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Brain amino acid concentration changes during status epilepticus induced by lithium and pilocarpine
EXPERIMENTAL
NEUROLOGY
108,61-70
(1990)
Brain Amino Acid Concentration Changes during Status Epilepticus Induced by Lithium and Pilocarpine NANCY Y. WALTON, SONNY GUNAWAN, AND DAVID M. TREIMAN Neurology
and Research Department
Services,
of Neurology,
Veterans UCLA
Administration West Los Angeles School of Medicine, Los Angeles,
Medical California
Center,
and
nobutyric acid (GABA) at times before and during the course of SE induced by lithium and pilocarpine as a first step in investigating the possible role these neurotransmitters play in the maintenance of late SE in this model. Serine, glutamine, and glycine were measured as part of the same assay. We have reported elsewhere (31) a sequence of changes in electroencephalographic (EEG) pattern which is seen in human generalized convulsive SE and in experimental SE in the rat. We used the EEG pattern to define the electrophysiologic stages of SE and determine the times at which brain samples were obtained in this experiment. Brain amino acid changes during SE, in experiments which included electrographic monitoring, have been reported only when SE was induced by mechanisms distinct from those involved in lithium/pilocarpine SE and only in paralyzed, artificially ventilated rats (5-7).
Amino acid concentrations were measured in specific structures from the brains of rats decapitated before and during the course of status epilepticus induced by lithium and pilocarpine, with the stages of status defined by the electroencephalographic (EEG) pattern displayed. Early status was marked by discrete seizures on EEG, mid status by continuous spiking, and late status by periodic epileptiform discharges. Aspartate levels were lower than control levels in most regions prior to the onset of status. The decline continued and reached statistical significance in different regions at times from early to late status. Glutamate concentrations were typically higher than control just prior to status onset and then decreased in a manner similar to aspartate, but with less percentage change. y-Aminobutyric acid increased during status, with the earliest statistically significant differences observed in mid status. These changes were observed in most forebrain structures studied, but the largest percentage changes in excitatory amino acid concentration were found in substantia nigra, where they fell to less than half of control. 0 1990 Academic Press, Inc.
METHOD Subjects. Adult, male, Sprague-Dawley rats (n = 42) were used as subjects in this experiment. Rats were implanted surgically with chronic epidural recording electrodes, as has been described previously (32). They were housed singly after surgery, with food and water available ad lib., room temperature maintained at 20°C and a 24-h diurnal lighting schedule (lights on from 0700 to 1900 each day). Rats were allowed to recover for 1 week prior to experimentation. Drugs. Ketamine (100 mg/ml, Parke-Davis), 87 mg/ kg, and xylazine (20 mg/ml, Haver), 13 mg/kg, given as a single intraperitoneal (ip) injection, were used for surgical anesthesia. Lithium chloride (Sigma) was dissolved in sterile water and injected ip at a dose of 3 mmol/kg. Pilocarpine (Sigma) was dissolved in saline and injected ip at a dose of 25 or 30 mg/kg. Experimental design. Six rats were assigned to each of seven groups, the characteristics of which are shown in Table 1. These groups were chosen to provide controls for each of the convulsant drugs used to induce SE and to allow us to seewhat, if any, changes were taking place
INTRODUCTION Systemic injection of pilocarpine to lithium-pretreated rats has been shown to produce a nearly universally fatal status epilepticus (SE) which lasts at least 2 h in nonparalyzed animals (13, 16). The induced SE is responsive to treatment with atropine or standard anticonvulsant drugs, provided the treatment is initiated early in the SE episode (21). We have reported that SE induced by lithium and pilocarpine becomes less responsive to treatment with large doses of diazepam the later in the SE episode that treatment is given (32). Induction of SE in this model requires cholinergic synaptic activity, but some other neurotransmitter system appears to be responsible for maintenance of the SE beyond about the first 30 min (15). Although changes in brain amino acid concentrations during SE might be either a cause or an effect of the ongoing seizure activity, we assayed the levels of aspartate, glutamate, and y-ami61
Copyright 0 1990 All rights of reproduction
0014-4&x/90 $3.00 by Academic Press, Inc. in any form reserved.
62
WALTON, TABLE
GUNAWAN.
1
Characteristics of Experimental and Control Groups Studied in This Experiment (n = 6 per Group) Group
First injection
Second injection
Control-l
Saline
Saline
Control-Z
Lithium
Saline
Control-3
Saline
Pilocarpine
Lithium
Pilocarpine
Early SE Mid SE
Lithium Lithium
Pilocarpine Pilocarpine
Late SE
Lithium
Pilocarpine
Before
Note.
SE onset
Second
injections
were given
Decapitation
time
60 min after second injection 60 min after second injection 60 min after second injection 15 min after second injection Discrete seizure on EEG 10 min continuous spiking on EEG Initial PEDs on EEG
24 h after
the first
injections.
prior to the onset of SE and during its earliest stage, as well as at the later stages which were of primary interest to us. EEG was monitored from the second injection until decapitation for all rats. Tissue processing. Brains were removed from the skull immediately after decapitation, placed into small plastic bags, and frozen by immersion in acetone and dry ice. This technique was chosen because the presence of metal electrodes in the skull precluded the use of microwave fixation (2) and the need to freeze the brains as quickly as possible once EEG criteria were met precluded use of Ponten’s (28) in situ freezing technique. Brains were stored at -70°C for up to 50 days prior to extraction. Regional microdissection was carried out on 300-pm frozen sections which had been cut in a cryostat at -15°C. The sections were placed on glass slides and brain structures were dissected out on a freezing stage (-15°C) under a microscope. Structures were chosen for microdissection and assay on the basis of our ability to rigorously define and reliably obtain the same tissue from all animals. One set of structures (nucleus accumbens, caudate-putamen, substantia nigra, hippocampus, septum, amygdala, inferior colliculus and cingulate, motor, pyriform, and entorhinal cortex) was selected because it has been suggested by others to be important in this or other experimental seizure models (1, 3, 8, 12, 18,19,29). Another set of structures (cerebellum, ponsmedulla, and superior colliculus) was chosen in hopes of
AND TREIMAN
finding at least one structure which showed little or no change in this model of SE. Cerebellum and pons-medulla were dissected away from the frozen brain prior to its being mounted in the cryostat. The following structures were dissected, guided by the atlas of Palkovits and Brownstein (24): cingulate cortex, from slices where the head of the caudate nucleus was identifiable to where the dorsal hippocampus was no longer connected across the hemispheres; pyriform cortex, from the second slice after the appearance of the corpus callosum to just before the first appearance of the ventral hippocampus; nucleus accumbens, from slices in which the head of the caudate nucleus was visible and the next 2100 pm; septum, from slices in which the lateral ventricles were clearly visible until the first appearance of the globus pallidus; frontal-parietal sensory-motor cortex, which will be hereafter referred to simply as motor cortex, from slices in which the head of the caudate nucleus was visible to those about 2100 pm past the initial appearance of the dorsal hippocampus; caudateputamen, from slices in which the head of the caudate was no longer completely surrounded by the corpus callosum to those about 600 pm beyond the first appearance of the dorsal hippocampus; hippocampus, both dorsal and ventral, including CAl, CA2, CA3, CA4, and the dentate gyrus; amygdala, from slices just posterior to the optic chiasm, continuing one or two slices beyond the first appearance of the ventral hippocampus; entorhinal cortex, from slices in which the ventral hippocampus was visible until the hemispheres disappeared just anterior to the cerebellum; substantia nigra, which could be seen in slices in which the dorsal and ventral hippocampi had merged to form a continuous, lateral structure, and continued for about 1200 pm; superior colliculus, which was readily identifiable about 600 pm after the appearance of substantia nigra and continued until the dentate gyrus was no longer seen; and inferior colliculus, from slices in which the dentate gyrus could no longer be seen until the appearance of the cerebellum. Dissected tissue was placed in frozen 1.5ml microcentrifuge tubes and weighed. Ice-cold 80% ethanol (18 ml/ g tissue wt) was added and the tissue was disrupted by ultrasonication. Homogenates were centrifuged at 15,OOOg,4”C, for 15 min. The supernatant was transferred to glass culture tubes maintained on ice and the extraction was repeated with more 80% ethanol. Supernatant from the second extraction was added to that from the first, the tube was vortexed, and the contents were measured and stored overnight at 4°C.
FIG. 1. Examples of EEGs recorded during this experiment, showing the patterns which defined the electrophysiological stages of SE: (a) nonseizing EEG recorded 14 min after injection of pilocarpine, before any electrographic or behavioral evidence of seizure activity was seen; (b) discrete electrographic seizure which ends abruptly and simultaneously in all channels (early SE), recorded 40 s after SE onset; (c) continuous, high-amplitude, rapid spiking, punctuated by brief flat periods (mid SE), recorded 30 min after SE onset; (d) periodic epileptiform discharges (late SE), recorded 90 min after SE onset.
AMINO
a F4-P4 W-P3
F3-F4 F4-P4
b F4-P4 W-P3 P3-F3 F3-F4 F4-P4 C F4-P4 W-P3 P3-F3 F3-F4 F4-P4
d F4-P4 W-P3 P3-F3 F3-F4
ACIDS
IN
STATUS
EPILEPTICUS
63
64
WALTON.
TABLE Regional
GUNAWAN,
RESULTS
acid concentration
Brain
structure
ASP
GLU
SER
GLN
GLY
GABA
Cingulate
cortex
Pyriform
cortex
2.36 (0.12) 2.37 (0.12) 2.66 (0.07) 2.28 (0.10) 1.71 (0.06) 1.62 (0.05) 2.09 (0.21) 1.95 (0.07) 2.21 (0.09) 1.62 (0.06) 2.49 (0.21) 2.29 (0.17) 2.30 (0.17) 2.89 (0.27)
9.19 (0.70) 9.27 (0.80) 9.79 (0.65) 9.86 (0.56) 7.84 (0.55) 8.41 (0.32) 4.43 (0.53) 9.68 (0.48) 8.33 (0.66) 6.59 (0.35) 5.54 (0.23) 5.40 (0.41) 8.88 (0.49) 5.12 (0.16)
0.76 (0.04) 0.92 (0.07) 0.77 (0.06) 1.02 (0.05) 0.80 (0.08) 0.96 (0.06) 0.47 (0.06) 1.00 (0.07) 0.74 (0.07) 0.77 (0.06) 0.43 (0.04) 0.44 (0.04) 0.69 (0.04) 0.56 (0.07)
3.61 (0.32) 4.36 (0.35) 3.98 (0.26) 4.73 (0.24) 4.88 (0.35) 4.54 (0.19) 2.74 (0.20) 4.13 (0.14) 4.50 (0.33) 3.98 (0.20) 3.31 (0.14) 3.37 (0.21) 4.81 (0.20) 2.88 (0.21)
0.66 (0.05) 0.60 (0.05) 0.65 (0.05) 0.70 (0.06) 0.71 (0.07) 0.74 (0.08) 0.88 (0.06) 0.92 (0.07) 0.69 (0.06) 0.66 (0.05) 1.39 (0.15) 1.12 (0.13) 1.12 (0.24) 3.53 (0.23)
1.50 (0.11) 1.89 (0.15) 1.60 (0.12) 1.52 (0.06) 2.67 (0.20) 1.81 (0.18) 5.09 (0.67) 1.94 (0.07) 3.45 (0.47) 3.02 (0.21) 2.77 (0.23) 3.37 (0.24) 1.73 (0.18) 1.58 (0.11)
Motor
cortex
Entorhinal Nucleus
cortex accumbens
Caudate-putamen Substantia
nigra
Hippocampus Amygdala Septum Inferior Superior
colliculus colliculus
Cerebellum Pons-medulla
TREIMAN
2
Amino Acid Concentrations from Rats Treated with Saline Only (n = 6) Amino
AND
Note. Concentrations are expressed in pmol/g tissue weight. Means are shown with standard deviations in parentheses below. Amino acid abbreviations: ASP, aspartate; GLU, glutamate; SER, serine; GLN, glutamine; GLY, glycine; GABA, y-aminobutyric acid.
Amino acid assay. Amino acids were assayed using a method developed in this laboratory (11). Briefly, norvaline (internal standard) was added to 50 ~1 of extraction supernatant and dried under vacuum. The residue was reacted with phenylisothiocyanate for 20 min at room temperature to form phenylthiocarbamyl derivatives of the amino acids. Excess reagent was removed under vacuum and the residue was dissolved in 300 ~1 0.1 M sodium acetate buffer (pH 5.7) and injected into the chromatograph. HPLC separation was carried out in a reversed-phase system with a Rainin Cl8 column as the stationary phase and a gradient system of 0.1 M sodium acetate buffer and acetonitrile/water (60/40) as the mobile phase. Absorbance was monitored by an ultraviolet detector set at 254 nm. Amino acid concentrations were calculated by linear regression analysis, comparing the peak area ratio of amino acid:internal standard to those obtained in standard curve determinations.
The progression of EEG changes which occurs in this model of SE has been described elsewhere (31, 32) and is illustrated in Fig. 1. Figure la shows the EEG as it appears after pilocarpine has been injected, but before any evidence of seizure activity appears. SE begins with discrete electrographic seizures in which spiking ceases simultaneously in all channels and is followed by a brief period of postictal low-voltage slow activity (Fig. lb). After several minutes the distinct seizure offset is no longer seen. Instead, irregular, l- to ~-HZ spike and wave activity follows episodes of rapid spiking, with spike amplitude increasing rapidly over time. Spiking eventually becomes relatively constant at a high amplitude and rate of occurrence, sometimes interspersed with brief flat periods (Fig. lc). Continuous spiking continues for l-2 h when SE is induced by lithium and pilocarpine and then PEDs, separated by periods of lower amplitude activity,
ASPARTATE
\
\
“s---q 1 *
8 100 -:: 90 -- -a,\
--I-
GLUTAMATE
.
.
Is
L -
b-N., *
100 90 f
iz ;T
.
*
\
80 70
g
I
-3
*
GLUTAMINE
2
is
120
5 a
110 100 90
GABA
---
=.*
i
I
I
Before Onset
00
0
0
Y-
k I
Discrete Seizures
I
I
Continuous Spiking
PEDs
FIG. 2. Amino acid concentration changes in motor cortex just before onset of and during SE induced by lithium and pilocarpine. In this and all figures to follow each point is the mean + standard error for six rats, and points marked + are statistically different from control at P < 0.05, while those marked * are different at P < 0.01.
AMINO
--- * \ ASPARTATE
ACIDS
IN
STATUS
Mean control concentrations of aspartate, glutamate, serine, glutamine, glycine, and GABA in all regions studied are given in Table 2. No statistically significant differences in concentrations of serine or glycine were found among the groups for any brain structure studied. Amino acid changes in neocortex, relative to control, are shown in Figs. 2-5. Aspartate decreased in all neocortical areas studied, with concentrations being lower before the onset of SE and reaching statistical significance in both the continuous spiking and PEDs groups (P < 0.01). Motor cortex (Fig. 2) also had significant changes in the concentrations of glutamate, glutamine, and GABA. Glutamate levels were significantly less than control in the continuous spiking and PEDs groups (P < 0.01). Concentrations of glutamine and GABA were significantly higher than control in the PEDs group in both motor and cingulate (Fig. 3) cortex (P < 0.01). Glutamate levels in cingulate cortex were significantly lower in the continuous spiking group (P < 0.05). Pyriform cortex (Fig. 4) showed no significant changes across groups for glutamate, glutamine, or GABA. GABA con-
\ \
b0 5
60
z? ;
110
F 8 0
loo 90 80
Ts 100
---
+-
---
I
0 z
\
\
\+
y-/-
GLUTAMINE
--
z
5<
\
GLUTAMATE
3’
4
,4 *
125 GABA
65
EPILEPTICUS
I l. -.
lOOf---
-L
,X0”
100
ASPAR‘CATE
--‘-)---y’
755
Before Onset
\ Discrete Seizures
FIG. 3. Amino acid concentration fore and during SE induced by lithium the same as in Fig. 2.
Continuous Spiking
* \
\
PEDs
changes in cingulate cortex and pilocarpine. Symbols
beare
begin to appear in runs of several seconds duration (Fig. Id). The PEDs gradually increase in frequency and duration of occurrence until the rat dies. Untreated SE is almost always fatal in this model (17,21,22,26). Data are given as means f standard deviation, unless noted otherwise. Latency of SE onset from the time of pilocarpine injection was 33.1 -t 13.0 min. Analysis of variance revealed no statistically significant differences among the experimental groups. Duration of SE at the time of decapitation was 2.5 ? 0.5 min for the group decapitated when the EEG showed discrete seizures, 30.2 -+ 13.7 min for the group decapitated at continuous spiking on EEG, and 111.3 f 20.7 min for the group decapitated at the first appearance of PEDs. The interval from decapitation to immersion of the brains in the freezing solution was 84.2 f 26.2 s, with no statistically significant differences among the groups. Evaluation of changes in brain amino acid concentrations was made by analysis of variance, with Dunnett’s t test used to compare differences between experimental group means and the mean of the control group which was given no drugs (9). Alpha was set at 0.05 for all tests.
/
\ GLUTAMATE
z
85’
8
1101
!k [r
1oo
2 z-
go
s
80 125 GABA
0 z
4--
f 100
,
---
/’
--k
751
Before Onset
Discrete Seizures
FIG. 4. Amino acid concentration fore and during SE induced by lithium -. the same as in Fig. 2.
Continuous Spiking
PEDs
changes in pyriform cortex and pilocarpine. Symbols
beare
100 52 2 tr’
66
WALTON.
\
ASPARTATE
g
\
75
\
\
73
0
90
2
80 J
8
100
*
* */-
GLUTAMATE
\
\
----*---
‘. ;
GUNAWAN,
90
z
GLUTAMINE
g
8o
5
140
a $
AND
TREIMAN
In hippocampus (Fig. 9), concentrations of aspartate were significantly decreased by the time of discrete seizures (P < 0.05) and decreased further by continuous spiking and PEDs (P < 0.01). Glutamate and glutamine levels were significantly lower only in the continuous spiking group (P < 0.05), while GABA rose significantly in the continuous spiking and PEDs groups (P < 0.01). Only aspartate changed significantly in amygdala (Fig. lo), being significantly lower than control in both the continuous spiking and PEDs groups (P < 0.01). In the septal nuclei (Fig. ll), aspartate concentrations were lower in the continuous spiking and PEDs groups (P < O.Ol), glutamate was lower at PEDs (P < 0.05), glutamine was lower at discrete seizures and continuous spiking (P < 0.05), and GABA was elevated at PEDs (P < 0.01). Fewer and smaller changes were observed in the cerebellum, inferior colliculus, and superior colliculus. Cerebellar aspartate was less than control at continuous spiking (P < 0.05) and PEDs (P < O.Ol), while glutamine was elevated at PEDs (P < 0.05). Only glutamine
% loot---f----,
I20 100
e
I
Before Onset
I
ASPARTATE
‘\.
‘\ \
I
Discrete Seizures
FIG. 5. Amino acid concentration before and during SE induced by lithium the same as in Fig. 2.
I
Continuous Spiking
P:Ds
changes in entorhinal cortex and pilocarpine. Symbols are
centrations rose significantly in entorhinal cortex in both the continuous spiking (P < 0.05) and PEDs (P < 0.01) groups (see Fig. 5). Nucleus accumbens (Fig. 6) showed significant declines in aspartate, glutamate, and glutamine in the continuous spiking and PEDs groups (P < O.Ol), while GABA levels increased in the PEDs group (P < 0.01). In caudate-putamen (Fig. 7), glutamate levels were significantly less than control in both the continuous spiking and PEDs groups (P < 0.01). Aspartate levels were significantly lower in the PEDs group (P < 0.01) while glutamine concentrations were decreased in the continuous spiking group (P < 0.01). GABA was increased significantly in the PEDs group (P < 0.01). Substantia nigra (see Fig. 8) showed the largest percentage decline of the excitatory amino acids of any structure studied here. Aspartate concentrations in the continuous spiking and PEDs groups were significantly lower than control (P < O.Ol), as were the concentrations of glutamate (P c 0.05). Glutamine did not change significantly, but GABA concentrations were significantly elevated at both continuous spiking (P < 0.05) and PEDs (P < 0.01).
+
///
-i
\* b”/
-N\ \
GLUTAMATE
\
\* --
w’
j
6 3 9
90 80
a
701
-\
\ f
---.+
* Y----i
GLUTAMINE
I Before Onset
I
Discrete Seizures
-4
0’/4*
I
Continuous Spiking
1 PEDs
FIG. 6. Amino acid concentration changes in nucleus accumbens before and during SE induced by lithium and pilocarpine. Symbols are the same as in Fig. 2.
AMINO
ASPARTATE
‘-e-----
ACIDS
\
\
\
IN
STATUS
We attempted to study an even later group, which was decapitated after a long duration of PEDs had occurred. These brains, however, did show evidence of general metabolic derangement, with concentrations of all amino acids measured rising at this time. These rats also appeared to be much more debilitated than any of our other animals. We believe the most parsimonious explanation for the pattern of changes which we observed in this experiment is that the excitatory amino acids, aspartate and glutamate, were being released rapidly enough during the time from onset of seizures to the stage during which the EEG pattern is one of continuous spiking to produce a significant decrease in the tissue concentrations of these amino acids. The rise in GABA concentrations is more difficult to explain. Although it is tempting to postulate that this increase represents a compensatory mechanism of the brain in response to continuous seizure activity, this explanation is difficult to defend. Increased release of GABA would lead to increased tissue levels only if
*
\ *
\
GLUTAMATE
\ \ \* s
--
140
-4
/ ‘*
100
.
67
EPILEPTICUS
4
GABA
\ ASPARTATE
t Be/ore
Onset
Disdrete Seizures
FIG. 7. Amino acid concentration before and during SE induced by lithium the same as in Fig. 2.
Conthuous Spiking
\
\
\
‘\
.
PiDs
changes in caudate-putamen and pilocarpine. Symbols
are
100 2 P
Z changed significantly in the superior and inferior colliculi, being elevated at PEDs in both regions (P < 0.01). None of the amino acids studied changed significantly in the pons-medulla.
.
70
GLUTAMATE
L_ -- s*
-s.. \
---4
a Ej
40 r
s
DISCUSSION
Changes in amino acid neurotransmitter brain concentrations are, of course, crude approximations of changes which may be occurring at the synaptic level. Furthermore, some of these amino acids have metabolic roles in brain, in addition to their neurotransmitter functions. The changes which we found do not appear to be secondary to any general derangement of amino acid metabolism, for several reasons. First of all, serine and glycine did not change in any of the structures we studied and glutamine changed significantly in only a few. In addition, while the concentrations of the excitatory amino acids were decreasing, the levels of GABA were rising. Finally, the extent of change for any of these amino acids differed widely across brain structures.
5 y
7oJ 160
a
140
z 3 a
120
0
I
GABA
c--
f *
100
800
4-
--
o-
Before Onset
Discrete Seizures
FIG. 8. Amino acid concentration fore and during SE induced by lithium the same as in Fig. 2.
Continuous Spiking
PEDs
changes in substantia nigra and pilocarpine. Symbols
beare
WALTON.
68
GUNAWAN,
-\. -\ -- +
loo*._
\
ASPARTATE
* \
i+!
1201 3
GLUTAMATE
/ /
. -\
0 \
k
0
\
/
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’
-6 -- -;’
/ / /
A
AND
TREIMAN
ine, Blennow et al. reported that cortical aspartate and glutamate decreased, but saw no change in GABA concentrations (6). Jope and colleagues reported that acetylcholine concentrations in both hippocampus and cortex decreased early and then rose dramatically later during SE induced by lithium and pilocarpine (17). The sampling times in their experiment were determined by the time elapsed after injection of pilocarpine, so only approximate comparisons can be made with our experimental groups. However, the time at which they found the highest levels of acetylcholine appears to be similar to that of our PEDs group. The anatomic distribution of changes in the concentrations of aspartate, glutamate, and GABA suggests extensive forebrain participation in late SE induced by lithium and pilocarpine. More posterior structures showed fewer and less extensive changes. Clifford et al. (8) reported that, in animals implanted with multiple depth electrodes, SE seemed to start simultaneously in many forebrain structures in some animals and in the ventral pallidum or nucleus accumbens in others. Jope
‘00-p.\
GASA /
a 80
I
I Before Onset
FIG. 9. Amino acid concentration and during SE induced by lithium same as in Fig. 2.
I Discrete Seizures
I Continuous Spiking
changes in hippocampus and pilocarpine. Symbols
1 PEDs
.
ASPARTATE
\ i
E g
before are the
75
L
8
a
\
50
100
a
GABA reuptake or metabolism were impaired. Synthesis of GABA has been shown to continue even after death (25), so it is more likely that release of GABA was impaired while synthesis continued, leading to an accumulation of GABA in brain tissue. It has been shown in other models of SE that GABAergic neurons are susceptible to early damage during uncontrolled SE (30). Although others have reported that lithium effects amino acid metabolism in brain (4,27), we found no significant changes in any of the amino acids measured in rats treated only with lithium or only with pilocarpine. This was not unexpected, since Jope and Morrisett (15) have elegantly demonstrated that cholinergic systems are primarily responsible for the onset of SE in this model. The reported effects of lithium on amino acid metabolism may require chronic treatment or single doses larger than those used here. When SE was induced by bicuculline, Chapman and her colleagues found the same pattern of changes in aspartate, glutamate, and GABA in neocortex as we have described here (7). With SE induced by homocyste-
8
80
s a
100
zcn
85
\
y+--+%~
0 0 a
\
GLUTAMINE
70 GABA
100
--
\
w
80
60
’
I
Beiore Onset
I
Discrete . Seizures
I
Continuous Spiking
FIG. 10. Amino acid concentration changes in amygdala and during SE induced by lithium and pilocarpine. Symbols same as in Fig. 2.
1
PEDs before are the
AMINO
ACIDS
IN
STATUS
.-)---a,\
function must be intact at these early times, because drugs which act by facilitating GABAergic inhibition (diazepam, phenobarbital) are effective in stopping SE in this and other SE models as well as in early human SE (20,21,23,32). If our interpretation of these results is valid, it suggests that treatment of human SE which has progressed to the point where it does not respond to the usual anticonvulsant therapies may require drugs with a completely different mechanism of action than the conventional GABA-enhancing anticonvulsants (20). Excitatory amino acid blockers may be required to halt ongoing seizure activity at this time, or compounds which limit the second messengers which are activated by the calcium influx associated with sustained NMDA receptor activation. The recently reported increase in survival rate following SE induced by lithium and pilocarpine when rats were treated with acepromazine raises the issue of catecholamine participation in late lithium/ pilocarpine SE, although these authors did not attempt to assessseizure control following treatment (26). The role of excitatory amino acid neurotransmitters in late SE requires much further research to be clarified and understood completely.
ASPARTATE
0 5 100 -8 85 T z
\ GLUTAMATE
-3---,,
-
Es 85 0 a 7n h
/
--
69
EPILEPTICUS
GLUTAMINE
‘-
ACKNOWLEDGMENTS Before Onset
Discrete Seizures
FIG. 11. Amino acid concentration before and during SE induced by lithium the same as in Fig. 2.
Continuous Spiking
PEDs
This work was supported by grants from the Veterans Administration and from the Epilepsy Foundation of America. The tireless and professional technical assistance of Mr. Barry Cole is gratefully acknowledged.
changes in the septal nuclei andpilocarpine. Symbols are
REFERENCES 1.
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PONTEN, U., R. A. RATCHESON, L. G. SALFORD, AND B. K. SIESJO. 1973. Optimum freezing conditions for cerebral metabolites in rats. J. Neurochem. 21: 1127-1138.
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ROBERTS, R. C., AND C. E. RIBAK. 1986. Anatomical changes of the GABAergic system in the inferior colliculus of the genetically epilepsy-prone rat. Life Sci. 39: 789-798.
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SLOVITER, R. S. 1987. Decreased hippocampal selective loss of interneurons in experimental 235: 73-76.
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TREIMAN, D. M., N. Y. WALTON, AND C. KENDRICK. 1990. A progressive sequence of electroencephalographic changes during generalized convulsive status epilepticus. Epilepsy Res. 5: 49-60.
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WALTON, epilepticus diazepam.
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S., N. Y. WALTON, AND D. M. TREIMAN. 1990. HPLC of selected amino acids in rat brain by precolumn with phenylisothiocyanate. Biomed. Chromatogr.,
AND F. LEEB-LUNDBERG. 1981. ConvuIsant and binding sites related to the GABA receptor/ionoIn Neurotransmitters, Seizures and Epilepsy K. G. Lloyd, W. Loscher, B. Meldrum;and E. H. Raven Press, New York.
IADAROLA, M. J., AND K. GALE. anticonvulsant activity mediated ence 218: 1237-1240.
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LINDVALL, O., M. INGVAR, AND F. H. GAGE. 1986. Short term status epilepticus in rats causes specific behavioral impairments related to substantia nigra necrosis. Exp. Brain Res. 64: 143148. LOTHMAN, E. W., AND R. C. COLLINS. 1981. Kainic acid induced limbic seizures: Metabolic, behavioral, electroencephalographic and neuropathological correlates. Bruin Res. 218: 299-318.
on rat brain rats studied
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glucose in vivo.
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N. Y., AND D. M. TREIMAN. 1988. Response of status induced by lithium and pilocarpine to treatment with Exp. Neurol. 101: 267-275.
NEUROLOGY
108,61-70
(1990)
Brain Amino Acid Concentration Changes during Status Epilepticus Induced by Lithium and Pilocarpine NANCY Y. WALTON, SONNY GUNAWAN, AND DAVID M. TREIMAN Neurology
and Research Department
Services,
of Neurology,
Veterans UCLA
Administration West Los Angeles School of Medicine, Los Angeles,
Medical California
Center,
and
nobutyric acid (GABA) at times before and during the course of SE induced by lithium and pilocarpine as a first step in investigating the possible role these neurotransmitters play in the maintenance of late SE in this model. Serine, glutamine, and glycine were measured as part of the same assay. We have reported elsewhere (31) a sequence of changes in electroencephalographic (EEG) pattern which is seen in human generalized convulsive SE and in experimental SE in the rat. We used the EEG pattern to define the electrophysiologic stages of SE and determine the times at which brain samples were obtained in this experiment. Brain amino acid changes during SE, in experiments which included electrographic monitoring, have been reported only when SE was induced by mechanisms distinct from those involved in lithium/pilocarpine SE and only in paralyzed, artificially ventilated rats (5-7).
Amino acid concentrations were measured in specific structures from the brains of rats decapitated before and during the course of status epilepticus induced by lithium and pilocarpine, with the stages of status defined by the electroencephalographic (EEG) pattern displayed. Early status was marked by discrete seizures on EEG, mid status by continuous spiking, and late status by periodic epileptiform discharges. Aspartate levels were lower than control levels in most regions prior to the onset of status. The decline continued and reached statistical significance in different regions at times from early to late status. Glutamate concentrations were typically higher than control just prior to status onset and then decreased in a manner similar to aspartate, but with less percentage change. y-Aminobutyric acid increased during status, with the earliest statistically significant differences observed in mid status. These changes were observed in most forebrain structures studied, but the largest percentage changes in excitatory amino acid concentration were found in substantia nigra, where they fell to less than half of control. 0 1990 Academic Press, Inc.
METHOD Subjects. Adult, male, Sprague-Dawley rats (n = 42) were used as subjects in this experiment. Rats were implanted surgically with chronic epidural recording electrodes, as has been described previously (32). They were housed singly after surgery, with food and water available ad lib., room temperature maintained at 20°C and a 24-h diurnal lighting schedule (lights on from 0700 to 1900 each day). Rats were allowed to recover for 1 week prior to experimentation. Drugs. Ketamine (100 mg/ml, Parke-Davis), 87 mg/ kg, and xylazine (20 mg/ml, Haver), 13 mg/kg, given as a single intraperitoneal (ip) injection, were used for surgical anesthesia. Lithium chloride (Sigma) was dissolved in sterile water and injected ip at a dose of 3 mmol/kg. Pilocarpine (Sigma) was dissolved in saline and injected ip at a dose of 25 or 30 mg/kg. Experimental design. Six rats were assigned to each of seven groups, the characteristics of which are shown in Table 1. These groups were chosen to provide controls for each of the convulsant drugs used to induce SE and to allow us to seewhat, if any, changes were taking place
INTRODUCTION Systemic injection of pilocarpine to lithium-pretreated rats has been shown to produce a nearly universally fatal status epilepticus (SE) which lasts at least 2 h in nonparalyzed animals (13, 16). The induced SE is responsive to treatment with atropine or standard anticonvulsant drugs, provided the treatment is initiated early in the SE episode (21). We have reported that SE induced by lithium and pilocarpine becomes less responsive to treatment with large doses of diazepam the later in the SE episode that treatment is given (32). Induction of SE in this model requires cholinergic synaptic activity, but some other neurotransmitter system appears to be responsible for maintenance of the SE beyond about the first 30 min (15). Although changes in brain amino acid concentrations during SE might be either a cause or an effect of the ongoing seizure activity, we assayed the levels of aspartate, glutamate, and y-ami61
Copyright 0 1990 All rights of reproduction
0014-4&x/90 $3.00 by Academic Press, Inc. in any form reserved.
62
WALTON, TABLE
GUNAWAN.
1
Characteristics of Experimental and Control Groups Studied in This Experiment (n = 6 per Group) Group
First injection
Second injection
Control-l
Saline
Saline
Control-Z
Lithium
Saline
Control-3
Saline
Pilocarpine
Lithium
Pilocarpine
Early SE Mid SE
Lithium Lithium
Pilocarpine Pilocarpine
Late SE
Lithium
Pilocarpine
Before
Note.
SE onset
Second
injections
were given
Decapitation
time
60 min after second injection 60 min after second injection 60 min after second injection 15 min after second injection Discrete seizure on EEG 10 min continuous spiking on EEG Initial PEDs on EEG
24 h after
the first
injections.
prior to the onset of SE and during its earliest stage, as well as at the later stages which were of primary interest to us. EEG was monitored from the second injection until decapitation for all rats. Tissue processing. Brains were removed from the skull immediately after decapitation, placed into small plastic bags, and frozen by immersion in acetone and dry ice. This technique was chosen because the presence of metal electrodes in the skull precluded the use of microwave fixation (2) and the need to freeze the brains as quickly as possible once EEG criteria were met precluded use of Ponten’s (28) in situ freezing technique. Brains were stored at -70°C for up to 50 days prior to extraction. Regional microdissection was carried out on 300-pm frozen sections which had been cut in a cryostat at -15°C. The sections were placed on glass slides and brain structures were dissected out on a freezing stage (-15°C) under a microscope. Structures were chosen for microdissection and assay on the basis of our ability to rigorously define and reliably obtain the same tissue from all animals. One set of structures (nucleus accumbens, caudate-putamen, substantia nigra, hippocampus, septum, amygdala, inferior colliculus and cingulate, motor, pyriform, and entorhinal cortex) was selected because it has been suggested by others to be important in this or other experimental seizure models (1, 3, 8, 12, 18,19,29). Another set of structures (cerebellum, ponsmedulla, and superior colliculus) was chosen in hopes of
AND TREIMAN
finding at least one structure which showed little or no change in this model of SE. Cerebellum and pons-medulla were dissected away from the frozen brain prior to its being mounted in the cryostat. The following structures were dissected, guided by the atlas of Palkovits and Brownstein (24): cingulate cortex, from slices where the head of the caudate nucleus was identifiable to where the dorsal hippocampus was no longer connected across the hemispheres; pyriform cortex, from the second slice after the appearance of the corpus callosum to just before the first appearance of the ventral hippocampus; nucleus accumbens, from slices in which the head of the caudate nucleus was visible and the next 2100 pm; septum, from slices in which the lateral ventricles were clearly visible until the first appearance of the globus pallidus; frontal-parietal sensory-motor cortex, which will be hereafter referred to simply as motor cortex, from slices in which the head of the caudate nucleus was visible to those about 2100 pm past the initial appearance of the dorsal hippocampus; caudateputamen, from slices in which the head of the caudate was no longer completely surrounded by the corpus callosum to those about 600 pm beyond the first appearance of the dorsal hippocampus; hippocampus, both dorsal and ventral, including CAl, CA2, CA3, CA4, and the dentate gyrus; amygdala, from slices just posterior to the optic chiasm, continuing one or two slices beyond the first appearance of the ventral hippocampus; entorhinal cortex, from slices in which the ventral hippocampus was visible until the hemispheres disappeared just anterior to the cerebellum; substantia nigra, which could be seen in slices in which the dorsal and ventral hippocampi had merged to form a continuous, lateral structure, and continued for about 1200 pm; superior colliculus, which was readily identifiable about 600 pm after the appearance of substantia nigra and continued until the dentate gyrus was no longer seen; and inferior colliculus, from slices in which the dentate gyrus could no longer be seen until the appearance of the cerebellum. Dissected tissue was placed in frozen 1.5ml microcentrifuge tubes and weighed. Ice-cold 80% ethanol (18 ml/ g tissue wt) was added and the tissue was disrupted by ultrasonication. Homogenates were centrifuged at 15,OOOg,4”C, for 15 min. The supernatant was transferred to glass culture tubes maintained on ice and the extraction was repeated with more 80% ethanol. Supernatant from the second extraction was added to that from the first, the tube was vortexed, and the contents were measured and stored overnight at 4°C.
FIG. 1. Examples of EEGs recorded during this experiment, showing the patterns which defined the electrophysiological stages of SE: (a) nonseizing EEG recorded 14 min after injection of pilocarpine, before any electrographic or behavioral evidence of seizure activity was seen; (b) discrete electrographic seizure which ends abruptly and simultaneously in all channels (early SE), recorded 40 s after SE onset; (c) continuous, high-amplitude, rapid spiking, punctuated by brief flat periods (mid SE), recorded 30 min after SE onset; (d) periodic epileptiform discharges (late SE), recorded 90 min after SE onset.
AMINO
a F4-P4 W-P3
F3-F4 F4-P4
b F4-P4 W-P3 P3-F3 F3-F4 F4-P4 C F4-P4 W-P3 P3-F3 F3-F4 F4-P4
d F4-P4 W-P3 P3-F3 F3-F4
ACIDS
IN
STATUS
EPILEPTICUS
63
64
WALTON.
TABLE Regional
GUNAWAN,
RESULTS
acid concentration
Brain
structure
ASP
GLU
SER
GLN
GLY
GABA
Cingulate
cortex
Pyriform
cortex
2.36 (0.12) 2.37 (0.12) 2.66 (0.07) 2.28 (0.10) 1.71 (0.06) 1.62 (0.05) 2.09 (0.21) 1.95 (0.07) 2.21 (0.09) 1.62 (0.06) 2.49 (0.21) 2.29 (0.17) 2.30 (0.17) 2.89 (0.27)
9.19 (0.70) 9.27 (0.80) 9.79 (0.65) 9.86 (0.56) 7.84 (0.55) 8.41 (0.32) 4.43 (0.53) 9.68 (0.48) 8.33 (0.66) 6.59 (0.35) 5.54 (0.23) 5.40 (0.41) 8.88 (0.49) 5.12 (0.16)
0.76 (0.04) 0.92 (0.07) 0.77 (0.06) 1.02 (0.05) 0.80 (0.08) 0.96 (0.06) 0.47 (0.06) 1.00 (0.07) 0.74 (0.07) 0.77 (0.06) 0.43 (0.04) 0.44 (0.04) 0.69 (0.04) 0.56 (0.07)
3.61 (0.32) 4.36 (0.35) 3.98 (0.26) 4.73 (0.24) 4.88 (0.35) 4.54 (0.19) 2.74 (0.20) 4.13 (0.14) 4.50 (0.33) 3.98 (0.20) 3.31 (0.14) 3.37 (0.21) 4.81 (0.20) 2.88 (0.21)
0.66 (0.05) 0.60 (0.05) 0.65 (0.05) 0.70 (0.06) 0.71 (0.07) 0.74 (0.08) 0.88 (0.06) 0.92 (0.07) 0.69 (0.06) 0.66 (0.05) 1.39 (0.15) 1.12 (0.13) 1.12 (0.24) 3.53 (0.23)
1.50 (0.11) 1.89 (0.15) 1.60 (0.12) 1.52 (0.06) 2.67 (0.20) 1.81 (0.18) 5.09 (0.67) 1.94 (0.07) 3.45 (0.47) 3.02 (0.21) 2.77 (0.23) 3.37 (0.24) 1.73 (0.18) 1.58 (0.11)
Motor
cortex
Entorhinal Nucleus
cortex accumbens
Caudate-putamen Substantia
nigra
Hippocampus Amygdala Septum Inferior Superior
colliculus colliculus
Cerebellum Pons-medulla
TREIMAN
2
Amino Acid Concentrations from Rats Treated with Saline Only (n = 6) Amino
AND
Note. Concentrations are expressed in pmol/g tissue weight. Means are shown with standard deviations in parentheses below. Amino acid abbreviations: ASP, aspartate; GLU, glutamate; SER, serine; GLN, glutamine; GLY, glycine; GABA, y-aminobutyric acid.
Amino acid assay. Amino acids were assayed using a method developed in this laboratory (11). Briefly, norvaline (internal standard) was added to 50 ~1 of extraction supernatant and dried under vacuum. The residue was reacted with phenylisothiocyanate for 20 min at room temperature to form phenylthiocarbamyl derivatives of the amino acids. Excess reagent was removed under vacuum and the residue was dissolved in 300 ~1 0.1 M sodium acetate buffer (pH 5.7) and injected into the chromatograph. HPLC separation was carried out in a reversed-phase system with a Rainin Cl8 column as the stationary phase and a gradient system of 0.1 M sodium acetate buffer and acetonitrile/water (60/40) as the mobile phase. Absorbance was monitored by an ultraviolet detector set at 254 nm. Amino acid concentrations were calculated by linear regression analysis, comparing the peak area ratio of amino acid:internal standard to those obtained in standard curve determinations.
The progression of EEG changes which occurs in this model of SE has been described elsewhere (31, 32) and is illustrated in Fig. 1. Figure la shows the EEG as it appears after pilocarpine has been injected, but before any evidence of seizure activity appears. SE begins with discrete electrographic seizures in which spiking ceases simultaneously in all channels and is followed by a brief period of postictal low-voltage slow activity (Fig. lb). After several minutes the distinct seizure offset is no longer seen. Instead, irregular, l- to ~-HZ spike and wave activity follows episodes of rapid spiking, with spike amplitude increasing rapidly over time. Spiking eventually becomes relatively constant at a high amplitude and rate of occurrence, sometimes interspersed with brief flat periods (Fig. lc). Continuous spiking continues for l-2 h when SE is induced by lithium and pilocarpine and then PEDs, separated by periods of lower amplitude activity,
ASPARTATE
\
\
“s---q 1 *
8 100 -:: 90 -- -a,\
--I-
GLUTAMATE
.
.
Is
L -
b-N., *
100 90 f
iz ;T
.
*
\
80 70
g
I
-3
*
GLUTAMINE
2
is
120
5 a
110 100 90
GABA
---
=.*
i
I
I
Before Onset
00
0
0
Y-
k I
Discrete Seizures
I
I
Continuous Spiking
PEDs
FIG. 2. Amino acid concentration changes in motor cortex just before onset of and during SE induced by lithium and pilocarpine. In this and all figures to follow each point is the mean + standard error for six rats, and points marked + are statistically different from control at P < 0.05, while those marked * are different at P < 0.01.
AMINO
--- * \ ASPARTATE
ACIDS
IN
STATUS
Mean control concentrations of aspartate, glutamate, serine, glutamine, glycine, and GABA in all regions studied are given in Table 2. No statistically significant differences in concentrations of serine or glycine were found among the groups for any brain structure studied. Amino acid changes in neocortex, relative to control, are shown in Figs. 2-5. Aspartate decreased in all neocortical areas studied, with concentrations being lower before the onset of SE and reaching statistical significance in both the continuous spiking and PEDs groups (P < 0.01). Motor cortex (Fig. 2) also had significant changes in the concentrations of glutamate, glutamine, and GABA. Glutamate levels were significantly less than control in the continuous spiking and PEDs groups (P < 0.01). Concentrations of glutamine and GABA were significantly higher than control in the PEDs group in both motor and cingulate (Fig. 3) cortex (P < 0.01). Glutamate levels in cingulate cortex were significantly lower in the continuous spiking group (P < 0.05). Pyriform cortex (Fig. 4) showed no significant changes across groups for glutamate, glutamine, or GABA. GABA con-
\ \
b0 5
60
z? ;
110
F 8 0
loo 90 80
Ts 100
---
+-
---
I
0 z
\
\
\+
y-/-
GLUTAMINE
--
z
5<
\
GLUTAMATE
3’
4
,4 *
125 GABA
65
EPILEPTICUS
I l. -.
lOOf---
-L
,X0”
100
ASPAR‘CATE
--‘-)---y’
755
Before Onset
\ Discrete Seizures
FIG. 3. Amino acid concentration fore and during SE induced by lithium the same as in Fig. 2.
Continuous Spiking
* \
\
PEDs
changes in cingulate cortex and pilocarpine. Symbols
beare
begin to appear in runs of several seconds duration (Fig. Id). The PEDs gradually increase in frequency and duration of occurrence until the rat dies. Untreated SE is almost always fatal in this model (17,21,22,26). Data are given as means f standard deviation, unless noted otherwise. Latency of SE onset from the time of pilocarpine injection was 33.1 -t 13.0 min. Analysis of variance revealed no statistically significant differences among the experimental groups. Duration of SE at the time of decapitation was 2.5 ? 0.5 min for the group decapitated when the EEG showed discrete seizures, 30.2 -+ 13.7 min for the group decapitated at continuous spiking on EEG, and 111.3 f 20.7 min for the group decapitated at the first appearance of PEDs. The interval from decapitation to immersion of the brains in the freezing solution was 84.2 f 26.2 s, with no statistically significant differences among the groups. Evaluation of changes in brain amino acid concentrations was made by analysis of variance, with Dunnett’s t test used to compare differences between experimental group means and the mean of the control group which was given no drugs (9). Alpha was set at 0.05 for all tests.
/
\ GLUTAMATE
z
85’
8
1101
!k [r
1oo
2 z-
go
s
80 125 GABA
0 z
4--
f 100
,
---
/’
--k
751
Before Onset
Discrete Seizures
FIG. 4. Amino acid concentration fore and during SE induced by lithium -. the same as in Fig. 2.
Continuous Spiking
PEDs
changes in pyriform cortex and pilocarpine. Symbols
beare
100 52 2 tr’
66
WALTON.
\
ASPARTATE
g
\
75
\
\
73
0
90
2
80 J
8
100
*
* */-
GLUTAMATE
\
\
----*---
‘. ;
GUNAWAN,
90
z
GLUTAMINE
g
8o
5
140
a $
AND
TREIMAN
In hippocampus (Fig. 9), concentrations of aspartate were significantly decreased by the time of discrete seizures (P < 0.05) and decreased further by continuous spiking and PEDs (P < 0.01). Glutamate and glutamine levels were significantly lower only in the continuous spiking group (P < 0.05), while GABA rose significantly in the continuous spiking and PEDs groups (P < 0.01). Only aspartate changed significantly in amygdala (Fig. lo), being significantly lower than control in both the continuous spiking and PEDs groups (P < 0.01). In the septal nuclei (Fig. ll), aspartate concentrations were lower in the continuous spiking and PEDs groups (P < O.Ol), glutamate was lower at PEDs (P < 0.05), glutamine was lower at discrete seizures and continuous spiking (P < 0.05), and GABA was elevated at PEDs (P < 0.01). Fewer and smaller changes were observed in the cerebellum, inferior colliculus, and superior colliculus. Cerebellar aspartate was less than control at continuous spiking (P < 0.05) and PEDs (P < O.Ol), while glutamine was elevated at PEDs (P < 0.05). Only glutamine
% loot---f----,
I20 100
e
I
Before Onset
I
ASPARTATE
‘\.
‘\ \
I
Discrete Seizures
FIG. 5. Amino acid concentration before and during SE induced by lithium the same as in Fig. 2.
I
Continuous Spiking
P:Ds
changes in entorhinal cortex and pilocarpine. Symbols are
centrations rose significantly in entorhinal cortex in both the continuous spiking (P < 0.05) and PEDs (P < 0.01) groups (see Fig. 5). Nucleus accumbens (Fig. 6) showed significant declines in aspartate, glutamate, and glutamine in the continuous spiking and PEDs groups (P < O.Ol), while GABA levels increased in the PEDs group (P < 0.01). In caudate-putamen (Fig. 7), glutamate levels were significantly less than control in both the continuous spiking and PEDs groups (P < 0.01). Aspartate levels were significantly lower in the PEDs group (P < 0.01) while glutamine concentrations were decreased in the continuous spiking group (P < 0.01). GABA was increased significantly in the PEDs group (P < 0.01). Substantia nigra (see Fig. 8) showed the largest percentage decline of the excitatory amino acids of any structure studied here. Aspartate concentrations in the continuous spiking and PEDs groups were significantly lower than control (P < O.Ol), as were the concentrations of glutamate (P c 0.05). Glutamine did not change significantly, but GABA concentrations were significantly elevated at both continuous spiking (P < 0.05) and PEDs (P < 0.01).
+
///
-i
\* b”/
-N\ \
GLUTAMATE
\
\* --
w’
j
6 3 9
90 80
a
701
-\
\ f
---.+
* Y----i
GLUTAMINE
I Before Onset
I
Discrete Seizures
-4
0’/4*
I
Continuous Spiking
1 PEDs
FIG. 6. Amino acid concentration changes in nucleus accumbens before and during SE induced by lithium and pilocarpine. Symbols are the same as in Fig. 2.
AMINO
ASPARTATE
‘-e-----
ACIDS
\
\
\
IN
STATUS
We attempted to study an even later group, which was decapitated after a long duration of PEDs had occurred. These brains, however, did show evidence of general metabolic derangement, with concentrations of all amino acids measured rising at this time. These rats also appeared to be much more debilitated than any of our other animals. We believe the most parsimonious explanation for the pattern of changes which we observed in this experiment is that the excitatory amino acids, aspartate and glutamate, were being released rapidly enough during the time from onset of seizures to the stage during which the EEG pattern is one of continuous spiking to produce a significant decrease in the tissue concentrations of these amino acids. The rise in GABA concentrations is more difficult to explain. Although it is tempting to postulate that this increase represents a compensatory mechanism of the brain in response to continuous seizure activity, this explanation is difficult to defend. Increased release of GABA would lead to increased tissue levels only if
*
\ *
\
GLUTAMATE
\ \ \* s
--
140
-4
/ ‘*
100
.
67
EPILEPTICUS
4
GABA
\ ASPARTATE
t Be/ore
Onset
Disdrete Seizures
FIG. 7. Amino acid concentration before and during SE induced by lithium the same as in Fig. 2.
Conthuous Spiking
\
\
\
‘\
.
PiDs
changes in caudate-putamen and pilocarpine. Symbols
are
100 2 P
Z changed significantly in the superior and inferior colliculi, being elevated at PEDs in both regions (P < 0.01). None of the amino acids studied changed significantly in the pons-medulla.
.
70
GLUTAMATE
L_ -- s*
-s.. \
---4
a Ej
40 r
s
DISCUSSION
Changes in amino acid neurotransmitter brain concentrations are, of course, crude approximations of changes which may be occurring at the synaptic level. Furthermore, some of these amino acids have metabolic roles in brain, in addition to their neurotransmitter functions. The changes which we found do not appear to be secondary to any general derangement of amino acid metabolism, for several reasons. First of all, serine and glycine did not change in any of the structures we studied and glutamine changed significantly in only a few. In addition, while the concentrations of the excitatory amino acids were decreasing, the levels of GABA were rising. Finally, the extent of change for any of these amino acids differed widely across brain structures.
5 y
7oJ 160
a
140
z 3 a
120
0
I
GABA
c--
f *
100
800
4-
--
o-
Before Onset
Discrete Seizures
FIG. 8. Amino acid concentration fore and during SE induced by lithium the same as in Fig. 2.
Continuous Spiking
PEDs
changes in substantia nigra and pilocarpine. Symbols
beare
WALTON.
68
GUNAWAN,
-\. -\ -- +
loo*._
\
ASPARTATE
* \
i+!
1201 3
GLUTAMATE
/ /
. -\
0 \
k
0
\
/
\
’
-6 -- -;’
/ / /
A
AND
TREIMAN
ine, Blennow et al. reported that cortical aspartate and glutamate decreased, but saw no change in GABA concentrations (6). Jope and colleagues reported that acetylcholine concentrations in both hippocampus and cortex decreased early and then rose dramatically later during SE induced by lithium and pilocarpine (17). The sampling times in their experiment were determined by the time elapsed after injection of pilocarpine, so only approximate comparisons can be made with our experimental groups. However, the time at which they found the highest levels of acetylcholine appears to be similar to that of our PEDs group. The anatomic distribution of changes in the concentrations of aspartate, glutamate, and GABA suggests extensive forebrain participation in late SE induced by lithium and pilocarpine. More posterior structures showed fewer and less extensive changes. Clifford et al. (8) reported that, in animals implanted with multiple depth electrodes, SE seemed to start simultaneously in many forebrain structures in some animals and in the ventral pallidum or nucleus accumbens in others. Jope
‘00-p.\
GASA /
a 80
I
I Before Onset
FIG. 9. Amino acid concentration and during SE induced by lithium same as in Fig. 2.
I Discrete Seizures
I Continuous Spiking
changes in hippocampus and pilocarpine. Symbols
1 PEDs
.
ASPARTATE
\ i
E g
before are the
75
L
8
a
\
50
100
a
GABA reuptake or metabolism were impaired. Synthesis of GABA has been shown to continue even after death (25), so it is more likely that release of GABA was impaired while synthesis continued, leading to an accumulation of GABA in brain tissue. It has been shown in other models of SE that GABAergic neurons are susceptible to early damage during uncontrolled SE (30). Although others have reported that lithium effects amino acid metabolism in brain (4,27), we found no significant changes in any of the amino acids measured in rats treated only with lithium or only with pilocarpine. This was not unexpected, since Jope and Morrisett (15) have elegantly demonstrated that cholinergic systems are primarily responsible for the onset of SE in this model. The reported effects of lithium on amino acid metabolism may require chronic treatment or single doses larger than those used here. When SE was induced by bicuculline, Chapman and her colleagues found the same pattern of changes in aspartate, glutamate, and GABA in neocortex as we have described here (7). With SE induced by homocyste-
8
80
s a
100
zcn
85
\
y+--+%~
0 0 a
\
GLUTAMINE
70 GABA
100
--
\
w
80
60
’
I
Beiore Onset
I
Discrete . Seizures
I
Continuous Spiking
FIG. 10. Amino acid concentration changes in amygdala and during SE induced by lithium and pilocarpine. Symbols same as in Fig. 2.
1
PEDs before are the
AMINO
ACIDS
IN
STATUS
.-)---a,\
function must be intact at these early times, because drugs which act by facilitating GABAergic inhibition (diazepam, phenobarbital) are effective in stopping SE in this and other SE models as well as in early human SE (20,21,23,32). If our interpretation of these results is valid, it suggests that treatment of human SE which has progressed to the point where it does not respond to the usual anticonvulsant therapies may require drugs with a completely different mechanism of action than the conventional GABA-enhancing anticonvulsants (20). Excitatory amino acid blockers may be required to halt ongoing seizure activity at this time, or compounds which limit the second messengers which are activated by the calcium influx associated with sustained NMDA receptor activation. The recently reported increase in survival rate following SE induced by lithium and pilocarpine when rats were treated with acepromazine raises the issue of catecholamine participation in late lithium/ pilocarpine SE, although these authors did not attempt to assessseizure control following treatment (26). The role of excitatory amino acid neurotransmitters in late SE requires much further research to be clarified and understood completely.
ASPARTATE
0 5 100 -8 85 T z
\ GLUTAMATE
-3---,,
-
Es 85 0 a 7n h
/
--
69
EPILEPTICUS
GLUTAMINE
‘-
ACKNOWLEDGMENTS Before Onset
Discrete Seizures
FIG. 11. Amino acid concentration before and during SE induced by lithium the same as in Fig. 2.
Continuous Spiking
PEDs
This work was supported by grants from the Veterans Administration and from the Epilepsy Foundation of America. The tireless and professional technical assistance of Mr. Barry Cole is gratefully acknowledged.
changes in the septal nuclei andpilocarpine. Symbols are
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