Received: 5 March 2019 Laboratory Instructor(s): Mr. Castillo and Ms.

Baysa

FORMAL REPORT

Experiment 2F: Specific Sensory Systems

K. Alquitran | S. Arda | C. Barrios | M. Bañares

Department of Biological Sciences, Summary

College of Science, University of Santo
Tomas, Philippines
Correspondence
C. Barrios, Department of Biological
Sciences, College of Science, University
of Santo Tomas
Email:
carylconcepcion.barrios.sci@ust.edu.ph
The nervous system is a complex network of nerves and cells that carry messages to and
from the brain and spinal cord to various parts of the body. It enables organisms to
receive sensory information from their external environment, process this information
and regulate neurosecretory and motor systems. Four activities were done to
understand the physiology of the nerve. Twitching was only observed through electrical
effect among others in testing the function of nerves. Applying anesthesia and locking
the pressure increases the threshold voltage needed to elicit response as time goes by
and had a minimum stimulus of 3 volts. The damaged brain and
spinal cord of the frog showed minimal to no response in comparison to the non-pithed
frog. For the action reflex and reflex time, it was observed that the 4% HCl gave the
fastest reflex time averaging 0.8 sec.

KEYWORDS
Sciatic nerve, nervous system, nervous system function, pithing

1 | INTRODUCTION they also conduct impulses and communicate with

The nervous system is a complex network of
nerves and cells that carry messages to and from the
brain and spinal cord to various parts of the body. It
enables organisms to receive sensory information
from their external environment, process this
information and regulate neurosecretory and motor
systems. (Je´kely, 2011). The brain evaluates and
store this input and generate adaptive responses
about the presence of resources and hazards in the
environment (Allman, 1999). The neuron is the
fundamental unit of the Nervous system. It s can be
stimulated by touch, light, sound and other stimuli,;
each other. It produces action potentials when they
receive electrical or chemical stimulation.
A nerves are cylindrical bundle of nerve in
the peripheral nervous system (PNS).It conveys
information in the form of electrochemical impulses
(known as nerve impulses or action potentials)
carried by the individual neurons that make up the
nerve. Nerves can be classified into two groups;
sensory or afferent, and motor or efferent. Sensory
neurons conduct sensory information from their
receptors to the central nervous system. Motor
neurons conduct signals from the central nervous

1 | © 2019 GROUP 10 4B1

ALQUITRAN, ARDA, BARRIOS, BAÑARES
system to muscle .The sciatic nerve is a large nerve
that originates from the distal spinal cord and
extends along nearly the entire length of the
hindlimb. As the case with many of the large nerves
of the vertebrate nervous system, the sciatic nerve is
a mixed function nerve, which means it is made up
of the axons of sensory and motor neurons. As the
sciatic nerve progresses distally along the hind limb,
it gives rise to branches. These branches contain
motor & sensory neurons involved in control of the
muscle groups of the upper leg, which is the
hamstring, and the lower leg (both flexors and
extensors). In addition, the sensory receptors in the
skin of the entire lower leg and the posteriolateral
surface of the upper leg transmit information to the
brain via sciatic nerve neurons.
This experiment aims to determine the
effect of mechanical, thermal, osmotic, and other
factors on nerve conduction, the effect of single and
double pithing on the different reflexes of a frog, to
determine the response of the nerves that are
exposed and not exposed to anesthetics, and to test
the time of reflex action of the frog in different
concentrations of acid.
. was determined at each stage. The cotton was
2 | MATERIALS AND METHODS
2.1 |Nerve Function
A muscle-nerve preparation was done by double
pithing the frog. To pith the frog, it was grasped
using the left hand, bending the head ventrally. A
dissection needle was used to insert into the
foramen magnum and pushed cranially to destroy
the brain. Afterwards, the needle was directed
caudally to destroy the spinal cord. The skin in the
abdomen and legs were removed to expose the
muscle and nerve. The urostyle was then lifted and
cut free to then expose the sciatic nerve. The sciatic
nerve was lifted using a glass hook and freed from
fascia attached to it. With the support of the glass
hook, different actions were done to the nerve. First,
it was mechanically pinched. Secondly, the free end
of the nerve was touched using a free glass hook. A
GROUP 1 | 4B8
small portion of the dead end of the nerve was then
cut and a few crystals of sodium chloride (NaCl) were
added to it. For the chemical effect, the free end of
the nerve was dipped in hydrochloric acid (HCl).
Lastly, a mild electrical shock of 3.0V was applied
using a nerve stimulator.
2.2 |SIGNAL BLOCKING IN THE NERVE
Anesthetic Solution
A long sciatic nerve was isolated and placed in a
moist glass plate. One end of the nerve was touched
with a stimulating electrode and the minimum
stimulus where it will elicit a response on the
gastrocnemius muscle was determined. Two small
pieces of cotton was soaked with anesthetic and
placed between the middle portions of the nerve. It
was then covered with filter paper that is moistened
with Amphibian Ringer’s solution. The free end of
the nerve was stimulated using the minimal stimulus
obtained previously. The stimulation was repeated
at two-minute intervals and the threshold stimulus
moistened with anesthesia periodically. The
anesthetic was then washed away with Amphibian
Ringer’s solution and the minimum stimulus that will
give a response to the gastrocnemius muscle was
determined at two-minute intervals.
2.3 |PRESSURE BLOCKING
The minimum stimulus of another nerve was
determined. With the use of a prong covered with
parafilm, pressure was added to the middle portion
of the nerve and after a few seconds, the pressure
was released immediately. The minimum stimulus
was again determined and the same procedure was
repeated at 1-minute intervals.
2.4 |FUNCTIONS OF THE CENTRAL NERVOUS
SYSTEM
ALQUITRAN, ARDA, BARRIOS, BAÑARES GROUP 1 | 4B8

A frog placed on a dissecting pan was observed. Its

qualities observed were the position of the head, The function of the nerve was examined using
eyes, legs, and the pulsation in the throat area. The chemical, mechanical, thermal, osmotic and
frog was then laid down in supine position, and the electrical stimulation. Mechanical, Osmotic and
righting position was observed. The pulsation in the Chemical Stimulation exhibited no response.
throat area was again determined when the frog was However, a response should be observed for all the
placed in a pail of water. The reaction of the frog was stimuli since frogs have sensory receptors that can
recorded after placing the frog on the dissecting pan recognize the stimuli and initiate a response.
and after making a loud noise. The firmness and
flaccidness of the frog’s legs were observed and after One of the probable reasons is that the threshold
gently pulling the hind leg of the frog, its resistance potential was not reached which created no
was determined. The reaction of the frog was response and the nerve was not kept moist.
recorded after pinching its toe, after its eye was
touched with a piece of cotton, and when its right 3.2 |Signal Blocking
foot was placed in a beaker with water being heated.
All these procedures were repeated after single Interval With Anesthetic Without Pressure
pithing the frog. All results were recorded. Anesthetic Block
1 (3.0 V) No (3.0 V) With No
Contraction Contraction Contraction
2.5 |REFLEX ACTION IN THE FROG 2 (4.5 V) No (4.5 V) With No
Contraction Contraction Contraction
After single pithing a frog, it was suspended by the 3 (6.0 V) No (6.0 V) With No
jaw with a wire hook attached to a clamp on an iron Contraction Contraction Contraction
4 (7.5 V) No (7.5 V) With No
stand. The long toe of the frog was immersed in 10%
Contraction Contraction Contraction
sodium bicarbonate and tap water, and then 5 (9.0 V) No (9.0 V) With No
immersed in 10% HCl. The time before the reflex Contraction Contraction Contraction
action was recorded. After 2 minutes, the procedure 6 (12.0 V) No (12.0 V) With No
was repeated using a different concentration of acid. Contraction Contraction Contraction

Another trial was made in a specific concentration Table 2 Using the minimum stimulus of 3.0 V, the
and the mean value of trials was computed. frog’s sciatic nerve was stimulated for a response
with and without the anesthetic.
3 | RESULTS
3.3 | Pressure Blocking
3.1 |Nerve Function
The second part would be examining the effects of
Stimulus Observations blocking the signals by applying pressure and
Mechanical No reaction. anesthetic solution. Using anesthetic solution would
inhibit depolarization of the nerve membrane by
Thermal Slight twitching of
interfering with Na+ and K+ currents.
gastrocnemius.
Osmotic No reaction.
Chemical No reaction.
Electrical Fast twitching of the
whole leg. (note: 3.0V)
Table 1 Results for Nerve Function
ALQUITRAN, ARDA, BARRIOS, BAÑARES GROUP 1 | 4B8

3.4 |Function of the Central Nervous System eyes open,

legs
Condition Observable Observation twitching
Traits (note:
Normal Position of Head Head throat
upright, eyes pulsation
open, legs very subtle
very active and bpm
(note: 96 not
bpm throat recordable)
pulsation) Righting Reflex Righting
Righting Reflex Righting Reflex
Reflex observed;
observed; frog’s legs
frog had the move to the
tendency to right but
turnover to failed to
its upright turn over
position In Water Throat
from the pulsation
right very subtle,
In Water Observed 53 not
bpm in recordable
water throat Reaction to No reaction
pulsation Noise
Reaction to Reflex Firmness of Legs Legs flaccid
Noise jumping Eye Response Eyelids
away from closed
the sound Table 4 Observations in a Single-Pithed frog
Firmness of Legs Legs very
firm
Eye Response Eyelids Condition Observable Observation
closed Traits
Table 3 Observations in a Normal Frog Double-pithed Position of Head Head flat on
frog pan, eyes
lucid (semi-
Condition Observable Observation closed), legs
Traits flaccid (jelly)
Single-Pithed Position of Head Head (note: no
frog downward throat
(flat with pulsation)
the pan), Righting Reflex Righting
ALQUITRAN, ARDA, BARRIOS, BAÑARES GROUP 1 | 4B8

Reflex not absorbs water and they breathe through their skin
observed; especially in areas where there is only a thin
no response covering, like the toes.
In Water Throat
pulsation 4 | DISCUSSION
not
observed In order to conduct these experiments, the frogs
Reaction to No Reaction were subjected to double-pithing procedure prior to
Noise the nerve dissection with the exception of procedure
Firmness of Legs Legs flaccid 2.4 (functions of the nervous system). The double
Eye Response Eyelids pithing procedure was done to ensure that the frog
closed would feel no pain once the nerve dissection begins.
Table 5 Observations in a Double-Pithed frog Double-pithing was performed by inserting a needle
on the cranial vault and moving it side to side. This
Normal response was observed in the non-pithed would destroy the brain and render the frog
frog after physiological tests are given. The double senseless and brain dead. To complete the double-
pithed frog however has no reflex actions responded pith, the needle was inserted into the vertebral
because the brain and spinal cord are fully damaged. canal. The spinal cord is destroyed if the frog’s legs
The single-pithed frog still has less response action go limp. No reflexes can be observed from the frog
compared to the non-pithed frog due to the intact after this is done. This will also make it easier to skin
spinal cord. the frog and dissect for the sciatic nerve.

During and after the dissection, the sciatic nerve

3.5 |Reflex Action of the Frog
must be kept moist using Ringer’s solution. This will
Time Acid Time Acid prevent the nerve from drying and becoming brittle.
(sec) Conc. (sec) Concentration The nerve must not be touched by any metal or
9 secs 1% 23 secs 6% hands to avoid depolarization which could affect the
2.79 secs 2% 4.01 7% result of the experiment, hence the use of ceramic
secs knife and forceps covered with parafilm.
1.8 secs 3% 1.63 8%
secs In the first part of the experiment (Nerve
0.8 secs 4% 2.16 9% Function), no response was observed from the all
secs the stimuli. There should be a response from the
0.66 secs 5% 2.27 10% frog due to the presence of the different sensory
secs receptors in the frog’s body such as the
Table 6 Reflex Action in a Frog mechanoreceptors and nociceptors which receives
and translates the stimuli.
In the activity, it was determined whether the The lack of response from the nerve may be
reaction time to a stimulus is influenced by the because the nerve was already depolarized. The
strength of the stimulus. The stimulus used was nerve has a resting potential of up to -70mV. Sodium
hydrochloric acid (HCl). Acidic solutions can channels when the nerve is stimulated causing an
therefore be felt more by the amphibian skin, since influx of sodium ions inside the nerve making it more
their skin is more sensitive to chemicals since it positive inside. If inside the nerve reaches the
ALQUITRAN, ARDA, BARRIOS, BAÑARES
threshold of -55mV, it will release an action potential
which will generate a response. However, if the
threshold of -55mV is not reached, no response will
be elicited which may have been what happened
with the nerve. It could also be that the nerve was
also not moist enough.
The effect of blocking signals was tested next
using the anesthetic solution and pressure blocking.
Based on the results, applying anesthesia resulted in
no contraction since using anesthesia increases the
threshold voltage needed to elicit a response.
However, if no anesthesia is applied, the threshold
voltage would also increase but very minimal
compared to adding an anesthetic solution since the
signals are not blocked. On the other hand, when
pressure is applied, the diameter of the nerve will be
reduced until no more signals are able to pass. Also,
the influence of pressure in blocking conduction is
entirely dependent upon deformation of the tissue
due to its unbalanced action (Causey, 1949).
In observing the function of the central nervous
system, the frog was observed in three ways: non-
pith, single-pith and doubled-pith. The non-pith frog
demonstrated normal results on the stimuli. The
single-pith frog showed little response. Lastly, the
double-pith frog showed no response at all.
The non-pith frog responded to all the stimuli
because it has a fully functioning central nervous
system. In the single-pith frog, only its brain was
destroyed therefore no response can be elicited
from stimuli that need to be processed by the brain
(non-reflex responses). Meanwhile, the double-pith
frog showed absolutely no response because the
entire central nervous system was already
destroyed.
Lastly, the reflex action of the frog was observed.
Acidic solutions can therefore be felt more by the
amphibian skin, since their skin is more sensitive to
chemicals since it absorbs water and they breathe
GROUP 1 | 4B8
through their skin especially in areas where there is
only a thin covering, like the toes.
Immersion of the toe in the acid was done from
the least concentration to the highest because
starting from the highest concentration might make
the skin more tolerant of the acid, therefore
affecting future results. After each immersion in
different concentrations, the skin or toe was washed
in alkali solution and water to restore is to its
previous condition prior to the immersion. This step
also readies the toe for the next concentration and
keeps it from drying. Dry skin may affect its
sensitivity.
REFERENCES
Allman, J. (1999). Evolving brains. New York, NY: Scientific
American Library.
Biopac. (2001). Frog Pith & Preparation. Retrieved February 21,
2016, from http://www.biopac.com/wp-
content/uploads/a01.pdf
Causey, G., & Palmer, E. (1949). The effect of pressure on nerve
conduction and nerve- fibre size. The Journal of Physiology,
109(1- 2), 22--231. Retrieved February 21, 2016,
fromhttp://www.ncbi.nlm.nih.gov/pmc/articl
es/PMC1392600/?page=11
input–output, and the skin brain thesis. Adaptive Behavior,
21(2), 67-85.
Je´kely, G. (2011). Origins and early evolution of neural circuits
for the control of ciliary
Keijzer, F., Van Duijn, M., & Lyon, P. (2013). What nervous
systems do: early evolution, locomotion. Proceedings of the
Royal Society B, 278, 914–922.
The Sciatic Nerve. Retrieved from
http://www.unm.edu/~toolson/sciatic_nerve.html.
Suckow, M., & Murthy, S. (2007). The IACUC Handbook (Second
ed.) (J. Silverman, Ed.). New York: CRC Press.