Probes for INS

Early brain development requires a tight orchestration between neural tube patterning and growth. How pattern formation and brain growth are coordinated is incompletely understood. Previously we showed that aristaless-related homeobox (ARX), a paired-like transcription factor, regulates cortical progenitor pool expansion by repressing an inhibitor of cell cycle progression. Here we show that ARX participates in establishing dorsoventral identity in the mouse forebrain. In Arx mutant mice, ventral genes, including Olig2, are ectopically expressed dorsally. Furthermore, Gli1 is upregulated, suggesting an ectopic activation of SHH signaling. We show that the ectopic Olig2 expression can be repressed by blocking SHH signaling, implicating a role for SHH signaling in Olig2 induction. We further demonstrate that the ectopic Olig2 accounts for the reduced Pax6 and Tbr2 expression, both dorsal specific genes essential for cortical progenitor cell proliferation. These data suggest a link between the control of dorsoventral identity of progenitor cells and the control of their proliferation. In summary, our data demonstrate that ARX functions in a gene regulatory network integrating normal forebrain patterning and growth, providing important insight into how mutations in ARX can disrupt multiple aspects of brain development and thus generate a wide spectrum of neurodevelopmental phenotypes observed in human patients.

The vestibular lamina (VL) forms the oral vestibule, creating a gap between the teeth, lips and cheeks. In a number of ciliopathies, formation of the vestibule is defective, leading to the creation of multiple frenula. In contrast to the neighbouring dental lamina, which forms the teeth, little is known about the genes that pattern the VL. Here, we establish a molecular signature for the usually non-odontogenic VL in mice and highlight several genes and signalling pathways that may play a role in its development. For one of these, the Sonic hedgehog (Shh) pathway, we show that co-receptors Gas1, Cdon and Boc are highly expressed in the VL and act to enhance the Shh signal from the forming incisor region. In Gas1 mutant mice, expression of Gli1 was disrupted and the VL epithelium failed to extend due to a loss of proliferation. This defect was exacerbated in Boc/Gas1 double mutants and could be phenocopied using cyclopamine in culture. Signals from the forming teeth, therefore, control development of the VL, coordinating the development of the dentition and the oral cavity.

Neurogenic lower urinary tract dysfunction typically develops after spinal cord injury. We investigated the time course and the anatomical changes in the spinal cord that may be causing lower urinary tract symptoms following injury. Rats were implanted with a bladder catheter and external urethral sphincter electromyography electrodes. Animals underwent a large, incomplete spinal transection at the T8/9 spinal level. At 1, 2-3, and 4 weeks after injury, the animals underwent urodynamic investigations. Urodynamic investigations showed detrusor overactivity and detrusor-sphincter-dyssynergia appearing over time at 3-4 weeks after injury. Lower urinary tract dysfunction was accompanied by an increase in density of C-fiber afferents in the lumbosacral dorsal horn. CRF-positive Barrington's and 5-HT-positive bulbospinal projections drastically decreased after injury, with partial compensation for the CRF fibers at 3-4 weeks. Interestingly, a decrease over time was observed in the number of GABAergic neurons in the lumbosacral dorsal horn and lamina X, and a decrease of glutamatergic cells in the dorsal horn. Detrusor overactivity and detrusor-sphincter-dyssynergia might therefore arise from a discrepancy in inhibitory/excitatory interneuron activity in the lumbosacral cord as well as input changes which develop over time after injury. The processes point to spinal plastic changes leading to malfunction of the important physiological pathway of lower urinary tract control.

The posterior intralaminar (PIL) complex of the thalamus is a multimodal nucleus that has been implicated in maternal behaviors and conspecific social behaviors in male and female rodents. Glutamatergic neurons are a major component of the PIL; however, their specific activity and role during social interactions has not yet been assessed.We used immunohistochemistry for the immediate early gene c-fos as a proxy for neuronal activity in the PIL of mice exposed to a novel social stimulus, a novel object stimulus, or no stimulus. We then used fiber photometry to record neural activity of glutamatergic neurons in the PIL in real-time during social and non-social interactions. Finally, we used inhibitory DREADDs in glutamatergic PIL neurons and tested social preference and social habituation-dishabituation.We observed significantly more c-fos-positive cells in the PIL of mice exposed to social versus object or no stimuli. Neural activity of PIL glutamatergic neurons was increased when male and female mice were engaged in social interaction with a same-sex juvenile or opposite-sex adult, but not a toy mouse. Neural activity positively correlated with social investigation bout length and negatively correlated with chronological order of bouts. Social preference was unaffected by inhibition; however, inhibiting activity of glutamatergic neurons in the PIL delayed the time it took female mice to form social habituation.Together these findings suggest that glutamatergic PIL neurons respond to social stimuli in both male and female mice and may regulate perceptual encoding of social information to facilitate recognition of social stimuli.

The central extended amygdala (CEA) and ventral pallidum (VP) are involved in diverse motivated behaviors based on rodent models. These structures are conserved, but expanded, in higher primates including human. Corticotropin releasing factor (CRF), a canonical 'stress molecule' associated with the CEA and VP circuitry across species, is dynamically regulated by stress and drugs of abuse and misuse. CRF's effects on circuits critically depend on its colocation with primary 'fast' transmitters, making this crucial for understanding circuit effects. We surveyed the distribution and colocalization of CRF-, VGluT2- (vesicular glutamate transporter 2) and VGAT- (vesicular GABA transporter) mRNA in specific subregions of the CEA and VP in young male monkeys. Although CRF-containing neurons were clustered in the lateral central bed nucleus (BSTLcn), the majority were broadly dispersed throughout other CEA subregions, and the VP. CRF/VGAT-only neurons were highest in the BSTLcn, lateral central amygdala nucleus (CeLcn), and medial central amygdala nucleus (CeM) (74%, 73%, and 85%, respectively). In contrast, lower percentages of CRF/VGAT only neurons populated the sublenticular extended amygdala (SLEAc), ventrolateral bed nucleus (BSTLP), and VP (53%, 54%, 17%, respectively), which had higher complements of CRF/VGAT/VGluT2 labeled neurons (33%, 29%, 67%, respectively). Thus, the majority of CRF-neurons at the 'poles' (BSTLcn and CeLcn/CeM) of the CEA are inhibitory, while the 'extended' BSTLP and SLEAc subregions, and neighboring VP, have a more complex profile with admixtures of 'multiplexed' excitatory CRF neurons. CRF's colocalization with its various fast transmitters is likely circuit-specific, and relevant for understanding CRF actions on specific target sites.SIGNIFICANCE STATEMENT:The central extended amygdala (CEA) and ventral pallidum (VP) regulate multiple motivated behaviors through differential downstream projections. The stress neuropeptide corticotropin releasing factor (CRF) is enriched in the CEA, and is thought to 'set the gain' through modulatory effects on co-expressed primary transmitters. Using protein and transcript assays in monkey, we found that CRF neurons are broadly and diffusely distributed in CEA and VP. CRF mRNA+ neurons colocalize with VGAT (GABA) and VGluT2 (glutamate) mRNAs in different proportions depending on subregion. CRF mRNA was also co-expressed in a subpopulation of VGAT/VGluT2 mRNA ('multiplexed') cells which were most prominent in the VP and 'pallidal'-like parts of the CEA. Heterogeneous CRF and fast transmitter co-expression across CEA/VP subregions implies circuit-specific effects.

The suprachiasmatic nucleus (SCN) drives circadian clock coherence through intercellular coupling, which is resistant to environmental perturbations. We report that primary cilia are required for intercellular coupling among SCN neurons to maintain the robustness of the internal clock in mice. Cilia in neuromedin S-producing (NMS) neurons exhibit pronounced circadian rhythmicity in abundance and length. Genetic ablation of ciliogenesis in NMS neurons enabled a rapid phase shift of the internal clock under jet-lag conditions. The circadian rhythms of individual neurons in cilia-deficient SCN slices lost their coherence after external perturbations. Rhythmic cilia changes drive oscillations of Sonic Hedgehog (Shh) signaling and clock gene expression. Inactivation of Shh signaling in NMS neurons phenocopied the effects of cilia ablation. Thus, cilia-Shh signaling in the SCN aids intercellular coupling.

This year in review on osteoarthritis biology summarizes a series of research articles published between the 2020 and 2021 Osteoarthritis Research Society International (OARSI) World Congress. Research hightlights were selected and discussed based on the new discoveries of OA's cellular molecular mechanism, anatomical signatures, potential therapeutic targets, and regenerative therapy. The recently developed potential therapeutic targets are summarized, and the research focuses on TGFβ and WNT signaling in joint tissue homeostasis, joint aging and the dynamic of synolytics in OA joint, and the roles of TRP2, LDHA, OSCAR in cartilage homeostasis and OA joints are highlighted. Subsquencially, new anatomical structures and OA features are introduced, such as synovitis-induced venous portal circulation, horiozontal fissures between cartilage and subchondral bone, the cellular derivation of osteophytes formation, OA subtypes, and subchondral remodeling and pain biology. Then, research on the possibility of tissue regeneration in OA joints are discussed; skeletal stem cells in OA cartilage regeneration, and preclinical results of regenerative therapy for meniscus tear and osteochondral tissue morphoghesis are included. At last, the clinical evidence of the importance of delivery site of bone marrow stem cells for OA treatment is discussed. These findings represent advances in our understanding of OA pathophysiology.

Coordination between the Hertwig’s Epithelial Root Sheath (HERS) and apical papilla (AP) is crucial for proper tooth root development. The Hedgehog (Hh) signaling pathway and Nfic are both involved in tooth root development, however their relationship has yet to be elucidated. Here we establish a time course of mouse molar root development by histological staining of sections and demonstrate that Hh signaling is active before and during root development in the AP and HERS using Gli1 reporter mice. The proper pattern of Hh signaling activity in the AP is critical for the proliferation of dental mesenchymal cells, because either inhibition with Hh inhibitors or constitutive activation of Hh signaling activity in transgenic mice leads to decreased proliferation in the AP and shorter roots. Moreover, Hh activity is elevated in Nfic-/- mice, a root defect model, whereas RNA sequencing and in situ hybridization show that Hh attenuator Hhip is down-regulated. ChIP and RNAscope analyses suggest that Nfic binds to the promoter region of Hhip. Treatment of Nfic-/- mice with Hh inhibitor partially restores cell proliferation, AP growth and root development. Taken together, our results demonstrate that an Nfic-Hhip-Hh signaling pathway is critical for apical papilla growth and proper root formation. This discovery provides insight into the molecular mechanisms regulating tooth root development.

Behavioral observations suggest a connection between anxiety and predator defense, but the underlying neural mechanisms remain unclear. Here we examine the role of the anterior hypothalamic nucleus (AHN), a node in the predator defense network, in anxiety-like behaviors. By in vivo recordings in male mice, we find that activity of AHN GABAergic (AHNVgat+) neurons shows individually stable increases when animals approach unfamiliar objects in an open field (OF) or when they explore the open-arm of an elevated plus-maze (EPM). Moreover, object-evoked AHN activity overlap with predator cue responses and correlate with the object and open-arm avoidance. Crucially, exploration-triggered optogenetic inhibition of AHNVgat+ neurons reduces object and open-arm avoidance. Furthermore, retrograde viral tracing identifies the ventral subiculum (vSub) of the hippocampal formation as a significant input to AHNVgat+ neurons in driving avoidance behaviors in anxiogenic situations. Thus, convergent activation of AHNVgat+ neurons serves as a shared mechanism between anxiety and predator defense to promote behavioral avoidance.

Infections induce a set of pleiotropic responses in animals, including anorexia, adipsia, lethargy and changes in temperature, collectively termed sickness behaviours1. Although these responses have been shown to be adaptive, the underlying neural mechanisms have not been elucidated2-4. Here we use of a set of unbiased methodologies to show that a specific subpopulation of neurons in the brainstem can control the diverse responses to a bacterial endotoxin (lipopolysaccharide (LPS)) that potently induces sickness behaviour. Whole-brain activity mapping revealed that subsets of neurons in the nucleus of the solitary tract (NTS) and the area postrema (AP) acutely express FOS after LPS treatment, and we found that subsequent reactivation of these specific neurons in FOS2A-iCreERT2 (also known as TRAP2) mice replicates the behavioural and thermal component of sickness. In addition, inhibition of LPS-activated neurons diminished all of the behavioural responses to LPS. Single-nucleus RNA sequencing of the NTS-AP was used to identify LPS-activated neural populations, and we found that activation of ADCYAP1+ neurons in the NTS-AP fully recapitulates the responses elicited by LPS. Furthermore, inhibition of these neurons significantly diminished the anorexia, adipsia and locomotor cessation seen after LPS injection. Together these studies map the pleiotropic effects of LPS to a neural population that is both necessary and sufficient for canonical elements of the sickness response, thus establishing a critical link between the brain and the response to infection.