Three-dimensional topography of rat trigeminal ganglion neurons using a combination of retrograde labeling and tissue-clearing techniques.

The trigeminal nerve is the sensory afferent of the orofacial regions and divided into three major branches. Cell bodies of the trigeminal nerve lie in the trigeminal ganglion and are surrounded by satellite cells. There is a close interaction between ganglion cells via satellite cells, but the function is not fully understood. In the present study, we clarified the ganglion cells' three-dimensional (3D) localization, which is essential to understand the functions of cell-cell interactions in the trigeminal ganglion. Fast blue was injected into 12 sites of the rat orofacial regions, and ganglion cells were retrogradely labeled. The labeled trigeminal ganglia were cleared by modified 3DISCO, imaged with confocal laser-scanning microscopy, and reconstructed in 3D. Histograms of the major axes of the fast blue-positive somata revealed that the peak major axes of the cells innervating the skin/mucosa were smaller than those of cells innervating the deep structures. Ganglion cells innervating the ophthalmic, maxillary, and mandibular divisions were distributed in the anterodorsal, central, and posterolateral portions of the trigeminal ganglion, respectively, with considerable overlap in the border region. The intermingling in the distribution of ganglion cells within each division was also high, in particular, within the mandibular division. Specifically, intermingling was observed in combinations of tongue and masseter/temporal muscles, maxillary/mandibular molars and masseter/temporal muscles, and tongue and mandibular molars. Double retrograde labeling confirmed that some ganglion cells innervating these combinations were closely apposed. Our data provide essential information for understanding the function of ganglion cell-cell interactions via satellite cells.

Musclin prevents depression-like behavior in male mice by activating urocortin 2 signaling in the hypothalamus.

Introduction: Physical activity is recommended as an alternative treatment for depression. Myokines, which are secreted from skeletal muscles during physical activity, play an important role in the skeletal muscle-brain axis. Musclin, a newly discovered myokine, exerts physical endurance, however, the effects of musclin on emotional behaviors, such as depression, have not been evaluated. This study aimed to access the anti-depressive effect of musclin and clarify the connection between depression-like behavior and hypothalamic neuropeptides in mice. Methods: We measured the immobility time in the forced swim (FS) test, the time spent in open arm in the elevated-plus maze (EPM) test, the mRNA levels of hypothalamic neuropeptides, and enumerated the c-Fos-positive cells in the paraventricular nucleus (PVN), arcuate nucleus (ARC), and nucleus tractus solitarii (NTS) in mice with the intraperitoneal (i.p.) administration of musclin. Next, we evaluated the effects of a selective corticotropin-releasing factor (CRF) type 1 receptor antagonist, selective CRF type 2 receptor antagonist, melanocortin receptor (MCR) agonist, and selective melanocortin 4 receptor (MC4R) agonist on changes in behaviors induced by musclin. Finally we evaluated the antidepressant effect of musclin using mice exposed to repeated water immersion (WI) stress. Results: We found that the i.p. and i.c.v. administration of musclin decreased the immobility time and relative time in the open arms (open %) in mice and increased urocortin 2 (Ucn 2) levels but decreased proopiomelanocortin levels in the hypothalamus. The numbers of c-Fos-positive cells were increased in the PVN and NTS but decreased in the ARC of mice with i.p. administration of musclin. The c-Fos-positive cells in the PVN were also found to be Ucn 2-positive. The antidepressant and anxiogenic effects of musclin were blocked by central administration of a CRF type 2 receptor antagonist and a melanocortin 4 receptor agonist, respectively. Peripheral administration of musclin also prevented depression-like behavior and the decrease in levels of hypothalamic Ucn 2 induced by repeated WI stress. Discussion: These data identify the antidepressant effects of musclin through the activation of central Ucn 2 signaling and suggest that musclin and Ucn 2 can be new therapeutic targets and endogenous peptides mediating the muscle-brain axis.

Malocclusion impairs cognitive behavior via AgRP signaling in adolescent mice.

Introduction: Occlusal disharmony induced by deteriorating oral health conditions, such as tooth loss and decreased masticatory muscle due to sarcopenia, is one of the causes of cognitive impairment. Chewing is an essential oral function for maintaining cognitive function not only in the elderly but also in young people. Malocclusion is an occlusal disharmony that commonly occurs in children. The connection between a decline in cognitive function and malocclusion in children has been shown with chronic mouth breathing, obstructive sleep apnea syndrome, and thumb/digit sucking habits. However, the mechanism of malocclusion-induced cognitive decline is not fully understood. We recently reported an association between feeding-related neuropeptides and cognitive decline in adolescent mice with activity-based anorexia. The aim of the present study was to assess the effects of malocclusion on cognitive behavior and clarify the connection between cognitive decline and hypothalamic feeding-related neuropeptides in adolescent mice with malocclusion. Methods: Four-week-old mice were randomly assigned to the sham-operated solid diet-fed (Sham/solid), sham-operated powder diet-fed (Sham/powder), or malocclusion-operated powder diet-fed (Malocclusion/powder) group. We applied composite resin to the mandibular anterior teeth to simulate malocclusion. We evaluated cognitive behavior using a novel object recognition (NOR) test, measured hypothalamic feeding-related neuropeptide mRNA expression levels, and enumerated c-Fos-positive cells in the hypothalamus 1 month after surgery. We also evaluated the effects of central antibody administration on cognitive behavior impairment in the NOR test. Results: The NOR indices were lower and the agouti-related peptide (AgRP) mRNA levels and number of c-Fos-positive cells were higher in the malocclusion/powder group than in the other groups. The c-Fos-positive cells were also AgRP-positive. We observed that the central administration of anti-AgRP antibody significantly increased the NOR indices. Discussion: The present study suggests that elevated cerebral AgRP signaling contributes to malocclusion-induced cognitive decline in adolescents, and the suppression of AgRP signaling can be a new therapeutic target against cognitive decline in occlusal disharmony.

Development of a system to analyze oral frailty associated with Alzheimer's disease using a mouse model.

The rapid aging of the population makes the detection and prevention of frailty increasingly important. Oral frailty has been proposed as a novel frailty phenotype and is defined as a decrease in oral function coexisting with a decline in cognitive and physical functions. Oral frailty has received particular attention in relation to Alzheimer's disease (AD). However, the pathomechanisms of oral frailty related to AD remain unknown. It is assumed that the mesencephalic trigeminal nucleus (Vmes), which controls mastication, is affected by AD pathology, and as a result, masticatory function may be impaired. To investigate this possibility, we included male 3 × Tg-AD mice and their non-transgenic counterpart (NonTg) of 3-4 months of age in the present study. Immunohistochemistry revealed amyloid-ß deposition and excessive tau phosphorylation in the Vmes of 3 × Tg-AD mice. Furthermore, vesicular glutamate transporter 1-immunopositive axon varicosities, which are derived from Vmes neurons, were significantly reduced in the trigeminal motor nucleus of 3 × Tg-AD mice. To investigate whether the AD pathology observed in the Vmes affects masticatory function, we analyzed electromyography of the masseter muscle during feeding. The 3 × Tg-AD mice showed a significant delay in masticatory rhythm compared to NonTg mice. Furthermore, we developed a system to simultaneously record bite force and electromyography of masseter, and devised a new method to estimate bite force during food chewing in mice. Since the muscle activity of the masseter showed a high correlation with bite force, it could be accurately estimated from the muscle activity. The estimated bite force of 3 × Tg-AD mice eating sunflower seeds was predominantly smaller than that of NonTg mice. However, there was no difference in masseter weight or muscle fiber cross-sectional area between the two groups, suggesting that the decreased bite force and delayed mastication rhythm observed in 3 × Tg-AD mice were not due to abnormality of the masseter. In conclusion, the decreased masticatory function observed in 3 × Tg-AD mice was most likely caused by AD pathology in the Vmes. Thus, novel quantitative analyses of masticatory function using the mouse model of AD enabled a comprehensive understanding of oral frailty pathogenesis.

Neuroprotective effect of oxytocin on cognitive dysfunction, DNA damage, and intracellular chloride disturbance in young mice after cranial irradiation.

Cranial radiation therapy (CRT) is an effective treatment for brain tumors; however, it also causes brain injuries. The pediatric brain is considered especially vulnerable compared to the adult brain; thus, brain injuries caused by CRT may severely affect their quality of life. In this study, we determined the neuroprotective effects of nasal oxytocin administration following cranial radiation in mice. We investigated the cognitive behavior of mice (novel object recognition test and novel object location test), phosphorylated histone H2AX (γ-H2AX) and K+-Cl- transporter (KCC2) by immunohistochemical analysis of the hippocampal sections, and neuronal cells by immunocytochemistry after radiation and oxytocin administration. We found that the number of γ-H2AX foci was increased, and the surface signal intensity of KCC2 immunofluorescence was decreased in cells that were irradiated with X-rays (1.5 Gy, for three consecutive days) compared with cells that were not. Furthermore, using MQAE, we found that the intracellular chloride ion concentration was downregulated in oxytocin-treated cells by increasing surface KCC2 expression. These results indicate that nasal oxytocin administration after cranial irradiation attenuates cognitive dysfunction in mice and exerts multifaceted neuroprotective effects on DNA damage and maintains chloride ion concentration in neuronal cells.

Tissue-resident M2 macrophages directly contact primary sensory neurons in the sensory ganglia after nerve injury.

BACKGROUND: Macrophages in the peripheral nervous system are key players in the repair of nerve tissue and the development of neuropathic pain due to peripheral nerve injury. However, there is a lack of information on the origin and morphological features of macrophages in sensory ganglia after peripheral nerve injury, unlike those in the brain and spinal cord. We analyzed the origin and morphological features of sensory ganglionic macrophages after nerve ligation or transection using wild-type mice and mice with bone-marrow cell transplants. METHODS: After protecting the head of C57BL/6J mice with lead caps, they were irradiated and transplanted with bone-marrow-derived cells from GFP transgenic mice. The infraorbital nerve of a branch of the trigeminal nerve of wild-type mice was ligated or the infraorbital nerve of GFP-positive bone-marrow-cell-transplanted mice was transected. After immunostaining the trigeminal ganglion, the structures of the ganglionic macrophages, neurons, and satellite glial cells were analyzed using two-dimensional or three-dimensional images. RESULTS: The number of damaged neurons in the trigeminal ganglion increased from day 1 after infraorbital nerve ligation. Ganglionic macrophages proliferated from days 3 to 5. Furthermore, the numbers of macrophages increased from days 3 to 15. Bone-marrow-derived macrophages increased on day 7 after the infraorbital nerve was transected in the trigeminal ganglion of GFP-positive bone-marrow-cell-transplanted mice but most of the ganglionic macrophages were composed of tissue-resident cells. On day 7 after infraorbital nerve ligation, ganglionic macrophages increased in volume, extended their processes between the neurons and satellite glial cells, and contacted these neurons. Most of the ganglionic macrophages showed an M2 phenotype when contact was observed, and little neuronal cell death occurred. CONCLUSION: Most of the macrophages that appear after a nerve injury are tissue-resident, and these make direct contact with damaged neurons that act in a tissue-protective manner in the M2 phenotype. These results imply that tissue-resident macrophages signal to neurons directly through physical contact.

Kamishoyosan (a Japanese traditional herbal formula), which effectively reduces the aggressive biting behavior of male and female mice, and potential regulation through increase of Tph1, Tph2, and Esr2 mRNA levels.

Kamishoyosan (KSS), a Japanese traditional herbal formula, is used to treat symptoms related to the autonomic nervous system in men and women; it is especially known for improving the symptoms of irritability (e.g., bad temper and persistent anger). Although clinical and ethological studies of KSS have been conducted, its efficacy in reducing irritability remains to be validated. In the present study, male and female ddY-strain mice were isolation-reared for 8 weeks (from the third postnatal week) to induce pathologically aggressive biting behavior (ABB), which was used as an indicator of irritability. The ABB of mice toward metal rods was measured using the Aggressive Response Meter. An intraperitoneal administration of KSS (100 mg/kg) effectively reduced ABB in male and female mice at 2 h after the administration; however, this effect was canceled by prior administration of WAY-100635 [a 5-hydroxytryptoamine (5-HT)-1A receptor antagonist; 0.5 mg/kg] and bicuculline (a type-A gamma-aminobutyric acid receptor antagonist; 1.0 mg/kg). Additionally, tamoxifen, ICI-182780, and G-15 (all estrogen receptor antagonists) inhibited the action of KSS in a dose-dependent manner. Furthermore, gene expression of tryptophan hydroxylase (Tph) 1 and Tph2 were increased and 5-HT immunofluorescence was slightly increased in the dorsal raphe nucleus (DRN) of isolation-reared mice administered with KSS. Collectively, these results indicate that KSS effectively reduces ABB in isolation-reared male and female mice through stimulation of 5-HT production in the DRN. Our findings also suggest that gene expression of estrogen receptor (Esr) 2 increased in the DRN might be associated with the reduction of ABB.

The Periodontium Damage Induces Neuronal Cell Death in the Trigeminal Mesencephalic Nucleus and Neurodegeneration in the Trigeminal Motor Nucleus in C57BL/6J Mice.

Proprioception from masticatory apparatus and periodontal ligaments comes through the trigeminal mesencephalic nucleus (Vmes). We evaluated the effects of tooth loss on neurodegeneration of the Vmes and trigeminal motor nucleus (Vmo). Bilateral maxillary molars of 2-month-old C57BL/6J mice were extracted under anesthesia. Neural projections of the Vmes to the periodontium were confirmed by injecting Fluoro-Gold (FG) retrogradely into the extraction sockets, and for the anterograde labeling adeno-associated virus encoding green fluorescent protein (AAV-GFP) was applied. For immunohistochemistry, Piezo2, ATF3, Caspase 3, ChAT and TDP-43 antibodies were used. At 1 month after tooth extraction, the number of Piezo2-immunoreactive (IR) Vmes neurons were decreased significantly. ATF3-IR neurons were detected on day 5 after tooth extraction. Dead cleaved caspase-3-IR neurons were found among Vmes neurons on days 7 and 12. In the Vmo, neuronal cytoplasmic inclusions (NCIs) formation type of TDP-43 increased at 1 and 2 months after extraction. These indicate the existence of neural projections from the Vmes to the periodontium in mice and that tooth loss induces the death of Vmes neurons followed by TDP-43 pathology in the Vmo. Therefore, tooth loss induces Vmes neuronal cell death, causing Vmo neurodegeneration and presumably affecting masticatory function.

Antagonism for NPY signaling reverses cognitive behavior defects induced by activity-based anorexia in mice.

Patients with AN often express psychological symptoms such as body image distortion, cognitive biases, abnormal facial recognition, and deficits in working memory. However, the molecular mechanisms underlying the impairment of cognitive behaviors in AN remain unknown. In the present study, we measured cognitive behavior using novel object recognition (NOR) tasks and mRNA expressions in hypothalamic neuropeptides in female C57BL/6J mice with activity-based anorexia (ABA). Additionally, we evaluated the effects of antagonists with intracerebroventricular (icv) administration on the impairment of cognitive behavior in NOR tasks. Our results showed that NOR indices were lowered, subsequently increasing mRNA levels of agouti-related peptide (AgRP) and neuropeptide Y (NPY), and c-Fos- and AgRP- or NPY-positive cells in the hypothalamic arcuate nucleus in ABA mice. We also observed that icv administration of anti-NPY antiserum (2 µl), anti-AgRP antibody (0.1 µg), and Y5 receptor antagonist CPG71683 (15 nmol) significantly reversed the decreased NOR indices. Therefore, our results suggest that increased NPY and AgRP signaling in the brain might contribute to the impairment of cognitive behavior in AN.

Neurodegeneration of Trigeminal Mesencephalic Neurons by the Tooth Loss Triggers the Progression of Alzheimer's Disease in 3×Tg-AD Model Mice.

BACKGROUND: The mesencephalic trigeminal nucleus (Vmes) is not only anatomically adjacent to the locus coeruleus (LC) but is also tightly associated with the function of the LC. The LC can be the first area in which Alzheimer's disease (AD) develops, although it is unclear how LC neuronal loss occurs. OBJECTIVE: We investigated whether neuronal death in the Vmes can be spread to adjacent LC in female triple transgenic (3×Tg)-AD mice, how amyloid-ß (Aß) is involved in LC neuronal loss, and how this neurodegeneration affects cognitive function. METHODS: The molars of 3×Tg-AD mice were extracted, and the mice were reared for one week to 4 months. Immunohistochemical analysis, and spatial learning/memory assessment using the Barnes maze were carried out. RESULTS: In 4-month-old 3×Tg-AD mice, aggregated cytotoxic Aß42 was found in granules in Vmes neurons. Neuronal death in the Vmes occurred after tooth extraction, resulting in the release of cytotoxic Aß42 and an increase in CD86 immunoreactive microglia. Released Aß42 damaged the LC, in turn inducing a significant reduction in hippocampal neurons in the CA1 and CA3 regions receiving projections from the LC. Based on spatial learning/memory assessment, after the tooth extraction in the 4-month-old 3×Tg-AD mice, increased latency was observed in 5-month-old 3×Tg-AD mice 1 month after tooth extraction, which is similar increase of latency observed in control 8-month-old 3×Tg-AD mice. Measures of cognitive deficits suggested an earlier shift to dementia-like behavior after tooth extraction. CONCLUSION: These findings suggest that tooth extraction in the predementia stage can trigger the spread of neurodegeneration from the Vmes, LC, and hippocampus and accelerate the onset of dementia.

Neuropeptide Y deficiency induces anxiety-like behaviours in zebrafish (Danio rerio).

Neuropeptide Y (NPY) controls energy homeostasis including orexigenic actions in mammalians and non-mammalians. Recently, NPY has attracted attention as a mediator of emotional behaviour and psychosomatic diseases. However, its functions are not fully understood. We established npy gene-deficient (NPY-KO) zebrafish (Danio rerio) to assess the relationship between NPY and emotional behaviours. The NPY-KO zebrafish exhibited similar growth, but pomc and avp mRNA levels in the brain were higher as compared to wild-type fish. NPY-KO zebrafish exhibited several anxiety-like behaviours, such as a decrease in social interaction in mirror test and decreased locomotion in black-white test. The acute cold stress-treated NPY-KO zebrafish exhibited anxiety-like behaviours such as remaining stationary and swimming along the side of the tank in the mirror test. Moreover, expression levels of anxiety-associated genes (orx and cck) and catecholamine production (gr, mr, th1 and th2) were significantly higher in NPY-KO zebrafish than in wild-type fish. We demonstrated that NPY-KO zebrafish have an anxiety phenotype and a stress-vulnerability like NPY-KO mice, whereby orx and/or catecholamine signalling may be involved in the mechanism actions.

Helicobacter pylori Vacuolating Cytotoxin A Causes Anorexia and Anxiety via Hypothalamic Urocortin 1 in Mice.

Helicobacter pylori (Hp) infection is related to the pathogenesis of chronic gastric disorders and extragastric diseases. Here, we examined the anorexigenic and anxiogenic effects of Hp vacuolating cytotoxin A (VacA) through activation of hypothalamic urocortin1 (Ucn1). VacA was detected in the hypothalamus after peripheral administration and increased Ucn1 mRNA expression and c-Fos-positive cells in the hypothalamus but not in the nucleus tractus solitarius. c-Fos and Ucn1-double positive cells were detected. CRF1 and CRF2 receptor antagonists suppressed VacA-induced anxiety and anorexia, respectively. VacA activated single paraventricular nucleus neurons and A7r5 cells; this activation was inhibited by phospholipase C (PLC) and protein kinase C (PKC) inhibitors. VacA causes anorexia and anxiety through the intracellular PLC-PKC pathway, migrates across the blood-brain barrier, and activates the Ucn1-CRF receptor axis.

Neuropeptides and ATP signaling in the trigeminal ganglion.

Peripheral nociceptive stimuli from orofacial structures are largely transmitted by the trigeminal nerve. According to the peripheral noxious stimuli, neurons in the trigeminal ganglion (TG) produce neuropeptides such as substance P, and calcitonin-gene-related peptide, etc. Beside the production of neuropeptides, there exists unique non-synaptic interaction system between maxillary and mandibular neurons in the TG. Neurons in the TG are surrounded by satellite glial cells (SGCs), which initially receive the signal from TG neurons. These activated SGCs secrete a transmitter to activate adjacent SGCs or TG neurons, thereby amplifying the signal, for example, from mandibular neurons to maxillary neurons in the TG. Similar to the dorsal root ganglion, in the TG, microglia/macrophage-like cells (MLCs) are activated by uptake of a transmitter from TG neurons or SGCs. This communication between neurons, SGCs, and MLCs results in responses such as ectopic pain, hyperesthesia, or allodynia. The focus of this review is the cooperative interaction of the maxillary and mandibular nerves in the TG by neuropeptides, and adenosine 3'-phosphate (ATP) signaling from neurons to SGCs and MLCs. Stimulated neurons either secrete ATP by means of vesicular nucleotide transporters, or secrete neuropeptides from the neuronal cell body to mediate signal transmission.

Neonatal maternal separation delays the GABA excitatory-to-inhibitory functional switch by inhibiting KCC2 expression.

The excitatory-to-inhibitory functional switch of γ-aminobutyric acid (GABA; GABA switch), which normally occurs in the first to the second postnatal week in the hippocampus, is necessary for the development of appropriate central nervous system function. A deficit in GABAergic inhibitory function could cause excitatory/inhibitory (E/I) neuron imbalance that is found in many neurodegenerative disorders. In the present study, we examined whether neonatal stress can affect the timing of the GABA functional switch and cause disorders during adolescence. Neonatal stress was induced in C57BL/6J male mouse pups by maternal separation (MS) on postnatal days (PND) 1-21. Histological quantification of K+-Cl- co-transporter (KCC2) and Ca2+ imaging were performed to examine the timing of the GABA switch during the MS period. To evaluate the influence of neonatal MS on adolescent hippocampal function, we quantified KCC2 expression and evaluated hippocampal-related behavioral tasks at PND35-38. We showed that MS delayed the timing of the GABA switch in the hippocampus and inhibited the increase in membrane KCC2 expression, with KCC2 expression inhibition persisting until adolescence. Behavioral tests showed impaired cognition, declined attention, hyperlocomotion, and aggressive character in maternally separated mice. Taken together, our results show that neonatal stress delayed the timing of the GABA switch, which could change the E/I balance and cause neurodegenerative disorders in later life.

Dorsal and ventral parts of thalamic nucleus submedius project to different areas of rat orbitofrontal cortex: A single neuron-tracing study using virus vectors.

The rodent orbitofrontal cortex is involved in a variety of cognitive and behavioral functions that require thalamic input to be successfully expressed. Although the thalamic nucleus submedius (Sm) is a major source of afferents to the orbitofrontal cortex, thalamocortical projection from the Sm has not been fully elucidated. In the present study, we first divided the rat Sm into dorsal and ventral parts according to the distribution of vesicular glutamate transporter 2-immunoreactive varicosities, which were somatosensory afferents from the brain stem. Subsequently we investigated dendritic and axonal arborizations of individual dorsal and ventral Sm neurons by visualizing the processes with Sindbis virus vectors expressing membrane-targeted fluorescent proteins. The number of dendritic processes of ventral Sm neurons was greater than that of dorsal Sm neurons. In the cerebral cortex, all the reconstructed Sm neurons sent axons primarily to layers 2-5. Interestingly, dorsal Sm neurons formed a single axon arbor exclusively within the ventrolateral orbital area, whereas ventral Sm neurons made two axon arbors in the lateral orbital and ventral orbital areas simultaneously. The spread of each axon arbor was 500-1000 µm in diameter in the direction tangential to the cortical surface. These results indicate that the dorsal and ventral Sm comprise two distinct thalamocortical pathways. The dorsal Sm pathway relay somatosensory information to the ventrolateral orbital area and may be involved in emotional and aversive aspects of nociceptive information processing, whereas the ventral Sm pathway seems to co-activate distant orbitofrontal cortical areas, and may link their functions under certain circumstances.

Dorsal and Ventral Parts of Thalamic Nucleus Submedius Project to Different Areas of Rat Orbitofrontal Cortex: A Single Neuron-Tracing Study Using Virus Vectors.

The rodent orbitofrontal cortex is involved in a variety of cognitive and behavioral functions that require thalamic input to be successfully expressed. Although the thalamic nucleus submedius (Sm) is a major source of afferents to the orbitofrontal cortex, thalamocortical projection from the Sm has not been fully elucidated. In the present study, we first divided the rat Sm into dorsal and ventral parts according to the distribution of vesicular glutamate transporter 2-immunoreactive varicosities, which were somatosensory afferents from the brain stem. Subsequently we investigated dendritic and axonal arborizations of individual dorsal and ventral Sm neurons by visualizing the processes with Sindbis virus vectors expressing membrane-targeted fluorescent proteins. The number of dendritic processes of ventral Sm neurons was greater than that of dorsal Sm neurons. In the cerebral cortex, all the reconstructed Sm neurons sent axons primarily to layers 2-5. Interestingly, dorsal Sm neurons formed a single axon arbor exclusively within the ventrolateral orbital area, whereas ventral Sm neurons made two axon arbors in the lateral orbital and ventral orbital areas simultaneously. The spread of each axon arbor was 500-1000 µm in diameter in the direction tangential to the cortical surface. These results indicate that the dorsal and ventral Sm comprise two distinct thalamocortical pathways. The dorsal Sm pathway relay somatosensory information to the ventrolateral orbital area and may be involved in emotional and aversive aspects of nociceptive information processing, whereas the ventral Sm pathway seems to co-activate distant orbitofrontal cortical areas, and may link their functions under certain circumstances. This article is protected by copyright. All rights reserved.

Individual mediodorsal thalamic neurons project to multiple areas of the rat prefrontal cortex: A single neuron-tracing study using virus vectors.

The prefrontal cortex has an important role in a variety of cognitive and executive processes, and is generally defined by its reciprocal connections with the mediodorsal thalamic nucleus (MD). The rat MD is mainly subdivided into three segments, the medial (MDm), central (MDc), and lateral (MDl) divisions, on the basis of the cytoarchitecture and chemoarchitecture. The MD segments are known to topographically project to multiple prefrontal areas at the population level: the MDm mainly to the prelimbic, infralimbic, and agranular insular areas; the MDc to the orbital and agranular insular areas; and the MDl to the prelimbic and anterior cingulate areas. However, it is unknown whether individual MD neurons project to single or multiple prefrontal cortical areas. In the present study, we visualized individual MD neurons with Sindbis virus vectors, and reconstructed whole structures of MD neurons. While the main cortical projection targets of MDm, MDc, and MDl neurons were generally consistent with those of previous results, it was found that individual MD neurons sent their axon fibers to multiple prefrontal areas, and displayed various projection patterns in the target areas. Furthermore, the axons of single MD neurons were not homogeneously spread, but were rather distributed to form patchy axon arbors approximately 1 mm in diameter. The multiple-area projections and patchy axon arbors of single MD neurons might be able to coactivate cortical neuron groups in distant prefrontal areas simultaneously. Furthermore, considerable heterogeneity of the projection patterns is likely, to recruit the different sets of cortical neurons, and thus contributes to a variety of prefrontal functions. J. Comp. Neurol. 525:166-185, 2017. © 2016 Wiley Periodicals, Inc.

The effect of repeated stress on KCC2 and NKCC1 immunoreactivity in the hippocampus of female mice.

K(+)-Cl(-) co-transporter (KCC2) and Na(+)-K(+)-2Cl(-) co-transporter (NKCC1) are the main regulators of neuronal intracellular chloride concentration; altered expression patterns of KCC2 and NKCC1 have been reported in several neurodegenerative diseases. In this paper, we show the effect of repeated stress on KCC2, NKCC1, and serine 940 phosphorylated KCC2 (pKCC2(ser940)) immunoreactivity. The data were obtained from the hippocampus of female mice using single-plane confocal microscopy images. The mean fluorescence intensity of the perisomatic area of neurons, defined as raw fluorescence intensity (RFI) was calculated. Repeated stress (RS) resulted in a decrease in perisomatic area of immunoreactive (IR)-KCC2 and an increase of the IR-NKCC1. In addition, RS decreased perisomatic IR-pKCC2(ser940), corresponding to that of KCC2. The data in this article support the results of a previous study [1] and provide the details of immunohistological methods. Interpretation of the data in this article can be found in "Repeated stress-induced expression pattern alterations of the hippocampal chloride transporters KCC2 and NKCC1 associated with behavioral abnormalities in female mice" by Tsukahara et al. [1].

Repeated stress-induced expression pattern alterations of the hippocampal chloride transporters KCC2 and NKCC1 associated with behavioral abnormalities in female mice.

The balance of cation-chloride co-transporters, particularly KCC2 and NKCC1, is critical for GABAergic inhibitory signaling. However, KCC2/NKCC1 balance is disrupted in many neurodegenerative diseases. Moreover, correlations between chronic stress, KCC2 and NKCC1 in the hippocampus remain poorly understood. Despite the fact that emotional disorders in humans are far more prevalent in women, there have been relatively few studies about female subjects. Here we investigated behaviors and expression patterns of KCC2 and NKCC1 in the hippocampi of female mice under chronic stress. Repeated stress (RS) was induced in experimental mice by repeated forced water administration. Then, expression patterns of GABAergic signaling molecules were identified by immunohistochemical analysis and performance was assessed using several behavioral tests. The results of semi-quantitative analysis showed that RS decreased KCC2 expression and increased NKCC1 expression in membranes of granular and pyramidal cells in the hippocampus. The novel object recognition (NOR) test and sociability test revealed that RS induced cognitive and sociability deficits, whereas RS increased the time spent in the open arms of the elevated plus maze test and induced attention deficits in other tests. In summary, RS induced alterations in membrane KCC2/NKCC1 balance in the hippocampus of female mice, which may contribute to GABAergic disinhibition associated with cognitional, sociability and attention deficits.

Patterning of mammalian heterodont dentition within the upper and lower jaws.

Mammalian heterodont dentition is differentiated into incisors, canines, premolars, and molars in the mesial-distal direction, in both the upper and lower jaws. Although all the lower teeth are rooted in the mandible, the upper incisors are rooted in the premaxilla and the upper canine and the teeth behind it are in the maxilla. The present study uncovers ontogenetic backgrounds to these shared and differing mesiodistal patterns of the upper and lower dentition. To this end, we examined the dentition development of the house shrew, Suncus murinus, instead of the rodent model animals because the dentition of this primitive eutherian species includes all the tooth classes, and no toothless diastema region. In the shrew, the upper incisor-forming region extended over the medial nasal prominence and the mesial part of the maxillary prominence. Consequently, the maxillary and mandibular prominences were in a mirror-image relationship in terms of the mesiodistally differentiated tooth-forming regions and of the complementary gene expression pattern, with Bmp4 in the mesial and Fgf8 in the distal regions. This suggests shared molecular mechanisms regulating tooth class differentiation between the upper and lower jaws. However, the premaxillary bone appeared within the mesenchyme of the medial nasal prominence, but grew distally beyond the former epithelial boundary with the maxillary prominence to form, finally, the incisive (premaxillary-maxillary) suture just mesial to the canine. Therefore, the developmental locations of the upper incisors are not inconsistent with the classical osteological criterion of the upper canine by comparative odontologists.