29]Excitatory postsynaptic potential amplitudes were depressed (23.4+/-8.5% mean+/-SD, n = 8) by concentrations of thiopental (100 micro Meter;Figure 5(A)) that produced isoelectric EEG activity in vivo [10]and in brain slices, whereas thiopental concentrations that evoked only burst suppression activity did not alter EPSP amplitudes (0.2 +/-3.2%; n = 5; at 50 micro Meter). Further evidence that depressed glutamatergic transmission contributed to isoelectric activity came from experiments showing that the glutamate receptor antagonists (plus/minus)-2-amino-5-phosphonovaleric acid (42 micro Meter, 3 of 3 slices;Figure 5(B)) or 6-cyano-7-nitroquinoxaline-2,3-dion (8 micro Meter, 5 of 5 slices) could force transitions from burst suppression to isoelectric activity in the presence of 50 micro Meter thiopental. This concentration of thiopental (50 micro Meter) by itself did not produce isoelectric activity (0 of 10 slices).

Anesthesia has been proposed to result from decreased excitatory transmission, [30–32]increased inhibitory transmission, [1,33–35]and/or inhibition of action potential generation via membrane hyperpolarization. [36–38]The current study demonstrated that each of these mechanisms could contribute to thiopental-induced alterations in synchronized neuronal activity. The threefold slowing in neocortical micro-EEG peak frequency from theta (7.3 Hz) to delta (2.5 Hz) activity, observed in the presence of 20 micro Meter thiopental, was associated with a threefold prolongation of inhibitory currents. Burst suppression activity occurred in the presence 50 micro Meter thiopental, and appeared to result from tonic GABA^A-mediated neuronal hyperpolarization, with intact excitatory transmission required to generate burst discharges. Isoelectric activity observed with 100 micro Meter thiopental appeared to result from depressed excitatory glutamatergic transmission, in addition to the aforementioned increases in both phasic and tonic inhibition. Thus, it is possible that the continuum of EEG effects associated with deepening states of thiopental-induced anesthesia (theta > delta > burst suppression > isoelectric) may be accounted for by a concentration-dependent recruitment of separate synaptic and membrane actions, as predicted by a multisite agent-specific theory of anesthetic action. [23,39–41].

Previous studies have shown that anesthetics prolong GABA-mediated inhibition while exerting little effect on IPSC amplitudes. [1,34,42]In the current study, inhibitory currents were prolonged by thiopental concentrations (20 micro Meter, Figure 3) that produced delta frequency slowing (Figure 2), whereas IPSC amplitudes remained unaltered. Thiopental-induced prolongation of inhibition may cause slowing by limiting neuronal discharge frequencies. To understand how limiting discharge frequencies could cause EEG slowing, it is necessary to think of the neuronal population dynamics underlying synchronous oscillatory EEG activity. The majority of neurons discharge preferentially during the peak negativity of EEG oscillations and are silent during peak positivities. [43]Thus, one full EEG oscillation (approximated by 360 degrees of a sine wave, (Figure 3(C)), likely represents the synchronous discharge, quiescence, and secondary discharge of a neuronal population. Stated another way, the length of time between neuronal population discharges determines the periodicity of an EEG oscillation. What then determines neuronal interdischarge time intervals? One likely candidate is the time course of recurrent GABA-mediated inhibition. In support of this, depression of GABA-mediated inhibition has been shown to elicit high-frequency discharge activity. [44]Conversely, high-frequency discharge activity associated with epilepsy can be suppressed by some barbiturates and other anesthetics [45]known to enhance GABAergic transmission. By prolonging the time course of inhibition, 20 micro Meter thiopental may limit neuronal discharge frequencies, allowing lower frequency delta activity while filtering out higher frequency theta activity.

Previous studies have shown that burst suppression activity occurs during surgical anesthesia with thiopental. [7,9,11,46–48]In the current study, thiopental-induced burst suppression-like activity was associated with increased tonic inhibition, evidenced by increased steady-state outward currents observed in the presence of 50 micro Meter thiopental (Figure 3(A)). The ability of muscimol to produce burst suppression activity (Figure 4) demonstrated that enhanced tonic GABAergic hyperpolarization was sufficient to produce this micro-EEG state. In the presence of muscimol, theta-like oscillations progressed directly to burst suppression activity without passing through a delta state. Delta activity should not and did not occur under these conditions because muscimol directly opens GABA^A-gated chloride channels without prolonging IPSC t¹/2 [49]: supporting the idea that delta slowing resulted from a prolongation of inhibitory currents, whereas burst suppression activity required enhanced tonic inhibition (hyperpolarization).

These results are consistent with previous in vivo findings that demonstrated that neocortical neurons hyperpolarize (approximately 10 mV) and increase their resting membrane conductance during anesthetic-induced burst suppression activity. [50]Increased tonic inhibition may contribute to burst suppression activity by hyperpolarizing EEG-generating neurons. Hyperpolarization would have three primary effects:(1) tonic cell discharge frequencies will decrease, reducing excitatory synaptic transmission between cortical neurons;(2) membrane potential-dependent inactivation will be removed from low threshold voltage activated calcium and sodium channels [51]; and (3) reduced cell discharge will leave fewer neurons refractory to firing. These conditions would favor a state in which EEG-generating neurons become both quiescent, leading to periods of suppressed EEG activity, and hyperexcitable, leading to large amplitude bursts in response to excitatory inputs.

In the current study, burst activity required excitatory inputs, and blockade of excitatory transmission with N-methyl-D-aspartate or (plus/minus)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate glutamate receptor antagonists caused a transition from sustained burst suppression activity to isoelectric activity (Figure 5). The use of a brain slice preparation ensured that ascending glutamatergic afferents were severed, suggesting that bursts were triggered by spontaneously active local excitatory neurons within the neocortex. In support of this, in vivo studies have demonstrated that thiopental-induced burst suppression activity persists in neocortex, which was isolated from ascending inputs by undercutting the white matter, while leaving the blood supply intact. [52,53]Intrinsic excitatory neurons must be capable of spontaneous discharge activity even in the presence of enhanced tonic and phasic inhibition produced by 50 micro Meter thiopental (Figure 3(A)).

Thiopental-induced delta, burst suppression, and isoelectric activity appear to come about by actions intrinsic to the neocortex because ascending projections were eliminated using isolated neocortical brain slices. However, there exists a possibility that damage caused by the slicing procedure could result in injury discharge activity from severed ascending tracts, and this activity could be modulated by thiopental. In the intact animal it is likely that subcortical structures also contribute to the production of these EEG patterns. For example, thalamocortical networks have been shown to be important in generating sustained delta activity during sleep. [54]One micro-EEG state not observed in neocortical brain slices was an early activation that preceded delta activity in vivo, and was characterized by increased power in theta, alpha, and beta frequencies. [10]Consistent with a subcortical loci for EEG activation, previous studies have demonstrated the ability of thalamocortical, [54,55]septocortical, and pontocortical afferents [43,56]to activate cortical EEG signals in vivo. In addition, manipulations that disconnect mesencephalic structures from the neocortex also block EEG activation. [57]Thus, subanesthetic concentrations of thiopental may directly excite ascending systems leading to EEG activation. Alternatively, low thiopental concentrations may depress cortical electrical activity resulting in a disinhibition of lower brain stem systems that feedback to excite cortical neurons, resulting in EEG activation. Thus, thiopental-induced EEG activation in vivo may reflect indirect effects of thiopental on brainstem activating systems, but EEG states associated with deepening levels of anesthesia (delta, burst suppression, and isoelectric activity) appear to be caused, at least partly, by direct effects of thiopental on cortical neurons.