Paraventricular nucleus neurons: important regulators of respiratory movement in mice with chronic intermittent hypoxia

Figures

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  Figure 1.. Increased expression of c-Fos in CIH mice. A: Schematic diagram of the experimental protocol: Mice were placed in an oxygen concentration control system for 8 weeks to establish a chronic intermittent hypoxia(CIH) mouse model. C-Fos staining was performed in brain regions including the paraventricular hypothalamic nucleus (PVN). B–I. Micrographs showing c-Fos-positive neurons (green) in four brain regions: PVN, Locus coeruleus (LC), pre-Bötzinger complex (preBötC), and nucleus tractus solitarius (NTS) from the 21% O2 group (n = 5) and CIH group (n = 5). J–N. Quantitative analyses comparing the density of c-Fos-positive neurons (normalized as neurons/mm2) per brain region between groups. There was a statistically significant increase in neuronal density was observed in all CIH-group regions compared to controls (21% O2 group) (****p < 0.0001, unpaired t-test), with the highest density in the PVN (****p < 0.0001, one-way ANOVA). Annotations: ‘4V’ denotes the 4th ventricle; ‘3V’ the 3rd ventricle; ‘AP’ the area postrema; ‘10N’ the dorsal motor nucleus of the vagus; ‘CC’ the central canal.

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  Figure 2.. Analysis of projection relationship between PVN and preBötC. A. Schematic diagram of the experimental approach: An anterograde non-transsynaptic tracing virus (AAV2/9-mCherry) was injected into the paraventricular hypothalamic nucleus (PVN), while a retrograde non-transsynaptic tracing virus (AAV2/R-EYFP) was injected into the ipsilateral pre-Bötzinger complex (preBötC). Brains were collected by cardiac perfusion after four weeks of viral transduction (n = 6). B. Schematic illustration of non-synaptic viral tracing: Anterograde tracing: projection from neuronal somata to nerve fiber terminals; retrograde tracing: projection from nerve fiber terminals to neuronal somata. C. Confocal micrographs (single channe): Anterogradely labeled red neuronal somata in the PVN; D. Single channel: Green retrogradely labeled neuronal somata in the PVN projecting from the preBötC. E. Co-localization in the PVN: Red anterogradely labeled neuronal somata with green retrogradely labeled neuronal somata from the preBötC; F. Magnified view of co-labeled neuronal somata in PVN (white arrows). G. Single channel: Retrograde green nerve fibers in the preBötC. H. Single channel: Red anterograde fibers from…

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  Figure 3.. Connection between the preBötC and the spinal phrenic nucleus. A. Schematic diagram of the experimental setup: An anterograde non-transsynaptic virus (AAV2/9-EYFP) was injected into the pre-Bötzinger complex (preBötC). Four weeks post-infection, the paraventricular hypothalamic nucleus (PVN) and the spinal phrenic nucleus were examined histologically (n = 5). B. Micrographs show no obvious anterogradely labeled EYFP-positive nerve fibers in the PVN. C–F. Photomicrographs reveal anterogradely labeled EYFP-positive nerve fibers in the phrenic motoneurons of lamina 9 (Ph9) region at C3–C6 spinal cord levels. Anatomical details of the C3–C5 Ph9 region: Left panels: Ph9 boundaries delineated by red dashed lines; Right panels: Magnified views of nerve fibers within Ph9(C, D, E). C6 Ph9 region: Left: Magnified nerve fibers in Ph9; Right: Ph9 contour outlined with red dashed line(F).

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  Figure 4.. Projections of the PVN to the spinal phrenic nucleus. A-B. Schematic diagram of the experimental setup: An anterograde non-transsynaptic virus (AAV2/9-mCherry) was injected into the paraventricular hypothalamic nucleus (PVN). Four weeks post-infection, histological examination of the spinal phrenic nucleus was performed (n = 5). C. Photomicrographs show: verification of PVN injection sites. D-G. Photomicrographs show no detectable red nerve fibers in the phrenic motoneurons of lamina 9 (Ph9) region of the C3∼C6 spinal cord.

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  Figure 5.. Impact of chemogenetic activation of PVN neurons on respiratory parameters in CIH mice. A. Schematic illustration of the Cre/LoxP expression strategy combined with non-transsynaptic viral tracing to validate the PVN-preBötC neural circuit. B-C. Experimental protocol: Chronic intermittent hypoxia(CIH) mice received bilateral injections of DIO-hM3Dq (n = 8) or DIO-mCherry (n = 8) into the paraventricular hypothalamic nucleus (PVN), and Retro-Cre + CTB555 (5:1 mixture) into the pre-Bötzinger complex (preBötC). Four weeks post-injection, mice were administered clozapine-N-oxide (CNO) followed by whole-body plethysmography (WBP) to measure respiratory frequency (RF), tidal volume (TV), and minute ventilation (MV). D. After 24 hours, experimental mice received saline injections followed by repeat respiratory testing. E. Schematic of bilateral PVN and preBötC virus injections. F-H. Chemogenetic stimulation effects on respiratory parameters: CNO activation in hM3Dq-expressing mice significantly increased RF and MV , without TV changes. No notable alterations in respiratory parameters were observed in the parallel-control (mCherry) and self-control (Saline) groups. (Statistical…

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  Figure 6.. Effects of chemogenetic inhibition of PVN neurons on basal ventilation in CIH mice. A. Schematic illustration of the Cre/LoxP expression strategy combined with non-transsynaptic viral tracing to validate the PVN-preBötC neural circuit. B-C. Experimental protocol: Chronic intermittent hypoxia(CIH) mice received bilateral paraventricular hypothalamic nucleus (PVN) injections of DIO-hM4Di (n = 8) or DIO-mCherry (n = 8), and pre-Bötzinger complex (preBötC) injections of Retro-Cre + CTB555 (5:1 mixture). After 4-week viral expression, mice underwent intraperitoneal clozapine-N-oxide (CNO) administration followed by whole-body plethysmography (WBP) to measure respiratory frequency (RF), tidal volume (TV), and minute ventilation (MV). D. At 24 hours post-CNO, experimental mice received saline injections with repeat WBP testing. E. Schematic of bilateral PVN and preBötC virus injections. F-H. Chemogenetic inhibition effects: CNO activation in hM4Di-expressing mice significantly decreased RF and MV, without TV changes. No alterations occurred in Parallel-control (mCherry) and self-control (Saline) groups. (Statistical significance indicated as *p < 0.05, **p < 0.005, ***p < 0.001, ****p <…

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  Figure 7.. Impact of chemogenetic activation of PVN neurons on blood gas levels and on c-Fos expression in preBötC of CIH mice. A. Experimental protocol: Chronic intermittent hypoxia (CIH) mice received bilateral paraventricular hypothalamic nucleus (PVN) injections of DIO-hM3Dq and pre-Bötzinger complex (preBötC) injections of Retro-Cre/CTB555 (5:1 mixture). Four weeks post-injection, mice received intraperitoneal saline or clozapine-N-oxide (CNO) (n = 8/group). B. Arterial blood gas analysis 60 minutes post-injection. C. 90 minutes post-injection, c-Fos staining and quantification on serial coronal sections of PVN and preBötC. D. No significant differences in arterial blood pH, PaO2, or oxygen saturation between hM3Dq-Saline (control, n = 8) and hM3Dq-CNO (experimental, n = 8) groups (p &gt; 0.05, unpaired t-test). E.a-b: Confocal micrographs showing bilateral preBötC injection sites and DIO-mCherry expression in PVN via PVN-preBötC circuit. c: c-Fos+/mCherry + co-localization in PVN of CNO-activated mice. d: mCherry + neurons in PVN (single channel). e: c-Fos + neurons in PVN (single channel). f: Magnified view of co-labeled neurons. g: Higher proportion of c-Fos+/mCherry + co-labeled…