युगपत Electroencephalography, लैक्टेट एकाग्रता और neuronal गतिविधि के Optogenetic हेरफेर के कृंतक प्रमस्तिष्क प्रांतस्था में वास्तविक समय मापन
Summary
गतिविधि मस्तिष्क cortical पिरामिड न्यूरॉन्स के optogenetically जबकि electroencephalogram, विद्युतपेशीलेख निगरानी कर रहे हैं, और मस्तिष्क लैक्टेट एकाग्रता जोड़ तोड़ के लिए एक प्रक्रिया में वर्णित है. प्रायोगिक रिकॉर्डिंग केबल सीमित चूहों पर प्रदर्शन कर रहे हैं, जबकि वे सहज नींद / जगा चक्र से गुजरना. Optogenetic उपकरण हमारी प्रयोगशाला में इकट्ठा किया जाता है, रिकॉर्डिंग उपकरण व्यावसायिक रूप से उपलब्ध है.
Abstract
Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.
Protocol
Representative Results
Discussion
तरीके यहाँ प्रस्तुत है एक एक समय संभव पहले नहीं पैमाने पर glycolytic मध्यवर्ती लैक्टेट की एकाग्रता मस्तिष्क में नींद और परिवर्तन के बीच संबंध को मापने के लिए अनुमति देते हैं. पशु मद्देनजर NREMS, और REMS के बीच सहज स?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
रक्षा विभाग (रक्षा एडवांस्ड रिसर्च प्रोजेक्ट्स एजेंसी, युवा संकाय पुरस्कार, अनुदान N66001-09-1-२,११७ संख्या) और NINDS (R15NS070734) द्वारा वित्त पोषित अनुसंधान.
Materials
| Component | Company | Catalogue number | Comments (optional) |
| BASi Mouse Guide Cannula | Pinnacle Technology/BASi Inc | 7032 | |
| Lactate Biosensor | Pinnacle Technology | 7004 | |
| Head Mount | Pinnacle Technology | 8402 | |
| Sleep/Biosensor Recording system | Pinnacle Technology | 8400-K1-SL | 2 EEG channels, 1 EMG channel, & 1 biosensor |
| Tethered Mouse in-vitro Calibration kit | Pinnacle Technology | 7000-K1-T | |
| Fiber Optic Guide Cannula | Plastics One | C312G | 21 Gauge Guide Cannula |
| Dummy Cannula | Plastics One | C312DC | 21 Gauge Dummy |
| Diamond Fiber Scribe | Thorlabs | S90W | |
| Fiber Connector Crimp Tool | Thorlabs | CT042 | |
| Furcation Tubing | Thorlabs | FT030 | 03.0 mm |
| Thorlabs | T10S13 | Max Dia. 0.012 | |
| Furcation Tube Stripper | Thorlabs | FTS3 | |
| Bare Hard Cladding Multimode Fiber | Thorlabs | BFL37-200 | 200 μm Core, 0.37 NA |
| Wire Snips/Kevlar Shears | Thorlabs | T865 | |
| Fiber Optic Epoxy | Thorlabs | F112 | |
| Fiber Stripper Tool | Thorlabs | ||
| Glass Polishing Plate | Thorlabs | CTG913 | |
| Rubber Polishing Pad | Thorlabs | NRS913 | |
| Eye Loupe | Thorlabs | JEL10 | |
| Kim Wipes | Thorlabs | KW32 | |
| Compressed Air | Thorlabs | CA3 | |
| Polishing Puck | Thorlabs | D50-xx | |
| Fiber Inspection scope | Thorlabs | CL-200 | |
| Polishing Films | Thorlabs | LFG5P, LFG3P, LFG1P, LFG03P | |
| FC/PC connector end | Thorlabs | 30126G2-240 | 240 μm Bore, SS Ferrule |
| MC Stimulus Unit | Multi-Channel Systems | STG-4002 | |
| MC Stimulus Software | Multi-Channel Systems | MC-Stimulus V 2.1.5 | |
| Blue Laser | CrystaLaser | CL473-050-0 | |
| Laser Power supply | CrystaLaser | CL2005 | |
| Fiber Optic Rotary Joint | Doric Lenses | FRJ-v4 | |
| Table 2. Supplies and equipment. |
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