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“Experimental design and methods applied” is described under pertinent

sub-headings of Subjects, Experimental designs and recording procedures,
Artificial neural network models, Training and testing protocol of MLPNN for
environmental stress classification.

3.1 Subjects and surgery: The experiments were carried out with male
Charles Foster rats of age 12-14 weeks and weight around 180-200 grams at the
beginning of the experiment. The rats were individually housed in polypropylene
cages (30 cm x 20 cm x 15 cm) with drinking water and food (Hindustan Liver
Limited, India) ad libitum on commercial laboratory food pellets. All rats were
kept in an ambient environment temperature of 23 ± 1 oC from birth and the animal
room was artificially illuminated with 12:12 hours Light: Dark cycle, changed at
07.00 hours and 19:00 hours IST.
Rats were chosen, as they are very well suited for neurophysiological
experiments because neurological developments in them are similar to human
development. The primitive cortex in rats is in a greater proportion and is more
superficial, so cortical electrodes can record the changes in primitive cortex, where
the stress affecting the mood is more pronounced. Moreover, the extensive sleep-
wake recordings could be done easily on the rats due to their nocturnal activity and
easy availability unlike the human subjects.
For the common grounding, midline Frontal stainless steel screw electrode,
1 mm in diameter, and two other similar screw electrodes were used for cortical
EEG. Four stainless steel loop electrodes, insulated, except at the tip (two for EOG
and two for EMG), were also used. Their socket contacts had earlier been prepared
to a seven-pin amphitronic connector. Screw and loop electrodes were connected
and soldered to the free pins of the connector, connected with thin flexible wires.
The EEG and grounding screw electrodes were kept free; however, four pins of
EOG and EMG electrodes were fixed in the amphitronics connector with the help
of dental acrylic, well before implantation. Screw electrodes were connected and

Experimental Design and Methods Applied

fixed to the socket contact by dental acrylic after fixing them on the skull. Such
separate connectors were used for each of the experimental animals for the
recording of electrophysiological signals. The procedure of electrodes
implantation in rats has been presented in Figures-3.1(a - d).

(a) (b)

(c) (d)

Figure-3.1: (a) The exposed skull of the anesthetized rat with positions
marked for attaching the screw electrodes. Two positions marked on bilateral
frontal region for EEG and one on the midline frontal region for grounding
electrodes. (b) Exposed skull with three screw electrodes. (c) After the fixation
of screws, two loop electrodes for EOG (left and right outer canthus muscles
respectively) and two loop electrodes for EMG (left and right cervical muscles
respectively) were sutured to keep them in position. (d) A rat after electrode

Experimental Design and Methods Applied

3.2 Experimental design and recording procedure

3.2.1 Heat stress model: The stress was produced in the rats, by subjecting
them in the Biological Oxygen Demand (BOD) incubator (Oceania, India) at
preset temperature of 38±1oC and relative humidity 45-50% (Sharma et al., 1998),
simulated with the environmental conditions of Varanasi (India) in the months of
May and June.
Acute heat stress: Rats were subjected to the BOD incubator for continuous
four hours of heat exposure from 8.00 a.m. to 12.00 p.m. for a single day, just
before the recording of electrophysiological signals.
Chronic heat stress: Rats were subjected to the BOD incubator for one hour
daily for 21 days of chronic heat exposure from 8.00 a.m. to 9.00 a.m. and
electrophysiological signals were recorded on 22nd day.
Control: Respective control groups of rats were placed in the incubator at room
temperature (23±1oC) and whole procedure was followed exactly similar to that of
their stressed groups.

3.2.2 Experimental protocol

Experimental protocol for acute and chronic stress group of animals has
been summarized in Tables-3.1 and 3.2 respectively.

Table – 3.1
Day Experimental protocol for acute stress group
1 Implantation of electrode measurement of body weight and body
2-7 Recovery and habituation to experimental chamber.
8 1. Measurement of body weight and the body temperature before stress
2. Animal subjected to the experimental chamber at high ambient
temperature (38±1oC) for 4 hours.
3. Measurement of core temperature after the stress.
4. Continuous recording of EEG, EOG and EMG for 4 hours.

Experimental Design and Methods Applied

Table 3.2

Day Experimental protocol for chronic stress group

1 1. Body weight and body temperature measured before the stress.
2. Animal subjected to the experimental chamber at high ambient temperature
(38±1oC) for 1 hour.
3. Measurement of body temperature after the stress.
4. Habituation of animals to the experimental chamber.
2 Animal subjected to the experimental chamber at high ambient temperature
(38±1oC) for 1 hour and habituated to the experimental chamber.
3,6,9,12 Same as day 1.
4,5,7-8, Same as day 2.
14 1. Same as day 2.
2. Implantation of electrode, measurement of weight and core temperature.
15. 1. Same as day 1.
2. Recovery from surgical stress.
16-17 Same as day 2.
18. Same as day 15.
19-20 Same as day 16-17.
21 Same as day 1.
22. Continuous recording of EEG, EOG and EMG for 4 hours.

3.3 Electrophysiological recordings

The test chamber (35 cm x 25 cm x 30 cm) was constructed entirely of
perspex and was located in a constantly illuminated (500-600 Lux white light),
sound insulated chamber (300 cm x 180 cm x 240 cm). Holes at regular distances

Experimental Design and Methods Applied

were made on the walls of test chamber for proper ventilation. The continuous
four hours of recordings of EEG, EOG and EMG were performed from 12.00 hour
to 16.00 hours IST (Indian Standard Time) on the recording day for chronic and
acute heat stressed rats through the 8 channels Electroencephalograph (EEG -8,
Recorders & Medicare Systems, India). The paper recordings were performed at
the chart speed of 7.5 mm/sec.
The recording setup along with the snapshot of running window have been
presented in Figure-3.2 and 3.3 and the parameters of amplifier setting for
different electrophysiological signals are given in Table-3.3.

Figure-3.2: The whole setup with the recording cage holding the rat whose
electrodes were connected to the EEG machine. The EEG machine was
connected to the AD card, which is fixed inside the computer. The analog
paper recording as well as, the digital recordings on the computer monitor, can
be seen.

Experimental Design and Methods Applied

Figure-3.3: The computer monitor with simultaneous three channels of

recordings of EEG, EOG and EMG respectively.
Table 3.3
The amplifier settings for recording of three electrophysiological signals
Signal Sensitivity in Low frequency High frequency 50 Hz filter
V/mm cut-off in Hz. cut -off in Hz
EEG 10 1 70 In
EOG 20 0.3 35 In
EMG 10 5 70 Out

Some examples of two minutes recordings of raw EEG signals have been given in

Experimental Design and Methods Applied

Figure-3.4: Raw EEG signals of length 2 minutes.

Experimental Design and Methods Applied

The digitized data was collected, stored and processed with the help of data
acquisition system (ADLiNK, 8112HG, NuDAQ, Taiwan) and processing
software (Visual Lab.-M, Version 2.0c, Blue Pearl Laboratory, USA). The
recordings were done with the sampling frequency of 256 Hz and selected data
were stored in hard disk in small segments (approximately 2 minutes) in separate
data files. Further, for ease of wavelet processing, recorded signals for all three
states were split into an epoch of two seconds length.
Two seconds long processed EEG signals for the three sleep
states - AWAKE, SWS and REM have been shown in Figure-3.5. Some examples of
two seconds epochs of unprocessed EEG recordings for AWAKE, SWS sleep and
REM conditions have been presented in Figures-3.6 (a-c). Figure-3.7 depicts
recordings of unprocessed sleep-EEG with its corresponding EMG and EOG signals
with baseline drift unadjusted.

Some examples of two-second long epochs of recorded unprocessed EEG signals

Amplitude (mv)

Amplitude (mv)
Amplitude (mv)

for AWAKE, SWS sleep and REM conditions have been shown in Figure-3.4.(a)
AWAKE data
SWS data
REM data

to 3.4(d). Figure-3.5 depicts three channels recordings of unprocessed sleep-EEG

0.1 0.4 0.1

with its corresponding EMG and EOG signals with Baseline drift unadjusted.
0.05 0.2 0.05

0 0 0

-0.05 -0.2 -0.05

-0.1 -0.4 -0.1

-0.15 -0.6 -0.15

-0.2 -0.8 -0.2

0 0.5 1 1.5 2 0 0.5 1 1.5 2 0 0.5 1 1.5 2

Time (s) Time (s) Time (s)

Figure-3.5: Processed EEG signals of AWAKE, SWS and REM states.

Experimental Design and Methods Applied


(b) SWS

(c) REM

Figure-3.6: Examples of some epochs of two seconds duration of unprocessed EEG

recordings for (a) AWAKE, (b) SWS sleep and (c) REM conditions.

Experimental Design and Methods Applied

3.4 EEG signal processing with wavelet

Having acquired the digital data for sleep staging from different subjects
belonging to acute, chronic, and control group, wavelet technique, which is based
on multiresolution analysis (MRA), was applied on each signal. The data
representing three sleep states such as AWAKE, REM and SWS were selected
from the raw EEG data recorded using VLM software and they were further
subdivided into two seconds long epoch. Since the sampling frequency being 256
Hz, one epoch comprises 512 data points. As total number of subjects for acute,
chronic and their control groups are each ten in numbers; number of data files
selected for each group are 600 and total number of data files considered for
analysis become 2400. Here, it should be noted that for each subject, 15 data files
belonging to AWAKE, REM, and SWS per hour of recording have been taken for
analysis, which turns out to be 60 for all the four hours of recording. Using
Matlab – 7 (The Mathworks Inc. with the wavelet and neural network toolbox), all
the epochs were loaded individually on Matlab’s editor and converted to MAT
files in Matlab’s workspace, which were further stored permanently in computer’s

3.4.1 Wavelet packet analysis

With the help of wavelet packet analysis, changes in frequency due to
stress for each signal (MAT files) were visually examined and analyzed. In the
orthogonal wavelet decomposition procedure, after splitting the signal into a
vector of approximation coefficients and detail coefficients, new approximation
coefficient vector is further split but successive details are never reanalyzed. The
wavelet packet offers a more complex and flexible analysis because
approximations as well as their respective details are split. So, it starts from a
scale-oriented decomposition and analyzes the obtained signals on frequency
bands. AWAKE, REM, and SWS signals are clearly distinguished.

3.4.2 Discrete wavelet transform (DWT)

Experimental Design and Methods Applied

It has been applied to extract the features in terms of coefficients of delta,

theta, alpha, and beta region by decomposing and subsampling operations, which
have to be used for training of neural networks. For decomposition of processed
EEG data of each epoch, different wavelets have been applied but finally,
Daubechies order-4 (db4) wavelet from the Daubechies family of orthogonal
wavelets with compact support was used. The signal was decomposed into five
levels of approximation and details. Using wavelet coefficients of above
mentioned levels, the approximation and detail records were reconstructed.
Approximation a4 is obtained by superimposing details d5 onto approximation a5.
Similarly, a3 is obtained by superimposing details d4 onto a4 and so on. Lastly, the
original signal is obtained by superimposing details d1 onto approximation a1.
Therefore, zooming into small scales and zooming out into large scales
respectively can reveal compactly spaced events in time and global waveform
patterns. Frequencies corresponding to different levels of decomposition with a
sampling frequency of 256 Hz are shown in Table-3.4. It can also be seen from
this table that the components of level 5 decomposition are within the delta range
(0.5-4 Hz), level 4 decomposition are within the theta range (4-8 Hz), level 3
decomposition are within the alpha range (8-14 Hz), level 2 decomposition are
within the beta range (14-30 Hz). Lower level decompositions corresponding to
higher frequencies have negligible magnitudes in a normal EEG. So, the selection
of the decomposition level is highly dependent on the frequency of the signal
components. The most suitable decomposition level for the analysis is the level
that corresponds to the frequency range, which contains those parts of the signal
that correlate well with the differences required for classification of the signal.
Table 3.4
Frequencies corresponding to different levels of decomposition
Level of decomposition 0 1 2 3 4 5
Scale (2) 1 2 4 8 16 32
Frequency (Hz) 128 64 32 16 8 4

Since the sampling frequency of time domain signal is 256 Hz

(256 samples per second), the highest frequency component in the original signal
according to Nyquist criteria is  radian in terms of angular frequency, which

Experimental Design and Methods Applied

corresponds to the linear frequency of 128 Hz. The above procedure of subband
coding is repeated for further decomposition. This decomposition halves the time
resolution since only half the number of samples now characterizes the entire
signal. However, this operation doubles the frequency resolution since the
bandwidth of the signal now spans only half the previous frequency band,
effectively reducing the uncertainty in the frequency by half.

3.4.3 Continuous wavelet transform

A Matlab program has been written to calculate all the coefficients from
scale 1 to 128 for each epoch containing 512 sample points and hence producing a
matrix of size [128, 512]. Continuous wavelet transform using Daubechies order-4
wavelet was applied to AWAKE, REM, and SWS sleep EEG data of size [512, 1]
over scales 1:128, which gives coefficients as a function of time and scale
(translation vector and scale). In order to know the frequency information
contained in the signal instead of scales [210], following formula has been used :
Fa 
where, Fa is the pseudo frequency corresponding to scale ‘a’ (in Hz), F c is
the center frequency or dominant frequency of a wavelet in Hz, defined as the
frequency with the highest amplitude in the Fourier transform of the wavelet
function, and  is the sampling period. Given below are the frequencies
corresponding to different levels of decomposition for db-4 wavelet on scales over
1 to 128.

Pseudo frequencies in (Hz) 3.697 7.935 16.59 30.42

Corresponding scales 46 23 11 6

Depending upon the desired frequency information, signals were grouped

together into delta (0.5 – 4 Hz), theta (4 – 8 Hz), alpha (8 – 14 Hz), and beta
(14 – 30 Hz) range. After extracting frequency information from the time domain

Experimental Design and Methods Applied

signal and categorizing into delta, theta, alpha, and beta frequency bands, powers
of all the four bands of AWAKE, REM and SWS were computed for each subject
belonging to stress group and control group, which have also been shown through
plots generated by Matlab (ref. Figures: 4.1-4.9). Graphs explained how frequency
changes with time for stress and control groups. The program is so written that
after calculating wavelet coefficients, frequencies and their powers, 4 basic plots
other than many subplots are generated projecting significant information
regarding time, frequency, and power. The first plot is a 3-D plot of time, scale,
and coefficients along X, Y, and Z axis respectively (Figure-4.3, Chapter-4). The
second plot contains four 3-D subplots each showing powers of the four frequency
bands separately with respect to time and frequency (Figures: 4.4 – 4.9). The third
plot (2-D) consisting of 4 subplots for delta, theta, alpha, and beta with time
[1, 512] along X-axis and power (≈ 10 -6 V2/10-7 V2) along Y – axis
(Figures: 4.10b – 4.12b). The fourth plot shows changes in power w.r.t. time for all
frequencies between 0 to 30 Hz in one epoch for AWAKE, SWS and REM
(Figure-4.14). Further, changes observed due to stress in EEG frequencies and
powers of all the four hours of continuous recording have been plotted and

Experimental Design and Methods Applied



60 61 62
Time (s) 0.2
100 200 300 400 500
Time (Samples)

100 200 300 400 500
Time (Samples)

Figure-3.7: 2 seconds long epoch of recorded unprocessed sleep-EEG with its

corresponding EMG and EOG signals with baseline drift unadjusted.

Experimental Design and Methods Applied

3.5 Data Organization

The EEG, EOG and EOG recordings were done with the sampling
frequency of 256 Hz and selected data were stored in hard disk in small segments
(approximately 2 minutes) in separate data files (.dat).MATLAB was used for
processing of these data files. These 2 min data files were further broken into 30
second samples for feature ex
traction. 30 sec sample is the prescribed sample length for visual sleep stage
Because of the offset in the measurement system, baseline removal was done and
the data was band-pass filtered for 0.5 Hz to 32 Hz. to remove all redundant
frequencies [9]. The following algorithm shows the step by step procedure that has
been followed to achieve the desired result.

Figure -3.8: Proposed step by step process

Experimental Design and Methods Applied

3.6 Feature Extraction

After the data is divided into segments of 30s interval, the feature
extraction algorithm is run on these datasets and corresponding features are stored
along with their label. Code is prepared in MATLAB 2014a software. The code is
prepared in a loop so that each data files are read , four set of features
corresponding to each 30 seconds interval is extracted and stored in an excel file
and then moves onto next data file.
Band Power and other time domain features that have been used are expounded
in the subsequent section.

3.6.1 Band Power

Relative band power of the given EEG signals in different frequency bands were
extracted [10]. Alpha (8-13Hz) and Beta (13-32Hz) frequencies constitutes a
major proportion of Awake state, Delta (0.5-4Hz) constitutes a major portion of
Slow wave sleep while Theta is present in very low proportion in Awake state. The
periodogram computes the power spectra for the entire input signal [11]:


Where F (signal) is the Fourier transform of the signal, and N is the

normalization factor, which Igor´s DSP Periodogram operation defaults to the
number of samples in the signal. Band power of a particular frequency range of a
signal is the area under the periodogram of the signal for that frequency band

Relative Band power is calculated for the four frequency bands on EEG (Fig 3.9
shows the power spectral density of a given frequency band) [8] [11] .Relative
band power is calculated by dividing the total power spectral density of a given
band to the total power spectral density of the entire frequency range. This relative
band power implies how much a particular frequency band constitutes in the
complete frequency band. This forms the basis of deciding whether a particular
frequency constitutes a large or small portion of the frequency band, which forms

Experimental Design and Methods Applied

the fuzzy sets for ANFIS. Apart from the relative band power total band power
was also extracted for the four frequency bands for the given data. Total band
power changes from control to Acute/Chronic state. The EOG and EMG activity is
also evaluated.

Figure-3.9: Periodogram power density of a given EEG sample

3.6.2 EOG & EMG Activity

EMG and EOG activity is required along with EEG to determine various sleep
stages [4] [6].We have calculated the mean of the absolute value of these signals in
the given sample. If EMG or EOG activity is high then mean of their absolute
value will also be high in the given time frame.


Where N represents the sample length or the time window for the signal x.

Experimental Design and Methods Applied

3.7 Fuzzy Logic Application for sleep stage and stress level
A Matlab program has been written to implement ANFIS for sleep stage
classification and analyze the results. ANFIS has been chosen because visual
inspection data was present to do the supervised learning. A sugeno type fuzzy
inference system is created first with 6 inputs and one output. The six inputs were
Alpha band power, Beta band power, Theta Band power, Delta band power, EOG
activity and EMG activity. Sleep stages classification has earlier been attempted
without using polygraphs but using only EEG signals. It dealt mostly with
identifying certain wave patterns and spindle and k-complexes detection to
classify sleep stages and the results were not very accurate but presence of other
event markers like EOG and EMG reinforces the sleep classification capabilities.

Figure-3.10: Overall ANFIS interface in MATLAB 2014

Figure 3.10 shows the ANFIS structure where six inputs to the Mamdani type
system can be seen with one output which represents the sleep stage. Also as far as
various methods used in AND and OR, min and max method is chosen. For
implication min method is chosen because it gives the best results in normal cases.

Experimental Design and Methods Applied

For aggregation max method is used and for defuzzification Centroid method,
most commonly used method, is used.
Regarding membership function for inputs the gbellmf function is used for
representing each fuzzy set low and high while for output triangular membership
function are used. We have chosen the Gbell membership function because of its
nature that it can take any values in any of its variables and it is differentiable all
throughout its range. Most of the membership function suffers this limitation e.g. a
triangular membership function has zero slopes at some points and is non-
differentiable while its variables cannot take certain values. Figure 3.11 shows a
generalized bell membership function which can take any variable for its
parameters a, b and c.

Figure-3.11: Generalized bell function

The output of the system is properly quantized to analyze the results. The training
has been done for 1000 epochs and system achieved satisfactory results with
learning algorithm converging in about 100 epochs.
After the sleep stages have been classified for each stress level, heat stress
classification is done separately for all the three stages because physiological
changes happens differently in each of the sleep stages. To analyze the changes in
total band power which happens predominantly when the subject is subjected to

Experimental Design and Methods Applied

thermal stress for each sleep stage. A measure which evaluates the difference of
the total band power for each input is coded. Since, these changes happen only in
the EEG, EOG and EMG data are not required.
Figure 3.12 shows a Mamdani type fuzzy inference system which has been applied
for tress level classification. tAlpha, tBeta, tTheta and tDelta are the changes in the
band power of respective frequency bands. The membership function for inputs
and outputs are of the form Triangular. The AND and OR methods are min and
max respectively. Implication and aggregation method are min and max
respectively. Defuzzification method is centroid as used for sleep stage
classifications as well.

Figure-3.12: Overall FIS interface in MATLAB 2014

Experimental Design and Methods Applied

The next section deals with the sleep stage detection and stress level detection

where use of fuzzy for these purposes has been expounded well.