Ethanol differentially modulates GABAA receptor-mediated chloride currents in hippocampal,cortical, and septal neurons in rat brain slices

Previous electrophysiological studies have reported conflicting results concerning the effects of ethanol on γ-aminobutyric acid-A (GABA_A) receptor-mediated responses in the brain. To examine the variables that might explain these inconsistencies, the present study was designed to determine whether ethanol modulation of synaptically evoked GABA responses is brain region dependent, to identify factors that might regulate ethanol sensitivity, and to investigate the mechanism(s) underlying ethanol modulation of GABA responses. Whole-cell voltage clamp methods were used to examine the effects of ethanol on synaptically evoked GABA_A inhibitory postsynaptic currents (IPSCs) recorded from neurons in hippocampus, cerebral cortex, and intermediate lateral and medial septum from rat brain slice preparations. Bicuculline-sensitive IPSCs elicited by local stimulation were pharmacologically isolated by pretreatment with the glutamate specific antagonists, DL-(?)-2-amino-5-phosphonovaleric acid (APV) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). Superfused ethanol (80 mM) potentiated evoked GABA_A IPSCs in cortical neurons and in intermediate lateral and medial septal neurons but not in CA1 hippocampal neurons. However, the mechanism by which ethanol enhanced GABA_A IPSC amplitudes differed between brain regions. In cortex, ethanol induced a hyperpolarizing shift in the GABA_A IPSC reversal potential (E_IPSC) without modifying the underlying GABA_A receptor-mediated conductance (G_IPSC). In contrast, ethanol enhanced GABA_A IPSC amplitudes in lateral and medial septal neurons by increasing the G_IPSC without modifying the E_IPSC These results suggest that ethanol differentially modulates responses to endogenous GABA released during synaptic activation and that important differences between various brain regions may reflect multiple mechanisms of ethanol action. © 1994 Wiley-Liss, Inc.     

Effects of insulin-induced hypoglycemia on somatostatin level and binding in rat cerebral cortex and hippocampus

The effects of severe insulin-induced hypoglycemia on somatostatin level and specific binding in the cerebral cortex and hippocampus were examined using 125I-Tyr11-somatostatin as a ligand. Severe insulin-induced hypoglycemia did not affect the level of somatostatin-like immunoreactivity in the brain areas studied. However, the number (but not the affinity) of specific somatostatin receptors was significantly decreased in membrane preparation from the hippocampus but not in the cerebral cortex at the onset of hypoglycemic coma (5-10 min). Administration of glucose at the onset of hypoglycemic coma brought about extensive recovery of hippocampal somatostatin receptor number. These results suggest that glucose modulates the somatostatin receptor in the rat hippocampus. The physiological significance of these findings remains to be clarified.     

Immunohisto- and cytochemical localization of cortical nicotinic cholinoceptors in rat and man

A monoclonal antibody (WF 6) raised against purified Torpedo nicotinic acetylcholine receptor was applied to study the cellular and subcellular receptor distribution in human and rat neocortex. In both species, immunostaining was most prominent in perikarya and dendrites of the projection neurons in layers III and V. In layer VI fusiform cells displayed immunoreactivity while in layers I, II and IV some round-shaped cells were immunostained. Subcellularly, immunoprecipitate was found in neuronal perikarya, dendrites and in the postsynaptic thickenings, indicating intracellular sites of synthesis, transport and membrane incorporation of receptor protein. The results suggest that WF 6-immunocytochemistry is a useful tool to label nicotinic cholinergic receptors rendering new information about the specific cell-type and subcellular receptor distribution hardly obtainable by using conventional receptor autoradiography.     

Correlation of glutamate-induced apoptosis with caspase activities in cultured rat cerebral cortical neurons

In cultured rat cortical neurons lactate dehydrogenase (LDH) activity in the medium, a cell-death marker, increased gradually after exposure to glutamate (100 microM to 1 mM) for 60 min and reached a plateau at 24 to 30 h. Neuronal death was mainly apoptotic as suggested by typical electron microscopic findings, fluorescent double staining with membrane-permeating and nonpermeating chromatin dyes, nick end labeling, and assessment of DNA fragmentation by agarose gel electrophoresis. After 1 mM glutamate exposure, a rise of interleukin-1beta converting enzyme (ICE)-like protease activity in neurons was parallel to cysteine protease p32 (CPP32)-like protease activity and declined before CPP32-like protease activity reached the peak (at 6 h). LDH activity in the medium of glutamate-exposed neurons was decreased by specific ICE and/or CPP32 inhibitors, acetyl-L-tyrosyl-L-valyl-L-alanyl-L-aspart-1-al (Ac-YVAD-CHO) and acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspart-1-al (Ac-DEVD-CHO), respectively, in a dose-dependent manner. Fluorescent double staining of nuclei also demonstrated that at 100 microM each inhibitor prevented neuronal apoptosis and that this effect was additive. Among agonists corresponding to various glutamate receptor subtypes, N-methyl-D-aspartate (NMDA) and kainate induced apoptosis in cortical neuronal cultures while alpha-amino-3-hydroxy-5-methylisoxazole-4-propinate (AMPA) did not. The metabotropic glutamate receptor agonist, 1-aminocyclopentane-1S, 3R-dicarboxylate (ACPD) prevented apoptosis. Interestingly, apoptosis at 24 h after agonist or antagonist exposure correlated closely with caspase activity 6 h after exposure.     

人与大鼠海马血管构筑的观察

目的比较分析人与大鼠海马构筑模式.方法本实验采用血管色素明胶灌注与甲酯铸型扫描电镜法,对人与大鼠海马血管来源、分布规律及超微结构进行观察,利用图像分析仪对其血管密度与管径进行测量并分析.结果人海马血管来源于大脑后动脉和脉络膜前动脉,而大鼠主要来源于大脑后动脉.人海马表面与内部血管呈规律性分布,而其内部血管分布不如大鼠海马明显.另外,见到人与大鼠海马内部均存在着丰富的血管吻合.同时观察到大鼠海马血管构筑与其神经组织结构呈明显的对应关系.结论大鼠海马血管分布与神经组织构筑相匹配,人与大鼠海马血管内部存在着广泛的血管吻合.     

Regulation of cortical acetylcholine release: Insights from in vivo microdialysis studies

Acetylcholine release links the activity of presynaptic neurons with their postsynaptic targets and thus represents the intercellular correlate of cholinergic neurotransmission. Here, we review the regulation and functional significance of acetylcholine release in the mammalian cerebral cortex, with a particular emphasis on information derived from in vivo microdialysis studies over the past three decades. This information is integrated with anatomical and behavioral data to derive conclusions regarding the role of cortical cholinergic transmission in normal behavioral and how its dysregulation may contribute to cognitive correlates of several neuropsychiatric conditions. Some unresolved issues regarding the regulation and significance of cortical acetylcholine release and the promise of new methodology for advancing our knowledge in this area are also briefly discussed.     

Consequences of perinatal hypoxia in developing brain: Changes in GABA transporter functioning in cortical,hippocampal and thalamic rat nerve terminals

Perinatal hypoxia leads to behavioral abnormalities, cognitive disabilities, and epilepsy resulting from alterations in neurodevelopment, maturation and construction of the network. Considering a particular role of γ-aminobutyric acid (GABA) for an immature brain, we analysed transporter-mediated [³H]GABA uptake in the cortical, hippocampal and thalamic nerve terminals isolated from rats of different age in the control and after perinatal hypoxia. The state of hypoxia was induced by exposure of rats at the age of 10 postnatal days (pd) (that corresponds approximately to the time of birth in humans) to a respiratory medium with low O₂ content (4% O₂ and 96%N₂) for 12 min (up to the initiation of clonico-tonic seizures). Here, we found that the initial rate of [³Н]GABA uptake was higher in the young rats (pd 17–19) as compared to the older ones (pd 24–26, 38–40 and 66–73) in both control and hypoxia groups. It decreased abruptly by 50% in the thalamus and by 25% in the cortex for the period from pd 17–19 to pd 66–73. In the hippocampus, a decrease in the rate during the same time interval was 25%. Exposure to hypoxia had no effect on the intensity of [³Н]GABA uptake by the cortical and thalamic nerve terminals, but caused a significant age-dependent attenuation (by 35%) of the uptake intensity in the hippocampal ones. Significant age-dependent hypoxia-independent decrease in [³Н]GABA uptake with step-like dynamics of changes was shown in the thalamus and cortex. Gradual age-dependent hypoxia-dependent decrease in [³Н]GABA uptake was revealed in the hippocampus, and so a particular vulnerability of the latest structure to hypoxia as compared to the cortex and thalamus was revealed.     

Improving effect of acetylcholine receptor agonists on a deficit of 2-deoxyglucose uptake in cerebral cortical and hippocampal slices in aged and AF64A-treated rats

The aim of the present study was to determine whether the facilitation of 2-deoxyglucose (2-DG) uptake in the cerebral and hippocampal slices by nicotinic and muscarinic receptor agonists is compromised in the aged rat brain. For this, the effects of the nicotinic receptor agonist nicotine, the muscarinic receptor agonists oxotremorine and McN-A-343, and the ACh esterase inhibitors physostigmine and NK247 on 2-DG uptake in the brain slices of young (2-month-old) and aged (24–26-month-old) rats were tested. The decrements of 2-DG uptake in the cortical slices of aged rats were significantly attenuated by treatment with oxotremorine, nicotine and amiridine. In contrast, the metabolic responsivity of hippocampal slices to these drugs was reduced. To assess whether age-related changes in 2-DG uptake may be due to deficits in cholinergic function, we tested these drugs on the decrements of 2-DG uptake in ethylcholine aziridinium (a neurotoxic analog of choline) injected rats. The reductions of 2-DG uptake by injection of ethylcholine aziridinium was attenuated by oxotremorine but not by physostigmine. The present results reveal that metabolic decrements in the cerebral cortex from aged or ethylcholine aziridinium-injected rats were attenuated by muscarinic and nicotinic receptor agonists, suggesting that the muscarinic and nicotinic receptor mechanism in the cerebral cortex may be involved in cholinergic drug-induced functional recovery in aged rats.     

The effect of muscarinic and nicotinic ACh antagonist on the facilitation of rat visual cortical responses

Combined blockade of both muscarinic and nicotinic receptors fully eliminated the effect of reticular facilitation in rat visual cortex. However, this effect lasted only 2 h, what can suggest that there may be other excitatory input to the cholinergic neurones in rat visual cortex which is activated after blockade of cholinergic transmission.     

Differential effects of M1 and M2 muscarinic drugs on septohippocampal, hippocampal and cortical neurons in the rat

The effects of muscarinic agonists (McN-A-343, pilocarpine, oxotremorine-M, carbachol) and antagonists (pirenzepine, gallamine) applied by iontophoresis were studied on several neuronal populations in the central nervous system of rats anesthetized with urethane. Septohippocampal neurons and neurons from hippocampus, subiculum and somatic sensory cortex were studied. Oxotremorine-M and carbachol had (almost exclusively) potent excitatory effects whereas pilocarpine had some and McN-A-343 had almost exclusively inhibitory effects on the 4 populations of neurons studied. Pirezepine blocked more easily the effects of pilocarpine and McN-A-343 than those of oxotremorine-M or carbachol. These results suggest (i) that many central neurons may bear different functional muscarinic receptors and (ii) that the various agonists studied might act through (at least partially) different mechanisms.     

Removal of the entorhinal cortex protects hippocampal CA-1 neurons from ischemic damage

Summary The excitatory (glutamatergic) innervation seems to determine a nerve cells vulnerability to complete, transient ischemia. Interruption of the excitatory afferents to the hippocampus by removal of the entorhinal cortex prior to ischemia allows examination of this hypothesis. Groups of adult male Wistar rats were subjected to 20 min of ischemia (fourvessel occlusion) 4 days following a sham procedure, unilateral or bilateral entorhinotomy. CA-1 pyramidal cell survival following ischemia was assessed by light microscopic examination (cell counts) 4 days after ischemia. Compared to control animals unilateral entorhinotomy protected 50% of the CA-1 pyramidal neurons ipsilateral to the lesion, whereas bilateral entorhinotomy resulted in 84% protection. The pathophysiology of ischemic brain damage is discussed, and it is suggested that the protection of CA-1 pyramidal neurons after entorhinotomy is due to interruption of the input to the dentate granule cells, which forms a link in the trisynaptic pathway from the entorhinal cortex to the CA-1.Supported by the NOVO foundation and by The Danish Medical Research Council grant no. 12-5285 and no. 12-5704     

Effects of T-588, a newly synthesized cognitive enhancer, on hippocampal CA1 neurons in rat brain tissue slices

The effects of a newly synthesized cognitive enhancer, (-)-R-alpha-[[2-(diethylamino) ethoxy] methyl] benzo [b] thiophene-5-methanol hydrochloride (T-588), on the membrane properties of hippocampal CA1 neurons were investigated in a rat brain slice preparation. T-588 produced a slow and long-lasting depolarization of CA1 neurons with an increase in membrane resistance; this action showed close similarity to that of acetylcholine (ACh). However, the action of T-588 was not affected by atropine, tetrodotoxin or DL-2-amino-5-phosphonovalerate, while the action of ACh was blocked by atropine. The estimated reversal potential of this T-588 effect was near -90 mV which is the reversal potential of potassium ions in CA1 neurons. In the whole-cell voltage-clamp study, T-588 produced a reversible block of the outward potassium current in CA1 neurons. T-588 did not block the afterhyperpolarization evoked by an intracellular current injection, while ACh suppressed it. These results suggest that T-588 has a direct effect on CA1 neurons independent of its cholinergic activity, resulting from blockade of a conductance carried predominantly by potassium ions.     

Postischemic alterations of spontaneous activities in rat hippocampal CA1 neurons

Changes of spontaneous impulse discharges in rat hippocampal neurons during and after transient forebrain ischemia were investigated electrophysiologically. Spontaneous impulse frequencies of CA1 neurons before ischemia were varied from 0.4 to 20.0 impulses/s and its average was5.8 ± 1.2 (means±S.E., n = 36). These spontaneous discharges were completely suppressed during forebrain ischemia exept for the transient hyperactivity observed just after the beginning of ischemia. Recovery of spontaneous discharges of CA1 neurons from suppression induced by 5 min ischemia started at 5 min, and neuronal activities were restored to pre-ischemic levels approximately 30 min after reperfusion. On the other hand, spontaneous impulse frequencies at all time points recorded after 20 min ischemia were less than 40% of the pre-ischemic levels. These continuous suppression of spontaneous activity after 20 min ischemia may suggest that neuronal function is impaired during and/or in the early stages of reperfusion, and functional disorders precede morphological degeneration.     

Human cortical neurons contain both nicotinic and muscarinic acetylcholine receptors: an immunocytochemical double-labeling study

Using immunofluorescence histochemistry, in the human cerebral cortex neurons immunoreactive for both nicotinic and muscarinic acetylcholine receptor proteins could be demonstrated. Vibratome sections of biopsy and autopsy specimens of human temporal and occipital lobes were incubated with monoclonal antibodies specific for muscarinic (M 35) and nicotinic (WF 6) acetylcholine receptor protein. Immunoreactive sites were visualized using a biotin-streptavidin-phycoerythrin system (M 35, red fluorescence) and fluorescein-conjugated immunoglobulins (WF 6, green fluorescence). Immunofluorescence of both antibodies was preponderant in pyramidal neurons located in layers II/III and V and their apical dendrites. Some round and ovoid immunolabeled cells were encountered in layers VI and IV. About 30% of the cholinoceptive cortical neurons, in particular the pyramidal cells, displayed immunoreactivity for both receptor types. The present investigation shows a subpopulation of human cortical neurons to contain both nicotinic and muscarinic receptors. The coexistence of acetylcholine receptors may provide the morphological basis of simultaneous impact of acetylcholine on both receptor types in the same neuron of the human cerebral cortex.     

Brainstem carbachol injections in the urethane anesthetized rat produce hippocampal theta rhythm and cortical desynchronization: a comparison of pedunculopontine tegmental versus nucleus pontis oralis injections

Previous research has demonstrated that brainstem injections of acetylcholine agonists (e.g., carbachol) produced electrophysiological indicators of rapid-eye-movement (REM) sleep in the cat. Recent reports now indicate that this phenomenon may hold true for rats as well. Relatively few reports, however, have examined the effect of these injections on REM indicators in the anesthetized rat, a preparation useful for elucidating underlying neurobiological mechanisms controlling REM sleep processes. The present study compared the effect of injections of carbachol (5 μg in 250 nl) into the pedunculopontine tegmental nucleus (PPTg) or the nucleus pontis oralis (NPO) on two tonic indicators of REM sleep in the urethane-anesthetized rat. Namely, changes in the hippocampal EEG and in the cortical EEG. Carbachol injections into either site produced a change in both the hippocampal EEG and cortical EEG to a REM-like state at short latencies. The length of these changes (duration of effect), however, was site-dependent. Thus, PPTg carbachol injections induced significantly longer lasting effects in both the hippocampal and cortical EEG than did NPO injections. The results suggest that brainstem carbachol injections in rats, as in cats, may provide a useful model for investigating tonic REM sleep processes.     

Long-lasting structural changes in CA3 hippocampal and layer V motor cortical pyramidal neurons associated with self-stimulation rewarding experience: a quantitative Golgi study

Self-stimulation (SS) rewarding experience induced structural changes in CA3 hippocampal and layer V motor cortical pyramidal neurons in adult male Wistar rats has been demonstrated. In the present study, whether these structural changes are transient or of a permanent nature was evaluated. Self-stimulation experience was provided for 1 h daily over a period of 10 days through bilaterally implanted bipolar electrodes in the lateral hypothalamus and the substantia nigra-ventral tegmental area. Following 10 days of SS experience, the rats were sacrificed after an interval of 30 and 60 days for the quantitative analysis of the dendritic morphology in Golgi stained CA3 hippocampal and layer V motor cortical pyramidal neurons. The results revealed a significant increase in the dendritic branching points and intersections in apical and basal dendrites in both types of neurons in 30 days post-SS group compared to sham control. The total number of apical and basal dendrites were significantly increased in both 30 and 60 days post-SS groups of rats. This study suggests that SS experience induced structural changes are sustainable, even in the absence of rewarding experience.     

Distribution of norepinephrine and dopamine in cerebral cortical areas of the rat

Concentrations of norepinephrine and dopamine were determined using enzyme isotope assay in 27 microdissected cerebral cortical areas of the rat. A detailed map is presented for microdissection of rat cerebral cortex. Norepinephrine was found in low but still measurable quantities throughout the cortex. Differences between cortical areas are also low. Relatively highest levels were demonstrated in the pyriform, insular and entorhinal cortices. The distribution of dopamine was found to be uneven with a maximal regional difference of 1:24. Concentration of dopamine was in all areas lower than that of norepinephrine. The highest dopamine concentration (2,4 ng/mg protein) was measured in the rostral pyriform cortex but other mesocortical (cingulate, frontal, insular and entorhinal) dopaminergic areas also contained relatively high amounts. Except for the caudal occipital and caudal entorhinal cortices all regions studied contained measurable quantities of dopamine. Its low concentration relative to norepinephrine (below 15%) suggests that in the cortical areas studied dopamine is present as the precursor of norepinephrine.     

In vivo and in vitro labelling of perineuronal nets in rat brain

Previous lectin-histochemical and immunocytochemical investigations using fixed tissue revealed perineuronal nets as lattice-like accumulations of extracellular matrix proteoglycans at the surface of several types of neurons. In the present study, perineuronal nets in the rat brain were labelled for the first time in vivo by stereotaxic injections of biotinylated Wisteria floribunda agglutinin (Bio-WFA), as well as in vitro, by incubation of unfixed brain slices with the same lectin. Six days after Bio-WFA injections into the parietal cortex, medial septum, reticular thalamic nucleus and red nucleus, the lectin remaining bound to perineuronal nets was detected by streptavidin/biotinylated peroxidase complexes or red fluorescent Cy3-streptavidin, respectively. Double-fluorescence labelling showed that Bio-WFA applied in vivo reacted with the chondroitin sulphate proteoglycan immunoreactive perineuronal nets in the injection zone. Labelling of perineuronal nets in unfixed slices was obtained with either Cy3-tagged WFA or Bio-WFA and subsequent visualization by Cy3-streptavidin which confirmed the region-dependent distribution patterns and the structural characteristics of perineuronal nets known from histochemical studies. These results provide support for the role of extracellular matrix proteoglycans to maintain a considerable chemical and, probably, spatial heterogeneity of the extracellular space in vivo. The ability of in vivo and in vitro labelling may promote the functional characterization of the extracellular matrix in various brain structures including its species-dependent neuronal association patterns.     

Effects of ethanol on rat somatosensory cortical neurons

In this study, we characterized the local effects of ethanol (EtOH) on postsynaptic potentials (PSPs) and membrane properties of layer II–III (L2–3) and layer V (L5) somatosensory cortical neurons. Intracellular recordings were done using the in vitro slice preparation of rat somatosensory cortex. Our results show that EtOH exerts local effects on cortical cell membrane at physiologically relevant concentrations. A predominant effect of EtOH was to reduce excitability of L2–3 and L5 neurons by increasing the rheobase, decreasing input resistance and repetitive firing, reducing PSPs amplitude and the probability of evoking action potentials. Early (6 ms) and late (18 ms) PSP components were affected differentially by EtOH, the late components being more suppressed. Overall, EtOH-mediated suppression of PSPs was stronger in L5 neurons. Cortical neurons were divided into three subtypes: regular spiking adapting (RS-A), regular spiking non-adapting (RS-NA) and bursting (D-IB) neurons. PSPs evoked in RS-A neurons were more sensitive to EtOH suppressant effects. EtOH effects on input resistance were distributed differentially among the three groups of neurons. These results support the notion that EtOH disrupts higher processing of somatosensory information via a differential alteration of cortical neuron's membrane properties and synaptic transmission.     

Effects of prenatal immune activation on hippocampal neurogenesis in the rat

Maternal infection during pregnancy has been associated with an increased risk for the development of schizophrenia, a disorder characterized by abnormalities in hippocampal morphology and function. Neurogenesis occurs in the hippocampus throughout development into adulthood and is believed to modulate hippocampal function. This study used a rat model in which bacterial endotoxin, lipopolysaccharide (LPS), is administered to pregnant dams, to test if prenatal immune activation has acute and/or long term effects on various phases of neurogenesis (proliferation, survival, differentiation) in the hippocampal dentate gyrus of offspring. When LPS was administered to dams on gestation days (GD) 15 and 16, there was decreased proliferation of dentate cells at postnatal day (PD) 14 and decreased survival of cells generated at PD14 in offspring. When prenatal exposure to LPS was later in pregnancy (GD 18 and 19), offspring showed decreased survival of cells generated both at the time of LPS exposure and at PD14. There was no change in cell proliferation or survival in adult offspring at PD60, with prenatal LPS exposure. Co-administration of the cyclo-oxygenase inhibitor, ibuprofen (IBU), together with prenatal LPS on GD 15 and 16, was unable to prevent the deficit in neuronal survival at PD14. IBU blocked LPS-induced fever but did not block LPS-induced increases in plasma cytokines and corticosterone in the pregnant dam. This indicates that deficits in neurogenesis caused by prenatal LPS are not mediated by LPS-induced fever or eicosanoid induction, but could be mediated by LPS-induced increases in maternal cytokines or corticosterone.