Correlation between hypermetabolism and neuronal damage during status epilepticus induced by lithium and pilocarpine in immature and adult rats.
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The correlation between seizure-induced hypermetabolism and subsequent neuronal damage was studied in 10-day-old (P10), 21-day-old (P21), and adult rats subjected to lithium-pilocarpine status epilepticus (SE). Local CMRglc (LCMRglc) values were measured by the [14C]2-deoxyglucose method for a duration of 45 minutes starting at 60 minutes after the onset of SE, and neuronal damage was assessed by cresyl violet staining at 6 days after SE. In P21 and adult rats, LCMRglc values were increased by 275 to 875% in all thalamic, cortical, forebrain, and hypothalamic regions plus the substantia nigra. In addition, at P21 there were also large increases in LCMRglc in brainstem regions. In P10 rats, metabolic increases were mostly located in cortical and forebrain regions plus the substantia nigra but did not affect hypothalamic, thalamic, or brainstem areas. In adult rats, there was an anatomical correlation between hypermetabolism and neuronal damage. At P21, although hypermetabolism occurred in regions with damage, the extent of damage varied considerably with the animals and ranged from an almost negligible to a very extended degree. Finally, in P10 rats, although quite pronounced hypermetabolism occurred, there was no neuronal damage induced by the seizures. Thus, in the present model of epilepsy, the correlation between marked hypermetabolism and neuronal damage can be shown in adult rats. Conversely, immature rats can sustain major metabolic activations that lead either to a variable extent of damage, as seen at P21, or no damage, as recorded at P10. (+info)
The functional topography of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor, revealed by scanning mutagenesis.
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Alanine-scanning mutagenesis has been applied to residues 100-121 in transmembrane domain 3 of the M1 muscarinic acetylcholine receptor. This study complements a previous investigation of the triad Asp122-Arg123-Tyr124 (Lu, Z-L., Curtis, C. A., Jones, P. G., Pavia, J., and Hulme, E. C. (1997) Mol. Pharmacol. 51, 234-241). The results demonstrate the alpha-helical secondary structure of the domain and suggest its orientation with respect to the other transmembrane domains. The C-terminal part of the helix appears to be largely buried within the receptor structure. On its surface, there is a patch of three residues, Val113, Leu116, and Ser120, which may form intramolecular contacts that help to stabilize the inactive ground state of the receptor. Mutagenic disruption of these increased agonist affinity and signaling efficacy. In two cases (L116A and S120A), this led to constitutive activation of the receptor. Parallel to the helix axis and spanning the whole transmembrane region, a distinct strip of residues on one face of transmembrane domain 3 forms intermolecular (acetylcholine-receptor, receptor-G protein) or intrareceptor bonds that contribute to the activated state. The binding of acetylcholine may destabilize the first set of contacts while favoring the formation of the second. (+info)
Latrunculin-A causes mydriasis and cycloplegia in the cynomolgus monkey.
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PURPOSE: To determine the effect of latrunculin (LAT)-A, which binds to G-actin and disassembles actin filaments, on the pupil, accommodation, and isolated ciliary muscle (CM) contraction in monkeys. METHODS: Pupil diameter (vernier calipers) and refraction (coincidence refractometry) were measured every 15 minutes from 0.75 to 3.5 hours after topical LAT-A 42 microg (approximately 10 microM in the anterior chamber [AC]). Refraction was measured every 5 minutes from 0.5 to 1.5 hours after intracameral injection of 10 microl of 50 microM LAT-A (approximately 5 microM in AC), with intramuscular infusion of 1.5 mg/kg pilocarpine HCl (PILO) during the first 15 minutes of measurements. Pupil diameter was measured at 1 and 2 hours, and refraction was measured every 5 minutes from 1 to 2 hours, after intravitreal injection of 20 microl of 1.25 mM LAT-A (approximately 10 microM in vitreous), with intramuscular infusion of 1.5 mg/kg PILO during the first 15 minutes of measurements (all after topical 2.5% phenylephrine), and contractile response of isolated CM strips, obtained <1 hour postmortem and mounted in a perfusion apparatus, to 10 microM PILO +/- LAT-A was measured at various concentrations. RESULTS: Topical LAT-A of 42 microg dilated the pupil without affecting refraction. Intracameral LAT-A of 5 microM inhibited miotic and accommodative responses to intramuscular PILO. Intravitreal LAT-A of 10 microM had no effect on accommodative or miotic responses to intramuscular PILO. LAT-A dose-dependently relaxed the PILO-contracted CM by up to 50% at 3 microM in both the longitudinal and circular vectors. CONCLUSIONS: In monkeys, LAT-A causes mydriasis and cycloplegia, perhaps related to its known ability to disrupt the actin microfilament network and consequently to affect cell contractility and adhesion. Effects of LAT-A on the iris and CM may have significant physiological and clinical implications. (+info)
Pilocarpine toxicity in retinal ganglion cells.
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PURPOSE: Muscarinic agents reduce intraocular pressure by enhancing aqueous outflow, probably by stimulating ciliary muscle contraction. However, pilocarpine is a well characterized neurotoxin and is widely used to generate animal seizure models. It was therefore investigated whether pilocarpine was also toxic to retinal ganglion cells. METHODS: Dissociated whole retinal preparations were prepared from postnatal day 16 to 19 rats. Retinal ganglion cells had been previously back-labeled with a fluorescent tracer. Retinal cells were incubated with pilocarpine, lithium, and inositol derivatives, and viability of the retrogradely labeled retinal ganglion cells was assayed after 24 hours. RESULTS: Pilocarpine was toxic to retinal ganglion cells in a dose-dependent fashion. This toxicity was potentiated by lithium and blocked by epi- and myo-inositol. CONCLUSIONS: Pilocarpine is toxic to retinal ganglion cells in a mixed culture assay. This toxicity appears to depend on the inositol pathway and is similar to its mode of action in other neurons. However, 0.4 mM pilocarpine (the lowest concentration that did not affect ganglion cell survival) is roughly 1000-fold higher than the vitreal concentration and 20-fold higher than the scleral concentration that can be obtained with topical administration of 2% pilocarpine in the rabbit eye. (+info)
Sites of reaction of pilocarpine.
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Analysis of the sites of reaction of a biologically important compound, pilocarpine, a molecule with imidazole and butyrolactone rings connected by a methylene bridge, has been accomplished in a quadrupole ion trap with the aim of characterizing its structure/reactivity relationships. Ion-molecule reactions of pilocarpine with chemical ionizing agents, dimethyl ether (DME), 2-methoxyethanol, and trimethyl borate (TMB), along with collision-activated dissociation elucidated the reaction sites of pilocarpine and made possible the comparison of structural features that affect sites of reaction. Based on MS/MS experiments, methylation occurs on the imidazole ring upon reactions with CH3OCH2+ or (CH3OCH2CH2OH)H+ ions but methylation occurs on the lactone ring for reactions with (CH3O)2B+ ions. Bracketing experiments with two model compounds, alpha-methyl-gamma-butyrolactone and N-methyl imidazole, show the imidazole ring to have a greater gas-phase basicity and methyl cation affinity than the lactone ring. The contrast of methylation by TMB ions on the lactone ring is explained by initial addition of the dimethoxyborinium ion, (CH3O)2B+, on the imidazole ring with subsequent collisional activation promoting an intramolecular transfer of a methyl group to the lactone ring with concurrent loss of CH3OBO. Semiempirical molecular orbital calculations are undertaken to further address the favored reaction sites. (+info)
Effect of pilocarpine on visual acuity and on the dimensions of the cornea and anterior chamber.
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The effect of pilocarpine on visual acuity and on the dimensions of the cornea, anterior chamber, and lens were studied in two groups of subjects. Significant changes in ocular tension, corneal curvature, anterior chamber depth, and lens anterior radius were found in a group of 55 glaucomatous eyes as a result of pilocarpine treatment, but there was no change in corneal thickness. Out of 102 glaucomatous eyes 78 became relatively myopic, and this appears to be due to changes in the dimensions of the lens of the eye similar to those occurring in accommodation, as a result of the effect of the drug on the ciliary muscle. The effect of pilocarpine on anterior chamber depth, area, and volume was studied in 125 eyes using a photographic method, and a significant reduction in the dimensions of the anterior chamber was observed as a result of the administration of pilocarpine. A significant correlation between depth and volume was also noted and the implications of this are discussed. (+info)
Recurrent mossy fiber pathway in rat dentate gyrus: synaptic currents evoked in presence and absence of seizure-induced growth.
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A common feature of temporal lobe epilepsy and of animal models of epilepsy is the growth of hippocampal mossy fibers into the dentate molecular layer, where at least some of them innervate granule cells. Because the mossy fibers are axons of granule cells, the recurrent mossy fiber pathway provides monosynaptic excitatory feedback to these neurons that could facilitate seizure discharge. We used the pilocarpine model of temporal lobe epilepsy to study the synaptic responses evoked by activating this pathway. Whole cell patch-clamp recording demonstrated that antidromic stimulation of the mossy fibers evoked an excitatory postsynaptic current (EPSC) in approximately 74% of granule cells from rats that had survived >10 wk after pilocarpine-induced status epilepticus. Recurrent mossy fiber growth was demonstrated with the Timm stain in all instances. In contrast, antidromic stimulation of the mossy fibers evoked an EPSC in only 5% of granule cells studied 4-6 days after status epilepticus, before recurrent mossy fiber growth became detectable. Notably, antidromic mossy fiber stimulation also evoked an EPSC in many granule cells from control rats. Clusters of mossy fiber-like Timm staining normally were present in the inner third of the dentate molecular layer at the level of the hippocampal formation from which slices were prepared, and several considerations suggested that the recorded EPSCs depended mainly on activation of recurrent mossy fibers rather than associational fibers. In both status epilepticus and control groups, the antidromically evoked EPSC was glutamatergic and involved the activation of both AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors. EPSCs recorded in granule cells from rats with recurrent mossy fiber growth differed in three respects from those recorded in control granule cells: they were much more frequently evoked, a number of them were unusually large, and the NMDA component of the response was generally much more prominent. In contrast to the antidromically evoked EPSC, the EPSC evoked by stimulation of the perforant path appeared to be unaffected by a prior episode of status epilepticus. These results support the hypothesis that recurrent mossy fiber growth and synapse formation increases the excitatory drive to dentate granule cells and thus facilitates repetitive synchronous discharge. Activation of NMDA receptors in the recurrent pathway may contribute to seizure propagation under depolarizing conditions. Mossy fiber-granule cell synapses also are present in normal rats, where they may contribute to repetitive granule cell discharge in regions of the dentate gyrus where their numbers are significant. (+info)
Role of proprioceptive signals from an insect femur-tibia joint in patterning motoneuronal activity of an adjacent leg joint.
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Interjoint reflex function of the insect leg contributes to postural control at rest or to movement control during locomotor movements. In the stick insect (Carausius morosus), we investigated the role that sensory signals from the femoral chordotonal organ (fCO), the transducer of the femur-tibia (FT) joint, play in patterning motoneuronal activity in the adjacent coxa-trochanteral (CT) joint when the joint control networks are in the movement control mode of the active behavioral state. In the active behavioral state, sensory signals from the fCO induced transitions of activity between antagonistic motoneuron pools, i.e., the levator trochanteris and the depressor trochanteris motoneurons. As such, elongation of the fCO, signaling flexion of the FT joint, terminated depressor motoneuron activity and initiated activity in levator motoneurons. Relaxation of the fCO, signaling extension of the FT joint, induced the opposite transition by initiating depressor motoneuron activity and terminating levator motoneuron activity. This interjoint influence of sensory signals from the fCO was independent of the generation of the intrajoint reflex reversal in the FT joint, i.e., the "active reaction," which is released by elongation signals from the fCO. The generation of these transitions in activity of trochanteral motoneurons barely depended on position or velocity signals from the fCO. This contrasts with the situation in the resting behavioral state when interjoint reflex action markedly depends on actual fCO stimulus parameters, i.e., position and velocity signals. In the active behavioral state, movement signals from the fCO obviously trigger or release centrally generated transitions in motoneuron activity, e.g., by affecting central rhythm generating networks driving trochanteral motoneuron pools. This conclusion was tested by stimulating the fCO in "fictive rhythmic" preparations, activated by the muscarinic agonist pilocarpine in the otherwise isolated and deafferented mesothoracic ganglion. In this situation, sensory signals from the fCO did in fact reset and entrain rhythmic activity in trochanteral motoneurons. The results indicate for the first time that when the stick insect locomotor system is active, sensory signals from the proprioceptor of one leg joint, i.e., the fCO, pattern motor activity in an adjacent leg joint, i.e., the CT joint, by affecting the central rhythm generating network driving the motoneurons of the adjacent joint. (+info)