Parental Behaviors in Rats and Mice - Wiley Online Library

Parental Behaviors in Rats and Mice

UNIT 8.15

The ultimate goal of reproductive behavior in mammals is the transmission of genetic information from one generation to the next. To succeed in this goal, adequate parental care and successful rearing of offspring to reproductive age are indispensable. Parental behavior is a complex category of activities that can be influenced by a multitude of internal and external factors (Weisner and Sheard, 1933; Rosenblatt and Lehrman, 1963). Laboratory rodents, such as rats and mice, are highly popular models used to study these factors, and their parental behavior can be assessed in numerous ways. The procedures described in this unit include a detailed description of each component of parental behavior in rats and mice (see Description of Parental Behaviors, below), followed by the methods used to evaluate these behaviors (see Basic Protocols 1 to 4). Since parental behaviors may also be induced in and displayed by nonlactating animals, the procedures used to induce maternal responding in virgin animals via continuous exposure to pups (sensitization; see Basic Protocol 5) and exogenous hormones (see Basic Protocol 6) are included. Finally, the objective of some experiments is to determine the extent to which an animal prefers or is motivated to respond to pups or to pup-associated cues without actually examining their maternal behaviors towards the pups. In these cases, tests are designed that do not involve the consummatory responses described above, but instead involve responses that reveal the animals’ preference among various pup-related cues, or between pup-related cues and cues that are not associated with pups (as in pup odors versus non-pup odors). These tests can also examine how strongly the cues associated with pups are reinforcing to the female. Numerous paradigms are described that have been used in the laboratory to examine the motivation of laboratory rodents to act parentally (see Basic Protocols 7 to 10), and information is provided on how to utilize these tests. NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals. NOTE: In all cases, animals should be housed under standard laboratory conditions, with an ambient temperature of ∼22°C, relative humidity of 40% to 50%, and a 12:12 light:dark photoperiod. Unless otherwise noted (e.g., Basic Protocol 9), standard laboratory rat or mouse chow and water should be available ad libitum. DESCRIPTION OF PARENTAL BEHAVIORS Parental behavior in rats and mice encompasses two general categories of responses: active behaviors and quiescent behaviors. Active parental behaviors include construction of a nest site, carrying pups from one place to another, and licking the pups. The performance of active parental behaviors and other nonparental activities is interspersed with relative quiescence, when parental rodents cease moving and quietly position themselves over the pups for prolonged periods of time. In lactating rodents, much of this quiescent time is spent nursing in distinct nursing postures. Although nonlactating rodents also quiescently position themselves over the litter, they cannot truly nurse because they do not have developed nipples upon which pups can suckle and they do not produce milk. These parental behaviors are defined below. Nest construction Parental rodents invariably have a single, central nest site in which they care for their young. Nest construction involves carrying of nest material with the mouth, or pushing it Contributed by Joseph S. Lonstein and Alison S. Fleming Current Protocols in Neuroscience (2001) 8.15.1-8.15.26 Copyright © 2001 by John Wiley & Sons, Inc.

Behavioral Neuroscience

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with the snout or paws, to the potential nest site. A tightly bound mass of material with a depression in the middle is usually created, in which the subject interacts with the pups. Retrieval Retrieval of displaced or scattered pups first involves the subject orienting to and moving towards the pup, often sniffing the pup before gently picking it up with the incisors, carrying it back to the nest site, and finally depositing it there. Retrieval typically indicates the carrying of a pup over a relatively long distance. In contrast, when a subject orally repositions pups for short distances within or around the nest, their behavior is sometimes referred to as “mouthing.” Licking of pups Parental rodents often spend a great deal of time licking neonates, a behavior that cleans the pups, increases their activity, and stimulates elimination of waste from the young. Licking also has long-term effects on the emotional, endocrinological, and sexual development of pups. Furthermore, lactating dams also obtain important resources from the pups’ urine. Therefore, it is often helpful to separate maternal licking into two categories, licking specifically of the pup’s anogenital region and licking of the rest of the pup’s body. Actively hovering over pups As indicated by the name of the behavior, the subject is positioned over some or all of the pups in the nest, but is not quiescent. Instead, the subject is relatively active, performing a variety of activities such as licking the pups, self-grooming, or moving nest material. In lactating rodents, hovering over the litter provides the pups access to the dam’s nipples. Quiescent positioning over pups In lactating rats, quiescence is typically induced by the suckling provided by a sufficient number (i.e., at least four) of pups. Once rendered quiescent, lactating dams will nurse their young for prolonged periods of time in a variety of nursing postures. One of the most frequent nursing postures displayed is kyphosis (upright crouch; Stern, 1996), which is characterized by the dam standing over the litter with rigidly splayed limbs and a dorsal arch of the spinal column. When the animal adopts a kyphosis posture, it does not lick the pups or engage in other active behaviors. A second common nursing response, laying supine, occurs when the animal is stretched out on its back or side with its nipples exposed. Other postures that may also be observed include laying prone on top of the litter with no limb support and the subject sitting hunched over the litter on its haunches with back limb support but no forelimb support. Nonlactating rats, and probably other nonlactating rodents as well, do not display kyphosis for prolonged periods of time and are very often found over the litter in the supine, prone, or hunched positions (Lonstein et al., 1999; Fig. 8.15.1). BASIC PROTOCOLS 1 TO 4: ASSESSMENT OF PARENTAL BEHAVIORS


Two commonly used methods to examine parental behaviors in rodents are periodic (spot-check) observations of the subject’s undisturbed behavior with the litter (see Basic Protocol 1), and continuous observation (see Basic Protocol 2)—either undisturbed or following varying periods of separation between the parent and litter (ranging from 5 min to 4 hr). Test durations can also range from 10 min to a number of hours. A shorter test using continuous observation is sometimes followed by periodic spot-checks throughout the day. The rationale for choosing a particular procedure depends on both the primary objective of the exercise and practical constraints. Procedures are also provided for assessing nest construction (see Basic Protocol 3) and retrieval of separated pups (see Basic Protocol 4).


Current Protocols in Neuroscience

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Figure 8.15.1 Maternal behaviors in postpartum mothers and maternally sensitized virgins. Differences between lactating and sensitized female rats are seen in the duration of time spent hovering over and nursing pups in each nursing posture and of retrieving and pup-licking during a 45-min observation following a 3-hr mother-litter separation on the fourth day after the onset of maternal behavior. *p < 0.05. Modified from Lonstein et al. (1999).

Assessment of Parental Behaviors Using Direct Periodic Spot-Check Observation This method typically involves pen-and-paper recording of the subjects’ behavior using a time-sampling procedure, where the presence or absence of each behavior is recorded in successive 5- to 20-sec bins or intervals. This procedure is advantageous because it requires little equipment and can provide a “snapshot” of the parent-litter interaction under undisturbed conditions at various periods throughout the day.

BASIC PROTOCOL 1

Materials Parental lactating or nonlactating rodent subject Litter of 1- to 8-day-old pups provided by a lactating “donor” animal (there are usually 6 to 8 pups in a culled litter, with equal numbers of males and females included) Clear glass or polypropylene home and testing cage (for rats: ∼48 × 28 × 16 cm; for mice: 21 × 16 × 13 cm) with wood chips or shavings for bedding (∼8 cups for rats, 4 cups for mice). Cotton balls or pads can be used for mice. Paper data sheets with each minute of the observation separated into 5-, 10- or 15-sec periods. Each sheet is divided into columns that represent time periods and rows that represent the individual behaviors. Stopwatch 1. If the subject’s home cage is opaque, transfer the subject and the pups into a clean transparent cage with fresh nesting material at least 24 hr prior to testing. It is best if the subject is tested in a familiar home cage.


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2. To begin the test, start the stopwatch and quietly note the subject’s behavior without disturbing her or the pups. Record the subject’s behavior (e.g., hovering over, nursing, licking, nesting) on the data sheet every 5, 10, or 15 sec until the conclusion of the test (tests usually last 10, 20, or 30 min). Record each behavior only once within any single interval. Take care not to disturb the subject when exiting the room at the end of testing. See Description of Parental Behaviors, above. Observations can be as short as 5 min or last as long as a few hours if necessary.

3. Repeat steps 1 and 2 as often as necessary throughout the day. BASIC PROTOCOL 2

Assessment of Parental Behaviors Using Continuous Observation Continuous observation of subject-litter interactions can be performed either without disturbing the subjects prior to testing or after a period of separation between the participants. Both require a computerized data acquisition system. This method is particularly useful because it allows one to obtain a level of detail that cannot be easily obtained with pen-and-paper collection of data. Following short 5- to 10-min separations, mothers will usually show all behaviors within a 10- to 15-min observation period, with the possible exception of nursing. However, if the subject and litter are separated for longer periods prior to testing (3 to 4 hr), the entire repertoire of parental behaviors is likely to be observed within a single 30-min observation. In most cases, the presence of an observer does not disturb maternal responding in rats and mice. If this is of particular concern, it is possible to videotape the subject and record the behavior at a later time (see Critical Parameters; discussion of influence of data collection procedure). Materials Parental lactating or nonlactating rodent subject Litter of 1- to 8-day-old pups provided by a lactating “donor” animal (there are usually 6 to 8 pups in a culled litter, with equal numbers of males and females included) Clear polypropylene pan cage (for rats: ∼48 × 28 × 16 cm; for mice: 21 × 16 × 13 cm) with wood chips or shavings for bedding (∼8 cups for rats, 4 cups for mice). Cotton balls or pads can be used for mice. Humid incubator set at nest temperature (∼34°C) Plastic or glass dish to hold pups Cotton swabs Small animal scale Laptop computer Data acquisition software that allows continuous recording of events at least once every second; available commercially (e.g., Noldus Observer, Noldus Information Technology; or Behavior Evaluation Strategies and Taxonomies software, Scolari, Sage Publications Software) although a rather simple data acquisition program may be generated in-house by a person with some computer programming experience Continuous observation using a subject-litter separation 1a. Remove pups from subject or surrogate lactating dam. Leave the subject alone in test cage. It is recommended, but not absolutely necessary, that investigators wear gloves while handling pups.




2a. Place pups in a small bowl or container and place them in a warm (34°C) environment for 5 to 10 min or in a humid 34°C incubator if separating pups for longer periods of 3 to 4 hr. 3a. Just before testing, remove pups from the bowl or incubator, again using gloves to prevent contamination by human odors. 4a. Gently hold each pup on its back and swab its anogenital region to stimulate urination. 5a. Place the entire litter on the scale and record the total weight to the nearest 0.1 g. This will provide the litter weight prior to any milk ingestion during interactions with the mother.

6a. Place pups back in the bowl and bring into the testing room. 7a. Turn on computer data acquisition system and prepare program to begin recording. Each behavior of interest should have a single key on the keyboard representing it (e.g., “R” could be used for retrieval of pups, “L” for licking the pups, etc.). For convenience, each key should be able to be toggled on so that individual keys do not have to be held down by the experimenter for the duration of each behavior.

8a. Quietly remove the top of the test cage and scatter the pups in the cage opposite to where the subject has built a nest, or where the subject is sitting if a nest has not been constructed. 9a. Start the computer and press the appropriate key each time that a particular behavior is observed. Stop the program after the necessary amount of time has passed. 10a. Remove the litter from the subject and again weigh the pups all together. Record the weight and return to the litter to either the subject or a lactating dam from the colony. Differences in litter weight between the beginning and conclusion of testing will reflect any amount of milk that the pups ingested during testing.

Continuous observation without a subject-litter separation This procedure is very similar to steps 1a to 10a above, except that pups are not removed from the subject before testing. 1b. Begin this test as one would a spot-check undisturbed observation (Basic Protocol 1, step 1). 2b. Without disturbing the subject or litter, quietly sit and start the data acquisition software. 3b. Continuously record each behavior displayed by the dam as it occurs by pressing the keys on the keyboard that represent each activity. 4b. After the desired period of time, conclude the test and exit the software program. 5b. Quietly leave the room without disturbing the subject and litter. Assessment of Nest Construction Many undisturbed observations of mothers interacting with their litters, as well as some observations made after a mother-litter separation, will not include the display of all parental behaviors. This is particularly true of nest construction (described here) and retrieval of pups (see Basic Protocol 4) because these behaviors occur infrequently in a relatively undisturbed environment. It is, therefore, often useful to conduct additional tests to assess these two parental behaviors.

BASIC PROTOCOL 3



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Materials Parental lactating or nonlactating rodent subject Litter of 1- to 8-day-old pups provided by a lactating “donor” animal (there are usually 6 to 8 pups in a culled litter, with equal numbers of males and females included) Clear glass or polypropylene home and testing cage (for rats: ∼48 × 28 × 16 cm; for mice: 21 × 16 × 13 cm) with wood chips or shavings for bedding (∼8 cups for rats, 4 cups for mice). Cotton balls or pads can be used for mice. Two-ply paper towels (16 × 25 cm) shredded into thin strips 1. Re-house subject and pups in the observation cage at least 24 hr before the first observation. Note that the pups do not necessarily have to be present during this test and are often removed immediately prior to performing step 2 below.

2. Scatter shredded paper towel strips over entire cage. 3. Four to 24 hr later, rate nest on 5-point scale: 0 = no nest, paper strips still scattered over entire floor of the cage 1 = poor nest, not all paper strips are used and the nest is flat 2 = fair nest, all paper is used but the nest is flat 3 = good nest, all paper is used and the nest has relatively low walls (5 cm) 4. If repeated measurements of nest construction are needed, destroy nests after scoring and repeat the procedure as many times as needed. ALTERNATE PROTOCOL

Assessment of Nest Construction—Mice Materials Parental lactating or nonlactating mouse subject Clear glass or polypropylene home and testing cage (21 × 16 × 13 cm) Cotton balls or pads (e.g., Nestlet squares) 1. Each day place a large number of cotton balls in the bottom of one side of the animals’ food bin and place food over the cotton. Allow the animal to pull the cotton through the cage wiring, just as it does for its food, and use the cotton for a nest. 2. The next day, check for the presence of a nest and rate it on the following scale: 1 = no nest 2 = saucer shaped nest 3 = nest with raised sides 4 = fully enclosed nest 3. After rating, weigh the nest. All cotton pulled into the cage is usually incorporated into the nest so nest weight is a reliable measure of nest construction.

4. Dispose of the nest and allow the subject to create another one, and rate the new nest the next morning. Repeat as necessary. Parental Behaviors in Rats and Mice



Assessment of Retrieval

BASIC PROTOCOL 4

Materials Parental lactating or nonlactating rodent subject Litter of 1- to 8-day-old pups provided by a lactating “donor” animal (there are usually 6 to 8 pups in a culled litter, with equal numbers of males and females included) Clear glass or polypropylene home and testing cage (rats: ∼48 × 28 × 16 cm; mice: 21 × 16 × 13 cm) with wood chips or shavings for bedding (∼8 cups for rats, 4 cups for mice), with floor of cage divided by 1-in. high plastic barriers into four equally-sized quadrants to prevent the pups from crawling to the subject. Cotton balls or pads can be used for mice. Stopwatch 1. Remove the pups from the subject and leave the subject alone in the test cage for at least 5 min. 2. Scatter the pups in the quadrant of the test cage opposite to the subject’s nest or sleeping corner. 3. Start stopwatch and monitor subject’s behavior for 10 min. 4. Note the time that the subject picks up the first pup, carries it back to the nest, and deposits it there. Note the time that the last pup is deposited into the nest. Note the number of pups retrieved within the 10-min test. 5. If more information is needed about retrieval of any stray pups that occurs after the 10-min test, use spot checks to note the position of dam and pups each hour after the 10-min retrieval test. Record the number of pups in the nest at each hour. INDUCTION AND ASSESSMENT OF PARENTAL BEHAVIOR IN NONLACTATING RATS AND MICE (SENSITIZATION)

BASIC PROTOCOL 5

Nonlactating adult rodents of many species do not spontaneously display parental behavior when presented with pups, and often actively avoid them. It is possible to overcome this inhibition of parental behavior by repeatedly or continuously exposing them to young pups, a process termed sensitization. Sensitization may sometimes be a useful method to examine factors regulating maternal behavior because the induction of parenting is presumably free of the influence of hormones. It should be noted that virgin or otherwise nonlactating adult mice of some strains may be spontaneously parental and may not require a sensitization procedure to induce parental responding. Materials Nonlactating rodent subject Unmanipulated lactating rats that can provide 1- to 8-day-old litters of test pups and that can foster any hungry pups (the total number of surrogate lactating dams necessary to provide recently fed pups each morning and feed hungry foster pups is approximately the same as the number of subjects that are actually being sensitized) Clear glass or polypropylene home and testing cage (for rats: ∼48 × 28 × 16 cm; for mice: 21 × 16 × 13 cm) with wood chips or shavings for bedding (∼8 cups for rats, 4 cups for mice). Cotton balls or pads can be used for mice. Stopwatch 1. Rehouse subjects individually in clean, clear polypropylene cages.



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2. At a time 48 hr after rehousing, place four to eight 1- to 8-day old pups in the cage opposite to the corner where the subject sleeps or has nested. 3. Observe the subject’s behavior continuously for 15 min. Note if the subject sniffs, retrieves, licks, or hovers over any of the pups. Occasionally, a retrieval or mouthing episode may involve the female biting too hard and breaking the skin of the pup. In this case, animals may start cannibalizing the pup. When this or any other injury to the pups occurs, immediately remove the pups from the cage.

4. Because the subjects are not lactating, remove and replace the pups numerous times each day (every 12 or 24 hr) with freshly fed pups obtained from a lactating “donor” dam. 5. Observe the subject’s behavior towards pups for 15, 30, or 60 min each morning when the pups are removed and then replaced. 6. If a subject is observed to sniff, retrieve, lick, and hover over all pups during each of two consecutive daily observations, consider them to be fully parental and conclude testing. Record the first day of the two consecutive observations of full parental behavior as the subject’s latency to become fully parental. 7. Continue exposing subjects to pups for 14 to 21 consecutive days. After this time, terminate testing and consider the remaining subjects not parental. Different strains of rats may take different lengths of time to become sensitized, ranging from an average of 4 days to 10 days or longer.

8. If necessary, assess ongoing parental behaviors in sensitized subjects as in lactating subjects, although nursing behavior will noticeably differ between virgin and lactating rats. BASIC PROTOCOL 6

HORMONAL INDUCTION OF MATERNAL BEHAVIOR IN VIRGIN RATS Although the virgin rat can be sensitized to show a pattern of maternal behavior similar to the postpartum mother, the intensity of maternal behavior exhibited is not identical in the two types of animals. Most obviously, the new mother responds maternally at the time of her first exposure to pups at parturition, whereas the virgin can take up to 15 days of contact with pups before responding maternally. This difference is due to the hormonal differences between the two types of animals. In some instances it may be desirable to assess maternal behavior in a hormonally primed virgin rat. There are a number of ways to hormonally prime the virgin animal. One very effective method was originally described by Bridges (1984). Most females undergoing the following treatment will typically respond maternally within 24 to 48 hr after the first introduction to pups, with a small minority (20% to 40%) being spontaneously maternal upon first exposure to the neonates. Materials Adult female rats Crystalline estradiol Crystalline progesterone Clear glass or polypropylene home and testing cage (for rats: ∼48 × 28 × 16 cm; for mice: 21 × 16 × 13 cm) with wood chips or shavings for bedding (∼8 cups for rats, 4 cups for mice). Cotton balls or pads can be used for mice.


Additional reagents and equipment for bilateral ovariectomy (UNIT 8.2, Support Protocol 2) and subcutaneous implant of hormone capsules (UNIT 8.2, Support Protocol 3)



1. Anesthetize and bilaterally ovariectomize adult virgin female rats (see Support Protocol 2).

UNIT 8.2,

2. Subcutaneously implant a 2-mm silastic capsule filled with estradiol in the nape of the subject’s neck (UNIT 8.2, Support Protocol 3). 3. Three days later, subcutaneously implant three 30-mm capsules filled with progesterone. 4. Ten days later, reanesthetize the subject and remove the progesterone capsules; do not remove the estrogen capsule. 5. Twenty-four hours after removal of the progesterone capsules, expose subjects to pups as described in the sensitization protocol (see Basic Protocol 5). ASSESSMENT OF MATERNAL PREFERENCES AND MOTIVATION IN RATS AND MICE Three tests are described here that can be used to test how salient and rewarding the pups or their sensory cues are to the subject. The first (see Basic Protocol 7) is a simple preference task that provides information on how attracted animals are to the pups or their sensory cues in the absence of learning or experience. The second (see Basic Protocol 8) is a conditioned place-preference task that uses pups as the reinforcing stimulus (Fleming et al., 1994) and involves forming an association between pups (the unconditioned stimuli) and the surrounding environment (the conditioned stimulus). The third (see Basic Protocol 9) is an operant task that uses pups as reinforcers for the instrumental bar-press response (Lee et al., 2000). This latter task is somewhat difficult to set up for a student project, but as a research tool it provides an interesting way of dissociating maternal behavior from maternal motivation, because both are tested in the same animals. Finally, a procedure is described that uses a T-maze extension of the subject’s home cage to assess maternal motivation (see Basic Protocol 10). There are numerous factors that can be investigated using these motivation procedures, including: 1. Parity: compare nulliparous versus primiparous versus multiparous female rats. 2. Maternal experience: compare maternally induced virgins versus maternally naïve virgins; compare primiparous females with varying amounts of postpartum experience with pups. 3. Hormonal status: compare ovariectomized virgin females with and without hormonal priming. 4. Deprivation condition: compare females tested after varying amounts of separation from the unconditioned stimulus (pups or pup-related cues). 5. Specificity of unconditioned stimulus: compare females exposed to pups, novel food pellets, Froot Loops, or other neutral stimuli. 6. Sensory features of pups: compare females exposed to pups of different ages, scent characteristics, or temperatures. 7. Sensory capacity of mother: compare females after experiencing a reduction in sensory capacity (e.g., olfactory denervations, etc.). Behavioral Neuroscience


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BASIC PROTOCOL 7

Test of the Unconditioned Preference for Pups or Pup-Related Cues There are many ways to assess an animal’s attraction to odors. One may use a Y-maze, a large arena, or a hole board with two different stimuli presented in opposite ends of the maze or arena, or in different holes. These are all essentially the same procedure. The following test is perhaps the simplest to conduct. In this test, pup and non-pup stimuli are placed in closed, perforated Plexiglas containers (for pups) or heavy glass bowls (for pup odors on nest-material) that are placed at opposite ends of a large arena (known as stimulus areas A and B; see Fig. 8.15.2A). Subjects are placed in the center of the arena equidistant from stimulus areas A and B. Two 5-min preference tests are then administered. In one test, pups or pup stimuli are placed in either stimulus area A or B and the other neutral or control stimuli (e.g., pink pencil erasers) are placed in the other area. In the second test, the positions of the two sets of stimuli are reversed. Placements of the pup stimuli on the first and second tests are counterbalanced between areas A and B across animals within a group. Materials Rat or mouse subject Pups (six 1- to 3-day old pups) or pup-related olfactory cues (4 ounces of clean nest-material on which pups have been placed for 6 to 12 hr, or onto which pup urine has been excreted); if nest material is used, use the same and standardized amounts of nest material during all tests Test arena (Fig. 8.15.2A): glass or Plexiglas aquarium (90 × 45 × 47 cm) divided into two goal areas (areas A and B; each 15 cm) at either end and two center areas, A1 (adjacent to A; 30 cm) and B1 (adjacent to B; 30 cm) Clear Plexiglas cube (8 × 8 × 8 cm) with perforated sides, top, and bottom (top removable to permit insertion of stimuli) Glass bowls (∼8 cm diameter × 3 cm high) to hold nest material Control stimuli: six small pink pencil erasers or control non-pup related olfactory cues (4 ounces of clean nest material on which juvenile animals or adult animals have been placed for a 6- to 12-hr period, or onto which urine has been excreted); if nest material is used, use the same and standardized amounts of nest material during all tests Data acquisition software that allows continuous recording of events at least once every second; available commercially (e.g., Noldus Observer, Noldus Information Technology; or Behavior Evaluation Strategies and Taxonomies software, Scolari, Sage Publications Software) although a rather simple data acquisition program may be generated in-house by a person with some computer programming experience Preparation of pup stimuli 1a. Place six 1- to 3-day old pups into one 8 × 8 × 8 cm Plexiglas box and place in either area A or area B. 2a. Place six pencil erasers into another Plexiglas box and place into either area A or B (whichever is opposite to the pup stimuli) as control stimuli. Preparation of pup-related olfactory stimuli 1b. Place 4 ounces of nesting material in a small cage and place pups onto the nesting material for 1 hr. Before and after nest material is removed, express pup urine by anogenital swabbing with a small cotton swab and place the swab into the nest material.




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Figure 8.15.2 (A) Schematic representation of the unconditioned preference chamber. (B) Representative data sheet.

Sometimes the entire olfactory complex associated with pups may be used as an olfactory stimulus, in which case, place clean nesting material into the cage of a day 1 or 2 postpartum lactating female overnight and remove it for testing the next morning.

2b. Place 4 ounces of clean nest-material for 6 to 12 hr in the cage of another conspecific animal, for example, a female in the diestrous stage of the estrous cycle or a juvenile animal, and then use this nest material during testing as a control stimulus.



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3. Place the two stimuli (i.e., test and control) in either area A or area B. For half of the test subjects within each group, place pup stimuli in area A. For the other half, place pup stimuli into area B. For the second preference test put pup stimuli into the area opposite to the area in which they were placed on the first test. 4. Place the subjects in the center of the arena between A1 and B1 and conduct a 5-min preference test. Using an event recorder (data acquisition system), continuously record: a. The time that subjects spend in each of area A and area B. b. The time spent in contact with the Plexiglas cube or the bowl containing nest material. c. The time spent sniffing, pawing and digging at the Plexiglas cube or nest material. d. The time spent in areas A1 or B1 of the arena. 5. To ensure that responses to either stimulus reflect attraction to or aversion from that particular stimulus, administer additional tests in which each stimulus is tested alone. Hence, in addition to testing pup stimuli versus a neutral stimulus (erasers), one may also test pup stimuli versus no stimulus, and a neutral stimulus versus no stimulus.

Perform statistical analyses of data 6. To determine preferences scores, compute the proportion of total test time that subjects spent in area A versus B and in areas A + A1 versus B + B1 (see Fig. 8.15.2A). The time spent sniffing the stimulus cubes or digging into the bowls can also be computed and used to assess preference. A proportional measure can be computed for each subject (time in preferred side/total time) and different groups compared by between-group tests. The proportion of animals (number showing a reference/total number) showing a preference in each group can be compared using χ2 tests. Alternately, single groups can be assessed for their preferences by means of Fisher’s exact probability test or paired t tests. BASIC PROTOCOL 8


Conditioned Place Preference Tests One procedure that involves learning processes to examine the reinforcing effects of pup stimulation is the conditioned place preference task (CPP; Fleming et al., 1994). In this paradigm, postpartum mothers are separated each day from pups for 23 hr and re-exposed to them three times for 1-hr periods each in one of two distinct chambers (the stimuluspaired chamber). This will occur at 48-hr intervals (training days 1, 3, and 5). On the alternate days (training days 2, 4, and 6), animals are placed for a 1-hr period into the alternate, distinct chamber (the non-paired chamber). The pups are absent during these non-paired days. On the seventh day, the test day, animals are given a 10-min free access to the two chambers, without the presence of the pups. The amount of time spent in each of the two chambers is recorded, and the proportion of time spent in the pup-associated chamber is computed. The CPP apparatus consists of two open-topped chambers of equal height and volume (chambers A and B) that differ distinctly in shape (triangular versus square), color (black versus white), slight odor (vanilla versus peppermint), and texture (smooth versus wire mesh floor). The two chambers are connected by a smaller rectangular central start box, from which the chambers can be closed off with dividers during the training phases. Different laboratories use different CPP apparatuses, but the objective is to have the chambers differ from one another as much as possible, while maintaining equally attractive properties when not paired with a reinforcer. Generally, Plexiglas construction permits easy cleaning between training sessions and tests (see Fig. 8.15.3 for one such



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box B vertical lines smooth floor

Figure 8.15.3 Schematic representation of conditioned place preference chamber.

apparatus, where chambers do not differ in shape, but do differ in orientation of black stripes, odor, and texture of the floors). Materials Pregnant female rat subjects housed individually in polypropylene cages (∼48 × 28 × 16 cm) with wood chips or shavings for bedding 70% ethanol Large plastic weighing containers (weigh boats) CPP apparatus (see above) 1. Days 0-2: When pregnant females give birth, adjust the litter to contain 8 pups. Leave dams with pups for 48 hr in the home cage. 2. Day 2: Wearing gloves, remove pups from the subject dam and place them with a surrogate lactating dam. Leave subject dam alone in the home cage with food and water freely available. 3. Day 3: After a 23-hr mother-pup separation, begin the CPP procedure. Training phase 4. On days 2, 4, and 6, give subjects a paired-chamber exposure to pups. Place six 1- to 3-day old pups into a weigh boat in the far corner of the paired chamber (either chamber A or B). Present the pups to half of the subjects in chamber A, the other half in chamber B. Make sure the chamber dividers are in place to prevent the subject from leaving the paired chamber. This counterbalancing procedure controls for individual preferences that may occur for specific chambers.

5. Remove the subjects from the home cage and place them in the appropriate paired chamber with the pups for 1 hr. 6. Observe the subjects’ behavior with pups by continuous observation or through periodic spot checks throughout the 1-hr period (see Basic Protocols 1 and 2). 7. Remove subjects from the CPP chamber and return them alone to their home cage. 8. Return pups to lactating surrogate mothers.



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Percent of animals

100

PP/EXP

90

V/EXP

80

PP/INEXP V/INEXP

70 60

no preference

50 40 30 20 10 0

PP/EXP 0.7

V/EXP PP/INEXP

Proportion of time

0.6

V/INEXP no preference

0.5 0.4 0.3 0.2 0.1 0 experienced

inexperienced

Figure 8.15.4 Percent of postpartum (PP) and virgin (V) animals showing a preference for the pup-paired box in a conditioned place preference task. (Bottom) Proportion of time spent by experienced and inexperienced virgin and lactating females in the chamber previously paired with pups. (Top) Percentage of animals showing a >50% preference.

9. Clean CPP apparatus with 70% ethanol and allow it to dry before next use. 10. On days 3, 5, and 7, give subjects a non-paired chamber exposure. Remove subjects from their home cage and place them in the previously empty (non-pup, either A or B) chamber for 1 hr. Remove the chamber dividers so that subjects can freely explore the CPP apparatus. Parental Behaviors in Rats and Mice



11. Observe the subjects’ behavior by continuous observation or spot checks and note which chamber they are in. Also note their nonmaternal behaviors such as self-grooming, sniffing cage, eating, drinking, and sleeping. 12. Return subjects to their home cage. 13. To insure that all animals receive 1 hr of daily interaction with pups, place six 1- to 3-day old pups into the subjects’ home cage beginning 1 hr after exposure to the non-paired chamber, and observe their behavior for 1 hr. 14. Remove pups and place with lactating surrogate mother. 15. Clean the CPP apparatus with 70% ethanol and allow it to dry. Test phase 16. The next day (day 8), test subjects for their preference in the CPP apparatus. Remove the dividers between central box and chambers A and B to give animal access to both chambers. 17. Place subjects into the central start box and observe their behavior continuously over a 10-min period. Note the frequency of entries into each chamber, the time spent in each chamber, the time spent in each corner of the chambers, and the time spent in the central start box. 18. Remove the subject after testing and return it to the home cage. 19. To analyze the data, compute the duration of time that subjects spent in each chamber as well as the proportion of time that they spent in the paired chambers. Also compute the percentage of subjects that showed more than 50% of their time in the paired chamber (Fig. 8.15.4). For example, if an animal spent 7 min in the paired chamber, 2 min in the unpaired chamber, and 1 min in the central box, its preference proportion is 7/(7 + 2) = 0.78 or 78% of the time spent in the two chambers.

Tests of Operant Responding In addition to the two tasks described above, an operant task may also be used to assess the reinforcing value of pups, and either natural lactating dams or previously sensitized rats (see Basic Protocol 5) can be tested with these methods. The operant task is different from the conditioned place preference task (Basic Protocol 8) in that it permits one to analyze the effects of the animal’s motivational state on the speed of acquiring a conditioned response to pups, as well as the effects of removing the pup reinforcement on extinction of the response. By changing the reinforcement schedule, this procedure also permits one to test the strength of pup reinforcement and to provide some quantitative estimate of that strength (i.e., which reinforcement schedules maintain bar-pressing for pups or which result in extinction). Animals are first trained to bar-press for a food reward to a certain criterion and then to bar-press for delivery of pups (see also Wilsoncroft, 1969). The age, gender, and sensory characteristics of the pups can vary depending on the goal of the exercise. It is also sometimes useful to observe the maternal behavior exhibited in the subjects’ home cage (see Basic Protocol 1 or 2) throughout the period that animals are being tested in the operant chamber. In that way, it is possible to compare the natural consummatory responses towards pups with their operant responses for them. The apparatus used here is a 26 cm × 26 cm × 30–cm modified operant chamber designed for rats, equipped with one retractable lever situated 6 cm above a solid Plexiglas floor (see Fig. 8.15.5). The wall furthest from the lever contains a door with an opening that is

BASIC PROTOCOL 9



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operant box

carousel for pup delivery

Figure 8.15.5 The operant conditioning apparatus.

8 cm in diameter. The operant box is fitted on the outside with a 12-compartment carousel type magazine that delivers either a rat pup or food reward (Kellogg’s brand Froot Loop breakfast cereal) via a chute (42° angle) to the dispenser following depression of the lever. The dispenser opening (5.5 cm in diameter) is located 2.25 cm from the lever and contains a removable drawer (8 × 9 × 10 cm). The carousel and delivery system were modified for this purpose in-house in the University of Toronto at Mississauga (UTM) shop and electronics department. For further information, contact A.S. Fleming ([email protected]). Parental Behaviors in Rats and Mice



Materials Rat test subject housed individually in polypropylene cages (∼48 × 28 × 16 cm) with wood chips or shaving for bedding (∼8 cups) Kellogg’s brand Froot Loop breakfast cereal, or similar food reward 1- to 3-day old rat pups White noise generator or radio Modified operant chamber (see description above) Computerized system to automate the operant schedule and deliver stimuli according to the specified response schedule Desk lamps with red light bulbs for nighttime observation NOTE: The initial training phase consists of habituation, shaping, and conditioning phases using the food reinforcer. Follow these by the pup test phase and an extinction phase. Habituation: Days 1 to 10 1. Handle animals for 5 min each day during the dark phase of the light/dark cycle with room illuminated by a red light bulb. Use a white noise generator or a radio set at low volume to mask extraneous noises in the environment. 2. On each day, place the subjects in an inactivated operant chamber (i.e., a chamber in which pressing the levers does not produce any effect) for 1 hr during which 10 Froot Loops are made available in the dispenser. This is done in order to familiarize subjects with handling, food, environment and to reduce freezing during the shaping/conditioning phase.

Shaping and food reward conditioning 3. Place animals on a food deprivation schedule that will achieve and maintain a body weight level of 85% of their baseline body weights, slowly, over ∼5 to 7 days. To maintain weight, provide animals with 1 to 4 pellets of standard rodent chow daily, depending on the daily recorded body weight. Provide water ad libitum during this time. Once 85% of baseline body weight is achieved, maintain this level of food deprivation during the shaping and conditioning phases of the experiment, which lasts ∼8 days. Shaping sessions 4. Carry out shaping sessions over an 8-day period with each session lasting ∼30 min. 5. Carry out all sessions under red light illumination using a white noise generator or radio set at low volume in order to mask any noise made by experimenters or the rotating carousel. 6. Clean the operant box and dispenser with 70% alcohol before each training session. 7. Begin each session by placing the subjects in the active operant chamber (i.e., bar presses produce food reward). 8. To accomplish shaping, reinforce behaviors that resemble bar-pressing behavior. For instance, reward the following behaviors with a 1/4 Froot Loop each time they occur: rearing in the chamber, rearing on the wall of the levers, sniffing the levers/dispenser, rearing over the levers, or placing forepaws on the levers. Initially reward subjects for behaviors that remotely resemble bar pressing (e.g., rearing anywhere in the chamber), then gradually shift to reinforcing actions most closely resembling a true bar press (e.g., rearing directly over the lever). Behavioral Neuroscience


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9. Continue 30-min shaping sessions until the subject begins to press the bar and retrieve the food reward. Once subjects acquire the ability to bar press independently, place them on an FR-1 reinforcement schedule (i.e., a single Froot Loop is delivered following each bar-press response). Consider subjects to have learned the bar press response once they achieve 50 bar presses within the 30-min testing session. 10. Once subjects reach this criterion, remove them from the operant box, place them back into their cages, and provide food and water ad libitum. 11. Following the last conditioning session on the eighth day, transfer all subjects that meet the conditioning criterion to a large observation cage, and provide two shredded paper towels and six freshly fed pups. Remove subjects that fail to reach the criterion from the study. Observe maternal behavior in the home cage using one of the observation procedures described above. Pup test phase 12. After the maternal behavior observations, remove pups from the cage and place them into a bowl with nesting material on a heating pad (30°C). Separate subjects from pups for 60 to 120 min before operant tests begin. 13. Transport subjects to the room containing the operant chamber. 14. Place 11 freshly nursed pups (1 to 10 days old) into individual compartments in the 12-compartment carousel. The one compartment over the chute is open into the chute and remains empty.

15. Place the subject into the operant chamber. During the first 10 min of the 30-min session, record the subjects’ behaviors using a paper and pencil procedure at 10-sec intervals during which several behaviors (both maternal and nonmaternal) are recorded. Divide data sheets into columns representing time intervals and rows representing individual behaviors. Note the presence or absence of each behavior at each 10-sec interval. Record the following behaviors: a. bar pressing: subject rears over one of the two levers, applies pressure on the bar using its forepaws resulting in the delivery of a single pup. b. pup retrieval: subject removes pup from the dispenser. c. lick pup: subject engages in licking the body of the pup. d. anogenital licking: female licks the genital region of the pup. e. hover over: rat positions herself over the pups. f. self-grooming. g. sniffing air. h. sniffing magazine. i. sniffing the operant box. j. sniffing the levers. k. settling: subject is inactive, staying in one particular portion of the apparatus. l. number of urinations/defecations. 16. Record the frequency of bar presses and pups retrieved for the next 20 min. The frequency of bar-presses and retrievals over the 30-min period are used as data for subsequent analyses.


17. To avoid the accumulation of pups in the hopper, whenever a group of six pups has been delivered following bar-presses, remove all pups from the operant chamber through a door in the hopper if pups have not been retrieved out of the hopper. If the



40

virgin postpartum

Bar presses/30 min

35 30 25 20 15 10 5 birth 0

−2

−1

Pregnancy

1

2

3

4

5

6

7

8

9

10

Days postpartum

Figure 8.15.6 The bar press frequency (“bar press rate”) increases after parturition in the new mother rat. Representative data showing lactating and virgin female rats bar pressing for pup reinforcement in the operant conditioning apparatus.

pups have been retrieved, remove them through a door in the back of the chamber. These pups are then “recycled” and placed back into the carousel. In this way the carousel always has an adequate supply of pups to accommodate bar-press responses.

18. Place subjects back in their home cage at the end of the 30-min session and immediately give them the pups that had been removed from them before testing began. Extinction phase 19. Employ the same procedures used during the operant chamber pup testing phase (steps 12 to 18) during the extinction phase of the experiment with the exception that bar pressing no longer delivers a pup/Froot Loop reinforcer. 20. Carry out each extinction phase for 5 to 7 days following both the pup testing phase and Froot Loop testing phase. Conduct 8-min behavioral observations with the exception that maternal behaviors are excluded. Behaviors that are included are press-presses, grooming, sniffing air, chamber, lever, magazine, and settling.

21. On the final day of testing, do not place pups back in the subject’s cage following testing in the operant chamber. See Figure 8.15.6 for an example of bar-press rates shown by postpartum and virgin female rats.



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home cage

32

37 cm

cm

10 cm

T-Maze

Figure 8.15.7 Schematic representation of T-maze extension of the home cage.

BASIC PROTOCOL 10

Using a T-Maze Extension of the Home Cage to Assess Maternal Motivation Another useful method to assess maternal motivation that is somewhat simpler to conduct assesses retrieval of pups within a Plexiglas T-maze extension of the subject’s home cage (Bridges et al., 1972). This test is based on the premise that subjects are fearful of the previously unfamiliar T-maze and only very motivated subjects will leave the home cage to locate the pup. Materials Rat test subject housed individually in a polypropylene cage (∼48 × 28 × 16 cm) with wood chips or shavings for bedding (∼8 cups) with 10 × 10–cm hole in wall to accommodate T-maze and which can be kept closed until testing (Fig. 8.15.7) Neutral stimulus (e.g., small pink pencil erasers) T-maze (10 cm width; 10 cm height; 33 cm stem length, 37 cm arm length) fitting into hole in subject’s home cage (Fig. 8.15.7) 1- to 8-day-old rat pups 1. Attach the T-maze (to the subject’s home cage through a 10 cm × 10 cm hole in the home cage wall that is closed until testing; Fig. 8.15.7). 2. Place a pup and a neutral stimulus in opposite arms of the maze. 3. Expose the opening in the home cage and record subjects’ behavior for 10 min. Record the time taken for the subject to emerge into the maze, as well as the time taken for them to locate the pup in the maze arm, and then retrieve the pup back to the home cage. 4. Return subject to home cage and close off the opening to the T-maze. Return stimulus pup to its lactating mother.




COMMENTARY Background Information As indicated in the extensive reviews by Numan (1994) and by Fleming and Corter (1995), the early work of Weisner and Sheard (1933) and then Rosenblatt and Lehrman (1963) provided the first, and among the most thorough, descriptions of maternal behavior in the laboratory rat. Probably the single most influential analytic or “invasive” study of the sensory control of maternal behavior was done by Beach and Jaynes in 1956; it involved observing maternal behavior in experienced mother rats after the systematic removal of different sensory systems, either singly or in combination. Although today we have a clearer understanding of the role of individual sensory systems in the control of maternal behavior, the importance of multisensory mechanisms established by Beach and Jaynes in 1956 is now well-established. The study of the hormonal control of maternal behavior, beginning with the early endocrine studies of Beach and Wilson in 1963, Lott and Fuchs in 1962, and Riddle et al. in 1935 (reviewed in Numan, 1994; Fleming and Corter, 1995) which did not find the endocrine effects that we now know to be important (see Numan, 1994), provided an approach to the analysis of the hormonal control of behavior using extirpation and replacement strategies that are still utilized today. Finally, prior to the seminal work by Numan in 1974 (reviewed in Numan, 1994) showing the importance of the medial preoptic area and its downstream projections in the regulation of maternal behavior, little was known about the neuroanatomy of maternal behavior. The early work by Beach in 1937 focused on neocortical structures and suggested that no one area of the cortex is crucial to the expression of maternal behavior. Other studies focused on the midline cortex and associated limbic structures, the hippocampus and septum (reviewed in Numan, 1994; Fleming and Corter, 1995). Through these and subsequent studies, we now know that the regulation of maternal behavior involves multiple brain systems that intersect with a “maternal circuit” and that mediate the multiple sensory and motivational systems that are activated when mother rats interact with their offspring.

Critical Parameters and Troubleshooting Training in the observation of behavior and establishing inter-observer reliability for behaviors Training in the observation of behavior and establishing inter-observer reliability on individual behaviors requires practice. A new trainee should be provided with videotapes of mother rats interacting with pups at different postpartum stages and should practice with an experienced observer. After a period of practice, reliabilities between two observers can be established by having a trainee and an experienced observer each record the first 10 min of the same segments of behavior on ten separate mother-litter pairs, and then compute reliability quotients for each major behavior. Inter-observer reliabilities are ideally >90%. Influence of data collection procedure There are a multitude of factors that should be considered when evaluating parental behaviors in rodents. As indicated briefly above (see Basic Protocol 1), the different methods of data collection can sometimes provide very different results and may influence how one interprets the effects of a particular manipulation. Although periodic spot-checks of a subject’s behavior are relatively easy and inexpensive, they may be disadvantageous because one cannot be sure that the specific “snapshot” taken is an accurate representation of the continuous, ongoing subject-litter interaction. Furthermore, it is unlikely that all parental behaviors— particularly nest construction and retrieval— will be displayed within a particular observation or series of observations. However, this latter problem can be ameliorated with the use of supplementary tests of nest construction and retrieval (see Basic Protocols 3 and 4). Most importantly, the use of periodic spot-checks will result in the loss of a great amount of detail because it is difficult, if not impossible, to accurately determine the total duration of time that behaviors are displayed or the exact duration of time between behaviors. Behaviors that can occur very quickly, such as mouthing of pups within the nest or a very brief bout of licking, may end up not being recorded at all. However, if the objective is to know simply whether or not animals are “maternal” and show all the components of behavior on a particular day, it may be adequate to combine a



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15-min continuous observation after a period of mother-litter separation with multiple 5-min spot-checks at 2-hr intervals throughout the day. Alternatively, considerably more information about the quality of behavior that is exhibited can easily be obtained with continuous direct observation of mother-litter interactions. Similar to the periodic spot-check observations, if a subject-litter separation is not used before testing, it is unlikely that the entire sequence of parental behaviors will be displayed in a single observation. The advantage of using a 3- to 4-hr separation of the subject and litter prior to testing is that the entire repertoire of behaviors is likely to be displayed during the one subsequent observation. However, there is heightened motivation for the subject and pups to remain in physical contact with one another, which increases the amount of time during the observation period that the subject will quiescently huddle or nurse when compared with undisturbed observations made over an entire 24-hr period (Lonstein et al., 1998). This may be advantageous when there is a particular interest in examining nursing behavior, but it may not provide an authentic representation of the continuous parent-litter interaction. Furthermore, the particular nursing postures observed may also be influenced by this procedure (Lonstein et al., 1998). Since none of these methods for collecting behavioral data are ideal by themselves, the most complete depiction of a subject’s behavior with pups may be obtained by using a combination of undisturbed continuous or spot-check observation plus continuous observation after a separation from pups. Another possibility is to make a continuous videotaped recording of a subject’s behavior with pups during an entire 24-hr period. Although this option is probably the ideal situation, it is rarely used because it requires a time-lapse video camera that can record during both the light and dark phases and a videotape recorder to play back the videotaped observation. Furthermore, a 24-hr time-lapse observation can take hours to transcribe with a data acquisition system.


Influence of the age of test pups The age of the pups affects the parental behavior of both lactating and nonlactating rodents. In lactating dams, many changes occur in their behavior as their litters age. These changes include decreased nest defense, decreased time spent in physical contact with the

litter, a lower propensity to retrieve displaced pups, a reduction in nest quality, and decreased time spent nursing in the kyphotic posture with a concomitant increase in supine nursing (Stern and Levine, 1972; Fleming and Rosenblatt, 1974). These changes are probably due both to developmental changes in the pups, such as increased motility and size or changes in ultrasonic vocalization type, as well as to physiological changes in the dam. In nonlactating virgin female rats, younger pups (