enkephalin--ala(2)-mephe(4)-gly(5)- and Epilepsy

A series of thirty N-(phenoxy)alkyl or N-{2-[2-(phenoxy)ethoxy]ethyl}aminoalkanols has been designed, synthesized and evaluated for anticonvulsant activity in MES, 6Hz test, and pilocarpine-induced status epilepticus. Among the title compounds, the most promising seems R-(-)-2N-{2-[2-(2,6-dimethylphenoxy)ethoxy]ethyl}aminopropan-1-ol hydrochloride (22a) with proved absolute configuration with X-ray analysis and enantiomeric purity. The compound is effective in MES test with ED50=12.92 mg/kg b.w. and its rotarod TD50=33.26 mg/kg b.w. The activity dose is also effective in a neurogenic pain model-the formalin test. Within high throughput profile assay, among eighty one targets, the strongest affinity of the compound is observed towards σ receptors and 5-HT transporter and the compound does not bind to hERG. It also does not exhibit mutagenic properties in the Vibrio harveyi test. Moreover, murine liver microsomal assay and pharmacokinetics profile (mice, iv, p.o., ip) indicate that the liver is the primary site of biotransformation of the compound, suggesting that both 22a and its metabolite(s) are active, compensating probably low bioavailability of the parent molecule.

Topics: Amino Alcohols; Animals; Anticonvulsants; Chemistry, Physical; Dose-Response Relationship, Drug; Drug Design; Epilepsy; Male; Mice; Microsomes, Liver; Molecular Structure; Pilocarpine

The abundance of synaptic connectivity in the cultured hippocampal slice preparation allows measurements of the unitary excitatory connection between pairs of pyramidal neurons using simultaneous presynaptic and postsynaptic intracellular recordings. However, the useful yield of these recordings can be greatly reduced by the presence of polysynaptic inhibition that occludes the measurement of the monosynaptic excitatory postsynaptic current (EPSC). We have found that the traditional method of eliminating contaminating synaptic inhibition with GABA receptor antagonists is of limited usefulness because the recurrent excitatory connections in organotypic slices cause epileptiform bursting in the absence of inhibitory function. This bursting obscures EPSCs to an even greater extent than the normally occurring polysynaptic inhibitory transmission. Here, we report a new method for isolating monosynaptic EPSCs using the mu-opioid agonist peptide DAMGO to reduce polysynaptic inhibition during these recordings. Activation of mu-opioid receptors is known to hyperpolarize inhibitory neurons. We found that DAMGO application reduces the amplitude and frequency of polysynaptic inhibition, allowing isolation of the excitatory connection between the two neurons being recorded. Furthermore, because inhibitory function is not completely eliminated by DAMGO application, epileptiform bursting very rarely develops. Therefore, the use of DAMGO to prevent polysynaptic inhibition without causing epileptiform bursting provides a useful tool to substantially increase the yield of experiments measuring the unitary excitatory connection between pyramidal neurons in the cultured hippocampal slice preparation.

Topics: Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Epilepsy; Excitatory Postsynaptic Potentials; GABA Antagonists; Hippocampus; Neural Inhibition; Neuronal Plasticity; Organ Culture Techniques; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptors, GABA; Receptors, Opioid; Synaptic Transmission

Mu-receptor binding changes were evaluated following the kainic acid (KA)-induced status epilepticus (SE) in young, adult, and elderly animals. Male Wistar rats were used as follows: young rats (15 days old) were treated with KA (7 mg/kg) and sacrificed 72 h (YKA3d) or 35 days (YKA35d) after SE; adult (90 days old) (AKA1d and AKA40d) and elderly rats (1-year-old) (EKA1d and EKA40d) were injected with KA (10 mg/kg) and then sacrificed 24 h or 40 days following SE. Their brains were processed for an autoradiography assay for mu-receptors. The YKA3d group showed increased values in dentate gyrus (39%) and a decrease in substantia nigra (26%); YKA35d animals had a reduction in caudate putamen (29%) and in substantia nigra (20%). The AKA1d group exhibited increased mu-receptors in caudate putamen (49%), cingulate (415%), frontal (52%), and temporal (53%) cortices: substantia nigra (56%), dentate gyrus (48%). and CA2 field of hippocampus (53%). The AKA40d group showed increased values in sensorimotor cortex (45%), anterior (39%), medial (65%), basolateral (202%), and central (32%) amygdaloid nuclei; dentate gyrus (80%) as well as CA2 (80%) and CA3 (49%) fields of hippocampus. The EKA1d group presented decreased mu-receptor binding in piriform (16%) and enthorinal (22%) cortices as well as in anterior amygdala nucleus (17%). The EKA40d group showed reduced values in sensorimotor cortex (14%) and substantia nigra (27%). The present results indicate that the mu-binding changes following SE depend on the rate of brain maturation.

Topics: Aging; Animals; Animals, Newborn; Binding Sites; Brain; Convulsants; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Epilepsy; Kainic Acid; Male; Neurons; Opioid Peptides; Radioligand Assay; Rats; Rats, Wistar; Receptors, Opioid, mu; Status Epilepticus; Up-Regulation

Application of 4-aminopyridine (4AP, 50 microM) to combined slices of adult rat hippocampus-entorhinal cortex-induced ictal and interictal epileptiform discharges, as well as slow field potentials that were abolished by the mu-opioid agonist [D-Ala2,N-Me-Phe4,Gly-ol5] enkephalin (DAGO, 10 microM) or the GABAA receptor antagonist bicuculline methiodide (BMI, 10 microM); hence, they represented synchronous GABA-mediated potentials. Ictal discharges originated in the entorhinal cortex and propagated to the hippocampus, whereas interictal activity of CA3 origin was usually recorded in the hippocampus. The GABA-mediated potentials had no fixed site of origin or modality of propagation; they closely preceded (0.2-5 sec) and thus appeared to initiate ictal discharges. Only ictal discharges were blocked by the antagonist of the NMDA receptor 3,3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP, 10 microM), whereas the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) abolished all epileptiform activities. The GABA-mediated potentials continued to occur synchronously in all regions even after concomitant application of CNQX and CPP. [K+]o elevations were recorded in the entorhinal cortex during the ictal discharge (peak values = 13.9 +/- 0.9 mM) and the synchronous GABA-mediated potentials (peak values = 4.2 +/- 0.1 mM); the latter increases were presumably attributable to postsynaptic GABAa-receptor activation because they were abolished by DAGO or BMI. Their role in initiating ictal activity was demonstrated by using DAGO, which abolished both GABA-mediated synchronous potentials and ictal discharges. These data indicate that NMDA-mediated ictal discharges induced by 4AP originate in the entorhinal cortex; such a conclusion is in line with clinical evidence obtained in temporal lobe epilepsy patients. 4AP also induces GABA-mediated potentials that spread within the limbic system when excitatory transmission is blocked and may play a role in initiating ictal discharge by increasing [K+]o.

Topics: 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Analgesics; Animals; Disease Models, Animal; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Entorhinal Cortex; Epilepsy; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Hippocampus; Limbic System; Male; Membrane Potentials; N-Methylaspartate; Nerve Fibers; Piperazines; Potassium; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Serotonin Receptor Agonists

1. The effects of enkephalins and enkephalinase inhibitors were studied in CA1 area in rat hippocampal slices. 2. The data demonstrate a prevalent involvement of mu opiate receptors in the epileptogenic properties of enkephalins. 3. A potentiation of the mu opiate receptor-mediated epileptogenic response by enkephalinase inhibitors has been shown. 4. The results also show an inability to affect basal CA1 field potentials by inhibition of endogenous endopeptidase.

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Methionine; Enkephalins; Epilepsy; Hippocampus; In Vitro Techniques; Male; Morphine; Neprilysin; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, mu

Cortical electroencephalographic (EEG) recordings were performed on rats after i.v. administration of morphine and specific mu- and delta-opioid peptides. DAGO (Tyr-D X Ala-Gly-N X Me X Phe-Gly-ol), the mu-selective peptide, produced repetitive paroxysmal discharges organized in a pattern analogous to that seen in tonic clonic seizures at doses which produced analgesia while DTLET (Tyr-D X Thr-Gly-Phe-Leu-Thr), the delta-selective peptide, produced 'petit-mal'-like seizures at doses which caused neither analgesia nor catatonia. It is suggested that the delta receptor is preferentially implicated in the epileptogenic spectrum of opioids.

Topics: Animals; Electroencephalography; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Epilepsy; Male; Morphine; Oligopeptides; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu