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Prenatal morphine exposure enhances seizure susceptibility but suppresses long-term potentiation in the limbic system of adult male rats
Brain Research 869 (2000) 186–193 www.elsevier.com / locate / bres
Research report
Prenatal morphine exposure enhances seizure susceptibility but suppresses long-term potentiation in the limbic system of adult male rats ´ˇ a,b,e , *, Patric K. Stanton a,b , Solomon L. Moshe´ a,b,c,e , Ilona Vathy b,d Libor Velısek a
Department of Neurology, K 312, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, New York, NY 10461, USA b Department of Neuroscience, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, New York, NY 10461, USA c Department of Pediatrics, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, New York, NY 10461, USA d Department of Psychiatry, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, New York, NY 10461, USA e Einstein /Montefiore Epilepsy Management Center, Bronx, New York, NY 10461, USA Accepted 4 April 2000
Abstract The present study examined the effects of prenatal morphine exposure on NMDA-dependent seizure susceptibility in the entorhinal cortex (EC), and on activity-dependent synaptic plasticity at Schaffer collateral and perforant path synapses in the hippocampus. During perfusion with Mg 21 -free ACSF, an enhancement of epileptiform discharges was found in the EC of slices from prenatally morphine-exposed male rats. A submaximal tetanic stimulation (2350 Hz / 1 s) in control slices elicited LTP at the Schaffer collateral-CA1 synapses, but neither LTP nor LTD was evoked at the perforant path-DG synapses. In slices from prenatally morphine-exposed adult male rats, long-term potentiation of synaptic transmission was not observed at Schaffer collateral-CA1 synapses, while the submaximal tetanus now elicited frank LTD of synaptic EPSPs at perforant path synapses. These data suggest that prenatal morphine exposure enhances the susceptibility of entorhinal cortex to the induction of epileptiform activity, but shifts long-term plasticity of hippocampal synapses in favor of LTD. 2000 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behaviour Topic: Drugs of abuse: opioids and others Keywords: Hippocampus; Entorhinal cortex; Brain slices; Long-term potentiation; Long-term depression; Prenatal morphine exposure; Seizure susceptibility; Synaptic plasticity
1. Introduction Opiates such as morphine, the active component of heroin, are frequently abused during pregnancy [21]. These substances can induce long-term psychological and behavioral alterations in exposed children [2]. Since longterm potentiation (LTP) is considered as a cellular and molecular basis for learning and memory [15] that are involved in behavioral and cognitive processes, it is of great concern to study the effects of prenatal morphine exposure on LTP. *Corresponding author. Tel.: 11-718-430-4277; fax: 11-718-4308899. ´ˇ E-mail address: [email protected] (L. Velısek)
There are reports that chronic morphine exposure, as well as morphine dependence, can interact with the induction of LTP. For example, chronic postnatal administration of morphine augments LTP induced by low-intensity primed-burst stimulation at Schaffer collateral-CA1 synapses in slices, suggesting that chronic morphine exposure may result in chemically induced shift in LTP threshold [17]. This effect of enhanced LTP in chronically morphine-exposed rats may be largely due to acute withdrawal effects seen in these slices since, initially, morphine was omitted in the bath [16]. However, very little is known about the long-term effects of prenatal morphine exposure on hippocampal LTP Additional reports show that morphine administration can interact with N-methyl-D-aspartate (NMDA) receptor-
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mediated neurotransmission which plays a central role in the LTP production. A single low dose of morphine inhibits NMDA-induced biting and seizures [1,37], while a single high dose potentiates NMDA-induced seizures in adult rats [1]. Morphine withdrawal also activates NMDA receptors and induces seizures [14]. Further, low systemic doses of morphine have anticonvulsant effects while high doses are proconvulsant [12]. Similarly, single intracerebral, intracerebroventricular or intrathecal administrations of morphine are proconvulsant [12]. Repeated administration of morphine increases susceptibility to seizures in adult rats, but these effects disappear within 5 days after morphine administration was terminated [23,25]. A sudden morphine withdrawal after repeated administration can also lead to short-term proconvulsant effects [30]. It is interesting that prenatal morphine exposure also alters NMDA receptor-mediated stereotypic behavior that may be regarded as a long-term effect of withdrawal [26]. Thus, the present study was designed to determine whether the exposure to morphine during mid to late gestation permanently alters two NMDA-dependent phenomena: long-term synaptic plasticity in the hippocampal formation and low Mg 21 -induced seizure susceptibility in the entorhinal cortex in adult male offspring.
2. Materials and methods Pregnant rats were randomly assigned to either a morphine-treated experimental or a saline-treated control group. Morphine or 0.9% saline were administered subcutaneously (s.c.) to female rats twice a day (08:00 and 20:00 h) on gestational days 11–18, as described previously [32]. Morphine sulfate was obtained from the National Institute on Drug Abuse (Research Technology Branch, Rockville, MD), and dissolved in 0.9% saline to a final injection volume of 0.1 ml. The dose of the first three morphine injections was 5 mg / kg, and the remaining injections 10 mg / kg [32]. On the first postnatal day (PN 1) pups were sexed, weighed, and tattooed for identification with black India ink on one foot pad. Morphine- and saline-exposed litters were cross-fostered such that each mother raised half of her own and half of the adopted pups of the opposite prenatal treatment. Litters were reduced to a maximum of 10 pups. Pups were weaned, weighed and housed individually on PN 25. All rats were kept on reversed light:dark cycle (14:10 h; lights on at 21:00 h) with access to food and water ad libitum. Only one salineand one morphine-exposed male from each litter were used in either seizure or synaptic plasticity experiments.
2.1. Slice preparation Saline- and morphine-exposed adult male rats were decapitated under deep ether anesthesia, brains removed, and hippocampi plus associated entorhinal cortex (EC)
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were quickly dissected. Horizontal, combined hippocampal-EC slices (400-mm thick) were cut with a vibratome (EMS) in ice-cold artificial cerebrospinal fluid (ACSF), containing (in mM): NaCl 126; KCl 5; NaH 2 PO 4 1.25; MgCl 2 2 or 0; CaCl 2 2; NaHCO 3 26; and glucose 10; pH 7.3–7.4. The slices were placed in an interface-type recording chamber perfused with 33–348C ACSF at 2 ml / min, oxygenated with 95% O 2 / 5% CO 2 . For determining seizure susceptibility, slices from one morphine- and one saline-exposed male rat were placed side by side in the recording chamber to maintain nearly identical experimental conditions. For the same reason, the order of cutting and the position of slices in the recording chamber were randomized. For synaptic plasticity experiments, simultaneous recordings were performed in one slice from either a morphine- or saline-exposed male rat dentate gyrus (DG) and CA1 subfield of the defined slice. After a 1-h incubation, the viability of slices was verified by stimulating stratum radiatum of CA1 subfield with bipolar stainless steel stimulating electrodes (Frederick Haer), and recording field potentials (field responses including population EPSPs and superimposed population spikes) in stratum pyramidale of area CA1, with an extracellular glass recording micropipette (filled with 2 M NaCl, 2–5 MV). Only slices responding to a single stimulus (intensity range 50–500 mA; inducing half-maximal response) with a single population spike at least 2 mV in amplitude and displaying paired-pulse inhibition at 20-ms interstimulus interval were used in the following experiments. Multiple population spikes as a response to single stimulus may indicate hyperexcitability of the slice due to cutting damage, hypoxia, dehydration, etc. Preserved paired-pulse inhibition at 20-ms interval indicated effective feed-forward inhibition. This parameter was used since inhibitory interneurons involved in the feed-forward inhibition are very vulnerable to any deteriorations. No more than two slices were used from any one rat.
2.2. Seizure susceptibility test To determine seizure susceptibility in the EC, a recording micropipette was placed in the deep layers (IV–V) of the medial EC (Fig. 1, top). After a stable DC baseline was recorded for at least 10 min, perfusion was switched to Mg 21 -free ACSF. Mg 21 -free ACSF regularly elicited two distinct types of discharges: (1) seizure-like events (SLE) which are long epileptiform discharges with a negative DC shift (duration 1–7 min each [10]); (2) SLEs progressed spontaneously into a second type of epileptiform activity, continuous status of short recurrent discharges (status-like activity; duration 1–10 s each discharge [10]). Short recurrent discharges continued for as long as Mg 21 -free ACSF was present. In these experiments, recording continued until short recurrent discharges developed in both slices [34]. The latency to onset of SLE, to onset of short recurrent discharges, and the pattern of initial discharges
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were recorded. Group means were compared with twotailed Student’s t-test for unpaired samples. The difference was considered significant at the level of P,0.05.
2.3. Synaptic plasticity test To assess synaptic plasticity at perforant path-DG granule cell and at Schaffer collateral-CA1 pyramidal cell synapses, recording electrodes were placed in the middle molecular layer of the upper blade of the DG and in stratum radiatum of the CA1 field. Test pulses were delivered to the perforant path (mixed perforant path stimulation; stimulating both lateral and medial portion between the outer and middle third of the molecular layer) and Schaffer collateral axons, alternating each 30 s at a stimulus intensity that elicited 50% of the maximal population EPSP slope. Then, high frequency, sub-maximal tetanization (2350 Hz / 1 s; 15-s intertrain interval) was delivered to one stimulation electrode, and 30 s later to the second electrode. As these initial experiments were intended to reveal hypothetical long-term difference in the DG synaptic plasticity between prenatally morphine- and saline-exposed offspring, lateral and medial perforant pathways were not tested separately. The authors are aware that several neuromodulators play differential roles in lateral versus medial perforant pathway (such as norepinephrine and opioids [3,6]) and will be investigated in upcoming studies. In all experiments, stable baseline population EPSPs were recorded for at least 10 min prior to high-frequency stimulation. A weak high-frequency stimulation paradigm was chosen because changes in synaptic plasticity in slices from morphine-exposed rats may be masked by a saturating supramaximal tetanus. Changes in population EPSP slope were measured 5 and 30 min after high frequency stimulation, which are time points typical for short- and long-term potentiation (STP and LTP), respectively. Three consecutive population EPSP slopes were averaged and then compared to baseline averages using a paired t-test. In all analyses, the significance level was preset to P,0.05.
3. Results In 14 of 16 EC slices, SLEs (discharges longer than 1 min) occurred as the initial epileptiform activity in Mg 21 free ACSF (Fig. 1a). In the remaining two slices (one from a morphine- and one from a saline-exposed rat), short discharges (7–30 s) represented the initial epileptiform activity (Fig. 1b). Both types of initial activity eventually developed into continuous status of short recurrent discharges (Fig. 1c). Initial epileptiform activity (i.e., combined SLEs and short discharges) as well as status-like activity occurred significantly earlier (Fig. 1d; *P,0.05) in the EC slices from morphine-exposed rats (n58) than in saline-exposed controls (n58).
Fig. 1. Effects of prenatal morphine exposure on epileptiform activity induced by Mg 21 -free medium in the EC. DC recording sites in medial EC (MEC) are hatched on a cartoon of ventral hippocampal formation [22]. (a) Typical seizure-like events (SLE; duration 1–7 min); (b) individual short discharges (duration less than 30 s); (c) typical status of recurrent discharges (status-like activity; duration of each 1–5 s); and (d) mean6S.E.M.(min) latency to onset of initial activity ((a) and (b) combined) and to onset of status activity (c) in the EC of slices from saline- (open bars) and morphine-exposed (solid bars) rats. *P,0.05 versus saline; Student’s t-test.
Analysis of the stimulation currents necessary to induce 50% of maximal EPSP slopes did not reveal any statistically significant differences in basal synaptic excitability in either the DG (saline, 167629 mA, n57; morphine, 217639 mA, n57; P.0.20) or field CA1 (saline, 278630 mA, n57; morphine, 307637 mA, n57; P.0.20). In the DG after the mixed perforant path stimulation, mean baseline EPSP slope was 2.1860.32 V/ s for saline-exposed (n57) and 1.7960.23 V/ s for morphine-exposed slices (n57; Student’s t-test; P.0.20). In the DG of slices from morphine-exposed rats, high frequency stimulation of both subdivisions of the perforant path using a sub-maximal tetanus (2350 Hz / 1 s) induced significant long-term depression (LTD) of perforant pathevoked EPSPs (Fig. 2a). This lasted at least 60 min
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Fig. 2. Effects of prenatal morphine exposure on the time course of synaptic potentiation at perforant path-DG and Schaffer collateral-CA1 synapses. (a) Mean (6S.E.M) EPSP slope plotted versus time, illustrating synaptic potentiation induced in the DG after the mixed perforant path sub-maximal stimulation (2350 Hz / 1 s) in slices from saline (open circles)- and morphine (closed circles)-exposed male rats. Inset: mean (6S.E.M) EPSP slopes (V/ s) for baseline, 5 min, and 30 min after high-frequency stimulation in slices from saline (open bars)- and morphine (solid bars)-exposed male rats. *P,0.05 versus baseline. (b) Mean (6S.E.M) synaptic potentiation induced by a sub-maximal tetanus (2350 Hz / 1 s) of Schaffer collaterals in the CA1 hippocampal area in slices from saline (open circles)- and morphine (closed circles)-exposed male rats. Details as in (a).
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(215.362.7% change; P,0.05, versus pre-tetanus baseline; n57). In contrast, the same high-frequency stimulation did not induce any short- or long-term changes in synaptic transmission in the DG slices from salineexposed rats (Fig. 2a; n57). In stratum radiatum of field CA1, mean baseline EPSP slope was similar (P.0.20) in slices from both morphineexposed rats (2.0560.15 V/ s; n57) and saline-exposed ones (2.1560.17 V/ s; n57). High-frequency stimulation induced significant LTP of Schaffer collateral synaptic transmission in slices from saline-exposed rats (11765.9% of baseline; P,0.05; Fig. 2b), while LTP was not observed in slices from morphine-exposed rats (102.768.1% of baseline; P.0.20). In addition, in slices from both salineand morphine-exposed rats, stimulation of Schaffer collaterals with test pulses evoked positive potentials in the non-stimulated DG between 10 and 30 ms after the stimulation. Moreover, after high-frequency stimulation, these positive potentials exhibited apparent potentiation in slices from morphine-exposed rats, but not in the slices from saline-exposed rats (Fig. 3). In contrast, there was no such potential in the non-stimulated CA1 after delivering of stimulus on perforant path.
4. Discussion The present study demonstrates that prenatal morphine exposure alters susceptibility to low Mg 21 -induced epileptiform activity, as well as activity-dependent synaptic plasticity in the limbic system in vitro. There is an increased susceptibility to low Mg 21 -induced epileptiform activity in hippocampal-EC slices of prenatally morphineexposed, adult male rats. The same prenatal morphine exposure impaired the expression of LTP in field CA1, and in the DG it induced a frank LTD in response to a weak tetanic stimulation. Thus, these experiments are initial indications of complex, long-term alterations in both synaptic plasticity and seizure susceptibility following a relatively short (E11–E18) prenatal exposure to morphine. As this initial study did not address underlying mechanisms for either synaptic plasticity or seizure susceptibility changes in prenatally morphine- and saline-exposed rats, we can only speculate about possibilities. While stimulation of Schaffer collateral-CA1 synapses induced significant LTP in slices from saline-exposed rats, LTP was not induced in the DG of control slices. There are several possible reasons: First, a recently communicated preliminary study suggests that both LTP or LTD may occur in the DG, with their occurrence being opioid receptor-dependent [9]. The breakpoint between LTP and LTD is at around 50% of the stimulus intensity that evokes a maximum response. This intensity was used in our study. Second, in these experiments, we stimulated both lateral and medial perforant pathways. It is possible that this mixed perforant path stimulation, combined with low
intensity stimulus contributed to sub-optimal LTP induction in the DG. Additionally, isolation, reversed dark:light cycle, and weaning on PN 25, may all interact with memory formation, and can modify the threshold for induction of LTP [19,20,35]. Nevertheless, although neither LTP nor LTD was observed in the DG in slices of prenatally saline-exposed rats, the same stimulus parameters did evoke significant LTD in the DG of slices from prenatally morphine-exposed rats, and LTP in the CA1 region of control slices. Studies are underway to further investigate this difference in response of the perforant pathway to threshold stimulation following prenatal morphine exposure, and to compare long-term plasticity in medial and lateral perforant pathways. It is possible that the effects of prenatal morphine exposure we observed are due to actions of such drug exposure on a variety of neurotransmitter and neuromodulator systems. Vathy and co-workers [31,33] have previously demonstrated that morphine administration to pregnant rats on gestation days 11–18 induces increases in norepinephrine (NE) content and turnover in the brain of prenatally morphine-exposed adult male offspring [31,33]. Stanton and colleagues have shown that NE can antagonize epileptiform activity in the EC, while simultaneously enhancing stimulus-evoked epileptiform discharges in the DG [27]. Interestingly, both NE depletion and blockade of NE b receptors markedly impaired the expression of LTP at perforant path-DG synapses [6,28,29]. Our results are consistent with the notion that morphine exposure could act by functionally impairing the actions of NE, resulting in a combination of enhanced seizure susceptibility in the EC and an impairment of LTP (and, thus, unmasking of LTD) in the DG and CA1. This seems to contradict our previous observations of an increase in NE content and turnover in prenatally morphine-exposed adult male rats [31,33]. However, in the current study, slices from prenatally morphine-exposed males are an acutely denervated preparation in which the removal of NE from a chronically overexpressing animal might reveal NE receptor downregulation. On the other hand, it is just as possible that prenatal morphine exposure directly modulate the expression of LTP in the DG by altering opioidergic tone [3,24]. Opioids are co-stored with glutamate at several hippocampal synapses, including the lateral perforant path input to DG granule cells and CA3 pyramidal neurons, as well as mossy fiber projections to CA3 [5,7,11,13]. In all these pathways, LTP induction is blocked by naloxone, a nonspecific antagonist of all major m, d, and k opioid receptor subtypes [4,8,18,36]. If exposure to morphine during development has widespread effects on brain opioid receptors, it could alter the regulatory mechanism(s) of opioid receptor-dependent LTP in the lateral perforant path, unmasking LTD in the medial perforant path. These possibilities are currently under investigation. The positive potentials we observed in the DG distal to the Schaffer collateral stimulation are of unknown origin.
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Fig. 3. Field EPSPs recorded 5 min before (left) and 5 min after (right) Schaffer collateral or perforant path high-frequency stimulation (HFS). Solid traces, extracellular responses, EPSP, in monosynaptic target layers; dotted traces, extracellular responses in the opposing, unstimulated area After Schaffer collateral stimulation, the solid trace was recorded in CA1 stratum radiatum, and the dotted trace is the response in stratum moleculare of the DG, and vice versa for perforant path stimulation. Positive potentials (dotted traces) of unknown origin are marked by arrows. (Stimulation artifacts were truncated.)
We can speculate that these positive potentials might be the return currents from distant inhibitory conductances amplified after prenatal morphine exposure. Moreover, the occurrence of these positive potentials in the DG after Schaffer collateral stimulation suggests a crosstalk between DG and CA1, although similar potentials in CA1 were not observed after the perforant path activation. Our results suggest that prenatal exposure to morphine may have complex long-term effects on NMDA receptormediated neurotransmission. While there was an increase
in epileptiform activity induced by low Mg 21 activation of NMDA receptors in the EC, induction of LTP appeared to be reduced in CA1 and the DG. Such effects could underlie memory and learning impairments in children that result from prenatal morphine exposure. While the connection between LTP in rodents and cognition in humans is not straightforward, our data may improve understanding of psychological and behavioral problems observed in children of mothers abusing opiates. It is of outmost importance to conduct long-term studies in animals to
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assess long-term alterations in the CNS of drug-exposed children.
Acknowledgements Supported by grants DA-05833 from NIDA (IV), the Whitehall and Klingenstein Foundations (PKS), and NS20253 from NINDS (SLM). SLM is the recipient of a Martin A. and Emily L. Fisher Fellowship in Neurology and Pediatrics. The procedures for animal experimentation utilized in this report were reviewed and approved by the AICUC.
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Research report
Prenatal morphine exposure enhances seizure susceptibility but suppresses long-term potentiation in the limbic system of adult male rats ´ˇ a,b,e , *, Patric K. Stanton a,b , Solomon L. Moshe´ a,b,c,e , Ilona Vathy b,d Libor Velısek a
Department of Neurology, K 312, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, New York, NY 10461, USA b Department of Neuroscience, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, New York, NY 10461, USA c Department of Pediatrics, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, New York, NY 10461, USA d Department of Psychiatry, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, New York, NY 10461, USA e Einstein /Montefiore Epilepsy Management Center, Bronx, New York, NY 10461, USA Accepted 4 April 2000
Abstract The present study examined the effects of prenatal morphine exposure on NMDA-dependent seizure susceptibility in the entorhinal cortex (EC), and on activity-dependent synaptic plasticity at Schaffer collateral and perforant path synapses in the hippocampus. During perfusion with Mg 21 -free ACSF, an enhancement of epileptiform discharges was found in the EC of slices from prenatally morphine-exposed male rats. A submaximal tetanic stimulation (2350 Hz / 1 s) in control slices elicited LTP at the Schaffer collateral-CA1 synapses, but neither LTP nor LTD was evoked at the perforant path-DG synapses. In slices from prenatally morphine-exposed adult male rats, long-term potentiation of synaptic transmission was not observed at Schaffer collateral-CA1 synapses, while the submaximal tetanus now elicited frank LTD of synaptic EPSPs at perforant path synapses. These data suggest that prenatal morphine exposure enhances the susceptibility of entorhinal cortex to the induction of epileptiform activity, but shifts long-term plasticity of hippocampal synapses in favor of LTD. 2000 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behaviour Topic: Drugs of abuse: opioids and others Keywords: Hippocampus; Entorhinal cortex; Brain slices; Long-term potentiation; Long-term depression; Prenatal morphine exposure; Seizure susceptibility; Synaptic plasticity
1. Introduction Opiates such as morphine, the active component of heroin, are frequently abused during pregnancy [21]. These substances can induce long-term psychological and behavioral alterations in exposed children [2]. Since longterm potentiation (LTP) is considered as a cellular and molecular basis for learning and memory [15] that are involved in behavioral and cognitive processes, it is of great concern to study the effects of prenatal morphine exposure on LTP. *Corresponding author. Tel.: 11-718-430-4277; fax: 11-718-4308899. ´ˇ E-mail address: [email protected] (L. Velısek)
There are reports that chronic morphine exposure, as well as morphine dependence, can interact with the induction of LTP. For example, chronic postnatal administration of morphine augments LTP induced by low-intensity primed-burst stimulation at Schaffer collateral-CA1 synapses in slices, suggesting that chronic morphine exposure may result in chemically induced shift in LTP threshold [17]. This effect of enhanced LTP in chronically morphine-exposed rats may be largely due to acute withdrawal effects seen in these slices since, initially, morphine was omitted in the bath [16]. However, very little is known about the long-term effects of prenatal morphine exposure on hippocampal LTP Additional reports show that morphine administration can interact with N-methyl-D-aspartate (NMDA) receptor-
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mediated neurotransmission which plays a central role in the LTP production. A single low dose of morphine inhibits NMDA-induced biting and seizures [1,37], while a single high dose potentiates NMDA-induced seizures in adult rats [1]. Morphine withdrawal also activates NMDA receptors and induces seizures [14]. Further, low systemic doses of morphine have anticonvulsant effects while high doses are proconvulsant [12]. Similarly, single intracerebral, intracerebroventricular or intrathecal administrations of morphine are proconvulsant [12]. Repeated administration of morphine increases susceptibility to seizures in adult rats, but these effects disappear within 5 days after morphine administration was terminated [23,25]. A sudden morphine withdrawal after repeated administration can also lead to short-term proconvulsant effects [30]. It is interesting that prenatal morphine exposure also alters NMDA receptor-mediated stereotypic behavior that may be regarded as a long-term effect of withdrawal [26]. Thus, the present study was designed to determine whether the exposure to morphine during mid to late gestation permanently alters two NMDA-dependent phenomena: long-term synaptic plasticity in the hippocampal formation and low Mg 21 -induced seizure susceptibility in the entorhinal cortex in adult male offspring.
2. Materials and methods Pregnant rats were randomly assigned to either a morphine-treated experimental or a saline-treated control group. Morphine or 0.9% saline were administered subcutaneously (s.c.) to female rats twice a day (08:00 and 20:00 h) on gestational days 11–18, as described previously [32]. Morphine sulfate was obtained from the National Institute on Drug Abuse (Research Technology Branch, Rockville, MD), and dissolved in 0.9% saline to a final injection volume of 0.1 ml. The dose of the first three morphine injections was 5 mg / kg, and the remaining injections 10 mg / kg [32]. On the first postnatal day (PN 1) pups were sexed, weighed, and tattooed for identification with black India ink on one foot pad. Morphine- and saline-exposed litters were cross-fostered such that each mother raised half of her own and half of the adopted pups of the opposite prenatal treatment. Litters were reduced to a maximum of 10 pups. Pups were weaned, weighed and housed individually on PN 25. All rats were kept on reversed light:dark cycle (14:10 h; lights on at 21:00 h) with access to food and water ad libitum. Only one salineand one morphine-exposed male from each litter were used in either seizure or synaptic plasticity experiments.
2.1. Slice preparation Saline- and morphine-exposed adult male rats were decapitated under deep ether anesthesia, brains removed, and hippocampi plus associated entorhinal cortex (EC)
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were quickly dissected. Horizontal, combined hippocampal-EC slices (400-mm thick) were cut with a vibratome (EMS) in ice-cold artificial cerebrospinal fluid (ACSF), containing (in mM): NaCl 126; KCl 5; NaH 2 PO 4 1.25; MgCl 2 2 or 0; CaCl 2 2; NaHCO 3 26; and glucose 10; pH 7.3–7.4. The slices were placed in an interface-type recording chamber perfused with 33–348C ACSF at 2 ml / min, oxygenated with 95% O 2 / 5% CO 2 . For determining seizure susceptibility, slices from one morphine- and one saline-exposed male rat were placed side by side in the recording chamber to maintain nearly identical experimental conditions. For the same reason, the order of cutting and the position of slices in the recording chamber were randomized. For synaptic plasticity experiments, simultaneous recordings were performed in one slice from either a morphine- or saline-exposed male rat dentate gyrus (DG) and CA1 subfield of the defined slice. After a 1-h incubation, the viability of slices was verified by stimulating stratum radiatum of CA1 subfield with bipolar stainless steel stimulating electrodes (Frederick Haer), and recording field potentials (field responses including population EPSPs and superimposed population spikes) in stratum pyramidale of area CA1, with an extracellular glass recording micropipette (filled with 2 M NaCl, 2–5 MV). Only slices responding to a single stimulus (intensity range 50–500 mA; inducing half-maximal response) with a single population spike at least 2 mV in amplitude and displaying paired-pulse inhibition at 20-ms interstimulus interval were used in the following experiments. Multiple population spikes as a response to single stimulus may indicate hyperexcitability of the slice due to cutting damage, hypoxia, dehydration, etc. Preserved paired-pulse inhibition at 20-ms interval indicated effective feed-forward inhibition. This parameter was used since inhibitory interneurons involved in the feed-forward inhibition are very vulnerable to any deteriorations. No more than two slices were used from any one rat.
2.2. Seizure susceptibility test To determine seizure susceptibility in the EC, a recording micropipette was placed in the deep layers (IV–V) of the medial EC (Fig. 1, top). After a stable DC baseline was recorded for at least 10 min, perfusion was switched to Mg 21 -free ACSF. Mg 21 -free ACSF regularly elicited two distinct types of discharges: (1) seizure-like events (SLE) which are long epileptiform discharges with a negative DC shift (duration 1–7 min each [10]); (2) SLEs progressed spontaneously into a second type of epileptiform activity, continuous status of short recurrent discharges (status-like activity; duration 1–10 s each discharge [10]). Short recurrent discharges continued for as long as Mg 21 -free ACSF was present. In these experiments, recording continued until short recurrent discharges developed in both slices [34]. The latency to onset of SLE, to onset of short recurrent discharges, and the pattern of initial discharges
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were recorded. Group means were compared with twotailed Student’s t-test for unpaired samples. The difference was considered significant at the level of P,0.05.
2.3. Synaptic plasticity test To assess synaptic plasticity at perforant path-DG granule cell and at Schaffer collateral-CA1 pyramidal cell synapses, recording electrodes were placed in the middle molecular layer of the upper blade of the DG and in stratum radiatum of the CA1 field. Test pulses were delivered to the perforant path (mixed perforant path stimulation; stimulating both lateral and medial portion between the outer and middle third of the molecular layer) and Schaffer collateral axons, alternating each 30 s at a stimulus intensity that elicited 50% of the maximal population EPSP slope. Then, high frequency, sub-maximal tetanization (2350 Hz / 1 s; 15-s intertrain interval) was delivered to one stimulation electrode, and 30 s later to the second electrode. As these initial experiments were intended to reveal hypothetical long-term difference in the DG synaptic plasticity between prenatally morphine- and saline-exposed offspring, lateral and medial perforant pathways were not tested separately. The authors are aware that several neuromodulators play differential roles in lateral versus medial perforant pathway (such as norepinephrine and opioids [3,6]) and will be investigated in upcoming studies. In all experiments, stable baseline population EPSPs were recorded for at least 10 min prior to high-frequency stimulation. A weak high-frequency stimulation paradigm was chosen because changes in synaptic plasticity in slices from morphine-exposed rats may be masked by a saturating supramaximal tetanus. Changes in population EPSP slope were measured 5 and 30 min after high frequency stimulation, which are time points typical for short- and long-term potentiation (STP and LTP), respectively. Three consecutive population EPSP slopes were averaged and then compared to baseline averages using a paired t-test. In all analyses, the significance level was preset to P,0.05.
3. Results In 14 of 16 EC slices, SLEs (discharges longer than 1 min) occurred as the initial epileptiform activity in Mg 21 free ACSF (Fig. 1a). In the remaining two slices (one from a morphine- and one from a saline-exposed rat), short discharges (7–30 s) represented the initial epileptiform activity (Fig. 1b). Both types of initial activity eventually developed into continuous status of short recurrent discharges (Fig. 1c). Initial epileptiform activity (i.e., combined SLEs and short discharges) as well as status-like activity occurred significantly earlier (Fig. 1d; *P,0.05) in the EC slices from morphine-exposed rats (n58) than in saline-exposed controls (n58).
Fig. 1. Effects of prenatal morphine exposure on epileptiform activity induced by Mg 21 -free medium in the EC. DC recording sites in medial EC (MEC) are hatched on a cartoon of ventral hippocampal formation [22]. (a) Typical seizure-like events (SLE; duration 1–7 min); (b) individual short discharges (duration less than 30 s); (c) typical status of recurrent discharges (status-like activity; duration of each 1–5 s); and (d) mean6S.E.M.(min) latency to onset of initial activity ((a) and (b) combined) and to onset of status activity (c) in the EC of slices from saline- (open bars) and morphine-exposed (solid bars) rats. *P,0.05 versus saline; Student’s t-test.
Analysis of the stimulation currents necessary to induce 50% of maximal EPSP slopes did not reveal any statistically significant differences in basal synaptic excitability in either the DG (saline, 167629 mA, n57; morphine, 217639 mA, n57; P.0.20) or field CA1 (saline, 278630 mA, n57; morphine, 307637 mA, n57; P.0.20). In the DG after the mixed perforant path stimulation, mean baseline EPSP slope was 2.1860.32 V/ s for saline-exposed (n57) and 1.7960.23 V/ s for morphine-exposed slices (n57; Student’s t-test; P.0.20). In the DG of slices from morphine-exposed rats, high frequency stimulation of both subdivisions of the perforant path using a sub-maximal tetanus (2350 Hz / 1 s) induced significant long-term depression (LTD) of perforant pathevoked EPSPs (Fig. 2a). This lasted at least 60 min
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Fig. 2. Effects of prenatal morphine exposure on the time course of synaptic potentiation at perforant path-DG and Schaffer collateral-CA1 synapses. (a) Mean (6S.E.M) EPSP slope plotted versus time, illustrating synaptic potentiation induced in the DG after the mixed perforant path sub-maximal stimulation (2350 Hz / 1 s) in slices from saline (open circles)- and morphine (closed circles)-exposed male rats. Inset: mean (6S.E.M) EPSP slopes (V/ s) for baseline, 5 min, and 30 min after high-frequency stimulation in slices from saline (open bars)- and morphine (solid bars)-exposed male rats. *P,0.05 versus baseline. (b) Mean (6S.E.M) synaptic potentiation induced by a sub-maximal tetanus (2350 Hz / 1 s) of Schaffer collaterals in the CA1 hippocampal area in slices from saline (open circles)- and morphine (closed circles)-exposed male rats. Details as in (a).
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(215.362.7% change; P,0.05, versus pre-tetanus baseline; n57). In contrast, the same high-frequency stimulation did not induce any short- or long-term changes in synaptic transmission in the DG slices from salineexposed rats (Fig. 2a; n57). In stratum radiatum of field CA1, mean baseline EPSP slope was similar (P.0.20) in slices from both morphineexposed rats (2.0560.15 V/ s; n57) and saline-exposed ones (2.1560.17 V/ s; n57). High-frequency stimulation induced significant LTP of Schaffer collateral synaptic transmission in slices from saline-exposed rats (11765.9% of baseline; P,0.05; Fig. 2b), while LTP was not observed in slices from morphine-exposed rats (102.768.1% of baseline; P.0.20). In addition, in slices from both salineand morphine-exposed rats, stimulation of Schaffer collaterals with test pulses evoked positive potentials in the non-stimulated DG between 10 and 30 ms after the stimulation. Moreover, after high-frequency stimulation, these positive potentials exhibited apparent potentiation in slices from morphine-exposed rats, but not in the slices from saline-exposed rats (Fig. 3). In contrast, there was no such potential in the non-stimulated CA1 after delivering of stimulus on perforant path.
4. Discussion The present study demonstrates that prenatal morphine exposure alters susceptibility to low Mg 21 -induced epileptiform activity, as well as activity-dependent synaptic plasticity in the limbic system in vitro. There is an increased susceptibility to low Mg 21 -induced epileptiform activity in hippocampal-EC slices of prenatally morphineexposed, adult male rats. The same prenatal morphine exposure impaired the expression of LTP in field CA1, and in the DG it induced a frank LTD in response to a weak tetanic stimulation. Thus, these experiments are initial indications of complex, long-term alterations in both synaptic plasticity and seizure susceptibility following a relatively short (E11–E18) prenatal exposure to morphine. As this initial study did not address underlying mechanisms for either synaptic plasticity or seizure susceptibility changes in prenatally morphine- and saline-exposed rats, we can only speculate about possibilities. While stimulation of Schaffer collateral-CA1 synapses induced significant LTP in slices from saline-exposed rats, LTP was not induced in the DG of control slices. There are several possible reasons: First, a recently communicated preliminary study suggests that both LTP or LTD may occur in the DG, with their occurrence being opioid receptor-dependent [9]. The breakpoint between LTP and LTD is at around 50% of the stimulus intensity that evokes a maximum response. This intensity was used in our study. Second, in these experiments, we stimulated both lateral and medial perforant pathways. It is possible that this mixed perforant path stimulation, combined with low
intensity stimulus contributed to sub-optimal LTP induction in the DG. Additionally, isolation, reversed dark:light cycle, and weaning on PN 25, may all interact with memory formation, and can modify the threshold for induction of LTP [19,20,35]. Nevertheless, although neither LTP nor LTD was observed in the DG in slices of prenatally saline-exposed rats, the same stimulus parameters did evoke significant LTD in the DG of slices from prenatally morphine-exposed rats, and LTP in the CA1 region of control slices. Studies are underway to further investigate this difference in response of the perforant pathway to threshold stimulation following prenatal morphine exposure, and to compare long-term plasticity in medial and lateral perforant pathways. It is possible that the effects of prenatal morphine exposure we observed are due to actions of such drug exposure on a variety of neurotransmitter and neuromodulator systems. Vathy and co-workers [31,33] have previously demonstrated that morphine administration to pregnant rats on gestation days 11–18 induces increases in norepinephrine (NE) content and turnover in the brain of prenatally morphine-exposed adult male offspring [31,33]. Stanton and colleagues have shown that NE can antagonize epileptiform activity in the EC, while simultaneously enhancing stimulus-evoked epileptiform discharges in the DG [27]. Interestingly, both NE depletion and blockade of NE b receptors markedly impaired the expression of LTP at perforant path-DG synapses [6,28,29]. Our results are consistent with the notion that morphine exposure could act by functionally impairing the actions of NE, resulting in a combination of enhanced seizure susceptibility in the EC and an impairment of LTP (and, thus, unmasking of LTD) in the DG and CA1. This seems to contradict our previous observations of an increase in NE content and turnover in prenatally morphine-exposed adult male rats [31,33]. However, in the current study, slices from prenatally morphine-exposed males are an acutely denervated preparation in which the removal of NE from a chronically overexpressing animal might reveal NE receptor downregulation. On the other hand, it is just as possible that prenatal morphine exposure directly modulate the expression of LTP in the DG by altering opioidergic tone [3,24]. Opioids are co-stored with glutamate at several hippocampal synapses, including the lateral perforant path input to DG granule cells and CA3 pyramidal neurons, as well as mossy fiber projections to CA3 [5,7,11,13]. In all these pathways, LTP induction is blocked by naloxone, a nonspecific antagonist of all major m, d, and k opioid receptor subtypes [4,8,18,36]. If exposure to morphine during development has widespread effects on brain opioid receptors, it could alter the regulatory mechanism(s) of opioid receptor-dependent LTP in the lateral perforant path, unmasking LTD in the medial perforant path. These possibilities are currently under investigation. The positive potentials we observed in the DG distal to the Schaffer collateral stimulation are of unknown origin.
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Fig. 3. Field EPSPs recorded 5 min before (left) and 5 min after (right) Schaffer collateral or perforant path high-frequency stimulation (HFS). Solid traces, extracellular responses, EPSP, in monosynaptic target layers; dotted traces, extracellular responses in the opposing, unstimulated area After Schaffer collateral stimulation, the solid trace was recorded in CA1 stratum radiatum, and the dotted trace is the response in stratum moleculare of the DG, and vice versa for perforant path stimulation. Positive potentials (dotted traces) of unknown origin are marked by arrows. (Stimulation artifacts were truncated.)
We can speculate that these positive potentials might be the return currents from distant inhibitory conductances amplified after prenatal morphine exposure. Moreover, the occurrence of these positive potentials in the DG after Schaffer collateral stimulation suggests a crosstalk between DG and CA1, although similar potentials in CA1 were not observed after the perforant path activation. Our results suggest that prenatal exposure to morphine may have complex long-term effects on NMDA receptormediated neurotransmission. While there was an increase
in epileptiform activity induced by low Mg 21 activation of NMDA receptors in the EC, induction of LTP appeared to be reduced in CA1 and the DG. Such effects could underlie memory and learning impairments in children that result from prenatal morphine exposure. While the connection between LTP in rodents and cognition in humans is not straightforward, our data may improve understanding of psychological and behavioral problems observed in children of mothers abusing opiates. It is of outmost importance to conduct long-term studies in animals to
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assess long-term alterations in the CNS of drug-exposed children.
Acknowledgements Supported by grants DA-05833 from NIDA (IV), the Whitehall and Klingenstein Foundations (PKS), and NS20253 from NINDS (SLM). SLM is the recipient of a Martin A. and Emily L. Fisher Fellowship in Neurology and Pediatrics. The procedures for animal experimentation utilized in this report were reviewed and approved by the AICUC.
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