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Neuroscience

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Published: April 30th, 2013

DOI:

10.3791/50312

1Department of Physiology, University of California, Los Angeles

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

1. Dissection of Brains from Adult Medaka

  1. Anesthetize adult male or female (Figure 1A) by immersing in 5 ml MS-222 (150 mg/L, pH 7.4); wait a couple of minutes after gills movements have ceased before decapitating. All procedures were approved by the Institutional Animal Care and Use Committee of University of California-Los Angeles.
  2. Decapitate the fish in fish saline at the caudal end of the operculum with scissors in a 60-mm diameter Petri dish.
  3. Transfer fish head to a 3.......

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An example of bilateral clusters of GFP-labeled TN-GnRH3 neurons from the excised brain of medaka fish are shown in Figures 1B and 1C. Each cluster contains about 8-10 GnRH neurons. The spontaneous neuronal activities of the target TN-GnRH3 were recorded in current-clamp mode (I=0) with typical firing rates of 0.5-6 Hz. The pattern of action potential firing is typically a tonic or beating pattern, with a fairly regular interspike interval. Sample traces are shown in Figure 2

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GnRH3:GFP transgenic fishes provide unique models to study the neurophysiological mechanisms underlying neuronal integration and regulation in central control of behaviors that are both directly and indirectly involved in reproduction3, 8-10. One of the significant advantages of this model system is that many GnRH3 neurons expressing GFP are close to the ventral surface of the brain, allowing for relatively easy access to the neurons for electrophysiological recording without disrupting neural circuit.......

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We thank Dr. Meng-Chin Lin and Ms. Yuan Dong for technical assistance. This work was supported by a grant from the National Institutes of Health HD053767 (subcontract to NLW), and by funds from the Department of Physiology and Office of the Vice Chancellor for Research, University of California-Los Angeles (NLW).

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Name Company Catalog Number Comments
Name Company Catalog Number Comments
Microscope Olympus BX50W (Upright)
Amplifier Axon Instruments Axoclamp 200B
A-D converter Computer Interference Corp. Digidata ITC-18
Cooled CCD camera PCO Computer Optics Sensicam
Xenon lamp Sutter Instruments Co.
GFP filter set Chroma Technologies
Imaging Software Intelligent Imaging Innovations Slidebook software
Electrophysiology Data Acquisition Software Axon Instruments Axograph software
Electrophysiology Data Acquisition Software AD Instruments Inc. PowerLab
Headstage for electrophysiology Axon Instruments CV 203BU
Micromanipulator Sutter Instrument Co MP-285
Recording Chamber Platform Warner Instrument Corp. P1
Recording Chamber Warner Instrument Corp. RC-26G
Electrode Puller Sutter instruments P87
Filament for electrode puller Sutter Instruments FB330B 3.0 mm wide trough filament
1.5 mm glass capillaries World Precision Instruments 1B150-4 Microelectrode for recording
Syringe Becton Dickinson 309586 3 ml
MS-222 Sigma E10521-10G Ethyl 3-aminobenzoate methanesulfonate salt
Fish saline mM: 134 NaCl; 2.9 KCl; 2.1 CaCl2; 1.2 MgCl2; 10 HEPES
Electrode solution (loose-patch) mM: 150 NaCl; 3.5 KCl; 2.5 CaCl2; 1.3 MgCl2; 10 HEPES; 10 glucose
Electrode solution (whole-cell patch) mM: 112.5 K-gluconate; NaCl; 17.5 KCl; 0.5 CaCl2; 1 MgCl2; 5 MgATP; 1 EGTA; 10 HEPES; 1 GTP; 0.1 leupeptin;10 phospho-creatine

  1. Kah, O., Lethimonier, C., Lareyre, J. J. Gonadotrophin-releasing hormone (GnRH) in the animal kingdom. J. Soc. Biol. 198 (1), 53-60 (2004).
  2. Ramakrishnan, S., Wayne, N. L. Social cues from conspecifics alter electrical activity of gonadotropin-releasing hormone neurons in the terminal nerve via visual signals. Am. J. Physiol. Regul. Integr. Comp. Physiol. 297 (1), R135-R141 (2009).
  3. Abe, H., Oka, Y. Mechanisms of neuromodulation by a nonhypophysiotropic GnRH system controlling motivation of reproductive behavior in the teleost. 57 (6), 665-674 (2011).
  4. Oka, Y. Tetrodotoxin-resistant persistent Na+ current underlying pacemaker potentials of fish gonadotrophin-releasing hormone neurones. J. Physiol. 482 (Pt. 1), 1-6 (1995).
  5. Zhao, Y., Wayne, N. L. Effects of Kisspeptin1 on Electrical Activity of an Extrahypothalamic Population of Gonadotropin-Releasing Hormone Neurons in Medaka. PLoS One. 7 (5), e37909 (2012).
  6. Wayne, N. L., et al. Whole-cell electrophysiology of gonadotropin-releasing hormone neurons that express green fluorescent protein in the terminal nerve of transgenic medaka (Oryzias latipes). Biol. Reprod. 73 (6), 1228-1234 (2005).
  7. Okubo, K., et al. Forebrain gonadotropin-releasing hormone neuronal development: insights from transgenic medaka and the relevance to X-linked Kallmann syndrome. Endocrinology. 147 (3), 1076-1084 (2006).
  8. Okubo, K., et al. A novel form of gonadotropin-releasing hormone in the medaka, Oryzias latipes. Biochem. Biophys. Res. Commun. 276 (1), 298-303 (2000).
  9. Ramakrishnan, S., et al. Acquisition of spontaneous electrical activity during embryonic development of gonadotropin-releasing hormone-3 neurons located in the terminal nerve of transgenic zebrafish (Danio rerio). Gen. Comp. Endocrinol. 168 (3), 401-407 (2010).
  10. Abraham, E., et al. Targeted gonadotropin-releasing hormone-3 neuron ablation in zebrafish: effects on neurogenesis, neuronal migration, and reproduction. Endocrinology. 151 (1), 332-340 (2010).
  11. Wayne, N. L., Kuwahara, K. Beta-endorphin alters electrical activity of gonadotropin releasing hormone neurons located in the terminal nerve of the teleost medaka (Oryzias latipes. Gen. Comp. Endocrinol. 150 (1), 41-47 (2007).
  12. Oka, Y. Three types of gonadotrophin-releasing hormone neurones and steroid-sensitive sexually dimorphic kisspeptin neurones in teleosts. J. Neuroendocrinol. 21 (4), 334-338 (2009).
  13. Molleman, A. . Patch Clamping: An Introductory Guide To Patch Clamp Electrophysiology. , (2003).

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