Tmem45b is essential for inflammation- and tissue injury-induced mechanical pain hypersensitivity.

Persistent mechanical pain hypersensitivity associated with peripheral inflammation, surgery, trauma, and nerve injury impairs patients' quality of life and daily activity. However, the molecular mechanism and treatment are not yet fully understood. Herein, we show that chemical ablation of isolectin B4-binding (IB4+) afferents by IB4-saporin injection into sciatic nerves completely and selectively inhibited inflammation- and tissue injury-induced mechanical pain hypersensitivity while thermal and mechanical pain hypersensitivities were normal following nerve injury. To determine the molecular mechanism involving the specific types of mechanical pain hypersensitivity, we compared gene expression profiles between IB4+ neuron-ablated and control dorsal root ganglion (DRG) neurons. We identified Tmem45b as one of 12 candidate genes that were specific to somatosensory ganglia and down-regulated by IB4+ neuronal ablation. Indeed, Tmem45b was expressed predominantly in IB4+ DRG neurons, where it was selectively localized in the trans Golgi apparatus of DRG neurons but not detectable in the peripheral and central branches of DRG axons. Tmem45b expression was barely detected in the spinal cord and brain. Although Tmem45b-knockout mice showed normal responses to noxious heat and noxious mechanical stimuli under normal conditions, mechanical pain hypersensitivity was selectively impaired after inflammation and tissue incision, reproducing the pain phenotype of IB4+ sensory neuron-ablated mice. Furthermore, acute knockdown by intrathecal injection of Tmem45b small interfering RNA, either before or after inflammation induction, successfully reduced mechanical pain hypersensitivity. Thus, our study demonstrates that Tmem45b is essential for inflammation- and tissue injury-induced mechanical pain hypersensitivity and highlights Tmem45b as a therapeutic target for future treatment.

Heterogeneous localization of muscarinic cholinoceptor M1 in the salivary ducts of adult mice.

We hypothesize variation in expression and localization, along the course of the glandular tubule, of muscarinic cholinergic receptor M1 which plays as a distinct contribution, though minor in comparison with M3 receptor, in saliva secretion. Localization of the M1 receptor was examined using immunohistochemistry in three major salivary glands. Although all glandular cells were more or less M1-immunoreactive, acinar cells were weakly immunoreactive, while ductal cells exhibited substantial M1-immunoreactivity. Many ductal cells exhibited clear polarity with higher immunoreactivity in their apical/supra-nuclear domain. However, some exhibited indistinct polarity because of additional higher immunoreactivity in their basal/infra-nuclear domain. A small group of cells with intense immunoreactivity was found, mostly located in the intercalated ducts or in portions of the striated ducts close to the intercalated ducts. In immuno-electron microscopy, the immunoreactive materials were mainly in the cytoplasm including various vesicles and vacuoles. Unexpectedly, distinct immunoreactivity on apical and basal plasma membranes was infrequent in most ductal cells. The heterogeneous localization of M1-immunoreactivity along the gland tubular system is discussed in view of possible modulatory roles of the M1 receptor in saliva secretion.

Deletion of exons encoding carboxypeptidase domain of Nna1 results in Purkinje cell degeneration (pcd) phenotype.

Purkinje cell degeneration (pcd) was first identified in a spontaneous mouse mutant showing cerebellar ataxia. In addition to cerebellar Purkinje cells (PCs), retinal photoreceptors, mitral cells in the olfactory bulb, and a discrete subpopulation of thalamic neurons also degenerate in the mutant brains. The gene responsible for the pcd mutant is Nna1, also known as ATP/GTP binding protein 1 or cytosolic carboxypeptidase-like 1, which encodes a zinc carboxypeptidase protein. To investigate pathogenesis of the pcd mutation in detail, we generated a conditional Nna1 allele targeting the carboxypeptidase domain at C-terminus. After Cre recombination and heterozygous crossing, we generated Nna1 knockout (KO) mice and found that the Nna1 KO mice began to show cerebellar ataxia at postnatal day 20 (P20). Most PCs degenerated until 4-week-old, except lobule X. Activated microglia and astrocytes were also observed in the Nna1 KO cerebellum. In the mutant brain, the Nna1 mRNA level was dramatically reduced, suggesting that nonsense-mediated mRNA decay occurs in it. Since the Nna1 protein acts as a de-glutamatase on the C-terminus of α-tubulin and ß-tubulin, increased polyglutamylated tubulin was detected in the Nna1 KO cerebellum. In addition, the endoplasmic reticulum stress marker, C/EBP homologous protein (CHOP), was up-regulated in the mutant PCs. We report the generation of a functional Nna1 conditional allele and possible mechanisms of PC death in the Nna1 KO in the cerebellum. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Early postnatal stress alters the extinction of context-dependent conditioned fear in adult rats.

Fear extinction is hypothesized to be a learning process based on a new inhibitory memory. The present study was conducted to elucidate the effect of early postnatal stress on the extinction of context-dependent fear memory in adult rats, with a focus on the serotonergic system. Extinction was estimated by the expression of freezing behavior during repeated extinction trials (i.e., repeated exposure to contextual fear conditioning) on consecutive days. The decrease in fear expression was attenuated in adult rats that had been subjected to footshock (FS) at the third postnatal week (3wFS), but not in those exposed to footshock at the second postnatal week (2wFS). The decreased attenuation of freezing behavior observed in 3wFS was abolished by repeated treatment with the partial N-methyl-D-aspartate receptor agonist D-cycloserine (15 mg/kg, i.p., for 4 days), which has been shown to facilitate cue-dependent extinction. Repeated treatment with the serotonin 5-hydroxytryptamine-1A (5-HT(1A)) receptor agonist tandospirone (1 mg/kg, i.p., for 4 days) prevented the expression of freezing behavior in 3wFS, whereas diazepam treatment (1 mg/kg, i.p., for 4 days) in 3wFS did not. These results suggest that exposure to early postnatal stress at the third week is responsible for attenuating extinction of contextual fear conditioning and is mediated by a serotonergic 5-HT(1A) receptor mechanism. In other words, exposure to traumatic events during the early postnatal period might precipitate long-lasting alterations in synaptic function that underlie extinction processes of context-dependent fear memory.