ELSEVIER Neuroscience Letters 188 (1995) 163-166

NEUROSCIINC[ IHl[iIS

Nootropic compound L-pyroglutamyl-D-alanine-amide restores hippocampal long-term potentiation impaired by exposure to ethanol in rats

Aisa N. Chepkova a,*, Nanuli V. Doreulee% Sergey S. Trofimov b, Tatjana A. Gudasheva b, Rita U. Ostrovskaya b, Vladimir G. Skrebitsky a

aBrain Research Institute, Russian Academy of Medical Sciences, per. Obucha, 5, 103064 Moscow, Russian Federation blnstitute of Pharmacology, Russian Academy of Medical Sciences, Baltiyskaja str., 8, 125315 Moscow, Russian Federation

Received 19 December 1994; revised version received 15 February 1995; accepted 22 February 1995

Abstract

The characteristics of long-term potentiation (LTP) in the hippocampus of rats prenatally exposed to ethanol and treated posmatally with nootropic compounds L-pyroglutamyl-D-alanine-amide (L-pGlu-D-AlaNH 2, PGA) or piracetam were studied using in vitro slice preparations. LTP was induced in the CA1 region by the orthodromic stimulation of the stratum radiatum with one train of 100 pulses (100 Hz, 1 s). The probability of LTP development in the hippocampus of young rats was significantly reduced by prenatal exposure to alcohol. This plasticity deficit was completely reversed by daily injections of PGA, 1 mg/kg for 12 days (8-19 days of postnatal development) but not of piracetam, 100 mg/kg. PGA (0.5/.tM) also prevented the inhibition of LTP development in hippocampal slices perfused with ethanol, 20 or 50 raM. The data indicate that PGA effectively restores synaptic plasticity after both prenatal and acute exposure to ethanol and suggest that impaired LTP may be a useful model for studying the mechanisms of action of nootropic compounds.

Keywords: Nootropics; Piracetam; L-Pyroglutamyl-D-alanine-amide; Long-term potentiation; Ethanol; Prenatal exposure; Hippocampal slices

Mental retardation and learning disabilities are com- mon consequences of fetal alcohol exposure in humans [1]. In laboratory animals, as in humans, prenatal expo- sure to alcohol results in learning and memory deficits that have been revealed in different learning paradigms and considered to be indicative of hippocampal dysfunc- tion [6,24 ].The selective damage of hippocampal cells and synaptic structures has been found as a result of pre- natal exposure to ethanol [29].

According to recent studies, nootropics or cognition enhancers may turn out 1:o be effective in the treatment of postalcohol deficiency. Piracetam, a standard nootropic agent has been shown to beneficially affect memory im- paired by perinatal alcohol exposure [21].We found that postnatal treatment with a stable peptide-like derivative of L-pyroglutamic acid L-pyroglutamyl-D-alanine amide (L-

* Corresponding author, Tel.: +7 095 917 84 52; Fax: +7 095 916 05 95; E-mail: aisa@synaptology.msk.su.

pGlu-D-AIaNH2, PGA) completely normalized the learn- ing capacity of rats disturbed by prenatal exposure to al- cohol [19]. This compound constructed as a peptide ana- logue of piracetam [9] was found to be much more potent as a cognitive enhancer than the prototype [ 18].

Despite well-documented clinical and experimental efficacy of nootropics, the mechanisms of their action remains largely unclear (see Ref. [7] for a recent review). One of the approaches to the mechanisms may be the study of the effects of nootropics on hippocampal long- term potentiation (LTP). LTP is a use-dependent form of synaptic plasticity that can be induced by high frequency stimulation (HFS) of synaptic inputs and is thought to underlie learning and memory processes 113]. Hippocam- pal LTP was reported to be impaired by prenatal [27], chronic [5] and acute [4,16] exposure to ethanol. It was of interest therefore to find out whether the postnatal treat- ment with a potent nootropic compound PGA restores synaptic plasticity in the hippocampus of offspring ex- posed to alcohol in utero.

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164 A.N. Chepkova et al. / Neuroscience Letters 188 (1995) I63-166

To address this issue, we analyzed LTP characteristics in the hippocampal CA1 field in normal rats and in rats exposed to ethanol in utero and subsequently treated with PGA or piracetam. Pregnant Wistar rats were daily intu- bated with 5 g/kg ethanol as a 25% water solution throughout the gestation period. Pregnant rats in the con- trol group received the identical volume of water daily. From the 8th to the 19th days after the delivery, the rat offspring was given subcutaneous injections of either saline or one of the nootropics in an equal volume of sa- line. Piracetam (Sigma) was administered in a dose of 100 mg/kg, and PGA (synthesized by dr. T.A. Gu- dasheva) in a dose of 1 mg/kg. After the last injection, the young male rats were separated from their mothers, housed by group and in 1-2 weeks used in blind electro- physiological experiments.

Electrophysiological experiments were performed on hippocampal slices. The slices were kept on a nylon net in a constant flow incubation chamber, submerged in a so- lution containing (in mM): NaC1 124, KC1 3, CaCI 2 2.4, MgSO 4 2.4, KH2PO4 1.25, NaHCO 3 26, D-glucose 10 and saturated with 95% 02/5% CO2 (t = 30°C, pH = 7.4). Ex- periments were started after pre-incubation of the slices for 1.5-2 h. Extracellular recordings were made from the pyramidal layer of the CA1 region using glass micropi- pettes filled with 1.5 M NaC1. To elicit synaptic re- sponses, Shaffer collateral/commissural afferents in stra- tum radiatum were stimulated through bipolar glass elec- trodes filled with a perfusion medium. Single pulses (0.1 Hz, 0.1 ms) ranging from subthreshold to su- pramaximal were used to obtain input-output curves (stimulus intensity versus pop-spike amplitude). The baseline was recorded for 1 h before LTP-inducing tetani- zation. To induce LTP, one train of high frequency stimulation (HFS; 100 Hz, 1 s) was used with pulse in- tensity adjusted so that evoked responses were approxi-

mately 1/2 maximal. Post-tetanic responses were tested in 15, 30, 45 and 60 rain after HFS.

Field responses were recorded, analyzed and stored on a computer. The magnitude of potentiation was calculated as a relative increase in the area under the linear part of an input-output curve. The data were statistically treated with the Mann-Whitney U-test and Fisher's exact prob- ability test.

A comparative analysis of response characteristics in slices from different groups revealed a small but signifi- cant decrease in pop-spike amplitudes in the fetal alcohol group (Table 1). Prenatal alcohol also markedly impaired the development of LTP. In the vast majority of control group slices, HFS produced LTP that is a persistent en- hancement of the pop-spike response which was main- tained for at least 1 h (Fig. 1A). In contrast, HFS induced LTP only in approximately half of the slices from fetal alcohol rats, while the remaining half demonstrated tran- sient potentiation decaying in 40-60 min (Fig. 1A, Table 1), which is classified as short-term potentiation (STP) [3]. The difference in the STP/LTP ratio between the control and the alcohol group was highly significant. The magnitude of proper LTP in the alcohol group slices was the same as in the control group, while STP magnitude was significantly reduced (Table 1).

The postnatal treatment of alcoholic offspring with PGA completely normalized both responsiveness and LTP development. Pop-spike amplitudes and the prob- ability of LTP development in the slices from fetal alco- hol rats treated with PGA tended to be slightly higher than in the control group. In contrast, piracetam treatment normalized responsiveness but did not reverse LTP deficit (Table 1, Fig. 1B).

The marked improvement of LTP development follow- ing postnatal treatment with PGA prompted us to study the effects of PGA on a well-documented LTP blockade

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Fig. 1. (A) The time course of the post-tetanic enhancement of a pop-spike amplitude in hippocampal slices from the control and fetal alcohol rats. The data indicate means and SEM. The difference between the control (n = 29) and alcohol (n = 29) group is significant at 45 and 60 min (P = 0.05, Mann- Whitney U-test). High frequency stimulation (HFS) is marked by an arrow. (B) The effects of the postnatal treatment of the alcoholic offspring with nootropic compounds L-pyroglutamyI-D-alanine-amide (PGA) and piracetam (PIR) on LTP development in the hippocampal slices. The data indicate the mean values and SEM of the post-tetanic enhancement of a pop-spike amplitude 60 min after HFS. (C) The time course of the post-tetanic changes in a pop-spike amplitude in the hippocampal slices pretreated with ethanol. The data indicate means and SEM. Slice perfusion with either ethanol alone (inverted closed triangles), or the mixture of ethanol with PGA (open triangles) is marked with a bar. HFS is indicated with arrows.

A.N. Chepkova et al. / Neuroscience Letters 188 (1995) 163-166 165

Table 1

Responsiveness and LTP development in hippocampal slices from fetal alcohol rats treated with L-pGlu-D-AIaNH 2 (PGA) or piracetam (PIR)

Group Pop--spike, PS STP/LTP Magnitude (%) Correlation, r

(mV) (n/n) STP LTP PS/LTP

Control + saline (32) 2.94 _+ 0.11 (32) 4/25 139.5 _+ 9.1 (4) 141.3 _+ 5.5 (25) -0.19 Alcohol + saline (33) 2.61 _+ 0.13"(33) 13116** 118.9 -+ 2.5"(13) 140.9 _+ 5.5 (16) -013 Alcohol + PGA (17) 3.28 _+ 0.19 (17) 0/16 - 142.2 _+ 5.8 (16) -0.49 Alcohol + PIR ( I l) 3.05 +_ 0.46 (11) 4/6 133.5 +- 2.5 (4) 141.8 _+ 11.0 (6) -0.45

The data on a pop-spike amplitude (a maximum value) and the magnitude of STP and LTP (as measured 15 min after HFS) are mean value _+ SEM; the number of slices (n) in parentheses. *, ** indicate different from control + saline (P < 0.(15, P < 0 .01) . Correlation r was eslimated between pop-spike amplitude at HFS and the magnitude of LTP as measured 60 min after HFS.

by ethanol in vitro [4,16]. These experiments were carried out on slices from young (1-1.5 months old) Wistar rats. Ethanol, 20 or 50 mM, was introduced into a perfusion medium 20 min before HFS, and washed out immediately after it. The data are summarized in Fig. 1C.

Acute ethanol exposure resulted in irreversible damage of the LTP development. HFS which was applied at the end of ethanol application induced no LTP in the majority of slices (6 out of 8). The second HFS applied 1 h follow- ing initial HFS produced depression instead of potentia- tion. When PGA, dissolw~d in the perfusion medium to the concentration of 0.5 ~M was administered in combi- nation with ethanol, the first HFS induced LTP in five out of nine slices tested. The second HFS produced LTP in all nine slices, The data analysis showed that PGA signifi- cantly (P < 0.001) increased the probability of LTP de- velopment in ethanol treated slices and accelerated the postalcohol recovery of their plastic properties.

Thus the results of the present study indicate that the nootropic compound PGA reverses the synaptic plasticity deficit, induced by both prenatal and acute eth.anol expo- sure. The mechanisms of PGA action are yet to be de- termined. The question is whether PGA treatment restores those very same mechanisms impaired by alcohol, or stimulates some other processes. Furthermore, one must ask whether the restoration of responsiveness and plastic- ity is due to the same mechanisms or different ones.

The primary cause of LTP deficit following in vitro ethanol exposure seems to be the blockade of N-methyl- D-aspartate (NMDA)-sensitive glutamate receptors [4,12, 16]. The activation of NMDA receptors and a consequent increase in the intracellular Ca 2+ concentration are con- sidered to be critical for the induction of LTP in the CA1 hippocampal region [3]. Ethanol in the concentrations used could also inhibit the activity of protein kinase C (PKC) [26], which is involved in both the induction and maintenance of LTP [3]. Therefore the reversal of LTP blockade by PGA could be explained by the modulation of glutamatergic transmission, calcium entry or intracellu- lar machinery.

There is increasing evidence that some piracetam-like nootropics (aniracetam, oxiracetam) may interact with

fast glutamatergic transmission, enhancing responses mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxa- zolepropionic acid (AMPA)- sensitive glutamate recep- tors [9,10,20,28] and facilitating Glu/Asp release [14]. They also positively modulate responses mediated by a metabotropic glutamate receptor [22] and activate PKC [13]. However, these nootropics leave unaffected re- sponses mediated by NMDA receptors [1 1,20] and LTP characteristics in the CA1 region of normal hippocampal slices [2,17,22,25]. Although PGA in the concentration used in our acute alcohol experiments (0.5,uM) did not affect field responses and LTP development in control slices (data not shown), it produced a marked (126.4+2.5%, n = 4 ) and wash-resistant (60rain) in- crease in pop-spike amplitude at 50~tM, suggesting an involvement of changes in intracellular machinery.

It should be noted that response amplitudes in slices from fetal alcohol rats treated with PGA tended to be slightly higher than in the control group. This increase in responsiveness could contribute to the restoration of syn- aptic plasticity. However, the absence of any positive correlation between the pop-spike amplitude: at HFS, and the magnitude of LTP (Table 1) makes it difficult to ex- plain the reversal of the plasticity deficit by increased responsiveness. The impairment resulting from prenatal exposure to alcohol is a reduced probability of STP con- version into LTP rather than a complete blockade of an induction phase. Although this reduced probability of LTP was mainly observed in slices with a low magnitude of STP, it does not imply the causality since a low magni- tude (<130%) LTP was present in both the control (n = 10, 40% of all slices with LTP) and alcohol (n = 50 31%) groups. A low magnitude of STP may be explained by the decreased sensitivity of NMDA receptors after prenatal alcohol exposure [15], while the failure to stabi- lize potentiation suggests the impairment of' LTP mainte- nance associated with the activation of calcium-dependent protein kinases and PKC in particular [3]. We can specu- late that PKC might be one of the targets of PGA action.

Whatever the mechanisms of PGA action may be, the data on LTP impairment following in utero exposure to alcohol and its restoration after postnatal treatment with

166 A.N. Chepkova et al. / Neuroscience Letters 188 (1995) 163-166

PGA are in close correlation with our behavioral findings [19] suggesting that the learning deficit caused by fetal alcohol may be intimately associated with the impairment of LTP. The use of an impaired LTP model may hold a lot of promise for the analysis of the mechanisms of nootropic action.

This work has partly been supported by grants from the Russian Foundation for Fundamental Sciences (94-04- 11430) and the International Science Foundation (MN 6OO0).

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