John D. Newman, PhD, Head, Unit on Developmental Neuroethology
Deborah Bernhards, BS, Biological Technician
Emily Aronoff, BA, Technical Training Fellow
Karen Ladd, Summer Student
Audrey Quinn, Summer Student

Crying is a universal act in infancy and an essential signal that activates care-giving behavior. Virtually nothing is known about the neural basis of crying or about why crying can be such a compelling stimulus to the listener. Our work is organized around three goals: (1) to determine the neural pathways that underlie cry production and cry perception; (2) to track the developmental course of crying in infancy, in particular to determine the roles of inheritance and experience in individual variability in crying behavior; and (3) to examine the interplay of cry acoustics and the hormonal and experiential status of care-givers in regulating the motivation to respond to a crying infant. We undertake behavioral experiments in nonhuman primates in order to define the critical features of infant crying that promote care-giving; acoustic analysis of cry sounds in the search for acoustic markers of developmental status, familial traits, environmental influences, and neurological risk factors; and functional neuroanatomical studies aimed at defining the neural populations activated during crying and cry perception.

The cry circuit in the brain

Newman, Aronoff, Bernhards, Quinn

A necessary step in understanding how the brain regulates crying and cry responding is the identification of the neural circuits that mediate such behaviors. We studied the brains of infant common marmosets by using immunocytochemical identification of Fos, the protein product of the immediate early gene c-fos, as a marker for functional activity of neurons following an extended bout of crying. Previously, we demonstrated that Fos is a reliable marker of functional activity of neurons in adult monkeys. Our current work is the first investigation that has applied this method to the brains of infant monkeys. Using a motorized stage microscope and the Life Science program from Bioquant, we are systematically digitizing thousands of images at 100x magnification of sections from throughout the brains of the infant monkeys at one, two, three, and four months of age. We subsequently collected the images into montages of each section, subjected them to quantification of the number of Fos-expressing neurons per image, and created a map of the distribution of regions within each montage with the greatest number of Fos-expressing neurons. We stained adjacent sections for Nissl granules, permitting detailed anatomical identification of the regions of greatest expression. Reference to a brain atlas produced in this laboratory assists in the ongoing construction of a "wiring diagram" of the structures making up the circuit underlying cry production at different ages during development.

Marmoset brain atlas

Newman, Aronoff, Bell, Silva

To describe the neuroanatomy of the cry circuit, it is obviously necessary to know what structures are under examination. A brain atlas provides the necessary identification of structures. There are two published brain atlases for the squirrel monkey, but the only atlas published for the marmoset has been out of print for many years. Therefore, we set out to create a marmoset brain atlas. Using a digital camera, we photographed frontal (coronal) sections cut through the brain of an adult common marmoset and stained for cell bodies (Nissl stain) and fiber tracts (Weil stain) and used the resulting images for the atlas. Rachel Bell used one of the existing squirrel monkey atlases as an aid in identifying and labeling the corresponding structures of the marmoset brain sections. Rather than using the actual photographs, we improved the usefulness of our atlas as a reference by creating and labeling schematic images of the photographed sections. In addition, we used a tablet monitor with an electronic stylus to create wire-frame tracings of the brain sections. The tracings may be stretched or otherwise manipulated to overlay MRI images in planned studies of marmosets that will use the MRI and functional MRI techniques. We plan to publish an atlas that will contain both the photographs and labeled schematic images.

The next stage in our work calls for the creation of an atlas of horizontal sections of the marmoset brain. Typically employed in MRI studies, the horizontal plane provides a view of a brain that spans much of the entire brain for a given slice. The difficulty is that no horizontal brain atlas for any New World non-human primate currently exists. Images of a brain of a macaque cut in the horizontal plane are available online and can provide some guidance in identifying structures in our horizontal series. In addition, a computer program is available that can rotate MRI images so that frontal sections can be rotated to the horizontal plane. Our plan is to use our existing frontal brain atlas to label the MRI images in the frontal plane and then rotate the images in the horizontal plane (retaining the labels), thereby providing a means to identify and label specific structures on the horizontal sections. In addition, we have stained and imaged an extensive series of frontal brain sections from infant marmosets for Nissl granules and will eventually use the sections to construct a brain atlas of the infant marmoset at different ages.

Cry characteristics of infant marmosets

Newman, Bernhards, Aronoff, Ladd

Our project employed Raven, a software program developed at Cornell University, to measure the acoustic structure of the cry sounds of infant marmosets. We made cry recordings by separating infants from their family group for periods of up to 15 minutes, during which extensive crying typically occurred. We then digitized the recordings and stored them on a computer for analysis, generating a sound spectrogram of each cry. Given that cries typically occur in bouts of two, a pair of cries produced eight time and frequency values. From these values, Karen Ladd calculated a total of 34 acoustic parameter measures (in the frequency and time domains) and saved the parameter values for statistical analysis. We recorded, digitized, and subjected to statistical analysis a total of 3,162 cries from infants at two to three months of age, offspring that had reached maturity (18 months-2 years of age), and the adult parents of these infants. Data from two unrelated groups (colonies) contributed to the study. We are interested in resolving the following issues: the degree of individuality (vocal signature) in marmoset cries in adulthood and during infancy; whether the cries of litter mates (fraternal twins) resemble each other more than the cries of age-matched unrelated individuals; whether there are familial traits that transcend specific litters (i.e., shared acoustic characteristics of offspring born to the same set of parents); and to what extent the cries of infants resemble the cries of their parents (i.e., calls made by parents when infants are briefly separated from their family groups). In addition, we wish to determine the developmental age at which sex differences in cries occur (a phenomenon in adults discovered our laboratory) as well as the age at which cry syntax (the relationship between the structure of the first and second element of a two-cry series) emerges. A summary of findings to date is too detailed to present here. However, we found that (1) most individuals, infants as well as adults, have strong individuality (as shown by discriminant function analyses); (2) litter differences are apparent for nearly all litters; (3) sex differences in cry structure emerge as early as two months of age; and (4) syntax in cry pair structure can be demonstrated at two months of age. These findings indicate tight regulation of marmoset cry structure starting early in infancy, suggestive of strong genetic control.

A paradigm for measuring cry responsiveness

Newman, Aronoff, Bernhards

One of the great mysteries of parenting is how compelling the cries of an infant are in activating care-giving behavior. In humans, it has long been assumed that the hormonal state of the mother plays a role in activating and maintaining infant retrieval and nurturance. However, fathers play an important role in infant care as well. The same is true in the common marmoset, the non-human primate species that we study. In addition, older marmoset brothers and sisters exhibit a great deal of interest in infants and gain care-giving experience by assisting in carrying the infants when they are not being fed. We wish to understand the mechanisms of infant care, particularly with respect to the role played by infant crying in such behavior. To this end, we developed a testing paradigm for evaluating subjects' interest in infants and infant crying. The paradigm is based on the orienting and approach behavior that a subject exhibits in a Y-maze when presented with a stimulus at the end of one of the arms. To date, we have used two stimulus paradigms, a live infant and an audio speaker emitting recorded cries from an infant. In the tests using live infant marmosets, subjects were brought into the test room in a small transfer cage and placed at the base of the Y-maze; a live infant was then brought in and placed at the end of one of the arms. After 15 seconds, the door to the transfer cage was raised, and the behavior of the subject evaluated. We considered approach to the end of the arm containing the infant as a positive response. Of eight adults with parenting experience, seven gave positive responses in at least one of two trials. Of seven older offspring in these groups (brothers and sisters of the infant stimulus animals), all failed to give a positive response (four failing to leave the transfer cage in both trials). In a second series of trials six months later with the same subjects (after new infants were born to one pair in the colony), we found that the parents gave positive responses while the older brothers and sisters did not. Unrelated individuals in the same colony room failed to show clear positive responses. The results suggest that further paradigm adjustments will be necessary to yield more consistently positive results. A second paradigm involved the use of an audio speaker emitting infant cries instead of live infant emitting cries. We used this procedure because too few infants are born in our colony to produce sufficient numbers of stimulus animals for regular testing. In testing during the period when live infants were also presented, five of seven adults with parenting experience approached the speaker emitting infant cries during one of two trials. We plan further testing with this paradigm as well as the use of a video playback of a crying infant. We plan to collect blood samples from subjects after testing to evaluate steroid and prolactin levels in animals giving positive and negative responses.

COLLABORATORS

Rachel Bell (Senior, Montgomery Blair High School), Laboratory of Functional and Molecular Imaging, NINDS, Bethesda, MD
Alfonso Silva, PhD, Laboratory of Functional and Molecular Imaging, NINDS, Bethesda, MD

For further information, contact newmanj@lce.nichd.nih.gov.