Key points
Perioral tactile signals are transmitted via the infraorbital nerve (ION) to trigeminal nuclei. Each cerebellar Purkinje cell (PC) receives this signal as complex spikes (CSs) via a ...climbing fibre (CF) emerging from the inferior olive (IO).
The anatomical pathway from trigeminal nuclei to the IO is not clearly identified.
In the present study, we examined candidate anatomical pathways for perioral sensory signalling by analysing CSs recorded from PCs in male mice by single unit recording.
CS generation by ION stimulation was inhibited by injection of a GABAA receptor agonist, muscimol, into the contralateral mesodiencephalic junction, which is referred to as the area parafascicularis prerubralis (PfPr). The number of CSs evoked by mechanical whisker stimulation was also decreased by contralateral PfPr inhibition.
These results suggest the existence of a sensory signalling pathway to the IO via the PfPr in mice.
Perioral tactile signals are transmitted via the infraorbital nerve (ION) to trigeminal nuclei. Each cerebellar Purkinje cell receives this signal as complex spikes (CSs) via a climbing fibre emerging from the inferior olive (IO). However, the anatomical pathway from the trigeminal nuclei to the IO is not clearly identified. In the present study, we recorded CSs from Purkinje cells in male mice by single unit recording, and examined the signal transduction pathway. CSs were evoked by electrical stimulation of the ipsilateral or contralateral ION with a latency of 20–70 ms. CS generation by ipsilateral ION stimulation was inhibited by injection of a GABAA receptor agonist, muscimol, into the contralateral mesodiencephalic junction, ranging from around the fasciculus retroflexus to the interstitial nucleus of Cajal, which is referred to as the area parafascicularis prerubralis (PfPr). CSs evoked by contralateral ION stimulation were also suppressed by muscimol injection into the PfPr, although the effective area was more restricted. Furthermore, CSs evoked by mechanical stimulation around the whisker region were suppressed by PfPr inhibition. We also found that the primary motor cortex plays a role to suppress this signalling pathway. These results indicate the existence of an anatomical pathway for conducting perioral sensory signals to the IO via the PfPr.
Key points
Perioral tactile signals are transmitted via the infraorbital nerve (ION) to trigeminal nuclei. Each cerebellar Purkinje cell (PC) receives this signal as complex spikes (CSs) via a climbing fibre (CF) emerging from the inferior olive (IO).
The anatomical pathway from trigeminal nuclei to the IO is not clearly identified.
In the present study, we examined candidate anatomical pathways for perioral sensory signalling by analysing CSs recorded from PCs in male mice by single unit recording.
CS generation by ION stimulation was inhibited by injection of a GABAA receptor agonist, muscimol, into the contralateral mesodiencephalic junction, which is referred to as the area parafascicularis prerubralis (PfPr). The number of CSs evoked by mechanical whisker stimulation was also decreased by contralateral PfPr inhibition.
These results suggest the existence of a sensory signalling pathway to the IO via the PfPr in mice.
Genetic engineering of non-human primates, which are most closely related to humans, has been expected to generate ideal animal models for human genetic diseases. The common marmoset (Callithrix ...jacchus) is a non-human primate species adequate for the production of genetically modified animals because of their small body size and high reproductive capacity. Autologous embryo transfer (AET) is routinely utilized in assisted reproductive technologies for humans but not for experimental animals. This study has developed a novel method for efficiently producing mutant marmosets using AET and CRISPR/Cas9 systems. The embryos were recovered from oviducts of naturally mated females, injected with Cas9/guide RNA, and transferred into the oviducts of the donors. This AET method can reduce the time for in vitro culture of embryos to less than 30 min. This method uses an embryo donor as the recipient, thus reducing the number of animals and allowing for "Reduction" in the 3R principles of humane experimental technique. Furthermore, this method can utilize nulliparous females as well as parous females. We applied our novel method and generated the 6 marmosets carrying mutations in the fragile X mental retardation 1 (FMR1) gene using only 18 females including 14 nulliparous females.
Nest-building behavior is a widely observed innate behavior. A nest provides animals with a secure environment for parenting, sleep, feeding, reproduction, and temperature maintenance. Since animal ...infants spend their time in a nest, nest-building behavior has been generally studied as parental behaviors, and the medial preoptic area (MPOA) neurons are known to be involved in parental nest-building. However, nest-building of singly housed male mice has been less examined. Here we show that male mice spent longer time in nest-building at the early to middle dark phase and at the end of the dark phase. These two periods are followed by sleep-rich periods. When a nest was removed and fresh nest material was introduced, both male and female mice built nests at Zeitgeber time (ZT) 6, but not at ZT12. Using Fos-immunostaining combined with double in situ hybridization of Vgat and Vglut2, we found that Vgat- and Vglut2-positive cells of the lateral preoptic area (LPOA) were the only hypothalamic neuron population that exhibited a greater number of activated cells in response to fresh nest material at ZT6, compared to being naturally awake at ZT12. Fos-positive LPOA neurons were negative for estrogen receptor 1 (Esr1). Both Vgat-positive and Vglut2-positive neurons in both the LPOA and MPOA were activated at pup retrieval by male mice. Our findings suggest the possibility that GABAergic and glutamatergic neurons in the LPOA are associated with nest-building behavior in male mice.
Neural circuits are shaped by elimination of early-formed redundant synapses during postnatal development. Retrograde signaling from postsynaptic cells regulates synapse elimination. In this work, we ...identified semaphorins, a family of versatile cell recognition molecules, as retrograde signals for elimination of redundant climbing fiber to Purkinje cell synapses in developing mouse cerebellum. Knockdown of Sema3A, a secreted semaphorin, in Purkinje cells or its receptor in climbing fibers accelerated synapse elimination during postnatal day 8 (P8) to P18. Conversely, knockdown of Sema7A, a membrane-anchored semaphorin, in Purkinje cells or either of its two receptors in climbing fibers impaired synapse elimination after P15. The effect of Sema7A involves signaling by metabotropic glutamate receptor 1, a canonical pathway for climbing fiber synapse elimination. These findings define how semaphorins retrogradely regulate multiple processes of synapse elimination.
The metabotropic glutamate receptor subtype 1 (mGluR1) is a major subtype of group I mGluRs, which contributes to the development and plasticity of synapses in the brain. In the sensory thalamus, the ...thalamocortical neuron receives sensory afferents and massive feedback input from corticothalamic (CT) fibers. Notably, mGluR1 is more concentrated in CT synapses in the sensory thalamus. In the visual thalamus, mGluR1 maintains mature afferent synaptic connectivity. However, it is unknown whether mGluR1 contributes to strengthening of immature synapses or weakening of excess synapses during development and whether mGluR1 at CT synapses heterosynaptically regulates the development or refinement of afferent synapses. Here we investigated the effects of knocking out the gene encoding mGluR1 or pharmacologically blocking cortical activity on the development and maintenance of lemniscal synapses, i.e., the somatosensory afferent synapses, in the ventral posteromedial somatosensory thalamus. mGluR1-knockout (KO) mice exhibited delayed developmental strengthening as well as incomplete elimination and remodeling after maturation of lemniscal synapses. Similar to the phenotypes exhibited by mGluR1-KO mice, pharmacological blockade of somatosensory cortical activity from P12 or P21 for 1 week in wild-type mice perturbed elimination or maintenance of lemniscal synapses, respectively. The same manipulation in mGluR1-KO mice failed to induce additional abnormalities in lemniscal synaptic connectivity. These results suggest that activation of mGluR1, driven by CT input, regulates multiple stages of the development of lemniscal synapses, including strengthening, refinement, and maintenance in the somatosensory thalamus.
AlkB homolog 1 (ALKBH1) is responsible for the biogenesis of 5‐formylcytidine (f5C) on mitochondrial tRNAMet and essential for mitochondrial protein synthesis. The brain, especially the hippocampus, ...is highly susceptible to mitochondrial dysfunction; hence, the maintenance of mitochondrial activity is strongly required to prevent disorders associated with hippocampal malfunction. To study the role of ALKBH1 in the hippocampus, we generated dorsal telencephalon‐specific Alkbh1 conditional knockout (cKO) mice in inbred C57BL/6 background. These mice showed reduced activity of the respiratory chain complex, hippocampal atrophy, and CA1 pyramidal neuron abnormalities. Furthermore, performances in the fear‐conditioning and Morris water maze tests in cKO mice indicated that the hippocampal abnormalities led to impaired hippocampus‐dependent learning. These findings indicate critical roles of ALKBH1 in the hippocampus.
Studying the non-human primate (NHP) brain is required for the translation of rodent research to humans, but remains a challenge for molecular, cellular, and circuit-level analyses in the NHP brain ...due to the lack of in vitro NHP brain system. Here, we report an in vitro NHP cerebral model using marmoset (Callithrix jacchus) embryonic stem cell-derived cerebral assembloids (CAs) that recapitulate inhibitory neuron migration and cortical network activity. Cortical organoids (COs) and ganglionic eminence organoids (GEOs) were induced from cjESCs and fused to generate CAs. GEO cells expressing the inhibitory neuron marker LHX6 migrated toward the cortical side of CAs. COs developed their spontaneous neural activity from a synchronized pattern to an unsynchronized pattern as COs matured. CAs containing excitatory and inhibitory neurons showed mature neural activity with an unsynchronized pattern. The CAs represent a powerful in vitro model for studying excitatory and inhibitory neuron interactions, cortical dynamics, and their dysfunction. The marmoset assembloid system will provide an in vitro platform for the NHP neurobiology and facilitate translation into humans in neuroscience research, regenerative medicine, and drug discovery.
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•An in vitro model of the marmoset cerebral cortex was developed using ES cells.•Fusion of cortical and ganglionic eminence organoids formed cerebral assembloids.•Inhibitory neurons from ganglionic eminence regions migrated to cortical regions.•Cortical organoids showed development-dependent changes in neural activity.•Cerebral assembloids showed mature neural activity with an unsynchronized pattern.
Development of the renal medulla continues after birth to form mature renal papilla and obtain urine-concentrating ability. Here, we found that a small GTPase, Rac1, plays a critical role in the ...postnatal development of renal papilla. Mice with distal tubule-specific deletion of Rac1 reached adulthood but showed polydipsia and polyuria with an impaired ability to concentrate urine. The elongation of renal papilla that occurs in the first weeks after birth was impaired in the Rac1-deficient infants, resulting in shortening and damage of the renal papilla. Moreover, the osmoprotective signaling mediated by nuclear factor of activated T cells 5, which is a key molecule of osmotic response to osmotic stress in renal medulla, was significantly impaired in the kidneys of the Rac1-deficient infants. These results demonstrate that Rac1 plays an important role in the development of renal papilla in the postnatal period, and suggested a potential link between Rac1 and osmotic response.
Abstract
In mammals, the circadian clock consists of transcriptional and translational feedback loops through DNA
cis
-elements such as E-box and RRE. The E-box-mediated core feedback loop is ...interlocked with the RRE-mediated feedback loop, but biological significance of the RRE-mediated loop has been elusive. In this study, we established mutant cells and mice deficient for rhythmic transcription of
Bmal1
gene by deleting its upstream RRE elements and hence disrupted the RRE-mediated feedback loop. We observed apparently normal circadian rhythms in the mutant cells and mice, but a combination of mathematical modeling and experiments revealed that the circadian period and amplitude of the mutants were more susceptible to disturbance of CRY1 protein rhythm. Our findings demonstrate that the RRE-mediated feedback regulation of
Bmal1
underpins the E-box-mediated rhythm in cooperation with CRY1-dependent posttranslational regulation of BMAL1 protein, thereby conferring the perturbation-resistant oscillation and chronologically-organized output of the circadian clock.
In Purkinje cells (PCs) of the cerebellum, a single “winner” climbing fiber (CF) monopolizes proximal dendrites, whereas hundreds of thousands of parallel fibers (PFs) innervate distal dendrites, and ...both CF and PF inputs innervate a narrow intermediate domain. It is unclear how this segregated CF and PF innervation is established on PC dendrites. Through reconstruction of dendritic innervation by serial electron microscopy, we show that from postnatal day 9–15 in mice, both CF and PF innervation territories vigorously expand because of an enlargement of the region of overlapping innervation. From postnatal day 15 onwards, segregation of these territories occurs with robust shortening of the overlapping proximal region. Thus, innervation territories by the heterologous inputs are refined during the early postnatal period. Intriguingly, this transition is arrested in mutant mice lacking the type 1 metabotropic glutamate receptor (mGluR1) or protein kinase Cγ (PKCγ), resulting in the persistence of an abnormally expanded overlapping region. This arrested territory refinement is rescued by lentivirus-mediated expression of mGluR1α into mGluR1-deficient PCs. At the proximal dendrite of rescued PCs, PF synapses are eliminated and free spines emerge instead, whereas the number and density of CF synapses are unchanged. Because the mGluR1-PKCγ signaling pathway is also essential for the late-phase of CF synapse elimination, this signaling pathway promotes the two key features of excitatory synaptic wiring in PCs, namely CF monoinnervation by eliminating redundant CF synapses from the soma, and segregated territories of CF and PF innervation by eliminating competing PF synapses from proximal dendrites.
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