Changes in benzodiazepine receptors in the limbic system following kindling of the olfactory bulb in rats

Repeated electrical stimulations of the olfactory bulb led to the progressive development of a generalized epilepsy (kindling effect). One week after the last stimulation eliciting a stage 5 seizure, diazepam-(3H) binding was studied in olfactory bulb-kindled rats. Numbers of benzodiazepine receptors were increased in kindled olfactory bulb and amygdala. No significant change was observed in hippocampus. This modification could be considered as a response of the inhibitory mechanisms to repeated seizures which is insufficient to counteract the installation of the kindling effect.     

Decreased benzodiazepine receptor binding in amygdala-kindled rat brains

3H-Flunitrazepam (3H-FLU) binding was measured in multiple brain regions of amygdala-kindled rats two weeks following the sixth Stage 5 convulsion. As compared to 'yoked' controls, the kindled animals displayed significant reductions in 3H-FLU binding in the ipsilateral cortex (20%) and in the hypothalamus (20%). Scatchard plots revealed that these reductions were due to changes in the maximal number of available binding sites (Bmax) rather than to alterations in receptor affinity (KD). No significant changes were found in the contralateral cortex, or in either the contralateral or ipsilateral amygdala, hippocampus or striatum. These data suggest that kindling is associated with long-lasting changes in the benzodiazepine receptor system and possibly with related changes in GABA-mediated neural inhibition.     

Modification of γ-Aminobutyric AcidA Receptor Binding and Function by N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline In Vitro and In Vivo: Effects of Aging

The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the gamma-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10(-4) M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 microM, or the agonist lorazepam, 10 microM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10(-4) M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations of Benzodiazepine Receptor Binding in Tremor Rats with Absence-Like Seizures

Tremor rats begin to exhibit clinical or electrical absence-like seizures after 6 weeks of age, and by 14 weeks of age, all have seizures. Central-type benzodiazepine receptor binding was investigated in tremor rats and control rats, aged 4 weeks and 16 weeks. Significantly lower benzodiazepine receptor density and no differences in affinity were found in the hippocampus of the tremor rats in comparison with that of control rats at both ages. This abnormality is considered to be due to a tremor gene and may be the cause of absence-like seizures in tremor rats. A significantly lower receptor density was found in the cerebellum at 4 weeks of age in the tremor rats than in the control rats. These changes may be related to tremorous movements in the tremor rats. Receptor density was significantly lower in the brainstems of tremor rats and control rats at 16 weeks of age than at 4 weeks of age, and the decrease was more marked in control rats. These facts may reflect a reduced decrease in the response to the dysfunction of gamma-aminobutyric acidergic neurons, or the function of the gamma-aminobutyric acid/benzodiazepine receptor system may be secondarily increased to suppress seizures in 16-week-old tremor rats.     

Lasting Increase in Serotonin 5-HT1A but Not 5-HT4 Receptor Subtypes in the Kindled Rat Dentate Gyrus: Dissociation from Local Presynaptic Effects

Abstract: We examined the effect of kindling on serotonergic neurotransmission in the hippocampus by measuring serotonin (5-HT) release and uptake in hippocampal synaptosomes and 5-HT_1A and 5-HT₄ receptor subtypes during and at different times after electrical kindling of the dentate gyrus. Using quantitative receptor autoradiography, we found that binding of 8-[³H]hydroxy-2-(di- n -propylamino)tetralin ([³H]8-OH-DPAT) to 5-HT_1A receptors was selectively increased by 20% on average ( p < 0.05) in the dentate gyrus of the stimulated and contralateral hippocampus 2 days after stage 2 (stereotypes and occasional retraction of a forelimb) and by 100% on average ( p < 0.05) 1 week after stage 5 (tonic-clonic seizures) compared with sham-stimulated rats. A 20% increase ( p < 0.05) was observed 1 month after the last generalized seizure. No changes were found after a single afterdischarge. 5-HT₄ receptors, which colocalize with 5-HT_1A receptors on hippocampal neurons, were not modified in kindled tissue. [³H]5-HT uptake and its release as well as the 5-HT_1B autoreceptor function did not differ from shams in hippocampal synaptosomes at stages 2 and 5. Systemic administration of 100 and 1,000 µg kg⁻¹ 8-OH-DPAT or 1,000 µg kg⁻¹ WAY-100,635, 30 min before each electrical stimulation, did not significantly alter kindling progression or the occurrence of stage 5 seizures in fully kindled rats. The changes in 5-HT_1A receptor density in the dentate gyrus are part of the plastic modifications occurring during kindling and may contribute to modulating tissue hyperexcitability.     

Amiodarone reduces the effect of T3 on beta adrenergic receptor density in rat heart

The effect of injection of 1 mg/kg triiodothyronine on cardiac beta-adrenoceptor state was investigated in hypothyroid rats and compared to the effect in hypothyroid rats pretreated with amiodarone (200 mg/kg/day for 8 days). The Kd values of iodocyanopindolol binding to the beta-receptors were not influenced by either T3 injection or by amiodarone treatment. In the absence of amiodarone, injection of triiodothyronine resulted in a small decrease in receptor density at 6 hr, followed by an increase at 24 hr. Rats treated with amiodarone showed a similar response pattern to hormone injection (i. e. a small decrease in receptor density at 6 hr, followed by an increase at 24 hr), but the amplitude of the response was significantly reduced. Moreover, in vehicle injected rats amiodarone treatment resulted in a decrease in receptor density when rats were mildly hypothyroid, but not when rats were severely hypothyroid. It is concluded that amiodarone interferes (directly or indirectly) with thyroid hormone action in the heart.     

Reactivity of brain benzodiazepine receptors and individual sensitivity of rats to pentylenetetrazole

The individual sensitivity of the male Wistar rats to acute pentylenetetrazole injection was studied, the density and the affinity of benzodiazepine receptors in the cerebellar cortex for 3H-diazepam was measured. It was demonstrated that the reactivity of benzodiazepine receptors underlies the individual sensitivity to pentylenetetrazole. The animals with higher sensitivity were characterized by more intensive reaction than the control and resistant animals, i.e., by an decrease in the receptors density (the initial receptors density being equal in the sensitive, resistant, and control animals). Daily injections of a subconvulsive dose of pentylenetetrazole during 24 days increase the animal sensitivity to this substance, and this was accompanied by an increase in the reactivity of benzodiasepine receptors. Later on, the produced high sensitivity became somewhat lower but persisted for 6 months. The receptors density in this period reduced almost by half. In sensitive rats, a single low dose of pentylenetetrazole injected 6 months after treatment increased the density of benzodiazepine receptors. The age-matched controls, the same acute dose of pentylenetetrazole decreased both the receptor density and affinity of their binding. It is suggested that the increase in reactivity of benzodiazepine receptors is actualized via the intracellular metabotropic feedback mechanism.     

Melatonin reverses pinealectomy-induced decrease of benzodiazepine binding in rat cerebral cortex

Pinealectomy of rats resulted in significant depression of benzodiazepine receptors (assessed by [³H]flunitrazepam binding) in cerebral cortex 3–14 days after surgery without affecting their affinity significantly. A single s.c. injection of melatonin (800 μg/kg body wt) restored the depressed brain benzodiazepine receptor sites. Single melatonin injections (up to 1600 μg/kg) to intact rats did not affect brain benzodiazepine binding when injected at either morning or evening hours. Daily melatonin treatment to intact rats for 5 days augmented benzodiazepine receptor density in brain (morning injections) or its dissociation constant (evening injections). Melatonin added in vitro to rat cerebral cortex membranes only slightly depressed [³H]flunitrazepam binding at 100 μM concentrations. These results point out a link between pineal activity and benzodiazepine receptor function in rats. They also indicate that pharmacological doses of melatonin affect benzodiazepine binding sites in rat cerebral cortex.     

Neurotransmitters and receptor binding in amygdaloid kindled rats: Serotonergic and noradrenergic modulatory effects

No significant difference could be found between 15 amygdaloid kindled rats and 15 implanted but non-stimulated control rats with respect to 10 receptor binding assays carried-out in various brain regions. In a further group of 33 kindled rats neurotransmitter agonists and antagonists were tested for their effects on the duration of the clonic forepaw component. ACh and DA agonists or antagonists had no significant effects. However, the serotonin antagonists and clonidine reduced, whereas 5HTP increased, the duration of clonus. Results are interpreted as an opposing modulatory effect of serotonin and noradrenaline in kindled seizures.     

Effect of amygdaloid kindling on rat striatal dopamine D1- and D2-receptors

The effect of kindling on dopaminergic (DA) neurotransmission was assessed by measuring dopamine D₁- and D₂-receptor binding in the dorsal and ventral striatum of rats either 2 hours (short-term) or 3–4 weeks (long-term) after the last kindled seizure. Kindling did not have any significant long-term effect on DA D₂-receptor K_d or B_max values in the dorsal or ventral striatum or on DA D₁-receptor parameters in the dorsal striatum. The short-term effect of kindled seizures was to abolish the asymmetry in DA D₂-receptor density observed in the dorsal striatum of control rats. DA D₁-receptor density was also increased in the dorsal striatum contralateral to the kindled amygdala of short-term rats. The short-term effects support the notion that limbic seizures can modify the lateral imbalance of DA activity in the striatum.     

Changes in [3H]flunitrazepam binding in the brain of rats made tolerant to and dependent upon pentobarbital

Changes in benzodiazepine binding sites labeled by [³H]flunitrazepam (FNZ) in twenty discrete brain regions of rats made tolerant to and dependent upon pentobarbital were examined. Animals were rendered tolerant by intracerebroventricular (i.c.v) infusion with pentobarbital (300 μg/ 10 μ1/ hr for six days) through pre-implanted cannulae connected to osmotic mini-pumps. The pentobarbital dependence was assessed 24 hr after abrupt withdrawal from pentobarbital. In the tolerant rats, a significant increase in [³H]FNZ binding sites was found in layer IV of frontal cortex and the molecular layer of olfactory bulb. [³H]FNZ binding sites in the pentobarbital dependent rats were significantly increased in layers I-III and V-VI of frontal cortex, caudate-putamen, olfactory tubercle, globus pallidus and ventral pallidum, in addition to those observed in the tolerant group. There was, however, no significant difference in the hippocampus and several regions in the hindbrain in either pentobarbital-treated group. Taken together with characteristics of subtypes of benzodiazepine receptors and changes in GABA-benzodiazepine receptor complexes elucidated in our previous studies, these findings suggest that both types of benzodiazepine receptors are involved in the development of pentobarbital intoxication mediated by GABA_A receptors.     

Cysteamine normalizes cerebral somatostatin level and binding in pentylenetetrazol-kindled rats

Rats were kindled by intraperitoneal injection of pentylenetetrazol (PTZ) (30 mg/Kg) every 48 h. Once kindled, some of the animals received a single injection of cysteamine (200 mg/Kg). Somatostatin-like immunoreactivity (SLI) and 125 I-Tyr11-somatostatin binding were measured in the frontoparietal cortex and hippocampus of the two experimental groups and the control rats. After PTZ kindling the following was observed: 1) SLI content was increased in the two areas; 2) Somatostatin receptor affinity decreased in the frontoparietal cortex and was unaltered in the hippocampus; 3) The number of somatostatin receptors decreased in the hippocampus and was unaltered in the frontoparietal cortex. Cysteamine, an agent which depletes brain somatostatin and suppresses kindled seizures in PTZ-treated rats, reversed the altered SLI levels and binding in these rats.     

Effects of bicuculline-induced seizures on benzodiazepine and adenosine receptors in developing rat brain

The effects of seizures induced by an acute administration of bicuculline have been investigated on the central benzodiazepine and adenosine receptors in developing rats and in adults. Generalized seizures rapidly increased the total number of both benzodiazepine binding sites and adenosine A1 receptors, without changes in receptor affinity (KD). It was concluded that such a phenomenon may facilitate the anticonvulsant action of benzodiazepine and adenosine via receptor binding and that it could be an adaptative process to protect subjects against recurrent seizures, especially in newborns.     

Postnatal development and GABA allosteric modulation of benzodiazepine receptor binding in the vitamin B-6 deficient rat brain

We have measured the postnatal development and GABA modulation of benzodiazepine receptors in neuronal membranes from vitamin B-6 deficient and normal rats. In rats fed vitamin B-6 adequate and deficient diets there were age-dependent changes in [³H]flunitrazepam binding site affinity and in the number of binding sites. Vitamin B-6 deficiency produced a significant reduction in the potency of GABA to enhance [³H]flunitrazepam binding to cortical membranes prepared from 14 day old rats. These results suggests an uncoupling of the GABAa/benzodiazepine receptor at a developmental period when the animals are most susceptible to spontaneous seizures.     

Subchronic treatment with cyclosporin A decreases the binding properties of the GABAA receptor in ovariectomized rats

In this study, we investigated the effect of cyclosporin A on the binding properties of the GABAA receptor in the hippocampus, known to be responsible for the induction of seizures, to clarify the mechanism of cyclosporin A-inhibited GABA neurotransmission in ovariectomized rats, as a climacterium model. The effects of single and subchronic treatments with cyclosporin A were examined on [3H]muscimol binding to hippocampal synaptosomal membranes in sham, ovariectomized, and estradiol/ovariectomized rats. A single treatment with cyclosporin A (40 mg/kg, i.p.) failed to change [3H]muscimol binding in the 3 groups, when compared with each corresponding vehicle-treated group. Subchronic treatment with cyclosporin A (40 mg/kg, i.p., once a day for 5 days) significantly decreased the amount of [3H]muscimol binding in ovariectomized rats. However, this inhibitory effect was not observed in sham or estradiol/ovariectomized rats. These results demonstrated that the binding activity of the GABAA receptor was decreased in ovariectomized rats after subchronic cyclosporin A treatment. This study supports the hypothesis that ovariectomy elevates the susceptibility to cyclosporin A-induced convulsions by accelerating the inhibitory actions of cyclosporin A on GABA neurotransmission in the hippocampus.     

Histamine H1 receptor binding capacities in the amygdalas of the amygdaloid kindled rat

The histamine H1 receptor binding capacity of the amygdalas of amygdaloid kindled rats was studied. In the kindled nonstimulated amygdala, significant decreases in K(D) and B(max) values compared with those of control amygdala were found 1 week after the last kindled seizure. One month after the last kindled seizure, the decreased K(D) value was sustained in the kindled nonstimulated amygdala. This decreased Bmax value 1 week after the last kindled seizure in nonstimulated amygdala may partly and transiently contribute to kindled seizure susceptibility. The decreased K(D) value in nonstimulated amygdala observed until 1 month after the last kindled seizure indicates the long-lasting increment of binding affinity of the pyrilamine binding site of the histamine H1 receptor in the steady state of kindled seizure susceptibility.     

Amygdala Kindling Potentiates Seizure-Stimulated Immediate-Early Gene Expression in Rat Cerebral Cortex

Kindling induces long-term adaptations in neuronal function that lead to a decreased threshold for induction of seizures. In the present study, the influence of amygdala kindling on levels of mRNA for the immediate-early genes (IEGs) c-fos, c-jun, and NGF1-A were examined both before and after an acute electroconvulsive seizure (ECS). Although amygdala kindling did not significantly influence resting levels of c-fos mRNA in cerebral cortex, ECS-stimulated levels of c-fos mRNA (examined 45 min after ECS) were approximately twofold greater in the cerebral cortex of kindled rats relative to sham-treated controls. The influence of kindling on IEG expression was dependent on the time course of kindling, as ECS-stimulated levels of c-fos mRNA were not significantly increased in stage 2 kindled animals. ECS-stimulated levels of c-jun and NGF1-A mRNA were also significantly increased in cerebral cortex of kindled rats relative to sham-treated controls. The influence of kindling on IEG expression was long-lasting because an acute ECS stimulus significantly elevated levels of c-fos and c-jun mRNA in the cerebral cortex of animals that were kindled 5 months previously. In contrast to these effects in cerebral cortex, kindling did not influence ECS-stimulated levels of c-fos mRNA in hippocampus. Finally, immunohistochemical studies revealed lamina-specific changes in the cerebral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monophosphoryl Lipid A and Pam3Cys Prevent the Increase in Seizure Susceptibility and Epileptogenesis in Rats Undergoing Traumatic Brain Injury

Five percent of all epilepsy cases are attributed to traumatic brain injury (TBI), which are known as post-traumatic epilepsy (PTE). Finding preventive strategies for PTE is valuable. Remarkable feature of TBI is activation of microglia and subsequent neuroinflammation, which provokes epileptogenesis. The toll-like receptor agonists monophosphoryl lipid A (MPL) and tri-palmitoyl-S-glyceryl-cysteine (Pam3Cys) are safe, well-tolerated and effective adjuvants existing in prophylactic human vaccines. We examined the impact of early injection of MPL and Pam3Cys to rats, on the rate of kindled seizures acquisition following TBI. Rats received a single dose (1 µg/rat) of MPL or Pam3Cys through intracerebroventricular injection. 5 days later, trauma was exerted to temporo-parietal cortex of rats by controlled cortical impact device. After 24 h, traumatic rats underwent amygdala kindling. Brain level of the inflammatory cytokine tumor necrosis factor-alpha (TNF-α) was also measured in traumatic rats by immunoblotting. Compared to non-traumatic (sham-operated) rats, traumatic rats showed three times lower seizure threshold (133?±?5 µA vs. 416.3?±?16 µA, p?<?0.001); about three times less number of stimuli to become kindled (5?±?1 vs. 14?±?2, p?<?0.01); longer duration of kindled seizure parameters including entire seizure behavior, generalized seizures, and afterdischarges (p?<?0.001); and a two times increase in the TNF-α level. MPL and Pam3Cys did not change kindling rate and the seizure parameters in sham-operated rats. The MPL- and Pam3Cys-pretreated traumatic rats displayed seizure threshold, speed of kindling, and duration of kindled seizure parameters, similar to the non-traumatic rats. Pretreatment by MPL and Pam3Cys prevented the increase in TNF-α level by trauma. Given that MPL and Pam3Cys currently have clinical use as well-tolerated vaccines with reliable safety, they have the potential to be used in prevention of PTE.     

Anticonvulsant effect of A1 but not A2A adenosine receptors of piriform cortex in amygdala-kindled rats

In this study, the effect of A1 and A2A adenosine receptor activity of the piriform cortex (PC) on amygdala-kindled seizures was investigated in rats. Animals were kindled by daily electrical stimulation of the amygdala. In fully kindled rats, N6-cyclohexyladenosine (CHA, a selective A1 agonist), 8-cyclopentyl-1,3-dimethylxanthine (CPT, a selective A1 antagonist), CGS21,680 hydrochloride (CGS, a selective A2A agonist), and ZM241,385 (ZM, a selective A2A antagonist) were microinjected bilaterally into the PC. Rats were stimulated 5 min post-drug microinjection and seizure parameters were measured. Results showed that intra-PC CHA (10 and 100 micromol/L) decreased the duration of both afterdischarge and stage 5 seizure and significantly increased the latency to stage 4 seizure. Intra-PC CPT increased afterdischarge and stage 5 seizure duration at the dose of 20 micromol/L. The anticonvulsant effect of CHA (100 micromol/L) was eliminated by CPT (10 micromol/L) pretreatment. On the other hand, neither intra-PC CGS nor ZM had a significant effect on kindled seizures. These results suggest that activity of A1, but not A2A, receptors of the PC have anticonvulsant effects on kindled seizures elicited from electrical stimulation of the amygdala.     

Evidence for diabetes-induced alterations in the sulfation of heparin sulfate intestinal epithelial cells

Two compounds with high affinity for the "peripheral type" benzodiazepine binding sites, PK 11195 (an isoquinoline derivative) and RO5-4864 (a benzodiazepine derivative) can modify the sensitivity of DBA/2J mice to audiogenic seizures. RO5-4864 (1-15 mg/kg) facilitates in a dose-dependent manner the audiogenic seizures and PK 11195 (2-5 mg/kg) antagonizes the RO5-4864 effects. At these doses PK 11195 alone does not modify the sensitivity to audiogenic seizures, but at doses between 20-80 mg/kg it protects DBA/2J mice against audiogenic seizures. By contrast PK 11195 is inactive against the facilitation of audiogenic seizures by ethyl-beta-carboline-3-carboxylate (a brain benzodiazepine receptor inverse agonist) and against the seizure elicited in absence of noise stimuli by RO5-4864 at doses between 20-40 mg/kg. These results suggest that facilitation by RO5-4864 of the audiogenic seizures and its antagonism by PK 11195 are mediated by the peripheral type benzodiazepine binding sites and agree with the thermodynamic analysis of the binding data which suggested that RO5-4864 might be an agonist and PK 11195 an antagonist. The good correlation between pharmacological effects and the occupancy degree of the binding sites as measured by the displacement of the "in vivo" [3H]-PK 11195 binding give an additional support to binding sites mediated effects.