N. Thomas

Effect of ascorbic acid on neurochemical, behavioral, and physiological systems mediated by catecholamines.

Abstract Ascorbic acid, at concentrations below that normally present in the brain, inhibited the dopamine-sensitive adenylate cyclase in vitro . Ascorbate had no effect on the norepinephrine-sensitive adenylate cyclase. To study the in vivo effect of ascorbic acid on central dopaminergic systems, mice (C57 B1/6J) were injected with pharmacological doses (2 g/kg) of ascorbate, which produced a significant elevation in brain ascorbate concentration. Injecting the mice with ascorbate (2 g/kg) blocked the amphetamine-induced (15 mg/kg) increase in stereotype behavior which has been reported to be mediated by dopaminergic neural systems. Ascorbate had no effect on the amphetamine-induced locomotor activity thought to be mediated by norepinephrine systems. Ascorbate (1 g/kg) attenuated apmorphine-induced hypothermia in this same strain of mice. This demonstrates the specific neurochemical, physiological, and behavioral alterations in dopaminergic systems produced by ascorbic acid and suggests possible therapeutic uses for ascorbate in conditions involving functional dopamine excess.

5-Hydroxytryptamine—A neurotransmitter of bovine retina

Abstract Bovine retina was found to contain appreciable amounts of 5-hydroxytryptamine (5-HT) and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA). In vitro incubation studies demonstrated a high affinity uptake system for [ 3 H]5-HT that is sodium and temperature dependent, ouabain-sensitive and saturable. Kinetic analysis of the uptake data demonstrates a high affinity transport system with an apparent K m of 4·3 × 10 −7 m . The uptake is blocked by chlorimipramine, an inhibitor of monoamine uptake. The 5-HT is presumably stored in storage vesicles as reserpine completely abolished the [ 3 H]5-HT uptake. Release studies demonstrated a K + stimulated, Ca 2+ dependent release of [ 3 H]5-HT from retina preloaded with [ 3 H]5-HT. The results are consistent with the suggestion that 5-HT is a transmitter in bovine retina.

Distribution of superoxide dismutase in rat brain.

THEENZYME, superoxidedismutase(EC.1.15.1.1),can provide a defense against toxic oxygen radicals which are produced by normal intracellular reactions in all aerobic cells (FRIDOVICH, 1 9 7 5 ; M c C o ~ ~ & FRIDOMCH, 1969a, b). The enzymevariously called cerebrocuprein (PORTER & FOLCH, 1957) or tetrazolium reductase inhibitor (FRIED et ul., 1970; 1973) has been demonstrated in mammalian brain (FRIED & FRIED, 1967; FRIED & MANDEL, 1975). The enzyme from mammalian liver and brain are identical in activity and properties (BEAUCHAMP & FRIDOVICH, I97 1). Since the neuron actively consumes oxygen, SOD may be involved in maintaining normal neuronal function. This study was designed to determine the regional distribution of SOD in rat brain, since it has been shown that certain brain regions are more susceptible to damage than others when exposed to hyperbaric oxygen (OGILVIE & BALENTINE. 1975). In order to gain information regarding the cellular localization of the enzyme, rat brain homogenates were fractionated into subcellular components. Of particular interest was the activity of SOD in synaptosomes since superoxide radicals can cause the oxidation of catecholamines (MISRA & FRIDOVICH, 1972), and these amines are present in certain nerve endings in the CNS. Of further interest is the demonstration that the superoxide radical is an intermediate in the oxidation of 6-hydroxydopamine and that this autooxidation can be inhibited by SOD (HEIKKILA & COHEN, 1973). The oxidation of 6-hydroxydopamine is believed to be the basis for its ability to destroy selectively catecholamine nerve terminals in experimental animals (UNGERSTEDT, 1971 ; BLOOM, 1973).

Ascorbate blocks amphetamine-induced turning behavior in rats with unilateral nigro-striatal lesions

Unilateral nigro-striatal lesions were produced in rats using 6-hydroxydopamine. Intraperitoneal injections of amphetamine induced circling behavior in these rats due to release of striatal dopamine contralateral to the lesion. Intraperitoneal injections of 1 g/kg of ascorbic acid elevated brain ascorbate. Ascorbate, like other drugs blocking dopamine receptors, attenuated the amphetamine-induced turning behavior. Thus, ascorbic acid might have a role in regulating dopaminergic transmission and could be of therapeutic value in disorders involving functional dopamine excess.

Isolation of synaptosomal fractions from rabbit retina

Homogenization of rabbit retina in isotonic sucrose and differential rate centrifugation yielded two morphologically distinct synaptosomal fractions. One fraction was enriched in photoreceptor cell synaptosomes; the second fraction contained small synaptosomes derived from conventional sized synapses most abundant in the inner plexiform layer. Attempts to further purify these fractions using a variety of density gradients proved unsuccessful due to poor viability of photoreceptor cell synaptosomes. The synaptosomes prepared by our method are functionally stable as they demonstrate high affinity uptake for putative retinal neurotransmitters, neurotransmitter-sensitive adenylate cyclase activity, and calcium-dependent, potassium-stimulated release of [14C]GABA and [3H]dopamine.

Prenatal maternal phenobarbital increases reactivity and retards habituation of mature offspring to environmental stimuli

Adult female offspring of C57 BL/6J mice injected daily with phenobarbital for the last third of pregnancy were more active than control offspring during a 3-min test period in an open field arena, thus confirming previous reports of lasting effects of prenatal exposure to phenobarbital. These offspring habituated less rapidly than control offspring to the open field and were more reactive to sudden changes in environmental stimuli. The behavioral changes were not accompanied by body or brain weight deficits. The maternal drug injections did not alter brain concentrations of dopamine or norepinephrine in the adult offspring or the degree of reduction in these transmitters produced by the synthesis inhibitors α-methyltyrosine. Although activity was reduced by the catecholamine synthesis inhibitor, the effect was similar for offspring of both drug-treated and control dams.

UPTAKE OF [14C]ASPARTIC ACID AND [14C]GLUTAMIC ACID BY RETINAL SYNAPTOSOMAL FRACTIONS

Abstract— Uptake systems for [14C]aspartate and [14C]glutamate were characterized in two distinct synaptosomal fractions solated from rabbit retina. The P, synaptosomal fraction was highly enriched in large photoreceptor cell synaptosomes but contained very few conventional sized synaptosomes from amacrine, horizontal or bipolar cells. In contrast, the P2 synaptosomal fraction contained numerous conventional sized synaptosomes and was virtually free of photoreceptor cell synaptosomes. Both synaptosomal fractions took up [14C]aspartate and [14C]glutamate with high affinity [Km= 1–2μM). Uptake characteristics were similar to those described for high affinity uptake systems in brain synaptosomes, i.e. saturation kinetics; temperature and Na+ dependence. Although the presence of a high affinity uptake system is not a definitive criterion for demonstration of functional neurotransmitter systems, it is an important and necessary prerequisite and can thus be considered as supportive evidence for the involvement of asparate and glutamate in neurotransmission in rabbit retina.

Serotonin uptake and release by subcellular fractions of bovine retina

Fractionation of bovine retina yielded two synaptosomal fractions of distinct morphology. TheP1 fraction was enriched in synaptic components of the outer plexiform layer, i.e. large photoreceptor terminals with post-synaptic horizontal and bipolar elements still attached. TheP2 fraction contained small brain-like synaptosomes primarily from amacrine, and bipolar terminals. TheP2 fraction demonstrated a high affinity uptake (Km = 4.58 × 10−7 M ) and K+ stimulated, Ca2+ dependent release of [3H]serotonin. The uptake system was saturable, sodium and temperature dependent, and demonstrated a high degree of pharmacologic specificity. TheP1fraction showed very little uptake and no evoked release. These results suggest that the majority of serotonin accumulating neurons have small terminals which sediment in theP2 fraction and which are virtually absent from theP1 fraction. Thus the serotonin systems appear to be localized to elements of the inner plexiform layer, i.e. bipolar or amacrine cells.

6-Methoxy-1, 2, 3, 4-tetrahydro-β-carboline effects on retinal serotonin

Abstract The compound 6-Methoxy-1, 2, 3, 4-tetrahydro-β-carboline (6-MeO-THβC) belonging to the group of hallucinogenic β-carbolines has been found to alter various aspects of serotonergic transmission in the brain. The action of 6-MeO-THβC on the newly identified 5-HT system of bovine retina was studied in vitro . The drug inhibited the high affinity uptake of [3H] 5-HT in a competitive manner and had no evident effect on the uptake of dopamine or GABA. In addition the compound increased the potassium evoked release of 5-HT from retina. Endogenous β-carbolines have been proposed to be involved in the modulation of serotonergic activity in the brain and a similar action is proposed in the retina. In addition these findings raise the possibility of retinal 5-HT system as a site of action for β-carbolines and similar hallucinogenic drugs.

Evidence for a serotonin neurotransmitter system in bovine retina

Amacrine cells are the interneurons of the retina which are responsible for much of the lateral integration of visual information as it passes through the inner plexiform layer. Recent studies suggest that amacrine cells represent a highly diversified class of cells which may be subdivided according to transmitter used, field of innervation, and specific function. Generally speaking, all major neurotransmitters found in the brain and spinal cord have also been found in retina, with the exception of norepinephrine (Bonting, 1976). In addition, each transmitter system has been localized autoradiographically to different subclasses of amacrine cells. To add to the complexity, substantial species variation in cell types has been noted in specific cell types, even within closely related species such as rat and rabbit. We have studied a specific set of amacrine cells in bovine retina which may use serotonin (5-hydroxytryptamine or 5-HT) as a transmitter. Although some slight differences were noted between the serotonin system in brain compared to retina, the similarities in the systems within these two neuronal tissues are striking. Of all the criteria which have been used to establish a compound as neurotransmitter within a specific region, perhaps the most essential is to demonstrate that the compound in question is present with the tissue. We have analyzed bovine retina for endogenous 5-HT using a spectrophotometric assay after initial purification of retinal supernatants on Sephadex and Amberlite columns (Karasawa ef al., 1974; Johnson and Lewander, 1970 and Maickel and Miller, 1966). The content of 5-HT and 5-HIAA in bovine retina was found to be 100.3 + 10.1 and 47.9 + 4.7 rig/g wet wt, respectively (Thomas and Redburn, 1979). Thus, one essential criterion has been met. While it is tempting to correlate 5-HT levels with the number of serotonergic neurons, such conclusions must be made with caution. Levels may vary tremendously with light exposure, postmortem time before assay, and activity of uptake systems and catabolic enzymes. Nevertheless, the retina1 pool of endogenous 5-HT appears to be relatively low (i.e. h l/3-1/10) compared to well-established levels in mammalian .brain (Costa and Rinaldi, 1958). One major mechanism for inactivation of serotonergic transmission in brain appears to be via a highaffinity uptake system (Fuller and Wong, 1978; Suzuki et al., 1978). We have demonstrated a sodium and temperature dependent uptake of 3H-5-HT with a K, of 0.4~M and V,., of 2.9 pmol/mg/min in retinal slices (Thomas and Redburn, 1979). We have developed procedures for isolating ? synaptosomal fraction from bovine retina which is enriched in terminals primarily from amacrine cells (Thomas and Redlturn, 1979). This fraction showed a 4-fold enrichment in uptake sites compared to homogenate fractions. The K, of retinal synaptosomal uptake was 0.4pM and the V,,, was 10.5 pmol/mg/ min. These parameters are roughly equivalent to those reported for rat brain synaptosomes: K, 5 0.2 PM, V,,, 16pmol/mg/min (Belin et al., 1976). The retinal uptake was blocked 90% or greater by 10e4 M ouabain (an inhibitor of sodium transport), by 2 x 10m5 M reserpine (an inhibitor of vesicular storage of indoleamines and catecholamines), by lo-’ FM chlorimipramine (a selective inhibitor of 5-HT uptake with much less effect on dopamine uptake), and by 10 PM 6-methoxy 1,2,3,4-tetrahydrofi-carboline (a blocker of 5-HT uptake with no effect on dopamine uptake). The bovine retina contains both type A and type B monamine oxidase (MAO). In brain, 5-HT is primarily metabolized by MAO-A whereas dopamine is metabolized by both MAO-A and B. The concentrations of MAO-A and B were approx. 4-fold higher in the synaptosomal fraction as compared to homogenate (Thomas et al., 1979A,B). Autoradiographic analysis demonstrated a specific neuronal accumulation of 3H-5-HT (Thomas et al., 1980). See Fig. 1 A-C. After incubation of bovine retina in low concentrations (4pM) 3H-5-HT in citro for 15 min, heavy accumulation of label was seen in two or three bands located near the center of the inner plexiform layer, and in cell bodies in the amacrine and ganglion cell layers. No label was observed in ganglion cell axons even after extended incubation periods, which suggests that the labeled cells in the ganglion cell layer may be displaced amacrine cells rather than ganglion cells. In further support of this interpretation, these labeled cells were often observed to extend a single thick process which merged with one of the bands of label in the inner plexiform layer. The autoradiographic pattern observed with 3H-dopamine was quite distinct from that observed