Department of Pharmacology and Therapeutics and Department of Anaesthesia, Faculty of Medicine, The University of British Columbia Vancouver, British Columbia V6T 1Z3, Canada
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ABSTRACT |
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Ries, Craig R. and Ernest Puil. Mechanism of anesthesia revealed by shunting actions of isoflurane on thalamocortical neurons. By using thalamic brain slices from juvenile rats and the whole cell recording technique, we determined the effects of aqueous applications of the anesthetic isoflurane (IFL) on tonic and burst firing activities of ventrobasal relay neurons. At concentrations equivalent to those used for in vivo anesthesia, IFL induced a hyperpolarization and increased membrane conductance in a reversible and concentration-dependent manner (ionic mechanism detailed in companion paper). The increased conductance short-circuited the effectiveness of depolarizing pulses and was the main cause for inhibition of tonic firing of action potentials. Despite the IFL-induced hyperpolarization, which theoretically should have promoted bursting, the shunt blocked the low-threshold Ca2+ spike (LTS) and associated burst firing of action potentials as well as the high-threshold Ca2+ spike (HTS). Increasing the amplitude of either the depolarizing test pulse or hyperpolarizing prepulse or increasing the duration of the hyperpolarizing prepulse partially reversed the blockade of the LTS burst. In voltage-clamp experiments on the T-type Ca2+ current, which produces the LTS, IFL decreased the spatial distribution of imposed voltages and hence impaired the activation of spatially distant T channels. Although IFL may have increased a dendritic leak conductance or decreased dendritic Ca2+ currents, the somatic shunt appeared to block initiation of the LTS and HTS as well as their electrotonic propogation to the axon hillock. In summary, IFL hyperpolarized thalamocortical neurons and shunted voltage-dependent Na+ and Ca2+ currents. Considering the importance of the thalamus in relaying different sensory modalities (i.e., somatosensation, audition, and vision) and motor information as well as the corticothalamocortical loops in mediating consciousness, the shunted firing activities of thalamocortical neurons would be instrumental for the production of anesthesia in vivo.
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INTRODUCTION |
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There is a common assumption that the
neurophysiological mechanisms underlying surgical anesthesia and sleep
are different. As if sleeping, anesthetized patients do not appreciate
auditory, olfactory, somatic, and visual sensations. The perceived
difference between anesthesia and sleep partly relates to the
well-known observation that intense stimuli can arouse sleeping but not
anesthetized animals. However, some anesthetics induce
electroencephalographic (EEG) activities (e.g., EEG spindling) that are
strikingly similar to those during certain sleep states
(Contreras and Steriade 1996). A study of anesthetic
effects on neurophysiological mechanisms involved in sleep states could
yield insight into drug mechanisms that result in unconsciousness.
Recent advances in sleep research delineated neuronal mechanisms
subserving sleep in neocortex, thalamus, basal forebrain, and brain
stem (see reviews by Steriade 1997; Steriade and
McCarley 1990
). During deep sleep, for example, neurons in the
corticothalamocortical system hyperpolarize and show spike-burst
rhythms. These slow oscillations have a thalamic origin and restrict
the transfer of incoming sensory information to neocortex. Anesthesia
in animals greatly decreases the electrical responses in the
corticothalamocortical system to the usual sensory signals or even
intense stimuli, signifying generally impaired communication between
thalamus and cortex (Angel 1991
).
There is an increasing recognition that thalamic nuclei have a critical
role in awareness and cognitive functions (Kinney et al.
1994). Even small increases in thalamocortical activity markedly enhance cortical activity, which to a considerable extent depends on the excitability of thalamocortical neurons (Steriade and McCarley 1990
). Anesthetics hyperpolarize neurons of
thalamic nuclei by increasing membrane conductance (Ries and
Puil 1993
; Sugiyama et al. 1992
) or increasing
responsiveness to inhibitory transmitter (Sykes and Thomson
1989
). Hence by reducing membrane excitability in both
thalamocortical and cortical neurons and their responsiveness to
synaptic input anesthetics would suppress corticothalamocortical
activity, producing unconsciousness (see review by
Krnjevi
and Puil 1997
).
In this first of two articles, we surveyed the effects of the ether
anesthetic isoflurane (IFL) on the excitability of thalamocortical neurons. The accompanying paper (Ries and Puil 1999)
describes the ionic mechanism of IFL action on these neurons.
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METHODS |
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Ethics approval for the experimental use of animals was obtained from The University of British Columbia. Sprague-Dawley rats of either gender (ages ranged from postnatal days P9-P22) were caged with their mother and littermates in a 12-h day-night cycle with food and water provided ad libitum.
At the beginning of each experiment, the animal was decapitated during
halothane-O2 anesthesia. In rapid sequence beginning with
surgical reflection of a skull cap, the brain was removed from the
cranial vault and submerged in cold aerated (95% O2-5% CO2) artificial cerebrospinal fluid (ACSF) maintained
<2°C by an external ice bath. After trimming the chilled brain, a
block containing thalamic tissue was glued with -cyanoacrylate onto a Teflon stage. With the block again submerged in cold aerated ACSF,
400- or 500-µmm-thick slices were sectioned with a Vibroslicer (Campden Instruments, London). Slices containing ventral-posterior thalamic nuclei (Palkovits and Brownstein 1988
) were
stored in aerated ACSF in a holding chamber at room temperature
[22 ± 2°C (means ± SD)] for
1 h.
Whole cell recordings from neurons were made with the slice submerged
in a Plexiglas chamber and maintained at either 22 or 31°C. Slices
were perfused by gravity with aerated ACSF at a flow rate of 2.5 ml/min
with inverted 60-ml plastic syringe barrels as reservoirs, Teflon
microtubing, and a Plexiglas manifold. By vascular-clamp removal, drug
solutions were washed into a chamber volume of 1.6 ml with a response
lag time of 30 s. Whole cell, patch-clamp recordings were
performed mostly in the current-clamp mode with an Axopatch-1C
amplifier (Axon Instruments, Foster City, CA). The recording pipettes
were pulled on a Narashige PP83 machine with thin-walled
borosilicate-glass tubing (1.5 mm OD, World Precision Instruments,
Sarasota, FL). Series-resistance compensation was routinely used and
adjusted before data acquisition. The membrane currents were low-pass
filtered (3 dB/octave) at a frequency of 2 kHz.
Data were collected >10 min after whole cell access to allow the internal pipette solution to equilibrate with the neuron. Current and voltage protocols were applied, and membrane time constants were measured with pCLAMP software (MS-DOS version 5.5, Axon Instruments). Data were stored on computer disk as well as on a chart recorder. In voltage-clamp studies, leak currents were subtracted with software created in our laboratory.
The ACSF had the following composition (in mM): NaCl 124, KCl 4, 1.25 KH2PO4, 2 CaCl2, 2 MgCl2, 10 dextrose, and 26 NaHCO3. The measured
osmolarity of this solution was 310 mosmol. On bubbling with 95%
O2-5% CO2, the solution had a pH of 7.32 at
22°C. Gas dispersion tubes were not used because their high
resistance reduced gas flow to <500 ml/min, which is insufficient for
proper function of the anesthetic vaporizers (Scurr and Feldman
1982). In some experiments, Mg2+ was substituted
for Ca2+.
The internal pipette solution contained (in mM) 140 KOH, 15 NaCl, 10 EGTA, 1 CaCl2, 10 Na HEPES, 2 Mg ATP, and 0.3 Na GTP. This
solution was balanced with D-gluconic acid to a pH of 7.30 at 22°C. The calculated [Ca2+] was 108 M
(Max Chelator software for Windows version 1.2, Pacific Grove). The
electrodes exhibited a series resistance of <30 M
. After recordings
of ~60 min, the average electrode offset was +1.1 ± 1.8 mV. Any
electrode offset as well as a measured junction potential of 12 mV was
subtracted from all recorded voltages (cf. Zhang and
Krnjevi
1993
). In this regard, expressions such as
"membrane potentials less than
65 mV" should be interpreted
mathematically (i.e., negative to
65 mV).
We utilized pharmacological blockers (Sigma-Aldrich Canada) and/or
voltage-clamp recordings in some experiments (see Ries and Puil
1999). Pharmacological blockade of voltage-gated
Na+, K+, and Ca2+ conductances
[TTX, 300 nM; 4-aminopyridine (4-AP), 3 mM; TEA, 10 mM;
Ba2+, 100 µM] was used to better isolate the T-type
Ca2+ current, which could be blocked by Ni2+
(0.5 mM). During voltage-clamp recordings, the external
[Ca2+] was decreased to 1 mM, and [Mg2+]
was increased to 3 mM to reduce an all-or-none dendritic
Ca2+ response during T-current activation.
IFL (Abbott Laboratories, Montreal, Canada), previously equilibrated
with ACSF at specified gas concentrations, was administered by
perfusion. Fluotec 3 halothane vaporizers (Cyprane, Keighley, UK),
previously dry aerated, were calibrated for IFL with an infrared gas
analyzer (Ohmeda Respiratory Gas Monitor 5250, Murray Hill). Because
the solutions required bubbling for 10-15 min for equilibration with
IFL gas (Miu and Puil 1989), three vaporizers were used
simultaneously to prepare separate solutions for concentration-effect
experiments. IFL was applied to only one neuron from each slice.
Experiments also were performed with IFL (1%) heated to 31°C with a specially constructed device. Three "resistive" heaters with DC power warmed the reservoir solutions, perfusion lines, and recording bath, allowing IFL equilibration at the experimental temperature. For the reservoir heater, an aluminum block was warmed with heat-sink resistors, whereas the perfusion lines were heated inside a brass tube by a nichrome wire. The recording chamber had an interface heater consisting of a gas hood flushed by warm moist O2-CO2 with or without IFL vapor. The interface gases were humidified inside a large-bore, thick-walled plastic ventilator hose by heating droplets of water with a plastic-coated nichrome wire. The droplets of water were injected by an electric syringe injector. Before slice placement in the bath, an electrical thermocouple was immersed with a micromanipulator to various depths and positions to document a constant bath temperature. Immediately after a recording session, the temperature was measured again to verify that it had not changed.
Statistical analysis was performed with GraphPad Prism software (version 2.0, San Diego, CA). Results were expressed as means ± SD. Regression lines were plotted with the method of least squares, linear associations were assessed with the Pearson correlation coefficient, and data were compared with 95% confidence intervals.
Experimental considerations of anesthetic potency
Age and temperature require consideration when determining IFL
concentrations that are equivalent to (not "relevant to")
concentrations used for in vivo anesthesia. The minimum alveolar
concentration (MAC), the concentration that prevents movement in
response to a noxious stimulus in 50% of subjects, is 30% higher in
juvenile rats than in adults (Cook et al. 1981). Hence
the adult MAC for IFL in rats of 1.46% (Mazze et al.
1985
) corresponds to a juvenile rat MAC of >1.9%. Because
solubility is twofold greater in ACSF at 22°C than at 37°C, the in
vitro equivalent IFL (gas) concentrations at 22 and 31°C are ~1 and
~1.5%, respectively.
Other considerations arise in interpreting in vitro results for in vivo
anesthesia. The potency of an inhalational anesthetic increases with
decreasing temperature. Body temperature does not impair consciousness
until <30°C (Halsey 1980). Thus the prevention of
movement with painful stimuli in goats maintained on life support at
22°C does not require anesthetic administration (Antognini 1993
). In the case of individual neurons, however, cooling from 30 to 20°C has a depolarizing effect on membrane potentials and increases input resistance (RI) (Shen and
Schwartzkroin 1988
). To offset this difficulty, experiments
often are performed with inhalational anesthetics at temperatures
>30°C (e.g., Miu and Puil 1989
). A detailed
description of the heating methods is critical for a comparative
analysis in such cases. Heating the perfusion line or a recording bath
that is distal to the site of the initial anesthetic equilibration with
ACSF can lead to a twofold greater than intended concentration. This
error is likely in investigations of inhalational anesthetics at
elevated temperatures (Eger 1986
).
The absorption by tubing and evaporation of an anesthetic or its uptake
into the slice present further difficulties. Anesthetic absorption by
tubing can decrease measured concentrations (Miu and Puil
1989). Significant evaporation of volatile anesthetics may
occur during the sampling procedure, especially in in vitro studies
with temperatures above the ambient. Similarly, the loss of
inhalational anesthetics under in vitro conditions is partly attributable to a decline in the concentration along the length of the
bath to approximately one-third at the site of the brain slice
(Pearce 1996
).
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RESULTS |
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Membrane properties of thalamic relay neurons
We investigated 83 neurons from the ventral-posterior thalamic
nucleus. On current-pulse injection, the neurons exhibited stable
membrane potentials (RMPs) and overshooting action potentials. The
average RMP was 67 ± 5 mV. The average
Ri was 220 ± 94 M
, as determined from
hyperpolarizing responses (<5 mV) to current pulses.
We did not often observe a depolarizing "sag" in the
hyperpolarizing response such as that produced by an
IH-like current (cf. McCormick and Pape
1990), presumably caused by inhibition by the internal
gluconate (Velumian et al. 1997
). Instead, a rapidly activating persistent rectifier, such as IKir
(Tennigkeit et al. 1996
), dominated the hyperpolarizing
voltage responses. Blockade of this type of rectifier with
Ba2+ application (100 µmM) (Sutor and Hablitz
1993
) in three neurons exposed a depolarizing
IH-like sag in response to hyperpolarizing current pulses.
Firing properties of thalamic relay neurons
Depolarizing current pulses elicited, at short latency, repetitive
(tonic) firing of action potentials in neurons held with DC at more
than or equal to 60 mV (Fig.
1A, bottom left).
We observed neither delayed nor accelerating spike firing such as that
produced by a slowly inactivating A-type K+ current
(Huguenard and Prince 1991
). When depolarized by current pulses from <
70 mV, the neurons showed a poorly graded slow
potential that generated burst firing of action potentials during the
pulse (Fig. 3A, left, control). In addition, the
burst response could be elicited from membrane potentials greater than
65 mV at the offset of a hyperpolarizing pulse (Fig. 1A,
bottom, and Fig. 3B, left). Burst
firing consisted of two to five action potentials (~300 Hz) on top of
the slow potential. Application of TTX (300 nM) eliminated the action
potentials, leaving a characteristic low-threshold Ca2+
spike (LTS). We confirmed a Ca2+ dependence by
demonstrating a total blockade of this slow potential on removal of
external Ca2+ (n = 6) or application of the
Ca2+ channel blocker Ni2+ (0.5 mM,
n = 4). On the other hand, application of 4-AP (3 mM, n = 3) increased the LTS amplitude. This implied that a
fast transient A current (Huguenard et al. 1991
)
controlled the LTS.
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IFL interference with tonic and burst modes of firing
The effects of IFL were assessed on voltage-dependent firing in
neurons at 22°C. To produce a tonic firing pattern, depolarizing current pulses were applied to neurons held at approximately 60 mV by
DC (Fig. 1A). Each depolarizing pulse was followed by a hyperpolarizing pulse for detection of changes in
Ri and generation of a rebound burst response.
The application of IFL (1 or 2%) induced a hyperpolarization after a
lag of 30 s (caused by "deadspace" in tubing) and suppressed
all firing (n = 5). Figure 1A shows the time
course of the hyperpolarization, elimination of tonic and burst firing,
and apparent decrease in Ri caused by an IFL application (2%). Despite the hyperpolarization, neurons did not fire
an LTS burst when depolarized with current-pulse injection. On
terminating an IFL application there was a delay for several minutes in
the neuron's ability to fire in either mode until the membrane
potential and Ri approached control values.
Because an attainment of a steady-state partial pressure at the site of
action was unlikely, the slow recovery may have reflected differences in the rates of IFL diffusion in the tissue and its elimination from
the bath. Presumably, the partitioning of IFL in the slice continued
during the initial washout. The tonic firing rate recovered slowly,
compared with the more rapid recovery of the bursting activity. The
inhibitory effect of IFL was entirely reproducible with subsequent applications.
Concentration-dependent effects of IFL on input conductance
An increased conductance was evident on DC compensation for the
IFL-induced hyperpolarization. During the compensation, the positive
holding current increased, implying that the increased conductance
produced an outward current (cf. Ries and Puil 1999). We
examined the concentration dependence of this conductance increase by
applying IFL for 5-10 min, only once to each neuron, to ensure a
maximal effect without previous IFL accumulation. We also applied IFL
in solutions at 31°C to approximate in vivo potencies (see METHODS). Like the control Ri value,
the change in Ri caused by an IFL application
varied widely for a given concentration at 22 and 31°C (i.e., 1 and
2% at 22°C and 1% at 31°C). However, IFL induced a larger change
in Ri in neurons that initially had a higher
control Ri. In plots of the change in
Ri against the control
Ri, there was a strong correlation at each IFL
concentration (Fig. 1B). The membrane time constant also
decreased (Fig. 1A) in parallel with the decrease in
Ri (r = 0.839, n = 19), implying little or no change in input capacitance. In the
remaining experiments, we compensated for the IFL-induced
hyperpolarization with either a manual or a computer-controlled voltage clamp.
IFL shunted Na+ spikes and tonic firing
While maintaining the holding potential near 60 mV with a manual
voltage clamp, IFL application (0.5, 1, and 2% at 22°C and 1% at
31°C; n = 22) eliminated tonic firing evoked by
control threshold current pulses (Fig.
2A, middle left).
The blockade was surmountable by increasing the amplitude of the
current pulse that produced one or more action potentials (Fig.
2A, middle right). For all neurons, the
IFL-induced percentage increase in current threshold was greater than
the percentage increase in current compensation for the
hyperpolarization. For example, IFL 1% at 31°C increased the current
threshold by 37 ± 12% compared with the DC compensation of
15 ± 3%.
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During IFL application, the proportionality diminished between the depolarizing pulse amplitude and the number of consecutive action potentials, despite DC- and current-pulse compensation. Fig. 2B shows current thresholds for single spikes and for frequency-matched repetitive firing at different IFL concentrations. Although a disparity between single and repetitive thresholds appeared to increase with increasing IFL concentration, the thresholds were not distinctly different at each concentration. The greater sensitivity of repetitive firing to IFL depression may have resulted from an increased voltage threshold for action potentials (Fig. 2C). It was apparent from these data that a relatively small conductance shunt markedly depressed the ability of thalamic relay neurons to respond to intrasomatic current-pulse injections.
IFL shunted LTSs and associated burst activity
We examined the effects of IFL on burst firing in 11 neurons
manually voltage clamped at less than 70 mV to remove T-current inactivation. First, IFL application at all concentrations (0.5, 1, and
2% at 22°C and 1% at 31°C) eliminated the LTS evoked by control
threshold current pulses (Fig.
3A, middle left).
Increases in the current-pulse amplitude partially overcame the
blockade, resulting in a more graded LTS (Fig. 3A,
middle right). In addition, IFL reduced the LTS duration and
number of spikes in the burst.
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We also tested the effects of IFL on the LTS burst at the offset of
hyperpolarizing pulses in neurons manually voltage clamped at
approximately 60 mV to allow T-current activation. At all concentrations, IFL application shunted the hyperpolarizing pulse and
suppressed the entire rebound response (Fig. 3, B and
C, middle left). To a small extent, an increase
in the amplitude (Fig. 3B, middle right) or
duration (Fig. 3C, middle right) of the
hyperpolarizing current pulse overcame the blockade of the rebound LTS
burst. In summary, IFL application produced marked reductions in the LTS burst evoked by depolarizing pulses and hyperpolarizing pulses, consistent with a conductance shunt mechanism.
IFL shunted T-current generation
We used voltage clamp to further assess the effects of IFL-induced
conductance shunt on the activation and inactivation of the T current
underlying the LTS. In neurons held near the resting potential, a
hyperpolarizing prepulse followed by a depolarizing command elicited a
transient inward current, or T current, with an activation threshold at
approximately 70 mV (Fig. 4,
A and B,
). The activation curve was steep,
and the current reached a maximum amplitude over a 5- to 10-mV range
(n = 17). As in another report of thalamic slices
(Crunelli et al. 1989
), the delayed all-or-none manner
of activation contrasted with reports of graded recordings from
dissociated neurons (cf. Huguenard 1996
) and presumably reflected a compromised voltage control as well as the rapid response from a large dendritic T-channel population.
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Cumulative applications of IFL (0.5, 1, and 2% at 22°C) reversibly decreased the peak amplitude of the current in a voltage-dependent manner (n = 7). Larger decreases were observed when the current was elicited near its threshold (Fig. 4A, middle) than from more positive test voltages (Fig. 4A, right). The all-or-none control response changed to a graded one with IFL, and the voltage-current curve shifted in a depolarizing direction along the voltage axis (Fig. 4B). The shift was greater with 2% than 1% IFL.
Before determining the effects of IFL on steady-state inactivation, we
observed that its voltage range was similar to that of dissociated
neurons (Huguenard 1996). The T current became available
for activation near RMP and smoothly increased in amplitude on
progressive hyperpolarization (Fig. 4C,
). The
application of IFL decreased the peak amplitude of the current,
expanded its voltage dependence, and shifted the inactivation curve in
a hyperpolarizing direction along the voltage axis (Fig. 4,
C and D). The application also delayed the
recovery from T-current inactivation (Fig. 4, C and
D). In view of the difficulty in voltage clamping a
transient dendritic conductance (Huguenard 1996
;
Müller and Lux 1993
), these curve shifts are
consistent with the suggestion that IFL decreased the T current and LTS
by causing an increase in somatic conductance.
IFL shunted high-threshold Ca2+ spikes
After blockade of Na+ channels with TTX, pulsed
depolarizations to more than 30 mV from a background depolarized
potential of approximately
60 mV produced a high-threshold
Ca2+ spike (HTS). A reversible elevation in threshold and
partial blockade of the HTS was evident in one neuron on applying the Ca2+ channel blocker Ni2+ (0.4 mM; cf.
neocortical neurons) (Kim and Connors 1993
), and we
assumed that such all-or-none spikes were Ca2+ dependent,
arising in dendrites (cf. medial geniculate thalamic neurons)
(Tennigkeit et al. 1996
). Application of IFL (1% at
22°C) shunted, in a reversible manner, the HTS (Fig.
5, n = 4). During IFL
application, the percentage increase in the amount of current required
for the HTS was twofold greater than that required to compensate for
the hyperpolarization (i.e., 76 ± 16% compared with 36 ± 18%, respectively). This difference may have related partly to the
increased voltage threshold for the HTS, which increased by 3 to 9 mV.
The IFL-induced changes reflect the effectiveness of the conductance
shunt in suppressing HTS generation.
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Effects of IFL on repolarizations
We determined the effects of IFL on the slow
afterhyperpolarization (AHP) and posttrain hyperpolarization while
manually maintaining a voltage clamp. Internal gluconate likely
inhibited the underlying Ca2+-activated K+
current (Velumian et al. 1997), as implied by the small
slow AHP amplitudes and infrequent posttrain hyperpolarizations. IFL application (1 and 2% at 22°C and 1% at 31°C; n = 14) did not affect the slow AHP amplitude but reduced the amplitude and
duration of the posttrain hyperpolarization (IFL 2% at 22°C,
n = 4).
Depolarizing afterpotentials (DAPs) were evoked with brief (5 ms)
depolarizing current-pulse injections in neurons held at various
potentials (55 to
75 mV). In contrast to the AHP amplitudes the
DAPs were large, especially in neurons held at hyperpolarized membrane
potentials. The DAP was strongly voltage dependent, progressively increased in size at holding voltages less than approximately
70 mV
and markedly decreased in amplitude when Ca2+ was omitted
from the external media (n = 1; cf. hippocampal
neurons) (Zhang et al. 1993
). Application of IFL (1 or
2%) reversibly decreased the amplitude and time course of the DAP
(n = 3).
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DISCUSSION |
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These investigations demonstrated that IFL markedly decreased
several aspects of membrane excitability in neurons of thalamic slices.
Because the in vitro membrane behavior of thalamocortical neurons
closely corresponds to in vivo observations (reviewed by
Steriade et al. 1990), our findings shed light on
reasons this anesthetic can mimic sleep states and produce
unconsciousness. We now can support the relevance of the long-held
assumption that the mechanism of anesthesia differs from sleep (see
INTRODUCTION).
Changes in membrane properties and firing modes
Although we may construe the increase in conductance,
hyperpolarization, and termination of tonic firing in thalamocortical neurons (see RESULTS) as sleeplike effects of IFL, the
elevation of Na+-spike threshold and suppression of LTS
bursts are distinct from the membrane events that occur during sleep
states (cf. Steriade et al. 1990). The IFL effects were
similar to the hyperpolarization induced by the hydrocarbon anesthetic
halothane on thalamic parafascicular neurons (Sugiyama et al.
1992
) and had several consequences. In thalamic ventrobasal
neurons, IFL hyperpolarized the membrane by several millivolts away
from the elevated threshold for Na+-spike firing, sometimes
into a voltage range for LTS bursting. We also found that the
concomitant increase in conductance was more significant because it
greatly reduced the effectiveness of membrane voltage transfer in the
generation of the LTS burst by somatic injection of currents. The
blockade was surmountable by large currents, implicating a widespread
current shunt of regions with the highest densities of
voltage-dependent, low-threshold Ca2+ and Na+
channels. The effects were likely due to a single mechanism of IFL
action, discussed in the accompanying paper (Ries and Puil 1999
).
Changes in LTS and HTS
The increase in stimulus requirement for the LTS and HTS as well
as tonic firing was always disproportionate (i.e., nonohmically related) to the IFL increase in conductance. Application of IFL during
limited spatial distribution of somatic step voltages shifted in
opposing directions, and in a concentration-dependent manner the
steady-state activation and inactivation curves for the T current that
produced the LTS. These shifts were consistent with a functional
disconnection of the dendritic activation of a T conductance from the
soma (cf. Müller and Lux 1993). The decreased electrotonic conduction may have also caused the observed IFL elimination of the dendritic HTSs. Although application of IFL may
increase the conductance over much of the dendritic tree, the somatic
shunt alone could hinder the initiation of the LTS and HTS caused by
somatic current injection as well as their electrotonic propagation to
the axon hillock. It remained unclear if actions of IFL directly on
Ca2+ channels (Herrington et al. 1991
;
McDowell et al. 1996
; Takenoshita and Steinbach
1991
) contributed to the observed susceptibilities of the LTS
and HTS.
Anesthetic concentrations and "relevance"
For relevance many investigators suggest that the responses
induced by an anesthetic under in vitro conditions must occur in a
concentration range that corresponds to the in vivo concentration range, i.e., the range in common clinical use. However, the question arises as to the extent of overlap in these concentrations. Does the
inhibition of a given physiological parameter indicate a relevant action or an important mechanism only if the graded
concentration-effect curve is identical to the in vivo quantal curve
for MAC (Franks and Lieb 1994)? In view of the current
results, it does not. Only a small perturbation may be required for the
cessation of function in a complex neuronal network. For example, a
small increase in leak conductance or a small shift in voltage
dependence of a membrane current with IFL at <1 MAC would greatly
attenuate the activities of thalamocortical neurons (cf.
RESULTS).
Significance of neurophysiological mechanism
Our in vitro findings imply that the mechanism of anesthesia is
distinct from sleep. During natural sleep, thalamocortical neurons
hyperpolarize because of cholinergic disfacilitation (Steriade and McCarley 1990). Normally this would initiate conditions for the intrinsic voltage-dependent burst firing mode that underlies slow
electrical oscillations in the corticothalamocortical system (Steriade 1997
). The slow oscillations synchronize
during sleep, thereby disrupting the continuous relay of sensory
information. In these experiments, the IFL-induced conductance
increases hyperpolarized the membrane potential yet prevented a shift
to either tonic or burst firing. Our previous in vitro investigations
demonstrated how auditory thalamic neurons lose their resonance as a
consequence of the IFL conductance increase (Tennigkeit et al.
1997
).
Under in vivo anesthesia a thalamic shunt would block the transfer of
somatic, auditory, and visual sensation as well as motor information
from the cerebellum and basal ganglia. A concentration-dependent decrease in information transfer to the cortex would contribute to the
functional spectrum of clinical anesthesia: analgesia, anxiolysis,
amnesia, and a loss of awareness as well as suppression of somatic
motor, cardiovascular, and hormonal responses to surgical injury.
Because the majority of thalamic inputs are corticothalamic projections
(Singer 1994), an anesthetic shunt also would prevent thalamocortical neurons from responding to the cortical activity that
is characteristic of wakefulness or engaging in intrathalamic oscillations. In summary, anesthetics would markedly impair the possibilities for bidirectional transmission at a "gateway" of corticothalamocortical operations that are essential for awareness.
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ACKNOWLEDGMENTS |
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We thank L. Corey for technical assistance.
The authors acknowledge the financial support of the Medical Research Council of Canada for a fellowship to C. R. Ries, for research grants to E. Puil, and the Department of Anaesthesia of The University of British Columbia for supporting C. R. Ries.
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FOOTNOTES |
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Address for reprint requests: C. R. Ries, Dept. of Pharmacology and Therapeutics, The University of British Columbia, 2176 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 June 1998; accepted in final form 14 December 1998.
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REFERENCES |
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