Neuroethology of Crying

John D. Newman, PhD, Head, Unit on Developmental Neuroethology
Michelle Becker, PhD, Guest Researcher
Deborah Bernhards, BS, Biological Technician
Katherine Garnett, BA, Technical Training Fellow
Shannon Fuhr, Summer Student
Molly Zametkin, Summer Student

Crying, a universal mammalian behavior in infancy, is an essential signal that activates care-giving behavior. Little is known about the neural basis of crying or about why crying can be such a compelling stimulus to the listener. Our goals are (1) to determine the neural pathways that underlie cry production and cry perception; (2) to track the developmental course of crying in infancy and, 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 non-human primates aimed at defining the critical features of infant crying that promote care-giving; perform acoustic analysis of cry sounds in the search for acoustic markers of developmental status, familial traits, environmental influences, and neurological risk factors; and conduct functional neuroanatomical studies aimed at defining the neural populations activated during crying and cry perception.

The cry circuit in the brain

Newman, Bernhards, Garnett, Fuhr

A necessary step in understanding how the brain regulates crying and cry responding is to identify the neural circuits that mediate crying and cry-response behaviors. Using an approach never before applied to the brains of infant monkeys, we studied the brains of infant common marmosets by using immunocytochemical identification of Fos as a marker for functional activity of neurons after an extended bout of crying; Fos is the protein product of the immediate-early gene c-fos. Using a Leica microscope with a motorized stage and the Life Science program from Bioquant, we are systematically digitizing thousands of images at 100x magnification from sections throughout the brains of infant monkeys at 1, 2, 3, and 4 months of age. We subsequently collect the images into montages of each section, subject them to quantification of the number of Fos-expressing neurons per image, and map the distribution of regions within each montage with the greatest number of Fos-expressing neurons. We stain adjacent sections for Nissl granules to permit detailed anatomical identification of the regions of greatest expression. Reference to a brain atlas produced in our laboratory assists in the construction of a “wiring diagram” of the structures making up the circuit underlying cry production at different ages during development.

Click for a larger version.
Figure 5.1
MRI image from female marmoset, with labeled brain structures.
Key: CA = caudate nucleus; CG = cingulate gyrus; CL = central lateral nucleus; CM = center median nucleus; DG = dentate gyrus; EW = Edinger-Westphal nucleus; HF = hippocampal formation; HI = lateral habenular nucleus; HM = medial habenular nucleus; III = oculomotor nucleus; Ins = insula; LD = lateral dorsal nucleus; LG = lateral geniculate nucleus; MD = mediodorsal nucleus; Pa = paraventricular nucleus; Pla = anterior nucleus of the pulvinar; Pli = inferior nucleus of the pulvinar; PSB = presubiculum; RN = red nucleus; RTP = reticulotegmental pontine nucleus; SB = subiculum; SNc = pars compacta of substantia nigra; SNr = pars reticulate of substantia nigra; STG = superior temporal gyrus; VLp = ventral lateral posterior nucleus. MRI image provided by Nicholas Bock.

Our latest findings are based on the digitization and measurement of Fos-labeled cells from eight infant brains, four at 1 month of age, two at 2 months of age, and two at 4 months of age (including one control brain). We digitized approximately 330 to 620 images (depending on the size of the original section) from each brain section and counted the labeled cells in each image. We created a grid overlay for each montage and entered the number of labeled cells from each image into the appropriate grid space. The result is an XY map of the distribution of counts for each montaged section. Examination of the map for each section clearly shows a differential distribution of subregions with high numbers of labeled cells, including periventricular grey in the diencephalon, septum, preoptic area, amygdala, and hippocampus in the rostral forebrain; anterior cingulate gyrus and gyrus rectus in the frontal cortical regions; superior temporal gyrus and periaqueductal grey of the midbrain. Studies of adult non-human primates indicate that most of these subregions are involved in auditory communication, but we demonstrated for the first time that the vocal production system is widely activated in such young animals. High counts in the superior temporal gyrus presumably reflect neurons in the auditory cortex activated during the infant’s vocalization. We are currently counting additional brain sections from the group of eight infants. In addition, we have developed a new method that overlays the actual counts (number of neurons containing Fos) on each frame of a software-created montage of the entire brain section. By color-coding the frames with the highest counts, we can immediately see where the greatest activity (hot spots) is located on the sections. Of particular interest, we note that the anterior cingulate gyrus and adjacent gyrus rectus of the youngest infants in the group contain large numbers of labeled neurons. This finding contradicts the prevailing model of infant crying, which posits a “midbrain model” and proposes that crying in early infancy is elaborated in the midbrain.

This year, we continued a study aimed at identifying neurons in the brains of individuals listening to infant cries. We exposed five adult marmosets to 30 minutes of recorded infant cries and then euthanized the animals in order to process their brains for Fos immunocytochemistry. Analysis of three brains from males demonstrated, for the first time in a non-human primate, that infant cries activate several defined areas of the brain, including the anterior cingulate gyrus and midline frontal cortex rostral to this area and the hippocampus, in addition to the expected temporal lobe auditory areas. All structures outside the temporal lobe auditory areas fall within the limbic system, as defined by Paul MacLean, suggesting that the limbic system may be a functionally integrated system for responding to cries, as originally proposed by MacLean for the thalamo-cingulate division of the limbic system.

Newman JD. Neural circuits underlying crying and cry responding in mammals. Behav Brain Res 2007;182:155-165.

Marmoset brain atlas

Newman, Bernhards, Zametkin, Winslow, Bock, Silva

Any effort to describe the neuroanatomy of the cry circuit requires identification of the appropriate structures for examination. A brain atlas aids in such identification. Over the past year, we used a brain atlas (created from the brain of an adult female) to identify structures in MRI images from a female marmoset. We selected MRI images that match as closely as possible the plates for the histological atlas created by our laboratory. We applied the same labels used for the histological atlas to the corresponding regions of the MRI images, thereby providing the equivalent of a histological atlas for MRI studies for use in identifying structures of interest in planned fMRI experiments (Figure 5.1).

Bock NA, Paiva FF, Nascimento GC, Newman JD, Silva AC. Cerebrospinal fluid to brain transport of manganese in a non-human primate revealed by MRI. Brain Res 2008;1198:160-170.

Collaborators

Nicholas A. Bock, PhD, Laboratory of Functional and Molecular Imaging, NINDS, Bethesda, MD
Afonso C. Silva, PhD, Laboratory of Functional and Molecular Imaging, NINDS, Bethesda, MD
James T. Winslow, PhD, Neurobiology Non-human Primate Core, NIMH, Bethesda, MD

For further information, contact newmanj@mail.nih.gov or visit http://udn.nichd.nih.gov