Behavioral Testing


Behavioral testing standard protocol (excel file)

Animal test results: All Coded (excel file)
 
 

Behavioral Testing
Open field activity
Crossing a wire mesh screen
Beam balance
Walking track analysis
Placing reaction
Righting response
Prehensile reflex
Nociceptive hot plate test
von Frey test
Staging
 
 

Behavioral Testing


A number of different test procedures intended to measure several behavioral aspects have been used. In general 20-30 animals were tested in each session, lasting for a period of 3 days. It should be noted that all tests were performed in most but not all cohorts.
 
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Open field activity

Explorative behavior was examined with the open field test (Dorce and Palermo-Neto, 1994; Peng et al., 1994; Drago et al., 1996), using a square area with walls (70 ´ 70 ´ 30 cm) in gray colored plastic. The floor was subdivided into 25 equally large squares. In dim light the animal was placed in the center of the arena and allowed to freely explore the field for 180 sec. During this period the following behavioral characteristics were evaluated: a) ambulation frequency (number of squares entered with all four feet); b) rearing frequency (number of times animals stood on hind limbs); c) immobility frequency (number of episodes of more than 3 sec without movement); d) the frequency of urination and defecation. In between animals, the open field was carefully washed with a water-ethanol solution to eliminate possible bias due to odors left by previous subjects.
   The effects of aging on locomotion, rearing and immobility frequencies and on urine and fecal deposits are for female rats summarized in Figure 2. As can be seen, aging is associated with a decline in ambulatory behavior and rearing, while the immobility frequency increase. In contrast, no effect of age on presumed stress responses, such as number of urine and fecal deposits, could be detected.
 
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Crossing a wire mesh screen


A 70 cm long, 2.5 cm-wire mesh screen was used. In dim light, the animal was placed at one end of the path and a 60W light source was directed to this spot. At the other end of the path, the “home cage” with litter mates was placed. Each animal was given 90 sec to cross the path. Records included distance, time, and number of errors, i.e. instances of misplaced hind paws (slips). Hind limb performance was evaluated since this test, like the beam walking test (Alexis et al., 1995), presumably examines hind limb sensorimotor function.
    All animals, except a few 30-month-old subjects, completed the 70 cm wire mesh within 90 sec. Among female rats between 3 to 22 months of age the time required to complete the task was on average 9 sec, with no major difference observed between the different age groups. However, aged rats (27-30 months old) needed on average 19 sec to cross the mesh. A similar pattern was also present with regard to the number of errors recorded, where aged rats had a substantially increased incidence of hind paw slips.
 
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Beam balance


A 2.5 cm-wide wooden beam was suspended 0.5 m above a soft surface. The rat was placed on the beam for a maximum of 60 sec, and the performance was ranked according to the following scale (adopted from Clifton et al., 1991):
1.balances with steady posture; paws on top of the beam;
2.grasps sides of beam and/or has shaky movement;
3.one ore more paw(s) slip off beam;
4.attempts to balance on beam but falls;
5.drapes over beam and/or hangs on beam and falls off;
6.falls off beam with no attempt to balance or hang on.

Each animal was subjected to three consecutive trials, and the mean score of these trials was calculated. As shown in Figure 4, three-month-old rats almost without exception manage to remain on the beam for 60 sec, while this ability rapidly declines with advancing age. Among the oldest rats, the average time on the beam was approximately 10 sec, and only quite few animals attempted to balance.  Figure 4. Female rat performance on the beam balance task. While young adults in most instances managed to remain on the beam for 60 sec (as indicated by a score of £ 3), aging female rats revealed a substantially decreased ability. Among the oldest rats, only a few individuals even attempted to balance. All data are presented as cohort mean +/- standard deviation.
 

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Walking track analysis


For this test, the animals feet were immersed in non-toxic acrylic color (fore paws with red and hind paws with black color) and they then had to walk through a 8.5 ´ 42 cm transparent plexiglass tunnel with the “home cage” at the other end. A stripe of high-quality paper was placed on the runway floor and taken out for analysis after the animal had crossed the path. The following records were made from the walking tracks: a) stride length (distance between fore paw-fore paw and hind paw-hind paw); b) gait width (distance between left and right hind paws), c) placement of hind paw relative to fore paw (distance between hind paw-fore paw in each step cycle).
    The stride length of female rats increases somewhat from 3 to 15 months of age, possibly relating to the growth of the animals, but then shows a substantial decline among the oldest rats. With regard to gait width and differences in the placement of hind paw relative to fore paw, both parameters show an increase with advancing
 
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Placing reaction


Tactile placing was evaluated by supporting the animals trunk while allowing the limbs to hang freely. The animal was then brought towards a table edge, and the dorsal and plantar surface of each foot were gently touched. For each test a score of 1 was given for normal, immediate placing; a score of 0.5 was given if the placing was delayed or incomplete; a score of 0 indicated absent placing (Gale et al., 1985; Alexis et al., 1995).
    All female rats, irrespective of age, had a normal placing reaction in their fore limbs. However, a decline in the hind limb placing reaction was observed among the oldest female animals.
 
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Righting response


The rat was held in the examiner’s hand approximately 20 cm above a soft surface, and the righting reflex was elicited by turning the rat over on its back upon release. The rat’s attempt to right itself was studied and a score of 2 was given if the animal showed a normal righting response, i.e. counter to the roll direction; a score of 1 was given if the righting response was weak, delayed or in the direction of the roll; a score of 0 indicated no righting attempt (Gale et al., 1985; von Euler et al., 1996).
The results are summarized in Figure 7, where it can be seen that although the oldest female rats show abnormal righting responses, most of them exhibit attempts
to right.
 
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Prehensile reflex


A taut horizontal steel wire was elevated approximately 50 cm above a soft surface. Individual rats were raised to this wire, which they grasped with their fore paws. The time each rat managed to hold onto the wire in three consecutive trials was recorded. The prehensile reflex refers to an animal’s ability to grasp a horizontal bar or wire with its fore paws and to remain suspended without dropping off, and has been reported to be a measure of muscle strength (Dean III et al., 1981). On the first trial, three-month old female rats were able to hold onto the wire for an average of 7 sec, while the corresponding figure for their 27-30 month-old counterparts was 4 sec. Moreover, while the third trial time was virtually unchanged for young adult rats, aged rats appeared more fatigable and only managed to remain suspended for an average of 1.5 sec.
 
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Nociceptive hot plate test


A plexiglas cylinder with a diameter of 20 cm was placed on a plate (30cm x 40cm), set to maintain a temperature of 52 ± 0.5°C. The animal was placed on the heated surface until it licked paws, jumped or vocalized. The time lapse between placement and reaction was recorded as response latency (Espejo and Mir, 1993; Langerman et al., 1995). The cut-off time was set at 30 sec to avoid tissue damage.
 
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von Frey test


Animals were put in a plexiglass container with a very fine mesh floor, and allowed to habituate for at least 10 min. von Frey hairs (a series of nylon monofilaments with logarithmically increasing stiffness that exert defined levels of force as they are pressed to the point where thy bend), were then applied perpendicular to the paw skin and depressed slowly until they bent. The hairs were tested in ascending order of stiffness, with each applied 5 times, and the threshold was defined as the first hair in a series evoking at least one response. The response criterion was a reflexive withdrawal that was clearly separate from stepping (Tal and Bennett, 1994). Thresholds were determined separately for hind limbs (mid-plantar region) and fore limbs (mid-palmar region). In 3-month-old female rats the tactile threshold was found to be higher in hind limbs (filament #15) than fore limbs (filament #14). This pattern was maintained in aged (30 months old) female rats that, moreover, disclosed a substantial increase in von Frey thresholds (hind limbs- filament #17; fore limbs- filament #16).
 
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Staging


In the initial phase of this project a staging protocol was set up allowing us to rank-order the aged animals according to their behavioral motor impairments. This was described in some detail in the early publications: “…All aged rats used in this study disclosed clinical signs of neuromuscular dysfunction. Mild cases (stage I) involved moderate muscular wasting of the hind limb, an adduction insufficiency causing the rats to stride with their hind limbs abducted compared to young adults, and with the lower part of the trunk closer to the supportive surface. Such rats were able to rise on their hind limbs -a common posture while a rat investigates e.g. a new environment-, but did so more infrequently and for shorter periods as compared to young adult rats. Aged rats, in general, also stride at a slower pace. With increasing symptoms (stage II) a clear clumsiness and a poorer co-ordination (ataxia-like) of the hind limbs became evident. Such cases showed a more pronounced atrophy of the hind limb musculature. At this stage there was often an asymmetry with respect to the severity of the symptoms between the two hind limbs. Still, rats at this stage managed to climb across a 1.5 inch wall but seemed less eager to search new environments and often reacted more slowly to audio-visual stimuli. Stage III involved a more or less complete paralysis of the hind limbs, again often engaging only one of the hind limbs first. These rats dragged themselves around using their fore limbs. The hind limbs were held in a flexed retracted position below the pelvis. When such rats were lifted from the supportive surface and then placed back, they did not extend their hind limbs on replacement. Furthermore, stage III rats usually did not manage to climb walls of one inch height and disclosed a severe atrophy of the hind limb muscles and sometimes also of the hip and low-back muscles. In the most severe cases, a tendency for both urinary and bowel emptying dysfunction began to show. At this stage the rats usually began losing weight rapidly and often appeared quite indifferent to the surrounding environment. Death usually followed from one-to-several weeks later. Clinical symptoms begin to develop in some rats at an age of 24-26 months, and progressive deterioration then occurred over a 4-8 month period. In the 30-month cohorts, about 90% of the surviving rats had some symptoms (stage I-III). About 40-70% were either stage II or stage III. In an earlier study we found that the median survival age of this rat strain under the standard condition in our department was 30 months, and this was also the rationale for using this group as "aged" rats (see Johnson et al., 1993)….”(from Johnson et al., 1995). This is essentially valid also for male and female SD rats used 1994-1999. In Figure 9, a compilation of stage-frequencies among female SD rats is shown at different survival times. Wistar rats show a similar frequency distribution, however, the number of observations is still quite small. We do not have experience enough to comment on stage-frequencies among Fischer 344 rats. Over-time the staging protocol has yielded consistent results, probably owing to its simplicity. In essence, two parameters guide the examinator to select a stage, namely:
  (a)is a limb weight bearing?
  (b)does a limb show a complete gait cycle coordinated with the other limb(s)?
Stage 0 animals show no signs of insufficiency concerning these two variables;
Stage 1 animals show some signs, including a lowering of the trunk towards the supportive surface and minor signs of gait cycle aberrations;
Stage 2 animals show clear gait cycle aberrations, often involving a poorer coordination between limbs. Body weight bearing is often clearly incapacitated in at least one limb;
Stage 3 animals disclose incomplete or complete paralysis of one or both hind limbs. Only minor efforts to initiate a gait cycle may be evident and the limb(s) can not support any body weight. In addition there are minor parameters such as the degree of muscle atrophy-speed-alertness, that may finally decide if an animal should be ranked e.g. stage 1 or stage 2.
 
 
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