1 Klinik für Anaesthesiologie, Technische Universität München, München, Germany. 2 Institut für Physiologie, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany*Corresponding author
Accepted for publication: February 22, 2002
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Abstract |
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Methods. TCNs (n=15) of the thalamic ventral posteromedial nucleus responding to mechanical stimulation of whiskers were investigated in rats anaesthetized with end-tidal concentrations of isoflurane of 0.9% (ISOlow, baseline) and
1.9% (ISOhigh). Response activity induced by controlled vibratory movement of single whiskers was recorded before, during and after iontophoretic administration of the GABAA receptor antagonist bicuculline to the vicinity of the recorded neurone.
Results. The increase in concentration of isoflurane induced a suppression of vibratory responses to 14 (4)% [mean (SEM)] of baseline activity. Blockade of GABAA receptors by bicuculline during ISOlow and ISOhigh caused increases in response activity to 259 (32)% and 116 (25)% of baseline activity, respectively. The increase in isoflurane concentration enhanced overall inhibitory inputs by 102 (38)%, whilst overall excitatory inputs were reduced by 54 (7)%.
Conclusions. These data suggest that doubling the concentration of isoflurane doubles the strength of GABAAergic inhibition and decreases the excitatory drive of TCNs by approximately 50%. The isoflurane-induced enhancement of GABAAergic inhibition led to a blockade of thalamocortical information transfer which was not accomplished by the effects of isoflurane on glutamatergic synaptic transmission alone. Thus, it appears that, with respect to transmission of information in the thalamus, the most prominent action of isoflurane is an enhancement of GABAAergic synaptic inhibition, and that effects on glutamatergic neurotransmission may contribute to a lesser extent.
Br J Anaesth 2002; 89: 294300
Keywords: anaesthetics, volatile; brain, thalamus; brain, somatosensory system
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Introduction |
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Thalamocortical relay neurones (TCNs) of the rat somatosensory thalamus were used for this study because their inputs, the neurotransmitters involved, as well as the biological relevance of information conveyed has been investigated extensively.1316 The trigeminal somatosensory pathway transmits tactile information from mechanoreceptors of the face to the cerebral cortex and consists of: the trigeminal nerve, the trigeminothalamic neurones of the brain stem, the TCNs of the ventral posteromedial nucleus (VPM) of the thalamus, and the primary somatosensory cortex.15 Discharges of TCN action potentials in response to stimulation of the receptive field (i.e. the TCN output) is the result of glutamatergic excitatory input (via N-methyl-D-aspartate [NMDA] as well as non-NMDA receptors) and GABAAergic inhibitory input (Fig. 1).1720
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Methods |
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Neuronal recording, stimulation and iontophoresis
Microelectrode-multibarrel assemblies consisting of a tungsten electrode (2 M impedance at 1 kHz) glued alongside a five-barrel micropipette (1020 µm diameter) were used to simultaneously record extracellular action potentials (spikes) of single neurones and to administer drugs to the vicinity of the recorded neurone by iontophoresis. The electrodes were inserted stereotaxically26 in dorsoventral penetrations and the recording sites in the thalamic VPM nucleus were confirmed by the neurones facial receptive fields and their characteristic response features.13 14 18 Following amplification, the neuronal activity was filtered and displayed on an oscilloscope (from which original recordings were photographed) as well as stored on a DAT recorder. Off-line analysis was performed using a window discriminator, an interface, and Spike2 software (Cambridge Electronic Design, UK).
Low-threshold mechanosensory TCNs responding with sustained spike discharges (under baseline anaesthesia) to vibration of whiskers (i.e. the receptive field) were selected.15 Controlled vibratory movement was attained by sinusoidal-shaped stimuli (30600 Hz; 200500 µm amplitude; 500 ms duration) applied to single whiskers glued to the probe of a feedback-controlled electromechanical stimulator (Somedic, Sweden). Stimulus parameters were adjusted according to the different sensitivities of the neurones, allowing for optimal and robust sustained responses during baseline conditions (i.e. occurring continuously during the entire duration of the stimulus). Peristimulus time histograms (PSTHs) were generated with a bin width of 1 ms from the neuronal responses to 20 consecutive stimulus repetitions delivered at 2.5 s intervals. From these PSTHs, neuronal activities were determined as mean discharge frequencies (spikes s1): ongoing activity was measured from the 1 s period preceding stimulus onset, and response activity (per stimulus) was determined from the discharge rate present during stimulus application (corrected for the underlying ongoing activity).
The barrels of the micropipette were filled with the competitive GABAA receptor antagonist bicuculline methochloride (510 mM in 165 mM NaCl, pH 3.0), the GABAA receptor/chloride channel blocker picrotoxin (5 mM in 165 mM NaCl, pH 3.5), GABA (0.5 M in distilled water, pH 3.5), and 0.9% NaCl. Drugs were ejected with positive currents, and negative currents were applied at all times outside periods of drug ejection (to prevent leakage of the drugs) using an iontophoresis device with a current balancing unit (NPI Electronic, Germany). GABAA receptor antagonists were ejected with incremental currents to achieve an optimal drug effect (i.e. a maximally obtainable increase in response activity without affecting ongoing activity or spike integrity). This approach is important because excessive doses of these drugs may induce spontaneous activity or high-frequency discharges, leading to spike decrement and failure by enrollment of non-synaptic mechanisms.28 In addition, doses of GABAA antagonists were tested under baseline anaesthesia against iontophoretic administration of GABA and only doses that antagonized the GABA-induced suppression of response activity were used. In this way, it was possible to obtain an estimate of the effective microiontophoretic dose (derived from the product of the intensity of the ejecting current and the duration of ejection) at which the antagonist could be used to ensure a receptor-selective effect. Currents of 40140 nA applied for 215 min were used for bicuculline and picrotoxin administration. Also, the tests with picrotoxin showed similar results as bicuculline administration; thus, potential effects of bicuculline on potassium channels28 29 were unlikely. Data were sampled during periods of reproducible, unchanged drug effects.
Experimental protocol
The experimental sequence consisted of recordings of neuronal activities before (control), during and after (recovery) iontophoresis of drugs under a low concentration of isoflurane (ISOlow) and after approximately doubling the concentration of isoflurane (ISOhigh). Following recordings under ISOhigh, neuronal activities were reassessed after return to baseline. After each change in concentration of isoflurane, an equilibration period of at least 20 min was allowed before data acquisition. Iontophoresis of drugs was followed by full recovery of response activity.
Baseline concentrations of isoflurane of 0.81.0% (ISOlow: mean concentration 0.9%) were used to take into account the individual rats susceptibility to isoflurane. Adequacy of baseline anaesthesia was based on the absence of haemodynamic reactions to noxious stimuli. The concentration of isoflurane was increased to 1.62.2% (ISOhigh: mean concentration 1.9%), aiming to almost completely suppress the response, with stimuli eliciting only a few response spikes (see Fig. 2A). This procedure allowed production of a clear effect of isoflurane while the spike under investigation could still be identified. The concentration of isoflurane was adapted according to its variant effects on neuronal activity in different rats.
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Results |
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Quantitative analysis (Fig. 2B top) shows that the control baseline response activity (F[ISOlow]) amounted to 15.5 spikes s1 and increased during bicuculline administration (F[ISOlow+bicuculline]) to 35.8 spikes s1. The inhibitory constant 1 of 0.57 [(35.815.5)/35.8=0.57] indicates that, under ISOlow, neuronal activity is attenuated via GABAAergic inhibition by 57%, resulting in a neuronal output of 43% of the excitatory input [output=(1
) x excitatory input: (10.57) x 35.8 spikes s1=15.5 spikes s1 (i.e. control response activity)]. Under ISOhigh, neuronal activity is attenuated by 99% (
2=0.99). The term
INHIBITION is calculated as 0.75[(
2
1)/
1: (0.990.57)/0.57=0.75], indicating that the increase in concentration of isoflurane enhanced GABAAergic inhibition by 75%. Using the values for the response activities during bicuculline administration under ISOlow (35.8 spikes s1) and ISOhigh (25.4 spikes s1), the term
EXCITATION is calculated as 0.29 [(25.435.8)/35.8=0.29], indicating that the increase in concentration of isoflurane caused a suppression of the neurones excitatory input by 29% (Fig. 2B bottom).
Figure 3 shows the population data for all 15 TCNs studied. The increase in concentration of isoflurane caused a suppression of the responses of TCNs, reflected by a decrease in response activity from 32.3 (5.0) spikes s1 (ISOlow) to 3.3 (0.7) spikes s1 (ISOhigh) (P<0.001). Administration of bicuculline significantly enhanced response activities under both isoflurane doses [74.6 (11.2) and 29.4 (4.7) spikes s1, respectively; P<0.001). Recovery data demonstrate the stability of the preparation over time (Fig. 3A). Pooled data analysis (Fig. 3B) revealed values for INHIBITION of 1.02 (0.38) and for
EXCITATION of 0.54 (0.07). These data indicate that the increase in concentration of isoflurane caused a suppression of response activity by 86 (4)% and this resulted from a concomitant mean enhancement of inhibitory mechanisms by 102% and a mean suppression of excitatory mechanisms by 54%.
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Discussion |
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Isoflurane-induced suppression of TCN response activity represents a thalamic block of information transfer regarding tactile events affecting the body surface, and this results in a functional deafferentation of the cortex. These data are in keeping with a functional magnetic resonance imaging study which showed that isoflurane impairs thalamocortical transmission of sensory signals in humans.30 The thalamic block of sensory signals may contribute to the state of anaesthesia, a view already put forward by Angel.21 This is further supported by a positron emission tomography study in humans suggesting that the isoflurane-induced suppression of thalamocortical output may underlie the loss of consciousness associated with the anaesthetic state.31 In the present study, the block of information transfer was reversed by antagonism of thalamic GABAA receptors, which is in agreement with a previous study.26 Considering the biological relevance of information processing, this underlines our notion that the effects of isoflurane on TCN inhibitory inputs are more important than the effects on excitatory inputs, since antagonism of GABAA receptors under ISOhigh restored thalamocortical information transfer despite prevailing suppression of excitatory inputs by 54%.
The increase in concentration of isoflurane caused roughly a halving of excitatory TCN input. This should be compared with an attenuation by 40% of the ascending afferent TCN input induced by an increase in concentration of isoflurane equivalent to the present study.22 In the previous study, TCN input was quantified using recordings from trigeminothalamic neurones, which constitute neurones immediately presynaptic to TCNs. Therefore, the slightly larger reduction of the excitatory TCN input demonstrated in the present study may involve effects additional to those on the ascending afferent input to TCNs. In our in vivo preparation the term
EXCITATION represents a composite estimate for the change in overall excitatory TCN input. First, it reflects the effects of isoflurane on the entire ascending subthalamic pathway of the somatosensory system, which consists of two glutamatergic synapses (one in the brain stem and one in the thalamus) and the associated modulatory networks involving GABAergic inhibition.15 Secondly,
EXCITATION represents an additonal excitatory input to TCNs which derives from descending corticothalamic projections. These projections exert facilitatory effects on thalamic response transmission,32 which may also be affected by direct and/or indirect actions of isoflurane on glutamatergic and/or GABAergic neurotransmission at the cortical level.
Our data are at variance with those of Stuth and colleagues,23 24 who introduced the experimental protocol for the quantification of neuronal inputs used here. Using a dog preparation with intact pathways, a similar reduction was noted of both the excitatory glutamatergic and inhibitory GABAAergic transmission to medullary expiratory neurones following a stepwise increase of halothane concentration from 0.9% to 1.8%.23 The authors concluded that the effect of the increase in halothane concentration was caused by a reduction of excitatory mechanisms and not by an enhancement of inhibitory mechanisms. In a subsequent study using a decerebrate dog preparation, the effects of 0.9% halothane were compared with a drug-free baseline.24 Here, the authors showed that halothane per se caused a reduction in excitatory input and an enhancement of inhibitory input. Differences in experimental models and neuronal networks involved and/or the anaesthetics studied may be reasons inter alia for the partly discrepant findings with our results. It is unknown to what degree interactions of isoflurane with intrinsic membrane properties (e.g. via actions on potassium channels)35 and/or other putative neuronal targets may have contributed to our findings. However, the importance of these actions, which have only been demonstrated in in vitro studies thus far, is still under debate.2 Our results are in line with a recent study showing that isoflurane produces its greatest effects on GABAergic synapses, with only small effects on glutamatergic synapses.7
Model assumptions and methodological considerations
The first assumption for an appropriate quantification of neuronal inputs is that TCN response activity is mainly driven by afferent excitatory and GABAAergic inhibitory inputs. It has been demonstrated that the response activity of TCNs in the rat VPM is mainly dependent on excitatory inputs via NMDA and non-NMDA receptors17 20 and that this afferent synaptic transmission is characterized by a high safety factor,18 33 indicating that information regarding tactile stimuli transmitted by brain stem neurones reaches TCNs largely unchanged. The output of TCNs is, however, shaped by GABAAergic inhibitory mechanisms18 19 originating almost exclusively in GABAAergic projections from the thalamic reticular nucleus, since the somatosensory thalamus of the rat is virtually devoid of inhibitory interneurones.14 15
Another assumption for the model is that the GABAAergic inhibitory mechanism operates in a multiplicative fashion. This concept of multiplication of excitatory and inhibitory inputs was first introduced by McCrimmon and colleagues34 for brainstem respiratory neurones. A similar tonically active GABAAergic mechanism governs the output of mechanosensory TCNs of the rat VPM;18 19 as Figure 2A exemplifies, bicuculline administration under ISOlow caused an amplification of the TCN response pattern which was a proportional replica of the initial response pattern. The finding that the response activity during blockade of GABAA receptors under ISOlow and ISOhigh is linearly related to control activity (P<0.02; factor 1.6 and 2.6, respectively) supports this concept.
The degree of GABAA receptor blockade will influence the accuracy of the quantification of changes in neuronal inputs. We are confident that our experimental protocol for drug administration caused a complete blockade of GABAA receptor-mediated inhibition since: (i) iontophoretically administered GABA was antagonized by the bicuculline doses used; and (ii) excessive doses of bicuculline were avoided because these may cause direct excitation of neurones in addition to the actions of bicuculline at GABAA receptors and may, for example, result in the appearance of spontaneous activity.28 35
As a result of the absence of a drug-free baseline (an inherent shortcoming of our acute rat preparation with intact pathways) we were not able to study the effects of isoflurane anaesthesia per se; instead, we studied the effects of an increase in concentration of isoflurane. This increase, however, produced a clear change in TCN function from transfer of information regarding sensory stimulation to its block. Influences of changes of haemodynamics on neuronal activity are considered to be negligible because decreases in arterial pressure exceeding those of the present study were shown not to affect TCN discharge activity.22
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