A simple integrator to process the electroencephalogram of small laboratory animals

A Simple Integrator to Process the Electroencephalogram of Small laboratory Animals

LORENZO BEANI, CLEMENTINA

BIANCHI,

PIETRO MARCHEII,

AND SERCIO TANGANELLI

A simple

integrator

This device quency

to process

simultaneously

(SF) and wave

the actual

the EEG of small laboratory

integrates,

From these

values

quanta1 component

of SA is calculated.

and

SA/SF,

induced

EEG activation,

different

seizures,

quantified.

Moreover,

parameters

(wave frequency of the

assumed

By measuring conditions,

wave

as a simplified

wave

area,

the average

allows one to correlate

fre-

index

SA/SF,

i.e.,

of the

wave frequency reaction,

drug-

can be easily distinguished

and SA/SF) with neurochemical

pharmacological

is described.

average

such as arousal

and anesthesia

this approach

animals

intervals,

the average

such as the release of neurotransmitters knowledge

short

area (SA, in p,V x set),

EEG power.

by calculating

at fixed

and

the fundamental

EEG

signs of brain activity,

from the brain. Thus, a more complete

profile

of neuroactive

compounds

is made

possible. Key Words:

Electroencephalogram;

alog digital

conversion

EEC waves area;

EEG waves frequency;

An-

of EEG

INTRODUCTION

A simple eters,

even

method

to quantify

without

computer

the essential facilities,

electroencephalogram

may be useful

param-

(EEG)

for many

pharmacological

purposes. In the past, measurements stein and Beck, 1965; al., 1974) or analysis Hjorth,

1970),

of the integrated

and average

Pirch and Rech, 1968; Etevenon of the integrated

and average

and of the individual

employed. Since these nitudes, i.e., frequency

approaches or voltage,

patterns, such as high voltage neglected.

frequency

wave amplitude

and Boissier, wave frequency

bands

(Neal

and Bond,

or high voltage

slow wave activity,

At present, methods based on the measurement of total voltage frequency spectrum distribution are preferred (Young et al., 1978;

From the Department

about

of Pharmacology,

(Byford,

et

1969;

1985) were

reflect only one of the two fundamental magrespectively, information regarding certain EEG

spiking

1981) so as to give information

(Gold-

1971; Dzoljit

amplitude/time

University

of Ferrara,

might

power and its Matthis et al.,

and amplitude/frequency

Via Fossato di Mortara,

be

23,441OO

pat-

Ferrara,

Italy. Address

reprint

23, 44100

Ferrara,

Received

requests

January

to: Professor

Lorenzo

Beani,

lstituto

di Farmacologia,

Via Fossato di Mortara,

Italy. 1986;

revised

and accepted

June 1986.

219 Journalof

Pharmacological

D 1’387 Elsevier

Science

Methods Publishing

17, 219-229 Co..

Inc.,

(1987)

52 Vanderbilt

Avenue,

New

York,

NY 10017

220

1. Beani et al. terns.

This

kind of elaboration,

not immediately effect of drugs

offer

on the EEG of groups

In this paper, a simple and area at fixed each moment. voltage

however,

requires

the few, essential

time,

digits

of small

apparatus is described

equipment

and does

to evaluate and compare the

laboratory

animals.

which integrates

the wave frequency

so as to calculate the average wave area and frequency

The value of the integrated

x time unit, which

is a simplified

of the arousal

reaction,

at

wave area (in ~.LV x set) is a dimensional, function

the average wave area may be considered approach makes it possible to discriminate and intensity

expensive

useful

of the voltage power @V2), while

the quanta1 component different EEG patterns,

and quantify

drug-induced

of such unit. This detail time course

changes in the EEG.

METHODS Animals Guinea

pigs of either

the experiment,

sex, weighing

the animals,

400-500

anesthetized

g, were used. At 2-3

with sodium

pentobarbital

were submitted to the implantation of steel screws in both frontal bones. A fifth screw in the nasal bones served as ground reference.

days before

(30 mg/kg i.p.) and parietal The screws,

insulated by means of dental cement, were connected to a small valve socket, soldered to the skull. The valve socket was used for the EEG records, performed with a four-channel direct-writing instrument (EEG Galileo, Model R 32 T). A connecting cable, provided with a swivel, ensured the freedom of the animal during the experiment. One hour after having set up the guinea pig in a soundproof chamber with dimmed light, the control EEG and the corresponding integrated tracing were recorded.

Drugs

were generally

injected

i.p., and their

effect was fol-

lowed in time.

Scheme of the Integrator The signals

of four monopolar

leads (parietal and frontal

by the EEG stages, were fed into a summing 1). The summed signals were subsequently

on each sides),

amplified

amplifier, through a buffer stage (Figure integrated for their frequency (SF) and

area (SA). The distinction of the EEG waves from the noise was made by a discriminator set at +I0 ~_LV,and the unification of the waves was made by a monostable multivibrator. the same time, their

Then,

the summation

of the signal

the waves were processed

area (SA) was carried

out.

The

by a full

outputs

number rectifier,

(SF) was performed.

At

and the summation

of

of SF and SA stages were fed into a

final stage of logical transfer and reset, controlled by a timer. This last device connected alternatively the pen of a direct-writing recorder with a) the integrator of the wave area for a preestablished interval of l-99 set and b) the integrator of the wave frequency for 0.1 sec. Repeating the whole cycle (a + b) produced a tracing composed of a sequence of right-angled triangles representing the SA, followed by a twitch of the pen in the opposite direction representing the SF of the preceding SA (top of Figure I). The height of both signals was proportional to the corresponding integrated

values,

whereas

the more

or less

regular

slope

of the triangle

hy-

221

SA

I

INPUT FIGURE 1. Scheme of the integrator used to process the tracing of four monopolar EEG records (input), in order to obtain the integrated values of wave frequency and wave area (WV x see) at fixed intervals. For explanation, see text. At the top of the drawing, integrated area and frequency values delivered from the EEG, fed with 10 pulses of lo-uV and 30-20lo-msec duration (left) and with 30-20-10 pulses of 10 JLVand lo-msec duration (right). Under each integration cycle of 1 set, the corresponding !&A/SFratio has been calculated. Calibration of SA = 4 uV X set; calibration of SF = 10 pulses.

I

FIGURE 2. Descriptive detailed scheme of the electronic components shown in Figure 1. 0, 1, 2, 3 = channel input; A, B, C, D, E, F = low noise operational amplifiers (O.A.); G, I = high performance O.A., low input current; H, 1 = general purposes O.A.; U, = output of summing amplifier; lJz = output of SA; U3 = output to pen recorder; 7413,74121 = digital circuits of lTL series; time constant of SA = 5 set; time constant of SF = 0.5 sec.

r

L

EEC Integrator for Small Animals potenuse 3, panel

depended 6). Details

on the more or less homogeneous of the electronic

circuit,

wave amplitude

component

specifications,

(see Figure and the time

constants of the integrators are given in Figure 2. This kind of electronic processing implies that: 1. The waveform

areas of each channel

are summed

in every

they are or are not synchronous.

Thus, the measured

of the four

and two frontal

records

2. The waveform

(two parietal

frequencies,

level discriminator

amplifier

finds the trigger

as one,

occurrences,

i.e.,

when

leads).

if the waves are synchronous

nels, are read by the summing

instance,

SA is the actual integration in two or more

single event.

chan-

Subsequently,

the

both for the single waveforms

of sufficient voltage (above IO FV) and for the synchronous, (summed) small waves reaching the threshold, so that parts of these waves are also counted. Consequently, the SF value represents overall events (single or synchronous), temporal sequence. It is worthy those

of note that the frequency

reported

depressed

in the literature

states of the brain

the average integrated frequency of the reaching the integrator according to a

values

for different

obtained

conditions

in this way fit well such as normal,

(see Results and Figure 4). In order

alert,

to define

with and

the SA

values in terms of PV x set and with the aim of easily calibrating the sequence EEG-integrator-pen recorder, the equivalence between the monopolar pulses delivered

from a square wave generator

battery

was tested.

set at 30 msec,

When

the time

the biphasic

same area as the monopolar wave generator.

and the biphasic constant

pulses of the EEG calibration

of the amplifier

pulses of 20 PV delivered

stages of the EEG was

by the EEG battery

pulses of 40 PV and 10 msec delivered

For EEG processing,

we found

it convenient

gave the

by the square

to adjust the sensitivity

of the integrators and of the whole system so as to obtain with 50 biphasic pulses of 20 ~J.Van SA “triangle” 1 cm high (corresponding to a total input of 80 FV x set, i.e. 20 FV

x set for each channel)

to 50 waves

simultaneously

and an SF “twitch”

delivered

1 cm high (corresponding

to each channel).

Calculations As a rule the sampling

of SA and SF, i.e.,

the reset of the integrated

area and

frequency values, was performed every 5 sec. This time interval proved to be the best to analyze the SF and SA patterns of the animals submitted to pharmacological treatments.

Only

in the case of arousal

reaction

time interval set at 1 sec. From 10 subsequent twitches) the average values of SA and SF were

by environmental samples obtained.

stimulus

was the

(i.e., 10 triangles and 10 Thus, the frequency/set,

the SA in 1 set, and the average wave area, i.e., the SNSF ratios were calculated. For example, if the mean of 10 subsequent SAs, taken every 5 set, was 20 mm (i.e., 160 kV, the calibrationi being set at 1 cm = 80 pV, see above) and the mean of 10 corresponding SFs was 10 mm (i.e., 50 waves), it follows that the SA (in 1 set) was 32 PV and the SF (in 1 set) was 10 waves. Thus, the SA/SF ratio in that period of 50 set was 3.2 FV

x

sec. To simplify,

the average

height

(in mm)

of 10 SA can be

223

a

EEG Integrator for Small Animals multiplied

by a factor

of 1.6 and

corresponding SF. Clearly, these tedious feeding

a second

determinations

calculations

pen of a double were

fully the changes

divided

made

by the average

can be avoided

pen recorder.

every 5-10

in the essential

min,

height

(in mm)

with a proper

electronic

For most purposes,

an interval

EEG parameters

sufficient

caused

of the device

however,

these

to describe

faith-

by different

drugs.

DRUGS Freshly used:

prepared

solutions

pentobarbital

sodium,

of the following phenobarbital

commercially

sodium,

available

pentylentetrazol,

drugs

were

y-aminobu-

tyric acid. RESULTS The EEG frequency wakeful

state were

frequencylsec Examples

and the integrated homogeneous:

= 23.2

+ 0.75;

SA (FV

of the EEG tracing

and average

the mean x 1 set)

wave area of guinea

pigs in

(r~:SEM) in 30 animals

values = 21.9

+ 1.2; %/SF

and of the corresponding

were:

= 0.91

integrated

+ 0.05.

records

taken

in three different conditions are shown in Figure 3. Clearly, 1) the state of wakefulness is associated with a regular sequence of high frequency and relatively low SA and W/SF are made

values;

evident

SF values;

2) the seizures

by an irregular

and 3) the slight anesthesia

acterized

by regular

sequences

induced

sequence

by pentylentetrazol

of high frequency

after pentobarbital

of low frequency

and the average

To check if this method rodepression and arousal the following 1. Some

animals

mg/kg been

tests were were

i.p.1 and then synchronized.

reaction

caused

wave area,

i.p.)

is char-

.%/SF values.

ratio always moved in the same taking into consideration only

i.e., the SA/SF ratio.

performed: injected

i.p. with

submitted

subanesthetic

to acoustic

doses of pentobarbital

stimulation

Figure 4A shows the time course by acoustic

pentylentetrazol,

i.p.)

could quantify the time course and intensity of the neureaction induced by chemicals or sensory stimulation,

stimulation,

creases average area modified 2. Other guinea pigs, anesthetized with

(20 mg/kg,

and very high SAand

Since the drug-induced changes in SAand the %/SF direction, the subsequent results will be described the frequency

(40 mg/kg,

and high SA and SA/

50 mg/kg

which

by the barbiturate. with phenobarbital,

after the EEG tracing and degree

increases

(20 had

of the arousal

frequency

and

de-

100 mg/kg i.p., were injected

i.v. In Figure 46 the frequency/set

and the SA/

FIGURE 3. Four monopolar frontal and parietal EEC records with the corresponding integrated tracings, showing wave frequency (SF), wave area (SA), and the calculated average wave area @A/SF). The records were taken from a guinea pig in: awake state (A), after pentylentetrazol 80 mg/kg i.p. (B), and after pentobarbital 20 mg/kg i.p. (0. Integration cycles of 5 sec. Calibration of SA = 80 PV x set, calibration of SF = 50 waves.

225

226

.

1

D

P

EEG Integrator for Small Animals SF ratio clearly describe

duration

and intensity

of the analeptic

effect of the latter

drug. 3. Still others effect

were

SF ratio reflects

changes

4. Finally,

some

intracerebroventricularly

transmitter

induced

the changes

tex (Tanganelli then,

injected

of this putative

by GABA

detected

with GABA,

was followed.

As shown

are polyphasic.

in the acetylcholine

50 Fmol,

and the

in Figure 4C, the SA/

Interestingly,

this pattern

release from the cerebral

cor-

et al., 1985). animals

the frequency/set

were

treated

with

and the average

different

of neurodepression.

In Figure 4D, the relationships

tween

doses and frequency

pentobarbital

doses

wave area were

of pentobarbital calculated

(of opposite

and average

i.p.;

at the peak

regression)

be-

wave area are shown.

DISCUSSION The above tervals

described

differs

from

integration

the analysis

that only the average

frequency/set

sumed as a simplified, quanta1 Consequently, our approach trum

distribution,

addition, chronous

but simply

this device waveforms

indicates

as the waves.

of the wave

than

measured

short

in-

(~LV’) in

area is asspec-

frequency.

In

This condition,

frequency

frequency

in the latter

by excluding

setting.

low voltage

condition Therefore,

the low voltage,

SF and the SNSF

the SA showed

however,

seems

to us

any noise of the

waves could be partly

the relevance of this loss, the integrated tracings by setting the discriminator level at 10 and 1 ~_LV.

in the standard

measuring the frequency to be rather limited. while

wave

average

was set at IO ~.LV(so as to exclude

from the SF. To check animals were compared

The average

and the average

shifts in the overall,

chain) the asynchronous,

The values

lationship,

is measured

and area at fixed,

bands and total power

index of the actual EEG power. does not evidentiate shifts in the frequency

Since the level discriminator recording-amplification

those

frequency

does not distinguish between sequences of long-lasting synof low voltage from sequences of generalized spiking having

the same area and frequency very infrequent.

canceled of some

of wave

of wave frequency

were

only 4-8%

the error

asynchronous

ratios showed

in

events seems

a dominating

the same kind and direction

higher

introduced

of changes

inverse

re-

as the SA/

4 FIGURE 4. (A) Wave frequency/set and average wave area (SASF) recorded and calculated in pentobarbital (PB)-pretreated (20 mg/kg) guinea pigs, submitted to acoustic stimulation. Time course and intensity of the changes induced by the stimulation (prevailing beta activity) are shown. Integration cycles of 1 sec. Mean f SEM of six experiments. (B) Awaking effect of pentylentetrazol (PTZ) 50 mg/kg i.v., versus anesthesia induced by phenobarbital (PH) 100 mgkg i.p. in the guinea pig. Integration cycles of 5 sec. Mean + SEM of five experiments. (C) Effect of CABA (50 Pmol) injected intracerebroventricularly (i.c.v.) on wave frequency/ set and average wave area in the unanesthetized guinea pigs. Integration cycles of 5 sec. Mean + SEM of five experiments. (D) Relationship between pentobarbital (PB) dosage, wave frequencylsec, and average wave area. Integration cycles of 5 sec. Mean * SEM of seven experiments for each dose.

227

228

1. Beani et al. SF ratios.

Since the SA is an indication

calculation

of WSF

This fact

may represent

may partly

synchronous

depend

waveforms

of the calibration as single events,

on the

delivered

procedure, although

of the total

a reliable kind

This IS clearly

shown

of electronic

see Methods)

counting,

because

by the summing

in terms of the %/SF activity.

a relatively

that the

itself. the

amplifier

Thus, the synchronous

B of Figure 3, where

SA and

it seems

the power

of the EEC (as in the case

were counted

their areas were integrated.

in panel

to a high

EEG power, replacing

to the four channels

(e.g., spiking sequences) were privileged be discriminated from the asynchronous corresponds

index

events

ratio and could well

a series of spikes

low SF (sixth signal

of the

in the EEC integration

tracing). In conclusion,

this approach

sleep and paroxysmal

spiking;

allows

one to: 1) distinguish

2) measure

time course

the awake

and intensity

state from

of the arousal

reaction and neurodepression induced by sensory stimuli or chemicals; and, consequently, 3) define the rank order of potency in a series of neuroactive compounds. The

most

changes tative

common

in behavior

transmitters

EEG patterns

can,

and in neurochemical from

the neocoftex

therefore, be parameters,

of freely

related with drug-induced such as the release of pu-

moving

animals

(Figures

3 and 4)

[see Beani et al. (1968, 19781, Antonelli et al. (1984), and Tanganelli et al. (1985)l. This last point is of particular interest in order to define the pharmacological profile

of a drug,

behavior,

depending

EEG, and brain

on whether

or not it causes

dissociation

between

biochemistry.

This work was supported by a C.N.R. comelli is gratefully acknowledged.

(No. 83/02036.04) grant. The technical assistance of Mr. A. Gia-

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