AAV9-mediated erythropoietin gene delivery into the brain protects nigral dopaminergic neurons in a rat model of Parkinson's disease.

We have recently shown that intrastriatal injection of recombinant human erythropoietin (EPO) protects dopaminergic (DA) neurons in the substantia nigra (SN) from 6-hydroxydopamine (6-OHDA) toxicity in a rat model of Parkinson's disease. However, systemic administration of EPO did not protect nigral DA neurons, suggesting that the blood-brain barrier limits the passage of EPO protein into the brain. In the present study, we used an adeno-associated viral (AAV) serotype 9 (AAV9) vector to deliver the human EPO gene into the brain of 6-OHDA-lesioned rats. We observed that expression of the human EPO gene was robust and stable in the striatum and the SN for up to 10 weeks. EPO-immunoreactive (IR) cells were widespread throughout the injected striatum, and EPO-IR neurons and fibers were also found in the ipsilateral SN. Enzyme-linked immunosorbent assay and western blot analyses exhibited dramatic levels of EPO protein in the injected striatum. As a result, nigral DA neurons were protected against 6-OHDA-induced toxicity. Amphetamine-induced rotational asymmetry and spontaneous forelimb use asymmetry were both attenuated. Interestingly, we also observed that intrastriatal injection of AAV9-EPO vectors led to increased numbers of red blood cells in peripheral blood. This highlights the importance of using an inducible gene delivery system for EPO gene delivery.

Rapid changes in synaptic vesicle cytochemistry after depolarization of cultured cholinergic sympathetic neurons.

Sympathetic neurons taken from rat superior cervical ganglia and grown in culture acquire cholinergic function under certain conditions. These cholinergic sympathetic neurons, however, retain a number of adrenergic properties, including the enzymes involved in the synthesis of norepinephrine (NE) and the storage of measurable amounts of NE. These neurons also retain a high affinity uptake system for NE; despite this, the majority of the synaptic vesicles remain clear even after incubation in catecholamines. The present study shows, however, that if these neurons are depolarized before incubation in catecholamine, the synaptic vesicles acquire dense cores indicative of amine storage. These manipulations are successful when cholinergic function is induced with either a medium that contains human placental serum and embryo extract or with heart-conditioned medium, and when the catecholamine is either NE or 5-hydroxydopamine. In some experiments, neurons are grown at low densities and shown to have cholinergic function by electrophysiological criteria. After incubation in NE, only 6% of the synaptic vesicles have dense cores. In contrast, similar neurons depolarized (80 mM K+) before incubation in catecholamine contain 82% dense-cored vesicles. These results are confirmed in network cultures where the percentage of dense-cored vesicles is increased 2.5 to 6.5 times by depolarizing the neurons before incubation with catecholamine. In both single neurons and in network cultures, the vesicle reloading is inhibited by reducing vesicle release during depolarization with an increased Mg++/Ca++ ratio or by blocking NE uptake either at the plasma membrane (desipramine) or at the vesicle membrane (reserpine). In addition, choline appears to play a competitive role because its presence during incubation in NE or after reloading results in decreased numbers of dense-cored vesicles. We conclude that the depolarization step preceding catecholamine incubation acts to empty the vesicles of acetylcholine, thus allowing them to reload with catecholamine. These data also suggest that the same vesicles may contain both neurotransmitters simultaneously.

6-Hydroxydopamine: evidence for superoxide radical as an oxidative intermediate.

Superoxide dismutase inhibited the autoxidation of 6-hydroxydopamine as measured by the rate of formation of a quinone and the rate of oxygen consumption. These observations demonstrate the formation of the superoxide radical during the autoxidation process. This finding may be relevant to the mechanism of adrenergic nerve terminal degeneration caused by 6-hydroxydopamine.

Major innervation of newborn rat cortex by monoaminergic neurons.

A major monoaminergic innervation in infant rat neocortex, predominantly in layer IV, has been demonstrated by ultrastructural and biochemical studies after the administration of exogenous catecholamine precursors and congeners. One-third of all cortical synapses have an uptake-storage mechanism for catecholamines. In newborn cortex, the storage capacity for catecholamines is tenfold greater than the endogenous levels, and the uptake-storage mechanism matures earlier than the ability to synthesize neurotransmitter.

Is the autoxidation of catecholamines involved in ischemia-reperfusion injury?

The autoxidation of catecholamines has been proposed to be a source of oxygen radicals in ischemia-reperfusion injury. However, this autoxidation per se is extremely slow at physiological pH and therefore is unlikely to be a primary source of oxygen radicals in ischemia-reperfusion injury. On the other hand, oxygen radicals from catecholamines are more likely to arise through catalyzed oxidations involving enzymatic systems and/or metal ions. It is these latter reactions that may be of interest with respect to damage associated with ischemia-reperfusion injury.

Intermediates in the aerobic autoxidation of 6-hydroxydopamine: relative importance under different reaction conditions.

Autoxidation of 6-hydroxydopamine (6-OHDA) proceeds through a balanced network of: transition metal ions, superoxide, hydrogen peroxide, hydroxyl radicals, and other species. The contribution of each to the reaction mechanism varies dramatically depending upon which scavengers are present. The contribution of each propagating intermediate increases when the involvement of others is diminished. Thus, superoxide (which is relatively unimportant when metal ions can participate) dominates the reaction when transition metal ions are bound (especially at higher pH), and it becomes essential in the simultaneous presence of catalase plus chelators. Transition metal ions participate more if superoxide is excluded; hydrogen peroxide becomes more important if both .O2- and metal ions are excluded; and hydroxyl radicals contribute more to the reaction mechanism if both H2O2 and .O2- are excluded. Superoxide dismutase inhibited strongly, by two distinct mechanisms: a high affinity mechanism (less than 13% inhibition) at catalytically effective concentrations, and a low affinity mechanism (almost complete inhibition at the highest concentrations) which depends upon both metal binding and catalytic actions. In the presence of DETAPAC catalytic concentrations of superoxide dismutase inhibited by over 98%. Conversely, metal chelating agents inhibited strongly in the presence of superoxide dismutase. When present alone they stimulated (like EDTA), inhibited (like desferrioxamine), or had little effect (like DETAPAC). Catalase which stimulated slightly but consistently (less than 5%) when added alone, inhibited 100% in the presence of superoxide dismutase + DETAPAC. However, in the absence of DETAPAC, catalase decreased inhibition by superoxide dismutase, yielding a 100% increase in reaction rate. Hydroxyl scavengers (formate, mannitol or glucose) alone produced little or no (less than 10%) inhibition, but inhibited by 30% in the presence of catalase + superoxide dismutase. Paradoxically, they stimulated the reaction in the presence of catalase + superoxide dismutase + DETAPAC.

Monoaminergic presynaptic axons and dendrites in rat locus coeruleus seen in reconstructions of serial sections.

Locus coeruleus was studied in rats pretreated with intraventricularly administered 5-hydroxydopamine 1/2 to 3 hours prior to conventional aldehydeosmium fixation. Presynaptic profiles in locus coeruleus neuropil were classified according to the characteristics of their vesicle populations and other features, as in our previous report. Similar categories of endings were observed, and the sites of postsynaptic innervation were identical to those described previously, that is, a majority of synapses were made with dendrites between 0.5 and 2.5 micrometers in cross-sectional diameter, a significant proportion was seen ending onto somatic and dendritic spines, with a relative paucity of synapses made with spine-free membrane of somata and large dendritic trunks. There were no significant differences between different morphological categories of afferent terminals and their spatial distribution onto various postsynaptic targets on locus coeruleus neurons. In addition to various membrane-bound compartments of the cytoplasm, three categories of synaptic endings were labelled by the synaptic marker, while all others were unlabelled. One of these was identified previously as containing small, rounded synaptic vesicles and an unusually large number of large, dense core vesicles. The synaptic vesicles were lightly labelled with scattered, small, eccentrically placed opaque cores inside the synaptic vesicles, apparently randomly distributed throughout the terminal. This terminal is thought to be serotonergic. A second category of labelled synapse has been previously identified as that derived from small, unmyelinated axons having clusters of pleomorphic synaptic vesicles in which the vesicles are heavily labelled by 5-hydroxydopamine. These are believed to represent catecholaminergic synaptic endings derived from recurrent collaterals as well as extrinsic catecholaminergic innervation of locus coeruleus. A final category of heavily labelled profile was identified as presynaptic dendrites, which, along with recurrent catecholaminergic axon collaterals, probably play an important part in the intrinsic regulation of nucleus locus coeruleus. When 59 labelled synapses were examined in adjacent serial sections, every vesicle-containing profile was associated with a synaptic contact having characteristic membrane specializations. A similar result was obtained when 132 other unlabelled terminals of different categories were examined in serial sections.

Effect of dopaminergic drugs on enhancement of the binding of quinuclidinyl benzilate by phencyclidine in vivo.

Phencyclidine has been shown to enhance the specific binding of [3H]quinuclidinyl benzilate (QNB) in the brain of the mouse when both compounds are given in vivo. Since a variety of studies indicate that dopaminergic (DA) systems are involved in the action of phencyclidine, it seemed that DA systems might mediate the enhancement of the binding of QNB by phencyclidine. The results of the present studies demonstrate that the neurotoxin 6-hydroxydopamine, the DA D1 antagonist (R)-8-[chloro]-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1-H-3-benzazepine-7- ol) (SCH23390), the DA D1 agonist p-dibenzylamino benzonitrile (SKF38393) and the DA D2 antagonist spiperone, all failed to modify the ability of phencyclidine to enhance the accumulation of QNB in brain. Quinpirole, a DA D2 agonist diminished accumulation of QNB after phencyclidine, however, this effect was confounded by quinpirole decreasing the non-specific binding of QNB, as well as increasing concentrations of QNB in plasma, when given with phencyclidine. In contrast, haloperidol, a DA antagonist, decreased the enhanced specific binding of QNB after phencyclidine without any apparent confounding influences. Since haloperidol is also known to strongly inhibit the binding of ligands to the sigma receptors, it was hypothesized that the actions of phencyclidine and haloperidol on the binding of QNB in vivo may be through sigma receptors rather than DA systems.

Depletion of catecholamines in the brain of rats differentially affects stimulation of locomotor activity by caffeine, D-amphetamine, and methylphenidate.

The purpose of this study was to assess the role of catecholamines in brain, in the stimulation of locomotor activity, induced by caffeine, as compared to the psychomotor stimulants D-amphetamine and methylphenidate. Adult male rats were pretreated with either (1) 2.5 mg/kg (i.p.) reserpine, 24 hr prior to testing of locomotor activity, (2) 50 mg/kg (i.p.) alpha-methyl-para-tyrosine (AMPT) 6 hr and 2 hr prior to testing of locomotor activity, (3) 200 micrograms/rat (i.c.v.) 6-hydroxydopamine (6-OHDA), or 25 mg/kg (i.p.) desmethylimipramine (DMI) and 200 micrograms/rat 6-OHDA (i.c.v.), 6-8 weeks prior to testing. Each treatment group had a matched control group. Levels of catecholamines in the forebrain were determined in each of the treatment and corresponding control groups. All rats were tested with doses of caffeine, D-amphetamine and methylphenidate (excluding the 6-OHDA-treated animals), administered in random order intraperitoneally 35 min before locomotor activity was measured for 30 min. Pretreatment with either reserpine or AMPT attenuated the stimulation of locomotor activity induced by caffeine and D-amphetamine but not that induced by methylphenidate. The dose-response curve for amphetamine was shifted downward and to the right by reserpine but was flattened by AMPT. The dose-response curve for caffeine was displaced downward in a similar manner by both reserpine and AMPT. Treatment with 6-OHDA or DMI + 6-OHDA produced the expected changes in the content of catecholamines in brain, but failed to modify dose-response curves for caffeine or amphetamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracyclization rates of 6-hydroxydopamine and 6-aminodopamine analogs under physiological conditions.

It is agreed that the neurotoxic action of 6-hydroxydopamine and 6-aminodopamine is related to their ease of oxidation. The initial oxidation products, the p-quinone and p-quinone imine, readily undergo 1,2-intracyclization. These reactions could represent an important loss of active neurotoxic agent available uptake. A variety of substituted 6-aminodopamine analogs was prepared and their formal potentials and cyclization rates were measured accurately. The effect of the balance of ease of oxidation vs. rate of cyclization on their neurotoxicity was examined. The results are in general accord with in vivo lifetimes for 6-hydroxydopamine and 6-aminodopamine in rat caudate nucleus.

Isolation and identificaton of an in vivo reaction product of 6-hydroxydopamine.

The product of oxidized 6-OHDA and GSH reacted in vitro has been identified by a variety of chemical and physical methods to be 2,4,5-trihydroxy-6-S-(glutathionyl)phenethylamine. Its chemical properties show it easily undergoes a variety of oxidative condensations and polymerization. Its oxidized form, the p-quinone, can be identified in small quantities in rat brain and mouse brain 1-3 h after 6-OHDA injection. This is believed to be the first report of a chemically identified species resulting from the in vivo interaction of 6-OHDA with CNS tissue.

Chemical and biological studies of 1-(2,5-dihydroxy-4-methylphenyl)-2-aminopropane, an analogue of 6-hydroxydopamine.

Autoxidation of the bis(O-demethyl)-p-hydroquinone metabolite of the psychotomimetic amine 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) at pH 7.4 leads exclusively to a bicyclic imino quinone. This imino quinone is a good alkylating agent, forming covalent adducts via 1,4 addition to thiols. The autoxidation appears to be dependent on trace metal catalysis and is dramatically inhibited by components of the 10000g supernatant fraction of rabbit liver homogenates. Incubation of tritium-labeled hydroquinone with bovine serum albumin under oxidizing conditions leads to significant amounts of nonextractable radioactivity which presumably is dependent on imino quinone alkylation of nucleophilic functionalities present on macromolecules. Incubation of tritium-labeled DOM with rabbit microsomes in the presence of NADPH leads to irreversible binding of the label to macromolecular components of the microsomes. Since this binding is NADPH dependent, it is likely that metabolic conversion of DOM to the hydroquinone is involved. The imino quinone oxidation product is highly lypophilic and is capable of crossing the blood-brain barrier. Intravenous administration of tritium-labeled imino quinone to rats resulted in significant nonextractable radioactivity in brain tissue. These properties of the hydroquinone metabolite parallel those reported for the structurally related sympatholytic compound 6-hydroxydopamine and have led to the hypothesis that the psychotomimetic properties of DOM may be mediated through 6-hydroxydopamine-type interactions of the hydroquinone with important macromolecules in the brain.

Noradrenergic and non-noradrenergic nerves containing small granular vesicles in Auerbach's plexus of the guinea-pig: evidence against the presence of noradrenergic synapses.

The appearance and distribution of varicosities containing small granular vesicles in Auerbach's plexus of the guinea-pig ileum, distal colon and rectum has been studied with the electron-microscope. Two types of varicosity were recognised. The first type was located predominantly at the surface of the plexus and did not form synapses on intrinsic neurons. This type became labelled with 5-hydroxydopamine, a specific marker for noradrenergic axons, and was destroyed by 6-hydroxydopamine and extrinsic denervation, procedures which lead to degeneration of noradrenergic nerves in the gut. The second type formed axodendritic and axosomatic synapses on intrinsic neurons and the morphology of its synaptic vesicles differed subtly from that of the first type. The second type was unaffected by 5-hydroxydopamine, 6-hydroxydopamine, or extrinsic denervation. It is concluded that the two types of small granular vesicle-containing varicosities belong to different neurons and that the first type is noradrenergic. Noradrenergic varicosities do not, therefore, form synapses in Auerbach's plexus. This conclusion is in accord with the electrophysiological findings. The second type of small granular vesicle-containing varicosity is not noradrenergic although it was formerly thought to be so. It is intrinsic to the gut and is resistant to the serotoninergic neurotoxin, 5,6-dihydroxytryptamine.

The effect of delta1-tetrahydrocannabinol on the release of (3H-(-)-noradrenaline from the isolated vas deferens of the rat.

1 The amount of tritium released upon transmural stimulation of the isolated vas of the rat incubated with [(3)H]-Delta(1)-tetrahydrocannabinol (THC) (2.8 muM) was significantly greater than the non-stimulated overflow.2 There was no difference between non-stimulated overflow and the effect of transmural stimulation of vasa equilibrated with [(14)C]-sorbitol (6.5 mg/ml).3 The difference between non-stimulated and stimulated efflux from vasa equilibrated with [(3)H]-Delta(1)-THC was abolished in rats pretreated for 24 h with 6-hydroxydopamine (250 mg/kg).4 The amount of tritium released upon transmural stimulation of the vas incubated with [(3)H]-noradrenaline (6 muM) was significantly greater than the non-stimulated overflow.5 Delta(1)-THC (560 nM and 14 muM) caused a significant dose-dependent reduction of both non-stimulated and stimulated tritium efflux, especially the latter.

6-Hydroxydopamine releases iron from ferritin and promotes ferritin-dependent lipid peroxidation.

Iron was released from ferritin by the catecholamine analog, 6-hydroxydopamine. Iron release was more efficient under nitrogen than in air, suggesting that the hydroquinone has the major role in the process. Superoxide dismutase, alone or in combination with catalase, strongly inhibited 6-hydroxydopamine oxidation and greatly enhanced the amount of ferritin iron release. Catalase alone had a similar, but lesser effect. Iron released from ferritin accelerated the autoxidation of 6-hydroxydopamine. This occurred by a mechanism that was inhibited by a combination of catalase and a chelator, and to a lesser extent by superoxide dismutase. 6-Hydroxydopamine was a good promoter of metal-catalysed lipid peroxidation, and ferritin-iron participated in the process. Superoxide dismutase, and to a lesser extent catalase, stimulated peroxidation catalysed by adventitious levels of iron, but in the presence of ferritin, each enzyme was inhibitory. It appears that the greatly enhanced iron release seen under these conditions accelerated the autoxidation of 6-hydroxydopamine so that less was available to participate in peroxidative reactions. However, when 6-hydroxydopamine autoxidation was prevented by a combination of superoxide dismutase and catalase, lipid peroxidation was also inhibited, suggesting that some intermediate of autoxidation is a further requirement for the process.

Patterns of destruction of mouse neuroblastoma cells by extracellular hydrogen peroxide formed by 6-hydroxydopamine and ascorbate.

The patterns of the cytolytic effects of 6-hydroxydopamine (6-OHDA), with/without ascorbate, on C-1300 and three other cloned mouse neuroblastoma cell lines (N1E-115, NS-20, N-18) were studied in vitro. The sensitivity to 6-OHDA differed and the three cloned cell lines were more sensitive than the wild type C-1300 cell line. Ascorbate synergistically potentiated the cytolytic effect of 6-OHDA to all four cell lines. The 6-OHDA cytotoxicity was eliminated by the addition of exogenous catalase but not by addition of other oxygen free radical scavengers, thereby suggesting that the hydrogen peroxide formed might influence the cells, extracellularly. In addition, the critical time for tumor cell lysis was the first 60 min of the reaction. The cytotoxicity induced by the unmasked cyclophosphamide, 4-hydroperoxycyclophosphamide, was synergistically enhanced in the presence of a nontoxic concentration of 6-OHDA and ascorbate. These data suggest that reactive oxygen intermediates may prove to be a good tool for destroying neuroblastoma cells.

Ultrastructural evidence of direct synaptic contact of catecholamine terminals with oxytocin-containing neurons in the parvocellular portion of the rat hypothalamic paraventricular nucleus.

Ultrastructural evidence of the direct interaction between catecholamine (CA) terminals and oxytocin (OX)-containing neurons in the parvocellular portion of the rat hypothalamic paraventricular nucleus was demonstrated using immunohistochemistry combined with false transmitter (5-hydroxydopamine, 5-OHDA) histochemistry. At the parvocellular portion, 5-OHDA labelled CA terminals make synaptic contact with proximal dendrites or somas of OX-positive cells, suggesting that the ascending CA system monosynaptically regulates the extrahypothalamic OX system.

Distribution of tritium label in the neonate rat brain following intracisternal or subcutaneous administration of [3H]6-OHDA. An autoradiographic study.

The present report describes the distribution of tritium label after injection of newborn rats with [3H]6-hydroxydopamine ([3H]6-OHDA). The animals were injected either intracisternally (i.c.) or subcutaneously (s.c.), with or without pretreatment with nomifensine, which blocks the high-affinity uptake of both noradrenaline (NA) and dopamine (DA), and sacrificed at intervals from 40 min to 24 h post-injection (p.i.). In i.c. injected animals, tritium label is demonstrable as early as 40 min p.i. in neurons of all known NA and DA cell groups. In NA neurons, it is taken up into cell body, dendrites, preterminal and terminal axons. The intensity of neuronal labeling is highest within the first 4 h p.i. and decreases in most neurons with longer postinjection intervals. A significant proportion of both NA and DA neurons degenerate beginning 6 h p.i., the majority show morphological signs of the axon reaction 24 h p.i. Uptake of [3H]6-OHDA into serotonergic and non-catecholaminergic neurons is not demonstrable. [3H]6-OHDA is accumulated by the following extraneuronal cells of the CNS: ependymal cells, epithelial cells of the choroid plexus, subependymal macrophages, smooth muscle cells in the wall of large intraparenchymal blood vessels, meningeal cells and glial cells. The time course of accumulation and disappearance of the label varies among these extraneuronal elements. The meningeal cells show the highest labeling intensity and degenerate within 24 h p.i. After pretreatment of the animals with nomifensine, the uptake of [3H]6-OHDA into NA and DA neurons is totally blocked; by contrast uptake of the labeled drug into extraneuronal cells is not prevented. These findings show that [3H]6-OHDA is not only accumulated by neurons possessing the high-affinity uptake for NA or DA, but by numerous other, extraneuronal cells which also participate in the metabolism of catecholamines.

Catecholaminergic axo-axonic synapses in the nucleus of the tractus solitarius (pars commissuralis) of the cat: possible relation to presynaptic regulation of baroreceptor reflexes.

A study was performed on adult cats to investigate synaptic structures of nerve terminals in the commissural portion of the nucleus of the tractus solitarius (NTS); a site where the cardiovascular afferent fibers terminate and where a dense plexus of adrenergic element also resides. Synaptic contacts observed in the commissural portion were predominantly axo-dendritic but a smaller number of axo-somatic synapses were also recognized. A third type of junction, axo-axonic, appears to be unique to the commissural portion of the nucleus, since no axo-axonic contacts were encountered in the medial and ventrolateral portions of the NTS. By labeling with the 'false' neurotransmitter 5-hydroxydopamine (5-OHDA), three types of axo-axonic contacts with different presynaptic components could be identified; namely, axon varicosities with small clear spherical vesicles, axon varicosities containing flat synaptic vesicles and others with small cord vesicles labeled with 5-OHDA. Postsynaptic components were always axon varicosities with clear spherical vesicles. After surgical denervation of the IXth and Xth cranial nerves, no degenerated axon varicosities were observed in the presynaptic side in the axo-axonic contact. The present study clearly demonstrates that the commissural portion of the NTS contains axo-axonic synapses and some of the adrenergic axons in this area provide the presynaptic components for this type of synapse. Possible roles of adrenergic nerves to presynaptic regulation of baroreceptor reflexes are discussed.

Spatial distribution of dopamine, methotrexate and antipyrine during continuous intracerebral microperfusion.

The spatial distributions of 3 model drugs, [14C]dopamine hydrochloride (DA), [3H]sodium methotrexate (MTX) and [14C]antipyrine (AP), were determined after 6 days continuous microperfusion of the diencephalon of rabbits. The basic pattern of distribution was the same for each drug: the tissue concentration, expressed as a function of the perfusate concentration, was maximal at the cannula tip, and declined sharply with radial distance from the tip. However, at any given distance, concentrations of radioisotopes derived from DA and MTX, both ionized, lipid-insoluble drugs, were one to two orders of magnitude higher than those derived from AP, a lipid-soluble drug. Although concentrations of isotopes derived from DA and MTX were in the same range, their distribution patterns were consistently different. The results demonstrate that intracerebrally microperfused drugs may have quantitatively different spatial distributions, related to their physicochemical characteristics and/or their binding and metabolism in brain tissue.