EXPERIMENTAL NEUROLOGY 97, 243-254 ( 1987)

Kindling by Repeated lntraperitoneal or lntracerebral lnjection

of Picrotoxin Transfers to Electrical Kindling

DONALD P. CAIN 1

Department ofPsychology, UniversityofWestem Ontario, London. Ontario, Canada N6A 5C2

Received June 25, 1986; revision received February 12, 1987

Picrotoxin kindling was examined in hooded rats by intraperitoneal or intracere-

bral injection in different groups. Repeated intraperitoneal injectíon resulted in the
progressive kindling of convulsions in a dose-related manner. Bidirectional transfer
to electrical kindling ofthe amygdala was also ohserved. Intraamygdala injection of
small doses through a chemitrode resulted in progressive kindling and subsequent
transfer to electrical kindling. Intraamygdala injection of large doses generally re-
sulted in status epilepticus and the subsequent inability to evoke afterdischarge during
transfer testing due to considerable tissue damage surrounding the chemítrode típ.
Picrotoxin kindling is similar to kindling by a variety of convulsant agents. However,
direct injection into the amygdala easily evokes status epilepticus and brain
damage. © 1987 Academic Press, Inc.

INTRODUCTION
The kindling of seizures occurs as a result ofthe repeated electrical stimula-
tion of the brain or injection of a variety of convulsant drugs in initially
subconvulsant amounts (7, 33). The available evidence suggests that a neces-
sary property of kindling treatments is the ability to evoke paroxysmal dis-
charge of neurons and associated afterdischarge (AD) (32, 35).
In principle it should be possible to kindle seizures using agents that antag-
onize the action of inhibitory neurotransmitters. Such agents might be ex-
pected to cause the disinhibition of neurons, allowing them to fire paroxys~
mally. Upon spaced repetition, the kindling of seizures should result.
Attempts have been made to kindle seizures using such agents, but these

Abbreviations: AD-afterdischarge, ADT-AD threshold.

1
Supported by a grant from the Natural Sciences and Engineering Research Council ofCan-
ada. The technical assistance ofSusan Smith and Kathy Desborough is gratefully acknowledged.

243
0014-4886/87 $3.00
Copyright © 1987 by Academic Press, !ne.
Ali rights ofreproduction in any forro reserved.
244 OONALD P. CAIN

have met with varying degrees of success (13). Morin et al. (24) injected {j-
carboline i.p. and observed the kindling of seizures, a result which appears
to be mediated through antagonism of the benzodiazepine receptor (23).
Nutt et al. (26) attempted to kindle seizures by the repeated i.p. injection of
picrotoxin, bicuculline, or pentylenetetrazol. These drugs are known to exert
their convulsant effect, in whole or in part, through the antagonism of GA-
BA-mediated inhibition (28). Picrotoxin and pentylenetetrazol kindled sei-
zures, but bicuculline did not. However, because the repeated i.p. injections
of picrotoxin and pentylenetetrazol did not decrease the threshold dose re-
quired for seizure when the subjects were subsequently challenged with a
single intravenous injection of the same drug, Nutt et al. (26) question the
straightforward interpretation of an increase in seizure susceptibility follow-
ing administration of drugs as evidence of kindling.
The present study reassessed the question of kindling by picrotoxin. An
attempt was made to kindle seizures using the i.p. route of administration,
with 48 h between administrations, as longer intervals were shown to result
in more reliable kindling than shorter intervals (30, 33). Our use of 48-h
intervals contrasts with the use of 24-h intervals by Nutt et al. (26). Direct
intraamygdala application ofthe drug was used in another group of subjects
to avoid problems in the interpretation of results obtained by repeated pe-
ripheral administration (4, 12). Both groups were then electrically kindled in
the amygdala in a test of transfer. For a bidirectional test of transfer, addi-
tional subjects electrically kindled in the amygdala were subsequently kin-
dled by repeated i.p. injection of picrotoxin.

METHODS
Ninety-five male hooded rats of the Royal Victoria strain weighing 350 to
450 g served as subjects.
Kindling with i.p. Picrotoxin. Forty-three rats were used in a <lose-response
study ofkindling by i.p. picrotoxin. Four doses were chosen on the basis of
pilot work: 1.5, 2.0, 2.5, and 3.0 mg/kg. The rats were divided into four
groups, each ofwhich received repeated, spaced (48 h) injections until a gen-
eralized convulsion occurred or 15 injections had been given. Picrotoxin
(Sigma) was dissolved in physiologic saline ata concentration of 3.0 mg/ml.
After each injection the rats were placed in an open-top clear acrylic chamber
(30 cm square) and videotaped for 1 h. The tape was later scored for convul-
sive behavior.
Transfer between i.p. Picrotoxin Kindling and Electrical Kindling. Ten rats
in the 2.0- and 2.5-mg/kg groups were used in a test of transfer to electrical
kindling. These groups were chosen because they developed progressively
stronger convulsions during the regimen of repeated injections (see Results).
 PICROTOXIN KINDLING 245

At the beginning ofthe experiment they were anesthetized with pentobarbi-

tal and received implantation of a bipolar electrode using standard stereo-
taxic techniques. The tip of the electrode was directed toward the basolateral
amygdala (29). Electrodes were constructed oftwo strands ofTeflon-coated
Nichrome wire twisted together and soldered to small contacts. These were
inserted into a miniature connector which was attached to the skull by dental
acrylic and small screws. One week after the last injection, the rats were elec-
trically kindled, as described elsewhere (4), by connecting them to a poly-
graph and Grass S88 stimulator. The EEG was monitored before and after
each stimulation. The afterdischarge threshold (ADT) was first determined
by applying an initial current of 40 µA base-to-peak, and gradually raising
the current in subsequent stimulations. The ADT was defined as the weakest
current that would evoke an AD of 4 sor longer. After determination of the
ADT, kindling stimulation at 200 µA was applied once daily untíl a stage 5
generalized convulsion (32) occurred. The current consisted of biphasic
square wave pulses, each 1.0 ms in duration, at 60 Hz, and a total duration
of 1 s. Six additional rats served as controls; they were treated similarly with
the exception that physiologic saline was substituted for picrotoxin.
Transfer between Electrical Kindling and i.p. Picrotoxin Kindling. For a
bidirectional test of transfer, 16 additional rats were electrically kindled in
the basolateral amygdala, and after a 1-week interval, rekindled by repeated,
spaced (48 h) i.p. injections of picrotoxin at 1.5 mg/kg. This dose was chosen
because it was found to result in very little seizure development in naive
rats (see Results). Five additional rats served as controls; they were treated
similarly with the exception that current was never passed through the
electrode.
Kindling with Intracerebral Picrotoxin. Fifteen rats received implantation
of a chemitrode for intracerebral injection of picrotoxin. The chemitrodes
were constructed by attaching a bipolar electrode to a 23-gauge guide can-
nula so that it protruded 1.0 mm beyond the tip of the cannula. The tip of
the guide cannula was placed 1.0 mm above the basolateral amygdala and
the 30-gauge injection cannula protruded 1.0 mm beyond the tip of the guide
cannula. The ADT was first determined as described above. Doses of picro-
toxin ranging from 1.0 to 10.0 µg (1.6 to 16.0 nmol) were then used in differ-
ent rats, and each rat received the same dose, repeated at 48-h intervals, until
a generalized convulsion occurred. Picrotoxin was dissolved in sterile dis-
tilled water at a concentration of 1O µg/ µI with the addition of a small
amount of NaOH. The pH was then adjusted to 7 .4 by adding a small
amount of acidic buffer, and sufficient NaCI was added to render the solution
iso-osmotic with tissue. The injections were delivered using a Sage infusion
pump, which was connected to the injection cannula by PElO tubing with
no dead space in the system. The maximal dose ( 1Oµg) was delivered in a
246 DONALD P. CAIN

volume of 1.0 µl during 34 s. Smaller doses of the drug were delivered by

injecting correspondingly smaller volumes of the same solution. The injec-
tion cannula remained in place for 15 s, then it was replaced by the obturator.
The EEG was monitored before, during, and for 1 h after each injection. The
rats were videotaped during this period, and the tapes were later scored for
convulsive behavior.
No vehicle control group was included, but observations in our laboratory
indicated that the repeated injection of an equivalent volume of vehicle using
the same techniques is completely without epileptogenic or kindling effect
(4, 5, 8).
Transfer between lntracerebra/ Picrotoxin Kindling and Electrical Kin-
dling. One week after the last drug injection, the rats in the intracerebral
picrotoxin group were electrically kindled through the chemitrode. The pro-
cedure was as described above, and included redetermination of ADT and
daily application of a 200-µA stimulation. An additional 1Ocontrol rats with
chemitrodes received spaced injections of physiologic saline corresponding
to the average number and volume of drug injections administered to the
kindled group. After a 1-week interval these rats were then electrically
kindled.
Histologica/ Analysis. At the end of testing all rats were anesthetized, per-
fused with Formalin-saline, and the brain was removed, frozen, and sec-
tioned for verification of electrode and cannula placements.

RESULTS
Kindling with i.p. Picrotoxin. The first sign of convulsive behavior was
immobility accompanied by weak twitching of the facial musculature. Sub-
sequent stronger responses consisted of sudden myoclonic jerks of the fore-
limbs, neck, and trunk. Maximal convulsions consisted of one or more
myoclonic jerks followed by a generalized tonic-clonic convulsion. The
groups that received repeated i.p. injection of picrotoxin displayed a steep
<lose-response effect in their rate of kindling to a generalized convulsion (Fig.
1). None of the rats in the 1.5 mg/kg group developed generalized convul-
sions, and only two rats developed weak convulsive signs (twitching). The
rats in the intermediate dose groups (2.0 and 2.5 mg/kg) developed general-
ized convulsions after a mean of 12.0 (SE: 1.25) and 4.0 (SE: 1.15) injections,
respectively, and rats in the 3.0-mg/kg group all displayed a generalized con-
vulsion after the first injection.
Transfer between i.p. Picrotoxin Kindling and Electrical Kindling. The
mean ADT ofthe rats in the 2.0 and 2.5 mg/kg groups used in the transfer
study was 92 µA (range: 50 to 250 µA), which was not significantly different
from that of the controls (88 µA, range: 40 to 200 µA, P > 0.05, Mann-
 PICROTOXIN KlNDLING 247

15

12

zo
¡:
u
w
z

1.5 2.0 2.5

DOSE (mg/kg)

F!G. 1. Kindling rate of groups receiving repeated i.p. injections of picrotoxin. Bars represent
means±SE.

Whitney U). They developed stage 5 convulsions after a mean of 6.3 ADs
(range: 3 to 10), which was significantly faster than the mean of 13.2 ADs
(range: 10 to 17) ofthe controls (p < 0.002, Mann-Whitney U).
Transfer between Electrical Kindling and i.p. Picrotoxin Kind/ing. Rats
that had first been electrically kindled displayed a stronger response to the
repeated injections of picrotoxin than did the control rats (Table 1). All elec-
trically kindled rats developed immobility and twitching, usually accompa-
nied by automatisms (chewing, head nodding), and myoclonicjerl<S that in-
volved the forelimbs, head, and upper trunk. One-quarter of them also devel-
oped a generalized convulsion. In contrast, 60% of the control rats developed
twitching, which was usually weak and not accompanied by automatisms,
and none developed myoclonic jerks or generalized convulsions. The differ-

TABLE 1
Percentage and Number ofRats Displaying Convulsive Signs
after 15 Repeated lnjections of Picrotoxin ª

Immobility and Generalized

twitching Myoclonic jerks convulsion

Group % N % N % N

Previously kindled 100 16/16 100* 16/16 25 4/16

Control 60 3/5 o 0/5 o 0/5

* Greater than control, P = 0.00004.

ª Injections were 1.5 mg/kg, i.p.
248 OONALD P. CAIN

10
9
8

6
5 •
4
3
a •
I• 1

1 2 3 4 5 6 7 8 9 w
DOSE (micrograms)

FIG. 2. Number of intraamygdala injections of picrotoxin required to kinclle convulsions as a

function of dose. Each point represents an experimental subject.

ence in incidence of myoclonic jerks between the experimental and control

groups was statistically significant (P = 0.00004, Fisher exact proba-
bility test).
Kindling with Intracerebral Picrotoxin. The rats that received intraamyg-
dala injections of picrotoxin all developed generalized convulsions, but there
was no clear relation between dose and rate of kindling (Fig. 2). Rats that
received doses of 1.0 to 2.5 µg kindled after 1 to 10 injections. Rats that
received doses of 3.0 µg or more always kindled in one trial, and usually
progressed into status epilepticus. After 1O to 30 min of seizure, diazepam
was administered to termínate the seizure.
Transfer between Intracerebral Picrotoxin Kindling and Electrical Kin-
dling. Redetermination ofthe ADT and attempts to electrically rekindle the
intracerebral picrotoxin rats resulted in mixed success, with sorne animals
responding to the electrical stimulation and others not respondling. Of the
15 rats that were initially kindled with picrotoxin, AD could be evoked in 7,
and the mean ADT was 178 µA (range: 50 to 500 µA). The mean ADT of
these same rats at the beginning ofthe experiment was 96 µA (range: 50 to
150). This difference was not statistically significant (P> 0.05, sign test), and
is primarily due to a high postpicrotoxin ADT in one rat. When these rats
were electrically rekindled at 200 µA (500 µA in the case of one rat) they
developed stage 5 convulsions after a mean of 6.3 ADs (range: 3 to 11), which
was significantly faster than the mean of 12.1 ADs (range: 9 to 20) required
by the control group (P < 0.02, Mann-Whitney U). These rats had received
injections of picrotoxin of from 1.0 to 2.0 µg. The other 8 rats could not be
rekindled because AD could not be evoked, even when stimulation intensi-
ties to 1500 µA were applied. The mean ADT of these rats at the beginning
ofthe experiment was 98 µA (range: 50 to 150), and they had received injec-
tions ofpicrotoxin offrom 2.0 to 10 µg.
 PICROTOXIN KINDLING 249

When the chemitrodes were removed and the brains of these rats were
examined histologically, the chemitrodes were found to be in good condi-
tion, but considerable damage was apparent near the chemitrode tip, suggest-
ing a lack of viable neurons in the vicinity of the electrode tip (Fig. 3c). In all
rats that received injections of 2.5 µg or larger there was considerable damage
in the temporal region surroundíng the electrode típ and additional damage
that extended along neuroanatomic pathways linked to the injected amyg-
dala. This damage often extended throughout the basal forebrain as far for-
ward as anterior olfactory structures (Fig. 3a, b ).

DISCUSSION
These results demonstrate that repeated, spaced i.p. or intraamygdaloid
injections of picrotoxin can kindle convulsions that are similar or identical
in form to those that can be kindled by repeated injection of a variety of
other convulsant drugs such as (3-carboline, pentylenetetrazol, carbachol, (3-
endorphin, and met-enkephalin (4-8, 23, 24).
The bidirectional transfer that was observed between i.p. picrotoxin and
electrical kindling is similar to that reported to occur between various phar-
macologic kindling agents and electrical kindling, and further supports the
conclusion that picrotoxin can kindle seizures (4, 5). Transfer from electrical
kindling to i.p. picrotoxin challenge has also been demonstrated ( 15, 16).
The occurrence of kindled seizures in response to intracerebral injection
ofpicrotoxin indicates that any peripheral mechanisms brought into play by
i.p. injection of the drug are not crucial for kindling. Similar results were
obtained with other pharmacologic kindling agents (4, 5, 8).
Subjects in the intracerebral group that received large doses of picrotoxin
kindled in one trial, and their brains were found to have considerable damage
near the chemitrode tip, which may have resulted from hyperexcitation or a
direct toxíc effect, or both. The absence of a measurable <lose-response effect
in this group and the lack of AD during transfer testing in subjects kindled
with large doses of picrotoxin are most likely the result of damage to neurons
near the chemitrode tip. The damage in and anterior to the pyriform cortex
observed in the present study (Fig. 3a and b) is similar to that observed pre-
viously after injectíon of comparable amounts of picrotoxin and after treat-
ments that produce seizures or status epilepticus (21, 22, 38, 39). The fact
that epileptogenic treatments can have severe neuropathologic consequences
that can potentíally ínterfere with subsequent transfer testing indicates that
caution should be exercised in the interpretation of failures of transfer of
kindling. Damage must be ruled out as a factor before it is concluded that a
genuine failure oftransfer occurred.
Nutt et al. (26) administered repeated i.p. injections of picrotoxin, bicucul-
line, and pentylenetetrazol to rats in an attempt to kindle seizures using
 N
Vo
o

8
~
B
'."C
~
z

A
FIG. 3. Brain ofrat kindled with 3.0 µg picrotoxin injected into amygdala. A, B-damage anterior to injection site in prepyrifonn and olfactory
regions. C-injection site.
 PICROTOXIN KINDLING 251

GABA antagonist drugs. They kindled seizures with picrotoxin and pentyl-
enetetrazol, but bicuculline produced only occasional mild jerking. In a sub-
sequent test of transfer to a challenge injection of the same drug administered
i. v., there was no evidence of a decrease in the threshold dose required for
seizure as a function ofthe previous i.p. drug injections. They therefore ques-
tioned whether kindling using these drugs was true pharmacological kindling
and, more generally, whether any increase in seizure susceptibility after re-
peated administration of drugs is kindling. However, as kindling with pentyl-
enetetrazol has been shown to transfer to electrical kindling and challenge
by a variety ofagents (4, 6, 7), the results that Nutt et al. obtained with this
drug are puzzling.
There are a number of features ofthe Nutt et al. study that argue against
acceptance oftheir conclusions. (i) The measure of transfer (threshold dose
of drug) is analogous to the threshold measure of current required to evoke
an AD in electrical kindling (ADT). However, the ADT-reduction effect in
electrical kindling <loes not transfer to the transfer site, even when evidence
ofpositive transfer, measured in rate of kindling, is obtained (6, 7, 31, 32).
Similarly, pharmacological kindling, subsequently confirmed as true kin-
dling by a test of positive transfer, does not cause a reduction in electrical
ADT (4, 36). Therefore, Nutt et al. (26) chose a dependent measure that
would not necessarily be expected to show transfer effects. (ii) Nutt et al.
administered the single transfer challenge by a different route from that used
during original kindling. Different routes of administration can yield very
different, and at times opposite, physiologic responses to the same drug due
to the different pharmacokinetics that may result ( 1, 40). (iii) A single chal-
lenge as a test of transfer has theoretical drawbacks that make it much less
desirable than full kindling by the repeated administration of an initially
subconvulsant transfertreatment [see (6, 7)] as was done in the original dem-
onstrations oftransfer (10, 32). (iv) Nutt et al. used a 24-h interval between
injections, which may be too short for the kindling method they chose (30,
33). Our use of a 48-h interval appears to be sufficiently long for successful
kindling using picrotoxin. In view of the above and the data reported here,
we conclude that kindling by repeated injection of picrotoxin is true pharma-
cological kindling. This conclusion is generally consistent with much earlier
evidence linking the GABA system with the antagonism ofkindling [e.g., (3,
14-20, 25, 34)].
The failure of Nutt et al. (26) to obtain kindling with bicuculline may be
due to the fact that bicuculline is unstable at physiologic pH, and is rapidly
converted to the less active bicucine (27). This may contribute to bicucul-
line's poor GABA-antagonist properties (38).
Norepinephrine is known to antagonize amygdala kindling, but severe de-
pletion of norepinephrine by 6-hydroxydopamine or antagonism of the in-
252 DONALD P. CAIN

hibitory effects of norepinephrine by drugs does not by itself kindle convul-

sions (9). The fact that picrotoxin readily kindles convulsions suggests that
GABA-mediated inhibition may be of greater importance in opposing kin-
dling than is norepinephrine-mediated inhibition.
Picrotoxin exerts its epileptogenic effect by blocking GABA-mediated
chloride conductance (11, 37). The bulk ofthe evidence suggests that picro-
toxin kindles seizures by reducing the inhibitory effect of GABA, which is
the most prominent inhibitory neurotransmitter in the mammalian brain,
thus allowing the evolution of repetitive excitatory events leading to paroxys-
mal depolarizing shifts ( 11, 38). However, evidence for direct excitatory
effects of picrotoxin has been reported (2), and it is not possible to exclude a
contribution ofthis kind in the kindling efficacy ofthis agent.

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