Depression of LTP in rat dentate gyrus by naloxone is reversed by GABAA blockade

BRAIN RESEARCH ELSEVIER

Brain Research 688 (1995) 56-60

Research report

Depression of LTP in rat dentate gyrus by naloxone is reversed by GABA A blockade Cui Wei Xie a, Darrell V. Lewis b, * a Department of Psychiatry and Biobehavioral Sciences, University of California at Los Angeles, School of Medicine, 760 Westwood Plaza, Los Angeles, CA 90024, USA b Department of Pediatrics (Neurology), Box 3430, Duke University Medical Center,Durham, NC 27710, USA Accepted 11 April 1995

Abstract

Long-term potentiation (LTP) of the lateral perforant path (LPP) to dentate granule cell (DGC) synapse is suppressed by the opioid antagonist, naloxone, and thus appears to be dependent upon the release of endogenous opioids from the LPP. It has been suggested that endogenous opioids enhance LTP by depressing GABA A inhibition. As one test of this hypothesis, we determined whether blockade of GABA A inhibition would alleviate the naloxone block of LTP in the LPP. Consistent with the hypothesis that endogenous opioids enable LTP by disinhibition of the DGCs, naloxone no longer blocked LTP in the presence of the GABA A antagonist, bicuculline methiodide. Furthermore, although blockade of/.~ receptors suppressed LTP of the slope of the population excitatory potential (pEPSP), blockade of both /z and /~ opioid receptors was needed to suppress LTP of both the pEPSP and the orthodromic population spike (OPS). Keywords: Long-term potentiation; Dentate gyms; Opioid; GABA A receptor; Disinhibition

1. Introduction

Endogenous opioids have been linked to LTP of excitatory neurotransmission in the lateral perforant path (LPP) projection to dentate gyrus and in the mossy fiber-CA3 synapses in the hippocampus. In the dentate, naloxone blocks LTP of the LPP-dentate granule cell (DGC) synapse [2,30] and both /.~ and 6 opioid receptors may be important for LPP-DGC LTP [1,3,30]. LTP in the LPP is also probably NMDA receptor dependent. Although one study of LPP-LTP in vivo found insensitivity to an NMDA antagonist [4], several other studies using the in vitro hippocampal slice have found LTP at the LPP-DGC synapses to be sensitive to NMDA receptor antagonists as well as sensitive to opioid receptor antagonists [9,14,30]. The mechanism whereby endogenous opioids augment LTP at the LPP-DGC synapse is unknown. However, one of the most striking effects of exogenously applied opioids in the hippocampus is disinhibition of both pyramidal neurons and DGCs [5,12,19,22,32]. We have shown that application of the /z agonist, PL017, reduces both GABA A and GABA B IPSCs (inhibitory post-synaptic currents) in

* Fax: (1) (919) 681-8943. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. Allrightsrese~ed

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DGCs [31]. Based on these findings, we have hypothesized that endogenous opioids released from the LPP during tetanic stimulation facilitate LTP by reducing GABAergic inhibition [30,31]. This hypothesis would predict that blockade of inhibition should relieve the block of LTP by naloxone. Accordingly, we have tested the effect of blockade of GABA A inhibition on the ability of naloxone to block LTP at the LPP-DGC synapse. In addition, LPP high frequency stimulation would be expected to release proenkephalin derived peptides acting primarily at /z and ~ receptors [7,13,26]. Therefore, if endogenous opioids are augmenting LTP by disinhibiting the dentate, blockade of LTP should be possible using either /x or 6 specific antagonists or both in combination. Accordingly, we have tested the ability of antagonists specific for these receptors to block LTP at the LPP-DGC synapse.

2. Materials and methods

2.1. Preparation of hippocampal slices Male Sprague-Dawley rats (175-280 g) were anesthetized with halothane and decapitated. Transverse hip-

C.W. Xie, D. V. Lewis/Brain Research 688 (1995) 56-60

pocampal slices 500 /xm thick were cut and placed in a holding chamber in 32°C artificial cerebrospinal fluid (ACSF) bubbled continuously with 95% 0 2 - 5 % CO 2. The ACSF consisted of (mM): NaC1 120, NaHCO 3 25, KC1 3.3, NaH2PO 4 1.23, CaCI 2 1.8, MgSO 4 1.2, and D-glucose 10 at pH 7.4. After at least 1 h of incubation in the holding chamber, the slices were transferred into a submerged 2 ml recording chamber, where they were perfused at a rate of 3 - 4 m l / m i n with warmed ACSF (32-34°C). Because the ventral portion of the hippocampus has a higher density of enkephalinergic innervation [13] and /x receptors [20], and is more susceptible to opioid effects [17], all experiments were performed using slices from the ventral hippocampus. 2.2. Stimulation and recording

A sharpened monopolar tungsten electrode was placed in the outer one-third of the dentate molecular layer to stimulate the LPP which was identified as previously described [30]. Constant-current rectangular stimulus pulses (0.1 ms, 0.04-1 mA) were delivered through the electrodes by an isolated stimulator. Extracellular responses were routinely monitored through recording pipettes (2 M NaC1, 1 - 8 M O ) placed in the granule cell layer to measure the orthodromic population spike (OPS) and in the outer molecular layer, in line with and 0.5-0.7 mm away from the stimulating electrode, to measure the population excitatory postsynaptic potential (pEPSP). Signals were digitized on a NIC-310 oscilloscope. 2.3. Experimental protocol

Twenty to 30 min after electrode placement, a baseline input/output ( I / O ) curve was established, and test stimulus intensities were set at the current that elicited the OPS of 50% maximal amplitude in control (0.1-0.3 mA). The test stimuli were delivered every other minute until stabilized responses were observed for at least 20 min. One second, 100 Hz trains of stimuli at the current level eliciting the 50% maximal response were used to tetanize the LPP. LTP was measured 30 min after the tetanus. All LTP measurements were done using the same stimulus current that elicited the 50% of maximal response prior to the tetanus. In the experiments testing the effect of GABA n blockade on LTP, four groups of slices were examined, each bathed in a different medium. Group 1 was in ACSF only; group 2 was in ACSF containing naloxone, 10/zM; group 3 was in ACSF containing bicuculline methiodide, 2 0 / x M (BMI); and group 4 was bathed in ACSF containing both naloxone (NX) (10 /xM) and BMI (20 /zM). In the experiments measuring the effects of specific opioid antagonists on LTP, five groups were used. Group 1 was tetanized in ACSF only, group 2 after exposure to the irreversible /x antagonist beta-funaltrexamine hydrochloride (13-FNA), group 3 in the 6 antagonist, ICI 174864

57

(ICI), group 4 in moderate concentrations of both antagonists and group 5 in higher concentrations of both antagonists. Exposure to I~-FNA was done by bathing the slices in 13-FNA (5 or 10 /zM) for 20 min and then washing in drug free ACSF for 1 h to remove unbound 13-FNA from the tissue before the experiment was started. 2.4. Data processing

The pEPSP slopes and OPS amplitudes were measured using a computer-assisted waveform analysis package (Waveform Basic, Nicolet). Slope was measured in the early phase of the pEPSP at its steepest portion. OPS amplitude was measured as the difference between the peak negativity and the averaged value of the two peak positivities. The amount of LTP was expressed as the 30 min post-train percent increase in the OPS amplitude or pEPSP slope relative to the baseline in ACSF only, which were designated 100%. Data were subjected to a one way analysis of variance to determine significance. Comparison of group means was made with a Students t-test and differences were considered significant at a level of P < 0.05.

3. Results When slices were bathed in BMI, the amplitude of the OPS elicited by the test stimuli increased markedly. This resulted in the response to our test stimuli at the 50% level being very close to the maximum dentate OPS amplitude. Because of this limitation, we used pEPSP slope as the sole measure of LTP in these experiments. The baseline pEPSP slopes were not affected by the application of BMI in agreement with other reports [29]. Relative to the ACSF control, slope of the pEPSP was 103.4_ 9.1% (mean + S.E.M.) in BMI (n = 10) and 97.6 + 9.3% in BMI and NX (n = 11). The results of the experiments (summarized in Fig. 1) using the GABA A antagonist, BMI, clearly demonstrated that NX did not reduce LPP-LTP when GABA A inhibition was blocked. One second trains to the LPP resulted in LTP of pEPSP slope in slices bathed in ACSF only, as we have previously reported [30]. Also, as previously reported [30], LTP was clearly suppressed in slices bathed in ACSF and NX. Slices which were tetanized in ACSF and BMI exhibited robust LTP which was slightly more pronounced than the LTP in ACSF alone, although the differences were not statistically significant. In addition, the group of slices tetanized in ACSF containing BMI and NX also showed robust LTP. Therefore, NX blocked LTP when GABA A inhibition was intact, but not when inhibition was reduced. The experiments using the/x and 6 specific antagonists demonstrated that LTP of the pEPSP was sensitive to /z blockade, but that to block LTP of the OPS required both /z and 6 blockade (summarized in Fig. 2). As before, in

C. W. Xie, D. V. Lewis/Brain Research 688 (1995) 56-60

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these experiments on the effects of 6 and /x antagonists on LTP, a control group of slices was tetanized in ACSF only. As expected, this control group of slices showed LTP of the OPS and pEPSP. The second group of slices was pretreated with 10 /xM 8-FNA to irreversibly block /z receptors and this treatment produced a significant reduction of the LTP of the pEPSP slope and a non-significant trend for less OPS LTP. Another group of slices, tetanized in the 6 antagonist, ICI (20 /zM), showed a small and not statistically significant reduction of LTP. The two antagonist treatments were combined in two other groups of slices. The first group, bathed in 5/.~M 13-FNA and 10/~M ICI, showed non-significant reductions in LTP. However,

A

CONTROL

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Fig. 2. Blockade of both /.t and 6 receptors suppresses LTP. A: examples of recorded OPS and pEPSP. The pretrain and 30 min posttrain responses are superimposed for easier comparison in each example. In ACSF alone (CONTROL), both the OPS amplitude and pEPSP slope increased after the train (arrows). In the presence of both 6-FNA and ICI, LTP of neither OPS amplitude nor pEPSP slope was observed. B: group data. LTP of both the OPS and pEPSP were observed in ACSF ( n = 9 ) . The p. antagonist 13-FNA produced a significant reduction of the pEPSP LTP (n = 8). Application of the 6 antagonist ICI showed a trend, though non-significant, to decreased LTP (n = 7). When both antagonists were applied to each slice at relatively lower concentrations, the trend was again seen (n =9). Finally, exposure to both antagonists at higher concentrations produced significant reductions in both OPS and pEPSP LTP (n = 9). * P < 0.05, * * * P < 0.01 as compared to ACSF group.

O3

the second group, treated with 10/xM 13-FNA and 2 0 / z M ICI, showed clear and significant blockade of LTP of both the OPS and the pEPSP slope.

120

I00 ACSF

NX

BMI

NX+BMI

Fig. 1. GABA A blockade prevents LTP suppression by naloxone. A: representative recordings. (1) pEPSP recorded in the outer molecular layer before (upper trace) and 30 min after (lower trace) the train in the presence of 10 kLM NX. The traces are superimposed in (2), indicating no LTP induced in the presence of NX alone. (3) pEPSP recorded from another slice in control medium (upper), in 50 /~M BMI + 10 /xM NX before the train (middle) and in BMI + NX 30 min after the train (lower). The traces are superimposed in (4). Upper: control vs. BMI + NX, both pretrain, showing no significant change in the pEPSP slop after applying BMI and NX. Lower: B M I + NX, pre-and posttrain. Note the evident potentiation of pEPSP slope after train. B: group data illustrates the LTP of the pEPSP slope. In ACSF (n = 10), LTP is clear, whereas in naloxone (NX), no LTP was observed (n = 8). Robust LTP was seen in either BMI alone ( n = 10) or the combination of N X + B M I (n = 11). Error bars represent S.E.M.s in this and the following figure. * P < 0.05, as compared with ACSF group.

4. Discussion

The mechanism whereby endogenous opioids facilitate LTP in the LPP is unknown. As first described by Bramham et al. in vivo [2], we also found [30], in rat hippocampus in vitro, that LTP of the LPP is reduced by NX. If opioids facilitate LTP in the LPP by producing disinhibition, then the opioid requirement for LTP induction would be absent with GABA A receptors blocked, as we have found. These findings are clearly compatible with, although not proof of, the disinhibition hypothesis. With regard to our findings, it is important to note that Hanse and Gustaffson have also reported that, in the presence of the GABA A antagonist,

C. W. Xie, D. V. Lewis/Brain Research 688 (1995) 56-60

picrotoxin, NX did not affect LTP in the guinea pig dentate [14]. However, these investigators did not state whether NX would block LTP in the guinea pig dentate under baseline conditions, which would be an important consideration, since the opioid receptor distribution [21] and pharmacology [16] may differ in the rat and guinea pig hippocampal formations. Pro-enkephalin derived peptides are found in the LPP terminals [7,13] and are released during high frequency stimulation [26]. The released opioids would bind primarily to 6 and /~ receptors [6], both of which are found in the dentate molecular layer [21]. Furthermore, both /x and 6 receptors reduce GABA release from GABAergic terminals in the hippocampus [18] and dentate [23,31]. Therefore, we tested the effects of /x and 6 antagonists on the LPP LTP. We found that the 6 antagonist alone in supramaximal concentrations did not by itself block LTP of either the OPS or the pEPSP in the in vitro slice, although the /x antagonist alone did block LTP of the pEPSP. Significant suppression of LTP of the OPS was obtained only by combining /z and 6 blockade. This is in keeping with the demonstrated ability of both /z and 6 agonists to produce disinhibition in the dentate [22,23] and may reflect the possibility that pro-enkephalin derived peptides from the LPP mediate effects through both 6 and /z receptors [6]. We did not test K antagonists because it seemed unlikely that K antagonists would reduce LPP-LTP since they have been reported to enhance perforant path LTP in the guinea pig [25,27]. Suppression of LPP-LTP in the slice preparation required high concentrations of both /z and 6 antagonists (ICI 20 /xM and 13-FNA 10 /zM) which would have been supramaximal in m a n y other preparations [8,10,11,15,24,28]. This could be partially due to the time dependency of the irreversible inactivation of /x receptors by 13-FNA. In the guinea pig ileum muscle preparation, 0.1 /xM 13-FNA incubation for 10 min produced only a small fraction of the maximum inhibition of morphine effects which could be obtained by incubation for over 80 min [24]. Thus, a much higher concentration (10 /zM) of 13-FNA was used in our study for rapid inactivation of /z receptors during a limited incubation period (20 min), and we have previously shown that this treatment completely blocks the ability of the /x agonist, PL017, to reduce evoked inhibitory postsynaptic currents in DGCs [31]. To avoid potential confounding agonist effects of 13-FNA at K receptors [28], slices were washed for one hour after 13-FNA application [24]. Although ICI also was used at high concentrations in our experiments, ICI has shown specificity for 6 receptors even in the micromolar range [10,11,15]. Our findings on LPP-LTP in the hippocampal slices disagree with those reported by Bramham, using in vivo preparations [1], in two areas. We and others [9,14,30] have found that LPP-LTP is entirely blocked in the slice preparation by NMDA antagonists, whereas Bramham and

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co-workers have found that an NMDA antagonist infused in vivo blocked only LTP of the OPS, but not of the pEPSP slope [4]. We suggest that the weight of the evidence favors NMDA receptor involvement in LPP-LTP. However, we also found that ICI alone did not significantly suppress LTP in vivo, whereas Bramham et al. [3] found in vivo that ICI blocked pEPSP LTP without affecting OPS LTP. We are not able to explain this discrepancy, and suggest that it could relate to differences between the in vivo and in vitro systems. In summary, our results are compatible with the hypothesis that endogenous opioids facilitate LTP in the LPP by producing disinhibition, which then increases NMDA receptor-mediated currents that contribute to the induction of LTP in this pathway. However, other effects are not excluded by these findings, such as opioids somehow augmenting excitatory amino acid transmission independent of inhibition. For instance, although we have shown that the /x agonist, PL017, does not alter non-NMDA EPSPs and can, in fact, reduce NMDA EPSPs in granule cells [31], it remains possible that 6 receptor activation could somehow directly augment NMDA currents or the second messenger systems engaged by calcium influx [1].

Acknowledgements This study was supported by NIDA Grants to D.V.L. (DA06735) and to C.W.X. (DA08571).

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