Free
Meeting Abstracts  |   May 2005
Sevoflurane Blocks Cholinergic Synaptic Transmission Postsynaptically but Does Not Affect Short-term Potentiation
Author Affiliations & Notes
  • Hiroaki Naruo, M.D.
    *
  • Shin Onizuka, M.D.
  • David Prince, M.Sc.
  • Mayumi Takasaki, M.D., Ph.D.
    §
  • Naweed I. Syed, Ph.D.
  • * Research Associate, § Professor, Department of Anesthesiology, Miyazaki Medical College. † Research Associate, ‡ Graduate Student, ∥ Professor, Cellular and Molecular Neurobiology Research Group, Faculty of Medicine, University of Calgary.
Article Information
Meeting Abstracts   |   May 2005
Sevoflurane Blocks Cholinergic Synaptic Transmission Postsynaptically but Does Not Affect Short-term Potentiation
Anesthesiology 5 2005, Vol.102, 920-928. doi:
Anesthesiology 5 2005, Vol.102, 920-928. doi:
THE inhalation anesthetics required during most surgical procedures affect both excitatory and inhibitory synaptic transmission in the nervous system.1 These effects involve either the suppression of presynaptic transmitter release2 or a modulation of postsynaptic receptors.3,4 In contrast to their well-defined actions on both γ-aminobutyric acid–mediated5 and glutamatergic6 synapses, less understood are their effects on cholinergic transmission,7,8 which is thought to be involved in learning and memory in the hippocampus.
Inhalation and intravenous anesthetics are both thought to impair memory and exhibit potent amnesic properties. For example, patients who followed instructions intraoperatively did not recall such events on recovery.9,10 Similarly, other studies have demonstrated that both implicit and explicit memory states in humans are affected to some degree, by a varying state of anesthesia.11 Reinsel et al.  12 have also demonstrated that memory is impaired during conscious sedation. From both psychologists’13 and anesthesiologists’14 points of view, memory for events during anesthesia has not been demonstrated.15 In contrast, numerous other studies have found no effect of anesthetics on various types of memories.16 For example, not only is the brain able to process auditory information during anesthesia,17–21 but also the cognitive functions required for memory remain unperturbed.22 
The above-cited examples provide ample reasoning to conclude that the issue whether anesthetics affect memory formation and retention at the cellular and network level remains polemical. This lack of fundamental knowledge in the field of anesthesiology vis-à-vis  synaptic plasticity stems from the complex nature of the mammalian brain, where cell–cell interactions between well-defined sets of functionally identified, presynaptic and postsynaptic neurons can not be studied directly.
Here, we demonstrate that the clinically relevant concentrations of sevoflurane affect cholinergic synaptic transmission between well-defined synaptic partners and that these effects involve postsynaptic acetylcholine receptors. Moreover, we provide direct evidence that despite its effects on synaptic transmission, sevoflurane does not prevent posttetanic potentiation (PTP) at this synapse. Similarly, sevoflurane application after the induction of synaptic plasticity (potentiation paradigm: tetanus in the presynaptic cell) did not prevent the subsequent expression of PTP. Taken together, our data provide the first direct evidence that despite their effects on synaptic transmission, an inhalation anesthetic does not affect synaptic plasticity seen at an excitatory cholinergic synapse.
Materials and Methods
Animals
Laboratory-raised stocks of the fresh water snail Lymnaea stagnalis  were maintained at room temperature (18°–20°C) in well-aerated aquaria and fed lettuces. Animals with shell lengths of 1–15 and 15–25 mm (approximate age, 2–6 months) were used for cell isolation and to prepare the brain conditioned medium, respectively. (Animal care certification is not required for invertebrate species such as L. stagnalis  at the University of Calgary Animal Resource Centre, Calgary, Alberta, Canada).
Neuronal Culture
Identified neurons were isolated from the intact ganglia according to previously, well-established procedures in the laboratory.23,24 In summary, snails were anesthetized with 10% Listerine (Pfizer Canada, Toronto, Ontario, Canada) (21.9% ethanol, 0.042% menthol) solution in normal Lymnaea  saline (containing 51.3 mm NaCl, 1–7 mm KCl, 4.1 mm CaCl2, and 1.5 mm MgCl2). HEPES was used to adjust the pH to 7.9. The central ring ganglia were dissected under sterile conditions as described previously25 and washed in a series of antibiotic saline (50 μg/ml gentamycine; three washes, 10 min each). The antibiotic-treated ganglia were then incubated in 0.2% trypsin (Sigma type III; Sigma Chemical Company, St. Louis, MO) for 20–22 min followed by 0.2% soybean trypsin inhibitor (Sigma type 1-S. Sigma Chemical Company) for 10 min, both in defined medium (DM). DM consisted of serum-free 50% L-15 medium with added inorganic salts at a concentration described above for saline, and the pH was adjusted to 7.9 with 1 N NaOH. As compared with the antibiotic saline, the gentamycine concentration in DM was reduced to 20 μg/ml. The enzyme-pretreated ganglia were pinned down to the bottom of a dissection dish containing 6–10 ml high-osmolarity DM (DM + 37.5 mm glucose). The connective tissue sheath surrounding the ganglia was removed with fine forceps, and the desired neurons were isolated by applying gentle suction to a fire-polished and Sigmacote (Sigma Chemical Company)–treated pipette. The individually extracted cells were plated on poly l-lysine–coated dishes containing brain conditioned medium, which was prepared by incubating central ring ganglia in DM (12 brains/6 ml DM for either 4 or 5 days). The isolated somata were juxtaposed in a soma–soma configuration.26 
Soma–Soma Synapse
Soma–soma synapses were prepared by juxtaposing the isolated somata of identified neurons as described previously.26 Specifically, identified presynaptic neuron (visceral dorsal 4 [VD4]) was isolated and paired with its postsynaptic partner (left pedal dorsal 1 [LPeD1]), and synapses were allowed to develop overnight in conditioned medium.
Electrophysiology
Well-established sharp electrode, intracellular recordings were made as described previously.24 Briefly, glass microelectrodes (1.5-mm internal diameter W/Fil; World Precision Instruments, Sarasota, FL) were fabricated on a vertical electrode puller (Kopf, 700 C; David Kopf Instruments, Tujunga, CA) and filled with a saturated solution of K2SO4(resistance 30–60 mΩ). Isolated neurons were viewed under a Zeiss (Telaval 31; Carl Zeiss Canada Ltd., North York, Ontario, Canada) inverted microscope and impaled using Narishigi micromanipulators (model MO-103; Narishigi Instruments, Tokyo Japan). The intracellular signals were amplified via  a preamplifier (Neurodata model IR-283; Cygus Technology Inc., Delaware, PA), displayed on a storage oscilloscope (Tektronix R5103N; Tektronix, Montreal, Quebec, Canada), and recorded on a chart recorder (Gould; Gould Instrument Systems, Babylon, NY). All experiments were performed at room temperature (18°–22°C). The tetanus comprised 8–10 action potentials, and the posttetanic action potential was delivered after 6 s of the tetanus.
Anesthetic Delivery
Sevoflurane (Maruishi Pharmaceuticals Inc., Osaka, Japan) was vaporized in 100% oxygen using a sevoflurane type-S MKIII-VIII (Acoma Medical Industry Co., Tokyo, Japan) vaporizer and bubbled for at least 15 min into the reservoirs containing Lymnaea  saline.27 To minimize gas loss over time, all anesthetic solutions were prepared fresh in sealed glass reservoirs. Precise anesthetic concentrations were determined by gas chromatographic analysis established previously in our laboratory.27 To minimize gas loss, Teflon tubing was used throughout the perfusion system, and sevoflurane was delivered directly to the somata using a fast perfusion system as described previously.28 Acetylcholine (1 μm) was pressure applied (80-ms pulses, 2–4 psi) directly onto the somata via  a pneumatic PicoPump (PV 800; World Precision Instruments).
FM1-43
The paired somata of presynaptic and postsynaptic neurons were incubated in 20 μm FM1-43 (Molecular Probes; Invitrogen Canada Inc., Burlington, Ontario, Canada) for 10 min before the addition of sevoflurane to the bath. The presynaptic neuron was impaled with a sharp electrode and stimulated to generate 100 action potentials (10 spikes/burst) to allow the uptake of the dye FM1-43 either in the presence or the absence of sevoflurane. FM1-43 and the anesthetic were then replaced with cold saline to prevent neuronal spiking and thus the subsequent loss of the dye, and to remove background fluorescence. Fluorescence images of the FM1-43–labeled cells were acquired using a Zeiss Axiovert 200 M inverted microscope (Carl Zeiss Canada Ltd.), and images were acquired and processed by excitation filters (480/30 nm), dichroic mirror (505 nm), and emission filters (570 LP nm or 610 nm). Both phase and fluorescent images were captured with a Photometrics Sensys (Photometrics, Tuscon, AZ) 1400 camera (1–100 ms exposure) connected to a computer running Axiovision 3.0 for Windows (Carl Zeiss Canada Ltd.).
Statistical Analysis
All parametric data are expressed as mean ± SE, and the significance was determined using analyses of variance with repeated measures. Nonparametric data are expressed as percents and were analyzed for significance using the t  test. Significance was assumed if P  was less than 0.05.
Results
Sevoflurane Suppresses Cholinergic Synaptic Transmission between VD4 and LPeD1
To test for the effects of inhalation anesthetic sevoflurane on synaptic transmission, specific excitatory synapses between VD4 and its postsynaptic partner LPeD1 were reconstructed in a soma–soma configuration. The isolated somata of VD4 and LPeD1 were extracted from visceral and left pedal ganglia, respectively, and paired overnight27 (fig. 1A). Excitatory, cholinergic synapses similar to those observed in vivo  29,30 developed between the paired cells. Specifically, induced action potentials in VD4 generated 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1 (n = 60; fig. 1B).
Fig. 1. Synapse formation between soma–soma paired  Lymnaea  neurons. (  A  ) Individually identifiable, presynaptic (visceral dorsal 4 [VD4]) and postsynaptic (left pedal dorsal 1 [LPeD1]) were soma–soma paired overnight. (  B  ) Specific synapses similar to those seen  in vivo  reformed overnight between the paired cells. An action potential in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials in LPeD1. 
Fig. 1. Synapse formation between soma–soma paired  Lymnaea  neurons. (  A  ) Individually identifiable, presynaptic (visceral dorsal 4 [VD4]) and postsynaptic (left pedal dorsal 1 [LPeD1]) were soma–soma paired overnight. (  B  ) Specific synapses similar to those seen  in vivo  reformed overnight between the paired cells. An action potential in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials in LPeD1. 
Fig. 1. Synapse formation between soma–soma paired  Lymnaea  neurons. (  A  ) Individually identifiable, presynaptic (visceral dorsal 4 [VD4]) and postsynaptic (left pedal dorsal 1 [LPeD1]) were soma–soma paired overnight. (  B  ) Specific synapses similar to those seen  in vivo  reformed overnight between the paired cells. An action potential in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials in LPeD1. 
×
To test whether sevoflurane affects synaptic transmission between VD4 and LPeD1, synapses were tested in either the absence or the presence of sevoflurane (0.5–3%). Sevoflurane delivered through a fast perfusion system directly at the contact site28 suppressed synaptic transmission between VD4 and LPeD1 in a concentration-dependent manner (percent of control: 0.5% = 68.81 ± 8.2, n = 9; 1% = 51.30 ± 8.1, n = 6; 1.5% = 43.35 ± 6.7, n = 5). Specifically, the amplitude of VD4-induced EPSPs in LPeD1 was significantly reduced by all sevoflurane concentrations used (fig. 2A). However, an almost complete blockage of synaptic transmission was achieved at a concentration of 3% (3.13 ± 4.4% of control, n = 5; fig. 2A). In all instances, the synaptic transmission was restored within a few minutes of washout with normal saline (fig. 2A, i–iii, and fig. 2B). Together, these data demonstrate that sevoflurane significantly and reversibly blocks synaptic transmission between VD4 and LPeD1 (percent of control: 0.5% = 94.54 ± 4.47; 1% = 94.06 ± 1.70; 1.5% = 85.53 ± 4.33; 3% = 78.73 ± 3.99; fig. 2B).
Fig. 2. Sevoflurane blocks synaptic transmission between visceral dorsal 4 (VD4) and left pedal dorsal 1 (LPeD1) in a concentration-dependent manner. VD4 and LPeD1 were soma–soma paired overnight, and synaptic transmission was tested electrophysiologically. Action potentials in VD4 (at  arrow  ) induced 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1 (  A  ). The amplitude of VD4-induced EPSP in LPeD1 was significantly suppressed by sevoflurane in a concentration-dependent manner (  A  ,  i    iii  show representative traces of sevoflurane (1.5%)–induced suppression of synaptic transmission and subsequently recovery after washout. Sevoflurane-induced effects were concentration dependent, and the synaptic transmission recovered fully after washout with normal saline (  B  ,  dark bars  ). The postsynaptic cell was current clamped at −80 mV throughout the experiment. 
Fig. 2. Sevoflurane blocks synaptic transmission between visceral dorsal 4 (VD4) and left pedal dorsal 1 (LPeD1) in a concentration-dependent manner. VD4 and LPeD1 were soma–soma paired overnight, and synaptic transmission was tested electrophysiologically. Action potentials in VD4 (at  arrow  ) induced 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1 (  A  ). The amplitude of VD4-induced EPSP in LPeD1 was significantly suppressed by sevoflurane in a concentration-dependent manner (  A  ,  i  –  iii  show representative traces of sevoflurane (1.5%)–induced suppression of synaptic transmission and subsequently recovery after washout. Sevoflurane-induced effects were concentration dependent, and the synaptic transmission recovered fully after washout with normal saline (  B  ,  dark bars  ). The postsynaptic cell was current clamped at −80 mV throughout the experiment. 
Fig. 2. Sevoflurane blocks synaptic transmission between visceral dorsal 4 (VD4) and left pedal dorsal 1 (LPeD1) in a concentration-dependent manner. VD4 and LPeD1 were soma–soma paired overnight, and synaptic transmission was tested electrophysiologically. Action potentials in VD4 (at  arrow  ) induced 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1 (  A  ). The amplitude of VD4-induced EPSP in LPeD1 was significantly suppressed by sevoflurane in a concentration-dependent manner (  A  ,  i    iii  show representative traces of sevoflurane (1.5%)–induced suppression of synaptic transmission and subsequently recovery after washout. Sevoflurane-induced effects were concentration dependent, and the synaptic transmission recovered fully after washout with normal saline (  B  ,  dark bars  ). The postsynaptic cell was current clamped at −80 mV throughout the experiment. 
×
Sevoflurane-induced Synaptic Suppression Does Not Involve Presynaptic Secretory Machinery
To test whether sevoflurane-induced suppression of synaptic transmission between VD4 and LPeD1 involved perturbation of presynaptic secretory machinery, cells were paired overnight (fig. 3). After 18–20 h of pairing, intracellular recordings were made from both cells in either the presence or the absence of sevoflurane plus the dye FM1-43. We reasoned that if sevoflurane affected exocytosis or endocytosis after the stimulation of the presynaptic cell, it would not uptake the dye. FM1-43 was added to the culture dish, and images were acquired first in the absence of the presynaptic activity (fig. 3A); no labeling was observed in VD4 under such control conditions (fig. 3B). VD4 was then stimulated by current injections (5 bursts, 100 action potentials), the dye was washed out with normal saline, and further images were acquired. We found fluorescently labeled puncta of VD4 neuritic processes either at the contact site or around the soma of LPeD1 (fig. 3C). Next, to test whether sevoflurane (3%) blocked exocytosis or endocytosis of cholinergic vesicles, images were first acquired in the presence of FM1-43 plus sevoflurane but in the absence of VD4 activity (figs. 3D and E). VD4 was then stimulated in the presence of sevoflurane plus FM1-43. Similar to the labeling observed under normal conditions (fig. 3C), FM1-43 labeling was discernable at the contact site (fig. 3F), suggesting that sevoflurane affected neither exocytosis nor the endocytotic process. Although these experiments (n = 6/case) do not reveal the qualitative differences between labeling during control (fig. 3C) and anesthetic conditions (fig. 3F), they do suggest that the sevoflurane-induced suppression of synaptic transmission seen previously (fig. 2) may not involve presynaptic machinery.
Fig. 3. Sevoflurane does not affect the presynaptic secretory machinery. To test whether sevoflurane-induced suppression of synaptic transmission between visceral dorsal 4 (VD4) and left pedal dorsal 1 (LPeD1) was due to the perturbation of presynaptic release machinery, synapses were tested in either the absence or the presence of 3% sevoflurane (depresses the synapse almost completely) and the dye FM1-43. Neurons were paired overnight and allowed to develop synapses. Synapses developed at the contact site between the pairs, and VD4 processes were seen often unsheathing the LPeD1 somata (  arrows  ,  A  and  D  ). To demonstrate the pattern of dye localization in a control VD4, the cell was impaled intracellularly, and the dye was added to the preparation. VD4 was prevented from spiking by injecting hyperpolarizing current (0.2 nA) for 10 min, and images were acquired. No staining was discernable in VD4 (  B  ). The presynaptic cell was then stimulated (100 action potentials) by current pulses, and the dye was replaced with normal, cold saline. Images were acquired again, which revealed punctate staining both at the contact site between the cells and also in VD4 processes encircling the LPeD1 somata (  arrows  ,  C  ). The above experiment was repeated in the presence of 3% sevoflurane (  D    F  ). Sevoflurane at a concentration that blocks synaptic transmission almost completely also failed to prevent the uptake of the dye FM1-43, and punctate staining (  F  ) similar to that seen under control (  C  ) conditions was clearly discernable in the presence of this anesthetic. 
Fig. 3. Sevoflurane does not affect the presynaptic secretory machinery. To test whether sevoflurane-induced suppression of synaptic transmission between visceral dorsal 4 (VD4) and left pedal dorsal 1 (LPeD1) was due to the perturbation of presynaptic release machinery, synapses were tested in either the absence or the presence of 3% sevoflurane (depresses the synapse almost completely) and the dye FM1-43. Neurons were paired overnight and allowed to develop synapses. Synapses developed at the contact site between the pairs, and VD4 processes were seen often unsheathing the LPeD1 somata (  arrows  ,  A  and  D  ). To demonstrate the pattern of dye localization in a control VD4, the cell was impaled intracellularly, and the dye was added to the preparation. VD4 was prevented from spiking by injecting hyperpolarizing current (0.2 nA) for 10 min, and images were acquired. No staining was discernable in VD4 (  B  ). The presynaptic cell was then stimulated (100 action potentials) by current pulses, and the dye was replaced with normal, cold saline. Images were acquired again, which revealed punctate staining both at the contact site between the cells and also in VD4 processes encircling the LPeD1 somata (  arrows  ,  C  ). The above experiment was repeated in the presence of 3% sevoflurane (  D  –  F  ). Sevoflurane at a concentration that blocks synaptic transmission almost completely also failed to prevent the uptake of the dye FM1-43, and punctate staining (  F  ) similar to that seen under control (  C  ) conditions was clearly discernable in the presence of this anesthetic. 
Fig. 3. Sevoflurane does not affect the presynaptic secretory machinery. To test whether sevoflurane-induced suppression of synaptic transmission between visceral dorsal 4 (VD4) and left pedal dorsal 1 (LPeD1) was due to the perturbation of presynaptic release machinery, synapses were tested in either the absence or the presence of 3% sevoflurane (depresses the synapse almost completely) and the dye FM1-43. Neurons were paired overnight and allowed to develop synapses. Synapses developed at the contact site between the pairs, and VD4 processes were seen often unsheathing the LPeD1 somata (  arrows  ,  A  and  D  ). To demonstrate the pattern of dye localization in a control VD4, the cell was impaled intracellularly, and the dye was added to the preparation. VD4 was prevented from spiking by injecting hyperpolarizing current (0.2 nA) for 10 min, and images were acquired. No staining was discernable in VD4 (  B  ). The presynaptic cell was then stimulated (100 action potentials) by current pulses, and the dye was replaced with normal, cold saline. Images were acquired again, which revealed punctate staining both at the contact site between the cells and also in VD4 processes encircling the LPeD1 somata (  arrows  ,  C  ). The above experiment was repeated in the presence of 3% sevoflurane (  D    F  ). Sevoflurane at a concentration that blocks synaptic transmission almost completely also failed to prevent the uptake of the dye FM1-43, and punctate staining (  F  ) similar to that seen under control (  C  ) conditions was clearly discernable in the presence of this anesthetic. 
×
Sevoflurane Blocks Postsynaptic Cholinergic Response in LPeD1
The synaptic transmission between VD4 and LPeD1 has previously been shown to be cholinergic.29,30 To test whether sevoflurane blocks cholinergic response in LPeD1, this neuron was cultured overnight. Intracellular recordings were made, and cholinergic responses were tested either in the presence or absence of various sevoflurane concentrations (0.5–3%). Specifically, cells were current clamped at −65 mV, and acetylcholine (10−6m) was pressure applied under a fast perfusion system28 in either the absence or the presence of sevoflurane. We found that sevoflurane blocked cholinergic responses in LPeD1 in a concentration-dependent (percent of control: 0.5% = 70.88 ± 4.43; 1% = 53.88 ± 4.56; 1.5% = 37.55 ± 4.89; 3% = 5.38 ± 4.83; fig. 4A) and reversible manner (washout percent of control: 0.5% = 95.03 ± 3.1; 1% = 91.72 ± 2.47; 1.5% = 87.46 ± 2.60; 3% = 79.43 ± 3.89; fig. 4A, i–iii, and fig. 4B). Either maximum or almost complete block was observed at a concentration of 3% (n = 11 for all concentrations; fig. 4). These data thus show that the sevoflurane-induced suppression of synaptic transmission between VD4 and LPeD1 likely involves postsynaptic cholinergic receptors.
Fig. 4. Sevoflurane blocks cholinergic response in left pedal dorsal 1 (LPeD1). To test whether sevoflurane-induced suppression of synaptic transmission involved cholinergic postsynaptic receptors, acetylcholine was tested on LPeD1 in either the absence or the presence of sevoflurane. Specifically, acetylcholine (10−5m) was pressure applied to a single or paired LPeD1, and its effects were monitored intracellularly, in either the presence or the absence of various sevoflurane concentrations (  A  ). The cholinergic responses in LPeD1 were significantly depressed by sevoflurane in a concentration-dependent and reversible manner (  A  ,  i    iii  ). These data are summarized in  B  , and the  darker bars  represent washout data. 
Fig. 4. Sevoflurane blocks cholinergic response in left pedal dorsal 1 (LPeD1). To test whether sevoflurane-induced suppression of synaptic transmission involved cholinergic postsynaptic receptors, acetylcholine was tested on LPeD1 in either the absence or the presence of sevoflurane. Specifically, acetylcholine (10−5m) was pressure applied to a single or paired LPeD1, and its effects were monitored intracellularly, in either the presence or the absence of various sevoflurane concentrations (  A  ). The cholinergic responses in LPeD1 were significantly depressed by sevoflurane in a concentration-dependent and reversible manner (  A  ,  i  –  iii  ). These data are summarized in  B  , and the  darker bars  represent washout data. 
Fig. 4. Sevoflurane blocks cholinergic response in left pedal dorsal 1 (LPeD1). To test whether sevoflurane-induced suppression of synaptic transmission involved cholinergic postsynaptic receptors, acetylcholine was tested on LPeD1 in either the absence or the presence of sevoflurane. Specifically, acetylcholine (10−5m) was pressure applied to a single or paired LPeD1, and its effects were monitored intracellularly, in either the presence or the absence of various sevoflurane concentrations (  A  ). The cholinergic responses in LPeD1 were significantly depressed by sevoflurane in a concentration-dependent and reversible manner (  A  ,  i    iii  ). These data are summarized in  B  , and the  darker bars  represent washout data. 
×
Sevoflurane Does Not Affect Posttatanic Potentiation at the VD4–LPeD1 Synapse
To test for the effects of sevoflurane on short-term synaptic plasticity, synapses were reconstructed overnight as described under the heading “Sevoflurane Blocks Postsynaptic Cholinergic Response in LPeD1.” Simultaneous intracellular recordings were made, and synapses were tested electrophysiologically. After a single action potential in VD4 that generated 1:1 EPSPs in LPeD1, a tetanus (8–10 action potentials) was delivered to VD4. Subsequent action potentials in VD4 delivered within a few seconds of the tetanus resulted in postsynaptic potentiation that only lasted for a few seconds (n = 20; fig. 5). To test whether sevoflurane affects this PTP, the above experiment was conducted in the presence of 1.5–3.0% sevoflurane. We discovered that although 1.5% sevoflurane reduced synaptic transmission between VD4 and LPeD1 to approximately 50%, the ratio between pretetanic and posttetanic EPSPs did not change (fig. 6), even when the synapse was tested in the presence of 3% sevoflurane (which almost completely blocked the synaptic transmission between the cells; fig. 6B). These data thus demonstrate that sevoflurane does not affect the genesis of PTP at this synapse.
Fig. 5. Visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse exhibits short-term potentiation. The soma–soma paired cells were simultaneously impaled intracellularly, and synaptic transmission was tested electrophysiologically. (  A  ) Action potentials in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1. The amplitude of first EPSP was measured, and a tetanus was delivered to VD4 (at  asterisk  , 10–10 action potentials), which resulted in a compound postsynaptic potential (PSP) in LPeD1. The subsequent action potential in VD4 (posttetanus) resulted in a few hundred percent enhancement of EPSPs amplitude in LPeD1, which gradually returned to its baseline within seconds. (  B  ) Summary data depicting the percent increase in the amplitude of posttetanic EPSPs in LPeD1. 
Fig. 5. Visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse exhibits short-term potentiation. The soma–soma paired cells were simultaneously impaled intracellularly, and synaptic transmission was tested electrophysiologically. (  A  ) Action potentials in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1. The amplitude of first EPSP was measured, and a tetanus was delivered to VD4 (at  asterisk  , 10–10 action potentials), which resulted in a compound postsynaptic potential (PSP) in LPeD1. The subsequent action potential in VD4 (posttetanus) resulted in a few hundred percent enhancement of EPSPs amplitude in LPeD1, which gradually returned to its baseline within seconds. (  B  ) Summary data depicting the percent increase in the amplitude of posttetanic EPSPs in LPeD1. 
Fig. 5. Visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse exhibits short-term potentiation. The soma–soma paired cells were simultaneously impaled intracellularly, and synaptic transmission was tested electrophysiologically. (  A  ) Action potentials in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1. The amplitude of first EPSP was measured, and a tetanus was delivered to VD4 (at  asterisk  , 10–10 action potentials), which resulted in a compound postsynaptic potential (PSP) in LPeD1. The subsequent action potential in VD4 (posttetanus) resulted in a few hundred percent enhancement of EPSPs amplitude in LPeD1, which gradually returned to its baseline within seconds. (  B  ) Summary data depicting the percent increase in the amplitude of posttetanic EPSPs in LPeD1. 
×
Fig. 6. Sevoflurane does not affect posttetanic potentiation (PTP) at the visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse. To test whether sevoflurane blocks VD4-induced PTP in LPeD1, synapses were tested electrophysiologically. (  A  ) Action potentials in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1 under control (  i  ) and various anesthetic conditions (  ii  ) and after washout (  iii  ). Although VD4-induced EPSPs were significantly suppressed by all sevoflurane concentrations used (  A  ), the ratio between the pretetanus and posttetanus EPSPs did not change significantly (  B  ), even for 3% sevoflurane, which blocked the synaptic transmission almost completely. 
Fig. 6. Sevoflurane does not affect posttetanic potentiation (PTP) at the visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse. To test whether sevoflurane blocks VD4-induced PTP in LPeD1, synapses were tested electrophysiologically. (  A  ) Action potentials in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1 under control (  i  ) and various anesthetic conditions (  ii  ) and after washout (  iii  ). Although VD4-induced EPSPs were significantly suppressed by all sevoflurane concentrations used (  A  ), the ratio between the pretetanus and posttetanus EPSPs did not change significantly (  B  ), even for 3% sevoflurane, which blocked the synaptic transmission almost completely. 
Fig. 6. Sevoflurane does not affect posttetanic potentiation (PTP) at the visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse. To test whether sevoflurane blocks VD4-induced PTP in LPeD1, synapses were tested electrophysiologically. (  A  ) Action potentials in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1 under control (  i  ) and various anesthetic conditions (  ii  ) and after washout (  iii  ). Although VD4-induced EPSPs were significantly suppressed by all sevoflurane concentrations used (  A  ), the ratio between the pretetanus and posttetanus EPSPs did not change significantly (  B  ), even for 3% sevoflurane, which blocked the synaptic transmission almost completely. 
×
We next sought to determine whether sevoflurane blocked the retention of PTP. The tetanus was delivered to VD4 under control saline conditions, and the preparation was then superfused with either normal saline (fig. 7A) or sevoflurane (fig. 7B) for 5 min. An action potential in VD4 generated an EPSP, whereas a burst produced compound PTP as shown previously (fig. 7). The perfusion was then switched to either normal saline (fig. 7A) or the anesthetic solution (fig. 7B) for an additional 5 min. After 2 min of washout with normal saline, the PTP was then tested as described above. We found that a 5-min exposure to sevoflurane (3%) did not prevent the “expression” of PTP at the VD4–LPeD1 synapse, which exhibited potentiation in a manner similar to that observed under control conditions (posttetanus EPSP amplitude = control: 28.5 ± 2.5 mV; sevoflurane: 27.8 ± 2.3 mV; fig. 7). The amplitudes of the PTP under control and sevoflurane conditions are compared in figure 7. Taken together, these data demonstrate that sevoflurane does not block PTP, nor does its application to a potentiated synapse eliminate short-term plasticity.
Fig. 7. Sevoflurane does not eliminate posttetanic potentiation (PTP) at the visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse. To test whether sevoflurane eliminates PTP, the soma–soma synapses between VD4 and LPeD1 were tested electrophysiologically. The PTP paradigm was used as described above. After the tetanus, VD4 was prevented from spiking, and the preparation was exposed to either control saline (  A  ) or sevoflurane (  B  ) for 5 min. The anesthetic solution was replaced with normal saline for 2 min, and the synaptic transmission was tested again under both experimental conditions. The posttetanic action potential in VD4 generated a potentiated excitatory postsynaptic potential in LPeD1 under both experimental conditions; the amplitudes of the posttetanic excitatory postsynaptic potentials were identical (  C  ; control: 28.5 ± 2.5 mV; sevoflurane: 27.8 ± 2.3 mV). NS = not significant. 
Fig. 7. Sevoflurane does not eliminate posttetanic potentiation (PTP) at the visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse. To test whether sevoflurane eliminates PTP, the soma–soma synapses between VD4 and LPeD1 were tested electrophysiologically. The PTP paradigm was used as described above. After the tetanus, VD4 was prevented from spiking, and the preparation was exposed to either control saline (  A  ) or sevoflurane (  B  ) for 5 min. The anesthetic solution was replaced with normal saline for 2 min, and the synaptic transmission was tested again under both experimental conditions. The posttetanic action potential in VD4 generated a potentiated excitatory postsynaptic potential in LPeD1 under both experimental conditions; the amplitudes of the posttetanic excitatory postsynaptic potentials were identical (  C  ; control: 28.5 ± 2.5 mV; sevoflurane: 27.8 ± 2.3 mV). NS = not significant. 
Fig. 7. Sevoflurane does not eliminate posttetanic potentiation (PTP) at the visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse. To test whether sevoflurane eliminates PTP, the soma–soma synapses between VD4 and LPeD1 were tested electrophysiologically. The PTP paradigm was used as described above. After the tetanus, VD4 was prevented from spiking, and the preparation was exposed to either control saline (  A  ) or sevoflurane (  B  ) for 5 min. The anesthetic solution was replaced with normal saline for 2 min, and the synaptic transmission was tested again under both experimental conditions. The posttetanic action potential in VD4 generated a potentiated excitatory postsynaptic potential in LPeD1 under both experimental conditions; the amplitudes of the posttetanic excitatory postsynaptic potentials were identical (  C  ; control: 28.5 ± 2.5 mV; sevoflurane: 27.8 ± 2.3 mV). NS = not significant. 
×
Discussion
This study has demonstrated that clinically relevant concentrations of sevoflurane block cholinergic, excitatory synaptic transmission postsynaptically. Moreover, using a model system approach, we have provided the first direct evidence that neither the expression of short-term plasticity nor its retention is affected by sevoflurane. Taken together, our data show that although sevoflurane significantly suppresses synaptic transmission at a cholinergic synapse, it does not affect presynaptic machinery mediating PTP, which, in many other systems, underlies working memory.31 This study thus provides direct physiologic evidence for the idea that short-term exposure of synapses to an anesthetic may not affect synaptic plasticity underlying PTP. However, these data should be treated with caution as learning and memory involve a larger population of neurons, often requiring interplay between complex cognitive information processing mechanisms in the brain. These data, at the level of a single synapse, do nevertheless demonstrate that at the cellular level, sevoflurane does not affect short-term synaptic plasticity between VD4 and LPeD1.
Anesthetics agents such as sevoflurane bring about a state of general anesthesia by affecting both excitatory and inhibitory synaptic transmission in the nervous system. For example, both glutamatergic and γ-aminobutyric acid–mediated synaptic transmissions are perturbed by intravenous and inhalation anesthetics.1 The anesthetic-induced changes in the efficacy of synaptic transmission involve either presynaptic or postsynaptic mechanisms or both. In contrast to their effects on glutamatergic and γ-aminobutyric acid–mediated synapses, less understood are the actions of anesthetics on cholinergic synaptic transmission, which, in the central nervous system, is thought to be involved in learning and memory.1 Similarly, nicotinic acetylcholine receptors in various other brain regions have been implicated in a variety of nervous system functions. For example, basal forebrain neurons involving nicotinic acetylcholine receptors regulate memory and arousal, whereas cholinergic pathway in pontomesencephalic area regulate sleep, memory, and locomotor patterned activity.1,32 Regardless of their location (presynaptic vs.  postsynaptic), most of these receptors are affected by anesthetics, although their precise sites of action have not yet been defined, because of the complexity of the vertebrate brain. A notable exception is a study on uniquely identified snail neurons where isoflurane was shown to directly inhibit nicotinic acetylcholine receptors with concentration dependencies that were similar to those of mice neurons.33,34 Together, the above studies on both vertebrate and invertebrate neurons suggest that anesthetics affect neuronal acetylcholine receptors though their direct actions on “synaptic receptors” have not yet been determined. In this study, we have provided direct evidence that sevoflurane suppresses the function of the synaptic acetylcholine receptors in a concentration-dependent and reversible manner.
Wu et al.  2 have recently demonstrated that isoflurane suppresses neurotransmitter release from glutamatergic, calyx-type synapse in the rat brainstem. These effects were shown to involve an anesthetic-induced reduction in the amplitude of the presynaptic action potential. Similarly, general anesthetics have also been shown to inhibit acetylcholine release in several other preparations,35,36 whereas other studies have not detected any affects of halothane, enflurane, or methoxyflurane on the secretion of this transmitter.37 Notwithstanding the fact that these discrepancies may arise from various different approaches or the model system used, this information is important in resolving the issue of whether anesthetics affect learning and memory, arousal, and pain, which often involves cholinergic synaptic transmission. The issue of whether anesthetics affect presynaptic or postsynaptic mechanisms by blocking cholinergic synaptic transmission is difficult to resolve in an intact preparation because cell–cell interactions between defined sets of presynaptic and postsynaptic neurons are often difficult to investigate directly. In this study, we took advantage of an invertebrate model system whose usefulness for various anesthetic studies has been well documented.27,33,38–41 Using the well-established soma–soma synapses between identified neurons,26–28,38,42 we have previously demonstrated that both inhalation (sevoflurane27) and intravenous (propofol) anesthetics block dopaminergic and cholinergic transmission between the soma–soma paired cells, respectively. In the current study, this model system approach was used to provide direct evidence that clinically relevant concentrations of sevoflurane also suppress synaptic transmission between Lymnaea  neurons paired in a soma–soma configuration. Previous studies on Lymnaea  have demonstrated that clinically relevant concentrations of halothane (1–2%) induce a state of complete “anesthesia.”43 Moreover, clinically relevant concentrations of enflurane blocked cholinergic synaptic transmission between Aplysia  neurons,44 whereas higher concentrations (4–6%) of sevoflurane were required to block dopaminergic, inhibitory synapses in Lymnaea  .27 In the current study, we have shown that clinically relevant concentrations of sevoflurane (1–3%) are sufficient to block cholinergic synaptic transmission between VD4 and LPeD1. Our data are thus consistent with previous studies on invertebrate models, and together, they demonstrate that clinically relevant concentrations effectively block/suppress synaptic transmission between neurons.
Fluorescent labeling of the presynaptic vesicles with the dye FM1-43 strongly suggests that sevoflurane most likely does not affect exocytotic or endocytotic processes—these data do not, however, provide unequivocal evidence to this effect. Specifically, we could not precisely quantify the extent of fluorescent labeling with FM1-43 in either the absence or the presence of sevoflurane. Nevertheless, these results demonstrate that sevoflurane does not significantly suppress exocytosis and endocytosis of cholinergic vesicles. These data are also consistent with our previously published studies in which, using FM1-43 dye, we demonstrated that propofol also did not affect both exocytosis and endocytosis between the soma–soma paired cells.38 Wu et al.  ,2 on the other hand, demonstrated that isoflurane-induced suppression of synaptic transmission at the calyx-type mammalian synapses involves presynaptic sites, such as the Na+channels. Because in our previous work and the data presented in this study, we did not observe an anesthetic-induced reduction in the amplitude of the presynaptic action potential, we are confident that in our model, sevoflurane does not affect presynaptic machinery such as the Na+channels or the vesicular endocytosis/exocytosis. Moreover, because extrasynaptic, cholinergic responses in LPeD1 neurons were completely and reversibly blocked by sevoflurane, it seems safe to infer that the suppression of synaptic transmission between VD4 and LPeD1 may have primarily involved postsynaptic mechanisms. Consistent with this notion are previous studies on unidentified Lymnaea  where acetylcholine receptors were also shown to be blocked by another inhalation anesthetic.33,34 However, whether these anesthetic-induced effects on cholinergic receptors involved any specific, postsynaptic ion channels or receptors remains unknown and will require further investigation. In vertebrate models, the neuronal nicotinic acetylcholine receptors have been shown to exhibit greater sensitivities to inhalation anesthetics as compared with their muscle counterparts.7,8 Although the mechanisms underlying these differential responses remain undefined, similar comparative data in invertebrates await further identification and characterization of various types of acetylcholine receptors.
In contrast to their actions on synaptic transmission, much less understood are the effects of anesthetics on synaptic plasticity that forms the basis for learning and memory in various animal models. For example, although inhalation anesthetics have been shown to block long-term potentiation at hippocampal synapses,45,46 their effects on short-term potentiation mediating working memory have not yet been fully defined. The data presented in this study thus provide the first direct evidence that clinically used concentrations of sevoflurane do not affect the “expression” of PTP, nor do they eliminate the short-term plasticity that is induced in the absence of this anesthetic. The synaptic transmission between the paired cells was significantly reduced, although the ratio between presynaptic and postsynaptic EPSPs remained unperturbed by sevoflurane. Because sevoflurane exposure of the synapse, after the PTP, had no effect on posttetanic EPSPs, our data provide direct evidence that this volatile anesthetic does not eliminate PTP, which had otherwise developed under normal conditions.
We have previously demonstrated that the PTP at VD4 and LPeD1 synapse primarily involves presynaptic mechanisms47 and is not time dependent but rather use dependent (Naruo et al.  , unpublished data). Specifically, if VD4 is stimulated to fire an action potential after the tetanus, the synapse depotentiates, and the synaptic transmission returns to its baseline. However, if VD4 is prevented from firing, the synapses remains potentiated for up to several hours.48 This synapse thus exhibits synaptic characteristics, which can account for working memory. Consistent with this notion are the data presented in figure 7, which shows that in the absence of VD4 activity, the synapse had remained potentiated for several minutes during sevoflurane exposure. Thus, PTP was shown here to be unaffected by sevoflurane. In this study, we have also shown that the sevoflurane-induced suppression of synaptic transmission between VD4 and LPeD1 primarily involves postsynaptic acetylcholine receptors, thus validating our hypothesis that sevoflurane-induced suppression of synaptic transmission between VD4 and LPeD1 involves postsynaptic but not presynaptic mechanisms. This model also provides us with an additional tool (sevoflurane) to decipher the cellular and synaptic mechanisms of synaptic plasticity in this and the other models.
The neurons used in the current study comprise the cardiorespiratory central pattern generator that underlies aerial respiration in Lymnaea  ,23 which exhibits various forms of memory.49–51 Because both the behavioral and the neuronal components of this memory have been extensively characterized at the level of single neurons, we believe that elucidating the mechanisms by which sevoflurane affects synaptic potentiation will elucidate the neuronal basis of behavioral plasticity at a resolution unapproachable elsewhere.
Excellent technical support was provided by Wali Zaidi (Technician, Department of Cell Biology and Anatomy, University of Calgary, Calgary, Alberta, Canada).
References
Antognini JF, Carstens EE, Raines DE: Neural Mechanisms of Anesthesia. Upper Saddle River, New Jersey, Humana Press, 2003Antognini, JF Carstens, EE Raines, DE Upper Saddle River, New Jersey Humana Press
Wu X-S, Sun J-Y, Evers AS, Crowder M, Wu L-G: Isoflurane inhibits transmitter release and the presynaptic action potential. Anesthesiology 2004; 100:663–70Wu, X-S Sun, J-Y Evers, AS Crowder, M Wu, L-G
Wakamori M, Ikemota Y, Akaike N: Effects of two volatile anesthetics and a volatile convulsant on the excitatory and inhibitory amino acid responses in dissociated CNS neurons of the rat. J Neurophysiol 1991; 66:2014–21Wakamori, M Ikemota, Y Akaike, N
Perouansky M, Baranov D, Salman M, Yaari Y: Effects of halothane on glutamate receptor–mediated excitatory postsynaptic currents. Anesthesiology 1995; 83:109–19Perouansky, M Baranov, D Salman, M Yaari, Y
Pistis M, Belelli D, Peters JA, Lambert JJ: The interaction of general anesthetics with recombinant GABAA and glycine receptors expressed in Xenopus laevis oocytes: A comparative study. Br J Pharmacol 1997; 122:1707–19Pistis, M Belelli, D Peters, JA Lambert, JJ
Kirson ED, Yaari Y, Perouansky M: Presynaptic and postsynaptic actions of halothane at glutamatergic synapses in the mouse hippocampus. Br J Pharmacol 1998; 124:1607–14Kirson, ED Yaari, Y Perouansky, M
Violet, Bownie DL, Nakisa RC, Lib WR, Franks NP: Differential sensitivities of mammalian neuronal and muscle nicotinic acetylcholine receptors to general anesthetics. Anesthesiology 1997; 86:866–74Violet, Bownie, DL Nakisa, RC Lib, WR Franks, NP
Flood P, Ramirez-Latorre J, Role L: α4β2neuronal nicotinic acetylcholine receptors in the central nervous system are inhibited by isoflurane and propofol, but α7-type nicotinic acetylcholine receptors are unaffected. Anesthesiology 1997; 86:859–65Flood, P Ramirez-Latorre, J Role, L
Nordstrom O, Sandin R: Recall during intermittent propofol anesthesia. Br J Anesth 1996; 76:699–701Nordstrom, O Sandin, R
Russell IF, and Wang M: Absence of memory for intraoperative information during surgery with total intravenous anesthesia. Br J Anesth 2001; 86:196–202Russell, IF Wang, M
Lubke GH, Kerssens C, Phaf H, Sebel PS: Dependence of explicit and implicit memory on hypnotic state in trauma patients. Anesthesiology 1999; 90:670–80Lubke, GH Kerssens, C Phaf, H Sebel, PS
Reinsel R, Veselis R, Wronski M, Marino P, Heino R, Alagesan R: Memory impairment during conscious sedation: A comparison of midazolam, propofol and thiopental, Memory and Awareness in Anesthesia. Edited by Sebel PS, Bonke B, Winograd E. Upper Saddle River, New Jersey, Prentice-Hall, 1993, pp 127–40Reinsel, R Veselis, R Wronski, M Marino, P Heino, R Alagesan, R Sebel PS, Bonke B, Winograd E Upper Saddle River, New Jersey Prentice-Hall
Merikle PM, Rondi G: Memory for events during anesthesia has not been demonstrated: A psychologist’s viewpoint, Memory and Awareness in Anesthesia. Edited by Sebel PS, Bonke B, Winograd E. Upper Saddle River, New Jersey, Prentice-Hall, 1993, pp 476–97Merikle, PM Rondi, G Sebel PS, Bonke B, Winograd E Upper Saddle River, New Jersey Prentice-Hall
Chortkoff BS, Bennett HL, Eger EI: Does nitrous oxide antagonize isoflurane-induced suppression of learning? Anesthesiology 1993; 79:724–32Chortkoff, BS Bennett, HL Eger, EI
Veselis RA: Gone but not forgotten—or was it? Br J Anesth 2004; 92:161–3Veselis, RA
Jelicic M, Bonke B: Learning during anesthesia: A survey of expert opinion, Memory and Awareness in Anesthesia III. Edited by Bonke B, Bovill JG, Moerman N. Assen, The Netherlands, Van Gorcum, 1996, pp 97–101Jelicic, M Bonke, B Bonke B, Bovill JG, Moerman N Assen, The Netherlands Van Gorcum
Lubke GH, Kerssens C, Gershon RY, Sebel PS: Memory formation during general anesthesia for emergency cesarean sections. Anesthesiology 2000; 92:1029–34Lubke, GH Kerssens, C Gershon, RY Sebel, PS
Plourde G, Joffe D, Villemure C, Trahan M: The P3a wave of the auditory event-related potential reveals registration of pitch change during sufentanil anesthesia for cardiac surgery. Anesthesiology 1993; 78:498–509Plourde, G Joffe, D Villemure, C Trahan, M
Schwender D, Kaiser A, Klasing S, Peter K, Poppel E: Midlatency auditory evoked potentials and explicit and implicit memory in patients undergoing cardiac surgery. Anesthesiology 1994; 80:493–501Schwender, D Kaiser, A Klasing, S Peter, K Poppel, E
Veselis RA: Memory function during anesthesia. Anesthesiology 1999; 90:648–50Veselis, RA
Veselis RA, Reinsel RA, Feshchenko VA, Dnistrian AM: A neuroanatomical construct for amnesic affects of propofol. Anesthesiology 2002; 97:329–37Veselis, RA Reinsel, RA Feshchenko, VA Dnistrian, AM
Merikle PM, Daneman M: Memory for unconsciously perceived events: Evidence from anesthetized patients. Conscious Cogn 1996; 5:524–41Merikle, PM Daneman, M
Syed NI, Bulloch AGM, Lukowiak K: In vitro reconstruction of the respiratory central pattern generator (CPG) of the mollusk Lymnaea  . Science 1990; 250:282–5Syed, NI Bulloch, AGM Lukowiak, K
Syed NI, Hasan Z, Lovell P: In vitro  reconstruction of neuronal circuits: A simple model system approach, Modern Techniques in Neuroscience Research. Edited by Windhorst U, Johnsson H. Berlin, Springer, 1999, pp 361–77Syed, NI Hasan, Z Lovell, P Windhorst U, Johnsson H Berlin Springer
Ridgway RL, Syed NI, Lukowiak K, Bulloch AGM: Nerve growth factor (NGF) induces sprouting of specific neurons of the snail, Lymnaea stagnalis  . J Neurobiol 1991; 22:377–90Ridgway, RL Syed, NI Lukowiak, K Bulloch, AGM
Feng Z-P, Klumperman J, Lukowiak K, Syed NI: In vitro synaptogenesis between the somata of identified Lymnaea neurons requires protein synthesis but not extrinsic growth factors or substrate adhesion molecules. J Neurosci 1997; 17:7839–49Feng, Z-P Klumperman, J Lukowiak, K Syed, NI
Hamakawa T, Feng Z-P, Grigoriev N, Inoue T, Takasaki M, Roth S, Lukowiak K, Hasan SU, Syed NI: Sevoflurane induced suppression if inhibitory synaptic transmission between soma-soma paired Lymnaea neurons. J Neurophysiol 1999; 82:2812–9Hamakawa, T Feng, Z-P Grigoriev, N Inoue, T Takasaki, M Roth, S Lukowiak, K Hasan, SU Syed, NI
Feng Z-P, Grigoriev N, Lukowiak K, Goldberg JI, Syed NI: Development of Ca2+hotspots during synaptogenesis between Lymnaea  neurons is target cell contact specific. J Physiol (London) 2002; 539:53–65Feng, Z-P Grigoriev, N Lukowiak, K Goldberg, JI Syed, NI
Woodin MA, Munno DW, Syed NI: Trophic factor-induced excitatory synaptogenesis involves postsynaptic modulation of nicotinic acetylcholine receptors. J Neurosci 2002; 22:505–14Woodin, MA Munno, DW Syed, NI
Munno DW, Prince DJ, Syed NI: Synapse number and synaptic efficacy are regulated by presynaptic cAMP and protein Kinase A. J Neurosci 2003; 23:4146–55Munno, DW Prince, DJ Syed, NI
Baddeley AD: Working memory. Science 1992; 255:556–9Baddeley, AD
Woolf NJ: Cholinergic systems in mammalian brain and spinal cord. Prog Neurobiol 1991; 37:475–524Woolf, NJ
Franks NP, Lieb WR: Selective effects of volatile general anesthetics on identified neurons. Ann N Y Acad Sci 1991; 625:54–70Franks, NP Lieb, WR
McKenzie D, Franks NP, Lieb WR: Actions of general anesthetics on a neuronal nicotinic acetylcholine receptor in isolated identified neurons of Lymnaea stagnalis  . Br J Pharm 1995; 115:275–82McKenzie, D Franks, NP Lieb, WR
Kanai T, Szerb JC: Mesencephalic reticular activating system and cortical acetylcholine output. Nature 1965; 205:80–2Kanai, T Szerb, JC
Keifer JC, Baghdoyan HA, Becker L, Lydic R: Halothane decrease pontine acetylcholine release and increase EEG spindles. Neuroreport 1994; 5:577–80Keifer, JC Baghdoyan, HA Becker, L Lydic, R
Bazil CW, Minneman KP: Anesthetic concentrations of enflurane and methoxyflurane in a rat brain in vitro  and in vivo  . J Pharm Pharmacol 1989; 41:835–9Bazil, CW Minneman, KP
Woodall AJ, Naruo H, Prince D, Feng Z-P, Winlow W, Takasaki M, Syed NI: Chronic anesthetic treatment blocks synaptogenesis but not neuronal regeneration cultured Lymnaea  neurons. J Neurophysiol 2003; 90:2232–9Woodall, AJ Naruo, H Prince, D Feng, Z-P Winlow, W Takasaki, M Syed, NI
Franks NP, Lieb WR: Volatile general anesthetics activate a novel neuronal K+current. Nature 1988; 333:662–4Franks, NP Lieb, WR
Franks NP, Lieb WR: Stereospecific effects of inhalational general anesthetic optical isomers on nerve ion channels. Science 1991; 254:427–30Franks, NP Lieb, WR
Franks NP, Lieb WR (1993) Selective actions of volatile general anesthetics at molecular and cellular levels. Br J Anesth 1993; 71:65–76Franks, NP Lieb, WR
Smit AB, Syed NI, Schapp D, Klumperman J, Kits KS, Lodder H, van der Schors RC, van Elk R, Sorgedrager B, Brejc K, Sixma T, Geraerts WPM: AchBP, a glia-derived acetylcholine-receptor-like modulation of cholinergic synaptic transmission. Nature 2001; 411:261–8Smit, AB Syed, NI Schapp, D Klumperman, J Kits, KS Lodder, H van der Schors, RC van Elk, R Sorgedrager, B Brejc, K Sixma, T Geraerts, WPM
Girdlestone D, McCrohan CR, Winlow W: The actions on halothane on spontaneous activity, action connections of the giant serotonin-containing neuron of Lymnaea  stagnalis. Comp Biochem Physiol C 1989; 93:333–9Girdlestone, D McCrohan, CR Winlow, W
Arimura H, Ikemoto Y: Action of enflurane on cholinergic transmission in identified Aplysia neurons. Br J Pharmacol 1986; 89:573–82Arimura, H Ikemoto, Y
MacIver MB, Tauck DL, Kendig JJ: General anesthetic modification of synaptic facilitation and long-term potentiation in hippocampus. Br J Anesth 1989; 62:301–10MacIver, MB Tauck, DL Kendig, JJ
Simon W, Hapfelmeier G, Kochs E, Zieglgänsberger W, Rammes G: Isoflurane blocks synaptic plasticity in the mouse hippocampus. Anesthesiology 2001; 94:1058–65Simon, W Hapfelmeier, G Kochs, E Zieglgänsberger, W Rammes, G
Naruo H, Munno DW, Takasaki M, Syed NI: Novel insights into mechanisms of post-tetanic potentiation in a model system. Soc Neurosci Abstr 2002; 552.8Naruo, H Munno, DW Takasaki, M Syed, NI
Prince D: A Novel Form of Post-tetanic Potentiation [thesis]. University of Calgary, Calgary, Alberta, Canada, 2004Prince, D University of Calgary Calgary, Alberta, Canada
Spencer GE, Kazmi MH, Syed NI, Lukowiak K: Changes in the activity of a CPG neuron after the reinforcement of an operantly conditioned behaviour in Lymnaea  . J Neurophysiol 2002; 88:1915–23Spencer, GE Kazmi, MH Syed, NI Lukowiak, K
Scheibenstock A, Krygier D, Haque Z, Syed NI, Lukowiak K: The soma of RPeD1 must be present for long-term memory formation of associative learning in Lymnaea. J Neurophysiol 2002; 88:1584–91Scheibenstock, A Krygier, D Haque, Z Syed, NI Lukowiak, K
Lukowiak K, Haque Z, Spencer G, Varshay N, Sangha S, Syed NI: Long-term memory survives nerve injury and the subsequent regeneration process. Learning Memory 2003; 10:44–54Lukowiak, K Haque, Z Spencer, G Varshay, N Sangha, S Syed, NI
Fig. 1. Synapse formation between soma–soma paired  Lymnaea  neurons. (  A  ) Individually identifiable, presynaptic (visceral dorsal 4 [VD4]) and postsynaptic (left pedal dorsal 1 [LPeD1]) were soma–soma paired overnight. (  B  ) Specific synapses similar to those seen  in vivo  reformed overnight between the paired cells. An action potential in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials in LPeD1. 
Fig. 1. Synapse formation between soma–soma paired  Lymnaea  neurons. (  A  ) Individually identifiable, presynaptic (visceral dorsal 4 [VD4]) and postsynaptic (left pedal dorsal 1 [LPeD1]) were soma–soma paired overnight. (  B  ) Specific synapses similar to those seen  in vivo  reformed overnight between the paired cells. An action potential in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials in LPeD1. 
Fig. 1. Synapse formation between soma–soma paired  Lymnaea  neurons. (  A  ) Individually identifiable, presynaptic (visceral dorsal 4 [VD4]) and postsynaptic (left pedal dorsal 1 [LPeD1]) were soma–soma paired overnight. (  B  ) Specific synapses similar to those seen  in vivo  reformed overnight between the paired cells. An action potential in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials in LPeD1. 
×
Fig. 2. Sevoflurane blocks synaptic transmission between visceral dorsal 4 (VD4) and left pedal dorsal 1 (LPeD1) in a concentration-dependent manner. VD4 and LPeD1 were soma–soma paired overnight, and synaptic transmission was tested electrophysiologically. Action potentials in VD4 (at  arrow  ) induced 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1 (  A  ). The amplitude of VD4-induced EPSP in LPeD1 was significantly suppressed by sevoflurane in a concentration-dependent manner (  A  ,  i    iii  show representative traces of sevoflurane (1.5%)–induced suppression of synaptic transmission and subsequently recovery after washout. Sevoflurane-induced effects were concentration dependent, and the synaptic transmission recovered fully after washout with normal saline (  B  ,  dark bars  ). The postsynaptic cell was current clamped at −80 mV throughout the experiment. 
Fig. 2. Sevoflurane blocks synaptic transmission between visceral dorsal 4 (VD4) and left pedal dorsal 1 (LPeD1) in a concentration-dependent manner. VD4 and LPeD1 were soma–soma paired overnight, and synaptic transmission was tested electrophysiologically. Action potentials in VD4 (at  arrow  ) induced 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1 (  A  ). The amplitude of VD4-induced EPSP in LPeD1 was significantly suppressed by sevoflurane in a concentration-dependent manner (  A  ,  i  –  iii  show representative traces of sevoflurane (1.5%)–induced suppression of synaptic transmission and subsequently recovery after washout. Sevoflurane-induced effects were concentration dependent, and the synaptic transmission recovered fully after washout with normal saline (  B  ,  dark bars  ). The postsynaptic cell was current clamped at −80 mV throughout the experiment. 
Fig. 2. Sevoflurane blocks synaptic transmission between visceral dorsal 4 (VD4) and left pedal dorsal 1 (LPeD1) in a concentration-dependent manner. VD4 and LPeD1 were soma–soma paired overnight, and synaptic transmission was tested electrophysiologically. Action potentials in VD4 (at  arrow  ) induced 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1 (  A  ). The amplitude of VD4-induced EPSP in LPeD1 was significantly suppressed by sevoflurane in a concentration-dependent manner (  A  ,  i    iii  show representative traces of sevoflurane (1.5%)–induced suppression of synaptic transmission and subsequently recovery after washout. Sevoflurane-induced effects were concentration dependent, and the synaptic transmission recovered fully after washout with normal saline (  B  ,  dark bars  ). The postsynaptic cell was current clamped at −80 mV throughout the experiment. 
×
Fig. 3. Sevoflurane does not affect the presynaptic secretory machinery. To test whether sevoflurane-induced suppression of synaptic transmission between visceral dorsal 4 (VD4) and left pedal dorsal 1 (LPeD1) was due to the perturbation of presynaptic release machinery, synapses were tested in either the absence or the presence of 3% sevoflurane (depresses the synapse almost completely) and the dye FM1-43. Neurons were paired overnight and allowed to develop synapses. Synapses developed at the contact site between the pairs, and VD4 processes were seen often unsheathing the LPeD1 somata (  arrows  ,  A  and  D  ). To demonstrate the pattern of dye localization in a control VD4, the cell was impaled intracellularly, and the dye was added to the preparation. VD4 was prevented from spiking by injecting hyperpolarizing current (0.2 nA) for 10 min, and images were acquired. No staining was discernable in VD4 (  B  ). The presynaptic cell was then stimulated (100 action potentials) by current pulses, and the dye was replaced with normal, cold saline. Images were acquired again, which revealed punctate staining both at the contact site between the cells and also in VD4 processes encircling the LPeD1 somata (  arrows  ,  C  ). The above experiment was repeated in the presence of 3% sevoflurane (  D    F  ). Sevoflurane at a concentration that blocks synaptic transmission almost completely also failed to prevent the uptake of the dye FM1-43, and punctate staining (  F  ) similar to that seen under control (  C  ) conditions was clearly discernable in the presence of this anesthetic. 
Fig. 3. Sevoflurane does not affect the presynaptic secretory machinery. To test whether sevoflurane-induced suppression of synaptic transmission between visceral dorsal 4 (VD4) and left pedal dorsal 1 (LPeD1) was due to the perturbation of presynaptic release machinery, synapses were tested in either the absence or the presence of 3% sevoflurane (depresses the synapse almost completely) and the dye FM1-43. Neurons were paired overnight and allowed to develop synapses. Synapses developed at the contact site between the pairs, and VD4 processes were seen often unsheathing the LPeD1 somata (  arrows  ,  A  and  D  ). To demonstrate the pattern of dye localization in a control VD4, the cell was impaled intracellularly, and the dye was added to the preparation. VD4 was prevented from spiking by injecting hyperpolarizing current (0.2 nA) for 10 min, and images were acquired. No staining was discernable in VD4 (  B  ). The presynaptic cell was then stimulated (100 action potentials) by current pulses, and the dye was replaced with normal, cold saline. Images were acquired again, which revealed punctate staining both at the contact site between the cells and also in VD4 processes encircling the LPeD1 somata (  arrows  ,  C  ). The above experiment was repeated in the presence of 3% sevoflurane (  D  –  F  ). Sevoflurane at a concentration that blocks synaptic transmission almost completely also failed to prevent the uptake of the dye FM1-43, and punctate staining (  F  ) similar to that seen under control (  C  ) conditions was clearly discernable in the presence of this anesthetic. 
Fig. 3. Sevoflurane does not affect the presynaptic secretory machinery. To test whether sevoflurane-induced suppression of synaptic transmission between visceral dorsal 4 (VD4) and left pedal dorsal 1 (LPeD1) was due to the perturbation of presynaptic release machinery, synapses were tested in either the absence or the presence of 3% sevoflurane (depresses the synapse almost completely) and the dye FM1-43. Neurons were paired overnight and allowed to develop synapses. Synapses developed at the contact site between the pairs, and VD4 processes were seen often unsheathing the LPeD1 somata (  arrows  ,  A  and  D  ). To demonstrate the pattern of dye localization in a control VD4, the cell was impaled intracellularly, and the dye was added to the preparation. VD4 was prevented from spiking by injecting hyperpolarizing current (0.2 nA) for 10 min, and images were acquired. No staining was discernable in VD4 (  B  ). The presynaptic cell was then stimulated (100 action potentials) by current pulses, and the dye was replaced with normal, cold saline. Images were acquired again, which revealed punctate staining both at the contact site between the cells and also in VD4 processes encircling the LPeD1 somata (  arrows  ,  C  ). The above experiment was repeated in the presence of 3% sevoflurane (  D    F  ). Sevoflurane at a concentration that blocks synaptic transmission almost completely also failed to prevent the uptake of the dye FM1-43, and punctate staining (  F  ) similar to that seen under control (  C  ) conditions was clearly discernable in the presence of this anesthetic. 
×
Fig. 4. Sevoflurane blocks cholinergic response in left pedal dorsal 1 (LPeD1). To test whether sevoflurane-induced suppression of synaptic transmission involved cholinergic postsynaptic receptors, acetylcholine was tested on LPeD1 in either the absence or the presence of sevoflurane. Specifically, acetylcholine (10−5m) was pressure applied to a single or paired LPeD1, and its effects were monitored intracellularly, in either the presence or the absence of various sevoflurane concentrations (  A  ). The cholinergic responses in LPeD1 were significantly depressed by sevoflurane in a concentration-dependent and reversible manner (  A  ,  i    iii  ). These data are summarized in  B  , and the  darker bars  represent washout data. 
Fig. 4. Sevoflurane blocks cholinergic response in left pedal dorsal 1 (LPeD1). To test whether sevoflurane-induced suppression of synaptic transmission involved cholinergic postsynaptic receptors, acetylcholine was tested on LPeD1 in either the absence or the presence of sevoflurane. Specifically, acetylcholine (10−5m) was pressure applied to a single or paired LPeD1, and its effects were monitored intracellularly, in either the presence or the absence of various sevoflurane concentrations (  A  ). The cholinergic responses in LPeD1 were significantly depressed by sevoflurane in a concentration-dependent and reversible manner (  A  ,  i  –  iii  ). These data are summarized in  B  , and the  darker bars  represent washout data. 
Fig. 4. Sevoflurane blocks cholinergic response in left pedal dorsal 1 (LPeD1). To test whether sevoflurane-induced suppression of synaptic transmission involved cholinergic postsynaptic receptors, acetylcholine was tested on LPeD1 in either the absence or the presence of sevoflurane. Specifically, acetylcholine (10−5m) was pressure applied to a single or paired LPeD1, and its effects were monitored intracellularly, in either the presence or the absence of various sevoflurane concentrations (  A  ). The cholinergic responses in LPeD1 were significantly depressed by sevoflurane in a concentration-dependent and reversible manner (  A  ,  i    iii  ). These data are summarized in  B  , and the  darker bars  represent washout data. 
×
Fig. 5. Visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse exhibits short-term potentiation. The soma–soma paired cells were simultaneously impaled intracellularly, and synaptic transmission was tested electrophysiologically. (  A  ) Action potentials in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1. The amplitude of first EPSP was measured, and a tetanus was delivered to VD4 (at  asterisk  , 10–10 action potentials), which resulted in a compound postsynaptic potential (PSP) in LPeD1. The subsequent action potential in VD4 (posttetanus) resulted in a few hundred percent enhancement of EPSPs amplitude in LPeD1, which gradually returned to its baseline within seconds. (  B  ) Summary data depicting the percent increase in the amplitude of posttetanic EPSPs in LPeD1. 
Fig. 5. Visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse exhibits short-term potentiation. The soma–soma paired cells were simultaneously impaled intracellularly, and synaptic transmission was tested electrophysiologically. (  A  ) Action potentials in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1. The amplitude of first EPSP was measured, and a tetanus was delivered to VD4 (at  asterisk  , 10–10 action potentials), which resulted in a compound postsynaptic potential (PSP) in LPeD1. The subsequent action potential in VD4 (posttetanus) resulted in a few hundred percent enhancement of EPSPs amplitude in LPeD1, which gradually returned to its baseline within seconds. (  B  ) Summary data depicting the percent increase in the amplitude of posttetanic EPSPs in LPeD1. 
Fig. 5. Visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse exhibits short-term potentiation. The soma–soma paired cells were simultaneously impaled intracellularly, and synaptic transmission was tested electrophysiologically. (  A  ) Action potentials in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1. The amplitude of first EPSP was measured, and a tetanus was delivered to VD4 (at  asterisk  , 10–10 action potentials), which resulted in a compound postsynaptic potential (PSP) in LPeD1. The subsequent action potential in VD4 (posttetanus) resulted in a few hundred percent enhancement of EPSPs amplitude in LPeD1, which gradually returned to its baseline within seconds. (  B  ) Summary data depicting the percent increase in the amplitude of posttetanic EPSPs in LPeD1. 
×
Fig. 6. Sevoflurane does not affect posttetanic potentiation (PTP) at the visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse. To test whether sevoflurane blocks VD4-induced PTP in LPeD1, synapses were tested electrophysiologically. (  A  ) Action potentials in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1 under control (  i  ) and various anesthetic conditions (  ii  ) and after washout (  iii  ). Although VD4-induced EPSPs were significantly suppressed by all sevoflurane concentrations used (  A  ), the ratio between the pretetanus and posttetanus EPSPs did not change significantly (  B  ), even for 3% sevoflurane, which blocked the synaptic transmission almost completely. 
Fig. 6. Sevoflurane does not affect posttetanic potentiation (PTP) at the visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse. To test whether sevoflurane blocks VD4-induced PTP in LPeD1, synapses were tested electrophysiologically. (  A  ) Action potentials in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1 under control (  i  ) and various anesthetic conditions (  ii  ) and after washout (  iii  ). Although VD4-induced EPSPs were significantly suppressed by all sevoflurane concentrations used (  A  ), the ratio between the pretetanus and posttetanus EPSPs did not change significantly (  B  ), even for 3% sevoflurane, which blocked the synaptic transmission almost completely. 
Fig. 6. Sevoflurane does not affect posttetanic potentiation (PTP) at the visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse. To test whether sevoflurane blocks VD4-induced PTP in LPeD1, synapses were tested electrophysiologically. (  A  ) Action potentials in VD4 (at  arrow  ) generated 1:1 excitatory postsynaptic potentials (EPSPs) in LPeD1 under control (  i  ) and various anesthetic conditions (  ii  ) and after washout (  iii  ). Although VD4-induced EPSPs were significantly suppressed by all sevoflurane concentrations used (  A  ), the ratio between the pretetanus and posttetanus EPSPs did not change significantly (  B  ), even for 3% sevoflurane, which blocked the synaptic transmission almost completely. 
×
Fig. 7. Sevoflurane does not eliminate posttetanic potentiation (PTP) at the visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse. To test whether sevoflurane eliminates PTP, the soma–soma synapses between VD4 and LPeD1 were tested electrophysiologically. The PTP paradigm was used as described above. After the tetanus, VD4 was prevented from spiking, and the preparation was exposed to either control saline (  A  ) or sevoflurane (  B  ) for 5 min. The anesthetic solution was replaced with normal saline for 2 min, and the synaptic transmission was tested again under both experimental conditions. The posttetanic action potential in VD4 generated a potentiated excitatory postsynaptic potential in LPeD1 under both experimental conditions; the amplitudes of the posttetanic excitatory postsynaptic potentials were identical (  C  ; control: 28.5 ± 2.5 mV; sevoflurane: 27.8 ± 2.3 mV). NS = not significant. 
Fig. 7. Sevoflurane does not eliminate posttetanic potentiation (PTP) at the visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse. To test whether sevoflurane eliminates PTP, the soma–soma synapses between VD4 and LPeD1 were tested electrophysiologically. The PTP paradigm was used as described above. After the tetanus, VD4 was prevented from spiking, and the preparation was exposed to either control saline (  A  ) or sevoflurane (  B  ) for 5 min. The anesthetic solution was replaced with normal saline for 2 min, and the synaptic transmission was tested again under both experimental conditions. The posttetanic action potential in VD4 generated a potentiated excitatory postsynaptic potential in LPeD1 under both experimental conditions; the amplitudes of the posttetanic excitatory postsynaptic potentials were identical (  C  ; control: 28.5 ± 2.5 mV; sevoflurane: 27.8 ± 2.3 mV). NS = not significant. 
Fig. 7. Sevoflurane does not eliminate posttetanic potentiation (PTP) at the visceral dorsal 4 (VD4)–left pedal dorsal 1 (LPeD1) synapse. To test whether sevoflurane eliminates PTP, the soma–soma synapses between VD4 and LPeD1 were tested electrophysiologically. The PTP paradigm was used as described above. After the tetanus, VD4 was prevented from spiking, and the preparation was exposed to either control saline (  A  ) or sevoflurane (  B  ) for 5 min. The anesthetic solution was replaced with normal saline for 2 min, and the synaptic transmission was tested again under both experimental conditions. The posttetanic action potential in VD4 generated a potentiated excitatory postsynaptic potential in LPeD1 under both experimental conditions; the amplitudes of the posttetanic excitatory postsynaptic potentials were identical (  C  ; control: 28.5 ± 2.5 mV; sevoflurane: 27.8 ± 2.3 mV). NS = not significant. 
×