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Several drugs of abuse are known to produce an array of deleterious effects, including alterations in neuronal circuitry and, ultimately, neuronal degeneration. For instance, methamphetamine was shown to induce substantial nigrostriatal dopaminergic terminal damage, including an increase in glial fibrillary acidic protein, a marker for astrocyte proliferation. Nevertheless, there was almost no attempt to define neurodegeneration by measuring the abundance of reactive microglia. In fact, some investigators fail to differentiate between astrocytes and microglia and claim glial fibrillary acidic protein to be a marker for gliosis. To date, there are numerous methods designed to assess brain neuropathologies resulting from a wide arsenal of insults. Regardless of the cause of neuronal damage, reactive glial cells always appear at and around the site of degeneration. These cells are distinguished by the exceptional abundance of peripheral benzodiazepine receptors (PBRs; omicron3 sites), particularly as compared to surrounding neurons. Measuring the binding of specific ligands to these PBRs (for example, [3H]PK 11195) offers a unique indirect marker for reliable impairment estimation in the central nervous system. Moreover, the availability of agents such as [11C]PK 11195 paved the road to in vivo animal and human brain positron emission tomography scanning, demonstrating inflammation-like processes in several diseases. Additionally, the measurement of increased binding of PBR ligands provides a faithful indicator for the behavioral and cognitive deficits accompanying neuronal injury.

There are numerous methods designed to monitor brain neuropathologies resulting from a wide arsenal of insults. Regardless of the cause of neuronal death, reactive glial cells always appear at and around the site of degeneration. These cells are distinguished by the exceptional abundance of peripheral benzodiazepine receptors, particularly compared with surrounding neurons. Measuring the binding of specific ligands to these peripheral benzodiazepine receptors offers a unique indirect marker for reliable damage assessment in the CNS and a faithful indicator for the accompanying cognitive deficits.

Employing [3H]Ro5-4864 as a radioligand, the existence of peripheral benzodiazepine binding sites in rat hind limb skeletal fibroblasts was explored. Saturable, high affinity binding of this ligand is described for fibroblasts and cell membrane preparations, demonstrating a pharmacological profile characteristic for such receptors. The binding of [3H]Ro5-4864 exhibited higher affinity and density in fibroblasts compared to membrane preparations. A quaternaryamine derivative of PK 11195 showed a similar potency as a displacer of [3H]Ro5-4864 in both preparations. The data suggest that peripheral benzodiazepine receptors are present in the plasma membrane of primary mammalian fibroblasts.

Nitrogen oxide-containing compounds displaced the peripheral benzodiazepine ligand [3H]Ro5-4864 from guinea pig membrane preparations. Sodium nitroprusside (SNP) was the most potent (IC50 = 5.61 +/- 1.72 x 10(-5) M). Moreover, its ability to bind to these receptors showed marked tissue variability (heart greater than kidney much greater than cerebral cortex). When tested on rat atrium, SNP by itself had no effect on basal inotropy or the increase in inotropy induced by (-)-S-BAY K 8644. In contrast, Ro5-4864 potentiated the marked increase in inotropy induced by (-)-S-Bay K 8644, and SNP completely abolished the potentiation of inotropy observed with Ro5-4864. Since peripheral benzodiazepine receptors are associated with calcium mobilization in the heart, these findings may indicate that some of the clinical effects of nitric oxide-generating drugs could be mediated by these receptors.

The effects of the peripheral-type benzodiapine receptor (PBR) ligands Ro 5-4864 and PK 11195 were studied in the spontaneously beating guinea pig atrium and in a model for myocardial ischemia in the rat. In the former, Bay K 8644 produced positive chronotropic and inotropic responses; intracarotid administration of this agonist (5 or 10 micrograms kg-1) to anesthetized rats elicited a transient increase in mean arterial blood pressure accompanied by alterations in the ECG pattern. Ro 5-4864 and PK 11195 (10 microM) completely blocked the positive chronotropic effect of Bay K 8644 in the atrium, PK 11209, a structural analog of PK 11195 with a low affinity for PBR, was inactive, and the central benzodiazepine receptor ligand clonazepam had a marginal effect. Ro 5-4864 potentiated whereas PK 11195 inhibited the myocardial ischemia produced by Bay K 8644 in the rat. Furthermore, PK 11195 blocked the combined response to Bay K 8644 and Ro 5-4864. Addition of Ro 5-4864 (10 microM) to the organ bath potentiated the inotropic effect of Bay K 8644 in the atria; PK 11195 at the same concentration inhibited this effect. Clonazepam and PK 11209 were both inactive in this regard. Nifedipine, a potent calcium channel antagonist, completely blocked the inotropic and chronotropic responses to Bay K 8644. PK 11195 and Ro 5-4864 did not affect this action. These findings strongly suggest that there is a functional association between PBR and voltage-operated calcium channels in the guinea pig atrium and rat cardiovascular system.

The density of "peripheral-type" binding sites for benzodiazepines (PBS) in the rat pineal gland was reduced by approximately 50% three weeks after superior cervical ganglionectomy or exposure to constant light; the apparent affinity of [3H]Ro 5-4864 (the prototype ligand for these sites) remained unchanged. In contrast, neither the density of PBS nor the apparent affinity of [3H]Ro 5-4864 for these sites was altered in cerebral cortex by these procedures. The demonstration that PBS density is under neural control in the pineal gland suggests that these sites may have a physiologic role. The high density of PBS in pineal (approximately 24 pmol/mg protein) suggests that this tissue may be a useful model to study both the physiologic and pharmacologic function of these sites.

Both barbiturates and ethanol have been reported to interact with the GABA-benzodiazepine receptor-chloride ionophore 'supramolecular complex'. These observations raise the possibility that some of the pharmacologic actions of barbiturates and ethanol may be mediated through this complex. In this study we have administered a series of drugs which bind to various components of the complex in an attempt to antagonize the lethality of sodium pentobarbital, and ethanol-induced loss of righting reflex in mice. It was found that isopropylbicyclophosphate (IPPO), a cage convulsant which binds at or near the chloride ionophore, greatly reduces the overall mortality (and increases latency to death) of animals pretreated with a lethal dose of pentobarbital. Picrotoxin also decreases pentobarbital lethality, but only at doses which were usually lethal when given alone. Picrotoxin shortened, rather than increased, latency to death. Strychnine did not prevent pentobarbital lethality, suggesting that the IPPO effect is not shared by convulsants in general. IPPO did not prevent ketamine-induced deaths, which supports the notion that the protective actions of IPPO are specific for depressant drugs which act at the chloride ionophore. IPPO also significantly reduced the duration of loss of righting reflex induced by ethanol. These observations suggest that the use of compounds which have a high affinity for the chloride ionophore in vitro might be fruitful in developing a clinical treatment for barbiturate or ethanol toxicity.

Previous studies have shown that Ro 5-4864 is a potent convulsant and increases the firing rate of substantia nigra zona reticulata neurons. The pharmacologic profile of compounds that antagonize these actions suggested that the effects of Ro 5-4864 were not mediated by "brain-type" benzodiazepine receptors. We examined a number of compounds that are structurally related to Ro 5-4864 for their capacities to displace [3H]Ro 5-4864 from "peripheral-type" binding sites and their potencies as convulsants (or as antagonists of Ro 5-4864-induced convulsions). It was observed that compounds such as KW 3600 (the N-desmethyl analog of Ro 5-4864), which have very low affinities for "peripheral-type" sites, are convulsants with a potency nearly equal to that of Ro 5-4864. In contrast, compounds such as Ro 5-6900 and PK 11195, which bind with very high affinities to "peripheral-type" binding sites, are neither convulsants nor do they antagonize the convulsant actions of Ro 5-4864. Within a series of compounds that are structurally related to Ro 5-4864 there is a good correlation (r = 0.93; p less than 0.01) between their potencies as convulsants and their capacities to displace [35S]t-butylbicyclophosphorothionate from sites that may be associated with the chloride ionophore. Thus, it appears that occupation of "peripheral-type" binding sites by high-affinity ligands may not be directly involved in the convulsant actions of Ro 5-4864 and related compounds.

Previous studies have shown that Ro 5-4864 is a potent convulsant. Investigation of a series of compounds structurally related to Ro-4864 revealed a good correlation (r = .93, p less than 0.01) between their potencies as convulsants and their abilities to displace [35S]t-butylbicyclophosphorothionate from sites associated with the chloride ionophore. In contrast, there appears to be no direct relationship between the convulsant potencies of these compounds and their affinities for "peripheral-type" binding sites for benzodiazepines. These data suggest that "peripheral-type" binding sites for benzodiazepines are not directly involved in the convulsant actions of Ro 5-4864 and related compounds nonetheless, several lines of evidence suggest that "peripheral-type" binding sites for benzodiazepines may be indirectly involved in the convulsant properties of Ro 5-4864.

The densities of dihydropyridine calcium antagonist binding sites were examined in brains and cardiac issues of representative species from 4 classes of non-mammalian vertebrates (aves, pigeon; amphibia, frog; reptilia, chameleon; and osteichthyes, trout) previously shown to have low or undetectable levels of 'peripheral-type' binding sites for benzodiazepines. Dihydropyridine binding sites were present in brain and cardiac tissue of these 4 classes of non-mammalian vertebrates. The apparent dissociation constants for [3H]nitrendipine in non-mammalian vertebrates were comparable to those found in mammalian (rat) tissues. The densities of [3H]nitrendipine binding sites in brain and cardiac tissues were highest in the pigeon, with lower densities in tissues from chameleon, frog and trout. The densities of dihydropyridine binding sites in the latter tissues were comparable to those observed in the rat. Thus, dihydropyridine binding sites are phylogenetically diverse yet have similar kinetic characteristics in mammals and non-mammalian vertebrates. Despite the pharmacologic evidence that links dihydropyridine binding sites and peripheral binding sites for benzodiazepines in mammals, the earlier evolutionary appearance of the former sites suggest that even though they may share one or more common effector mechanisms, they are discrete entities.

Recent observations have shown that "peripheral-type" binding sites for benzodiazepines (PBS) are under neural and/or hormonal control in the pineal gland, olfactory bulb, and kidney. These studies resulted in a search for endogenous substances which might physiologically subserve PBS. Acidified methanol or trichloroacetic acid extraction of both peripheral tissues and brain followed by ultrafiltration and/or gel filtration and high performance liquid chromatography revealed the presence of both high (Mr greater than 10,000) and low (Mr less than 500) molecular weight substances which inhibit the binding of [3H] Ro 5-4864 to PBS while only slightly inhibiting the binding of [3H] diazepam to classical "brain-type" benzodiazepine receptors.

Four classes of non-mammalian vertebrates were examined for the presence of both 'brain-specific' and 'peripheral-type' binding sites for benzodiazepines in the central nervous system. 'Brain-specific' binding sites for benzodiazepines were found in the central nervous systems of all non-mammalian vertebrates studied. However, in contrast to mammals, either very low or undetectable levels of 'peripheral-type' binding sites for benzodiazepines were observed in the central nervous systems of these non-mammalian vertebrates. Furthermore, the density of 'peripheral-type' binding sites for benzodiazepines in non-mammalian vertebrate heart was less than or equal to 2% of that found in mammalian cardiac tissue. These findings suggest a very late evolutionary appearance of 'peripheral-type' binding sites for benzodiazepines, implying that these sites may have (a) highly specialized function(s) in both peripheral tissues and the central nervous system.

Buspirone, a clinically effective anxiolytic, has not shown robust effects consistently in procedures used traditionally with rodents and nonhuman primates to evaluate potential antianxiety activity. When key pecking by pigeons was maintained by food and was punished alternately under one component of a multiple schedule by the presentation of electric shock (conflict procedure), buspirone (0.03-10.0 mg/kg i.m.) produced increases in punished responding that were up to 30 times those of the control response rate. These doses did not affect or decreased unpunished responding. A buspirone analog, MJ 13805 (gepirone) produced effects similar to buspirone, although unpunished responding was slightly more sensitive to the rate-decreasing effects of MJ 13805 than to those of buspirone. A metabolite of buspirone, 1-pyrimidinyl piperazine (1-PP; MJ 13653), did not affect key pecking across a wide dose range (0.01-3.0 mg/kg i.m.), although slight decreases in both punished and unpunished responding occurred at the highest dose. Increases in punished responding with buspirone were not affected by the benzodiazepine receptor antagonist Ro 15-1788 (0.01-0.1 mg/kg i.m.). [3H]Diazepam binding to pigeon cerebrum or cerebellum in vivo was not altered by buspirone, or did buspirone, MJ 13805, or 1-pyrimidinyl piperazine displace [3H]flunitrazepam binding in vitro at pharmacologically relevant concentrations. These findings confirm previous work demonstrating marked rate-increasing effects of buspirone on punished responding in the pigeon, extend such effects to the buspirone analog MJ 13805 and indicate that the effects of buspirone are not mediated through the benzodiazepine receptor complex.

The density of [3H]flunitrazepam and [3H]Ro 5-4864 binding sites was examined 19-21 and 27-31 days after surgical isolation of the olfactory bulbs. While statistically significant reductions in [3H]flunitrazepam binding were observed in the olfactory bulbs at both intervals, a significant reduction in [3H]Ro 5-4864 binding was observed only at 27-31 days after surgery. No consistent changes in the binding of either radioligand were observed in cerebella. Extirpation of the olfactory bulbs resulted in a significant reduction in the convulsant potency (but not efficacy) of Ro 5-4864, while neither the potency nor efficacy of pentylenetetrazole was affected by this procedure. These results suggest that benzodiazepine receptors and 'peripheral-type' binding sites for benzodiazepines are regulated independently, and that at least a subpopulation of peripheral-type binding sites are associated with neuronal elements in the olfactory bulb. These data also suggest that peripheral-type binding sites for benzodiazepines in the olfactory bulbs may influence the convulsant actions of Ro 5-4864.

'Peripheral' binding sites for benzodiazepines are under neural or homonal control in the pineal gland, olfactory bulb, and kidney. These observations prompted a search for an endogenous substance which could modulate these sites under physiological conditions. Acidified methanol extracts from several tissues (e.g. stomach, kidney, lung) were found to inhibit the binding of [3H]Ro 5-4864 to 'peripheral' binding sites, but did not significantly affect the binding of [3H]diazepam to 'brain' benzodiazepine receptors. Fractionation of a crude extract prepared from antral stomach by either ultrafiltration or gel filtration chromatography yielded high (Mr greater than 10 000) and low (Mr less than 1000) Mr fractions which competitively inhibited [3H]Ro 5-4864 binding to 'peripheral' sites. These observations suggest the presence of endogenous substances in several rat tissues which may represent physiologically important ligands for 'peripheral' binding sites for benzodiazepines.

The effects of chemically and electrically-induced convulsions on the binding of [3H]Ro 5-4864 to peripheral benzodiazepine receptors (PBR) was studied in both peripheral tissues and the central nervous system (CNS). Acute, maximal electroshock (MES) increased the density of PBR in mouse cerebral cortex as evidenced by a 30% increase in the Bmax of this archetypic ligand. These values returned to control levels by 60 minutes after MES treatment. In contrast, thirty and sixty minutes after convulsions induced by Ro 5-4864, strychnine, or pentylenetetrazol, neither the Bmax nor Kd of [3H]Ro 5-4864 binding to mouse cerebral cortical membranes was altered. The increase in [3H]Ro 5-4864 binding to cortex observed 30 minutes after MES was blocked by anticonvulsant doses of phenobarbital, phenytoin and clonazepam. No changes in the characteristics of [3H]Ro 5-4864 binding was observed in cerebellar or hippocampal membranes 30 minutes following acute MES. Further, after long-term MES administration (1 treatment/day, 5 days), no change in PBR density could be detected 30 minutes after the last MES. Finally, while no change in PBR density was noted in the kidneys 30 minutes after the MES, a significant increase in PBR density was seen in the cardiac ventricles. These results demonstrate a selective modulation of PBR density by MES, suggesting that the PBR could be involved in either the generation of seizures or in postictal compensatory processes.