Explore The Role Of Glutamatergic Neuron Within The Hypothalamic Parventricular Nitcleus In Blood Pressure Modulation Via Optogenetics

Objective: It has been proved that the glutamatergic inputs in the hypothalamicparaventricular nucleus (PVN) contribute to heighted sympathetic outflow inhypertension. Since the glutamatergic synaptic inputs to the PVN neurons are fromglutamatergic interneurons within the PVN, subnuclei of hypothalamus, andtelencephalic regions, it is impossible to determine which origins of glutamatergicinputs play a major role in the contribution of heighted sympathetic by traditionalmethods such as electrical stimulation and pharmacological tools, resulting from lackingof cell-type specific resolution. By combining the use of light and genetically encodedlight-sensitive proteins, optogenetics allow cell type-specific targeted, millisecond-scalefast control of precisely defined events in complex biological systems, thereby thisbreakthrough method will help us to isolate the glutmatergic neuron in the PVN, toidentify its role in blood pressure modulation and furthermore to explore the underlingmechanism of neural regulation of endocrine and autonomic mechanism.Materials and methods: In vitro experiment lentiviral vectors carrying theChR2/eNpHR3.0-YFP fusion gene under control of the CamKâ…¡ α promoter, to targetglutamatergic neuron in PVN, were constructed using standard cloning techniques.Male SD, WKY and spontaneous hypertension rats (SHRs) were subjected to theseexperiments, concentrated lentivial vectors were stereotactically injected into the ratPVN (anteroposterior=-1.5mm from bregma; lateral=±0.35; dorsal-ventral=-7.8mm). For electrophysiological experiments,2weeks post-injection,250μmthickness PVN slices were prepared in ice-cold cutting solution using vibratome. After arecovery period of30mins in ACSF at34℃or so, the slices transferred to a storagechamber to incubate in ACSF continuously gassed with95%O2+5%CO2at least1 hour before electrophysiological recording at room temperature (23-26℃). And thenslices were removed to recording chamber mounted on upright microscope andcontinuously perfused with carbonated ACSF (3ml min^-1) ventilated with95%O2+5%CO2. PVN slices were visualized by standard transmission optical on an uprightfluorescent microscope, and images were recorded with CCD camera.Electrophysiological recordings in neurons were performed at room temperature,membrane currents were measured with the patch-clamp technique in the whole cellvoltage-clamp configuration. For ChR2/eNpHR3.0activation, blue/yellow light pulseswere generated using the DG-4high-speed optical switch system. The CamKⅡα andGAD67were detected by immunohistochemistry using antibodies against CamKâ…¡ α andGAD₆₇,2weeks later after the fixed slices with the virus vectors injection had beencollected. In vivo experiment The surgeries of implantation of optical neuronal-interface were performed under aseptic condition, the fiber guide was inserted beforethe operation of stereotatactic injection and then the fiber guide system was anchored bydental cement. After the finish of viral vectors injection, the internal cannula waswithdrawn, and a dummy cannula was inserted to keep the fiber guide patent. Theradiotelemetry techniques were applied to monitoring the blood pressure and ECG inconscious freely moving laboratory animals after the implantment of DSI transmitter tomatch with the optica-neuronal interface. An initial method of long-term sampling ofcerebral venous blood in conscious rats was also introduced by implanting a cannulainto the confluence of sinuses around the site of lambda.Results: Acute rat brain slices that10days post-injection through the fiber guide withlentivirus carrying the ChR2/eNpHR3.0-YFP fusion protein showed a robust expressionin the PVN. At higher magnification, the YFP fluorescence was found to bepreferentially localized to the neuron membrane. Whole cell recording obtained fromthe neurons ChR2⁺/NpHR⁺in acute brain slices, indicated robust depolarizing orhypopolarizing photocurrent required for action potential generation or inhibition.The outcomes of immunohistochemistry verified that the ChR2/eNpHR3.0wasexpressed specifically in the glutamatergic neurons within the PVN, as hypothesized from our use of lentiviral vectors that carrying ChR2/eNpHR3.0under the control ofCamKâ…¡ αpromoter. In vivo experiment, via neuronal-interface, photostimulation ofeNpHR3.0-transfected PVN glutamatergic neurons with593.5nm wave-length yellowlight decreased the mean arterial blood pressure by20mmHg or so and lasted for about20miniuts in SHRs.Conclusions: We have succeeded in building up a whole set of the optogeneticsplatform, which aims to explore the role of glutamatergic neuron within the PVN inblood pressure modulation. This platform includes3main parts: the viral vectors toolsthat can deliver the opsins to cell-type specific neurons; the light deliver technologiesthat can photostimulate the target brain regions with user friendly; a well-designedreadout system matching with the optical-neuronal interface. Our results provide themost driect evidence that the glutamatergic neurons within the PVN have a bloodmodulating function, yet furthermore works will needed to elucidate the underlingmechanism of neural regulation of endocrine and autonomic mechanism.