A COMPARATIVE STUDY OF ANAESTHETIC AGENTS ON HIGH VOLTAGE ACTIVATED CALCIUM
CHANNEL CURRENTS IN IDENTIFIED MOLLUSCAN NEURONS
Terrence J. Morris
1
, Philip M. Hopkins
2,3
and William Winlow
4,5
1
Department Science and Technology - Biology, Douglas College, 700 Ryal Avenue, New Westminster, British Columbia,
Canada;
2
Leeds Institute of Medical Research at St James’s, School of Medicine, University of Leeds, Leeds, United Kingdom;
3
Malignant Hyperthermia Investigation Unit, Leeds Institute of Molecular Medicine, St. James’s University Hospital, Leeds,
LS9 7TF, United Kingdom;
4
Department of Biology, University of Naples Federico II, Via Cintia 26, 80126, Naples, Italy;
5
Institute of Ageing and Chronic Diseases, University of Liverpool, Liverpool, United Kingdom.
Corresponding author: William Winlow
Key Words: General anaesthetic, calcium channels, Lymnaea, light yellow cells
SUMMARY
1. Using the two electrode voltage clamp configuration, a high voltage activated whole-cell Ca
2+
channel current (I
Ba
) was recorded from a cluster of neurosecretory ‘Light Yellow’ Cells (LYC) in the
right parietal ganglion of the pond snail Lymnaea stagnalis.
2. Recordings of I
Ba
from LYCs show a reversible concentration-dependent depression of current
amplitude in the presence of the volatile anaesthetics halothane, isoflurane and sevoflurane, or the
non-volatile anaesthetic pentobarbitone at clinical concentrations.
3. In the presence of the anaesthetics investigated, I
Ba
measured at the end of the depolarizing test
pulse showed proportionally greater depression than that at measured peak amplitude, as well as
significant decrease in the rate of activation or increase in inactivation or both.
4. Within the range of concentrations used, the concentration-response plots for all the
anaesthetics investigated correlate strongly to straight line functions, with linear regression R
2
values
> 0.99 in all instances.
5. For volatile anaesthetics, the dose-response regression slopes for I
Ba
increase in magnitude, in
order of gradient: sevoflurane, isoflurane and halothane, a sequence which reflects their order of
clinical potency in terms of MAC value.
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INTRODUCTION
General anaesthetics form a chemically diverse group of agents which have in common the
property of inducing narcosis and analgesia to varying degrees. They tend to depress neuronal
excitability and synaptic transmission, though detailed responses vary for the particular drug and are
often cell specific. Work with both vertebrate and invertebrate neuronal preparations has shown both
[Ca
2+
]
i
(intracellular calcium concentration) and Ca
2+
influx to be dramatically affected by a number of
anaesthetics. Increases in [Ca
2+
]
i
in the presence of volatile anaesthetics has been shown in CA1
hippocampal cells in rats (Mody et al., 1991) and cultured molluscan neurons in the absence of
extracellular Ca
2+
(Ahmed et al, 2020; Winlow et al., 1995), implying an anaesthetic triggered release
of Ca
2+
from intracellular stores. Voltage-gated calcium channels within the cell membranes also
appear to be important targets for these agents and calcium influx has been shown to decrease in a
dose-dependent manner in their presence (Yar & Winlow, 2016). T-type calcium currents (see below)
have also been shown to be differentially sensitive to volatile anaesthetics at clinical concentrations in
various cell types (McDowell et al., 1999). These are interesting findings but the dual action of Ca
2+
adds complexity to understanding global effects on cell activity. Ca
2+
is also a major second messenger
and plays a central role in the control of a variety of cell processes, so that alterations of calcium influx
and [Ca
2+
]
i
have the potential to modify numerous, interrelated neuronal functions. Transient
increases in [Ca
2+
]
i
may be initiated by release from intracellular storage sites associated with the
endoplasmic reticulum (Yamaguchi, 2019) and/or an increase in Ca
2+
permeability of the plasma
membrane associated with neural activity or the actions of neurotransmitters (Miller, 1991) and may
operate by a calcium-induced calcium release mechanism (Sandler and Barbara, 1999; Petrou et al,
2017).
In vertebrate tissues, calcium channels have been classified by their electrophysiological and
biochemical properties. Nowycky et al. (1985) identified three types of Ca
2+
channel in whole-cell
currents recorded from chick dorsal root ganglion cells, called L-type, T-type and N-type. They have
become the prototypes that form the basis of a widely accepted system of calcium channel
classification (Catterall, 2011; Catterall, et al, 2019, 2020) . The transient T-type current is activated
by small depolarisations, termed Low-Voltage-Activated (LVA), whilst sustained L-type currents are
activated by large depolarisations - High-Voltage-Activated (HVA) - and blocked by organic calcium
channel antagonists. N-type currents are also included in the HVA category but can be transient or
sustained. An increasing number of calcium channels from both vertebrate (Hoehn et al., 1993) and
non-vertebrate systems (Pearson et al., 1993) have proven difficult to identify within this scheme.
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Molluscan neurons in particular can display HVA Ca
2+
currents with conflicting pharmacological and
electrophysiological profiles (Kits & Mansvelder, 1996), but L-type currents have clearly been
identified as the sole HVA current in isolated, cultured pedal I cluster neurons of Lymnaea stagnalis
(Yar and Winlow, 2016).
The molluscan CNS (central nervous system) has several specific advantages for the
neurobiologist exemplified in Lymnaea (Kerkut, 1989; Leake & Walker, 1980); nerve cells are
accessible, relatively simply organised, easily identified and in many cases large enough for two
electrode work. Moreover, volatile anaesthetics produce many changes in behavior and neuronal
activity in Lymnaea that equate well to those found in mammals (McCrohan et al, 1987; Winlow &
Girdlestone, 1988; Girdlestone et al 1989) and facilitate its use as a single “model” system in studies on
anaesthetic mechanisms at behavioral and cellular levels (Winlow, 1984; Winlow, et al, 2018;
Moghadam et al, 2019).
The HVA calcium currents recorded from the cultured pedal I cluster neurons of Lymnaea (Yar
and Winlow, 2016) using single electrode voltage clamp showed a reversible, dose-dependent
suppression of Ba
2+
mediated current by halothane at concentrations ranging between 05 to 40
percent. Here we report on data using the two-electrode voltage clamp technique to investigate
calcium channel currents recorded from the Light Yellow Cell (LYC) group of neurosecretory neurons
in the right parietal ganglion of Lymnaea in the intact brain, which play a general role in body fluid
regulation (Benjamin and Kemenes, 2020). They lie on the ventral lobe of the right parietal ganglion
and can be observed from either the dorsal or ventral surface of the ganglion. LYCs have large somata
and fire spontaneous bursts of spikes (van Swigchem, 1979). Their action potentials have a
prominent shoulder or pseudoplateau phase, believed to be largely Ca
2+
driven (Aldrich, Jr. et al., 1979;
van Swigchem, 1979).
Studies of the effects of general anaesthetics on voltage gated Ca
2+
channels have often involved
the use of a variety of preparations and cell type, frequently at concentrations outside the clinical
range. The purpose of this study was, therefore, to determine the action of a number of general
anaesthetics, at clinical concentrations, on calcium currents recorded from the same identified cell
group in a single model system. In this way the dose-response profile and relative potency of each
agent can be directly compared. Here, we characterize the electrophysiology and pharmacology of the
LYC Ca
2+
currents, and the effects upon them of clinical concentrations of the volatile anaesthetics
halothane, isoflurane and sevoflurane and the systemic anaesthetic sodium pentobarbital, which is
now mostly used in veterinary anesthesia (Lester et al, 2012).
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METHODS
Snail brains were prepared according to the methods of Benjamin & Winlow (1981) in a HEPES
buffered snail saline (see below) at room temperature, approximately 20 C. Briefly, intact central
ganglia were pinned out in the perfusion chamber, ventral surface uppermost, and the sheath of
connective tissue removed from the right parietal ganglion with fine ground forceps. The inner cell
integument was softened with several drops of protease solution (Pronase from Streptomyces griseus,
Boehringer Mannheim Biochemica, in a solution of 4 mg/ml of snail saline) applied directly to the
ganglia, and washed off thoroughly with saline after about 3 minutes. The preparation was then
perfused continuously at 3.5 ml per minute with aerated saline. After 10-15 minutes the perfusing
medium was switched to the recording solution, also at room temperature, after which a cell within
the LYC cluster was selected for microelectrode penetration.
Solutions - Preliminary dissection and perfusion prior to recording was carried out in a snail saline
(Benjamin & Winlow, 1981) containing (mmol l
-1
): Na
+
594, K
+
20, Mg
2+
20, Ca
2+
40, Cl
−
380,
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 500, and glucose 03. The pH was
corrected to 7.8 with 2M NaOH.
All current measurements were made from cells in the intact brain, bathed in recording solution
which contained (mmol l
-1
): Na
+
65, Cs
+
20, Mg
2+
15, Ba
2+
10, Cl
−
23, Br
−
30, tetraethylammonium
(TEA) 30, 4-aminopyridine 10, HEPES 10, glucose 5 and pyruvate 10. The whole cell currents
measured were Ba
2+
currents rather than Ca
2+
currents per se. Ba
2+
was used as the charge carrying
ion since no currents were detectable when Ba
2+
was replaced by Ca
2+
. Possible explanations for this
are first, that the calcium channels in these cells are much more permeant to Ba
2+
than to Ca
2+
,
thereby increasing the signal to noise ratio to a resolvable level and, second, Ca
2+
entering the cell
might produce Ca
2+
-dependent inactivation of the channels being studied. Ba
2+
also has the advantage
that it blocks contaminating K
+
currents, which were further blocked by the presence of TEA
+
and Cs
+
in the recording solution. [Na
+
]
out
was kept low to exclude voltage-dependent Na
+
currents. The
addition of pyruvate was found to increase the recording time of cells considerably. Organic Ca
2+
-
channel blockers, verapamil (Sigma) and nifedipine (Sigma), were dissolved initially in ethanol to give
stock solutions of 1 mg ml
-1
before being diluted in the recording solution to the required
concentration.
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The pipette filling solution, modified from Orchard et al. (1991), contained
(mmol l
-1
): KCl 2500, calcium buffer ethyleneglycol-bis (-amino-ethyl ether) N,N'-tetra-acetic acid
(EGTA) and K-ATP 10.
Administration of anaesthetics - Volatile anaesthetics were vaporized into air using Ohmeda vaporizers
set to the desired percentage mixture. The anaesthetic-air mixture was bubbled at 1 litre per minute
into a small volume (about 150 ml) of recording solution in a 250 ml flask and vented though a closed
extraction system. Percentage vapour concentration was routinely calibrated using a Normac
anaesthetic agent monitor. Equilibration time was taken as 15 -20 minutes (Girdlestone et al., 1989)
after which a stopper containing an underwater seal air inlet was inserted and the solution used
immediately via a closed delivery system. Millimolar concentrations of isoflurane at 1, 2 and 4% were
determined by gas-liquid chromatography as (mean SD for n = 3): 044 0.05, 076 018 and 1.89
0.37 respectively (in an ideally equilibrated solution a 1% vol/vol concentration of gas represents 042
mM). These values approximate those for equilibrated halothane solutions (Winlow et al., 1998) and
are very close to the normal clinical concentration of halothane in arterial blood (Davies et al., 1972).
The clinical use of pentobarbitone in humans is nowadays rare, although it may be used in
veterinary anesthesia (Lester et al, 2012). Therefore, most uptake studies are on barbiturates used in
contemporary anaesthesia, thiopentone sodium in particular. Thiopentone sodium has the same
clinical potency as pentobarbitone (Dundee, 1974) but possesses contrasting and more desirable
pharmacokinetic properties (Lant, 1982). The concentration range of pentobarbitone used in this
investigation was 200 to 800 M, which equates to approximately 50 to 200 g per ml of recording
solution. The median dose of thiopentone required to induce anaesthesia is about 3.5 mg/kg body
weight, equivalent to approximately 100g/ml plasma concentration (Dundee et al., 1982).
Recording from cells - Micropipettes (resistance 8−10 M) were pulled on a one stage vertical puller
using filamented borosilicate glass (Clarke Electromedical Instruments). An Axoclamp 2A amplifier
was used to clamp cells and acquire data via proprietary software (PClamp 6.0) running on a 486 DX
33MHz IBM clone PC through an Axoclamp Digidata 1200 digital-analogue interface. Data were
sampled at 5 kHz, with automatic leak subtraction, estimated electronically before each test pulse as
the sum of currents developed during a series of four inverted pulse steps at ¼ amplitude of the test
pulse itself. A Gaussian filter with 500 Hz cut-off was applied to the raw data after acquisition to
attenuate noise.
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![Figure 2 Steady state inactivation curve for Isus and Itrans, recorded from 8 cells. Currents are expressed as a percentage of the corresponding transient and sustained currents at the holding potential of −100mV for a test potential of −15mV. Holding potentials were (mV) −100, −80, −60, −40, −30, and −20. No significant differences were found between transient and sustained values at each holding potential, using a paired t-test. A single curve was fitted to all data points by the least squares method using the Boltzmann distribution: I/Imax = 1/[1+exp(Vh−V1/2)/k] where V1/2 = −4088mV, Vh is the holding potential and k = 708 mV.](/figures/figure-2-steady-state-inactivation-curve-for-isus-and-itrans-2dpcnced.webp)