चूहा midbrain डोपामिनर्जिक गतिशील दबाना तकनीक का प्रयोग न्यूरॉन्स में NMDA रिसेप्टर प्रवाहकत्त्व के आवेदन
Summary
इस वीडियो में, हम का प्रदर्शन कैसे एक डोपामिनर्जिक चूहा मस्तिष्क स्लाइसें में पूरे सेल विन्यास में दर्ज न्यूरॉन में एक प्रवाहकत्त्व लागू करने के लिए. इस तकनीक को गतिशील दबाना कहा जाता है.
Abstract
Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.
Protocol
Discussion
गतिशील दबाना यहाँ प्रदर्शन तकनीक experimenter एक रिसेप्टर के सक्रियण के बिजली के प्रभावों की नकल करने की अनुमति देकर प्रत्यक्ष वर्तमान इंजेक्शन की पारंपरिक तकनीक के सुधार पर है. इस वीडियो में, हम पता चला है कि एक प्रभाव जोड़ने के डोपामिनर्जिक न्यूरॉन का सहज गतिविधि के लिए एक NMDA रिसेप्टर के सक्रियण के कार्रवाई क्षमता की एक फट अर्थात् कर रहे हैं पैदा कर सकते हैं.
हार्डवेयर / सॉफ्टवेयर कार्यान्वयन के लचीलेपन के कारण, एक्सटेंशन की एक किस्म का इस्तेमाल किया जा सकता है. नकारात्मक से सकारात्मक इंजेक्शन वर्तमान का संकेत बंद किया जा सकता है, एक परिदृश्य जहां सक्रिय रिसेप्टर के प्रभाव एक न्यूरॉन से निकाल दिया जाता है का प्रतिनिधित्व. मॉडल न्यूरॉन्स, के फार्म का अंतर समीकरणों की एक श्रृंखला में प्रतिनिधित्व भी संख्यानुसार हल किया जा सकता है और experimenter छोटे नेटवर्क की जांच के लिए अनुमति देते हैं.
Disclosures
The authors have nothing to disclose.
Acknowledgements
यह काम MH084494 CJL (), और MH079276 और NS060658 (कैप) द्वारा समर्थित किया गया.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| K-gluconate anhydrous | Reagent | Sigma-Aldrich | ||
| HEPES | Reagent | Fisher Scientific | ||
| CaCl2 X 2H2O | Reagent | Fisher Scientific | ||
| Ethylene glycol-bis(B-aminoethyl ether)-N,N,N’,N’-tetraacetic acid | Reagent | EGTA; Sigma-Aldrich | ||
| MgATP | Reagent | MP Biomedicals | ||
| NaGTP | Reagent | MP Biomedicals | ||
| MgCl2 | Reagent | Sigma-Aldrich | ||
| NaHCO3 | Reagent | Sigma-Aldrich | ||
| KCl | Reagent | Fisher Scientific | ||
| NaH2PO4, Anhydrous | Reagent | Fisher Scientific | ||
| Glucose | Reagent | Acros Organics | ||
| NaCl | Reagent | Fisher Scientific | ||
| CholCl | Reagent | Sigma-Aldrich | ||
| Sodium Pyruvate | Reagent | Fisher Scientific | ||
| Ascorbic Acid | Reagent | Acros Organics | ||
| Glutathione | Reagent | Sigma-Aldrich | ||
| Olympus BX51WI Microscope (with 40x objective) | Microscope | Olympus | ||
| 2 A/D converters | Equipment | e.g. Heka Instruments Inc. ITC-18, National Instruments BNC-2090A | ||
| Multiclamp 700B with CV-7B headstage | Equipment | Molecular Devices | ||
| P-97 Flaming/Brown Micropipette Puller | Equipment | Sutter Instrument Company | ||
| Microfil syringe needles | Equipment | World Precision Instruments | ||
| Micromanipulator | Equipment | Siskiyou, Inc. | ||
| Monitor | Equipment | Triview |
References
- Robinson, H. P., Kawai, N. Injection of digitally synthesized synaptic conductance transients to measure the integrative properties of neurons. J Neurosci Methods. 49, 157-165 (1993).
- Sharp, A. A., O’Neil, M. B., Abbott, L. F., Marder, E. The dynamic clamp: artificial conductances in biological neurons. Trends Neurosci. 16, 389-394 (1993).
- Sharp, A. A., O’Neil, M. B., Abbott, L. F., Marder, E. Dynamic clamp: computer-generated conductances in real neurons. J Neurophysiol. 69, 992-995 (1993).
- Prinz, A. A., Abbott, L. F., Marder, E. The dynamic clamp comes of age. Trends Neurosci. 27, 218-224 (2004).
- Deister, C. A., Teagarden, M. A., Wilson, C. J., Paladini, C. A. An intrinsic neuronal oscillator underlies dopaminergic neuron bursting. J Neurosci. 29, 15888-15897 (2009).
- Lobb, C. J., Wilson, C. J., Paladini, C. A. A dynamic role for GABA receptors on the firing pattern of midbrain dopaminergic neurons. J Neurophysiol. 104, 403-413 (2010).
