Strychnine-sensitive glycine receptors in rat caudatoputamen are expressed by cholinergic interneurons

Strychnine-sensitive glycine receptors are ligand-gated anion channels widely expressed in spinal cord and brainstem. Recent functional studies demonstrating glycine-induced release of [(3)H]acetylcholine in rat caudatoputamen suggested the existence of excitatory glycine receptors in that region. Since the expression of glycine receptors in the caudatoputamen had not been reported earlier, we studied the glycine receptor-like immunoreactivity in this structure using a monoclonal antibody (mAb4a) recognizing an epitope common to all of the ligand-binding alpha-subunit variants of the glycine receptor. [Becker et al. (1993) Brain Res. 11, 327-333; Nicola et al. (1992) Neurosci. Lett. 138, 173-178]. Immunohistochemistry with mAb4a disclosed a specific staining of sparsely distributed large neurons in rat caudatoputamen, displaying an immunoreactive signal of lower intensity than that observed in motoneurons in spinal cord. Fluorescent dual labelling demonstrated that glycine receptor-like immunoreactivity co-localizes with choline acetyltransferase-like immunoreactivity in rat caudatoputamen. All neurons with glycine receptor-like immunoreactivity in the caudatoputamen studied were immunoreactive with choline acetyltransferase, and represented a subpopulation of cholinergic neurons (approximately 90% of the somata with choline acetyltransferase-like immunoreactivity).These results suggest that strychnine-sensitive glycine receptors are present on cholinergic interneurons in rat caudatoputamen, supporting the hypothesis that glycine receptors inducing striatal release of [(3)H]acetylcholine may be localized to cholinergic neurons.     

Metabotropic glutamate receptors modulate feedback inhibition in a developmentally regulated manner in rat dentate gyrus

We investigated group II metabotropic glutamate receptor (mGluR) modulation of glutamatergic input onto hilar-border interneurones and its regulation of feedback inhibition in the dentate gyrus. Selective activation of group II mGluRs with (2 S ,2' R ,3' R )-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) depressed mossy fibre (MF)-evoked excitatory drive to these interneurones with significantly greater depression in juvenile than adult rats. During 20 Hz MF stimulus trains, EPSCs became depressed. Depression during the early, but not later part of the train was significantly greater in juvenile than adult rats and was blocked by the mGluR antagonist (2 S )-2-amino-2-[(1 S ,2 S )-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495). In dentate granule cells from juvenile rats polysynaptic feedback IPSCs, but not monosynaptic IPSCs, were strongly suppressed by DCG-IV. DCG-IV also suppressed feedback inhibition of perforant path-evoked population spikes. In contrast, in adult animals DCG-IV did not significantly depress feedback inhibition. During 20 Hz stimulus trains in juvenile animals the summation of polysynaptic, but not monosynaptic IPSCs was suppressed by synaptically activated group II mGluRs. Blockade of these mGluRs with LY341495 significantly increased the area and duration of the summated IPSC, causing greater feedback inhibition of granule cell firing. In contrast, in adult animals LY341495 did not alter feedback inhibition following the stimulus train. These findings indicate that group II mGluRs modulate excitatory drive to interneurones in a developmentally regulated manner and thereby modulate feedback inhibition in the dentate gyrus.     

Phaclofen antagonizes post-tetanic disinhibition in the rat dentate gyrus

Tetanic mossy fiber stimulation transiently reduced recurrent inhibition in the rat dentate gyrus. The post-tetanic depression of inhibition was maximal 200 ms after the tetanus and typically lasted for about 2 s. Phaclofen, a selective gamma-aminobutyric acid-B (GABAB) receptor antagonist, significantly increased the post-tetanic level of inhibition. These results suggest that GABAB receptor activation is important for the development of post tetanic disinhibition. We suggest that GABA released during repetitive firing acts on GABAB receptors on inhibitory interneurons to suppress recurrent inhibition.     

Activation of NMDA receptors in rat dentate gyrus granule cells by spontaneous and evoked transmitter release

Activation of N-methyl-D-aspartate (NMDA) receptors by synaptically released glutamate in the nervous system is usually studied using evoked events mediated by a complex mixture of AMPA, kainate, and NMDA receptors. Here we have characterized pharmacologically isolated spontaneous NMDA receptor-mediated synaptic events and compared them to stimulus evoked excitatory postsynaptic currents (EPSCs) in the same cell to distinguish between various modes of activation of NMDA receptors. Spontaneous NMDA receptor-mediated EPSCs recorded at 34 degrees C in dentate gyrus granule cells (DGGC) have a frequency of 2.5 +/- 0.3 Hz and an average peak amplitude of 13.2 +/- 0.8 pA, a 10-90% rise time of 5.4 +/- 0.3 ms, and a decay time constant of 42.1 +/- 2.1 ms. The single-channel conductance estimated by nonstationary fluctuation analysis was 60 +/- 5 pS. The amplitudes (46.5 +/- 6.4 pA) and 10-90% rise times (18 +/- 2.3 ms) of EPSCs evoked from the entorhinal cortex/subiculum border are significantly larger than the same parameters for spontaneous events (paired t-test, P < 0.05, n = 17). Perfusion of 50 microM D(-)-2-amino-5-phosphonopentanoic acid blocked all spontaneous activity and caused a significant baseline current shift of 18.8 +/- 3.0 pA, thus identifying a tonic conductance mediated by NMDA receptors. The NR2B antagonist ifenprodil (10 microM) significantly reduced the frequency of spontaneous events but had no effect on their kinetics or on the baseline current or variance. At the same time, the peak current and charge of stimulus-evoked events were significantly diminished by ifenprodil. Thus spontaneous NMDA receptor-mediated events in DGGC are predominantly mediated by NR2A or possibly NR2A/NR2B receptors while the activation of NR2B receptors reduces the excitability of entorhinal afferents either directly or through an effect on the entorhinal cells.     

M1-like muscarinic acetylcholine receptors regulate fast-spiking interneuron excitability in rat dentate gyrus

Cholinergic transmission through muscarinic acetylcholine receptors (mAChRs) plays a key role in cortical oscillations. Although fast-spiking (FS), parvalbumin-expressing basket cells (BCs) are proposed to be the cellular substrates of gamma oscillations, previous studies reported that FS nonpyramidal cells in neocortical areas are unresponsive to cholinergic modulation. Dentate gyrus (DG) is an independent gamma oscillator in the hippocampal formation. However, in contrast to other cortical regions, the direct impact of mAChR activation on FS BC excitability in this area has not been investigated. Here, we show that bath-applied muscarine or carbachol, two mAChR agonists, depolarize DG BCs in the acute brain slices, leading to action potential firing in the theta-gamma bands in the presence of blockers of ionotropic glutamate and γ-aminobutyric acid type A receptors at physiological temperatures. The depolarizing action persists in the presence of tetrodotoxin, a voltage-gated Na⁺ channel blocker. In voltage-clamp recordings, muscarine markedly reduces background K⁺ currents. These effects are mimicked by oxotremorine methiodide, an mAChR-specific agonist, and largely reversed by atropine, a non-selective mAChR antagonist, or pirenzepine, an M₁ receptor antagonist, but not by gallamine, an M_2/4 receptor antagonist. Interestingly, in contrast to M₁-receptor-mediated depolarization, M₂ receptor activation by the specific agonist arecaidine but-2-ynyl ester tosylate down-regulates GABA release at BC axons—the effect is occluded by gallamine, an M₂ receptor antagonist. Overall, muscarinic activation results in a net increase in phasic inhibitory output to the target cells. Thus, cholinergic activation through M₁-like receptor enhances BC activity and promotes the generation of nested theta and gamma rhythms, thereby enhancing hippocampal function and associated performance.     

Fast network oscillations in the rat dentate gyrus in vitro

The dentate gyrus is a prominent source of gamma frequency activity in the hippocampal formation in vivo. Here we show that transient epochs of gamma frequency network activity (67 +/- 12 Hz) can be generated in the dentate gyrus of rat hippocampal slices, following brief pressure ejections of a high-molarity potassium solution onto the molecular layer. Oscillatory activity remains synchronized over distances >300 microm and is accompanied by a modest rise in [K(+)](o). Gamma frequency oscillations were abolished by a GABA(A) receptor antagonist demonstrating their dependence on rhythmic inhibition. However, in many cases, higher frequency oscillations (>80 Hz) remained in the absence of synaptic transmission, thus demonstrating that nonsynaptic factors may underlie fast oscillatory activity.     

Early life trauma decreases glucocorticoid receptors in rat dentate gyrus upon adult re-stress: reversal by escitalopram

Early exposure to adverse experiences may lead to specific changes in hippocampal glucocorticoid function resulting in abnormalities within the hypothalamic-adrenal axis. Given interactions between the neuroendocrine and central serotonergic systems, we hypothesized that exposure to early trauma would lead to abnormal hypothalamic-adrenal axis activity that would be normalized by pretreatment with a selective serotonin re-uptake inhibitor. Hypothalamic-adrenal axis function was assessed by determining basal corticosterone levels and hippocampal glucocorticoid receptor immunoreactivity. Rats were subjected to a triple stressor on postnatal day 28, and again to a single swim re-stress session on postnatal day 35 and postnatal day 60. On postnatal day 61 i.e. 24 h after the last re-stress, trunk blood was collected for serum corticosterone determinations and hippocampal tissue was collected for immunohistochemistry of glucocorticoid receptors. Escitalopram (5mg/kg) or saline vehicle was administered from postnatal day 47-postnatal day 60 via osmotic mini-pumps. Animals exposed to early life trauma showed an increase in basal corticosterone levels, and a significant decrease in the ratio of glucocorticoid receptor positive cells to total cells in the hilus, granule cell layer and the dentate gyrus. Both the increase in basal corticosterone and decrease in glucocorticoid receptor immunoreactivity were reversed by escitalopram pretreatment. These data confirm alterations in hypothalamic-adrenalaxis function that may stem from decreases in glucocorticoid receptor levels, in response to early adverse experiences, and demonstrate that these alterations are reversed by serotonin re-uptake inhibitor pretreatment.     

Vitamin E affects cell death in adult rat dentate gyrus

We have previously reported the presence of dying cells in the granule cell layer (GCL) of adult rat dentate gyrus (DG), where neurogenesis occurs. In particular, we found that cell death in the GCL increased in vitamin E deficiency and decreased in vitamin E supplementation. These findings were regarded as related to changes in neurogenesis rate, which in turn was influenced by vitamin E availability; a neuroprotective effect of vitamin E on cell death was also proposed. In order to verify this latter hypothesis, we have studied cell death in all layers of DG in vitamin E-deficient and vitamin E-supplemented rats and in control rats at different ages, using TUNEL and nick translation techniques. The phenotype of TUNEL-positive cells was characterized and the existence of dying BrdU-positive cells was investigated. Dying cells with neuronal phenotype were observed throughout the DG in all experimental groups. The number of TUNEL-positive cells decreased from juvenile to adult age. A higher number of TUNEL-positive cells in vitamin E-deficient rats and a lower number in vitamin E-supplemented rats, with respect to age-matched controls, were found; moreover, in these groups, TUNEL-positive cells had a different percentage distribution in the different layers of the DG. Our results confirm the occurrence of cell death in DG, demonstrate that cell death affects neuronal cells and support the hypothesis that the effect of vitamin E on cell death is not related to neurogenesis.     

Postischemic exercise decreases neurogenesis in the adult rat dentate gyrus

Running exercise enhances neurogenesis in the normal adult and aged hippocampus. However, the effect of exercise on neurogenesis in the ischemic hippocampus is unclear. Here, we show that running exercise has different effects on ischemic and non-ischemic brain. Young (3-4-month-old) normotensive Wistar rats were used for this study. We administered bromodeoxyuridine (BrdU) to rats 7 days after the induction of transient forebrain ischemia or sham operation. BrdU-labeled cells were increased in the ischemic subgranular zone (SGZ) and granule cell layer (GCL) and double immunofluoresence showed approximately 80% of BrdU-labeled cells expressed neuronal markers. To assess the effect of running exercise on neurogenesis, BrdU-labeled cells in these regions were quantified after 1 day and 14 days. In sham-operated rats, the numbers of BrdU-labeled cells were significantly increased (2.2-fold) in the SGZ and GCL in response to running exercise. The numbers of BrdU-labeled cells were increased in response to ischemia, however, they were decreased 14 days after BrdU administration and running exercise accelerated the reduction in BrdU-labeled cells in ischemic rats. These findings suggest that running exercise has a negative effect on neurogenesis in the ischemic hippocampus. This may be important with respect to assessment of therapeutic approaches for functional recovery after stroke.     

Dendritic atrophy in the dentate gyrus of the senescent rat

Quantitative electron microscopic analysis of the supragranular zone of the dentate gyrus molecular layer has shown that the number, volume fraction and surface area of dendritic shaft profiles are significantly decreased in senescent rats, relative to young adults. These modifications of dendritic morphology, which are not associated with age-related changes in dimensions of the molecular layer or in numbers of granule cells, may result from a decrease in the number and/or length of dendrites. In either case, the decreases in the number, volume fraction and surface area of dendritic shaft profiles found in the dentate gyrus of senescent rats signify an age-related atrophy of dendrites. Comparison of changes in the number and volume fraction of dendritic shaft profiles has demonstrated that age-related dendritic atrophy involves predominantly smaller dendritic branches.     

Alpha-tocopherol controls cell proliferation in the adult rat dentate gyrus

The effect of alpha-tocopherol on cell proliferation and proliferated cell survival was investigated in the dentate gyrus of adult rats. Adult rats were supplemented with alpha-tocopherol, injected with 5-bromo-2'-deoxyuridine (BrdU), that is incorporated into DNA during the S-phase, and killed at different time after BrdU injection. The number of newborn cells decreased after alpha-tocopherol supplementation, confirming the hypothesis that alpha-tocopherol is able to depress cell proliferation in vivo. Most newborn cells die within few days; more newborn cells survive in alpha-tocopherol-treated rats, suggesting the hypothesis that alpha-tocopherol decreases cell death.     

齿状回层的形成

中枢神经系统大部分结构内的神经元及其纤维成层排列,尤以大脑皮质、小脑皮质和海马(hippocampus)最为明显.这种层状结构对中枢神经系统完成其功能非常重要,皮质神经元排列紊乱的突变小鼠表现出功能上的障碍.我们曾以联合器官型脑片培养技术(organotypic slice co-culture)为主要手段对海马结构内齿状回(dentate gyrus)层的形成进行了深入细致的研究,结果表明,齿状回内细胞和纤维层的形成受不同因子的控制和调节.     

Enkephalin analogues depress synaptic potentials in rat dentate granule cells recorded intracellularly in vitro

Enkephalin analogues were superfused onto hippocampal slices during intracellular recording of dentate granule cells. The enkephalins elicited either weak depolarizations, weak hyperpolarizations, or no effect on membrane potential, in about equal numbers of cells tested. Similarly, input resistance either decreased, increased or did not change, and was not well correlated with the potential changes. However, at all concentrations tested (2-10 microM) the enkephalins reduced evoked depolarizing synaptic potentials by up to 70%. We speculate that a major function of endogenous enkephalin-containing fibers projecting to the dentate is to dampen afferent synaptic transmission.     

A role for N-methyl-D-aspartate receptors in norepinephrine-induced long-lasting potentiation in the dentate gyrus

Summary Mechanisms of action of norepinephrine (NE) on dentate gyrus granule cells were studied in rat hippocampal slices using extra- and intracellular recordings and measurements of stimulus and amino acid-induced changes in extracellular Ca²⁺ and K⁺ concentration. Bath application of NE (10–50 M) induced long-lasting potentiation of perforant path evoked potentials, and markedly enhanced high-frequency stimulus-induced Ca²⁺ influx and K⁺ efflux, actions blocked by -receptor antagonists and mimicked by agonists. Enhanced Ca²⁺ influx was primarily postsynaptic, since presynaptic [Ca²⁺]₀ in the stratum moleculare synaptic field was not altered by NE. Interestingly, the potentiation of both ionic fluxes and evoked population potentials were antagonized by the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (APV). Furthermore, NE selectively enhanced the [Ca²⁺]₀, [K⁺]₀ and extracellular slow negative field potentials elicited by iontophoretically applied NMDA, but not those induced by the excitatory amino acid quisqualate. These results suggest that granule cell influx of Ca²⁺ through NMDA ionophores is enhanced by NE via -receptor activation. In intracellular recordings, NE depolarized granule cells (4.8±1.1 mV), and increased input resistance (R_N) by 34±6.5%. These actions were also blocked by either the -antagonist propranolol or specific ₁-blocker metoprolol. Moreover, the depolarization and R_N increase persisted for long periods (93±12 min) after NE washout. In contrast, while NE, in the presence of APV, still depolarized granule cells and increased R_N, APV made these actions quickly reversible upon NE washout (16±9 min). This suggested that NE induction of long-term, but not short-term, plasticity in the dentate gyrus requires NMDA receptor activation. NE may be enhancing granule cell firing by some combination of blockade on the late Ca²⁺-activated K⁺ conductance and depolarization of granule cells, both actions that can bring granule cells into a voltage range where NMDA receptors are more easily activated. Furthermore, NE also elicited activity-independent long-lasting depolarization and R_N increases, which required functional NMDA receptors to persist.     

N-methyl-d-aspartate receptor expression during adult neurogenesis in the rat dentate gyrus

N-methyl-d-aspartate (NMDA) receptors play a crucial role in the regulation of neuronal development during embryogenesis and they also regulate the rate of neurogenesis and proliferation in the adult dentate gyrus. However, the mechanism by which they influence these processes is not fully understood. NMDA receptors seem to be functional in hippocampal precursor cells and recently generated granule neurons, although there is no anatomical correlate of these physiological observations. We have analyzed the expression of the NMDA receptor subunits NR1 and NR2B in precursor cells and recently generated granule neurons of the adult rat dentate gyrus, using 5'bromodeoxyuridine, green fluorescent protein-retrovirus and immunohistochemistry. Our results indicate that NR1 and NR2B are expressed in some proliferating cells of the adult subgranular zone. These receptors are absent from transiently amplifying progenitors (type 2-3 cells) but they are found in glial fibrillar acidic protein expressing cells in the subgranular zone, suggesting its presence in bipotential (type-1) precursor cells. NR1 and NR2B are rarely found in granule cells younger than 60 h. By contrast, many granule cells generated 14 days before killing express both NMDA receptor subunits. These results demonstrate that adult hippocampal neurogenesis may be regulated by NMDA receptors present in precursor cells and in differentiating granule neurons, although these receptors are probably not located on synapses. However, an indirect effect through NMDA receptors located in other cell types should not be excluded.     

Electrophysiological properties of neurones in cultures from postnatal rat dentate gyrus

Electrophysiological properties of neurofilament-positive neurones in dissociated cell cultures were prepared at postnatal days 4–5 from rat dentate gyrus and studied using the whole-cell patch-clamp technique. These cells expressed a fast-inactivating, 0.5 M tetrodotoxin-sensitive Na⁺ current; a high-voltage-activated (HVA) Ca²⁺ current, which was 30 M Cd²⁺- and partially 2 M nicardipine-sensitive; and an inward rectifier current, which was sensitive to extracellularly applied 1 mM Cs⁺. The outward current pattern was composed of a delayed rectifier-like outward current sensitive to 20 mM tetraethylammonium (TEA) and a fast-inactivating, Ca²⁺-dependent outward current. This transient Ca²⁺-dependent K⁺ outward current was identified by a subtraction procedure. K⁺ currents recorded under conditions of blocked Ca²⁺ currents (after rundown of the HVA Ca²⁺ current or blocked by extracellularly applied Cd²⁺) were subtracted from control currents. By comparison with the current pattern of identified dentate granule cells, it is concluded that the investigated cell type originated from interneurones or projection neurones of the dentate hilus.     

Lesion-induced alterations of lectin binding sites in the rat dentate gyrus

The pattern of binding of horseradish peroxidase conjugated lectins (Concanavalin A, fucose binding protein, Ricinus communis agglutinin) was examined in the rat hippocampal formation both prior to and following a lesion of the entorhinal cortex. In normal animals, Concanavalin A binding sites were concentrated around the granular and pyramidal cell bodies. Receptors were less concentrated in the stratum radiatum, stratum oriens and the dentate molecular layer. Receptors of fucose binding protein were concentrated in the granular and pyramidal cells, the boundary between the first and second quarters of the molecular layer and at the hippocampal fissure. Ricinus communis agglutinin binding sites were highest in the first 1/4 of the molecular layer and lowest in the stratum lacunosum-moleculare. Three days after an entorhinal lesion, the binding of Concanavalin A and fucose binding protein in the molecular layer was increased and while Ricinus communis agglutinin binding was unchanged. At thirty days post-lesion there was an increase in the binding of Concanavalin A and fucose binding protein in the molecular layer and stratum lacunosum-moleculare, whereas Ricinus communis agglutinin binding sites increased only in the molecular layer.The extensive alterations of lectin receptors that occur simultaneously with reactive synaptogenesis may indicate that membrane-bound glycoconjugates have an important role in this process.     

Loss of synapses in the dentate gyrus of the senescent rat

Synapses were counted in electron micrographs of the middle third of the molecular layer of the dentate gyrus of Fischer 344 rats, 3 months and 25 months of age. A 27% decrease in the number of synapses was found in senescent animals compared with young adults. This loss of synapses could not be correlated with changes in synaptic size, tissue volume or number of postsynaptic granule cells.