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Unité de Neurosciences Intégratives et Computationnelles, Centre National de la Recherche Scientifique, Gif sur Yvette, France
Submitted 14 April 2006; accepted in final form 22 December 2006
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ABSTRACT |
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INTRODUCTION |
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), changes of the balance of activity within other neural networks have also been observed in, for instance, the hippocampus and cerebellum (Antkowiak and Heck 1997; Dickinson et al. 2003; Faulkner et al. 1998). However, most of the studies on the effects of anesthetics have been devoted to their interactions with GABAA receptors at the molecular level, and their actions on network and cellular activity remain only poorly understood. To obtain a better understanding of anesthetic action on integrative function, we explored how the anesthetic etomidate influences sensory processing and synaptic plasticity in a cerebellum-like network.
Etomidate is an imidazole, nonbarbiturate hypnotic agent that is increasingly used in procedural sedation (Rothermel 2003) and that, at anesthetic doses, potentiates the activity of GABA at GABAA receptors (Yang and Uchida 1996) with selectivity for 
2 and 3 subunit–containing receptors (Reynolds et al. 2003). Separate sedative and hypnotic effects of etomidate have been distinguished. The sedative effect of etomidate seems to act through the 2 subunit of the GABAA receptor (Reynolds et al. 2003) and is mediated principally by enhancement of tonic conductance. This has been described in thalamocortical neurons of the ventrobasalis complex that have been implicated in the generation of sleep (Belelli et al. 2005), but the effect of etomidate on different neurons is varied, depending on the subunit composition of synaptic and extrasynaptic receptors. Belelli et al. (2005) also described potentiation of miniature inhibitory postsynaptic potentials (mIPSPs) in both ventrobasalis neurons and neurons of the thalamic nucleus reticularis, although the latter neurons are not tonically hyperpolarized by etomidate.
Another of the few available reports on the effects of etomidate on neural networks shows that, in cortical networks, etomidate either depresses or enhances theta wave oscillations, again depending on the type of GABA receptor -subunit that is present (Drexler et al. 2005). It has also been shown that etomidate reduces cortical cell responsiveness to incoming sensory transmission in the dorsal column nucleus in rats (Angel and Arnott 1999), although the animals in this study also received baseline anesthesia with urethane.
These experiments have been designed to examine the effects of etomidate on sensory processing and integration with descending modulation signals and on synaptic plasticity involving GABAergic microcircuits, using as an experimental model the electrosensory lobe of the mormyrid electric fish. The electrosensory lateral line lobe (ELL) contains a cerebellum-like network in which primary afferent input is integrated with descending corollary discharge signals that provide an active filtering mechanism, enabling the network to remove predictable features from reafferent sensory input. The intrinsic circuitry of the ELL is well known (Bell et al. 2005; Grant et al. 1996; Meek et al. 1996, 1999) and closely resembles that of the dorsal cochlear nucleus, with a number of features also common to mammalian cortical structures. Much of the integrative physiology of the electrosensory system has been described in vivo and in vitro, and it has been shown that spike timing–dependent plasticity of synaptic responses to parallel fiber input serves to form and update ongoing central predictions of electrosensory input (Bell et al. 1997; Han et al. 2000b). Plasticity results from repetitive association of pre- and postsynaptic events and is most clearly expressed in GABAergic interneurons known as medium ganglionic layer neurons (MG), in which it depends on the dendritic backpropagation of characteristically broad action potentials through the apical dendritic tree in the molecular layer (Bell et al. 1997; Gómez et al. 2005; Grant et al. 1998; Han et al. 2000b). This study of the network mechanisms and cellular phenomena underlying etomidate anesthesia complements a recent exploration of the effects of etomidate anesthesia in vivo on responses to electrosensory stimuli and modulation of the structure of receptive fields (Engelmann et al. 2006).
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METHODS |
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The procedures to isolate the brain and to prepare 400-µm-thick slices have been described previously by Grant et al. (1998). Briefly, the ELL was collected in ice-cold low-sodium artificial cerebrospinal fluid (ACSF), containing (in mM) 0 NaCl, 2.5 KCl, 1.25 NaH2PO4·H2O, 24 NaHCO3, 2 CaCl2, 2 MgSO4·7 H2O, 10 glucose, and 210 sucrose. The tissue was sectioned transversally under ice-cold low-sodium ACSF with a sapphire knife (DDK, Wilmington, DE) in a Leica 2000 vibratome (Leica, Wetzlar, Germany); the cutting plane was tilted 10° from horizontal. The slices were collected in low-sodium ACSF at room temperature (23–25°C) and transferred to an interface recording chamber. Here they were superfused with a medium-sodium/high-magnesium solution containing (in mM) 58 NaCl, 2.5 KCl, 1.25 NaH2PO4·H2O, 24 NaHCO3, 2 CaCl2, 2 MgSO4·7 H2O, 10 glucose, and 105 sucrose. After a further 20 min, the superfusion solution was changed to one in which the sucrose was replaced by 116 mM NaCl for 45 min. Finally, the slices were superfused with normal ACSF composed of (in mM) 116 NaCl, 2.5 KCl, 1.25 NaH2PO4·H2O, 24 NaHCO3, 2 CaCl2, 1.2 MgSO4·7 H2O, and 10 glucose. All solutions were saturated with a 95% O2-5% CO2 gas mixture. The pH was 7.3–7.4, and osmolarity was 
280 mOsm. Perfusion was 1–2 ml/min by gravity flow.
Stimulation
The ELL molecular layer or the deep fiber layer was stimulated using paired gold-plated tungsten electrodes (Frederic Haer and Co.; Fig. 1). Constant current stimulation strength was 3–20 µA, and the stimulus cycle repetition interval was 4 s.
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Field potential recordings were made with glass microelectrodes containing 3 M NaCl, with resistances of 7–10 M
. For current source density analysis (CSD), field potential recordings were made serially at points separated by 25-µm steps through the ELL, starting at the dorsal margin of the molecular layer, through the layers to the deep fiber layer (Fig. 1, dotted line). Current sinks (produced by current flowing into the cells) and sources (produced by current flowing out of the cells) were calculated from averages of 15 field potential traces recorded at each site to assess current flow through the layers of the ELL. Because the ELL is a laminated structure and is activated homogeneously with respect to the laminar planes (stimulation of primary afferents or parallel fibers induces current flow through the layers in one plane only), the one-dimensional CSD method can be applied (Gómez et al. 2005; Mitzdorf 1985). CSD is calculated as the second spatial derivative and is experimentally approximated by 
2V(t)/x2 
(Vd – 2V0 + Vv)/
x2 where V0 is the field potential for which CSD is calculated at time (t), Vd is the field potential at time (t) for the point 25 µm more dorsal, and Vv is the field potential at time (t) for the point 25 µm more ventral to V0. x is the distance between two field potential recording sites: 25 µm in these experiments. Because we did not measure extracellular conductivity, these CSD analyses give a qualitative rather than a quantitative estimation of current flow. To highlight and compare events of low-amplitude, the CSD data were multiplied by an amplification factor of 3,000 and subsequently transformed by applying an arctangent function (f). This is a sigmoid function where f0 = 0, and limits are fx
-infinite = –
/2, fx+infinite = /2. The result of this transformation is that the resolution of low-amplitude events is enhanced. High-amplitude events, such as those generated by stimulus artifacts, are not affected, because they were already saturated and cannot increase further (Gómez et al. 2004). In CSD plots, the color code conversion of current values is made with a modified version of the standard contourf function of MATLAB (Mathworks, Natick, MA).
Intracellular recordings were made with sharp microelectrodes filled with 2 M potassium methyl sulfate (Sigma, Steinheim, Germany) containing 2% biocytin (resistance, 140–240 M; Sigma) to label the cells that were recorded. Neuron morphology was matched with electrophysiological characteristics of the cell types: efferent cells fire large narrow spikes, whereas MG cells fire both large broad spikes and small narrow spikes (Bell et al. 1997; Grant et al. 1998).
Etomidate (Janssen-Cilag, Issy les Moulineaux, France) was bath-applied at 4.1 µM, which is the concentration that has been used to produce deep surgical anesthesia of the fish for in vivo experiments (Gómez et al. 2004). The action of GABA at GABAA receptors was blocked using bicuculline (30 µM) or picrotoxin (20 µM; Sigma). D-2-amino-5-phosphonopentanoate (AP-5; Sigma) was bath-applied at 40 µM to block N-methyl-D-aspartate (NMDA) receptor activation in some experiments.
To analyze whether etomidate modulates spike timing–dependent plasticity, the protocol described by Han et al. (2000b) was used to induce depression at parallel fiber synapses with the apical dendrites of ganglionic layer neurons. Briefly, a synaptically evoked, subthreshold EPSP was paired with backpropagating action potentials evoked by an intracellular stimulus delivered after a delay of 25 ms. The stimulation frequency was 0.2 Hz and pairing lasted for 6 min.
Data acquisition and analysis
Data were stored after amplification (Axoclamp 2B amplifier, Axon Instuments, Foster City, CA) using Elphy acquisition software (G. Sadoc, CNRS, France) and analyzed off-line. CSD plots were made using Matlab software (Mathworks). Statistical comparisons were made with the paired Student's t-test, unless indicated otherwise.
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RESULTS |
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Stimulation in the deep fiber layer of the ELL evokes a single large negative field potential recorded in the layer itself that most likely corresponds to the activation of primary afferent fibers (Fig. 2, trace e, "n pre") (Grant et al. 1998), being very similar to the primary afferent volley recorded in vivo in response to the fish's electric organ discharge (Engelmann et al. 2006). Primary afferent fibers terminate in a restricted, topographically organized manner in the overlying granular layer, and here, the response to the same stimulation is a two-peaked negative field potential that probably reflects the presynaptic activity of the primary afferents (n pre) followed by the postsynaptic response of their target granular cells (Fig. 2, trace d, n post).
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80% of the total cell population in the ganglionic cell layer. LG, LF, and MG cells all extend apical dendrites in a regular array into the overlying molecular layer, as shown in Fig. 1.
The negative waves originating in the plexiform and ganglionic layers, termed "early" (ne) and "late" (nl), propagate outward through the molecular layer (Fig. 2, traces a and b). It was suggested previously that nl corresponds to the synchronous backpropagation of action potentials through the apical dendritic tree of MG cells, which have characteristically broad action potentials lasting 10 ms (Gómez et al. 2005; Grant et al. 1998). The amplitude of both nl and of the corresponding backpropagating current sink (Fig. 3) diminishes with distance, although it extends to the outer limit of the molecular layer. This corresponds to the anatomy of ganglionic layer neurons, and in particular MG neurons, whose apical dendrites extend across the whole depth of the molecular layer (Fig. 1).
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When successive stimuli are delivered to the deep fiber layer with an interval of 50 ms, backpropagation of activity in the molecular layer is markedly depressed in response to the second stimulus (Fig. 3, A and F). The example shown in Fig. 3F shows that, after the second stimulus, the amplitude of the nl field potential (measured at the level indicated by the dotted white line in Fig. 3A) was reduced to 70.1 ± 12.7% of the response to the first stimulus (P < 0.05; n = 4). The duration of the backpropagating sink (again measured at the level of the arrow in Fig. 3A) was also reduced from 5.6 ± 0.8 ms after the first stimulus to 4.1 ± 0.3 ms after the second stimulus (P < 0.05; n = 4). As a consequence of the paired-pulse depression seen in the field potentials, the sink corresponding to the propagation (nl) in the CSD is strongly attenuated (Fig. 3A), and the latter part of the current source in the ganglionic cell layer was essentially absent after the second stimulus, although the sink in the granular and lower layers remained unchanged (P = 0.89; n = 4, Fig. 3A). This confirms that this later ganglionic/plexiform layer current source is coupled to the backpropagating molecular layer sink and not to the sink in the deep fiber and granular cell layers.
Etomidate applied in the bath solution, at a concentration that produces deep anesthesia in vivo (4.1 µM), markedly reduced backpropagating activity in the molecular layer (Fig. 3B). The nl-wave amplitude was depressed by 88 ± 6% after the first stimulus (Fig. 3F; P < 0.05; n = 4), and only a very small response was obtained to the second stimulus. The corresponding late source in the plexiform and upper granular layer also disappeared. However, the early source and sink associated with primary afferent activity in the deeper layers and the synaptic response of granular cells were not affected. Paired-pulse depression persisted in the presence of etomidate, especially evident for the backpropagating sink in the molecular layer, showing that paired-pulse depression does not depend on GABAA receptor activity.
The GABAA receptor antagonists bicuculline or picrotoxin, applied in addition to etomidate, restored backpropagation, suggesting strongly that etomidate had blocked backpropagating events through potentiation of inhibition acting at GABAA receptors on the soma or apical dendrites of ganglionic layer neurons (Fig. 3C).
Interestingly, in the deep fiber layer, bicuculline increased the duration of the primary afferent-related current sink (white arrow) from 3.4 ± 0.3 to 10.2 ± 0.7 ms (P < 0.05; n = 4) and after low-amplitude activity that was still visible 30 ms after the stimulus, suggesting that postsynaptic responses to the primary afferent volley might be prolonged. This indicates that the initial excitatory response to deep layer stimulation is normally followed rapidly by strong GABAergic inhibition. Because this primary afferent-related current sink was not altered under etomidate alone (Fig. 3B), it can be inferred that GABAergic inhibition is normally maximal in the deep fiber layer under control conditions. Picrotoxin similarly increased the duration of this sink, confirming that the effect evoked by bicuculline can be attributed to its action on GABAA receptors rather then to interaction with calcium- and voltage-gated potassium channels (data not shown; n = 2). However, it is also possible that the GABAA receptor subunit composition of primary afferents or granular cells excludes any inhibitory effects of etomidate.
Paired-pulse depression persisted in the presence of bicuculline or picrotoxin in both the deep fiber layer and the molecular layer. Because, under these conditions, GABAA receptors are blocked, it must be concluded that paired-pulse depression is not the direct result of inhibition acting through GABAA receptors, confirming the results of Han et al. (2000a). Washout of bicuculline (Fig. 3D) and then etomidate (Fig. 3E) shows that the effects induced by etomidate and bicuculline are reversible, although we observed that it often took several hours to obtain good etomidate washout in this isolated preparation in contrast with the rather rapid recovery observed in vivo (Engelmann et al. 2006).
While these results are most readily observed in CSD plots that show an ensemble view of network activity, the same effects of etomidate, and etomidate with bicuculline, can be seen in the field potentials shown in Fig. 3F, recorded in the molecular layer at the level indicated by a white dotted line in Fig. 3A.
The reduced amplitude of the backpropagating sink could reflect either reduced initiation of backpropagating spikes, or failure of broad spikes to backpropagate in MG cells. To test this, a weak deep fiber layer stimulus was applied initially, sufficient to produce a backpropagating sink (Fig. 4 A) but below the level of response saturation. The backpropagating sink was reduced under etomidate (Fig. 4B) but could be restored by doubling the stimulus intensity (Fig. 4C; n = 3). This shows that backpropagation was not blocked per se but that the threshold for initiation of backpropagating events was increased under etomidate.
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Stimulation in the ELL molecular layer (Figs. 5 and 6) activates parallel fibers running through the molecular layer that synapse with the apical dendritic arborization of ganglionic layer neurons and with GABAergic stellate cells intrinsic to the molecular layer (Fig. 1). This stimulation evokes three negative field potentials in the molecular layer (Fig. 5, trace a), accompanied by a positive wave in the ganglionic and plexiform layers containing the somata and basal dendrites of the LG, MG, and LF cells (Fig. 5, traces b and c). It has been suggested by previous authors (Gómez et al. 2005; Grant et al. 1998) that these waves represent the parallel fiber volley (n1), activation of synapses on the apical dendrites of ganglionic layer neurons (n2), and synchronously generated backpropagating broad spikes fired by MG cells (n3). Paired-pulse stimulation produced marked facilitation of these field potentials, in particular n3. The amplitude of n2 increased to 132 ± 15% (P < 0.01; n = 8), and n3 became clearly visible.
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Etomidate essentially abolished backpropagation of dendritic events from the ganglionic layer through the molecular layer, and thus also the n3 field potential, although the initial genesis of a late current sink in the ganglionic cell layer was maintained and continued to show paired pulse facilitation (Fig. 6B). The earlier strong sink corresponding to synaptic activation (n2) in the molecular layer became a little longer, possibly because it was no longer masked by the source/sink/source constellation characteristic of the backpropagating action potentials (see Mitzdorf 1985).
Bicuculline restored backpropagation of dendritic events from the ganglionic layer through the molecular layer, and indeed amplified synaptically induced current sinks in both the ganglionic and molecular layer beyond control levels (Fig. 6C; n = 4). Bath-application of picrotoxin yielded similar results (n = 2; data not shown). This confirms that the action of etomidate was mediated through GABAA receptors and also underlines the mixed excitatory and inhibitory nature of synaptic responses to parallel fiber stimulation under normal conditions.
Apical dendritic trees in the molecular layer receive many GABAergic synapses (Meek et al. 1996). Thus the effect of etomidate in reducing backpropagation in response to molecular layer stimulation might be produced by both an increase in the threshold for action potential initiation and increased shunting inhibition of synaptic origin at the level of the proximal dendrites. This possibility was addressed by applying a weak stimulus, sufficiently strong to elicit backpropagation (Fig. 7 A) but without saturation of the response. Etomidate reduced the backpropagating sink (Fig. 7B), but this could be at least partially restored by doubling the stimulus strength (Fig. 7C, response to second stimulus; n = 3). This suggests that the threshold for backpropagation was increased under etomidate but shows that etomidate did not completely block the mechanism for backpropagation.
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When the parallel fiber stimulus was stronger and evoked a larger postsynaptic response in the distal dendrites, the flanking sources were also stronger. The intensity of the source separating the sinks in the inner and outer molecular layer may be sufficient to mask any backpropagating sink. An alternative explanation is that potentiation of inhibitory synaptic input to the apical dendritic tree increases shunting inhibition at the level of the proximal dendrites and thus action potentials fail to backpropagate. Shunting inhibition of this type has been described in cerebellar Purkinje cells, where inhibitory input from stellate cells reduces the amplitude of calcium spikes (Callaway et al. 1995). If this is the case, the increased amplitude of the molecular layer sink "on beam" with the parallel fiber input must be simply the consequence of prolonged distal synaptic activity, especially of the NMDA receptor component. The duration of the NMDA component of the postsynaptic response was shown by bath-application of AP-5 (40 µM), which strongly reduced the late phase of the current sink in the outer molecular layer (Fig. 8, white arrow).
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Cellular responses to parallel fiber stimulation
Resting membrane potential was similar (P = 0.94) for MG cells and for efferent neurons, in the range of 64.5 ± 3.4 (SE) mV (n = 19). All MG and efferent cells (n = 19) showed an EPSP after parallel fiber stimulation, and this was followed by a visible IPSP in 12 cells (Fig. 9).
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on average), tested in one MG cell and three LG cells. In the single LF cell recorded, input resistance decreased only very slightly (2%), consistent with the absence of tonic hyperpolarization. Thus it seems that GABA is constantly present in the slice preparation and that etomidate potentiated tonic GABAergic activity in all LG and MG cells tested.
Etomidate markedly potentiated IPSPs in all cells in which they were visible initially and also unmasked inhibitory responses in cells where no IPSP was initially apparent. This shows that etomidate also potentiates phasic GABA receptors in both interneurons and projection neurons. In five MG and seven LG cells in which an EPSP–IPSP sequence was already apparent, etomidate prolonged the IPSP 300 ms (Fig. 9C). An IPSP developed under etomidate in four MG, one LF, and two LG cells in which only an EPSP was visible in control conditions (Fig. 9, A and B). In some cases the earlier EPSP seemed to be shunted by the increase after IPSP, as shown for the LF and LG cells in Fig. 9, B and C. These results show that efferent cells and inhibitory interneurons with apical dendrites projecting through the molecular layer all receive both excitatory and inhibitory input after parallel fiber stimulation and that the resulting postsynaptic potential is a composite event, in which the inhibitory component is amplified under etomidate.
Bicuculline antagonized the potentiated inhibition induced by etomidate and effectively blocked IPSPs generated by parallel fiber stimulation (Fig. 9D). This confirms that the inhibitory input is mediated through GABAA receptors. The threshold for synaptically evoked postsynaptic action potential generation was also reduced under bicuculline, suggesting that, even in control conditions, spiking threshold is controlled by tonic inhibition. Compared with the normal condition, the pharmacological blockade of GABAA receptors removed shunting inhibition and allowed the membrane to reach the threshold for spiking faster, resulting in earlier action potential firing (a shift of 0.6 ± 0.07 ms). Action potentials were also larger under bicuculline, no longer being shunted by the following disynaptic IPSP (amplitude of the 1st spike increased from 61 ± 2 to 67 ± 3 mV), and the cell was able to fire more than one action potential in response to molecular layer stimulation (Fig. 9D; tested in 2 MG and 4 LG cells).
Intracellular records showed that reduced neuron excitability under etomidate was caused by both increased tonic hyperpolarization and potentiation of synaptic inhibition and that increased shunting inhibition at the level of the proximal dendrites very probably played a role in preventing the backpropagation of dendritic action potentials.
This was explored in four MG and three LG cells, looking at responses to both intracellular depolarization and parallel fiber synaptic input. Figure 10 A1 shows an MG cell in which parallel fiber stimulation evoked an EPSP, giving rise to a spikelet followed by a broad action potential and a following IPSP. Intracellular stimulation evoked a spikelet followed by a broad action potential. Note that spikelets have been identified as axonal spikes in a previous publication (Grant et al. 1998).
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The impact of tonic hyperpolarization and of the potentiation of synaptic IPSPs on action potential firing was also noted in LG efferent neurons (Fig. 10C). In the control situation, an intracellular depolarization after synaptic activation at a delay of 20 ms generated a burst of four action potentials (Fig. 10C1). Under etomidate, only a single action potential was generated by the same stimulus (Fig. 10C2). However, when the delay between the parallel fiber stimulus and the intracellular depolarization was increased to 55 ms, the neuron again fired a burst of four action potentials (Fig. 10C3). This shows that the tonic hyperpolarization induced by etomidate was not sufficient, alone, to completely block action potential initiation in all three cells tested and shows the significance of the increased duration of the parallel fiber IPSP under etomidate.
Plasticity
Associative, spike timing–dependent, inverse Hebbian plasticity at the parallel fiber to MG cell synapse has been documented in detail in previous studies (Bell et al. 1997; Han et al. 2000b). Briefly, expression of spike timing–dependent synaptic depression requires that a postsynaptic backpropagating action potential occur within a window of 60 ms after activation of the parallel fiber input. The plastic change in synaptic efficacy involves the NMDA component of the parallel fiber postsynaptic response and also depends on postsynaptic intracellular calcium. Functionally, this is a mechanism by which an internal expectation driven by the corollary discharge pathway is updated in the case of a predictable sensory input (Bell 2001). A previous study has shown reduced expression of plasticity of corollary discharge input to MG neurons in vivo in animals anesthetized with etomidate (Engelmann et al. 2006). Because this in vitro study has shown that etomidate reduces the initiation of backpropagating action potentials, it was particularly interesting to test for plasticity to better understand its action related to anesthetic properties.
Spike timing–dependent depression of excitatory input was induced in MG cells by pairing a subthreshold parallel fiber EPSP with a postsynaptic broad spike that was evoked by an intracellular depolarizing pulse 25 ms after EPSP onset, using the protocol published in several previous studies (Bell et al. 1997; Han et al. 2000b). As a control to show that plastic change involved principally the excitatory component of the synaptic response, the associative pairing protocol was first applied under etomidate with bicuculline to block the disynaptic inhibitory component of the response (Fig. 11 A). After pairing, EPSP amplitude was reduced (n = 2), giving the same result as in previous studies (Bell et al. 1997; Han et al. 2000b). However, because etomidate acts on GABAA receptors, it is necessary to carry out the experiment in the absence of bicuculline, using a protocol similar to that published in Bell et al. (1997). In the absence of bicuculline, the postsynaptic response to the parallel fiber stimulus is a mixed excitatory/inhibitory sequence. Figure 11B (black trace) shows a control response to parallel fiber stimulation under etomidate, before any pairing protocols. After pairing, the excitatory component of the response was reduced by 26 ± 6% (n = 5 cells from 5 fish; P < 0.05; Kolmogorov-Smirnov; gray trace). This unmasked the early onset of the disynaptic IPSP. In the absence of further pre- and postsynaptic association, the response to parallel fiber input gradually returned toward control values over 10 min (Fig. 11C), as has been described in the previous studies cited above. Thus spike timing–dependent plasticity at the parallel fiber synapse with MG neurons can be shown in the presence of etomidate, as in the control situation (Bell et al. 1997).
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DISCUSSION |
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60 ms after presynaptic activation (Bell et al. 1997; Han et al. 2000b). Our recent in vivo studies showed that etomidate anesthesia indeed affects plasticity, but this effect may have been influenced by the slower electric organ discharge rate under etomidate, which reduces the number and frequency of pairings during a given period (Engelmann et al. 2006).
These results indicate that etomidate indeed affects plasticity, acting directly within the ELL by inhibiting firing of backpropagating spikes, rather than by blocking the underlying molecular mechanisms of plastic change at parallel fiber-to-MG cell synapses. Intracellular records have shown that association of parallel fiber input with backpropagating action potentials, generated by imposed depolarization, can still produce spike timing–dependent synaptic depression under etomidate. The exact mechanism involved in spike timing–dependent plasticity has not yet been completely identified in the ELL but is known to depend on NMDA receptor activation and increased postsynaptic calcium (Han et al. 2000b). This CSD analysis showed local amplification of distal dendritic events, generated by coincidence of the backpropagating action potentials with the NMDA component of the dendritic postsynaptic response. This suggests that a strong local depolarization at this level, sufficient to unblock voltage-dependent block of NMDA receptor channels by Mg2+ (Kampa et al. 2004), may be the necessary trigger for plastic change.
The effects of etomidate were blocked by either bicuculline or picrotoxin, confirming that its action is mediated through GABAA receptor channels. Previous studies have shown that etomidate binds to GABAA receptors, increasing the probability of Cl– channel opening and prolonging their open state (Yang and Uchida 1996). At anesthetic concentrations, etomidate potentiates the inhibitory activity of GABA but does not exert any direct effect by itself. At higher concentrations, which have been reported in the range of <10 µM (Hong and Wang 2005) to 80 µM (Yang and Uchida 1996), etomidate has GABAmimetic effects and activates chloride currents directly. The concentration that was used in these experiments (4.1 µM), corresponding to the dose that produces deep surgical anesthesia in vivo (Gómez et al. 2004), is very probably below the threshold for GABAmimetic effects, considering that the chloride current induced by application of 10 µM of etomidate is very small (Hong and Wang 2005). Therefore it is our interpretation that most, if not all, of the effects observed in this study were caused by potentiation of the inhibitory action of GABA.
In these experiments, intracellular records showed that, under etomidate, almost all ELL neurons became tonically hyperpolarized and that input resistance decreased, suggesting an action on tonic GABAA receptors (Farrant and Nusser 2005), increasing chloride ion permeability and moving the membrane potential closer to the chloride equilibrium potential. By removing the membrane potential further from the action potential firing threshold, this affected the probability of firing, especially of high-threshold backpropagating dendritic spikes, in response to excitatory synaptic input.
In the ELL, excitatory input, whether of sensory or central origin, is almost always followed by short latency inhibition, and these results show that, in addition to a tonic hyperpolarizing action, etomidate also markedly increased the amplitude and duration of synaptic inhibitory potentials, indicating a parallel action on postsynaptic phasic GABAA receptors. Because the somata of ELL efferent neurons in the ganglionic and plexiform layers and the spiny apical dendrites of LG, MG, and LF neurons in the molecular layer are densely covered with GABAergic terminals (Grant et al. 1996; Meek et al. 1996), potentiation of IPSPs by etomidate would have the effect of clamping the network in a hyperpolarized state for much of the time and effectively regulating backpropagation of dendritic action potentials by shunting dendritic current. Without dendritic backpropagation, incoming parallel fiber input no longer generated an active event in the distal dendritic region, and it is likely that this would significantly change the integration of synaptic input in the apical dendritic tree.
A number of studies have dissociated the amnestic and sedative-hypnotic effects of etomidate, showing that they are mediated through different subunits of the GABAA receptor (Belelli et al. 2005; Cheng et al. 2006; Reynolds et al. 2003). The sedative effects of etomidate require the presence of 2 or 3 subunits while amnestic effects are associated with potentiation of tonic currents through the 
5 receptor subunit, but this action also requires the presence of 2 or 3 subunits for etomidate to bind to the receptor (Jurd et al. 2003; Wafford et al. 2004). Because both tonic and phasic effects of etomidate are seen in LG efferent cells and MG inhibitory interneurons, it seems likely that GABAA receptors in these cells contain both the 5 subunit and the 2 or 3 subunits, resulting in both tonic hyperpolarization and increased synaptic inhibition observed under etomidate anesthesia.
In terms of integrative function and the mechanism of sensory attenuation in anesthetic action, the effects of etomidate would be to increase response threshold and to constrain the timing of responses to excitatory input by potentiation of the following short-latency inhibition. While this would apparently increase the temporal precision of spike timing, it would also reduce the probability that either sensory input or descending activity alone could produce a network output. The overall effect would be to increase the "temporal contrast," transmitting only the stronger network responses caused by coincidence of the highly synchronized, reafferent sensory input generated by the fish's own electric organ discharge with the precisely timed descending corollary discharge-driven excitatory input.
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. H. van den Burg, Unité de Neurosciences Intégratives et Computationnelles, CNRS, 1 Avenue de la Terrasse, 91190 Gif sur Yvette, France (E-mail: vandenburg{at}inaf.cnrs-gif.fr)
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INTRODUCTION
, SD) evokes a negative wave (n pre) in the deep fiber and intermediate layers (traces d and e) that reflects activity of primary afferents, followed by a 2nd negative wave (n post) appearing in the granular layer (traces c and d) that corresponds to excitation of postsynaptic granular cells. Early (ne) and late (nl) negative waves propagate outwards through molecular layer (traces a and b). df, deep fiber layer; int, intermediate layer; d gran, deep granular cell layer; s gran, superficial granular cell layer; plex, plexiform layer; ggl, ganglionic cell layer; mol, molecular layer.
) generate a sink in deep fiber, intermediate, and deep granular layers that is associated with n pre and n post waves in overlying field potentials. A double current source develops in superficial granular, plexiform, and ganglionic layers. A backpropagating source/sink/source constellation appears several milliseconds later in the molecular layer; this sink is associated with nl. Backpropagating activity is markedly depressed when a 2nd, identical stimulus is given 50 ms after the 1st one. Dotted white line in the molecular layer indicates recording site of field potentials shown in F. B: etomidate strongly reduces backpropagating activity, in parallel to loss of associated 2nd part of double current source in the granular layer. Etomidate has no effect on the sink in deep layers. C: bicuculline antagonizes effects of etomidate. Backpropagation is restored and the sink in deep layers is prolonged (white arrow), although etomidate alone had not reduced it previously. D: washout of bicuculline. E: washout of etomidate shows that effects of etomidate are reversible. F: comparison of field potentials recorded in the molecular layer show strong reduction of nl in the presence of etomidate and that this effect is blocked by addition of bicuculline.
