Spatial transcriptomics reveals a role for sensory nerves in preserving cranial suture patency through modulation of BMP/TGF-β signaling

: The patterning and ossification of the mammalian skeleton requires the coordinated actions of both intrinsic bone morphogens and extrinsic neurovascular signals, which function in a temporal and spatial fashion to control mesenchymal progenitor cell (MPC) fate. Here, we show the genetic inhibition of tropomyosin receptor kinase A (TrkA) sensory nerve innervation of the developing cranium results in premature calvarial suture closure, associated with a decrease in suture MPC proliferation and increased mineralization. In vitro, axons from peripheral afferent neurons derived from dorsal root ganglions (DRGs) of wild-type mice induce MPC proliferation in a spatially restricted manner via a soluble factor when cocultured in microfluidic chambers. Comparative spatial transcriptomic analysis of the cranial sutures in vivo confirmed a positive association between sensory axons and proliferative MPCs. SpatialTime analysis across the developing suture revealed regional-specific alterations in bone morphogenetic protein (BMP) and TGF-β signaling pathway transcripts in response to TrkA inhibition. RNA sequencing of DRG cell bodies, following direct, axonal coculture with MPCs, confirmed the alterations in BMP/TGF-β signaling pathway transcripts. Among these, the BMP inhibitor follistatin-like 1 (FSTL1) replicated key features of the neural-to-bone influence, including mitogenic and anti-osteogenic effects via the inhibition of BMP/TGF-β signaling. Taken together, our results demonstrate that sensory nerve-derived signals, including FSTL1, function to coordinate cranial bone patterning by regulating MPC proliferation and differentiation in the suture mesenchyme

Interglomerular Connectivity within the Canonical and GC-D/Necklace Olfactory Subsystems.

The mammalian main olfactory system contains several subsystems that differ not only in the receptors they express and the glomerular targets they innervate within the main olfactory bulb (MOB), but also in the strategies they use to process odor information. The canonical main olfactory system employs a combinatorial coding strategy that represents odorant identity as a pattern of glomerular activity. By contrast, the "GC-D/necklace" olfactory subsystem-formed by olfactory sensory neurons expressing the receptor guanylyl cyclase GC-D and their target necklace glomeruli (NGs) encircling the caudal MOB-is critical for the detection of a small number of semiochemicals that promote the acquisition of food preferences. The formation of these socially-transmitted food preferences requires the animal to integrate information about two types of olfactory stimuli: these specialized social chemosignals and the food odors themselves. However, the neural mechanisms with which the GC-D/necklace subsystem processes this information are unclear. We used stimulus-induced increases in intrinsic fluorescence signals to map functional circuitry associated with NGs and canonical glomeruli (CGs) in the MOB. As expected, CG-associated activity spread laterally through both the glomerular and external plexiform layers associated with activated glomeruli. Activation of CGs or NGs resulted in activity spread between the two types of glomeruli; there was no evidence of preferential connectivity between individual necklace glomeruli. These results support previous anatomical findings that suggest the canonical and GC-D/necklace subsystems are functionally connected and may integrate general odor and semiochemical information in the MOB

Using Intrinsic Flavoprotein and NAD(P)H Imaging to Map Functional Circuitry in the Main Olfactory Bulb.

Neurons exhibit strong coupling of electrochemical and metabolic activity. Increases in intrinsic fluorescence from either oxidized flavoproteins or reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] in the mitochondria have been used as an indicator of neuronal activity for the functional mapping of neural circuits. However, this technique has not been used to investigate the flow of olfactory information within the circuitry of the main olfactory bulb (MOB). We found that intrinsic flavoprotein fluorescence signals induced by electrical stimulation of single glomeruli displayed biphasic responses within both the glomerular (GL) and external plexiform layers (EPL) of the MOB. Pharmacological blockers of mitochondrial activity, voltage-gated Na+ channels, or ionotropic glutamate receptors abolished stimulus-dependent flavoprotein responses. Blockade of GABAA receptors enhanced the amplitude and spatiotemporal spread of the flavoprotein signals, indicating an important role for inhibitory neurotransmission in shaping the spread of neural activity in the MOB. Stimulus-dependent spread of fluorescence across the GL and EPL displayed a spatial distribution consistent with that of individual glomerular microcircuits mapped by neuroanatomic tract tracing. These findings demonstrated the feasibility of intrinsic fluorescence imaging in the olfactory systems and provided a new tool to examine the functional circuitry of the MOB

GABA_AR antagonist enhances lateral flavoprotein signal spread in the GL and EPL.

<p>(<b>A and B</b>) Representative stimulus-dependent flavoprotein response before treatment (A) and during bath application of 10 μM GBZ (B). (top) Representative overlaid, pseudo-colored ΔF/F images at 2.5s after stimulus initiation (stimulation initiated at 0s). Representative point ROIs (regions of interest; white squares) from stimulated glomerulus, central EPL and neighboring rostral/caudal areas. ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GrL, granule cell layer; R, rostral; C, caudal. Asterisk, tip of the stimulating electrode. Targeted glomerulus indicated by dashed circle. Scale bars = 400 μm. (A and B, right) Heat map of the vertical ROI signal response over time. The late flavoprotein signal seen at the top of the image comes from the targeted glomerulus as visualized through the lateral aspect of the MOB slice. (A and B, bottom) Heat maps of the horizontal ROI signal response from the GL and EPL over time. <b>(C and D)</b> Mean signal peak of the (C) GL (F_(2,16) = 49.2, p = 0.002, n = 9) and (D) EPL (F_(2,16) = 5.4, p<0.001, n = 9) at 360 μm rostral (black), center (white), and 360 μm caudal (gray) areas as indicated in (A). <b>(E and F)</b> Following GBZ treatment, the mean FWHM lateral spread in the (E) GL (F_(2,8) = 14.0, p = 0.018, n = 9) and (F) EPL (F_(2,8) = 21.2, p = 0.009, n = 9) increased when compared to pretreated slices. Differences in the duration of the flavoprotein signal in the rostral and caudal dimensions were not quantified.</p

NAD(P)H signal response to changes in stimulation parameters.

<p><b>(A-C)</b> Mean NAD(P)H signal peak (black) and trough (gray) in the GL (solid) and EPL (dash) as a function of train stimulus (A) strength, (B) frequency, and (C) duration. (A) Increasing the stimulation strength from 10μA to 100μA while holding the other stimulation parameters constant (50 Hz, 2 s train, 100 μs pulse) resulted in increased dark phase amplitudes in the GL (R² = 0.324, F_(1,18) = 8.64, p<0.01, n = 6) and EPL (R² = 0.266, F_(1,18) = 6.51, p<0.05, n = 6) amplitudes. (B) Increasing the stimulation frequency from 10Hz to 50Hz resulted in increased dark phase amplitudes in the EPL (R² = 0.352, F_(1,14) = 7.61, p<0.02, n = 6). (C) Increasing the stimulation duration from 0.5s to 6.0s resulted in increased GL dark phase (R² = 0.520, F_(1,18) = 19.49, p<0.01, n = 6) and light phase (R² = 0.303, F_(1,18) = 7.81, p<0.02, n = 6) amplitudes. <b>(D)</b> NAD(P)H signal FDHM duration in the GL increases as a function of train stimulus duration (R² = 0.267, F_(1,18) = 6.56, p = 0.02, n = 6). <b>(E)</b> GL (R² = 0.619, F_(1,18) = 29.19, p<0.01, n = 6) and EPL (R² = 0.287, F_(1,18) = 7.23, p<0.02, n = 6) signals exhibit increases in time-to-trough as a function of train stimulation duration.</p

Flavoprotein signal profile in the glomerular and external plexiform layers.

<p><b>(A)</b> (top) Flavoprotein response at 2.5s post-stimulus initiation. ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GrL, granule cell layer; R, rostral; C, caudal. Asterisk, tip of the stimulating electrode. Targeted glomerulus indicated by dashed circle. Scale bars = 150 μm; (right) Vertical ROI heat map across the MOB layers exhibiting translaminar stimulus-dependent spread over time; (bottom) Horizontal ROI heat maps from the GL and EPL exhibiting stimulus-dependent lateral spread over time. <b>(B and C)</b> Representative ROI individual (gray) and mean (black) traces from the activated (B) glomerular and (C) EPL, indicated in (A). Line: stimulus train delivery and duration. <b>(D)</b> Mean signal amplitude of the light and dark phases between the GL and EPL areas. <b>(E)</b> Mean FDHM between the GL and EPL. <b>(F and G)</b> Flavoprotein signal (F) time-to-peak and (G) time-to-trough between the GL and EPL.</p

Lateral signal spread is dependent on interglomerular pathway.

<p><b>(A)</b> Schematic of the MOB circuitry with GL surgical microcut, and preservation of the mitral-granule-mitral pathway (red highlights). ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GrL, granule cell layer; PG, periglomerular cell; SA, short axon cell; ET, external tufted cell; MC, mitral cell; GC, granule cell. <b>(B)</b> Fluorescence image of MOB slice. Asterisk, tip of the stimulating electrode. Targeted glomerulus indicated by dashed circle. Representative point ROIs from neighboring rostral/caudal areas (squares). Scale bar = 400 μm. <b>(C)</b>. Overlaid pseudo-colored ΔF/F stimulus-dependent responses before (top) and during (bottom) GBZ treatment at 2.5s after stimulus initiation. <b>(D and E)</b> Mean signal peak of the GL (D) (F_(2,6) = 10.02, p = 0.012, Bonferroni t-test, n = 4) and EPL (E) (F_(2,6) = 9.72, p = 0.013, Bonferroni t-test, n = 6) at 360 μm rostral (black) and 360 μm caudal (gray) of the stimulated glomerulus. <b>(F and G)</b> Normalized horizontal signal traces in the GL (F) and EPL (G), before (black) and during (red) GBZ treatment. <b>(H and I)</b> Mean FWHM signal spread in the GL (H) (F_(2,5) = 7.2, p<0.001, n = 6) and EPL (I) (F_(2,5) = 0.7, p = 0.02, n = 6).</p

Quantification of lateral flavoprotein signal spread.

<p><b>(A)</b> Resting light fluorescent (top) and ΔF/F flavoprotein response image at 2.5 s post-stimulus initiation (bottom) from a representative MOB slice. ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GrL, granule cell layer; R, rostral; C, caudal. Asterisk, tip of the stimulating electrode. Targeted glomerulus indicated by dashed circle. Scale bar = 400 μm <b>(B and C)</b> Horizontal ROI heat maps of the (B) GL and (C) EPL taken from (A). Mean (black) and individual (gray) traces determined from the horizontal ROIs depicting lateral spread of signal averaged across the first 2.5 s of stimulus. The “0” indicates the central midline of the stimulated glomerulus, with the negative and positive values indicating rostral and caudal directions, respectively. <b>(D)</b> Mean full-width at half-maximum (FWHM) of the signal exhibited a symmetrical lateral spread perpendicular from the y-axis, where the FWHM signal spread is larger in the EPL compared to the GL.</p

GABA_AR antagonist enhanced flavoprotein signal response.

<p><b>(A)</b> Stimulus-dependent flavoprotein response (left) before treatment, (middle) during bath-applied 10 μM GBZ, and (right) after wash at 1.5 s post-stimulation initiation. ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GrL, granule cell layer; R, rostral; C, caudal. Asterisk, tip of the stimulating electrode. Targeted glomerulus indicated by dashed circle. GBZ treatment increased translaminar and lateral signal spread across the GL and EPL. Scale bars = 150 μm. <b>(B and C)</b> ROI traces from the (B) GL and (C) EPL from all treatment conditions indicated in (A). Traces were generated from central ROIs within the GL and EPL, as depicted in (A). Line: stimulus train delivery and duration. <b>(D)</b> Mean light phase (black) and dark phase (gray) signals from central GL (solid) and EPL (dash) ROIs exhibit increases in amplitude following GBZ treatment. <b>(E)</b> Mean light phase (black) and dark phase (gray) signal amplitudes from the rostral and the caudal ROIs of the GL (solid) and EPL (dash) as depicted in (A). Both rostral (left) and caudal (right) areas exhibited amplitude increases following GBZ treatment. <b>(F)</b> Additional treatment with 10 μM NBQX and 50 μM AP-V abolished the flavoprotein response in the GL and EPL, indicating postsynaptic activation.</p

Gabazine treatment enhances lateral signal spread following necklace glomerulus stimulation.

<p><b>(A)</b> Fluorescence image of coronal MOB slice with stimulus electrode on a single NG (asterisk). Scale bar = 400μm. <b>(B and C)</b> Gabazine (GBZ)-treated slices exhibit increased stimulus-dependent flavoprotein (B) and NAD(P)H (C) signal response and spread. <b>(D and E)</b> Mean intrinsic signal peak of the (D) GL (F_(1,2) = 18.8, p<0.004) and (E) EPL (F_(1,2) = 21.4, p<0.001) from the stimulated necklace (NG) and canonical glomeruli (CG).</p