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Anesth Analg 2007; 105:993-997
© 2007 International Anesthesia Research Society
doi: 10.1213/01.ane.0000282023.42639.b2
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ANESTHETIC PHARMACOLOGY

Section Editor:
Marcel E. Durieux Section Editor Tony Gin

Low Concentrations of Pentobarbital Enhance Excitability of Rat Hippocampal Neurons

David P. Archer, MD*, and Sheldon H. Roth, PhD*{dagger}

From the Departments of *Anesthesia and {dagger}Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada.

Address correspondence to David P. Archer, MD, Department of Anesthesia, Foothills Medical Center, 1403 29th St, Calgary, Alberta, Canada T2N 2T9. Address e-mail to darcher{at}ucalgary.ca.


   Abstract
Top
 Abstract
Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACKGROUND: Although the excitation phase observed during anesthetic induction and emergence is familiar to anesthesiologists, the cellular mechanisms of this phenomenon are not well understood. At anesthetic concentrations approximately one-tenth those required for surgical anesthesia, subjects demonstrate increased responsiveness to noxious stimulation. We previously estimated that the decrease in nociceptive reflex threshold is maximal at pentobarbital concentrations of approximately 5 µM. Here we used the rat hippocampal slice preparation to examine whether 5 µM pentobarbital increases the excitability of neurons.

METHODS: Intracellular recordings were obtained from CA1 neurons during stimulation of the Schaffer collateral pathway. We examined the effect of pentobarbital on resting intrinsic membrane properties and stimulus-response relationships. Excitability was evaluated with the relationship between the synaptic signal strength, as indicated by the excitatory postsynaptic potential slope, and the probability of spiking (E-S relationship).

RESULTS: Pentobarbital increased the excitability of hippocampal neurons, as shown by an increased probability of spiking at any given synaptic signal strength (P = 0.002), an effect known as "E-S potentiation." Pentobarbital was associated with an increase in the input resistance of the neuron and a shift of the action potential threshold towards more negative values. Pentobarbital did not increase the excitatory postsynaptic potential slope at any given stimulus strength.

CONCLUSIONS: At a 5 µM concentration, pentobarbital increased E-S coupling by enhancing the excitability of the postsynaptic neurons. Pentobarbital induced changes in intrinsic membrane properties that may contribute to increased excitability.


   Introduction
Top
 Abstract
 Introduction
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Patients exhibit excitatory phenomena at anesthetic concentrations that are approximately 10%–30% of those that are useful for general anesthesia (1–3). During the period of excitation, the patient may manifest such undesirable effects as hypertension, tachycardia, laryngospasm, and delirium. The causes of the excitement stages of anesthesia are not well understood. It is not clear to what extent increases in intrinsic cellular excitability or imbalances between excitatory and inhibitory pathways contribute to anesthetic-induced excitation. Development of a pharmacological strategy to prevent or blunt excitatory phenomena during sedation or light levels of anesthesia has been hampered by an incomplete understanding of the cellular mechanisms responsible. At a cellular level, subanesthetic concentrations of nitrous oxide and volatile anesthetics have been reported to block inhibitory responses (disinhibit) in midbrain reticular neurons (4) and in hippocampal neurons (5).

Low concentrations of pentobarbital (1–5 µM) reduce the threshold for nociceptive withdrawal responses in vivo (6) and enhance paired-pulse facilitation of synaptic transmission in CA1 hippocampal neurons in vitro (7). The clinical equivalent of this concentration of pentobarbital is likely to be an IV dose of 5 mg/kg (3).

Here we used hippocampal slices to study the effects of a low concentration of pentobarbital (5 µM) on the strength of the synaptic stimulus required to generate an action potential in CA1 neurons. The experimental paradigm in the present study resembles our previous in vivo studies in which we examined the effect of anesthetics on the intensity of the noxious stimulus required to activate paw or tail withdrawal (6,8,9).

Enhancement of a stimulus-response relationship can be explained by two mechanisms: decreased inhibition (disinhibition), and increased responsiveness of the neuron (increased excitability). With techniques that examine individual neurons, synaptic disinhibition is accompanied by an increase in the excitatory postsynaptic potential (EPSP) evoked by a given level of afferent stimulation (5,10,11). An increase in excitability is characterized by an increase in the probability of action potential generation (spiking) at any given strength of synaptic stimulation [EPSP-spiking (E-S) potentiation] (10,11). In the present study, we used intracellular recording of membrane potentials in CA1 hippocampal neurons to examine the effect of pentobarbital on the size of the EPSPs evoked by stimulation of the Schaffer collateral pathway and the ability of the EPSP to generate an action potential.


   METHODS
Top
 Abstract
 Introduction
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental procedures were approved by the Animal Care Committee, Faculty of Medicine, University of Calgary. Experiments were performed on transverse slices of hippocampus prepared from 20- to 30-day-old male Sprague-Dawley rats as previously described (7). Each animal was anesthetized with ether and decapitated immediately after apnea. The brain was rapidly removed and immersed in cold (8°C–10°C) physiologic solution containing NaCl, 124 mM; KCl, 5 mM; NaHCO3, 26 mM; CaCl2, 2 mM; MgSO4, 2 mM; glucose, 10 mM; and NaH2PO4, 1.25 mM. Four hundred micrometer thick slices were cut using a tissue chopper (Stoelting, IL) and placed on a nylon mesh screen at the gas–liquid interface surface of a recording chamber. Slices were continuously perfused with physiologic solution (1 mL/min) and a prewarmed, humidified gas mixture (95% oxygen to 5% carbon dioxide, 1.5 L/min). Slices were incubated without stimulation for 1 h at room temperature and thereafter maintained at 35°C ± 1°C.

Intracellular recording of membrane potentials was performed in neurons in the stratum pyramidale of the CA1 impaled blindly with sharp glass microelectrodes (resistance, 50–100 M{Omega}, filled with 3 M potassium acetate). Membrane potentials were recorded using an Axoclamp® 2B amplifier in bridge mode, stored digitally and analyzed with Clampfit® analysis software (both from Axon Instruments Inc., Union City, CA). Input resistance of the impaled neuron (RN) was measured at each condition (control, drug exposure) during the experiment by negative current injection (amplitude, 0.5–3.6 nA) through the intracellular electrode. Cells were accepted for study when spiking behavior was elicited by current injection, resting membrane potential was more than –55 mV and RN exceeded 20 M{Omega}.

We designed a protocol to assess the relationship between synaptic stimulation strength and neuronal excitability. During the first 30 min of intracellular recording, the EPSP strength-spiking probability (E-S) relationship was characterized by applying a range of stimulus strengths to the Schaffer collateral pathway at 0.1 Hz while recording the EPSP slopes and amplitudes of responses in the impaled CA1 neuron. We also recorded the presence and threshold values for action potentials (spikes) that were evoked. Stimuli were generated by a Grass S88 stimulator and SIU5 isolation unit (Grass Corp., Quincy, MA) and applied with a bipolar tungsten electrode. The stimulation sequence was then repeated after a 30 min exposure of the slice to perfusate containing 5 µM pentobarbital.

In the present study, we examined the effects of pentobarbital as a prototype anesthetic, in a low concentration (5 µM). The latter concentration has been shown to have excitatory effects characterized by enhanced nociceptive reflexes in vivo (6) and facilitation of hippocampal synaptic transmission in vitro (7). Clinically, the effects of this concentration of pentobarbital would likely correspond to the lightly sedated state observed after IV doses of 5 mg/kg (3). For each group of experiments, control studies of 60 min duration, but without any added pentobarbital, were performed to identify any time-dependent changes.

The relationships between stimulus intensity and EPSP slope, and EPSP slope and amplitude were characterized for each neuron by the slope of the linear regression fit to the recorded data. The relationships under control conditions were compared with those obtained in the presence of pentobarbital by paired Student's t test. The EPSP responses for each neuron were sorted according to ascending values of EPSP slope and then collected into bins of 0.5–1.0 mV/ms. Within each bin, the probability of generating an action potential (spiking probability) was calculated. The E-S curves were constructed from the probability of spiking versus the normalized EPSP slope (the maximum response was defined as 1) (10) and fit to a sigmoid function by nonlinear regression analysis. For each neuron, nonlinear regression analysis calculated a value for the EPSP slope that was associated with a 50% probability of spiking (EPSPslope50).

Differences between values obtained under experimental conditions (30 min exposure to pentobarbital or artificial cerebrospinal fluid [aCSF]) were compared with those obtained under control conditions by paired Student's t test for single comparisons and by two-factor analysis of variance for the analysis of the EPSPslope50 values. Statistical regression analyses and curve fitting were performed with Sigmastat® and Sigmaplot® software (both from SPSS, Chicago, IL). Statistical significance was inferred when P < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Stimulation of the Schaffer collateral pathway evoked EPSPs (Fig. 1). The main finding of the study was that exposure to pentobarbital increased the probability of action potential generation (spiking) at any given strength of synaptic stimulus as measured by the EPSP slope. Representative data for a single neuron are shown in Figure 2. Pooled data from several neurons are shown in Figure 3. Note that in these experiments the input voltage was adjusted to produce spiking at approximately the middle of the input range. Therefore, if a cell became more excitable, the investigator reduced the stimulator intensity (Figs. 2A, C, and D). As seen in the inset tracing of Figure 2B, pentobarbital exposure was associated with spiking at lower EPSP slopes and lower threshold potentials (Table 1). The increased responsiveness of the postsynaptic neuron to afferent stimulation was due to E-S potentiation (Figs. 2B, 3A and B). Pentobarbital did not change the size of the EPSP as measured by either the relationship between stimulus intensity and EPSP slope (n = 12, P = 0.80), or the relationship between EPSP slope and EPSP height (n = 12, P = 0.49) (Figs. 2C and D). Pentobarbital did not alter the resting membrane potential (Vm), but did decrease the membrane potential at which action potentials were generated (Fig. 2E).


Figure 117
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  		Figure 1. (A) Schematic diagram of the hippocampal slice. Sharp electrodes were used to obtain intracellular recordings from CA1 pyramidal neurons. (B) Representative tracing (black) of an excitatory postsynaptic potential (EPSP) evoked by stimulation of the Schaffer collateral pathway. In the subsequent tracing (gray), the EPSP size was sufficient to generate an action potential. The horizontal dashed line represents the resting membrane potential.

 

Figure 217
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  		Figure 2. Results from a single CA1 neuron showing that exposure to pentobarbital increases the probability of action potential generation (spiking) without increasing the strength of the excitatory postsynaptic potential (EPSP). The strength of the input stimulus to the Schaffer collateral pathway was adjusted to produce spiking at approximately the middle of the input range. Therefore, if a cell became more excitable, stimulus intensity was reduced, in the present example from 7–9.5 V (control) to 7–9 V during pentobarbital exposure. The responses of the neuron (EPSP strength and spiking) were recorded during control conditions, and after 30 min exposure to pentobarbital (gray bar). (A) The EPSP slopes (circles) of responses after pentobarbital exposure were less than during control conditions. The presence of a cross within the circle indicates spiking. (B) Exposure to pentobarbital shifted the EPSP slope–spiking probability (E-S) curve to the left, indicating increased excitability of the CA1 neuron. Filled and open circles represent control and pentobarbital conditions, respectively. The inset shows representative tracings of spiking responses. (C, D) Pentobarbital did not alter the relationships between synaptic stimulus-EPSP slope relationship (C) or EPSP slope-EPSP amplitude (D). (E) Examining only responses that generated action potentials, pentobarbital made the spike threshold (squares, right axis) more negative, but did not alter the resting membrane potential, Vm (triangles, left axis).

 

Figure 317
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  		Figure 3. Exposure to pentobarbital resulted in E-S potentiation of the postsynaptic CA1 neuron. Excitatory postsynaptic potential (EPSP) slope was normalized by defining the maximal EPSP slope recorded during each period of the experiment (control or 30 min) as 1. Curves were fitted to pooled data by nonlinear regression analysis. (A) Five neurons perfused with artificial cerebrospinal fluid (aCSF). There was no time-related shift in the relationship between EPSP slope and probability of spiking. (B) Pooled data from five neurons treated with pentobarbital showing the shift in the EPSP slope–spiking probability relationship toward lower values of EPSP slope. (C) The EPSP slope (determined by curve fitting each neuron separately) associated with a 50% probability of spiking was decreased after treatment with pentobarbital (two-factor analysis of variance, P = 0.002), but not after perfusion with aCSF. Mean values ± sd for five neurons in each treatment group.

 

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  		Table 1. Characteristics of Minimum Action Potential Responses

 

The excitability of each neuron was characterized by the EPSP slope that was associated with a 50% probability of spiking. In absolute values, pentobarbital was associated with a decrease in the EPSPslope50 from 3.2 ± 0.4 mV/ms under control conditions to 2.3 ± 0.3 mV/ms after exposure to pentobarbital (n = 5 slices, P < 0.01). Analysis of normalized data confirmed that the significant factor in the increase in E-S coupling was the exposure to pentobarbital and not the duration of the experiment (Fig. 3C).

We also analyzed the influence of pentobarbital at a second point in the stimulus-response relationship, the "minimum response." For each experiment, we defined the "minimum response" as the stimulus with the smallest synaptic strength, as defined by the smallest EPSP slope that was capable of generating an action potential. Exposure to pentobarbital resulted in a decrease in the size (slope and amplitude) of the EPSP that was just sufficient to generate a spike (Table 1).

Exposure to pentobarbital was associated with an increase in RN and a decrease in action potential threshold (Table 1). These changes in intrinsic membrane properties of the postsynaptic neuron would contribute to increased excitability.


   DISCUSSION
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results show that pentobarbital increased the responsiveness of CA1 neurons to synaptic stimulation from the Schaffer collateral afferent pathway. The increased responsiveness was characterized by an increased probability of spiking for a given value of the EPSP slope. Pentobarbital also reduced the minimum action potential threshold and increased the RN of the neurons. These changes in the intrinsic electrical properties of the neurons could contribute to increased excitability. Taken together, these findings support the hypothesis that a low concentration of pentobarbital was associated with E-S potentiation mediated by an increase in CA1 neuron excitability. Although pentobarbital has been shown to enhance synaptic transmission (12,13), these are the first data of which we are aware that demonstrate enhanced excitability of hippocampal neurons by pentobarbital.

A prominent action of barbiturates, including pentobarbital, is to enhance the action of {gamma}-aminobutyric acid (GABA) at the type A GABA (GABAA) receptor (14). Increased synaptic GABAA-ergic activity enhances the size of inhibitory postsynaptic potentials (15), whereas in extrasynaptic regions, the effect is to augment a tonic inhibitory conductance (16). Since decrease in inhibitory postsynaptic potentials size would be expected to result in enhanced EPSPs, our present findings do not support synaptic disinhibition. Our finding of increased membrane resistance could reflect a reduction of tonic extrasynaptic inhibitory conductance demonstrated by Cheng et al. (16), although we are unaware of evidence that such an effect occurs at the concentrations used here. Wan and Puil (17) reported an association between excitatory effects of pentobarbital (0.1–50 µM) and increased membrane resistance in thalamic neurons. The results of the present study add to the body of evidence that the excitatory effects of pentobarbital in central neurons are associated with increases in membrane resistance.

From a pharmacological standpoint, the study design suffered from two shortcomings. The first was that we were unable to provide a traditional dose-response relationship for the excitatory effects of pentobarbital because the concentration range for excitatory effects is so narrow (1–5 µM) (7) that distinction between these doses is difficult to achieve with the drug administration strategy used here. The second shortcoming was that we were unable to perform washout studies to confirm the reversibility of the excitatory effects. In our hands, the blind sharp electrode technique was time-consuming and tedious. Frequent loss of contact with the cell precluded doing washout studies to demonstrate reversibility of the changes that we observed, since removal of drugs from the slice may require hours (18). Instead, we relied on studies performed during perfusion with aCSF to control for any possible effects of time.

In summary, the findings of the present study show that a low concentration of pentobarbital was associated with EPSP-spike potentiation characterized by increased excitability of the postsynaptic neurons. Changes in the intrinsic electrical properties of the postsynaptic neuron were observed that may contribute to the increased excitability. The results do not support the hypothesis that pentobarbital caused a reduction in synaptic inhibition.


   Footnotes
 
Accepted for publication June 18, 2007.

Supported by a grant from the Canadian Institutes of Health Research and the Canadian Neurotrauma Research Program, Ottawa, Canada.

Reprints will not be available from the author.


   REFERENCES
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2007 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press