Presynaptically Released Cbln1 Induces Dynamic Axonal Structural Changes by Interacting with GluD2 during Cerebellar Synapse Formation

SummaryDifferentiation of pre- and postsynaptic sites is coordinated by reciprocal interaction across synaptic clefts. At parallel fiber (PF)-Purkinje cell (PC) synapses, dendritic spines are autonomously formed without PF influence. However, little is known about how presynaptic structural changes are induced and how they lead to differentiation of mature synapses. Here, we show that Cbln1 released from PFs induces dynamic structural changes in PFs by a mechanism that depends on postsynaptic glutamate receptor delta2 (GluD2) and presynaptic neurexin (Nrx). Time-lapse imaging in organotypic culture and ultrastructural analyses in vivo revealed that Nrx-Cbln1-GluD2 signaling induces PF protrusions that often formed circular structures and encapsulated PC spines. Such structural changes in PFs were associated with the accumulation of synaptic vesicles and GluD2, leading to formation of mature synapses. Thus, PF protrusions triggered by Nrx-Cbln1-GluD2 signaling may promote bidirectional maturation of PF-PC synapses by a positive feedback mechanism

Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution changes

ホルマリン漬けから着想した小分子可視化法 --医薬品開発効率化につながる新たな戦略--. 京都大学プレスリリース. 2022-12-05.Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/excretion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein, we report a new method termed “fixation-driven chemical cross-linking of exogenous ligands (FixEL), ” which traps and images exogenously administered molecules of interest (MOIs) in complex tissues. This method relies on protein-MOI interactions and chemical cross-linking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOIs such as the scaffold of PET tracer derivatives or drug-like small molecules

Optogenetic Control of Synaptic AMPA Receptor Endocytosis Reveals Roles of LTD in Motor Learning

Long-term depression (LTD) of AMPA-type glutamate receptor (AMPA receptor)-mediated synaptic transmission has been proposed as a cellular substrate for learning and memory. Although activity-induced AMPA receptor endocytosis is believed to underlie LTD, it remains largely unclear whether LTD and AMPA receptor endocytosis at specific synapses are causally linked to learning and memory in vivo. Here we developed a new optogenetic tool, termed PhotonSABER, which enabled the temporal, spatial, and cell-type-specific control of AMPA receptor endocytosis at active synapses, while the basal synaptic properties and other forms of synaptic plasticity were unaffected. We found that fiberoptic illumination to Purkinje cells expressing PhotonSABER in vivo inhibited cerebellar motor learning during adaptation of the horizontal optokinetic response and vestibulo-ocular reflex, as well as synaptic AMPA receptor decrease in the flocculus. Our results demonstrate that LTD and AMPA receptor endocytosis at specific neuronal circuits were directly responsible for motor learning in vivo

The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum

金沢大学医薬保健研究域保健学系Endocannabinoids work as retrograde messengers and contribute to short-term and long-term modulation of synaptic transmission via presynaptic cannabinoid receptors. It is generally accepted that the CB1 cannabinoid receptor (CB1) mediates the effects of endocannabinoid in inhibitory synapses. For excitatory synapses, however, contributions of CB1, "CB3," and some other unidentified receptors have been suggested. In the present study we used electrophysiological and immunohistochemical techniques and examined the type(s) of cannabinoid receptor functioning at hippocampal and cerebellar excitatory synapses. Our electrophysiological data clearly demonstrate the predominant contribution of CB1. At hippocampal excitatory synapses on pyramidal neurons the cannabinoid-induced synaptic suppression was reversed by a CB1-specific antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl- 1H-pyrazole-3-carboxamide (AM251), and was absent in CB1 knock-out mice. At climbing fiber (CF) and parallel fiber (PF) synapses on cerebellar Purkinje cells the cannabinoid-dependent suppression was absent in CB1 knock-out mice. The presence of CB1 at presynaptic terminals was confirmed by immunohistochemical experiments with specific antibodies against CB1. In immunoelectron microscopy the densities of CB1-positive signals in hippocampal excitatory terminals and cerebellar PF terminals were much lower than in inhibitory terminals but were clearly higher than the background. Along the long axis of PFs, the CB1 was localized at a much higher density on the perisynaptic membrane than on the extrasynaptic and synaptic regions. In contrast, CB1 density was low in CF terminals and was not significantly higher than the background. Despite the discrepancy between the electrophysiological and morphological data for CB1 expression on CFs, these results collectively indicate that CB1 is responsible for cannabinoid-dependent suppression of excitatory transmission in the hippocampus and cerebellum. Copyright © 2006 Society for Neuroscience

An Invertebrate Hyperglycemic Model for the Identification of Anti-Diabetic Drugs

The number of individuals diagnosed with type 2 diabetes mellitus, which is caused by insulin resistance and/or abnormal insulin secretion, is increasing worldwide, creating a strong demand for the development of more effective anti-diabetic drugs. However, animal-based screening for anti-diabetic compounds requires sacrifice of a large number of diabetic animals, which presents issues in terms of animal welfare. Here, we established a method for evaluating the anti-diabetic effects of compounds using an invertebrate animal, the silkworm, Bombyx mori. Sugar levels in silkworm hemolymph increased immediately after feeding silkworms a high glucose-containing diet, resulting in impaired growth. Human insulin and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, decreased the hemolymph sugar levels of the hyperglycemic silkworms and restored growth. Treatment of the isolated fat body with human insulin in an in vitro culture system increased total sugar in the fat body and stimulated Akt phosphorylation. These responses were inhibited by wortmannin, an inhibitor of phosphoinositide 3 kinase. Moreover, AICAR stimulated AMPK phosphorylation in the silkworm fat body. Administration of aminoguanidine, a Maillard reaction inhibitor, repressed the accumulation of Maillard reaction products (advanced glycation end-products; AGEs) in the hyperglycemic silkworms and restored growth, suggesting that the growth defect of hyperglycemic silkworms is caused by AGE accumulation in the hemolymph. Furthermore, we identified galactose as a hypoglycemic compound in jiou, an herbal medicine for diabetes, by monitoring its hypoglycemic activity in hyperglycemic silkworms. These results suggest that the hyperglycemic silkworm model is useful for identifying anti-diabetic drugs that show therapeutic effects in mammals

Development and validation of questionnaires for eating‐related distress among advanced cancer patients and families

Background: Eating‐related distress (ERD) is one type of psychosocial distress among advanced cancer patients and family caregivers. Its alleviation is a key issue in palliative care; however, there is no validated tool for measuring ERD. Methods: The purpose of this study was to validate tools for evaluating ERD among patients and family caregivers. The study consisted of a development and validation/retest phase. In the development phase, we made preliminary questionnaires for patients and family caregivers. After face validity and content validity, we performed an exploratory factor analysis and discussed the final adoption of items. In the validation/retest phase, we examined factor validity with an exploratory factor analysis. We calculated Pearson's correlation coefficients between the questionnaire for patients, the Functional Assessment of Anorexia/Cachexia Therapy Anorexia Cachexia Subscale (FAACT ACS) and the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire‐Cachexia 24 (EORTC QLQ‐CAX24) and Pearson's correlation coefficients between the questionnaire for family caregivers and the Caregiver Quality of Life Index‐Cancer (CQOLC) for concurrent validity. We calculated Cronbach's alpha coefficients (Cronbach's alpha) and intraclass correlation coefficients (ICCs) for internal consistency and test–retest reliability. We performed the Mann–Whitney U test between the questionnaires and cancer cachexia based on criteria from the international consensus for known‐group validity. Results: In the development phase, 162 pairs of patients and family caregivers were asked to participate, and 144 patients and 106 family caregivers responded. In the validation/retest phase, 333 pairs of patients and family caregivers were asked to participate, and 234 patients and 152 family caregivers responded. Overall, 183 patients and 112 family caregivers did the retest. Seven conceptual groups were extracted for the ERD among patients and family caregivers, respectively. Patient factors 1–7 correlated with FAACT ACS (r = −0.63, −0.43, −0.55, −0.40, −0.38, −0.54, −0.38, respectively) and EORTC QLQ‐CAX24 (r = 0.58, 0.40, 0.60, 0.49, 0.38, 0.59, 0.42, respectively). Family factors 1–7 correlated with CQOLC (r = −0.34, −0.30, −0.37, −0.37, −0.46, −0.42, −0.40, respectively). The values of Cronbach's alpha and ICC of each factor and all factors of patients ranged from 0.84 to 0.96 and 0.67 to 0.83, respectively. Those of each factor and all factors of family caregivers ranged from 0.84 to 0.96 and 0.63 to 0.84, respectively. The cachexia group of patients had significantly higher scores than the non‐cachexia group for each factor and all factors. Conclusions: Newly developed tools for measuring ERD experienced by advanced cancer patients and family caregivers have been validated

Segmental and complementary expression of L-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase and neutral amino acid transporter ASCT1 in the mouse kidney.

3-Phosphoglycerate dehydrogenase (Phgdh) is the initial step enzyme in the phosphorylated pathway of L-serine biosynthesis. We have previously revealed in the brain that Phgdh is preferentially expressed in glial cells, but not in neurons, and that glia-borne L-serine exerts strong neurotrophic actions to neuronal survive, differentiation, and development. To investigate whether such an L-serine-meditated intercellular relationship is constructed in peripheral organs and tissues, we examined the kidney, which is one of the organs with the highest expression of Phgdh mRNA in the body. We found that Phgdh was distributed highly in the renal papilla and inner layer of the outer zone and moderately in the cortex, whereas it was almost negative in the outer layer of the outer zone. This heterogeneous distribution was due to selective expression in distinct tubular segments, i.e., the Bowman's capsule, proximal tubule, and thin limbs of the Henle's loop. Interestingly, neutral amino acid transporter ASCT1, which preferentially transports alanine, serine, cysteine, and threonine, was selectively expressed in Phgdh-negative tubular segments, i.e., the distal tubule and collecting duct. Therefore, either Phgdh or ASCT1 is provided to each segment of renal tubules, suggesting that metabolic interplay mediated by L-serine biosynthesis and supply may exist in the kidney too