1. Animal behavior testing in research
Dr. S Voiculescu

2. To see the world in a grain of sand
And a heaven in a wildflower
Hold infinity in the palm of your hand
And eternity in an hour
John Donne

3. Behavior 1 a : the manner of conducting oneself
b :  anything that an organism does involving action and response to stimulation
c :  the response of an individual, group, or species to its environment
2 :  the way in which something functions or operates

4. Behavior The ultimate assay of neural function
Most of the behavioral methodology comes from research on rats
Ethograms of rodents are similar enough to allow for generalization of the methods

5. Uses
What parts of the brain are used for a certain behavior (how does a trained rat's performance change after manipulation of different parts of its brain, like the hippocampus)
How does performance change after a rat receives certain research protocol (meds, neurotox, gene manipulation)
Neurodevelopment

6. What kind of behavior (I) general appearance
(II) sensorimotor behavior
(III) immobility and its reflexes
(IV) locomotion
(V) skilled movement
(VI) species-specific behaviors
(VII) learning: short-term memory (frontal lobe), object memory (rhinal cortex), emotional memory (amygdala), implicit/explicit memory (rhinal resp hippocampus)

7. Is behavior sex-dependent?
Behavioral testing is usually done on male rats
Female testing may be done- needs estrum phases correlation- vaginal smear

8. Methods
End-point measures- measures of the consequences of actions- a bar is pressed, an arm of the maze was entered…)
Kinematics- Cartesian representations of actions- distance, velocity, trajectories
Movement description- Eshkol Wachmann Movement Notation

9. End-point: a. normal rat/b. brain injury
Movement notation
Cartesian- trajectory of the tip of the third digit

10. Observational tests
Quick, home cage, no special equip

11. Importance of observational tests- example- fur color
Pregnant rats- injected with bromodeoxyuridine E11-E21
BrdU labels mitotic cells immunohistological techniques
Cortical neurons development
Common embyo origin of melanocytes and neurons (altered migration)

12. Open field behavior
Ignores food in order to make exploratory movements
Rats eat outside the cage after habituation
!habituation period is very important while behavior testing- usually a few days- one week (depending on protocol)

14. Sensorymotor tests- Rotarod
based on a rotating rod with forced motor activity
evaluates balance and sensorymotor coordination of the subjects
rat is placed on a horizontally oriented, rotating cylinder (rod) suspended above a cage floor, (not high enough to injure the animal, but high enough to induce avoidance of fall)

15. The length of time that the rat stays on this rotating rod is a measure of their balance, coordination, physical condition, and motor-planning.
The speed of the rotarod is motorically driven, and may either be held constant, or accelerated
frequently used in early stages of drug development to screen-out drugs that might later cause impairment in human driving, etc

17. Beam walking
assesses motor coordination, particularly of the hindlimbs
rats have to traverse an elevated narrow beam which is suspended between a start platform and their home cage
difficulty of this task can be varied by using beams with different shapes and widths

19. Footprint analysis
evaluates the walking pattern of rats and thereby allows to detect gait abnormalities
footprint patterns of rats are analyzed after the rat was walking along a narrow corridor
hind and fore paws are coated with non-toxic paints of different colours

21. Motor reflexes Postural and righting reflexes
Visual, vestibular, surface body senses, proprioception
Immobility- with or without postural support- both normal (cold, scared/ hot, asleep, resting)
Animal hypnosis= tonic immobility

22. Posture Animals defend immobility when placed in an instable condition
Haloperidol- dopamine antagonist- immobility with postural support
bracing

23. Tail suspension Assymetry- unilateral injury
Cortical- turn contralateral to lesion
Dopamine depletion-turn ipsilateral
Flexion of forelimbs towards the body (including grasping on each other)- pyramidal/extrapyramidal lesions

24. Righting response
When placed in a condition of instable equillibrum- will attempt to regain upright posture in relation to gravity
Visual, vestibular, tactile, proprioceptive
Adjusts posture when released on a cushion from 1 m

25. Specific behaviors Aggression
Used to establish social hierarchies, defend territories
Widely used for human aggression
Test- dominance tube test

26. Dominance tube test
Habituation 5 min
Testing phase- max 2 min

27. Aggression Primitive behavior
Humoral regulation- testosterone, progesterone, estrogens, melatonin, ADH, cortisol, ghrelin
Nervous structures- olphactory bulbs, amygdala, lateral septum, anterior hypothalamus
Central serotonin depletion increased offensive aggression

28. Learning and memory Short- term Long- term
Working- on the last trial I have found food here
Procedural- escape platform somewhere in the swimming pool

29. Novel object recognition test
proposed by Ennanceur and Delacour (1988)
assesses the natural preference for novel objects, while the rat explores its environment (the open field arena).

30. NOR The test consists of three phases:
Habituation freely explore the open-field arena for 5 minutes in the absence of any objects
Familiarization one rat at a time is placed back into the arena with two identical sample objects (1+11), and allowed to explore for 5 minutes;

31. NOR
Testing (5 min) occurs after a variable retention period, depending on the type of memory to be tested (5 minutes for short memory and 24 hours for long memory assessment). One of the two objects from the previous phase is replaced by a novel object (1+NO).

32. NOR time spent to explore a familiar object (Tf)
time spent exploring a novel one (Tn)
discrimination index, DI=(Tn-Tf)/(Tn+Tf)
DI can vary between +1 and -1, a positive score indicates more time spent with the novel object (Tn), while a negative score indicates more time spent with the familiar object (Tf). Zero score indicates a null preference.

33. T maze spontaneous alternation test
The T-maze is shaped like a T.
The test animal starts at the base of the T.
A reward may be placed in one arm of the maze, or different rewards may be placed in each arm. The rat walks foward and chooses the left or right arm of the maze.
Or- NO reward- spontaneous alternation- working memory

35. Morris water maze Circular pool, diam= 1.5 m
Opaque water (powdered milk)
Platform 10 sq cm- 1 cm under water surface
In order to escape- must find platform
Succesive trials- different start points- visual cues in the room
Same platform location= procedural memory
Moving platform= spatial working memory

37. Anxiety testing- Open field test (OFT)
originally created by Calvin Hall as a measure of fearfulness
assesses the response of lab animal subjects to an inescapable arena (anxiety), exploratory behavior and emotionality
used in pharmacological studies for anxiety assessment, as a measure of anxiolytic drug efficacy

38. Interpretation
rats spend more time in the centre zone and less time near the walls of the arena if they are less anxious
rats were allowed to freely explore the testing arena for 10 minutes
recorded variables are: the time spent in the central zone, the number of central area crossings and the number of defecations.

40. Anxiety testing- Elevated plus maze EPM
protocol of Walf et al., 2007
animals are first placed in the central area of the maze and are allowed to freely explore the arena
anxiety is defined by a decreased time spent in the open arms and decreased no of crossings

43. Depression assessment tests
Last to be performed
Change the behavior of the rats
Despair tests- forced swim test and tail suspension test

44. Forced swim test modified Porsolt et al. protocol (1977)
2-day procedure in which rats swim under conditions in which escape is not possible
Day 1-rats were placed in a 35 cm tall, 30 cm diameter cylinder, filled up to 21.5 cm with water at 24±0.5C. After 15 min the rats are removed from water, dried with towels and placed in a warmed enclosure.
Day 2- 5-min test sessions were succeeded at 24 h and videotaped from above of the cylinders.

45. frequency of mobility and immobility of the animals, the presence of differences in the active moves
two observers, by manually recording rat behavior once at 5 seconds during the 5 minutes test trial.
Three behaviors
(1) immobility
(2) climbing
(3) swimming

47. Tail suspension test
conceptually related to the forced swimming test, except that immobility is induced by suspending the rats by the tail.
After initially trying to escape by vigorous movements, at some point, rats will become immobile
The duration of immobility is reduced by antidepressant medication - used to test different drugs’ efficacy.
Advantages over the forced swim procedure: no hypothermia is induced, no special post-experimental treatment (rubbing down, a warmed environment) is required.

48. Rats are individually suspended from the tip of the tail.
Duration of mobility and immobility are determined, as well as variations of the active moves of the rats during testing (presence or absence of certain types of movements).
Each rat was tested in a single trial that has lasted for 6 minutes.

49. 1) swinging - keeping its body straight, the rat continuously moves its paws in a vertical position and/or moves its body from side to side
2) curling - the rodent engages in active twisting movements.

51. Gestational chronodisruption and neurodevelopment

52. Gestational chronodisruption
Disruption of the maternal environment during pregnancy is a key contributor to offspring diseases that develop in adult life
Miscarriage, preterm delivery, low birth weight

53. Aim
impact of altered chronodisruption and melatonin signalling on the adult metabolism and behaviour is extensively investigated
effect of prenatal chronodisruption in the second half of the pregnancy on the brains of adult male Wistar rats

54. Functional pinealectomy
Continuous light exposure of pregnant Wistar rats, between days 12-21(functional pinealectomy)
Control dams exposed to normal light/dark cycle (06:00–18:00 hours).
Two groups of male offspring resulted from the pregnancies:
control group (C) (n = 8)
prenatal chronodisrupted (PCD)  group (n = 8)

55. Behavioral testing
After postnatal day 60, male offspring were tested for:
anxiety behavior (using Elevated plus maze test and Open field test)
memory impairment (using Novel object recognition test)
depression (using Tail suspension test and Forced swim test)
recorded using EthoVision XT software

56. OFT mean time spent in central area

57. OFT- no of crossings

58. EPM

59. EPM

60. EPM

61. NOR short-term memory

62. NOR short-term memory

63. FST

64. TST

65. Behavioral changes Anxiety behavior
Short-term memory impairment but normal long-term memory
Depressive behavior when assessed by TST but a normal FST

66. Molecular evidence of neural insult
Melatonin and serotonin
Brain oxidative stress
Apoptosis assessment
Clock gene expression

67. Melatonin level

68. AANAT and MTR (hypothalamus)

69. Serotonin

72. Melatonin and serotonin
Model efficiency (P0)
Slc6a4- serotonin reuptaker- higher expression levels in PCD
Melatonin and serotonin depletion- anxiety behavior, impairment in short-term memory and depression

73. FST- Dopamine
TST- Dopamine + Serotonin

74. Free ROS

75. Oxidative enzymes

76. Oxidative stress Melatonin- antioxidant molecule
At pharmacological levels melatonin was shown to stimulate gene expression of antioxidant enzymes in the brain, or to increase their activity (Sod1, Gpx1,Gss)
Chronic oxidative stress
low melatonin-scavenger
loss of activity of antioxidant enzymes

77. Apoptosis gene expression
While Bcl2 showed no change in expression levels, Bax had a significant down-regulation in the PCD subjects (translated to a 1.5 fold decrease)

78. Bcl2/Bax Chronic oxidative stress leads to apoptosis resistance
Bcl2 – antiapoptotic
Bax- proapoptoic
In our model Bcl2/Bax is higher (downregulation of Bax) antiapoptotic effect

79. Clock genes
Gene expression levels were determined for three genes: Rora, Clock, and Arntl1 in four areas of the brain. The only significant detected difference was in the expression levels for Rora in the amygdala (1.66 fold increase in the PCD subjects). *p<0.05, compared to control adult rats.

80. Rora Upregulated in the amygdala Rora- melatonin nuclear receptor
Rora protects neurons against oxidative stress

81. Gestational chronodisruption leads to
Altered behavior in adult offspring produced by the following possible mechanisms
Low melatonin and serotonin levels and signaling in the brain
Chronic oxidative stress and/or the underlying adaptative responses produced by this state may explain behavioral changes from a molecular perspective
Impaired circadian system development
Basis of degenerative diseases?

82. Thank you Ana Maria Zagrean Leon Zagrean Diana Le Duc Adrian Rosca
Vlad Zeca

83. Thank you!!!