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The HeartMate 3 risk score (HM3RS) was developed from the Multicenter Study of MagLev Technology in Patients Undergoing Mechanical Circulatory Support Therapy with HeartMate 3 (MOMENTUM 3) clinical trial to predict 1 and 2 year mortality after left ventricular assist device implantation. However, it has not been validated in a real-world population, especially after the heart transplant allocation system change on October 18, 2018. In this multicenter retrospective analysis, we found that HM3RS did not predict 1 and 2 year outcomes in the contemporary era, highlighting the need to revise this risk prediction tool in the real-world setting., Competing Interests: Disclosure: The authors have no conflicts of interest to report., (Copyright © ASAIO 2024.)

The aim of this study was to analyze if the 2-pyrrolidinone rich fraction of Brassica oleracea var. capitata exhibiting antioxidant and in vitro anticancer activities. 2-Pyrrolidinone is an active compound present in Brassica oleracea var. capitata. Our findings explored the potential use of 2-pyrrolidinone in cancer treatment. This compound was identified and isolated by gas chromatography-mass spectrometry and high-performance liquid chromatography from the leaf of Brassica oleracea var. capitata. The resultant rich active compound exhibited in vitro cytotoxicity in HeLa and PC-3 human cancer cell lines, and it also exhibited antioxidant activity in cell free assays. DAPI staining, an apoptotic analysis and cell cycle analysis were performed to evaluate the anticancer activity of 2-pyrrolidinone against the above cell lines. The IC50 value of 2-pyrrolidinone was determined to be of 2.5 µg/ml for HeLa, 3 µg/ml for PC-3 cells at 24 h and 1.5 µg/ml for HeLa and 2 µg/ml for PC-3 cells at 48 h, respectively. However, cell cycle analysis revealed that the anti-proliferative effects of the 2-pyrrolidinone were mediated through cell cycle arrest in the G0/G1 phase. These results from the current study suggest that the 2-pyrrolidinone have potential anticancer effects, which will lead to the development of new anticancer agents for arresting cancer cells growth in vitro., (Copyright © 2012 John Wiley & Sons, Ltd.)

The Bordeaux Neuroscience Institute brings together all the disciplines that constitute the clinical and experimental neurosciences. Outside of the Paris region, the Institute represents the largest community of researchers working on the nervous system. The aim of this brief historical piece is to describe how neuroscientists in Bordeaux are the heirs to a long neuropsychiatric tradition established by pioneers of national and international renown. This tradition has been maintained, without interruption, through many generations. The careers and scientific work of these great neurologists and psychiatrists are briefly evoked, and particularly those of A. Pitres, E. Régis and E. Azam in the 19th century; and, in the 20th century, J. Abadie, H. Verger and R. Cruchet. The determining influence of P Delmas-Marsalet (1898-1977), Professor of Neuropsychiatry, on the development of modern neurosciences in Bordeaux is recalled through his work, his teachings, and his numerous students.

Altricial mammals use olfaction long before the olfactory bulb has reached its anatomically mature state. Indeed, while audition and vision are still not functional, the olfactory system of newborn animals can clearly process distinct odorant molecules. Although several previous studies have emphasized the important role that olfaction plays in early critical functions, it has been difficult to develop a sensitive and reliable test to precisely quantify olfactory ability in pups. One difficulty in determining early sensory capabilities is the rather limited behavioral repertory of neonates. The present study examines the use of ultrasonic vocalizations emitted by isolated rodent pups as a potential index of odor detection in newborn mice. As early as postnatal day 2, mice reliably decrease their emission of ultrasonic calls in response to odor exposure to the bedding of adult male mice but not in response to clean bedding odors or to non-social odorant molecules. A toxin known to damage the olfactory epithelium in adult, the 3-methylindole, impairs the ultrasonic call responses triggered by exposure to male bedding, thus confirming the efficiency of this olfactotoxin on mice pups. The administration of 3-methylindole severely reduced the life expectancy of the majority of subjects. This result is discussed according to the critical role of olfaction in nipple-seeking behavior in mouse pups.

Recently, modern neuroscience has made considerable progress in understanding how the brain perceives, discriminates, and recognizes odorant molecules. This growing knowledge took over when the sense of smell was no longer considered only as a matter for poetry or the perfume industry. Over the last decades, chemical senses captured the attention of scientists who started to investigate the different stages of olfactory pathways. Distinct fields such as genetic, biochemistry, cellular biology, neurophysiology, and behavior have contributed to provide a picture of how odor information is processed in the olfactory system as it moves from the periphery to higher areas of the brain. So far, the combination of these approaches has been most effective at the cellular level, but there are already signs, and even greater hope, that the same is gradually happening at the systems level. This review summarizes the current ideas concerning the cellular mechanisms and organizational strategies used by the olfactory system to process olfactory information. We present findings that exemplified the high degree of olfactory plasticity, with special emphasis on the first central relay of the olfactory system. Recent observations supporting the necessity of such plasticity for adult brain functions are also discussed. Due to space constraints, this review focuses mainly on the olfactory systems of vertebrates, and primarily those of mammals.

Dopamine, one of main modulatory neurotransmitters of the nervous system acts on target cells through two classes of G protein-coupled receptors, D1 and D2. The two dopamine receptor classes display different structures, interact with different regulatory partners (including heterotrimeric G proteins) and, accordingly, have independent evolutionary origins. In vertebrates, each of these receptor classes comprises several subtypes, generated by two steps of gene duplications, early in vertebrate evolution. In the D1 receptor class, the D1A, D1B, D1C and D1D subtypes, and in the D2 class, the D2, D3 and D4 receptor subtypes have been conserved in most vertebrate groups. This conservation has been driven by the acquisition, by each receptor subtype, of a small number of specific properties, which were selected for adaptive purpose in vertebrates. Among these properties, affinity for dopamine, the natural ligand, intrinsic receptor activity, and agonist-induced desensitization clearly distinguish the receptor subtypes. In addition, each dopamine receptor subtype is addressed to a specific location within neuronal networks, although detailed information is lacking for several receptor subtypes. Receptors localization at diverse subcellular places in neurons may also differ from one subtype to another, resulting in different ways of regulating cell signalisation. One challenge for future research on dopamine and its receptors would be to identify the nature of the protein partners and the molecular mechanisms involved in localizing receptors to the neuronal plasma membrane. In this respect, the evolutionary approach we have undertaken suggests that, due to gene duplications, a reasonable degree of freedom exists in the tight organisation of dopamine receptors in neurons. This "evolvability" of dopamine systems has been instrumental to adapt the vertebrate species to nearly all the possible environments.

In the mammalian forebrain, most neurons originate from proliferating cells in the ventricular zone lining the lateral ventricles, including a discrete area of the subventricular zone (SVZ). In this region, neurogenesis continues into adulthood. Most of the cells generated in the SVZ are neuronal precursors with progeny that migrate rostrally along a pathway known as the rostral migratory stream before they reach the main olfactory bulb (MOB) where they differentiate into local interneurons. The olfactory system thus provides an attractive model to investigate neuronal production and survival, processes involving interplay between genetic and epigenetic influences. The present study was conducted to investigate whether exposure to an odor-enriched environment affects neurogenesis and learning in adult mice. Animals housed in either a standard or an odor-enriched environment for 40 d were injected intraperitoneally with bromodeoxyuridine (BrdU) to detect proliferation among progenitor cells and to follow their survival in the MOB. The number of BrdU-labeled neurons was not altered 4 hr after a single BrdU injection. In contrast, the number of surviving progenitors 3 weeks after BrdU injection was markedly increased in animals housed in an enriched environment. This effect was specific because enriched odor exposure did not influence hippocampal neurogenesis. Finally, we showed that adult mice housed in odor-enriched cages display improved olfactory memory without a change in spatial learning performance. By maintaining a constitutive turnover of granule cells subjected to modulation by environmental cues, ongoing bulbar neurogenesis could be associated with improved olfactory memory.

Olfaction was long considered to belong more to the realm of art than to that of science. As a result, how the brain perceives, discriminates, and recognizes odorant molecules is still a mystery. Recent progress has nonetheless been made at early stages of the olfactory pathway when olfactory studies entered into the molecular era to elucidate the first contact of an odor molecule with a receptor. Our group focuses on the analysis of odor information in the olfactory bulb, the first processing relay in the mammalian brain. Using this model, we are attempting to decipher the code for odorant information. Furthermore, the olfactory bulb also provides an attractive model to investigate neuronal proliferation, differentiation, migration, and neuronal death, processes involving an interplay between genetic and epigenetic influences. Finally, our goal is to explore the possible consequences of the olfactory bulb plasticity, in olfactory performance. For these purposes, we aim to combine morphological, electrophysiological and behavioral approaches to investigate: (1) how the olfactory bulb processes odor molecule information, (2) how neural precursors differentiate into olfactory bulb interneurons, (3) how these newly-generated neurons integrate into an operational neural network, (4) what role they play in the adult olfactory bulb, and (5) how are basic olfactory functions maintained in such a sensory system subjected to continuous renewal of a large percentage of its neuronal population. These questions should provide new fuel for the molecular and cellular bases of sensory perception and shed light onto cellular bases of learning and memory.

In this review, we discuss some of the neural processes involved in the perception of odors that, together with audition and vision, provide essential information for analyzing our surroundings. We shall see how odor detection and learning induce substantial structural and functional changes at the first relay of the olfactory system, i.e., the main olfactory bulb. Among the mechanisms that participate in these modifications is the persistence of a high level of interneuron neurogenesis within the adult olfactory bulb. Our goal is to present some observations related to the neurogenesis that may aid in understanding the neural mechanisms of sensory perception and shed light on the cellular basis of olfactory learning. We summarize the current ideas concerning the molecular mechanisms and organizational strategies used by the olfactory system to transduce, encode, and process information at various levels in the olfactory sensory pathway. Due to space constraints, this review focuses exclusively on the olfactory systems of vertebrates and primarily those of mammals.

The dopamine D(2) receptor exists as a long (D(2a)) and a short (D(2b)) isoform generated by alternative splicing of the corresponding transcript, which modifies the length of the third cytoplasmic loop implicated in heterotrimeric G-protein-coupling. Anatomical data suggested that this segment regulates the intracellular traffic and localization of the receptor. To directly address this question we used a combination of tagging procedures and immunocytochemical techniques to detect each of the two D(2) receptor isoforms. Surprisingly, most of the newly synthesized receptors accumulate in large intracellular compartments, the plasma membrane being only weakly labeled, without significant difference between the two receptor isoforms. Double labeling experiments showed that this localization corresponded neither to endosomal compartments nor to the Golgi apparatus. The D(2) receptor is mostly retained in the endoplasmic reticulum (ER), the long isoform more efficiently than the short one. It is accompanied by a striking vacuolization of the ER, roughly proportional to the expression levels of the two receptor isoforms. This phenomenon is partly overcome by treatment with pertussis toxin. In addition, an intrinsic activity of the D(2) receptor isoforms is revealed by [(35)S]-GTP gamma S binding and cAMP assay, which suggested that expression of weakly but constitutively active D(2) receptors promotes activation of heterotrimeric G protein inside the secretory pathway. This mechanism may participate in the regulation of the cellular traffic of the D(2) receptors isoforms.

E3, E4, and E3-4 are naturally occurring estrogen receptor (ER) isoforms, generated through differential splicing of the ERalpha primary transcript and abundantly expressed in embryonic rat pituitary. Studies in COS cells transfected with full-length ERalpha or its three splice variants fused to green fluorescent protein (GFP), revealed a different subcellular localization for each isoform. In the absence of estradiol, full-length ERalpha-GFP was predominantly nuclear, and E3-GFP and E4-GFP were present both in cytoplasm and nucleus, whereas E3-4-GFP was predominantly cytoplasmic. Upon hormone treatment, a dramatic redistribution of full-length ERalpha-GFP and E3-GFP, from a diffuse to punctate pattern, occurred within the nucleus. In contrast, the distribution of E4-GFP and E3-4-GFP was unaffected. Nuclear fractionation studies showed that full-length ER-alpha and E3 displayed the same hormone-induced ability to tether to nuclear matrix, whereas nuclear E4 appeared to remain loosely associated to functional nuclear constituents. When cotransfected with an estrogen-inducible reporter plasmid (VIT-TK-CAT) in ER-negative (CHO k1) and ER-positive pituitary (GH4 C1) cells, E3-4 exhibited a very weak estrogen-dependent transactivation activity, whereas E3 had an inhibitory effect on full-length ER action. Conversely, E4 displayed estrogen-independent transcriptional activity in ER-negative cells, and in ER-positive cells, enhanced the estrogen-induced gene expression as efficiently as full-length ERalpha. In a gel mobility shift assay, phosphorylated E4 was able to form a specific complex with a consensus ERE, while E3 and E3-4 never did bind by themselves. The observed inhibitory action of E3 on estrogen-dependent transcription would rather involve protein-protein interactions such as formation of heterodimers with full-length ERalpha, as suggested by immunoprecipitation followed by Western blotting. These data suggest that E3 and E4 may play a physiologically relevant role as negative or constitutively positive modulators of transcription, in the developing rat pituitary.

Neuronal synchronization in the olfactory bulb has been proposed to arise from a diffuse action of glutamate released from mitral cells (MC, olfactory bulb relay neurons). According to this hypothesis, glutamate spills over from dendrodendritic synapses formed between MC and granule cells (GC, olfactory bulb interneurons) to activate neighboring MC. The excitation of MC is balanced by a strong inhibition from GC. Here we show that MC excitation is caused by glutamate released from bulbar interneurons located in the GC layer. These reciprocal synapses depend on an unusual, 2-amino-5-phosphonovaleric acid-resistant, N-methyl-d-aspartate receptor. This type of feedback excitation onto relay neurons may strengthen the original sensory input signal and further extend the function of the dendritic microcircuit within the main olfactory bulb.

Disruption of both alleles of the prion protein gene, Prnp, has been shown repeatedly to abolish the susceptibility of mice to developing prion diseases. However, conflicting results have been obtained from phenotypic analyses of prion protein (PrP)-deficient (Prnp0/0) mice lines. To explore the possible neurophysiological properties associated with expression or absence of the normal isoform of the cellular prion protein (PrPC), we used conventional in vitro extracellular field potential recordings in the hippocampal CA1 area of mice from two independently-derived Prnp0/0 strains. Basal synaptic transmission and a short-term form of synaptic plasticity were analysed in this study. Results were compared with animals carrying a wild-type mouse PrP transgene to investigate whether PrP expression levels influence glutamatergic synaptic transmission in the hippocampus. There was a clear correlation between excitatory synaptic transmission and PrP expression; i.e. the range of synaptic responses increased with the level of PrPC expression. On the other hand, the probability of transmitter release, as assessed by paired-pulse facilitation, appeared unchanged. Interestingly, whereas the overall range for synaptic responses was still greater in older mice over-expressing PrPC, this effect in these animals appeared to be due to better recruitment of fibres rather than facilitation of synaptic transmission per se. Taken together, these data are strong evidence for a functional role for PrPC in modulating synaptic transmission.

Within the brain, HIV-1 targets the microglia and astrocytes. Previous studies have reported that viral entry into astrocytes is independent of CD4, in contrast to microglia. We aimed to determine whether chemokine receptors play a role in mediating CD4-independent HIV-1 entry into astrocytes. We found that embryonic astrocytes and microglial cells express CCR5, CCR3, and CXCR4 transcripts. Intracellular calcium levels in astrocytes were found to increase following application of RANTES, MIP-1beta (CCR5-agonist), SDF-1alpha (CXCR4-agonist), but not eotaxin (CCR3-agonist). In microglial cells, eotaxin was also able to modulate internal calcium homeostasis. CD4 was not present at the cell surface of purified astrocytes but CD4 mRNA could be detected by RT-PCR. Neither HIV-1(9533) (R5 isolate) nor HIV-1(LAI) (X4 isolate) penetrated into purified astrocytes. In contrast, mixed CNS cell cultures were infected by HIV-1(9533) and this was inhibited by anti-CD4 mAb in 4/4 tested cultures and by anti-CCR5 mAb in 2/4. Thus, the HIV-1 R5 strain requires CD4 to penetrate into brain cells, suggesting that CCR5 cannot be used as the primary receptor for M-tropic HIV-1 strains in astrocytes. Moreover, inconstant inhibition of HIV-1 entry by anti-CCR5 mAb supports the existence of alternative coreceptors for penetration of M-tropic isolates into brain cells., (Copyright 2001 Wiley-Liss, Inc.)

It has been shown recently that in mitral cells of the rat olfactory bulb, N-methyl-D-aspartate (NMDA) autoreceptors are activated during mitral cell firing. Here we consider in more details the mechanisms of mitral cell self-excitation and its physiological relevance. We show that both ionotropic NMDA and non-NMDA autoreceptors are activated by glutamate released from primary and secondary dendrites. In contrast to non-NMDA autoreceptors, NMDA autoreceptors are almost exclusively located on secondary dendrites and their activation generates a large and sustained self-excitation. Both intracellularly evoked and miniature NMDA-R mediated synaptic potentials are blocked by intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) and result from a calcium-dependent release of glutamate. Self-excitation can be produced by a single spike, and trains of spikes result in frequency facilitation. Thus activation of excitatory autoreceptors is a major function of action potentials backpropagating in mitral cell dendrites, which results in an immediate positive feedback counteracting recurrent inhibition and increasing the signal-to-noise ratio of olfactory inputs.

In this review, we discuss some of the neural processes involved in the perception of odors which, together with audition and vision, provide essential information for analyzing our surroundings. We shall see how odor detection and learning induce substantial structural and functional changes at the first relay of the olfactory system, i.e., the main olfactory bulb. Among the mechanisms which participate in these modifications are changes in the cell's responses to a transmitter and the persistence of a high level of interneuron neurogenesis within the adult olfactory bulb. Our goal is to present some observations related to these two phenomena that may aid in understanding the neural mechanisms of sensory perception and shed light on the cellular basis of olfactory learning. To this purpose, we summarize the current ideas concerning the molecular mechanisms and organizational strategies used by the olfactory system to transduce, encode, and process information at various levels in the olfactory sensory pathway. Due to space constraints, this review focuses exclusively on the olfactory systems of vertebrates and primarily those of mammals.

Polysialic acid-neural cell adhesion molecule (PSA-NCAM) expression in the adult nervous system is restricted to regions retaining a capacity for morphological plasticity. For the female rat hypothalamoneurohypophysial system (HNS), we have previously shown that lactation induces a dramatic decrease in PSA-NCAM, while leaving the level of total NCAM protein unchanged. Here, we wanted to elucidate the molecular mechanisms leading to a downregulation of PSA, thereby stabilizing newly established synapses and neurohemal contacts that accompany the increased activity of oxytocinergic neurons. First, we show that the overall specific activity of polysialyltransferases present in tissue extracts from supraoptic nuclei decreases by approximately 50% during lactation. So far, two polysialyltransferase enzymes, STX and PST, have been characterized for their capacity to transfer PSA onto NCAM in vitro. Using a competitive RT-PCR on RNA extracts from the HNS, we demonstrate furthermore a significant decrease in the expression levels of both STX and PST mRNAs in lactating versus virgin animals. Interestingly, this downregulation of NCAM polysialylation is not correlated with the post-transcriptional regulation of variable alternative spliced exon splicing, in contrast to neural development. The control of polysialylation via a regulation of both enzyme activity and expression underlines the important role of this post-translational modification of NCAM in morphofunctional plasticity in adult brain.

In adult rodents, neurons are continually generated in the subventricular zone of the forebrain, from where they migrate tangentially toward the olfactory bulb, the only known target for these neuronal precursors. Within the main olfactory bulb, they ascend radially into the granule and periglomerular cell layers, where they differentiate mainly into local interneurons. The functional consequences of this permanent generation and integration of new neurons into existing circuits are unknown. To address this question, we used neural cell adhesion molecule-deficient mice that have documented deficits in the migration of olfactory-bulb neuron precursors, leading to about 40% size reduction of this structure. Our anatomical study reveals that this reduction is restricted to the granule cell layer, a structure that contains exclusively gamma-aminobutyric acid (GABA)ergic interneurons. Furthermore, mutant mice were subjected to experiments designed to examine the behavioral consequences of such anatomical alteration. We found that the specific reduction in the newly generated interneuron population resulted in an impairment of discrimination between odors. In contrast, both the detection thresholds for odors and short-term olfactory memory were unaltered, demonstrating that a critical number of bulbar granule cells is crucial only for odor discrimination but not for general olfactory functions.

Adenosine can influence dopaminergic neurotransmission in the basal ganglia via postsynaptic interaction between adenosine A2A and dopamine D2 receptors. We have used a human neuroblastoma cell line (SH-SY5Y) that was found to express constitutively moderate levels of adenosine A1 and A2A receptors (approximately 100 fmol/mg of protein) to investigate the interactions of A2A/D2 receptors, at a cellular level. After transfection with human D2L receptor cDNA, SH-SY5Y cells expressed between 500 and 1,100 fmol of D2 receptors/mg of protein. In membrane preparations, stimulation of adenosine A2A receptors decreased the affinity of dopamine D2 receptors for dopamine. In intact cells, the calcium concentration elevation induced by KCI treatment was moderate, and dopamine had no effect on either resting intracellular free Ca2+ concentration ([Ca2+]i) or KCI-induced responses. In contrast, pretreatment with adenosine deaminase for 2 days dramatically increased the elevation of [Ca2+]i evoked by KCI, which then was totally reversed by dopamine. The effects induced by 48-h adenosine inactivation were mimicked by application of adenosine A1 antagonists and could not be further reversed by acute activation of either A1 or A2A receptors. Acute application of the selective A2 receptor agonist CGS-21680 counteracted the D2 receptor-induced [Ca2+]i responses. The present study shows that SH-SY5Y cells are endowed with functional adenosine A2A and A1 receptors and that A2A receptors exert an antagonistic acute effect on dopamine D2 receptor-mediated functions. In contrast, A1 receptors induce a tonic modulatory role on these dopamine functions.

Dopamine is a widespread neurotransmitter which exerts numerous neuromodulatory actions in the vertebrate central nervous system. This pleiotropic activity relies on the organisation of dopamine-synthesizing neuronal pathways and on a multiplicity of specific membrane receptors. A comparative approach has been undertaken to gain clues on the genetic events which took place during evolution to devise the dopamine systems of modern vertebrates. The localisation and phenotype of dopamine-synthesizing neurones is determined by different gene networks in each of the dopaminergic nuclei. However, despite this amazing diversity, the overall organisation of the dopaminergic nuclei is strinkingly conserved in the main vertebrates groups. In sharp contrast, the number of dopamine receptors subtypes has been multiplied by two major steps of gene duplications during vertebrates evolution. The first one occurred in the lineage leading to agnathans, whereas the second was concomitant to the emergence of cartilaginous fish. Accordingly, three subtypes exist in D1 receptor class (D1A, D1B, D1C) in all the jawed vertebrates, with two exceptions: eutherian mammals where only two D1 subtypes are found (D1A, D1B) and archosaurs where a fourth subtype is present (D1D). Comparisons of the pharmacological and biochemical characteristics of the dopamine receptors in vertebrate groups revealed homologous features that define each of the receptor subtypes and that have been fixed after gene duplications. The comparison of the distribution of the D1 receptor transcripts in the brain of teleosts and mammals points to significant conserved or derived expression territories, revealing previously neglected aspects of dopamine physiology in vertebrates.

Rhythmic patterns of neuronal activity have been found at multiple levels of various sensory systems. In the olfactory bulb or the antennal lobe, oscillatory activity exhibits a broad range of frequencies and has been proposed to encode sensory information. However, the neural mechanisms underlying these oscillations are unknown. Bulbar oscillations might be an emergent network property arising from neuronal interactions and/or resulting from intrinsic oscillations in individual neurons. Here we show that mitral cells (output neurons of the olfactory bulb) display subthreshold oscillations of their membrane potential. These oscillations are mediated by tetrodotoxin-sensitive sodium currents and range in frequency from 10 to 50 Hz as a function of resting membrane potential. Because the voltage dependency of oscillation frequency was found to be similar to that for action potential generation, we studied how subthreshold oscillations could influence the timing of action potentials elicited by synaptic inputs. Indeed, we found that subthreshold oscillatory activity can trigger the precise occurrence of action potentials generated in response to EPSPs. Furthermore, IPSPs were found to set the phase of subthreshold oscillations and can lead to "rebound" spikes with a constant latency. Because intrinsic oscillations of membrane potential enable very precise temporal control of neuronal firing, we propose that these oscillations provide an effective means to synchronize mitral cell subpopulations during the processing of olfactory information.

The main olfactory bulb is a critical relay step between the olfactory epithelium and the olfactory cortex. A marked feature of the bulb is its massive innervation by cholinergic inputs from the basal forebrain. In this study, we addressed the functional interaction between cholinergic inputs and intrinsic bulbar circuitry. Determining the roles of acetylcholine (ACh) requires the characterization of cholinergic effects on both neural excitability and synaptic transmission. For this purpose, we used electrophysiological techniques to localize and characterize the diverse roles of ACh in mouse olfactory bulb slices. We found that cholinergic inputs have a surprising number of target receptor populations that are expressed on three different neuronal types in the bulb. Specifically, nicotinic acetylcholine receptors excite both the output neurons of the bulb, i.e., the mitral cells, as well as interneurons located in the periglomerular regions. These nicotine-induced responses in interneurons are short lasting, whereas responses in mitral cells are long lasting. In contrast, muscarinic receptors have an inhibitory effect on the firing rate of interneurons from a deeper layer, granule cells, while at the same time they increase the degree of activity-independent transmitter release from these cells onto mitral cells. Cholinergic signaling thus was found to have multiple and opposing roles in the olfactory bulb. These dual cholinergic effects on mitral cells and interneurons may be important in modulating olfactory bulb output to central structures required for driven behaviors and may be relevant to understanding mechanisms underlying the perturbations of cholinergic inputs to cortex that occur in Alzheimer's disease.

Both observations in humans with disorders of dopaminergic transmission and molecular studies point to an important role for dopamine in olfaction. In this study we found that dopamine receptor activation in the olfactory bulb causes a significant depression of synaptic transmission at the first relay between olfactory receptor neurons and mitral cells. This depression was found to be caused by activation of the D2 subtype of dopamine receptor and was reversible by a specific D2 receptor antagonist. A change in paired-pulse modulation during the depression suggests a presynaptic locus of action. The depression was found to occur independent of synaptic activity. These results provide the first evidence for dopaminergic control of inputs to the main olfactory bulb. The magnitude and locus of dopamine's modulatory capabilities in the bulb suggest important roles for dopamine in odorant processing.

The expression time course of estrogen receptor alpha (ER alpha) was analyzed by RT-PCR in fetal and newborn rat pituitaries. In addition to the classical ER alpha messenger RNA (mRNA), three shorter transcripts were detected and subsequently cloned. Sequence analysis showed that they corresponded to ER alpha mRNAs lacking exon 3 (which encodes a zinc finger in the DNA-binding domain), exon 4 (which encodes the nuclear localization signal and part of the steroid-binding domain), or both exons 3 and 4. As analyzed by RT-PCR and ribonuclease protection assay, the respective expression levels of the different transcripts varied dramatically during pituitary development; short forms appeared 4 days before full-length ER alpha mRNA. On Western blots from rat pituitaries of different ages, an ER alpha-specific antiserum labeled four protein bands of the expected molecular weights, revealing that all four ER alpha mRNAs are translated in vivo. Immunocytochemistry, using the same antiserum, showed the ER alpha to be present first in the cytosol of intermediate lobe cells (around embryonic day 16). Only 5 days later, nuclear staining became detectable in the anterior lobe. We argue that the observed cytosolic staining will be essentially due to short ER alpha isoforms, which are indeed more abundantly expressed in the intermediate lobe. These data suggest that during pituitary development, the activity of the ER alpha might be specifically regulated by differential splicing of its primary transcript, resulting in a differential subcellular localization of the isoforms.

The Raf kinases play an important and specific role in the activation of extracellular signal-regulated kinases (ERK) cascade. Beside its role in the control of proliferation and differentiation, the ERK cascade has also been implicated in neuron-specific functions. In order to gain clues on the function of Raf kinases in the adult central nervous system (CNS), we performed a comparative analysis of the distribution and subcellular localization of the different Raf kinases in rat brain with antibodies specific for the different Raf kinases. We show that B-Raf and Raf-1 proteins are present in most brain areas, whereas A-Raf is not detected. Interestingly, the two Raf proteins have an approximately similar pattern of distribution with a rostro-caudal decreasing gradient of expression. These two kinases are colocalized in neurons but they are differentially located in subcellular compartments. Raf-1 is localized mainly in the cytosolic fraction around the nucleus, whereas B-Raf is widely distributed in the cell bodies and in the neuritic processes. In addition, we demonstrated that numerous B-Raf isoforms are present in the brain. These isoforms have a differential pattern of distribution, some of them being ubiquitously expressed whereas others are localized to specific brain areas. These isoforms also have a clear differential subcellular localization, specially in Triton-insoluble fractions, but also in synaptosomal, membrane and cytosolic compartments. Altogether these results suggest that each Raf protein could have a distinct signalling regulatory function in the brain with regard to its subcellular localization.

Cell adhesion molecules (CAMs) are known to be involved in a variety of developmental processes that play key roles in the establishment of synaptic connectivity during embryonic development, but recent evidence implicates the same molecules in synaptic plasticity of the adult. In the present study, we have used neural CAM (NCAM)-deficient mice, which have learning and behavioral deficits, to evaluate NCAM function in the hippocampal mossy fiber system. Morphological studies demonstrated that fasciculation and laminar growth of mossy fibers were strongly affected, leading to innervation of CA3 pyramidal cells at ectopic sites, whereas individual mossy fiber boutons appeared normal. Electrophysiological recordings performed in hippocampal slice preparations revealed that both basal synaptic transmission and two forms of short-term plasticity, i.e., paired-pulse facilitation and frequency facilitation, were normal in mice lacking all forms of NCAM. However, long-term potentiation of glutamatergic excitatory synapses after brief trains of repetitive stimulation was abolished. Taken together, these results strongly suggest that in the hippocampal mossy fiber system, NCAM is essential both for correct axonal growth and synaptogenesis and for long-term changes in synaptic strength.

The two isoforms of the D2 dopamine receptor are generated by alternative splicing of the exon 6 of the premessenger RNA (pre-mRNA), changing the length of the third cytoplasmic loop involved in the coupling to G proteins. In the MMQ PRL cell line, sex steroid hormones modulated the proportion of the two D2 receptor isoforms. Under controlled culture conditions, 17beta-estradiol (E2) strongly favored the production of the long isoform of D2 mRNA over the short one, whereas both isoforms were equally abundant when culture medium was hormone depleted. In the presence of progesterone (P), E2 action was inhibited, and equal amounts of each D2 receptor isoform were produced in the cells. Hormone treatments never modified either the total amount of D2 receptor mRNA and D2 receptor binding sites or D2 receptor-mediated inhibition of adenylyl cyclase. Specific antagonists demonstrated that the activity of each hormone depended on their nuclear receptors. Inhibitors of gene transcription or translation also showed that their activity required protein synthesis. The expression of the short D2 receptor isoform was never prominent, even at the single cell level. Analysis of the intron sequence flanking alternative exon 6 showed that only the upstream intron presented two sequence tracts known to be targets for splicing factors. Taken together, these results provide converging evidence for a physiologically relevant mechanism by which sex steroid receptors could regulate the expression of a splicing factor favoring the production of the long dopamine D2 receptor isoform.

Ca2+ ions trigger the release of hormones and neurotransmitters and contribute to making the secretory vesicles competent for fusion. Here, we present evidence for the involvement of the GTP-binding protein Rab3a in the sensitivity of the exocytotic process to internal [Ca2+]. The secretory activity of bovine adrenal chromaffin cells was elicited by Ca2+ dialysis through a patch-clamp pipette and assayed by monitoring changes in cell membrane capacitance. Microinjection of antisense oligonucleotides directed to rab3a mRNA increased the secretory activity observed at low (0.2-4 microM) [Ca2+], but did not change the maximal activity observed at 10 microM free [Ca2+]. Moreover, after a train of depolarizing stimuli, the secretory activity of antisense-injected cells dialyzed with 10 microM [Ca2+] was increased significantly compared with that of control cells. This result suggests that the activity of either Rab3a or its partners might change upon stimulation. We conclude that Rab3a, together with its partners, participates in the Ca2+ dependence of exocytosis and that its activity is modulated further in a stimulus-dependent manner. These findings should provide some clues to elucidate the role of Rab3a in synaptic plasticity.

The time course of D2 receptor expression assessed by the levels of the corresponding binding sites and mRNA was studied in rat striatum during ontogenesis and in primary cultures of cells taken at embryonic day (E) 17 and postnatal day (P) 4. In the two experimental situations, the amount of D2 receptor mRNA and number of binding sites increased regularly from E16 to P15, indicating that expression of D2 receptors in striatal neurons occurs independently from a dopaminergic input. Incubation of striatal primary cultures with 10(-5) M retinoic acid significantly increased the level of D2 receptor mRNA, whereas thyroid hormone, vitamin D3, and steroid hormones (estradiol, testosterone, and corticosterone) had no effect. The transcriptional activity of the rat D2 receptor gene promoter region, which bears a retinoic acid-responsive element, was increased by retinoic acid in transfected C6 glioma cells but not in transfected MMQ prolactin cells. Thyroid hormone and vitamin D3 were not effective in either cell line. Finally, mutations of the putative retinoic acid-responsive element inhibited the transcriptional effect of retinoic acid. These results suggest that retinoic acid is a key factor in regulation of the embryonic onset of the dopaminergic D2 receptor.

We report the isolation of a full-length eel tyrosine hydroxylase (TH) cDNA that is characterized by a long 3' untranslated region and by a diversity restricted to the 3' end owing to the differential use of three polyadenylation signals. The longest eel TH mRNA was distinctive in the presence of four pentameric elements (AUUUA) in the AU-rich 3' noncoding region. Such a diversity could provide the basis of posttranscriptional or translational regulation of eel TH gene expression. Comparison of the eel TH sequence with those of other aromatic amino acid hydroxylases (TH, tryptophan hydroxylase, and phenylalanine hydroxylase) and phylogenetic analysis confirmed that the N-terminal regulatory domain is highly divergent, contrasting with the conservation of the catalytic core of the enzyme. Molecular phylogenies including the available sequences of the three hydroxylase genes suggested that the duplication of their common ancestor occurred before the emergence of arthropods. The regional expression of the eel TH mRNA was studied by semiquantitative PCR, northern blots, and in situ hybridization and compared with the immunocytochemical localization of TH protein. The data showed that TH mRNA is mostly expressed in the olfactory and hypothalamic areas, whereas sparse TH-expressing cell bodies are present in the telencephalic region and brainstem. No labeling was detected in the mesencephalic area, in striking contrast with that found in amphibians and amniotes.

We have previously shown that the phosphorylated form of microtubule-associated protein 1B (MAP1B-P), which is located in growing axons during development and regeneration, remains detectable in the adult central nervous system only in areas that undergo morphologic plasticity (Nothias et al. [1996] J. Comp. Neurol. 368:317-334). Our objective in the present study was to determine whether lesion-induced axonal remodeling, in the adult rat, is associated with reinduction of MAP1B phosphorylation. MAP1B-P was not detectable in intact adult thalamic ventrobasal complex (VB), although low levels of MAP1B and its mRNA were present. A neuron-depletion of VB by in situ injection of kainic acid was followed by an induction of MAP1B phosphorylation by 24 hours postlesion. MAP1B-P was detected in fibers originating from undamaged neurons that were not located in the lesion, as demonstrated by the absence of hybridized MAP1B-mRNA. Ultrastructural analysis confirmed the exclusive location of MAP1B-P in axons in a proximodistal gradient. MAP1B phosphorylation appeared to be regulated by posttranslational modification of existing protein because the levels of MAP1B-mRNA did not change. The number of MAP1B-P-labeled fibers increased during the first month postlesion and remained high for a long period. Double staining by using axonal tracing with dextran-biotin and tyrosine hydroxylase immunohistochemistry, showed the presence of MAP1B-P in VB afferents from somatosensory relays and the locus coeruleus. This study supports the hypothesis that MAP1B, at a particular state of phosphorylation, is correlated with axonal remodeling in the adult central nervous system (CNS). We suggest that the interaction of MAP1B-P with microtubules allows the modulation of their dynamic properties during periods of increased axonal plasticity.

DARPP-32 is a cyclic adenosine monophosphate-regulated inhibitor of protein phosphatase 1, highly enriched in striatonigral neurons. Stimulation of dopamine D1 receptors increases phosphorylation of DARPP-32, whereas glutamate acting on N-methyl-D-aspartate receptors induces its dephosphorylation. Yet, to date, there is little direct evidence for the function of DARPP-32 in striatal neurons. Using a whole cell patch-clamp technique, we have studied the role of DARPP-32 in the regulation of voltage-gated sodium channels in rat striatal neurons maintained in primary culture. Injection of phospho-DARPP-32, but not of the unphosphorylated form, reduced the sodium current amplitude. This effect was similar to those induced by okadaic acid, with which there was no additivity and by tautomycin. Our results indicate that, in striatal neurons, sodium channels are under dynamic control by phosphorylation/dephosphorylation, and that phospho-DARPP-32 reduces sodium current by stabilizing a phosphorylated state of the channel or an associated regulatory protein. We propose that the DARPP-32-mediated modulation of sodium channels, via inhibition of phosphatase 1, contributes to the regulation of these channels by D1 receptors and other neurotransmitters which influence the state of phosphorylation of DARPP-32.

The evolving vertebrate nervous system was accompanied by major gene duplication events generating novel organs and a sympathetic system. Vertebrate neural pathways synthesizing catecholamine neurotransmitters (dopamine and noradrenaline), were subsequently recruited to process increased information demands by mediating psychomotor functions such as selective attention/predictive reward and emotional drive via the activation of multiple G-protein linked catecholamine receptor subtypes. Here we show that the evolution of these receptor-mediated events were similarly driven by forces of gene duplication, at the cephalochordate/vertebrate transition. In the cephalochordate Amphioxus, a sister group to vertebrates, a single catecholamine receptor gene was found, which based on molecular phylogeny and functional analysis formed a monophyletic group with both vertebrate dopamine D1 and beta adrenergic receptor classes. In addition, the presence of dopamine but not of noradrenaline was assayed in Amphioxus. In contrast, two distinct genes homologous to jawed vertebrate dopamine D1 and beta adrenergic receptor genes were extant in representatives of the earliest craniates, lamprey and hagfish, paralleling high dopamine and noradrenaline content throughout the brain. These data suggest that a D1/beta receptor gene duplication was required to elaborate novel catecholamine psychomotor adaptive responses and that a noradrenergic system specifically emerged at the origin of vertebrate evolution.

As a general philosophical framework, the author presents a theoretical position according to which 'opposition' is at the heart of the workings of the living being and the very origins of life. He then deals with 'opposing processes' and neural mechanisms which appear to underlie them. The theory rests on the premise that a great number of our actions are derived from our emotions, and that the coupling of pleasure/suffering controls the expression of our desires. Developed in the 1970s, this theory is founded on the observation of dependence phenomena (addictions), but its value extends far beyond drugs, to all types of human conduct, including sexuality, play, alimentary behaviours, etc. Inconsistent with classical theories, it subordinates the act to an affective state. The author finally examines the phenomenon of anxiety in the light of these considerations.

Detection of apoptotic neurons and microglial cells in the brains of human immunodeficiency virus type 1 (HIV-1)-infected patients has suggested that programmed cell death may be implicated in the physiopathology of HIV-1 encephalopathy. To analyze in vitro the intracellular signals induced by HIV-1 in human neurons and the associated neuronal death, we tested cultured human central nervous system (CNS) cells for apoptosis induced by HIV-1 and gp120 and for signaling pathways activated by gp120. HIV-1 and gp120 induced apoptosis of neurons and microglial cells but not of astrocytes or transformed microglial cells. Gp120 activated c-Jun N-terminal kinase (JNK) and p42 extracellular-regulated kinase (ERK) in primary CNS cells, with an early peak of activation at 2 to 5 minutes that was not present when pure microglial or astrocyte cultures were tested, followed by a late and sustained activation (10 and 60 minutes) in primary and enriched glial cell cultures as well as in transformed microglial cells. This demonstrates that gp120 could be an effector of HIV-1-induced apoptosis in the CNS and act directly on neuronal and glial cells.

Post-transcriptional modification of the neural cell adhesion molecule (NCAM) by polysialic acid significantly decreases NCAM adhesiveness and more generally modifies cell-cell interactions. Polysialic acid-NCAM (PSA-NCAM) is mainly expressed in the developing nervous system. In the adult, its expression is restricted to regions that retain morphological plasticity, such as the hypothalamo-neurohypophysial system during lactation in rats. Since cell-cell interactions and synaptic contacts in the hypothalamo-neurohypophysial system are greatly increased during lactation, we examined whether PSA-NCAM expression is modified during this period. Immunohistochemistry and immunoblotting showed that, compared with virgin rats, PSA-NCAM dramatically decreased during lactation in both the supraoptic nuclei and the neurohypophysis, and returned to its initial level only after weaning. This decrease was progressive and became significant only at the end of the first week of lactation. By contrast, modifications in the level of NCAM protein or changes in the splicing pattern of NCAM mRNAs could not be detected. The decline in polysialic acid on the NCAM molecule could strengthen membrane appositions, thereby stabilizing the newly established synapses and neurohaemal contacts in the hypothalamo-neurohypophysial system that accompany the increased neuronal activity that occurs during lactation. We also studied the regulation of the phosphorylated microtubule-associated protein-1B (MAP1B-P), whose distribution pattern largely overlaps with that of PSA-NCAM in the adult brain. Expression of MAP1B-P was greatly increased during lactation in the hypothalamic axons projecting into the neurohypophysis. Thus, the expression patterns of both PSA-NCAM and MAP1B-P may reflect the permanent structural plasticity characterizing the hypothalamo-neurohypophysial system in the adult.