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 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).
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, 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.
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.
2. Multibarrel Glass Pipette Manufacturing
3. Drugs for Microiontophoresis
Note: The drugs used for microiontophoresis must have an electrical charge when dissolved in water. Check the literature to see if the drug of interest is appropriate for this procedure. Here, the procedure for gabazine, an antagonist of the GABAA receptor, is described.
4. Surgery and Headpost Implantation
5. Electrophysiological Recording and Microiontophoresis
6. Data Analysis
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 increment in the evoked response was also evident in the accumulated peri-stimulus time histograms obtained from the neural response to all the frequencies and intensities presented (Figure 4B).
One or two pairs of frequencies within the neuron's FRA (crosses in Figure 4A) were chosen to be presented as repetitive or rare sounds in an oddball paradigm, to study the level of SSA in their responses. For two example neurons, the sound-evoked responses to each frequency (f1 and f2) before and during the gabazine injection are shown as dot rasters in Figure 5A,B. For the two example neurons, there was a clear change in the sound-evoked response as observed in the dot raster and PSTHs.
Likewise, the local blockade of the GABAA-receptors increased the neuronal response to the great majority of rare and repetitive tones (Figure 5C) in agreement with our previous study16. Different levels of SSA were observed in the neural responses to f1 and f2, which was reflected in positive CSI (CSI interval: -0.26 to 0.43, 0.25 ± 0.23) and SI values (SIs interval: -0.41 to 0.83, 0.25 ± 0.23). For most of the cases, the increased response to the repetitive tone caused a drop in those indices as observed in Figure 5D.
Figure 1. Material for the Manufacture of Tungsten Electrodes. (A) Electrode alignment tool, with some tungsten wires in place. The lighter piece on the left is a stop for the wires. The scissor tip indicates the side used as a guide for cutting the wires to the desired length. (B) Empty spindle. (C) Spindle with a batch of electrodes attached, and resting on a holder. Please click here to view a larger version of this figure.
Figure 2. Accessories for Awake Mice Recordings. (A) Headpost. (B) Custom-made cushioned foam restrainer. Place the mouse in between both pieces, leaving the head outside. (C) Modifications to the stereotaxic frame. The headpost holder (top bar) assists during headpost implantation and restrains the head during the recordings. The bite piece (lower bar) is only used during the headpost implantation. Please click here to view a larger version of this figure.
Figure 3. Example of Recording in the Awake Mouse. (A) Frequency response area of a neuron from the mouse IC. (B) Waveforms of the spikes recorded from this neuron. (C, D) Dot raster and peristumulus-time histogram recorded from this neuron using an oddball paradigm at the frequencies indicated by the dots in (A). Redrawn from data published in Duque and Malmierca26. Please click here to view a larger version of this figure.
Figure 4. Effect of the Blockade of the GABAA-receptors on the Spectral and Temporal Response Properties. (A) Frequency response area of the four IC recorded neurons before and during the microiontophoretic injection of gabazine. The black crosses in (A) indicate the frequencies chosen to be presented as rare and repetitive sounds. (B) Accumulated peri-stimulus time histograms of the neural response to all the frequencies and intensities presented to construct the response area before and during the injection of gabazine. The black bars indicate the sound duration. Please click here to view a larger version of this figure.
Figure 5. Effect of the Blockade of GABAA receptors on the Response to Repetitive and Rare Sounds. (A, B) Dot rasters of the spiking response to a pair of frequencies presented as rare (red, 10%) and repetitive stimulus (blue, 90%) before and during the application of gabazine. Each frequency is played in two sequences such that each one was presented as rare- and repetitive. The shaded background indicates the duration of the stimulus. (C). Single-neuron responses to seven pairs of frequencies presented as the rare and as the repetitive sound before and during the application of gabazine. (D) SSA indices (circles: CSI, crosses: SI) of the neural response to rare and repetitive sounds measured before and during the application of gabazine. The responses of the same neuron are indicated with the same color. Please click here to view a larger version of this figure.
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), the temporal response patterns (Figure 4B) and the SSA (Figure 5).
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 on the same animal. Using microiontophoresis it is unclear how far the applied neuroactive substances diffuse into the surrounding tissue. Therefore, a possible effect on neuronal network activity can be elicited by affecting not only the recorded neuron but also the surrounding glial and neuronal cells. For example, Candy and colleagues42 have shown that certain iontophoretically delivered molecules, which are not rapidly removed, can diffuse up to 600 µm. In the mice IC this range would cover most of the extent of the dendritic arbors but also affect the neuronal response of adjacent cells. In summary, the drug released by iontophoresis is not as spatially restricted as intracellular drug dialysis, which allows the dissection in finer detail of the synaptic inputs to the target neuron and test of local network processes16,43, it is more specific than other experimental procedures used to manipulate the level of neurotransmission or neuromodulation, such as systemic injections, the use of optogenetic techniques, or push-pull dialysis.
The tip diameter of the glass pipettes and the magnitude of the retention and injection currents are key factors to monitor in order to avoid nonspecific drug leakage into the tissue. The low injection currents (of the order of tens of nA) limit the spread of the drug allowing recording of neurons close to one another. For example, three of the four neurons used as examples (neurons 1 – 3) were recorded along the same track with distances of 16 and 800 µm between them. Despite the short distance of 16 µm between neuron 1 and 2, a clearly different response of neuron 2 was observed (Figure 4).
Some alternatives to the proposed piggy-back configuration have been used previously. One of them consists of the use of one of the multibarrel pipettes for recording, instead of a separate electrode. While the construction of the ensembles is less complicated in this case, there are drawbacks. First, all the channels will have the same opening diameter at the tip, which may not be optimal for both recording and drug delivery. Also, the protruding electrode tip in the piggy-back configuration prevents damage to the target cell likely caused by the wider tip of the multibarrel. The second common alternative is the use of single-barrel glass pipettes for recording15,44 instead of tungsten electrodes. Glass electrodes are easy to manufacture to the required dimensions, but tend to clog during long recordings or when travelling deep into the brain, so in our experience tungsten electrodes provide more reliable recordings. Moreover, by using tungsten electrodes it is possible to produce electrolytic lesion to locate the recording sites, without the further elaborate histological procedures required when a neural tracer injection is used45. The use of multibarrel glass pipettes allows the release of multiple agonists and/or antagonists very close to the recording site, which is a tremendous advantage when the interaction between neurotransmitter systems on sensory processing is under study.
The procedures described in the present protocol for the manufacturing of the piggy-back electrodes make it possible to produce recording electrodes according to the specific requirements of the experiment and of the characteristics of the target area of interest in a systematic, yet customized manner. In addition, the materials for the headpost fixation are conventionally used in dentistry so they are readily available. Thus, the critical steps in the protocol are the animal habituation and the etching of the tungsten electrodes, which are the main factors contributing to well-isolated neurons and stable electrophysiological recordings. Overall, this technique is relatively simple, economical and very reliable as a means to study the role of multiple neuroactive substances on single or multi-unit neuronal activity in awake, head-restrained mice.
The authors have nothing to disclose.
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.
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 |