Vasoactive Intestinal Peptide (VIP) Regulates Activity-Dependent Neuroprotective Protein (ADNP) Expression In Vivo

Vasoactive intestinal peptide (VIP) is an important mediator of development during the neural tube closure period of embryogenesis and may regulate, in part, the expression of activity-dependent neuroprotective protein (ADNP), which is essential for neural tube closure and embryogenesis. To evaluate the impact of VIP expression in vivo on ADNP and the related protein ADNP2 the current study examined gene expression in adult wild-type (VIP +/+) and VIP null (VIP −/−) offspring of VIP deficient mothers (VIP+/−) comparing them to wild-type offspring of wild-type mothers. Quantitative real time polymerase chain reaction (PCR), using an ABI Prisma cycler revealed regionally specific reductions of ADNP mRNA in the brains of VIP null mice compared with the brains of wild-type offspring of a wild-type mother. ADNP was significantly reduced in the cortex and hypothalamus of VIP null mice, but not in the hippocampus or thalamus. ADNP2 exhibited a similar pattern but reached a statistically significant reduction only in the hypothalamus. The mRNA for ADNP and ADNP2 also tended to be reduced in the cortex and hippocampus of the wild-type littermates of the VIP null mice, indicating that the VIP genotype of the mother may have had an impact on the ADNP expression of her offspring, regardless of their own VIP genotype. These results showed that VIP regulated brain ADNP expression in a regionally specific manner and indicated that both maternal and offspring VIP genotype may influence ADNP expression in the brain.     

Differential Regulation of Vasoactive Intestinal Peptide (VIP) in the Dentate Gyrus and Hippocampus via the NO-cGMP Pathway Following Kainic Acid-Induced Seizure in the Rat

We have previously shown that kainic acid (KA) increases nitric oxide (NO) synthase (NOS) production in the rat dentate gyrus (DG) and hippocampus (CA3), and NOS inhibition [(by N^G-nitro-L-arginine methylester (L-NAME)] modulates the vasoactive intestinal peptide (VIP)-responsive gene, activity-dependent neuroprotective protein, and alters neuro- and astrogliogenesis (Cosgrave et al. in Neurobiol Dis 30(3):281–292 2008, J Mol Neurosci 39(1–2):9–21, 2009, 2010). In the present study, using the same model we demonstrate that VIP synthesis is differentially regulated by the NO-cyclic guanosine monophosphate (cGMP) pathway in the DG and CA3 at 3 h and 3 days post-KA. At 3 h post-KA: In L-NAME+KA/7-nitroindazole (7-NI)+KA, stratum granulosum (SG) and subgranular zone (SGZ) cells were intensely stained for VIP when compared with L-NAME/7-NI/KA alone. Soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, blocks cGMP production), suppressed astrocytic activation (glial fibrillary acidic protein) but other cell types were VIP⁺; however, ODQ+KA suppressed overall VIP synthesis in the DG. At 3 days post-KA: In L-NAME+KA/7-NI+KA, SGZ and SG cells continued to express VIP, while in the KA alone, only SGZ cells were VIP⁺. ODQ increased VIP⁺ cells in the SG, and in contrast to 3 h, VIP-containing nNOS⁺ cells increased in ODQ+KA when compared to vehicle+KA. In the hippocampus, 7-NI/ODQ had no effect on VIP at 3 h/3 days, while L-NAME+KA at 3 days increased VIP⁺ cells, but reduced VIP-like immunoreactivity in astrocytes. These results suggest that the NO-cGMP pathway differentially regulates VIP in the DG and hippocampus during seizure.     

Rapid Aging in the Perforant Path Projections to the Rodent Dentate Gyrus

Why layers II/III of entorhinal cortex (EC) deteriorate in advance of other regions during the earliest stages of Alzheimer''s disease is poorly understood. Failure of retrograde trophic support from synapses to cell bodies is a common cause of neuronal atrophy, and we accordingly tested for early-life deterioration in projections of rodent layer II EC neurons. Using electrophysiology and quantitative imaging, changes in EC terminals during young adulthood were evaluated in male rats and mice. Field excitatory postsynaptic potentials, input/output curves, and frequency following capacity by lateral perforant path (LPP) projections from lateral EC to dentate gyrus were unchanged from 3 to 8–10 months of age. In contrast, the unusual presynaptic form of long-term potentiation (LTP) expressed by the LPP was profoundly impaired by 8 months in rats and mice. This impairment was accompanied by a reduction in the spine to terminal endocannabinoid signaling needed for LPP-LTP induction and was offset by an agent that enhances signaling. There was a pronounced age-related increase in synaptophysin within LPP terminals, an effect suggestive of incipient pathology. Relatedly, presynaptic levels of TrkB—receptors mediating retrograde trophic signaling—were reduced in the LPP terminal field. LTP and TrkB content were also reduced in the medial perforant path of 8- to 10-month-old rats. As predicted, performance on an LPP-dependent episodic memory task declined by late adulthood. We propose that memory-related synaptic plasticity in EC projections is unusually sensitive to aging, which predisposes EC neurons to pathogenesis later in life.SIGNIFICANCE STATEMENT Neurons within human superficial entorhinal cortex are particularly vulnerable to effects of aging and Alzheimer''s disease, although why this is the case is not understood. Here we report that perforant path projections from layer II entorhinal cortex to the dentate gyrus exhibit rapid aging in rodents, including reduced synaptic plasticity and abnormal protein content by 8–10 months of age. Moreover, there was a substantial decline in the performance of an episodic memory task that depends on entorhinal cortical projections at the same ages. Overall, the results suggest that the loss of plasticity and related trophic signaling predispose the entorhinal neurons to functional decline in relatively young adulthood.     

Vulnerability of the Dentate Gyrus to Aging and Intracerebroventricular Administration of Kainic Acid

The hippocampal formation is highly vulnerable to the aging process, demonstrating functional alterations in circuitry with aging. Aging may also change the sensitivity of the hippocampal formation to excitotoxic lesions. In this study, using young adult, middle aged, and aged Fischer 344 rats, we evaluated morphometric changes in the dentate gyrus as a function of age and also in response to an administration of an excitotoxin (kainic acid) into the right lateral ventricle. The dentate gyrus was measured for changes in the area of dentate hilus and the dentate granule cell layer, alterations in the width of the dentate granule cell layer, and degree of dentate hilar cell loss. With aging, the hilar area increased in size while the area and width of the dentate granule cell layer remained constant. However, the most striking change with aging was a significant reduction in the number of dentate hilar neurons. Intracerebroventricular kainic acid produced consistent lesions in the entire ipsilateral CA3 region, and the size of CA3 lesion was identical in all three ages of animals. Following the lesion, areas of both the dentate hilus and the granule cell layer were significantly decreased in only young adult and middle aged animals whereas the width of the dentate granule cell layer was significantly increased only in the middle aged group. In contrast, dentate hilar neurons were significantly reduced in all ages of animals with the maximum reductions in neuron number observed in the aged group. Thus, aging in the dentate gyrus is characterized by a significantly decreased number of dentate hilar neurons and also a significantly increased susceptibility of dentate hilar neurons to excitotoxic damage.     

NADPH-Diaphorase (Nitric Oxide Synthase) Staining in the Rat Supraoptic Nucleus is Activity-Dependent: Possible Functional Implications

NADPH-diaphorase has recently been shown to be the enzyme nitric oxide (NO) synthase, and to be present in the rat supraoptic nucleus (SON) and posterior pituitary. Investigations were carried out to assess whether there is any difference in the extent to which this enzyme is present, as assessed by light-microscopic histochemistry, in SON of normal and dehydrated male Wistar rats. In normal rats there was clear cellular heterogeneity; cells located in the ventral and caudal areas of the SON stained only weakly or not at all, while cells in the rostro-dorsal areas of the nucleus stained strongly. Dehydration of rats for 12 h caused a large and rapid increase in staining intensity of the nucleus, particularly of cells in its ventral and caudal parts. On the basis of its known biological actions, and the kinetics of its induction, it is suggested that NO would be a strong candidate as a modulator of SON and posterior pituitary morphology and function, with the potential to rapidly modulate blood flow, neuronal activity, and possibly astrocyte morphology, in response to changes in neuronal activity.     

Effect of Prenatal Protein Deprivation on Postnatal Granule Cell Generation in the Hippocampal Dentate Gyrus

The effect of prenatal malnutrition, produced by protein deprivation, on postnatal neurogenesis of granule cells in the fascia dentata of the rat hippocampal formation was examined by injecting tritiated thymidine on P8 and P15 and sacrificing the pups on P30, or by injecting on P30 and sacrificing on P90. The number of labeled granule cells was significantly decreased in prenatally malnourished rats injected on P8, and unaffected in those injected on P15. In contrast, the number of labeled granule cells in prenatally malnourished rats was significantly increased in animals injected on P30. The study shows that prenatal malnutrition significantly alters the postnatal pattern of granule cell neurogenesis in rat hippocampal formation and that the effect persists despite nutritional rehabilitation at birth. Copyright © 1996 Elsevier Science Inc.     

Relationship Between GAP-43 Expression in the Dentate Gyrus and Synaptic Reorganization of Hippocampal Mossy Fibres in Rats Treated with Kainic Acid

Kainic acid-induced seizures in adult rats produce neurodegeneration in the hippocampus followed by sprouting of the mossy fibres in the inner molecular layer of the dentate gyrus and changes in GAP-43 expression in the granule cells. In the present study we observed that 4 days after kainic acid injection a dense plexus of silver impregnated degenerating terminals detected by Gallyas's method and a decrease of GAP-43 immunostaining was observed in the inner molecular layer of the dentate gyrus indicating deafferentiation of this region. This was associated with the formation of an intense GAP-43 immunostained band in the supragranular layer. MK-801, a non-competitive inhibitor of the NMDA receptor, which partially inhibited the behavioural seizures induced by KA, also protected from the inner molecular layer deafferentation and markedly reduced the expression of GAP-43 mRNA in the granule cells and the intense GAP-43 immunostained band in the supragranular layer, suggesting a relationship among these events. Two months after kainic acid injection the intense supragranular GAP-43 positive band was no longer evident but the whole inner molecular layer appeared more labelled in association with the formation of the collateral sprouting of the mossy fibres in the inner molecular layer as detected by Timm's staining. These effects were also markedly reduced by the pretreatment with MK-801. Taken together, these experiments indicate for the first time a direct relationship between the increase of GAP-43 immunostaining in the inner molecular layer of the dentate gyrus and the collateral sprouting of mossy fibres in this district in response to kainic acid induced seizures. This further supports the hypothesis that the early induction of GAP-43 in granule cells may be one of the molecular mechanisms required for the synaptic reorganization of the mossy fibres.     

Novelty-elicited, Noradrenaline-dependent Enhancement of Excitability in the Dentate Gyrus

In order to relate noradrenaline-dependent potentiation in the dentate gyrus to behavioural events, rats were made to explore an environment in which their encounters with novel stimuli could be strictly controlled and monitored. Previous experiments have shown that an encounter with novel objects in a holeboard elicits a burst response in a large population of noradrenergic neurons of the locus coeruleus. Such a burst response has been demonstrated to produce a large and transient potentiation of the population spike in the dentate gyrus. In the present series of experiments, rats were chronically implanted with stimulating electrodes in the perforant pathway and recording electrodes in the dentate gyrus. Evoked potentials were monitored in the awake rat, first while it was resting quietly in a familiar environment and then while it was exploring the holeboard containing a novel object in a specific hole. There was a tonic increase in population spike amplitude when the rat was placed in the novel holeboard environment, but this effect gradually dissipated. This increase was partly blocked by the β-noradenergic antagonist propranolol. In addition there was a robust phasic increase in spike amplitude when the rat encountered a novel stimulus. This phasic response lasted ˜T50–75 s and was absent in animals treated with propranolol. These results show that a behavioural encounter with a novel stimulus can transiently enhance information transmission through the hippocampus, and suggest that activation of the noradrenergic system by the novel stimulus mediates this behaviour-dependent gating.     

Mossy Cells of the Rat Dentate Gyrus are lmmunoreactive for Calcitonin Gene-related Peptide (CGRP)

The neuropeptide calcitonin gene-related peptide (CGRP) was localized in the hippocampus and dentate gyrus of the rat by immunocytochemistry at the light and electron microscopic levels. Without colchicine treatment only faint neuropil labelling was found in the inner molecular layer of the dentate gyrus. Following colchicine treatment, a large number of neurons with numerous complex spines along the proximal dendrites were visualized in the hilus of the dentate gyrus, particularly in the ventral areas, and, in addition, staining of the inner molecular layer became stronger. Several CA3c pyramidal cells located adjacent to the hilar region in the ventral hippocampus also appeared to be faintly positive, although in most cases only their axon initial segments were labelled. Outside this region, the subicular end of the CA1 subfield contained occasional CGRP-positive non-pyramidal cells. The hilar CGRP-positive neurons were negative for parvalbumin, calretinin, cholecystokinin and somatostatin, whereas most of them were immunoreactive for GluR2/3 (the AMPA-type glutamate receptor known to be expressed largely by principal cells). Correlated electron microscopy showed that the spines along the proximal dendritic shafts indeed correspond to thorny excrescences engulfed by large complex mossy terminals forming asymmetrical synapses. Pre-embedding immunogold staining demonstrated that CGRP immunoreactivity in the inner molecular layer was confined to axon terminals that form asymmetrical synapses, and the labelling was associated with large dense-core vesicles. The present data provide direct evidence that CGRP is present in mossy cells of the dentate gyrus and to a lesser degree in CA3c pyramidal cells of the ventral hippocampus. These CGRP-containing principal cells terminate largely in the inner molecular layer of the dentate gyrus, and may release the neuropeptide in conjunction with their 'classical' neurotransmitter, glutamate.     

Oestradiol Regulates Neuropeptide Y Release and Gene Coupling with the GABAergic and Glutamatergic Synapses in the Adult Female Rat Dentate Gyrus

Neuropeptide Y (NPY) is an endogenous modulator of neuronal activity affecting both GABAergic and glutamatergic transmission. Previously, we found that oestradiol modifies the number of NPY immunoreactive neurones in the hippocampal dentate gyrus. In the present study, we investigated which oestrogen receptor type is responsible for these changes in the number of NPY‐positive neurones. Furthermore, we determined the effects of oestrogen receptor activation on NPY release. Finally, we examined the contribution of oestrogen toward the remodelling of the GABAergic and glutamatergic gene networks in terms of coupling with Npy gene expression in ovariectomised rats. We found that activation of either oestrogen receptor type (ERα or ERβ) increases the number of NPY‐immunopositive neurones and enhances NPY release in the dentate gyrus. We also found that, compared to oestrogen‐lacking ovariectomised rats, oestrogen replacement increases the probability of synergistic/antagonistic coupling between the Npy and GABAergic synapse genes, whereas the glutamatergic synapse genes are less likely to be coupled with Npy under similar conditions. The data together suggest that oestrogens play a critical role in the regulation of NPY system activity and are also involved in the coupling/uncoupling of the Npy gene with the GABAergic and glutamatergic synapses in the female rat dentate gyrus.     

Ketamine Affects the Neurogenesis of the Hippocampal Dentate Gyrus in 7-Day-Old Rats

Ketamine has been reported to cause neonatal neurotoxicity via a neuronal apoptosis mechanism; however, no in vivo research has reported whether ketamine could affect postnatal neurogenesis in the hippocampal dentate gyrus (DG). A growing number of experiments suggest that postnatal hippocampal neurogenesis is the foundation of maintaining normal hippocampus function into adulthood. Therefore, this study investigated the effect of ketamine on hippocampal neurogenesis. Male Sprague–Dawley rats were divided into two groups: the control group (equal volume of normal saline), and the ketamine-anesthesia group (40 mg/kg ketamine in four injections at 1 h intervals). The S-phase marker 5-bromodeoxyuridine (BrdU) was administered after ketamine exposure to postnatal day 7 (PND-7) rats, and the neurogenesis in the hippocampal DG was assessed using single- or double-immunofluorescence staining. The expression of GFAP in the hippocampal DG was measured by western blot analysis. Spatial reference memory was tested by Morris water maze at 2 months after PND-7 rats exposed to ketamine treatment. The present results showed that neonatal ketamine exposure significantly inhibited neural stem cell (NSC) proliferation, decreased astrocytic differentiation, and markedly enhanced neuronal differentiation. The disruptive effect of ketamine on the proliferation and differentiation of NSCs lasted at least 1 week and disappeared by 2 weeks after ketamine exposure. Moreover, the migration of newborn neurons in the granule cell layer and the growth of astrocytes in the hippocampal DG were inhibited by ketamine on PND-37 and PND-44. Finally, ketamine caused a deficit in hippocampal-dependent spatial reference memory tasks at 2 months old. Our results suggested that ketamine may interfere with hippocampal neurogenesis and long-term neurocognitive function in PND-7 rats. These findings may provide a new perspective to explain the adult neurocognitive dysfunction induced by neonatal ketamine exposure.     

Kainic Acid-Induced Seizures Modulate Akt (SER473) Phosphorylation in the Hippocampus of Dopamine D2 Receptor Knockout Mice

Dopamine D2 receptor (D2R) signalling has been shown to modulate seizure-induced hippocampal cell death. D2R knockout (D2R?/?) mice are more susceptible to kainic acid (KA)-induced excitotoxicity, displaying cell death in the CA3 subfield of the hippocampus at KA doses not damaging in wild-type (WT) animals. Absence of D2R signalling in the hippocampus leads to activation (dephosphorylation) of glycogen synthase kinase 3β (GSK-3β) after KA (20 mg/kg), which is not associated with a change in the phosphorylation of the GSK-3β regulator Akt at the canonical threonine 308 residue. In the present study, we investigated alternative pathways responsible for the activation of GSK-3β in the hippocampus of the D2R?/? mice 24 h following KA-induced seizures. Here, we show that phosphorylation of Akt occurs at serine 473 (Ser473) in the CA3 region of WT but not D2R?/? mice following KA. Moreover, the CA1 subregion, which does not undergo neurodegeneration in either WT or D2R?/? mice, displays a strong induction of Akt (Ser473) phosphorylation after KA. Additionally, the vulnerability in the CA3 is not associated with changes to p38MAPK and Dishevelled activation, and β-catenin does not appear to be a downstream target of the GSK-3β. Thus, we propose that GSK-3β phosphorylation-mediated hippocampal cell survival may depend on Akt (Ser473) phosphorylation; loss of D2R-mediated signalling in the CA3 region of D2R?/? mice leads to reduced Akt (Ser473) phosphorylation rendering neurons more vulnerable to apoptosis. Further investigation is required to fully elucidate the GSK-3β targets involved in D2R-dependent response to excitotoxicity.     

The Neuroprotective Effects of Carnosine in Early Stage of Focal Ischemia Rodent Model

Objective

This study was conducted to elucidate neuroprotective effect of carnosine in early stage of stroke.

Methods

Early stage of rodent stroke model and neuroblastoma chemical hypoxia model was established by middle cerebral artery occlusion and antimycin A. Neuroprotective effect of carnosine was investigated with 100, 250, and 500 mg of carnosine treatment. And antioxidant expression was analyzed by enzyme linked immunosorbent assay (ELISA) and western blot in brain and blood.

Results

Intraperitoneal injection of 500 mg carnosine induced significant decrease of infarct volume and expansion of penumbra (p<0.05). The expression of superoxide dismutase (SOD) showed significant increase than in saline group in blood and brain (p<0.05). In the analysis of chemical hypoxia, carnosine induced increase of neuronal cell viability and decrease of reactive oxygen species (ROS) production.

Conclusion

Carnosine has neuroprotective property which was related to antioxidant capacity in early stage of stroke. And, the oxidative stress should be considered one of major factor in early ischemic stroke.     

Target Selectivity and Neurochemical Characteristics of VIP-immunoreactive Interneurons in the Rat Dentate Gyrus

Vasoactive intestinal polypeptide (VIP) has been shown to be present in a morphologically heterogeneous subpopulation of interneurons in the dentate gyrus, but the relationship between their input and output characteristics and neurochemical features has not been established. Three types of VIP-immunoreactive cells have been identified on the basis of these criteria: (i) cells forming a dense axonal plexus in the hilus have always coexisted with the calcium binding protein calretinin (CR), but never with the neuropeptide cholecystokinin (CCK). The postsynaptic targets of these VIP-positive cells were neurons visualized by immunostaining for substance P receptor, which is known to label different hilar non-principal cells. (ii) VIP-immunoreactive basket cells, innervating predominantly the somata and proximal dendrites of granule cells, were found in the stratum moleculare and stratum granulosum. They contained CCK, but not CR. (iii) Cells projecting to the stratum moleculare were found to have dendrites and axons restricted to this layer. In 75% of these cells VIP coexisted with CR but not with CCK, and they established multiple contacts largely with non-principal cells. GABA was shown to be present but the calcium-binding proteins calbindin D28K and parvalbumin were absent in all three types of VIP-containing interneuron. On the basis of these observations we conclude that three different types of VIP-positive neuron are present in this area, and are likely to subserve different inhibitory functions: cells with a hilar projection as well as those projecting to the stratum moleculare may synchronize the activity of hilar and other interneurons, or disinhibit granule cells by specific interneuron-to-interneuron connections. In contrast, basket cells control the activity of granule cells directly, via perisomatic inhibition.     

S-Nitrosoglutathione Mimics the Beneficial Activity of Endothelial Nitric Oxide Synthase-Derived Nitric Oxide in a Mouse Model of Stroke

Background: The nitric oxide (NO)-producing activity of endothelial nitric oxide synthase (eNOS) plays a significant role in maintaining endothelial function and protecting against the stroke injury. However, the activity of the eNOS enzyme and the metabolism of major NO metabolite S-nitrosoglutathione (GSNO) are dysregulated after stroke, causing endothelial dysfunction. We investigated whether an administration of exogenous of GSNO or enhancing the level of endogenous GSNO protects against neurovascular injury in wild-type (WT) and eNOS-null (endothelial dysfunction) mouse models of cerebral ischemia-reperfusion (IR). Methods: Transient cerebral ischemic injury was induced by middle cerebral artery occlusion (MCAO) for 60 minutes in male adult WT and eNOS null mice. GSNO (0.1 mg/kg body weight, intravenously) or N6022 (GSNO reductase inhibitor, 5.0 mg/kg body weight, intravenously) was administered 30 minutes before MCAO in preinjury and at the reperfusion in postinjury studies. Brain infarctions, edema, and neurobehavioral functions were evaluated at 24 hours after the reperfusion. Results: eNOS-null mice had a higher degree (P< .05) of injury than WT. Pre- or postinjury treatment with either GSNO or N6022 significantly reduced infarct volume, improved neurological and sensorimotor function in both WT and eNOS-null mice. Conclusion: Reduced brain infarctions and edema, and improved neurobehavioral functions by pre- or postinjury GSNO treatment of eNOS knock out mice indicate that GSNO can attenuate IR injury, likely by mimicking the eNOS-derived NO-dependent anti-ischemic and anti-inflammatory functions. Neurovascular protection by GSNO/N6022 in both pre- and postischemic injury groups support GSNO as a promising drug candidate for the prevention and treatment of stroke injury.     

NMDA and Kainate-evoked Release of Nitric Oxide and Classical Transmitters in the Rat Striatum: In Vivo Evidence that Nitric Oxide May Play a Neuroprotective Role

The effects of N-methy-d -aspartate (NMDA), kainate, S-α-amino-3-hydroxyd-5-methyl-4-isoxazole propionate (AMPA) and KCI on striatal nitric oxide (NO), acetylcholine (ACh), dopamine (DA), serotonin (5-HT), aspartate (ASP), glutamate (GLU) and γ-aminobutyric acid (GABA) release were measured in anaesthetized rats in vivo by microdialysis and in vitro in organotypic slice cultures. Local NMDA (1–100 μM) infusion by retrodialysis dose-dependently increased levels of classical transmitters, NO₂-, NO₃-, ctrulline and arginine at similar thresholds (10 γM) Similar patterns of NMDA-evoked (50 μM) release were seen in striatal cultures. NMDA-evoked changes were all calcium-dependent and blocked by NMDA (APV or MK-801) but not AMPN/kainate (DNQX) receptor antagonists, excepting DA which could be prevented by both. In vivo, kainate increased NO₂^-, NO₃^-, CIT and ARG levels at 50 and 100 μM but was less potent than NMDA. Kainate also evoked significant Ach₁ DA and GLU release dose-dependently starting at 1–10 μM whereas 5-HT, ASP and GABA required 50 or 100 μM doses. Kainate effects were inhibited by DNQX, but not by APV, and were calcium-dependent. AMPA failed to alter NO₂^-, NO₃^-, CIT or ARG levels at 50 or 100 μM doses but dose-dependently increased ACh and DA. Similar results were seen with kainate (50 μM) and AMPA (50 μM) in vitro KCI evoked NO₂^-, NO₃^-, CIT and ARG release as well as that of the classical transmitters in vivo and in vitro. In vivo administration of the NO synthase inhibitor L-nitroarginine (L-NARG; 100 μM) significantly reduced NO₂^-, NO₃^- and CIT levels and prevented NMDA, kainate or KCI-evoked increases. It also potentiated ACh, ASP, GLU and GABA release and reduced that of DA in response to 50 μM NMDA whereas treatment with an NO-donor (SNAP; 10 μM) significantly reduced evoked ACh, ASP and GLU release. The NO synthase inhibitor L-NARG potentiated kainate-evoked ACh release and reduced that of DA, although less potently than NMDA, but it had no effect on KCI-evoked transmitter release. Overall, these results show that both NMDA and kainate increase striatal NO release at similar dose-thresholds as for classical transmitter release suggesting that NO is dynamically released under physiological and not just pathological conditions. Reduction of striatal NO levels also potentiates calcium-dependent transmitter release in response to NMDA and, to a lesser extent, kainate, whereas increasing them reduces it. This is consistent with a role for NO as a neuroprotective agent in this region acting to desensitize NMDA receptors.     

Subdivisions in the Multiple GABAergic Innervation of Granule Cells in the Dentate Gyrus of the Rat Hippocampus

The sources of GABAergic innervation to granule cells were studied to establish how the basic cortical circuit is implemented in the dentate gyrus. Five types of neuron having extensive local axons were recorded electrophysiologically in vitro and filled intracellularly with biocytin (Han et al., 1993). They were processed for electron microscopy in order to reveal their synaptic organization and postsynaptic targets, and to test whether their terminals contained GABA. (1) The hilar cell, with axon terminals in the commissural and association pathway termination field (HICAP cell), formed Gray's type 2 (symmetrical) synapses with large proximal dendritic shafts (n= 18), two-thirds of which could be shown to emit spines, and with small dendritic branches (n= 6). Other boutons of the HICAP neuron were found to make either Gray's type 1 (asymmetrical) synapses (n= 4) or type 2 synapses (n= 6) with dendritic spines. Using a highly sensitive silver-intensified immunogold method for the postembedding visualization of GABA immunoreactivity, both the terminals and the dendrites of the HICAP cell were found to be immunopositive, whereas its postsynaptic targets were GABA-immunonegative. The dendritic shafts of the HICAP cell received synapses from both GABA-negative and GABA-positive boutons; the dendritic spines which densely covered the main apical dendrite in the medial one-third of the molecular layer received synapses from GABA-negative boutons. (2) The hilar cell, with axon terminals distributed in conjunction with the perforant path termination field (HIPP cell), established type 2 synapses with distal dendritic shafts (n= 17), most of which could be shown to emit spines, small-calibre dendritic profiles (n= 2) and dendritic spines (n= 6), all showing characteristics of granule cell dendrites. The sparsely spiny dendrites of the HIPP cell were covered with many synaptic boutons on both their shafts and their spines. (3) The cell with soma in the molecular layer had an axon associated with the perforant path termination field (MOPP cell). This GABA-immunoreactive cell made type 2 synapses exclusively on dendritic shafts (n= 20), 60% of which could be shown to emit spines. The smooth dendrites of the MOPP cell were also restricted to the outer two-thirds of the molecular layer, where they received both GABA-negative and GABA-positive synaptic inputs. (4) The extensive axonal arborization of the dentate basket cell terminated mainly on somata (n= 26) and proximal dendrites (n= 9) in the granule cell layer, and some boutons made synapses on somatic spines (n= 6); all boutons established type 2 synapses. (5) The dentate axo-axonic cell established type 2 synapses (n= 14) exclusively on axon initial segments of granule cells in the granule cell layer, and on initial segments of presumed mossy cells in the hilus. The results demonstrate that granule cells receive inputs from the local circuit axons of at least five distinct types of dentate neuron terminating in mutually exclusive domains of the cell's surface in four out of five cases. Four of the cell types (HICAP cell, MOPP cell, basket cell, axo-axonic cell) contain GABA, and the HIPP cell may also be inhibitory. The specific local inhibitory neurons terminating in conjunction with particular excitatory amino acid inputs to the granule cells (types 1 – 3) are in a position to interact selectively with the specific inputs on the same dendritic segment. This arrangement provides a possibility for the independent regulation of the gain and long-term potentiation of separate excitatory inputs, through different sets of GABAergic local circuit neurons. The pairing of excitatory and inhibitory inputs may also provide a mechanism for the downward reseating of excitatory postsynaptic potentials, thereby extending their dynamic range.