Wataru Matsunaga

Metabolic mapping of the brain in pregnant, parturient and lactating rats using fos immunohistochemistry.

The present study was designed to investigate Fos-positive neurons of the female rat brain at various reproductive states in order to analyze the metabolic map connected with pregnancy, parturition and lactation. The number of Fos-positive neurons in each brain nucleus was analyzed with a quantitative immunohistochemical method in virgin, pregnant, parturient, lactating and arrested lactating rats. In parturient rats, a significant number of Fos-positive neurons was observed as compared to virgin or pregnant females in the following brain regions; the bed nucleus of the stria terminalis (BST), lateral septal nucleus (LS), medial preoptic area (MPA), periventricular hypothalamic nucleus (Pe), parvocellular paraventricular hypothalamic nucleus (PaPVN), magnocellular paraventricular hypothalamic nucleus (MaPVN), supraoptic nucleus (SON), paraventricular thalamic nucleus (PV), anterior hypothalamic area (AHA), lateral hypothalamic area (LH), amygdaloid nucleus (AM), supramammillary nucleus (SuM), substantia nigra (SN), central grey (CG), microcellular tegmental nucleus (MiTg), subparafascicular thalamic nucleus (SPF), posterior hypothalamic area (PH), dorsal raphe nucleus (DR), locus coeruleus (LC), dorsal parabrachial nucleus (DPB), nucleus of solitary tract (Sol), and ventrolateral medulla (VLM). Significant differences were found in the number of Fos-positive neurons between parturient and lactating females, although localization of Fos-positive neurons in lactating females was quite similar to parturient ones. Between parturient and lactating rats: (1) In the MPA, PaPVN, AHA, arcuate hypothalamic nucleus (Arc), ventromedial hypothalamic nucleus (VMH), MLT, and Ge, the number of Fos-positive neurons of lactating females were significantly higher than those of parturient ones; (2) In the LS, Pe, PV, LH, AM, SuM, CG, MiTg, SPF, PH, DR, LC, and VLM, there was no significant differences in the number of Fos-positive neurons; (3) In the BST, MaPVN, SON, SN, DPB and Sol, the number of Fos-positive neurons of lactating rats were significantly lower than those of parturient ones. These different patterns of Fos expression among many brain regions may be owing to the functional differences in each region. Fos expression in lactating rats was apparently induced by suckling stimulation because the removal of their litters immediately after parturition completely eliminated expression of Fos protein in each nucleus. These results suggest that the localization of Fos-positive neurons in a number of neural populations throughout the brain may be revealing the neural circuits in response to parturition or lactation.

LPS-induced Fos expression in oxytocin and vasopressin neurons of the rat hypothalamus.

The aim of this study was to examine the involvement of the hypothalamic oxytocin (OXT) and vasopressin (AVP) neurons in acute phase reaction using quantitative dual-labeled immunostaining with Fos and either OXT and AVP in several hypothalamic regions. Administration of low dose (5 microg/kg) and high dose (125 microg/kg) of LPS induced intense nuclear Fos immunoreactivity in many OXT and AVP neurons in all the observed hypothalamic regions. The percentage of Fos-positive nuclei in OXT magnocellular neurons was higher than that of AVP magnocellular neurons in the supraoptic nucleus (SON), the magnocellular neurons in the paraventricular nucleus (magPVN), rostral SON (rSON), and nucleus circularis (NC), whose axons terminate at the posterior pituitary for peripheral release. The percentage of Fos-positive nuclei in AVP parvocellular neurons in the paraventricular nucleus (parPVN) was higher than that of OXT parvocellular neurons, whose axons terminate within the brain for central release. Moreover, the percentage of Fos-positive nuclei in AVP magnocellular neurons of the SON and rSON was significantly higher than that of the magPVN and NC when animals were given LPS via intraperitoneal (i.p.)-injection. This regional heterogeneity was not observed in OXT magnocellular neurons of i.p.-injected rats or in either OXT or AVP magnocellular neurons of intravenous (i.v. )-injected rats. The present data suggest that LPS-induced peripheral release of AVP and OXT is due to the activation of the magnocellular neurons in the SON, magPVN, NC, and rSON, and the central release of those hormones is in part derived from the activation of parvocellular neurons in the PVN. It is also suggested that the activation of AVP magnocellular neurons is heterogeneous among the four hypothalamic regions, but that of OXT magnocellular neurons is homogenous among these brain regions in response to LPS administration.

Time course of Fos and Fras expression in the hypothalamic supraoptic neurons during chronic osmotic stimulation.

The Fos family comprises Fos and several subtypes of Fos-related proteins (Fras) such as FosB, Fra-1, Fra-2, DeltaFosB, and chronic Fras. Changes in the expression of Fos family proteins with time are not well elucidated, particularly during chronic stimulation. In the present experiments, we investigated quantitatively the time course changes in Fos, FosB and Fras immunoreactivity in the magnocellular neurons of the supraoptic nucleus (SON) during acute and chronic osmotic stimulation. A small number of Fos- and FosB-positive neurons were observed in the SON of control rats, while many Fras-positive neurons were seen in control animals. Significant increases in the numbers of Fos-, FosB-, and Fras-positive neurons were observed 2 h after acute osmotic stimulation by intraperitoneal (i.p.) injection of 3% NaCl solution. Although the number of Fos-positive neurons returned to the control level 4 h after i.p. injection, a significant number of FosB- and Fras-positive neurons were still observed 8 h after i.p. injection. During chronic osmotic stimulation by giving 2% NaCl solution for 2 and 5 days, a large number of Fos-positive neurons were observed, but the cessation of chronic osmotic stimulation by normal water drinking immediately decreased the number of Fos-positive neurons to the control level within 2 h. The number of FosB-positive neurons was increased with period of chronic osmotic stimulation, and a significant number were observed 2-8 h after the cessation of the stimulation. The number of Fras-positive neurons was also significantly higher during chronic osmotic stimulation, and this number was significantly high 2-8 h after the cessation of the stimulation. RT-PCR analysis demonstrated the persistent expression of c-fos mRNA in the SON during chronic osmotic stimulation. These results suggest that c-fos mRNA and Fos protein are constitutively elevated during chronic osmotic stimulation and the time course changes in Fos are different from those seen in FosB and Fras.

Comparison of the expression of two immediate early gene proteins, FosB and Fos in the rat preoptic area, hypothalamus and brainstem during pregnancy, parturition and lactation

Medial preoptic area (MPA), supraoptic nucleus (SON), magnocellular (MaPVN) and parvocellular (PaPVN) paraventricular hypothalamic nuclei, and mesencephalic lateral tegmentum (MLT) are involved in maternal behavior, parturition and lactation. This study investigated the FosB and Fos immunoreactivity in these regions of virgin, pregnant, parturient, lactating, and lactating-arrested rats. The patterns of FosB and Fos expression were compared between the sections taken from the same animals. Quantitative immunohistochemistry revealed a significant increase in the numbers of FosB-positive neurons in the MPA, SON, MaPVN, and MLT of parturient and lactating females as compared with pregnant or virgin animals. In lactating rats, the numbers of FosB-positive neurons in the MPA, PaPVN, and MLT were increased, but the numbers in the SON and MaPVN were decreased as compared with parturient females. Many Fos-positive neurons were also seen in parturient and lactating rats, and the patterns of Fos expression in each region were quite similar to those of FosB. Moreover, double-labeling immunohistochemistry revealed that: (1) many FosB-positive nuclei were observed in oxytocin and vasopressin neurons of the SON and PVN in parturient rats; (2) within FosB-positive neurons, 89.5% in the MPA, 86.8% in the MLT of parturient rats, and 92% in the MPA and 90.8% in the MLT of lactating animals were also Fos-positive. Only a small number of FosB and Fos-positive neurons were seen in females that were killed in the early stage of parturition. Removal of the litters immediately after parturition completely eliminated FosB and Fos expression in each region in the dams. Taken together, the present results suggest that FosB expression is co-involved with Fos in the neural activation during parturition and lactation in rats.

Expression of the IgLON cell adhesion molecules kilon and OBCAM in hypothalamic magnocellular neurons

The vasopressin (AVP) and oxytocin (OXT) magnocellular neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) display reversible structural plasticity of neurons and glial cells under different conditions of neuropeptide secretion. In the present study, we investigated the expression of two immunoglobulin superfamily (IgSF) proteins, Kilon and OBCAM, in the magnocellular neurons by using monoclonal antibodies. Anti‐Kilon antibody reacted specifically with the bacterially expressed recombinant Kilon but not with the recombinant OBCAM, and similarly anti‐OBCAM antibody specifically recognized the recombinant OBCAM. Western blotting analysis revealed the specific expression of Kilon and OBCAM in the SON homogenates. Although Kilon and OBCAM of the SON homogenates were present as the insoluble form, most Kilon was present in the Triton‐insoluble fraction, and OBCAM was localized mainly in the Triton‐soluble fraction. Immunocytochemistry revealed Kilon and OBCAM immunoreactivity in the magnocellular neurons of the SON and PVN of the rat hypothalamus compared with outside of the SON and PVN in the hypothalamus. The double‐labeling study with confocal microscopy further demonstrated that Kilon immunoreactivity was observed mainly in the dendrites of AVP‐secreting neurons and also occasionally OXT‐secreting neurons. However, OBCAM immunoreactivity was exclusively seen in the dendrites of AVP‐secreting magnocellular neurons. Chronic physiological stimulation by 2% NaCl had no effect on the expression levels of either IgLON protein in the SON. Our study thus demonstrated specific expression of Kilon and OBCAM in the hypothalamic magnocellular neurons, particularly in dendrites, suggesting that they confer on magnocellular neurons the ability to rearrange dendritic connectivity. J. Comp. Neurol. 424:74–85, 2000.

Redistribution of MAP2 immunoreactivity in the neurohypophysial astrocytes of adult rats during dehydration

The low-molecular-weight microtubule-associated protein-2 (LMW MAP2) is expressed in immature and developing brains, and decreases its content dramatically along with maturation of the central nervous system. In our previous studies, we demonstrated through western blots and dual-labeling immunohistochemistry that LMW MAP2 is expressed in the pituicytes, modified astrocytes of the neurohypophysis in adult rats. The present study aimed to examine changes in the MAP2 immunoreactivity within pituicyte in adult rats under various hydration states using quantitative morphometrical analysis to demonstrate in vivo shape conversion of the pituicyte morphology. In well-hydrated control rats, light microscopic observation revealed that MAP2-stained pituicytes ramified long and well-branched processes. At electron microscopic level, MAP2 immunoreactivity was found in the fine process and cell body of all pituicyte cytoplasm, but not in the axonal terminals containing neurosecretory vesicles. The quantitative analysis demonstrated that the cell size and perimeter of MAP2-stained pituicytes were significantly greater as compared with those of cells stained with glial fibrillary acidic protein (GFAP). When the rats were dehydrated with water deprivation or drinking of 2% saline solution, the process of MAP2-stained pituicytes was less branched due to retracting their cellular processes as compared with those of well-hydrated control and rehydrated rats. The quantitative analysis further demonstrated that water deprivation significantly reduced the cell size, perimeter and length of cellular processes of MAP2-stained pituicytes as compared with those of control. The present finding indicates that MAP2 staining is better method for investigating in vivo shape conversion of the pituicyte morphology than GFAP one. Moreover, the finding that hydration states significantly and reversibly alter in vivo pituicyte shape supports the hypothesis that the plastic shape conversion of pituicyte morphology is responsible for morphological plasticity in the neurohypophysis.

Microtubule-associated protein-2 in the hypothalamo-neurohypophysial system: low-molecular-weight microtubule-associated protein-2 in pituitary astrocytes

Microtubule-associated protein-2 is the most abundant microtubule-associated protein in the brain and is responsible for morphogenesis and maintenance of the nervous system. In the present experiments, we have examined the localization of microtubule-associated protein-2 in the hypothalamo-neurohypophysial system of the rat using western blots and immunohistochemistry. Two monoclonal antibodies against microtubule-associated protein-2, antibody C and AP20, were used: antibody C recognizes both the high- and low-molecular-weight isoforms of microtubule-associated protein-2; antibody AP20 specifically detects high-molecular-weight microtubule-associated protein-2 only. Western blot analysis revealed expression of high-molecular-weight microtubule-associated protein-2 in the whole brain, hippocampus and whole hypothalamus. While the supraoptic nucleus expressed only high-molecular-weight microtubule-associated protein-2, the adult posterior pituitary predominantly expressed low-molecular-weight microtubule-associated protein-2, which was also seen in the embryonic whole brain. Light microscopic immunohistochemistry revealed that both antibody C and AP20 intensely stained dendrites of the dendritic and somatic zones in the supraoptic nucleus. Double labeling with antibodies against microtubule-associated protein-2 and oxytocin (or vasopressin) demonstrated that microtubule-associated protein-2 was localized in dendrites of magnocellular neurons in the supraoptic nucleus. In the posterior pituitary, however, antibody C stained fine processes and cell bodies of astrocytes, which were identified by an antibody against glial fibrillary acidic protein. Antibody AP20 also stained fine processes of some astrocytes in the posterior pituitary, but the intensity of immunoreactivity with antibody AP20 was weaker than that with antibody C. This result suggests that microtubule-associated protein-2 in astrocytes of the posterior pituitary is predominantly of the low-molecular-weight type. Moreover, western blots revealed low-molecular-weight microtubule-associated protein-2 of the posterior pituitary at a molecular weight slightly higher than embryonically expressed low-molecular-weight microtubule-associated protein-2, indicating that low-molecular-weight microtubule-associated protein-2 in the posterior pituitary is possibly the isoform microtubule-associated protein-2d. The present results demonstrate that astrocytes in the posterior pituitary of adult rats still retain the ability to express the immature variant of microtubule-associated protein-2, low-molecular-weight microtubule-associated protein-2, and its expression is probably linked to structural plasticity.

Involvement of neurotrophic factors in aging of noradrenergic innervations in hippocampus and frontal cortex

In the present study, we investigated the age-dependent changes in the axon terminals of the locus coeruleus (LC) neurons in the frontal cortex and hippocampus, in which a high degree of axonal branching in the middle-aged brain was suggested to occur in our previous electrophysiological study. We used 6-, 13- and 25-month-old male F344/N rats, and performed Western blot analysis of the norepinephrine transporter (NET), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). NET expression level increased in the 13-month-old hippocampus, but was not altered by aging in the frontal cortex. BDNF expression level increased in the hippocampus, but did not change with age in the frontal cortex. On the other hand, GDNF expression level was increased with age in the frontal cortex, but was not in the hippocampus. These results suggest that the LC noradrenergic innervations may be locally regulated by different neurotrophic factors that exert their trophic actions at different target sites.

Changes in the localization of NAP-22, a calmodulin binding membrane protein, during the development of neuronal polarity

NAP-22, a neuronal tissue-enriched acidic membrane protein, is a Ca(2+)-dependent calmodulin binding protein and has similar biochemical characteristics to GAP-43 (neuromodulin). Recent biochemical studies have demonstrated that NAP-22 localizes in the membrane raft domain with a cholesterol-dependent manner. Since the raft domain is assumed to be important to establish and/or to maintain the cell polarity, we have investigated the changes in the localization of NAP-22 during the development of the neuronal polarity in vitro and in vivo, using cultured hippocampal neurons and developing cerebellum neurons, respectively. Cultured hippocampal neurons initially extended several short processes, and at this stage NAP-22 was distributed more or less evenly among them. During the maturation of neuronal cells, NAP-22 was sorted preferentially into the axon. Throughout the developmental stages of hippocampal neurons, the localization change of NAP-22 was quite similar to that of tau, an axonal marker protein, but not to that of microtubule-associated protein-2 (MAP-2), a dendritic marker protein. Further confocal microscopic observation demonstrated the colocalization of NAP-22 and either tau or vesicle-associated protein-2 (VAMP-2). A comparison of the time course of the axonal localization of NAP-22 and GAP-43 showed that NAP-22 localization was much later than that of GAP-43. The correlation between the expression of NAP-22 and synaptogenesis in the cerebellar granular layer, particularly in the synaptic glomeruli, was also investigated. There existed many VAMP-2 positive synapses but no NAP-22 positive ones in 1-week-old cerebellum. On sections of 2-week-old cerebellum, accumulation of NAP-22 to the synaptic glomeruli was clearly observed and this accumulation became clearer during the maturation of the synaptic structure. The present results suggest the possibility that NAP-22 plays an important role in the maturation and/or the maintenance of synapses rather than in the process of the axonal outgrowth, by controlling cholesterol-dependent membrane dynamics.

Effect of AV3V lesions on Fos expression and cell size increases in magnocellular neurons of the rat hypothalamus during chronic dehydration

The effects of osmotic stimulation on Fos expression and cell size increase in the supraoptic nucleus were evaluated in intact, sham-operated, and AV3V-lesioned rats. Fos-positive neurons were found mainly in the AV3V regions and the hypothalamic magnocellular neurons in the forebrain in dehydrated intact rats. Intraperitoneal injection of hypertonic saline and chronic dehydration induced a significant increase in number of Fos-positive neurons in the supraoptic nucleus of intact and sham-operated rats. AV3V lesions completely abolished the expression of Fos in SON neurons of rats that were intraperitoneally injected with hypertonic saline and were chronically dehydrated. Chronic dehydration increased significantly cell size of the OXT and AVP magnocelluar neurons in intact and sham-operated rats. However, there was no increase in cell size of those in the AV3V-lesioned rats. These results demonstrate that neural input derived from AV3V regions plays a significant role in causing Fos expression and structural changes such as cell size increase in the hypothalamic magnocellular neurons with osmotic stimulation.