Name: extracellular recording of neuronal activity combined with microiontophoretic application of neuroactive substances in awake mice
Uploaded: 2016-05-21T15:00:00
Description: Watch this Scientific Journal Video about Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice at JoVE.com

This project was funded by the MINECO grants BFU201343608-P and PSI2013-49348-EXP, and the JCYL grant SA343U14 to MSM and MRC core funding to ARP. YAA held a CONACyT (216106) and a SEP fellowship.

All experimental procedures were carried out at the University of Salamanca with the approval of, and using methods conforming to the standards of, the University of Salamanca Animal Care Committee as well as the standards of the European Union (Directive 2010/63/EU) for the use of animals in neuroscience research.&#160;
1. Tungsten Electrodes
Note: The manufacture of tungsten electrodes is based on the original technique described in Merrill and Ainsworth27 and Ainsworth et al.28</su...

The microiontophoresis of neuroactive substances in awake animals is a powerful technique to probe and dissect the role of specific synaptic inputs on the activity of single neurons40,41. More importantly, this procedure allows the determination of the impact of the neurotransmitters and neuromodulators on neural circuits without the potential interference of anesthetics. Here, we demonstrate that the application of gabazine in the IC of awake mice robustly changed the frequency tuning (Figure 4A</stro...

The interplay of neural excitation and inhibition is fundamental for the processing of sensory information1. It is also known that anesthesia has a strong impact on the dynamics of cortical activation and the temporal pattern of synaptic inputs2,3. For example, it has been observed that anesthetics alter the duration of visually-evoked responses in cortical neurons3,4. Moreover, the ratio between excitatory and inhibitory synaptic inputs is different in anesthetized and awake animals4,5, altering both evoked and spontaneous activity rates6,7. By measuring the synaptic conductances, Haider and colleagues4 found that inhibition matched excitation in amplitude under anesthesia whereas during wakefulness, inhibition was stronger than excitation. These findings prompt the development of experimental procedures to study the impact of specific synaptic inputs on sensory processing in awake animals.
The controlled ejection of charged neuroactive substances by applying small current injections (on the order of nA) has been extensively used to study the contribution of synaptic inputs and the role of putative cell receptors in sensory processing8-13. This technique, known as microiontophoresis, allows the application of drugs in the vicinity of the recorded neuron, which contributes to a rapid and confined effect. This procedure is more suitable for studying local effects of neuroactive substances, compared to the widespread effect elicited by other experimental manipulations such as systemic injections, microdialysis or the use of optogenetic techniques. Usually, a piggy-back electrode configuration14,15 is used to simultaneously record the target neuron and deliver the neuroactive substances of interest. It consists of a recording electrode attached to a multibarrel pipette that carries the neuroactive substances. Modifications of the original procedure described by Havey and Caspary14 have been implemented. For example, a tungsten electrode, instead of a glass one,&#160;can be used to record the neural activity16. Previously published methods for the manufacture of tungsten electrodes17,18 involve three general steps: electrolytic etching of tungsten wire tips, glass insulation, and adjustment of the tip exposure to meet recording requirements.
An interesting and emergent field in auditory neuroscience is the study of stimulus-specific adaptation (SSA19). SSA is a specific decrease in the neural response to repetitive sounds that does not generalize to other, rarely presented sounds. The importance of SSA resides in its potential role as a neural mechanism underlying deviance detection in the auditory brain, as well as a possible neuronal correlate for the late mismatch negativity component of the auditory evoked potential20,21. SSA occurs from the IC up to the auditory cortex19,22-24. GABAA-mediated inhibition has been demonstrated to act as a gain control mechanism on SSA7,16,25, which has also been shown to be affected by anesthesia26. Here we present a protocol that combines previously described methods for recording the single-unit activity of IC neurons before and during the application of a selective antagonist of the GABAA-receptors in awake mice. First, we describe the manufacture of piggy-back electrodes and next, the surgical and recording methods. To test for the efficacy of drug release, we compared the receptive field as well as the level of SSA of IC neurons before and during the microiontophoretic ejection of gabazine.

<table>
	<tbody>
		<tr>
			<td>Tungsten wire</td>
			<td>Harvard Apparatus LTD</td>
			<td>33-0099</td>
			<td>0.005 inches x 3 inches</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary&#160;</td>
			<td>Harvard Apparatus LTD</td>
			<td>30-0053</td>
			<td class="xl66" height="21" style="height:15.75pt;width:494pt" width="658">Borosilicate standard wall without filament, 1.5 mm OD, 0.86 mm ID, 100 mm long</td>
		</tr>
		<tr>
			<td>Multibarrel glass capillaries&#160;</td>
			<td>World Precision Instruments</td>
			<td>5B120F-4&#160;</td>
			<td>5-barrel capillary, 4 inches long, 1.2 mm OD, with filament</td>
		</tr>
		<tr>
			<td>Diaplus</td>
			<td>DiaDent</td>
			<td>2001-2101</td>
			<td>Light-curing adhesive, used to attach the tungsten electrode to the glas multibarrel pipette</td>
		</tr>
		<tr>
			<td>G-Bond</td>
			<td>GC Corporation</td>
			<td>2277</td>
			<td>Light-curing adhesive, used to attach the headpost to the animal&#39;s skull</td>
		</tr>
		<tr>
			<td>Charisma</td>
			<td>Heraeus Kulzer</td>
			<td>66000087</td>
			<td>Light-curing composite, used to reinforce the bond of the headpost with the skull</td>
		</tr>
		<tr>
			<td>Araldit Cristal</td>
			<td>Ceys</td>
			<td></td>
			<td>2-component expoxy, used to further secure the attachment of the tungsten electrode to the glass multibarrel pipette</td>
		</tr>
		<tr>
			<td>Heating blanket</td>
			<td>Cibertec</td>
			<td>RTC1</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>Narishige</td>
			<td>SR-6N</td>
			<td>Modified for mice</td>
		</tr>
		<tr>
			<td>Microiontophoretic device</td>
			<td>Harvard Apparatus LTD</td>
			<td>Neurophore BH-2</td>
			<td>Including IP-2 iontophoresis pumps (one for each drug delivery channel) and a balance module</td>
		</tr>
		<tr>
			<td>Multibarrel glass pipette puller</td>
			<td>Narishige</td>
			<td>Model PE-21</td>
			<td></td>
		</tr>
		<tr>
			<td>LED lamp</td>
			<td>Technoflux</td>
			<td>CV-215</td>
			<td>5 W, 430-485 nm</td>
		</tr>
		<tr>
			<td>MicroFil</td>
			<td>World Precision Instruments</td>
			<td>MF34G-5</td>
			<td>Flexible plastic needle, 34 AWG</td>
		</tr>
		<tr>
			<td>Imalgene</td>
			<td>Merial</td>
			<td></td>
			<td>Ketamine, 100 mg/mL</td>
		</tr>
		<tr>
			<td>Rompun</td>
			<td>Bayer</td>
			<td></td>
			<td>Xylazine, 20 mg/mL</td>
		</tr>
		<tr>
			<td>Gabazine / SR-95531</td>
			<td>Sigma</td>
			<td>S106</td>
			<td>Prepare ~ 1000&#181;l of 20 mM gabazine in distilled water and adjust the pH to 4</td>
		</tr>
	</tbody>
</table>

extracellular recording of neuronal activity combined with microiontophoretic application of neuroactive substances in awake mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We recorded the single-unit activity of 4 well-isolated neurons of the IC. Typical signal-to-noise ratios obtained during extracellular recordings in awake mice are shown in Figure 3B. Figure 4A shows the frequency response area (FRA) of each neuron before and during the blockade of the GABAA-receptors with gabazine. An increment in the response strength (spikes/stimulus) as well as a broadening of the spectral tuning was observed. The incremen...

Watch this Scientific Journal Video about Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice at JoVE.com

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

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