Brain Research, 522 (1990) 69-75

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Elsevier BRES 15642

Dopaminergic antagonists prevent long-term maintenance of posttetanic LTP in the CA1 region of rat hippocampal slices Uwe Frey 1, Helmut Schroeder 2 and Hansjiirgen Matthies L2 tlnstitute of Neurobiology and Brain Research, Academy of Sciences, Magdeburg (G.D.R.) and 2Institute of Pharmacology and Toxicology, Medidal Academy Magdeburg, Magdeburg (G.D.R.) (Accepted 2 January 1990)

Key words: Long-term potentiation; Maintenance of long-term potentiation; Hippocampal CA1; Dopamine; Neuroleptics

The involvement of dopaminergic mechanisms in the induction and maintenance of posttetanic long-term potentiation (LTP) was investigated on CA1 cells of rat hippocampal slices. The presence of the dopamine receptor blocker domperidone in a concentration of 1/zM during tetanization with 3 trains of 100 impulses (100 Hz) and a train interval of 10 rain influences neither the synaptic transmission nor the induction of LTP. However, the potentiation of both the population spike and the population EPSP gradually decreases, thus significantly differing from control LTP about 4 h after initiation and reaching the level of non-tetanized controls about 7-8 h after tetanization. The simultaneous presence of 1 ~M apomorphine during tetanization abolishes this effect of domperidone indicating the specific dopaminolytic nature of its action. Also the presence of the dopamine antagonists sulpiride and flupenthixol, respectively, in a concentration of 1/zM during tetanization likewise prevents the occurrence of the late LTP maintenance. The determination of [14C]dopamine in 2 min fractions from the superfused slices after preloading during a preincubation period revealed that a low frequency stimulation of the Schaffer collaterals with 0.33 Hz does not influence the spontaneous efflux of dopamine, whereas the tetanization with an impulse train of 100 Hz produces a significantly enhanced release. The observations suggest that dopaminergic influences during and immediately after tetanization at least additionally contribute to the induction of postsynaptic mechanisms subserving a late, long-lasting maintenance of potentiation. The results also support the assumed existence of different subsequent stages of LTP.

INTRODUCTION High frequency stimulation of distinct afferents to principal hippocampal cells produces a prolonged increase of synaptic efficacy. The duration of this long-term potentiation (LTP), lasting for several days or weeks in vivo suggested a role of LTP as a mnemonic device or at least gave rise to use of this p h e n o m e n o n as a model of m e m o r y formation 4°. The induction of LTP obviously requires the depolarization of the postsynaptic neurons as well as the glutamate-dependent activation of their N-methyi-o-aspartate ( N M D A ) binding sites coupled to Ca 2+ channels (for review see ref. 7). The increase of intraneuronal Ca 2+ seems to be responsible for a chain of events resulting in an enhanced synaptic efficacy. However, this N M D A - m e d i a t e d synaptic potentiation is decrementa117 and seems to require additional mechanisms for a longer lasting maintenance of several hours or even days. There exist different results and suggestions explaining the realization of an early LTP maintenance

occurring the first 2 - 4 h after tetanization 6,21,23,29"32. However, the mechanisms enabling LTP to persist for longer than 5 - 6 h are still waiting for elucidation. The involvement of aminergic influences can be considered with regard to the modulating action, particularly of noradrenaline in the dentate gyrus 5'9'16'19'36'38'39 and of dopamine in the CA1 region of the hippocampus. A production of a LTP-like potentiation by dopamine in the CA1 region 15 as well as the inhibition of LTP-induction in the dentate gyrus 25 were obtained. We could recently report the first observations demonstrating a prevention of the late maintenance of LTP in hippocampal CA1 cells in vitro due to the presence of the dopamine antagonist domperidone during tetanization 12. In the present study, we confirm these results using other antagonists and also demonstrate that dopamine is actually released with tetanization. The observations suggest that dopaminergic transmission at least contributes to the induction of postsynaptic processes during or immediately after tetanization which are required for a late, long-lasting maintenance of

Correspondence: U. Frey, Institute of Neurobiology and Brain Research, Academy of Sciences, GDR, Brennecke-Str. 6, 3090 Magdeburg, G.D.R. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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field-EPSP record

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8 hour

Fig. 1. Upper part: schematic representation of a hippocampal lamella showing the location of stimulating electrodes in the stratum radiatum (S1, tetanized input; $2, non-tetanized input) and recording electrodes in the CA1 cell body layer (PS) and CA1 dendritic layer (SF of the population EPSP). Lower part: representative example of an averaged potential. The two markers indicate the steepest 400/~s segment from which 8 successive points were used for calculation of SE

LTP d e p e n d i n g o n n e w l y synthesized p r o t e i n s according to o u r hypothesis of a multi-stage n a t u r e of LTP TM

to 8 h. The responses were averaged on-line, stored on tape and subsequently analyzed using a PDP 11/34 laboratory computer. The slope function of the field EPSP (SF; mV/ms) was calculated from 8 successive points of the steepest 400/~s segment, whereas the amplitude of the population spike was evaluated from the voltage difference between onset and peak. LTP was induced only by one stimulation electrode (input S1) applying three trains of 100 pulses (100 Hz, duration 0.2 ms per polarity) with 10 min train interval. This particular schedule of tetanization was used, because it has been found to produce a sufficient and very stable long-lasting maintenance of potentiation in vitro for longer than 8 h 11'31. Altogether, 6 experimental groups were investigated: (1) posttetanic control LTP (n = 9), (2) tetanization with 1/~M domperidone (DOM (n = 11), (3) tetanization with 1/~M sulpiride (SUL) (n = 8), (4) tetanization with the solvent for DOM and SUL (n = 9), (5) tetanization with 1/~M apomorphine (APO) and I pM DOM (n = 8) and (6) tetanization with 1/~M flupenthixol (FPT) (n = 7). All dopamine receptor antagonists as well as the agonist apomorphine (APO) were applied to the bath medium 15 min before LTP induction. DOM and SUL were solved in 50 pl acetic acid, which had neither effects on the recorded potentials in nontetanized control slices nor during LTP up to 8 h after tetanization (data not shown). The dopamine antagonist FPT was solved in 50 /~1 DMSO which had likewise no effects in the used concentration. The drugs were washed out immediately after the last tetanization. The data were statistically evaluated with the two-tailed MannWhitney U-test and the Wilcoxon matched-pairs signed-rank test, respectively. For determination of dopamine release, 66 slices from 11 rats were prepared according to the procedure described for electrophysioiogical recording. On each slice, a stimulation electrode was positioned in the stratum radiatum and a recording electrode in the CA1 pyramidal cell layer to verify the vitality and function of each preparation. After 2 h preincuhation, flupenthixol and domperidone (1/~M) were added to the medium 5 min before the incubation with [t4C]dopamine to prevent its direct action. The slices were incubated with [i4C]dopamine (spec. act. 2.07 GBq/mmol, Radiochemical Centre Amersham, U.K.) for 10 min. Then the slices were superfused with an oxygenated modified Krebs-Ringer solution at a flow rate of 1.3 ml/min to remove the incubation medium and the surface radioactivity. The slices were stimulated 50, 60 and 70 min after beginning of superfusion, either with an impulse train of 100 Hz (100 pulses, 0.2 ms, 70/~A), or with 4 stimuli of 0.33 Hz (0.2 ms, 70/~A) as control stimulation. The released radioactive material was collected in fractions of 2 min and determined using a liquid scintillation counter. The values were expressed as percent of total released radioactivity, which contained 81% specific [14C]dopamine, as determined in aliquots by HPLC.

13,20,22,23,34

RESULTS MATERIALS AND METHODS Fifty-two transversely chopped hippocampal slices (400/~m) from 52 seven-week-old rats were prepared and incubated in a microcirculation chamber4] for extracellular recordings as previously described 11'31. Monopolar electrolytically sharpened and laquercoated stainless-steel electrodes were positioned in the stratum radiatum of the CA1 region for stimulation and in the CA1 dendritic and cell body layer, respectively, for recording (Fig. 1). The two stimulating electrodes were placed at adequate distances to stimulate separate inputs (for detail see ref. 25; the criteria for separate stimulation were used according to ref. 9). One hour prior to tetanization, a stimulation strength was determined for further testing which elicited a population spike of 25% of its maximal amplitude. The baseline was recorded for 30-60 min before LTP induction. Four biphasic constant current pulses (0.2 Hz, 0.1 ms per polarity) were used for testing at 1, 11, 21, 30 and 60 min as well as every following hour after first tetanization up

T h e p o t e n t i a l s e v o k e d by the test stimuli f r o m the first stimulating e l e c t r o d e (S1) also u s e d for p r o d u c t i o n of LTP by high f r e q u e n c y s t i m u l a t i o n were n o t i n f l u e n c e d in control e x p e r i m e n t s by the low c o n c e n t r a t i o n s of dopam i n e a n t a g o n i s t s used in this study. A f t e r t e t a n i z a t i o n in the p r e s e n c e of 1/~M D O M , the s a m e initial p o t e n t i a t i o n of the p o p u l a t i o n spike a m p l i t u d e (PS) o c c u r r e d as in drug-free control slices (Fig. 2). H o w e v e r , after the last t e t a n i z a t i o n , the PS p o t e n t i a t i o n a l r e a d y e x h i b i t e d a slight d e p r e s s i o n which g r a d u a l l y d e v e l o p e d t h u s app r o a c h i n g the b a s e l i n e values of test p o t e n t i a l s after 6 - 7 h. A significant difference b e t w e e n the PS of c o n t r o l LTP a n d of the d r u g - t r e a t e d groups could be o b s e r v e d f r o m 4

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Dopaminergic antagonists prevent long-term maintenance of posttetanic LTP in the CA1 region of rat hippocampal slices.

The involvement of dopaminergic mechanisms in the induction and maintenance of posttetanic long-term potentiation (LTP) was investigated on CA1 cells ...
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