MATERIALS AND METHODS - Information and Library...
Transcript of MATERIALS AND METHODS - Information and Library...
MATERIALS AND
METHODS
The details of the materials and methods used in this
otudy for the collection and analysis of the data can,
conveniently, be described under the followins headings :
[A] MntorJnlo unod to oolloot the data
[B] Methodology used to acquire the data
[C] Statistical analysis of the data
LAl HA.IERIALS. llSJill. IQ. COI.LKCT IllK nAIA
[I] EXPERIMENTAL ANIMAL:
Malo wlotar rats, wolshins in the range of 250-350 sm were
used. Rats were chosen as experimental animals for the
follNring reaoons
(1) Easy availability.
(2) Eaoe of handling.
(3) Rats are polycyclic animals and hence are suitable for
this type of study.
(4) Medial and lateral preoptic area are better demarcated in
rats.
(5) Sufficient literature were available.
(6) Availability of stereotaxic apparatus and atlas.
Experimental
House Facility,
polyethylene cages
rats were obtained from the Central Animal
JNU. They were kept, individually, in
(15 em x 9.5 em x 6.2 em) with food and
water ad libitum and under 14:10 dark:light cycle.
[11] KQOIPMKNTS:
(1) UtorootllllisJ.. A.ppnrntuo: Thie instrument (Type
Narishinge Scientific Instrument Lab., Japan) was used
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SR-6,
for
implanting guide cannulae precisely into mPOA and lPOA without
major damage to other areaa.
(2) Polygraph:
It io an inetrument used for recording on chart
electrical elgnale after their amplification. To record
electrophysiological parameters viz. EEG, EMG and EOG,
paper
the
a 4
channel Grass Polygraph (model 79 D) was used.· It consisted of
the following components
(i) 4 channela,
( i 1) A mn.\ n Hw itch for rutting off and on the power supply to
the instrument,
(iii) A ewltch for selectively driving the recording pens along
with the chart paper,
(iv) A opoed regulator (Puoh buttons): for the regulation of
the recording paper speed. The paper speed could be set at
apeede of either 2.5, 5, 10, 25, 50 and 100 mm/sec6nd or
rom/minute by preeelng respective push buttons. In this study
paper opeed was maintained mostly at 2.5 mm/sec. Occasionally,
the speed was increased to 25 mm/sec or 50 mm/sec for the
clarity of the waves and subsequent analysis of the signals,
(v) A timer : It gave a tiny mark at every second, a bigger
mark at every 5th oecond and still a ~isser mark at every
minute.
Further, each channel of the polygraph consisted of (a)
Differential AC Preamplifier Grass Model 7P5 and 7P3
interchangeable preamplifiers were used. The preamplifier had
the following adjustments :
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1ft>_ A.C. l~r.cllllP.llllor. : It conoiated of following components
( i) A. ruUr. Q.f. Input Selector Switches ~ G.1 and G.z.l.
Each Knob with 5 option pointe helped in selecting
desired combination of electrodes without disturbing
th1) an :\.mo lo.
(ii) Cal-Use-Cal Switch : This switch selected the function
to be performed by 7P5 i.e. calibration and recording
by CAL or USE positiono respectively. This switch selected the
signal from tho calibrator and connected it to the input of the
preamplifier. The pV setting on the left provided calibration
voltages of 5, 50 fV whereas on the right provided calibration
voltages of 0.5 and 5 MV.
(iii) Cal G1 Nes. Switch : This switch produced the actual
calibration signal, once the value of this signal was set on
tho Cnl-tloo-Cal switch. When thio owitch wao depressed, a
negative calibration signal was applied to G1 with respect to
G2 and tho pen deflected upwards. Calibration voltages were
derived from a mercury cell battery.
(iv) lLZ. t...uuL... Lmi Froa. -Time Constant Switch! It was used
to adjust low frequency response. This 5 position switch
eliminated the waveo of frequency (time constant) range in
steps of either 0.15, 0.3, 1.0, 3.0 or 10 Hz having a.
corresponding decay time constants of 0.45, 0.24, 0.1, 0.04,
0.015 oec respectively.
(v) SJmaitivitv Switches: This switch together with adjacent
Multiply by switch determined the sensitivity of the pen
dofleotlot\. The amplification of the preamplifier could be
oolectod between 20 ~V/om to 150 MV/cm with the help of a 6
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stepa (20, 30, 50, 75, 100 and 150) switch and a 3 step
Multiply knob (X1, X10, X100).
(vi) Ad.L... CaL.. K.nQb.: Proper adjustment of this switch permited
the sensitivity settings of the fV/cm and Multiply by switches
to read correctly.
!fa~ Preamplifier: It consisted of following components :
(i) Function Switch: This switch determined whether the
drl V()l' amplifier was connected to 7P3 amplifier or to
integrator circuits.
(ii) Cnlibrat.lon. fud.t.uh: This switch selected the voltage of
the calibration signal and connected the input to the amplifier
circuit, once calibration hoa been peiformed. The uV setting on
the left of the switch provided calibration voltage of 10, 20,
60, 100, 200 pV. Tho MV setting on the right of the switch
provided oa'libration voltage of 0.5, 1, 2, 5, 10 and 20 MV.
(iii) CJU..... Switch: It produced tho· actual signal, the value of
which depended on the setting of the calibrator switch. When
depressed, a negative DC calibration voltage was applied to G1
with respect to G2 . When released, it produced the equivalent
of the oppooito polarity.
(iv) Senaltivity HI-LO Slide Switch: This switch together with
the adJ ooent · oensi ti v 1 ty · vernier control, determined the
sensitivity of pen deflection as related to input voltage. HI
was for the signal range of 10-500 pV, whereas LO for the
signal range of 0.5-250 MV.
(v) Scnoitlvity Yernior Control: This switch provided a
continuous adjustment of sensitivity, as well as allowed
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prooiAo oolibrntion. Ita olookwiee turning increased
sensitivity.
the
5 {vi) 112 ~ LQR Ereauenoy CTim~ Constant) Switch: This
position switch eliminated the waves of frequency (time
or 10 conotant) range in otepe of either 0.15, 0.3, 1.0, 3.0
Hz/eec, having a corresponding time constants of 0.45, 24, 0.1,
0.04, 0.015 sec reopectively.
(b) Driver Amplifier: It amplified the power of the
ampllflod olsttnl, proportionate to the signal, to euoh a level
that tho pen could be moved. It comprised of following
oomponente
(i) PolaritY K.n.Qh: It determined .the movement of the
upward or downward to that of the baae line.
(ii) · Batl$l L..in.e. Kn.Qb.: It enabled to adjust the pen
horl::Jontnl llno ond thuo neutralized the stray
any,
pen,
along a
DC, if
( 111) ~-"- C.Y...o.lo. EJ.lt.o.r. Knob.: It eliminated, when on, selectively
~aves of line frequenci, 50 Hz in this situation.
(iv) Driver Sonoltivlty Switcheo Thooo owitohes determined
the eeneitivity of pen deflection.
(v) lll.gh Frequency G.u..t. Q.f.f. K.rulb.: It filtered off waves of
frequency beyond 40, 3, 0.5 KHz, and 75, 35, 15, 3, 0.5, 0.1 Hz
from tho oisnal.
(vi) Driver Sensitivity K.n.Qb. This knob provided a
continuouo adjuotment of sensitivity·of pen deflection.
The adjustments of different knobs for recording EEG, EOG
and EMG in thie study were ae·follows:
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Parameter
1~1\G
EMG EOG
1/2 Amp. Low Freq.
1.0 Hz 3.0 Hz 0. 3 Hz
1/2 Amp. Time High Freq. Constant
75 Hz '75 Hz 35 Hz
0.1 0.04 0.24.
50 Cycle Filter
Out Out In
-------~----------------------------------------------------
(3) Digital Thermometer : It (model 802, Century, India) was
ueed for recording rectal temperature (Tree). This instrument
coneieted of a flexible probe with a thermocouple (Chrom-
Alumol) oonnot· nt tho t:lp. It wao inoorted into the rectum and
the Tree could be read from digital display. It was battery (9
volta) operated and wae eoneitive to a range of temperature
from 0.00-100.00 °C with a resolution of 0.1 °C.
III. ACCESSORIES:
(1) Klootrodco:
Two types of electrodes were used for picking up the
electrical activity from the animal.
(i) Screw Electrodes: These were prepared by soldering radio
wires to small stainless steel aorewa (Fig, MF.1A) for picking
up electrical activity of the brain (EEG).
(ii) Wire Electrodes: These were prepared by stripping the
insulation at the tip of the flexible radio wires and then
making a loop at the otripped end and were used for recording
EMG and EOG (Fig. MF.1B).
(2) Cannulae Aeaembly;
It wae uoed for mlcroinJectlon of a definite volume of a
chemical in solution into the mPOA/lPOA. Each assembly
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A B
I
Fig. KF.l The figure shows; A: Screw Electrode; B. Wire Electrodes C: "Bilateral mPOA Guide Cannula; D: Bilateral lPOA Guide Cannula E. ~lockers; F: Guide Cannula with Blockers; G: Injector; H: Guide Cannula with injector; I: Operated rat, ~-Ti th nine pin plug
consisted of outer guide cannulae, blocker and inner injector
cannu.la.
(i) Guide Cannulae:
For the purpose of bilateral injection into m- and lPOA
two typeo of guide cannulae were used. Separate guide cannulae
wcr<-' unod ln onoh oxpor lmcnt.
(a) Bilateral m£QA Guide Cannula It was used for
approaching mPOA bilaterally and prepared by soldering two, 2
em long, 24 G stainless steel tubing at a height of 1-1.2 em
from one end. The midpoints of the two tubings were separated
laterally by 0.6 to 1.0 mm (Fig. MF. lC).
(b) Dilat~ lfQA Guide CannulaL It was used for approaching
lPOA bilaterally and was prepared by soldering two, 2 em long,
24 ll at.oiuleeo stool tubing at a height of 1-1.2 ems from one
end. However, the tubings were separated by 2.6-3.0 mm (Fig.
MF. lD).
While preparing these cannulae, care was
main taln t.he two tubings parallel and their ends on
plt.HH~.
(11) Blockero:
taken
the
to
same
TheBe were used as stylets for the guide cannulae to avoid
blocking of the guide cannulae by brain tissue while
introducing them into brain and also to prevent oozing out of
cerebrospinal fluid (CSF) after the introduction of the guide
cannulae. These were prepared by bending a 26 G stainless steel
wire at one end and putting some dental cement on it (Fig. MF.
lE). The length of the blocker was exactly equal to the length
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of tho guido onnnulno (Fig. MF. lF).
(iii) Injector=
It wao uoed for microinjeotion of a definite quantity of
chemical in solution. 30 G atainleaa steel tubing (injector
oonnulo) attached to a 1 ul mloroayringe (llamilton, U.K.)
through a small (10-12 erne) polyethylene tubing, was used as
injector (llig. MF. 1G). Tho length of tho injector cannula was
adjusted (with a bead) in such a way that ita tip projected
about 1-1.5 mm beyond the guide cannulae (Fig. MF.lH). The
guide cannulae along with blockers were implanted and fixed
with the skull whereas injector waa used for injection.
[IV] CHEMICALS:
The cl1emioala, uoed in this study, their chemical formulae
nature of action and their sources, were as follows :
No. Chemicals
1.
2.
3.
4.
5.
6.
1.
8.
9.
Maroa in (Bupivacaine HCl)
Clonidlne HCl
Yohimbine HCl
Methoxamine HCl
Prazonin HCl
L-lsoproteronol HCl
Propranolol HCl
N,N,Dimethyl Acetamide
Salino {0.9%)
Formula
c11n18ClN03
c 19n22c1N5o4
c11 H18o3NC1
c 16n22c1No2
c4H9NO
0.9% NaCl
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Function
Local Anaesthetic
a2 antagonist
a 1 agonist
a 1 antagonist
~ agonist
~ antagonist
Vehicle for Prazosin HCl
Control
Source
Sarabhai Chern., India
Sigma Chern., U.S.A.
-do-
-do-
-do-
-do-
-do-
-do-
Local
[V] MICROTOME:
A rotary microtome was used to out paraffin sections of
required thickness.
[1 V ] llRAlN Ail,AS;
Brain atlas provided three dimensional coordinates with
histological and reconstructed photographs which aided in
approaching a target area precisely, Stereotaxic atlas of rat
brain by different authors viz. Konig and Klippel (1963),
Oawaldo-Cruz and Rocha-Miranda (1968), .Pellegrino et al.
(1979), Paxinos and Watson (1982) are available
"The Hat Brain in Stereotaxic Coordinates" by
Watson (1982) was used in this study.
[B) HETHODQLQGY USKU IQ ACQUIRE Ill& nAIA
Thio aspect comprised of 3 events :
[I] Preparation of animals
[II] Recording and collecth)n of data
[Ill] 111Bt.ological verification of sites
[ 1] J»HI\1'1\HATlON OJt' ANlMAMl:
This phase
implantation of
included acclimatization of
electrodes and bilateral guide
their respective sites.
(1) Acolimntlzation;
now-a-daye.
Paxinos and
animals and
cannulae to
After bringing rata from the Central Animal House, JNU,
firstly they were acclimatized to the recording environment and
the rootnl probo for a minimum of 2 sesoion6 of 3 hours each.
For acclimatization, rats with rectal probe, inserted 6 ems
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d<H~i' lnnldo the reo tum, wore kopt i.n the recording chamber
(Ambient temperature 26 ~ 1°C).
{ 2) s-tereotaxic Implantation:
Stereotaxic implantation of guide cannulae and different
electrodes were done under surgical anaesthesia and aseptic
conditionn. All the ourgical instruments and materials were
sterilized properly. Rata were anaesthetized by intraperitonial
injootlon of Bodium pentobarbitone (Loba-Chemie Indo-Auatranal
Co., India) at the rate of 35 mg/kg. After anaesthetization,
akin over the head was shaved off hairs and the rat was fixed
in the stereotaxic apparatus. The skull of the rat was exposed
by making a longitudinal incision on the skin over the skull.
The muscles over skull were then scrapped, exposing bregma and
lnmbda.
(i) Implantation of Klectrodeo:
Two ecrew electrodoe wero implanted bilaterally for
recording cortical EEG. Each of the electrodes was fixed at a
diBt.ance of + 2.00 mm from bregma and 4 mm lateral to the mid
sagittal suture. Another screw electrode was fixed in the
midline overlying the frontal sinus which acted as animal
ground. The EMG electrodes were fixed to the dorsal cervical
neck muscles bilaterally. The EOG electrodes were fixed
bilaterally to the muscles near external canthus. Two more
anchorage screws were also screwed to the skull.
(11) Implantation of Guide Cannulae:
A Bmall oval window (2.5-3.0 mm x 2 mm for mPOA and 4-4.6
66
mm x 2 mm for lPOA) wao made around the bregma with the help of
a dental drill. The window exposed the brain covered with
duramater. The duramater was punctured carefully with a fine
needle. There after, the bilateral guide cannulae along with
the blockers, previously held straight, was implanted
approaching either mPOA or lPOA, according to the following
ooord iiH\tf,o (Paxinos and Wa taon, 1982):
Brain roll{ ion
mPOA
lPOA
A.P. referred to brflgma
(mm)
-0.3 to -0.8
-0.3 to -0.8
H.L. referred to sagittal plane
(mm)
0.5 ±. 0.2
1.5 ±. 0.2
D - V referred to the dura
(mm)
*For targeting. these pointe, guide cannulae were kept 1.00 to 1.5 mm above the actual target, for the injector cannulae were longer than guide cannulae by 1.00 to 1.6 mm.
The implanted guide cannulae, EEG and ground electrodes
were fixed to the akull with dental acrylic. The free ends of I
all the electrode wires were soldered to a 9 pin female plug
which was fixed to the akull with dental acrylic {Fig. MF.11).
After the surgery, the rat was transferred to a cage with
ood and water ad libitum. The operated rat was maintained with
adequate post-operative care for recovery. Recording of this
pre[ltlrod rat was done after at least 4 days of recovery from
post~operative trauma. On 3rd recovery day onwards, rate with
roctal probe inserted into the rectum and recording plug
connected to the polygraph, was kept in the.recording chamber,
for at lonot two oooolona of at least 3 hours each, for further
acclimatization. EEG, EOG, EHG and Tree were recorded
66
Fig. !1¥.2.
0 0 0 0 0 0 0 0
0
The figure shows schematic representation of the experimental set up.
Gimultaneously. The experimental set up is shown in Figure, MF.
2. Only those rata which showed normal variation in the Tree
were selected while those showing abnormal Tree were not used
for further experimentation.
[II] RKCORDIKG AND COLLKCTIOK OF DATA:
Electrophyoiologioal parameters (viz. EEG, EMG and EOG)
signifying S-W were recorded simultaneously in three different
channelo of a Grass polygraph (model 790). Rata are polycyclic
animals having predominance of sleep during the day and
predominance of wakefulness at niaht. Normally, the Tree of
rats are 37.80 ± 0.01 and 38.3 ± 0.1 °C during day and night,
respectively, i.e. a lower Tree durina day and higher Tree at
night (Heller and Glotzbach, 1977i Mohan Kumar et al., 1984,
1985). llonce, the experiments were conducted both during the
day (09.30 hr to 18.00 hr) and the night (21.30 hr to 5.00 hr).
Recording of eleotrophysioloaioal parameters (viz. EEG, EMG and
EOG) and Tree was done continuously except for an interruption
(otoppod temporarily) for injootlon of ohemloalB into
mPOA/lPOA. Hence for convenience, the reoordina procedure is
diocuo~ed under two headings:
[1] Pre-injection recording
[2] Poet-injection recording
(l)fro-lnJoatlon Raaordlna:
Experimental rat, with recording plug connected to the
polygraph 8nd tho rootnl probe connected to the thermometer was
left in tl1e recording cage at least one hour before the start
67
of actual recording. EEG, EHG and EOG wore simultaneously
recorded in three different channels of the polygraph at least
for 30 min. Tree of the rat was also recorded simultaneously
every 5 min. The behavior of the rat viz. body posture,
movomcnto etc. were continuouoly monitored and noted. All the
chnnnclo of the polygraph were calibrated meanwhile.
Polygrapl1ic recording was done at a paper speed of 2.5 mm/sec.
Ilowov<w, for better visualization and counting of the waves,
occasionally the speed was increased to 25 mm/sec and 50
rom/sec.
InJection Qf Chemicals:
After pre-injection recording, 0.2 or 0.4 ~1 of saline or
~hemical was injected into mPOA or lPOA with the help of an
injector cannula and microoyringe. The chemicals (in aolution)
were injected bilaterally at the rate of 0.1 pl/min, so as to
provido onough time for the diffuaion of chemicals and not much
distortions in the tissue. The injector cannula was retained in
the same position for at least one minute on each side after
the injection to prevent backward flow of the injected
chemicals due to capillary action. Injector cannula was then
removed and blockers were replaced. The whole injection
procedure took around 7-6 min for 0.2 pl bilateral injection
and 11-12 min for 0.4 pl of bilateral injection. Injection w~s
never repeated in any of the experimental rats to avoid any
discrepancy in the results due to chemical or morphological
changes in tho inJection oite (Routtenberg, 1972). However, in
some of the experiments in combination studies, second
68
injection waa made within 10 min of the firat injection. In
these cases also, experiments were never repeated on the same
rnt.
( 2) Poat-lnleatlon Recording:
Aft.er inJection, nll tho four parameters (EEG, EMG, EOG
and Tree) were recorded till the effects lasted. EEG, EMG and
EOG were recorded for 1.6 - 2.6 hr while Tree for 4.00- 7.6 hr
depending upon the effectiveneao of the chemical injected.
[III].IIISTOLOGICAL VERIFICATION OF INJECTION SITES:
'l'ho inJection oitoo nnd spread of injected chemicals were
identified by the presence and extension of Prussian blue
coloration in the histological sections (Bagga et al., 1981,
1984). At the end of experiments, under deep ether anaesthesia,
0. 2/0.4 ).11 (tho nmotmt: o~~m'l to tho omount or chemical injected
in the same rat during experiment) of 2% FeC1 3 solution was
:\nJootod into tho fH:tmo site and in the same manner where
chemicals were injected. After about 20-30 min the brain of the
unlmn l wnn 1-)orfunod .\r1 trnourd lnlly with 25-30 ml of aaline
followed by 70-80 ml of 10% formol-saline containing 3%
potassium ferrocyanide. After perfusion, the brain was taken
out and preserved in 10% formaldehyde solution. A small piece
of the fixed brain tlsoue (6 x 6 mm) including the site of
injection and ito surrounding area was cut and embedded in
paraffin wax. Serial ooctiono of 30 p thickness were cut on
rotary microtome. Sections were stained with Eosin to provide
contrast to the site of injection visible as Prussian blue
69
£ CXJ
Bregma 0.2 mm
~ 54321 0 1 2 3 4 5 7 ~ 8
9
J 7
8
9
10
~. 8
9
10 Bregma-1 .8mm
7 6 5 4 3 2 0 5 6 7
• j
Fig. ft¥.3. Reconstruct i on diagram of the histological sections of rat brain, through med ial and lateral preoptic areas, according to the atlas of Paxinos and Watson. The filled areas show the extension of the sites where injections of chemicals were effective in Jinfluencing S-W/Trec ,, whereas hatched areas represent ineffective sites. Inse t shows the phot ... ")micrographs of the histological sections. Abbrevi ations as in the text.
colour rspot .. St.ainod oeotiono wore mounted in DPX and examined
under the microscope. The presence and the extension of
Prusaian blue coloration represented the site and spread of the
injected chemical. The spread of the Prussian blue spot was
marked by tracing the blue spots on drawings {histological
mapa) from brain atlas of Paxinos and Watson {Fig. MF. 3). Only
thooe rats where the Prussian blue colour spots were within the
range of m- and lPOA were considered for analysi~.
[C] STATISTICAL ANAI,YSKS QE. IllK DATA
[I] CLASSIFICATION OF SLKKP-WAKKFU~NKSS:
Firotly, the whole result was scanned visually to know the
general pattern and presence of unusual EEG, EMG and EOG waves,
nrt.lfno t.n, i\lo t;urbnnootJ t' t.o. which wore noted while record ina.
The whole record was divided into blocks of 5 min and every
block was analyzed in terms of 5 stages of sleep-wakefulness
viz. awake movement {AM), Awake quiet {AQ), slow wave sleep
(SWS), deop oleep (DS) and rapid eye movement {REM) sleep
according to the following criteria:
Stt\to of s-w.
KEG EMG EOG Remarks (muscle tone) (Eye movements)
-------------------------------------------~---------------------1. Active
Awake
2. AIH\ke Quiet
3. Slow W:wo ~n c'N'
4. Deep Sleep
5. REM Sleep
Deaynchronized Voltaget:: 30 pV Frequency= 30-40 Hz or more Desynchronized, oplndlo mny be present but lese than 25% of t.he time
EEG eynohroni?Jatlon* between 2f>- M11% of \".h(' rc0ordlng tlmo
Movements artifacts
Low muscle to no
Lower than AQ
Synchronization Very low more than 50% of the recording time
EEG desynchroni- No muscle zation, higher tone in voltns~ (40 pV) than W
Frequent/ irregular
Few or aboent
Fig. MF.4
Fig, MF.4
Absent *High amplt. (50- 300 P._V) and low freq.
(6-24, Avg. 12~16) EEG waves
(Fig. MF.4)
Absent
Fig. MF.4
Frequent and and monophasic eye movements
lh.Ho~<wor, in t.h lo n t.udy, t.1w o tagoo o.f wnkofulneoo (awake
quiet. nnd nwake movement) were taken together under
"HakefulneoB, .. whereas all the three stags of sleep viz. SWS,
DS and REM sleep wore taken together under "sleep'', It was then
calculated that how much time the animal has spent in different
stages in each of the blocks of 5 min and during total
recording period. For analysis of the data "wakefulness" was
taken into consideration to avoid zero readings for sleep
during tho post-injection period.
71
AC TIVE AWA KE
EEG ·¥~Jit •• t,:tJ·~~··-·--·j;:'f~IMh.,., .. ~r·kW EMG ----------------------------~ EOG~~-. , I ,,. I I
QU IE T AWAKE
fl ...... ~~~
SLOW SLEEP ....... "~"~~~~ DEEP SLEEP
-------- -I 20 Sec.
Fig. M¥.4. : The figure sho ws polygraphic traces of EEG, EMG and E(~ characterizing sleep-wakefulness . Calibration : vertical bar for EEG, 100 pV; EMG, 50 pV and EOG, 200 pV. Speed calibration : 20 s ec.
[II] QUANTIFICATION OF RECTAL TKMPKRATURK:
During experimentation, Tree was recorded every
However, for statistical analyses, depending upon the
5 min.
duration
of recording, Tree at every 10, 15 or 30 min interval was taken
into 1 consideration. To avoid more number of pointe I Tree from
long term (6.00-'7 .5 hr) I medium (2.5 - 3.5 hr) and short term
recording (1.00- 2.00) were analyzed at every 30 min, 15 min
and 10 min interval, respectively. Tree for 101 15 and 30 min
intervals were calculated by averaging the recorded Tree for 10
(two 5 min intervale), 15 (three 5 min intervals) and 30 min
(six 5 min intervals), respectively. Effectiveness of chemicals
in influencing Tree was determined by comparing the Tree at
every 101 15 or 30 min pre-injection intervale with Tree for
identical intervale in the poet-inJection period. The rate of
change of Tree wao c8lculated by comparing the actual Tree at
different post-inJection intervals with the average Tree of the
whole pre-·lnJootion period ( i. o. the baoal temperature).
[III] llAIA ANALYSIS;
(1) ~ AnnlYalo:
Dn t.a ann lysis was done in the following steps:
i) The effect of saline/chemical on S-W was calculated by
comparing the extent of W at different pre-inJection intervals
with the extents of W at different intervals during post
inJection period.
(il) The extent of W before and after saline inJection for all
tho ohomtoolt.,, t)XO(')pt prn.lllonin whore N~N-DA WtltJ uood, into m
and lPOA were taken as control.
72
(ill) The extent of W during pre- and :post-saline-injection
period were compared with W for respective intervals before and
after injection of each of the chemical into m- and lPOA.
(iv) A significant difference between the extent of W during
rcapoctlvo po~t-aallne and poat-chemlcal injedtion intervale
indicated about the effectiveness of the chemical in
influencing W after its injection into m-/lPOA.
(v) J\nd finally, tho differential influence of mPOA and lPOA
wno dctormlnod by comparing the pre- and poet-injection (saline
and chemicals) values of W at different intervals for mPOA with
the extents of W at identical intervals for lPOA.
( 2 ) I.r.rul Annlyu is:
Same procedure and steps were followed for a) determining
tho effeot. of oallno/chomioal on 'l'reo and b) for determining
the difference between the effectiveness of mPOA and lPOA in
l nfl \lt'HW lnll 1' r<'o .
[IV] STATISTICAL, HKTIIODS:
The data obtained in this study did not show a normal
distribution which is the essential precondition fo~ many of
the parametric tests like t test, F test etc. used for testing
the validity of difference between two conditions (e.g. W
during pre-injection and post-injection period). On the other
hand tho use of non-parametric test ia advantageous in the
senae that there is no such precondition about the normality of
t,ho ditJ trlbution. In thio study, therefore, non-parametric
statistical methods were applied for testing the statistical
73
slgnl.ficance (Siegel, 1956; Conover, 1971).
The tests applied for analyzing the data included :
(1) Wilcoxon matched pair signed rank test for small samples.
(2) Mann-Whitney test for small samples.
( 1) tUlcoxun Hatched rn.1.r. Uls.ncd llnn.k. IcJl.t. !.ul:. Small SampleR;
This test was applied for comparing the extent of W in
pt·o-lnjcc t.ion period w 1 th that of poet- injection values for the
aamo rat. By this method, following steps were followed to
determine the significance of the difference:
(i) Differenceo between the scores of the matched pair, under
two conditions wore calculated.
(ii) The differences were ranked, without regard to sign, in a
manner that rank one (1) was allotted to the emalleat
difference and two (2) to the next smalleat and so on.
(iii) The sign of the difference waa affixed to each rank.
(iv) Polro l1ovlng no difference wore dropped from the analysia.
In thn t. cnoo N Wt\o oalcula ted no, N = the number of matched
pnlro mlnuo the number of pairs whooe difference = 0.
(v) Teat statistic (T), which is equal to the smaller sum of
like signed ranks, waa calculated.
(vi) T calculated wao finally compared with the tabulated
value of T ~iven at different level of significance, according
to the following rules :
11 0 (Null hypotheois) reJected if T .i Ta for observed value of
• N •.
H0 ooo<n)t.od if T > Ta for oboerved value of N.
(Here, Ta = tabulated value of T at a level of
74
aignificonce). Therefore, the difference in W/Treo between pre
and poot-lnjection period woe accepted to be significant when
the calculated value of T waa < the tabulated value of Ta at a
given level of aignificance.
( 2) Hu.nn::.tihilno~ !Ddt. !o..r. fiiUlll 1io.mllwt.:...
Tl1io method is also known as Wilcoxon teat of independent
nnmpl<''' ( CoiWV<}r, l fi'T 1). 1 t; Wt\O uo<H' for tmalyoio of data
comins from two independent samples (from different group of
rn t.1:1) . I~' or c.:'xomple, the oompar loon between ex ten to of W during
post-injection period after saline injection into mPOA and
lPOA, or the comparison of the extents of Tree before and after
saline injection into lPOA with that of the Tree before and
after clonidine injection into lPOA etc., etc.
In this method,aignificance of the difference was
calculated in the following stope
{i) All the scoreo, irrespective of their nature, were ranked
in o mt.\IHHH' that omalleot ocoro wao allotted a rank of one ( 1)
and nt~xt higher to amnlleot as two ( 2) and so on.
(ii) Tl\en, T (Toot Stotiotlca) was calculated by the formula:
T = S - n(n+l)/2 Where,
ll = olze of the population
s = the sum of the ranks assigned to observations
from one population.
(iii) The calculated value of T was compared with the table
value of T and and level of oisnlficance was
folh')I.JS!
calculated as
{a) In t.1w tnllod tetJt reject 110 at a level of significance a
75
if T is leas than a/2 quantile Wa/2 or if T is greater than 1 -
a/2 quantile Wl-a/2. Accept 11 0 if T is between or equal to the
two quantiles.
(b) ln one-tailed toot, omoll vuluoo of T indicate that Ill is
true. Therefore reject 11 0 a level of significance a if T is
loou t.hon t.ho a t.h quan tllo Wa. Aooept. 11 0 if T is greater than
or equal toW.
(c) In one-tailed teet, largo valuec of T, or small valuea of
T, indicate that Hl is true. Therefore reject H0 at the level
of oisnlfioonoo a if T io greater than Wl-a, or (equivalently)
if T. io leoo than Wa. Ac6ept H0 if T is less than or equal to
Wl-a.
I.r.o.ll Analyoio:
Same statistical teats (Wilcoxon matched pair signed rank
toot and Mann-Whl t.ney t.eot.) wore applied for determining the
level of significance of the difference in Tree after injection
of dlfferot\t chemicals into m/lPOA, and in determining the
differential influence of mPOA and lPOA in the regulation of
Tree.
76