Mu opioid receptor-effector coupling and trafficking in dorsal root ganglia neurons.

Morphine induces profound analgesic tolerance in vivo despite inducing little internalization of the mu opioid receptor (muOR). Previously proposed explanations suggest that this lack of internalization could either lead to prolonged signaling and associated compensatory changes in downstream signaling systems, or that the receptor is unable to recycle and resensitize and so loses efficacy, either mechanism resulting in tolerance. We therefore examined, in cultured neurons, the relationship between muOR internalization and desensitization in response to two agonists, D-Ala2, N-MePhe4, Gly5-ol-enkephalin (DAMGO) and morphine. In addition, we studied the chimeric mu/delta opioid receptor (mu/ partial differentialOR) which could affect internalization and desensitization in neurons. Dorsal root ganglia neurons from muOR knockout mice were transduced with an adenovirus expressing either receptor and their respective internalization, desensitization and trafficking profiles determined. Both receptors desensitized equally, measured by Ca2+ current inhibition, during the first 5 min of agonist exposure to DAMGO or morphine treatment, although the mu/partial differentialOR desensitized more extensively. Such rapid desensitization was unrelated to internalization as DAMGO, but not morphine, internalized both receptors after 20 min. In response to DAMGO the mu/partial differentialOR internalized more rapidly than the muOR and was trafficked through Rab4-positive endosomes and lysosomal-associated membrane protein-1-labeled lysosomes whereas the muOR was trafficked through Rab4 and Rab11-positive endosomes. Chronic desensitization of the Ca2+ current response, after 24 h of morphine or DAMGO incubation, was seen in the DAMGO, but not morphine-treated, muOR-expressing cells. Such persistence of signaling after chronic morphine treatment suggests that compensation of downstream signaling systems, rather than loss of efficacy due to poor receptor recycling, is a more likely mechanism of morphine tolerance in vivo. In contrast to the muOR, the mu/partial differentialOR showed equivalent desensitization whether morphine or DAMGO treated, but internalized further with DAMGO than morphine. Such ligand-independent desensitization could be a result of the observed higher rate of synthesis and degradation of this chimeric receptor.

Functional coupling, desensitization and internalization of virally expressed mu opioid receptors in cultured dorsal root ganglion neurons from mu opioid receptor knockout mice.

Although mu opioid receptors desensitize in various cell lines in vitro, the relationship of this change in signaling efficacy to the development of tolerance in vivo remains uncertain. It is clear that a system is needed in which functional mu opioid receptor expression is obtained in appropriate neurons so that desensitization can be measured, manipulated, and mutated receptors expressed in this environment. We have developed a recombinant system in which expression of a flag-tagged mu opioid receptor is returned to dorsal root ganglia neurons from mu opioid receptor knockout mice in vitro. Flow cytometry analysis showed that adenoviral-mediated expression of the amino-terminal flag-tagged mu opioid receptor in neurons resulted in approximately 1.3x10(6) receptors/cell. Many mu opioid receptor cell lines express a similar density of receptors but this is approximately 7x greater than the number of endogenous receptors expressed by matched wild-type neurons. Inhibition of the high voltage-activated calcium currents in dorsal root ganglia neurons by the mu agonist, D-Ala(2), N-MePhe(4), Gly(5)-ol-enkephalin (DAMGO), was not different between the endogenous and flag-tagged receptor at several concentrations of DAMGO used. Both receptors desensitized equally over the first 6 h of DAMGO pre-incubation, but after 24 h the response of the endogenous receptor to DAMGO had desensitized further than the flag- tagged receptor (71+/-3 vs 29+/-7% respectively; P<0.002), indicating less desensitization in neurons expressing a higher density of receptor. Using flow cytometry to quantify the percentage of receptors remaining on the neuronal cell surface, the flag-tagged receptor internalized by 17+/-1% after 20 min and 55+/-2% after 24 h of DAMGO. These data indicate that this return of function model in neurons recapitulates many of the characteristics of endogenous mu opioid receptor function previously identified in non-neuronal cell lines.

Deficient long-term memory and long-lasting long-term potentiation in mice with a targeted deletion of neurotrophin-4 gene.

We examined the learning and memory of neurotrophin-4 (NT4)-/- mice by using fear conditioning. In both cue and context conditioning, we found significant deficits in the NT4 mutants at 2 and 24 h after training but not at 30 min. Hippocampal slices from the mutant mice showed normal basal synaptic transmission, short-term plasticity, and decremental long-term potentiation (LTP) at the Schaffer collateral-CA1 synapses. These findings, together with the normal short-term memory, suggest that the hippocampal development of NT4-/- mice is largely unaffected. However, consistent with the long-term memory defects, the long-lasting LTP at the same synapses was attenuated significantly in the mutant mice. Our results suggest that NT4 plays a physiological role essential for hippocampus- and amygdala-dependent long-term memory and hippocampal long-lasting LTP and that NT4 may be useful in the therapy of acquired disorders of learning and memory.

Impairment of hippocampal long-term potentiation by Alzheimer amyloid beta-peptides.

Although it is generally believed that amyloid beta (Abeta) peptides contribute to the pathogenesis of Alzheimer's disease, the precise role of these peptides in the development of memory loss of Alzheimer's disease, has not been fully understood. The present study examined the effect of several synthetic Abeta peptides on long-term potentiation (LTP), a cellular model of learning and memory, in rat hippocampal slices. Brief perfusion of slices with low concentrations (200 nM or 1 microM) of Abeta(1-42), Abeta(1-40) or their active fragment Abeta(25--35) significantly inhibited LTP induction without affecting the basal synaptic transmission and posttetanic potentiation in the dentate medial perforant path. A similar effect of Abeta(25-35) was also observed in the Schaffer collateral-CA1 pathway. When comparing actions of several Abeta variants derived from Abeta(25-35), the N-terminal sequence of Abeta(25-35) was found necessary for inhibiting LTP. In addition, Abeta variants lacking neurotoxic action and aggregating property were also able to block LTP, suggesting that this effect was neurotoxicity independent. Our findings demonstrated that subneurotoxic concentrations of Abeta peptides could strongly suppress long-term synaptic plasticity in the hippocampus. Such an effect might underlie the memory deficits seen in Alzheimer's disease before neuronal cell loss.

Orphanin FQ suppresses NMDA receptor-dependent long-term depression and depotentiation in hippocampal dentate gyrus.

We reported previously that orphanin FQ (OFQ) inhibited NMDA receptor-mediated synaptic currents and consequently suppressed induction of long-term potentiation (LTP) in the hippocampal dentate gyrus. This study examines the effect of OFQ on several other forms of long-term synaptic plasticity in the lateral perforant path of mouse hippocampal dentate gyrus. (1) Long-term depression (LTD): a low frequency stimulation (1 Hz, 15 min) applied to the lateral perforant path induced a long-lasting reduction in the dentate field potentials in slices from 22- to 30-day-old mice. This LTD was sensitive to the NMDA receptor blocker D-AP5, and could be significantly attenuated by bath application of OFQ (1 microM, 25 min). (2) Primed LTD: induction of LTD in slices from 50- to 65-day-old mice required a priming procedure consisting of multiple high frequency stimulus trains delivered in the presence of D-AP5 before the low-frequency stimulation. OFQ applied during the low-frequency stimulation, but not during the priming trains, blocked induction of primed LTD. (3) Depotentiation: high-frequency train-induced dentate LTP could be reversed by a subsequent low-frequency stimulation. This depotentiation was also attenuated by either OFQ or D-AP5 applied during low-frequency stimulation. These results, together with our previous findings, suggest that OFQ inhibits bidirectional changes in synaptic strength in the dentate; and its multiple actions on NMDA receptor-dependent, long-term synaptic plasticity might work in tandem to regulate hippocampus-dependent learning and memory.

Orphanin FQ/nociceptin inhibits synaptic transmission and long-term potentiation in rat dentate gyrus through postsynaptic mechanisms.

Orphanin FQ/nociceptin (OFQ), a recently characterized natural ligand for the opioid receptor-like 1 (ORL1) receptor, shares structural similarity to the endogenous opioids. Our previous study found that OFQ, like classical opioids, modulated synaptic transmission and long-term potentiation (LTP) in the hippocampal CA1 region, suggesting a modulatory role for OFQ in synaptic plasticity involved in learning and memory. In the present study we investigated the action of OFQ in the dentate gyrus and explored possible underlying cellular mechanisms. Field potential recordings showed that OFQ significantly inhibited excitatory synaptic transmission and LTP induction in the dentate lateral perforant path. In the presence of OFQ, the excitatory postsynaptic potential (EPSP) slope-population spike (E-S) curve was shifted to the right, and no significant change was found in paired-pulse facilitation, suggesting a postsynaptic mechanism responsible for the inhibition of synaptic transmission. Under whole cell voltage-clamp conditions, bath application of OFQ activated K+ currents in most granule cells tested at a holding potential of -50 mV, suggesting that OFQ could reduce the excitability of dentate granule cells by hyperpolarizing cell membranes. OFQ also inhibited the amplitude of N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs) without affecting alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated EPSCs. This inhibition was not blocked by opioid receptor antagonists. Furthermore, the inward currents evoked by focal application of NMDA to granule cells were suppressed by OFQ in a dose-dependent manner, suggesting that OFQ may suppress LTP by inhibiting the function of postsynaptic NMDA receptors. These results demonstrate that OFQ may negatively modulate synaptic transmission and plasticity in the dentate gyrus through postsynaptic mechanisms, including hyperpolarization of granule cells as well as inhibition of the function of postsynaptic NMDA receptors/channels in dentate granule cells.

Involvement of cAMP-dependent protein kinase in mu-opioid modulation of NMDA-mediated synaptic currents.

We have previously reported dual effects of mu-opioids on N-methyl-D-aspartate (NMDA)-receptor-mediated synaptic events in the hippocampal dentate gyrus: an indirect facilitating effect via suppression of GABAergic interneurons (disinhibition) and a direct inhibitory effect in the presence of gamma-aminobutyric acid-A (GABA(A)) antagonists. The cellular mechanism underlying the inhibitory effect of mu-opioids remains to be determined. In the present study we examine the role of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) in mu-opioid-induced inhibition of NMDA currents in rat hippocampal slices. NMDA-receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) were evoked by stimulating the lateral perforant path and were recorded from dentate granule cells with the use of whole cell voltage-clamp techniques in the presence of the GABA(A) antagonist and a non-NMDA type of glutamate receptor antagonist. Two selective mu-agonists, [N-MePhe3, D-Pro4]-morphiceptin and [D-Ala2, N-MePhe4, Gly-ol5]-enkephalin, induced dose-dependent inhibition of NMDA EPSCs in a concentration range of 0.3-10 microM. This inhibitory effect could be completely reversed by the opioid antagonists naloxone or prevented by a selective mu-antagonist cyprodime, but was not affected by removal of Mg2+ from the external perfusion medium. Intracellular application of pertussis toxin (PTX) into the granule cell via whole cell recording pipettes completely prevented mu-opioid-induced reduction in NMDA currents, suggesting that a postsynaptic mechanism involving PTX-sensitive G proteins might be responsible for the inhibitory action of mu-opioids. Further studies were conducted to identify the intracellular messengers that coupled with G proteins and transduced the effect of mu-opioids in granule cells. The adenylate cyclase activator forskolin was found to enhance NMDA-receptor-mediated synaptic responses and to reverse the inhibitory effect of mu-opioids. Sp-cAMPS, a specific PKA activator, also enhanced NMDA EPSCs, whereas the PKA inhibitor Rp-cAMPS reduced NMDA EPSCs and occluded further inhibition of the current by mu-opioids. These findings strongly suggest that NMDA receptor function is subject to the modulation by PKA, and that mu-opioids can inhibit NMDA currents through suppression of the cAMP cascade in the postsynaptic neuron. Combined with our previous findings, the present results also indicate that mu-opioids can modulate NMDA-receptor-mediated synaptic activity in a complex manner. The net effect of mu-opioids in the dentate gyrus may depend on the interplay between its disinhibitory action, which facilitates NMDA-receptor-mediated responses, and its inhibitory action on the cAMP cascade.

Orphanin FQ inhibits synaptic transmission and long-term potentiation in rat hippocampus.

It is known that opioid peptides acting on opioid receptors can modulate hippocampal synaptic functions. Although a novel member of the opioid receptor family, ORL1 receptors, that displays high-sequence homology with classical opioid receptors is abundant in the hippocampus, little is known regarding its role in synaptic function. The present study was designed to investigate whether activation of the ORL1 receptor by its natural ligand, orphanin FQ, could modulate synaptic transmission and synaptic plasticity in the hippocampus. The actions of orphanin FQ in the CA1 and dentate gyrus were examined by field potential recordings in response to stimulation of Schaffer collaterals and perforant path, respectively. Our results showed that orphanin FQ, but not the inactive analog des-Phe1-orphanin FQ, reduced both the slope of the excitatory postsynaptic potentials and population spike amplitude. The inhibitory effect of orphanin FQ is dose dependent and probably involves a presynaptic mechanism, as suggested by the significantly increased paired-pulse facilitation evoked in the presence of orphanin FQ. In addition, orphanin FQ was found to inhibit the induction of long-term potentiation at the Schaffer collateral-CA1 synapse. These results demonstrate that orphanin FQ can function as an inhibitory modulator regulating synaptic transmission and synaptic plasticity in the hippocampus, suggesting that activation of ORL1 receptors may play an important role in synaptic plasticity involved in learning and memory.

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

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 GABAA inhibition. As one test of this hypothesis, we determined whether blockade of GABAA 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 GABAA antagonist, bicuculline methiodide. Furthermore, although blockade of mu receptors suppressed LTP of the slope of the population excitatory potential (pEPSP), blockade of both mu and delta opioid receptors was needed to suppress LTP of both the pEPSP and the orthodromic population spike (OPS).

Endogenous opioids regulate long-term potentiation of synaptic inhibition in the dentate gyrus of rat hippocampus.

Long-term potentiation (LTP) of excitatory transmission in the hippocampus has been extensively studied as a synaptic model of learning and memory. Here we report a new form of LTP in which inhibitory synaptic signals are potentiated following tetanic stimulation of an opioid-containing excitatory pathway in the presence of opioid antagonists. The lateral perforant path (LPP) was stimulated at the dentate outer molecular layer of hippocampal slices. Evoked synaptic currents were recorded from dentate granule cells using whole-cell voltage-clamp techniques. A high-frequency stimulus train (100 Hz, 1 sec) delivered to the LPP in the presence of naloxone (1 microM) was found to induce a long-lasting potentiation (20 min to 2 hr) in the amplitude of gamma-aminobutyric acidA (GABAA) receptor-mediated inhibitory postsynaptic currents (IPSCs) of granule cells. Such a potentiation was not observed when tetanizing the LPP in control medium. Naloxone-revealed LTP of LPP-evoked IPSCs did not depend upon the presence of granule cell discharge, and was not accompanied by potentiation of mossy fiber-evoked IPSCs, indicating that feedforward, but not feedback, inhibitory circuits were involved. Induction of this LTP could be completely blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonopentanoic acid (D-APV). However, it was not significantly affected by hyperpolarization of granule cells. These results suggest that LTP may occur at the excitatory synapses between LPP terminals and GABAergic interneurons, rather than at the inhibitory synapses between interneurons and granule cells. Further examination using selective opioid antagonists demonstrated that blocking delta, but not mu and kappa, receptors is critical for inducing LTP of IPSCs in granule cells.

GABAB autoreceptors mediate activity-dependent disinhibition and enhance signal transmission in the dentate gyrus.

1. Activity-dependent depression (fading) of polysynaptic inhibition and the effects of this disinhibition on signal transmission were studied in the dentate gyrus of the rat hippocampal slice with the use of intracellular and extracellular recordings. 2. Polysynaptic inhibitory postsynaptic potentials/currents (IPSP/Cs) were evoked in dentate granule cells by stimulation of mossy fibers in stratum lucidum of area CA3b/c. These mossy fiber-evoked IPSP/Cs consisted of an early GABAA receptor-mediated component (IPSP/CA) and a late GABAB receptor-mediated component (IPSP/CB). 3. When paired stimuli were delivered 200 ms apart under voltage clamp, the amplitude of the IPSCA and IPSCB evoked by the second stimulus was reduced by 37.0 +/- 4.0 and 61.6 +/- 7.8% (mean +/- SE), respectively. Paired-pulse depression of both IPSCA and IPSCB was greatest at interstimulus intervals of 100-400 ms with a maximal effect when stimuli were delivered 200 ms apart. 4. (+/-) Baclofen, a GABAB receptor agonist, suppressed both components of the mossy fiber-evoked IPSP in a concentration-dependent fashion. At a concentration that only partially suppressed the initial IPSP, baclofen occluded paired-pulse depression of IPSPA. In addition, paired-pulse depression of IPSPA was blocked in a concentration-dependent fashion by 2-hydroxy-saclofen (10-400 microM), a GABAB receptor antagonist. 5. The contribution of the IPSPB conductance increase to paired-pulse depression of IPSPA was evaluated. Paired-pulse depression of IPSPA was significantly greater than was the depression of the response to a current pulse delivered 200 ms after the mossy fiber stimulus. In addition, injection of granule cells with GTP gamma S, a nonhydrolyzable guanosine triphosphate (GTP) analogue, occluded both IPSPB as well as the effects of baclofen on the granule cell membrane by activating G proteins but did not reduce paired-pulse depression of IPSPA or suppression of IPSPA by baclofen. Finally, examination of the first and second IPSCA evoked by paired stimuli 200 ms apart revealed no significant differences in response kinetics. Taken together, these results indicate that postsynaptic GABAB receptors on the granule cells are not responsible for paired-pulse depression of IPSPA. 6. Monosynaptic IPSPs were evoked by direct stimulation of inhibitory neurons in the inner molecular layer of the dentate gyrus during pharmacological blockade of excitatory transmission with D(-)-2-amino-5-phosphonovaleric acid (D-APV), an N-methyl-D-aspartate (NMDA) receptor antagonist and 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA glutamate receptor antagonist.(ABSTRACT TRUNCATED AT 400 WORDS)

Mu opioid receptor-mediated modulation of synaptic currents in dentate granule cells of rat hippocampus.

1. The effect of a selective mu opioid agonist, [N-MePhe3-D-Pro4]morphiceptin (PL017), on synaptic transmission in the dentate gyrus was examined in hippocampal slices. Synaptic currents were evoked by stimulation of the outer molecular layer and recorded from granule cells using whole-cell voltage-clamp techniques. 2. Monosynaptic inhibitory postsynaptic currents (IPSCs) were evoked in the presence of D(-)-2-amino-5-phosphonovaleric acid (D-APV), and N-methyl-D-aspartate (NMDA) receptor antagonist, and 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA type of glutamate receptor antagonist. The IPSCs consisted of a gamma-aminobutyric acid (GABA)A receptor-mediated early component and a GABAB receptor-mediated late component. 3. Bath application of PL017 (0.3-3 microM) induced a dose-dependent reduction in the amplitude of both early IPSCs (21-56%) and late IPSCs (43-81%). These effects could be reversed by the opiate antagonist naloxone (1 microM) or prevented by the selective mu antagonist beta-funaltrexamine hydrochloride (10 microM). 4. NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) were revealed in the presence of DNQX and the GABAA antagonist bicuculline methiodide. PL017 (3 microM) caused a 35% reduction in the amplitude of NMDA EPSCs. NMDA receptor-mediated population EPSPs recorded extracellularly were also inhibited by 3 microM PL017 to a similar degree. 5. Non-NMDA receptor-mediated EPSCs were demonstrated in the presence of D-APV and bicuculline methiodide. The amplitude of non-NMDA EPSCs was not affected by PL017.(ABSTRACT TRUNCATED AT 250 WORDS)

A glutamate antagonist blocks perforant path stimulation-induced reduction of dynorphin peptide and prodynorphin mRNA levels in rat hippocampus.

Stimulation of the perforant path elicits a behavioral response, wet dog shakes (WDS), and reduction in hippocampal dynorphin A(1-8) immunoreactivity (DYN-IR) and prodynorphin mRNA (DYN mRNA) in rats. This study examined whether glutamate, the proposed endogenous transmitter released by perforant fibers, mediated the above responses. A glutamate antagonist, gamma-D-glutamylglycine (DGG, 25 micrograms/0.5 microliters), or artificial cerebrospinal fluid (ACSF, 0.5 microliters) was injected into the ventral hippocampus 10-20 min prior to acute or daily stimulation of the left perforant path in rats. In acute stimulation experiments, 4 consecutive stimulation trials elicited a total of 73 +/- 4 WDS at an average threshold intensity of 0.46 +/- 0.03 mA in ACSF-treated rats. The hippocampal DYN-IR in these animals decreased by more than 40% in both dorsal and ventral hippocampus relative to sham-stimulated rats. DGG injections significantly elevated the threshold for WDS (0.78 +/- 0.05 mA, P less than 0.01), reduced the number of WDS (45 +/- 6, P less than 0.01), and partially antagonized stimulation-induced reduction of DYN-IR in the ventral, but not dorsal, hippocampus. In daily stimulation experiments, rats received a single trial of stimulation once per day for 6 days. Daily DGG pretreatment almost completely abolished WDS at control threshold intensities, and significantly inhibited stimulation-induced decrease of DYN-IR in both dorsal and ventral hippocampus. In situ hybridization using a 35S-labeled oligodeoxyribonucleotide probe demonstrated a clear depletion of DYN mRNA signal in the dentate granule cell layer of ACSF-treated animals. This depletion was completely prevented in DGG-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Opioid-mediated facilitation of long-term potentiation at the lateral perforant path-dentate granule cell synapse.

Opioid effects on the development of long-term potentiation (LTP) were investigated at the lateral perforant path (LPP)-dentate granule cell synapse of the hippocampal slice. High frequency stimuli were delivered to the outer molecular layer of the dentate to tetanize the LPP. Significant LTP was induced in the amplitude of the orthodromic population spike and the slope of the population excitatory postsynaptic potential recorded from the granule cell layer and molecular layer. Bath application of naloxone (0.1-10 microM), an opioid antagonist, induced a dose-dependent reduction in the potentiation of both orthodromic population spike and excitatory postsynaptic potential evoked in the LPP, but not in adjacent medial perforant path. PLO17 ([N-MePhe3-D-Pro4]morphiception; 0.3 or 1 microM), a mu opioid agonist, reduced the threshold for LTP and increased the amount of LTP in the LPP. PLO17 also reduced recurrent inhibition and enhanced an N-methyl-D-aspartate (NMDA) receptor-mediated component in single pulse-evoked field potentials in the LPP. The effects of PLO17 were antagonized by 1 microM naloxone and the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (100 microM). These findings suggest that endogenous opioids released during high frequency stimulation play an important role in the induction of LTP at the LPP synapses. A mu receptor-mediated disinhibition which increases current flow through NMDA channels may contribute to the opioid enhancement of LTP in the dentate gyrus.

Opioid-induced epileptiform bursting in hippocampal slices: higher susceptibility in ventral than dorsal hippocampus.

The disparity between the seizure sensitivity of the dorsal and ventral hippocampus to opioid peptides was studied by an in vitro electrophysiological method. Slices taken from the ventral (temporal) and dorsal (septal) regions of rat hippocampi were perfused in artificial cerebrospinal fluid bubbled continuously with 95% O2-5% CO2 at 34 degrees C. A stimulating electrode was placed in the stratum radiatum of CA3 region and electrical activity was recorded from the pyramidal cell body layer of the CA3b region. Paired dorsal and ventral hippocampal slices were perfused with [N-Me-Phe3-D-Pro4]morphiceptin (PL017), a specific mu opioid receptor agonist. Application of 0.05 microM PL017 produced triggered and spontaneous bursting in 20% of ventral hippocampal slices, but no such effect was observed in dorsal hippocampal slices. At 0.5 microM PL017, 80% of ventral hippocampal slices developed spontaneous bursting, whereas only 10% of dorsal hippocampal slices had spontaneous bursting. Slices from the ventral hippocampus consistently produced greater degrees of bursting at lower doses relative to the dorsal hippocampus. The addition of 0.1 microM naloxone before or after PL017 inhibited the triggered response but could not block the spontaneous bursting. Perfusion of ACSF for 1 hr also eliminated the triggered response but could only reduce the frequency of the spontaneous bursting. These results suggest that the ventral hippocampus has a higher susceptibility to PL017-induced epileptiform bursting, and this effect is mediated, at least in part, through mu opioid receptors.

Perforant path stimulation differentially alters prodynorphin mRNA and proenkephalin mRNA levels in the entorhinal cortex-hippocampal region.

The regulatory effect of the perforant path on opioid gene expression in the entorhinal cortex-hippocampal region was investigated. The left perforant path was electrically stimulated at the angular bundle under conditions which elicit wet dog shakes but no motor seizures in rats. Animals were given either an acute stimulation composed of several consecutive stimulation trials, or daily stimulations with a single trial every day for 6 days. Rats were then sacrificed at 24 h or 6 days after the last trial. The amounts of prodynorphin mRNA (DYN mRNA) and proenkephalin A mRNA (EK mRNA) in the hippocampus and entorhinal cortex were measured by RNA blot analysis. Dynorphin A(1-8) and [Met5]enkephalin immunoreactivities were determined by radioimmunoassay. A decrease in DYN mRNA level of approximately 50-80% was found on both sides of the hippocampus 24 h after both acute and daily stimulation. Hippocampal dynorphin A(1-8) immunoreactivity was also reduced at 24 h, and persisted for at least 6 days. In contrast, bilateral increases in EK mRNA level were observed in the hippocampus (54-101%) and entorhinal cortex (97-165%) 24 h after the acute stimulation. Also, [Met5]enkephalin immunoreactivity in the hippocampus tended to be increased at this time. These results indicate that activation of the perforant path inhibits the gene expression of prodynorphin, but enhances that of proenkephalin in the entorhinal cortex-hippocampal region.

Changes of proenkephalin and prodynorphin mRNAs and related peptides in rat brain during the development of deep prepyriform cortex kindling.

The effects of deep prepyriform cortex (DPC) kindling on the amount of proenkephalin and prodynorphin mRNAs, Met5-enkephalin (ME) and dynorphin (DYN) in rat brain were examined. Animals received electrical stimulation of the DPC until two consecutive stage 2 seizures (S2) or stage 5 seizures (S5) were attained. The proenkephalin mRNA and ME contents in the entorhinal cortex were increased 24 h after S2 and also 5 min and 24 h post S5. In the hippocampus, the proenkephalin mRNA level was reduced 24 h after S2 but increased 5 min and 24 h after S5. Elevated hippocampal ME concentration was observed 24 h after S2 and S5. Similarly, the ME level in the frontal cortex was increased 24 h after S2 and S5 but the proenkephalin mRNA content was only elevated at S5. In the striatum, the proenkephalin mRNA level was slightly increased 24 h after S2 and S5, but no change in ME content was found. The amount of prodynorphin mRNA in the hippocampus was attenuated only at 24 h after S5, whereas DYN concentration was reduced 5 min after S5. No change in striatal DYN concentration was observed despite a slight elevation of prodynorphin mRNA 24 h post S2 and S5. Six weeks after the last seizure, no difference in ME and DYN was found between kindled and control animals. These findings indicate that the enkephalin-containing perforant pathway in the entorhinal cortex-hippocampal region is particularly sensitive to electrical stimulations applied to the DPC. Its role and importance in the development of kindling are discussed.

Deep prepyriform cortex kindling differentially alters the levels of prodynorphin mRNA in rat hippocampus and striatum.

The effect of deep prepyriform cortex (DPC) kindling on the levels of prodynorphin mRNA (DYN mRNA) in rat hippocampus and striatum was examined under two different stimulation paradigms. Electrical stimulations were delivered to rats twice per day (slow kindling) or once every hour (fast kindling) until two consecutive stage 5 kindled seizures occurred. Animals were decapitated 24 h after reaching the second stage 5 seizure, and DYN mRNA levels in the brain were determined by RNA blot analysis. In the slow kindling model, the level of DYN mRNA in the hippocampus was reduced by 57%, whereas the level of striatal DYN mRNA was increased by 34% compared to sham-operated controls. Fast kindling induced a similar decrease in the DYN mRNA level in the hippocampus, but did not alter that in the striatum. These results, together with the previous report that kindling decreased dynorphin A(1-8) level in the hippocampus, suggest that electrical kindling decreases the biosynthesis of dynorphin peptides in the hippocampus and, in the slow DPC kindling model, also increases the gene expression of dynorphin in the striatum.

Single or repeated electroconvulsive shocks alter the levels of prodynorphin and proenkephalin mRNAs in rat brain.

The effects of single or repeated electroconvulsive shocks (ECS) on the abundance of mRNAs coding for prodynorphin (DYN mRNA) and proenkephalin (EK mRNA) in rat brain were investigated. Rats were given either a single ECS and sacrificed at 0.5, 2, 6, or 12 h post-shock (expt. I), or were subjected to ECS for 1, 3 or 6 days and killed 24 h after the last shock (expt. II). The amounts of DYN mRNA and EK mRNA were measured in several brain regions using RNA blot analysis. In expt. I, a biphasic change in the DYN mRNA level was found in the hippocampus, with an initial 49% decrease at 0.5 h followed by a 51% increase at 6 h after a single ECS. The EK mRNA content in the entorhinal cortex started to increase at 0.5 h, and continued to rise, reaching 260% of the control level at 12 h post-shock. A smaller increase in the EK mRNA level was found also in the hippocampus at 2 to 12 h post-shock. No significant increase in [Met5]-enkephalin or dynorphin A(1-8) immunoreactivity was detected by radioimmunoassay in either area. In expt. II, a dramatic reduction in the DYN mRNA level was observed in the hippocampus 24 h after a single or repeated ECS. In contrast, elevated DYN mRNA levels were seen in the striatum and hypothalamus. The EK mRNA level remained elevated in the entorhinal cortex after 6 daily ECS.(ABSTRACT TRUNCATED AT 250 WORDS)