```markdown
# Goal/Experiment:
Analysis of the time evolution of auditory steady-state responses (ASSR) recorded in rats.

## Analysis of the time evolution of auditory steady-state responses (ASSR) recorded in rats

#### Version 2

**Authors:**
1. Pavel Prado - Advanced Center for Electrical and Electronic Engineering (AC3E), Universidad Técnica Federico Santa María, Chile
2. Eduardo Martínez-Montes - Cuban Neuroscience Center
3. Matías Zañartu - Department of Electronic Engineering, Universidad Técnica Federico Santa María, Valparaíso, Chile

dx.doi.org/10.17504/protocols.io.wejfbcn

## Abstract

Auditory steady-state responses (ASSRs) are brain oscillations locked to the periodic properties of acoustic stimuli. Audiological tests based on the acquisition of ASSR are useful for estimating hearing sensitivity, mainly because multiple hearing frequencies can be simultaneously assessed, and the auditory response can be objectively detected using statistical tests.

Typically, the extraction of the auditory response from the measured signal relies on averaging epochs of the EEG, time-locked to the stimulus. This assumes that the auditory response is steady over time and that averaging increases the signal-to-noise ratio of the measurement.

Since time-domain averaging of epochs within a recording does not allow distinction between methodological and physiological related variations in the amplitude of the ASSR, we designed a protocol for analyzing the dynamics of the auditory response during the acquisition procedure. The protocol allows us to compute the ASSR amplitude at a given time window without being compromised by the segments of the preceding EEG. 

## Guidelines

The study must be performed under the approval of the local Animal Research and Ethics Committee. Specifically, this study followed the guidelines of the Cuban Neuroscience Center and the National Center for Animal Breeding of Cuba. 

## Safety Warnings

Handle animals following standard safety procedures. Standard safety procedures should also be adhered to when handling disposable needles and syringes. National and local electrical safety regulations must be followed.

## Before Starting

Care, feeding, breeding, and maintenance of animals should follow standard local guidelines. Animals should be housed in a standard bio-clean animal room under a 12-hour light/dark cycle at 22-24°C, with free access to food and tap water.

## Preparation

1. **Anesthesia:**
    - Administer ketamine (75.0 mg/kg, intraperitoneal) and diazepam (5.0 mg/kg, intraperitoneal).

2. **Supplemental anesthesia:**
    - Maintain the animal in an areflexic state with supplemental doses during the experiment.

3. **Atropine sulfate:**
    - Administer 0.06 mg/kg intramuscularly to decrease mucosal secretions.

4. **Temperature control:**
    - Maintain body temperature at 37.0±0.1°C using a body temperature control system.

5. **Post-experiment care:**
    - Return animals to the colony after recovery from anesthesia—animal sacrifice is not required.

## Acoustic Stimulation and EEG Recording

6. **Presentation:**
    - Acoustic stimuli are presented monaurally via an ER-3A Etymotic Research Insert Earphone.

7. **Custom ear molds:**
    - Use custom-fitted ear molds to replace the original foam for coupling the earphone to the rat’s ear.

8. **Stimulation system calibration:**
    - Refer acoustic levels to a Brüel & Kjær artificial ear (type 4152). Calibrate with a Brüel & Kjær 2250 sound level meter and type 4144 microphone.

9. **Acoustic stimuli generation:**
    - Generate stimuli using standard hardware/software. Example: continuous tones of 8 kHz sinusoidally-modulated in amplitude at 115 Hz are generated using the ASSR software module of the AUDIX system (Havana, Cuba). Stimulus intensity is fixed at 50 dB SPL.

10. **Electrophysiological responses:**
    - Record responses differentially using stainless-steel needle electrodes inserted subdermally (vertex positive; neck negative; thorax reference).
    - Amplify recordings with a gain of 1.2x10^4 and band-pass filter frequencies from 10 to 300 Hz.

12. **Digitization:**
    - Digitize the output at 16-bit resolution and sample at 920 Hz.

13. **Artifact rejection:**
    - Reject segments with electrical oscillations exceeding 50 mV online.

14. **Data acquisition:**
    - Complete 60 artifact-free epochs of 4.45 s duration each (4096 time-points each). Allow 10 minutes between consecutive recordings. Thirty recordings are acquired from each animal.

## Data Processing

15. **Software Processing:**
    - Perform data processing using in-house MATLAB codes (MathWorks, USA).

16. **Data Matrix Arrangement:**
    - Rearrange the 60 sequential epochs of the 30 recordings offline into a data matrix of 30 rows and 60 columns (one matrix per animal).

17. **Noise Influence Reduction (Optional):**
    - Modify the dataset to reduce noise influence on auditory response computation.

18. **Column-wise Averaging:**
    - Average the 30 epochs column-wise for each time window to reduce EEG background noise and detect the ASSR amplitude.

19. **ASSR Amplitude Computation:**
    - Compute the amplitude for each group of epochs using Fast Fourier Transform (FFT). Use an FFT length of 4096 time-points, aligning with the length of an epoch (4.45 s). With sampling at 920 Hz, the FFT resolution is 0.22 Hz. Windowing technique is not implemented.

20. **Spectral Amplitude:**
    - Define amplitude as the spectral amplitude at 115 Hz. Vector average the amplitude of 30 spectral components to calculate residual noise level (RNL).

21. **Statistical Comparison:**
    - Compare ASSR amplitudes with corresponding RNL using Hotelling’s T2 multivariate test in the AUDIX system, considering both amplitude and phase oscillations.

22. **Time Evolution:**
    - Plot ASSR amplitudes as a function of time. Fit to time courses using negative exponential functions if R^2>0.85 and p<0.05.

23. **Statistical Tests:**
    - Apply statistical tests, such as One-way ANOVAs (p<0.05) and post-hoc analyses (Tukey test, p<0.05), as needed to analyze the stability of the ASSR amplitude and RNL.

### Calculation of Adaptive Behavior Index:

When an adaptive behavior is detected, the adaptation index (P_adapt) of the response is calculated using the equation:

\[ P_{\text{adapt}} = 100 \left( \frac{A_{\text{mp}\max} - A_{\text{mpadapt}}}{A_{\text{mp}\max}} \right) \]

Where:
- \( A_{\text{mp}\max} \) represents the maximum amplitude of the fitted curve.
- \( A_{\text{mpadapt}} \) represents its asymptotic value (defined as the amplitude estimated when the recording length was three times the time constant of the fitted exponential function).

## Summary

A summary of the protocol:
1. Presenting acoustic stimuli modulated in amplitude at 115 Hz.
2. Organizing the dataset (matrix with 60 columns and 30 rows).
3. Spectral analysis of the averaged measurement.
4. Graphical representation of ASSR dynamics.

![Protocol Diagram](https://example.com/path/to/protocol-diagram.png)

This protocol is distributed under the terms of the Creative Commons Attribution License.

endofoutput
```