MATERIALS AND METHODS - Information and Library …shodhganga.inflibnet.ac.in/bitstream/10603/24472/9/09_chapter 3.pdf · Parameter 1~1\G EMG EOG 1/2 Amp. Low Freq. 1.0 Hz 3.0 Hz

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

56

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 :

57

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

58

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

59

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:

60

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

61

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

62

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

63

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

64

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