The Effects of Buthotus schach Scorpion Venom on Electrophysiological Properties of Magnocellular Neurons of Rat Supraoptic Nucleus
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Iranian Journal of Pharmaceutical Research (2018), 17 (1): 184-192 Copyright © 2018 by School of Pharmacy
Received: September 2016 Shaheed Beheshti University of Medical Sciences and Health Services
Accepted: May 2017
Original Article
The Effects of Buthotus schach Scorpion Venom on Electrophysiological
Properties of Magnocellular Neurons of Rat Supraoptic Nucleus
Akram Aboutorabia, Nima Naderib, Hamid Gholami pourbadiec, Hossein Zolfaghariand and Hossein
Vatanpour b*
a
Student Research Committee, School of Pharmacy, Shahid Beheshti University of Medical
Sciences, Tehran, Iran. bDepartment of Toxicology and Pharmacology, School of Pharmacy,
Shahid Beheshti University of Medical Sciences, Tehran, Iran. cDepartment of physiology
and pharmacology, Pasteur Institute of Iran, Tehran, Iran. dDepartment of venomous animals
and antivenom production Razi vaccine and serum research institute Agricultural research
education and extension organization ( AREEO), Karaj, Iran.
Abstract
Bothutous Schach (BS) scorpion venom consists of several polypeptides that could
modulate ion channels. In this study, the effects of BS crude venom on passive and active
electrophysiological properties of rat neurons in supraoptic nucleus (SON) of hypothalamus
was investigated using whole-cell patch clamp technique. The results showed that bath
application of BS venom produced significant change in passive properties of SON neurons,
namely a decrease in resting membrane potential and an increase in input resistance of the cells.
Also, significant change in active properties of SON neurons was shown after bath application
of BS venom; including a decrease in the number of evoked action potential along with an
increase in half width and decay time of action potential and a significant decrease in after-
hyperpolarization amplitude. Finally, a decreased latency to the first spike accompanied by
a lower current threshold to elicit the first spike was shown compared with the values before
venom application. These effects are possibly through blocking different ion channels including
potassium channels. Further experiments using different fractions of the venom is required to
specify venom effects on various ion channels.
Keywords: Buthotus schach; Ion channels; Whole-cell patch clamp; Scorpion venom.
Introduction and cytolytic peptides; some of them may serve
as leading compounds for drug design with
Approximately 1500 various species of therapeutic value (3, 4). In excitable and non-
scorpions divided into thirteen families are excitable cells, the scorpion neurotoxins block or
reported throughout the world (1); from which modify ion channel functions (4). The scorpion
Buthidae family is considered to be the largest and venoms are generally classified into major
the most medically important (2). The scorpion groups of neurotoxins. The first group is formed
venom possesses a heterogeneous collection of by short-chain peptides, consist of 20 to 43
pharmacologically active polypeptides such as amino acid residues and usually act as potassium
neurotoxins, antimicrobial peptide, proteases, channels blocker. The other group with long-
chain peptides scorpion toxins, containing 58 to
* Corresponding author: 76 amino acid residues is modifier of the sodium
E-mail: vatanpour.hossein@gmail.com channels (5). In excitable cells, it is well accepted
The Effects of Scorpion Venom on Potassium Channels
that pharmacological agents interacting with ion and was continuously bubbled with O2 (95%)
channels significantly change the physiological and CO2 (5%). Coronal slices containing
properties of the cells. After development of hypothalamic region were prepared using a
cellular electrophysiology, the physiological vibrating microtome (Campden Instruments,
roles of different ion channels were discovered UK). The Slices were then transferred to an
in a variety of cells (6). Buthotus Schach (BS) or incubation chamber, where they were maintained
hottentotta zagrosensis is known as one of the most submerged at 32-34 °C for one hour in a CSF
dangerous scorpions, with limited distributed solution (described later) and then kept at room
species of scorpion from Buthidae family in the temperature until time of recording.
middle west of Iran (7). BS venom can cause
convulsion, arrhythmia, respiratory depression, Whole cell patch-clamp recordings
and cardiac arrest in human. Previous studies The slices were transferred to recording
indicated that BS venoms can cause paralytic chamber set on the stage of an upright
effects on nerve-muscle preparations (8, 9). microscope (Olympus BX51W1, Japan). Patch
The purpose of this study was to investigate the clamp recording was done on MCNs of SON
effects of BS scorpion crude venom on intrinsic that were identified based on method described
and active electrophysiological properties of the by Hirasawa et al. (11). The slices were perfused
magnocellular neurons (MCNs) of supraoptic by artificial cerebrospinal fluid (aCSF) solution
nucleus (SON) in rat hypothalamus, using patch- containing (in mM) 124 NaCl, 2.8 KCl, 2 CaCl2,
clamp techniques. 2 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, and 10
D-glucose (Osmolarity = 290 mOsm) bubbled
Experimental with a mixture of O2 (95%) and CO2 (5%) at room
temperature (25 ± 2 ºC). Whole cell recordings
Venom were done under visual control using infrared
Crude BS venom was provided by the difference interference contrast (IR-DIC) optics
Department of Poisonous Animals, Razi Vaccine (Hamamatsu, Japan). Whole cell recordings were
and Serum Research Institute, Karaj, Iran. made using Multiclamp 700B amplifier (Axon
The crude venom was obtained by electrically Instruments, USA) equipped with Digidata
stimulating the telson of scorpions and then was 1320 A/D converter (Axon Instruments, USA).
lyophilized (10). Recordings were made using borosilicate glass
pipettes (1.2 mm O.D., 0.9 mm I.D.) with 4–7
Animals MΩ resistance when they were filled with
Male wistar rats (60–80 g) were obtained intracellular solution consisting (in mM): 90
from Pasteur Institute (Tehran, Iran), were potassium gluconate, 30 KCl, 2 MgCl2, 2 EGTA,
allowed free access to water and the pellet diet 5 NaCl, 10 HEPES (pH = 7.25; osmolarity = 290
under standardized housing conditions with mOsm). Recordings were accepted if the series
a 12 h:12 h light:dark cycle and at 22 ± 2 ºC resistance was less than 25 MΩ, and if it did not
temperature with a relative humidity of 40%. vary by 20% during the experiment.
All experiments were carried out according After establishing the whole-cell recording
to the guidelines of the National Institutes of configuration in current-clamp condition, the
Health (NIH Publications No. 80-23, revised cell was permitted to stabilize for 1–2 min to
1996), and approved by the Ethics Committee at allow equilibration between the cell interior and
Shahid Beheshti University of Medical Sciences. the micropipette solution. The resting membrane
potential was then measured. The membrane
Preparation of slices input resistance (Rin) was determined by applying
Rats were anesthetized with isoflorane, the 1000 ms hyperpolarizing current pulses (0 to
brain was immediately removed and placed in -200 pA; -50 pA increment) and calculating the
ice-cold slicing solution containing (in mM): 206 slope of the resultant current-voltage (I-V) curve
sucrose, 2.8 KCl, 1 MgCl2, 2 MgSO4, 1 CaCl2, within the linear portion. The action potential
1.25 NaH2PO4, 26 NaHCO3, and 10 D-glucose (AP) half-width was measured at one half of
185
(p
width of APs at 50 pA (p < 0.05), 100 pA (p
The Effects of Scorpion Venom on Potassium Channels
on calcium-activated potassium channels.
Consistent with our results, bath application
of putative BK channel blockers, iberiotoxin
(IBTX), and paxillin caused an increase in half-
width and a decrease in fast AHP amplitude of
spikes in brainstem neurons (20-22). It should
be noted that the depolarization of membrane
potential could affect the shape of action
potential through blockade of voltage gated
sodium and potassium channels, thus give rise
to changes in firing rate and half-width of action
potentials. After BS venom-induced membrane
depolarization, inactivation of voltage gated
Figure. 4. Effect of bath application of BS venom on evoked sodium channels would occur that eventually
action potentials by depolarizing currents. Changes in Decay
Figure.
time of evoked4. Effect of bath
action potentials application
per pulses were assessedof BS decreases
venom on firing
evokedrate. action
Similarpotentials
situation was by depolarizing
before and after bath application of venom at 0.3 (A), 1 (B), 3 shown when potassium channel blocker such
(C), and 10 µg/mL (D). Data were shown as mean ± SEM (N =
currents. Changes in Decay time of evoked action as cesium chloride
potentials perorpulses
tetraethyl
wereammonium
assessed was before and after
5-8 cells). *pAboutorabi A et al. / IJPR (2018), 17 (1): 184-192
Figure.6. Changes in firing properties of SON cells in response to ramp currents. (A) Representative spike firings of SON cells in
Figure.6.
response to ramp Changes
current clamp in after
before and firing properties
venom of Bath
application. SON cells inofresponse
application to ramp
venom induced currents.
significant (A)
decrease Representative
in latency of
the first AP (B), a significant decrease of current required to evoke the first AP (C), and a significant decrease in AP numbers (D). Data
spike
are shown as mean firings
± SEM. of SON
*pThe Effects of Scorpion Venom on Potassium Channels
evaluated and further experiments is required to to evaluate the specific effects of different
clarify the effects of BS venom on IA currents. components of the venom on ion channels
There is a discrepancy between occurrence of subtypes. Also, the effects of BS venom/toxins
first AP and number of APs after BS venom on different subtypes of potassium channel could
application. As we found here, BS venom be evaluated using single channel recording.
increased the input resistance. Cells with higher Furthermore, use of BS venom, or its specific
input resistance reach to the AP threshold by components, in controlled low doses to modulate
applying less amount of current which could be of potassium channels, could provide potential
interpreted that they have lower threshold. On therapeutic approaches to control diseases of
the other hand, as demonstrated on the traces, the excitable membranes such as seizure.
decreased number of APs is because of failure
to AP generation which might be due to use Acknowledgment
dependently inactivation of voltage gated sodium
channels by BS venom. Another explanation for This study was funded by grant from National
this discrepancy is that the venom may block Institute for Medical Research Development
some leak potassium channels resulting in (NIMAD) (No: 943728). Also aided by
depolarization of the cells. Cells with depolarized Neuroscience Research Center, Shahid Beheshti
membrane potential have lower threshold and at University of Medical Sciences.
the same time may produce lower APs due to
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