Chaos analysis of EEG during isoflurane-induced loss of righting in rats.

It has long been known that electroencephalogram (EEG) signals generate chaotic strange attractors and the shape of these attractors correlate with depth of anesthesia. We applied chaos analysis to frontal cortical and hippocampal micro-EEG signals from implanted microelectrodes (layer 4 and CA1, respectively). Rats were taken to and from loss of righting reflex (LORR) with isoflurane and behavioral measures were compared to attractor shape. Resting EEG signals at LORR differed markedly from awake signals, more similar to slow wave sleep signals, and easily discerned in raw recordings (high amplitude slow waves), and in fast Fourier transform analysis (FFT; increased delta power), in good agreement with previous studies. EEG activation stimulated by turning rats on their side, to test righting, produced signals quite similar to awake resting state EEG signals. That is, the high amplitude slow wave activity changed to low amplitude fast activity that lasted for several seconds, before returning to slow wave activity. This occurred regardless of whether the rat was able to right itself, or not. Testing paw pinch and tail clamp responses produced similar EEG activations, even from deep anesthesia when burst suppression dominated the spontaneous EEG. Chaotic attractor shape was far better at discerning between these awake-like signals, at loss of responses, than was FFT analysis. Comparisons are provided between FFT and chaos analysis of EEG during awake walking, slow wave sleep, and isoflurane-induced effects at several depths of anesthesia. Attractors readily discriminated between natural sleep and isoflurane-induced "delta" activity. Chaotic attractor shapes changed gradually through the transition from awake to LORR, indicating that this was not an on/off like transition, but rather a point along a continuum of brain states.

Determination of diffusion and partition coefficients of propofol in rat brain tissue: implications for studies of drug action in vitro.

BACKGROUND: Propofol (2,6-diisopropylphenol) is a widely used general anaesthetic that modulates gamma-aminobutyric acid type A (GABA(A)) receptors, the major inhibitory neurotransmitter receptor in the brain. Previous studies have found that the concentration of propofol that is required to affect synaptic inhibition in brain slices is much higher than the free concentration that is achieved clinically and that modulates isolated receptors. We tested whether this is accounted for by slow equilibration in brain tissue, and determined the concentration that must be applied to achieve appropriate brain levels. METHODS: Rat brain slices 300-microm thick were placed in a solution of 100 microM propofol in artificial cerebrospinal fluid for times ranging from 7.5 to 480 min. Concentrations in these slices were measured by HPLC to determine diffusion and partition coefficients. Electrophysiological measurements of the rate at which effects of 5 microM propofol developed were compared with the calculated rate of increase in tissue concentration. RESULTS: The diffusion coefficient was approximately 0.02x10(-6) cm2 s(-1), and the brain:artificial cerebrospinal fluid partition coefficient was 36. Diffusion times in brain slices agreed well with time course measurements of propofol-induced depression of synaptic responses, which continued to increase over 5 h. This depression was reversed by blocking GABA inhibition with picrotoxin (100 microM). CONCLUSIONS: Propofol does enhance inhibition in brain slices at a concentration of 0.63 microM in the superfusate, which produces brain concentrations corresponding with those achieved in vivo, but equilibration requires several hours. It is likely that slow diffusion to GABA receptors accounts for the high concentrations (>10 microM) that were needed to depress evoked responses in previous investigations.

Anesthetic properties of 4-iodopropofol: implications for mechanisms of anesthesia.

BACKGROUND: Positive modulation of gamma-aminobutyric acid type A (GABAA) receptor function is recognized as an important component of the central nervous system depressant effects of many general anesthetics, including propofol. The role for GABAA receptors as an essential site in the anesthetic actions of propofol was recently challenged by a report that the propofol analog 4-iodopropofol (4-iodo-2,6-diisopropylphenol) potentiated and directly activated GABAA receptors, yet was devoid of sedative-anesthetic effects in rats after intraperitoneal injection. Given the important implications of these findings for theories of anesthesia, the authors compared the effects of 4-iodopropofol with those of propofol using established in vivo and in vitro assays of both GABAA receptor-dependent and -independent anesthetic actions. METHODS: The effects of propofol and 4-iodopropofol were analyzed on heterologously expressed recombinant human GABAA alpha1beta2gamma2 receptors, evoked population spike amplitudes in rat hippocampal slices, and glutamate release from rat cerebrocortical synaptosomes in vitro. Anesthetic potency was determined by loss of righting reflex in Xenopus laevis tadpoles, in mice after intraperitoneal injection, and in rats after intravenous injection. RESULTS: Like propofol, 4-iodopropofol enhanced GABA-induced currents in recombinant GABAA receptors, inhibited synaptic transmission in rat hippocampal slices, and inhibited sodium channel-mediated glutamate release from synaptosomes, but with reduced potency. After intraperitoneal injection, 4-iodopropofol did not produce anesthesia in mice, but it was not detected in serum or brain. However, 4-iodopropofol did produce anesthesia in tadpoles (EC50 = 2.5 +/- 0.5 microM) and in rats after intravenous injection (ED50 = 49 +/- 6.2 mg/kg). CONCLUSIONS: Propofol and 4-iodopropofol produced similar actions on several previously identified cellular and molecular targets of general anesthetic action, and both compounds induced anesthesia in tadpoles and rats. The failure of 4-iodopropofol to induce anesthesia in rodents after intraperitoneal injection is attributed to a pharmacokinetic difference from propofol rather than to major pharmacodynamic differences.

Agent-selective effects of volatile anesthetics on GABAA receptor-mediated synaptic inhibition in hippocampal interneurons.

BACKGROUND: A relatively small number of inhibitory interneurons can control the excitability and synchronization of large numbers of pyramidal cells in hippocampus and other cortical regions. Thus, anesthetic modulation of interneurons could play an important role for the maintenance of anesthesia. The aim of this study was to compare effects produced by volatile anesthetics on inhibitory postsynaptic currents (IPSCs) of rat hippocampal interneurons. METHODS: Pharmacologically isolated gamma-aminobutyric acid type A (GABAA) receptor-mediated IPSCs were recorded with whole cell patch-clamp techniques in visually identified interneurons of rat hippocampal slices. Neurons located in the stratum radiatum-lacunosum moleculare of the CA1 region were studied. The effects of clinically relevant concentrations (1.0 rat minimum alveolar concentration) of halothane, enflurane, isoflurane, and sevoflurane were compared on kinetics of both stimulus-evoked and spontaneous GABAA receptor-mediated IPSCs in interneurons. RESULTS: Halothane (1.2 vol% approximately 0.35 mm), enflurane (2.2 vol% approximately 0.60 mm), isoflurane (1.4 vol% approximately 0.50 mm), and sevoflurane (2.7 vol% approximately 0.40 mm) preferentially depressed evoked IPSC amplitudes to 79.8 +/- 9.3% of control (n = 5), 38.2 +/- 8.6% (n = 6), 52.4 +/- 8.4% (n = 5), and 46.1 +/- 16.0% (n = 8), respectively. In addition, all anesthetics differentially prolonged the decay time constant of evoked IPSCs to 290.1 +/- 33.2% of control, 423.6 +/- 47.1, 277.0 +/- 32.2, and 529 +/- 48.5%, respectively. The frequencies of spontaneous IPSCs were increased by all anesthetics (twofold to threefold). Thus, the total negative charge transfer mediated by GABAA receptors between synaptically connected interneurons was enhanced by all anesthetics. CONCLUSIONS: Volatile anesthetics differentially enhanced GABAA receptor-mediated synaptic inhibition in rat hippocampal interneurons, suggesting that hippocampal interneuron circuits are depressed by these anesthetics in an agent-specific manner.

Membrane and synaptic actions of halothane on rat hippocampal pyramidal neurons and inhibitory interneurons.

A relatively small number of inhibitory interneurons can control the excitability and synchronization of large numbers of pyramidal neurons in hippocampus and other cortical regions. Thus, anesthetic modulation of interneurons could play an important role during anesthesia. The aim of this study was to investigate effects of a general anesthetic, halothane, on membrane and synaptic properties of rat hippocampal interneurons. GABA receptor-mediated IPSCs were recorded with whole-cell patch-clamp techniques in visually identified CA1 pyramidal cells and interneurons located at the border of stratum lacunosum-moleculare and stratum radiatum. Halothane (0.35 mm congruent with 1.2 vol%) depressed evoked IPSC amplitudes recorded from both pyramidal cells and inhibitory interneurons. Also, halothane considerably prolonged the decay time constant of evoked IPSCs in pyramidal cells and interneurons. The frequencies of miniature IPSCs were increased by halothane (two- to threefold) in both types of neuron. On the other hand, halothane effects on resting membrane potentials were variable but minimal in both types of neurons. In current-clamp recordings, halothane depressed EPSP amplitudes and increased IPSP amplitudes recorded from both types of neurons. In addition, halothane increased the failure rate of synaptically evoked action potentials. Taken together, these data provide evidence that halothane increases GABA(A) receptor-mediated synaptic inhibition between synaptically connected interneurons and depresses excitatory transmission, similar to effects observed in pyramidal neurons.

Hypoglycemic and hypoxic modulation of cortical micro-EEG activity in rat brain slices.

OBJECTIVE: Electroencephalogram (EEG) recordings exhibit stereotypic alterations during transient ischemia in mammals. One disadvantage of using in vitro models for ischemia studies is the lack of a sensitive electrophysiological measure for the degree of ischemic damage to a large population of neurons. The present study examined effects of hypoglycemia, hypoxia or both on an in vitro micro-EEG model, to determine whether this model provides a sensitive measure. METHODS: Theta frequency (4-8 Hz) micro-EEG oscillations were evoked in rat neocortical brain slices using the cholinergic agonist carbachol (100 microM) and the GABA(A) antagonist bicuculline (10 microM). Extracellular field micro-EEG signals and whole cell patch clamp recordings were used to monitor electrical activity. RESULTS: Upon removal of oxygen and/or glucose, theta oscillation amplitudes progressively declined to isoelectric levels. Low frequency delta oscillations (0.5-3.0 Hz) and burst suppression discharges were prominent during hypoglycemic episodes and upon recovery. Time to onset of isoelectric activity was faster in slices deprived of both glucose and oxygen (7.0 +/- 1.8 min) and oxygen alone (5.0 +/- 1.5 min) compared to hypoglycemia alone (25.6 +/- 3.8 min, P < 0.01, ANOVA). Hypoxia and hypoglycemia-induced isoelectric activity occurred prior to significant population spike depression from control levels (87.7 +/- 16.9% control amplitude, P > 0.35 (t test compared with control) for hypoglycemia; 93.6 +/- 27.0%, P > 0.72 for hypoxia). Spreading depression (SD) was observed in 11/12 (91.7%) slices deprived of both sugar and oxygen, but not in hypoxic (0/4) or hypoglycemic (0/5) slices. In all cases, SD occurred later than isoelectric activity. Theta oscillations recovered within 10 min in 12/13 (92.3%) slices that did not undergo SD, but slices that underwent SD failed to recover theta activity (0/4), though all (4/4) at least partially recovered the population spike (>40%). CONCLUSIONS: These results suggest that synchronized micro-EEG activity may be a useful and sensitive indicator of early-onset and possibly reversible ischemic damage.

Excitatory synaptic transmission mediated by NMDA receptors is more sensitive to isoflurane than are non-NMDA receptor-mediated responses.

BACKGROUND: Effects of volatile anesthetic agents on N-methyl-D-aspartate (NMDA) receptor-mediated excitatory synaptic transmission have not been well characterized. The authors compared effects produced by halothane and isoflurane on electrophysiologic properties of NMDA and non-NMDA receptor-mediated synaptic responses in slices from the rat hippocampus. METHODS: Field excitatory postsynaptic potentials (fEPSPs) in the CA1 area were recorded with extracellular electrodes after electrical stimulation of Schaffer-collateral-commissural fiber inputs. NMDA or non-NMDA receptor-mediated fEPSPs were pharmacologically isolated using selective antagonists. Clinically relevant concentrations of halothane or isoflurane were applied to slices in an artificial cerebrospinal fluid perfusate. Paired pulse facilitation was used as a measure of presynaptic effects of the anesthetic agents. RESULTS: Clinically relevant concentrations of halothane (1.2 vol% approximately 0.35 mM) depressed fEPSP amplitudes mediated by NMDA receptors and non-NMDA receptors to a similar degree (mean +/- SD: 63.3 +/- 14.0% of control, n = 5; 60.2 +/- 7.3% of control, n = 7, respectively). In contrast, isoflurane (1.4 vol% approximately 0.50 mM) preferentially depressed fEPSP amplitudes mediated by NMDA receptors (44.0 +/- 7.4% of control, n = 6, P < 0.001) compared with those for non-NMDA receptors (68.7 +/- 5.4% of control n = 6), indicating a selective, additional postsynaptic effect. Paired pulse facilitation of fEPSPs was increased significantly by both anesthetic agents from 1.37 +/- 0.13 to 1.91 +/- 0.25 (n = 5, P < 0.05 for halothane) and from 1.44 +/- 0.04 to 1.64 +/- 0.08 (n = 5, P < 0.01 for isoflurane), suggesting that presynaptic mechanisms are also involved in fEPSP depression produced by the anesthetic agents. Neither rise times nor decay times of fEPSPs were changed in the presence of the anesthetic agents. CONCLUSIONS: These results indicate that fEPSPs mediated by postsynaptic NMDA receptors are more sensitive to clinically relevant concentrations of isoflurane than are non-NMDA receptor-mediated responses, but this selective effect was not observed for halothane. Both agents also appeared to depress release of glutamate from nerve terminals via presynaptic actions.

Halothane depresses action potential conduction in hippocampal axons.

General anesthetics are thought to depress the central nervous system (CNS) by acting at synapses; however, only a few studies have compared effects on axonal conduction with effects on synaptic responses using mammalian CNS preparations. The present study used glutamate receptor antagonists (CNQX/APV) or low calcium to block synaptic transmission, allowing Schaffer-collateral axon fiber volleys to be recorded from rat hippocampal brain slices. Since fiber volleys are compound action potentials, they provide a measure of axonal conduction in Schaffer-collateral fibers. Clinical concentrations of the inhalational anesthetic, halothane (1 rat MAC, 1.2 vol.%), produced an 18 +/- 2.3% depression of fiber volley amplitudes (mean +/- S.D.; p < 0.001 ANOVA, n = 10). Depression of action potential conduction accounted for approximately 30% of the overall depression of synaptic transmission produced by halothane at this concentration. Halothane-induced fiber volley depression occurred with little change in conduction velocity, similar to the effect seen with decreased stimulus intensity, but significantly different from the decreased velocity produced by tetrodotoxin (100 nM, p < 0.005). The results indicate that halothane can depress axonal conduction at clinically relevant concentrations and that this depression could contribute to the CNS depression that is associated with anesthesia.

Concentration measures of volatile anesthetics in the aqueous phase using calcium sensitive electrodes.

Volatile anesthetic concentrations have been difficult to measure, but are an important experimental parameter for in vitro studies of anesthetic actions. Calcium sensitive electrodes were investigated as a means of continuously monitoring anesthetic concentrations in artificial cerebrospinal fluids (ACSF). Anesthetic-induced Ca2+ electrode signals were compared at room (22 degrees C) and physiological (35 degrees C) temperatures. Electrophysiological measures of anesthetic effects on synaptic potentials provided a bioassay. Halothane and isoflurane produced negative changes in calcium electrode potentials which were linearly related to concentrations over a clinically useful range (0.5-1.5 MAC). Anesthetic-induced voltages persisted in nominally zero Ca2+ ACSF and even in deionized water. A good correlation (r>0.9) was found for calcium electrode measures of anesthetic concentration and synaptic response depression produced by halothane, at both 22 and 35 degrees C. These results support three conclusions: (1) calcium sensitive electrodes provide a useful measure of volatile anesthetic concentrations in aqueous solution. (2) Care must be taken when using these electrodes for Ca2+ concentration measurements, if a volatile anesthetic is also to be used, since the anesthetic could introduce an appreciable error (>50%). (3) A temperature change of 13 degrees C had surprisingly little effect on Ca2+ electrode responses or on synaptic depression produced by anesthetics.

G-force induced alterations in rat EEG activity: a quantitative analysis.

A major physical limitation affecting pilots is G-force (+Gz, head-to-foot inertial load) induced loss of consciousness. Previous studies have shown that +Gz produces qualitatively similar effects on human and rat EEG activity. The present study sought to quantitatively correlate changes in rat EEG activity with increasing +Gz levels. A frontal-parietal differential electrode recorded rat EEG data during +Gz exposures (30 s) ranging from +0.5 to +25.0 Gz. Acceleration levels < or = +10 Gz had little effect on EEG activity. Acceleration levels of +15 to +20 Gz were associated with increased EEG slowing, depression and sharp waves. Acceleration levels > or = +17.5 Gz evoked burst suppression followed by isoelectric activity. Times to first onset of delta, depressed, and isoelectric EEG activity were approximately 12, 14 and 18 s, respectively. Acceleration effects on delta (1-4 Hz), theta (5-8 Hz), alpha (9-12 Hz), beta (13-30 Hz) and total (1-30 Hz) EEG powers were examined using Fourier transform analysis. EEG measures with the most predictive value at the following post-acceleration onset times (PAOT) were as follows (in s); increasing theta power: PAOT 0-2, decreasing delta power: PAOT 3-9, and decreasing beta power: PAOT > or = 12. This study provides a quantitative description of +Gz-induced alterations in EEG magnitude, time course and spectral content. Additionally, several EEG measures were identified which correlated with acceleration level at specific post-acceleration onset times.

Voltage-clamp analysis of halothane effects on GABA(A fast) and GABA(A slow) inhibitory currents.

Voltage-clamped GABA(A fast) and GABA(A slow) inhibitory postsynaptic currents (IPSCs) were selectively elicited in hippocampal area CA1 pyramidal neurons. Clinically relevant concentrations of halothane (1.2 vol.%) prolonged both GABA(A fast) and GABA(A slow) IPSC decay times approximately 2.5 fold, while having little to no effect on current amplitudes or rise times. Current-voltage analysis revealed that IPSC reversal potentials (-70 to -75 mV) remained constant in the presence of halothane. Under control conditions, GABA(A slow) IPSC decay times increased linearly with membrane depolarization, and this IPSC decay time voltage dependence was not significantly altered by halothane. These results confirm the existence of separable GABA(A fast) and GABA(A slow) IPSCs in hippocampus, and further elucidate the effects of halothane on these currents.

Physiology, pharmacology, and topography of cholinergic neocortical oscillations in vitro.

Rat neocortical brain slices generated rhythmic extracellular field [microelectroencephalogram (micro-EEG)] oscillations at theta frequencies (3-12 Hz) when exposed to pharmacological conditions that mimicked endogenous ascending cholinergic and GABAergic inputs. Use of the specific receptor agonist and antagonist carbachol and bicuculline revealed that simultaneous muscarinic receptor activation and gamma-aminobutyric acid-A (GABA(A))-mediated disinhibition were necessary to elicit neocortical oscillations. Rhythmic activity was independent of GABA(B) receptor activation, but required intact glutamatergic transmission, evidenced by blockade or disruption of oscillations by 6-cyano-7-nitroquinoxaline-2,3-dione and (+/-)-2-amino-5-phosphonovaleric acid, respectively. Multisite mapping studies showed that oscillations were localized to areas 29d and 18b (Oc2MM) and parts of areas 18a and 17. Peak oscillation amplitudes occurred in layer 2/3, and phase reversals were observed in layers 1 and 5. Current source density analysis revealed large-amplitude current sinks and sources in layers 2/3 and 5, respectively. An initial shift in peak inward current density from layer 1 to layer 2/3 indicated that two processes underlie an initial depolarization followed by oscillatory activity. Laminar transections localized oscillation-generating circuitry to superficial cortical layers and sharp-spike-generating circuitry to deep cortical layers. Whole cell recordings identified three distinct cell types based on response properties during rhythmic micro-EEG activity: oscillation-ON (theta-ON) and -OFF (theta-OFF) neurons, and transiently depolarizing glial cells. Theta-ON neurons displayed membrane potential oscillations that increased in amplitude with hyperpolarization (from -30 to -90 mV). This, taken together with a glutamate antagonist-induced depression of rhythmic micro-EEG activity, indicated that cholinergically driven neocortical oscillations require excitatory synaptic transmission. We conclude that under the appropriate pharmacological conditions, neocortical brain slices were capable of producing localized theta frequency oscillations. Experiments examining oscillation physiology, pharmacology, and topography demonstrated that neocortical brain slice oscillations share many similarities with the in vivo and in vitro theta EEG activity recorded in other brain regions.

Riluzole anesthesia: use-dependent block of presynaptic glutamate fibers.

BACKGROUND: Riluzole (RP 54274) is an experimental benzothiazole with anesthetic properties, but little is known about its synaptic or cellular actions. METHODS: The authors investigated riluzole effects on synaptic response of CA 1 pyramidal neurons in rat hippocampal brain slices. Electrophysiologic recordings of population spikes (PS), excitatory postsynaptic potentials (EPSP), and fiber volleys were studied. Paired pulse stimulation (120 ms interpulse interval) was used to measure effects on gamma-amino butyric acid (GABA)-mediated synaptic inhibition, and stimulus trains (33 Hz) were used to test for use-dependent effects. RESULTS: Synaptically evoked PS discharge was blocked in a concentration-dependent manner by riluzole (2.0-20 microM), similar to effects produced by other anesthetics. Paired pulse inhibition was not altered by riluzole. In contrast, 20 microM thiopental produced a marked increase in paired pulse inhibition. Riluzole (5.0 microM) produced a 46.6 +/- 19.8% depression of glutamate-mediated EPSPs, which could account for most of the mate-mediated EPSPs, which could account for most of the depression of PS discharge (54.2 +/- 12.6%) produced by this concentration. Riluzole produced a 36 +/- 17% depression of fiver volley amplitudes, which, based on input/output analysis, could completely account for the depression of EPSPs. The depression of fiber volley amplitudes showed a marked use-dependence; the second and subsequent action potentials in a train were progressively depressed by riluzole to a greater extent than the first action potential. CONCLUSIONS: Riluzole produced a potent block of excitatory synaptic transmission via depression of presynaptic conduction in glutamatergic nerve fibers. The use-dependent depression observed resembled that produced by some local anesthetics on nerve conduction and sodium channels. The presynaptic action, together with a lack of effect on gamma-amino butyric acid-mediated inhibition, provides a unique mechanism of action for a general anesthetic.

Thiopental uncouples hippocampal and cortical synchronized electroencephalographic activity.

BACKGROUND: Thiopental produces a concentration-dependent continuum of effects on the cortical electroencephalogram (EEG) that has been linked to behavioral measures of anesthetic depth. The complexity of the response, however, limits a clear insight into the neurophysiologic actions of thiopental. The current study investigated thiopental actions on cortical EEG and hippocampal electrical activity, to determine whether similar effects occur on both structures and to compare synchronized activity between these structures. METHODS: Thiopental was administered intravenously via an implanted catheter in freely moving rats. Arterial blood oxygen/carbon dioxide concentration, thiopental concentrations, and temperature were monitored and controlled. Neocortical EEG was recorded from implanted dural surface electrodes and hippocampal neuron electrical activity was recorded from stereotaxically placed microelectrodes. Pharmacokinetic models were used to determine effect site concentrations. RESULTS: Thiopental produced an increase in EEG frequency and amplitude at low concentrations (15-20 micrograms/ml total plasma, approximately 10 microM unbound), which produced a loss of righting reflex. This was followed by a frequency decrease and burst suppression activity at higher concentrations (50-80 micrograms/ml, approximately 60 microM), which produced a loss of tail pinch and corneal reflexes. Higher concentrations of thiopental ( > 60 micrograms/ml) uncoupled synchronized burst discharges recorded in hippocampus and cortex. Isoelectric EEG activity was associated with concentrations of 70-90 micrograms/ml (approximately 80 microM) and a deep level of anesthesia; motor reflexes were abolished, although cardiovascular reflexes remained. In all frequency bands, similar concentration-EEG effect relationships were observed for cortical and hippocampal signals, only differing in the magnitude of response. A reversed progression of effects was observed on recovery. CONCLUSIONS: The results confirm earlier findings in humans and animals and demonstrate that both the hippocampus and neocortex exhibit burst suppression and isoelectric activity during thiopental anesthesia. Thiopental-induced synchronized burst activity was depressed by progressively higher concentrations. The lost synchronization suggests a depression of synaptic coupling between cortical structures contributes to anesthesia.

Synaptic mechanisms of thiopental-induced alterations in synchronized cortical activity.

BACKGROUND: Anesthetic depth after barbiturate administration has been correlated with distinct electroencephalogram (EEG) patterns. The current study used a rat neocortical brain slice micro-EEG preparation to investigate synaptic mechanisms underlying thiopental-induced transitions in synchronized neuronal activity. METHODS: Concentration-dependent cellular actions of thiopental were investigated in brain slices using specific pharmacologic probes, whole cell patch clamps, and extracellular field recordings. Theta-Like micro-EEG oscillations were elicited in neocortical slices by mimicking subcortical cholinergic and gamma-aminobutyric acid (GABA) afferent input with carbachol (100 microM), a cholinergic agonist, and bicuculline (10 microM) a GABAA antagonist. RESULTS: In the presence of 20 microM thiopental, micro-EEG slowing from theta (7.3 +/- 0.9 Hz, mean +/- SD, n = 19) to delta frequencies (2.5 +/- 0.5 Hz, n = 11) was associated with a threefold prolongation of inhibitory currents. Burst suppression activity occurred at 50 microM thiopental, and appeared to result from direct activation of GABAA-gated chloride currents, observed with voltage clamp recordings, and mimicked with a direct acting GABAA agonist, muscimol (1 microM). Isoelectric activity occurred at 100 microM thiopental, and likely resulted from reduced glutamatergic transmission, evidenced by depressed excitatory postsynaptic potentials. Glutamatergic excitation was required for burst suppression activity, because glutamate receptor antagonists blocked thiopental-induced bursts; forcing a transition to isoelectric activity. CONCLUSIONS: Thiopental produced a continuum of EEG-like states in brain slices similar to those observed in vivo. The progression of thiopental-induced effects appear to have resulted from specific cellular actions that were recruited in a concentration-dependent manner. Progressive enhancement of synaptic inhibition followed by depression of excitatory transmission led to micro-EEG frequency slowing, burst suppression, and isoelectric activity.

Diltiazem spares corneal A delta mechano and C fiber cold receptors and preserves epithelial wound healing.

Recent interest in the corneal analgesic properties of diltiazem prompted the present study examining concentration-dependent effects of this calcium channel blocker on C fiber cold receptors and A delta mechanoreceptors. Both afferent fiber types mediate an eye blink reflex, important for protecting the corneal surface. The effects of neuroactive concentrations of diltiazem on corneal would healing were also studied. An in vitro rabbit cornea preparation was used for both electrophysiological recording and wound healing, allowing precise concentration-response analysis. Diltiazem produced a concentration-dependent depression of cold fiber discharge activity (10 to 250 microM), but did not affect mechanoreceptor afferents. In addition, the broad spectrum Ca2+ channel blockers, Ni2+ and Cd2+, did not cause a significant reduction in A delta mechano or C fiber discharge activity. Diltiazem had no effect on corneal epithelial wound healing to a concentration of 50 microM. This is important if diltiazem is to be used for therapeutic control of pain following corneal injury or surgery, because sparing of the eye blink reflexes and wound healing are desirable properties for a corneal analgesic.

Structural and functional specialization of A delta and C fiber free nerve endings innervating rabbit corneal epithelium.

An in vitro preparation of rabbit cornea was used to compare anatomical specialization and electrophysiological responses of A delta and C fiber sensory afferents which terminate as free nerve endings. Living nerve endings were visualized using epifluorescence microscopy and the vital dye 4-di2-ASP, and response properties were determined using microstimulation and recording of fiber discharge activity. Fiber type was determined based on conduction velocity measurement and preferred stimulus energy (modality) of each fiber. Four modality-specific fiber populations were identified: (1) slowly adapting C fiber cold receptors (conduction velocity of 0.25-1.6 m/sec), (2) C fiber chemosensitive units with mixed phasic and tonic activity (1.1-1.8 m/sec), (3) rapidly adapting mechanosensitive A delta fibers (1.5-2.8 m/sec), and (4) high-threshold mechano/heat (> 350 dyne or > 40 degrees C) phasic A delta afferents (3.5-4.4 m/sec). In addition to these physiological differences, anatomical specialization was also noted. A delta fiber nerve endings were distinguished from those of C fibers by thin, elongated sensory endings that ran parallel to the corneal surface; C fiber endings formed short, branching clusters that ran mostly perpendicular to the surface. The elongated structure of A delta nerve endings was associated with directional selectivity for mechanical stimuli. These results substantiate previous suggestions that free nerve endings can exhibit both structural and functional specialization.

Free nerve ending terminal morphology is fiber type specific for A delta and C fibers innervating rabbit corneal epithelium.

1. A delta and C fibers are the smallest diameter and most numerous axons in peripheral nerve bundles. They have been thought to terminate as "free" nerve endings lacking organized structure. The present study used a vital fluorescent dye to selectively visualize living free nerve endings innervating rabbit corneal epithelium, allowing structure to be correlated with electrophysiological and functional characteristics. 2. Conduction velocity measurement of visually identified nerve endings were used to discriminate between C and A delta fibers. C fiber sensory endings terminated as short (< 50 microns) vertically directed processes clustered within the epithelium. A delta fibers terminated as long (0.1-1.2 mm) horizontal processes running parallel to the epithelial surface. 3. Only A delta fiber endings were mechanoreceptive, and the unique elongated structure imparted directional selectivity. Comparison of physiological and electrical activation indicated that mechanical stimuli were transduced in < 600 microseconds. This study confirms previous suggestions of structural and functional specialization for "free" nerve endings.