Long-term exposure of cortical cell cultures to clonazepam reduces benzodiazepine receptor binding

To determine if reduced drug efficacy after long-term exposure to clonazepam may be a consequence of benzodiazepine receptor alterations, cerebral cortical cell cultures were exposed to the drug (200 nM) for 14 days. Receptor binding was assayed on living cells in situ. After drug exposure, binding in experimental cultures differed markedly from controls with respect to total, specific, and clonazepam-displaceable (neuronal) benzodiazepine binding (60%, 53%, and 6% of control values, respectively) but recovered within 96 h of drug removal. RO5-4864-displaceable (nonneuronal) binding was modestly reduced at 0 time (72% of control), but returned to control values in 24 h. The differences in binding could be attributable to a relatively reduced affinity of the high-affinity binding site (Kd approximately 18 nM for controls and approximately 30 nM for drug-exposed cultures) but not to changes in the low-affinity binding site or to reduced numbers of receptors.     

Characteristics of benzodiazepine receptor binding in living cultures of mouse cerebral cortex at physiologic temperature

Benzodiazepine (BDZ) receptor binding was assayed in situ in dispersed cultures of fetal mouse cerebral cortex at 37 degrees C and at 0 degrees C and compared. Contrary to data obtained from disrupted tissues, receptor binding at physiologic temperature was reduced only to 40% of that observed in the cold when intact tissues were used for assay. Neuronal (clonazepam-displaceable) receptor binding was essentially unaffected by changes in temperature; however, non-neuronal (Ro5-4864-displaceable) binding was reduced to 25% of that of 0 degrees C. A relatively high-affinity (Kd approximately 24 nM) as well as a low-affinity (Kd approximately 200 nM) binding site were identified; the numbers of binding sites were only modestly reduced at physiologic temperature and probably reflect a predominant reduction in non-neuronal sites.     

Bi-directional alterations of LTP after acute homocysteine exposure

Homocysteine (HCY) is a known risk factor for neuronal diseases. We here report that HCY (10–1000 μM) interfered bi-directionally with long-term potentiation (LTP) in hippocampal slices, causing an impairment at concentrations <100 μM, and enhancement ≥500 μM. By comparison, NMDA unidirectionally reduced LTP, whereas l-cysteine led to facilitated LTP. Such HCY-induced alterations in neuronal communication may contribute to cognitive failure in dementia.     

Astroglial alterations in rat hippocampus during chronic lead exposure.

The present study was performed in order to follow the response of astroglial cells in the rat hippocampus to chronic low-level lead exposure. The experiments combined immunohistochemistry using anti-glial fibrillary acidic protein (GFAP) antibody and conventional transmission electron microscopy (EM). Chronic administration with drinking water [1 g% w/v (subclinical dose) of lead acetate dissolved in distilled water] was started through the mother's milk when pups were 7 days old. Following weaning, experimental offspring were treated for 3 months with the same concentration of adulterated water. The group of intoxicated animals and their controls were sacrificed by perfusion-fixation at 30, 60, and 90 days of exposure. After 60 days of lead treatment, staining of GFAP-positive cells demonstrated an astroglial transformation from the quiescent to the reactive state, characterized by an increase in GFAP. In control rats no changes in GFAP immunostaining were observed. The intensity of the astroglial response was enhanced after 90 days of lead intoxication, showing an increment of GFAP immunoreactivity. Quantification of these changes was made by computerized image analysis, confirming that the sectional areas of the astroglia in lead-exposed animals were larger than those in controls. These results are consistent with the ultrastructural alterations. Simultaneously with the increment in gliofilaments, intranuclear inclusions were seen in some astrocytes. The mechanisms by which lead affects astrocytes are unknown. Probably the astroglial changes induced by lead intoxication produce microenvironmental modifications that may disturb the neuronal function.     

GABA receptor alterations after chronic lithium administration. Comparison with carbamazepine and sodium valproate.

1. Effects of lithium, carbamazepine, sodium valproate and baclofen on GABA receptors were examined in several regions of the rat brain. 2. [3H]Muscimol (MUS) and [3H] (-)baclofen (BAC) were used to label GABAA and GABAB receptors, respectively, in synaptic membranes from rat brain. 3. Single treatment with lithium chloride, carbamazepine or sodium valproate did not change [3H]MUS or [3H]BAC binding in the frontal cortex, hippocampus and thalamus. 4. Following chronic treatment with lithium, carbamazepine or sodium valproate, [3H]BAC binding was significantly increased in the hippocampus but not in the frontal cortex, thalamus or striatum. 5. [3H]Muscimol binding did not change in any region examined after chronic treatment with lithium, carbamazepine or sodium valproate. 6. Single and chronic administration of baclofen did not change [3H]MUS or [3H]BAC binding. 7. One common mechanism of action of mood stabilizers may be mediated by GABAB receptors in the hippocampus.     

Neural and behavioral alterations after early exposure to phenobarbital

Mice who were exposed to phenobarbital prenatally (B mice) had at adulthood deficits in the hippocampal eight-arm maze, spontaneous alternations, and water maze behaviors. Morphological studies revealed neuronal losses in the hippocampus. The surviving neurons had reductions from control in the number of dendritic branches, area and spine density, but wider fission angle than control. Neurochemical studies on the hippocampus revealed the following alterations: (a) decrease in NE level and the number of the NE cell bodies (b) no change in the serotonergic system (c) an increase in muscarinic receptors Bmax in the hippocampus; (d) no changes in GABA and benzodiazepine receptors. However, neonatal phenobarbital exposure caused an increase in the Bmax of GABA and benzodiazepine receptors. Transplantation of fetal septal cholinergic neurons into the hippocampus of B mice reversed most of the deficits in eight-arm maze behavior, while transplantation of noradrenergic cells did not affect the performance of B mice. In further studies on cholinergic mechanisms, the dopaminergic innervations in the septum (originating from A10), which are known to indirectly inhibit the activity of the septohippocampal cholinergic pathways, were destroyed by 6-OHDA. B mice treated with 6-OHDA had an increase in hippocampal ChAT activity and improved their eight-arm maze performance. Thus, understanding of the mechanism of a particular behavioral deficit enables one to correct it despite the nonspecific action of the neuroteratogen.     

Benzodiazepine receptor binding by membranes from brain cell cultures following chronic treatment with diazepam

Murine brain cells differentiated for 19 days in culture before treatment for 5 days with 10 microM diazepam. Radioligand binding to the benzodiazepine receptors on membranes obtained from these cultures was determined by the filtration assay method. Decreased binding was observed in the membranes from treated cultures relative to untreated cells. However, this decrease appears to be due, at least in part, to competition from residual drug in the assay system despite extensive cell and membrane rinses. These data emphasize the difficulty in ascribing the mechanism of benzodiazepine tolerance observed clinically with chronic treatment to receptor down-regulation as determined by binding assays.     

Selective alterations in glutamate receptor subtypes after unilateral orbital enucleation

Glutamate is the major excitatory neurotransmitter in the rat visual system. Using quantitative autoradiography the effect of unilateral orbital enucleation on [³H]kainate, [³H]-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid ([³H]AMPA) and [³H]glutamate binding to kainate, quisqualate and NMDA receptors respectively has been examined within anatomical components of the visual pathway at 4 time points up to 20 days post-lesion. The time course for the degeneration of retinal projection fibres was assessed in a separate group of animals by quantifying [³H]cyclohexyladenosine ([³H]CHA) binding to presynaptic adenosine A₁ receptors. Over the first 5 days after orbital enucleation, there were no significant alterations in glutamate or adenosine A₁ receptor binding in visual structures of the visually deprived hemisphere. However, at 10 days post-lesion [³H]AMPA binding was significantly reduced (30%) in the visually deprived superior colliculus but unaltered in other visual structures. At this time point there was also a significant reduction (50%) in [³H]CHA binding in the visually deprived superior colliculus but not in other retino-recipient nuclei. There were similar changes in [³H]AMPA and [³H]CHA binding at 20 days post-enucleation. [³H]Kainate binding was significantly increased in the visually deprived superior colliculus only at 20 days post-enucleation. Saturation analysis of [³H]kainate and [³H]AMPA binding at this time point indicated a selective increase in the b_max value for the high affinity [³H]kainate binding site and a concomitant decrease in the b_max value for the high affinity [³H]AMPA binding site in the visually deprived superior colliculus. There were, however, no significant alterations in [³H]AMPA or [³H]kainate binding in other primary projection areas or in secondary visual areas (e.g. visual cortex) at any time point. NMDA sensitive [³H]glutamate binding was unaltered in the visually deprived hemisphere up to 20 days post-enucleation. These results suggest an upregulation of kainate receptors in the visually deprived superior colliculus after orbital enucleation and a loss of presynaptic quisqualate receptors on degenerating retinal fibres. The plastic alterations in kainate receptors in the superior colliculus are supportive of electrophysiological data suggesting a physiological role for these sites in mediating excitatory postsynaptic potentials in tectal neurons.     

Pharmacological alterations after clot removal in monkey chronic cerebral vasospasm

An autologous blood clot was placed bilaterally around cerebral arteries in the basal subarachnoid space to mimic subarachoid haemorrhage (SAH). Cynomolgus monkeys were randomised into five groups: sham operated groups, clot removal group at 48, 72 or 96 h after. SAH and clot group. The proximal parts of the middle cerebral arteries were cut into rings for isometric tension measurements at 7 days after SAH. Potassium chloride, 5-hydroxytryptamine (5-HT), norepinephrine (NE), adenosine triphosphate (ATP), prostaglandin F(2alpha) (PGF(2alpha)) and haemoglobin all induced dosed-dependent contractions. There was a progressive attenution of the contractions in response to the agonists in the clot removal and the clot groups, which reached statistical significance at 48 h after SAH in the case of 5-HT, and at 72 h in the cases of NE, ATP and PGF(2alpha) as compared with the sham operated group. These pharmacological results suggest that clot removal should within the first two days after SAH to prevnt cerebral vasospasm.     

Hippocampal cholinergic alterations and related behavioral deficits after early exposure to ethanol

The present study was designed to ascertain septohippocampal cholinergic alterations and their related behavioral deficits after early exposure to ethanol. Mouse pups were exposed to ethanol, 3 g/kg by daily subcutaneous injection on postnatal days 2–14. At age 50 days, the ethanol-exposed mice had significant reductions from control levels in eight-arm maze performance. For example, on the fourth testing day, the number of correct entries in the ethanol group was 21% below control levels (P < 0.05) and the number of trials needed to enter all arms was 48% above control (P < 0.001). It took the ethanol-exposed mice twice the time to reach criterion than it did control (P < 0.01). A 33% increase from control level in muscarinic receptor number (β_max) was found in the treated mice of age 22 days and a 64% increase at age 50 days (P < 0.001). However, no differences between control and treated groups could be detected in the presynaptic component of the cholinergic innervation, choline acetyltransferase activity. The results suggest that early ethanol exposure acts on hippocampal function similarly to phenobarbital, probably via alterations in postsynaptic processes in the septohippocampal cholinergic pathways.     

Length of continuous cocaine exposure determines the persistence of muscarinic and benzodiazepine receptor alterations

The effects of varied durations of cocaine (1, 3 or 5 days) on muscarinic (MSC) and benzodiazepine (BZD) binding sites in striatum and hippocampus were investigated using homogenate receptor binding. The progressive alterations in these receptor sites from a 5 day cocaine administration were also examined 12 h, 2 days or 21 days after drug exposure. Neither a one nor a three day exposure to cocaine produced any long-term alteration in BZD binding in either structure whereas a 5 day administration produced significant increases in binding. Decreases in MSC receptor binding were apparent in striatum from either a 3 or 5 day cocaine exposure and in hippocampus from a 5 day exposure. The 5 day cocaine exposure produced immediate increases in striatal and hippocampal BZD binding which persisted for 21 days. Conversely, 5 days of cocaine produced a short-term increase in MSC receptor binding im both structures which then became significantly decreased 21 days later. Based on the divergent pattern of changes in BZD and MSC receptor types over time in these structures, it appears that cocaine may induce such changes via separate mechanisms. In addition, it is apparent that changes in the numbers of these receptor sites after cocaine exposure can be quite dynamic, changing rapidly over time.     

Noradrenaline receptor sensitivity after chronic ethanol administration

Summary The effects of the central noradrenaline receptor stimulating agent clonidine on blood pressure, heart rate, and flexor reflex activity were studied in intact rats and in rats chronically treated with ethanol. The clonidine-induced changes were similar in ethanol-treated animals and in animals never subjected to ethanol. The results suggest that the sensitivity of central noradrenaline receptors involved in the mediation of the circulatory changes and of the flexor reflex activity after clonidine is not altered by chronic administration of ethanol.     

Decreased Beta2*‐nicotinic acetylcholine receptor availability after chronic ethanol exposure in nonhuman primates

Ethanol associated behaviors have been linked to the β₂‐subunit containing nicotinic acetylcholine receptors (β₂*‐nAChR); however, there is conflicting evidence on ethanol‐induced changes in nAChR expression during and after chronic ethanol consumption. In this study, five male animals orally self‐administered ethanol for 18 ± 1 weeks. Animals were scanned with [¹²³I]5‐IA‐85380 and SPECT prior to ethanol self‐administration, and at 24 h and 5‐13 wks withdrawal. β₂*‐nAChR availability was not significantly different from baseline at 24 h withdrawal, but was significantly decreased compared to baseline at 5‐13 wks withdrawal throughout the cortex and in the thalamus, but not the midbrain. The percent decrease in β₂*‐nAChR availability from baseline to 5‐13 wks withdrawal in the parietal cortex was negatively correlated with total grams of ethanol consumed in lifetime and in the midbrain was negatively correlated with average daily ethanol consumption (g/kg). Prolonged withdrawal from chronic ethanol consumption is associated with a decrease in β₂*‐nAChR availability. The decrease in β₂*‐nAChR availability is influenced by alcohol consumption, suggesting the chronicity and severity of alcohol consumption may underlie persistent changes in β₂*‐nAChR availability. Synapse 64:729–732, 2010. © 2010 Wiley‐Liss, Inc.     

Hippocampal cholinergic alterations and related behavioral deficits after early exposure to phenobarbital.

Mice were exposed to phenobarbital (PhB) prenatally and neonatally. Prenatal exposure was accomplished by feeding the mother PhB (3 g/kg milled food) on gestation days 9-18. Neonatal exposure was accomplished by daily injections of 50 mg/kg sodium PhB directly to the pups on days 2-21. Long-term biochemical alterations in the pre- and postsynaptic septohippocampal system, as well as related behavioral deficits, were assessed in the treated animals. Significant increase in B(max) values for binding of [3H]QNB to muscarinic cholinergic receptors was obtained on both ages 22 and 50 in prenatally (40-90%, respectively, p less than 0.001) and neonatally exposed (58-89%, p less than 0.001) mice whereas Kd remained normal. Similarly, a significant increase of inositol phosphate (IP) formation in response to carbachol was found after both prenatal and neonatal exposure to PhB (p less than 0.05). No alterations in choline acetyltransferase (ChAT) activity were observed in the prenatally or neonatally treated animals. The early exposed mice showed deficits in the performance in Morris water maze, a behavior related to the septohippocampal pathway. The results suggest that early exposure to PhB induces alterations in postsynaptic components of the hippocampal cholinergic system and concomitantly to impairment in hippocampus-related behavior.     

Regionally specific alterations in the low-affinity GABAA receptor following perinatal exposure to diazepam

Alterations in a low affinity form of the GABA_A receptor were examined with [³H]bicuculline methylchloride in the adult rat following perinatal exposure to diazepam. Perinatal exposure resulted in a significant reduction in [³H]bicuculline binding in the cingulate cortex. A significant decrease in the ability of GABA to displace bound [³H]bicuculline was observed only in the hypothalamus. The results suggest that the effects of perinatal exposure to diazepam are regionally specific and that benzodiazepine receptors and low affinity GABA_A receptors are functionally linked during the perinatal period.     

Functional alterations in CNS catecholamine systems in adolescence and adulthood after neonatal chlorpyrifos exposure

Chlorpyrifos (CPF), one of the most widely used pesticides, is a neurobehavioral teratogen in animals. We administered CPF to neonatal rats on postnatal days (PN) 1-4 (1 mg/kg) or PN11-14 (5 mg/kg), regimens devoid of overt systemic toxicity. We then examined the impact on catecholaminergic systems in adolescence (PN30) and adulthood (PN60), assessing basal neurotransmitter content and transmitter utilization rates (turnover) in brain regions comprising the major noradrenergic and dopaminergic projections. Although CPF had only sporadic effects on basal norepinephrine and dopamine content, it profoundly suppressed norepinephrine turnover across multiple regions, indicative of net reductions in presynaptic activity. Dopamine turnover showed less consistent effects, with subnormal turnover in some regions and activation in others. We also evaluated whether CPF exposure altered the ability of catecholamine systems to respond to acute cholinergic stimulation, elicited by administration of a single challenge dose of nicotine. In the normal brain, nicotine increases the utilization of norepinephrine and dopamine. With only a few exceptions, animals receiving neonatal CPF exposure showed lasting desensitization of the nicotine response; not only was the activation by nicotine blunted in the CPF group, but in some regions the nicotine response was reversed, eliciting a reduction in transmitter turnover. These results indicate that neonatal CPF exposure produces widespread deficiencies in catecholaminergic synaptic function that persist into adulthood, and that are best revealed by dynamic measures of synaptic activity and responsiveness, as opposed to static markers like basal transmitter levels. The effects seen here are likely to contribute to alterations in behavioral performance that persist or emerge long after the termination of CPF exposure.     

Long term alterations in neuroimmune responses of female rats after neonatal exposure to lipopolysaccharide

Male and female rats display significant gender-associated differences in their responses to an immune challenge, and gender-specific alterations in many aspects of physiology are seen after a variety of interventions during the neonatal period. It is well-established that neonatal exposure to an immune challenge can alter centrally mediated inflammatory responses in adult male rats and yet little is known about female responses after a similar challenge. We therefore asked if neonatal exposure to lipopolysaccharide (LPS) would alter febrile and hypothalamic cyclooxygenase (COX)-2 responses to an adult immune challenge in female rats. Female Sprague-Dawley rats were administered a single injection of the bacterial endotoxin LPS at postnatal day 14 and were examined as adults for febrile, COX-2 and activity changes to LPS, as well as responses to interleukin (IL)-1beta. Adult female rat responses were similar to those we have seen previously for the males in that febrile and hypothalamic COX-2 responses to adult LPS were attenuated in neonatally LPS-treated animals. Responses to adult IL-1beta were unaffected. Interestingly, females did not display the elevated basal hypothalamic COX-2 that was previously seen in males. Thus we demonstrate that, like in the males, neonatal exposure to LPS has a powerful effect on adult responses to further LPS challenge in the female rats.     

Pre- and postsynaptic alterations in the septohippocampal cholinergic innervations after prenatal exposure to drugs

The present study was designed to evaluate possible presynaptic and postsynaptic alterations in the hippocampal cholinergic innervations that account for the hippocampus-related behavioral deficits found after prenatal drug exposure. Mice were prenatally exposed to either phenobarbital or heroin. On postnatal day 50, the hippocampi were removed and protein kinase C (PkC) activity, the amounts of Gi, Go, and Gq guanosine 5’-triphosphate binding proteins (G-proteins), and choline transports were determined. Basal PkC activity was higher than control levels in both phenobarbital and heroin treated mice, by 41% and 35%, respectively. The increase of PkC activity in response to carbachol was impaired in both treatment groups: in control mice, membrane PkC activity in hippocampal slices increased by 40%–50%, while no such response, or even slight reduction in PkC activity, was observed in the drug-exposed offspring. A significant increase was found in Gi and Gq G-proteins (18%–21%) in mice exposed to phenobarbital or to heroin compared with control levels. The amount of choline transporters, determined by hemicholinium binding, increased by 70% compared with the control level in mice prenatally exposed to heroin, and increased by 71% in mice prenatally exposed to phenobarbital. The alterations in basal and carbachol-stimulated hippocampal PkC activity after prenatal drug exposure may be related to an impairment in long-term potentiation (LTP); which plays an important role in hippocampal related behavioral abilities, changes in which are caused by prenatal drug exposure.