Caspase-3 activity is reduced after spinal cord injury in mice lacking dynorphin: differential effects on glia and neurons.

Dynorphins are endogenous opioid peptide products of the prodynorphin gene. An extensive literature suggests that dynorphins have deleterious effects on CNS injury outcome. We thus examined whether a deficiency of dynorphin would protect against tissue damage after spinal cord injury (SCI), and if individual cell types would be specifically affected. Wild-type and prodynorphin(-/-) mice received a moderate contusion injury at 10th thoracic vertebrae (T10). Caspase-3 activity at the injury site was significantly decreased in tissue homogenates from prodynorphin(-/-) mice after 4 h. We examined frozen sections at 4 h post-injury by immunostaining for active caspase-3. At 3-4 mm rostral or caudal to the injury, >90% of all neurons, astrocytes and oligodendrocytes expressed active caspase-3 in both wild-type and knockout mice. At 6-7 mm, there were fewer caspase-3(+) oligodendrocytes and astrocytes than at 3-4 mm. Importantly, caspase-3 activation was significantly lower in prodynorphin(-/-) oligodendrocytes and astrocytes, as compared with wild-type mice. In contrast, while caspase-3 expression in neurons also declined with further distance from the injury, there was no effect of genotype. Radioimmunoassay showed that dynorphin A(1-17) was regionally increased in wild-type injured versus sham-injured tissues, although levels of the prodynorphin processing product Arg(6)-Leu-enkephalin were unchanged. Our results indicate that dynorphin peptides affect the extent of post-injury caspase-3 activation, and that glia are especially sensitive to these effects. By promoting caspase-3 activation, dynorphin peptides likely increase the probability of glial apoptosis after SCI. While normally beneficial, our findings suggest that prodynorphin or its peptide products become maladaptive following SCI and contribute to secondary injury.

Glial-restricted precursors: patterns of expression of opioid receptors and relationship to human immunodeficiency virus-1 Tat and morphine susceptibility in vitro.

Recent evidence suggests that human immunodeficiency virus (HIV)-induced pathogenesis is exacerbated by opioid abuse and that the synergistic toxicity may result from direct actions of opioids in immature glia or glial precursors. To assess whether opioids and HIV proteins are directly toxic to glial-restricted precursors (GRPs), we isolated neural stem cells from the incipient spinal cord of embryonic day 10.5 ICR mice. GRPs were characterized immunocytochemically and by reverse transcriptase-polymerase chain reaction (RT-PCR). At 1 day in vitro (DIV), GRPs failed to express mu opioid receptors (MOR or MOP) or kappa-opioid receptors (KOR or KOP); however, at 5 DIV, most GRPs expressed MOR and KOR. The effects of morphine (500 nM) and/or Tat (100 nM) on GRP viability were assessed in GRPs at 5 DIV by examining the apoptotic effector caspase-3 and cell viability (ethidium monoazide exclusion) at 96 h following continuous exposure. Tat or morphine alone or in combination caused significant increases in GRP cell death at 96 h, but not at 24 h, following exposure. Although morphine or Tat caused increases in caspase-3 activity at 4 h, this was not accompanied with increased cleaved caspase-3 immunoreactive or ethidium monoazide-positive dying cells at 24 h. The results indicate that prolonged morphine or Tat exposure is intrinsically toxic to isolated GRPs and/or their progeny in vitro. Moreover, MOR and KOR are widely expressed by Sox2 and/or Nkx2.2-positive GRPs in vitro and the pattern of receptor expression appears to be developmentally regulated. The temporal requirement for prolonged morphine and HIV-1 Tat exposure to evoke toxicity in glia may coincide with the attainment of a particular stage of maturation and/or the development of particular apoptotic effector pathways and may be unique to spinal cord GRPs. Should similar patterns occur in vivo then we predict that immature astroglia and oligodendroglia may be preferentially vulnerable to HIV-1 infection or chronic opiate exposure.

Genetic background regulates semaphorin gene expression and epileptogenesis in mouse brain after kainic acid status epilepticus.

The host response to neural injury, which can include axonal sprouting and synaptic reorganization is likely to be under tight genetic regulatory control at the level of the genome and may be implicated in epileptogenesis. Despite its importance, however, the molecular basis of synaptic reorganization is unclear. We have studied the development of synaptic reorganization, semaphorin gene expression, and epileptogenesis in hippocampus of epileptogenic sensitive (FVB/NJ) and epileptogenic resistant (C57BL/6J) mice (i.e. distinct genetic backgrounds) after kainic acid-induced status epilepticus. Our results support the hypothesis that disruption of transcriptional regulation of axon guidance genes leads to a differential loss of tonic neuropilin-2 dependent activation of semaphorin 3F receptors on hippocampal neurons on distinct genetic backgrounds. This results in rearranged synaptic circuitry and thus promotes epileptogenesis. These findings may define biologic principles underlying the role of semaphorin signaling which may broadly apply to other systems undergoing neural regeneration.

Differential involvement of p38 and JNK MAP kinases in HIV-1 Tat and gp120-induced apoptosis and neurite degeneration in striatal neurons.

The role of p38 and c-jun-N-terminal kinases 1/2, members of the mitogen-activated protein kinase family, in mediating the toxic effects of human immunodeficiency virus-1 transactivator of transcription (Tat) and gp120 were explored in primary mouse striatal neurons in vitro. Both Tat and gp120 caused significant increases in p38 and c-jun-N-terminal kinase mitogen-activated protein kinase phosphorylation, caspase-3 activity, neurite losses and cell death in striatal neurons. Tat-induced increases in caspase-3 activity were significantly attenuated by an inhibitor of c-jun-N-terminal kinase (anthra[1,9-cd]pyrazol-6(2H)-one), but not by an inhibitor of p38 ([4-(4-fluorophenyl)-2-(4-methylsul-finylphenyl)-5-(4-pyridyl)1 H-imidazole]), mitogen-activated protein kinase. However, despite preventing increases in caspase-3 activity, c-jun-N-terminal kinase inhibition failed to avert Tat-induced neuronal losses suggesting that the reductions in caspase-3 activity were insufficient to prevent cell death caused by Tat. Alternatively, gp120-induced increases in caspase-3 activity, neurite losses and neuronal death were prevented by p38, but not c-jun-N-terminal kinase, mitogen-activated protein kinase inhibition. Our findings suggest that gp120 induces neuronal dysfunction and death through actions at p38 mitogen-activated protein kinase, while Tat kills neurons through actions that are independent of p38 or c-jun-N-terminal kinase mitogen-activated protein kinase, or through the concurrent activation of multiple proapoptotic pathways.

Molecular targets of opiate drug abuse in neuroAIDS.

Opiate drug abuse, through selective actions at mu-opioid receptors (MOR), exacerbates the pathogenesis of human immunodeficiency virus-1 (HIV-1) in the CNS by disrupting glial homeostasis, increasing inflammation, and decreasing the threshold for pro-apoptotic events in neurons. Neurons are affected directly and indirectly by opiate-HIV interactions. Although most opiates drugs have some affinity for kappa (KOR) and/or delta (DOR) opioid receptors, their neurotoxic effects are largely mediated through MOR. Besides direct actions on the neurons themselves, opiates directly affect MOR-expressing astrocytes and microglia. Because of their broad-reaching actions in glia, opiate abuse causes widespread metabolic derangement, inflammation, and the disruption of neuron-glial relationships, which likely contribute to neuronal dysfunction, death, and HIV encephalitis. In addition to direct actions on neural cells, opioids modulate inflammation and disrupt normal intercellular interactions among immunocytes (macrophages and lymphocytes), which on balance further promote neuronal dysfunction and death. The neural pathways involved in opiate enhancement of HIV-induced inflammation and cell death, appear to involve MOR activation with downstream effects through PI3-kinase/Akt and/or MAPK signaling, which suggests possible targets for therapeutic intervention in neuroAIDS.

Sensitization of enteric neurons to morphine by HIV-1 Tat protein.

BACKGROUND: Gastrointestinal (GI) dysfunction is a major cause of morbidity in acquired immunodeficiency syndrome (AIDS). HIV-1-induced neuropathogenesis is significantly enhanced by opiate abuse, which increases proinflammatory chemokine/cytokine release, the production of reactive species, glial reactivity, and neuronal injury in the central nervous system. Despite marked interactions in the gut, little is known about the effects of HIV-1 in combination with opiate use on the enteric nervous system. METHODS: To explore HIV-opiate interactions in myenteric neurons, the effects of Tat ± morphine (0.03, 0.3, and 3 µM) were examined in isolated neurons from doxycycline- (DOX-) inducible HIV-1 Tat(1-86) transgenic mice or following in vitro Tat 100 nM exposure (>6 h). KEY RESULTS: Current clamp recordings demonstrated increased neuronal excitability in neurons of inducible Tat(+) mice (Tat+/DOX) compared to control Tat-/DOX mice. In neurons from Tat+/DOX, but not from Tat-/DOX mice, 0.03 µM morphine significantly reduced neuronal excitability, fast transient and late long-lasting sodium currents. There was a significant leftward shift in V(0.5) of inactivation following exposure to 0.03 µM morphine, with a 50% decrease in availability of sodium channels at -100 mV. Similar effects were noted with in vitro Tat exposure in the presence of 0.3 µM morphine. Additionally, GI motility was significantly more sensitive to morphine in Tat(+) mice than Tat(-) mice. CONCLUSIONS & INFERENCES: Overall, these data suggest that the sensitivity of enteric neurons to morphine is enhanced in the presence of Tat. Opiates and HIV-1 may uniquely interact to exacerbate the deleterious effects of HIV-1-infection and opiate exposure on GI function.

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction.

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.

Autoradiographic studies of cerebellar histogenesis in the premetamorphic bullfrog tadpole: I. Generation of the external granular layer.

This study examines the time of origin of cells in the external granular layer (EGL) in the frog cerebellum during early stages of development. Premetamorphic bullfrog tadpoles were given multiple intraperitoneal injections of 3H-thymidine (10 microCi/g body weight per injection) at developmental stages ranging from 4 weeks to 1 year and were killed at either 6 or 12 months of age. Autoradiograms were analyzed to determine the time when cells of the EGL were generated by an examination of the labeling pattern in the neuroepithelial cap where EGL cells were presumably formed and in the EGL into which they migrated. The developmental stage of the cerebellum in the 6-month-old tadpole was essentially the same as that of the 12-month-old animal except for an increased size in the older tadpole. The cerebellum in both age groups contained a distinct neuroepithelial cap and an EGL, which was somewhat better formed in the 12-month-old tadpole. Some heavily labeled cells were found in the neuroepithelial caps of 6-month-old tadpoles from injection times of 6 weeks to 6 months. In the cerebella of 12-month-old tadpoles, however, heavily labeled cells were found in the neuroepithelial cap only with the injection time of 12 months; with injection times from 7 to 11 months, the cells were labeled lightly. Labeled EGL cells were found in the cerebella of 6-month-old tadpoles from an injection time of 6 weeks on; with injection times from 10 weeks to 6 months some EGL cells contained heavy amounts of label. In the cerebella of 12-month-old tadpoles, labeling of EGL cells was not detectable with injection times of 7-9 months; they contained light to medium labeling with injection times of 10 and 11 months and heavy labeling when injected at 12 months. These results indicate that EGL cells are generated continuously in premetamorphic tadpoles from the age of 6 weeks to 12 months. Furthermore, these results suggest that the rate of EGL cell formation is faster during the second half-year of development than during the first.

Stellate cell development in the frog cerebellum during spontaneous and thyroxine-induced metamorphosis.

Stellate cell development was studied in the bullfrog cerebellum during spontaneous and thyroxine-induced metamorphosis using the Golgi-Kopsch method and electron microscopy. Cells that possessed axosomatic synapses and resembled stellate cells were present even in the incipient molecular layer of the cerebellum in the premetamorphic tadpole. These cells may have originated from the early, transient wave of external granule cells that have been reported in the cerebellum of premetamorphic tadpoles in the first 6 months of development, and may constitute the variant population of stellate cells that are present later during development or the degenerating cells that have been observed during metamorphosis as scattered dying cells in the molecular layer. Typical stellate cells, whose development resembled the genesis and differentiation of stellate cells in birds and mammals, were initially observed at the outer border of the molecular layer that is adjacent to the external granular layer during the onset of metamorphosis. These stellate cells were bipolar with processes extending in a plane perpendicular to elongating parallel fibers, and with progressive development, became multipolar with dendrites oriented in various directions with respect to the pia. Stellate cell axons innervate the dendrites and somata of Purkinje cells and other stellate cells, and can be categorized into two types: (1) axons with extensive branching near the soma of origin, and (2) long axons with few branches that occasionally terminate in the Purkinje cell layer. Atypical neurons that did not resemble typical stellate cells were also present in the molecular layer; these might be classified as a stellate cell variant. The generation and differentiation of stellate cells can be induced 1 to 2 years prematurely by administering thyroid hormone to premetamorphic tadpoles. Like most events of cerebellar histogenesis in the frog, stellate cell development also appears to be largely a thyroid-dependent phenomenon.

Autoradiographic studies of cerebellar histogenesis in the premetamorphic bullfrog tadpole: II. Formation of the interauricular granular band.

This study examines the origin of cells in the interauricular granular band (iagb) in the cerebellum of the frog tadpole during early stages of development by means of histological and autoradiographic methods. Premetamorphic bullfrog tadpoles were exposed to multiple doses of 3H-thymidine (10 microCi/g body weight per exposure) at developmental stages ranging from 1 week to 1 year and were killed at either 6 or 12 months of age. The autoradiographic data were examined to determine the time when cells of the iagb were generated. Our findings show that initial generation of iagb cells begins at week 3 and that a peak in the formation of postmitotic neurons is reached at the age of 10 weeks. This is followed by other peaks of cell generation at the ages of 16 weeks, 10 months, and 11.5 months. The generation cycles of iagb cells are interrupted by periods of quiescence when label cannot be detected in any of the cells. These quiescent periods occur at the ages of 20-26 weeks, 7 months, and 12 months. These findings indicate that cells of the iagb are generated in a cyclical manner over the entire 1-year period which was studied. Comparison of our present data on iagb cell formation with the generation of cells in the EGL shows that the production of these two groups of cells is overlapping, but cells of the iagb begin and cease production before those of the EGL. On the basis of our findings we propose that the cells of the iagb and the EGL belong in separate cell groups which are generated by distinct subpopulations of germinal cells in the neuroepithelial cap.

Granule cell development in the frog cerebellum during spontaneous and thyroxine-induced metamorphosis.

Granule cell maturation in the cerebellum of bullfrog tadpoles was studied during both spontaneous and thyroxine-induced metamorphosis by using electron microscopy and Golgi-impregnated preparations. The production of cerebellar microneurons, a majority of which are granule cell precursors, was quantitatively compared during spontaneous and thyroxine-induced metamorphosis by using stereological methods and biochemical measurements of DNA. Granule cell migration and differentiation appeared morphologically similar during spontaneous and thyroxine-induced metamorphosis. In both instances, granule cells migrated tangentially along the pial surface, migrated into the internal granular layer, developed dendritic arbors, and formed synaptic contacts with the processes of Golgi cells and with mossy fibers. These events are similar to developmental processes that have been described in detail in other animals. Quantitative stereological measurements demonstrated similar overall patterns of change during spontaneous and thyroxine-induced metamorphosis. Most notably, increases in the volume of the external granule layer correlated with increases in the relative and total amounts of DNA. However, measurements of total DNA were consistently reduced during the period of accelerated change that occurs in thyroxine-induced metamorphosis, although external granular layer volume was greater in these tadpoles after 2 and 3 weeks of thyroxine treatment than in spontaneously metamorphosing tadpoles. While granule cell development in the frog is largely dependent on thyroid hormone, differences between thyroid-hormone-induced and spontaneously metamorphosing tadpoles suggest that normal patterns of cerebellar development are also dependent on events that occur in premetamorphic tadpoles in the absence of thyroid hormone.

Endogenous opioid systems and the regulation of dendritic growth and spine formation.

The role of endogenous opioid systems (endogenous opioids and opioid receptors) in neuronal development was examined in 10- and 21-day-old rats by utilizing an opioid antagonist (naltrexone) paradigm. Throughout the first 3 weeks of life, Sprague-Dawley rats were given daily subcutaneous injections of either 50 mg/kg naltrexone, a dosage that invoked a complete (24 hours/day) receptor blockade, or 1 mg/kg naltrexone, a dosage which intermittently blocked (4-6 hours/day) opioid receptors and exacerbated opioid action; animals injected with sterile water served as controls. Pyramidal cells from the frontoparietal cortex (layer III) and hippocampal field CA1, and cerebellar Purkinje cells, were impregnated by using the Golgi-Kopsch method; total and mean dendrite segment length, branch frequency, and spine concentration were analyzed morphometrically. Perturbations of endogenous opioid systems caused region-dependent alterations in dendrite complexity and/or spine concentration in all brain areas. Continuous opioid receptor blockade resulted in dramatic increases in dendrite and/or spine elaboration compared to controls at 10 days in all brain regions; however, these increases were only evident in the hippocampus at 21 days. With intermittent blockade, dendrite and/or spine growth were often subnormal, being predominant at day 21. Our results indicate that endogenous opioid systems are critical regulators of neuronal differentiation, and they control growth through an inhibitory mechanism. Considering previous findings demonstrating that neurobehavioral ontogeny is dependent on endogenous opioid-opioid receptor interactions, the present results suggest an opioid-dependent, structure-function relationship between neuronal and behavioral maturation.

Purkinje cell maturation in the frog cerebellum during thyroxine-induced metamorphosis.

Purkinje cell maturation during thyroxine-induced metamorphosis in premetamorphic bullfrog tadpoles was studied using electron microscopy and Golgi (silver-impregnated) preparations. Cerebella from tadpoles were examined following 1, 2, or 3 weeks of thyroxine treatment. Particular attention was paid to possible differences between the two populations of Purkinje cells previously described, i.e. (i) the smaller population located in the dorsal part of the cerebellum, where the Purkinje cells show dendritic arborization long before the appearance of the external granular layer, and (ii) the larger population located in the middle and ventral regions of the cerebellum, where the Purkinje cells begin to undergo maturation during metamorphosis when the external granular layer is established. Following thyroxine treatment, both populations of Purkinje cells showed rapid maturational change. In the mature (dorsal) group, dendritic growth resumed in the presence of an external granular layer increasing the complexity of their dendritic arbors. Moreover, climbing fiber synapses translocated from contacts on the soma to the thorns of growing dendrites, and somatic processes often disappeared. The immature (ventral) group showed dramatic differentiation of the perikaryon including polarization of cytoplasm with subsequent dendritic outgrowth and formation of somatic processes in the presence of climbing fibers. Stellate cell contacts appeared on the smooth portion of the soma of many Purkinje cells. Dendritic growth during thyroxine-induced metamorphosis was characterized by growth (elongation) with minimal branching, which is initially observed during spontaneous metamorphosis. Typically, these growing dendrites ended in growth cones, some with one or several filopodia. Developing Purkinje cell dendritic spines formed synapses with parallel fibers. The present study has provided an example of the dramatic nature of thyroxine's action in inducing the complex series of detailed maturational changes in the cerebellum 1-2 yr ahead of schedule. In addition, the results show that thyroxine-induced Purkinje cell maturation is more rapid and synchronous than that seen during spontaneous metamorphosis. It is concluded that Purkinje cell maturation during metamorphosis is largely dependent on thyroid hormone.

Dynorphin A toxicity in striatal neurons via an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor mechanism.

Dynorphin A (1-17) is an endogenous opioid peptide that is antinociceptive at physiological concentrations, but in excess can elicit a number of pathological effects. Both kappa-opioid and N-methyl-D-aspartate receptor antagonists modulate dynorphin toxicity, suggesting that dynorphin is acting directly or indirectly through these receptor types. We found in spinal cord neurons that the neurotoxic effects of dynorphin A and several dynorphin-derived peptide fragments are largely mediated by N-methyl-D-aspartate receptors. Despite these findings, aspects of dynorphin A toxicity could not be accounted for by opioid or N-methyl-D-aspartate receptor mechanisms. To address this issue, neurons enriched in kappa-opioid, N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors were isolated from embryonic day-15 mouse striata and the effects of extracellularly administered dynorphin A (1-17) and (13-17) on neuronal survival were examined in vitro. Unlike spinal cord neurons, N-methyl-D-aspartate receptors mature later than alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors in striatal neurons, thus providing a strategy to elucidate non-N-methyl-D-aspartate receptor-mediated mechanisms of toxicity. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. Dynorphin A (1-17 or 13-17; 10 microM) caused significant neuronal losses after 48 to 72 hours versus untreated controls. Dynorphin A or A (13-17) toxicity was unaffected by the opioid receptor antagonist naloxone (10 microM) or by dizocilpine (10 microM). In contrast, the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline- 2,3-dione (10 microM) significantly attenuated only dynorphin A (1-17)-induced neuronal losses and not that induced by dynorphin A (13-17). Dynorphin A (1-17) toxicity was accompanied by a proportional loss of R2 and R3 subunits of the AMPA receptor complex, but not non-N-methyl-D-aspartateR1, expressing neurons and was mimicked by the ampakine 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine. Although it is unclear whether dynorphin A activates alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors directly or indirectly via glutamate release, our culture conditions do not support glutamate retention or accumulation. Our findings suggest that dynorphin A (1-17) can exert toxic effects on striatal neurons via an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor mechanism.

Chronic nicotine exposure reduces N-methyl-D-aspartate receptor-mediated damage in the hippocampus without altering calcium accumulation or extrusion: evidence of calbindin-D28K overexpression.

Neuronal accumulation of excess Ca2+ has been implicated in cellular death following several forms of physical and chemotoxic insult. Recent studies have suggested that exposure to agonists at brain nicotinic acetylcholine receptors reduces cytotoxic consequences of increased intracellular Ca2+ following some insults. In the present study, the ability of chronic exposure to (-)-nicotine to reduce cytotoxicity and attenuate increases in intracellular Ca2+ caused by exposure to N-methyl-D-aspartate were examined in organotypic cultures of rat hippocampus. Cultures were exposed to nicotine (0.1-10.0 microM) for five days prior to excitotoxic insult with N-methyl-D-aspartate. Exposure to N-methyl-D-aspartate produced concentration-dependent increases in both accumulation of 45Ca and in early and delayed cell death in the CA1, CA3 and dentate gyrus regions of cultures. The CA1 region of the hippocampus displayed the greatest sensitivity to cytotoxic effects of N-methyl-D-aspartate exposure; however, this regional difference was not associated with increased accumulation of 45Ca. Prior exposure to nicotine markedly attenuated N-methyl-D-aspartate-induced early and delayed cell death in each hippocampal region at concentrations as low as 0.1microM. However, nicotine did not alter the initial N-methyl-D-aspartate-stimulated influx of 45Ca or enhance extrusion of accumulated 45Ca measured at several time-points after insult. Five days of exposure to nicotine markedly increased immunoreactivity of the Ca2+ binding protein calbindin-D28K in each region of hippocampal cultures, effects reduced by mecamylamine co-exposure. These findings suggest that the potent protective effects of chronic nicotine exposure against neuronal overexcitation are not likely attributable to attenuations of Ca2+ accumulation, but are likely related to increased buffering of accumulated Ca2+.

Synergistic neurotoxicity of opioids and human immunodeficiency virus-1 Tat protein in striatal neurons in vitro.

Human immunodeficiency virus (HIV) infection selectively targets the striatum, a region rich in opioid receptor-expressing neural cells, resulting in gliosis and neuronal losses. Opioids can be neuroprotective or can promote neurodegeneration. To determine whether opioids modify the response of neurons to human immunodeficiency virus type 1 (HIV-1) Tat protein-induced neurotoxicity, neural cell cultures from mouse striatum were initially characterized for mu and/or kappa opioid receptor immunoreactivity. These cultures were continuously treated with morphine, the opioid antagonist naloxone, and/or HIV-1 Tat (1-72) protein, a non-neurotoxic HIV-1 Tat deletion mutant (TatDelta31-61) protein, or immunoneutralized HIV-1 Tat (1-72) protein. Neuronal and astrocyte viability was examined by ethidium monoazide exclusion, and by apoptotic changes in nuclear heterochromatin using Hoechst 33342. Morphine (10nM, 100nM or 1microM) significantly increased Tat-induced (100 or 200nM) neuronal losses by about two-fold at 24h following exposure. The synergistic effects of morphine and Tat were prevented by naloxone (3microM), indicating the involvement of opioid receptors. Furthermore, morphine was not toxic when combined with mutant Tat or immunoneutralized Tat. Neuronal losses were accompanied by chromatin condensation and pyknosis. Astrocyte viability was unaffected. These findings demonstrate that acute opioid exposure can exacerbate the neurodegenerative effect of HIV-1 Tat protein in striatal neurons, and infer a means by which opioids may hasten the progression of HIV-associated dementia.

Dynorphin A (1-17) induces apoptosis in striatal neurons in vitro through alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor-mediated cytochrome c release and caspase-3 activation.

Dynorphin A (1-17), an endogenous opioid neuropeptide, can have pathophysiological consequences at high concentrations through actions involving glutamate receptors. Despite evidence of excitotoxicity, the basic mechanisms underlying dynorphin-induced cell death have not been explored. To address this question, we examined the role of caspase-dependent apoptotic events in mediating dynorphin A (1-17) toxicity in embryonic mouse striatal neuron cultures. In addition, the role of opioid and/or glutamate receptors were assessed pharmacologically using dizocilpine maleate (MK(+)801), a non-equilibrium N-methyl-D-aspartate (NMDA) antagonist; 6-cyano-7-nitroquinoxaline-2,3-dione, a competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate antagonist; or (-)-naloxone, a general opioid antagonist. The results show that dynorphin A (1-17) (>or=10 nM) caused concentration-dependent increases in caspase-3 activity that were accompanied by mitochondrial release of cytochrome c and the subsequent death of cultured mouse striatal neurons. Moreover, dynorphin A-induced neurotoxicity and caspase-3 activation were significantly attenuated by the cell permeable caspase inhibitor, caspase-3 inhibitor-II (z-DEVD-FMK), further suggesting an apoptotic cascade involving caspase-3. AMPA/kainate receptor blockade significantly attenuated dynorphin A-induced cytochrome c release and/or caspase-3 activity, while NMDA or opioid receptor blockade typically failed to prevent the apoptotic response. Last, dynorphin-induced caspase-3 activation was mimicked by the ampakine CX546 [1-(1,4-benzodioxan-6-ylcarbonyl)piperidine], which suggests that the activation of AMPA receptor subunits may be sufficient to mediate toxicity in striatal neurons. These findings provide novel evidence that dynorphin-induced striatal neurotoxicity is mediated by a caspase-dependent apoptotic mechanism that largely involves AMPA/kainate receptors.

Estrogen protects against the synergistic toxicity by HIV proteins, methamphetamine and cocaine.

BACKGROUND: Human immunodeficiency virus (HIV) infection continues to increase at alarming rates in drug abusers, especially in women. Drugs of abuse can cause long-lasting damage to the brain and HIV infection frequently leads to a dementing illness. To determine how these drugs interact with HIV to cause CNS damage, we used an in vitro human neuronal culture characterized for the presence of dopaminergic receptors, transporters and estrogen receptors. We determined the combined effects of dopaminergic drugs, methamphetamine, or cocaine with neurotoxic HIV proteins, gp120 and Tat. RESULTS: Acute exposure to these substances resulted in synergistic neurotoxic responses as measured by changes in mitochondrial membrane potential and neuronal cell death. Neurotoxicity occurred in a sub-population of neurons. Importantly, the presence of 17beta-estradiol prevented these synergistic neurotoxicities and the neuroprotective effects were partly mediated by estrogen receptors. CONCLUSION: Our observations suggest that methamphetamine and cocaine may affect the course of HIV dementia, and additionally suggest that estrogens modify the HIV-drug interactions.

Alterations within the endogenous opioid system in mice with targeted deletion of the neutral endopeptidase ('enkephalinase') gene.

The biological inactivation of enkephalins by neutral endopeptidase (enkephalinase, NEP, EC3.4.24.11) represents a major mechanism for the termination of enkephalinergic signalling in brain. A pharmacological blockade of NEP-activity enhances extracellular enkephalin concentrations and induces opioid-dependent analgesia. Recently, knockout mice lacking the enzyme NEP have been developed [Lu et al., J. Exp. Med. 1995;181:2271-2275]. The present study investigates the functional consequences and biochemical compensatory strategies of a systemic elimination of NEP activity in these knockout mice. Using biochemical and behavioural tests we found that the lack of NEP activity in brain is not compensated by enhanced activities of alternative enkephalin-degrading enzymes. Also no change in enkephalin biosynthesis was detectable by in situ methods quantifying striatal proenkephalin-mRNA levels in NEP-deficient mice compared with wildtype. Only a 21% reduction of mu receptor density in crude brain homogenates of NEP knockout mice was observed, while delta- and kappa-opioid receptor densities were unchanged. This receptor downregulation was also confirmed functionally in the hot-plate paradigm. NEP knockouts developed normally, but showed enhanced aggressive behaviour in the resident-intruder paradigm, and altered locomotor activity as assessed in the photobeam system. Thus, although NEP plays a substantial role in enkephalinergic neurotransmission, the biochemical adaptations within the opioid system of NEP-deficient mice are of only modest nature.

Co-localization of proenkephalin mRNA using cRNA probes and a cell-type-specific immunocytochemical marker for intact astrocytes in vitro.

To determine whether cultured astrocytes express opioid gene mRNA, a method was developed for co-localizing a cell-type specific immunocytochemical marker for astrocytes, glial fibrillary acidic protein (GFAP), and proenkephalin mRNA in situ hybridization signal using high affinity cRNA probes. GFAP immunoreactivity and proenkephalin mRNA hybridization reaction were examined in intact glial cell preparations from neonatal mice that were cultured for 4-6 days prior to fixation. The double labelling method described herein permits the unambiguous identification of mRNA expression in specific populations of intact cultured cells using cell type-specific markers.