Effect of forced motility on the noradrenaline and 5-hydroxytryptamine metabolism in different parts of the rat brain

The levels of 5-HT and 5-HIAA in the rat brain were measured after 1, 6, 24 and 36 h of forced motility in a treadwheel. The results showed that 5-HT metabolism had already increased in the forebrain after 1 h of exercise but later also increased in the brain stem, especially in the mesencephalon-pons-medulla regions. These changes had almost entirely disappeared after 50 min of rest. The NA levels with and without monoamine oxidase inhibitor were measured in animals after 36 h of forced motility. The results indicated an increase in NA metabolism in the brain stem.     

Conclusive evidence for distinct transporters for 5-hydroxytryptamine and noradrenaline in pulmonary endothelial cells of the rat

The aims of this study were to obtain conclusive evidence about the roles of a 5-hydroxytryptamine [5-HT] transporter and uptake, in the dissipation of 5-HT in the lungs of the rat and to compare the properties of the 5-HT transporter in rat lungs with that in other tissues, including brain and platelets. In the first part of the study, the IC₅₀ values of a range of selective inhibitors and substrates of the 5-HT transporter or uptake₁ were determined for inhibition of uptake of 5-HT or noradrenaline in intact perfused lungs of rats. Monoamine oxidase was inhibited and, in experiments with noradrenaline, catechol-O-methyltransferase was also inhibited. Initial rates of uptake of 5-HT or noradrenaline were measured in lungs perfused with 2 nmol/l ³H-5-HT or ³H-noradrenaline for 2 min, in the absence or presence of at least three concentrations of paroxetine, citalopram, fluoxetine, 7-methyltryptamine, tryptamine, nisoxetine, imipramine, 5-HT, desipramine, (+)-oxaprotiline, cocaine or tyramine. The results showed that pharmacologically distinct transporters are involved in the uptake of 5-HT and noradrenaline in rat lungs, since there was no significant correlation between the IC₅₀ values for inhibition of 5-HT and noradrenaline uptake in the lungs. However, there were significant correlations between the IC₅₀ values for (a) inhibition of 5-HT uptake in rat lungs and of uptake by the 5-HT transporter in rat brain and (b) inhibition of noradrenaline uptake₁ in rat lungs and of uptake, in rat phaeochromocytoma PC-12 cells. The results support the conclusion that 5-HT uptake in rat lungs occurs, at least predominantly, by a 5-HT transporter which is very similar to or the same as that in other tissues, such as the brain, and provide further evidence for transport of noradrenaline by uptake₁.Further experiments were carried out to determine whether there is any transport of 5-HT by uptake₁ or of noradrenaline by the 5-HT transporter in rat lungs. Lungs were perfused with 2 nmol/1 ³H-5-HT or ³H-noradrenaline for 2 min in the absence or presence of 1 mol/l citalopram, desipramine, or citalopram and desipramine. The results showed that there was no evidence of any transport of 5-HT in the lungs by uptake₁ or of noradrenaline by the 5-HT transporter, in that desipramine had no effect on 5-HT uptake (in the absence or presence of citalopram) and citalopram had no effect on noradrenaline uptake (in the absence or presence of desipramine).The final series of experiments was carried out to determine whether, at high concentrations of the amine, there is any interaction of 5-HT with uptake₁ or of noradrenaline with the 5-HT transporter. Noradrenaline, at a concentration of 10 mol/l, did not affect 5-HT uptake in lungs perfused with 2 nmol/l ³H-5-HT for 2 min (uptake₁ inhibited), but 50 mol/l 5-HT inhibited noradrenaline uptake by 56% in lungs perfused with 2 nmol/l ³H-noradrenaline for 2 min (5HT transporter inhibited). These and the above results show that the 5-HT transporter appears to be exclusively responsible for 5-HT uptake in rat lungs, despite the possible interaction of 5-HT at high concentrations with the uptake, transporter in the cells. On the other hand, noradrenaline is transported exclusively by uptake₁ in the lungs, and there is no evidence that it interacts with the 5-HT transporter, even at high concentrations.Preliminary results of this study were presented to the December 1993 meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (Paczkowski and Bryan-Lluka 1993).     

Effects of ketamine, midazolam, thiopental, and propofol on brain ischemia injury in rat cerebral cortical slices

AIM: To compare the effects of ketamine, midazolam, thiopental, and propofol on brain ischemia by the model of oxygen-glucose deprivation (OGD) in rat cerebral cortical slices. METHODS: Cerebral cortical slices were incubated in 2 % 2,3,5-triphenyltetrazolium chloride (TTC) solution after OGD, the damages and effects of ketamine, midazolam, thiopental, and propofol were quantitativlye evaluated by ELISA reader of absorbance (A) at 490 nm, which indicated the red formazan extracted from slices, lactic dehydrogenase (LDH) releases in the incubated supernate were also measured. RESULTS: Progressive prolongation of OGD resulted in decreases of TTC staining. The percentage of tissue injury had a positive correlation with LDH releases, r=0.9609, P<0.01. Two hours of     

Involvement of different calcium channels in K+- and veratridine-induced increases of cytosolic calcium concentration in rat cerebral cortical synaptosomes

Intracellular calcium ion concentrations ([Ca²⁺]_i) in rat cerebral cortical synaptosomes were measured, using the calcium chelating fluorescence dye fura-2. The synaptosomes were depolarized by elevation of the extracellular K⁺ concentration or by addition of veratridine, which opens voltage-dependent Na⁺-channels and prevents their inactivation. Both enhancement of the concentration of extracellular K⁺ (up to 60 mM) and veratridine (1–100 μM) increased the [Ca²⁺]_i in a concentration-dependent manner. In the absence of extracellular Ca²⁺, the K⁺- and veratridine-induced increases in [Ca²⁺]_i were abolished, indicating that the increase in [Ca²⁺]_i was due to an influx of extracellular Ca²⁺. Tetrodotoxin (TTX), a blocker of the voltage-dependent Na⁺ channel, inhibited the veratridine-induced (10 μM) Ca²⁺ influx by more than 80%, while the K⁺-evoked (30 mM) increase of [Ca²⁺]_i was TTX-resistant. Both the K⁺- and the veratridine-induced Ca²⁺ influx were not reduced by nifedipine (1 μM), a blocker of L-type Ca²⁺ channels. Blockade of the voltage dependent N-type Ca²⁺ channels with ω-conotoxin GVIA (ω-CTx GVIA; 0.1 μM) and of the voltage-dependent P/Q-type channels with ω-agatoxin IVA (ω-AgaTx IVA; 0.2 μM) inhibited the K⁺-induced increase in [Ca²⁺]_i by about 30 and 55%, respectively; these effects were additive. ω-Conotoxin MVIIC (ω-CTx MVIIC) at a concentration of 0.2 μM, which may be assumed to block predominantly the Q-type Ca²⁺ channel, inhibited the K⁺-induced increase in [Ca²⁺]_i by 50%. The veratridine-induced increase in [Ca²⁺]_i was reduced by about 25% by ω-CTx GVIA (0.1 μM), but was resistant to ω-AgaTx IVA (0.2 μM) and ω-CTx MVIIC (0.2 μM). Mibefradil (former designation Ro 40-5967), a Ca²⁺ antagonist which blocks all types of voltage-dependent Ca²⁺ channels including the T and R channels, led to a concentration-dependent inhibition of the K⁺- and veratridine-induced increase in [Ca²⁺]_i (abolition at 10 μM mibefradil). Ifenprodil, another non-specific blocker of voltage-dependent Ca²⁺ channels, also inhibited the K⁺- and veratridine-induced increase in [Ca²⁺]_i in concentration-dependent manner and abolished it at 320 μM ifenprodil. In contrast, KB-R 7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate; 1 and 3 μM), a highly potent and selective inhibitor of the Na⁺/Ca²⁺ exchanger (NCX1), failed to inhibit the K⁺- and veratridine-induced increase in [Ca²⁺]_i. It is concluded that the K⁺-induced increase in free cytosolic Ca²⁺ results from Ca²⁺ influx through voltage-dependent N- and, above all, Q-type Ca²⁺ channels. N-type Ca²⁺ channels also play a minor role in the veratridine-induced increase in [Ca²⁺]_i, but P/Q-type channels do not appear to be involved at all. The inhibition of the veratridine-induced, ω-CTx GVIA- and ω-AgaTx IVA-resistant increase in [Ca²⁺]_i by mibefradil and the failure of KB-R 7943 to inhibit this response are compatible with the suggestion that in rat cerebral cortical synaptosomes, Ca²⁺ influx via the R-type Ca²⁺ channel and/or another so far uncharacterized Ca²⁺ channel may substantially contribute to the veratridine-induced increase in [Ca²⁺]_i. Received: 7 March 1997 / Accepted: 9 September 1997     

The effect of the selective 5-HT1A agonists alnespirone (S-20499) and 8-OH-DPAT on extracellular 5-hydroxytryptamine in different regions of rat brain

1. We have examined the effects of the systemic administration of the selective 5-HT_1A agonist alnespirone (S-20499) on in vivo 5-hydroxytryptamine (5-HT) release in the dorsal raphe nucleus, the median raphe nucleus and four forebrain areas innervated differentially by both (dorsal striatum, frontal cortex, ventral hippocampus and dorsal hippocampus).
2. Alnespirone (0.1–3 mg kg⁻¹, s.c.) dose-dependently reduced extracellular 5-HT in the six areas examined. In forebrain, the maximal reductions occurred in striatum and frontal cortex (maximal reduction to 23 and 29% of baseline, respectively). Those in dorsal and ventral hippocampus were more moderate (to ca 65% of baseline). In contrast, the decrease in 5-HT elicited in the median raphe nucleus was more marked than that in the dorsal raphe nucleus (to ca 30 and 60% of baseline, respectively). The selective 5-HT_1A antagonist WAY-100635 (0.5 mg kg⁻¹, s.c.) prevented the decrease in 5-HT induced by alnespirone (0.3 mg kg⁻¹, s.c.) in frontal cortex.
3. 8-OH-DPAT (0.025, 0.1 and 0.3 mg kg⁻¹, s.c.) also reduced extracellular 5-HT in a regionally-selective manner (e.g., to 32% of baseline in striatum and to 69% in dorsal hippocampus at 0.1 mg kg⁻¹, s.c.). In midbrain, 8-OH-DPAT reduced the dialysate 5-HT slightly more in the median than in the dorsal raphe nucleus at all doses examined.
4. Doses of both compounds close to their respective ED₅₀ values (0.3 mg kg⁻¹ alnespirone, 0.025 mg kg⁻¹ 8-OH-DPAT) reduced 5-HT to a comparable extent in all regions examined. However, the reductions attained at higher doses were more pronounced for 8-OH-DPAT.
5. These data show that the reduction of 5-HT release elicited by alnespirone and 8-OH-DPAT is more important in forebrain areas innervated by 5-hydroxytryptaminergic neurones of the dorsal raphe nucleus. This regional selectivity seems unlikely to be accounted for by differences in the sensitivity of 5-HT_1A autoreceptors controlling 5-HT release, given the dissimilar effects of these two 5-HT_1A agonists in regions rich in cell bodies and nerve terminals. This suggests the presence of complex mechanisms of control of 5-HT release by 5-HT_1A receptors.

     

The influence of the long-term administration of Curcuma longa extract on learning and spatial memory as well as the concentration of brain neurotransmitters and level of plasma corticosterone in aged rats

The effects of chronic pre-treatment with a standardised extract of Curcuma longa on learning and spatial memory in aged 24-month old male Wistar rats were estimated in a Morris water maze paradigm. Animals received the extract orally for two months in prepared rodent chow to obtain the doses 10 and 50 mg/kg/day. At the end of behavioural trials the concentration of neurotransmitters, their metabolites and amino acids in selected brain regions were estimated.There was a significant decrease in escape latency over four days of training in both treated groups in comparison to the control group. In a probe trial on the 5th day the C10 group crossed the target area more often and spent more time in the SE quadrant than control group.Significant differences in brain monoamines and amino acid levels between groups were noticed. The increase in the 5-HT (5-hydroxytryptamine) level in the prefrontal cortex correlated positively with the number of crossings over the target area during the first probe trial in both pre-treated groups. The plasma corticosterone level was lower in both pre-treated groups than in the control group. This suggests enhanced learning ability and spatial memory after C.longa extract treatment with the modulation of central serotoninergic system activity, and may be linked with an increased tolerance to stress conditions.A decrease in hippocampal glutamate in animals given plant extract compared to control rats was observed. It is possible that extract may influence a reduction in glutamate-induced excitotoxicity and consequently the neurodegeneration processes in the hippocampus.     

8-Hydroxy-2-(di-n-propylamino) tetralin is devoid of activity at the 5-hydroxytryptamine autoreceptor in rat brain. Implications for the proposed link between the autoreceptor and the [3H] 5-HT recognition site

Summary Experiments have been carried out to provide direct evidence for the proposed presynaptic 5-HT autoreceptor agonist activity of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) a compound with selectivity for the 5-HT_1A subtype of the 5-HT₁ binding site. Rat brain frontal cortex slices were preincubated with [³H] 5-hydroxytryptamine and continuously stimulated with Krebs solution containing paroxetine and elevated K⁺ ions (25 mmol/l). The elevated efflux of tritium caused by exposure to K⁺ Krebs was inhibited in a dose related manner by 5-hydroxytryptamine and this inhibition was attenuated in the presence of quipazine and methiothepin.In slices of the rat frontal cortex, 8-OH-DPAT was without agonist or antagonist activity at the 5-HT autoreceptor at concentrations up to 1 mol/l. Higher concentrations caused an increase in basal efflux of tritium. 8-OH-DPAT (1 mol/l) was also without inhibitory activity in the piriform cortex, striatum and the hippocampus.These experiments have therefore failed to provide direct evidence for agonist activity of 8-OH-DPAT at the 5-HT autoreceptor and alternative explanations must be sought for its biochemical and behavioural effects in vivo. Moreover, the fact that 8-OH-DPAT is inactive at the autoreceptor at concentrations selective for the 5-HT_1A recognition site suggests that this subtype of the 5-HT_1A binding site may not correspond to the 5-HT autoreceptor.Part of this work was presented at the Joint Meeting of the French and German Pharmacological and Toxicological Societies, Freiburg, September 1983     

N-methyl-d-aspartate (NMDA) receptor-mediated stimulation of noradrenaline release,but not release of other neurotransmitters,in the rat brain cortex: receptor location,characterization and desensitization

Summary Rat brain cortex slices and synaptosomes (in a few experiments also hippocampal synaptosomes) preincubated with ³H-noradrenaline, ³H-5-hydroxytryptamine, ³H-choline, ³H-glutamate or ³H--aminobutyric acid were used to investigate the ³H-transmitter release in response to exposure to N-methyl-d-aspartate (NMDA) and other excitatory amino acids. The slices and synaptosomes were superfused with Mg²⁺-free, otherwise physiologically composed salt solution.In cortical slices preincubated with ³H-noradrenaline, NMDA concentration-dependently stimulated ³H overflow, whereas no such effect occurred in slices preincubated with ³H-5-hydroxytryptamine, ³H-choline, ³H-utamate or ³H--aminobutyric acid. In cortical slices preincubated with ³H-noradrenaline, the NMDA-evoked ³H overflow was abolished by tetrodotoxin, presence of Mg²⁺ 1.2 mmol/l or absence of Ca²⁺. 2-Amino-5-phosphonovaleric acid produced a parallel shift to the right of the NMDA concentration-response curve, whereas (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine hydrogen maleate (MK-801) not only shifted the concentration-response curve to the right but also reduced the maximum effect of NMDA. Other excitatory amino acid receptor agonists also stimulated ³H overflow, yielding the following rank order of potency: NMDA > l-glutamate > l-aspartate. Kainate and, in particular, quisqualate exhibited only low potencies and/or intrinsic activities. Prolonged (25 min) exposure of ³HNA-preincubated cortical slices to a high NMDA concentration produced a short-lasting peak of ³H overflow, followed by a second phase lasting as long as the compound was present; in this phase, ³H overflow was clearly less pronounced and gradually decreased with time. The stimulatory effect of a high NMDA concentration was concentration-dependently reduced by 20 min of pre-superfusion with NMDA or l-glutamate at concentrations which by themselves produced either no or, at the most, moderate increase in ³H efflux in the two 5-min periods before application of the NMDA stimulus; in contrast, the veratridine-evoked ³H overflow was increased by pre-exposure to these NMDA concentrations. Neither in 3H-noradrenaline-preincubated synaptosomes prepared from the cortex or hippocampus nor in cortical synaptosomes preincubated with ³H-5-hy-droxytryptamine did NMDA evoke ³H overflow. The veratridine-evoked ³H overflow from ³H-noradrenaline-preincubated cortical synaptosomes was not affected by simultaneous administration of NMDA.It is concluded that NMDA selectively stimulates noradrenaline release in the rat brain cortex via NMDA receptors which appear not to be located on the noradrenergic nerve terminals. The NMDA receptor is rapidly desensitized in response to continuous application of NMDA (tachyphylaxis) or l-glutamate (cross-tachyphylaxis). Send offprint requests to M. Göthert at the above address