Emiko Senba

Chronic stress, as well as acute stress, reduces BDNF mRNA expression in the rat hippocampus but less robustly.

Daily restraint for 3 weeks was shown to atrophy dendrites of hippocampal pyramidal neurons in rats. Brain-derived neurotrophic factor (BDNF), which maintains neuronal survival and morphology, has been shown to decrease in response to acute stress. Plasma glucocorticoid (GC) and serotonergic projections from the raphe nuclei play major roles in reducing BDNF synthesis in the hippocampus. We investigated BDNF mRNA levels there, together with plasma GC levels, GC receptors in the hippocampus/hypothalamus and 5-HT synthesizing enzyme, tryptophan hydroxylase in the raphe nuclei, in animals chronically stressed for 1-3 weeks, using in situ hybridization and immunohistochemistry. In these animals, BDNF mRNA levels were significantly decreased in the hippocampus after 6 h of restraint, but the ability of restraint to reduce BDNF synthesis seemed less robust than that seen in acute stress models. HPA axis response to stress in these animals assessed by plasma GC levels was delayed and sustained, and the GC receptor in the paraventricular hypothalamic nucleus was increased at 1 week. Tryptophan hydroxylase immunoreactivity was increased in the median raphe nucleus at 2-3 weeks. Repetitive stress-induced reduction of BDNF may partly contribute to the neuronal atrophy/death and reduction of hippocampal volume observed both in animals and humans suffering chronic stress and/or depression.

5-HT2A receptor subtype in the peripheral branch of sensory fibers is involved in the potentiation of inflammatory pain in rats

&NA; One of the major serotonin (5‐HT) receptor subtypes expressed in the rat dorsal root ganglion (DRG) neurons is the 5‐HT2A receptor. We have previously shown that 5‐HT2A receptors in the peripheral sensory terminals are responsible for 5‐HT‐induced pain and hyperalgesia. In the present study, we characterized neurons expressing 5‐HT2A receptors in the rat DRG neurons by means of in situ hybridization, immunohistochemistry, reverse transcription‐polymerase chain reaction (RT‐PCR) and behavioral tests. In situ hybridization on consecutive sections revealed that 5‐HT2A receptor mRNA is colocalized with calcitonin‐gene related peptide (CGRP) mRNA (100/104; 96.2%) but not with c‐Ret mRNA (1/115; 0.9%). Signals for 5‐HT2A receptor mRNA were found in 9.4±2.2% of normal DRG (L5) neurons, most of which were small to medium in size. Four days of complete Freunds adjuvant‐induced inflammation of the hindpaw doubled the incidence of 5‐HT2A receptor mRNA‐expressing neurons to 19.3±2.8%. The level of 5‐HT2A receptor mRNA in DRGs of normal and various pathological conditions was then determined by RT‐PCR. The level was up‐regulated by peripheral inflammation, but not by axotomy or chronic constriction of the peripheral nerve. Systemic administration of 5‐HT2A receptor antagonist (Sarpogrelate HCI) produced analgesic effects on thermal hyperalgesia caused by peripheral inflammation, but failed to attenuate thermal hyperalgesia in chronic constriction injury model. These findings suggest that 5‐HT2A receptors are mainly expressed in CGRP‐synthesizing small DRG neurons and may be involved in the potentiation of inflammatory pain in the periphery.

Complete overlap of interleukin-31 receptor A and oncostatin M receptor β in the adult dorsal root ganglia with distinct developmental expression patterns

Interleukin-31 receptor A (IL-31RA) is a newly identified type I cytokine receptor, that is related to gp130, the common receptor of the interleukin (IL) -6 family cytokines. Recent studies have shown that IL-31RA forms a functional receptor complex for IL-31 together with the beta subunit of oncostatin M receptor (OSMRbeta). However, little is known about the target cells of IL-31 because it remains unclear which types of cells express IL-31RA. In our previous reports, we demonstrated that OSMRbeta is expressed in a subset of small-sized nociceptive neurons of adult dorsal root ganglia (DRGs). In the present study, we investigated the IL-31RA expression in the adult and developing DRGs. From a northern blot analysis and in situ hybridization histochemistry, IL-31RA mRNA was found to be expressed in the adult DRGs. According to reverse-transcriptase polymerase chain reaction, IL-31RA mRNA was detected in the DRGs and trigeminal ganglia, while no expression of IL-31RA mRNA was observed in the CNS. Double immunofluorescence staining revealed IL-31RA to be expressed in a subset of small-sized neurons, all of which colocalized with OSMRbeta. In addition, the expression of IL-31 RA was detected in afferent fibers in the spinal cord and the dermis of the skin. We also found that the developmental expression pattern of IL-31RA was different from that of OSMRbeta; IL31RA-positive neurons in DRGs first appeared at postnatal day (PN) 10 and reached the adult level at PN14, whereas OSMRbeta-positive neurons were observed at PN0 for the first time. We previously demonstrated OSMRbeta-expressing neurons to decrease, however, they were not found to disappear in oncostatin M (OSM) -deficient mice. These findings suggest that IL-31 and OSM may thus have redundant functions in the development of OSMRbeta-expressing neurons.

The effects of acute and chronic restraint stress on activation of ERK in the rostral ventromedial medulla and locus coeruleus

&NA; Extracellular signal‐regulated kinase (ERK) is a key molecule in numerous cellular and physiological processes in the CNS. Exposure to stressors causes substantial effects on the perception and response to pain. The rostral ventromedial medulla (RVM) and the locus coeruleus (LC) play crucial roles in descending pain modulation system. In the present study, the activation of ERK in the RVM and the LC in rats following acute and chronic restraint stress was examined in order to characterize the mechanisms underlying stress induced analgesic and hyperalgesic responses. Rats were stressed by restraint 6 h daily for 3 weeks. The acute and chronic restraint stresses produced analgesic and hyperalgesic reactions, respectively, to thermal stimuli applied to the tail. The phospho‐ERK‐immunoreactive (p‐ERK‐IR) neurons were observed in the nucleus raphe magnus (NRM), nucleus reticularis gigantocellularis pars alpha (GiA) and LC. In the RVM, the number of p‐ERK‐IR neurons per section in the 3‐week restraint rats (14.3±1.2) was significantly higher than that in the control rats (8.9±0.7) [P<0.01]. About 75% of p‐ERK‐IR neurons in the RVM in the 3‐week restraint rats were serotonergic neurons. Protein levels of tryptophan hydroxylase were significantly increased in the RVM region in the 3‐week restraint rats. On the other hand, the chronic restraint stress significantly decreased p‐ERK‐IR in the LC [P<0.05]. These findings suggest that chronic restraint stress‐induced activation of ERK in the RVM and the suppression in the LC may be involved in the modulation of the pain threshold by chronic stress.

Induction of brain-derived neurotrophic factor by leptin in the ventromedial hypothalamus

Leptin, an adipocyte-derived hormone, reduces food intake by regulating orexigenic and anorexigenic factors in the hypothalamus. Although brain-derived neurotrophic factor is an important anorexigenic factor in the hypothalamus, little is known about the regulation of brain-derived neurotrophic factor expression by leptin in the hypothalamus. In the present study, we examined the effect of leptin on the expression of brain-derived neurotrophic factor in the hypothalamus. I.V. administration of leptin (10 microg/g) led to the increase in the expression of brain-derived neurotrophic factor mRNA, which was observed in the dorsomedial part of the ventromedial hypothalamic nucleus. The increased expression of brain-derived neurotrophic factor mRNA was detected in phosphorylated signal transducer and activator of transcription 3-positive neurons, suggesting that leptin induced brain-derived neurotrophic factor expression in neurons of the dorsomedial part of the ventromedial hypothalamic nucleus. In addition, the expression of brain-derived neurotrophic factor was increased at the protein level in the ventromedial hypothalamic nucleus of leptin-injected mice. Interestingly, brain-derived neurotrophic factor-positive fibers also increased in the ventromedial hypothalamic nucleus and dorsomedial hypothalamic nucleus of leptin-injected mice, which were in close apposition to tyrosine kinase receptor B-immunoreactive neurons and colocalized with synaptophysin, a marker of presynaptic terminals. These results suggest that leptin induces brain-derived neurotrophic factor expression in the dorsomedial part of the ventromedial hypothalamic nucleus and brain-derived neurotrophic factor may exert as anorexigenic factors possibly through the activation of tyrosine kinase receptor B in the ventromedial hypothalamic nucleus and dorsomedial hypothalamic nucleus.

The forkhead transcription factors, Foxp1 and Foxp2, identify different subpopulations of projection neurons in the mouse cerebral cortex.

Foxp1 and Foxp2, which belong to the forkhead transcription factor family, are expressed in the developing and adult mouse brain, including the striatum, thalamus, and cerebral cortex. Recent reports suggest that FOXP1 and FOXP2 are involved in the development of speech and language in humans. Although both Foxp1 and Foxp2 are expressed in the neural circuits that mediate speech and language, including the corticostriatal circuit, the functions of Foxp1 and Foxp2 in the cerebral cortex remain unclear. To gain insight into the functions of Foxp1 and Foxp2 in the cerebral cortex, we characterized Foxp1- and Foxp2-expressing cells in postnatal and adult mice using immunohistochemistry. In adult mice, Foxp1 was expressed in neurons of layers III-VIa in the neocortex, whereas the expression of Foxp2 was restricted to dopamine and cyclic adenosine 3,5-monophosphate-regulated phosphoprotein, 32 kDa (DARPP-32)(+) neurons of layer VI. In addition, Foxp2 was weakly expressed in the neurons of layer V of the motor cortex and hindlimb and forelimb regions of the primary somatosensory cortex. Both Foxp1 and Foxp2 were expressed in the ionotropic glutamate receptor (GluR) 2/3(+) neurons, and colocalized with none of GluR1, gamma-aminobutyric acid, calbindin, and parvalbumin, indicating that expression of Foxp1 and Foxp2 is restricted to projection neurons. During the postnatal stages, Foxp1 was predominantly expressed in Satb2(+)/Ctip2(-) corticocortical projection neurons of layers III-V and in Tbr1(+) corticothalamic projection neurons of layer VIa. Although Foxp2 was also expressed in Tbr1(+) corticothalamic projection neurons of layer VI, no colocalization of Foxp1 with Foxp2 was observed from postnatal day (P) 0 to P7. These findings suggest that Foxp1 and Foxp2 may be involved in the development of different cortical projection neurons during the early postnatal stages in addition to the establishment and maintenance of different cortical circuits from the late postnatal stage to adulthood.

Foxp1 gene expression in projection neurons of the mouse striatum

The developmental processes of maturation in the CNS are the result of specific events including mitogenesis, differentiation, and cell death which occur in a precise spatial and temporal manner. It has been reported that many transcription factors, including forkhead transcription factors, play a key role in these processes. First, we examined the expression pattern of the forkhead transcription factor Foxp1 in the adult CNS. Foxp1 was highly expressed in the striatum and moderately in the cerebral cortex, CA1/2 subfields of the hippocampus, and several thalamic nuclei. In situ hybridization combined with immunohistochemistry in the striatum of adult mice revealed that Foxp1 mRNA was detected in a subset of projection neurons, not in interneurons. In addition, the expression of Foxp1 mRNA was observed in the developing basal ganglia with the exception of the globus pallidus. Thus, Foxp1 mRNA was expressed in a subset of striatal projection neurons, probably the matrix neurons. The expression pattern of Foxp1 mRNA suggests that Foxp1 may play a role in the development and formation of a circuit in the basal ganglia, which is involving the matrix neurons.

Fasting-Induced Activation of Mitogen-Activated Protein Kinases (ERK/p38) in the Mouse Hypothalamus

Activity‐dependent changes in neuronal plasticity depend critically on gene regulation. To understand how fasting‐induced stimulation leads to gene regulation through intracellular signalling pathways, we investigated the effect of fasting on activation of the mitogen‐activated protein kinase (MAPK) family, the extracellular signal‐regulated kinase (ERK) and the p38 MAPK (p38) in mouse hypothalamus. In the hypothalamic arcuate nucleus, phosphorylation of ERK significantly increased during fasting, spatially coincident with phosphorylation of cAMP response element binding protein (CREB), induction of c‐Fos, and expression of neuropeptide Y (NPY). In the paraventricular nucleus (PVN) of fasted mice, activation of p38 in addition to ERK was also observed. In the arcuate nucleus of ob/ob mice, phosphorylations of ERK and CREB were decreased during fasting, whereas the expression of NPY was increased. In the PVN, increased activation of p38 was observed in spite of decreased activation of ERK. These results suggest that ERK and p38 are differentially activated by fasting in distinct regions of the hypothalamus depending on the condition of energy balance.

Expression of oncostatin M receptor β in a specific subset of nociceptive sensory neurons

Oncostatin M belongs to the interleukin‐6 family of cytokines and acts as a multifunctional cytokine during murine embryogenesis and in inflammatory reactions. Although it has been demonstrated that oncostatin M has biological activities on many types of cells, including hepatocytes, dermal fibroblasts and endothelial cells, the roles of oncostatin M in the murine peripheral nervous system remain unclear. Here, we investigated the expression of specific β‐subunit of oncostatin M receptor in the dorsal root ganglia of adult mice. In the adult dorsal root ganglia, β‐subunit of oncostatin M receptor was exclusively expressed in small‐sized neurons. Approximately 13% of total dorsal root ganglia neurons in mice contained β‐subunit of oncostatin M receptor. The double‐immunofluorescence method revealed that approximately 28% of β‐subunit of oncostatin M receptor‐positive neurons contained TrkA immunoreactivity, 63% expressed Ret immunoreactivity and 58% bound isolectin B4. No neuropeptides, including substance P and calcitonin gene‐related peptide, were contained in the neurons. In addition, all β‐subunit of oncostatin M receptor‐positive neurons expressed both vanilloid receptor 1 and P2X3 purinergic receptor. These neurons projected to the inner portion of lamina II in the dorsal horn of spinal cord and the dermis of skin. Seven days after sciatic nerve axotomy, the expression of β‐subunit of oncostatin M receptor was down‐regulated in the lumbar dorsal root ganglia of the injured side. Our study demonstrated that β‐subunit of oncostatin M receptor was expressed in both cell bodies and processes of nonpeptidergic nociceptive neurons in adult mice, suggesting that oncostatin M may affect the nociceptive function of the neurons through the modulation of vanilloid receptor 1 and P2X3 expression.

TRPV2, a capsaicin receptor homologue, is expressed predominantly in the neurotrophin-3-dependent subpopulation of primary sensory neurons

TRPV2, a member of transient receptor potential ion channels, responds to high-threshold noxious heat, but neither to capsaicin nor to proton. Although TRPV2 is expressed in medium- to large-sized dorsal root ganglion (DRG) neurons with myelinated fibers in adult rodents, little is known about the neurotrophin dependence of TRPV2-positive neurons in the developing and adult DRGs of mice. In the present study, using immunohistochemistry, we found that TRPV2 was first expressed in DRG neurons at embryonic day (E) 11.5, when neither TRPV1 nor TRPM8 was detected yet. Double-immunofluorescence staining revealed that tyrosine kinase receptor C (TrkC) was expressed in most of TRPV2-positive DRG neurons at E11.5 and E13.5. In addition, the percentage of TRPV2-positive neurons in the total DRG neurons at E13.5 reached the same as that of adulthood. In adult DRGs, TrkC and Ret were expressed in 68% and 25% of TRPV2-positive neurons, respectively. These results suggest that TRPV2 is expressed predominantly in the NT-3-dependent subpopulation of DRG neurons throughout development and in adult mice.