Effect of chronic stress on cholinergic transmission in rat hippocampus

We previously demonstrated that chronic stress impaired prefrontal cortex-sensitive working memory, but not reference memory. Since the hippocampal cholinergic system is also involved in these memories, we examined the effects of chronic stress on cholinergic transmission in the rat hippocampus. A microdialysis study revealed that the stress did not affect the basal acetylcholine release, but enhanced the KCl-evoked response. These results suggest that cholinergic transmission in the chronically stressed hippocampus does not contribute to working memory impairment, but it may be involved in maintenance of reference memory.     

Regulation of glucocorticoid receptor immunoreactivity in the rat hippocampus by andronergic-anabolic steroids

To determine whether androenic-anabolic steroids (AAS) regulate glucocorticoid receptors (GR), the effects of a mixture of three commonly abused AAS (testosterone cypionate, nandrolone decanoate and boldenone undecylenate) on GR immunoreactivity (ir), were examined in hippocampi of adrenalectomized male rats. Treatment with AAS for 1 week increased nuclear GRir in pyramidal cells of CA1, and granule cells of the dentate gyrus. These findings suggest a role for GR in the mediation of some of the CNS effects of androgenic-anabolic steroids.     

创伤性脑损伤后大鼠海马区糖皮质激素受体mRNA的表达

目的研究创伤性脑损伤(TBI)后大鼠海马区糖皮质激素受体(GR)mRNA表达的变化及其对大鼠认知功能的影响。方法建立大鼠头颅侧向旋转加速脑创伤模型,应用逆转录酶-聚合酶链式反应(RT-PCR)和Morris水迷宫检测伤后大鼠海马区GR mRNA的表达与学习记忆功能的关系。结果伤后4～7 d大鼠海马区GR持续低表达;Morris水迷宫检测伤后大鼠出现认知功能障碍。结论 TBI大鼠海马区GR mRNA的降低影响大鼠认知功能。     

In vivo microdialysis measures of extracellular serotonin in the rat hippocampus during sleep–wakefulness

We investigated extracellular 5-hydroxytryptamine (5-HT) levels in rat hippocampus during different stages of the sleep–waking cycle using in vivo microdialysis. The extracellular 5-HT level was highest in active waking (AW) and, when compared to AW, 5-HT level was progressively lower in quiet waking (QW; 78%), quiet sleep (QS; 50%) and REM (which we termed active sleep (AS); 40%). Functional implications of AS related-decreased 5-HT in the hippocampus are discussed.     

Glucocorticoids modulate the NGF mRNA response in the rat hippocampus after traumatic brain injury

Nerve growth factor (NGF) expression in the rat hippocampus is increased after experimental traumatic brain injury (TBI) and is neuroprotective. Glucocorticoids are regulators of brain neurotrophin levels and are often prescribed following TBI. The effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on the expression of NGF mRNA in the hippocampus after TBI has not been investigated to date. We used fluid percussion injury and in situ hybridisation to evaluate the expression of NGF mRNA in the hippocampus 4 h after TBI in adrenal-intact or adrenalectomised rats (with or without CORT replacement). TBI increased expression of NGF mRNA in sham-ADX rats, but not in ADX rats. Furthermore, CORT replacement in ADX rats restored the increase in NGF mRNA induced by TBI. These findings suggest that glucocorticoids have an important role in the induction of hippocampal NGF mRNA after TBI.     

Demonstration of the facilitatory role of 8-OH-DPAT on cholinergic transmission in the rat hippocampus using in vivo microdialysis

The role of the serotonin (5-HT)_1A receptor in the regulation of acetylcholine (ACh) release in the hippocampus was investigated using an in vivo microdialysis technique and a sensitive radioimmunoassay specific for ACh. The mean (±S.E.M.) basal ACh contents in the hippocampal perfusate of conscious, freely moving rats was 60 ± 4 (n = 29) and 3691 ± 265 fmol/30 min (n = 31), respectively, in the absence and presence of physostigmine (Phy) in the perfusion fluid. Systemic administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 0.5 mg/kg, s.c.), a 5-HT_1A agonist, significantly enhanced ACh release both in the presence and absence of Phy. Local application of 8-OH-DPAT (3–30 μM) into the hippocampus through the microdialysis probe significantly potentiated ACh release only in the presence of Phy, whereas no significant effect was observed in its absence. Pretreatment with NAN-190 (3 mg/kg, i.p.), a 5-HT_1A antagonist, eliminated the increasing effect of systemically applied 8-OH-DPAT on ACh release, while NAN-190 alone had no effect on basal ACh release either in the absence or presence of Phy. Consistent with the time course of ACh release, systemic administration of 8-OH-DPAT evoked hyperlocomotion, which was reversed by NAN-190. However, local hippocampal application of 8-OH-DPAT did not affect the locomotor activity of the rats. These findings suggest that at least two different sites are involved in the 8-OH-DPAT-induced increase in the release of ACh in the rat hippocampus in vivo.     

Enhanced localization of amyloid β precursor protein in the rat hippocampus following long-term adrenalectomy

Using various antibodies to the amyloid ß precursor protein (APP) associated with Alzheimer's disease, we investigated changes in the distribution of APP in the hippocampus and neocortex of adrenalectomized (ADX) rats. In contrast to sham-operated controls, ADX rats euthanised after a survival period of 5 months showed striking APP reactivity in the CA1–CA4 fields and in the surviving cells in the dentate gyrus. Our results suggest the enhanced APP reactivity in hippocampal neurons may pertain to previous observations on the accumulation of APP fragments in the neocortex during ischemic or traumatic injury. Thus, long-term hormone deprivation would be another factor, which may influence the expression of APP in brain.     

Cellular reactions to implantation of a microdialysis tube in the rat hippocampus

Summary Microdialysis tubes, used for measurements of extracellular neurotransmitter concentrations, were implanted in rat dorsal hippocampus to study the adjacent tissue reaction. The brain was examined 1–60 days after the implantation. Within the first 2 days, normal neuropil and only occasional hemorrhage surrounded the microdialysis tube. Three days following the implantation astrocytes close to the dialysis tube, hypertrophied. Hypertrophic astrocyte processes invaded the spongy fiber wall. There was no increase in the number of astrocytes. Fourteen days after the fiber insertion layers of reticulin-positive fibers separated astrocytes and the remaining neuropil from the fiber wall. Late tissue changes (1 and 2 months) consisted of collagen deposits and occasional granuloma formation. These results can be used to predict the optimal time for commencing microdialysis after the fiber implantation.Supported by grants 12-5705 and 12-6077 from the Danish Medical Research Council and by the Novo foundation     

Adrenalectomy-induced granule cell death is predicated on the disappearance of glucocorticoid receptor immunoreactivity in the rat hippocampal granule cell layer

In this study, we observed the changes of glucocorticoid receptor (GR)-immunoreactivity (ir) and cell death in the rat hippocampal granule cell layer at various periods after adrenalectomy (ADX). Our results revealed that all of the rats shortly after ADX showed a rapid loss of GR-ir and subsequent appearance of degenerating cells in the granule cell layer. One month after ADX, however, about 80% of the rats displayed a restoration of GR-ir and the absence of degenerating cells in the granule cell layer, and this phenomenon was successively noted for 6 months. Hippocampal structural destruction 3 and 6 months after ADX was found in about 20% of the rats with loss of GR-ir in the granule cell layer; the ADX rats with even weak GR-ir in this area had a normal hippocampus. The treatment of rats with synthetic GR agonist, dexamethasone, immediately after ADX prevented the loss of GR-ir and significantly reduced the number of degenerating cells in the granule cell layer. Our results clarified that granule cell death after ADX was necessarily accompanied by the disappearance of GR-ir in the granule cell layer, suggesting that ADX-induced granule cell death is predicated on the loss of GR-ir and that the presence of GR-ir in this area may be important for granule cell survival.     

Temporal changes in extracellular acetylcholine and CA1 pyramidal cells in gerbil hippocampus following transient cerebral ischemia

Temporal changes in cholinergic functions following transient cerebral ischemia (10 min) were studied in the hippocampus of awake unrestrained gerbils using in vivo microdialysis. These data were compared with the results for temporal change in the area of each CA1 cell soma, measured with a microcomputer imaging device. KCl-induced release of acetylcholine (ACh) tended to be lower within 1 day after recirculation, and was significantly lower on the 4th, 7th and 14th days. Atropine-induced release of ACh gradually decreased over the test period. In histological estimation, no differences were observed within the 1st day, but a significant decrease of the area of CA1 cell soma was observed from the 4th to 14th days. Moreover, ischemia over 2 min decreased KCl- and atropine-induced ACh release on the 14th day without significant changes of hippocampal CA1 pyramidal cell. From these results, it is clear that ischemia produced dysfunction of hippocampal cholinergic neurons, and that dysfunction of the hippocampal cholinergic system following transient ischemia precedes pyramidal cell damage in the hippocampal CA1 subfield.     

Baseline and 8-OH-DPAT-induced release of acetylcholine in the hippocampus of aged rats with different levels of cognitive dysfunction

During aging, neurotransmission systems such as the cholinergic and serotonergic ones are altered. Using rats aged 3 or 24-26 months, this study investigated whether the well-described 8-OH-DPAT-induced increase of hippocampal acetylcholine release was altered in aged rats and whether it may vary according to the magnitude of age-related cognitive deficits. Long-Evans female rats aged 24-26 months were classified as good or bad performers on the basis of their reference-memory performance in a Morris water-maze task. Subsequently, the efficiency of 5-HT(1A) receptor agonist 8-OH-DPAT (0.5 mg/kg, s.c.) in triggering hippocampal acetylcholine release was evaluated by in vivo microdialysis and high performance liquid chromatography analysis. Besides a reduced baseline release in aged rats and a correlation between the baseline release and probe-trial performance in all rats, the results demonstrated that 8-OH-DPAT produced a significant increase of hippocampal acetylcholine release (peak value) in all rats, whether aged or young. While significant in bad performers (+56%), this increase did not reach significance in good performers (+32%). The results suggest that (i) some aspects of cognitive alterations related to aging might be linked to the baseline release of acetylcholine in the hippocampus, and (ii) the cholinergic innervation of the hippocampus of aged rats responds almost normally to systemic activation of 5-HT(1A) receptors, and (iii) differential alterations of cholinergic/serotonergic interactions assessed by determination of the 8-OH-DPAT-induced release of acetylcholine in the hippocampus could not be linked with clarity to the cognitive status of aged rats.     

Distribution of glucocorticoid and mineralocorticoid receptor messenger RNA expression in human postmortem hippocampus

Corticosteroids bind to hippocampal glucocorticoid (GR) and mineralocorticoid (MR) receptors, thereby affecting behaviour and neurochemical transmission. Rat hippocampus has high levels of both receptors and their messenger RNAs (mRNA), but there is little information on receptors in human brain. We used in situ hybridization to determine the distribution of GR and MR mRNA expression in human hippocampus. Frozen sections of human postmortem hippocampus (5 patients, 58-88 years old, without cerebral pathology) were postfixed in paraformaldehyde and hybridized with 35S-UTP-labelled cRNA probes (transcribed in vitro from human cDNA subclones) under stringent conditions. Control included hybridization with sense probes and heterologous cRNA competition studies. GR mRNA was highly expressed in dentate gyrus, CA3 and CA4, but levels were significantly lower in CA1 and CA2. MR mRNA was also very highly expressed in hippocampus, with significantly higher levels in dentate gyrus and CA2, CA3 and CA4 than CA1. Controls confirmed the specificity of hybridization and there was little hybridization of sense probes. High GR and MR mRNA expression is found in both rat and human hippocampus but the subregional distributions clearly differ between the species.     

Inhibition of glutamate release in rat hippocampus by kynurenic acid does not protect CA1 cells from forebrain ischemia

We assessed the effect of a broad spectrum glutamatergic receptor antagonist, kynurenic acid (500 mg/kg) on ischemia-induced hippocampal glutamate release and neuronal damage. Kynurenic acid significantly decreased glutamate release during ischemia but had no effect on the hippocampal lesion. Some protection was observed in the cortex and in the striatum. These data suggested that the extracellular accumulation of glutamate during forebrain ischemia does not play a major role in the hippocampus.     

Facilitation by arachidonic acid of acetylcholine release from the rat hippocampus

We investigated the effect of arachidonic acid (AA) on the release of [3H]acetylcholine ([3H]ACh) from the rat hippocampus. AA (3-30 microM) increased the basal tritium outflow and the field-electrically evoked release of [3H]ACh from hippocampal slices in a concentration-dependent manner. AA (30 microM) produced a 69+/-7% facilitation of the evoked and a 36+/-3% facilitation of basal tritium outflow. The effect of AA (30 microM) on the evoked tritium release was prevented by bovine serum albumin (BSA, 1%), which quenches AA, and was unaffected by the cyclooxygenase inhibitor, indomethacin (100 microM), and the lipooxygenase inhibitor, nordihydroguaiaretic acid (50 microM). Phospholipase A2 (PLA2, 2 U/ml), an enzyme that releases AA from the sn-2 position of phospholipids, mimicked the facilitatory effect of AA on the evoked tritium release (86+/-14% facilitation), an effect prevented by BSA (1%). The PLA2 activator, melittin (1 microM), enhanced the evoked tritium release by 98+/-11%, an effect prevented by the PLA2 inhibitor, arachidonyl trifluromethylketone (AACOCF3, 20 microM), and by BSA (1%). AA (30 microM), but not arachidic acid (30 microM), also facilitated (72+/-9%) the veratridine (10 microM)-evoked [3H]ACh release from superfused hippocampal synaptosomes, whereas PLA2 (2 U/ml) and melittin (1 microM) caused a lower facilitation (46+/-1% and 38+/-5%, respectively). The present results show that both exogenously added and endogenously produced AA increase the evoked release of [3H]ACh from rat hippocampal nerve terminals. Since muscarinic activation triggers AA production and we now observed that AA enhances ACh release, it is proposed that AA may act as a facilitatory retrograde messenger in hippocampal cholinergic muscarinic transmission as it has been proposed to act in glutamatergic transmission.     

Corticosterone alters G protein α-subunit levels in the rat hippocampus

The hypothalamic-pituitary-adrenal axis regulates the synthesis and secretion of corticosteroid hormones. The hippocampus, a component of the limbic system, contains the highest concentration of corticosteroid receptors in the brain and may play an important role in regulating hypothalamic-pituitary-adrenal axis activity and mediating physiological responses to stress. The corticosteroid hormone corticosterone alters the response elicited by activation of several different G protein-linked neurotransmitter receptors in the hippocampus. In the present study we used Western blot and immunohistochemical techniques to determine the effects of chronic adrenalectomy (ADX), low basal (CT) and high (HCT) corticosterone treatments on G_s, G_{i1 and 2} and G_o α-subunit levels and intracellular location in the rat hippocampus. CT treatment increased G_s α-subunit levels and HCT treatment increased the levels of G_s, G_{i1 and 2} and G_o α-subunits when compared to sham as detected on Western blots. No change in the intracellular location of the G protein α-subunits was detected using immunohistochemistry. Based on our results, we conclude that corticosterone alters G protein α-subunit levels in the rat hippocampus without altering their intracellular location. These results provide an important piece of information towards understanding how corticosteroids alter G protein-linked neurotransmitter receptor-mediated responses.     

Acetylcholine release in the hippocampus: regulation by monoaminergic afferents as assessed by in vivo microdialysis

The role of monoamines in the functional regulation of the septo-hippocampal cholinergic system was studied using in vivo microdialysis of acetylcholine (ACh) release in the hippocampus of awake unrestrained rats. Systemic administration of the dopamine receptor agonist apomorphine (2.0 mg/kg) resulted in a 170% increase in hippocampal ACh overflow. Similarly the catecholamine-releasing agent amphetamine (2.5 mg/kg) produced a 400% increase in ACh overflow. The effect induced by amphetamine, but not that of apomorphine, was blocked in animals pretreated with the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine (AMPT). The effect of amphetamine on ACh release was reduced by 75% after a 6-hydroxydopamine (6-OHDA) lesion of the ventral tegmental area (VTA) but was not affected by 6-OHDA lesions of the noradrenergic dorsal and ventral bundles. However, baseline ACh overflow was increased by 130% by the dorsal and ventral bundle lesions. The serotonin-releasing agent p-chloroamphetamine (2.5 mg/kg) produced a 160% increase in hippocampal ACh release, and this effect was enhanced after a 5,7-dihydroxytryptamine (5,7-DHT) lesion of the serotonin projection system. The results show that surgical or pharmacological manipulations of the ascending brainstem monoaminergic systems, which innervate wide areas of the forebrain, including the septum and the hippocampal formation, have pronounced effects on septo-hippocampal cholinergic activity. Thus, the present data provide support for the view that information regarding behavioral state and arousal is conveyed to the septo-hippocampal system via ascending monoaminergic systems.     

Corticosteroids alter G protein inwardly rectifying potassium channels protein levels in hippocampal subfields

Corticosterone or cortisol, stress hormones in rat and human, respectively, alter neurotransmitter receptor-mediated responses in the brain. Corticosterone could alter these responses by modifying any component of the receptor-effector pathway. Many of these receptors are linked to guanine nucleotide regulatory proteins (G proteins) which, in turn, can activate second messenger systems and/or ion channels, such as G protein inwardly rectifying potassium channels (GIRK). The aim of these experiments was to determine whether corticosterone treatment altered the levels of GIRK proteins in rat hippocampus. Corticosterone treatment selectively altered the levels of GIRK1 and GIRK2 (measured on immunoblots) depending on the subfield of the hippocampus examined. These data lend credence to the hypothesis that corticosterone differentially alters neurotransmitter receptor-mediated responses dependent on the brain area.     

Inhibition of cholinesterase elicits muscarinic receptor-mediated synaptic transmission in the rat adrenal medulla

To determine the role of acetylcholinesterase in cholinergic synaptic transmission in the adrenal medulla in vivo, we applied a dialysis technique to the adrenal medulla of anesthetized rats and examined the effect of acetylcholinesterase inhibitor on the contribution of nicotinic and muscarinic receptors to catecholamine release. Exogenous acetylcholine-induced epinephrine release was inhibited by atropine (a muscarinic receptor antagonist) as well as hexamethonium (a nicotinic receptor antagonist). Endogenous acetylcholine (nerve stimulation)-induced epinephrine release was inhibited by hexamethonium but not atropine. In the presence of neostigmine (an acetylcholinesterase inhibitor), both exogenous and endogenous acetylcholine-induced catecholamine release was enhanced. In either case, epinephrine release was inhibited by atropine as well as hexamethonium. In the presence of eserine (another acetylcholinesterase inhibitor), endogenous acetylcholine-induced epinephrine release was also inhibited by atropine. Exogenous or endogenous acetylcholine-induced norepinephrine release was primarily inhibited by hexamethonium regardless of whether neostigmine was absent or present. In the rat adrenal medulla, the inhibition of acetylcholinesterase not only enhanced cholinergic synaptic transmission but also elicited muscarinic receptor-mediated synaptic transmission for epinephrine release.     

Raphe cells grafted into the hippocampus can ameliorate spatial memory deficits in rats with combined serotonergic/cholinergic deficiencies

The ability of embryonic raphe cells grafted into the hippocampus to restore spatial learning ability was tested in rats with combined serotonergic/cholinergic deficits. Embryonic raphe cells (E14) were transplanted into the hippocampus of serotonin-depleted rats. Two to 3 months after transplantation, control, lesioned and grafted rats were tested in a spatial memory task (a water maze) with and without the addition of atropine. All 3 groups could negotiate the water maze equally well, in non-drug conditions. The injection of atropine caused a severe disruption of performance only in the serotonin depleted rats. The presence of an active serotonergic graft was examined in the intact rat hippocampus using the serotonin releasing drug fenfluramine (FFA). A pronounced depression of hippocampal EEG was observed in control and grafted but not in lesioned rats 15 min after the injection of FFA. These results suggest the involvement of serotonin in cognitive functions in the rat. Furthermore, it is suggested that an interaction between serotonergic and cholinergic neurotransmission occurs in the hippocampus.     

Dexamethasone enhances serum deprivation-induced necrotic death of rat C6 glioma cells through activation of glucocorticoid receptors

Glucocorticoids have been shown to be neurotoxic and appear to play a role in neuronal cell loss during aging and following neuropathological insults. However, very little is known about the effects of these steroid hormones on glial cells. The effect of the synthetic glucocorticoid dexamethasone (DEX) on glial cell viability was therefore examined by measuring neutral red uptake into rat C6 glioma cells. Serum deprivation markedly reduced cell viability, and this effect was significantly enhanced by DEX. Electrophoretic analysis showed that the cell damage induced by either serum deprivation alone or in combination with DEX was not accompanied by the degradation of DNA into nucleosomic fragments. Electron microscopic studies confirmed that serum deprivation and glucocorticoid treatment caused necrotic cell death. Furthermore, the effect of DEX on cell viability could be mimicked by the glucocorticoid receptor agonist RU28362, and completely prevented by the glucocorticoid receptor antagonist RU38486. These results indicate that dexamethasone can enhance the necrotic death of glioma cells induced by serum deprivation, suggesting that glucocorticoids may be involved in the chronic alteration of brain function arising from neuropathological damage to glial cells.