Anthony S. Basile

Peripheral benzodiazepine receptors and mitochondrial function.

For over 20 years, numerous investigations have focused on elucidating the function of the peripheral benzodiazepine receptor (PBR). This relatively small protein (18kDa) arouses great interest because of its association with numerous biological functions, including the regulation of cellular proliferation, immunomodulation, porphyrin transport and heme biosynthesis, anion transport, regulation of steroidogenesis and apoptosis. Although the receptor was first identified as a binding site for the benzodiazepine, diazepam, in peripheral organ systems, the PBR was subsequently found to be distinct from the central benzodiazepine receptor (CBR) in terms of its pharmacological profile, structure, subcellular localization, tissue distribution and physiological functions. The PBR is widely expressed throughout the body, with high densities found in steroid-producing tissues. In contrast, its expression in the CNS is restricted to ependymal cells and glia. The benzodiazepine Ro5-4864 and the isoquinoline carboxamide PK11195 exhibit nanomolar affinity for the PBR, and are the archtypic pharmacological tools for characterizing the receptor and its function. Primary among these functions are its regulation of steroidogenesis and apoptosis, which reflect its mitochondrial localization and involvement in oxidative processes. This review will evaluate the basic pharmacology and molecular biology of the PBR, and highlight its role in regulating mitochondrial function, the mitochondrial transmembrane potential and its sensitivity to reactive oxygen species (ROS), and neurosteroid synthesis, processes relevant to the pathogenesis of a number of neurological and neuropsychiatric disorders.

Early increases in TNF-α, IL-6 and IL-1β levels following transient cerebral ischemia in gerbil brain

Abstract The effects of transient global ischemia using bilateral carotid artery occlusion on regional cytokine levels in gerbil brain were investigated using enzyme-linked immunoassay techniques. Brain concentrations of interleukin-6 (IL-6), interleukin-lβ (IL-1β), and tumor necrosis factor-α (TNF-α) were increased during the early recirculation period (

Activation of microglia by secreted amyloid precursor protein evokes release of glutamate by cystine exchange and attenuates synaptic function.

Microglial activation as part of a chronic inflammatory response is a prominent component of Alzheimers disease. Secreted forms of the β‐amyloid precursor protein (sAPP) previously were found to activate microglia, elevating their neurotoxic potential. To explore neurotoxic mechanisms, we analyzed microglia‐conditioned medium for agents that could activate glutamate receptors. Conditioned medium from primary rat microglia activated by sAPP caused a calcium elevation in hippocampal neurons, whereas medium from untreated microglia did not. This response was sensitive to the NMDA receptor antagonist, aminophosphonovaleric acid. Analysis of microglia‐conditioned by HPLC revealed dramatically higher concentrations of glutamate in cultures exposed to sAPP. Indeed, the glutamate levels in sAPP‐treated cultures were substantially higher than those in cultures treated with amyloid β‐peptide. This sAPP‐evoked glutamate release was completely blocked by inhibition of the cystine–glutamate antiporter by α‐aminoadipate or use of cystine‐free medium. Furthermore, a sublethal concentration of sAPP compromised synaptic density in microglia–neuron cocultures, as evidenced by neuronal connectivity assay. Finally, the neurotoxicity evoked by sAPP in microglia‐neuron cocultures was attenuated by inhibitors of either the neuronal nitric oxide synthase (NG‐propyl‐l‐arginine) or inducible nitric oxide synthase (1400 W). Together, these data indicate a scenario by which microglia activated by sAPP release excitotoxic levels of glutamate, probably as a consequence of autoprotective antioxidant glutathione production within the microglia, ultimately causing synaptic degeneration and neuronal death.

M2 Muscarinic Acetylcholine Receptor Knock-Out Mice Show Deficits in Behavioral Flexibility, Working Memory, and Hippocampal Plasticity

Muscarinic acetylcholine receptors are known to play key roles in facilitating cognitive processes. However, the specific roles of the individual muscarinic receptor subtypes (M1-M5) in learning and memory are not well understood at present. In the present study, we used wild-type (M2+/+) and M2 receptor-deficient (M2-/-) mice to examine the potential role of M2 receptors in learning and memory and hippocampal synaptic plasticity. M2-/- mice showed significant deficits in behavioral flexibility and working memory in the Barnes circular maze and the T-maze delayed alternation tests, respectively. The behavioral deficits of M2-/- mice were associated with profound changes in neuronal plasticity studied at the Schaffer-CA1 synapse of hippocampal slices. Strikingly, short-term potentiation (STP) was abolished, and long-term potentiation (LTP) was drastically reduced after high-frequency stimulation of M2-/- hippocampi. Treatment of M2-/- hippocampal slices with the GABAA receptor antagonist, bicuculline, restored STP and significantly increased LTP. Whole-cell recordings from CA1 pyramidal cells demonstrated a much stronger disinhibition of GABAergic than glutamatergic transmission in M2-/- hippocampi, which was particularly prominent during stimulus trains. Increased strength of GABAergic inhibition is thus a likely mechanism underlying the impaired synaptic plasticity observed with M2-/- hippocampi. Moreover, the persistent enhancement of excitatory synaptic transmission in CA1 pyramidal cells induced by the transient application of a low concentration of a muscarinic agonist (referred to as LTPm) was totally abolished in M2-/- mice. Because impaired muscarinic cholinergic neurotransmission is associated with Alzheimers disease and normal aging processes, these findings should be of considerable therapeutic relevance.

Deletion of the M5 muscarinic acetylcholine receptor attenuates morphine reinforcement and withdrawal but not morphine analgesia

Little is known about the physiological roles of the M5 muscarinic receptor, the last member of the muscarinic receptor family (M1–M5) to be cloned. In the brain, the M5 receptor subtype is preferentially expressed by dopaminergic neurons of the substantia nigra and the ventral tegmental area. Dopaminergic neurons located in the ventral tegmental area are known to play important roles in mediating both the rewarding effects of opiates and other drugs of abuse and the manifestations of opiate/drug withdrawal symptoms. We therefore speculated that acetylcholine-dependent activation of M5 receptors might modulate the manifestations of opiate reward and withdrawal. This hypothesis was tested in a series of behavioral, biochemical, and neurochemical studies using M5 receptor-deficient mice (M5−/− mice) as novel experimental tools. We found that the rewarding effects of morphine, as measured in the conditioned place preference paradigm, were substantially reduced in M5−/− mice. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M5−/− mice. In contrast, the analgesic efficacy of morphine and the development of tolerance to the analgesic effects of morphine remained unaltered by the lack of M5 receptors. The finding that M5 receptor activity modulates both morphine reward and withdrawal processes suggests that M5 receptors may represent a novel target for the treatment of opiate addiction.

Subcellular localization of "peripheral-type" binding sites for benzodiazepines in rat brain.

Abstract: The binding of [3H]Ro 5–4864, a specific ligand for “peripheral‐type” benzodiazepine binding sites and [3H]Ro 15–1788, a specific ligand for the central benzodiazepine receptors, was determined in subcellular fractions of rat brain. As previously reported, the highest levels of “peripheral‐type” benzodiazepine binding sites and benzodiazepine receptors were found in the crude P1, and P2 fractions, respectively. Purification of these crude fractions revealed that high levels of both [3H]Ro 5–4864 and [3H]Ro 15–1788 binding were present in the mitochondrial and synaptosomal fractions. In contrast, the purified nuclei and myelin contained low levels of both [3H]Ro 5–4864 and [3H]Ro 15–1788 binding.

Role for M5 muscarinic acetylcholine receptors in cocaine addiction.

Muscarinic cholinergic receptors of the M5 subtype are expressed by dopamine‐containing neurons of the ventral tegmentum. These M5 receptors modulate the activity of midbrain dopaminergic neurons, which play an important role in mediating reinforcing properties of abused psychostimulants like cocaine. The potential role of M5 receptors in the reinforcing effects of cocaine was investigated using M5 receptor‐deficient mice in a model of acute cocaine self‐administration. The M5‐deficient mice self‐administered cocaine at a significantly lower rate than wild‐type controls. In the conditioned place preference procedure, a classic test for evaluating the rewarding properties of drugs, M5‐deficient mice spent significantly less time in the cocaine‐paired compartment than control mice. Moreover, the severity of the cocaine withdrawal syndrome (withdrawal‐associated anxiety measured in the elevated plus‐maze) was significantly attenuated in mice lacking the M5 receptor. These results demonstrate that M5 receptors play an important role in mediating both cocaine‐associated reinforcement and withdrawal.

Mice transgenically overexpressing sulfonylurea receptor 1 in forebrain resist seizure induction and excitotoxic neuron death

The ability of the sulfonylurea receptor (SUR) 1 to suppress seizures and excitotoxic neuron damage was assessed in mice transgenically overexpressing this receptor. Fertilized eggs from FVB mice were injected with a construct containing SUR cDNA and a calcium-calmodulin kinase IIα promoter. The resulting mice showed normal gross anatomy, brain morphology and histology, and locomotor and cognitive behavior. However, they overexpressed the SUR1 transgene, yielding a 9- to 12-fold increase in the density of [3H]glibenclamide binding to the cortex, hippocampus, and striatum. These mice resisted kainic acid-induced seizures, showing a 36% decrease in average maximum seizure intensity and a 75% survival rate at a dose that killed 53% of the wild-type mice. Kainic acid-treated transgenic mice showed no significant loss of hippocampal pyramidal neurons or expression of heat shock protein 70, whereas wild-type mice lost 68–79% of pyramidal neurons in the CA1–3 subfields and expressed high levels of heat shock protein 70 after kainate administration. These results indicate that the transgenic overexpression of SUR1 alone in forebrain structures significantly protects mice from seizures and neuronal damage without interfering with locomotor or cognitive function.

Internucleosomal DNA fragmentation in gerbil hippocampus following forebrain ischemia.

Internucleosomal DNA fragmentation, the characteristics feature of programmed cell death, was demonstrated in gerbil hippocampus following 10 min of forebrain ischemia. Quantitative analysis revealed the presence of DNA fragments as early as 12 h after ischemia, reaching a maximum at 48 h. Measurable DNA fragmentation was still present in 3/3 subjects 96 h after the ischemic insult. In situ staining of hippocampus demonstrated pronounced DNA fragmentation that was localized in the CA1 region. The localization of fragmented DNA to the CA1 is consistent with the vulnerability of this layer to ischemic insult, and indicates that DNA fragmentation may be associated with the delayed loss of CA1 neurons in this model of forebrain ischemia.

Characterization of the hypnotic properties of oleamide.

While preliminary studies associated oleamide with sleep regulation, we now characterize the involvement of oleamide in sleep using a number of techniques. Peripheral administration of oleamide to rats dose dependently suppressed motor activity in the open field, with an ED50 of 17+/-1.5mg/kg for the decrease in distance traveled. Moreover, endogenous oleamide concentrations increased 3- to 4-fold in the cerebrospinal fluid of rats sleep-deprived for 6 h or longer. Oleamide also decreased sleep latency to 44-64% of control values without altering other sleep parameters. Unlike many putative endogenous sleep-inducing agents, oleamide potently induces behavioral and electroencephalographic manifestations of sleep. Moreover, its endogenous concentrations and temporal associations are consistent with previous reports of its enhancement of serotonergic and GABAergic neurotransmission, which may be involved in sleep induction.