Sachine Yoshida

Functional, anatomical, and neurochemical differentiation of medial preoptic area subregions in relation to maternal behavior in the mouse

In rodents, previous findings indicate critical involvement of the medial preoptic area (MPOA) in the neural control of maternal behavior. However, the specification of the particular MPOA subregions involved in maternal behavior and the identification of the neurochemical phenotype(s) of the essential neurons demands additional study. Therefore, we investigated the chemical neuroanatomy of the essential MPOA subregion for maternal behavior in C57BL/6J female mice. Using the oxytocinergic neurons in the dorsal MPOA as a primary regional marker, we first assessed the distribution of c‐Fos‐expressing neurons in the MPOA during maternal behavior using immunohistochemistry. Results showed that non‐oxytocinergic neurons in the dorsal and ventral MPOA prominently expressed c‐Fos during maternal behavior. Then using excitotoxic lesion studies, we determined the specific MPOA area that is necessary for maternal behavior. Bilateral lesions of the central MPOA, where c‐Fos was expressed only moderately, effectively disrupted maternal behavior, although lesions to the dorsal and ventral MPOA regions were ineffective. These centrally lesioned females were highly infanticidal irrespective of their previous maternal experience. Neurochemical investigations showed that more than 75% of the c‐Fos‐expressing neurons in central MPOA were GABAergic. Many of them also expressed galanin, neurotensin, and/or tachykinin2 mRNAs. Finally, the central MPOA was populated by numerous glutamatergic neurons, although only a small percentage of these neurons colocalized with c‐Fos. To conclude, the central MPOA is the indispensable subregion for mouse maternal behavior, and GABAergic and/or peptidergic neurons in this area were transcriptionally activated during maternal behavior. J. Comp. Neurol. 521:1633–1663, 2013.

Cajal–Retzius cells and subplate neurons differentially express vesicular glutamate transporters 1 and 2 during development of mouse cortex

In the light of the various neurobiological effects of glutamate in brain development, although some embryonic cells are a probable source of glutamate involved in the development of precursor cells and/or immature neurons, little is known about when and where glutamate plays its crucial roles during corticogenesis. To investigate these roles, we focused on the developmental expression of vesicular glutamate transporter (VGLUT)1 and VGLUT2, which are regarded as the best markers for verifying glutamatergic neuron identity, especially the spatiotemporal distributions of their transcripts and proteins in the developing mouse cortex and hippocampus. In situ hybridization studies revealed that VGLUT1 mRNA is expressed in preplate and marginal zone cells at embryonic day (E)10 and in subplate cells by E13, whereas VGLUT2 mRNA is expressed in preplate and marginal zone cells at E10 and in cells of the subventricular zone by E13. Reverse transcriptase–polymerase chain reaction analysis detected full‐length VGLUT1 and VGLUT2 gene transcripts in the embryonic brain. By dual labeling combined with immunostaining for microtubule‐associated protein 2 (MAP2) or reelin, we showed that MAP2‐positive preplate and marginal zone neurons and subplate neurons express VGLUT1, while reelin‐positive preplate and marginal zone cells and MAP2‐negative subventricular zone cells express VGLUT2. The present study is the first to provide morphologically reliable evidence showing that Cajal–Retzius cells and subplate neurons are glutamatergic, and that the two cells differentially express VGLUT1 and VGLUT2, respectively, as the specific transport system of glutamate in some events orchestrated by these cells during the cortical development of mice.

Infant Calming Responses during Maternal Carrying in Humans and Mice

BACKGROUND Mother-infant bonding is the earliest and most critical social relationship of mammalian infants. To promote this bond, infants have innate behaviors to seek maternal proximity and protest upon separation via communication with the mother vocally and through body movement. However, the physiological mechanisms regulating these infant behaviors remain largely undefined. RESULTS Here we show a novel set of infant cooperative responses during maternal carrying. Infants under 6 months of age carried by a walking mother immediately stopped voluntary movement and crying and exhibited a rapid heart rate decrease, compared with holding by a sitting mother. Furthermore, we identified strikingly similar responses in mouse pups as defined by immobility and diminished ultrasonic vocalizations and heart rate. Using pharmacologic and genetic interventions in mouse pups, we identified the upstream and downstream neural systems regulating the calming response. Somatosensory and proprioceptive input signaling are required for induction, and parasympathetic and cerebellar functions mediate cardiac and motor output, respectively. The loss of the calming response hindered maternal rescue of the pups, suggesting a functional significance for the identified calming response. CONCLUSIONS Our study has demonstrated for the first time that the infant calming response to maternal carrying is a coordinated set of central, motor, and cardiac regulations and is a conserved component of mammalian mother-infant interactions. Our findings provide evidence for and have the potential to impact current parenting theory and practice, since unsoothable crying is the major risk factor for child abuse.

Two distinct subtypes of serotonergic fibers classified by co-expression with vesicular glutamate transporter 3 in rat forebrain

Although virtually all of serotonin (5-HT) neurons in the midbrain raphe nuclei of rats are known to express vesicular glutamate transporter 3 (VGLUT3), VGLUT3-positive 5-HT fibers have been identified only in the cerebral cortex and hippocampus. Thus, our understanding of forebrain sites where 5-HT-glutamate interaction may be potentially managed by such possible glutamatergic 5-HT fibers themselves, is still largely fragmentary from a morphological point of view. To address this issue, we analyzed the rat forebrain by immunohistochemistry and chemical lesion experiment of 5-HT neurons by intracerebroventricular injection of a neurotoxin, 5,7-dihydroxytriptamine. Contrary to expectation, the double-label immunofluorescence staining revealed that the incidence of VGLUT3-positive 5-HT fibers is generally low over the forebrain, demonstrating occasional fibers with one or two double-labeled varicosities. The most extreme example was the nucleus of the lateral olfactory tract (LOT), which seemed to be devoid of double-labeled fibers despite high densities of 5-HT fibers and VGLUT3-positive fibers. In sharp contrast, robust plexuses of VGLUT3-positive 5-HT fibers were found in the dorsal, but not ventral, part of the lateral septum. The lesion experiment carried out to explore whether VGLUT3 exists in 5-HT fibers showed that in lesioned rats VGLUT3-positive fibers almost completely disappear from the septal region but seemed unchanged in the LOT. The present study shows that midbrain raphe-derived 5-HT fibers can be classified into two subtypes depending on co-expression with VGLUT3 staining in the forebrain.

Upregulated expression of neuropeptide Y in hypothalamic–pituitary system of rats by chronic dexamethasone administration

To study the effect of adrenal steroids on neuropeptide Y (NPY) synthesis in the hypothalamic-pituitary system, we examined NPY expression in rats treated with dexamethasone (a synthetic glucocorticoid) by in situ hybridization and immunohistochemistry. Rats were injected daily with dexamethasone (0.2mg/100g/day for 10 days, sc) or sesame oil (vehicle control), or non-injected (intact control). Relative staining area for corticotropin-releasing hormone or neurophysin II, a vasopressin carrier protein, was increased in the external zone of the median eminence in vehicle control, but was equivalent to that of intact control in the dexamethasone-injected group. Density of NPY-stained fiber varicosities was drastically increased in the external, but not the internal, zone of dexamethasone-injected group, coinciding with the increased NPY hybridization signal level in the arcuate nucleus. Dual-labeling experiments revealed no colocalization of NPY with hypophysiotropic or other peptides examined in single fibers of the median eminence. In the dexamethasone-injected group, expressions of NPY mRNA and peptide were detectable in a few pituitary cells, with some being corticotropes. These results suggest that NPY plays hormonal roles in the hypothalamic-pituitary-adrenal axis.

Postnatal changes in expression of vesicular glutamate transporters in the main olfactory bulb of the rat.

Olfactory information is initially processed through intricate synaptic interactions between glutamatergic projection neurons and GABAergic interneurons in the olfactory bulb. Although bulbar neurons and networks have been reported to develop even postnatally, much is yet unknown about the glutamatergic neuron development. To address this issue, we studied the postnatal ontogeny of vesicular glutamate transporters (VGLUT1 and VGLUT2) in the main olfactory bulb of rats, using in situ hybridization, immunohistochemistry, and their combination. In situ hybridization data showed that VGLUT1 mRNA is intensely expressed in differentiating mitral cells and smaller cells of the mitral cell layer (MCL) on postnatal day 1 (P1), and also at lower levels in small- and medium-sized cells, presumably tufted cell populations, of the external plexiform layer (EPL) from P5 onward. VGLUT2 mRNA was expressed in many MCL cell populations on P1, also in small- and medium-sized cells of the EPL at almost the same level as MCL cells between P5 and P7, and became apparently less intense in the MCL than in the EPL from P10 onward. The expression, unlike VGLUT1 mRNA, was also found in small-sized cells of the interglomerular region. In partial agreement with these data, immunohistochemical analyses demonstrated that subsets of mitral and EPL cells are stained for VGLUT1 or VGLUT2, with the former cells coexpressing both subtypes until P5. Moreover, a combined fluorescence in situ hybridization-immunohistochemical dual labeling of the P10 bulb revealed that neither VGLUT1 nor VGLUT2 mRNA is expressed in GABAergic or dopaminergic periglomerular cells, implying their expression in other periglomerular cell subclasses, external tufted cells and/or short-axon cells. Thus, the present study suggests that early in the postnatal development distinct glutamatergic bulbar neurons of rats express spatiotemporally either or both of the two VGLUT subtypes as a specific vesicular transport system, specifically contributing to glutamate-mediated neurobiological events.

The ontogenic expressions of multiple vesicular glutamate transporters during postnatal development of rat pineal gland.

The pineal gland expresses vesicular glutamate transporters 1 and 2 (VGLUT1 and VGLUT2), which are thought to transport glutamate into synaptic-like microvesicles in the pinealocytes. Recently, we reported that the rat pineal gland also expresses VGLUT1v which is a novel variant of VGLUT1 during the perinatal period. To explore the biological significance of these VGLUT expressions in pineal development, we studied the ontogeny of VGLUT in this gland by in situ hybridization, immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (RT-PCR) using rats. Histological analysis revealed that intensities of VGLUT1 hybridization signal and immunostaining drastically increase by postnatal day (P) 7, whereas VGLUT2 expression exhibits high levels of mRNA and protein at birth and decreases gradually from P7 onward. Quantitative RT-PCR analysis supported these histological observations, showing that expressions of VGLUT1 and VGLUT2 exhibit opposite patterns to each other. Coinciding with VGLUT1-upregulation, RT-PCR data showed that expressions of dynamin 1 and endophilin 1, which are factors predictably involved in the endocytotic recovery of VGLUT1-associated vesicle, are also increased by P7. Quantitative RT-PCR analysis of VGLUT1v demonstrated that its mRNA expression is upregulated by P7, kept at the same level until P14, and apparently decreased at P21, suggesting its functional property required for a certain developmental event. Moreover, a comparison of mRNA expressions at daytime and nighttime revealed that neither VGLUT1 nor VGLUT1v shows any difference in both P7 and P21 glands, whereas VGLUT2 is significantly lower at daytime than at nighttime at P21 but not P7, the time point at which the melatonin rhythm is not yet generated. The present study shows that expressions of these VGLUT types are differentially regulated during postnatal pineal development, each presumably participating in physiologically distinct glutamatergic functions.

Transport Response is a filial-specific behavioral response to maternal carrying in C57BL/6 mice

BackgroundA mother carries her young in many altricial mammals, such as cats, lions, rats and mice. During maternal carrying, the transported young assume a compact posture. We have recently shown that, in both humans and mice, the carried infants immediately calmed down and showed reductions in heart rate, distress vocalizations, and voluntary movement. The loss of the calming response in mouse pups hindered maternal retrieval efficacy. These findings suggested that the infant calming response functioned to reduce the maternal burden of carrying and was therefore conserved in a variety of mammalian species. However, it remains unclear how and when each component of this calming response develops and whether it is a filial-specific behavior.ResultsWe dissected various components of the carrying-induced responses in mouse pups, collectively called the “Transport Response” herein. We showed that during the second postnatal week, pups exhibited characteristic compact posture with limb ventroflexion. The body trunk remained paradoxically pliable, suggesting complex neural regulation throughout the body. Pups also showed an increased pain tolerance to a tail pinch during the Transport Response. Analyses of the developmental courses of distinct components of the Transport Response revealed the independent regulation of each component: in the first postnatal week, the cessation of ultrasonic vocalizations was exhibited prominently; in the second postnatal week, immobilization reached its peak; and toward the third postnatal week, the postural component became fully matured. At the end of the third postnatal week, when the pups are able to transport by themselves, the pups no longer exhibited the Transport Response.ConclusionsThis study has revealed the mouse Transport Response as a complex set of behavioral and physiological components, each of which has a specific postnatal time window but is orchestrated in a well-matched manner with the maturation of ambulatory ability in the pups. These findings collectively indicate that the Transport Response is a filial-specific, innate behavioral reaction and is distinct from a simple reflex or defensive freezing response. The Transport Response could be a novel index of primitive filial attachment behaviors, acting to smooth mother-infant interaction.

Three lessons from Philip Teitelbaum and their application to studies of motor development in humans and mice

In this paper we present a number of studies, some with humans and some with animals that have been directly influenced by the work of Philip Teitelbaum. As appropriate for a festschrift, we integrate a number of contributions from different fields (specifically, clinical psychopathology and neurobiology) and present the studies, not so much chronologically, but in ways that illustrate some of the key lessons that we have learned from Philip Teitelbaum. We would summarize these lessons as follows: (1) Complex and precise tools are required to assess the complexity of specific behaviors; using simplified tools will bias our understanding of the behavior itself. (2) Studying a specific behavior in atypical brain conditions is required to fully understand that behavior and how the brain controls it. (3) Considering the evolutionary basis of human behavior, studying corresponding behaviors in other mammalian species is required to strengthen and deepen our knowledge of specific human behaviors. In this paper we describe studies on motor development in humans and mice and their implication for the diagnosis of autism and early parental care that illustrate these three lessons.

The calming effect of maternal carrying in different mammalian species

Attachment theory postulates that mothers and their infants possess some basic physiological mechanisms that favor their dyadic interaction and bonding. Many studies have focused on the maternal physiological mechanisms that promote attachment (e.g., mothers’ automatic responses to infant faces and/or cries), and relatively less have examined infant physiology. Thus, the physiological mechanisms regulating infant bonding behaviors remain largely undefined. This review elucidates some of the neurobiological mechanisms governing social bonding and cooperation in humans by focusing on maternal carrying and its beneficial effect on mother–infant interaction in mammalian species (e.g., in humans, big cats, and rodents). These studies show that infants have a specific calming response to maternal carrying. A human infant carried by his/her walking mother exhibits a rapid heart rate decrease, and immediately stops voluntary movement and crying compared to when he/she is held in a sitting position. Furthermore, strikingly similar responses were identified in mouse rodents, who exhibit immobility, diminished ultra-sonic vocalizations and heart rate. In general, the studies described in the current review demonstrate the calming effect of maternal carrying to be comprised of a complex set of behavioral and physiological components, each of which has a specific postnatal time window and is orchestrated in a well-matched manner with the maturation of the infants. Such reactions could have been evolutionarily adaptive in mammalian mother–infant interactions. The findings have implications for parenting practices in developmentally normal populations. In addition, we propose that infants’ physiological response may be useful in clinical assessments as we discuss possible implications on early screening for child psychopathology (e.g., autism spectrum disorders and perinatal brain disorders).