BACKGROUND
a longstanding controversy in the anesthesia literature concerns whether general anesthetics exert their actions primarily by depression of synaptic transmission or by blocking nerve conduction like local anesthetics.
thesleff  <cit>  first proposed that general and local anesthetics share a common mechanism to block na conductance .
alternatively, other early studies yielded evidence that volatile anesthetics preferentially depress synaptic potentials rather than axonal conduction  <cit> .
at present, most studies of volatile anesthetic molecular targets are directed toward synaptic ligand-gated ion channels, as these sites appear more sensitive to volatile anesthetics than voltage-gated channels  <cit> .
however, other studies have shown that volatile anesthetics depress axonal conduction or block voltage-gated na channels.
berg-johnsen and langmoen  <cit>  reported that  <dig> mac isoflurane  produced an 18% depression of fiber volley amplitudes recorded from small unmyelinated fibers in the hippocampal ca <dig> region.
more recently, mikulec et al.
<cit>  reported that  <dig> mac halothane depressed schaffer-collateral axon fiber volleys by 18%.
these findings and others support a presynaptic action of volatile anesthetics to depress axonal excitability.
glutamatergic synapses are of particular interest with regard to anesthetic mechanisms.
these excitatory synapses are distributed across the neural axis and are especially abundant in telencephalic structures such as hippocampus and cortex  <cit> .
volatile anesthetics have long been recognized to depress glutamatergic neurotransmission, with evidence for both pre- and postsynaptic sites of action, including actions to depress excitability  <cit> .
some specialized glutamatergic synapses have enabled investigators to specifically look at anesthetic actions in electrically isolated presynaptic structures.
wu et al.
<cit>  reported that isoflurane at clinical concentrations produced small reductions in spike amplitudes in the calyx of held.
the nonlinearities in coupling of excitability to ca2+ influx and vesicle fusion produced a proportionally larger action of isoflurane to reduce transmitter release.
ouyang et al.
<cit>  found that isoflurane inhibited na channels in isolated neurohypophyseal nerve terminals.
in addition, sandstrom  <cit>  reported that isoflurane reduced presynaptic excitability in the phylogenetically distant larval drosophila glutamatergic neuromuscular junction – an effect that is also consistent with na channel inhibition.
we sought to address the question of whether isoflurane inhibits voltage-gated na channels to depress glutamatergic neurotransmission in the hippocampus.
earlier investigations found that isoflurane depressed glutamate release from synaptosomes by inhibiting voltage-gated ca channels  <cit> , although p/q and n-type ca channels don't appear to be directly targeted  <cit> .
volatile anesthetics may directly act on presynaptic na channels to depress neurotransmission.
studies in the hippocampus will help to ascertain whether volatile anesthetics generally inhibit na+ conductance in the cns or if this action is more specifically associated with the specialized synapses where it has been best documented  <cit> .
RESULTS
tetrodotoxin depresses neurotransmission without effect on facilitation
fiber volley  and epsp amplitudes were measured with a minimum-to-baseline  algorithm and compared with a measure of the rate of change for the initial waveform slope .
figure 1a shows an averaged epsp with the two methods represented by dashed lines  and solid lines .
the two measures were compared for variability by linear fits when plotted as functions of the stimulus voltage over a  <dig> v range .
in both cases the data obtained by the mtb algorithm had a higher correlation coefficient than the slope data, especially at low voltages as in the case of fvs.
this trend is also evident in fig.
1a,  that shows a greater scatter of slope measurements at lower stimulus voltages at the start and end of the experiment.
facilitation was plotted in the same panel  and remained constant across large changes in epsp amplitudes.
isoflurane produced a reversible depression of epsp amplitudes, accompanied by an increase in facilitation as previously reported  <cit> .
figure 1b shows the time course of a typical experiment in which  <dig> μm isoflurane  depressed epsp amplitudes.
the conditioning  epsp was depressed by ~60%.
the test epsp  was depressed by a smaller amount, which increased facilitation by ~20% .
the actions of isoflurane on glutamatergic neurotransmission follow the same pattern as when extracellular ca2+ is reduced from  <dig> mm to  <dig>  mm .
low external ca2+ depressed both conditioning and test epsps while facilitation increased.
by contrast, ttx  depressed epsps to about the same level as isoflurane or low ca2+, however ttx did not change facilitation.
isoflurane depressed glutamatergic epsps over a wide range of stimulus voltages .
epsps uniformly decreased in amplitude after exposure of the slice to  <dig> μm isoflurane .
figure 2a  shows the relation between fv and epsp amplitudes.
fv-epsp relations were obtained over a  <dig> v stimulus range in  <dig>  v increments.
the results show isoflurane depressed epsps independently of the fv amplitudes .
isoflurane shifted the apparent slope of the fv-epsp relation , which would be expected for depression of epsp amplitudes with no or little action on fv amplitudes.
this trend was also observed for experiments with reduced external ca2+  in which epsps were reduced in amplitude and the fv-epsp slope was shifted in a similar manner .
however, the fv-epsp relation obtained with ttx  was entirely dependent on fv amplitudes .
while epsps were depressed by ttx, the fv-epsp slope relation in the presence of ttx was unchanged  since all of the epsp depression produced by ttx was due to depression of fv inputs.
isoflurane significantly increased paired-pulse facilitation  as did low ca2+  when compared with ttx .
by comparison, facilitation was largely unaffected by  <dig> μm cnqx or reduced stimulus voltage.
isoflurane  depressed the first epsp by about the same amount as when external ca2+ was reduced to  <dig>  mm .
however, in the same experiments , isoflurane's action to enhance facilitation was significantly less than low ca2+ .
because facilitation is increased when synaptic release probability is reduced  <cit> , the actions of isoflurane appear to involve additional sites besides those related to excitation-release coupling or ca2+ ion influx.
partial reversal of isoflurane depression of epsps by high external calcium
since low external ca2+ produced a depressant effect like isoflurane on glutamatergic neurotransmission, we were interested in whether raising external ca2+ would reverse the actions of isoflurane.
we recorded epsps during perfusion of isoflurane in acsf with high ca2+ .
figure  <dig> shows that isoflurane depression of the first epsp was reversed in high ca2+ acsf.
the reversal was significant .
however, isoflurane delivered in high ca2+ acsf still exerted a depressant action on the second epsp and facilitation was significantly decreased from isoflurane in standard acsf .
minimal fiber volley and somatic na spike depression by isoflurane
fiber volleys represent a summation of many action potentials that arrive at the ca <dig> region following schaffer collateral stimulation.
due to the difficulty of patching nerve terminal fibers to study action potentials directly, we decided to study isoflurane actions on somatic na currents recorded from whole cell patches on cells in the ca <dig> pyramidal cell layer.
the perforated patch method allowed us to reliably obtain access to the cell interior while patching blind  <cit> .
currents were activated by a voltage step to - <dig> mv from a holding potential of - <dig> mv.
a  <dig> ms depolarizing pulse was delivered every  <dig> s. representative currents in fig.
<dig>  left show a decrease in amplitude and a slight delay after a  <dig> minute exposure of the slice to  <dig> nm ttx.
in a separate experiment, currents recorded  <dig> minutes after exposure to  <dig> μm isoflurane were indistinguishable from controls .
these results are in general agreement with our field recording experiments, although somatic na channels may have different properties than those in terminal fibers.
because fiber volleys are partially occluded by epsps , the dose-dependent action of isoflurane on fiber volleys was investigated following block of glutamate receptors with 2-amino-5-phosphonovaleric acid  and 6-cyano-7-nitroquinoxaline- <dig> -dione .
this treatment eliminated epsps leaving only fvs .
observable block of fvs only became apparent with high  or supraclinical  isoflurane .
at these concentrations isoflurane produced a reduction of fv amplitudes in a manner comparable to, but not as great as  <dig> nm ttx .
fv amplitudes were depressed in a dose-dependent manner  with an ec <dig> of ~ <dig>  mm  estimated from a least-squares fit extrapolation.
but at clinical levels,  <dig> μm  isoflurane only depressed fv amplitudes by  <dig>  ±  <dig> % .
this is in contrast to the volatile anesthetic halothane, which depressed fv amplitudes by ~18% at  <dig> mac  <cit> , using identical recording conditions.
discussion
volatile anesthetics are known to depress glutamatergic synaptic transmission, although the molecular mechanisms are unclear.
wu et al.
<cit>  reported that isoflurane inhibited presynaptic action potentials in the calyx of held to reduce glutamatergic neurotransmission.
we were interested in further study of the presynaptic action of isoflurane, as wu et al.
<cit>  did not compare isoflurane and ttx effects, nor did they look at effects on facilitation.
to test the generality of isoflurane depression of action potentials, we studied the effects of isoflurane in comparison with ttx on epsps in the ca <dig> region of the rat hippocampus and on fvs recorded from presynaptic schaffer-collateral fibers.
ttx blocks all brain na channels, including those in the calyx of held  <cit> .
we found that clinical concentrations of isoflurane had little to no inhibitory action on fv amplitudes, although epsps were strongly depressed and facilitation was increased.
somatic na currents were also unaffected by  <dig> μm isoflurane.
on the other hand, ttx reduced fv amplitudes and epsp amplitudes but had no effect on facilitation.
ttx also had no effect on the slope of the fv-epsp relation.
the actions of ttx likely resulted in part from nerve fiber conduction block.
fewer conducting fibers would reduce responsiveness to the stimulating electrode and account for the similar actions of ttx and reduced stimulus voltage on paired-pulse facilitation and the fv-epsp relation .
ttx could also reduce na spike amplitudes and decrease fiber conduction velocity  <cit>  .
unlike ttx, isoflurane at clinical concentrations doesn't appear to inhibit na channel activity in hippocampal schaffer-collateral fibers.
depression of epsps and enhancement of facilitation by isoflurane likely results from actions downstream from na channels at sites that contribute more directly to excitation-release coupling, as supported by the insensitivity of the fv-epsp slope to ttx .
fiber volleys in schaffer collateral fibers represent a summation of closely-timed multiple action potentials.
their close temporal coordination is reflected by the short duration of the fiber volley, which is typically about 5– <dig> ms. fv-epsp relations typically show an asymptotic decrease at higher fv amplitudes , which contrasts to the accelerating relation of the action potential – epsc relation in the calyx of held  <cit> .
however, wu et al  <cit>  obtained their epsc data by use of a scaled action potential as a voltage command while we obtained epsp data by changing the stimulus voltage.
our methodology reflects an experimental focus on the properties of a population of synapses, with results that show a broad insensitivity of terminal fiber na channels to isoflurane.
further study is needed at individual ca <dig> synapses to determine whether a subpopulation may be more sensitive to conduction block by isoflurane.
elevated external ca2+ reversed isoflurane depression of the first epsp.
this is likely related to the enhanced release of transmitter by increased ca2+ influx during nerve terminal depolarization.
elevated external ca2+ may also enhance nmda receptor currents.
however, this contribution to the epsp is likely to be small, as the proportion of the nmda receptor current carried by ca2+ is fractional, e.g.
17% of the total nr1/nr2a current  <cit> .
voltage-gated p/q type ca channels are localized at synaptic terminals in many central neurons  <cit> , including the mature calyx of held where they closely associate with synaptic vesicles  <cit>  and appear to have an important role in paired-pulse facilitation  <cit> .
high external  will increase ca2+ influx when ca channels open in response to depolarization.
the result would likely increase the synaptic release probability  <cit>  and tend to reduce facilitation, as we observed .
while p/q ca channels are not strongly inhibited by volatile anesthetics  <cit>  there remains the possibility that volatile anesthetics may reduce calcium-induced calcium release from internal stores, as described in dendrites of hippocampal pyramidal cells  <cit>  and suggested as an anesthetic mechanism for pentobarbitone  <cit> .
another possible site includes the n-ethylmaleimide-sensitive factor attachment protein receptor  complex.
the snare complex is involved in vesicle docking and fusion at most synapses.
disruption of snare proteins with botulinum toxins a and f can each reduce the initial release probability.
however, botox f doesn't produce facilitation unless external  is lowered  <cit> .
our results showing partial reversal of isoflurane effects on neurotransmission with high external  suggest the snare complex may warrant further study as targets of volatile anesthetics.
in fact, isoflurane and halothane have recently been reported to bind to syntaxin and the snare complex at clinical concentrations  <cit> , although the binding sites on the snare proteins have not yet been characterized.
it is important to note that not all volatile anesthetics may target the same molecular sites.
our results with isoflurane differ from the reported actions of halothane on synaptic transmission.
like isoflurane, halothane depresses epsp amplitudes in rat hippocampal slices at clinical concentrations  <cit> .
however, halothane also depresses schaffer-collateral axon fiber volley amplitudes  <cit> .
unlike ttx, halothane has no effect on conduction velocity but does increase facilitation of epsps  <cit> .
while the dissimilar pharmacological profiles for halothane and isoflurane are inconsistent with a unitary model of anesthesia, both can be understood within a multi-site model of volatile anesthetic action  <cit> .
the weak depressant action of isoflurane on schaffer-collateral fvs indicates that presynaptic actions of isoflurane are not homogeneous in the brain – especially when contrasted to the results reported by ouyang et al.
<cit>  and wu et al.
<cit> .
furthermore, baudoux et al  <cit> , who studied ca2+ transients in ca <dig> pyramidal cell axons, concluded that general anesthetics are unlikely to interfere with action potential propagation even in fine axon collaterals.
factors that could affect regional variability in action potential depression include the degree of terminal fiber myelination  as well as the diversity of voltage-gated na channel expression and subcellular distribution  <cit> .
five ttx-sensitive na channel α-subunits have been reported to be expressed in hippocampal neurons: nav <dig> , nav <dig> , nav <dig> , nav <dig>  and nav <dig>   <cit> , with complex patterns of expression even between different neurons.
on the other hand, leão et al.
<cit>  reported that na channels are absent from the calyx of held.
their immunocytochemical data show nav <dig>  but not nav <dig>  was expressed at the axonal heminodes from which action potentials passively invade the calyx  <cit> .
these data taken together suggest that the expression pattern of na channel α-subunits in neurons can influence anesthetic sensitivity.
in addition, α-subunits are frequently associated with a β <dig> subunit that can modulate na channel gating  <cit> .
so it appears that there are numerous possible isoforms of na channels that are expressed heterogeneously in the cns.
the degree to which these isoforms differ with respect to volatile anesthetic sensitivity is presently unknown.
CONCLUSIONS
we conclude that for glutamatergic hippocampal neurons, isoflurane depresses transmission downstream from na channels.
unlike ttx, isoflurane simultaneously increased facilitation and depressed epsps with only a minimal reduction of fiber volley amplitudes.
consistent with previous studies, a likely mechanism of isoflurane action is to reduce presynaptic ca2+ levels following nerve terminal depolarization and/or to disrupt the vesicle release process.

 a longstanding controversy in the anesthesia literature concerns whether general anesthetics exert their actions primarily by depression of synaptic transmission or by blocking nerve conduction like local anesthetics. thesleff first proposed that general and local anesthetics share a common mechanism to block na conductance. alternatively, other early studies yielded evidence that volatile anesthetics preferentially depress synaptic potentials rather than axonal conduction. at present, most studies of volatile anesthetic molecular targets are directed toward synaptic ligand-gated ion channels, as these sites appear more sensitive to volatile anesthetics than voltage-gated channels. however, other studies have shown that volatile anesthetics depress axonal conduction or block voltage-gated na channels. berg-johnsen and langmoen reported that mac isoflurane produced an 18% depression of fiber volley amplitudes recorded from small unmyelinated fibers in the hippocampal ca region. more recently, mikulec et al. reported that mac halothane depressed schaffer-collateral axon fiber volleys by 18%. these findings and others support a presynaptic action of volatile anesthetics to depress axonal excitability. glutamatergic synapses are of particular interest with regard to anesthetic mechanisms. these excitatory synapses are distributed across the neural axis and are especially abundant in telencephalic structures such as hippocampus and cortex. volatile anesthetics have long been recognized to depress glutamatergic neurotransmission, with evidence for both pre- and postsynaptic sites of action, including actions to depress excitability. some specialized glutamatergic synapses have enabled investigators to specifically look at anesthetic actions in electrically isolated presynaptic structures. wu et al. reported that isoflurane at clinical concentrations produced small reductions in spike amplitudes in the calyx of held. the nonlinearities in coupling of excitability to ca2+ influx and vesicle fusion produced a proportionally larger action of isoflurane to reduce transmitter release. ouyang et al. found that isoflurane inhibited na channels in isolated neurohypophyseal nerve terminals. in addition, sandstrom reported that isoflurane reduced presynaptic excitability in the phylogenetically distant larval drosophila glutamatergic neuromuscular junction – an effect that is also consistent with na channel inhibition. we sought to address the question of whether isoflurane inhibits voltage-gated na channels to depress glutamatergic neurotransmission in the hippocampus. earlier investigations found that isoflurane depressed glutamate release from synaptosomes by inhibiting voltage-gated ca channels, although p/q and n-type ca channels don't appear to be directly targeted. volatile anesthetics may directly act on presynaptic na channels to depress neurotransmission. studies in the hippocampus will help to ascertain whether volatile anesthetics generally inhibit na+ conductance in the cns or if this action is more specifically associated with the specialized synapses where it has been best documented.  tetrodotoxin depresses neurotransmission without effect on facilitation fiber volley and epsp amplitudes were measured with a minimum-to-baseline algorithm and compared with a measure of the rate of change for the initial waveform slope. figure 1a shows an averaged epsp with the two methods represented by dashed lines and solid lines. the two measures were compared for variability by linear fits when plotted as functions of the stimulus voltage over a v range. in both cases the data obtained by the mtb algorithm had a higher correlation coefficient than the slope data, especially at low voltages as in the case of fvs. this trend is also evident in fig. 1a, that shows a greater scatter of slope measurements at lower stimulus voltages at the start and end of the experiment. facilitation was plotted in the same panel and remained constant across large changes in epsp amplitudes. isoflurane produced a reversible depression of epsp amplitudes, accompanied by an increase in facilitation as previously reported. figure 1b shows the time course of a typical experiment in which μm isoflurane depressed epsp amplitudes. the conditioning epsp was depressed by ~60%. the test epsp was depressed by a smaller amount, which increased facilitation by ~20%. the actions of isoflurane on glutamatergic neurotransmission follow the same pattern as when extracellular ca2+ is reduced from mm to mm. low external ca2+ depressed both conditioning and test epsps while facilitation increased. by contrast, ttx depressed epsps to about the same level as isoflurane or low ca2+, however ttx did not change facilitation. isoflurane depressed glutamatergic epsps over a wide range of stimulus voltages. epsps uniformly decreased in amplitude after exposure of the slice to μm isoflurane. figure 2a shows the relation between fv and epsp amplitudes. fv-epsp relations were obtained over a v stimulus range in v increments. the results show isoflurane depressed epsps independently of the fv amplitudes. isoflurane shifted the apparent slope of the fv-epsp relation, which would be expected for depression of epsp amplitudes with no or little action on fv amplitudes. this trend was also observed for experiments with reduced external ca2+ in which epsps were reduced in amplitude and the fv-epsp slope was shifted in a similar manner. however, the fv-epsp relation obtained with ttx was entirely dependent on fv amplitudes. while epsps were depressed by ttx, the fv-epsp slope relation in the presence of ttx was unchanged since all of the epsp depression produced by ttx was due to depression of fv inputs. isoflurane significantly increased paired-pulse facilitation as did low ca2+ when compared with ttx. by comparison, facilitation was largely unaffected by μm cnqx or reduced stimulus voltage. isoflurane depressed the first epsp by about the same amount as when external ca2+ was reduced to mm. however, in the same experiments, isoflurane's action to enhance facilitation was significantly less than low ca2+. because facilitation is increased when synaptic release probability is reduced, the actions of isoflurane appear to involve additional sites besides those related to excitation-release coupling or ca2+ ion influx. partial reversal of isoflurane depression of epsps by high external calcium since low external ca2+ produced a depressant effect like isoflurane on glutamatergic neurotransmission, we were interested in whether raising external ca2+ would reverse the actions of isoflurane. we recorded epsps during perfusion of isoflurane in acsf with high ca2+. figure shows that isoflurane depression of the first epsp was reversed in high ca2+ acsf. the reversal was significant. however, isoflurane delivered in high ca2+ acsf still exerted a depressant action on the second epsp and facilitation was significantly decreased from isoflurane in standard acsf. minimal fiber volley and somatic na spike depression by isoflurane fiber volleys represent a summation of many action potentials that arrive at the ca region following schaffer collateral stimulation. due to the difficulty of patching nerve terminal fibers to study action potentials directly, we decided to study isoflurane actions on somatic na currents recorded from whole cell patches on cells in the ca pyramidal cell layer. the perforated patch method allowed us to reliably obtain access to the cell interior while patching blind. currents were activated by a voltage step to - mv from a holding potential of - mv. a ms depolarizing pulse was delivered every s. representative currents in fig. left show a decrease in amplitude and a slight delay after a minute exposure of the slice to nm ttx. in a separate experiment, currents recorded minutes after exposure to μm isoflurane were indistinguishable from controls. these results are in general agreement with our field recording experiments, although somatic na channels may have different properties than those in terminal fibers. because fiber volleys are partially occluded by epsps, the dose-dependent action of isoflurane on fiber volleys was investigated following block of glutamate receptors with 2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline- -dione. this treatment eliminated epsps leaving only fvs. observable block of fvs only became apparent with high or supraclinical isoflurane. at these concentrations isoflurane produced a reduction of fv amplitudes in a manner comparable to, but not as great as nm ttx. fv amplitudes were depressed in a dose-dependent manner with an ec of ~ mm estimated from a least-squares fit extrapolation. but at clinical levels, μm isoflurane only depressed fv amplitudes by ± %. this is in contrast to the volatile anesthetic halothane, which depressed fv amplitudes by ~18% at mac, using identical recording conditions. discussion volatile anesthetics are known to depress glutamatergic synaptic transmission, although the molecular mechanisms are unclear. wu et al. reported that isoflurane inhibited presynaptic action potentials in the calyx of held to reduce glutamatergic neurotransmission. we were interested in further study of the presynaptic action of isoflurane, as wu et al. did not compare isoflurane and ttx effects, nor did they look at effects on facilitation. to test the generality of isoflurane depression of action potentials, we studied the effects of isoflurane in comparison with ttx on epsps in the ca region of the rat hippocampus and on fvs recorded from presynaptic schaffer-collateral fibers. ttx blocks all brain na channels, including those in the calyx of held. we found that clinical concentrations of isoflurane had little to no inhibitory action on fv amplitudes, although epsps were strongly depressed and facilitation was increased. somatic na currents were also unaffected by μm isoflurane. on the other hand, ttx reduced fv amplitudes and epsp amplitudes but had no effect on facilitation. ttx also had no effect on the slope of the fv-epsp relation. the actions of ttx likely resulted in part from nerve fiber conduction block. fewer conducting fibers would reduce responsiveness to the stimulating electrode and account for the similar actions of ttx and reduced stimulus voltage on paired-pulse facilitation and the fv-epsp relation. ttx could also reduce na spike amplitudes and decrease fiber conduction velocity. unlike ttx, isoflurane at clinical concentrations doesn't appear to inhibit na channel activity in hippocampal schaffer-collateral fibers. depression of epsps and enhancement of facilitation by isoflurane likely results from actions downstream from na channels at sites that contribute more directly to excitation-release coupling, as supported by the insensitivity of the fv-epsp slope to ttx. fiber volleys in schaffer collateral fibers represent a summation of closely-timed multiple action potentials. their close temporal coordination is reflected by the short duration of the fiber volley, which is typically about 5– ms. fv-epsp relations typically show an asymptotic decrease at higher fv amplitudes, which contrasts to the accelerating relation of the action potential – epsc relation in the calyx of held. however, wu et al obtained their epsc data by use of a scaled action potential as a voltage command while we obtained epsp data by changing the stimulus voltage. our methodology reflects an experimental focus on the properties of a population of synapses, with results that show a broad insensitivity of terminal fiber na channels to isoflurane. further study is needed at individual ca synapses to determine whether a subpopulation may be more sensitive to conduction block by isoflurane. elevated external ca2+ reversed isoflurane depression of the first epsp. this is likely related to the enhanced release of transmitter by increased ca2+ influx during nerve terminal depolarization. elevated external ca2+ may also enhance nmda receptor currents. however, this contribution to the epsp is likely to be small, as the proportion of the nmda receptor current carried by ca2+ is fractional, e.g. 17% of the total nr1/nr2a current. voltage-gated p/q type ca channels are localized at synaptic terminals in many central neurons, including the mature calyx of held where they closely associate with synaptic vesicles and appear to have an important role in paired-pulse facilitation. high external will increase ca2+ influx when ca channels open in response to depolarization. the result would likely increase the synaptic release probability and tend to reduce facilitation, as we observed. while p/q ca channels are not strongly inhibited by volatile anesthetics there remains the possibility that volatile anesthetics may reduce calcium-induced calcium release from internal stores, as described in dendrites of hippocampal pyramidal cells and suggested as an anesthetic mechanism for pentobarbitone. another possible site includes the n-ethylmaleimide-sensitive factor attachment protein receptor complex. the snare complex is involved in vesicle docking and fusion at most synapses. disruption of snare proteins with botulinum toxins a and f can each reduce the initial release probability. however, botox f doesn't produce facilitation unless external is lowered. our results showing partial reversal of isoflurane effects on neurotransmission with high external suggest the snare complex may warrant further study as targets of volatile anesthetics. in fact, isoflurane and halothane have recently been reported to bind to syntaxin and the snare complex at clinical concentrations, although the binding sites on the snare proteins have not yet been characterized. it is important to note that not all volatile anesthetics may target the same molecular sites. our results with isoflurane differ from the reported actions of halothane on synaptic transmission. like isoflurane, halothane depresses epsp amplitudes in rat hippocampal slices at clinical concentrations. however, halothane also depresses schaffer-collateral axon fiber volley amplitudes. unlike ttx, halothane has no effect on conduction velocity but does increase facilitation of epsps. while the dissimilar pharmacological profiles for halothane and isoflurane are inconsistent with a unitary model of anesthesia, both can be understood within a multi-site model of volatile anesthetic action. the weak depressant action of isoflurane on schaffer-collateral fvs indicates that presynaptic actions of isoflurane are not homogeneous in the brain – especially when contrasted to the results reported by ouyang et al. and wu et al.. furthermore, baudoux et al, who studied ca2+ transients in ca pyramidal cell axons, concluded that general anesthetics are unlikely to interfere with action potential propagation even in fine axon collaterals. factors that could affect regional variability in action potential depression include the degree of terminal fiber myelination as well as the diversity of voltage-gated na channel expression and subcellular distribution. five ttx-sensitive na channel α-subunits have been reported to be expressed in hippocampal neurons: nav, nav, nav, nav and nav, with complex patterns of expression even between different neurons. on the other hand, leão et al. reported that na channels are absent from the calyx of held. their immunocytochemical data show nav but not nav was expressed at the axonal heminodes from which action potentials passively invade the calyx. these data taken together suggest that the expression pattern of na channel α-subunits in neurons can influence anesthetic sensitivity. in addition, α-subunits are frequently associated with a β subunit that can modulate na channel gating. so it appears that there are numerous possible isoforms of na channels that are expressed heterogeneously in the cns. the degree to which these isoforms differ with respect to volatile anesthetic sensitivity is presently unknown.  we conclude that for glutamatergic hippocampal neurons, isoflurane depresses transmission downstream from na channels. unlike ttx, isoflurane simultaneously increased facilitation and depressed epsps with only a minimal reduction of fiber volley amplitudes. consistent with previous studies, a likely mechanism of isoflurane action is to reduce presynaptic ca2+ levels following nerve terminal depolarization and/or to disrupt the vesicle release process. 
to further investigate this question we examined the actions of isoflurane, ttx, extracellular ca2+, cnqx and stimulus voltage on glutamate-mediated transmission at hippocampal excitatory synapses. consistent with previous studies, these results indicate a presynaptic site of action that involves reduced excitation-release coupling. recent studies suggest that isoflurane reduces glutamate release by block of na channels. isoflurane appears to depress ca synapses at presynaptic sites downstream from na channels, as evident by the increased facilitation that accompanies epsp depression. fiber volleys did not exhibit depression by isoflurane, as has been reported for other brain regions. isoflurane reversibly depressed epsp amplitudes by ~60% while facilitation increased ~20%. similarly, partial antagonism of postsynaptic glutamate receptors with cnqx depressed epsp amplitudes with no change in facilitation. isoflurane depression of epsp amplitudes could also be partly reversed by high external ca2+ that also decreased facilitation. however, epsp depression by low external ca2+ was accompanied by an increase in facilitation comparable to isoflurane. by contrast, ttx didn't alter the fv-epsp curve slope, indicating that epsp depression resulted from fv depression. anesthetic-induced cns depression is thought to involve reduction of glutamate release from nerve terminals. the ec for isoflurane depression of fvs was ~ mm.
 anesthetic-induced cns depression is thought to involve reduction of glutamate release from nerve terminals. recent studies suggest that isoflurane reduces glutamate release by block of na channels. to further investigate this question we examined the actions of isoflurane, ttx, extracellular ca2+, cnqx and stimulus voltage on glutamate-mediated transmission at hippocampal excitatory synapses. epsps were recorded from ca neurons in rat hippocampal brain slices in response to schaffer-collateral fiber stimulation.  isoflurane reversibly depressed epsp amplitudes by ~60% while facilitation increased ~20%. consistent with previous studies, these results indicate a presynaptic site of action that involves reduced excitation-release coupling. epsps were depressed to comparable levels by ttx or lowered stim, but facilitation was not changed, indicating a simple failure of axonal conduction. similarly, partial antagonism of postsynaptic glutamate receptors with cnqx depressed epsp amplitudes with no change in facilitation. however, epsp depression by low external ca2+ was accompanied by an increase in facilitation comparable to isoflurane. isoflurane depression of epsp amplitudes could also be partly reversed by high external ca2+ that also decreased facilitation. isoflurane or low ca2+ markedly reduced the slopes of fiber volley -epsp input-output curves, consistent with little or no effect on fvs. by contrast, ttx didn't alter the fv-epsp curve slope, indicating that epsp depression resulted from fv depression. fvs were remarkably resistant to isoflurane. somatic spike currents were unaffected by μm isoflurane as well. the ec for isoflurane depression of fvs was ~ mm.  isoflurane appears to depress ca synapses at presynaptic sites downstream from na channels, as evident by the increased facilitation that accompanies epsp depression. fiber volleys did not exhibit depression by isoflurane, as has been reported for other brain regions. 