METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO - A.A. 2017-2018 Farmacologia Speciale - Elearning
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METODI SPERIMENTALI PER LO
STUDIO DEL COMPORTAMENTO
Farmacologia Speciale
A.A. 2017-2018
SPERIMENTAZIONE ANIMALE METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO A.A. 2017-2018
SPERIMENTAZIONE ANIMALE
Utilizzo scientifico a scopo di studio e ricerca di animali
Animali ed essere umano condividono geni, meccanismi e
funzioni biologiche che fanno dei primi dei buoni modelli per
capire quel che accade anche nella nostra specie, consentendo di
fare deduzioni e predizioni
RATTO TOPO
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018
PRINCIPIO DELLE 3 “R”
1959
Reduction Riduzione Incoraggia alternative alla
sperimentazione animale
Replacement Sostituzione Mira a migliorare il benessere
degli animali e la qualità
scientifica dove l’uso di
Refinement Affinamento animali non può essere evitato
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018
NEUROSCIENZE COMPORTAMENTALI Locomotor behavior Socio-Sexual interaction Emotional reactivity Learning and memory abilities Models of depression Motor coordination and balance METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO A.A. 2017-2018
1) LOCOMOTOR BEHAVIOR
Open-field: it consists of a square or circular platform where a rat or a
mouse is released for exploration. An observer will record the number
of square limit crossings with both forepaws; number of fecal boli;
thigmotaxis (time spent near the walls); frequency and duration of
rearing (standing with the body inclined vertically, forequarters raised),
wall-rearing (standing on the hind-limbs and touching the walls of the
apparatus with the forelimbs), grooming (rubbing the body with paws or
mouth and rubbing the head with paws) and immobility.
Di Giovanni et al. Brain Res, 1993;
Candland and Nagy. Ann NY Acad Sci, 1969
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018
1) LOCOMOTOR BEHAVIOR
The open-field is useful for detecting any defect on spontaneous
locomotor activity due to drug treatment, lesion in the brain, strain
differences or genetic manipulation.
The open-field can also be used for testing anxiety in animals. The
measure of latency and the patterns of exploration of the field are
examined. Anxious animal will take longer time to adventure in the centre
part of the arena. Another way of measuring anxiety is to introduce a
novel object in the open-field and measure the time it takes for an animal
to approach the novel object. This task is valuable in early phases of
genetic phenotyping or drug screening.
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO A.A. 2017-2018
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO A.A. 2017-2018
2) SOCIO-SEXUAL INTERACTION
Maternal behavior Sexual patterns
Adult
aggressiveness
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018MATERNAL BEHAVIOR
Maternal care in rats and mice:
– Licking Chantrey and Jenkins. Anim Behav, 1982;
– Nursing positions Noirot. Anim Behav, 1982;
Laviola et al. Psychoparmacology, 1990
– Nest building
– Retrieving
Passive Nursing Arched Nursing
No Maternal Behavior
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018SOCIAL INTERACTION AND ADULT AGGRESSIVENESS
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018SOCIAL INTERACTION AND ADULT AGGRESSIVENESS
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018SOCIAL INTERACTION AND ADULT AGGRESSIVENESS
sostanze dopanti (steroidi
androgeni anabolizzanti)
psicostimolanti (cocaina ed
amfetaminici)
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-20183) EMOTIONAL REACTIVITY Ultrasonic vocalization test Elevated zero or plus-maze test Social interaction test METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO A.A. 2017-2018
ULTRASONIC VOCALIZATION TEST
The ultrasonic vocalization test, performed recording ultrasonic calls emitted by
pups removed from their nest, is considered a reliable tool to investigate the
anxiolytic and anxiogenic activity of drugs.
One male per litter for each treatment will be randomly removed from the nest
and, after axillary’s temperature measure, placed in a shallow glass dish (15 cm
diameter and 10 cm deep) 15 sec before the test. This will confine the pup
relative to the microphone, which will be supported vertically 15 cm above the
dish and thus will avoid handling it during the recording session. Ultrasonic calls
will be recorded for 15 sec to collect the baseline levels, then each pup will be
injected with the drug or its vehicle, and returned to the nest. 30 min after the
injection, ultrasonic emission will be recorded for 15 sec. The number of calls
will be counted by listening to the audible output of the bat-detector. Data will
be expressed as percent change from baseline.
Gardner. J Pharmacol Methods, 1985;
Insel et al. Pharmacol Biochem Behav, 1986
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO A.A. 2017-2018
Flat call Frequency steps call
3-Syl call 2-Syl call
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018Effects of Diazepam METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO A.A. 2017-2018
ELEVATED ZERO OR PLUS- MAZE TEST
Zero-maze test: this test, based on the natural aversion and avoidance of
rodents for open spaces, is a modification of the elevated plus-maze model of
anxiety which incorporates both traditional (% of time in the open arms) and
novel ethological measures (number of head dipping over the edge and
stretched-attend postures from the closed to the open quadrant). The design
comprises an elevated annular platform with two opposite enclosed
quadrants and two open, removing any ambiguity in interpretation of time
spent on the central square of the traditional design and allowing
uninterrupted exploration.
Shepherd et al.
Psychopharmacology, 1994;
Bickerdike et al., Eur. Journal
Pharmacology, 1994
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018ELEVATED ZERO-MAZE TEST
Zero-maze test. Adult rats will be weighed and injected with the drug
or its vehicle. 30 min after the injection rats will be individually placed
on a closed quadrant and a 5-min test period will be recorded on
videotape for subsequent analysis.
Behavioural measures will be calculated as follows:
(a) % time spent on the “open” quadrants (% TO);
(b) number of exploratory head dips (HDIPS) made over the edge of the
platform, either from the exit of the “closed” quadrant, or whilst on the
“open” quadrant;
(c) number of stretched-attend postures (SAP) made from the exit of a
“closed” quadrant towards an “open” quadrant. This exploratory
posture can be described as a forward elongation of the body, with
static hind-quarters, followed by a retraction to the original position.
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018ELEVATED PLUS-MAZE TEST METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO A.A. 2017-2018
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO A.A. 2017-2018
Effetto delle benzodiazepine nell’elevated plus-maze test
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018SOCIAL INTERACTION TEST
The social interaction test was developed in the 1978 as the first animal test of
anxiety that endeavoured to use ethologically relevant sources of anxiety, and to
use a natural form of behaviour as the dependent measure. The dependent
measure is the time that pairs of male rats or mice spend in social interaction.
This time decreases when rats/mice are anxious. This test can be performed at
different rat/mouse ages and with different conditions such as familiar or
unfamiliar partner and arena.
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018SOCIAL INTERACTION TEST
Adolescent male rats will be individually housed for 24 hours before the
test; then they will be weighed and injected with drug or its vehicle. 30 min
after the injection each rat will be tested in a 30 min period of social
interactions with a same sex, age and strain unfamiliar partner.
The unfamiliar pair will be placed in a cage identical to the home-cage with
new sawdust as bedding. The behaviour of each pair will be video-recorded
and scored. Scored social behaviours will be: Play behaviour (Wrestling,
Pinning); Affiliative behaviours (Social inactive, Social rest, Allogrooming);
Soliciting behaviours (Push, Crawl); Investigative behaviours (Follow; Mutual
circle and Social sniffing).
Non-social behaviours will be: Inactive, Exploring, Jumping, Digging and Self-
grooming.
File and Hyde. Br J Pharmacol, 1978;
File and Seth. Eur J Pharmacol, 2003
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018Social Play METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO A.A. 2017-2018
4) LEARNING AND MEMORY ABILITIES METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO A.A. 2017-2018
4) LEARNING AND MEMORY ABILITIES
Object recognition task. Object recognition, which is assessed from the
preference normal rodents display for investigating novel rather than
familiar complex objects can be used to provide a valid and relatively pure
measure of "working memory“. Indeed, advantages associated with this
class of measure include the fact that performance does not depend on
the retention of a rule, nor is influenced by changes in responsivity to
reward.
Ennaceur and Delacour. Behav.
Brain Res, 1988;
Ennaceur et al. Exp. Brain Res.,
1997;
Bartolini et al. Pharmacol.
Biochem. Behav., 1996
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO A.A. 2017-2018
SPATIAL MEMORY: MORRIS WATER MAZE
The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test
hippocampal-dependent learning, including acquisition of spatial memory and long-term spatial memory. The
MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the
differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions. In addition, the
MWM testing environment reduces odor trail interference. This has led the task to be used extensively in the study
of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in
the validation of rodent models for neurocognitive disorders such as Alzheimer's Disease.
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018SPATIAL MEMORY: MORRIS WATER MAZE METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO A.A. 2017-2018
5) MODELS OF DEPRESSION: FORCED
SWIMMING TEST
In the forced swimming test, a rodent
is placed in a cylindrical box containing
cold (22-24 °C) water. Antidepressants
reduce floating and increase swimming
and struggling. These parameters are
considered to be an indicator of
antidepressant activity.
Porsolt. Nature, 1977
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-20186) MOTOR COORDINATION AND BALANCE
The Rotarod technique has proved to be of great value in
research involving screening of drugs which are potentially
active on balance and motor coordination. It is used to asses
the ability of an animal to balance on a rotating rod.
Dunham et al. J Am Pharamc Assoc Sci Ed, 1957;
Carter et al. J Neurosci, 1999.
In the beam walking test rodents are trained to traverse a
series of elevated, narrow beams to reach an enclosed
platform. The protocol measures foot slips and latency to
traverse the beams.
Gentile et al. Behav Biol, 1999
A number of different parameters of gait can be analyzed
simply and effectively using footprint patterns. To obtain
footprints, a rodent’s paws are coated with paints and the
rodent is allowed to walk along a narrow, paper-covered
corridor, leaving a track of footprints.
Golani. Brain Resl, 1979
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018Rotarod Raised beam test
These tests are most frequently
used in conjunction with
unilateral 6-hydroxydopamine (6-
OHDA) lesions as a model of
Parkinson’s disease.
Footprint analysis. The paws are dipped in ink or
paint, so that the animals leave a trail of footprints
as they walk or run along a corridor to a goal box.
Measurements of stride length, base width, and fore
and hind paw overlap give an indication of gait.
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
A.A. 2017-2018Mouse behavioral assays for the major symptom categories of schizophrenia. Positive symptoms. The psychomotor agitation and hyper-
responsivity to psychotomimetic drugs found in schizophrenia can be modeled in mice by testing locomotor responses in novel and stressful
environments (A) and locomotor hyperactivity-inducing effects of psychotomimetics, such as NMDAR antagonists (B), respectively. Negative
symptoms. Schizophrenia is often associated with blunted affect, social withdrawal, and loss of pleasure in normally rewarding activities
(anhedonia). Social interactions with other mice (C) and decreased preference or motivation to obtain rewarding substances, such as
sucrose (D), can be assessed as a means to model these negative symptoms. Cognitive/executive symptoms. Abnormalities in cognition and
executive functions are a prominent feature of schizophrenia and range from deficits in episodic memory, poor attention and sensorimotor
gating, to impaired reversal learning and set-shifting. Learning and memory can be tested in mice using, e.g., the reference memory version of
the Morris water maze (E), while prepulse inhibition of the startle response provides a ready measure of sensorimotor gating (F).
METODI SPERIMENTALI PER LO STUDIO DEL COMPORTAMENTO
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