The in situ perfused spinal cord of the rat

The In Situ Perfused Spinal Cord of the Rat Applicability of Drugs and Chemicals, Sodium-Lithium Exchange, and Calcium Reduction to Functional Intact Central Nervous System Tissue

WALTER

DEUTSCHMANN AND HANS H. WELLH~NER

A new forequarters

preparation,

adult rat, is described icological

with

investigations.

The

mercuriphenylsulfonate, acid ethyl

ester

exchange

the intent

medium

were using

advantages

spinal

2) the oxygen function,

Key Words: Dinitrophenol;

in the perfusion

central

effects,

actions

the flow

medium.

of substances

rate, the sodium

and the pCOz of the perfusion

forequarters

has a number

of advantages over an experimental

supply

dependent

forequarters

from

ions,

preparation;

acid ethyl

circulation.

dependent

ester;

These is under

on the cardio-

under

each animal;

5) the action of substances of drugs,

including

the preparation

by the substance

may be obtained

concentrations

(-)-Nipecotic

supplying

to the cord is no longer

curve

preparation,

on its physiological

of fluids

may be impaired

may be performed;

Perfused

and (-)-nipecotic

The study was extended

new perfused

which

of extreme

and toxp-chloro-

The

dose-response

experiments presence

reduction primary

metabolic

cord of the

cord,

an intact animal

vascular

bicuculline,

the pH, the pOz,

are: 1) the composition

control; complete

secondary

measured.

the intact cervical design

between

spinal

of strychnine,

were studied.

and calcium

concentrations,

cervical

for pharmacological

actions

dinitrophenol,

discharges

to discriminate

and ion changes and their and potassium

perfused

to its suitability

dose-dependent

ouabain,

on mass reflex

to sodium-lithium With

the in situ

respect

study;

3) a

4) washout

can be studied

in the

etc.

Spinal

cord in situ;

Ouabain;

Bicuculline;

Strychnine

INTRODUCTION

Drugs

as well as changes in the ionic composition

the study of synaptic transmission

in dissociated

of media can serve as tools

central nervous

in

system (CNS) prep-

arations (homogenates, synaptosomes, slices, and tissue and cell cultures). These procedures used on in vitro preparations may have undesirable actions that preclude their systemic application in the intact animal. A perfused forequarters preparation, including the intact cervical spinal cord, could surmount the limitations of

From the Abteilung Toxikologie im Zentrum Pharmakologie schule Hannover, Hannover, Federal Republic of Germany.

und Toxikologie der Medizinischen

Hoch-

Address reprint requests to: Dr. W. Deutschmann, Abteilung Toxikologie, Zentrum Pharmakologie und Toxikologie, Medizinische Hochschule Hannover, Konstanty-Gutschow-StraBe 8, D-3000 Hannover 61, Federal Republic of Germany. Received August 1984; revised and accepted August 5, 1985. 143 journalof

Pharmacological

0 1986 Elsevier

Methods

Science Publishing

15, 143-155.

0160-5402/86/$03.50

(1986)

Co., Inc.. 52 Vanderbilt

Avenue, New York, NY lM)17

144

and H. H. Wellhiiner

W. Deutschmann experiments perfusion portion

in vivo.

We

have recently

of the rat cervical spinal of the CNS

mann et al., 1983). to the perfused

in an excellent However,

pharmacological

functional

a method

for

for many hours,

and histological

the

intraarterial

maintains

condition

a large

(Deutsch-

it cannot be taken for granted that drug application

forequarters

resent a valid experimental onstrate that the perfused

described

cord, which,

preparation

or changes of its ionic environment

rep-

design. Therefore, an attempt has been made to demforequarters, and with it the cervical spinal cord, allow

experiments

with

functionally

intact CNS

tissue,

which

hitherto

have been reserved to other isolated organs or to in vitro CNS preparations. thermore, it is the aim of this work to present an uptake-inhibiting technique can be applied to the perfused afflicted

with

MATERIALS

unwanted

AND

forequarters

preparation

Furthat

and that is not too seriously

actions.

METHODS

Chemicals Bicuculline,

dinitrophenol,

ouabain,

and strychnine

sulfate

were from

chloromercuriphenylsulfonate (pCMS) and gallamine triethiodide perfluorotributylamine was from 3M Company, and the emulsifier from

BASF

Wyandotte.

(-)-Nipecotic

acid ethyl

ester

tartrate

Serva,

p-

were from Sigma, Pluronic F68 was

was synthesized

by

Dr. P. Krogsgaard-Larsen, Copenhagen, and was a gift of Dr. W. Loscher, Berlin. Saccharose and all inorganic salts were from Merck, Darmstadt. Solutions of pCMS and (- )-nipecotic acid ethyl ester in saline were freshly prepared before use, and 0.02 mmol of ouabain were dissolved with 1 ml of absolute ethanol. This stock solution

was added to the perfusion

medium

was 0.87 x lop6 M with the lowest

medium.

(lop4 M) ouabain concentration. Drugs have to cross an intact blood-brain medium through

to the in situ the barrier

perfused

was estimated

for pCMS and dinitrophenol The pK value of (-)-nipecotic Krogsgaard-Larsen. Animals

spinal

Other

and Perfusion

The

ethanol

concentration

barrier cord.

when applied with the perfusion

The

for a particular

fraction drug from

available

for

diffusion

its pK value. pK values

were determined by titration in aqueous acid ethyl ester was kindly communicated

pKvalues

in the

(1O-6 M) and 87 x lop6 with the highest

were taken from the literature

solutions. by Dr. P.

(Windholz,

1976).

Technique

Experiments were performed in male Sprague-Dawley rats weighing 350-450 g (Charles River Wiga, Sulzfeld). Their cervical spinal cord was perfused intraarterially with a Krebs-Henseleit solution containing microdispersed perfluorotributylamine FC 43 (20% v/v) as an oxygen carrier and the polyol Pluronic F 68 (5% w/v) as an emulsifier with additional colloid-osmotic action. The preparation of this medium (FC medium),

the surgery,

the perfusion

technique,

the measurements

of the ion

concentrations and of pH, pOz, and pC0, in the affluent and effluent medium, determination of the venous outflow, and the details of neuronal stimulation recording were essentially as described by Deutschmann et al. (1983).

the and

Applicability of Drugs to Spinal Cord In Situ Briefly, after abdominal incision, the aorta and the inferior caval vein were cannulated. The tips of the inlet and outlet catheters were located below the aortic arch and the right atrium, respectively. The entire animal anterior to the diaphragm was perfused. Filling of the arteries supplying the cervical spinal cord, the brain, and the nerves and muscles of the forelimbs could be demonstrated radiographitally. Before extracorporal circulation was started, 100 ml of FC medium were passed through the preparation to remove all blood from the vascular system. The perfusion loop was then closed and the 100 ml of FC medium remaining in the system were recirculated. The pH, the pOz, and the pCOz in the FC medium were determined with electrodes in a flow cuvette placed in shunts parallel to either the affluent or the effluent perfusion line. The venous outflow was determined intermittently with a graduated tube. Sodium and potassium were measured in the supernatant of the centrifuged FC medium with a flame photometer. Stimulation

and Recording

Mass reflex discharges were evoked and recorded previous to perfusion and continuously under perfusion. In conditioning experiments the N. radialis was stimulated with variable delay before producing the test reflex (stimulus at N. medianus, recorded from N. ulnaris; the exposed nerves were mounted on platinum electrodes for stimulation and recording, and were covered with warm paraffin oil). The amplitude of the conditioned test reflex was set in relation to the amplitude of the noninhibited test reflex. Conditioning experiments were performed during the latter 10 min of an incubation period. All reflex discharges were stored on tape, and groups of ten reflex discharges were averaged off line.

Application

of Drugs during Perfusion

The procedure for drug application was as follows. Every 30 min the concentration of a particular substance was raised in the medium by stepwise addition of 0.5 ml of an appropriately concentrated stock solution. Ion concentrations were changed in the following way. The perfusion was switched from medium A to 200 ml of medium B containing the new ion concentration. The first 100 ml of medium B were discarded, and the remaining 100 ml were recirculated. An analogous procedure was used to wash out drugs. The concentrations given for drugs and ions are their concentrations in the perfusion medium. The incubation period of each new concentration or composition was 30 min. RESULTS Strychnine Convulsions evoked by an electric or acoustic stimulus occurred at a strychnine concentration of lO-‘j M (without gallamine). Mass reflex discharges, recorded in the presence of gallamine (0.9 to 1.8 x lop4 M), were increased by 2 x 10e6 M strychnine (Figure 1) and spontaneous discharges with long latencies (100 msec) occurred. These effects were hardly reversible on washing. Neither strychnine nor

145

I

0.05 mV

FIGURE 1. Influence of strychnine on mass reflex discharges, recorded from the N. ulnaris of the perfused forequarters preparation. The stimulus was applied to the N. medianus (upper line), to the N. radialis (middle line), and to both nerves simultaneously (lower line). left column: discharges registered prior to the administration of drugs. Center column: discharges in the presence of gallamine triethiodide (1.8 x 10m4 M) in the perfusion medium. Right column: discharges after addition of strychnine (2 x 10e6 M) to the perfusion medium. test reflex amplitude in X of initial value

inhibition uncon ditioned reflex H

-0 0

lo”

lo’ pCMS

10”

lo’

10”

10” wash

mol/l

FIGURE 2. Amplitude of an unconditioned medianus-ulnaris test reflex and its maximal inhibition by a conditioning stimulus to the radial nerve as a function of p-chloromercuriphenylsulfonate (pCMS) concentration in the perfusion medium. At all concentrations, the maximal inhibition occurred at a delay of 20 msec between the conditioning and the test stimulus. (O....... 0) Unconditioned text reflex amplitudes; (O---O) maximally inhibited reflex as a percentage of the unconditioned reflex.

Applicability of Drugs to Spinal Cord In Situ gallamine showed an influence medium and on its flow rate.

on the ion concentrations

in the recirculating

FC

p-Chloromercuriphenylsulfonate The pK values of 1.3 and 7.1 were measured for &MS. The reflex amplitude declined with increasing concentrations of the substance. The conditioned inhibition of the test reflex was progressively reduced by pCMS concentrations of IO-‘, IO-‘, 10P6, and 1O-5 M, respectively (Figure 2). The cornea1 reflex was abolished at concentrations between 10e5 and IOU3 M. The depression by pCMS of the test reflex was partly reversible by washing; the cornea1 reflex and the spontaneous respiration reappeared in two of four experiments. Virtually no changes of the flow, of the sodium and potassium concentrations, and of the pH, pOz, and pCOz occurred or could be related to @MS when the data were compared with data from longtime experiments (Deutschmann et al., 1983). Ouabain Ouabain at a concentration of 5 x 1O-5 M reduced the reflex amplitude by about 50%. No influence on the conditioned inhibition was detected at ouabain concentrations of 5 x 10-6, 10e5, and 5 x 10e5 M, and the spontaneous respiration as well as the corneal. reflex remained intact. When the ouabain concentration was elevated to lop4 M, no further decrease of the reflex amplitudes occurred, but the conditioned inhibition, the spontaneous respiration, and the cornea1 reflex were abolished and did not appear on washing. When the ouabain concentration was increased through IO- 6, 10P5, and 5 x lop5 M to lop4 M, the potassium concentration in the recirculating medium increased from 5.48 mM through 6.50, 8.50, and 9.33 mM to 10.64 mM, while the sodium concentration, the arteriovenous oxygen difference, the pCOz, the pH, and the flow rate remained virtually unchanged. Dinitrophenol A pK value of 3.65 was measured for this acid. The substance in a concentration of lop4 M led to a 50% reduction of the reflex amplitude and to a considerable reduction of the conditioned inhibition (Figure 3). The spontaneous respiration and the cornea1 reflex were abolished. When the perfusion was changed to fresh FC medium, the cornea1 reflex did not recover, the spontaneous respiration set in after 3 min, the reflex amplitude recovered to only 60% of its initial value, and the conditioned inhibition recovered completely. When the concentration of dinitrophenol was increased to 5 x 1O-4 M, all reflex activity was irreversibly abolished within 2 min. At 10m4 M, the sodium concentration remained virtually constant, potassium rose from 6.4 to 8.3 mM, the flow increased from 21.4 to 25.0 ml/min, and the arteriovenous oxygen difference increased from 510 to 605 torr. (-)-Nipecotic

Acid Ethyl Ester, Bicuculline

of (-)-nipecotic acid ethyl ester at a concentration of 5 x lop6 of all reflex components decreased moderately and simultaneously but not markedly faster than in the longtime experiments we have described In the presence

M, the amplitudes

147

148

W. Deutschmann

and H. H. Wellhiiner

o-l.. , . 0

40

220 100 300ms delay of conditioning stimulus

FIGURE 3. Action of dinitrophenol on the inhibition of the medianus-ulnaris test reflex by a conditioning stimulus to the radial nerve. (0) Before addition of dinitrophenol; (M) after 30 min of perfusion with dinitrophenol, 10m4 M; (0) after a washout period of 1 hr. The reflex amplitudes are expressed as a fraction of the unconditioned reflex.

et al., 1983). More important, the addition of the drug to the FC medium led to a reduction of the inhibition (evoked by a stimulus to the radial nerve) of a test reflex (evoked by stimulation of the median nerve and recorded from the ulnar nerve) (Figure 4A,B). The disinhibited test reflex components had a latency of about 5 msec. Test reflex components with a latency longer than 5 msec were not disinhibited. The disinhibition was virtually absent at a drug concentration of lO-‘j M; it was apparent at 5 x lO-~‘j M, complete at 1O-5 M, and reversible on washing. It could not be antagonized with bicuculline at 10m6 M. In the absence of (- )-nipecotic acid ethyl ester, bicuculiine itself at a concentration of 10m6 M left unimpaired only the test reflex component with a latency of about 5 msec but reduced markedly the components with longer latencies (Figure 5). At the higher concentrations of bicuculline (5 x low6 M, lop5 M), spontaneous discharges from the ulnar nerve and convulsions were observed. Virtually no changes occurred in the flow rate of the medium, in the concentration of sodium and potassium, in the blood gases, in the spontaneous respiration, and in the cornea1 reflex, when the animal was under the influence of (-)-nipecotic acid ethyl ester or bicuculline. fDeutschmann

Sodium-lithium

Exchange

When sodium was replaced with lithium (Li) in the FC medium, the osmolarity dropped from 335 to 275 mosm but was readjusted with saccharose. When the isoosmolar Li medium was substituted for the FC medium, the amplitudes of the

I

C--

FIGURE 4. (A) Influence of (- )-nipecotic acid ethyl ester on mass reflex discharges recorded from the N. ulnaris of the perfused forequarters preparation. Capital letters: The test stimulus was applied to the N. medianus. Small letters: An additional conditioning stimulus was applied to the N. radialis, 40 msec before the test stimulus. (A,a) Before addition of (-)-nipecotic acid ethyl ester; (B,b) after perfusion with (-)-nipecotic acid ethyl ester, 10m5 M, for 30 min; (C,c) After a washout period of 30 min. The arrows indicate the application of the test stimulus, and the dotted lines bridge the stimulus artifacts (see also Figure 48).

100

50

0

40

100

160

220

300 ms

delay of conditioning stimulus acid ethyl ester on the conditioned FIGURE 4. (B) Dose-dependent action of (-)-nipecotic inhibition (evoked from the radial nerve) of a test reflex (evoked from the median nerve and recorded from the ulnar nerve). (0) Before addition (-)-nipecotic acid ethyl ester; (A) 5 x 10e6 M nipecotic acid ethyl ester; (m) 1 x lop5 molar nipecotic acid ethyl ester; (0) After a washout period of 30 min. The reflex amplitudes are expressed as percentages of the unconditioned test reflex. For a direct illustration of waveforms, before drug addition, in the presence of (-)-nipecotic acid ethyl ester (1 x 1O-5 M), and after the washout period, see Figure 4A.

150

W. Deutschmann

and H. H. Wellhoner

FIGURE 5. Influence of bicuculline on mass reflex discharges recorded from the N. ulnaris of the perfused forequarters preparation. The stimulus was applied to the N. medianus. (A) Before addition of bicuculline; (B) after perfusion with bicuculline, 1O-6 M, for 30 min; (C) After a washout period of 1 hr. The arrows indicate the application of the stimulus, and the dotted lines bridge the stimulus artifacts.

0.1 mV 10 ms

+Y

‘:lic F

G

i-l

I

K

FIGURE 6. Influence of lithium-sodium exchange on the medianus-ulnaris reflex. (A) Before exchange; (B-F) after perfusion with lithium-containing medium for 4, 4.5, 5, 10, and 20 min, respectively; (C-I,K) washout with sodium-containing medium for 2, 3, 5, and 20 min, respectively.

Appli~bili~

of Drugs to Spinal Cord In Situ

test reflex declined fast and had virtually disappeared after 20 min. The short latency components were less sensitive than the long latency components. Whereas the former recovered readily when the perfusion was switched back to FC medium, the latter did not (Figure 6). Conditioned inhibition of the test reflex could be evoked to the same extent before and after perfusion with the Li medium. Due to the fast changes of the reflex amplitudes, no reliable studies on the conditioned inhibition could be done during the lithium perfusion or the washout period. During the lithium perfusion of 30 min, the spontaneous respiration and the cornea1 reflex disappeared within IO min, the flow increased from 18.8 to 27.0 ml/min, the arteriovenous oxygen difference decreased from 510 to 225 torr, the pC0, remained constant, the potassium concentration rose from 6.2 to 9.9 mM, and the sodium concentration rose from 0 to 30 mM during the initial 5 min and then remained at this value. Spontaneous respiration and the cornea1 reflex reappeared in the washout period. Reduction

of the Calcium Concentration

When FC medium was replaced with a medium containing no calcium, the reflex amplitudes declined, and spontaneous respiration ceased after 5 min, but the corneal reflex remained intact, and virtually no change was observed in the blood gases, in the concentration of sodium and potassium, and in the flow rate. After IO min of Ca-deficient perfusion, the calcium concentration in the medium was 0.4 mM (as compared to 1.4 mM in the FC medium at the end of the preperfusion period). On switching back to FC medium, the respiration, but not the reflex amplitudes, recovered after 1 min. DISCUSSION Strychnine This alkaloid fpK values 2.3 and 8.0 (Windholz, 1976)] easily crosses the bloodbrain barrier. Its convulsive action results from disinhibition (Bradley et al., 1953) due to a glycine antagonism (Curtis et al., 1971b). In the perfused forequarters preparation, strychnine produced the expected motoric convulsions and enhanced the spinal reflex activities just like in an intact animal. The handicap in relation to dissociated CNS preparations of motoric activities could easily be overcome with a neuromuscular blocking agent.

This substance and the related p-chloromercuribenzoic acid are thiol reagents that have been employed as inhibitors of transmitter uptake [@MS 10e4 M: in rat CNS homogenates and slices (Johnston and Iversen, 1971; Johnston et al., 1976a); in cat spinal cord slices (Balcar and Johnston, 1973); in the perfused central canal of the cat spinal cord (Fagg et al., 1978); in isolated spinal roots of rats (Davies and Johnston, 1974); pCMS IO-’ M: micropipette iontophoresis experiments: (Curtis et al., 1970)]. The &MS showed toxic systemic actions, even when applied locally in the perfused cat central canal (Fagg et al., 1978). In the present experiments, at

151

152

W. Deutschmann

and H. H. Wellhiiner

a pH value of 7.4 in the perfusion be ionized

completely.

seems to penetrate ically. Actions became the

into the CNS in an adequate

of @MS

apparent

representing flexes.

formerly

the that

on the reflex

at concentrations

concentrations

sumption

percentage

At a concentration

of IO-”

processes

In biochemical

The

(Figure

are more

to have the following

be considered acceptable

should @MS

action

1973;

preferable

pharmacological

of the

be explained than

than curve

on the excitatory as well

asre-

as on

effect. spinal cord, @MS

It does not seem to change

the flow

appears rate of the

or the pH, pOz, and pCOZ values, but candidates (Johnston and Iversen, 1971;

Fagg et al., 1978). to induce

lower

course

on inhibitory

its maximal

system-

spinal cord already

to pCMS

based on the in situ perfused

perfusion medium, the ion concentrations, it can inhibit the uptake of many transmitter Balcar and Johnston,

2) may

M, the pCMS

advantages.

it is applied

of magnitude

bell-shaped

sensitive

seems to have reached

studies

when

of the perfused

to four orders

in vitro.

inhibition

reflexes

amount

response three

used

inhibitory

excitatory

medium, the acidic phenyl sulfonate group in reverse to the local application,

Nevertheless,

a more

However, selective

in most instances uptake

inhibition

it might

by a more

agent.

Ouabain Ouabain

has been

used as an inhibitor

M (Johnston

et al., 1976a);

et al., 1973;

Davies

present

5 x lop4

and Johnston,

experiments

for both glycine

M (Davidoff 1974);

only moderate

lop5

changes

and GABA

and Adair, M (Iversen

uptake

[IO-’

1975); 10e4 M (Bennett and Neal,

in the electrical

196811.In the

excitability

of the per-

fused spinal cord have been observed at the lower concentration range of IO-’ M. The observed steep rise in the potassium concentration corresponds with the general

inhibition

suitable

of the

Na/K-ATPase

for use in a recirculating

by ouabain.

Therefore,

the

drug

appears

un-

system.

Dinitrophenol Dinitrophenol

has been

shown

[lop3 M (Iversen

and Neal,

1968; Davies and Johnston,

5 x 10e4 M (Davidoff

and Adair,

to inhibit 1975);

the uptake

both of glycine 1974; Johnston

10e4 M (Beart and Johnston,

and GABA

et al., 1976a); 197311. In the

present investigations it suppressed the electrical activity in the spinal cord already at a concentration of lop4 M. This drug uncouples the oxidative phosphorylation and thus impairs energy-dependent processes such as the reuptake of neurotransmitters. Its use as a tool in the in situ perfused spinal cord is limited, because the entire forequarters preparation suffered under the diminished utilization of oxygen due to dinitrophenol. The oxygen consumption rose from 1.1 ml oxygen/min and 100 g of body weight to 1.6 ml of oxygen/min and 100 g of body weight under the influence of lop4 M dinitrophenol. This impairment in turn leads to an increase of potassium

release

( - )-Nipecotic

and a decrease

1975)

resistance.

Acid Ethyl Ester, Bicuculline

The pK value of the piperidine Johnston,

of the vascular

N is 10.28 in the free acid (Krogsgaard-Larsen

but 9.3 in its ethyl

ester

(Krogsgaard-Larsen,

personal

and

communi-

Appli~b~ii~

of Drugs to Spinal Cord In Situ

cation). From the Henderson-Hasselbalch equation, it can be calculated that at the pH value of 7.4 in the perfusion medium, only about 1% of the ester exists in the nonionized form. Frey et al. (1979), after intraperitoneal injection of the ester, could detect only the free acid in the CNS of mice. Therefore, it seems reasonable to assume that the nonesterified (--)-nipecotic acid was also the active agent in the present experiment. Because (--)-nipecotic acid has proved to be a potent inhibitor of the GABA uptake (Johnston et al., 1976b), a decrease of the nonconditioned test reflex or an increase in the effectiveness of the conditioning inhibitor stimulus was to be expected; however, neither occurred. On the contrary, the conditioned inhibition of a short latency test reflex component was reduced by about 50% at an ester concentration of 5 x lO-‘j M. This concentration is reasonable. In brain slices the I& for the inhibition of GABA uptake was 5 x lop6 M (Johnston et al., 1976b). The action of (-)-nipecotic acid on the conditioned inhibition may be explained by a postsynaptic antagonist action of the substance at glycine-operated synapses, as described by Krogsgaard-Larsen et al. (1975). This hypothesis is consistent with our finding that in the presence of (-)-nipecotic acid ethyl ester in the perfusion medium, the conditioned inhibition is removed from the short latency rather than from the long latency test reflex components (Figure 4A); it also agrees with our observation that this disinhibition cannot be antagonized with bicuculline, which is a GABA antagonist both at presynaptic and postsynaptic receptors (Curtis et al., *1971a, 1977; Evans, 1978). Bicuculline [pKvalue 9.16 (Windholz, 1976)], after addition to the perfusion medium, readily crossed the blood-brain barrier, which was evident from its action on the long-latency components of the test reflex (Figure 5) and from its convulsive action at higher concentrations. Lithium-Sodium

Exchange

In the experiments of Bennett et al. (1973), lithium could not satisfy the sodium requirement of the uptake mechanism for several amino acids. Such a reduced uptake of transmitter substances can hardly be held responsible for the fast decrease of the test reflex amplitude in the present experiments. It seems more likely that the depolarizing postsynaptic action of (excitato~) transmitters declines when sodium is exchanged for lithium. Evans et al. (1977), recording from the ventral roots of the isolated frog spinal cord, found a reduction of the depolarizing action of several amino acids when they replaced sodium with lithium, sucrose, or choline chloride. Their results are more suitable to explain our finding in the in situ perfused spinal cord. low Calcium The transmitter release depends on the extracellular calcium concentration (Kelly et al., 1979). With reduced calcium concentration, one expects a reduced release of transmitters. In isolated hemisected spinal cords of kittens (Shapovalov et al., 1979) or of mice (Bagust and Kerkut, 1979), a reduction of the calcium concentration (to 25% of its initial value in the experiments of Bagust and Kerkut) in the superfusion medium did not impair axonal conduction but did depress synaptic activity. Our findings, with a calcium concentration lowered to 28% of its initial value, are in line

153

154

W. Deutschmann with

these

and H. H. Wellhiiner

results.

The

lowering the calcium mitter substance. Drugs

decrease

as well as alterations

as tools

of many of these procedures present

application work

to inhibit

is restricted

unwanted

ethyl

can be successfully

rations

with

spinal

CNS

on

of transare used

The systemic

toxicity

to an intact CNS in vivo,

preparations

response

(for instance,

studied

the intact cervical

reuptake.

Dose-CNS

effects

release

pH, and ionic environment

to dissociated

systemic

that we observed

an impaired

their administration

is offered.

with serious

amplitude

from

transmitter

precludes

an alternative

ester)

may result

of temperature,

in CNS pharmacology

and their

in the test reflex

concentration

in vitro.

relationships

pCMS

In the

of drugs

and (- )-nipecotic

in the new perfused

cord in it. Compared

forequarters

with

acid

prepa-

the intact animal,

yet another advantage of this preparation is apparent. It also allows for removal of drugs by perfusing the system with a drug-free medium (see section on washout of ( - )-nipecotic and substantial

acid ethyl ester and, in part, of dinitrophenol). changes in the ionic composition

may be accomplished

in a perfusion

exchange and reduction

of calcium

ouabain and dinitrophenol that not only forequarters could

could

primary

preparations,

be kept under

vented or clearly The present

central

indicated

isolated

Thus,

perfused

brain,

tinguish

effects

metabolic

reuptake with

be identified

inhibitors

intent

to show

in the perfused

effects were also detectable and

malnutrition

of the spinal

that the perfused

heart,

kidney, this

cord is either

pre-

can be taken.

forequarters

and liver

preparation

the early glycinergic

the later GABAergic

investigation

preparation

allows

with functionally intact CNS tissue, experiments that to isolated perfused organ preparation (such as the

In addition,

between

rapid milieu

on sodium-lithium

The potent

and adequate countermeasures

work demonstrates

preparations.

(see section

in this

nervous

but secondary

control.

pharmacological experiments hitherto have been reserved CNS

preparation concentration).

were included

Furthermore,

of the medium-extracellular

components

preparations

components

(see section

etc.) or to in vitro

appears to offer the ability of conditioned

on (-)-nipecotic

to dis-

inhibition

and

acid ethyl ester and

bicuculline). This work

was supported

We gratefully AG, Hannover,

determined

ische Hochschule ish School synthesized cology,

of Pharmacy,

pk values, measured

Copenhagen,

the substance Berlin.

of the Deutsche

the help we received

Hannover,

Universitat

the figures.

by a grant

acknowledge

Dr. W.E.

the calcium

received

Mrs. A. Kroger

Mrs.

K. Erdogan

ische Hochschule

Hannover).

Kulpmann,

prepared

from

provided

Department

Loscher,

technical

are indebted

to H.H.W.

Dr. R. Huschens, of Clinical

Kali-Chemie

Chemistry,

Medizin-

Dr. P. Krogsgaard-Larsen,

the pk value of Dr. W.

the manuscript

The authors

sources:

concentrations,

communicated

that we

Forschungsgemeinschaft

from various

(-

)-nipecotic

Department

assistance

and Mrs.

(all from the Department to Dr. G. Erdmann

Royal Dan-

acid ethyl ester and

of Veterinary C. Hotopp

of Toxicology,

for critical

remarks

Pharma-

helped

with

Medizinand helpful

suggestions.

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Fagg GE, Jordan CC, Webster RA (1978) Descending fibre-mediated release of endogenous glutamate and glycine from the perfused cat spinal cord in vivo. Brain Res 158:159-170. Frey HH, Popp C, Loscher W (1979) fnfluence of inhibitors of the high affinity GAEA uptake on seizure thresholds in mice. Neuropharmacology 18:581-590. lversen LL, Neal MJ (1968) The uptake of (‘H)GABA by slices of rat cerebral cortex. / Neurochem 15:1141-1149. Johnston GAR, lversen LL (1971) Clycine uptake in rat central nervous system slices and homogenates: evidence for different uptake systems in spinal cord and cerebral cortex. J Neurochem 18:1951-1961. Johnston CAR, Stephanson AL, Twitchin B (1976a) Uptake and release of nipecotic acid by rat brain slices. I Neurochem 26:83-87. Johnston CAR, Krogsgaard-Larsen P, Stephanson AL, Twitchin ET (1976b) Inhibition of the uptake of GABA and related amino acids in rat brain slices by the optical isomers of nipecotic acid. ] Neurochem 26:1029-1032. Kelly RB, Deutsch JW, Carlson SS, Wagner JA (1979) Biochemistry of neurotransmitter release. Annu Rev Neurosci 3: 399-446. Krogsgaard-Larsen P, Johnston GAR (1975) lnhibition of GABA uptake in rat brain slices by nipecotic acid, various isoxazoles and related compounds. f Neurochem 25:797-802. Krogsgaard-Larsen P, Johnston GAR, Curtis DR, Game CJA, McCulloch KU (1975) Structure and biological activity of a series of conformationally restricted analogues of GABA. / Neurochem 25:803-809. Shapovalov Al, Shiriaev Bl, TamarovaZA (1979) Synaptic activity in motoneurons of the immature cat spinal cord in vitro. Effects of manganese and tetrodotoxin. Brain Res 160:524-5X3. Windholz E, Ed (1976) The Merck NJ: Merck & Co, Inc.

Index.

Rahway,

155