The molecular components of store-operated Ca2+ influx channels (SOCs) in proliferative and migratory vascular smooth muscle cells (VSMCs) are quite intricate with many channels contributing to SOCs. They include the Ca2+-selective Orai1 and members of the transient receptor potential canonical (TRPC) channels, which are activated by the endoplasmic reticulum Ca2+ sensor STIM1. The scaffolding protein Homer assembles SOC complexes, but its role in VSMCs is not well understood. Here, we asked whether these SOC components and Homer1 are present in the same complex in VSMCs and how Homer1 contributes to VSMC SOCs, proliferation, and migration leading to neointima formation. Homer1 expression levels are upregulated in balloon-injured vs. uninjured VSMCs. Coimmunoprecipitation assays revealed the presence and interaction of all SOC components in the injured VSMCs, where Homer1 interacts with Orai1 and various TRPC channels. Accordingly, knockdown of Homer1 in cultured VSMCs partially inhibited SOCs, VSMC migration, and VSMC proliferation. Neointimal area was reduced after treatment with an adeno-associated viral vector expressing a short hairpin RNA against Homer1 mRNA (AAV-shHomer1). These findings stress the role of multiple Ca2+ influx channels in VSMCs and are the first to show the role of Homer proteins in VSMCs and its importance in neointima formation.
Chronic intermittent hypoxia (CIH), an animal model of sleep apnea, has been shown to alter the activity of second order chemoreceptor neurons in the caudal nucleus of the solitary tract (cNTS). While numerous studies have focused on excitatory plasticity, few studies have explored CIH-induced plasticity impacting inhibitory inputs to NTS neurons, and the roles of GABAergic and glycinergic inputs on heightened cNTS excitability following CIH are unknown. In addition, changes in astrocyte function may play a role in cNTS plasticity responses to CIH. This study tested the effects of a 7-day CIH protocol on miniature inhibitory postsynaptic currents (mIPSCs) in cNTS neurons receiving chemoreceptor afferents. Normoxia treated rats primarily displayed GABA mIPSCs while CIH treated rats exhibited a shift toward combined GABA/glycine mediated mIPSCs. CIH increased glycinergic mIPSC amplitude and area. This shift was not observed in dorsal motor nucleus of the vagus neurons or cNTS cells from females. Immunohistochemistry showed that strengthened glycinergic mIPSCs were associated with increased glycine receptor protein and were dependent on receptor trafficking in CIH treated rats. Additionally, CIH altered astrocyte morphology in the cNTS and inactivation of astrocytes following CIH reduced glycine receptor mediated mIPSC frequency and overall mIPSC amplitude. In cNTS, CIH produced changes in glycine signaling that appear to reflect increased trafficking of glycine receptors to the cell membrane. Increased glycine signaling in cNTS associated with CIH also appears to be dependent on astrocytes. Additional studies will be needed to determine how CIH influences glycine receptor expression and astrocyte function in cNTS.
Studies have shown that the median preoptic nucleus (MnPO) contributes to hypertension associated with chronic intermittent hypoxia (CIH), a model of the hypoxemia associated with sleep apnea. Virally mediated lesions of MnPO neurons prevent the development of a sustained hypertension associated with CIH. To gain more insight into the properties of excitatory transmission to MnPO neurons develops in CIH, whole cell patch clamp recordings were from MnPO neurons brain slices from rats exposed to 7 days of CIH (6 minute cycles; 3 minutes of 21% oxygen room air pumped in, 90 seconds of nitrogen pumped in to lower the chamber O2 to 10% oxygen, and then 90 seconds of maintenance at 10% oxygen) for 8 h during the light phase or were normoxic controls. Slices were perfused with oxygenates artificial cerebrospinal fluid and the MnPO was visually identified. Miniature excitatory postsynaptic currents (mEPSCs) were recorded in the presences of 25 µm SR95531 (GABAA antagonist) and 1 µm TTX (block Na+ channel‐mediated action potentials) using a conventional whole cell patch clamp configuration. The mEPSCs were collected over a 4 min period and amplitude and frequency were compared between cells obtained from slice prepared from normoxic control rats (NORM) or rats exposed to CIH. CIH was associated with a significant increase in mean amplitude (CIH 17.15 ± 0.65 pA, n=19 cells; NORM (15.6 ± 0.6 pA; n=18 cells, P <0.05). Similarly, mean frequency was significantly elevated after CIH (CIH 4.05 ± 0.5 Hz, n=19 vs NORM 2.56 ± 0.37 Hz, n=18; p<0.05) and associated with a significant leftward shift in the cumulative probability of inter event intervals. The results suggest that CIH simultaneously increases enhances spontaneous presynaptic neurotransmitter release while also increasing postsynaptic responsiveness in MnPO. These presynaptic and postsynaptic changes in MnPO that are associated with CIH may represent mechanism contributing to CIH hypertension.
The paraventricular nucleus of the hypothalamus (PVN) is an important autonomic control center. It receives afferent inputs from many brain regions, including the median preoptic nucleus (MnPO). The connection between the MnPO and the PVN is particularly important in generating chronic intermittent hypoxia phenotypes such as increased sympathetic activity leading to the development of hypertension. PVN‐projecting MnPO neurons discharge frequency is <5Hz. To gain more insight into the acute change in PVN neuron properties after low‐frequency stimulation, whole‐cell patch‐clamp recordings were performed on PVN neurons. Adult 250‐350g male rats were injected bilaterally in the PVN with 100 nL retrogradely transported adeno‐associated virus encoding Channelrhodopsin (AAVrg‐CaMKIIa‐hChR2(H134R)‐mCherry) or with AAVrg‐CAG‐tdTomato as control. Three weeks after the injections, the rats were anesthetized with isoflurane (2‐3%) and sacrificed to prepare horizontal brain slices containing the PVN and MnPO. Postsynaptic currents were recorded from PVN neurons in whole‐cell voltage clamp configuration (Vhold=‐60 mV) under constant bath perfusion of normal aCSF (2‐3 ml/min). Axon terminals in the PVN were stimulated with 5Hz LED‐generated blue light (470nM) pulses of 50‐ms duration, 5 seconds off/on for a total of 1 min, delivered using optical fiber directed to the MnPO. Photo‐evoked currents were measured during 5 min before and after the photo‐stimulation train. Amplitude and frequency of spontaneous currents were compared before and after stimulation as well. At the end of each recording, cells were characterized as type I (magnocellular) or type II (parvocellular) PVN neurons based on the presence of transient outward rectification. Data were analyzed offline using Easy Electrophysiology v2.3.3 software. For both cell types, 5 Hz optogenetic stimulation caused a significant reduction in the optogenetic‐evoked current amplitude during the first 15 minutes after stimulation (before stimulation 94.88±2.85 %, n=9 cells vs. 15 minutes after stimulation 67.52±6.15 %, n=9. P=0.0027) before returning to baseline values. Post‐stimulation spontaneous postsynaptic current amplitude and frequency decreased in type II PVN neurons from retrograde ChR2 animals (pre‐stimulation amplitude 100±0.62 % vs. post‐stimulation amplitude 86.50±0.54 %, P=0.0371; pre‐stimulation frequency 3.82±1.7 Hz, post‐stimulation frequency 2.396±1.2 Hz, P= 0.0385) but not in type I PVN neurons from retrograde ChR2 animals or cells from control animals. Kinetic profiles of spontaneous current in type I and II PVN neurons did not change after 5 Hz optogenetic stimulation. The results suggest that low‐frequency stimulation discriminately alters spontaneous presynaptic neurotransmitter release and possibly postsynaptic responsiveness in PVN neurons. Additional experiments will be needed to specifically stimulate PVN afferents from the MnPO.
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