Length of postovariectomy interval and age, but not estrogen replacement, regulate N-methyl-D-aspartate receptor mRNA levels in the hippocampus of female rats

Estrogens and N-methyl-D-aspartate (NMDA) receptors regulate multiple aspects of morphological and functional plasticity in young animals. For example, estrogens increase spine density in the hippocampus, and NMDA antagonists block these effects. Few studies have examined the effects of age, postovariectomy interval, and duration of estrogen replacement in the hippocampus and more specifically on NMDA receptor subunits. Therefore, the present study was designed to investigate the effects of short- and long-term estrogen replacement or deprivation on mRNA levels of three NMDA receptor subunits, NR1, NR2A, and NR2B, in the hippocampus of aging female Sprague-Dawley rats. Young (3- to 4-month-old) and middle-aged (12- to 13-month-old) rats were ovariectomized for 1 month and then treated with estrogen or vehicle for either 2 days or 2 weeks. Another set of middle-aged and aged (24-to 25-month-old) animals were ovariectomized for 6 months and treated with estrogen or vehicle for 2 days or 2 weeks. RNase protection assay was used to assess changes in the NMDA receptor subunit mRNA levels. Our results demonstrated significant effects of age and length of ovariectomy on NMDA receptor mRNA levels, with little effect of the estrogen status of the animals on these parameters. The largest effect was seen for the length of the postovariectomy interval, with the results demonstrating that rats with a short-term ovariectomy have substantially higher NMDA receptor subunit mRNA levels than animals with long-term ovariectomy. The most dramatic effects of aging were seen for NR1 and NR2B mRNAs in ventral hippocampus, with large age-related increases. These data suggest that age and duration of ovariectomy impact NMDA receptor mRNA levels in the hippocampus, potentially affecting the stoichiometry and/or function of these receptors. These findings have important implications for postmenopausal or hysterectomy/oophorectomy estrogen depletion and replacement in humans.     

Phencyclidine-induced discriminative stimulus is mediated via phencyclidine binding sites on the N-methyl-D-aspartate receptor-ion channel complex, not via sigma(1) receptors

The effects of several N-methyl-D-aspartate (NMDA) receptor- and sigma receptor-related compounds on the discriminative stimulus effects of phencyclidine (PCP) were examined in rats trained to discriminate PCP (1.5 mg/kg, i.p.) from saline under a two-lever fixed ratio 20 schedule of food reinforcement. PCP produced a dose-dependent increase in PCP-appropriate responding. A non-competitive NMDA receptor antagonist, dizocilpine (0.2 mg/kg, i.p.) and a putative sigma(1) receptor agonist, (+)-SKF-10047 (10 mg/kg, i.p.) fully substituted for PCP in every rat tested. Neither a competitive NMDA receptor antagonist, CGS-19755 (0.1-3 mg/kg, i.p.), sigma(1) receptor agonist, (+)-pentazocine (10-30 mg/kg, i.p.) nor dextromethorphan (10-20 mg/kg, i.p.) produced PCP-like discriminative stimulus effects. The discriminative stimulus effects of PCP (1.5 mg/kg, i.p.), dizocilpine (0.2 mg/kg, i.p.) and (+)-SKF-10047 (10 mg/kg, i.p.) were significantly attenuated by CGS-19755 (1 mg/kg, i.p.), but not by sigma(1) receptor antagonist BMY-14802 (10 mg/kg, i.p.) and NE-100 (5 mg/kg, i.p.). These results suggest that the discriminative stimulus effects of PCP are predominantly mediated via PCP binding sites on the NMDA receptor-ion channel complex, not via sigma(1) receptors. In addition, the PCP-like discriminative stimulus effects of (+)-SKF-10047 were demonstrated to be mediated via PCP binding sites.     

Decreased central GABA B receptor binding sites in diabetic rats

Little is known about the implication of central GABAergic neurons. However, there is evidence suggesting a growing importance of GABAergic function in the action of antidepressants. Since streptozotocin (STZ)-diabetic rats have been shown to be resistant to the action of various antidepressants, we were interested in evaluating the density of GABAergic receptor binding sites in the cortex of STZ-diabetic rats on day 15 and day 30 of diabetes. A specific and marked decrease in GABA B receptor density was observed with no change in GABA A. Although no clear relationship could be demonstrated, it may be suggested that a central GABAergic dysfunction of diabetic rats may contribute to explain their resistance to antidepressants.     

Autoradiographic detection of vasopressin binding sites, but not of oxytocin binding sites, in the sheep olfactory bulb

Oxytocin facilitates maternal behaviour in sheep. In the present study, we searched for the presence of oxytocin and vasopressin binding sites in the sheep olfactory bulb, a brain area which is thought to be involved in specific bond formation between the ewe and its lamb. Using in vitro autoradiography, we observed binding of tritiated vasopressin to the glomerular layer of the olfactory bulb. Competition studies performed with structural analogues and the use of a 125I-labelled linear vasopressin antagonist suggested that sites which bind vasopressin are V1 type receptors. In contrast, specific binding sites for oxytocin in the olfactory bulb could be detected neither in control females, nor in ovariectomized females treated with estradiol nor in postparturient ewes, although such sites were present in the uterus.     

The N-methyl-D-aspartate receptor complex in Alzheimer's disease: reduced regulation by glycine but not zinc

The binding of [3H]MK-801 to the N-methyl-D-aspartate receptor complex of well-washed cortical membranes from brains of examples of Alzheimer's disease and controls has been determined in incubations containing either glutamate or glycine plus glutamate. No changes were detected in the IC50 values for inhibition by zinc in the Alzheimer's samples compared to control although 'glycine-dependent' binding of the [3H]-ligand was significantly reduced in Alzheimer's disease.     

Depression: reduced number of granule cells in the hippocampus of female,but not male,rats due to prenatal restraint stress

It has been hypothesized that decreased neurogenesis in the dentate gyrus may be involved in mediating depressive disorders, which are 1.5-3 times more frequent in women than in men. Additionally, prenatal stress may increase the risk of developing depression in adulthood. However, the interrelations between prenatal stress and the development of depression in adulthood, preferentially in females, are not understood. Here, we subjected pregnant rats to a single 20-min period of restraint stress on day 18 after mating. When the offspring were 75 days of age, the numbers of granule cells and pyramidal cells (area CA1-3) in the hippocampus were analyzed with the optical fractionator. The Cavalieri's principle was applied to analyze the volumes of both granule cell layer and pyramidal cell layer in the hippocampus. Prenatally stressed females, but not males, had reduced numbers of hippocampal granule cells compared to their non-prenatally stressed counterparts. This is the first report of a sex-specific difference concerning the reduction of the number of hippocampal granule cells due to prenatal stress. In humans, prenatal stress may induce cell loss in the granule cells of the hippocampus preferentially in females compared to males, and this may be a sex-specific predisposing factor for the development of depression in adulthood.     

Selective loss of cerebral cortical sigma, but not PCP binding sites in schizophrenia.

Drugs such as phencyclidine (PCP) that interact with PCP and sigma binding sites can produce psychotomimetic effects that resemble some symptoms of schizophrenia. Therefore, it has been suggested that PCP and sigma receptors may be important in the clinical manifestations of schizophrenia. Assays of these two binding sites in human postmortem brains showed consistent significant reductions in the density of sigma, but not PCP sites, in schizophrenics as compared with age-matched and postmortem interval-matched normal and suicide controls. Reductions in the density of sigma binding sites in schizophrenia were most prominent in temporal cerebral cortex, and were accompanied by a small increase in affinity for the ligand [3H]haloperidol. These data provide the first evidence for alterations in sigma binding sites in schizophrenia, and suggest that selective sigma ligands may be useful in the treatment of the disorder.     

Divergent ontogeny of sigma and phencyclidine binding sites in the rat brain

The postnatal developmental patterns of sigma (sigma) and phencyclidine (PCP) binding sites were compared in the rat brain. The results show diametrically different ontogenic patterns for the sites. While both the affinity and the density of sigma sites remain constant throughout the developmental period tested (postnatal day 1 to 1 year), the density of PCP binding sites increases from the time of birth, reaching the adult level by postnatal day 14. The differences in developmental patterns provide evidence for distinctive properties of cerebral sigma and PCP binding sites.     

Gestational exposure to phencyclidine (PCP) in rats decreases PCP binding sites in term fetal brain

Pregnant Sprague-Dawley rats were treated with 5 mg/kg body weight of phencyclidine (PCP) injected at 1 ml/kg subcutaneously on three consecutive days at four different stages of gestation. Within 10–30 min after treatment, dams showed some lack of motor coordination and became lethargic. On gestational day 21, all rats were killed by decapitation and brains were dissected and stored from mother and fetus for neurochemical analysis. PCP, dopamine and muscarinic cholinergic receptor binding was measured in membranes prepared from maternal and fetal whole brain. Neurotransmitter concentrations were also measured in the fetal brain homogenates. There was a significant decrease in PCP binding sites in fetal but not maternal brains after maternal PCP injection at gestational days 12–14, 15–17 and 18–20, but not at 9–11 days. Dopamine and muscarinic cholinergic receptor binding was not significantly altered in fetal or maternal brain when compared with vehicle control animals. The whole brain dopamine, 3,4-dihydroxyphenylacetic acid, serotonin, and 5-hydroxyindoleacetic acid concentrations did not show significant change in any group studied. These data indicate that gestational exposure to PCP decreases high affinity binding of PCP in term fetal brain at doses which do not alter maternal PCP receptor binding.     

Developmental study of [3H]TCP and [3H]glycine binding sites in the rat hippocampus

The postnatal development of [3H]thienylphencyclidine ([3H]TCP) sites in rat hippocampus has been studied autoradiographically and with membrane preparations. [3H]TCP binding increased progressively from birth to adulthood; this is due to a change in the maximal number of sites (Bmax) but not in the affinity (Kd). A different developmental pattern was found for strychnine-insensitive [3H]glycine binding which also increased after birth, but reached adult levels earlier than [3H]TCP binding. The ontogenesis of TCP or glycine sites also differed from that previously described for N-methyl-D-aspartate (NMDA) sites in the hippocampus. In neonatal, as in adult hippocampus, [3H]TCP binding was enhanced by NMDA or glycine and reduced by Mg2+. We suggest that TCP sites are functionally coupled to the NMDA receptor-ion channel complex in developing as in mature hippocampus, but that there are developmental changes in the receptor channel complex.     

Cholecystokinin antagonists proglumide, lorglumide and benzotript, but not L-364,718, interact with brain opioid binding sites

It has been reported that proglumide and L-364,718 potentiate opioid-induced antinociception. However, their mode of action in pain modulation is still not understood. To evaluate a possible interaction with opioid receptors, we determined the affinities of the CCK antagonists proglumide, lorglumide, benzotript and L-364,718 on mu, delta and kappa binding sites, using guinea pig brain crude synaptosome preparations. These affinities were compared to that of the central CCK binding site, using rat brain slide-mounted sections. At 100 microM, proglumide competed for 13% and 17% of mu and kappa binding sites, but did not interact with delta and CCK sites. At this concentration, lorglumide reduced mu, delta, kappa and CCK specific binding by 44%, 69%, 35% and 88%, whereas benzotript diminished it by 16%, 13%, 38% and 48%, respectively. L-364,718 did not interact with opioid receptors (assay limit of solubility, 10 microM) but had a high affinity for CCK binding sites (IC50, 126nM). The lack of selectivity of proglumide, lorglumide and benzotript for CCK receptors suggests that their reported ability to potentiate morphine analgesia may be related to an interaction with opioid receptors.     

Decreased neprilysin immunoreactivity in Alzheimer disease, but not in pathological aging

Although evidence suggests that extensive cortical beta-amyloid (Abeta) deposition is essential in Alzheimer disease (AD), it is also detected in nondemented elderly individuals with pathologic aging (PA). Given evidence that neutral endopeptidase (NEP) or neprilysin, a key enzyme for clearance of Abeta, is decreased in AD, the goal of the present study was to determine if NEP was also decreased in PA. We measured NEP immunoreactivity in frontal cortex of 12 AD and six PA cases and compared this with 10 normal (N) elderly individuals. None had any significant other pathology, and they were similar with respect to age, sex, and postmortem delay. In addition, Abeta1-40 and Abeta1-42 were measured by enzyme-linked immunosorbent assay (ELISA), whereas tau, synaptophysin, and alpha-synuclein were measured on Western blots. The AD cases had more neuritic plaques, neurofibrillary tangles, higher Braak stage, and more tau immunoreactivity in frontal cortex than both PA and N. In contrast, both PA and AD had more senile plaques and Abeta1-42 than N. NEP immunoreactivity was decreased in AD but not in PA. The decrease was unlikely the result of neuronal or synaptic loss because NEP immunoreactivity in frontotemporal degeneration with comparable degrees of synaptic loss as the AD cases was not different from control subjects. Although NEP enzyme activity was decreased in approximately half the AD cases, on average, it was not decreased compared with N or PA. The results add further evidence that PA is distinct from AD and indicate that decreased Abeta degradation by NEP is unlikely to contribute significantly to amyloid deposition in PA or, in many cases, of AD.     

Fall in density, but not number of myenteric neurons and circular muscle nerve fibres in guinea-pig colon with ageing

Abstract  In guinea-pig ileum, ageing has been associated with a decrease in enteric neurons. This study examined guinea-pig colon and measured changes in gut dimensions, neuron size, density and ganglionic area. Changes in motor nerve fibres in the circular muscle were also measured. Myenteric neurons in whole-mount preparations of mid-colon from 2-week, 6-month, and 2-year-old guinea-pigs were labelled immunohistochemically with the neuronal marker human neuronal protein HuC/HuD, and numbers of neurons mm⁻², neuronal size, ganglionic area mm⁻², gut length, circumference and muscle thickness were measured. Corrected numbers of neurons mm⁻² and ganglionic area mm⁻² accounting for growth of the colon were calculated. Additionally, nerve fibres in circular muscle cross-sections were labelled with antibodies against nitric oxide synthase (NOS) and substance P (SP) and the density of nerve fibres in circular muscle was measured. The numbers of neurons mm⁻² decreased by 56% (from 2 weeks to 2 years) with no change in neuron size. Total neuron numbers decreased by 19% ( P  = 0.14) when adjusted for changes in length and circumference with age. The percentage area of NOS- and SP-immunoreactive (IR) nerve fibres in the circular muscle decreased ( P  < 0.001), but the total area of NOS and SP-IR nerve fibres increased ( P  < 0.01) due to an age-related increase in muscle thickness. The density of myenteric neurons in guinea-pig mid-colon halved from 2 weeks to 2 years, but when the increase in colon dimensions was considered, the number of neurons decreased by only 19%. The percentage area of motor nerve fibres in the circular muscle decreased with no change in total volume of nerve fibres.     

Post-stress facilitation of serotonergic, but not noradrenergic, neurotransmission in the dorsal hippocampus prevents learned helplessness development in rats

Recent pieces of evidence suggest that the dorsal hippocampus may mediate adaptation to severe and inescapable stress, possibly by the facilitation of serotonergic and/or noradrenergic neurotransmission. Chronic social stress and high corticosteroid levels would impair this coping mechanism, predisposing animals to learned helplessness. To test the hypothesis that increasing serotonin or noradrenaline levels in the dorsal hippocampus would attenuate the development of learned helplessness (LH), rats received inescapable foot shock (IS) and were tested in a shuttle box 24 h latter. Prestressed animals showed impairment of escape responses. This effect was prevented by bilateral intrahippocampal injections of zimelidine (100 nmol/0.5 microl), a serotonin reuptake blocker, immediately after IS. This effect was not observed when zimelidine was administered before or 2 h after IS. Bilateral intrahippocampal injections of desipramine (3 or 30 nmol/0.5 microl), a noradrenaline reuptake blocker, before IS or immediately after it did not prevent LH development. Desipramine (30 nmol) enhanced LH development when injected before IS. These data suggest that poststress facilitation of hippocampal serotonergic, but not noradrenergic, neurotransmission in the dorsal hippocampus facilitates adaptation to severe inescapable stress. Antidepressant effects of noradrenaline-selective drugs seem to depend on other structures than the dorsal hippocampus.     

Oral administration of glycine increases extracellular serotonin but not dopamine in the prefrontal cortex of rats

Aim: Glycine, one of the non‐essential amino acids, has been reported to be effective in reducing negative symptoms of schizophrenia. Recently, we found that glycine improves subjective sleep quality in humans. The aim of this study was to investigate the effects of oral glycine administration on endogenous 5‐hydroxytryptamine (5‐HT) and dopamine in the prefrontal cortex (PFC) of living rats. Methods: Microdialysis probes were inserted stereotaxically into the rat prefrontal cortex. Cortical levels of 5‐HT and dopamine were measured following oral administration of 1 or 2 g/kg glycine, 2 g/kg d ‐serine, or 2 g/kg L‐serine. Results: Both glycine and d ‐serine significantly increased extracellular 5‐HT levels for 10 min, whereas dopamine levels remained unchanged. L‐serine, in contrast, had no significant effects on 5‐HT levels. Conclusions: It is possible that the increase in 5‐HT in response to glycine and d ‐serine was mediated by N‐methyl‐D‐aspartate receptors. The transient increase in 5‐HT in the PFC might be associated with the alleviation of negative symptoms in patients with schizophrenia and the amelioration of sleep quality in patients with insomnia.     

Decreased experimental anxiety and voluntary ethanol consumption in rats following central but not basolateral amygdala lesions

A long-debated ‘tension reduction' hypothesis postulates anti-anxiety effects to be important for ethanol reward, and states that elevated anxiety levels might predispose for ethanol consumption and addiction. Human data are contradictory, possibly due to heterogeneity of patient samples. In rats, baseline levels of experimental anxiety have been reported to correlate with voluntary ethanol consumption. Here, we addressed the possibility that mechanisms underlying experimental anxiety might be causally related to regulation of voluntary ethanol intake. Rats were bilaterally lesioned in central amygdala using microinjections of ibotenic acid. This resulted in a robust release of punished drinking in a modified Vogel conflict test, an effect typically seen with anxiety reducing drugs. This effect was specific, as unpunished drinking was unaffected by the lesion. On the elevated plus-maze, central amygdala lesions did not affect behaviour under baseline conditions, but attenuated the anxiogenic effect of restraint stress. Measures of locomotor activity were not affected. Voluntary ethanol consumption was examined in a two-bottle, free choice paradigm. Ethanol intake was markedly decreased in the lesion group. Total fluid intake was not affected. Basolateral amygdala lesions, which did not affect conflict behaviour, also left ethanol intake unaffected. These results are consistent with previous reports of an important role for central amygdala in anxiety related behaviours, and suggest that cell bodies located in central amygdala might be important in this context. Further, our results support a relation between experimental anxiety and voluntary ethanol consumption.     

Folic acid,but not folate,regulates different stages of neurogenesis in the ventral hippocampus of adult female rats

Folate is an important regulator of hippocampal neurogenesis, and folic acid is needed prenatally to reduce the risk of neural tube defects. Both high levels of folic acid and low levels of folate can be harmful to health because low levels of folate have been linked to several diseases while high folic acid supplements can mask a vitamin B₁₂ deficiency. Depressed patients exhibit folate deficiencies, lower levels of hippocampal neurogenesis, elevated levels of homocysteine and elevated levels of the stress hormone, cortisol, which may be inter‐related. In the present study, we were interested in whether different doses of natural folate or synthetic folic acid diets can influence neurogenesis in the hippocampus, levels of plasma homocysteine and serum corticosterone in adult female rats. Adult female Sprague‐Dawley rats underwent dietary interventions for 29 days. Animals were randomly assigned to six different dietary groups: folate deficient + succinylsulphathiazole (SST), low 5‐methyltetrahydrofolate (5‐MTHF), low 5‐MTHF + (SST), high 5‐MTHF + SST, low folic acid and high folic acid. SST was added to a subset of the 5‐MTHF diets to eliminate folic acid production in the gut. Before and after dietary treatment, blood samples were collected for corticosterone and homocysteine analysis, and brain tissue was collected for neurogenesis analysis. High folic acid and low 5‐MTHF without SST increased the number of immature neurones (doublecortin‐expressing cells) within the ventral hippocampus compared to folate deficient controls. Low 5‐MTHF without SST significantly increased the number of immature neurones compared to low and high 5‐MTHF + SST, indicating that SST interfered with elevations in neurogenesis. Low folic acid and high 5‐MTHF + SST reduced plasma homocysteine levels compared to controls, although there was no significant effect of diet on serum corticosterone levels. In addition, low folic acid and high 5‐MTHF + SST reduced the number of mature new neurones in the ventral hippocampus (bromodeoxyuridine/NeuN‐positive cells) compared to folate deficient controls. Overall, folic acid dose‐dependently influenced neurogenesis with low levels decreasing but high levels increasing neurogenesis in the ventral hippocampus, suggesting that this region, which is important for regulating stress, is particularly sensitive to folic acid in diets. Furthermore, the addition of SST negated the effects of 5‐MTHF to increase neurogenesis in the ventral hippocampus.     

β-Adrenergic, but not benzodiazepine, binding sites are reduced in dystrophic mouse brain

We have examined brain tissue from dystrophic mice (male 129/ReJ-dy and female 129 B6F₁/J-dy) to determine whether CNS lesions may accompany the known muscular defects. In all brain regions examined we found a significant reduction in the numbers of β-adrenergic binding sites when compared to brain from control mice. In contrast, the number of benzodiazepine binding sites appeared normal. These changes in adrenergic sites may be related to the known abnormally high excretion of catecholamines. Our results also emphasise the need to study extra-muscular sites in this disease.