Administer 1.5 g/kg urethane (20% solution, i.p., Sinopharm Chemical Reagent Co., Shanghai) for analgesia initially and 0.5 g/kg/h urethane throughout surgery and recording.
Administer atropine sulphate (0.05 mg/kg, s.c.) 15 min before induction of anesthesia to inhibit tracheal secretion.
Liberally apply a local anesthetic (xylocaine, 2%) to the wound.
After mounted the animal in a stereotaxic device, make a midline incision and exposed the top scalp.
Perform a craniotomy to vertically access the MGB and the auditory sector of the TRN, and then remove the dura mater.
Throughout the experiment, keep the rat on a heating blanket, and maintained the body temperature at 37 – 38°C with a feedback switching circuit5-9.
Implant stereotaxically tungsten microelectrodes with impedances of 2 – 7 M? (Frederick Haer & Co., Bowdoinham, ME) into the TRN and MGB from the top of the brain, according to a rat brain atlas10.
Measure the vertical coordinate of the electrode array from a point slightly above the cortical surface.
Position the recording electrodes with a stepping-motor microdrive, which is controlled from outside the soundproof room.
Amplify the signal recorded by the microelectrode, together with the acoustic stimulus signal, and stored them using TDT software (OpenEX, TDT) and Axoscope software (Axon Instruments, Sunnyvale, CA).
Calculate the time of spike occurrence relative to stimulus delivery using Matlab software (Mathworks, Inc, Natick, MA).
1 Once the electrode is lowered into the auditory sector of the TRN, neurons would show responses to noise-burst stimuli. Then randomly present pure tones of varying frequencies (500Hz – 48 KHz) to construct the frequency response function as one of them is shown in the following Figure 1. Present each frequency at least for 15 trials. Define the best frequency (f0) based on the frequency response function.
Set a deviance paradigm which consists of the following sequence of stimuli: f1, f1, f1, f1, f1, f1, f1, f1, f1, f1, f2, and p.
Set a control paradigm, in which f2 is replaced with f1 to generate the following sequence: f1, f1, f1, f1, f1, f1, f1, f1, f1, f1, f1, and p. The rational for the paradigms is that the TRN neurons would be activated by f2 in the deviance paradigm, but not by the final f1 in the control paradigm.
Compared MGB neuronal responses to p under the deviance and control paradigms. Differences would show the modulatory effect of the TRN deviance preference.
Choose frequencies of f1 and f2 that evoked no response from the MGB neuron being recorded in order to exclude the last adaptation effect of the responses to the preceding sequence of tones (f1, …, f1 or f1,…, f2) on the responses to p,.
Set the ISI at 150 ms between consecutive f1 stimuli and between f1 and f2.
Separate the f2 stimulus from p by 50 ms. The p is followed f2 without any delay, as the duration of all stimuli (f1, f2, and p) is 50 ms.
Set the inter-block stimulus at 3 s.
Randomly present blocks of the deviance and control paradigms based on a computer program. Figure 3 shows one of the results in which the MGB neuron showed no responses to f1 and f2 but responded to the probe stimulus, p. As shown in Figure 3, the preceding deviance paradigm modulates the auditory response of the MGB neuron (see the results in Figure 5 of Ref5).
Figure 3 near here
Define an index to measure the modulatory effect on the auditory response of the MGB neurons by the preceding deviance paradigm. The index IDC is defined as (RD – RC) / (RC+ RD), where RD and RC are the responses to the probe stimulus in the deviance and control paradigms, respectively. A negative value indicates a suppressive effect, while a positive value indicates an enhanced effect.
Set the threshold for determining a modulatory effect, IDCth, on a neuron-by-neuron basis.
Calculated the IDCth for each individual neuron using the above equation with responses from the odd (30 trials) and even number (30 trials) of trials in the control paradigm. When the absolute value of IDC exceeds the absolute value of IDCth, the neuron is considered to have undergone modulation and included in the statistics. Refer to Figure 5 in Ref5 for the results of IDC distribution of the MGB neurons.
Combination of light and sound stimuli:
Present a combination of a light stimulus (50 ms) and a noise-burst with a 50-ms interval (LS paradigm).
Present only the sound stimulus (S paradigm) in the control paradigm.
Deliver white light through an array of light diodes, which is placed inside the sound-proof chamber and controlled electronically outside the chamber.
Compare MGB neuronal responses to the sound stimulus under the LS paradigm and S paradigm.
Perform an initial examination of the neuronal response to the visual stimulus only to exclude the adaptation effect.
Randomly present the LS and S paradigms in every 3 s.
Sort neuronal response data for each paradigm using a homemade program. Figure 4 shows a result (more results could be found at Figure 7 in Ref5).
Figure 4 near here
Try the different tones f1 and f2
Make the sound level of the probe stimulus as low as possible to extract the maximal modulation effect.
TRN inactivation suppresses deviance modulation of MGB:
Glue a tungsten microelectrode to the injection glass pipette to monitor the activity of the TRN. The distance between the two tips was approximately 200 ?m.
Monitor MGB neuron’s deviance modulation effect.
Injected lidocaine (0.3 microL, 20 mg/ml; Sigma, St. Louis, MO) in the TRN using a microinjection syringe (Hamilton, Reno, NV), when there is modulation to the MGB.
Monitor neuronal activities in the TRN before, during and after the injection (Figure 5).
Repeat deviance detection paradigm at 2-3 minutes after the lidocaine injection.
Figure 5 near here
Compare the above results with that before the TRN inactivation (Figure 6, and Figure 6 in Ref5).
Figure 6 near here