Katsuya Uchida

Stimulatory effects of prostaglandin E2 on neurogenesis in the dentate gyrus of the adult rat.

Abstract Neurogenesis in the dentate gyrus of adult rodents is elicited by transient global ischemia. Cyclooxygenase (COX) -2, a rate-limiting enzyme for prostanoid synthesis, is also induced by ischemia. We recently found that the administration of a non-selective COX inhibitor to ischemic animals suppressed cell proliferation in the subgranular zone (SGZ) at the dentate gyrus of the hippocampus. To clarify whether prostaglandin E2 (PGE2) synthesis by COXs is involved in neurogenesis, sulprostone, an analogue of PGE2, was injected into the rat hippocampus. Sulprostone injection increased the number of 5-bromo-2′-deoxyuridine (BrdU)-positive cells in the SGZ. BrdU-positive cells also expressed polysialylated isoforms of neural cell adhesion molecule and neuronal nuclear antigen. These results suggest that PGE2 plays an important role in the proliferation of cells in the SGZ.

Acetylsalicylic acid reduces ischemia-induced proliferation of dentate cells in gerbils.

Transient global ischemia causes neurogenesis in the dentate gyrus of adult rodents. Ischemic insults to rodents also induce cyclooxygenase-2 (COX-2), an isoform of cyclooxygenases (COXs) and a rate-limiting enzyme for prostanoid synthesis. In the present experiments, adult Mongolian gerbils were chronically treated with acetylsalicylic acid (ASA), a non-selective COX inhibitor, and the proliferation of cells in the dentate gyrus was examined under ischemia. It was proved that BrdU-labeled cells in the dentate gyrus were significantly reduced in number following ASA treatment after 10 min global ischemia. The result strongly suggests that COX, probably COX-2, and prostanoids play an important role in the proliferation of neural cells after ischemia in gerbils.

Differential effects of forced swim-stress on the corticotropin-releasing hormone and vasopressin gene transcription in the parvocellular division of the paraventricular nucleus of rat hypothalamus.

Corticotropin-releasing hormone (CRH) and vasopressin (AVP) colocalize in the parvocellular division of the paraventricular nucleus of the hypothalamus (PVN). We examined the effect of forced swim-stress on the CRH and AVP primary transcript (hnRNA) levels in the rat PVN by semi-quantitative in situ hybridization. CRH hnRNA increased markedly following 10-min swim-stress and returned to the basal level by 2 h. AVP hnRNA in the parvocellular division of the PVN, where AVP colocalizes with CRH, did not change significantly immediately after the swim-stress, but it did increase significantly 2 h after the stress. Pretreatment with dexamethasone abolished the increases in CRH and AVP hnRNA levels after the swim-stress. The present results demonstrate the differential effects of forced swim-stress on the CRH and AVP gene transcription in the parvocellular PVN, confirming the diverse response of the dual peptide-containing system in the face of acute stressful events.

Impaired neurogenesis in the growth-retarded mouse is reversed by T3 treatment.

Triiodothyronine (T3) regulates neuronal development, and T3 deficiency impairs the formation of the central nervous system during childhood. Here, we used the hypothyroidal growth-retarded (grt) mouse to investigate whether the generation of new neurons is affected by T3 deficiency. Cell proliferation in the hippocampus, as measured by 5-bromo-2′-deoxyuridine (BrdU) incorporation, was markedly decreased in the grt mouse while the number of BrdU-positive cells in T3-treated grt mice was equivalent to that of wild type mice. The cellular differentiation rate did not differ among the experimental groups. These results suggest that T3 plays an important role during neurogenesis.

Participation of the Prolactin-Releasing Peptide-Containing Neurones in Caudal Medulla in Conveying Haemorrhagic Stress-Induced Signals to the Paraventricular Nucleus of the Hypothalamus

The prolactin‐releasing peptide (PrRP) has been proposed to be a co‐transmitter or modulator of noradrenaline (NA) because it colocalises with NA in the A1 (in the ventrolateral reticular formation) and A2 (in the nucleus of the solitary tract; NTS) cell groups in the caudal medulla. The baroreceptor signals, originating from the great vessels, are transmitted primarily to the NTS, and then part of the signals is conveyed to the hypothalamic neuroendocrine neurones via the ascending NA neurones. The hypotensive haemorrhagic paradigm was employed to examine whether the PrRP‐containing neurones in the caudal medulla participate in conveying signals to the hypothalamic neuroendocrine neurones. Among the caudal medullary A1 or A2 neurones, the majority of the PrRP‐immunoreactive (‐ir) neurones became c‐Fos‐ir at 2 h after hypotensive haemorrhage. Hypothalamic corticotrophin‐releasing hormone‐ir neurones and vasopressin‐ir neurones became c‐Fos positive in parallel with the activation of medullary PrRP‐ir neurones. After delivery of retrograde tracer fluorogold (FG) to the paraventricular nucleus of the hypothalamus (PVN), part of the PrRP/FG double‐labelled neurones in the A1 and A2 became c‐Fos‐ir after haemorrhage, demonstrating that PrRP‐ir neurones participate in conveying the haemorrhagic stress‐induced signals from the medulla to the PVN. PrRP and/or NA were microinjected directly to the PVN of conscious rats, and they presented a synergistic action on arginine vasopressin release, whereas an additive action was observed for adrenocorticotrophin release. These results suggest that the PrRP‐containing NA neurones in the caudal medulla may relay the haemorrhagic stress‐induced medullary inputs to the hypothalamic neuroendocrine neurones.

Targeting of Locus Ceruleus Noradrenergic Neurons Expressing Human Interleukin-2 Receptor α-Subunit in Transgenic Mice by a Recombinant Immunotoxin anti-Tac(Fv)-PE38: A Study for Exploring Noradrenergic Influence upon Anxiety-Like and Depression-Like Behaviors

The noradrenergic (NA) neurons in the locus ceruleus (LC) were ablated with a high degree of selectivity by immunotoxin-mediated neuronal targeting. Transgenic mice were used in which the human interleukin-2 receptor-α subunit (hIL-2Rα; Tac) is expressed under the promoter of dopamine β-hydroxylase. The recombinant immunotoxin, which is composed of the Fv fragment of an anti-hIL-2Rα monoclonal antibody fused to a truncated form of Pseudomonas exotoxin [anti-Tac(Fv)-PE38], was injected bilaterally into the LC of the mouse. As a result, the LC-NA neurons disappeared almost completely, and tissue noradrenaline was depleted in brain regions that receive NA inputs from the LC. The decrement of tissue noradrenaline content was more profound compared with that in mice treated with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), a neurotoxin capable of ablating axons originating from the LC-NA neurons. Mice treated with either the immunotoxin or DSP-4 presented increased anxiety-like behaviors; in contrast, only the immunotoxin-treated mice, and not the DSP-4-treated mice, showed increased depression-like behavior. The immunotoxin-mediated neuronal targeting may provide a means for further unraveling the links between the LC and pathological manifestations of neurological disorders.

Visualization of Corticotropin-Releasing Factor Neurons by Fluorescent Proteins in the Mouse Brain and Characterization of Labeled Neurons in the Paraventricular Nucleus of the Hypothalamus

Corticotropin-releasing factor (CRF) is the key regulator of the hypothalamic-pituitary-adrenal axis. CRF neurons cannot be distinguished morphologically from other neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) without immunostaining. Thus, we generated a knock-in mouse that expresses modified yellow fluorescent protein (Venus) in CRF neurons (CRF-Venus), and yet its expression is driven by the CRF promoter and responds to changes in the interior milieu. In CRF-Venus, Venus-expressing neurons were distributed in brain regions harboring CRF neurons, including the PVH. The majority of Venus-expressing neurons overlapped with CRF-expressing neurons in the PVH, but many neurons expressed only Venus or CRF in a physiological glucocorticoid condition. After glucocorticoid deprivation, however, Venus expression intensified, and most Venus neurons coexpressed CRF. Conversely, Venus expression was suppressed by excess glucocorticoids. Expression of copeptin, a peptide encoded within the vasopressin gene, was induced in PVH-Venus neurons by glucocorticoid deprivation and suppressed by glucocorticoid administration. Thus, Venus neurons recapitulated glucocorticoid-dependent vasopressin expression in PVH-CRF neurons. Noradrenaline increased the frequency of glutamate-dependent excitatory postsynaptic currents recorded from Venus-expressing neurons in the voltage clamp mode. In addition, the CRF-iCre knock-in mouse was crossed with a CAG-CAT-EGFP reporter mouse to yield the Tg(CAG-CAT-EGFP/wt);CRF(iCre/wt) (EGFP/CRF-iCre) mouse, in which enhanced green fluorescent protein (EGFP) is driven by the CAG promoter. EGFP was expressed more constitutively in the PVH of EGFP/CRF-iCre mice. Thus, CRF-Venus may have an advantage for monitoring dynamic changes in CRF neurons and CRF networks in different glucocorticoid states.

Glucocorticoid Dependency of Surgical Stress-Induced FosB/ΔFosB Expression in the Paraventricular and Supraoptic Nuclei of the Rat Hypothalamus

FosB is a member of the Fos family transcription factors. To determine whether FosB expression is regulated by glucocorticoids (GCs) in the hypothalamus, rats underwent sham adrenalectomy (sham‐ADX) or bilateral ADX, and FosB/ΔFosB (ΔFosB, a truncated splice variant of FosB)‐immunoreactivity (ir) was determined in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). In the parvocellular division of the PVN (paPVN) and SON, FosB/ΔFosB‐immunoreactivity (ir) increased significantly following sham‐ADX compared to naive rats, which was suppressed with either corticosterone (CORT) or dexamethasone (DEX). Following ADX, the increase in FosB/ΔFosB‐ir was much more prominent than that in the sham‐ADX group, and the ADX‐induced robust increase was suppressed by CORT or DEX, but not by aldosterone. Stressless removal of CORT from drinking water did not induce FosB/ΔFosB‐ir in either the PVN or SON, and thus the up‐regulation of FosB/ΔFosB‐ir following ADX was dependent on the systemic stress associated with surgery. In the paPVN, the majority of corticotrophin‐releasing hormone (CRH) neurones co‐expressed FosB/ΔFosB‐ir following ADX, whereas, in the magnocellular division of the PVN, vasopressin (AVP) and oxytocin (OXT) neurones did not express FosB/ΔFosB‐ir. In the SON, approximately 40% of the AVP neurones co‐expressed FosB/ΔFosB‐ir following ADX, but the OXT neurones were devoid of FosB/ΔFosB‐ir. In concert with these results obtained in vivo, DEX suppressed the forskolin‐induced increase in FosB gene promoter activity in a homologous hypothalamic cell line. These results suggest that GCs may be a potent regulator of FosB/ΔFosB expression, which is induced by stress, in hypothalamic neuroendocrine neurones.

Distribution of corticotropin-releasing factor neurons in the mouse brain: a study using corticotropin-releasing factor-modified yellow fluorescent protein knock-in mouse

We examined the morphological features of corticotropin-releasing factor (CRF) neurons in a mouse line in which modified yellow fluorescent protein (Venus) was expressed under the CRF promoter. We previously generated the CRF-Venus knock-in mouse, in which Venus is inserted into the CRF gene locus by homologous recombination. In the present study, the neomycin phosphotransferase gene (Neo), driven by the pgk-1 promoter, was deleted from the CRF-Venus mouse genome, and a CRF-Venus∆Neo mouse was generated. Venus expression is much more prominent in the CRF-Venus∆Neo mouse when compared to the CRF-Venus mouse. In addition, most Venus-expressing neurons co-express CRF mRNA. Venus-expressing neurons constitute a discrete population of neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) that project to the median eminence. Venus-expressing neurons were also found in brain regions outside the neuroendocrine PVH, including the olfactory bulb, the piriform cortex (Pir), the extended amygdala, the hippocampus, the neocortices, Barrington’s nucleus, the midbrain/pontine dorsal tegmentum, the periaqueductal gray, and the inferior olivary nucleus (IO). Venus-expressing perikarya co-expressing CRF mRNA could be observed clearly even in regions where CRF-immunoreactive perikarya could hardly be identified. We demonstrated that the CRF neurons contain glutamate in the Pir and IO, while they contain gamma-aminobutyric acid in the neocortex, the bed nucleus of the stria terminalis, the hippocampus, and the amygdala. A population of CRF neurons was demonstrated to be cholinergic in the midbrain tegmentum. The CRF-Venus∆Neo mouse may be useful for studying the structural and functional properties of CRF neurons in the mouse brain.

Quantitative analysis of thyroid-stimulating hormone messenger RNA and heterogeneous nuclear RNA in hypothyroid rats.

Thyroid-stimulating hormone (TSH) stimulates the synthesis and release of thyroid hormones including triiodothyronine (T3) and thyroxine (T4). Semiquantitative analyses using northern blot and in situ hybridization suggested that TSH gene transcription is upregulated under conditions of hypothyroidism. However, no quantitative analysis of TSH gene expression using real-time polymerase chain reaction (PCR) has been reported. In this study, we quantitated the TSHbeta messenger ribonucleic acid (mRNA) level as well as the TSHbeta heterogeneous nuclear ribonucleic acid (hnRNA) level in the anterior pituitary of hypothyroid rats, by real-time PCR using the LightCycler system. The hnRNA is the primary deoxyribonucleic acid (DNA) transcript, which reflects the transcription rate more reliably than the mRNA because of its short half-life. In the anterior pituitary of rats with methimazol-induced chronic hypothyroidism, both mRNA and hnRNA expression of TSHbeta were upregulated fourfold relative to normal rats (n=4). Our method provides a rapid and accurate measure of gene transcription. In the present report, we described a technique for accurate measurement of TSHbeta hnRNA level.