Persistent extrasynaptic hyperdopaminergia in the mouse hippocampus induces plasticity and recognition memory deficits reversed by the atypical antipsychotic sulpiride.

Evidence suggests that subcortical hyperdopaminergia alters cognitive function in schizophrenia and antipsychotic drugs (APD) fail at rescuing cognitive deficits in patients. In a previous study, we showed that blocking D2 dopamine receptors (D2R), a core action of APD, led to profound reshaping of mesohippocampal fibers, deficits in synaptic transmission and impairments in learning and memory in the mouse hippocampus (HP). However, it is currently unknown how excessive dopamine affects HP-related cognitive functions, and how APD would impact HP functions in such a state. After verifying the presence of DAT-positive neuronal projections in the ventral (temporal), but not in the dorsal (septal), part of the HP, GBR12935, a blocker of dopamine transporter (DAT), was infused in the CA1 of adult C57Bl/6 mice to produce local hyperdopaminergia. Chronic GBR12935 infusion in temporal CA1 induced a mild learning impairment in the Morris Water Maze and abolished long-term recognition memory in novel-object (NORT) and object-place recognition tasks (OPRT). Deficits were accompanied by a significant decrease in DAT+ mesohippocampal fibers. Intrahippocampal or systemic treatment with sulpiride during GBR infusions improved the NORT deficit but not that of OPRT. In vitro application of GBR on hippocampal slices abolished long-term depression (LTD) of fEPSP in temporal CA1. LTD was rescued by co-application with sulpiride. In conclusion, chronic DAT blockade in temporal CA1 profoundly altered mesohippocampal modulation of hippocampal functions. Contrary to previous observations in normodopaminergic mice, antagonising D2Rs was beneficial for cognitive functions in the context of hippocampal hyperdopaminergia.

Absence of VGLUT3 Expression Leads to Impaired Fear Memory in Mice.

Fear is an emotional mechanism that helps to cope with potential hazards. However, when fear is generalized, it becomes maladaptive and represents a core symptom of posttraumatic stress disorder (PTSD). Converging lines of research show that dysfunction of glutamatergic neurotransmission is a cardinal feature of trauma and stress related disorders such as PTSD. However, the involvement of glutamatergic co-transmission in fear is less well understood. Glutamate is accumulated into synaptic vesicles by vesicular glutamate transporters (VGLUTs). The atypical subtype, VGLUT3, is responsible for the co-transmission of glutamate with acetylcholine, serotonin, or GABA. To understand the involvement of VGLUT3-dependent co-transmission in aversive memories, we used a Pavlovian fear conditioning paradigm in VGLUT3-/- mice. Our results revealed a higher contextual fear memory in these mice, despite a facilitation of extinction. In addition, the absence of VGLUT3 leads to fear generalization, probably because of a pattern separation deficit. Our study suggests that the VGLUT3 network plays a crucial role in regulating emotional memories. Hence, VGLUT3 is a key player in the processing of aversive memories and therefore a potential therapeutic target in stress-related disorders.

Functional Connectivity between Hippocampus and Lateral Septum is Affected in Very Young Alzheimer's Transgenic Mouse Model.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-ß and tau proteins, which are believed to lead to neural damage that translates into brain dysfunction and cognitive deficits. Brain dysfunction can be evaluated by measuring single-neuron activity (spikes), global neural activity (local field potentials, LFPs) and the interaction between them. Considering that the dynamic interactions between the hippocampal pyramidal cells and lateral septum are important for proper structure function, we used the complete septo-hippocampal preparation from 30-day-old controls and J20-AD transgenic mice to record changes in spiking activity from the lateral septum and its relationship with LFP activity from the CA1 area. The cross-correlation analysis revealed that young J20 transgenic mice exhibit a significant reduction in coupling between lateral septum single-cell activity and neural network activity from the hippocampal CA1. Consistently, phase-lock analysis between lateral septum single-cell activity and CA1 neural network activity showed lower values in J20 transgenic mice. Similarly, the LFP- LFP coherence between CA1 and septum in the theta range showed lower values in J20 animals. Importantly, alterations were found before any detectable signs of cognitive deficits. Our data indicate that the disruption in the communication between hippocampus and rostral lateral septum is an early event in AD pathology and may contribute to the deficits observed during AD.

Regulation of the Hippocampal Network by VGLUT3-Positive CCK- GABAergic Basket Cells.

Hippocampal interneurons release the inhibitory transmitter GABA to regulate excitation, rhythm generation and synaptic plasticity. A subpopulation of GABAergic basket cells co-expresses the GABA/glycine vesicular transporters (VIAAT) and the atypical type III vesicular glutamate transporter (VGLUT3); therefore, these cells have the ability to signal with both GABA and glutamate. GABAergic transmission by basket cells has been extensively characterized but nothing is known about the functional implications of VGLUT3-dependent glutamate released by these cells. Here, using VGLUT3-null mice we observed that the loss of VGLUT3 results in a metaplastic shift in synaptic plasticity at Shaeffer's collaterals - CA1 synapses and an altered theta oscillation. These changes were paralleled by the loss of a VGLUT3-dependent inhibition of GABAergic current in CA1 pyramidal layer. Therefore presynaptic type III metabotropic could be activated by glutamate released from VGLUT3-positive interneurons. This putative presynaptic heterologous feedback mechanism inhibits local GABAergic tone and regulates the hippocampal neuronal network.

Distribution of vesicular glutamate transporters in the human brain.

Glutamate is the major excitatory transmitter in the brain. Vesicular glutamate transporters (VGLUT1-3) are responsible for uploading glutamate into synaptic vesicles. VGLUT1 and VGLUT2 are considered as specific markers of canonical glutamatergic neurons, while VGLUT3 is found in neurons previously shown to use other neurotransmitters than glutamate. Although there exists a rich literature on the localization of these glutamatergic markers in the rodent brain, little is currently known about the distribution of VGLUT1-3 in the human brain. In the present study, using subtype specific probes and antisera, we examined the localization of the three vesicular glutamate transporters in the human brain by in situ hybridization, immunoautoradiography and immunohistochemistry. We found that the VGLUT1 transcript was highly expressed in the cerebral cortex, hippocampus and cerebellum, whereas VGLUT2 mRNA was mainly found in the thalamus and brainstem. VGLUT3 mRNA was localized in scarce neurons within the cerebral cortex, hippocampus, striatum and raphe nuclei. Following immunoautoradiographic labeling, intense VGLUT1- and VGLUT2-immunoreactivities were observed in all regions investigated (cerebral cortex, hippocampus, caudate-putamen, cerebellum, thalamus, amygdala, substantia nigra, raphe) while VGLUT3 was absent from the thalamus and cerebellum. This extensive mapping of VGLUT1-3 in human brain reveals distributions that correspond for the most part to those previously described in rodent brains.

miR-1202 is a primate-specific and brain-enriched microRNA involved in major depression and antidepressant treatment.

Major depressive disorder (MDD) is a prevalent mood disorder that is associated with differential prefrontal brain expression patterns. Treatment of MDD includes a variety of biopsychosocial approaches. In medical practice, antidepressant drugs are the most common treatment for depressive episodes, and they are among the most prescribed medications in North America. Although antidepressants are clearly effective, particularly for moderate to severe depressive episodes, there is variability in how individuals respond to antidepressant treatment. Failure to respond has individual, economic and social consequences for patients and their families. Several lines of evidence demonstrate that genes are regulated through the activity of microRNAs (miRNAs), which act as fine-tuners and on-off switches of gene expression. Here we report on complementary studies using postmortem human brain samples, cellular assays and samples from clinical trials of patients with depression and show that miR-1202, a miRNA specific to primates and enriched in the human brain, is differentially expressed in individuals with depression. Additionally, miR-1202 regulates expression of the gene encoding metabotropic glutamate receptor-4 (GRM4) and predicts antidepressant response at baseline. These results suggest that miR-1202 is associated with the pathophysiology of depression and is a potential target for new antidepressant treatments.

Dopamine facilitates dendritic spine formation by cultured striatal medium spiny neurons through both D1 and D2 dopamine receptors.

Variations of dopamine (DA) levels induced by drugs of abuse or in the context of Parkinson's disease modulate the number of dendritic spines in medium spiny neurons (MSNs) of the striatum, showing that DA plays a major role in the structural plasticity of MSNs. However, little is presently known regarding early spine development in MSNs occurring before the arrival of cortical inputs and in particular about the role of DA and D1 (D1R) and D2 (D2R) DA receptors. A cell culture model reconstituting early cellular interactions between MSNs, intrinsic cholinergic interneurons and DA neurons was used to study the role of DA in spine formation. After 5 or 10 days in vitro, the presence of DA neurons increased the number of immature spine-like protrusions. In MSN monocultures, chronic activation of D1R or D2R also increased the number of spines and spinophilin expression in MSNs, suggesting a direct role for these receptors. In DA-MSN cocultures, chronic blockade of D1R or D2R reduced the number of dendritic spines. Interestingly, the combined activation or blockade of both D1R and D2R failed to elicit more extensive spine formation, suggesting that both receptors act through a mechanism that is not additive. Finally, we found increased ionotropic glutamate receptor responsiveness and miniature excitatory postsynaptic current (EPSC) frequency in DA-MSN co-cultures, in parallel with a higher number of spines containing PSD-95, suggesting that the newly formed spines present functional post-synaptic machinery preparing the MSNs to receive additional glutamatergic contacts. These results represent a first step in the understanding of how dopamine neurons promote the structural plasticity of MSNs during the development of basal ganglia circuits.

Somatodendritic dopamine release requires synaptotagmin 4 and 7 and the participation of voltage-gated calcium channels.

Somatodendritic (STD) dopamine (DA) release is a key mechanism for the autoregulatory control of DA release in the brain. However, its molecular mechanism remains undetermined. We tested the hypothesis that differential expression of synaptotagmin (Syt) isoforms explains some of the differential properties of terminal and STD DA release. Down-regulation of the dendritically expressed Syt4 and Syt7 severely reduced STD DA release, whereas terminal release required Syt1. Moreover, we found that although mobilization of intracellular Ca(2+) stores is inefficient, Ca(2+) influx through N- and P/Q-type voltage-gated channels is critical to trigger STD DA release. Our findings provide an explanation for the differential Ca(2+) requirement of terminal and STD DA release. In addition, we propose that not all sources of intracellular Ca(2+) are equally efficient to trigger this release mechanism. Our findings have implications for a better understanding of a fundamental cell biological process mediating transcellular signaling in a system critical for diseases such as Parkinson disease.

The endocannabinoid 2-arachidonoylglycerol inhibits long-term potentiation of glutamatergic synapses onto ventral tegmental area dopamine neurons in mice.

Drugs of abuse cause changes in the mesocorticolimbic dopamine (DA) system, such as a long-term potentiation (LTP)-like phenomenon at glutamatergic synapses onto ventral tegmental area (VTA) DA neurons. Abolishing this LTP interferes with drug-seeking behavior. Endocannabinoids (ECs) can be released by DA neurons in response to repetitive activation, which can inhibit glutamate release. Therefore, we hypothesized that ECs may act as negative regulators of LTP. Here we tested the induction of LTP in DA neurons of the VTA in mice expressing enhanced green fluorescent protein under the control of the tyrosine hydroxylase promoter. Immunohistochemistry showed colocalization of CB1 receptors with vesicular glutamate transporter (VGLUT)1 in terminals near DA neuron dendrites, with less extensive colocalization with VGLUT2. In addition, a CB1 receptor agonist, as well as trains of stimulation leading to EC production, decreased glutamate release onto DA neurons. We found that blocking CB1 receptors or synthesis of the EC 2-arachidonoylglycerol (2-AG) was without effect on basal excitatory postsynaptic potential amplitude; however, it facilitated the induction of LTP. As previously reported, antagonizing γ-aminobutyric acid (GABA)(A) transmission also facilitated LTP induction. Combining GABA(A) and CB1 receptor antagonists did not lead to larger LTP. LTP induced in the presence of CB1 receptor blockade was prevented by an N-methyl-d-aspartate receptor antagonist. Our observations argue in favor of the hypothesis that 2-AG acts as a negative regulator of LTP in the VTA. Understanding the factors that regulate long-term synaptic plasticity in this circuit is critical to aid our comprehension of drug addiction in humans.

Neuroinflammation is associated with changes in glial mGluR5 expression and the development of neonatal excitotoxic lesions.

It has been hypothesized that neuroinflammation triggered during brain development can alter brain functions later in life. We investigated the contribution of inflammation to the alteration of normal brain circuitries in the context of neuroexcitotoxicity following neonatal ventral hippocampal lesions in rats with ibotenic acid, an NMDA glutamate receptor agonist. Excitotoxic ibotenic acid lesions led to a significant and persistent astrogliosis and microglial activation, associated with the production of inflammatory mediators. This response was accompanied by a significant increase in metabotropic glutamate receptor type 5 (mGluR5) expression within two distinct neuroinflammatory cell types; astrocytes and microglia. The participation of inflammation to the neurotoxin-induced lesion was further supported by the prevention of hippocampal neuronal loss, glial mGluR5 expression and some of the behavioral perturbations associated to the excitotoxic lesion by concurrent anti-inflammatory treatment with minocycline. These results indicate that neuroinflammation significantly contributes to long-lasting excitotoxic effects of the neurotoxin and to some behavioral phenotypes associated with this model. Thus, the control of the inflammatory response may prevent the deleterious effects of excitotoxic processes that are triggered during brain development, limiting the risk to develop some of the behavioral manifestations related to these processes in adulthood.

Fluoxetine treatment abolishes the in vitro respiratory response to acidosis in neonatal mice.

BACKGROUND: To secure pH homeostasis, the central respiratory network must permanently adapt its rhythmic motor drive to environment and behaviour. In neonates, it is commonly admitted that the retrotrapezoid/parafacial respiratory group of neurons of the ventral medulla plays the primary role in the respiratory response to acidosis, although the serotonergic system may also contribute to this response. METHODOLOGY/PRINCIPAL FINDINGS: Using en bloc medullary preparations from neonatal mice, we have shown for the first time that the respiratory response to acidosis is abolished after pre-treatment with the serotonin-transporter blocker fluoxetine (25-50 µM, 20 min), a commonly used antidepressant. Using mRNA in situ hybridization and immunohistology, we have also shown the expression of the serotonin transporter mRNA and serotonin-containing neurons in the vicinity of the RTN/pFRG of neonatal mice. CONCLUSIONS: These results reveal that the serotonergic system plays a pivotal role in pH homeostasis. Although obtained in vitro in neonatal mice, they suggest that drugs targeting the serotonergic system should be used with caution in infants, pregnant women and breastfeeding mothers.

[Recent discoveries on the function and plasticity of central dopamine pathways]. / Découvertes récentes sur la fonction et la plasticité des voies dopaminergiques du cerveau.

Despite the fact that the neurotransmitter dopamine was discovered more than 50 years ago, we still have limited knowledge of its physiological and pathological roles. Recent work has unveiled novel and surprising properties of dopamine neurons and of other key players involved in regulating the dopamine system. For example, the integration of the dopamine signal by its receptors depends on many proteins of diverse signaling pathways and also of other types of receptors that interact with and regulate dopamine receptors: many new promising interactions have been reported during the past few years. Also, we are beginning to discover that chronic treatment with dopamine receptor ligands or other molecules affecting dopaminergic pathways induce long-term molecular, structural and functional rearrangements that could ultimately force us to revisit the mechanism of action of established therapeutic agents. Finally, the discovery of glutamate co-release by dopamine neurons is leading us to reconsider some keys aspects of dopamine neuron physiology.

The mammalian sympathetic prevertebral ganglia: models for the study of neuronal networks and basic neuronal properties.

The mammalian sympathetic prevertebral ganglia regulate various visceral functions and in particular the digestive tract motility. Several integrative properties of these ganglia have been described: convergence of central inputs, projection of visceral inputs at the pre- and post synaptic level and pacemaker activity of the neurones. This review presents the results obtained on another integrative property which has been widely studied over the last 10 years: the modulation of the fast central inputs by neuromodulators such as nitric oxide, ceramide and GABA. These substances facilitate or inhibit the fast central inputs through complex interrelated actions. We also present striking results recently obtained during the study of a regulatory reflex of the digestive tract motility organized by the prevertebral ganglia: the gastro-duodenal inhibitory reflex. During this reflex, the neurotransmitter released by the visceral afferent fibres to activate the ganglionic neurones is gaseous: nitric oxide. Moreover, the mechanism conducting the excitation along the afferent and efferent fibres is independent of action potentials. This mechanism requires the integrity of the membrane lipid rafts and the activation in cascade of the following second messenger sequence: ceramide, calcium, nitric oxide and guanosine 3', 5'-cyclic monophosphate. The existence of this mechanism gives grounds for rethinking one of the central dogmas in neuroscience according to which excitation is only conducted along nerves by an electrical phenomenon, the action potential. All these results strengthen the role of the prevertebral ganglia as a model for the study of neuronal networks and basic neuronal properties.

Culture of postnatal mesencephalic dopamine neurons on an astrocyte monolayer.

This unit presents a protocol for primary culture of postnatal mesencephalic dopamine neurons grown on an astrocyte monolayer, which can be used to investigate cellular and molecular mechanisms regulating dopamine neuron function. Using this in vitro approach, dopamine neurons survive for an extended period of time and establish functional axon terminals and dendrites that display properties similar to those observed in vivo and in brain slices. An alternate protocol is provided for a microculture system in which astrocytes are grown on a spatially limited surface where single or small groups of dopamine neurons develop. Under such conditions, isolated neurons establish synaptic contacts, or autapses, onto their own somatodendritic compartment, thus facilitating morphological and physiological experiments.

Neuronal conduction of excitation without action potentials based on ceramide production.

BACKGROUND: Action potentials are the classic mechanism by which neurons convey a state of excitation throughout their length, leading, after synaptic transmission, to the activation of other neurons and consequently to network functioning. Using an in vitro integrated model, we found previously that peripheral networks in the autonomic nervous system can organise an unconventional regulatory reflex of the digestive tract motility without action potentials. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we used combined neuropharmacological and biochemical approaches to elucidate some steps of the mechanism that conveys excitation along the nerves fibres without action potentials. This mechanism requires the production of ceramide in membrane lipid rafts, which triggers in the cytoplasm an increase in intracellular calcium concentration, followed by activation of a neuronal nitric oxide synthase leading to local production of nitric oxide, and then to guanosine cyclic monophosphate. This sequence of second messengers is activated in cascade from rafts to rafts to ensure conduction of the excitation along the nerve fibres. CONCLUSIONS/SIGNIFICANCE: Our results indicate that second messengers are involved in neuronal conduction of excitation without action potentials. This mechanism represents the first evidence-to our knowledge-that excitation is carried along nerves independently of electrical signals. This unexpected ceramide-based conduction of excitation without action potentials along the autonomic nerve fibres opens up new prospects in our understanding of neuronal functioning.

Proteomic of lipid rafts in the exocrine pancreas from diet-induced obese rats.

In the present work, we induced obesity in rats with high-energy-starch diet and studied exocrine pancreas response. The zymogen granule (ZG) or purified plasma membrane (PM) from the exocrine pancreas was used for the isolation of the detergent-resistant membranes (DRMs). Based on high content of cholesterol, GM1, the bile salt dependent lipase (BSDL), and GP2 enrichment, the low-density fractions were defined as lipid rafts. Additionally, the rafts vesicles were determined by immunogold labeling with anti BSDL. By combining MALDI-TOF/MS and nano-LC ESI Q-TOF MS/MS proteomic identification we have selected 33 proteins from the lipid rafts which were classified into at least four functional families. Our data suggest that the acinar PM from the diet-induced obesity rats may be organized into lipid rafts, and characterization of rafts proteome can contribute to improve our understanding of food digestion under obesity.

[Sphingolipids, vehicle for pathogenic agents and cause of genetic diseases]. / Les sphingolipides: vecteurs d'agents pathogènes et cause de maladies génétiques.

Sphingolipids are present in all eukaryotic cells and share a sphingoid base : sphingosine. They were first discovered in 1884 and for a long time they were thought to participate to membrane structure only. Recently it has been established that they are mainly located in particular areas of the membrane called rafts which are signalling platforms. It has also been demonstrated that sphingolipids are receptors and second messengers. They play a crucial role in cellular functioning and are necessary to maintenance and developing of living organisms. However due to their receptor properties, they are also gateway for penetration of pathogenic agents such as virus (Ebola, HIV) or toxins (botulinium, tetanus). These agents first bind to glycosphingolipids or proteins mainly located in rafts. The complex so formed is required for the crossing of the membrane by the pathogenic agent. Sphingolipids metabolism is regulated by numerous enzymes. A failure in the activity of one of them induces an accumulation of sphingolipids known as sphingolipidoses. These are genetic diseases having severe consequences for the survival of the organism. The precise mechanisms of the sphingolipidoses are still mainly unknown which explains why few therapeutic strategies are available. These particular properties of lipids rafts and sphingolipids explain why a growing number of studies in the medical and scientific fields are devoted to them.