A 'long-term-potentiation-like' facilitation of hippocampal synaptic transmission induced by the nootropic nefiracetam

Nefiracetam, a nootropic agent, enhanced the slope of field excitatory postsynaptic potentials in the CA1 region of rat hippocampal slices to about 170% of basal levels, being evident still at 4-h washing-out of the drug. A similar sustained enhancement (>/=16 h after i.m. injection with nefiracetam) was observed in the population spikes recorded from the granular cell layer of the intact mouse hippocampus. Saturation of the enhancement in the synaptic strength occluded potentiation obtained with long-term potentiation (LTP) induced by high-frequency (tetanic) stimulation, and vice versa. Interestingly, the facilitatory action of nefiracetam was blocked by either the nicotinic acetylcholine (ACh) receptor antagonists, alpha-bungarotoxin and mecamylamine, or the selective protein kinase C (PKC) inhibitor, GF109203X, but in contrast, it was not affected by D-2-amino-5-phosphonovaleric acid (APV), a selective N-methyl-D-aspartate (NMDA) receptor antagonist. The results of the present study suggest that nefiracetam, whereas the action is independent of NMDA receptors, induces an 'LTP-like' facilitation of hippocampal synaptic transmission as a consequence of modulation of nicotinic ACh receptors and PKC. This may represent a likely mechanism underlying the cognition-enhancing actions of nefiracetam.     

Antisense mapping KOR-1: evidence for multiple kappa analgesic mechanisms

In binding assays, both dynorphin B and alpha-neoendorphin are relatively selective for the kappa1b site, unlike U50,488H which has high affinity for both kappa1a and kappa1b sites. In vivo, U50,488H, dynorphin B and alpha-neoendorphin analgesia are reversed by the kappa1-selective antagonist, nor-binaltorphimine (norBNI). Antisense mapping the three exons of KOR-1 revealed that probes targeting all three exons blocked U50,488H analgesia, as expected. However, the selectivity profile of dynorphin B and alpha-neoendorphin analgesia towards the various antisense oligodeoxynucleotides differed markedly from U50,488H, implying a different receptor mechanism of action.     

Effects of opioid receptor antagonists on the effects of i.v. morphine on carrageenin evoked c-Fos expression in the superficial dorsal horn of the rat spinal cord

This study performed in freely moving rats evaluated the ability of specific opioid receptor antagonists to reverse the inhibitory effects of morphine on carrageenin-induced c-Fos expression in the spinal cord. Our study focused on the superficial dorsal horn (laminae I-II), which is the main termination site of nociceptive primary afferent fibers and is rich in opioid receptors. In order to replicate clinical routes of administration, all agents were administered intravenously (i.v.). As previously demonstrated, pre-administered i.v. morphine (3 mg/kg) produced a marked decrease (58+/-5%) in the number of Fos-LI neurones measured at 2 h after intraplantar (i.pl.) carrageenin (6 mg/150 microl) and yet was without influence on peripheral oedema. This decrease in c-Fos expression was completely blocked by combined administration of morphine with the mu-opioid receptor antagonist, [D-Phe-Cys-Tyr-D-Orn-Thr-Pen-Thr-NH2] (CTOP-1+1 mg/kg). Naltrindole (NTI-1+1 mg/kg), a delta-opioid receptor antagonist partially blocked the effects of systemic morphine, so that the inhibitory effects of morphine after NTI injection are now 40+/-4%. However, this effect of NTI was weak since the depressive effects of morphine were still highly significant (p<0.001). In contrast, nor-binaltorphimine (nor-BNI-1+1 mg/kg), a kappa-opioid receptor antagonist, had no significant effect on the effects of morphine. These results indicate the major contribution of mu-opioid receptors to the antinociceptive effects of systemic morphine at the level of the superficial dorsal horn. The observed effect of NTI is not necessarily related to a direct action of morphine on delta-opioid receptors and some possible actions of this antagonist are discussed.     

Ginkgo biloba extract EGb761 reduces the development of amphetamine-induced behavioral sensitization: effects on hippocampal type II corticosteroid receptors

Pretreatment of rats with the extract of Ginkgo biloba termed EGb761 reduced the behavioral sensitization induced by successive -amphetamine administrations (0.5 mg/kg) as estimated by increasing values of locomotor activity. EGb761 pretreatment also prevented the reduced density of [³H]dexamethasone binding sites in the dentate gyrus and the CA1 hippocampal regions of -amphetamine treated animals. These observations suggest that EGb761, by reducing glucocorticoid levels, could modulate the activity of the neuronal systems involved in the expression of the behavioral sensitization.     

Cellular distribution of the NMDA receptor subunit NMDAR1 in fetal ventral mesencephalon transplants in the dopamine-depleted striatum of a rat

Immunohistochemistry was performed to demonstrate the cellular distribution of N-methyl-D-aspartate (NMDA) receptor subunit NMDAR1 in the intrastriatal grafts of a rat model of Parkinson's disease. Unilateral 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal pathway were produced in young adult female rats. Neural transplantation was performed with fetal ventral mesencephalon (VM) tissue (at embryonic day 15) 3 weeks after the 6-OHDA lesions. In the fetal VM in which the tyrosine hydroxylase (TH) immunoreactivity was intensely observed, no NMDAR1 subunit immunoreactivity was detected. Immunopositive cells of NMDAR1 were densely distributed in the intact SNc contralateral to the lesions, in which intense immunoreactivity for TH was observed. In contrast, the cells positive for NMDAR1 in the SNr were scattered. The immunoreactivity for NMDAR1 was markedly decreased in the SNc, but not in the SNr on the lesioned side. Double immunostaining revealed that most TH-positive cells in the SNc showed moderate NMDAR1 immunoreactivity. Within the intrastriatal fetal VM grafts containing TH-positive cells, NMDAR1-positive cells tended to locate homogeneously within the grafts. These were composed of various cell sizes and shapes, but they were mainly medium-sized and aspiny cells. Double immunostaining revealed that a part of the TH-positive cells in the grafts was also immunopositive for NMDAR1. Taken together with our previous studies, it is suggested that both dopaminergic neurons and nondopaminergic neurons in the VM transplants appear to be modified functionally by glutamatergic afferents via various glutamate receptors, including NMDAR1.     

Acetylcholine suppresses the spread of excitation in the visual cortex revealed by optical recording: possible differential effect depending on the source of input

Optical recording with a voltage-sensitive dye was performed in visual cortical slices of the rat to determine the effect of acetylcholine (ACh) on the spread of excitation. In the presence of ACh, the spread of excitation initiated by stimulation at the white matter/layer VI (WM/VI) was greatly suppressed throughout the cortex, with less suppression in the middle layers. By comparing the effect of ACh with that of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the fraction of the synaptic component that was sensitive to ACh was evaluated. ACh suppressed approximately 40-50% (maximum 55.8%, n = 11) of the initial synaptic component in the superficial and deep layers. In the middle, however, the effect was weakest and only approximately 20-30% (minimum 20.9%, n = 11) of the initial synaptic component was suppressed. On the basis of histological analysis, the region with the weakest ACh effect extended from upper V to lower II/III. To identify the site of ACh action in terms of pre- versus postsynaptic localization, exogenous glutamate was applied. Because ACh did not suppress the excitation induced by glutamate, the site of the ACh action was indicated to be presynaptic. When layer II/III was stimulated instead of WM/VI, the suppression was uniform throughout the cortex. A muscarinic receptor antagonist, atropine, blocked the suppression by ACh. In conclusion, our results indicate the following two points. First, ACh strongly suppresses intracortical connectivity through presynaptic muscarinic receptors. Secondly, in contrast to the intracortical connection, some group(s) of fibres, possibly thalamocortical afferents that arise from white matter and terminate in the middle cortical layers are suppressed much less by ACh. While ACh has been reported to have confusingly diverse effects, e.g. direct depolarization and hyperpolarization as well as synaptic facilitation and suppression, its effect on the propagation of excitation is very clear; suppression on intracortical connection, leaving thalamocortical inputs rather intact. We postulate that cholinergic innervation enables the afferent input to have a relatively dominant effect in the cortex.     

Previous maternal experience potentiates the effect of parturition on oxytocin receptor mRNA expression in the paraventricular nucleus

In sheep, central oxytocin release at parturition induces maternal behaviour which is thought to be mediated by changes in the expression of central oxytocin receptors. The distribution, effects of parturition, previous maternal experience and hormonal status on the distribution of an oxytocin receptor was investigated using immunocytochemistry and in situ hybridization. In ewes with no previous maternal experience, parturition induced significant increases in oxytocin receptor mRNA expression in the anterior olfactory nucleus, medial preoptic area, ventromedial hypothalamus, lateral septum, medial amygdala, bed nucleus of the stria terminalis and diagonal band of Broca. In maternally experienced ewes, parturition induced additional increases in two areas, the paraventricular nucleus and the Islands of Calleja. The changes in progesterone and oestrogen that occur during late pregnancy and parturition appear to contribute to increases in expression in the anterior olfactory nucleus, Islands of Calleja, medial preoptic area, ventromedial hypothalamus, bed nucleus of the stria terminalis and diagonal band of Broca, but not in the paraventricular nucleus, lateral septum and medial amygdala. These results demonstrate that progesterone and oestrogen priming enhance oxytocin receptor mRNA expression in a number of regions in the olfactory system, hypothalamus and limbic brain. These effects appear to be independent of maternal experience. Parturition increases oxytocin receptor mRNA expression in all the areas influenced by hormonal priming and the lateral septum, medial amygdala and paraventricular nucleus. Maternal experience also enhances expression of oxytocin receptor mRNA in the paraventricular nucleus and the Islands of Calleja. Because the paraventricular nucleus is the main source of oxytocin release in the brain, this upgrading of autoreceptors as a result of maternal experience may serve to enhance release of this peptide in projection sites regulating maternal behaviour.     

Cells defective in sphingolipids biosynthesis express low amounts of muscle nicotinic acetylcholine receptor

The properties of the nicotinic acetylcholine receptor (AChR) are modulated by its lipid microenvironment. Studies of such modulation are hampered by the cell's homeostatic mechanisms that impede sustained modification of membrane lipid composition. We have devised a novel strategy to circumvent this problem and study the effect of changes in plasma membrane lipid composition on the functional properties of AChR. This approach is based on the stable transfection of AChR subunit cDNAs into cells defective in a specific lipid metabolic pathway. In the present work we illustrate this new strategy with the successful transfection of a temperature-sensitive Chinese hamster ovary (CHO) cell line, SPB-1, with the genes corresponding to the four adult mouse AChR subunits. The new clone, SPB-1/SPH, carries a mutation of the gene coding for serine palmitoyl transferase, the enzyme that catalyses the first step in sphingomyelin (Sph) biosynthesis. This defect causes a decrease of Sph de novo synthesis at non-permissive temperatures. The IC50 for inhibition of alpha-BTX binding with the agonist carbamoylcholine exhibited values of 3.6 and 2.7 microm in the wild-type and Sph-deficient cell lines, respectively. The corresponding IC50 values for the competitive antagonist D-tubocurarine (D-TC) were 2.8 and 3.4 microm, respectively. No differences in single-channel properties were observed between wild-type and mutant cell lines grown at the non-permissive, lipid defect-expressing temperature using the patch-clamp technique. Both cells exhibited two open times with mean values of 0.35 +/- 0.05 and 1.78 +/- 0.2 ms at 12 degrees C. Taken together, these results suggest that the AChR is expressed as the complete heteroligomer. However, only 10-20% of the total AChR synthesized reached the surface membrane in the mutant cell line and exhibited a higher metabolic turnover, with a half-life about 50% shorter than the wild-type cells. When control CHO-K1/A5 cells were treated with fumonisin B1, an inhibitor of sphingosine (sphinganine) N-acetyltransferase (ceramide synthase), a 45.5% decrease in cell surface AChR expression was observed. The results suggest that sphingomyelin deficiency conditions AChR targeting to the plasma membrane.     

Sexually dimorphic expression of sst1 and sst2 somatostatin receptor subtypes in the arcuate nucleus and anterior pituitary of adult rats

The pattern of growth hormone (GH) secretion and rate of somatic growth are markedly sexually dimorphic, but the underlying neuroendocrine mechanisms are far from clear. In the present study, we tested the hypothesis that the sexual dimorphism of GH secretion may be due to gender-related differences in the transduction of somatostatin's actions in brain and/or pituitary. To accomplish this, we compared the distributional pattern and level of expression of two somatostatin receptor subtypes, sst1 and sst2, in the brain and pituitary of adult male and female rats by in-situ hybridization using 35S-labelled antisense riboprobes. In the brain, the hybridization pattern and labelling density of sst1 and sst2 mRNA-expressing cells, as revealed by computer-assisted image analysis, in areas including the cerebral cortex, medial habenula (MHb) and ventromedial hypothalamic nucleus (VMN), were similar in male and female rats. In contrast, there was a marked sex-related difference in sst1 expression in the arcuate nucleus of the hypothalamus; both the number and labelling density of sst1 mRNA-expressing cells were two- to threefold greater in males than in females and this significant increase was homogenous throughout the rostrocaudal extent of the nucleus. No gender-related differences in arcuate sst2 mRNA levels were found. At the level of the anterior pituitary, the labelling density of sst2 mRNA in males was significantly higher than that of females. No sex-related difference in pituitary sst1 mRNA was observed. These results demonstrate a sexual dimorphism in the expression of two somatostatin receptor subtypes, sst1 and sst2, at the level of the arcuate nucleus and anterior pituitary, respectively. Such dimorphism suggests a differential involvement of sst1 and sst2 in GH regulation with respect to gender, and may imply roles for sst2 and sst1 in transducing somatostatin's actions on pituitary somatotrophs and GH-releasing hormone-containing arcuate neurones, respectively, to generate the lower basal and higher GH pulse levels characteristic of the male rat.     

Screening for mutations in candidate genes for hypospadias

Hypospadias, a condition with a frontally placed urethral orifice on the penis, is the most common malformation in males. During fetal development several components are necessary for normal male genital development. Testosterone and dihydrotestosterone act via the androgen receptor but a defective receptor function results in different degrees of genital malformations. Testosterone-5α-reductase converts testosterone to dihydrotestosterone, which is crucial for normal differentiation, and a total lack of this enzyme results, in syndromes with hypospadias. The Wilms' tumour 1 (WT1) gene is expressed in the fetal gonad and genital malformations can occur due to WT1 gene mutations. These genes are therefore strong candidate genes for hypospadias. We have analysed 35 boys with hypopadias and one girl diagnosed as with complete androgen insensitivity syndrome, using exon by exon polymerace chain reaction (PCR) amplification of the AR, WT1 and 5α-reductase genes and screened for point mutations and performed subsequent DNA sequencing. No mutations in any of these genes were found in the 26 patients with isolated hypospadias. Two patients with severe hypospadias with cryptorchidism were found to carry mutations in the androgen receptor gene. Also the girl with clinically diagnosed complete androgen insensitivity was found to be homozygous for a splice mutation in the 5α-reductase gene. In summary, mutations in the WT1, AR and 5α-reductase genes are not common causes of isolated hypospadias. Received: 1 October 1997 / Accepted: 4 May 1998