Ultrastructural Evidence for a Role of Astrocytes and Glycogen-Derived Lactate in Learning-Dependent Synaptic Stabilization.

Long-term memory formation (LTM) is a process accompanied by energy-demanding structural changes at synapses and increased spine density. Concomitant increases in both spine volume and postsynaptic density (PSD) surface area have been suggested but never quantified in vivo by clear-cut experimental evidence. Using novel object recognition in mice as a learning task followed by 3D electron microscopy analysis, we demonstrate that LTM induced all aforementioned synaptic changes, together with an increase in the size of astrocytic glycogen granules, which are a source of lactate for neurons. The selective inhibition of glycogen metabolism in astrocytes impaired learning, affecting all the related synaptic changes. Intrahippocampal administration of l-lactate rescued the behavioral phenotype, along with spine density within 24 hours. Spine dynamics in hippocampal organotypic slices undergoing theta burst-induced long-term potentiation was similarly affected by inhibition of glycogen metabolism and rescued by l-lactate. These results suggest that learning primes astrocytic energy stores and signaling to sustain synaptic plasticity via l-lactate.

Peripheral administration of lactate produces antidepressant-like effects.

This corrects the article DOI: 10.1038/mp.2016.179.

Peripheral administration of lactate produces antidepressant-like effects.

In addition to its role as metabolic substrate that can sustain neuronal function and viability, emerging evidence supports a role for l-lactate as an intercellular signaling molecule involved in synaptic plasticity. Clinical and basic research studies have shown that major depression and chronic stress are associated with alterations in structural and functional plasticity. These findings led us to investigate the role of l-lactate as a potential novel antidepressant. Here we show that peripheral administration of l-lactate produces antidepressant-like effects in different animal models of depression that respond to acute and chronic antidepressant treatment. The antidepressant-like effects of l-lactate are associated with increases in hippocampal lactate levels and with changes in the expression of target genes involved in serotonin receptor trafficking, astrocyte functions, neurogenesis, nitric oxide synthesis and cAMP signaling. Further elucidation of the mechanisms underlying the antidepressant effects of l-lactate may help to identify novel therapeutic targets for the treatment of depression.

Disrupting astrocyte-neuron lactate transfer persistently reduces conditioned responses to cocaine.

A central problem in the treatment of drug addiction is the high risk of relapse often precipitated by drug-associated cues. The transfer of glycogen-derived lactate from astrocytes to neurons is required for long-term memory. Whereas blockade of drug memory reconsolidation represents a potential therapeutic strategy, the role of astrocyte-neuron lactate transport in long-term conditioning has received little attention. By infusing an inhibitor of glycogen phosphorylase into the basolateral amygdala of rats, we report that disruption of astrocyte-derived lactate not only transiently impaired the acquisition of a cocaine-induced conditioned place preference but also persistently disrupted an established conditioning. The drug memory was rescued by L-Lactate co-administration through a mechanism requiring the synaptic plasticity-related transcription factor Zif268 and extracellular signal-regulated kinase (ERK) signalling pathway but not the brain-derived neurotrophic factor (Bdnf). The long-term amnesia induced by glycogenolysis inhibition and the concomitant decreased expression of phospho-ERK were both restored with L-Lactate co-administration. These findings reveal a critical role for astrocyte-derived lactate in positive memory formation and highlight a novel amygdala-dependent reconsolidation process, whose disruption may offer a novel therapeutic target to reduce the long-lasting conditioned responses to cocaine.

Metabolic gene expression changes in astrocytes in Multiple Sclerosis cerebral cortex are indicative of immune-mediated signaling.

Emerging as an important correlate of neurological dysfunction in Multiple Sclerosis (MS), extended focal and diffuse gray matter abnormalities have been found and linked to clinical manifestations such as seizures, fatigue and cognitive dysfunction. To investigate possible underlying mechanisms we analyzed the molecular alterations in histopathological normal appearing cortical gray matter (NAGM) in MS. By performing a differential gene expression analysis of NAGM of control and MS cases we identified reduced transcription of astrocyte specific genes involved in the astrocyte-neuron lactate shuttle (ANLS) and the glutamate-glutamine cycle (GGC). Additional quantitative immunohistochemical analysis demonstrating a CX43 loss in MS NAGM confirmed a crucial involvement of astrocytes and emphasizes their importance in MS pathogenesis. Concurrently, a Toll-like/IL-1ß signaling expression signature was detected in MS NAGM, indicating that immune-related signaling might be responsible for the downregulation of ANLS and GGC gene expression in MS NAGM. Indeed, challenging astrocytes with immune stimuli such as IL-1ß and LPS reduced their ANLS and GGC gene expression in vitro. The detected upregulation of IL1B in MS NAGM suggests inflammasome priming. For this reason, astrocyte cultures were treated with ATP and ATP/LPS as for inflammasome activation. This treatment led to a reduction of ANLS and GGC gene expression in a comparable manner. To investigate potential sources for ANLS and GGC downregulation in MS NAGM, we first performed an adjuvant-driven stimulation of the peripheral immune system in C57Bl/6 mice in vivo. This led to similar gene expression changes in spinal cord demonstrating that peripheral immune signals might be one source for astrocytic gene expression changes in the brain. IL1B upregulation in MS NAGM itself points to a possible endogenous signaling process leading to ANLS and GGC downregulation. This is supported by our findings that, among others, MS NAGM astrocytes express inflammasome components and that astrocytes are capable to release Il-1ß in-vitro. Altogether, our data suggests that immune signaling of immune- and/or central nervous system origin drives alterations in astrocytic ANLS and GGC gene regulation in the MS NAGM. Such a mechanism might underlie cortical brain dysfunctions frequently encountered in MS patients.

Exploring neural cell dynamics with digital holographic microscopy.

In this review, we summarize how the new concept of digital optics applied to the field of holographic microscopy has allowed the development of a reliable and flexible digital holographic quantitative phase microscopy (DH-QPM) technique at the nanoscale particularly suitable for cell imaging. Particular emphasis is placed on the original biological information provided by the quantitative phase signal. We present the most relevant DH-QPM applications in the field of cell biology, including automated cell counts, recognition, classification, three-dimensional tracking, discrimination between physiological and pathophysiological states, and the study of cell membrane fluctuations at the nanoscale. In the last part, original results show how DH-QPM can address two important issues in the field of neurobiology, namely, multiple-site optical recording of neuronal activity and noninvasive visualization of dendritic spine dynamics resulting from a full digital holographic microscopy tomographic approach.

Induction of brain aquaporin 9 (AQP9) in catecholaminergic neurons in diabetic rats.

Aquaporin 9 facilitates the diffusion of water but also glycerol and monocarboxylates, known as brain energy substrates. AQP9 was recently observed in catecholaminergic neurons that are implicated in energy homeostasis and also possibly in neuroendocrine effects of diabetes. Recently it has been observed that the level of AQP9 expression in hepatocytes is sensitive to the blood concentration of insulin. Furthermore, insulin injection in the brain is known to be related to the energy homeostasis. Based on these observations, we investigated if the concentration of insulin affects the level of brain AQP9 expression and if so, in which cell types. This study has been carried out, in a model of the diabetic rat generated by streptozotocin injection and on brainstem slices. In diabetic rats showing a decrease in systemic insulin concentration, AQP9 is only increased in brain areas containing catecholaminergic neurons. In contrast, no significant change is detected in the cerebral cortex and the cerebellum. Using immunocytochemistry, we are able to show that the increase in AQP9 expression is specifically present in catecholaminergic neurons. In brainstem slice cultures, 2 microM insulin induces a significant decrease in AQP9 protein levels 6 h after application, suggesting that brain AQP9 is also regulated by the insulin. These results show that the level of expression of brain AQP9 is affected by variations of the concentration of insulin in a diabetic model and in vitro.

Dual action of L-Lactate on the activity of NR2B-containing NMDA receptors: from potentiation to neuroprotection.

L-Lactate is a positive modulator of NMDAR-mediated signaling resulting in plasticity gene induction and memory consolidation. However, L-Lactate is also able to protect neurons against excito-toxic NMDAR activity, an indication of a mitigating action of L-Lactate on NMDA signaling. In this study, we provide experimental evidence that resolves this apparent paradox. Transient co-application of glutamate/glycine (1 µM/100 µM; 2 min) in primary cultures of mouse cortical neurons triggers a NMDA-dependent Ca2+ signal positively modulated by L-Lactate (10 mM) or DTT (1 mM) but decreased by Pyruvate (10 mM). This L-Lactate and DTT-induced potentiation is blocked by Ifenprodil (2 µM), a specific blocker of NMDARs containing NR2B sub-units. In contrast, co-application of glutamate/glycine (1 mM/100 µM; 2 min) elicits a NMDAR-dependent excitotoxic death in 49% of neurons. L-Lactate and Pyruvate significantly reduce this rate of cell death processes (respectively to 23% and 9%) while DTT has no effect (54% of neuronal death). This L-Lactate-induced neuroprotection is blocked by carbenoxolone and glibenclamide, respectively blockers of pannexins and KATP. In conclusion, our results show that L-Lactate is involved in two distinct and independent pathways defined as NMDAR-mediated potentiation pathway (or NADH pathway) and a neuroprotective pathway (or Pyruvate/ATP pathway), the prevalence of each one depending on the strength of the glutamatergic stimulus.

Glucose and lactate are equally effective in energizing activity-dependent synaptic vesicle turnover in purified cortical neurons.

This study examines the role of glucose and lactate as energy substrates to sustain synaptic vesicle cycling. Synaptic vesicle turnover was assessed in a quantitative manner by fluorescence microscopy in primary cultures of mouse cortical neurons. An electrode-equipped perfusion chamber was used to stimulate cells both by electrical field and potassium depolarization during image acquisition. An image analysis procedure was elaborated to select in an unbiased manner synaptic boutons loaded with the fluorescent dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43). Whereas a minority of the sites fully released their dye content following electrical stimulation, others needed subsequent K(+) depolarization to achieve full release. This functional heterogeneity was not significantly altered by the nature of metabolic substrates. Repetitive stimulation sequences of FM1-43 uptake and release were then performed in the absence of any metabolic substrate and showed that the number of active sites dramatically decreased after the first cycle of loading/unloading. The presence of 1 mM glucose or lactate was sufficient to sustain synaptic vesicle cycling under these conditions. Moreover, both substrates were equivalent for recovery of function after a phase of decreased metabolic substrate availability. Thus, lactate appears to be equivalent to glucose for sustaining synaptic vesicle turnover in cultured cortical neurons during activity.

Regulation of neuron-astrocyte metabolic coupling across the sleep-wake cycle.

Over the last thirty years, a growing number of studies showed that astrocytes play a pivotal role in the energy support to synapses. More precisely, astrocytes adjust energy production to neuronal energy needs through different mechanisms grouped under the term "neurometabolic coupling" (NMC). In this review we describe these mechanisms of coupling and how they involve astrocytes. From a physiological point of view, these mechanisms of coupling are particularly important to ensure normal synaptic functioning when neurons undergo rapid and repetitive changes in the firing rate such as during the sleep/wake transitions. Investigations into brain energy metabolism during the sleep/wake cycle have been mainly focused on glucose (Gluc) consumption and on glycogen metabolism. However, the recent development of substrate-specific biosensors allowed measurements of the variation in extracellular levels of glutamate, Gluc and lactate (Lac) with a time resolution compatible with sleep stage duration. Together with gene expression data these experiments allowed to better define the variations of energy metabolite regulation across the sleep/wake cycle. The aim of this review is to bring into perspective the role of astrocytes and NMC in the regulation of the sleep/wake cycle. The data reviewed also suggest an important role of the astrocytic network. In addition, the role of astrocytes in NMC mechanisms is consistent with the "local and use dependent" sleep hypothesis.

L-Lactate protects neurons against excitotoxicity: implication of an ATP-mediated signaling cascade.

Converging experimental data indicate a neuroprotective action of L-Lactate. Using Digital Holographic Microscopy, we observe that transient application of glutamate (100 µM; 2 min) elicits a NMDA-dependent death in 65% of mouse cortical neurons in culture. In the presence of L-Lactate (or Pyruvate), the percentage of neuronal death decreases to 32%. UK5099, a blocker of the Mitochondrial Pyruvate Carrier, fully prevents L-Lactate-mediated neuroprotection. In addition, L-Lactate-induced neuroprotection is not only inhibited by probenicid and carbenoxolone, two blockers of ATP channel pannexins, but also abolished by apyrase, an enzyme degrading ATP, suggesting that ATP produced by the Lactate/Pyruvate pathway is released to act on purinergic receptors in an autocrine/paracrine manner. Finally, pharmacological approaches support the involvement of the P2Y receptors associated to the PI3-kinase pathway, leading to activation of KATP channels. This set of results indicates that L-Lactate acts as a signalling molecule for neuroprotection against excitotoxicity through coordinated cellular pathways involving ATP production, release and activation of a P2Y/KATP cascade.

Protein targeting to glycogen is a master regulator of glycogen synthesis in astrocytes.

The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the neurotransmitter noradrenaline. To achieve further insight into the role of PTG in the regulation of astrocytic glycogen, its levels of expression were manipulated in primary cultures of mouse cortical astrocytes using adenovirus-mediated overexpression of tagged-PTG or siRNA to downregulate its expression. Infection of astrocytes with adenovirus led to a strong increase in PTG expression and was associated with massive glycogen accumulation (>100 fold), demonstrating that increased PTG expression is sufficient to induce glycogen synthesis and accumulation. In contrast, siRNA-mediated downregulation of PTG resulted in a 2-fold decrease in glycogen levels. Interestingly, PTG downregulation strongly impaired long-term astrocytic glycogen synthesis induced by insulin or noradrenaline. Finally, these effects of PTG downregulation on glycogen metabolism could also be observed in cultured astrocytes isolated from PTG-KO mice. Collectively, these observations point to a major role of PTG in the regulation of glycogen synthesis in astrocytes and indicate that conditions leading to changes in PTG expression will directly impact glycogen levels in this cell type.

Involvement of the agmatinergic system in the depressive-like phenotype of the Crtc1 knockout mouse model of depression.

Recent studies implicate the arginine-decarboxylation product agmatine in mood regulation. Agmatine has antidepressant properties in rodent models of depression, and agmatinase (Agmat), the agmatine-degrading enzyme, is upregulated in the brains of mood disorder patients. We have previously shown that mice lacking CREB-regulated transcription coactivator 1 (CRTC1) associate behavioral and molecular depressive-like endophenotypes, as well as blunted responses to classical antidepressants. Here, the molecular basis of the behavioral phenotype of Crtc1(-/-) mice was further examined using microarray gene expression profiling that revealed an upregulation of Agmat in the cortex of Crtc1(-/-) mice. Quantitative polymerase chain reaction and western blot analyses confirmed Agmat upregulation in the Crtc1(-/-) prefrontal cortex (PFC) and hippocampus, which were further demonstrated by confocal immunofluorescence microscopy to comprise an increased number of Agmat-expressing cells, notably parvalbumin- and somatostatin-positive interneurons. Acute agmatine and ketamine treatments comparably improved the depressive-like behavior of male and female Crtc1(-/-) mice in the forced swim test, suggesting that exogenous agmatine has a rapid antidepressant effect through the compensation of agmatine deficit because of upregulated Agmat. Agmatine rapidly increased brain-derived neurotrophic factor (BDNF) levels only in the PFC of wild-type (WT) females, and decreased eukaryotic elongation factor 2 (eEF2) phosphorylation in the PFC of male and female WT mice, indicating that agmatine might be a fast-acting antidepressant with N-methyl-D-aspartate (NMDA) receptor antagonist properties. Collectively, these findings implicate Agmat in the depressive-like phenotype of Crtc1(-/-) mice, refine current understanding of the agmatinergic system in the brain and highlight its putative role in major depression.

VIP neurons in the cerebral cortex.

VIP-containing cells in the neocortex are intrinsic neurons of the bipolar type, which release VIP through mechanisms that involve Ca2+ and lipoxygenase metabolites. VIP receptors are coupled to cAMP-generating systems that are amplified by various neurotransmitters such as noradrenaline, histamine and GABA. Pierre Magistretti reviews the evidence that VIP neurons play an important role in the local regulation of metabolism in the cerebral cortex by stimulating glycogenolysis and altering cortical blood flow.

Astrocytes as a predominant cellular site of (99m)Tc-HMPAO retention.

Technetium-99m-d,l-hexamethylpropylene amine oxime ((99m)Tc-HMPAO) retention in the brain monitored by single photon emission computed tomography (SPECT) is currently used as a marker of cerebral blood flow. The purported mechanism by which (99m)Tc-HMPAO accumulates in the brain is through its intracellular conversion from a lipophilic form to more hydrophilic derivatives within the brain parenchyma. The issue of the contribution of different cell types on (99m)Tc-HMPAO retention was investigated in vitro by studying the accumulation of (99m)Tc-HMPAO in primary cultures of mouse cortical astrocytes and neurons. Results show that (99m)Tc-HMPAO retention predominates in astrocytes over neurons by a factor of approximately 2.5 (0.26 +/- 0.05 vs. 0.095 +/- 0.042 fmol/mg protein after 120 minutes, respectively). Diethyl maleate (60 micromol/L), ethacrynic acid (1 mmol/L) and buthionine sulfoximine (1 mmol/L), 3 agents which significantly reduced glutathione levels also decreased (99m)Tc-HMPAO retention in both astrocytes (29%, 3%, and 46% of control, respectively) and neurons (69%, 11% and 63% of control). Decrease did not always correlate with glutathione levels, however, which suggests that other factors could be involved. The possibility that cell energy status determines (99m)Tc-HMPAO retention was also assessed. Agents that activate (glutamate, azide) or inhibit (cytochalasin B) glucose utilization in astrocytes, as measured by the (3)H-2-deoxyglucose method, were without effect on (99m)Tc-HMPAO retention. In conclusion, the data presented indicate that astrocytes may constitute a prominent site of (99m)Tc-HMPAO retention and most likely contribute significantly to the SPECT signal. In addition, the data also suggest that specific alterations in glial cell metabolism could explain flow-independent changes in (99m)Tc-HMPAO retention in the brain as observed by SPECT in some pathologies.

Selective distribution of lactate dehydrogenase isoenzymes in neurons and astrocytes of human brain.

In vertebrates, the interconversion of lactate and pyruvate is catalyzed by the enzyme lactate dehydrogenase. Two distinct subunits combine to form the five tetrameric isoenzymes of lactate dehydrogenase. The LDH-5 subunit (muscle type) has higher maximal velocity (Vmax) and is present in glycolytic tissues, favoring the formation of lactate from pyruvate. The LDH-1 subunit (heart type) is inhibited by pyruvate and therefore preferentially drives the reaction toward the production of pyruvate. There is mounting evidence indicating that during activation the brain resorts to the transient glycolytic processing of glucose. Indeed, transient lactate formation during physiological stimulation has been shown by 1H-magnetic resonance spectroscopy. However, since whole-brain arteriovenous studies under basal conditions indicate a virtually complete oxidation of glucose, the vast proportion of the lactate transiently formed during activation is likely to be oxidized. These in vivo data suggest that lactate may be formed in certain cells and oxidized in others. We therefore set out to determine whether the two isoforms of lactate dehydrogenase are localized to selective cell types in the human brain. We report here the production and characterization of two rat antisera, specific for the LDH-5 and LDH-1 subunits of lactate dehydrogenase, respectively. Immunohistochemical, immunodot, and western-blot analyses show that these antisera specifically recognize their homologous antigens. Immunohistochemistry on 10 control cases demonstrated a differential cellular distribution between both subunits in the hippocampus and occipital cortex: neurons are exclusively stained with the anti-LDH1 subunit while astrocytes are stained by both antibodies. These observations support the notion of a regulated lactate flux between astrocytes and neurons.

Astrocyte-specific expression of aquaporin-9 in mouse brain is increased after transient focal cerebral ischemia.

Aquaporin-9 (AQP9) is a new member of the aquaporin family of water-selective channels mainly expressed in liver and testis, presenting the characteristic of also being permeable to various solutes, particularly lactate. Recent data have shown the presence of AQP9 on tanycytes in the rat brain. In the current study, the authors show the expression of AQP9 in astrocytes in the mouse brain and changes in its expression after cerebral ischemia. Indeed, in control mouse, the AQP9 immunolabeling is present on astrocytic processes bordering the subarachnoid space and ventricles. The labeling also is observed on astrocytes in the white matter, hippocampus, hypothalamus, and lateral septum. After focal transient ischemia, an increase of the immunolabeling is detected on astrocytes in periinfarct areas. This AQP9 distribution study in mouse brain suggests a role of AQP9 in water homeostasis in the central nervous system. Furthermore, the overexpression of AQP9 on astrocytes surrounding an ischemic lesion suggests that AQP9 may also play a role in the regulation of postischemia edema and, in view of its permeability to monocarboxylates, in the clearance of lactate from the ischemic focus.

Modulation of VIP-Stimulated cAMP Formation by Excitatory Amino Acids in Mouse Cerebral Cortex.

We have investigated the modulatory action of excitatory amino acids (EAA) on vasoactive intestinal polypeptide (VIP)-stimulated cAMP formation in mouse cerebral cortical slices. Glutamate and aspartate potentiate in a concentration-dependent manner the effect of VIP. In order to characterize the type of receptor involved, we have used three prototypical EAA receptor agonists, that is, kainate, N-methyl-d-aspartate (NMDA) and quisqualate. Kainate mimicked the effect of glutamate, NMDA was inactive and quisqualate displayed an inhibitory action. Furthermore, ibotenate also potentiated the effect of VIP on cAMP formation, while l-homocysteate exhibited an inhibitory action. Ibotenate was 4-fold more potent and 2.5 times more effective than glutamate. However, the effects of kainate and ibotenate were not additive, suggesting the activation of a common receptor. Thus, based on this metabotropic action, EAA can be categorized into the following classes: (i) those that potentiate the effect of VIP, such as glutamate, aspartate, kainate and ibotenate; (ii) those that inhibit the effect of VIP, such as l-homocysteate and quisqualate; and (iii) those that are ineffective, such as NMDA and d-homocysteate. The effects of glutamate or ibotenate on VIP-stimulated cAMP formation were completely inhibited by l-phosphoserine and only partially by kynurenate. In a low chloride medium, or in the presence of 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate-hydrochloride (TMB-8), an inhibitor of calcium release from internal stores, EAA did not potentiate the effect of VIP, thus stressing the importance of these ions for the transduction of the glutamatergic signal. Our results indicate the existence of marked interactions between EAA and VIP on cAMP formation; the pharmacology of these interactions is, however, clearly distinct from the classical pharmacology of EAA which is mainly based on electrophysiological and binding studies.

Vasoactive intestinal peptide binding sites and fibers in the brain of the pigeon Columba livia: an autoradiographic and immunohistochemical study.

The distribution of vasoactive intestinal peptide (VIP) binding sites in the pigeon brain was examined by in vitro autoradiography on slide-mounted sections. A fully characterized monoiodinated form of VIP, which maintains the biological activity of the native peptide, was used throughout this study. The highest densities of binding sites were observed in the hyperstriatum dorsale, archistriatum, auditory field L of neostriatum, area corticoidea dorsolateralis and temporo-parieto-occipitalis, area parahippocampalis, tectum opticum, nucleus dorsomedialis anterior thalami, and in the periventricular area of the hypothalamus. Lower densities of specific binding occurred in the neostriatum, hyperstriatum ventrale and nucleus septi lateralis, dorsolateral area of the thalamus, and lateral and posteromedial hypothalamus. Very low to background levels of VIP binding were detected in the ectostriatum, paleostriatum primitivum, paleostriatum augmentatum, lobus parolfactorius, nucleus accumbens, most of the brainstem, and the cerebellum. The distribution of VIP-containing fibers and terminals was examined by indirect immunofluorescence using a polyclonal antibody against porcine VIP. Fibers and terminals were observed in the area corticoidea dorsolateralis, area parahippocampalis, hippocampus, hyperstriatum accessorium, hyperstriatum dorsale, archistriatum, tuberculum olfactorium, nuclei dorsolateralis and dorsomedialis of the thalamus, and throughout the hypothalamus and the median eminence. Long projecting fibers were visualized in the tractus septohippocampalis. In the brainstem VIP immunoreactive fibers and terminals were observed mainly in the substantia grisea centralis, fasciculus longitudinalis medialis, lemniscus lateralis, and in the area surrounding the nuclei of the 7th, 9th, and 10th cranial nerves. The correlation between the distribution of VIP binding sites and immunoreactive fibers and terminals was assessed in a restricted number of regions. A qualitatively good matching was found in the area corticoidea dorsolateralis, hyperstriatum dorsale, hyperstriatum accessorium, nucleus septi lateralis, nuclei dorsomedialis and dorsolateralis thalami, and in some hypothalamic areas. A striking mismatch occurred in the hyperstriatum ventrale, neostriatum, tectum opticum (high to moderate density of binding sites but only few immunoreactive profiles), and in the tuberculum olfactorium, median eminence, and spinal cord (lower density of binding sites but abundant immunoreactive profiles). The paleostriatum, lobus parolfactorius, and ectostriatum were virtually devoid of both binding sites and immunoreactive profiles. The results are discussed in relation to the known actions of VIP in the rodent and avian brain and are compared with previous observations on the distribution of VIP binding sites in the central nervous system of other vertebrates.

High potency of the orally-active NMDA-receptor antagonist CGP 40 116 in inhibiting excitatory postsynaptic potentials of rat basolateral amygdala neurones in vitro.

Conventional intracellular recordings were used to monitor postsynaptic potentials of basolateral amygdala neurones (BLA) in brain slices comprising the BLA, the entorhinal cortex (EC) and the hippocampus, in which the EC-BLA connections were preserved. Stimulation of the BLA with a bipolar electrode elicited complex postsynaptic potentials consisting of alpha-amino-3-hydroxy-5-methyl-isoxazoleproprionic acid (AMPA) receptor-mediated fast excitatory postsynaptic potentials (fast EPSPs), gamma-amino-butyric acid [GABA(A)] receptor-mediated fast inhibitory postsynaptic potentials (fast IPSPs) and GABAB receptor-mediated slow IPSPs. Bath application of 10 microM of the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and of 10 microM of the GABA(A) receptor antagonist bicuculline methiodide (BMI) revealed a N-methyl-D-aspartate (NMDA) receptor-mediated slow EPSPs, which was occasionally followed by a GABAB receptor-mediated slow IPSPs. Under these conditions, the log concentration-response curve for D-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 40 116), a newly developed drug with proposed NMDA-receptor antagonist properties, was compared to that obtained with the 'classic' antagonist D(-)-2-amino-5-phosphonopentanoic acid (D-AP5) in inhibiting the NMDA-mediated postsynaptic potentials. CGP 40 116 (IC50: 130 nM) was over 30 times more potent than D-AP5 (IC50: 4100 nM) in reducing NMDA-mediated slow EPSP. In conclusion, the present study indicates that CGP 40 116, a new orally-active NMDA antagonist, shows a very high potency on NMDA receptors in the amygdala and may therefore be a valuable tool for studying the behavioural effect of NMDA-receptor mediated transmission in this structure.