Dietary uridine-5′-monophosphate supplementation increases potassium-evoked dopamine release and promotes neurite outgrowth in aged rats

Membrane phospholipids like phosphatidylcholine (PC) are required for cellular growth and repair, and specifically for synaptic function. PC synthesis is controlled by cellular levels of its precursor, cytidine-5'-diphosphate choline (CDP-choline), which is produced from cytidine triphosphate (CTP) and phosphocholine. In rat PC12 cells exogenous uridine was shown to elevate intracellular CDP-choline levels, by promoting the synthesis of uridine triphosphate (UTP), which was partly converted to CTP. In such cells uridine also enhanced the neurite outgrowth produced by nerve growth factor (NGF). The present study assessed the effect of dietary supplementation with uridine-5'-monophosphate disodium (UMP-2Na+, an additive in infant milk formulas) on striatal dopamine (DA) release in aged rats. Male Fischer 344 rats consumed either a control diet or one fortified with 2.5% UMP for 6 wk, ad libitum. In vivo microdialysis was then used to measure spontaneous and potassium (K+)-evoked DA release in the right striatum. Potassium (K+)-evoked DA release was significantly greater among UMP-treated rats, i.e., 341+/-21% of basal levels vs. 283+/-9% of basal levels in control rats (p<0.05); basal DA release was unchanged. In general, each animal's K+-evoked DA release correlated with its striatal DA content, measured postmortem. The levels of neurofilament-70 and neurofilament-M proteins, biomarkers of neurite outgrowth, increased to 182+/-25% (p<0.05) and 221+/-34% (p<0.01) of control values, respectively, with UMP consumption. Hence, UMP treatment not only enhances membrane phosphatide production but also can modulate two membrane-dependent processes, neurotransmitter release and neurite outgrowth, in vivo.     

Effects of transient forebrain ischemia on long-term enhancement of dopamine release in rat striatal slices

We studied the effects of transient forebrain ischemia in vivo on long-term enhancement of dopamine (DA) release from rat striatal slices. One hour after the high-frequency tetanic stimulation (HFTS) or l-glutamate (10⁻⁶ M) application in Mg²⁺-free medium to striatal slices, the high concentration of KCl (high K⁺)-evoked DA release was measured. Tetanic stimulation or l-glutamate application significantly potentiated the high-K⁺-evoked DA release. When striatal slices were prepared from rats exposed to 3 min of ischemia followed by 24-h survival, the enhancement of DA release by HFTS was unaffected by ischemia. In contrast, the enhancement of DA release by HFTS was impaired in rats exposed to 5 min or 10 min of ischemia. In addition, high K⁺-evoked DA release per se was significantly impaired by 10 min of ischemia. The enhancement of DA release elicited by pretreatment with l-glutamate was also impaired in the rats exposed to 5 min of ischemia. When striatal slices were prepared from rats exposed to 5 min of ischemia with 7-day survival, the enhancement of DA release by HFTS was still impaired. The present results indicate that the neuronal mechanisms of the enhancement of DA release may be more sensitive to impairement from short periods of ischemia. Furthermore, the results suggest that an impairment of long-term enhancement of DA release by ischemia may be related the dysfunction of motor performance in rats exposed to ischemia.     

Alterations in muscarinic control of striatal dopamine autoreceptors in senescence: a deficit at the ligand-muscarinic receptor interface?

Research has indicated that the release of striatal dopamine (DA) is controlled by inhibitory DA autoreceptors which are in turn regulated by inhibitory muscarinic inhibitory cholinergic heteroreceptors (HTRs) located in close vicinity to the autoreceptors. Muscarinic activation enhances K⁺-evoked release of DA from striatal slices from mature but not senescent rats. Since it has been shown that age-dependent declines in Ca²⁺ mediated acetylcholine release can be restored by the ionophore A23187, it was of interest to determine if age-related decrements in Ca²⁺ mobilization might contribute to the alterations in muscarinic control of the striatal DA autoreceptors seen in senescence. Cross-cut striatal tissue slices obtained from two age-groups (6 and 24 months) of Wistar rats were superfused with a modified Krebs-Ringer medium containing 2.5 mM KCl. After a 30-min equilibration period, a 5-min baseline fraction was collected. The medium was then switched to one which contained 30 mM KCl and, depending upon the experiment, the muscarinic agonists carbachol, or oxotremorine or the Ca²⁺ mobilizing agents A23187 or inositoltrisphosphate (IP₃) and enhancement of K⁺-evoked release of DA was examined. Six 5-min fractions were collected. DA release was determined by HPLC coupled to electrochemical detection. Results indicated that although deficits were seen in oxotremorine and carbachol enhancement of K⁺-evoked release of DA, these decrements were not observed when either A23187 or IP₃ were utilized to enhance the K⁺-evoked release of DA. Other control experiments indicated that these Ca²⁺ mobilizing agents were not effective in promoting DA release from striatal tissue from either young or old animals in the presence of 2.5 mM KCl. Thus, the observed enhancement of K⁺-evoked release of DA by these agents only occurred during depolarization, and was probably not the result of direct toxic effects allowing DA to ‘leak’ from the terminal. Since age-differences in enhancement of K⁺-evoked release of DA were only seen with the muscarinic agonists and not with either IP₃ or A23187, these findings indicate that there may be an age-related deficit in Ca²⁺ mobilization that may find its locus at the muscarinic receptor-ligand interface. Once this interface is bypassed, and Ca²⁺ mobilized intracellularly the age-differences in enhancement are not seen.     

Release of endogenous catecholamines from slices of hypothalamus of rats

The release of endogenous catecholamines from superfused slices of rat hypothalamus was studied under basal conditions and during release evoked by 40 mM K⁺. Catecholamines in superfusates, and in extracts of the tissue after stimulation, were isolated by column chromatography and quantitated by liquid chromatography with electrochemical detection. Norepinephrine (NE) was not consistently demonstrable in superfusate collected under basal conditions, but 40 mM K⁺ caused the release of from 2 to 4 ng/g of tissue per min. The addition of cocaine to the superfusate caused increases in basal and evoked release of NE. Epinephrine (E) could be measured in superfusates of slices from male but not female rats and then only when cocaine was added to the superfusate. Accordingly, the concentration of E in hypothalamus was greater in male rats than in female rats. Dopamine (DA) was not consistently measurable in the spontaneous overflow from slices either in the presence or absence of cocaine. K⁺-evoked release of DA could be demonstrated in slices from female rats. The addition of cocaine increased the evoked release of DA from slices from both sexes. Corticosterone, added to cocaine, had no effects on the efflux of any of the catecholamines. The experiments suggest that neuronal reuptake of all catecholamines is very efficient in the hypothalamus both under basal conditions and during evoked release.     

Differential regulation of dopamine release by N-methyl-d-aspartate receptors in rat striatum after partial and extreme lesions of the nigro-striatal pathway

The participation of N-methyl-d-aspartate (NMDA) receptors on dopamine (DA) efflux in the striatum of anaesthetized rats, which had their DA nigrostriatal pathway previously lesioned with different doses of 6-hydroxydopamine (6-OH-DA), was assessed by in vivo microdialysis methodology. In addition, the in vivo basal DA and dihydroxy-phenyl-acetic acid (DOPAC) effluxes and the effect of local K⁺-depolarization on DA release were also evaluated in the striatum of these 6-OH-DA treated rats. Lesioned rats were divided in three groups corresponding to animals with 25–75%, 75–95% and >95% of striatum tissue DA depletion, respectively. Striatal DA tissue depletion between 25–75% occurred in parallel with a 30% reduction in DA extracellular levels, with a moderate 10% increase in basal fractional DA efflux, and with no statistical changes in the fractional DA efflux induced by NMDA (500 μM) receptor stimulation by reverse dialysis. Rats with higher DA tissue depletion (between 75–95%) exhibited a 60% reduction in DA extracellular levels in the striatum and this reduction occurred in parallel with a modest rise in basal fractional DA efflux, but with a striking decrease in the NMDA-induced fractional DA efflux. In rats with extreme or >95% of striatal DA tissue depletion, basal fractional DA efflux in the striatum increased quite substantially along with a recovery in the ability of NMDA receptor stimulation to induce fractional DA release. The >95% striatal DA-depleted rats also exhibited a significant decrease in tissue and extracellular DOPAC/DA ratio when compared to sham and partially DA-depleted rats. In contrast to the previous results, fractional DA efflux induced by reverse dialysis with K⁺ (40 mM) remained the same in the striatum of sham and all groups of DA-tissue depleted rats. The present findings suggest the existence of at least three features associated to the regulation of basal and NMDA-induced extracellular levels of DA in the striatum of rats as a function of striatal tissue DA depletion produced by 6-OH-DA. They also support the view that a differential regulation of basal and NMDA-induced DA extracellular levels occur in partial and extreme DA-depleted striatum after 6-OH-DA treatment. Such findings may have implications as regard to the participation of the NMDA receptor in the compensatory mechanisms associated to the progress of Parkinson's disease, as well as in the therapeutic treatment of this neurological disorder.     

β-Endorphin Inhibition of Endogenous Norepinephrine Release From the A2 Noradrenergic Nucleus In Vitro: Role of Mu Opiate Receptors and Na Ion Permeability

An in vitro approach was used to determine the opioid receptor subtype mediating β-endorphin inhibition of endogenous norepinephrine release from the A2 nucleus in the caudal dorsomedial medulla of rats. The voltage-sensitive Na⁺ channel blocker tetrodotoxin was used to investigate the role of Na⁺-dependent action potentials in β-endorphin inhibition of K⁺-evoked norepinephrine release. Human β-endorphin_1–31 inhibited K⁺-evoked norepinephrine release in a concentration-dependent fashion. Activation of δ- and χ-opioid receptors had no effect on endogenous norepinephrine release. The inhibitory effect of β-endorphin was blocked in a concentration-dependent manner by the μ-opioid receptor antagonist CTOP (Cys², Tyr³, Orn⁵, Pen⁷ amide). Tetrodotoxin (TTX) inhibited norepinephrine release evoked by 25 mM K⁺ in a concentration-dependent manner and blocked the inhibitory effects of β-endorphin. These results indicate that β-endorphin acts on μ-opioid receptors to inhibit K⁺-evoked norepinephrine release from A2 neurons and suggest that the receptors involved are not located on noradrenergic nerve terminals.     

Changes in high K+-evoked serotonin release and serotonin2A/2C receptor binding in the frontal cortex of rats with thioacetamide-induced hepatic encephalopathy

Summary. Serotonergic systems were investigated in the frontal cortex of rats with thioacetamide (TAA)-induced acute hepatic encephalopathy (HE). Extracellular basal levels of 5-HT showed no difference between control and HE animals, whereas the levels of 5-HIAA were significantly increased in HE rats. Unlike basal levels, high K⁺-evoked 5-HT release was significantly higher in HE rats than controls. B_max of (±)-1-(2,5-dimethoxy-4- [¹²⁵I] iodophenyl)-2-aminopropane ([¹²⁵I] DOI) binding, mainly labeling postsynaptic 5-HT_2A receptors, was significantly decreased without any change in K_d in HE rats. These results suggest that there is no change in basal 5-HT release in the cortex of rats with TAA-induced HE despite the increase in intraneuronal 5-HT metabolism and in the size of releasable 5-HT pool, and that serotonergic neurotransmission via 5-HT_2A receptor is altered in the brain area of rats with HE. Accepted October 21, 1997 / Received April 29, 1997     

Properties of the voltage-gated calcium channels mediating dopamine and acetylcholine release from the isolated rat retina

We examined the properties of voltage-gated calcium channels mediating endogenous dopamine (DA) and acetylcholine (ACh) release in the isolated rat retina. Application of 30 mM KCl elicited the release of DA and ACh, and these releases were abolished in Ca²⁺-free medium. The high K⁺-evoked DA release was largely blocked by both of ω-agatoxin IVA and ω-conotoxin MVIIC, P- and Q-type calcium channel antagonists, and partly blocked by isradipine, an L-type calcium channel antagonist, and ω-conotoxin GVIA, an N-type calcium channel antagonist, ω-Agatoxin IVA at a small dose, sufficient to block P-type channels alone, was however without effect. On the other hand, the high K⁺-evoked ACh release was partly blocked by ω-agatoxin IVA and ω-conotoxin MVIIC, but was resistant to isradipine and ω-conotoxin GVIA. Flunarizine, a non-selective T-type calcium channel antagonist, did not inhibit the release of DA and ACh. Cd²⁺ markedly blocked the release of both DA and ACh, Co²⁺ and Ni²⁺ slightly blocked the release of DA, and the release of ACh was not blocked by these two divalent cations. These results suggest that the high K⁺-evoked release of retinal DA is largely mediated by ω-agatoxin IVA and ω-conotoxin MVIIC sensitive calcium channels (probably Q-type channels), while the release of retinal ACh is largely mediated by as yet uncharacterized Cd²⁺ sensitive calcium channels. The properties of voltage-gated calcium channels involved in the release of ACh in the rat retina differ from those of DA.     

Modulation of dopamine uptake and release in rat striatal synaptosomes by glycine and glutamate

The uptake and release of dopamine (DA) by rat brain striatal synaptosomes were studied in presence of glycine or glutamate (0.5–20 mM) in incubation media containing 5 or 55 mM KCl. In low K⁺ medium glycine, up to 5 mM had little effect on DA uptake, but higher concentrations of the amino acid inhibited the uptake. High K⁺ medium resulted in a decrease in DA uptake as compared to that of basal K⁺ medium. Glycine at 1 μM, but not at higher concentrations, prevented the inhibition induced by high K⁺ depolarization. However, glutamate in the low K⁺ medium and at a 0.5 mM concentration, stimulated DA uptake, but at higher concentrations it inhibited the uptake. In the high K⁺ medium, glutamate in all concentrations potentiated the inhibition of DA uptake induced by K⁺ depolarization. The DA release response of the synaptosomes to glycine concentrations (1–20 mM) in low K⁺ medium was a biphasic pattern, with a stimulation at 1 mM and an inhibition at higher concentrations. This pattern was reversed when DA release was measured in the high K⁺ medium. The pattern of DA release in the presence of glutamate concentrations (1–20 mM) was a triphasic one, with an inhibition at 1 mM, stimulation at 5 mM, and a less effective inhibition at higher concentrations. In the high K⁺ medium, glutamate at 1 mM concentration prevented the stimulation induced by K⁺ depolarization, but at 5 mM reversed the rate of release to the depolarization state. Results of this study suggest that glycine and glutamate have more than a simple inhibitory or excitatory transmitter role in the striatum, respectively. The identical effects of glycine with that of glutamate in certain concentrations is consistent with previous reports that glycine andN-methyld-aspartate (NMDA) act as coagonists of a common excitatory amino acid receptor.     

Chronic administration of DHA and UMP improves the impaired memory of environmentally impoverished rats

Living in an enriched environment (EC) during development enhances memory function in adulthood; living in an impoverished environment (IC) impairs memory function. Compounds previously demonstrated to improve memory among IC rats include CDP-choline and uridine monophosphate (UMP). Brain phosphatidylcholine (PC) synthesis utilizes both the uridine formed from the metabolism of exogenous CDP-choline and UMP, and the choline formed from that of CDP-choline. It also uses the polyunsaturated fatty acid (PUFA) DHA, a precursor for the diacylglycerol incorporated into PC. DHA administration also improves cognition in young and aged rodents and humans; its effects on cognitively impaired IC rats have not been characterized. We have thus examined the consequences of administering DHA (300 mg/kg) by gavage, UMP (0.5% in the diet), or both compounds on hippocampal- and striatal-dependent forms of memory among rats exposed to EC or IC conditions for 1 month starting at weaning, and consuming a choline-containing diet. We observe that giving IC rats either dietary UMP or gavaged DHA improves performance on the hidden version of the Morris water maze (all P<0.05), a hippocampal-dependent task; co-administration of both phosphatide precursors further enhances the IC rats' performance on this task (P<0.001). Neither UMP nor DHA, nor giving both compounds, affects the performance of EC rats on the hidden version of the Morris water maze (P>0.05), nor the performance by IC or EC rats on the visible version of the Morris water maze (all P>0.05), a striatal-dependent task. We confirm that co-administration of UMP and DHA to rats increases brain levels of the phosphatides PC, PE, SM, PS, PI, and total brain phospholipid levels (all P<0.05), and show that rearing animals in an enriched environment also elevates brain PC, PS, and PI levels (all P<0.01) and total brain phospholipids (P<0.01) compared with their levels in animals reared in an IC environment. These findings suggest that giving DHA plus UMP can ameliorate memory deficits associated with rearing under impoverished conditions, and that this effect may be mediated in part through enhanced synthesis of brain membrane phosphatides.