Neuroscience
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/30817
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Browsing Neuroscience by Author "Cunningham, Joseph"
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Item Changes in PVN Neurons after Low-Frequency Acute Optogenetic Stimulation(2022) Paundralingga, Obed; Jia, Shuping; Cunningham, JosephThe 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, rats were anesthetized with isoflurane (2-3%) and sacrificed to prepare horizontal brain slices containing the PVN and MnPO. With the bath perfused with normal aCSF (2-3 ml/min), postsynaptic currents were recorded from PVN neurons in whole-cell voltage clamp configuration (Vhold=-60 mV). 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. Evoked currents were measured every 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 MnPO photostimulation differentially might reduce spontaneous presynaptic neurotransmitter release and possibly postsynaptic responsiveness in PVN neurons. Additional experiments will be needed to specifically stimulate PVN afferents from the MnPO.Item Sex Specific Effects of Salt Loading on GABAA Responses in Oxytocin Neurons from the Supraoptic Nucleus(2022) Brock, Courtney; Bachelor, Martha E.; Little, Joel; Farmer, George; Balapattabi, Kirthikaa; Cunningham, JosephArginine Vasopressin (AVP) and oxytocin (OXY) contribute to body fluid balance homeostasis. Salt loading (2% NaCl for 7 days) increases both AVP and OXY release in rats. The chronic increase in AVP release is associated with a change in the sensitivity of AVP neurons in the supraoptic nucleus (SON) to GABA so that GABAA receptor activation becomes excitatory. It is not clear if a similar mechanism is associated with chronic OXY release in this model. Our hypothesis is that changes in chloride homeostasis associated with salt loading occur in OXY neurons. To test this hypothesis, we used a chloride imaging approach with a ratio metric chloride sensitive dye, ClopHensorN (Addgene #50758) combine with an AAV with an oxytocin specific promoter (pFBOT563, Addgene # 40864). Adult, intact, Sprague Dawley rats of both sexes were anesthetized with isoflurane (2-3%) and were bilaterally injected with the AAV2-pFBOT--ClophensorN virus directly into the SON. Rats of both sexes were salt loaded by providing them with only 2% NaCl to drink for 7 days. Later, the animals were sacrificed and the brains were rapidly removed. The SON was dissected away from the brain and the cells were dissociated, plated on cover slips, and incubated for two hours. After incubation, recordings were taken using ratiometric live cell imaging on an inverted microscope. Selected neurons were sequentially excited at 445nm and 556nm and then emission data was collected between 500-550nm and 580-653nm respectively. After 40 cycles of 3-second recordings, muscimol (100nM), a GABAA receptor agonist was transiently applied to the cells and then allowed to wash off. Background fluorescence was subtracted. In cells from males, muscimol resulted in chloride influx in 70% OXY of the cells tested while chloride influx was observed in all OXY cells from females. The results suggest that salt loading may influence GABA responses of OXY neurons in males but not females.