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

Summary

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.

Abstract

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 GABA_A 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).

Introduction

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 inputs^2,3. For example, it has been observed that anesthetics alter the duration of visually-evoked responses in cortical neurons^3,4. Moreover, the ratio between excitatory and inhibitory synaptic inputs is different in anesthetized and awake animals^4,5, altering both evoked and spontaneous activity rates^6,7. By measuring the synaptic conductances, Haider and colleagues⁴

Protocol

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. 

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 and performed using the workstation setup described in Bullock et al.¹⁷.

1. To record single-unit extracellular acti....

Results

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 GABA_A-receptors with gabazine. An increment in the response strength (spikes/stimulus) as well as a broadening of the spectral tuning was observed. The incremen.......

Discussion

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 neurons^40,41. 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

Materials

List of materials used in this article

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