Memory consolidation in senescence: effects of CO2, amphetamine and morphine.

Alterations in memory storage processes that occur in senescence were investigated by challenging young and old female "small Wistar" rats with posttraining administration of CO2, amphetamine or morphine, and measuring retention performance. Neither duration of CO2 immersion, nor the time of CO2 immersion after training had a differential amnestic effect with age on retention of a one-trial, shock-motivated inhibitory avoidance task. These results indicate that the times during which memory is susceptible to disruption for old and young rats are similar. Challenge with drugs, however, did reveal age-related alterations in memory storage processes. Amphetamine attenuated CO2-induced amnesia in young rats, but had no effect in old rats. This could not be attributed to a general decline in response to amphetamine in old rats because amphetamine increased open field activity of both young and old animals. Morphine also had a differential effect on memory with age: it caused amnesia in old rats trained in a one-trial hot plate escape task, while having no effect on retention performance of young rats. Thus, the modulatory influence of catecholamine and opioid systems on memory processes is probably altered in senescence.

Preferential release of newly synthesized 3 H-acetylcholine from rat cerebral cortex slices in vitro.

1. Slices of rat cerebral cortex after treatment with the irreversible cholinesterase inhibitor soman, were incubated for 5 min in a Krebs-Henseleit solution containing 25 mM KCl and (3)H-choline. Subsequently incubation was continued in a medium containing non-radioactive choline and this medium was replaced at 5 min intervals. The amounts of labelled and total acetylcholine (ACh) released into the medium and extracted from the slices were determined at intervals.2. After the initial 5 min contact with (3)H-choline, 44% of the newly synthesized ACh contained a choline moiety originating from the choline in the medium. During the initial 5 min and the subsequent incubation part of the labelled ACh was released. While the rate of total ACh release remained constant, that of the release of labelled ACh was highest in the 5 min period following the initial incubation with (3)H-choline and then declined exponentially.3. The ratio of labelled ACh/total ACh in the ACh released during the initial 5 min incubation with (3)H-choline and during the subsequent 5 min was about three times as high as that in the ACh extracted from the slices at the end of these incubation periods.4. The ratio of labelled ACh/total ACh in superficial layers of the slices was not higher than that in the total slices.5. The rates of release of labelled and unlabelled ACh decreased when calcium was omitted from the incubation medium and were restored when the calcium was added. This suggests that both labelled and unlabelled ACh were released from nerve endings. The efflux of (3)H-choline was not calcium dependent.6. It is concluded that labelled ACh newly synthesized from externally applied (3)H-choline does not exchange immediately with all other ACh in the tissue and has a greater chance of being released than unlabelled ACh.

Differences in presynaptic alpha-blockade, noradrenaline uptake inhibition, and potential antidepressant activity between (+)- and (-)mianserin.

The effect of racemic mianserin on K+-evoked tritium release from rat brain cortex slices previously incubated with 3H-L-noradrenaline was studied. Racemic mianserin (10(-9)--10(-5) M) increased stimulation-induced release dose-dependently. As methysergide, metiamide, and cyproheptadine failed to do so, it was concluded that this effect was probably not caused by the antihistamine or antiserotonin activity of racemic mianserin, but due to its alpha-adrenolytic effect. Evaluation of the effects of the enantiomers (+)(S)mianserin and (-)(R)mianserin showed that the alpha-adrenolytic effect resided in the (+)isomer, whereas the (-)isomer was inactive at a concentration of 10(-6) M. Inhibition of noradrenaline into rat hypothalamic synaptosomes also showed stereospecificity in that (+)mianserin was about 300-times more active than(-)mianserin. Inhibition of rat muricidal behavior, a test for potential antidepressant activity, showed a similar dissociation in the effects of the two enantiomers, in that (+)mianserin was active, whereas (-)mianserin was not.

Response changes after repeated low apomorphine: dopamine autoreceptor desensitization or learning?

Repeated injection of rats with low doses of apomorphine (APO), which selectively interact with dopamine (DA) autoreceptors, caused a change in yawning responses that suggests initial low-APO-induced desensitization of DA autoreceptors, followed by a long-lasting rebound hypersensitivity. Repeated treatment with low APO followed by open-field testing, however, yielded totally different results. APO accelerated intrasession response decrement and upon repeated administration enhanced the intersession response decrement. Both for yawning and open-field behavior, the response change after the second dose of APO was only evident when the first as well as the second APO injection were followed by exposure of the rat to the same test situation. These results indicate that response changes after repeated treatment with low APO are not due to a simple DA-agonist-induced change in receptor sensitivity but that drug experience combined with environmental influences play a decisive role.

Synaptosomal uptake studies on recombinant inbred mice; neurotransmitter interaction and behavioral correlates.

The uptake of noradrenaline, choline and GABA into whole brain synaptosomes from the mice inbred strains C57BL/6By, BALB/cBy, their reciprocal crosses and seven recombinant inbred strains derived from the F2 crosses was measured in order to study the relation between different neurotransmitter systems and their possible correlations with behavioral parameters. Analysis of variance demonstrated significant differences in both Vmax and Km for each substrate between the inbred strains studied. Only the Km for GABA uptake did not reach significance although a strong trend (P = 0.07) was apparent. For none of the uptake parameters clear strain distribution patterns were found. A positive phenotypic as well as genotypic correlation was found between the Vmax and Km for noradrenaline, choline and GABA uptake. A genetic analysis showed a positive correlation between the Vmax for noradrenaline and choline uptake. Since prior to these neurochemical studies, all subjects were tested in a variety of behavioral paradigms, phenotypic and genotypic correlations between behavioral and neurochemical parameters could be calculated. The most consistent correlations were found between noradrenaline and choline uptake and water consumption and the preference ratio as measured in a conditioned taste aversion test.

A Parkinson-like neurologic deficit in primates is caused by a novel 4-substituted piperidine.

A neurologic deficit characterized by hypokinesia, postural flexion, and to a lesser extent, rigidity, tremor and myoclonus, has been observed in cynomolgus monkeys following administration of 1-methyl-4-(1-methylpyrrol-2-yl)-4-piperidinol (MMPP), a novel 4-substituted piperidine. The syndrome, similar to that described for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), developed within 3-7 days after oral or i.v. dosing, and was accompanied by lesions in the substantia nigra. The behavioral syndrome was seen to a lesser extent in dogs but not in rats. MMPP contains a hydroxyl group on the 4-position of the pyridine ring; the corresponding dehydration product was inactive.

Detailed analysis of the effects of apomorphine and d-amphetamine on spontaneous locomotor behaviour of rats as measured in a TV-based, automated open-field system.

Rat open field behaviour was measured in a TV-based, automated system. Habituation was evident in saline-treated rats. Most variables measured declined over a 10 min period. Apomorphine affected rat open-field behaviour bimodally, i.e. low doses of apomorphine (0.02-0.08 mg/kg, s.c.) decreased most aspects of rat open-field behaviour, whereas at higher doses (0.2 and 0.5 mg/kg, s.c.) various aspects of open-field behaviour were stimulated. Rearing and average speed, however, were monotonically depressed. Amphetamine (1 and 2 mg/kg) stimulated most aspects of rats open-field behaviour, including rearing. Speed was not affected by amphetamine. Habituation was more pronounced after low doses of apomorphine than after saline treatment and was absent after high doses of apomorphine and after amphetamine. The results represent a detailed analysis of apomorphine and amphetamine effects on rat open-field behaviour and show that the distinct components of this behaviour are affected differentially.

On the oscillatory decay of the myotonic action of 9-anthroic acid. Influence of cholinesterase inhibition.

The effects of the myotonic agent, 9-anthroic acid (ANCA), and the acetylcholinesterase (AChE) inhibitor, soman, on the isolated phrenic nerve--diaphragm preparation of the rat have been studied. ANCA induced after-contractions which followed the twitches evoked by either direct or indirect stimulation. In the intermittently stimulated muscle the height of the after-contractions decreased rather rapidly and in an oscillatory fashion. The maximum height of each after-contraction was attained after the twitch had reached its peak. AChE inhibition changed the shape of these after-contractions in the indirectly, but not in the directly stimulated diaphragm. After AChE inhibition, the after-contractions decreased more slowly and in a non-oscillatory manner. Similar phenomena were observed in vivo in the gastrocnemius--soleus muscles. The effects of ANCA in the AChE-inhibited and in the non-inhibited diaphragm could be mimicked by incubation in low chloride media. Addition of ouabain to the non-inhibited diaphragm treated with ANCA, caused an immediate and striking enhancement followed by a rapid loss of the after-contractions, whereas the twitches remained fairly constant. In the AChE-inhibited diaphragm treated with ANCA, ouabain caused no increase but only a rapid and complete decay of the after-contractions with a decrease of the twitches. It is suggested that after inhibition of the AChE in the ANCA-treated muscle fibre, the site of initiation of the repetitive action potentials which cause the after-contractions shifts from the transverse tubular system to the motor end-plate. Moreover, it is suggested that the sodium pump is involved in the gradual decay of the myotonic action of ANCA.

Further studies on the therapy of organophosphorous anti-cholinesterase intoxication with veratrinic compounds; the role of calcium.

Experiments were carried out to investigate the role of calcium in the therapy of soman intoxication with 9-anthroic acid (ANCA), a compound with veratrine-like pharmacological properties. The effects of ANCA on the respiratory paralysis and on the calcium content of the blood and that of the hindleg muscles were determined in anaesthetized, atropinized rats injected with 4 times LD50 soman. The respiratory paralysis which in control animals occurs within a few min after the injection of soman can be delayed about 2.5 hr by treatment with ANCA. It was found that ANCA causes a small decrease of the blood calcium content, an effect which is potentiated by soman. A comparison was made between the calcium accumulation in the indirectly stimulated gastrocnemius-soleus muscles in these animals with that in the non-stimulated muscles on the other side. Whereas the injection of soman or ANCA alone caused no change, the combination of the two drugs induced a two-fold increase in the accumulation of calcium in the stimulated muscles. The non-stimulated muscles remained unaffected. The accumulation of calcium in the stimulated muscles induced by soman and ANCA could be partly antagonized by lowering the free calcium concentration of the blood by EDTA. Moreover, treatment with EDTA improved the therapeutic effects of ANCA. It is concluded that the therapy of soman poisoning with ANCA falls short in completely preventing respiratory failure since ANCA causes an accumulation of calcium in the stimulated muscles of soman-poisoned animals.

Age-related acquisition deficits and activity in rats.

Spontaneous motor activity and physically undemanding conditioned suppression of drinking behavior were assessed in rats of 3, 12, 18 or 30 months (M) age. Initial spontaneous motor activity of 12 M rats was as low as that of 30 M rats and both were significantly lower than that of 3 M rats. The performance of 3, 12 and 18 M rats were similar to each other, whereas 30 M rats started to perform poorer after one week of training. It is concluded that the acquisition deficits observed here in aged rats occur only after 18 months age and appear to be independent of the subjects' reactivity to punishment and their activity prior to training.

Changes in rat brain norepinephrine levels and turnover after olfactory bulbectomy.

After bilateral olfactory bulbectomy in rats a significant increase of norepinephrine (NE) level in the hypothalamus was found. However, no difference was observed between hypothalamic NE turnover of bulbectomized and sham operated animals in the amygdaloid cortex the NE level was not affected by bulbectomy. In this area, however, the NE turnover appeared to be decreased after bulbectomy. The latter finding may be related to the deficits in passive avoidance behaviour as found in bulbectomized rats.

Interaction between dopamine influx and efflux in rat striatal synaptosomes.

Exchange release has previously been proposed as the mechanism by which dopamine is transported over the synaptosomal membrane. An increase in carrier-mediated dopamine release should therefore result in an enhanced rate of synaptosomal dopamine uptake. In order to test this hypothesis, rat striatal synaptosomes were incubated with 14C-dopamine until equilibrium. Then, the 14C-dopamine concentration in the medium was reduced in order to elicit various rates of net dopamine efflux, while influx was monitored using tracer amounts of 3H-dopamine. Dopamine release was concentration dependent and saturable and thus may be carrier-mediated. In contrast to that expected for a mechanism of exchange release, dopamine influx was depressed as compared to equilibrium conditions. Furthermore, nomifensine, a specific inhibitor of dopamine influx, did not attenuate dopamine efflux. It is concluded that dopamine transport over the synaptosomal membrane may not be via a mechanism of strict exchange release.

Presynaptic alpha-block and inhibition of noradrenaline and 5-hydroxytryptamine reuptake by a series of compounds related to mianserin.

A structure-activity relationship study was undertaken for a variety of structural analogues of the tetracyclic antidepressant mianserin. Presynaptic alpha-blocking activity in vitro was evaluated measuring the potentiation of depolarization-induced noradrenaline (NA) release from rat cerebral cortex slices. Inhibition of NA and 5-hydroxytryptamine reuptake was measured in rat hypothalamic or striatal synaptosomes, respectively. Presynaptic alpha-blockade was only found in molecules with an overall bent shape. Flat rigid molecules or flexible ones were not active. Six-membered, chair-formed D-rings (containing the -NCH3 moiety) appeared better than 5- or 7-membered ones. Heteroatom substitution, but not hydroxylation or methylation, of the bridge between the two aromatic rings left presynaptic alpha-blockade unaffected. N-Demethylation and aromatic methyl- or chlorine-substitution reduced presynaptic alpha-blockade. In pyridine ring-substituted analogues the localization of the heteroatom appeared to be crucial. 5-Hydroxytryptamine reuptake inhibitory activity was only found in desmethylmianserin. NA uptake inhibition was found in many mianserin analogues, especially those with an exocyclic-N(CH3)2 moiety. Structure activity relationships for NA reuptake inhibition differed from those for presynaptic alpha-blockade and were generally less stringent. For both properties simple additivity relationships appeared to be absent.