Intracerebroventricular Administration of l-Buthionine Sulfoximine: A Method for Depleting Brain Glutathione

Sprague-Dawley rats (200-260 g) were anesthetized with chloral hydrate (400 mg/kg) and polyethylene cannulae were permanently implanted into the lateral ventricles. One or two days later, L-buthionine-[S,R]-sulfoximine (L-BSO), an apparently selective inhibitor of gamma-glutamylcysteine synthetase, was administered intracerebroventricularly through the cannulae. The brain content of glutathione (GSH) was determined by HPLC with electrochemical detection (gold/mercury electrode) using N-acetylcysteine as internal standard. A time-course study of the changes in the striatum following a single dose of L-BSO (3.2 mg) revealed a maximal depletion of GSH (-60%) approximately 48 h after the administration. The effects of various doses of L-BSO on GSH in the striatum, in the limbic region, and in the cortex were assessed at 24 h and 48 h after the administration. L-BSO (0.02-3.2 mg) produced dose-dependent reductions of GSH in all brain regions studied at both time intervals. In a long-term experiment L-BSO (3.2 mg) was administered every second day. After 4 days, i.e., after two injections, striatal GSH was reduced by approximately 70%. No further depletion of GSH was obtained by additional injections of L-BSO, but GSH was maintained at this low level for the 12 days studied. These results suggest that L-BSO, administered intracerebroventricularly, would serve as a useful tool for evaluation of the biological role of GSH in the CNS.     

Changes induced by 6-hydroxydopamine in the paraventricular nucleus

Summary Remodelling of catecholaminergic (CA) fibers after cerebral intraventricular 6-hydroxydopamine (6-OH-DA) administration was evaluated quantitatively in the paraventricular nucleus (PAR) of young adult rats, using fluorescence microscopy (FM) and electron microscopy (EM). Fluorescent CA varicosities and CA boutons (marked with 5-OH-DA) were counted after survival periods of 4, 21, 56 or 180 days. Four days after 6-OH-DA treatment, the number of fluorescent varicosities dropped to 45% of control numbers but was restored to 79% of control values by 180 days. In the EM study, marked boutons had dropped more dramatically: to 12% of control numbers, after 4 days and 54% by 180 days post-neurotoxin. These data provide strong evidence that substantial but incomplete restoration of CA terminals occurred in PAR. It is of interest that, in all survival intervals, percentage reductions in numbers of CA terminals were more extreme when EM was used for quantification. Nevertheless, the trends indicating partial restoration of terminal numbers with time were parallel in the FM and EM studies. Structures identified as CA growth cones in PAR contained a feltwork of fine filaments together with mitochondria, granular vesicles (often with electron-dense cores marked by the 5-OH-DA label), vacuoles and smooth-surfaced reticulum. The presence of growth cones, some of which persisted 11 months after neurotoxin administration, further supports the inference that a regenerative response of CA elements was evoked in PAR by the 6-OH-DA treatment.Presented in part at IV International Catecholamine Symposium in California, September 1978     

Differential regulation of glutamate, aspartate and gamma-amino-butyrate release by N-methyl-D-aspartate receptors in rat striatum after partial and extensive lesions to the nigro-striatal dopamine pathway.

The in vivo microdialysis methodology was used to assess the effect of N-methyl-D-aspartate (NMDA) receptor ligands on glutamate (GLU), aspartate (ASP) and gamma-aminobutyrate (GABA) extracellular levels in the striatum of anaesthetized rats, after damage to the dopamine (DA) nigrostriatal pathway by injections of different doses of 6-hydroxydopamine (6-OH-DA) seven days earlier. The 6-OH-DA treated rats were divided into two groups, corresponding to animals with 20-80% (partial) and 85-99% (extensive) striatal DA tissue depletion, respectively. In rats with partial DA depletion, the striatal extracellular ASP levels significantly increased after intrastriatal dialysis perfusion with MK-801 (100 microM), an antagonist of NMDA receptors. In addition, a change in the pattern of local NMDA (500 microM)- induced efflux of ASP was observed in the striatum of these rats. However, in these partially DA-depleted striata no changes were found in basal extracellular levels of GLU, ASP and GABA or in NMDA- and MK-801-mediated effluxes of GLU and GABA relative to striata from sham rats. In contrast, rats with extensive striatal DA depletion exhibited a significant increase in ASP and GABA extracellular striatal levels, after intrastriatal dialysis perfusion with NMDA. In addition, the MK-801-mediated stimulation of extracellular ASP levels was accentuated along with the appearance of a MK-801 mediated increase in extracellular striatal GLU. Finally, basal extracellular levels of ASP, but not of GLU and GABA, were found to increase in extensive DA-depleted striata when compared to sham and partially DA-depleted striata. Thus, a differential regulation of basal and NMDA receptor-mediated release of transmitter amino acids occur seven days after partial and extensive DA-depleted striatum by 6-OH-DA-induced lesions of the nigrostriatal DA pathway. These findings may have implications as regards the participation of NMDA receptors in the compensatory mechanisms associated with the progress of Parkinson's disease, as well as in the treatment of this neurological disorder.     

Oxidative stress and pro-apoptotic conditions in a rodent model of Wilson's disease

Wilson's disease (WD) is an inherited disorder, characterized by selective copper deposition in liver and brain, chronic hepatitis and extra-pyramidal signs. In this study, we investigated changes of biochemical markers of oxidative stress and apoptosis in liver, striatum and cerebral cortex homogenates from Long-Evans Cinnamon (LEC) rats, a mutant strain isolated from Long Evans (LE) rats, in whom spontaneous hepatitis develops shortly after birth. LEC and control (LE) rats at 11 and 14 weeks of age were used. We determined tissue levels of glutathione (GSH/GSSG ratio), lipid peroxides, protein-thiols (P-SH), nitric oxide metabolites, activities of caspase-3 and total superoxide-dismutase (SOD), striatal levels of monoamines and serum levels of hepatic amino-transferases. We observed a decrease of protein-thiols, GSH/GSSG ratio and nitrogen species associated to increased lipid peroxidation in the liver and striatum - but not in the cerebral cortex - of LEC rats, accompanied by dramatic increase in serum amino-transferases and decrease of striatal catecholamines. Conversely, SOD and caspase-3 activity increased consistently only in the cortex of LEC rats. Hence, we assume that enhanced oxidative stress may play a central role in the cell degeneration in WD, at the main sites of copper deposition, with discrete pro-apoptotic conditions developing in distal areas.     

Depletion of Brain Glutathione in Preweanling Mice by L-Buthionine Sulfoximine

Previous studies indicated that DL-buthionine sulfoximine (DL-BSO), an agent that inhibits the biosynthesis of GSH in liver and other peripheral organs, fails to suppress levels of GSH in the CNS. In the current study, preweanling mice responded to repeated injections of L-BSO with marked declines (79.6-86.5%) of GSH content in brain and spinal cord. In adult mice, the same treatment schedule produced only modest declines (17.8-29.2%) of GSH content in brain and a 55.9% decline in spinal cord. Pretreatment of preweanling mice with L-BSO represents a tool for studying the role of GSH in the CNS.     

Glutathione status in constituted physiological fluids containing albumin

1. Glutathione (GSH) and cysteine, added to the constituted incubation medium, rapidly disappeared from the medium in the presence of bovine serum albumin (BSA). The major portions of added GSH and cysteine were oxidized. Only a fraction was recovered as cysteine-GSH mixed disulfide in case of GSH. About 15-30% cysteine or GSH were not recovered in the media. 2. The rate of GSH oxidation was linear with time, however, GSH disappearance was not linear with GSH concentrations. 3. Oxidation of GSH to GSSG in the albumin supplemented media was greater under O2 atmosphere, but was significantly decreased under N2 atmosphere. 4. Catalase, a peroxy radical scavenger, but not dimethyl pyroline N-oxide (DMPO), N-tertbutyl-2(-2 sulfophenyl)-nitrone (NTBSPN), mannitol or superoxide dismutase (SOD), decreased BSA mediated GSH oxidation. 5. GSH oxidation was abolished when mono- or divalent metal ions were absent in the BSA supplemented media. 6. Alkaline pH favored and acidic pH inhibited GSH oxidation. GSH oxidation was maximum above pH 7.4. GSH oxidation was minimal in the media containing boiled BSA. 7. A reaction mechanism involving the mixed GSH-BSA disulfide formation, followed by the reduction of these disulfides by GSH and subsequent release of GSSG is proposed.     

Role of vicinal protein thiols in radiation and cytotoxic responses

Glutathione (GSH) and more recently protein thiols (P-SH) have been found to play a major role in cellular radiation response. However, the effects of protein vicinal thiols, which are important for the functions of several major enzymes, on cellular responses to radiation have not been clearly delineated. Here we investigated the effects of depleting GSH and protein vicinal thiols (HS-P-SH) and P-SH on cell toxicity and radiation response. We used hydroxyethyldisulfide (HEDS, beta-mercaptoethanol-disulfide) alone and in combination with phenylarsine oxide (PAO) to alter P-SH, HS-P-SH and GSH. HEDS, a direct substrate for thioredoxin reductase and an indirect substrate for glutaredoxin (thioltransferase), did not alter protein vicinal thiols in cells. However, PAO, which specifically forms a covalent adduct with vicinal thiols, blocked bioreduction of HEDS; there was a concomitant and yet unexplained decrease in K1 cell GSH in the presence of HEDS and PAO. G6PD+ (K1) and G6PD- (E89) cells treated with L-buthionine sulfoximine (L-BSO) for 72 h to deplete GSH followed by PAO showed an increased cytotoxic response. However, the surviving E89 cells showed a 10,000-fold greater radiation lethality than the K1 cells. The effects of rapid depletion of GSH by a combination of L-BSO and dimethyfumarate (DMF), a glutathione-S-transferase substrate, were also investigated. Under these conditions, PAO radiosensitized the E89 cells more than 1000-fold over the K1 cells. The potential mechanisms for the altered response may be related to the inhibition of thioredoxin reductase and glutaredoxin. Both are key enzymes involved in DNA synthesis, protein homeostasis and cell survival. With GSH removed, vicinal thiols appear to play a critical role in determining cell survival and radiosensitivity. Decreasing P-SH and removing GSH and vicinal thiols is extremely toxic to K1 and E89 cells. We conclude that radiation sensitivity and cell survival are dependent on vicinal thiol and GSH. In the former and latter cases, the protein thiols are also important.     

The possible role of estrogen and selective estrogen receptor modulators in a rat model of Parkinson's disease

AimThe aim of the present study was to assess and compare the effect of 17β-estradiol and two different selective estrogen receptor modulators (SERMs), tamoxifen and raloxifene, as well as a selective estrogen receptor alpha agonist, propyl-pyrazole-triol (PPT) and a selective estrogen receptor beta agonist, diarylpropionitrile (DPN), on behavioral and biochemical alterations in 6-hydroxydopamine (6-OHDA)-induced nigral dopaminergic cell death in rats.Main methods80 female Wister rats were used. Animals were divided into eight equal groups: Group I; Sham operated, Group II; subjected to ovariectomy (OVX), Group III; OVX rats received striatal injection of 6-OHDA, Groups IV–VIII; OVX rats received striatal injection of 6-OHDA and were injected daily with 17β-estradiol, tamoxifen, raloxifene, PPT and DPN respectively for 5 days before 6-OHDA and continued for further 2 weeks.Key findingsResults showed that striatal injection of 6-OHDA produced significant behavioral alteration suggestive of PD, together with significant decrease in striatal dopamine, homovanillic acid (HVA) and 3,4-dihydroxyphenyl acetic acid (DOPAC) concentrations. 6-OHDA-induced nigral dopaminergic cell death was characterized by oxidative stress, evidenced by significant decrease in striatal glutathione peroxidase activity, as well as apoptosis, evidenced by significant increase in nigral caspase-3 activity. Treatment with 17β-estradiol, raloxifene, PPT, but neither tamoxifen nor DPN, resulted in significant amelioration of the behavioral and biochemical alterations induced by 6-OHDA.SignificanceThese findings suggest that estrogen and some SERMs having estrogenic agonist activity in the brain, like raloxifene, might exert beneficial effect in PD.     

Chronic depletion of glutathione (GSH) and minimal modification of LDL in vivo: its prevention by glutathione mono ester (GME) therapy

A decline in reduced glutathione (GSH) level is associated with aging and free radical mediated diseases. The objective of this study was to determine whether the chronic depletion of extra cellular GSH causes oxidative damage to the circulating macromolecules such as lipoproteins. Decreased concentrations of plasma glutathione, vitamin E and ascorbic acid were recorded in the rats treated with buthionine sulfoximine (BSO), a selective GSH inhibitor. In LDL isolated from BSO-treated animals, the concentration of malondialdehyde (MDA) and conjugated dienes were significantly increased (P<0.01), whereas the levels of vitamin E were decreased (P<0.01). The analysis of total and LDL cholesterol revealed significant changes between the control and experimental groups. Of interest, altered concentrations of lyso-phosphatidyl choline (Lyso-PC) and phosphatidyl choline (PC) were recorded from the BSO mediated minimally modified LDL. A negative correlation between LDL-BDC/MDA and its antioxidant capacity was noted. Upon in vitro oxidation with CuSO(4), the electrophoretic behavior of purified LDL-apoprotein-B on agarose gel showed an increased mobility in BSO-treated rats, indicative of in vivo modification of LDL to become susceptible for in vitro oxidation. The increased mobility of LDL (after in vitro oxidation) isolated from the BSO-treated animals correlates with a decrease in its amino groups, as determined by the trinitrobenzene sulfonic acid (TNBS) reactants. However, the mobility of LDL molecule was not altered due to BSO treatment in vivo. Interestingly, the minimal modification on LDL does not lead to any vascular damage in the dorsal aorta of the rats injected with BSO. The administration of glutathione monoester (GME), at a dose of 5 mmol/kg body weight, twice a day, for 30 days, to animals treated with l-buthionine-SR-sulfoximine (BSO, 4 mmol/kg body weight, twice a day, for 30 days) normalized the antioxidant status and prevented the minimal modifications on LDL. Thus, increasing the cellular GSH levels may trigger beneficial effects against oxidative stress.     

Glutathione loading prevents free radical injury in red blood cells after storage

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity^[1]. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1–6°C for 0, 42 and 84 days in a conventional additive solution (Adsol®) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4–6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (¹⁴C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (～ 50 %), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.     

Glutathione loading prevents free radical injury in red blood cells after storage

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity^[1]. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1-6°C for 0, 42 and 84 days in a conventional additive solution (Adsol®) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4-6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (¹⁴C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (∼ 50 %), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.     

Toxic and Protective Effects of l-DOPA on Mesencephalic Cell Cultures

Abstract: The autoxidation of L-DOPA or dopamine (DA) and the metabolism of DA by monoamine oxidase generate a spectrum of toxic species, namely, hydrogen peroxide, oxy radicals, semiquinones, and quinones. When primary dissociated cultures of rat mesencephalon were incubated with L-DOPA (200 μ M ) for 48 h, the number of tyrosine hydroxylase-positive neurons (DA neurons) was reduced to 69.7% of control values, accompanied by a decrease in [³H]DA uptake to 42.3% of control values; the remaining DA neurons exhibited reduced neurite length and overall deterioration. Lack of simultaneous change in the number of neurons stained with neuron-specific enolase indicated that toxicity was relatively specific for DA neurons. At the same time, the level of GSH, a major cellular antioxidant, rose to 125.2% of control values. Thus, exposure of mesencephalic cultures to L-DOPA results in both damaging and antioxidant actions. Ascorbate (200 μ M ), an antioxidant, prevented the rise in GSH. The effect of ascorbate on GSH points to an oxidative signal to initiate the rise in GSH content. On the other hand, neither inhibition of monoamine oxidase with pargyline nor addition of superoxide dismutase or catalase to the culture medium prevented the rise in GSH level or the loss in [³H]DA uptake. The latter results tend to exclude the products of monoamine oxidase activity or the presence of hydrogen peroxide or superoxide in the medium as responsible agents for the rise in GSH or neuronal toxicity. In cultures treated with L-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, l-DOPA prevented cell death by L-BSO.     

Oxidation of DJ-1 induced by 6-hydroxydopamine decreasing intracellular glutathione

DJ-1, the causative gene of a familial form of Parkinson's disease (PD), has been reported to undergo preferential oxidation of the cysteine residue at position 106 (Cys-106) under oxidative stress; however, details of the molecular mechanisms are not well known. In the present study, mechanisms of DJ-1 oxidation induced by 6-hydroxydopamine (6-OHDA) were investigated by using SH-SY5Y cells. The treatment of these cells with 6-OHDA caused an obvious acidic spot sift of DJ-1 due to its oxidation. However, when catalase, which is an hydrogen peroxide (H(2)O(2))-removing enzyme, was added during the treatment, it failed to prevent the oxidation induced by 6-OHDA, suggesting that electrophilic p-quinone formed from 6-OHDA, but not H(2)O(2), was responsible for the DJ-1 oxidation. Benzoquinone, another electrophilic p-quinone, also induced DJ-1 oxidation. The intracellular glutathione (GSH) levels were significantly decreased by 6-OHDA, irrespective of the presence or absence of catalase. The inhibition of GSH synthesis by buthionine sulfoximine resulted in a decrease in GSH levels and enhancement of DJ-1 oxidation. The pretreatment of cells with N-acetyl-cysteine prevented the loss of intracellular GSH and subsequently DJ-1 oxidation induced by 6-OHDA. Collectively, these results suggest that electrophilic p-quinone formed from 6-OHDA induces DJ-1 oxidation by decreasing intracellular GSH.     

Lipid Peroxidation Scavengers Prevent the Carbonylation of Cytoskeletal Brain Proteins Induced by Glutathione Depletion

In this study, we investigated the possible link between lipid peroxidation (LPO) and the formation of protein carbonyls (PCOs) during depletion of brain glutathione (GSH). To this end, rat brain slices were incubated with the GSH depletor diethyl maleate (DEM) in the absence or presence of classical LPO scavengers: trolox, caffeic acid phenethyl ester (CAPE), and butylated hydroxytoluene (BHT). All three scavengers reduced DEM-induced lipid oxidation and protein carbonylation, suggesting that intermediates/products of the LPO pathway such as lipid hydroperoxides, 4-hydroxynonenal and/or malondialdehyde are involved in the process. Additional in vitro experiments revealed that, among these products, lipid hydroperoxides are most likely responsible for protein oxidation. Interestingly, BHT prevented the carbonylation of cytoskeletal proteins but not that of soluble proteins, suggesting the existence of different mechanisms of PCO formation during GSH depletion. In pull-down experiments, beta-actin and alpha/beta-tubulin were identified as major carbonylation targets during GSH depletion, although other cytoskeletal proteins such as neurofilament proteins and glial fibrillary acidic protein were also carbonylated. These findings may be important in the context of neurological disorders that exhibit decreased GSH levels and increased protein carbonylation such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis.     

Regeneration of monoamine-containing axons in the developing and adult spinal cord of the rat following intraspinal 6-OH-dopamine injections or transections

Summary Growth of descending noradrenaline (NA) and 5-hydroxytryptamine (5-HT) axons in the rat spinal cord during ontogenesis and following mechanical or chemical, 6-hydroxydopamine (6-OH-DA) induced, axotomy, was studied with the Falck-Hillarp histochemical fluorescence method for monoamines.The major NA and 5-HT axon bundles and terminal innervation areas are present already at birth and an essentially mature pattern of innervation is reached after two weeks.Complete degeneration of both 5-HT and NA nerves in the distal segment is obtained by a transection of the spinal cord. Sprouting of the cut monoamine fibers into the necrotic zone and scar tissue is vigorous in both immature and mature animals, but regeneration into the distal segment is very poor.Selective degeneration of the descending NA axons and terminals is obtained by a localized intraspinal 6-OH-DA injection. Thus, the 5-HT fiber systems as well as all other parts of the spinal cord are left intact. The method should therefore prove useful for evaluating the exact functional role of the NA and 5-HT neuron systems in the spinal cord.Reinnervation of the distal part of the spinal cord by new NA fibers following 6-OH-DA induced denervation is described. This process is faster in younger animals but takes place also in adult animals. The present evidence suggests that reinnervation mainly is the result of downgrowth of the axotomized fibers, but growth in the form of collateral sprouting from a few possibly surviving fibers in the distal region may also contribute. Reinnervation lead to a normal innervation pattern within 1–2 months in the various age groups.It is suggested that the poor regeneration of many spinal nerve tracts often reported in the literature following transection of the spinal cord is due to extraneuronal factors such as scar tissue and impaired circulation rather than to the nerves per se since reinnervation by NA nerves was very poor following mechanical transection but good following chemical, 6-OH-DA-induced axotomy.     

Effects of systemic administration of 6-hydroxydopamine on the circumventricular organs in nonhuman primates

6-Hydroxydopamine (6-OH-DA) has been shown to produce degenerative changes in noradrenergic nerve terminals and preterminals in the CNS following intracisternal, intraventricular or direct injection into the brain parenchyma. Systemic injection of 6-OH-DA is known to result in degenerative changes in noradrenergic terminals in the peripheral nervous system. However, only a few studies have been carried out on the effects of systemic injections of 6-OH-DA on noradrenergic terminals in the CNS. In the present study cynomolgus and squirrel monkeys were injected intravenously on two successive days with total doses of 350 mg/kg and 150 mg/kg of 6-OH-DA, respectively, and sacrificed at 2 and 24 h following the second injection. Degenerative changes in the area postrema (AP) neurons in all injected animals were characterized by a generalized increase in electron density of cytoplasmic elements in axonal terminals and preterminals. Multilamellar bodies, clusters of clear and dense core vesicles, increased numbers of secondary lysosomes, and an increase in the number of glycogen increased markedly in injected animals, but no other glial alterations were observed. The number of mast cells in the AP was greater in injected than in noninjected animals, both in the perivascular spaces (PVS) and in parenchymal locations. Cell processes in the PVS were occasionally observed to contain electron dense bodies, and degenerative changes were seen in supraependymal processes in some injected animals.     

Actions of 6-hydroxydopamine quinones on catecholamine neurons.

—The effect of the para-(PQ) and the ortho-(OQ) quinones of 6-hydroxydopamine (6-OH-DA) on transmitter uptake-storage mechanisms of catecholamine neurons in mouse and rat has been investigated. After the administration of PQ and OQ there was a dose-dependent and long-lasting disappearance of noradrenaline (NA) nerve terminals as demonstrated by fluorescence histochemistry and a reduction of the in vitro uptake of [³H]NA in mouse atrium and iris. These effects could be completely counteracted by blockade of the ‘membrane pump’ transport mechanism with desipramine, while monoamine oxidase inhibition, by nialamide and administration of ascorbic acid potentiated the effects produced by the two quinones. The results obtained after PQ and OQ were largely identical with those seen after administration of 6-OH-DA, well-known for its neurotoxic action on catecholamine neurons. It is therefore concluded that PQ and OQ are able to produce an acute and selective degeneration of NA nerve terminals similar to that of 6-OH-DA. The results obtained after intraventricular injection of the quinones into rat brain were also in agreement with this view. Neonatal administration of PQ or OQ to mice caused a permanent and marked decrease in [³H]NA uptake in the cerebral cortex and the spinal cord, whereas the uptake was markedly increased in the pons-medulla, similar to that seen after 6-OH-DA. The PQ and the OQ were equally potent in most experiments although clearly less potent than 6-OH-DA itself. The quinones were also found to be equally or slightly less potent than 6-OH-DA in affecting [³H]NA uptake and retention in vitro in atrium and cerebral cortex from untreated mice. It may be concluded that PQ and OQ exert their neurotoxic action on NA neurons after transition to 6-OH-DA, after a rapid extraneuronal equilibration. 6-OH-DA thus formed can thereafter be taken up and accumulated intraneuronally by use of the ‘membrane pump’ and the specific degenerative action is elicited. The lower neurotoxic potency of the quinones may be attributed to their known ability to undergo covalent binding with proteins and/or formation of 5,6-dihydroxyindole.     

Effects of Suanzaorentang on behavior changes and central monoamines

Previously, it was found that the ancient Chinese remedy of Suanzaorentang could be a promising anxiolytic drug (Chen and Hsieh, 1985a, Chen and Hsieh, 1985b). To understand the mechanism of the action of Suanzaorentang, the effects of Suanzaorentang on behavior changes and central monoamines and their metabolites were studied in rats. It was found that Suanzaorentang significantly (1) prolonged the period from the onset of clonic to tonic convulsions induced by pentylenetetrazol or picrotoxin, (2) prolonged the sleep duration induced by hexobarbital, (3) reduced locomotor activity, (4) enhanced the hypomotility induced by alpha-MT, (5) reduced the locomotor stimulation produced by levodopa plus benserazide, and (6) reduced central HVA, VMA, and 5-HIAA, but had no significant effects on central DA, NA, and 5-HT. These facts implied that Suanzaorentang decreased the turnover rate of central monoamines and central catecholaminergic activity.     

Studies on the specificity of uptake and release of radiolabelled histamine in rat brain slices

Previously it has been shown that radiolabelled histamine is taken up by brain slices and may subsequently be released by depolarizing stimuli in a calcium-dependent manner, indicating the involvement of neurons in uptake and release of histamine.The present study demonstrates that after incubation of brain slices with low (nM) concentrations of [³H]histamine the amine may be taken up by (and released from) dopaminergic and serotonergic neurons (nerve terminals). Thus 6-hydroxydopamine- and 5,7-dihydroxytryptamine-induced lesions not only reduced the uptake of [³H]dopamine (in striatal slices) and [³H]serotonin (in hippocampal slices), but also, though to a lesser extent, that of [³H]histamine. Immunocytochemical findings revealed that the neurotoxins did not visibly affect histaminergic neurons. Lesioning of noradrenergic neurons appeared not to alter significantly the uptake of [³H]histamine. Further, various drugs acting on either catecholamine-, serotonin- or opioid-receptors and known to cause presynaptic inhibition of the release of [³H]dopamine or [³H]wrotonin from striatal or hippocampal slices also inhibited [³H]histamine release.It is concluded that incubation of brain slices with low concentrations of [³H]histamine does not result in a selective labelling of histaminergic neurons. The possibility that, unlike other monoamines, histamine is not subject to high-affinity uptake by the nerve terminals from which it was released, is discussed.