Cyclic adenosine monophosphate: selective increase in caudate nucleus after administration of L-dopa.

Treatment with the dopamine precursor L-dopa produced a significant accumulation of adenosine 3',5'-monophosphate (cyclic AMP) in the caudate nucleus of the rat. In contrast, there was no change in the amount of cyclic AMP in the cerebellum. Accumulation of cyclic AMP in the caudate nucleus after administration of L-dopa was prevented by prior treatment with the decarboxylase inhibitor RO 4-4602. These observations and those in other laboratories support the assumption that dopamine formed from L-dopa selectively activates striatal adenylate cyclase. The in vivo activation of adenylate cyclase after treatment with L-dopa may be a useful model for studying neurological and psychiatric disorders that are thought to involve the dopaminergic system of the brain.

Activation of the nigrostriatal dopaminergic pathway by injection of cholera enterotoxin into the substantia nigra.

Twenty-four hours after unilateral injection of cholera enterotoxin into the rat substantia nigra there is an increase, in the striatum on the injected side, of basal adenylate cyclase activity, 3,4-dihydroxyphenylacetic acid, and 3-methoxy-4-hydroxyphenylacetic acid. Moreover, there is an increase of motor activity, and rats tend to circle contralateral to the side of the injection. Injection of cholera enterotoxin into brain nuclei may be a useful procedure for pharmacologically activating selected neuronal systems of brain and for studying the pharmacology of drugs that are suspected of interacting with these systems.

Light stimulates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine neurons.

Retinal dopamine-containing amacrine neurons are rapidly activated by light, as shown by an increase in the rate of dopamine formation in vivo and a concomitant increase in the activity of tyrosine hydroxylase, measured in vitro with a subsaturating concentration of pteridine cofactor. Activation of tyrosine hydroxylase also occurs when isolated eyes from rats killed in the dark are exposed to a strobe light. Studies of amacrine neurons should provide basic data about the biochemical processing of visual information, as well as the physiological presynaptic regulatory mechanisms of dopamine-containing neurons.

GM1 and NGF synergism on choline acetyltransferase and choline uptake in aged brain.

In the brain of aged rats high affinity choline uptake (HAChU) of the striatum, hippocampus, and frontal cortex is lower than in young rats, while choline acetyltransferase (ChAT) activity is lower in striatum and frontal cortex. Infusion into the lateral cerebral ventricle with nerve growth factor (NGF) enhances the low values of these cholinergic markers in a dose- and region-dependent manner. GM1 ganglioside infused into the lateral ventricle, at a dose that is ineffective alone, together with NGF synergistically enhances the effect of NGF on ChAT and HAChU activities in the brain of aged animals. The pharmacology of this GM1/NGF synergism suggests potentiation of response.

Distribution of aromatic L-amino acid decarboxylase mRNA in mouse brain by in situ hybridization histology.

Aromatic L-amino acid decarboxylase (AAAD) is the second enzyme in the sequence leading to the synthesis of catecholamines or serotonin. Antisense riboprobes for aromatic L-amino acid decarboxylase mRNA were used to map the gene in mouse brain by in situ hybridization. The substantia nigra, the ventral tegmental nucleus, the dorsal raphe nucleus, the locus coeruleus, and the olfactory bulb contained the highest signal for AAAD mRNA. After treatment with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the signal disappeared in the substantia nigra, decreased somewhat in the ventral tegmental area, and remained unchanged in the dorsal raphe nucleus. Hypothalamic and cerebellar Purkinje neurons known to contain histidine decarboxylase or glutamic acid decarboxylase, respectively, were unlabeled by the probes. However, neurons in the deep layers of the frontal cortex, many thalamic nuclei, and the pyramidal neurons of the hippocampus were lightly to moderately labeled for mouse AAAD mRNA. The presence of AAAD message in these neurons suggests that the enzyme has functions other than that for the synthesis of the classical biogenic amine neurotransmitters.

Is dopamine a transmitter in the periphery?

There is now substantial experimental evidence supporting the hypothesis that a dopaminergic neuronal system is present in peripheral tissues. This evidence includes identified and characterized dopaminergic receptors, the presence of relatively large concentrations of dopamine and DOPAC in some neurons and organ systems, and the differential loss of norepinephrine or dopamine following treatment with catecholaminergic neurotoxins. There are still many studies that remain to be completed, however, existing evidence is consistent with a peripheral dopaminergic neuronal system.

Correlation between histamine-induced neuronal excitability and activation of adenylate cyclase in the guinea pig hippocampus.

In a homogenate of guinea pig hippocampus histamine activated adenylate cyclase and in a hippocampal slice preparation it increased the firing rate of pyramidal cells in the CA3 region. Both activities were apparently mediated by H2 receptors. The concentration of histamine and of the H2 receptor agonist, impromidine, required to stimulate activity was similar in each test preparation with impromidine being about 100-fold more potent than histamine. Moreover, the H2 receptor antagonists, cimetidine and ICIA 5165, each reversed the activation by histamine of the two test preparations, with ICIA 5165 being about 100-fold more potent than cimetidine. Thus, there is a correlation between activation of cyclase and neuronal excitability induced by histamine. These observations support a large body of evidence suggesting that histamine is a neurotransmitter or modulator in the CNS.

Intracerebroventricular administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its metabolite 1-methyl-4-phenylpyridinium ion (MPP+) decrease dopamine and increase acetylcholine in the mouse neostriatum.

We found that both MPTP and its metabolite MPP+ decrease dopamine and increase acetylcholine content of mouse neostriatum when administered intracerebroventricularly (ICV). These observations support the notion that MPP+ may be the active neurotoxin formed in brain after MPTP administration. They also suggest that cholinergic mechanisms may be a target of the neurotoxin.

Administration of GM1 ganglioside restores the dopamine content in striatum after chronic treatment with MPTP.

The dopamine (DA) content of the mouse striatum thirty days after treatment with MPTP is significantly reduced. GM1 ganglioside administration during the thirty day period results in a partial restoration of the striatal DA content.

Immunohistochemical evidence for epinephrine-containing retinal amacrine cells.

The enzyme for the synthesis of epinephrine, phenylethanolamine-N-methyltransferase, has been localized, by an indirect immunofluorescent staining method, to a subpopulation of amacrine cells in the rat retina. The immunoreactive cells are located primarily in the inner nuclear layer and send a single process to the inner plexiform layer. Most of the immunoreactivity is found in the center of the inner plexiform layer. A small percentage of immunoreactive cell bodies were found in the inner plexiform layer and occasionally cells were observed in the ganglion cell layer. These epinephrine-containing amacrine cells are morphologically distinct from the dopamine-containing amacrine cells previously described by formaldehyde fluorescence and we speculate from reports in the literature that epinephrine-containing amacrine cells may play a role in modulating the activity of dopamine-containing amacrine cells.

GM1 ganglioside restores dopaminergic neurochemical and morphological markers in aged rats.

The monosialoganglioside GM1 exerts neurotrophic-like activity in vitro and in vivo. In particular, it improves cholinergic neuron morphology and chemistry and learning abilities of cognitively impaired aged rats and young animals with cholinergic lesions, and restores neurochemical, pharmacological, morphological and behavioral parameters in animal models of Parkinson's disease. Our studies present evidence that GM1 reverses dopaminergic deficits in the nigrostriatal neurons of aged rats. GM1 administered to aged Sprague-Dawley rats for 30 days reversed the decreased activity of tyrosine hydroxylase in the midbrain and striatum, elevated the reduced protein content and mRNA levels of the enzyme in the midbrain, and reversed the decrements of dopamine and 3,4-dihydroxyphenylacetic acid content in both the midbrain and striatum. Tyrosine hydroxylase activity of the midbrain, but not of the striatum, remained elevated for 15 days after discontinuing GM1. The count profiles of tyrosine hydroxylase-immunopositive neurons, the size of tyrosine hydroxylase-immunopositive neurons and the number of tyrosine hydroxylase-immunopositive fibers were decreased in the substantia nigra pars compacta and the ventral tegmental area of aged rats. GM1 corrected the morphology of dopaminergic neurons in the substantia nigra pars compacta and partially improved it in the ventral tegmental area. These findings support the notion that the aged striatal dopaminergic neurons respond to GM1, and strengthen the utility of using this compound for combating age-associated neuronal deficits.

GM1 ganglioside enhances cholinergic parameters in the brain of senescent rats.

GM1 ganglioside and nerve growth factor both promote the recovery of injured central cholinergic neurons in young animals. Brain cholinergic activity declines with aging and nerve growth factor has been shown to correct cholinergic deficits in senescent animals. We have administered GM1, to young (three months old) or senescent (22-24 months old) rats and evaluated acetylcholine and choline content, choline acetyltransferase and acetylcholinesterase activity as well as choline uptake in striatum, hippocampus and frontal cortex. For some studies, nerve growth factor was administered alone or together with GM1. Our results indicate that cholinergic neurochemical parameters are decreased in some brain areas of senescent animals and that both GM1 and nerve growth factor can enhance their recovery.

Fluorometric estimation of 4-hydroxy-3-methoxyphenylethyleneglycol sulphate in brain.

1. A simple, sensitive, and selective method was developed for the fluorimetric estimation of 4-hydroxy-3-methoxyphenylethyleneglycol sulphate (MOPEG-SO(4)) in rat brain.2. MOPEG-SO(4) was isolated on a DEAE Sephadex column. Fluorescence was measured at 465 nm (excitation at 320 nm) after heating the column eluate with acid and reheating after adding ethylene diamine.3. The concentration in brain of MOPEG-SO(4) increased after blockade of its transport from brain by treatment with probenecid and decreased after inhibiting its synthesis by treatment with alpha-methyltyrosine or pargyline.4. Our studies suggest that measurement in changes in the rate of formation of MOPEG-SO(4) may be a useful index of noradrenaline turnover.

Monoamine oxidase in sympathetic nerves: a transmitter specific enzyme type.

1. When rat brain or superior cervical ganglion monoamine oxidase was incubated with increasing concentrations of clorgyline, using tyramine as substrate, the inhibition of the enzyme could be represented by a pair of sigmoidal curves joined by a horizontal region where inhibition was constant. Tyramine appeared to be metabolized by two enzymes, one of which was highly sensitive to clorgyline, designated A, whereas the other enzyme, designated B, was less sensitive to clorgyline.2. The ratio of A/B activity for brain was 6/4 while in the ganglion it was 9/1.3. When the experiments were repeated using noradrenaline as the substrate, the inhibition of the enzyme followed a simple sigmoidal curve where deamination was inhibited by low concentrations of clorgyline as observed with enzyme A.4. We conclude that tyramine is deaminated by both A and B enzymes whereas noradrenaline is deaminated only by enzyme A, the enzyme which is most active in the ganglion. Our observations are consistent with the hypothesis that a specific intraneuronal monoamine oxidase plays an important role in the catabolism of noradrenaline in sympathetic nerves.

Regulation of metaraminol efflux from rat heart and salivary gland.

1. The turnover rate of noradrenaline (NA) in heart and submaxillary salivary gland was studied in rats exposed to 4 degrees C or maintained at room temperature (22 degrees C). Cold exposure increased the turnover of the NA store in heart but not in salivary gland.2. In another series of experiments the decline of metaraminol (M) from heart and submaxillary salivary gland was studied in rats exposed to 4 degrees C or maintained at room temperature. Cold exposure accelerated the efflux of M from heart, but not from salivary gland. It is concluded that the accelerated decline of M from heart is the consequence of selective activation of the sympathetic nerves that innervate the heart.3. The turnover of NA was studied in rat heart after the administration of M (100 mug/kg intravenously) or its precursor alpha-methyl-meta-tyrosine (200 mg/kg intraperitoneally). Turnover remained essentially normal after these drugs.4. The administration of desipramine (DMI, 20 mg/kg intraperitoneally) 1 hr after M (100 mug/kg intravenously) induced a rapid sustained efflux of M from heart and salivary gland. The results of this study suggest that the slow decline of M from heart is the result of the great affinity of the amine retrieval mechanism in sympathetic nerve endings for M. DMI inhibits the retrieval mechanism, thus accelerating the efflux of M.

Monoamine oxidase in rat arteries: evidence for different forms and selective localization.

1. Two forms of monoamine oxidase activity were differentiated in rat mesenteric and femoral artery by means of substrate and inhibitor specificities: one form deaminated tyramine, 5-hydroxytryptamine and noradrenaline and was highly sensitive to pargyline and clorgyline but resistant towards carbonyl reagents. This form resembled type A monoamine oxidase previously described. The other deaminated tyramine but not 5-hydroxytryptamine or noradrenaline and was inhibited by carbonyl reagents but not by clorgyline or pargyline.2. About one third of the total monoamine oxidase in homogenates of rat mesenteric artery was recovered in a 10(5)g supernatant. Both forms were partially soluble, but relatively less of the type A activity was recovered in the soluble fraction.3. Chemical sympathectomy with 6-hydroxydopamine resulted in a loss of 59% of monoamine oxidase activity in the mesenteric artery. There was a selective loss of type A activity, as revealed by the 70% decrease in 5-hydroxytryptamine deaminating ability and by the marked decrease in clorgyline sensitivity. The second monoamine oxidase species was resistant to 6-hydroxydopamine. The soluble activity was not affected by chemical sympathectomy. Most of the transmitter-specific monoamine oxidase of the arterial wall was localized within the adrenergic nerve endings. Our observations are consistent with the hypothesis that extraneuronal monoamine oxidase plays only a minor role in metabolizing noradrenaline in sympathetically innervated tissues.4. Plasma amine oxidase might originate from the arterial wall since it has similar characteristics to that found in the mesenteric artery.

Aromatic L-amino acid decarboxylase modulation and Parkinson's disease.

Aromatic L-amino acid decarboxylase (AAAD) is the second enzyme in the sequence leading to the synthesis of the catecholamines and serotonin, and it is the rate-limiting enzyme for the synthesis of the trace amines. In the striatum AAAD activity is increased by neuronal firing and diminished or enhanced by activation or blocking dopamine (DA) D1 or D2 receptors, respectively. At least two biochemical mechanisms appear responsible for modulation, short-term involving second messengers and possible phosphorylation, and long-term involving protein synthesis. In Parkinson's disease AAAD is the rate-controlling enzyme for the synthesis of DA when L-DOPA is administered and any change of AAAD activity could have clinical consequences. Indeed, the "on-off phenomenon" where there are fluctuations between off-periods of marked akinesia over several hours with on-periods of improved motility may be related to oscillating or poorly modulated AAAD activity and conversion of L-DOPA to DA. Studies are presented demonstrating how AAAD activity can be enhanced in an animal model of Parkinson's disease and how rapid fluctuations of AAAD can be provoked via second messenger system activation.

Differentiation of noradrenergic and dopaminergic neurons in the cardiovascular system.

Dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and norepinephrine (NE) are present in the rat cardiovascular system. All of the catechols can be partially depleted by administering 6-hydroxydopamine (6-HODA). When animals are pretreated with desipramine before 6-HODA, there is a selective partial depletion of DA and DOPAC. NE can be partially depleted with minimal effects on DA and DOPAC by administering N-(2-chloroethyl)N-ethyl-2-bromobenzylamine (DSP-4). These results are consistent with the hypothesis that independent dopaminergic and noradrenergic elements are present in the rat cardiovascular system and that DA is not solely a precursor for NE. NE, DA and DOPAC were assayed in human vessels and the pattern of distribution of the catechols is consistent with the results reported for animals.

c-fos mRNA in mouse brain after MPTP treatment.

The neurotoxin, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induces a transient increase of mRNA for the immediate-early gene c-fos in the mouse brain. The c-fos mRNA level is MPTP dose-dependent and is evident in all brain regions tested including striatum, hypothalamus, cortex, hippocampus, cerebellum and midbrain. There are regional differences in the time-course for the rise of c-fos mRNA. Pretreatment with deprenyl, a selective monoamine oxidase B inhibitor, pargyline, a nonselective monoamine oxidase inhibitor, or mazindol, a dopamine uptake transport inhibitor, does not prevent the c-fos mRNA increase, suggesting that the elevation is due to the action of MPTP and not its neurotoxic metabolite MPP+.

Expression of cloned aromatic L-amino acid decarboxylase in Xenopus laevis oocytes.

Sense mRNA coding for bovine adrenal medulla aromatic L-amino acid decarboxylase (AADC) was expressed following microinjection into Xenopus laevis oocytes. The expressed enzyme activity was stereoselective for L-5-hydroxytryptophan and L-DOPA and blocked by NSD-1015 an inhibitor of AADC. Heating the expressed enzyme at 55 degrees C resulted in a parallel loss of activity towards both substrates. Our findings are consistent with the prevailing notion that a single enzyme is able to decarboxylate both substrates in vivo.