Background Anesthesia is produced by a depression of central nervous system function, however, the sites and mechanisms of action underlying this depression remain poorly defined. The present study compared and contrasted effects produced by five general anesthetics on synaptic circuitry in the CA1 region of hippocampal slices.
Background
Anesthesia is produced by a depression of central nervous system function, however, the sites and mechanisms of action underlying this depression remain poorly defined. The present study compared and contrasted effects produced by five general anesthetics on synaptic circuitry in the CA1 region of hippocampal slices.Results At clinically relevant and equi-effective concentrations, presynaptic and postsynaptic anesthetic actions were evident at glutamate-mediated excitatory synapses and at GABA-mediated inhibitory synapses. In addition, depressant effects on membrane excitability were observed for CA1 neuron discharge in response to direct current depolarization. Combined actions at several of these sites contributed to CA1 circuit depression, but the relative degree of effect at each site was different for each anesthetic studied. For example, most of propofol's depressant effect (> 70 %) was reversed with a GABA antagonist, but only a minor portion of isoflurane's depression was reversed (< 20 %). Differences were also apparent on glutamate synapses-pentobarbital depressed transmission by > 50 %, but thiopental by only < 25 %.
Results
At clinically relevant and equi-effective concentrations, presynaptic and postsynaptic anesthetic actions were evident at glutamate-mediated excitatory synapses and at GABA-mediated inhibitory synapses. In addition, depressant effects on membrane excitability were observed for CA1 neuron discharge in response to direct current depolarization. Combined actions at several of these sites contributed to CA1 circuit depression, but the relative degree of effect at each site was different for each anesthetic studied. For example, most of propofol's depressant effect (> 70 %) was reversed with a GABA antagonist, but only a minor portion of isoflurane's depression was reversed (< 20 %). Differences were also apparent on glutamate synapses-pentobarbital depressed transmission by > 50 %, but thiopental by only < 25 %.Conclusions These results, in as much as they may be relevant to anesthesia, indicate that general anesthetics act at several discrete sites, supporting a multi-site, agent specific theory for anesthetic actions. No single effect site (e.g. GABA synapses) or mechanism of action (e.g. depressed membrane excitability) could account for all of the effects produced for any anesthetic studied.
Conclusions
These results, in as much as they may be relevant to anesthesia, indicate that general anesthetics act at several discrete sites, supporting a multi-site, agent specific theory for anesthetic actions. No single effect site (e.g. GABA synapses) or mechanism of action (e.g. depressed membrane excitability) could account for all of the effects produced for any anesthetic studied.

Background General anesthetics have been shown to depress neuronal responses in virtually all brain areas studied and this depression has been proposed to result from actions at GABA A -mediated inhibitory synapses and postsynaptic chloride channels [ 1 - 4 ], potassium channels [ 5 - 7 ], or calcium channels [ 8 - 11 ], and/or at glutamate-mediated excitatory synapses [ 12 - 17 ]. The last decade has seen a major shift in our understanding of general anesthetic mechanisms of action, away from a non-specific Unitary theory of action, towards a detailed view of anesthetic actions at membrane receptor and ion channel targets for these agents [ 18 , 19 ]. It is likely that several anesthetic actions occurring at independent sites contribute in additive ways to depress neuronal circuits in higher brain structures. Alternatively, anesthetic effects could result from actions at only a few sites and this should become evident by studying overall effects on the CA1 neural circuit and 'chasing down' the underlying actions. In the present study, the effects produced by five general anesthetics were studied at several possible sites of action within the well characterized Schaffer-collateral to CA1 neuron circuit using electrophysiological recordings from rat hippocampal slices. The CA1 circuit has previously been shown to be depressed by anesthetics from several chemical classes [ 20 - 26 ] at concentrations which alter hippocampal electrical activity in chronically instrumented rats during anesthesia [ 27 - 29 ]. The five agents chosen for this study are all clinically used anesthetics and provide a good representation from unique chemical classes: a halocarbon (halothane), halogenated ether (isoflurane), barbiturate (pentobarbital), sulfonated-barbiturate (thiopental), and a newer di-isopropylphenol compound, propofol.
Background
General anesthetics have been shown to depress neuronal responses in virtually all brain areas studied and this depression has been proposed to result from actions at GABA A -mediated inhibitory synapses and postsynaptic chloride channels [ 1 - 4 ], potassium channels [ 5 - 7 ], or calcium channels [ 8 - 11 ], and/or at glutamate-mediated excitatory synapses [ 12 - 17 ]. The last decade has seen a major shift in our understanding of general anesthetic mechanisms of action, away from a non-specific Unitary theory of action, towards a detailed view of anesthetic actions at membrane receptor and ion channel targets for these agents [ 18 , 19 ]. It is likely that several anesthetic actions occurring at independent sites contribute in additive ways to depress neuronal circuits in higher brain structures. Alternatively, anesthetic effects could result from actions at only a few sites and this should become evident by studying overall effects on the CA1 neural circuit and 'chasing down' the underlying actions. In the present study, the effects produced by five general anesthetics were studied at several possible sites of action within the well characterized Schaffer-collateral to CA1 neuron circuit using electrophysiological recordings from rat hippocampal slices. The CA1 circuit has previously been shown to be depressed by anesthetics from several chemical classes [ 20 - 26 ] at concentrations which alter hippocampal electrical activity in chronically instrumented rats during anesthesia [ 27 - 29 ]. The five agents chosen for this study are all clinically used anesthetics and provide a good representation from unique chemical classes: a halocarbon (halothane), halogenated ether (isoflurane), barbiturate (pentobarbital), sulfonated-barbiturate (thiopental), and a newer di-isopropylphenol compound, propofol.Results and discussion Anesthetics enhance GABA-mediated inhibition All five anesthetics depressed synaptically evoked discharge, measured as a block of population spike (PS) responses recorded from CA1 neurons (Fig.  1 ). The two volatile anesthetics, halothane and isoflurane, produced a nearly complete depression (to 3.3 ± 3.5 and 5.6 ± 7.1 % of control respectively) at clinically effective concentrations: halothane (1.0 rat MAC; 1.25 vol % ~ 250 μM) and isoflurane (1.0 rat MAC; 1.55 vol % ~ 350 μM; for Sprague-Daley rats [ 30 ]; Minimum Alveolar Concentration – the expired anesthetic gas concentration for a 50 % loss of a tail clamp response – motor reflex in rats). The three intravenous agents, pentobarbital (400 μM), thiopental (80 μM) and propofol (30 μM), also depressed PS responses to a comparable degree: 1.7 ± 3.1, 3.4 ± 2.8 and 6.2 ± 5.8 % of control responses, respectively (p < 0.001, n ≥ 5 slices from individual rats, for all five agents compared with pre-anesthetic control responses, using ANOVA-Tukey). All anesthetic effects were reversible on washout of the agent with drug free ACSF. It should be noted that the more lipophilic intravenous anesthetics produce lower effect site concentrations in these brain slices than the applied concentrations shown, especially for these short time periods of application, because it can take several hours for these agents to diffuse 200 to 300 μm into brain slices and achieve steady-state levels [ 31 ]. For example, an applied concentration of 30 μM propofol would be expected to produce only ~ 1.0 to 3.0 μM at a recording depth of 250 microns within 30 minutes [ 32 ]. The volatile anesthetics, in contrast, rapidly equilibrate throughout the brain slice due to their relatively high aqueous solubility. These equi-effective applied concentrations for PS depression were used in subsequent experiments to determine whether this depression resulted from enhanced GABA A -mediated inhibition. A GABA A  receptor antagonist, bicuculline, was applied in the continued presence of each anesthetic to attempt to reverse the anesthetic-induced PS depression. Bicuculline (10 μM) reversed anesthetic-induced PS depression to varying degrees for each agent: isoflurane – 16.2 ± 7.4 %, halothane – 22.3 ± 18.4 %, pentobarbital – 56.2 ± 12.4 %, thiopental – 64.9 ± 12.9 % and propofol – 69.5 ± 14.3 %. Similar degrees of reversal were observed using the GABA-chloride channel blocker, picrotoxin (100 μM; a supra-maximal blocking concentration). A GABA B  receptor antagonist, CGP 55845A (10 μM) did not reverse PS depression for any of the anesthetics studied (see Table  1 ). None of the anesthetics produced a significant depression of antidromically stimulated PS responses (± 5 % depression, p > 0.15) indicating that CA1 neuron axonal conduction was not appreciably altered. Thus, enhanced GABA A -mediated inhibition appeared to play a major role for the PS depression produced by propofol and thiopental (~ 75 %), less so for pentobarbital (~ 50 %), and contributed only partially to the depressant effects of the volatile anesthetics (< 25 %; Fig.  1E ). Anesthetics depress glutamate-mediated excitatory synapses To determine whether anesthetic-induced PS depression resulted from depressed glutamate-mediated excitatory synaptic inputs to CA1 neurons, field excitatory postsynaptic potentials (EPSPs) were recorded from dendritic regions in stratum radiatum. All five anesthetics depressed EPSP responses (e.g. Fig.  1C  and  1D ): isoflurane to 52.2 ± 7.6 (p < 0.001), halothane 61.3 ± 8.4 (p < 0.001), pentobarbital 54.5 ± 4.8 (p < 0.001), thiopental 75.5 ± 9.8 (p < 0.01) and propofol 72.7 ± 23.5 (p < 0.05) % of control responses. Bicuculline did not reverse volatile anesthetic-induced EPSP depression, but did partially reverse the effect for pentobarbital (11.4 ± 3.6 %) and completely reversed the EPSP depression produced by thiopental and propofol (Fig.  1F ). Thus, depressed glutamate-mediated synaptic excitation appeared to play an important role for PS depression produced by isoflurane, halothane and pentobarbital. The thiopental and propofol-induced EPSP depression would also contribute to PS depression for these agents, but appeared to occur via enhanced GABA-mediated inhibition at a dendritic level, since this depression was reversed by bicuculline. Pre- and postsynaptic sites of action at GABA A  synapses Whole cell voltage clamp recordings from CA1 neurons were used to examine more closely anesthetic effects on membrane currents at GABA synapses. Spontaneous GABA-mediated inhibitory postsynaptic currents (IPSCs) were observed in all CA1 neurons studied (n = 15) and were completely blocked by bicuculline (10 μM; Fig.  2A ). In the presence of glutamate receptor antagonists (CNQX 17.2 μM and APV 100 μM) the anesthetics produced agent-specific effects on holding currents needed to clamp neurons at the control resting membrane potentials (-60 to -70 mV). Propofol was most effective at increasing holding currents (376 ± 83 pA, n = 3), followed by thiopental (320 ± 72 pA, n = 4) and pentobarbital (127 ± 65 pA, n = 3). Halothane (n = 6) and isoflurane (n = 3) produced weaker and more variable responses (< 50 pA). The changes in holding currents produced by propofol and the barbiturates were reversed by bicuculline (10 μM) or picrotoxin (100 μM), indicating that they involved activation of GABA A -mediated chloride channels. The most dramatic effect produced by all five anesthetics was observed on IPSCs (e.g. Fig.  2B ). Membrane charge transfer, for example, was increased by 3 to 4 fold in the presence of halothane and came about by at least two separate mechanisms. The first mechanism was a prolongation of IPSC time course (Fig.  2C ) resulting in nearly a 3 fold increase in charge transfer for each IPSC (284 ± 33 % of control, p < 0.005, n = 6). This result was in good agreement with previous findings showing that anesthetics prolong IPSCs by increasing the open time of GABA-gated channels in the postsynaptic membrane [ 33 - 35 ]. The second mechanism appeared to involve presynaptic sites, observed as an increase in frequency of IPSCs (143 ± 28 % of control, p < 0.005, n = 6 neurons from separate slices) and occurred with a small, but significant, depression in IPSC amplitudes (92 ± 6 % of control, p < 0.05, n = 6). The anesthetic-induced IPSC frequency increase was also observed in the presence of tetrodotoxin, used to block action potentials (n = 5 for halothane, n = 4 for propofol), indicating a direct action on GABA nerve terminals. This confirms earlier findings that anesthetics can increase IPSC frequency and the release of GABA from nerve terminals [ 36 - 39 ]. This presynaptic effect combines with postsynaptic prolongation of IPSCs to account for the marked increase in membrane charge transfer observed, and would contribute to the anesthetic-induced postsynaptic hyperpolarization of CA1 neurons previously reported [ 4 , 40 - 42 ]. All of the anesthetics studied increased inhibitory charge transfer and the degree of enhancement corresponded well with the ability of bicuculline to reverse the anesthetic-induced depression of population spike responses (Fig.  1  and Table  1 ). For halothane and isoflurane, this enhanced inhibitory charge transfer played a relatively minor role in population spike depression compared with their ability to depress glutamate-mediated excitatory inputs to the CA1 neurons. Anesthetics increase paired-pulse facilitation To determine whether presynaptic actions also contribute to anesthetic effects at glutamate synapses, paired pulse (120 ms) facilitation of Schaffer-collateral evoked EPSPs were studied. In the presence of either halothane or isoflurane no apparent change in EPSP rise time or decay kinetics were observed (Fig.  3A ), contrasting with the marked prolongation of IPSC decay time produced by the anesthetics. Facilitation was increased to nearly 115 % of control and this effect was independent of GABA A -mediated actions, since they persisted in the presence of the antagonist – bicuculline (Fig.  3B ). This increase in facilitation is consistent with a presynaptic depression of glutamate release from nerve terminals, perhaps via depressant actions on voltage activated calcium or sodium channels which couple axon spike depolarization to the release of transmitter [ 8 , 9 , 17 , 43 , 44 ]. Anesthetics increase paired-pulse inhibition Agent-specific effects were observed for paired pulse inhibitory responses (120 ms separation) recorded from CA1 neurons (Fig.  3C ). Halothane and isoflurane produced no apparent change in paired pulse responses, both the first and second population spike following a pair of stimuli were depressed to a similar degree by these anesthetics. In contrast, propofol, thiopental and pentobarbital increased paired pulse inhibition, evident in a greater degree of depression for the second of a pair of responses. To quantify these increases in paired pulse inhibition, effects on second pulse responses were compared at concentrations that produced a half maximal depression of first spike responses. At a level of 50 % depression of first pulse responses, pentobarbital produced a 134 ± 8 % increase in second pulse inhibition, thiopental produced a 156 ± 15 % increase and propofol produced a 149 ± 13% increase (p < 0.001, n = 5 for each agent compared to first pulse responses). This effect is consistent with  in vivo  findings [ 45 ] and is thought to reflect a greater degree of GABA-mediated inhibition contributing to the second of a pair of stimuli, via recurrent (feedback) activation of inhibitory interneurons caused by the first pulse [ 26 ]. Anesthetics depress CA1 neuron excitability To determine whether the anesthetics could alter postsynaptic membrane excitability, effects on action potentials evoked by direct current injection into CA1 neurons were studied. Differences in effect were apparent across anesthetic agents – hardly any effect was evident for halothane and isoflurane, but the barbiturates and propofol produced a significant depression of action potential discharge (Fig.  3D ). When measured as a reduction in the number of action potentials produced in response to a one second long depolarizing current step, halothane produced an 8.2 ± 2.2 % depression and isoflurane an 11.6 ± 6.1 % depression (p < 0.01 for both agents compared to control responses). Propofol was much more effective at depressing CA1 discharge, producing a 93.5 ± 6.1 % depression (p < 0.001). Thiopental produced a 90.3 ± 9.9 % depression and pentobarbital a 79.5 ± 7.4 % depression (p < 0.001 for both anesthetics compared with control). The anesthetic-induced depression of spike discharge activity was accompanied by decreases in membrane resistance and to a lesser extent by small changes in membrane resting potential. In spite of the marked depressant effects observed for the intravenous anesthetics on spike discharge, none of the anesthetics appeared to alter action potential amplitude, rise time or decay profiles (Fig.  3D ), suggesting that the major depressant effect was accounted for by actions on spike threshold – not on the sodium currents which underlie action potentials  per se .
Results and discussion
Anesthetics enhance GABA-mediated inhibition All five anesthetics depressed synaptically evoked discharge, measured as a block of population spike (PS) responses recorded from CA1 neurons (Fig.  1 ). The two volatile anesthetics, halothane and isoflurane, produced a nearly complete depression (to 3.3 ± 3.5 and 5.6 ± 7.1 % of control respectively) at clinically effective concentrations: halothane (1.0 rat MAC; 1.25 vol % ~ 250 μM) and isoflurane (1.0 rat MAC; 1.55 vol % ~ 350 μM; for Sprague-Daley rats [ 30 ]; Minimum Alveolar Concentration – the expired anesthetic gas concentration for a 50 % loss of a tail clamp response – motor reflex in rats). The three intravenous agents, pentobarbital (400 μM), thiopental (80 μM) and propofol (30 μM), also depressed PS responses to a comparable degree: 1.7 ± 3.1, 3.4 ± 2.8 and 6.2 ± 5.8 % of control responses, respectively (p < 0.001, n ≥ 5 slices from individual rats, for all five agents compared with pre-anesthetic control responses, using ANOVA-Tukey). All anesthetic effects were reversible on washout of the agent with drug free ACSF. It should be noted that the more lipophilic intravenous anesthetics produce lower effect site concentrations in these brain slices than the applied concentrations shown, especially for these short time periods of application, because it can take several hours for these agents to diffuse 200 to 300 μm into brain slices and achieve steady-state levels [ 31 ]. For example, an applied concentration of 30 μM propofol would be expected to produce only ~ 1.0 to 3.0 μM at a recording depth of 250 microns within 30 minutes [ 32 ]. The volatile anesthetics, in contrast, rapidly equilibrate throughout the brain slice due to their relatively high aqueous solubility. These equi-effective applied concentrations for PS depression were used in subsequent experiments to determine whether this depression resulted from enhanced GABA A -mediated inhibition. A GABA A  receptor antagonist, bicuculline, was applied in the continued presence of each anesthetic to attempt to reverse the anesthetic-induced PS depression. Bicuculline (10 μM) reversed anesthetic-induced PS depression to varying degrees for each agent: isoflurane – 16.2 ± 7.4 %, halothane – 22.3 ± 18.4 %, pentobarbital – 56.2 ± 12.4 %, thiopental – 64.9 ± 12.9 % and propofol – 69.5 ± 14.3 %. Similar degrees of reversal were observed using the GABA-chloride channel blocker, picrotoxin (100 μM; a supra-maximal blocking concentration). A GABA B  receptor antagonist, CGP 55845A (10 μM) did not reverse PS depression for any of the anesthetics studied (see Table  1 ). None of the anesthetics produced a significant depression of antidromically stimulated PS responses (± 5 % depression, p > 0.15) indicating that CA1 neuron axonal conduction was not appreciably altered. Thus, enhanced GABA A -mediated inhibition appeared to play a major role for the PS depression produced by propofol and thiopental (~ 75 %), less so for pentobarbital (~ 50 %), and contributed only partially to the depressant effects of the volatile anesthetics (< 25 %; Fig.  1E ).
Anesthetics enhance GABA-mediated inhibition
All five anesthetics depressed synaptically evoked discharge, measured as a block of population spike (PS) responses recorded from CA1 neurons (Fig.  1 ). The two volatile anesthetics, halothane and isoflurane, produced a nearly complete depression (to 3.3 ± 3.5 and 5.6 ± 7.1 % of control respectively) at clinically effective concentrations: halothane (1.0 rat MAC; 1.25 vol % ~ 250 μM) and isoflurane (1.0 rat MAC; 1.55 vol % ~ 350 μM; for Sprague-Daley rats [ 30 ]; Minimum Alveolar Concentration – the expired anesthetic gas concentration for a 50 % loss of a tail clamp response – motor reflex in rats). The three intravenous agents, pentobarbital (400 μM), thiopental (80 μM) and propofol (30 μM), also depressed PS responses to a comparable degree: 1.7 ± 3.1, 3.4 ± 2.8 and 6.2 ± 5.8 % of control responses, respectively (p < 0.001, n ≥ 5 slices from individual rats, for all five agents compared with pre-anesthetic control responses, using ANOVA-Tukey). All anesthetic effects were reversible on washout of the agent with drug free ACSF. It should be noted that the more lipophilic intravenous anesthetics produce lower effect site concentrations in these brain slices than the applied concentrations shown, especially for these short time periods of application, because it can take several hours for these agents to diffuse 200 to 300 μm into brain slices and achieve steady-state levels [ 31 ]. For example, an applied concentration of 30 μM propofol would be expected to produce only ~ 1.0 to 3.0 μM at a recording depth of 250 microns within 30 minutes [ 32 ]. The volatile anesthetics, in contrast, rapidly equilibrate throughout the brain slice due to their relatively high aqueous solubility.These equi-effective applied concentrations for PS depression were used in subsequent experiments to determine whether this depression resulted from enhanced GABA A -mediated inhibition.A GABA A  receptor antagonist, bicuculline, was applied in the continued presence of each anesthetic to attempt to reverse the anesthetic-induced PS depression. Bicuculline (10 μM) reversed anesthetic-induced PS depression to varying degrees for each agent: isoflurane – 16.2 ± 7.4 %, halothane – 22.3 ± 18.4 %, pentobarbital – 56.2 ± 12.4 %, thiopental – 64.9 ± 12.9 % and propofol – 69.5 ± 14.3 %. Similar degrees of reversal were observed using the GABA-chloride channel blocker, picrotoxin (100 μM; a supra-maximal blocking concentration). A GABA B  receptor antagonist, CGP 55845A (10 μM) did not reverse PS depression for any of the anesthetics studied (see Table  1 ). None of the anesthetics produced a significant depression of antidromically stimulated PS responses (± 5 % depression, p > 0.15) indicating that CA1 neuron axonal conduction was not appreciably altered. Thus, enhanced GABA A -mediated inhibition appeared to play a major role for the PS depression produced by propofol and thiopental (~ 75 %), less so for pentobarbital (~ 50 %), and contributed only partially to the depressant effects of the volatile anesthetics (< 25 %; Fig.  1E ).Anesthetics depress glutamate-mediated excitatory synapses To determine whether anesthetic-induced PS depression resulted from depressed glutamate-mediated excitatory synaptic inputs to CA1 neurons, field excitatory postsynaptic potentials (EPSPs) were recorded from dendritic regions in stratum radiatum. All five anesthetics depressed EPSP responses (e.g. Fig.  1C  and  1D ): isoflurane to 52.2 ± 7.6 (p < 0.001), halothane 61.3 ± 8.4 (p < 0.001), pentobarbital 54.5 ± 4.8 (p < 0.001), thiopental 75.5 ± 9.8 (p < 0.01) and propofol 72.7 ± 23.5 (p < 0.05) % of control responses. Bicuculline did not reverse volatile anesthetic-induced EPSP depression, but did partially reverse the effect for pentobarbital (11.4 ± 3.6 %) and completely reversed the EPSP depression produced by thiopental and propofol (Fig.  1F ). Thus, depressed glutamate-mediated synaptic excitation appeared to play an important role for PS depression produced by isoflurane, halothane and pentobarbital. The thiopental and propofol-induced EPSP depression would also contribute to PS depression for these agents, but appeared to occur via enhanced GABA-mediated inhibition at a dendritic level, since this depression was reversed by bicuculline.
Anesthetics depress glutamate-mediated excitatory synapses
To determine whether anesthetic-induced PS depression resulted from depressed glutamate-mediated excitatory synaptic inputs to CA1 neurons, field excitatory postsynaptic potentials (EPSPs) were recorded from dendritic regions in stratum radiatum. All five anesthetics depressed EPSP responses (e.g. Fig.  1C  and  1D ): isoflurane to 52.2 ± 7.6 (p < 0.001), halothane 61.3 ± 8.4 (p < 0.001), pentobarbital 54.5 ± 4.8 (p < 0.001), thiopental 75.5 ± 9.8 (p < 0.01) and propofol 72.7 ± 23.5 (p < 0.05) % of control responses. Bicuculline did not reverse volatile anesthetic-induced EPSP depression, but did partially reverse the effect for pentobarbital (11.4 ± 3.6 %) and completely reversed the EPSP depression produced by thiopental and propofol (Fig.  1F ). Thus, depressed glutamate-mediated synaptic excitation appeared to play an important role for PS depression produced by isoflurane, halothane and pentobarbital. The thiopental and propofol-induced EPSP depression would also contribute to PS depression for these agents, but appeared to occur via enhanced GABA-mediated inhibition at a dendritic level, since this depression was reversed by bicuculline.Pre- and postsynaptic sites of action at GABA A  synapses Whole cell voltage clamp recordings from CA1 neurons were used to examine more closely anesthetic effects on membrane currents at GABA synapses. Spontaneous GABA-mediated inhibitory postsynaptic currents (IPSCs) were observed in all CA1 neurons studied (n = 15) and were completely blocked by bicuculline (10 μM; Fig.  2A ). In the presence of glutamate receptor antagonists (CNQX 17.2 μM and APV 100 μM) the anesthetics produced agent-specific effects on holding currents needed to clamp neurons at the control resting membrane potentials (-60 to -70 mV). Propofol was most effective at increasing holding currents (376 ± 83 pA, n = 3), followed by thiopental (320 ± 72 pA, n = 4) and pentobarbital (127 ± 65 pA, n = 3). Halothane (n = 6) and isoflurane (n = 3) produced weaker and more variable responses (< 50 pA). The changes in holding currents produced by propofol and the barbiturates were reversed by bicuculline (10 μM) or picrotoxin (100 μM), indicating that they involved activation of GABA A -mediated chloride channels. The most dramatic effect produced by all five anesthetics was observed on IPSCs (e.g. Fig.  2B ). Membrane charge transfer, for example, was increased by 3 to 4 fold in the presence of halothane and came about by at least two separate mechanisms. The first mechanism was a prolongation of IPSC time course (Fig.  2C ) resulting in nearly a 3 fold increase in charge transfer for each IPSC (284 ± 33 % of control, p < 0.005, n = 6). This result was in good agreement with previous findings showing that anesthetics prolong IPSCs by increasing the open time of GABA-gated channels in the postsynaptic membrane [ 33 - 35 ]. The second mechanism appeared to involve presynaptic sites, observed as an increase in frequency of IPSCs (143 ± 28 % of control, p < 0.005, n = 6 neurons from separate slices) and occurred with a small, but significant, depression in IPSC amplitudes (92 ± 6 % of control, p < 0.05, n = 6). The anesthetic-induced IPSC frequency increase was also observed in the presence of tetrodotoxin, used to block action potentials (n = 5 for halothane, n = 4 for propofol), indicating a direct action on GABA nerve terminals. This confirms earlier findings that anesthetics can increase IPSC frequency and the release of GABA from nerve terminals [ 36 - 39 ]. This presynaptic effect combines with postsynaptic prolongation of IPSCs to account for the marked increase in membrane charge transfer observed, and would contribute to the anesthetic-induced postsynaptic hyperpolarization of CA1 neurons previously reported [ 4 , 40 - 42 ]. All of the anesthetics studied increased inhibitory charge transfer and the degree of enhancement corresponded well with the ability of bicuculline to reverse the anesthetic-induced depression of population spike responses (Fig.  1  and Table  1 ). For halothane and isoflurane, this enhanced inhibitory charge transfer played a relatively minor role in population spike depression compared with their ability to depress glutamate-mediated excitatory inputs to the CA1 neurons.
Pre- and postsynaptic sites of action at GABA A  synapses
Whole cell voltage clamp recordings from CA1 neurons were used to examine more closely anesthetic effects on membrane currents at GABA synapses. Spontaneous GABA-mediated inhibitory postsynaptic currents (IPSCs) were observed in all CA1 neurons studied (n = 15) and were completely blocked by bicuculline (10 μM; Fig.  2A ). In the presence of glutamate receptor antagonists (CNQX 17.2 μM and APV 100 μM) the anesthetics produced agent-specific effects on holding currents needed to clamp neurons at the control resting membrane potentials (-60 to -70 mV). Propofol was most effective at increasing holding currents (376 ± 83 pA, n = 3), followed by thiopental (320 ± 72 pA, n = 4) and pentobarbital (127 ± 65 pA, n = 3). Halothane (n = 6) and isoflurane (n = 3) produced weaker and more variable responses (< 50 pA). The changes in holding currents produced by propofol and the barbiturates were reversed by bicuculline (10 μM) or picrotoxin (100 μM), indicating that they involved activation of GABA A -mediated chloride channels.The most dramatic effect produced by all five anesthetics was observed on IPSCs (e.g. Fig.  2B ). Membrane charge transfer, for example, was increased by 3 to 4 fold in the presence of halothane and came about by at least two separate mechanisms. The first mechanism was a prolongation of IPSC time course (Fig.  2C ) resulting in nearly a 3 fold increase in charge transfer for each IPSC (284 ± 33 % of control, p < 0.005, n = 6). This result was in good agreement with previous findings showing that anesthetics prolong IPSCs by increasing the open time of GABA-gated channels in the postsynaptic membrane [ 33 - 35 ]. The second mechanism appeared to involve presynaptic sites, observed as an increase in frequency of IPSCs (143 ± 28 % of control, p < 0.005, n = 6 neurons from separate slices) and occurred with a small, but significant, depression in IPSC amplitudes (92 ± 6 % of control, p < 0.05, n = 6). The anesthetic-induced IPSC frequency increase was also observed in the presence of tetrodotoxin, used to block action potentials (n = 5 for halothane, n = 4 for propofol), indicating a direct action on GABA nerve terminals. This confirms earlier findings that anesthetics can increase IPSC frequency and the release of GABA from nerve terminals [ 36 - 39 ]. This presynaptic effect combines with postsynaptic prolongation of IPSCs to account for the marked increase in membrane charge transfer observed, and would contribute to the anesthetic-induced postsynaptic hyperpolarization of CA1 neurons previously reported [ 4 , 40 - 42 ]. All of the anesthetics studied increased inhibitory charge transfer and the degree of enhancement corresponded well with the ability of bicuculline to reverse the anesthetic-induced depression of population spike responses (Fig.  1  and Table  1 ). For halothane and isoflurane, this enhanced inhibitory charge transfer played a relatively minor role in population spike depression compared with their ability to depress glutamate-mediated excitatory inputs to the CA1 neurons.Anesthetics increase paired-pulse facilitation To determine whether presynaptic actions also contribute to anesthetic effects at glutamate synapses, paired pulse (120 ms) facilitation of Schaffer-collateral evoked EPSPs were studied. In the presence of either halothane or isoflurane no apparent change in EPSP rise time or decay kinetics were observed (Fig.  3A ), contrasting with the marked prolongation of IPSC decay time produced by the anesthetics. Facilitation was increased to nearly 115 % of control and this effect was independent of GABA A -mediated actions, since they persisted in the presence of the antagonist – bicuculline (Fig.  3B ). This increase in facilitation is consistent with a presynaptic depression of glutamate release from nerve terminals, perhaps via depressant actions on voltage activated calcium or sodium channels which couple axon spike depolarization to the release of transmitter [ 8 , 9 , 17 , 43 , 44 ].
Anesthetics increase paired-pulse facilitation
To determine whether presynaptic actions also contribute to anesthetic effects at glutamate synapses, paired pulse (120 ms) facilitation of Schaffer-collateral evoked EPSPs were studied. In the presence of either halothane or isoflurane no apparent change in EPSP rise time or decay kinetics were observed (Fig.  3A ), contrasting with the marked prolongation of IPSC decay time produced by the anesthetics. Facilitation was increased to nearly 115 % of control and this effect was independent of GABA A -mediated actions, since they persisted in the presence of the antagonist – bicuculline (Fig.  3B ). This increase in facilitation is consistent with a presynaptic depression of glutamate release from nerve terminals, perhaps via depressant actions on voltage activated calcium or sodium channels which couple axon spike depolarization to the release of transmitter [ 8 , 9 , 17 , 43 , 44 ].Anesthetics increase paired-pulse inhibition Agent-specific effects were observed for paired pulse inhibitory responses (120 ms separation) recorded from CA1 neurons (Fig.  3C ). Halothane and isoflurane produced no apparent change in paired pulse responses, both the first and second population spike following a pair of stimuli were depressed to a similar degree by these anesthetics. In contrast, propofol, thiopental and pentobarbital increased paired pulse inhibition, evident in a greater degree of depression for the second of a pair of responses. To quantify these increases in paired pulse inhibition, effects on second pulse responses were compared at concentrations that produced a half maximal depression of first spike responses. At a level of 50 % depression of first pulse responses, pentobarbital produced a 134 ± 8 % increase in second pulse inhibition, thiopental produced a 156 ± 15 % increase and propofol produced a 149 ± 13% increase (p < 0.001, n = 5 for each agent compared to first pulse responses). This effect is consistent with  in vivo  findings [ 45 ] and is thought to reflect a greater degree of GABA-mediated inhibition contributing to the second of a pair of stimuli, via recurrent (feedback) activation of inhibitory interneurons caused by the first pulse [ 26 ].
Anesthetics increase paired-pulse inhibition
Agent-specific effects were observed for paired pulse inhibitory responses (120 ms separation) recorded from CA1 neurons (Fig.  3C ). Halothane and isoflurane produced no apparent change in paired pulse responses, both the first and second population spike following a pair of stimuli were depressed to a similar degree by these anesthetics. In contrast, propofol, thiopental and pentobarbital increased paired pulse inhibition, evident in a greater degree of depression for the second of a pair of responses. To quantify these increases in paired pulse inhibition, effects on second pulse responses were compared at concentrations that produced a half maximal depression of first spike responses. At a level of 50 % depression of first pulse responses, pentobarbital produced a 134 ± 8 % increase in second pulse inhibition, thiopental produced a 156 ± 15 % increase and propofol produced a 149 ± 13% increase (p < 0.001, n = 5 for each agent compared to first pulse responses). This effect is consistent with  in vivo  findings [ 45 ] and is thought to reflect a greater degree of GABA-mediated inhibition contributing to the second of a pair of stimuli, via recurrent (feedback) activation of inhibitory interneurons caused by the first pulse [ 26 ].in vivoAnesthetics depress CA1 neuron excitability To determine whether the anesthetics could alter postsynaptic membrane excitability, effects on action potentials evoked by direct current injection into CA1 neurons were studied. Differences in effect were apparent across anesthetic agents – hardly any effect was evident for halothane and isoflurane, but the barbiturates and propofol produced a significant depression of action potential discharge (Fig.  3D ). When measured as a reduction in the number of action potentials produced in response to a one second long depolarizing current step, halothane produced an 8.2 ± 2.2 % depression and isoflurane an 11.6 ± 6.1 % depression (p < 0.01 for both agents compared to control responses). Propofol was much more effective at depressing CA1 discharge, producing a 93.5 ± 6.1 % depression (p < 0.001). Thiopental produced a 90.3 ± 9.9 % depression and pentobarbital a 79.5 ± 7.4 % depression (p < 0.001 for both anesthetics compared with control). The anesthetic-induced depression of spike discharge activity was accompanied by decreases in membrane resistance and to a lesser extent by small changes in membrane resting potential. In spite of the marked depressant effects observed for the intravenous anesthetics on spike discharge, none of the anesthetics appeared to alter action potential amplitude, rise time or decay profiles (Fig.  3D ), suggesting that the major depressant effect was accounted for by actions on spike threshold – not on the sodium currents which underlie action potentials  per se .
Anesthetics depress CA1 neuron excitability
To determine whether the anesthetics could alter postsynaptic membrane excitability, effects on action potentials evoked by direct current injection into CA1 neurons were studied. Differences in effect were apparent across anesthetic agents – hardly any effect was evident for halothane and isoflurane, but the barbiturates and propofol produced a significant depression of action potential discharge (Fig.  3D ). When measured as a reduction in the number of action potentials produced in response to a one second long depolarizing current step, halothane produced an 8.2 ± 2.2 % depression and isoflurane an 11.6 ± 6.1 % depression (p < 0.01 for both agents compared to control responses). Propofol was much more effective at depressing CA1 discharge, producing a 93.5 ± 6.1 % depression (p < 0.001). Thiopental produced a 90.3 ± 9.9 % depression and pentobarbital a 79.5 ± 7.4 % depression (p < 0.001 for both anesthetics compared with control). The anesthetic-induced depression of spike discharge activity was accompanied by decreases in membrane resistance and to a lesser extent by small changes in membrane resting potential. In spite of the marked depressant effects observed for the intravenous anesthetics on spike discharge, none of the anesthetics appeared to alter action potential amplitude, rise time or decay profiles (Fig.  3D ), suggesting that the major depressant effect was accounted for by actions on spike threshold – not on the sodium currents which underlie action potentials  per se .per seConclusions Two conclusions can be drawn from these results: 1) for a given anesthetic, like halothane, multiple sites of action contributed in an additive manner to produce an overall depression of transmission through the CA1 neuronal circuitry (Fig.  3E ); 2) for each anesthetic the degree of effect was agent specific at some of these sites. Together the results support a Multisite Agent Specific (MAS) mechanism of action for general anesthetics. This represents a departure from traditional Unitary theories of action in several important respects. Unitary theories posit that all anesthetics act via a common molecular mechanism, such as to change the fluidity of nerve cell membranes, or to enhance a potassium current, or most recently to enhance GABA-mediated inhibition [ 2 , 3 ]. With the MAS theory, no common site of action is required (nor apparent) for anesthetics. This is consistent with observations at the molecular, [ 46 - 50 ] cellular [ 22 , 51 ] and behavioral levels [ 52 - 55 ]. Differing degrees of action (efficacy) were evident at both glutamate and GABA synapses for each anesthetic. For example, our results demonstrate that the two barbiturates studied appear to have differing degrees of effect at GABA synapses since thiopental's depressant effects were reversed ~ 65 % by a GABA antagonist, but pentobarbital's effects were only reversed by ~ 55 %. Similarly, these two barbiturates exhibited differing degrees of depression for glutamate-mediated excitatory inputs to the CA1 neurons, pentobarbital produced a 45 % depression in contrast to thiopental with only a 25 % depression. It was interesting that opposite actions were seen at presynaptic sites (GABA release was increased by anesthetics, while glutamate release was depressed) and at postsynaptic sites (GABA-mediated synaptic currents were prolonged, glutamate-mediated currents were not). The MAS theory can readily account for the unique agent-specific profiles of effects observed in various experimental models, and also seen clinically – a long standing weakness of Unitary theories [ 56 ]. Finally, the MAS theory predicts that agents which selectively target GABA and glutamate synapses could lead to the design of safer and more effective therapeutic agents for anesthesia, that exhibit fewer undesirable side effects. Glutamate and GABA synapses in the hippocampus are among the best characterized synapses in the brain and appear to utilize receptor subtypes which are similar to those in neocortex, thalamus and other higher brain regions. Thus, the effects described in the present study would be expected to occur in these other brain regions as well, but it should be noted that different GABA and glutamate receptor subtype distributions are known to occur in cerebellum, spinal and some brain stem nuclei, and it remains to be determined whether anesthetics alter these synapses in a similar manner to their hippocampal counterparts. Ted Eger's group at UCSF has recently found that enhanced GABA-mediated synaptic transmission at the spinal level plays an important role for propofol-induced immobility in response to a noxious stimulus [ 54 ], but this was not the case for isoflurane-induced immobility [ 57 ]. This agrees well with our findings that the volatile anesthetic-induced depression of synaptic signaling involves mechanisms other than enhanced GABA inhibition (see also [ 58 ]), while the depression produced by the barbiturates and propofol are more dependent on enhanced GABA-mediated inhibition. Additional  in vivo  support comes from studies utilizing a GABA beta 3 receptor mutant mouse model – proprofol-induced anesthesia was blocked in these mice, while volatile anesthetic effects were not [ 59 ]. Taken together with these  in vivo  findings, our results indicate that effects on GABA synapses play a role in anesthetic actions, especially for propofol, thiopental and pentobarbital; but the results also indicate that effects on glutamate synapses and postsynaptic membrane excitability contribute to the CNS depression produced by all anesthetics. Given the multiple effects observed for anesthetic actions on the two types of synapses studied here, it is likely that effects on other neurotransmitter systems also contribute to anesthetic-induced depression of the CNS.
Conclusions
Two conclusions can be drawn from these results: 1) for a given anesthetic, like halothane, multiple sites of action contributed in an additive manner to produce an overall depression of transmission through the CA1 neuronal circuitry (Fig.  3E ); 2) for each anesthetic the degree of effect was agent specific at some of these sites. Together the results support a Multisite Agent Specific (MAS) mechanism of action for general anesthetics. This represents a departure from traditional Unitary theories of action in several important respects. Unitary theories posit that all anesthetics act via a common molecular mechanism, such as to change the fluidity of nerve cell membranes, or to enhance a potassium current, or most recently to enhance GABA-mediated inhibition [ 2 , 3 ]. With the MAS theory, no common site of action is required (nor apparent) for anesthetics. This is consistent with observations at the molecular, [ 46 - 50 ] cellular [ 22 , 51 ] and behavioral levels [ 52 - 55 ].Differing degrees of action (efficacy) were evident at both glutamate and GABA synapses for each anesthetic. For example, our results demonstrate that the two barbiturates studied appear to have differing degrees of effect at GABA synapses since thiopental's depressant effects were reversed ~ 65 % by a GABA antagonist, but pentobarbital's effects were only reversed by ~ 55 %. Similarly, these two barbiturates exhibited differing degrees of depression for glutamate-mediated excitatory inputs to the CA1 neurons, pentobarbital produced a 45 % depression in contrast to thiopental with only a 25 % depression. It was interesting that opposite actions were seen at presynaptic sites (GABA release was increased by anesthetics, while glutamate release was depressed) and at postsynaptic sites (GABA-mediated synaptic currents were prolonged, glutamate-mediated currents were not). The MAS theory can readily account for the unique agent-specific profiles of effects observed in various experimental models, and also seen clinically – a long standing weakness of Unitary theories [ 56 ]. Finally, the MAS theory predicts that agents which selectively target GABA and glutamate synapses could lead to the design of safer and more effective therapeutic agents for anesthesia, that exhibit fewer undesirable side effects.Glutamate and GABA synapses in the hippocampus are among the best characterized synapses in the brain and appear to utilize receptor subtypes which are similar to those in neocortex, thalamus and other higher brain regions. Thus, the effects described in the present study would be expected to occur in these other brain regions as well, but it should be noted that different GABA and glutamate receptor subtype distributions are known to occur in cerebellum, spinal and some brain stem nuclei, and it remains to be determined whether anesthetics alter these synapses in a similar manner to their hippocampal counterparts. Ted Eger's group at UCSF has recently found that enhanced GABA-mediated synaptic transmission at the spinal level plays an important role for propofol-induced immobility in response to a noxious stimulus [ 54 ], but this was not the case for isoflurane-induced immobility [ 57 ]. This agrees well with our findings that the volatile anesthetic-induced depression of synaptic signaling involves mechanisms other than enhanced GABA inhibition (see also [ 58 ]), while the depression produced by the barbiturates and propofol are more dependent on enhanced GABA-mediated inhibition. Additional  in vivo  support comes from studies utilizing a GABA beta 3 receptor mutant mouse model – proprofol-induced anesthesia was blocked in these mice, while volatile anesthetic effects were not [ 59 ]. Taken together with these  in vivo  findings, our results indicate that effects on GABA synapses play a role in anesthetic actions, especially for propofol, thiopental and pentobarbital; but the results also indicate that effects on glutamate synapses and postsynaptic membrane excitability contribute to the CNS depression produced by all anesthetics. Given the multiple effects observed for anesthetic actions on the two types of synapses studied here, it is likely that effects on other neurotransmitter systems also contribute to anesthetic-induced depression of the CNS.in vivoin vivoMethods Male Sprague-Dawley rats were anesthetized with ether (22 vol % in air) and the brain was rapidly removed and placed in ice cold (5°C) and pregassed (95/5 % O 2 /CO 2 , carbogen) artificial cerebral spinal fluid (ACSF). The ACSF had the following composition (in mM): Na 151.25; K 2.5; Ca 2.0; Mg 2.0; Cl 131.5; HCO 3  26.0; SO 4  2.0; H 2 PO 4  1.25; and glucose 10. Whole brain coronal slices (450 μm) were cut using a vibratome (Campden Instruments), following careful removal of the dura and pia membranes. Hemisected brain slices were equilibrated for at least one hour at room temperature in an incubation chamber filled with ACSF and continually bubbled with carbogen. Individual slices were transferred to a recording chamber and equilibrated for an additional 10 minutes prior to electrophysiological recording. Oxygenated ACSF solution was continuously perfused through the chamber at a flow rate of 3.0 ml/min and maintained at 22 ± 1°C. The present studies were carried out at room temperature because synaptic responses recorded from cooler brain slices exhibit considerably better baseline stability and the tissue remains viable for many more hours  in vitro  compared to slices maintained at physiological temperatures. Room temperature also facilitates the use of submerged preparations (oxygen solubility and delivery to slices is increased), which allows the use of 60× optics to visualize single neurons for the patch clamp recordings used in some experiments. Previous studies comparing both volatile and intravenous anesthetic effects at physiological and cooler temperatures in brain slices found that there were no apparent differences in effects [ 43 , 61 , 66 ]. The most important effect of lower temperature is to increase the aqueous solubility of the volatile anesthetics and previous work from our laboratory has described in detail the solubility changes observed at 22  vs . 35°C and our methods for measuring and compensating for changed aqueous solubility, as well as the remarkably similar physiological responses recorded from brain slices at these two temperatures [ 43 , 66 ]. To measure population spikes, bipolar tungsten microelectrodes were placed on Schaffer-collateral fibers to electrically stimulate inputs to hippocampal CA1 pyramidal neurons. Glass recording electrodes filled with ACSF (2 to 5 KOhm) were placed in stratum pyramidale to record stimulus-evoked population spike field potentials, or in stratum radiatum to record field EPSPs. Single stimulus pulses (0.01 to 0.05 ms duration; 10 to 80 μA @ 1.0 to 5.0 V) were delivered via constant current isolation units (Grass Instruments, SIU 6D) from a Grass S8800 two channel stimulator; at stimulus rates of 0.05 Hz. Field potential signals were amplified (× 1000), filtered (1 Hz to 10 KHz, bandpass), conditioned (DC offset), and digitally stored for later analysis (A/D with 20 μs resolution on a 486, and 50 MHz microcomputer using Data Wave Systems Corp. or Strathclyde Electrophysiological software). Whole cell patch-clamp recordings were made using thin-walled borosilicate capillaries (1.5 mm O.D.) pulled in two stages on a Narishige PP83 pipette puller. Patch electrodes were filled with the following intracellular solution (in mM): potassium gluconate or CsCl2 – 100, EGTA – 10, MgCl2 – 5, HEPES free acid – 40, ATP disodium salt – 0.3, and GTP sodium salt -0.3. The electrode solution also contained the local anesthetic QX 314 (1.0 mM) in some experiments, to prevent action potential discharge that would contaminate recordings of IPSCs. Electrode solutions were filtered and pH adjusted to 7.2 using KOH or CsOH and had a final osmolarity of 260 to 270 mOSM. Patch electrodes with a DC resistance of 4 to 5 MOhm were used. Recordings were made using an Axoclamp 2A preamplifier (Axon Instruments) in single electrode voltage clamp mode with > 80 % series resistance compensation and > 5 GOhm seals. Patch-clamp current signals were filtered (0.1 Hz to 10 KHz, bandpass), amplified (× 100) and digitized (10 KHz) for storage and analysis. Frequency and amplitudes of IPSCs were analyzed using Data Wave Technologies and Strathclyde Electrophysiological software in a continuous data recording configuration. The intravenous anesthetics (propofol and pentobarbital) were made fresh for each experiment, solubilized using 0.5% dimethyl sulfoxide (DMSO) and sonicated immediately prior to test administration in stock solutions and serially diluted into ACSF to achieve the final concentrations for testing. Volatile anesthetics (halothane and isoflurane) were applied in the perfusate at equilibrated concentrations, delivered from calibrated vaporizers and bubbled into the perfusate for at least 10 min prior to switching from control ACSF, to ensure steady-state concentrations were achieved. The concentration of volatile anesthetics in the gas phase were continually measured using a Puritan-Bennett anesthetic monitor. Only a single concentration of a given anesthetic was tested on each brain slice. Data are expressed as the mean ± standard deviation and statistical analysis (ANOVA with post Tukey test) was performed using Instat from GraphPad Software. For drug effects on paired pulse inhibition, the percent change was first calculated as: Drug 1st/Control 1st = (0.5 × 100) - 100 % = 50 % depression; and Drug 2nd/Control 2nd = (X × 100) - 100 % = X % depression. Then the percent increase in paired pulse inhibition was = (X / 50) × 100 %. This approach has the advantage of normalizing paired responses with respect to varying degrees of population spike facilitation observed during control recordings, i.e. differing degrees of EPSP facilitation on a background of differing degrees of inhibition from preparation to preparation.
Methods
Male Sprague-Dawley rats were anesthetized with ether (22 vol % in air) and the brain was rapidly removed and placed in ice cold (5°C) and pregassed (95/5 % O 2 /CO 2 , carbogen) artificial cerebral spinal fluid (ACSF). The ACSF had the following composition (in mM): Na 151.25; K 2.5; Ca 2.0; Mg 2.0; Cl 131.5; HCO 3  26.0; SO 4  2.0; H 2 PO 4  1.25; and glucose 10. Whole brain coronal slices (450 μm) were cut using a vibratome (Campden Instruments), following careful removal of the dura and pia membranes. Hemisected brain slices were equilibrated for at least one hour at room temperature in an incubation chamber filled with ACSF and continually bubbled with carbogen. Individual slices were transferred to a recording chamber and equilibrated for an additional 10 minutes prior to electrophysiological recording. Oxygenated ACSF solution was continuously perfused through the chamber at a flow rate of 3.0 ml/min and maintained at 22 ± 1°C. The present studies were carried out at room temperature because synaptic responses recorded from cooler brain slices exhibit considerably better baseline stability and the tissue remains viable for many more hours  in vitro  compared to slices maintained at physiological temperatures. Room temperature also facilitates the use of submerged preparations (oxygen solubility and delivery to slices is increased), which allows the use of 60× optics to visualize single neurons for the patch clamp recordings used in some experiments. Previous studies comparing both volatile and intravenous anesthetic effects at physiological and cooler temperatures in brain slices found that there were no apparent differences in effects [ 43 , 61 , 66 ]. The most important effect of lower temperature is to increase the aqueous solubility of the volatile anesthetics and previous work from our laboratory has described in detail the solubility changes observed at 22  vs . 35°C and our methods for measuring and compensating for changed aqueous solubility, as well as the remarkably similar physiological responses recorded from brain slices at these two temperatures [ 43 , 66 ].in vitrovsTo measure population spikes, bipolar tungsten microelectrodes were placed on Schaffer-collateral fibers to electrically stimulate inputs to hippocampal CA1 pyramidal neurons. Glass recording electrodes filled with ACSF (2 to 5 KOhm) were placed in stratum pyramidale to record stimulus-evoked population spike field potentials, or in stratum radiatum to record field EPSPs. Single stimulus pulses (0.01 to 0.05 ms duration; 10 to 80 μA @ 1.0 to 5.0 V) were delivered via constant current isolation units (Grass Instruments, SIU 6D) from a Grass S8800 two channel stimulator; at stimulus rates of 0.05 Hz. Field potential signals were amplified (× 1000), filtered (1 Hz to 10 KHz, bandpass), conditioned (DC offset), and digitally stored for later analysis (A/D with 20 μs resolution on a 486, and 50 MHz microcomputer using Data Wave Systems Corp. or Strathclyde Electrophysiological software).Whole cell patch-clamp recordings were made using thin-walled borosilicate capillaries (1.5 mm O.D.) pulled in two stages on a Narishige PP83 pipette puller. Patch electrodes were filled with the following intracellular solution (in mM): potassium gluconate or CsCl2 – 100, EGTA – 10, MgCl2 – 5, HEPES free acid – 40, ATP disodium salt – 0.3, and GTP sodium salt -0.3. The electrode solution also contained the local anesthetic QX 314 (1.0 mM) in some experiments, to prevent action potential discharge that would contaminate recordings of IPSCs. Electrode solutions were filtered and pH adjusted to 7.2 using KOH or CsOH and had a final osmolarity of 260 to 270 mOSM. Patch electrodes with a DC resistance of 4 to 5 MOhm were used. Recordings were made using an Axoclamp 2A preamplifier (Axon Instruments) in single electrode voltage clamp mode with > 80 % series resistance compensation and > 5 GOhm seals. Patch-clamp current signals were filtered (0.1 Hz to 10 KHz, bandpass), amplified (× 100) and digitized (10 KHz) for storage and analysis. Frequency and amplitudes of IPSCs were analyzed using Data Wave Technologies and Strathclyde Electrophysiological software in a continuous data recording configuration.The intravenous anesthetics (propofol and pentobarbital) were made fresh for each experiment, solubilized using 0.5% dimethyl sulfoxide (DMSO) and sonicated immediately prior to test administration in stock solutions and serially diluted into ACSF to achieve the final concentrations for testing. Volatile anesthetics (halothane and isoflurane) were applied in the perfusate at equilibrated concentrations, delivered from calibrated vaporizers and bubbled into the perfusate for at least 10 min prior to switching from control ACSF, to ensure steady-state concentrations were achieved. The concentration of volatile anesthetics in the gas phase were continually measured using a Puritan-Bennett anesthetic monitor. Only a single concentration of a given anesthetic was tested on each brain slice.Data are expressed as the mean ± standard deviation and statistical analysis (ANOVA with post Tukey test) was performed using Instat from GraphPad Software. For drug effects on paired pulse inhibition, the percent change was first calculated as: Drug 1st/Control 1st = (0.5 × 100) - 100 % = 50 % depression; and Drug 2nd/Control 2nd = (X × 100) - 100 % = X % depression. Then the percent increase in paired pulse inhibition was = (X / 50) × 100 %. This approach has the advantage of normalizing paired responses with respect to varying degrees of population spike facilitation observed during control recordings, i.e. differing degrees of EPSP facilitation on a background of differing degrees of inhibition from preparation to preparation.Authors' contributions SP and AMH contributed equally to this study by conducting most of the experimental work and they also contributed to data analysis and helped with figure preparation. MBM conceived of the study and contributed to experimental design, data analysis and interpretation of results; as well as wrote and prepared the manuscript.
Authors' contributions
SP and AMH contributed equally to this study by conducting most of the experimental work and they also contributed to data analysis and helped with figure preparation. MBM conceived of the study and contributed to experimental design, data analysis and interpretation of results; as well as wrote and prepared the manuscript.


Reinforcement - a process that helps prevent interbreeding between hybridising populations - is an important and little understood mechanism driving the completion of speciation

Reinforcement, like sympatric speciation (see  Box 1 ), has charisma. Evolutionary biologists are still deeply uncertain about how often these processes take place, and hence how important they are in explaining the biological diversity we see today. Empirical and theoretical support for both ideas has waxed and waned over recent decades. Yet both ideas have consistently garnered an unusual amount of attention.Box 1. Glossary
 Sympatry —Area(s) of overlap in the ranges of populations, enabling potential interbreeding.
 Allopatry —Area(s) of population ranges that do not overlap with one another, preventing interbreeding.
 Sympatric speciation —Speciation that occurs within a range of sympatry.
 Reinforcement —The evolution of mechanisms that prevent interbreeding between newly interacting incipient species, as a result of selection against hybrids (narrow definition) or interspecific matings (broad definition) (See  Figure 1 ).
 Conspecific sperm precedence —Disproportional fertilization of a female by sperm of a conspecific male, when that female has mated with both conspecific and heterospecific males.
Box 1. Glossary
Sympatry —Area(s) of overlap in the ranges of populations, enabling potential interbreeding.SympatryAllopatry —Area(s) of population ranges that do not overlap with one another, preventing interbreeding.AllopatrySympatric speciation —Speciation that occurs within a range of sympatry.Sympatric speciationReinforcement —The evolution of mechanisms that prevent interbreeding between newly interacting incipient species, as a result of selection against hybrids (narrow definition) or interspecific matings (broad definition) (See  Figure 1 ).ReinforcementConspecific sperm precedence —Disproportional fertilization of a female by sperm of a conspecific male, when that female has mated with both conspecific and heterospecific males.Conspecific sperm precedenceMuch of the appeal of both reinforcement and sympatric speciation lies in the way they unite micro- and macroevolution. Reinforcement, a concept popularized by  Dobzhansky (1937) , is a process by which speciation, a macroevolutionary process, can be driven directly by natural selection, one of the primary microevolutionary forces. Sympatric speciation can make the same claim. Because of this close linkage between the concepts, the study of one can tell us a great deal about the other (see  Kirkpatrick and Ravigné 2002 ). Such studies can also reveal a lot about the general role of microevolution in species divergence.Reinforcement provides a pathway toward the completion of the speciation process. Imagine that two divergent populations (potentially even classified as separate species) come into contact after a period of allopatry ( Figure 1 ). If the populations have been apart for a long time, evolved differences between them will cause a certain degree of incompatibility when the populations come together. Often, this incompatibility comes in the form of low hybrid fitness (postzygotic isolation) or mismatched mating characteristics (premating isolation). The degree of the development of these isolating mechanisms is roughly proportional to the genetic distance between the populations, reflecting the fact that incompatibilities accumulate over time ( Coyne and Orr 1989 ).If the isolating mechanisms between these populations are only partially complete, extensive hybridization may occur. This can result in fusion back into a single population, or in the swamping of one population's gene pool by the genes of the other (extinction). But there is another possibility, one that can cause the speciation between the two populations to proceed. Remember that if the populations have been separated for long enough, it is likely that hybrids between them will have relatively low fitness. Individuals who mate with members of the opposing population will therefore produce offspring of poor quality, and hence have lower fitness than individuals that mate within their own population. This favors the evolution (or further divergence) of characteristics that cause mating within, rather than between, populations ( Figure 1C ). Speciation between the populations is driven further towards completion through this increase in premating isolation.This process, the evolution of premating isolation after secondary contact due to selection against hybrids, is reinforcement sensu  Dobzhansky (1937) . Recent authors have broadened the definition of reinforcement to include as a driving force any form of selection against mating between populations (e.g.,  Servedio and Noor 2003 ). This could include, for example, lower fertility, or higher mortality of females that mate with members of other populations. In all definitions, however, the microevolutionary process of selection is essential for reinforcement. In fact, in reinforcement, speciation itself can be thought of as an adaptive response to selection. It is little wonder that this causal linking of micro- and macroevolution has appeal for many evolutionary biologists.Reinforcement in the 21st Century Despite the substantial progress in our understanding of reinforcement that has been achieved over the last few decades, many questions remain about the process. These questions lend themselves to exploration by a broad variety of disciplines (evolution, ecology, behavior, phylogenetics, phylogeography, genetics), approaches (experimental, observational, comparative, theoretical) and taxonomic systems. Doubtless, the most important unanswered question about reinforcement is how often it occurs. It is very difficult to prove that reinforcement is occurring, or has occurred, between two species. Reinforcement occasionally leaves a signature, called reproductive character displacement, in which mating characteristics have diverged between populations in areas of sympatry but not areas of allopatry ( Figure 2 ) (the relationship between reinforcement and reproductive character displacement, and controversy over the definition of the latter, is reviewed in  Howard 1993 ). In sympatric areas, populations are capable of producing hybrids, which drives reinforcement, while in allopatry hybrid production, and hence the selection for reinforcement, is absent. Reproductive character displacement has been found to be common, suggesting to some that reinforcement may be common as well ( Howard 1993 ). It is universally acknowledged, however, both that reproductive character displacement can be caused by processes other than reinforcement, and that reinforcement can occur without leaving this signature (e.g., when population ranges are completely sympatric). Proving that reinforcement has occurred requires the ruling out of several alternative hypotheses, which are themselves difficult to assess ( Noor 1999 ;  Coyne and Orr 2004 ). Figure 2 The Pattern of Reproductive Character Displacement (A) Reproductive character displacement due to the presence of an area of overlap between two populations (differences in mating characteristics are represented by color changes, with the hatched area showing divergence in sympatry). Reproductive character displacement can also occur when the sympatric and allopatric areas are not contiguous. (B) Reproductive character displacement can appear as a cline in mating cues or mating preferences (y-axis), with divergence originating in the area of sympatry and spreading into areas of allopatry. Several isolated examples of reinforcement between specific pairs of species have been demonstrated, fairly conclusively, in a variety of taxa including  Drosophila pseudoobscura  and  D. persimilis  ( Noor 1995 ), flycatchers ( Sætre et al. 1997 ), sticklebacks (e.g.,  Rundle and Schluter 1998 ), spadefoot toads ( Pfennig 2003 ), and walking-stick insects ( Nosil et al. 2003 ) (see also reviews of  Noor 1999 ;  Coyne and Orr 2004 ). These studies involve a variety of behavioral tests of mate choice, analyses of hybrid fitness and the production of hybrids in the wild, and controls for alternative explanations. While examples such as these provide essential information about reinforcement, their slow rate of compilation and biased reporting do not provide efficient ways to assess how often reinforcement occurs in general. Comparative approaches, which examine patterns across a broader taxonomic group, can also provide support for reinforcement without these detailed mechanistic analyses (review in  Coyne and Orr 2004 ). The revival of reinforcement in the late 1980s began with one such study in the genus  Drosophila  ( Coyne and Orr 1989 ). By comparing patterns across a wide number of species, such studies can give a better assessment of the potential frequency with which reinforcement occurs—without, however, providing conclusive evidence for reinforcement between specific species pairs. Another area where further research is essential is the determination of which biological factors promote reinforcement, as opposed to population fusion. Theoretical studies, using mathematical models and computer simulations, are proving useful in pinpointing the effects of many factors such as migration rates and patterns, the type of selection against interspecific mating, and the genetic basis of premating isolation (reviews in  Turelli et al. 2001 ;  Servedio and Noor 2003 ). Fortunately, some of the cases of reinforcement in specific species pairs are now being developed to the point where they can address similar questions (e.g., sex linkage of mating genes;  Sætre et al. 2003 ). Both theoretical studies and these well developed empirical systems are also starting to address a third important area of research: how reinforcement interacts with other forces, such as ecological selection pressures, that promote speciation (e.g.,  Servedio 2004 ;  Nosil et al. 2003 ). These integrated studies are essential to the correct placement of reinforcement within the bigger context of speciation processes. In recent years, exciting developments have started to take place in the analysis of the genetics of reinforcement (reviewed in  Servedio and Noor 2003 ). These developments both parallel and overlap with progress made on the genetics of speciation and species differences in general. For example, significant progress has recently been made in identifying the genetic control of hybrid incompatibilities (e.g.,  Presgraves et al. 2003 ;  Barbash et al. 2003 ). This progress has been accompanied by a new understanding of how chromosomal rearrangements may allow these incompatibilities to be maintained despite hybridization in sympatry ( Rieseberg 2001 ;  Navarro and Barton 2003 ;  Brown et al. 2004 ). Sympatric maintenance of incompatibilities, of course, has profound implications for reinforcement, which requires these incompatibilities as the force driving divergence ( Noor et al. 2001 ). Genetic analysis is also allowing a new understanding of the mechanisms by which reinforcement might be taking place in specific cases. Work by  Ortiz-Barrientos et al. (2004)  in this issue of  PLoS Biology  illustrates the extent of the insights that can be made with this approach. Using high-resolution genetic mapping the authors have identified the locations of genes that cause increased discrimination against  Drosophila persimilis  males by  D. pseudoobscura  females, due to reinforcement in sympatry. Surprisingly, these genes map to very different areas of the chromosomes than do genes that cause a basal level of mating discrimination between the species in allopatry. Among other insights, the position of these genes suggests that the reinforced discrimination is based on odor, not on the mechanism used in allopatry, male song. This leads to the novel conclusion that reinforcement is not just increasing the strength of an already existing mechanism of species discrimination, but is occurring through the development of a new discrimination system. These kinds of developments can also motivate more realistic theoretical models of the reinforcement process.
Reinforcement in the 21st Century
Despite the substantial progress in our understanding of reinforcement that has been achieved over the last few decades, many questions remain about the process. These questions lend themselves to exploration by a broad variety of disciplines (evolution, ecology, behavior, phylogenetics, phylogeography, genetics), approaches (experimental, observational, comparative, theoretical) and taxonomic systems.Doubtless, the most important unanswered question about reinforcement is how often it occurs. It is very difficult to prove that reinforcement is occurring, or has occurred, between two species. Reinforcement occasionally leaves a signature, called reproductive character displacement, in which mating characteristics have diverged between populations in areas of sympatry but not areas of allopatry ( Figure 2 ) (the relationship between reinforcement and reproductive character displacement, and controversy over the definition of the latter, is reviewed in  Howard 1993 ). In sympatric areas, populations are capable of producing hybrids, which drives reinforcement, while in allopatry hybrid production, and hence the selection for reinforcement, is absent. Reproductive character displacement has been found to be common, suggesting to some that reinforcement may be common as well ( Howard 1993 ). It is universally acknowledged, however, both that reproductive character displacement can be caused by processes other than reinforcement, and that reinforcement can occur without leaving this signature (e.g., when population ranges are completely sympatric). Proving that reinforcement has occurred requires the ruling out of several alternative hypotheses, which are themselves difficult to assess ( Noor 1999 ;  Coyne and Orr 2004 ).Several isolated examples of reinforcement between specific pairs of species have been demonstrated, fairly conclusively, in a variety of taxa including  Drosophila pseudoobscura  and  D. persimilis  ( Noor 1995 ), flycatchers ( Sætre et al. 1997 ), sticklebacks (e.g.,  Rundle and Schluter 1998 ), spadefoot toads ( Pfennig 2003 ), and walking-stick insects ( Nosil et al. 2003 ) (see also reviews of  Noor 1999 ;  Coyne and Orr 2004 ). These studies involve a variety of behavioral tests of mate choice, analyses of hybrid fitness and the production of hybrids in the wild, and controls for alternative explanations.While examples such as these provide essential information about reinforcement, their slow rate of compilation and biased reporting do not provide efficient ways to assess how often reinforcement occurs in general. Comparative approaches, which examine patterns across a broader taxonomic group, can also provide support for reinforcement without these detailed mechanistic analyses (review in  Coyne and Orr 2004 ). The revival of reinforcement in the late 1980s began with one such study in the genus  Drosophila  ( Coyne and Orr 1989 ). By comparing patterns across a wide number of species, such studies can give a better assessment of the potential frequency with which reinforcement occurs—without, however, providing conclusive evidence for reinforcement between specific species pairs.Another area where further research is essential is the determination of which biological factors promote reinforcement, as opposed to population fusion. Theoretical studies, using mathematical models and computer simulations, are proving useful in pinpointing the effects of many factors such as migration rates and patterns, the type of selection against interspecific mating, and the genetic basis of premating isolation (reviews in  Turelli et al. 2001 ;  Servedio and Noor 2003 ). Fortunately, some of the cases of reinforcement in specific species pairs are now being developed to the point where they can address similar questions (e.g., sex linkage of mating genes;  Sætre et al. 2003 ). Both theoretical studies and these well developed empirical systems are also starting to address a third important area of research: how reinforcement interacts with other forces, such as ecological selection pressures, that promote speciation (e.g.,  Servedio 2004 ;  Nosil et al. 2003 ). These integrated studies are essential to the correct placement of reinforcement within the bigger context of speciation processes.In recent years, exciting developments have started to take place in the analysis of the genetics of reinforcement (reviewed in  Servedio and Noor 2003 ). These developments both parallel and overlap with progress made on the genetics of speciation and species differences in general. For example, significant progress has recently been made in identifying the genetic control of hybrid incompatibilities (e.g.,  Presgraves et al. 2003 ;  Barbash et al. 2003 ). This progress has been accompanied by a new understanding of how chromosomal rearrangements may allow these incompatibilities to be maintained despite hybridization in sympatry ( Rieseberg 2001 ;  Navarro and Barton 2003 ;  Brown et al. 2004 ). Sympatric maintenance of incompatibilities, of course, has profound implications for reinforcement, which requires these incompatibilities as the force driving divergence ( Noor et al. 2001 ).Genetic analysis is also allowing a new understanding of the mechanisms by which reinforcement might be taking place in specific cases. Work by  Ortiz-Barrientos et al. (2004)  in this issue of  PLoS Biology  illustrates the extent of the insights that can be made with this approach. Using high-resolution genetic mapping the authors have identified the locations of genes that cause increased discrimination against  Drosophila persimilis  males by  D. pseudoobscura  females, due to reinforcement in sympatry. Surprisingly, these genes map to very different areas of the chromosomes than do genes that cause a basal level of mating discrimination between the species in allopatry. Among other insights, the position of these genes suggests that the reinforced discrimination is based on odor, not on the mechanism used in allopatry, male song. This leads to the novel conclusion that reinforcement is not just increasing the strength of an already existing mechanism of species discrimination, but is occurring through the development of a new discrimination system. These kinds of developments can also motivate more realistic theoretical models of the reinforcement process.PLoS BiologyImplications and Extensions of Reinforcement What if, when our assessment of the frequency of reinforcement is improved, it turns out to have been a rare occurrence in the generation of current biological diversity? The study of reinforcement is broad and varied enough that many of our findings about the process would still have wide-reaching implications. First, recall the claim, at the start of this article, that studying reinforcement reveals much about the role of microevolution in the macroevolutionary process of speciation. Knowledge gained about this relationship is not only directly applicable to the very similar process of sympatric speciation, but can also tell us a great deal about speciation caused by ecological adaptation and sexual selection, which are critical components of reinforcement in many systems (e.g.,  Nosil et al. 2003 ;  Haavie et al. 2004 ). Studies looking for reinforcement have also led to insights into the formation and maintenance of hybrid zones (e.g.,  Butlin 1998 ;  Britch et al. 2001 ). Situations where reinforcement fails to occur likewise teach a lesson, elucidating possible mechanisms of extinction when secondary contact occurs between species. Second, analysis of reinforcement clarifies the interactions between levels of reproductive isolation that occur at different stages in the life cycle. Reinforcement, broadly defined, can be driven by isolation at the postzygotic level or by incompatibilities that occur between mating and zygote production (postmating-prezygotic incompatibilities;  Servedio 2001 ). Postzygotic isolation can likewise cause divergence at the premating stage (reinforcement) or potentially at the postmating-prezygotic stage, through the evolution of conspecific sperm precedence ( Marshall et al. 2002 ). These various stages of isolation have different degrees of importance among plants, free-spawning marine invertebrates, and other internally and externally fertilizing animals ( Bernasconi et al. 2004 ). Analysis of these stages of isolation, their interactions, and the evolutionary pressures they are under therefore has broad implications for comparative reproductive biology across these varied groups. Finally, regardless of whether reinforcement has been a common pathway in speciation, its relevance may be increasing. Reinforcement is a possible outcome anytime species that are capable of hybridization come into contact. Human activity is increasing the incidence of secondary contact by altering habitat and introducing invasive species. This contact often results in hybridization (reviews in  Rhymer and Simberloff 1996 ;  Mooney and Cleland 2001 ). It is important to identify and understand the properties of species pairs that make extensive introgression, extinction, stable hybrid zones, or reinforcement likely outcomes of such contact. If reinforcement has played a small role in the generation of current diversity, it may be because secondary contact itself has historically been a rare occurrence. It is the frequency of reinforcement among incidences of secondary contact that will determine its importance in the near future.
Implications and Extensions of Reinforcement
What if, when our assessment of the frequency of reinforcement is improved, it turns out to have been a rare occurrence in the generation of current biological diversity? The study of reinforcement is broad and varied enough that many of our findings about the process would still have wide-reaching implications.First, recall the claim, at the start of this article, that studying reinforcement reveals much about the role of microevolution in the macroevolutionary process of speciation. Knowledge gained about this relationship is not only directly applicable to the very similar process of sympatric speciation, but can also tell us a great deal about speciation caused by ecological adaptation and sexual selection, which are critical components of reinforcement in many systems (e.g.,  Nosil et al. 2003 ;  Haavie et al. 2004 ). Studies looking for reinforcement have also led to insights into the formation and maintenance of hybrid zones (e.g.,  Butlin 1998 ;  Britch et al. 2001 ). Situations where reinforcement fails to occur likewise teach a lesson, elucidating possible mechanisms of extinction when secondary contact occurs between species.Second, analysis of reinforcement clarifies the interactions between levels of reproductive isolation that occur at different stages in the life cycle. Reinforcement, broadly defined, can be driven by isolation at the postzygotic level or by incompatibilities that occur between mating and zygote production (postmating-prezygotic incompatibilities;  Servedio 2001 ). Postzygotic isolation can likewise cause divergence at the premating stage (reinforcement) or potentially at the postmating-prezygotic stage, through the evolution of conspecific sperm precedence ( Marshall et al. 2002 ). These various stages of isolation have different degrees of importance among plants, free-spawning marine invertebrates, and other internally and externally fertilizing animals ( Bernasconi et al. 2004 ). Analysis of these stages of isolation, their interactions, and the evolutionary pressures they are under therefore has broad implications for comparative reproductive biology across these varied groups.Finally, regardless of whether reinforcement has been a common pathway in speciation, its relevance may be increasing. Reinforcement is a possible outcome anytime species that are capable of hybridization come into contact. Human activity is increasing the incidence of secondary contact by altering habitat and introducing invasive species. This contact often results in hybridization (reviews in  Rhymer and Simberloff 1996 ;  Mooney and Cleland 2001 ). It is important to identify and understand the properties of species pairs that make extensive introgression, extinction, stable hybrid zones, or reinforcement likely outcomes of such contact. If reinforcement has played a small role in the generation of current diversity, it may be because secondary contact itself has historically been a rare occurrence. It is the frequency of reinforcement among incidences of secondary contact that will determine its importance in the near future.