Hyperbaric pressure effects on voltage-dependent Ca+2 channels: Relevance to HPNS

Abstract

Known and unpublished data regarding hyperbaric pressure (HP) effects on voltage dependent-Ca2+ channels (VDCCs) were reviewed in an attempt to elucidate their role in the development of high-pressure neurological syndrome (HPNS). Most postulated effects from studies performed in the last two decades (e.g., depressed maximal current) rely on indirect findings, derived from extracellular [Ca2+] manipulation or by observing Ca(2+)-dependent processes. More recent experiments have tried to directly measure Ca2+ currents under high pressure conditions, some of which are potentially challenging previous indirect findings on one hand, but support findings from work done on neuronal behavior on the other. Additional support for some of the recent findings is provided by computer simulation of pressure effects on a spinal motor neuron activity. HP effect on different types of VDCCs seems to be selective - i.e., HP may suppress, facilitate or not change their activity. Thus, the specific distribution of the various types of the channels in each synaptic terminal or throughout the neuron will determine their function and will influence the neuronal network behavior under HP. Further research is needed in order to fully understand the HPNS etiology.

Full text

UHM 2010, VOL. 37, NO. 5 – VOLTAGE-DEPENDENT CA2+ CHANNELS AND HYPERBARIC PRESSURE
245
Hyperbaric pressure effects on voltage-dependent
Ca+2 channels: Relevance to HPNS
BEN AVINER1, YEHUDIT GNATEK1, GIDEON GRADWOHL2, YORAM GROSSMAN1
1 Department of Physiology and Neurobiology, Faculty of Health Sciences and Zlotowski Center
of Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, Israel;
2 Medical Engineering Unit, Department of Physics, The Jerusalem College of Technology, Jerusalem, Israel
CORRESPONDING AUTHOR: Ben Aviner – aviner@bgu.ac.il
AbstrAct
Known and unpublished data regarding hyperbaric pressure (HP) effects on voltage dependent-Ca2+
channels (VDCCs) were reviewed in an attempt to elucidate their role in the development of high-
pressure neurological syndrome (HPNS). Most postulated effects from studies performed in the last
two decades (e.g., depressed maximal current) rely on indirect ndings, derived from extracellular
[Ca2+] manipulation or by observing Ca2+-dependent processes. More recent experiments have tried
to directly measure Ca2+ currents under high pressure conditions, some of which are potentially
challenging previous indirect ndings on one hand, but support ndings from work done on neuronal
behavior on the other. Additional support for some of the recent ndings is provided by computer
simulation of pressure effects on a spinal motor neuron activity. HP effect on different types of
VDCCs seems to be selective – i.e., HP may suppress, facilitate or not change their activity. Thus,
the specic distribution of the various types of the channels in each synaptic terminal or throughout
the neuron will determine their function and will inuence the neuronal network behavior under HP.
Further research is needed in order to fully understand the HPNS etiology.
v
INTRODUCTION
Mankind has conquered soil more than 376,000 km
above sea level, landing humans on the Earth’s moon
repeatedly, yet the farthest descent accomplished
with a manned submarine, the Trieste, in January 1960,
was the sole attempt, reaching 10.9 km below
the ocean surface.
Whereas many species have been adapted to life
under great pressures in their search of new browses
in the continuum of evolution, humans have remained
quite limited in that sense. On a planet covered 70%
by oceans, about 70% of which are deeper than
2-3 km (20-30 MPa), if humans are ever to explore
the abyss – even with the aid of supreme technical
support – our pressure susceptibility must be studied
in order to remove restrictions that prevent us
from entering the frontiers of the deep oceans.
Neurophysiological effects of pressure
Hyperbaric environments present many physiological
challenges, especially affecting the lungs, hollow
viscera and the nervous system. Under pressure,
soft tissues of the body behave as a uid and rapidly
transmit any pressure applied against the surface of
the body to the adjacent uid compartments. This
results in hydrostatic compression of the cerebral spinal
uid, cerebral circulation, and extracellular and
intracellular uid compartments of the mammalian
CNS. Thus, practically every cell is exposed to
the ambient pressure.
Common neurological problems associated with
hyperbaric environments included oxygen toxicity,
which is thought to occur through increased oxidative
stress, as well as nitrogen narcosis (inert-gas narcosis)
and high-pressure neurological syndrome (HPNS)
[1,2]. Of these neurological problems, all but HPNS
can be alleviated and even eliminated by controlling
partial pressures of absorbed tissue gases at normal
values while under pressure, leading to the notion
Copyright © 2010 Undersea and Hyperbaric Medical Society, Inc.
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UHM 2010, VOL. 37, NO. 5 – VOLTAGE-DEPENDENT CA2+ CHANNELS AND HYPERBARIC PRESSURE
that HPNS occurs due to effects of pressure per se
[3]. HPNS signs and symptoms include vision and
auditory disturbances, dizziness, nausea, reduction
of cognitive functions, decreased motor coordination,
sleep disorders and electroecephalogram (EEG)
changes.
Although muscle performance at HP was altered
[4], HPNS signs and symptoms are generally associated
with signs of CNS hyperexcitability and EEG changes
[5]. These affect the performance of deep sea divers
exposed to pressures above 1.0 MPa [6] in a manner
that risks their lives and health. At greater pressures
(as in deeper diving), serious signs such as tremors,
convulsions and seizures leading to death may
occur [1].
An individual susceptibility to the hyperbaric
environment was found in both human and animal
experiment [7,8]. The pressure threshold for HPNS
also seems species-dependent, with an inverse relation
to the complexity of their central nervous system.
Complete seizures have been seen in sh at 5-13
MPa, in reptiles at 10-13 MPa, rodents at about 9
MPa, and in primates at 6-10 MPa [9]. Tremors
became apparent in humans exposed to pressures of
2.5 MPa, which progressed to myoclonus at 5 MPa
[10].
It is conceivable that this constellation of signs
and symptoms arises from brain malfunction that
probably reects changes in intrinsic neuronal
properties and disturbances in network synaptic
activity.
Molecular effects of pressure
Effects on synaptic transmission
The synapse is an interface between two cells where
intercellular communication takes place, thereby
enabling the formation of neuronal networks. Transmis-
sion across the chemical synapse is attained by the
release of neurotransmitter molecules from the
presynaptic terminal that bind to the postsynaptic
membrane receptors of the target cell and produce
synaptic potential. Pressure profoundly depressed
synaptic transmission at all synapses examined so
far, including individual synapse [11], neuromuscular
junction (NMJ) [12,13], excitatory and inhibitory
synapse [14,15] and in vertebrates and invertebrates
[10]. A 50-70% depression of glutamatergic excitatory
post-synaptic potential (EPSP) at 10 MPa was demon-
strated in the crustacean neuromuscular synapses
[12,15,16] and in the squid giant synapse [17], while
a more modest effect of pressure was observed in
cholinergic responses: nicotinic transmission in
mammalian NMJ [18], muscarinic response in
cervical sympathetic ganglion [19], and in cholinergic
synapses in mollusks [11]. Pressure has also been
shown to reduce population eld EPSP (pEPSP) in rat
hippocampal [20,21] and dentate gyrus [22,23] brain
slices, and in guinea pig cerebellar Purkinje cells [24].
The latter study also suggested for the rst time that
this reduction could be attributed to a specic Ca2+
channel-dependent component of the pEPSP (N-type).
The obvious question is what stage of synaptic
transmission is the pressure-sensitive one? Several
lines of evidence suggest that pressure predominantly
affects presynaptic mechanisms. First, since trans-
mitter release has common properties across various
synapses whereas post-synaptic responses differ
considerably, the given uniformity of the pressure
effect at all synapses suggests a presynaptic site.
Second, several changes induced by pressure at
synapses are of properties associated with events
at the presynaptic terminal:
a. HP markedly and reversibly depressed
spontaneous miniature end-plate potentials
frequency in the frog NMJ, without a noticeable
change in its mean amplitude (probably due to its
dual effect of reducing the amplitude and
lengthening the decay time of the miniature end-
plate currents; thus the receptor’s charge transfer
remains the same) [25];
b. HP increased facilitation and tetanic potentiation
[15].
c. Evidence from synaptosomes (sealed vesicles
from broken nerve terminals, containing Ca+2
channels and the synaptic release apparatus)
showed slowed release and in some cases a
moderate reduction in the maximal release [26],
with the exception of the aspartate synapse [27].
d. When the presynaptic mechanisms were bypassed
by direct application of the neurotransmitter ACh,
pressure had no effect on the response in helix
neurons [11].
However, it is important to note that there are changes
in the kinetics of excitatory post synaptic potentials
(EPSPs) and excitatory post-synaptic currents (EPSCs)
in most synapses, as well as pressure modulation of
specic ligand-gated ion-channels, that will contribute
to the depression mechanisms through post-synaptic
effect [10].
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Overall, most evidences point towards a presyn-
aptic mechanism for pressure depression of synaptic
transmission, and many of the effects can be explained
by depression of Ca2+ inux into the presynaptic
terminal, through voltage-dependent Ca2+ channels
(VDCCs), which is the trigger for the subsequent steps
of synaptic transmission. Furthermore, low [Ca2+]o
mimics the effects of HP [13,28], leading together to
the notion that the VDCCs are indeed involved in
this depression.
Effects on voltage-dependent ion channels
Ion channels are transmembranal proteins, the function
of which is associated with conformational changes.
The specic ion (negative or positive) inux or efux
across the membrane (depending on the ion electro-
chemical gradient) determines its effect on the
membrane potential. Voltage-dependent channels are
mainly modulated by the membrane potential, usually
activated by membrane depolarization, and deactiv-
ated when the potential recovers to resting level.
Many of the channels also exhibit voltage-dependent
inactivation that occurs during maintenance of
membrane depolarization. Voltage-dependent Na+ and
K+ channels are responsible for the generation and
conduction of action potential (AP) along neuronal
axons and muscle bers, and evidence has accumu-
lated to show that AP duration is lengthened
at HP [29-32].
Pressure effect on voltage-gated Na+ channels
varies between relatively moderate [32,33] to
signicant [34] reduction of action potential Na+
current amplitude and slowed its activation and
inactivation. When voltage-dependent K+ channels
were examined at HP, most studies have shown K+
currents to be enhanced [35-38], while others have
suggested their depression [36,39]. In the follow-
ing paragraphs we will discuss in detail HP effect
on VDCCs.
Voltage dependent Ca2+ channels
VDCCs mediate Ca2+ inux in response to membrane
depolarization. This transient Ca2+ inux serves as
the second messenger of electrical signaling, initiating
intracellular events such as neurotransmitter release
from presynaptic terminals, neuronal excitability,
excitation-contraction coupling in cardiac muscles,
hormone secretion, ciliary movement and gene
expression.
General structure
VDCCs are members of a gene super family of trans-
membranal ion channel proteins that includes voltage-
gated K+ and Na+ channels [40,41]. Various VDCC
types exist, composed of four or ve distinct subunits
(α1, α2δ, β, γ) that are encoded by multiple genes [42].
Their general organization is illustrated in Figure 1 (see
Page 248).
α1 subunit: The largest subunit (190-250 kDa)
that holds the ion conduction pore, the voltage sensor,
the channel gating area and most of the known sites
of channel regulation by second messengers, drugs,
and toxins [43]. Like the α subunit of the sodium
channel, it is organized in four homologous domains
(I-IV), each consisting of six transmembranal helices
(S1-S6) and a P-loop between S5 and S6 that together
form the channel’s pore. This loop determines the chan-
nel ion conductance and selectivity. Upon membrane
depolarization the positively charged S4 segment,
which functions as the voltage sensor for activation,
moves outward and rotates, thus initiating a
conformational change that opens the pore.
 β subunit: An intracellular protein, 52-78 kDa,
that can interact with and modulate α1 subunit [42, 44].
α2δ subunits: Transmembranal disulde-linked
proteins (175 kDa). The δ section is anchored to the
membrane, while the α2 subunit is entirely extra-
cellular [44].
 γ subunit: Composed of four transmembranal
helices (33 kDa). No evidence was available as to the
exact role of this subunit in trafcking or regulating of
the channel complex for most channel types. However,
a recent study has shown that it does have a role in
modulating the Cav1.1 channel [45] [see the
following subheads: “Nomenclature” (below) and
“Physiological and pharmacological properties”
(Page 248)].
Ten α1, four β, four α2δ and eight γ subunits iso-
forms are known to date, attesting to the wide diversity
of the VDCCs and their functional properties. Although
these supporting subunits modulate the properties of
the channel complex, the pharmacological and physio-
logical diversity of Ca2+ channels arises primarily
from the existence of multiple α1 subunits [46].
Nomenclature
In 2000, a systematic nomenclature was adopted [43],
based on the α1 various isoforms. Ca2+ channels were
named using the chemical symbol of the principal
permeating ion (Ca) with the principal physiological
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FIGUrE 1 – Spatial organization of the subunits constructing the VDCC
regulator (voltage) indicated as a subscript (Cav). The
numerical identier relates to the gene subfamily of the
α1 subunit (1 to 3 at present) and the order of discovery
of the α1 subunit within that subfamily (1 through n).
These three subfamilies correspond with the distinct
classes of Ca2+ currents (see below), previously used as
the classier parameter. The Cav1 subfamily (Cav1.1-
Cav1.4) includes the L-type Ca2+ currents. The Cav2
subfamily (Cav2.1- Cav2.3) includes the P/Q-type,
N-type and R-type Ca2+ currents. The Cav3 subfamily
(Cav3.1- Cav3.3) includes the T-type Ca2+ currents
(see Table 1, facing page).
Physiological and pharmacological properties
The different Ca2+ currents were dened by physiolog-
ical and pharmacological properties [47-49] (Table 1).
L-type currents (Cav1) require high voltage for
activation (HVA), have high single-channel
conductance and inactivate slowly during depolariza-
tion. They are the main Ca2+ currents recorded in muscle
and endocrine cells, where they initiate contraction
and secretion [50]. L-type currents can also be found
in cardiac muscle and neuronal dendrites and soma
[51], where they are involved in regulation of gene
expression and in integration of synaptic input
[47]. This family is blocked by organic antagonists,
including dihydropyridine (DHP) and is regulated pri-
marily by protein phosphorylation through a second
messenger-activated kinase pathway [42].
N-type, P/Q-type, and R-type currents (Cav2.1,
Cav2.2 and Cav2.3 respectively) are HVA channels,
insensitive to organic L-type channel blockers but
are blocked by specic polypeptide toxins from snail
and spider venoms [49]. This family is predominantly
expressed in the neurons, where they initiate neuro-
transmission and mediate Ca2+ entry into cell bodies
and dendrites. However they can also be found in the
heart, pituitary, pancreas and testes [50]. Cav2 chan-
nels are regulated by direct binding of soluble NSF
attachment receptor (SNARE) proteins and GTP bind-
ing proteins, and that primary mode of regulation is itself
regulated by protein phosphorylation pathways [42].
T-type currents (Cav3) require low voltage for
activation (LVA), inactivate rapidly, deactivate slowly,
have small single-channel conductance [52] and are
resistant to Ca2+ channel antagonists. They are
expressed in a variety of cell types, including neuronal
cell bodies and dendrites, where they are involved in
shaping the AP and controlling pattern of repetitive
ring [50]. The molecular mechanisms of the Cav3
channel regulation are currently unknown.
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249
________________________________________________________________________________
chAnnEl cUrrEnt locAlIzAtIon spEcIFIc cEllUlAr
AntAGonIst FUnctIon
________________________________________________________________________________
CaV1.1 L Skeletal muscle; Dihydropyridines; Excitation-contraction coupling;
transverse tubules phenylalkylamines; Excitation-coupled-Ca2+ entry *
benzothiazepines
________________________________________________________________________________
CaV1.2 L cardiac myocytes; Dihydropyridines; Excitation-contraction coupling;
smooth muscle myocytes; phenylalkylamines; hormone release;
endocrine cells; benzothiazepines regulation of transcription;
neuronal cell bodies; synaptic integration
proximal dendrites
________________________________________________________________________________
CaV1.3 L endocrine cells; neuronal Dihydropyridines; Hormone release; regulation of
cell bodies and dendrites; phenylalkylamines; transcription; synaptic regulation;
cardiac atrial myocytes benzothiazepines cardiac pacemaking; hearing;
and pacemaker cells; neurotransmitter release from
cochlear hair cells sensory cells
________________________________________________________________________________
CaV1.4 L retinal rod and bipolar Dihydropyridines; Neurotransmitter release
cells; spinal cord; phenylalkylamines; from photoreceptors
adrenal gland; benzothiazepines
mast cells
________________________________________________________________________________
CaV2.1 P/Q nerve terminals and ω – Agatoxin IVA Neurotransmitter release;
dendrites; dendritic Ca2+ transients;
neuroendocrine cells hormone release
________________________________________________________________________________
CaV2.2 N nerve terminals and ω – Conotoxin Neurotransmitter release;
dendrites; GVIA dendritic Ca2+ transients;
neuroendocrine cells hormone release
________________________________________________________________________________
CaV2.3 R neuronal cell bodies SNX-482 Repetitive ring;
and dendrites dendritic Ca2+ transients
________________________________________________________________________________
CaV3.1 T neuronal cell bodies None Pacemaking; repetitive ring
and dendrites;
cardiac and smooth
muscle myocytes
________________________________________________________________________________
CaV3.2 T neuronal cell bodies None Pacemaking; repetitive ring
and dendrites;
cardiac and smooth
muscle myocytes
________________________________________________________________________________
CaV3.3 T neuronal cell bodies None Pacemaking; repetitive ring
and dendrites
________________________________________________________________________________
tAblE 1: Subunit composition and function of Ca2+ channel types, modied from [50]; * [53] added.
tAblE 1 – Ca2+ channel types
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Pressure effects on voltage-dependent
Ca2+ channels
Synaptic release is a multistep mechanism. The
rst crucial stage is Ca2+ inux into the presynaptic
terminal and elevation of cytosolic Ca2+ concentration
([Ca2+]i) following membrane depolarization by
the invading AP. Increased [Ca2+]i leads to fusion of
docked vesicles with the terminal plasma membrane,
ending in evoked neurotransmitter release. As noted
above, most evidence support presynaptic mechanisms
as the underling cause of pressure depression of
synaptic transmission. Decreased Ca2+ inux into
the presynaptic terminal appears to be a good
explanation for many of these effects.
Indirect evidence
Most available data on VDCCs under pressure are
indirect evidence, acquired by manipulating extra-
cellular Ca2+ concentrations ([Ca2+]o) or by observing
Ca2+-dependent functions. Such a function was stud-
ied in the Paramecium, where the brief reversal of
swimming direction is Ca2+-dependent. Normally the
reversal occurs when the protozoan encounters the
container wall. Under 10 MPa hydrostatic pressure
this brief reversal of swimming direction was
inhibited [54]. Furthermore, spontaneous reversals
induced by Ba2+ were blocked during pressuriza-
tion, suggesting that pressure decreases Ca2+ inux
through the Paramecium’s unclassied VDCC. This
was supported by studies comparing pressure ef-
fects with the responses under different [Ca2+]o.
A theoretical model for transmitter release in
crustaceans has been developed by Parnas et al. (1982)
[55] in which the release process is divided into three
main steps: 1) Ca2+ entry; 2) neurotransmitter release;
and 3) removal of intracellular Ca2+, each step with
its specic characterizations. Studies on crustacean
neuromuscular synapses examined the relationship
between [Ca2+]o, EPSC amplitude and facilitation
using this model [13,15,56]. The analysis indicated
that pressure was acting to reduce Ca2+ inux, rather
than to affect intracellular removal of Ca2+ or the
release process. In addition, decreased [Ca2+]o
mimicked the pressure effect on EPSC’s amplitude,
while increasing [Ca2+]o above normal levels an-
tagonized its effect. Furthermore, application of vari-
ous Ca2+ channel blockers aggravated the depressant
effect of pressure on crustacean EPSCs, supporting
the notion that HP depresses synaptic response by
impeding Ca2+ inux [57]. Similar effect of [Ca2+]o
was reported for CNS single pEPSPs in the hippo-
campal dentate gyrus [58]. In contrast, HP had little
effect on the curve relating [Ca2+]o and single spinal
cord monosynaptic reex response (a measurement
of dorsal root compound AP) in newborn rats and did
not change its saturation level [59]. The slow after-
hyperpolarization (sAHP) amplitude of the AP was
reduced by HP in rat CA1 [39], a reduction which
could be explained by a depression of the SK
potassium channel, responsible for the sAHP. But
this channel is activated by the rise of [Ca2+]i during
each AP, potentially pointing to a reduction in
Ca2+ inux through VDCCs.
Previous studies have demonstrated colocalization
of different VDCCs in single motor nerve terminals
of frog [60], mouse [61] and CNS terminals [62] as
well as the presence of various VDCCs involved in
transmission in the CNS [63,64]. This non-homoge-
neous expression of VDCCs is probably manifested in
different responses to HP among various species and
different synapses in a given species, according to the
channels sensitivity to pressure. Indeed, at crustacean
neuromuscular synapses, the Ca2+ channel involved in
transmission resembles the vertebrate N-type channel
and, as mentioned above, this transmission is
depressed under pressure conditions, probably due
to reduction of Ca2+ inux through the VDCC [57].
A study by Etzion and Grossman (2000) [24] in
cerebellar Purkinje cells support these ndings. When
non-selective reduction in Ca2+ inux was employed
(Cd2+ application or low [Ca2+]o), partial synaptic
depression occurred, and pressure substantially added
to this depression. However, following a similar partial
block by a selective N-type Ca2+ channel blocker
(CTX), pressure had almost no additional effect,
strengthening the hypothesis that pressure blocks
mainly the N-type channel.
HP slightly increased the apparent synaptic
delay, partially due to a decrease in axonal
conduction velocity [35]. However, simultaneous
measurement of the nerve terminal current and EPSCs
uncovered a pressure effect on synaptic delay per se
[56]. Under normal conditions [Ca2+]o does not affect
synaptic delay. Yet, at 10.1 MPa, decreasing [Ca2+]o
increased synaptic delay. The apparent activation
volume of the pressure sensitive reaction is reminiscent
of the pressure effect on ionic channels, but also of
the exocytosis mechanism itself, which seems to be
depressed by HP [25]. Endocytotic membrane
retrieval, another presynaptic Ca2+ inux-dependent
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process [65, 66], is also inhibited by HP [67], further
supporting the hypothesis that impeded Ca2+ ux has
a substantial role in the synaptic transmission
malfunction at HP.
In the context of indirect studies, it is important
to note that pressure might interfere with Ca2+ action
within the terminal rather than decreasing ux (e.g.,
vesicle fusion and exocytosis). Furthermore, reducing
[Ca2+]o can also have postsynaptic effects – e.g.,
on the glutamate receptor [26].
Direct evidence
Although only a few works performed direct measure-
ments of Ca2+ currents and uptake, the available studies
reinforce the ndings mentioned above.
Ca2+ uptake by synaptosomes
Early measurements of voltage-dependent radiolabeled
Ca2+ uptake into brain synaptosomes, revealed that
HP depresses its uptake [68], supporting the concept
of decreased Ca2+ inux due to HP. To further test this
concept, Gilman et al. (1991) [69] used articially
added Ca2+ ionophore (A23187) to bypass Ca2+
channels and examined pressure effects on Ca2+ inux
through the ionophore and consequent radiolabled
GABA release. HP slightly increased the Ca2+ inux,
but depressed the release. These results indicate that,
although pressure probably diminishes Ca2+ inux
through VDCCs, it also affects processes subsequent
to Ca2+ entry, such as vesicle fusion [25,70] and
endocytotic membrane retrieval [67].
Ca2+ current measurements
In bovine chromafn cells, direct measurements of
Ca2+ currents did not show any signicant alteration
(only a very small increase in some experiments) after
pressurization to 40 MPa [70]. The channel in these
cells has a similar kinetic behavior to the L- and
P/Q-type channels in other neurons, suggesting that,
unlike the N-type channel, these channels are resistant
to pressurization. It has also been reported that
similar resistance to pressure is obtained for P-type
Ca2+ action potentials in guinea pig cerebellar Purkinje
cells [71]. On the other hand, Ca2+ current measured
in a rat skeletal L-type channel following decom-
pression from HP (20 MPa) was reported to be af-
fected by the treatment, with reduced peak amplitude,
prolonged time-to-peak and slower current decay [72].
The effect of pressure on two types of colocalized
Ca2+ currents was rst tested in the frog motor nerve
[26,34]. In addition to the action potential Na+ current
of the axons, blocking K+ channels using tetraethyl-
ammonium (TEA) revealed a slower Ca2+-dependent
current comprised of fast (ICaF) and slow (ICaS)
components [73] that reect the Ca2+ inward current
at the terminals. Both phases were blocked by Cd2+ and
ω-conotoxin (N- and L-type blockers), but only ICaS
was diminished by nifedipine and nitrendipine (L-type
blockers). Pressurization to 6.9 MPa suppressed ICaF
by about 87% , whereas ICaS was much less sensitive
to pressure (29% reduction) and was partially restored
by increased [Ca2+]o [34]. These results could
theoretically be derived from a reduction in nerve
terminal depolarization by the invading AP. To verify
that the decline in current is a direct effect of pres-
sure on the VDCCs, the terminal was depolarized di-
rectly via the electrode. Similar results were obtained
(Aviner et al., unpublished data). These results further
strengthen the concept that pressure exerts a
differential effect on various types of VDCCs at the
nerve terminal.
Studies in oocytes
A widely utilized expression system of ion channels is
the Xenopus oocyte, which has the ability to synthesize
exogenous protein when injected with foreign mRNA
[74]. In this preparation, along with the possibility to
directly measure the channel currents, one can express
a certain channel type from a chosen species, down to
the specic isoforms composing it. Consequently, the
responses are an exclusive result of the overexpressed
channel almost without interfering “noise.” Further-
more, this setup enables a more detailed and systematic
study of the channel’s kinetics in addition to
its maximal current.
In preliminary studies by Aviner et al. [75,76],
a rabbit’s Cav3.2 T-type Ca2+ channel (TTCC) and
Cav1.2 L-type Ca2+ channel (LTCC) were expressed
separately in oocytes. HP signicantly reduced the
maximal current of the Cav3.2 at relatively low
pressures (1.0 MPa), suggesting high sensitivity to
HP. Surprisingly, HP (5.0 MPa) almost doubled the
maximal currents generated by the Cav1.2. This
nding may be in contrast with previous works
reporting the LTCCs to be quite resistant to pressure
application in frog (Rana pipiens) NMJ [34], in bovine
adrenal chromafn cells [70] and guinea pig Purkinje
cells [57]. However, a possible explanation may be de-
rived from the variety of VDCCs and the difculty in
their identication in each preparation. Furthermore,
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more recent studies have shown that approximately
half of Ca2+ currents in bovine chromafn cells are
mediated by Cav2.1 channels (which seems pressure-
resistant, as mentioned above), and only 15-20% by
Cav1.2 [77-80], which may explain the slight increase
of current at HP. These results demonstrate again, on
a molecular level, that HP has differential effects on
various VDCCs. HP did not affect the inactivation of
both Cav1.2 and Cav3.2 channels, supporting the
contemporary concept of different activation and
inactivation mechanisms of voltage-gated ionic
channels.
FIGUrE 2 – Simulation of motoneuron ‘38’ spike boosting by pressure exposure
FIGUrE 2: Details of the model are described in [81]. Membrane potential is shown at the soma. Na+ and
K+ channels are incorporated in the initial segment-soma, and dendrites. LTCCs are located at the proximal
dendrites 0 – 400 μm from the soma and are distributed by an exponential decay function. The included
conductances (gNMDA , gAMPA and gLTCC) of the model reect the macroscopic conductances, since single-
channel conductance is generally believed to be unaltered by HP [84-86].
A – control, action potential is evoked by a single AMPA/ NMDA EPSP.
b – pressure-induced 30% increase of gNMDA and 50% longer τ decay, while gAMPA was decreased by 30%.
c – pressure-induced 100% increase of dendritic gLTCC alone.
D – combining both pressure effects on NMDA/AMPA and LTCC (B and C). See text for results.
Computer simulations
The possibility that the increased current in specic
LTCC (Cav1.2) may explain the previously observed
boosting effect of HP on depressed synaptic potential
in generating population spikes in CNS neurons
[20,23] is quite intriguing (see Figure 3, facing page).
In order to examine this hypothesis we used a
computer model simulation of “realistic” spinal motor
neuron utilizing NEURON software, which was
developed in our laboratory [81]. We studied the effect
of HP-induced increase of NMDA receptor activity
at the synaptic input [82,83] and/or increased Cav1.2
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253
FIGUrE 3 – Postulated HP effects, based on neuronal VDCCs distribution
FIGUrE 3 – Top, schematic representation of two adjacent CNS neurons; dashed squares point to sections
of the neuron in which VDCC types are known to be expressed.
Center, a ow chart describing anticipated function of signal transfer for each section.
Bottom, VDCC distribution and known HP effects on VDCC types are indicated.
activity embedded at the neuronal dendrites on the
intracellularly “recorded” ring pattern of the motor-
neuron in response to a single glutamatergic
EPSP (see Figure 2, facing page).
Under the model morphological and physiological
“realistic” conditions, the enhanced glutamatergic
NMDA synaptic potential, concomitantly with
moderate reduction in AMPA synaptic potential,
increased the number of evoked spikes (Figure 2B).
In contrast, increased gLTCC alone, did not contrib-
ute to the number of evoked spikes (Figure 2C),
although the “hump” following the rst action
potential was enhanced due to the increase of gLTCC
(inset of Fig 2C) relative to the “hump” of the control
conditions (inset of Figure 2A). However, the
combination of changes in both synaptic input and
LTCC (B+C) increased the number of spikes to an
even greater extent (Figure 2D).
It is worth noticing that the gLTCC, which is
partially responsible for the “hump,” in Figure 2C, is ac-
tivated by normal, relatively short-time gAMPA and gNM-
DA, while in Figure 2D it is activated by much greater –
and especially longer – EPSP that optimize its response.
We therefore suggest that specic LTCC (Cav1.2) may
boost glutamatergic EPSPs under pressure conditions.
DISCUSSION
From the available data, it is clear that pressure ef-
fects on VDCCs are selective and depend on their
specic family and, possibly, sub-family. It appears
that one of the more pressure-susceptible Ca2+ chan-
nels is the N-type channel, shown to be depressed un-
der pressure [24, 34, 57]. This channel is known to be
expressed in nerve terminals (see Table 1, Page 249),
suggesting its participation in pressure effect on syn-
aptic depression. Nevertheless, the identication of
the N-type channel in these studies was either by its
similarity to known N-type channel characteristics
or by pharmacological means. More direct measure-
ments are required to establish these ndings. Another
channel expressed in nerve terminals is the P/Q-type
channel, which, as mentioned above, was associated
with pressure resistance in guinea pig cerebellar
Purkinje cells [70,71,80]. Hence, it is conceivable that
synapses in which transmission involves predominantly
P-type channels will be much less sensitive to pressure
than those involving N-type channels.
Terminal button Distal Proximal Soma
dendrite dendrite
Input Synaptic Signal Signal Signal Output
signal transmission integration integration transfer signal
L (1.3) – unknown L (1.3) – L (1.2) – L (1.2) – augmentation Depends on
L (1.4) – unknown unknown augmentation L (1.3) unknown specific types’
T – reduction T – reduction density of
N – reduction N – reduction expression
P/Q – unaffected P/Q – unaffected
R – unknown R – unknown
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TTCC (Cav3.2), that presented high sensitivity to
pressure [76], is found mainly in neuronal soma and
dendrites, and is known to be involved especially in
generating bursting behavior and rhythmic activity
in pacemaker neurons [87]. Accordingly, the current
reduction of this channel is expected to slow and
impair the neuronal “clock” functions. HP depres-
sion of the Cav3.2 seems to be maximal at a pressure
of 1.0 MPa, at which professional divers begin to
experience mild HPNS. This may indicate the
channel’s involvement in this state of HPNS. We may
speculate that the contribution of such a channel will
depend on its distribution in the brain regions. For
example, TTCCs are expressed at the reticular thalamic
nucleus, hence disturbances of its neuronal activ-
ity could lead to changes in EEG. This indeed was
demonstrated by Rostain et al. (1997) [88] in human
divers. The thalamus is also responsible for sleep,
awareness and activity periods. Therefore, inter-
ference with its performance could lead to sleep
disorders on one hand, or drowsiness on the other.
TTCCs are also expressed in the striatum, which
has a role in executive functions, movement
planning and modulation, as well as transmitting
sensory inputs to the cortex. Disruption of their
activity could lead, respectively, to reduced cognitive
performance, impaired coordination, and vision and
auditory disturbances – which, in fact, are all part
of HPNS.
As mentioned above, pressure effects on the
LTCCs are contradicting [34,70]. However, the
Cav1.2 channel, which was augmented at HP [75], is
present in the cell bodies and proximal dendrites of
neurons in the dentate gyrus and hippocampus [51]
(see Table 1). Based on the known localization of the
channel and our computer simulation, we suggest that
pressure-potentiated L-type currents in the proximal
dendrite may boost pressure-depressed subthreshold
synaptic potentials to generate action potentials (see
Figure 3), as in fact observed in hippocampal brain
slices [20,23]. Such increase in dendritic excitability
could contribute to the generation of the network
hyperexcitability in HPNS, by a non-synaptic
mechanism.
This is a good example for another way through
which pressure-selective effects on VDCC might impact
neuronal networks, other than synaptic transmission.
Although analysis of synaptic release in crusta-
ceans indicated that pressure acts to reduce Ca2+ inux,
rather than to affect intracellular removal of Ca2+ or
the release process [13,15,56], there is evidence for
pressure depression of other presynaptic mechanisms,
mainly exocytosis [70]. However, it may not play
a major role in fast transmission but rather in slow
secretion of neuromodulators and neurohormones.
Most evidence linking [Ca2+]o and Ca2+ inux to
the effect of HP are in single or twin responses [13,15,
56-58]. When frequency responses of different CNS
synapses were examined, changing [Ca2+]o did not
always align with the effect of pressure and
occasionally had an opposite effect [22, 89]. These
studies indicate that hyperbaric pressure probably
interferes with additional mechanisms of release such
as exocytosis [70].
The complexity of the CNS function, the variety
of VDCCs and the selective effect of pressure makes
it even more challenging to point to the potential role
of VDCCs in HPNS. Systematic and detailed study
of the different VDCCs, in parallel to other possible
pressure-affected molecules and mechanisms, will
shed more light and increase our understanding of
the underling processes of HPNS. This will certainly
increase our ability to explore the abyss of the
oceans and exploit its resources in the future.
ACKNOWLEDGEMENT
This study was partially supported by a grant
from the USA Ofce of Naval Research (ONR)
No. N000141010163 to Y.G.
n
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o
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