Autonomic nervous system, ANS, Screening techniques, pharmacological screening.

2. Drugs that exerts pharmacological effects stimulating
activation, intensification or inhibition of either the
sympathetic or parasympathetic nervous system have
been historically referred to as autonomic drugs.
 Sympathomimetic
 Sympatholytic
 Para sympathomimetic
 Para sympatholytic
 Ganglionic blockers and stimulants

4. Sympathomimetic agent is meant a compound whose activity
imitates that of epinephrine in the periphery.
Epinephrine or electrical stimulation of sympathetic nerves may
evoke any of the following responses:
 mydriasis in the eye
 acceleration of the heart
 dilatation of the coronary arteries
 relaxation of bronchial muscle
 inhibition of gastric secretion
 glycogenolysis in the liver
 elevation of the blood pressure

5. During a program of blind screening, a sympathomimetic
agent may sometimes be recognized by the presence of
effects such as:
 mydriasis,
 increase in the depth and rate of respiration,
 increase in motor activity,
 piloerection,
 and sensitivity to handling.

6.  After intravenous injection of 0.04 mg/kg epinephrine
into rats, a brief mydriasis occurs. The peak of the effect is
an enlargement in the pupillary diameter of 2 mm,
which appears in 30 seconds and endures for 8 minutes.
 Study of mydriasis can also be done in rabbits more
precisely because of size of pupil is big.
 Sympathomimetic agents dilates pupil by acting on radial
muscle and para-sympatholytic agents dilate pupil by
acting on sphincter muscles.

7.  The rat uterus is considered to be sensitive to epinephrine, but
liable to show excessive spontaneous activity, which is
diminished by lowering the temperature and decreasing the
calcium in the Ringer solution
 Acetylcholine (0.5 to 1.0 μg) is added to the bath every 2
minutes, and washed out when the effect is maximal (30 to 40
seconds). Doses of epinephrine, in 0.21 ml of solution or less,
are added to the bath 1 minute before a dose of acetylcholine.
 Another dose of epinephrine is given as soon as the response
to acetylcholine is normal. The departure from the usual
response to acetylcholine is a measure of the activity of
epinephrine or of a similar sympathomimetic agent.
Norepinephrine is comparatively inactive on this preparation.

8.  A similar technique can be applied to the first 3 cm of the
rat ascending colon. The temperature of the bath is
changed, and the Ringer solution is the same as that used
for the uterus.
 The lowest dose of epinephrine or test substance causing
the response to acetylcholine to be reduced by one-half, is
taken as a measure of the epinephrine-like activity.

9.  The narrow part of the rectal cecum is removed from a hen or
cock.
 A length of 4 to 5 cm is suspended in a tissue bath of 3 to 5 ml
volume. The movements of the longitudinal muscle are recoded
with an amplification of 15, and a tension of 1 to 5 gm.
 Drugs are allowed to act for 30 seconds before rinsing.
Ephedrine, diluted 1.5 X 106, relaxes the muscle and inhibits
spontaneous movements.
 The preparation is forty times more sensitive to epinephrine
than to norepinephrine.

10.  Spleens are obtained from cats under Nembutal (30 mg/kg)
anaesthesia. After a day in a refrigerator at 4°C, the spleen is
dissected to yield strips of tissue 4 cm long and 0.5 cm wide.
 The strip is fixed in a glass tissue bath, one end attached to the
bath, the other attached to a lever, weighing 2 gm.
 The contractions are recorded on a kymograph with
magnification of twenty times. After a relaxation period of 2
hours, the spleen strip is tested by adding drugs to the bath.
 The following substances caused a contraction: epinephrine,
1:107 (with some rhythmic activity superimposed on a tonic
contraction).

12.  Substances that inhibit the mediation of adrenaline, nor
adrenaline or mixture of catecholamine transmission are
called adrenergic-blocking agents, epinephrine
antagonists, adrenolytic agents, and sympatholytic agents.
 Sympatholytic activity may be recognized in a program of
blind screening, or in a broad program of screening,
usually by the effect of the test substance on the blood
pressure of the cat.

13.  Test is administered in rats and after one hour the epinephrine
is given.
 Antagonism of epinephrine will be proven if rats do not die
more than 50%.
 Control group of rats will die due to pulmonary oedema by
LD67 dose of epinephrine.
 If there is decrease in mortality, it shows sympatholytic
activity.

14.  For screening of sympatholytic agents, particularly
hypotensive agents.
 Cats are given oral dose of test substance, the degree and
duration of prolapse is calculated.
 For control group, guanithidine can be taken to compare
response of prolapse.

15.  Rats are anesthetized with urethane.
 BP is measured with manometer connected to cannula
in carotid artery.
 Test is given and again blood pressure is measured.
 Significant reduction in BP is seen if the test substance
posses sympatholytic activity.
 ADC: activity dose co-efficient (vaso-depressive
response measurement)
ADC =
Maximal of depression in BP (mm) x time interval of depression (mins)
2 x (dose of test in mg/kg)

16.  Spleen of cat contains 3 type of receptors, one for Ach,
one for Histamine and one for Adrenaline & Serotonin.
 Cat is anesthetized and spleen is isolated, and cut into
strips of specific size.
 It is mounted in organ bath and phenoxybenzamine i.e.
central sympatholytic is added.
 After 5 mins Ach, His, Epinephrine and 5-HT are added
respectively in each strip.
 There shows contraction of spleen muscle due to
sympatholytic activity.

17.  Isolated organ system
1. Vas deferens
2. Vascular smooth muscle
3. Intestine
4. Uterus
 Intact animal system
1. Atrial BP
2. Nictitating membrane

18.  Isoprenaline induced tachycardia
 Pressor depressor responses (ethyl norepinephrine)
 Hemodynamic measurements (blood flow )
 Responses of rat’s uterus
 Dog’s submaxillary gland
VD, tc

20. Substances that show an affinity for parasympathetic
receptors are muscarinic or para-sympathomimetic
agents.
There are three kinds of these agents:
 those causing a change in acetylcholine
concentration in the vicinity of the receptors,
 those reacting with the receptor,
 and those reacting with other receptors, that is,
receptors in the system which are not affected by
acetylcholine.

21.  Rats, mice, or guinea pigs with a pigmented iris are selected. Just
before use, the animal is sacrificed, and the eyeballs are quickly
enucleated with the aid of blunt, curved surgical scissors. The retro
bulbar structures are cleanly and carefully separated from the
eyeball.
 The cornea is transacted and cut free along the limbal margin.
 The eyes are briefly washed in Krebs-Ringer solution and then
mounted in appropriately sized, hemispherical sockets, set in black
Lucite trays.
 The trays are submerged in 20 ml bicarbonate-buffered Krebs-
Ringer solution, for which a chamber of clear Lucite was designed so
that it fitted the standard mechanical stage of a microscope.
 The pupillary diameter is read by means of a microscope in which
the ocular has a micrometre disk; the magnification is approximately
30 times

22.  The eyes are placed in the chamber, and, after a 30-minute
period for stabilization of the preparation, the pupillary
diameter is measured as a control, and the Krebs solution is
replaced with fresh Krebs solution containing the
predetermined concentration of the test substance. The solution
is allowed to exert its effect for 30 minutes. The pupillary
diameter is then measured. The response is expressed as a
ratio of this diameter to the control diameter. The plot of this
ratio against the logarithm of the molar concentration of the
test substance is a straight line.
 The pupillary diameter decreased from 100% of the control to
40% with 104 M methacholine, with 106 M carbamylcholine,
and with 103 M pilocarpine

23.  The trachea is removed from a freshly sacrificed rat. It is placed in
Krebs-Henseleit solution and dissected free of extraneous tissue.
 One end is attached to a U-tube, the other end to a capillary tube, 1
mm in internal diameter.
 The apparatus is placed in a 10 ml organ bath containing Krebs-
Henseleit solution at 37°, and aerated with 95% oxygen-5% carbon
dioxide.
 The capillary tube, 200 mm long, is mounted in a vertical position
above the vertical trachea, and it is fitted with a millimetre scale.
 The fluid in the capillary is flushed through from the U-tube and
then adjusted to be 70 to 100 mm above the level in the bath.
 The U-tube is then closed off from the fluid in the trachea with a
stop-cock, so that changes in the height of the fluid in the capillary
are due to tracheal contractions or relaxations.
 After 10 minutes, a steady level is read. The organ bath fluid is
changed from below, thus avoiding exposure of the tissue to air.

24.  Consider what happens when the trachea constricts. Let D be its
diameter at rest; C, its diameter in a constricted state; L, its
length; and V, its volume. Let h be the height of liquid in the
capillary, having a diameter of 1 mm. D is assumed to be
constant. Then:-
 Thus the value of C, the constricted diameter, is proportional to
the square root of the change in height of the fluid in the capillary.
A contact time of 1.5 to 3.0 minutes is used with constrictor
agents, and successive doses are applied every 5 to 8 minutes. The
organ bath is flushed out at least twice between doses.

26.  Substances that antagonize the action of acetylcholine
in terminal synapses are called parasympatholytic
(anticholinergic, antimuscarinic) agents.
 Their activity may be evidenced by mydriasis and by
inhibition of the secretions of the alimentary tract
(saliva, gastric juice, etc.).

27.  White mice are injected intraperitoneally with a solution of
the test compound.
 For the first hour after injection the pupil diameters, expressed
in 0.04 mm units, are measured every 10 minutes, and again at
90 and 120 minutes, by means of the eyepiece of a dissecting
microscope having a graduated scale.
 The quantal mydriatic effect is positive if the pupil diameter
exceeds 30/25 mm at any time after injection. For quantitative
results, a minimum of 4 doses and 60 mice are used.

28.  Mice of a single strain, of either sex, and weighing
15.5 to 16.5 gm are used. The mice are placed singly
in glass jars on a white table, beneath a fluorescent
light in an otherwise dark room. After half hour, the
pupil diameter of the mice is measured by means of
a graduated scale in the eyepiece of a dissecting
microscope.
 The animals are then injected with a drug, the
intraperitoneal dose being given in 0.01 ml per gram
of body weight. The pupil diameter is again
determined after 10, 20, and 30 minutes. A group of 5
mice is used for each dose level.

29.  The method of Edwards et al. (1960) has been applied to the
rabbit. Into the left eye of 3 rabbits are instilled two drops of
0.1% aqueous solution of atropine sulfate; into the left eye of 3
other rabbits are instilled two drops (0.2 ml) of a 1% aqueous
solution of the test compound. The right eye of each rabbit
serves as a control.
 The pupillary diameters of both eyes in all of the rabbits are
measured at intervals, and the average mydriasis is expressed as
the percent increase or decrease of the diameter of the test pupil in
comparison with that of the control pupil. Atropine sulfate effects
a 42 % mydriasis, persisting for 50 hours.
 This test incidentally may show whether the test substance is
irritating to the rabbit eye. Therefore the treated eye and the
control eye should be compared for swelling, redness, or other
signs of irritation. If irritation is found, it may be desirable to test
the substance for irritation to the skin and mucous membrane, as
well as for sensitizing action.

30. The blockade of salivation in the rabbit with graded responses is used in the method of
von Issekutz (1917). It has been shown that a quantal procedure may be used, since a
linear relation was found when the probit of the rabbits showing 50% inhibition of
pilocarpine- induced salivation was plotted against the logarithm of the doses of
atropine.
Brown and Quinton (1957) have developed a quantitative method with rabbits. White
rabbits weighing at least 1.5 kg are used. An oral dose of 500 to 625 mg/kg urethane
is given to effect sedation. The dose is contained in 25 ml of saline. Each rabbit is
placed in a stock, so that the head remains above a funnel, in which the animal saliva
is collected, after it has been given a subcutaneous dose of 5 mg/kg pilocarpine
nitrate, administered 30 minutes later than the urethane. Drugs antagonizing
acetylcholine are given up to 70 minutes before the pilocarpine, although this interval is
too long for atropine.
If a rabbit in preliminary experiments does not show a 50% reduction in salivation
when 25 Mg/kg atropine is given 15 minutes before the pilocarpine, the animal is not
used. It is presumed that the animals insensitive to atropine have large quantities of
plasma atropinesterase.

31.  Groups of 20 fasted, Swiss Webster mice of either sex are
given various doses of the drugs by stomach tube. The animals
are anesthetized by intraperitoneal injection of sodium
pentobarbital (2 mg contained in 0.2 ml). When the animals no
longer respond to a vigorous tail pinch, the intestines are
exposed by cutting a flap in the abdominal wall.
 Methacholine, 0.25 μg contained in 0.2 ml, is applied topically
to the exposed intestines, which are observed for a carefully
timed 3-second period. Two points are scored if the
methacholine spasm is completely prevented; one point is
scored if the spasm is reduced in intensity or occurs only in a
limited area.

32. This test is designed to show parasympathetic blockade, and its
reversal, as demonstrated by lacrimation (de Jongh et al., 1955).
Wistar rats weighing 150 to 225 gm are used. The animals are kept at
36° for 10 minutes before use in order to cause vasodilatation.
The test compounds are then injected into the tail vein, the volume of
solution being 1 ml/kg of body weight, and the duration of the injection
being 20 seconds. A dose of 0.5 mg/kg carbamylcholine is given
intraperitoneally after 15, 45, 75, and 105 minutes, or until a positive
response is obtained. The appearance of a coloured spot on a piece of
cotton pressed against the eye, within 3 minutes after the injection of
carbamyl-choline, is taken as a positive response. Failure to obtain a
coloured spot at the proper time interval is considered
parasympatholytic activity.

33. A. The rectus muscle of the frog
B. The isolated ileum of the mouse
C. The isolated ileum of the rabbit

34. Female albino mice, 2 to 4 months of age, are fasted overnight. They are injected
intraperitoneally with the drug contained in 0.1 ml/10 gm of body weight. One
hour later the animals are given by stomach tube 0.3 ml of an aqueous suspension
of 10% charcoal and 5% gum acacia. Two hours after the charcoal meal the
mice are sacrificed, and the intestines are excised from the cardia to the anus. On
a clean white glass the extended intestine is measured from the pylorus to the
cecum. Control mice have black ceca. (In 300 control mice given saline, the ceca
were filled with charcoal).
Antispasmodics cause the ceca to be free of charcoal, that is, to have natural
colour. The all-or-none criterion is believed to be superior to measuring the
distance traversed by the charcoal meal in ratio to the intestinal length, because
these measurements are inaccurate, and because the dose-effect curves are very
flat.
Antispasmodics, analgesics, and other classes of drugs such as antihistaminics
cause a decrease in intestinal propulsion, probably by interference with cholinergic
receptors in the alimentary tract. Thus positive activity shown by a charcoal-free
cecum may indicate parasympatholytic activity, extensive nervous depression, etc.

35. A modification of this procedure (Witkin et al., 1961) is as
follows: Mice are deprived of food for 17 to 22 hours but are
allowed to have water. At a specified time after subcutaneous
injection of the drug, the mice are given orally 0.25 ml of a 5 %
suspension of carbon black in 5 % gum tragacanth. Thirty
minutes after this administration the animals are sacrificed with
chloroform. The stomach and small intestine are removed and
stretched by hanging a 3-gm weight from one end for 20 seconds.
The percentage of the small intestine which the carbon has
traversed is then recorded.
There is no explained correlation between analgesia and
gastrointestinal propulsion. Nevertheless all known analgesics
significantly inhibit the propulsion of a charcoal meal in mice

37.  Ganglion-blocking drugs are of two kinds:
 Those which cause initial stimulation of the ganglion are
called depolarizing agents;
 Those which cause no initial stimulation and thus block in
the absence of any depolarization are called competitive
blockers.
 Acetylcholine depolarizes the neuromuscular endplate, as
decamethonium does, whereas d-tubocurarine does not.

38.  Take female mice and give intraperitoneal dose of test
drug. Followed by iv injection of nicotine after some time
interval.
 Mice are immediately placed on elevated wooden
platform.
 Mice treated only with nicotine will show stimulant
activity, in 5 seconds such as clonic seizure, tonic seizure
and death.
 Mice treated with blocking agents will prevent death and
seizures, will antagonize nicotine.

39.  Animals are anesthetized and are kept in supine position. The
cervical sympathetic trunk is dissected out under a dissecting
microscope, is tied peripherally and cut. The nerve is kept
immersed in warm paraffin. Stimulation is by Ag-AgCl
electrodes.
 A surgical silk thread is tied through the lower eyelid, and is
fixed by plasticine to a mirror, suspended by a fine wire. A light
slit is focused on the mirror, and the leading edge of the
reflected light beam is allowed to fall on a photocell. When the
eyelid retracts, the beam of reflected light sweeps over a
surface of the photocell proportionally to the lid's movement.

40.  The blood pressures of white rabbits weighing 1.5 to 2.0 kg are
recorded with a modified Grant ear-capsule. The rabbits are
placed in an electrically heated box maintained at body
temperature.
 The minimal pressure required to occlude the central artery of
the right ear is recorded once or twice a minute until steady
readings are obtained. The drug in 0.3 ml saline is injected into
the marginal vein of the left ear. Injections of potent drugs are
made every 3 days.
 Besides finding the effect of a compound on the blood
pressure, the animal may be observed for mydriasis, defecation,
urination, and salivation. The method may be used to test
ganglion-blocking agents like hexamethonium.