Atsushi Funahashi

Developmental alteration of serotonin neurons in the raphe nucleus of rats with methylazoxymethanol-induced microcephaly

SummaryPrenatal exposure of pregnant rats to methylazoxymethanol acetate (MAM), an anti-mitotic agent, on day 15 of gestation induces severe microcephaly in the offspring. The present study first investigated a developmental alteration of serotonin (5HT) neurons immunohistochemically in the dorsal and median raphe nuclei in serial sections in both control and microcephalic rats (MAM-rats) at 35 days of age. 5HT-immunoreactive neurons in the MAM-rats were reduced in number and irregularly distributed in the dorsal and median raphe nuclei compared with those in the control. The dendrites of neurons in these nuclei in the MAM-rats were very short and twisted. A follow-up observation on the development of the cerebral cortex at 5, 9 and 28 days of age was performed using Nissl-stained preparations, which revealed a disorganized cell arrangement in the cerebral cortex of the MAM-rats at the very early postnatal period. Furthermore, the distribution of 5HT-immunoreactive fibers into the cerebral cortex was also examined using brains of 28 days of age. In MAM-rats of this age, abnormally tortuous 5HT-immunoreactive fibers were observed in the cerebral cortex. 5HT neurons in the raphe nuclei are known to project their ascending axons widely into the entire cortical area during the 1st postnatal week. Thus, the association of disorganized cortical cell arrangement and the hyperdense and tortuous distribution of 5HT-immunoreactive fibers in the cerebral cortex support the idea of target-dependent secondary degeneration of 5HT neurons in the dorsal and median raphe nuclei of the MAM-rats.

Distribution of Corticogeniculo-Neurons and Projection of Geniculocortical Fibers in Methylazoxymethanol-lnduced Microcephalic Rats

Abstract In the rat with methylazoxymethanol (MAM)‐induced microcephaly, the distribution of corticogeniculate neurons and the projection of genicurocorti‐cal fibers were examined using the horseradish peroxidase (HRP) tracing technique. In agreement with the previous studies, HRP‐labeled corticogeniculate neurons in the MAM‐rats were irregularly distributed and reduced in number in the occipital cortex and neurons in the LGNd also showed developmental deficits such as reduced cell number and abnormal distribution. It was first demonstrated that in the MAM‐rats, the density of HRP‐labeled geniculocortical fibers observed in the corpus calosum was remarkably low than the control. Moreover, HRP‐labeled fiber plexus in the MAM‐rats revealed a layer‐like appearance similar to the control. However, its major location did not coincide with layer IV because of the obscure lamination pattern in the superficial and intermediate layers. Besides, in the MAM‐rats, the density of the HRP‐labeled geniculocortical afferents in the plexus was abnormally high and these afferent terminals showed uneven distribution compared with more uniform distribution in the control. From these observations, it is suggested that there is a possibility of abnormal neuronal contacts between the geniculocortical afferent and the target cortical neurons in the visual cortex.

Abnormal development of serotonin nerve fibers in the visual cortex in rats with methylazoxymethanol-induced microcephaly

Abstract The postnatal development of serotonin (5HT)-immunoreactive axons was studied in the visual cortex of the cerebrum in both normal and microcephalic rats during early postnatal and young adult stages. Severe microcephaly in rat offspring was induced by prenatal exposure to methylazoxymethanol acetate (MAM), an anti-mitotic agent, on day 15 of gestation. From postnatal day 1 (PND 1) to PND 5, fine and short 5HT fibers were irregularly dispersed throughout the occipital cortex in both the control and MAM-treated rats (MAM-rats). A conspicuous aggregation of dot-like 5HT terminals was found in controls, but not in MAM-rats, in a shallow layer of the dorsomedial region of the occipital cortical plate. On PND 7, such an aggregation of 5HT terminals was found in both groups. The density of the aggregation increased up to PND 9, but then decreased gradually, finally becoming unrecognizable at around PND 15 in both groups. MAM-rats, however, always showed hyperaggregation of 5HT terminals when compared with controls on the same PND. The density of 5HT fibers gradually increased, and finally made up a network-like formation at PND 28 in both groups, its pattern was essentially identical to the abnormal distribution of 5HT fibers during the later stage. As a result, the network-like formation of 5HT fibers in the MAM-rats at PND 28 was markedly twisted and somewhat hyperdense. In Nissl-stained preparations from PND 9 to 15, the 5HT terminal aggregation in the control rats was precisely confined to the newly forming layer IV of the visual cortex. In the MAM-rats, on the other hand, the aggregation of 5HT terminals was not associated with a specific cortical layer because of a disarranged cytoarchitecture of the microcephaly.

Immunohistochemical Examination of Developmental Brain Defects

Recent advances in immunohistochemistry have revealed the precise distribution of various neurotransmitter systems, their projections and ultrastructure in the brain. Based on these data, the structure and function of specific neurons or a group of neurons in the brain can be understood in terms of the dynamics of the neurotransmitters they utilize. This strategy of neuroscience may be utilized to explore higher brain functions such as learning, memory, cognition and sleep. Prenatal exposure of pregnant rats to methylazoxymethanol acetate (MAM) on day 15 of gestation produces offspring with severe microcephaly (MAM‐rats). These MAM‐rats show various behavioral abnormalities in learning, memory and cognition as well as hyperactivity and reduction of paradoxical sleep. Developmental abnormalities of serotonin (5HT) fibers in the cerebral cortex and 5HT neurons in raphe nuclei are examined. The cell deficits of cortical target neurons and irregularly arranged cortical layers were observed. Developmental abnormalities of 5HT neurons are discussed in terms of target dependent secondary degeneration. Vasoactive intestinal polypeptide (VIP) has an important role in behavioral modification. VIP is suggested to have a functional correlation with 5HT in the brain. The reduction of VIP neurons and hypo‐aggregation of their axonal boutons were observed in the cerebral cortex and hippocampus of infant MAM‐rats. However, in adult MAM‐rats, VIP axonal boutons showed layer‐dependent hyper‐ or hypo‐aggregation compared with the age‐matched control. The difference in the number of VIP neurons became small between the controls and MAM‐rats. These immunohistochemical observations of brain defects have an important implication for understanding the abnormal behavior of MAM‐rats.

Vasoactive Intestinal Polypeptide-containing Neurons and Processes in the Developing Hippocampus of Rats Prenatally Exposed to Methylazoxymethanol Acetate

Developmental alteration of the dendritic arborization and axonal boutons of vasoactive intestinal polypeptide (VIP) neurons in the CA1 hippocampus was immunohistochemically examined in both control and microcephalic rats at postnatal days 15 and 60. Microcephaly was induced by prenatal exposure to methylazoxymethanol acetate (MAM; 20 mg/kg) on day 15 of gestation (MAM rats). In the control at postnatal day 15, VIP‐immunoreactive axonal boutons were densely aggregated specifically in the stratum pyramidale. At postnatal day 60, these axonal boutons in the stratum pyramidale of the controls were somewhat less dense. Another aggregation of axonal boutons was also observed along the upper part of the stratum oriens near the alveus border (area A). In the MAM rats at postnatal day 15, on the other hand, VIP‐immunoreactive neurons in the CA1 hippocampus were decreased in number and the dendritic arborization of these neurons was very poor compared with the controls. The density of VIP‐immunoreactive axonal boutons was remarkably lower than the controls. In the MAM‐rats at postnatal day 60, density of the axonal boutons was almost similar with that of MAM‐rats at postnatal day 15 in the stratum pyramidale, whereas a drastic increase was observed in area A, which made the bouton aggregation more dense than in the controls at postnatal day 60. These observations suggest that there is an area‐dependent difference in the development of VIP neurons in the CA1 hippocampus which may be related to the area‐dependent heterogeneous vulnerability of VIP neurons to MAM exposure.