210
Brain Research, 557 (1991) 210-216 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 0(0899391169132
BRES 16913
Changes in pre- and postsynaptic components of noradrenergic transmission in hippocampal kindling in rats A. Vezzani, M. Rizzi, R. Serafini, G. Vigan6 and R. Samanin lstituto di Ricerche Farmacologiche 'Mario Negri', Milan (Italy)
(Accepted 9 April 1991) Key words: Noradrenaline; Isoproterenol; al-Adrenoceptor; fll-Adrenoceptor; Release; Phosphatidyl inositol turnover; Cyclic adenosine monophosphate; Kindling; Hippocampus; Rat
We investigated whether modifications in noradrenergic neurotransmission occurred during the development of hippocampal kindling in rats. We measured the release of [3H]norepinephrine (NE) induced by field-electrical stimulation, NE-stimulation of inositol phosphates ([3H]IP) accumulation in the presence of LiCI and isoproterenol-induced accumulation of cAMP in hippocampal slices taken from rats electrically kindled at stages 2 and 5 in the dorsal hippocampus. One week after the last of at least 3 consecutive stage 5 seizures or 48 h after the last stage 2 stimulation, 2 min electrical stimulation of stratum pyramidale CA1-CA3 or dentate gyrus (DG) slices from kindled and contralateral hippocampi induced frequency-dependent NE release (respectively 2, 4 and 8 times spontaneous release measured at 2, 5 and 10 Hz) which did not significantly differ from that observed in shams (implanted with electrodes but not stimulated). Basal release of NE from kindled and sham-treated rats did not differ either. Isoproterenol induced a dose-dependent increase above basal cAMP concentration ranging from 40% at 0.01 ~M to 180% at 10/~M (P < 0.01, Dunnett's test) which did not differ between stages 2 and 5 and sham-hippocampi. NE (1-1000 #,M) induced a dose-dependent, prazosin-sensitive increase in [3H]IP accumulation in the hippocampal slices. A significantly higher increase was found at stages 2 (P < 0.05, Tukey's test) and 5 (P < 0.05 and P < 0.01, Tukey's test) compared to shams at all doses studied. No differences were observed in the basal cAMP or [3H]IP concentrations in kindled versus sham animals. No differences were observed in [3H]IP accumulation induced by carbachol (10, 100, 1000/~M) in stage 5-kindled rats and shams. These changes have various possible implications for kindling-induced plasticity. INTRODUCTION Kindling is an animal model of neuronal plasticity and epileptogenesis. It develops over time in response to r e p e a t e d electrical or chemical stimuli leading to progressively m o r e intense limbic seizures and eventually clonic m o t o r seizures 12. Once established, this p h e n o m enon is a p p a r e n t l y irreversible. T h e biochemical mechanisms underlying the development of kindling and its persistence are mostly unknown but recent observations suggest that it is associated with increased activity of glutamate ( G L U ) - c o n t a i n i n g neurons T M and enhanced postsynaptic N-methyl-Daspartate ( N M D A ) r e c e p t o r sensitivity26'35. A m o n g the neurotransmitters studied so far, norepinephrine ( N E ) has been shown to have p r o f o u n d effects in this model. D e p l e t i o n of forebrain N E facilitates the d e v e l o p m e n t of kindling 7 without affecting kindling once established 39. Locus coeruleus stimulation 15'38 and activation of a2-adrenoceptors 1° inhibit kindling development. Interactions b e t w e e n N E and G L U have also been
r e p o r t e d in the hippocampus: N E enhances the excitation induced by G L U on h i p p o c a m p a l neurons by blocking the slow CaE+-dependent K + currents 29 and it increases the K+-induced G L U release in the d e n t a t e gyrus ( D G ) but not in stratum p y r a m i d a l e CA1-CA323. Thus noradrenergic neurotransmission a p p e a r s to be modified in kindling and this might affect G L U - c o n t a i n i n g neurons contributing to the e n h a n c e d N M D A - r e c e p t o r sensitivity which underlies kindling. In vivo N E release increases in the h i p p o c a m p u s after an electrical stimulus inducing an afterdischarge ( A D ) la' 19. D u r i n g rapid h i p p o c a m p a l kindling the in vivo release of N E in response to the first electrical stimulation gradually d r o p p e d back to baseline levels with further stimulations TM. This suggests t h e r e is only a transient initial increase of N E release during kindling. O t h e r authors 16A7 have found no significant changes of N E release in a m y g d a l a - k i n d l e d rats. D o w n - r e g u l a t i o n of txEand f l : a d r e n o c e p t o r s has b e e n r e p o r t e d after amygdala kindling 6'25'3~. In this study we used biochemical indices to confirm that changes in n o r a d r e n e r g i c neurotransmission occur during kindling and to clarify the type of
Correspondence: A. Vezzani, Istituto di Ricerche Farmacologiche 'Mario Negri', via Eritrea 62, 20157 Milano, Italy. Fax: (39) (2) 354 6277.
211 receptors
mainly involved. We studied
the effect of
k i n d l i n g o n t h e basal and field e l e c t r i c a l - s t i m u l a t e d N E release from stratum pyramidale CA1-CA3
and D G
slices as p r e s y n a p t i c m a r k e r s o f n o r a d r e n e r g i c activity, and o n N E - s t i m u l a t e d PI t u r n o v e r and i s o p r o t e r e n o l i n d u c e d c A M P a c c u m u l a t i o n as p o s t s y n a p t i c m a r k e r s o f t h e f u n c t i o n a l status o f a 1- and f l l - a d r e n o c e p t o r s . T h e s e parameters stage
w e r e assessed at t h e early, p r e c o n v u l s i v e
of kindling
(stage
2)
and
once
kindling
was
e s t a b l i s h e d (stage 5). A p r e l i m i n a r y r e p o r t o n t h e s e d a t a has a p p e a r e d as an abstract 36. MATERIALS AND METHODS Male Sprague-Dawley rats (250-280 g, Charles River, Italy) were used. The animals were housed at constant temperature (23 °C) and relative humidity (60%) with a fixed 12 h light-dark cycle and free access to food and water.
Implantation procedure The surgical procedure for electrode implantation in the dorsal hippocampus under Equitensin anesthesia (9.7 mg/ml sodium pentobarbitai in saline; 42.6 mg/mi chloral hydrate in propyleneglycol, 21.2 mg/ml MgSO 4 in ethanol; 3.5 ml/kg, i.p.) was as previously described 37. For implantation the coordinates from bregma were: (nose bar -2.5) AP 3.5, L 2.3, H 2.9. In addition, staiuless-steel screw electrodes were placed bilaterally over the sensorimotor cortex and a ground lead was screwed over the nasal sinus. The electrodes were connected to a multipin socket (March Electronics, New York, NY) and were secured to the skull by acrylic dental cement. Electroencephalographic (EEG) recordings were made in unanesthetized, freely moving animals as previously described 37.
Kindling procedure Kindling was started after a postoperative period of 7 days, when the animals showed no behavioral signs of pain or discomfort. The rats were allowed to acclimatize in a Plexiglas cage and an EEG recording (4-channel EEG polygraph, model B8P, Battaglia Rangoni, Bologna, Italy) was made for at least 10 min to assess the spontaneous EEG pattern. Constant current stimuli were delivered to the left hippocampus (granule cells of the dentate gyms) through a bipolar electrode (recording electrode) twice daily for 5 days and once daily for 2 days (week-end), at intervals of at least 6 h. The parameters of stimulation were 50 Hz, 2 ms monophasic rectangular wave pulses for 1 s, the current intensity ranging between 60 and 200 /~V. Behavior was observed and AD duration measured in the stimulated hippocampus after each stimulation, for every animal. Before starting electrical stimulation, the rats were randomly assigned to two groups and received 12 _+ I and 24 _+ 2 stimuli (mean _+ S.E.M.) to reach, respectively, stages 2 and 5 of kindling according to Racine's classification3°. Previous experiments had shown that after 12 stimuli the animals displayed stereotypies and occasional retraction of a forelimb (stage 2). Stage 5 was characterized by tonic-clonic seizures with rearing and falling. Animals were considered fully kindled when they experienced at least 3 consecutive stage 5 seizures. Controls were implanted with electrodes and handled in the same way as the experimental rats but were not electrically stimulated. Light microscopic analysis of Nissl-stained 40/~m cryostat sections was made 37 on completion of kindling to verify the electrode placement in the granule cells of the DG.
Biochemical assays For biochemical determinations, rats kindled at stages 2 and 5, and their controls, were killed, respectively, 48 h and one week after the last stimulation.
L-[3H]NE release Experimental conditions for the uptake and release of L-[3H]NE from brain slices were chosen according to the methods described by Ziance and Rutledge 4° and Nelson et ai.z7, with some modifications. The dorsal hippocampi (left and right separately) were mechanically sliced in the transverse plane at a thickness of 1 mm with a McIlwain chopper. The slices were pooled and suspended in 5 ml of oxygenated Krebs-bicarbonate buffer (OKB), pH 7.6, containing ascorbic acid (10 rag/100 mi) and pargyline (5 rag/100 mi). Slices were then microdissected in CA1-CA3 and DG subregions. After preincubation for 15 rain at 37 °C, 0.1 /~M L-[3H]NE (13 Ci/mmol; radiochemical purity 90%; NEN, Boston, MA, U.S.A.) was added and the slices were incubated for another 20 rain. The supernatant was decanted off and the slices were washed 3 times with fresh OKB. The slices were mounted on superfusion chambers (about 8 mg wet weight tissue/chamber) and superfused at 0.5 ml/min at 37 °C with OKB. Samples were collected every 5 min. A baseline recording was made after a 30-min washout period. After 60 min baseline the slices were electrically stimulated for 2 min at 2, 5 and 10 Hz at interstimulus intervals of 60 min. Radioactivity was assessed in the various fractions by scintillation spectrometry. Slices were recovered at the end of the experiment and dissolved in 0.5 mi of Soluene (Packard Instrument, Downers Grove, IL, U.S.A.) and the 3H content was determined in the same way. Fractional release was calculated as 3H released into the medium during each 5 min fraction as a percentage of the 3H content of the slices during that interval. The effect of the stimulation was calculated as the fractional release during electrical stimulation ($2) divided by the baseline spontaneous release (S1), the latter being the average of 3 samples before drug application. Stimulated release was assessed as the average of the sample collected during stimulation and the one after.
cAMP accumulation cAMP accumulation was measured according to the method of Magistretti and Schorderet 24. Rats were decapitated, the brains removed rapidly and placed on ice. The dorsal hippocampi of both hemispheres were dissected out and pooled in an oxygenated modified Krebs-HEPES (OKH) buffer containing (in raM): NaCI 125; KCI 5; CaCI 2 2; MgSO 4 1.2; KH2PO 4 1.2; glucose 3; HEPES 25, pH 7.4. The hippocampi were chopped in miniprisms (350 x 350 /zm) and resuspended in 5 ml OKH. After replacing the buffer once, the slices were incubated in a vial containing 5 ml OKH at 37 °C with mild shaking for 30 rain. The slices were then washed twice, the second wash with OKH containing 0.5 mM isobutyl methylxanthine (IBMX). Test tubes containing 50/~l OKH + 0.5 mM IBMX _+ isoproterenol (10-9-10 -5 M) were incubated at 37 °C for 5 rain then 50/~l of densely packed slices were distributed into individual test tubes and incubated for another 10 rain. Incubation was terminated by boiling the samples for 5 rain followed by sonication for 5 s. The samples were then rapidly centrifuged (16,000 g for 5 rain), the supernatant was separated and stored at -20 °C until assayed. cAMP present in the samples was determined by a cAMP assay kit (Amersham International, Ltd., Buckinghamshire, U.K.). Experimental values were calculated from a standard curve ranging from 0 to 16 pmol of cAMP. Proteins were measured in the pellets according to the method of Lowry et al. 22.
PI turnover Phosphatidylinositol (PI) hydrolysis was measured according to Brown et al.4. Dorsal hippocampi of both hemispheres were chopped into 350 x 350/~m miniprisms. The cubes were pooled and washed 3 times in OKB, pH 7.4. After 45 mln preincubation in OKB at 37 °C the slices were incubated for 60 rain with 0.2/~M myo-[3H]inositol (CEA, Gif-sur-Yvette, Cedex, France) in the presence of 5 mM LiCI then washed 3 times with OKB. The prelabeled miniprisms (40 /zl of densely packed cubes) were incubated at 37 °C for 60 rain with OKB-LiCI + agonists in a total
212 volume of 310 #l. When antagonists were used, they were added 5 min before the agonists. Incubation was terminated by addition of 930 #l of ice-cold methanol-chloroform-6N HCI (2:1:0.01). The slices were then sonicated and centrifuged and the supernatants used for determination of [3H]inositoi phosphates (IP). The supernatants were subjected to 4 diethylether extraction cycles. After neutralization with 10 mM sodium tetraborate, the extracts were applied to
Dowex AG 1-X8 100-200 mesh formate columns and the various [aH]IP (inositol mono-, hi- and tri-phosphates) were eluted together in a pooled fraction according to Berridge et al. 2. Materials
NE was purchased from Fluka Chemie AG (Bucks, Switzerland); isoproterenol sulfate was purchased from Boehringer Ingelheim (Ingelheim, ER.G.); carbachol was obtained from Bracco S.p.A. (Milano, Italy) and prazosin HCI from Pfizer (Latina, Italy). Chemicals used for Krebs-buffers were of the highest grade commercially available. Statistical analysis
One way ANOVA followed by Tukey's test and Dunnetts' test (two-tailed comparisons) were used for, respectively, single and multiple comparisons between the experimental groups and their controls. RESULTS Electrical stimulation of the dorsal hippocampus initially triggered a high-frequency, high-amplitude A D . The length and complexity of the A D were significantly
1st
AFTER
increased at stage 5 compared to the beginning of kindling (Fig. 1). Fig. 2 depicts the effect of electrical stimulation at various frequencies (2, 5 and 10 Hz) on the fractional release of N E from stratum pyramidale C A 1 - C A 3 and D G slices at stages 2 (left side) and 5 (right side) of hippocampal kindling. After 60 min perfusion, spontaneous L-[3H]NE release was stable at a rate of about 1-2% of the total 3H in the slice/min. The electrically stimulated N E release from C A 1 - C A 3 and D G slices was >80% Ca2+-dependent as measured by omitting Ca 2+ and adding 1 mM E G T A to the O K B p e r f u s i n g solution (data not shown). NE was released to a similar extent from C A 1 - C A 3 and D G slices, as indicated by the non-significant differences in the basal (data not shown) and electrically stimulated output from the two subfields (Fig. 2). NE release was directly related to the frequency of stimulation, as shown by the about 2-, 4- and 8-fold increases above spontaneous release measured, respectively, at 2, 5 and 10 Hz. No significant differences were observed in the release of N E from C A 1 - C A 3 and D G slices from the stimulated and contralateral hippocampus at stages 2 (Fig. 2, left panels) and 5 (Fig. 2, right panels) of kindling as compared to shams. No significant differ-
DISCHARGE
RCTX LCTX RHP •
L
LHP
22 nd AFTER DISCHARGE ( s t . 5 ) .... --
l,L ~ u
. . . . . L
.....
J,,L,..l.,I ...... ",LL,J,...a;I.
I.,mhldid, dll|lJjLa,~,,JuL
; JL,J~,J.,,.~d~.~,~i~lSH,~iuj~JJ~l,J~..," F
,J~,LJ,J ....
.u.,~a,~.~.
~t.,i h .lJ.~.]~, ................ fl
5 sec
Fig. 1. Representative EEG tracings from a rat after the first stimulation of kindling and after the first stage 5 seizure. RCTX and LC'IIK are fight and left cortex; RHP and LHP are right and left hippocampus. The stimulus was delivered to the left dorsal hippocampus (upper blade of DG). Vertical calibration: 100/~V.
213 S T R A T U M P Y R A M I D A L E CA1-CA3
A
~
20 18
14
14
1
Kindled ipsi Kindled contra
10
~
4
0
2Hz
5Hz
'
10Hz
2Hz
5Hz
10Hz
DENTATE GYRUS
12 ~" O3
==
_=
10
s 6
,_o
4
u.
2 2Hz
5Hz
10Hz
2Hz
5Hz
10Hz
Fig. 2. L-[aH]NE release from stratum pyramidale CA1-CA3 and dentate gyrus slices of stage 2 (left) and stage 5 (fight) kindled rats. Data are mean _+ S.E.M., in fractional release during stimulation ($2) divided by the baseline of spontaneous release ($1) from 3 experiments (3 rats/group/expefiment). 'Sham' indicates rats implanted with electrodes but not stimulated. Kindled 'ipsi' and 'contra' refer, respectively, to the stimulated and contralateral hippocampus. Slices were stimulated at the indicated frequencies for 2 rain; samples were collected every 5 min. The speed of perfusion was 0.5 ml/min. Rats were killed 48 h after the last stimulation (stage 2) or 1 week after the last seizure (stage
5).
ences between kindled and sham animals were observed on stimulating NE release with 20 and 40 mM KCl (data not shown). Fig. 3 shows the isoproterenol-stimulated cAMP accumulation in hippocampi at stages 2 and 5 of kindling and in sham-treated animals. A dose-related increase above basal value in cAMP accumulation was seen in shams, ranging from about 40% at 0.01/tM to 180% at 10 ~M (P < 0.01, Dunnett's test). No significant differences were observed in the basal cAMP concentration (see legend to Fig. 3) or in the effect of NE (0.001-10/~M) on cAMP accumulation in hippocampi of kindled rats compared to shams (Tukey's test). Fig. 4 shows the effect of various concentrations of NE (1-1000/~M) on PI turnover in hippocampi of kindled rats as compared to shams. NE induced a dose-dependent increase in PI turnover as measured by [3H]IP accumulation in the slices in the presence of LiCl. The maximal stimulation, about 350% above basal values, was reached at 100/tM (P < 0.01; Dunnett's test). The
300
b
~
abb
~ 100 Basal
~ 0.001
• [] []
Sham Kindled st. 2 Kindled st.5
I 0,01
0.1
1
10
(Isoproterenol) (~LM)
Fig. 3. Isoproterenol-induced cAMP accumulation in hippocampi of stage 2 and stage 5 kindled rats. Data are mean + S.E.M. (10 rats/group) expressed as % increase above basal values (pmol/mg protein) sham, 5.03 + 0.3; stage 2 , 4 . 7 4 + 0.15; stage 5, 5.3 + 0,4). Rats were killed 48 h after the last stimulation (stage 2) or 1 week after the last seizure (stage 5). Statistical analysis was done on absolute values, a, P < 0.05; b, P < 0.01 vs respective basal (Dunnett's test).
214 effect of NE on PI turnover was mediated by stimulation of ax-adrenocepters as demonstrated by the fact that 1 /aM prazosin abolished the effect of 10 and 1000/aM NE in slices taken from naive rats (% of basal: NE 10/aM, 185 -+ 3; NE 1000/~M, 382 _+ 6; prazosin + NE 10/aM, 116 + 6; prazosin + NE 1000/aM, 154 + 2 (n = 7). A significantly larger increase in [3H]IP accumulation was seen in stage 2 (P < 0.05, Tukey's test) and stage 5 kindled rats (P < 0.05 and P < 0.01, Tukey's test) compared to shams at the same doses (Fig. 4A,B). No significant differences were found in the basal concentrations of IP between rats kindled at stages 2 and 5 and their respective shams (data not shown). The enhanced accumulation of [3H]IP in kindled rats was maintained 1 month after stage 5 acquisition (% of basal: NE 100/aM, sham 446 + 17; kindling 511 _+ 21 (n = 5), P < 0.01, Tukey's test). To investigate whether the enhanced rate of PI turnover in kindled rats was selective for phospholipase C coupled to al-adrenoceptors we investigated the effect of carbachol, a muscarinic agonist, on [3H]IP
A 6O0
b,c
b,c
• Sham [ ] Kindled
50O
400 r-
.o
300 ¢
E
.= 8
O. "r
200 d
~
100
2:
e~
0 Bssal
1
10
100
B
b,c
5O0
J=
1000
b,d
b,d
400
300
-5
E o
o. "T -r
200
,0o ] 0 Basal
1
10
100
1000
(NE)(pM)
Fig. 4. Norepinephrine (NE)-stimulated PI turnover in hippocampi of stage 2 (A) and stage 5 (B) kindled rats. Data are mean _+S.E.M. of 3 experiments (6 rats/group/experiment). [3H]IP accumulation was expressed as % increase above basal values. Rats were killed 48 h after the last stimulation (stage 2) or 1 week after the last seizure (stage 5). Statistical analysis was done on absolute values, a, P < 0.05; b, P < 0.01 vs respective basal (Dunnett's test); c, P < 0.05; d, P < 0.01 vs sham at the same dose (Tukey's test).
accumulation in stage 5 kindled rats. Increases above basal levels were, respectively, 60, 150 and 250% at 10, 100 and 1000 /aM carbachol in shams (P < 0.01; Dunnett's test). Basal values and the effect of carbachoi were the same for sham- and kindled-rats at all doses studied (data not shown). DISCUSSION The main finding of this study is that selective modifications in the functional status of al-adrenoceptors occur during kindling in the absence of changes in NE release from presynaptic neurons under basal and stimulated conditions. We found no significant changes in basal or in electrically and K+-stimulated NE release in C A 1 - C A 3 and D G at stages 2 and 5. These results are in agreement with the report by Kant et al. 17 that spontaneous and K+-stimulated release of endogenous NE was not affected in various brain regions of amygdala-kindled rats. Along the same line, Jones and Johnson 16 found no difference in the ability of NMDA to release NE from amygdala or hippocampus slices after amygdaloid kindling. The reported depletion of NE in hippocampus of kindled rats 5 might therefore depend on factors other than changes in the release mechanism. fl~-Adrenoceptor-mediated cAMP accumulation was not altered either during the development or after the acquisition of kindling. A down-regulation of fll-adrenoceptors has been reported in the hippocampus after amygdala kindling25'31; should this happen in our conditions too the unmodified cAMP response could result from a compensatory enhancement in the coupling of the receptors to the second messenger system. This aspect is currently under investigation in our laboratory. Stimulation of PI turnover by NE was enhanced in stages 2 and 5 of kindling, and remained higher than controls for at least 1 month after the last stage 5 seizure. The fact that the effect occurred at the early preconvulsive stage (stage 2) and was long-lasting makes it unlikely that it was a mere consequence of seizure activity. Furthermore, the lack of changes in carbacholstimulated PI hydrolysis at stage 5 suggests that the enhanced response induced by NE was not due to a non-specific increase in the activity of phospholipase C. The action of NE may reflect an enhanced coupling of al-adrenoceptors with phospholipase C or an increase in the density and/or affinity of al-binding sites. This hypothesis is currently under investigation in our laboratory, al-Adrenoceptors have been reported to mediate the inhibitory effects of endogenous NE released by locus coeruleus stimulation on hippocampal pyramidal neurons 8. Suppressant effects of NE through al-adrenoce p-
215 tors on Ca2+-dependent regenerative potentials in D G 33 and on low Mg2÷-induced epileptiform activity in the entorhinal cortex slices 32 have also been reported. Amplification of the al-receptor-linked second messenger system may, therefore, be aimed at blunting the enhanced neuronal excitation in the kindled hippocampus 3. The more pronounced enhancement of PI turnover in stage 5 than in stage 2 and the persistence of this effect seems to support this. Previous studies on NE-stimulated PI hydrolysis in kindling found contrasting results. Iadarola et a1.13 found a significant increase in the basal but no changes in the 100/~M NE-stimulated IP formation in the hippocampus of stage 5 amygdala-kindled rats. The different site of kindling may explain the different results. Stelzer et al. 34 reported that D G kindling significantly reduced both basal and NE-induced accumulation of IP in hippocampal slices. These authors used a much shorter interval (24-48 h) after the last stage 5 seizure than in the present study (1 week). In view of the finding that epileptic discharges inhibit a~-adrenoceptor-mediated stimulation of PI turnover 9, the presence of interictal spike activity in the early intervals after stage 5 seizures 21 might account for the results from Stelzer's group. In summary, two lines of evidence emerged from our study. First, hippocampal kindling caused no significant changes in N E release in stratum pyramidale C A 1 - C A 3 and D G at stages 2 and 5. The transient increase of N E
release in vivo in the hippocampus following induction of an A D 1a'19 might perhaps help start up the p h e n o m e n o n but has no role in its development and maintenance. Second, there was clear-cut enhancement of the PI response to al-adrenoceptors during various phases of kindling with no modification of the c A M P response to fll-adrenoceptor stimulation. The effect on NE-induced PI turnover is similar to that observed for ibotenate-dependent stimulation although it lasted longer L13 suggesting that IP hydrolysis and the consequent cascade of intracellular events are part of the mechanisms that accompany permanent changes in the excitability of the hippocampus following kindling. The mechanism by which al-adrenoceptor stimulated PI turnover is up-regulated by kindling is unknown. Besides changes in the density and/or affinity of a 1receptor sites, other factors such as an enhanced coupling to phospholipase C, a failure in protein kinase Cmediated desensitization of the receptors 2° or a reduced ability of glutamate to inhibit the NE-mediated response 28 can be involved. Further studies are needed to clarify this issue and the functional significance of this phenomenon.
REFERENCES
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Acknowledgements. R.S. and M.R. are recipients of a fellowship from the Lions Club Milano 'Alessandro Manzoni'. This work was supported by the CNR (National Research Council, Rome, Italy), Contract 89.01272.04.
216 19 Kokaia, M., Kal~n, P., Bengzon, K.J. and Lindvall, O., Noradrenaline and 5-hydroxytryptamine release in the hippocampus during seizures induced by hippocampal kindling stimulation: an in vivo microdialysis study, Neuroscience, 32 (1989) 647-656. 20 Labarca, R., Janowsky, A., Patel, J. and Paul, S.M., Phorbol esters inhibit agonist-induced [3H]inositol-l-phosphate accumulation in rat hippocampal slices, Biochem. Biophys. Res. Commun., 123 (1984) 703-709. 21 Leung, L.S., Spontaneous hippocampal interictal spikes following local kindling: time-course of change and relation to behavioral seizures, Brain Research, 513 (1990) 308-314. 22 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 23 Lynch, M.A. and Bliss, T.V.P., Noradrenaline modulates the release of [14C]glutamate from dentate but not from CA1/CA3 slices of rat hippocampus, Neuropharmacology, 25 (1986) 493-498. 24 Magistretti, P.J. and Schorderet, M., VIP and noradrenaline act synergistically to increase cyclic AMP in cerebral cortex, Nature, 308 (1984) 280-282. 25 McNamara, J.O., Selective alterations of regional fl-adrenergic receptor binding in the kindling model of epilepsy, Exp. Neurol., 61 (1978) 582-591. 26 Mody, I. and Heinemann, U., NMDA receptors of dentate gyrus granule cells participate in synaptic transmission following kindling, Nature, 326 (1987) 701-704. 27 Nelson, M.E, Zaczeck, R. and Coyle, J.T., Effects of sustained seizures produced by intrahippocampal injection of kainic acid on noradrenergic neurons: evidence for local control of norepinephrine release, J. Pharmacol. Exp. Ther., 214 (1980) 694-702. 28 Nicoletti, F., Iadarola, M.J., Wrobleski, J.T. and Costa, E., Excitatory amino acid recognition sites coupled with inositol phospholipid metabolism: developmental changes and interaction with al-adrenoceptors , Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 1931-1935. 29 Nicoll, R.A., Madison, D.V. and Lancaster, B., Noradrenergic modulation of neuronal excitability in mammalian hippocampus. In H.V. Meltzer (Ed.), Psychopharmacology. The Third Generation of Progress, Raven, New York, 1987, pp. 105-112. 30 Racine, R.J., Modification of seizure activity by electrical
31 32
33
34
35 36
37
38
39 40
stimulation. II. Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294. Stanford, S.C. and Jefferys, J.G.R., Down-regulation of a 2- and fl-adrenoceptor binding sites in rat cortex caused by amygdalar kindling, Exp. Neurol., 90 (1985) 108-117. Stanton, P.K., Jones, R.S.G., Mody, I. and Heinemann, U., Epileptiform activity induced by lowering extraceilular [Mg2÷] in combined hippocampal-entorhinal cortex slices: modulation by receptors for norepinephrine and N-methyl-o-aspartate, Epilepsy Res., 1 (1987) 53-62. Stanton, P.K., Mody, I. and Heinemann, U., Down-regulation of norepinephrine sensitivity after induction of long-term neuronal plasticity (kindling) in the rat dentate gyrus, Brain Research, 476 (1989) 367-372. Stelzer, A., Feasey, K.J., Moneta, M.E., Sincini, E., Bruggencate, G. and Noble, E.P., Inositol l-phosphate formation in long-term potentiation and kindling, Brain Research, 490 (1989) 41-47. Stelzer, A., Slater, N.T. and Bruggencate, G., Activation of NMDA receptors blocks GABAergic inhibition in an in vitro model of epilepsy, Nature, 326 (1987) 698-701. Vezzani, A., Serafini, R., Bendotti, C., Rizzi, M., Vigan6, G. and Samanin, R., Modulation of the presynaptic and postsynaptic components of noradrenergic transmission by kindling, Neurochem. Int., 16 Suppl. 1 (1990) 72. Vezzani, A., Wu, H.Q., Tullii, M. and Samanin, R., Anticonvulsant drugs effective against human temporal lobe epilepsy prevent seizures but not neurotoxicity induced in rats by quinolinic acid: electroencephalographic, behavioral and histological assessments, J. Pharmacol. Exp. Ther., 239 (1986) 256-262. Weiss, G.K., Lewis, J., Jimenez-Rivera, C., Vigil, A. and Corcoran, M.E., Antikindling effects of locus coeruleus stimulation: mediation by ascending noradrenergic projections, Exp. Neurol., 108 (1990) 136-140. Westerberg, V., Lewis, J. and Corcoran, M.E., Depletion of noradrenaline fails to affect kindled seizures, Exp. Neurol., 84 (1984) 237-241. Ziance, R.J. and Rutledge, C.O., A comparison of the effects of fenfluramine and amphetamine on uptake, release and catabolism of norepinephrine in rat brain, J. Pharmacol. Exp. Ther., 180 (1972) 118-126.
Brain Research, 557 (1991) 210-216 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 0(0899391169132
BRES 16913
Changes in pre- and postsynaptic components of noradrenergic transmission in hippocampal kindling in rats A. Vezzani, M. Rizzi, R. Serafini, G. Vigan6 and R. Samanin lstituto di Ricerche Farmacologiche 'Mario Negri', Milan (Italy)
(Accepted 9 April 1991) Key words: Noradrenaline; Isoproterenol; al-Adrenoceptor; fll-Adrenoceptor; Release; Phosphatidyl inositol turnover; Cyclic adenosine monophosphate; Kindling; Hippocampus; Rat
We investigated whether modifications in noradrenergic neurotransmission occurred during the development of hippocampal kindling in rats. We measured the release of [3H]norepinephrine (NE) induced by field-electrical stimulation, NE-stimulation of inositol phosphates ([3H]IP) accumulation in the presence of LiCI and isoproterenol-induced accumulation of cAMP in hippocampal slices taken from rats electrically kindled at stages 2 and 5 in the dorsal hippocampus. One week after the last of at least 3 consecutive stage 5 seizures or 48 h after the last stage 2 stimulation, 2 min electrical stimulation of stratum pyramidale CA1-CA3 or dentate gyrus (DG) slices from kindled and contralateral hippocampi induced frequency-dependent NE release (respectively 2, 4 and 8 times spontaneous release measured at 2, 5 and 10 Hz) which did not significantly differ from that observed in shams (implanted with electrodes but not stimulated). Basal release of NE from kindled and sham-treated rats did not differ either. Isoproterenol induced a dose-dependent increase above basal cAMP concentration ranging from 40% at 0.01 ~M to 180% at 10/~M (P < 0.01, Dunnett's test) which did not differ between stages 2 and 5 and sham-hippocampi. NE (1-1000 #,M) induced a dose-dependent, prazosin-sensitive increase in [3H]IP accumulation in the hippocampal slices. A significantly higher increase was found at stages 2 (P < 0.05, Tukey's test) and 5 (P < 0.05 and P < 0.01, Tukey's test) compared to shams at all doses studied. No differences were observed in the basal cAMP or [3H]IP concentrations in kindled versus sham animals. No differences were observed in [3H]IP accumulation induced by carbachol (10, 100, 1000/~M) in stage 5-kindled rats and shams. These changes have various possible implications for kindling-induced plasticity. INTRODUCTION Kindling is an animal model of neuronal plasticity and epileptogenesis. It develops over time in response to r e p e a t e d electrical or chemical stimuli leading to progressively m o r e intense limbic seizures and eventually clonic m o t o r seizures 12. Once established, this p h e n o m enon is a p p a r e n t l y irreversible. T h e biochemical mechanisms underlying the development of kindling and its persistence are mostly unknown but recent observations suggest that it is associated with increased activity of glutamate ( G L U ) - c o n t a i n i n g neurons T M and enhanced postsynaptic N-methyl-Daspartate ( N M D A ) r e c e p t o r sensitivity26'35. A m o n g the neurotransmitters studied so far, norepinephrine ( N E ) has been shown to have p r o f o u n d effects in this model. D e p l e t i o n of forebrain N E facilitates the d e v e l o p m e n t of kindling 7 without affecting kindling once established 39. Locus coeruleus stimulation 15'38 and activation of a2-adrenoceptors 1° inhibit kindling development. Interactions b e t w e e n N E and G L U have also been
r e p o r t e d in the hippocampus: N E enhances the excitation induced by G L U on h i p p o c a m p a l neurons by blocking the slow CaE+-dependent K + currents 29 and it increases the K+-induced G L U release in the d e n t a t e gyrus ( D G ) but not in stratum p y r a m i d a l e CA1-CA323. Thus noradrenergic neurotransmission a p p e a r s to be modified in kindling and this might affect G L U - c o n t a i n i n g neurons contributing to the e n h a n c e d N M D A - r e c e p t o r sensitivity which underlies kindling. In vivo N E release increases in the h i p p o c a m p u s after an electrical stimulus inducing an afterdischarge ( A D ) la' 19. D u r i n g rapid h i p p o c a m p a l kindling the in vivo release of N E in response to the first electrical stimulation gradually d r o p p e d back to baseline levels with further stimulations TM. This suggests t h e r e is only a transient initial increase of N E release during kindling. O t h e r authors 16A7 have found no significant changes of N E release in a m y g d a l a - k i n d l e d rats. D o w n - r e g u l a t i o n of txEand f l : a d r e n o c e p t o r s has b e e n r e p o r t e d after amygdala kindling 6'25'3~. In this study we used biochemical indices to confirm that changes in n o r a d r e n e r g i c neurotransmission occur during kindling and to clarify the type of
Correspondence: A. Vezzani, Istituto di Ricerche Farmacologiche 'Mario Negri', via Eritrea 62, 20157 Milano, Italy. Fax: (39) (2) 354 6277.
211 receptors
mainly involved. We studied
the effect of
k i n d l i n g o n t h e basal and field e l e c t r i c a l - s t i m u l a t e d N E release from stratum pyramidale CA1-CA3
and D G
slices as p r e s y n a p t i c m a r k e r s o f n o r a d r e n e r g i c activity, and o n N E - s t i m u l a t e d PI t u r n o v e r and i s o p r o t e r e n o l i n d u c e d c A M P a c c u m u l a t i o n as p o s t s y n a p t i c m a r k e r s o f t h e f u n c t i o n a l status o f a 1- and f l l - a d r e n o c e p t o r s . T h e s e parameters stage
w e r e assessed at t h e early, p r e c o n v u l s i v e
of kindling
(stage
2)
and
once
kindling
was
e s t a b l i s h e d (stage 5). A p r e l i m i n a r y r e p o r t o n t h e s e d a t a has a p p e a r e d as an abstract 36. MATERIALS AND METHODS Male Sprague-Dawley rats (250-280 g, Charles River, Italy) were used. The animals were housed at constant temperature (23 °C) and relative humidity (60%) with a fixed 12 h light-dark cycle and free access to food and water.
Implantation procedure The surgical procedure for electrode implantation in the dorsal hippocampus under Equitensin anesthesia (9.7 mg/ml sodium pentobarbitai in saline; 42.6 mg/mi chloral hydrate in propyleneglycol, 21.2 mg/ml MgSO 4 in ethanol; 3.5 ml/kg, i.p.) was as previously described 37. For implantation the coordinates from bregma were: (nose bar -2.5) AP 3.5, L 2.3, H 2.9. In addition, staiuless-steel screw electrodes were placed bilaterally over the sensorimotor cortex and a ground lead was screwed over the nasal sinus. The electrodes were connected to a multipin socket (March Electronics, New York, NY) and were secured to the skull by acrylic dental cement. Electroencephalographic (EEG) recordings were made in unanesthetized, freely moving animals as previously described 37.
Kindling procedure Kindling was started after a postoperative period of 7 days, when the animals showed no behavioral signs of pain or discomfort. The rats were allowed to acclimatize in a Plexiglas cage and an EEG recording (4-channel EEG polygraph, model B8P, Battaglia Rangoni, Bologna, Italy) was made for at least 10 min to assess the spontaneous EEG pattern. Constant current stimuli were delivered to the left hippocampus (granule cells of the dentate gyms) through a bipolar electrode (recording electrode) twice daily for 5 days and once daily for 2 days (week-end), at intervals of at least 6 h. The parameters of stimulation were 50 Hz, 2 ms monophasic rectangular wave pulses for 1 s, the current intensity ranging between 60 and 200 /~V. Behavior was observed and AD duration measured in the stimulated hippocampus after each stimulation, for every animal. Before starting electrical stimulation, the rats were randomly assigned to two groups and received 12 _+ I and 24 _+ 2 stimuli (mean _+ S.E.M.) to reach, respectively, stages 2 and 5 of kindling according to Racine's classification3°. Previous experiments had shown that after 12 stimuli the animals displayed stereotypies and occasional retraction of a forelimb (stage 2). Stage 5 was characterized by tonic-clonic seizures with rearing and falling. Animals were considered fully kindled when they experienced at least 3 consecutive stage 5 seizures. Controls were implanted with electrodes and handled in the same way as the experimental rats but were not electrically stimulated. Light microscopic analysis of Nissl-stained 40/~m cryostat sections was made 37 on completion of kindling to verify the electrode placement in the granule cells of the DG.
Biochemical assays For biochemical determinations, rats kindled at stages 2 and 5, and their controls, were killed, respectively, 48 h and one week after the last stimulation.
L-[3H]NE release Experimental conditions for the uptake and release of L-[3H]NE from brain slices were chosen according to the methods described by Ziance and Rutledge 4° and Nelson et ai.z7, with some modifications. The dorsal hippocampi (left and right separately) were mechanically sliced in the transverse plane at a thickness of 1 mm with a McIlwain chopper. The slices were pooled and suspended in 5 ml of oxygenated Krebs-bicarbonate buffer (OKB), pH 7.6, containing ascorbic acid (10 rag/100 mi) and pargyline (5 rag/100 mi). Slices were then microdissected in CA1-CA3 and DG subregions. After preincubation for 15 rain at 37 °C, 0.1 /~M L-[3H]NE (13 Ci/mmol; radiochemical purity 90%; NEN, Boston, MA, U.S.A.) was added and the slices were incubated for another 20 rain. The supernatant was decanted off and the slices were washed 3 times with fresh OKB. The slices were mounted on superfusion chambers (about 8 mg wet weight tissue/chamber) and superfused at 0.5 ml/min at 37 °C with OKB. Samples were collected every 5 min. A baseline recording was made after a 30-min washout period. After 60 min baseline the slices were electrically stimulated for 2 min at 2, 5 and 10 Hz at interstimulus intervals of 60 min. Radioactivity was assessed in the various fractions by scintillation spectrometry. Slices were recovered at the end of the experiment and dissolved in 0.5 mi of Soluene (Packard Instrument, Downers Grove, IL, U.S.A.) and the 3H content was determined in the same way. Fractional release was calculated as 3H released into the medium during each 5 min fraction as a percentage of the 3H content of the slices during that interval. The effect of the stimulation was calculated as the fractional release during electrical stimulation ($2) divided by the baseline spontaneous release (S1), the latter being the average of 3 samples before drug application. Stimulated release was assessed as the average of the sample collected during stimulation and the one after.
cAMP accumulation cAMP accumulation was measured according to the method of Magistretti and Schorderet 24. Rats were decapitated, the brains removed rapidly and placed on ice. The dorsal hippocampi of both hemispheres were dissected out and pooled in an oxygenated modified Krebs-HEPES (OKH) buffer containing (in raM): NaCI 125; KCI 5; CaCI 2 2; MgSO 4 1.2; KH2PO 4 1.2; glucose 3; HEPES 25, pH 7.4. The hippocampi were chopped in miniprisms (350 x 350 /zm) and resuspended in 5 ml OKH. After replacing the buffer once, the slices were incubated in a vial containing 5 ml OKH at 37 °C with mild shaking for 30 rain. The slices were then washed twice, the second wash with OKH containing 0.5 mM isobutyl methylxanthine (IBMX). Test tubes containing 50/~l OKH + 0.5 mM IBMX _+ isoproterenol (10-9-10 -5 M) were incubated at 37 °C for 5 rain then 50/~l of densely packed slices were distributed into individual test tubes and incubated for another 10 rain. Incubation was terminated by boiling the samples for 5 rain followed by sonication for 5 s. The samples were then rapidly centrifuged (16,000 g for 5 rain), the supernatant was separated and stored at -20 °C until assayed. cAMP present in the samples was determined by a cAMP assay kit (Amersham International, Ltd., Buckinghamshire, U.K.). Experimental values were calculated from a standard curve ranging from 0 to 16 pmol of cAMP. Proteins were measured in the pellets according to the method of Lowry et al. 22.
PI turnover Phosphatidylinositol (PI) hydrolysis was measured according to Brown et al.4. Dorsal hippocampi of both hemispheres were chopped into 350 x 350/~m miniprisms. The cubes were pooled and washed 3 times in OKB, pH 7.4. After 45 mln preincubation in OKB at 37 °C the slices were incubated for 60 rain with 0.2/~M myo-[3H]inositol (CEA, Gif-sur-Yvette, Cedex, France) in the presence of 5 mM LiCI then washed 3 times with OKB. The prelabeled miniprisms (40 /zl of densely packed cubes) were incubated at 37 °C for 60 rain with OKB-LiCI + agonists in a total
212 volume of 310 #l. When antagonists were used, they were added 5 min before the agonists. Incubation was terminated by addition of 930 #l of ice-cold methanol-chloroform-6N HCI (2:1:0.01). The slices were then sonicated and centrifuged and the supernatants used for determination of [3H]inositoi phosphates (IP). The supernatants were subjected to 4 diethylether extraction cycles. After neutralization with 10 mM sodium tetraborate, the extracts were applied to
Dowex AG 1-X8 100-200 mesh formate columns and the various [aH]IP (inositol mono-, hi- and tri-phosphates) were eluted together in a pooled fraction according to Berridge et al. 2. Materials
NE was purchased from Fluka Chemie AG (Bucks, Switzerland); isoproterenol sulfate was purchased from Boehringer Ingelheim (Ingelheim, ER.G.); carbachol was obtained from Bracco S.p.A. (Milano, Italy) and prazosin HCI from Pfizer (Latina, Italy). Chemicals used for Krebs-buffers were of the highest grade commercially available. Statistical analysis
One way ANOVA followed by Tukey's test and Dunnetts' test (two-tailed comparisons) were used for, respectively, single and multiple comparisons between the experimental groups and their controls. RESULTS Electrical stimulation of the dorsal hippocampus initially triggered a high-frequency, high-amplitude A D . The length and complexity of the A D were significantly
1st
AFTER
increased at stage 5 compared to the beginning of kindling (Fig. 1). Fig. 2 depicts the effect of electrical stimulation at various frequencies (2, 5 and 10 Hz) on the fractional release of N E from stratum pyramidale C A 1 - C A 3 and D G slices at stages 2 (left side) and 5 (right side) of hippocampal kindling. After 60 min perfusion, spontaneous L-[3H]NE release was stable at a rate of about 1-2% of the total 3H in the slice/min. The electrically stimulated N E release from C A 1 - C A 3 and D G slices was >80% Ca2+-dependent as measured by omitting Ca 2+ and adding 1 mM E G T A to the O K B p e r f u s i n g solution (data not shown). NE was released to a similar extent from C A 1 - C A 3 and D G slices, as indicated by the non-significant differences in the basal (data not shown) and electrically stimulated output from the two subfields (Fig. 2). NE release was directly related to the frequency of stimulation, as shown by the about 2-, 4- and 8-fold increases above spontaneous release measured, respectively, at 2, 5 and 10 Hz. No significant differences were observed in the release of N E from C A 1 - C A 3 and D G slices from the stimulated and contralateral hippocampus at stages 2 (Fig. 2, left panels) and 5 (Fig. 2, right panels) of kindling as compared to shams. No significant differ-
DISCHARGE
RCTX LCTX RHP •
L
LHP
22 nd AFTER DISCHARGE ( s t . 5 ) .... --
l,L ~ u
. . . . . L
.....
J,,L,..l.,I ...... ",LL,J,...a;I.
I.,mhldid, dll|lJjLa,~,,JuL
; JL,J~,J.,,.~d~.~,~i~lSH,~iuj~JJ~l,J~..," F
,J~,LJ,J ....
.u.,~a,~.~.
~t.,i h .lJ.~.]~, ................ fl
5 sec
Fig. 1. Representative EEG tracings from a rat after the first stimulation of kindling and after the first stage 5 seizure. RCTX and LC'IIK are fight and left cortex; RHP and LHP are right and left hippocampus. The stimulus was delivered to the left dorsal hippocampus (upper blade of DG). Vertical calibration: 100/~V.
213 S T R A T U M P Y R A M I D A L E CA1-CA3
A
~
20 18
14
14
1
Kindled ipsi Kindled contra
10
~
4
0
2Hz
5Hz
'
10Hz
2Hz
5Hz
10Hz
DENTATE GYRUS
12 ~" O3
==
_=
10
s 6
,_o
4
u.
2 2Hz
5Hz
10Hz
2Hz
5Hz
10Hz
Fig. 2. L-[aH]NE release from stratum pyramidale CA1-CA3 and dentate gyrus slices of stage 2 (left) and stage 5 (fight) kindled rats. Data are mean _+ S.E.M., in fractional release during stimulation ($2) divided by the baseline of spontaneous release ($1) from 3 experiments (3 rats/group/expefiment). 'Sham' indicates rats implanted with electrodes but not stimulated. Kindled 'ipsi' and 'contra' refer, respectively, to the stimulated and contralateral hippocampus. Slices were stimulated at the indicated frequencies for 2 rain; samples were collected every 5 min. The speed of perfusion was 0.5 ml/min. Rats were killed 48 h after the last stimulation (stage 2) or 1 week after the last seizure (stage
5).
ences between kindled and sham animals were observed on stimulating NE release with 20 and 40 mM KCl (data not shown). Fig. 3 shows the isoproterenol-stimulated cAMP accumulation in hippocampi at stages 2 and 5 of kindling and in sham-treated animals. A dose-related increase above basal value in cAMP accumulation was seen in shams, ranging from about 40% at 0.01/tM to 180% at 10 ~M (P < 0.01, Dunnett's test). No significant differences were observed in the basal cAMP concentration (see legend to Fig. 3) or in the effect of NE (0.001-10/~M) on cAMP accumulation in hippocampi of kindled rats compared to shams (Tukey's test). Fig. 4 shows the effect of various concentrations of NE (1-1000/~M) on PI turnover in hippocampi of kindled rats as compared to shams. NE induced a dose-dependent increase in PI turnover as measured by [3H]IP accumulation in the slices in the presence of LiCl. The maximal stimulation, about 350% above basal values, was reached at 100/tM (P < 0.01; Dunnett's test). The
300
b
~
abb
~ 100 Basal
~ 0.001
• [] []
Sham Kindled st. 2 Kindled st.5
I 0,01
0.1
1
10
(Isoproterenol) (~LM)
Fig. 3. Isoproterenol-induced cAMP accumulation in hippocampi of stage 2 and stage 5 kindled rats. Data are mean + S.E.M. (10 rats/group) expressed as % increase above basal values (pmol/mg protein) sham, 5.03 + 0.3; stage 2 , 4 . 7 4 + 0.15; stage 5, 5.3 + 0,4). Rats were killed 48 h after the last stimulation (stage 2) or 1 week after the last seizure (stage 5). Statistical analysis was done on absolute values, a, P < 0.05; b, P < 0.01 vs respective basal (Dunnett's test).
214 effect of NE on PI turnover was mediated by stimulation of ax-adrenocepters as demonstrated by the fact that 1 /aM prazosin abolished the effect of 10 and 1000/aM NE in slices taken from naive rats (% of basal: NE 10/aM, 185 -+ 3; NE 1000/~M, 382 _+ 6; prazosin + NE 10/aM, 116 + 6; prazosin + NE 1000/aM, 154 + 2 (n = 7). A significantly larger increase in [3H]IP accumulation was seen in stage 2 (P < 0.05, Tukey's test) and stage 5 kindled rats (P < 0.05 and P < 0.01, Tukey's test) compared to shams at the same doses (Fig. 4A,B). No significant differences were found in the basal concentrations of IP between rats kindled at stages 2 and 5 and their respective shams (data not shown). The enhanced accumulation of [3H]IP in kindled rats was maintained 1 month after stage 5 acquisition (% of basal: NE 100/aM, sham 446 + 17; kindling 511 _+ 21 (n = 5), P < 0.01, Tukey's test). To investigate whether the enhanced rate of PI turnover in kindled rats was selective for phospholipase C coupled to al-adrenoceptors we investigated the effect of carbachol, a muscarinic agonist, on [3H]IP
A 6O0
b,c
b,c
• Sham [ ] Kindled
50O
400 r-
.o
300 ¢
E
.= 8
O. "r
200 d
~
100
2:
e~
0 Bssal
1
10
100
B
b,c
5O0
J=
1000
b,d
b,d
400
300
-5
E o
o. "T -r
200
,0o ] 0 Basal
1
10
100
1000
(NE)(pM)
Fig. 4. Norepinephrine (NE)-stimulated PI turnover in hippocampi of stage 2 (A) and stage 5 (B) kindled rats. Data are mean _+S.E.M. of 3 experiments (6 rats/group/experiment). [3H]IP accumulation was expressed as % increase above basal values. Rats were killed 48 h after the last stimulation (stage 2) or 1 week after the last seizure (stage 5). Statistical analysis was done on absolute values, a, P < 0.05; b, P < 0.01 vs respective basal (Dunnett's test); c, P < 0.05; d, P < 0.01 vs sham at the same dose (Tukey's test).
accumulation in stage 5 kindled rats. Increases above basal levels were, respectively, 60, 150 and 250% at 10, 100 and 1000 /aM carbachol in shams (P < 0.01; Dunnett's test). Basal values and the effect of carbachoi were the same for sham- and kindled-rats at all doses studied (data not shown). DISCUSSION The main finding of this study is that selective modifications in the functional status of al-adrenoceptors occur during kindling in the absence of changes in NE release from presynaptic neurons under basal and stimulated conditions. We found no significant changes in basal or in electrically and K+-stimulated NE release in C A 1 - C A 3 and D G at stages 2 and 5. These results are in agreement with the report by Kant et al. 17 that spontaneous and K+-stimulated release of endogenous NE was not affected in various brain regions of amygdala-kindled rats. Along the same line, Jones and Johnson 16 found no difference in the ability of NMDA to release NE from amygdala or hippocampus slices after amygdaloid kindling. The reported depletion of NE in hippocampus of kindled rats 5 might therefore depend on factors other than changes in the release mechanism. fl~-Adrenoceptor-mediated cAMP accumulation was not altered either during the development or after the acquisition of kindling. A down-regulation of fll-adrenoceptors has been reported in the hippocampus after amygdala kindling25'31; should this happen in our conditions too the unmodified cAMP response could result from a compensatory enhancement in the coupling of the receptors to the second messenger system. This aspect is currently under investigation in our laboratory. Stimulation of PI turnover by NE was enhanced in stages 2 and 5 of kindling, and remained higher than controls for at least 1 month after the last stage 5 seizure. The fact that the effect occurred at the early preconvulsive stage (stage 2) and was long-lasting makes it unlikely that it was a mere consequence of seizure activity. Furthermore, the lack of changes in carbacholstimulated PI hydrolysis at stage 5 suggests that the enhanced response induced by NE was not due to a non-specific increase in the activity of phospholipase C. The action of NE may reflect an enhanced coupling of al-adrenoceptors with phospholipase C or an increase in the density and/or affinity of al-binding sites. This hypothesis is currently under investigation in our laboratory, al-Adrenoceptors have been reported to mediate the inhibitory effects of endogenous NE released by locus coeruleus stimulation on hippocampal pyramidal neurons 8. Suppressant effects of NE through al-adrenoce p-
215 tors on Ca2+-dependent regenerative potentials in D G 33 and on low Mg2÷-induced epileptiform activity in the entorhinal cortex slices 32 have also been reported. Amplification of the al-receptor-linked second messenger system may, therefore, be aimed at blunting the enhanced neuronal excitation in the kindled hippocampus 3. The more pronounced enhancement of PI turnover in stage 5 than in stage 2 and the persistence of this effect seems to support this. Previous studies on NE-stimulated PI hydrolysis in kindling found contrasting results. Iadarola et a1.13 found a significant increase in the basal but no changes in the 100/~M NE-stimulated IP formation in the hippocampus of stage 5 amygdala-kindled rats. The different site of kindling may explain the different results. Stelzer et al. 34 reported that D G kindling significantly reduced both basal and NE-induced accumulation of IP in hippocampal slices. These authors used a much shorter interval (24-48 h) after the last stage 5 seizure than in the present study (1 week). In view of the finding that epileptic discharges inhibit a~-adrenoceptor-mediated stimulation of PI turnover 9, the presence of interictal spike activity in the early intervals after stage 5 seizures 21 might account for the results from Stelzer's group. In summary, two lines of evidence emerged from our study. First, hippocampal kindling caused no significant changes in N E release in stratum pyramidale C A 1 - C A 3 and D G at stages 2 and 5. The transient increase of N E
release in vivo in the hippocampus following induction of an A D 1a'19 might perhaps help start up the p h e n o m e n o n but has no role in its development and maintenance. Second, there was clear-cut enhancement of the PI response to al-adrenoceptors during various phases of kindling with no modification of the c A M P response to fll-adrenoceptor stimulation. The effect on NE-induced PI turnover is similar to that observed for ibotenate-dependent stimulation although it lasted longer L13 suggesting that IP hydrolysis and the consequent cascade of intracellular events are part of the mechanisms that accompany permanent changes in the excitability of the hippocampus following kindling. The mechanism by which al-adrenoceptor stimulated PI turnover is up-regulated by kindling is unknown. Besides changes in the density and/or affinity of a 1receptor sites, other factors such as an enhanced coupling to phospholipase C, a failure in protein kinase Cmediated desensitization of the receptors 2° or a reduced ability of glutamate to inhibit the NE-mediated response 28 can be involved. Further studies are needed to clarify this issue and the functional significance of this phenomenon.
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