Responsiveness of the Zurich Claudication Questionnaire in Patients with Lumbar Spinal Stenosis Undergoing Nonsurgical Treatment: A Secondary Analysis of a Randomized Controlled Trial.

STUDY DESIGN: Secondary analysis of a randomized controlled trial. OBJECTIVE: We investigated the ability to distinguish patients with lumbar spinal stenosis (LSS) who improved from those who did not after receiving nonsurgical treatment. We used the disorder-specific Zurich Claudication Questionnaire (ZCQ) satisfaction subscale as an external anchor and estimated the minimal clinically important differences (MCIDs) for the ZCQ symptom severity and physical function subscales. SUMMARY OF BACKGROUND DATA: The ZCQ satisfaction subscale effectively distinguishes surgical patients who improved from those who did not for LSS. However, its responsiveness in nonsurgical treatment has not been evaluated yet. METHODS: Eighty-four patients with LSS who received supervised physical therapy or a home exercise program were included. Patients were classified as responders or nonresponders according to the cutoff of 2.5 for the ZCQ satisfaction subscales at 6 weeks and 1 year. The external responsiveness of the ZCQ satisfaction subscale was assessed using correlational and receiver-operating characteristic (ROC) curve analyses. MCIDs for the ZCQ symptom severity and physical function subscales were estimated using anchor and distribution approaches. RESULTS: Pearson's correlation coefficients between the changes in outcomes and the ZCQ satisfaction subscale at 6 weeks and 1 year were 0.37-0.58 (symptom severity) and 0.40-0.45 (physical function subscales) (>0.30 is considered a good anchor). The area under the ROC curve values were 0.66-0.72 and 0.63-0.71 for the symptom severity and physical function subscales, respectively (>0.7 is considered acceptable). The MCIDs at 6 weeks and 1 year estimated from anchor-based approaches were -0.64 to -0.13 (symptom severity) and -0.39 to 0.10 (physical function), and those from the distribution-based approaches were -0.31 to -0.30 and -0.29 to -0.27, respectively. CONCLUSION: The findings of this study suggest that the ZCQ satisfaction subscale has less ability to distinguish patients with LSS who improved in the ZCQ symptom severity and physical function subscales from those who did not after nonsurgical treatment, compared to those after surgical treatment.

Suspected renal interstitial cell tumor causing polycythemia in two dogs.

Here we report a case series of two dogs diagnosed as renal interstitial cell tumor (RICT) accompanied by elevated serum erythropoietin level and marked polycythemia. RICT is a rare tumor in dogs, originating from renal interstitial cells. While several renal tumors such as renal lymphoma, adenocarcinoma, carcinoma, sarcoma, fibrosarcoma and nephroblastoma may cause polycythemia, polycythemia caused by RICT has never been reported in dogs. The tumors in both dogs were solitary and lied within cortex or cortico-medullary junction. Histopathology revealed spindle-shaped cells suggesting mesenchymal origin, with no mitotic figures suggesting that the tumors in both dogs were benign. Following surgical removal of the affected kidney, serum erythropoietin level and polycythemia normalized in both dogs.

Immunohistochemical localization of P2Y12 purinoceptors in the rat carotid body.

The present study investigated the localization of the adenosine 5'-diphosphate (ADP)-selective P2Y12 purinoceptors in the rat carotid body using multilabeling immunofluorescence. Punctate immunoreactive products for P2Y12 were distributed in chemoreceptive type I cells immunoreactive to vesicular nucleotide transporter (VNUT) or dopamine beta-hydroxylase, but not in S100B-immunoreactive glial-like type II cells. P2Y12 immunoreactivity was localized in cell clusters containing VNUT-immunoreactive type I cells surrounded by the perinuclear cytoplasm and cytoplasmic processes of type II cells immunoreactive for ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and NTPDase3, which hydrolyze extracellular nucleotide tri- and/or di-phosphates. In ATP bioluminescence assays using carotid bodies, the degradation of extracellular ATP was attenuated in the presence of the selective NTPDases inhibitor ARL67156, suggesting ATP-degrading activity by NTPDases in the tissue. These results suggest that ATP released from type I cells is degraded into ADP and adenosine 5'-monophosphate by NTPDases expressed in type II cells, and that ADP modulates type I cells via P2Y12 purinoceptors.

Origin and spatial population structure of Malagasy native chickens based on mitochondrial DNA.

Since Malagasy human culture became established in a multi-layered way by genetic admixture of Austronesian (Indonesia), Bantu (East Africa) and West Asian populations, the Malagasy native livestock should also have originated from these regions. While recent genetic studies revealed that Malagasy native dogs and goats were propagated from Africa, the origin of Malagasy native chickens is still controversial. Here, we conducted a phylogeographic analysis of the native chickens, focusing on the historical relationships among the Indian Ocean rim countries and based on mitochondrial D-loop sequences. Although previous work suggested that the rare Haplogroup D occurs with high frequencies in Island Southeast Asia-Pacific, East Africa and Madagascar, the major mitochondrial lineage in Malagasy populations is actually not Haplogroup D but the Sub-haplogroup C2, which is also observed in East Africa, North Africa, India and West Asia. We demonstrate that the Malagasy native chickens were propagated directly from West Asia (including India and North Africa), and not via East Africa. Furthermore, they display clear genetic differentiation within Madagascar, separated into the Highland and Lowland regions as seen in the human genomic landscape on this island. Our findings provide new insights for better understanding the intercommunion of material/non-material cultures within and around Madagascar.

Minimal clinically important differences in walking capacity and physical activity after nonsurgical treatment in patients with lumbar spinal stenosis: a secondary analysis of a randomized controlled trial.

BACKGROUND CONTEXT: Little information is available about the minimal clinically important differences (MCIDs) for objective physical measurements in people with lumbar spinal stenosis (LSS). PURPOSE: To use disorder-specific anchor and, multiple anchor-, and distribution-based approaches to determine the MCIDs for walking capacity and physical activity in patients with LSS receiving nonsurgical treatment. STUDY DESIGN/SETTING: Secondary analysis of a randomized controlled trial. PATIENT SAMPLE: Sixty-nine patients with neurogenic claudication caused by LSS receiving outpatient physical therapy. OUTCOME MEASURES: Zurich claudication questionnaire (ZCQ), self-paced walking test (SPWT), and number of daily steps measured by pedometry. METHODS: All patients completed the ZCQ, SPWT, and pedometry at the baseline and after 6 weeks. For the anchor-based approach, ZCQ symptom severity, physical function, and satisfaction subscales were used as the external anchors. Using the receiver-operating characteristic (ROC) curve, the MCIDs were determined based on the optimal cutoff points for changes in the SPWT or daily steps. For the distribution-based approach, the MCIDs were estimated from the standard deviations (SDs) of the baseline scores of the SPWT and daily steps. RESULTS: In the anchor-based approach, only the ZCQ satisfaction subscale for the SPWT (0.73), and ZCQ symptom severity subscale for daily steps (0.71) exceeded the area under the ROC curve value of 0.7, which is considered acceptable. When using these subscales as anchors, the ROC curves and optimal cutoff points indicated MCIDs of 151 m for the SPWT and 1,149 steps for daily steps. The distribution-based approach estimated the MCIDs as 280 m for the SPWT and 1,274 steps for daily steps, and had a moderate effect size (0.5 SD). CONCLUSIONS: The anchor-based approach had limited external responsiveness when the ZCQ was used as the anchor. However, this information may be helpful for interpreting walking capacity and physical activity in patients with LSS receiving nonsurgical treatment and for estimating power and sample size when planning new trials.

ADP-mediated Modulation of Intracellular Calcium Responses in Chromaffin Cells: The Role of Ectonucleoside Triphosphate Diphosphohydrolase 2 on Rat Adrenal Medulla Function.

The present study investigated the localization and the adenosine 5'-triphosphate (ATP)-degrading function of the plasma membrane-bound ecto-nucleotidase, ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2), in the rat adrenal medulla. The effect of ATP degradation product, adenosine 5'-diphosphate (ADP), on carbachol (CCh)-induced intracellular Ca2+ ([Ca2+]i) responses in adrenal chromaffin cells was examined using calcium imaging. NTPDase2-immunoreactive cells were distributed between chromaffin cells. NTPDase2-immunoreactive cells were immunoreactive for glial fibrillary acidic protein and S100B, suggesting that they were sustentacular cells. NTPDase2-immunoreactive cells surrounded chromaffin cells immunoreactive for vesicular nucleotide transporter and P2Y12 ADP-selective purinoceptors. In ATP bioluminescence assays using adrenal medullary slices, ATP was rapidly degraded and its degradation was attenuated by the NTPDase inhibitors sodium polyoxotungstate (POM-1) and 6-N, N-diethyl-d-ß,γ-dibromomethylene ATP (ARL67156). ADP inhibited CCh-induced [Ca2+]i increases of chromaffin cells in adrenal medullary slices. The inhibition of CCh-induced [Ca2+]i increases by ADP was blocked by the P2Y12 purinoceptor antagonist AZD1283. CCh-induced [Ca2+]i increases were also inhibited by the P2Y1, P2Y12, and P2Y13 purinoceptor agonist 2-methylthioadenosine diphosphate trisodium (2MeSADP), in combination with the P2Y1 purinoceptor antagonist MRS2179. These results suggest that sustentacular cells express NTPDase2 to degrade ATP released from adrenal chromaffin cells, and ADP modulates the excitability of chromaffin cells via P2Y12 purinoceptors to regulate catecholamine release during preganglionic sympathetic stimuli. (J Histochem Cytochem 72: 41-60, 2024).

Morphological Analysis of the Olfactory System of the Pig-Nosed Turtle, Carettochelys insculpta.

The turtle olfactory organ consists of the upper (UCE) and lower (LCE) chamber epithelium, projecting to the ventral and dorsal parts of the olfactory bulbs, respectively. The UCE is associated with glands, contains ciliated olfactory receptor neurons, and is assumed to detect odorants primarily in air, while the LCE is devoid of glands, contains microvillous olfactory receptor neurons, and is assumed to detect odorants primarily in water. Examining the olfactory system of the pig-nosed turtle, Carettochelys insculpta, this study found that both the upper and lower chambers of the nasal cavity were lined with sensory epithelium devoid of associated glands and contained ciliated olfactory receptor neurons. Moreover, the olfactory bulbs were not divided into dorsal and ventral parts. These results suggest that the olfactory system of the pig-nosed turtle is a single system specialized for detecting odorants in water.

In situ hybridization analysis of odorant receptor expression in the olfactory organ of the pig-nosed turtle Carettochelys insculpta.

The turtle olfactory organ consists of upper (UCE) and lower (LCE) chamber epithelium, which send axons to the ventral and dorsal portions of the olfactory bulbs, respectively. Generally, the UCE is associated with glands and contains ciliated olfactory receptor neurons (ORNs), while the LCE is devoid of glands and contains microvillous ORNs. However, the olfactory organ of the pig-nosed turtle Carettochelys insculpta appears to be a single olfactory system morphologically: there are no associated glands; ciliated ORNs are distributed throughout the olfactory organ; and the olfactory bulb is not divided into ventral and dorsal portions. In this study, we analyzed the expression of odorant receptors (ORs), the major olfactory receptors in turtles, in the pig-nosed turtle olfactory organ, via in situ hybridization. Of 690 ORs, 375 were classified as class I and 315 as class II. Some class II ORs were expressed predominantly in the posterior dorsomedial walls of the nasal cavity, while other class II ORs and all class I ORs examined were expressed in the remaining region. These results suggest that the pig-nosed turtle olfactory organ can be divided into two regions according to the expression of ORs.

Three-dimensional architecture of the subepithelial corpuscular nerve ending in the rat epiglottis reconstructed by array tomography with scanning electron microscopy.

In the rat laryngeal mucosa, subepithelial corpuscular nerve endings, called laminar nerve endings, are distributed in the epiglottis and arytenoid region and are activated by the pressure changes of the laryngeal cavity. They are also suggested to play a role in efferent regulation because of secretory vesicles in the axoplasm. In the present study, the laminar nerve endings in the rat laryngeal mucosa were analyzed by 3D reconstruction from serial ultrathin sections in addition to immunohistochemistry for synapsin 1. In the light microscopy, synapsin 1-immunoreactive flattened or bulbous terminal parts of the laminar endings were also immunoreactive with VGLUT1, and were surrounded by S100- or S100B-immunoreactive Schwann cells and vimentin-immunoreactive fibroblasts. In the electron microscopy, 3D reconstruction views showed that laminar endings were composed of flattened terminal parts sized 2-5 µm in longitudinal length, overlapping in three to five multiple layers. The terminal parts of the endings were incompletely wrapped by flat cytoplasmic processes of the Schwann cells. In addition, the fibroblast network surrounded the complex of nerve endings and the Schwann cells. Several terminal parts entered through the basement membrane into the epithelial layer and attached to the basal epithelial cells, suggesting that interaction between epithelial cells and laminar nerve endings plays an important role in sensing the pressure changes in the laryngeal cavity. Secretory vesicles were unevenly distributed throughout the terminal part of the laminar nerve endings. The secretory vesicles were frequently observed in the peripheral limb of the terminal parts. It suggests that the laminar nerve endings in the larynx may release glutamate to maintain continuous discharge during the stretching of the laryngeal mucosa.

Expression patterns of the transcription factors Fezf1, Fezf2, and Bcl11b in the olfactory organs of turtle embryos.

Many tetrapod vertebrates have two distinct olfactory organs, the olfactory epithelium (OE) and vomeronasal organ (VNO). In turtles, the olfactory organ consists of two types of sensory epithelia, the upper chamber epithelium (UCE; corresponding to the OE) and the lower chamber epithelium (LCE; corresponding to the VNO). In many turtle species, the UCE contains ciliated olfactory receptor cells (ORCs) and the LCE contains microvillous ORCs. To date, several transcription factors involved in the development of the OE and VNO have been identified in mammals. Fez family zinc-finger protein 1 and 2 (Fezf1 and 2) are expressed in the OE and VNO, respectively, of mouse embryos, and are involved in the development and maintenance of ORCs. B-cell lymphoma/leukemia 11B (Bcl11b) is expressed in the mouse embryo OE except the dorsomedial parts of the nasal cavity, and regulates the expression of odorant receptors in the ORCs. In this study, we examined the expression of Fezf1, Fezf2, and Bcl11b in the olfactory organs of embryos in three turtle species, Pelodiscus sinensis, Trachemys scripta elegans, and Centrochelys sulcata, to evaluate their involvement in the development of reptile olfactory organs. In all three turtle species, Bcl11b was expressed in the UCE, Fezf2 in the LCE, and Fezf1 in both the UCE and LCE. These results imply that the roles of the transcription factors Fezf1, Fezf2, and Bcl11b in olfactory organ development are conserved among mammals and turtles.

Expression of type 1 vomeronasal receptors in the olfactory organ of the African lungfish, Protopterus dolloi.

The vomeronasal organ is an olfactory organ found in amphibians and higher vertebrates. Type 1 vomeronasal receptors, one of the major olfactory receptors in vertebrates, are expressed in the vomeronasal organ in mammals. In amphibians and fish, they are expressed in the olfactory epithelium. The lungfish, which is the species of fish most closely related to amphibians, has a primitive vomeronasal organ: the recess epithelium. Expression of type 1 vomeronasal receptors has been reported in both the olfactory epithelium and the recess epithelium in three species of African lungfish and one species of South American lungfish. However, a previous study suggested that in the African lungfish Protopterus dolloi these receptors are expressed only in the olfactory epithelium. In this study, we identified 21 type 1 vomeronasal receptor genes in P. dolloi and examined the expression sites in the olfactory organ. In P. dolloi, most cells expressing the type 1 vomeronasal receptor were distributed in the olfactory epithelium, but a few were also found in the recess epithelium. This implies that the functions of the olfactory epithelium and the primitive vomeronasal organ are incompletely separated, and that all extant African and South American lungfish share this trait.

Lumbar paraspinal muscle morphology is associated with spinal degeneration in patients with lumbar spinal stenosis.

BACKGROUND CONTEXT: Lumbar spinal stenosis (LSS) has been reported to induce changes in paraspinal muscle morphology, but objective physical function and degenerative spine conditions are rarely assessed. PURPOSE: To identify factors associated with paraspinal muscle morphology using objective physical and degenerative spine assessments in patients with LSS. STUDY DESIGN/SETTING: Cross-sectional design. PATIENT SAMPLE: Seventy patients with neurogenic claudication caused by LSS, receiving outpatient physical therapy. OUTCOME MEASURES: Cross-sectional area (CSA) and functional CSA (FCSA) of the multifidus, erector spinae, and psoas muscles, the severity of stenosis, disc degeneration, and endplate abnormalities were evaluated by magnetic resonance imaging, as well as sagittal spinopelvic alignment by X-ray. Objective physical assessments included pedometry and claudication distance. Patient-reported outcomes included the numerical rating scale of low back pain, leg pain, and leg numbness, and the Zurich Claudication Questionnaire. METHODS: To assess the impact of LSS on paraspinal muscles, FCSA and FCSA/CSA were compared between the dominant and nondominant sides based on the patients' neurogenic symptoms, and multivariable regression analyses adjusted for age, sex, height, and weight were performed; p<.05 was considered significant. RESULTS: Seventy patients were analyzed. At one level below the maximum stenotic level, erector spinae FCSA on the dominant side was significantly lower than that on the nondominant side. In the multivariable regression analyses, at one level below the symptomatic level, disc degeneration, endplate abnormalities, and lumbar spinopelvic alignment, such as decreased lumbar lordosis and increased pelvic tilt, were negatively associated with multifidus FCSA and FCSA/CSA ratio. A significant association was observed between dural sac CSA and erector spinae FCSA. Throughout L1/2 to L5/S, disc degeneration, endplate abnormalities, and lumbar spinopelvic alignment were negatively associated with multifidus and erector spinae FCSA or FCSA/CSA. CONCLUSIONS: Lumbar paraspinal muscle asymmetry caused by LSS was observed only in erector spinae. Disc degeneration, endplate abnormalities, and lumbar spinopelvic alignment, rather than spinal stenosis and LSS symptoms, were more associated with paraspinal muscle atrophy or fat infiltration.

Immunohistochemical analysis of glutamatergic and serotonergic signaling pathways in chemosensory cell clusters in the pharynx and larynx of rats.

The present study examined cellular components and the localization of vesicular glutamate transporter (VGLUT) 1 and 2 and serotonin (5-HT) in chemosensory cell clusters in the rat pharynx and larynx. Triple immunolabeling for guanine nucleotide-binding protein G (t), subunit ⍺3 (GNAT3) and nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) with synaptotagmin-1 (Syt1) revealed NTPDase2-immunoreactive type I-like cells in addition to GNAT3-immunoreactive type II-like and Syt1-immunoreactive type III-like cells in pharyngolaryngeal chemosensory cell clusters. Therefore, these clusters appear to comprise similar cell types to those in the lingual taste buds with slight morphological modifications. An immunofluorescence analysis of VGLUT1 or VGLUT2 and GNAT3 with P2X3 purinoceptors revealed that VGLUTs co-localized to P2X3-immunoreactive spherical nerve terminals closely associated with GNAT3-immunoreactive type II-like cells. Moreover, triple immunolabeling for Syt1/synaptosomal-associated protein, 25 kDa (SNAP25) and P2X3 with VGLUT1 or VGLUT2 revealed punctate immunoreactive products for VGLUT1 and VGLUT2 within P2X3-immunoreactive flat axon terminals wrapped around Syt1/SNAP25-immunoreactive type III-like cells. The afferent nerve fibers innervating cell clusters may contain glutamate and release it by exocytosis. On the other hand, immunoreactive products for 5-HT and dopa decarboxylase were detected in Syt1-immunoreactive cells, indicating the release of 5-HT by these cells. The present results suggest that chemosensory cell clusters in the pharynx and larynx may be modulated by intrinsic glutamate and 5-HT.

In situ hybridization analysis of olfactory receptor expression in the sea turtle olfactory organ.

The olfactory organ of turtles consists of an upper chamber epithelium (UCE) with associated glands, and a lower chamber epithelium (LCE) devoid of glands. The UCE and LCE are referred to as the air-nose and the water-nose, respectively, because the UCE is thought to detect airborne odorants, while the LCE detects waterborne odorants. However, it is not clear how the two are used in the olfactory organ. Odorant receptors (ORs) are the major olfactory receptors in turtles; they are classified as class I and II ORs, distinguished by their primary structure. Class I ORs are suggested to be receptive to water-soluble ligands and class II ORs to volatile ligands. This study analyzed the expression of class I and II ORs in hatchlings of the green sea turtle, Chelonia mydas, through in situ hybridization, to determine the localization of OR-expressing cells in the olfactory organ. Class I OR-expressing cells were distributed mainly in the LCE, implying that the LCE is receptive to waterborne odorants. Class II OR-expressing cells were distributed in both the UCE and LCE, implying that the entire olfactory organ is receptive to airborne odorants. The widespread expression of class II ORs may increase opportunities for sea turtles to sense airborne odorants.

Immunohistochemical distribution of Ca2+/calmodulin-dependent protein kinase II subunits in the rat carotid body.

Carotid body (CB) activity stimulated by a lower partial oxygen pressure in rats is enhanced by exposure to chronic intermittent hypoxia. However, the mechanisms that modulate CB activity remain unclear. In the present study, the expression and distribution of one of the candidate molecules to modulate reactivity, Ca2+/calmodulin-dependent protein kinase II (CaMKII) were examined in the rat CB using reverse transcriptional polymerase chain reaction and immunofluorescence with isoform-specific antibodies. CaMKIIγ and CaMKIIδ were distributed in CB chemoreceptor cells, and exhibited intense immunoreactivity in dopamine ß-hydroxylase-positive chemoreceptor cells. CaMKIIß and CaMKIIγ were distributed in sensory nerve endings attached to chemoreceptor cells of the CB. In the petrosal ganglion, immunoreactivities for CaMKIIα, CaMKIIß, CaMKIIγ, and CaMKIIδ were detected in the perinuclear region of ganglion cells. The present results indicate that CaMKIIγ and CaMKIIδ in chemoreceptor cells and CaMKIIß and CaMKIIγ in sensory nerve endings enhanced reciprocal synaptic transmission, i.e., noradrenaline and ATP for cells to neurons and glutamate for neurons to cells.

Branched-chain amino acids plus vitamin D supplementation promote increased muscle strength following lumbar surgery for lumbar spinal stenosis: a randomized trial.

BACKGROUND CONTEXT: Adequate nutrition is essential to address the surgical stress response and mitigate loss of muscle mass, strength, and functionality in older adults with lumbar spinal stenosis (LSS). However, it is unknown whether amino acids and/or vitamin D are beneficial in older adults following lumbar surgery for LSS. PURPOSE: To evaluate whether branched-chain amino acids (BCAA) plus vitamin D supplementation could attenuate the loss of muscle mass and strength, accelerate the return of functional mobility, and improve clinical outcomes following lumbar surgery for LSS. STUDY DESIGN/SETTING: A single-center, single-blind randomized controlled trial. PATIENT SAMPLE: Eighty patients who received lumbar surgery for LSS. OUTCOME MEASURES: The primary outcome was the Zurich claudication questionnaire (ZCQ), and secondary outcomes included knee muscle strength, muscle mass measured by bioelectrical impedance analysis, gait speed and a timed up-and-go test (TUG) at 12 weeks postoperatively. Follow-up assessment was performed for the ZCQ at 52 weeks postoperatively. METHODS: Patients ingested the supplementation (BCAA group: BCAA plus vitamin D, Nonamino acid group: nonamino acid) twice daily for 3 weeks from the day after surgery, and received two hours of postoperative inpatient rehabilitation 5 times a week. RESULTS: No significant differences were observed in the mean changes on the ZCQ between the two groups at 12 weeks and 52 weeks. At 2 weeks postoperatively, the nonamino acid group showed significant deterioration compared with the BCAA group for strengths of knee extensor and knee flexor (p < .01). At 12 weeks, the BCAA group showed significant improvements in knee extensor strength and knee flexor strength compared with the nonamino acid group (p <.01). There were no significant differences in mean changes of muscle mass, maximum gait speed, and TUG at 12 weeks between two groups. CONCLUSIONS: BCAA plus vitamin D supplementation did not improve LSS-related clinical outcomes after lumbar surgery for LSS, even though muscle strength increased. Future studies should focus on long-term outcomes for muscle mass and physical function, including development of sarcopenia and frailty.

Type 1 vomeronasal receptor expression in juvenile and adult lungfish olfactory organ.

Lungfish are the most closely related fish to tetrapods. The olfactory organ of lungfish contains lamellae and abundant recesses at the base of lamellae. Based on the ultrastructural and histochemical characteristics, the lamellar olfactory epithelium (OE), covering the surface of lamellae, and the recess epithelium, contained in the recesses, are thought to correspond to the OE of teleosts and the vomeronasal organ (VNO) of tetrapods. With increasing body size, the recesses increase in number and distribution range in the olfactory organ. In tetrapods, the expression of olfactory receptors is different between the OE and VNO; for instance, the type 1 vomeronasal receptor (V1R) is expressed only in the OE in amphibians and mainly in the VNO in mammals. We recently reported that V1R-expressing cells are contained mainly in the lamellar OE but also rarely in the recess epithelium in the olfactory organ of lungfish of approximately 30 cm body length. However, it is unclear whether the distribution of V1R-expressing cells in the olfactory organ varies during development. In this study, we compared the expression of V1Rs in the olfactory organs between juveniles and adults of the African lungfish Protopterus aethiopicus and South American lungfish, Lepidosiren paradoxa. The density of V1R-expressing cells was higher in the lamellae than in the recesses in all specimens evaluated, and this pattern was more pronounced in juveniles than adults. In addition, the juveniles showed a higher density of V1R-expressing cells in the lamellae compared with the adults. Our results imply that differences in lifestyle between juveniles and adults are related to differences in the density of V1R-expressing cells in the lamellae of lungfish.

Immunolocalization of vesicular glutamate transporter 2 and exocytosis-related proteins in afferent nerve endings innervating taste buds in the rat incisive papilla.

The present study aimed to investigate the immunolocalization of vesicular glutamate transporter (VGLUT) 1 and 2, and proteins associated with exocytosis, i.e., core components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex (synaptosomal-associated protein of 25 kDa, syntaxin 1, and vesicle-associated membrane protein 2) and synaptotagmin-1 (Syt1), in incisive papillary taste buds of rats using double-indirect immunofluorescence. No VGLUT1 immunoreactivity was observed, whereas VGLUT2-immunoreactive punctate products were closely associated with guanine nucleotide-binding protein G(t) subunit α3-immmunoreactive cells in taste buds. VGLUT2 was immunolocalized in P2X3 purinoceptor-expressing afferent nerve endings. Synaptosomal-associated protein of 25 kDa, syntaxin 1, and vesicle-associated membrane protein 2 were immunolocalized in nerve endings containing VGLUT2-immunoreactive products as well as a few cells in taste buds. VGLUT2 was co-immunolocalized in some intragemmal nerve endings immunoreactive for Syt1, a calcium sensor implicated in vesicle membrane fusion. The present results suggest that afferent nerve endings innervating incisive taste buds release glutamate by exocytosis to modulate taste cell function.

Type 1 vomeronasal receptors expressed in the olfactory organs of two African lungfish, Protopterus annectens and Protopterus amphibius.

Lungfish are the fish related most closely to tetrapods. The olfactory organ of lungfish contains two distinct sensory epithelia: the lamellar olfactory epithelium (OE) and the recess epithelium (RecE). Based on their ultrastructural and histological characteristics, the lamellar OE and the RecE are considered to correspond respectively to the teleost OE and a primitive vomeronasal organ (VNO). In tetrapods, the OE and VNO have been shown to express different families of olfactory receptors; for example, in mammals, the OE expresses odorant receptors and trace amine-associated receptors, while the VNO expresses type 1 (V1Rs) and type 2 (V2Rs) vomeronasal receptors. In the present study, we examined the expression of V1Rs in the olfactory organs of two African lungfish, Protopterus annectens and Protopterus amphibius. RNA sequencing and phylogenetic analyses identified 29 V1R genes in P. annectens and 50 V1R genes in P. amphibius. Most V1Rs identified in these lungfish were classified as the tetrapod-type V1Rs initially found in tetrapods and distinct from fish-type V1Rs. In teleost, which all lack a VNO, all olfactory receptors are expressed in the OE, while in Xenopus V1Rs are expressed exclusively in the OE, and not in the VNO. In situ hybridization analysis indicated that lungfish V1Rs were expressed mainly in the lamellar OE and rarely in the RecE. These results imply that V1R expression in lungfish represents an intermediate step toward the complete segregation of V1R expression between the OE and VNO, reflecting the phylogenetic position of lungfish between teleosts and amphibians.

Distribution of proteins for synaptic release in nerve endings associated with the trachealis muscle of rats.

The immunohistochemical localization of proteins for synaptic release was examined in smooth muscle-associated sensory nerve endings using whole-mount preparations of the rat trachea. Plant-like smooth muscle-associated nerve endings with immunoreactivity for Na+-K+-ATPase, α3-subunit were identified in the trachealis muscle. VGLUT1, synapsin1, t-SNARE proteins (SNAP25 and syntaxin1), v-SNARE proteins (VAMP1 and VAMP2), and a presynaptic active zone-related protein (piccolo) were detected in the terminal parts of these endings. These results suggest that smooth muscle-associated nerve endings secrete glutamate to modulate sensorimotor functions in the lung deflation reflex.