Attractive action of FGF-signaling contributes to the postnatal developing hippocampus.

During brain development neural cell migration is a crucial, well-orchestrated, process, which leads to the proper whole brain structural organization. As development proceeds, new neurons are continuously produced, and this protracted neurogenesis is maintained throughout life in specialized germinative areas inside the telencephalon: the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus. In the anterior SVZ, newly generated neurons migrate through long distances, along the rostral migratory stream (RMS), before reaching their final destinations in the olfactory bulb (OB). Intriguingly, recent observations pointed out the existence of other postnatal tangential routes of migration alternative to the RMS but still starting from the SVZ. The presence of such dynamic and heterogeneous cell movements contributes to important features in the postnatal brain such as neural cell replacement and plasticity in cortical regions. In this work, we asked whether a caudal migratory pathway starting from the caudal SVZ continues through life. Strikingly, in vivo analysis of this caudal migration revealed the presence of a postnatal contribution of SVZ to the hippocampus. In vitro studies of the caudal migratory stream revealed the role of FGF signaling in attracting caudally the migrating neuroblasts during postnatal stages. Our findings demonstrate a postnatal neuronal contribution from the caudal ganglionic eminence (CGE) CGE-SVZ to the hippocampus through an FGF-dependent migrating mechanism. All together our data emphasizes the emerging idea that a developmental program is still operating in discrete domains of the postnatal brain and may contribute to the regulation of neural cell replacement processes in physiological plasticity and/or pathological circumstances.

Postsynaptic membrane fusion and long-term potentiation.

The possibility that membrane fusion events in the postsynaptic cell may be required for the change in synaptic strength resulting from long-term potentiation (LTP) was examined. Introducing substances into the postsynaptic cell that block membrane fusion at a number of different steps reduced LTP. Introducing SNAP, a protein that promotes membrane fusion, into cells enhanced synaptic transmission, and this enhancement was significantly less when generated in synapses that expressed LTP. Thus, postsynaptic fusion events, which could be involved either in retrograde signaling or in regulating postsynaptic receptor function or both, contribute to LTP.

Differential G protein-mediated coupling of D2 dopamine receptors to K+ and Ca2+ currents in rat anterior pituitary cells.

In anterior pituitary cells, dopamine, acting on D2 dopamine receptors, concomitantly reduces calcium currents and increases potassium currents. These dopamine effects require the presence of intracellular GTP and are blocked by pretreatment of the cells with pertussis toxin, suggesting that one or more G protein is involved. To identify the G proteins involved in coupling D2 receptors to these currents, we performed patch-clamp recordings in the whole-cell configuration using pipettes containing affinity-purified polyclonal antibodies raised against either Go alpha, Gi3 alpha, or Gi1,2 alpha. Dialysis with Go alpha antiserum significantly reduced the inhibition of calcium currents induced by dopamine, while increase of potassium currents was markedly attenuated only by Gi3 alpha antiserum. We therefore conclude that in pituitary cells, two different G proteins are involved in the signal transduction mechanism that links D2 receptor activation to a specific modulation of the four types of ionic channels studied here.

Stable transfection of calbindin-D28k into the GH3 cell line alters calcium currents and intracellular calcium homeostasis.

Previous work demonstrating the presence and differential distribution of Ca(2+)-binding proteins in the CNS has led to the proposal that cytosolic proteins, such as calbindin-D28k (CB), may play a pivotal role in neurons. We have used a retrovirus containing the full-length cDNA for CB to transfect the pituitary tumor cell line GH3, to generate CB-expressing GH3 cells and to investigate whether ionic channel activities as well as the concentration of intracellular free Ca2+ ([Ca2+]i) homeostasis could be altered by the presence of this Ca(2+)-binding protein. We show that CB-transfected GH3 cells exhibited lower Ca2+ entry through voltage-dependent Ca2+ channels and were better able to reduce [Ca2+]i transients evoked by voltage depolarizations than the wild-type parent cell line. These observations provide a mechanism by which CB may protect tissues against Ca(2+)-mediated excitotoxicity.

Rab3 proteins: key players in the control of exocytosis.

Although some mechanistic aspects of exocytosis, such as fusion events, have been well documented by the technique of time-resolved membrane-capacitance measurement, it was only recently that new insights into the molecular mechanisms involved in the traffic of secretory vesicles were provided by the convergence of different lines of research. In this review Lledo et al. present some of the recent findings concerning small GTPases of the Rab3 subfamily which regulate hormone release, triggered by entry of Ca2+, in endocrine and neuroendocrine cells. In view of these new results, Rab proteins might be considered as candidates for inhibition or stimulation of specific steps involved in vesicle traffic.

Control of action potential timing by intrinsic subthreshold oscillations in olfactory bulb output neurons.

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.

Multiple and opposing roles of cholinergic transmission in the main olfactory bulb.

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.

Rab3A and Rab3B carboxy-terminal peptides are both potent and specific inhibitors of prolactin release by rat cultured anterior pituitary cells.

Chimeric polypeptides composed of the homeodomain of Antennapedia and of the C-terminus of several low molecular weight GTP-binding proteins of the rab family have been found to translocate through the membrane of cells in culture and to accumulate in the cytoplasm and nucleus. We have used these chimeric peptides to study, in intact endocrine cells, a putative role for the C-terminal domain of rab proteins in the exocytotic process. We show that the internalization of 33- and 32-amino acid polypeptides corresponding to the C-terminal domains of rab3A and rab3B blocks calcium-triggered PRL release from adult rat anterior pituitary cells maintained in primary culture. This effect is specific to rab3 since it is not observed after internalization of either rab1 or rab2 C-terminal peptides. In addition, we demonstrate that this inhibition does not require the geranylgeranylation of the internalized C-termini. As rab3B normally shows a permissive effect on exocytosis in PRL-secreting cells, we suggest that the C-terminal domains of rab3A and rab3B contain structural elements that compete with endogenous rab3 necessary for calcium-induced exocytosis.

Dialysis of lactotropes with antisense oligonucleotides assigns guanine nucleotide binding protein subtypes to their channel effectors.

This article describes a new approach for determining the role of endogenous guanine nucleotide binding (G) protein subunits in signal transduction. Sequential patch-clamping was applied to BSA gradient-enriched cultured lactotropes from lactating rats, first to dialyze antisense oligodeoxyribonucleotides (AS) directed against G alpha protein mRNAs and 48 h later to record ion-current responses to the PRL release inhibitor, dopamine. The effectiveness and specificity of action of six types of AS were determined by their effects on the in vitro translation of alpha o, alpha i1, alpha i2, alpha i3, and alpha s. The specificity of AS could be enhanced by replacing guanine by cytosine bases within the center core of AS and by maximizing the number of mismatches against nontargeted mRNAs within the extremities of AS. A total of 59 out of 240 cells could be investigated using the sequential patch clamp procedure in the absence of antibiotics. The typical decrease of the voltage-activated calcium current in response to 10 nM dopamine was diminished or abolished by AS, in correlation with the inhibition of in vitro translation of the alpha o subunit. The typical increase of the voltage-activated potassium current in response to dopamine was abolished by AS directed against alpha i3 but not alpha o mRNA. Control experiments showed that culture conditions or loss of receptor affinity for dopamine were not responsible for the loss of response. The results suggest that dopamine D2 receptors are linked via alpha o to calcium channels and via alpha i3 to potassium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of ultrasonic vocalizations to assess olfactory detection in mouse pups treated with 3-methylindole.

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.

Dopamine inhibits two characterized voltage-dependent calcium currents in identified rat lactotroph cells.

The effects of dopamine (DA) on voltage-dependent Ca2+ currents were investigated in cultured rat lactotroph cells using the patch clamp recording technique. Each recorded cell was identified by the reverse hemolytic plaque assay. In the whole-cell configuration, two types of Ca2+ currents, L and T, were characterized on the basis of their kinetics, voltage sensitivity, and pharmacology. The L component had a threshold of -25 mV, showed little inactivation during a 150-msec voltage step, and was maximal at +10 mV. Cadmium ions (100 microM) significantly reduced its amplitude (75%). The T component was activated at a membrane potential close to -50 mV, was maximal at -10 mV, and showed a voltage-dependent inactivation between -90 and -30 mV. It was quickly inactivated during a maintained depolarization (time constant, 27 ms at -30 mV) and was strongly reduced (80%) by nickel ions (100 microM). Bath application of DA (10 nM) caused a markedly general depression of inward Ca2+ currents, acting differently on the T- and L-type currents. DA application shifted the voltage-dependence of the L-type current activation toward depolarization values (8 mV) without modifying its time- and voltage-dependent inactivation. In contrast, DA enhanced the inactivation of the T-type current by accelerating its time-dependent inactivation (25% decrease in the time constant of inactivation) and by shifting the voltage-dependence of the T-type current inactivation toward hyperpolarizing values (-63 mV in control vs. -77 mV in the presence of DA). These effects of DA were dose-dependent and involved the activation of a D2 receptor type. They were mimicked by bromocriptine application (10 nM), whereas sulpiride (100 nM) blocked the DA-evoked response. The D1 antagonist SCH 23390 was ineffective up to 100 microM. All of these DA-induced modifications in Ca2+ currents were abolished using a GTP-free pipette solution or after pretreatment of cells with pertussis toxin, suggesting that DA can regulate the function of Ca2+ channels through GTP-binding proteins (G-proteins). Our results show that DA acts simultaneously by reducing both voltage-dependent Ca2+ currents on lactotroph cells. Thus, DA reduces the entry of Ca2+ ions across the surface membrane and thereby influences electrical activity and the cytosolic free Ca2+ concentration involved in both basal and evoked PRL release.

Reduction of dopamine D2 receptor transduction by activation of adenosine A2a receptors in stably A2a/D2 (long-form) receptor co-transfected mouse fibroblast cell lines: studies on intracellular calcium levels.

A stably co-transfected mouse fibroblast cell line, which expresses the long form of the human dopamine D2 receptor and the dog adenosine A2a receptor, was used to analyse the mechanism underlying changes in the cytosolic free calcium concentration ([Ca2+]i) induced by activation of D2 (long-form) receptors and its modulation by the A2a receptor agonist CGS 21680 by means of fura-2 imaging. Quinpirole (1-1000 nM), a D2 receptor agonist, caused a concentration-dependent increase in [Ca2+]i. Haloperidol, a preferential D2 receptor antagonist, completely blocked this [Ca2+]i response to quinpirole. Preincubation with Ca(2+)-free medium containing 2 mM EGTA or a medium containing 320 mM ethanol, an inositol 1,4,5-triphosphate receptor antagonist, substantially diminished the increase in [Ca2+]i evoked by quinpirole. Furthermore, quinpirole totally failed to elevate [Ca2+]i in a medium containing both 2 mM EGTA and 320 mM ethanol. CGS 21680 (1-500 nM) did not, by itself, exert any significant effect on [Ca2+]i. However, 100 nM of CGS 21680 substantially counteracted the [Ca2+]i responses to quinpirole (10-1000 nM). Moreover, this counteraction still occurred after blocking Ca2+ mobilization from intracellular stores with ethanol, but disappeared when the cells were pretreated with the Ca(2+)-free medium containing 2 mM EGTA. Our findings imply that the D2 (long-form) receptors in the present fibroblast cell line can raise [Ca2+]i both via Ca2+ influx from the extracellular medium and Ca2+ release from intracellular stores via activation of inositol 1,4,5-triphosphate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Exocytosis in excitable cells: a conserved molecular machinery from yeast to neuron.

One of the basic cellular functions of nearly every cell type is the exocytotic release of synthesized molecules, stored and packaged into intracellular vesicles or granules. A variety of approaches has been used to identify and characterize the molecules that mediate vesicular trafficking along the secretory pathway. The findings obtained with these approaches suggest that common mechanisms may underlie a wide variety of vesicle-mediated transport steps. This review presents some of the recent findings regarding the study of the cellular mechanisms which control neurotransmitter and hormone release from neurons and endocrine cells respectively, and focuses on regulation of these mechanisms. The similarities between these two cell types can be seen as evidence to support the hypothesis according to which the regulated exocytosis apparatus could have evolved from a constitutive fusion machinery to which some key modulators have been added. Insight into secretory vesicles will be relevant not only to the understanding of vesicular trafficking or cell polarity but also to the understanding of higher nervous functions resulting from synaptic plasticity.

Chronic stimulation of D2 dopamine receptors specifically inhibits calcium but not potassium currents in rat lactotrophs.

The present study examines the effect of chronic dopamine treatment, known to inhibit prolactin release from anterior pituitary, on two Ca2+ and K+ currents in cultured rat lactotrophs. K+ and Ca2+ currents were recorded using the whole-cell mode of the patch-clamp technique. The two types of voltage-dependent Ca2+ currents are called SD and FD (slowly deactivating and fast deactivating current component, respectively) and the two types of voltage-dependent K+ currents, IA and IK. All current types were isolated by tail current analysis. The amplitude of both normalized calcium components depended on the length of the culture (n = 48) while normalized amplitudes of both potassium currents remained constant (n = 9). Incubation of cells during 72 h with 50 microM of Actinomycin D, an inhibitor of mRNA synthesis, suggested that this increase in Ca2+ currents involved the synthesis of proteins. Long-lasting D2 receptor stimulation (8 days; 10 nM RU 24213) prevented this selective effect through activation of a pertussis toxin-sensitive G protein. We also examined whether cyclic adenosine-3',5'-cyclic-monophosphate (cyclic AMP) or Ca2+/phospholipid-dependent protein kinase (protein kinase C) could affect this development of channel activity.(ABSTRACT TRUNCATED AT 250 WORDS)

A guanine nucleotide-binding protein mediates the inhibition of voltage-dependent calcium currents by dopamine in rat lactotrophs.

The present study examines the effect of dopamine (DA), known to inhibit prolactin (PRL) release, on voltage-activated calcium currents in identified rat lactotrophs. Two types of voltage-dependent Ca2+ currents were recorded using the whole-cell mode of the patch-clamp technique. Both were reversibly inhibited by DA application. The inhibitory action of DA was reduced by (i) sulpiride (D2 antagonist), (ii) preincubation of the cells with pertussis toxin (PTX), and (iii) inclusion of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) in the pipette solution, whereas it was potentiated by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). This DA-induced response could not be overcome by changing the adenosine 3',5'-cyclic monophosphate level. These findings suggest that DA can inhibit Ca2+ entry through voltage-activated Ca2+ channels via a PTX-sensitive G protein(s) pathway thereby affecting PRL release from rat lactotrophs.

PSA-NCAM: an important regulator of hippocampal plasticity.

The Neural Cell Adhesion Molecule (NCAM) serves as a temporally and spatially regulated modulator of a variety of cell-cell interactions. This review summarizes recent results of studies aimed at understanding its regulation of expression and biological function, thereby focussing on its polysialylated isoforms (PSA-NCAM). The detailed analysis of the expression of PSA and NCAM in the hippocampal mossy fiber system and the morphological consequences of PSA-NCAM deficiency in mice support the notion that the levels of expression of NCAM are important not only for the regulation and maintenance of structural changes, such as migration, axonal growth and fasciculation, but also for activity-induced plasticity. There is evidence that PSA-NCAM can specifically contribute to a presynaptic form of plasticity, namely long-term potentiation at hippocampal mossy fiber synapses. This is consistent with previous observations that NCAM-deficient mice show deficits in spatial learning and exploratory behavior. Furthermore, our data points to an important role of the hypothalamic-pituitary-adrenal axis, which is the principle adaptive response of the organism to environmental challenges, in the control of PSA-NCAM expression in the hippocampal formation. In particular, we evidence an inhibitory influence of corticosterone on PSA-NCAM expression.

[Neuronal connectivity and chemical mediators involved in olfactory message transmission]. / Connectivité neuronale et médiateurs chimiques impliqués dans la transmission du message olfactif.

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.

[Experimental study of mechanisms of neuronal death in the course oh HIV infection]. / Etude expérimentale des mécanismes de la mort neuronale au cours de l'infection à VIH.

The HIV1 virus and its envelope glycoprotein gp120 are toxic for human neurones in vitro. This neurotoxicity is, at least partially, of an apoptotic nature, resulting from the interaction of gp120 with the neuronal membrane which leads to perturbations of intracellular signaling systems. These latter bring about on the one hand a raising of [Ca2+]i partly due to the potentiation of the NMDA receptor response to endogenous glutamate and on the other hand the activation of certain MAP kinases (ERK and JNK) which lead to the initiation of the cell death program.

Dynamics of olfactory and hippocampal neurogenesis in adult sheep.

Although adult neurogenesis has been conserved in higher vertebrates such as primates and humans, timing of generation, migration, and differentiation of new neurons appears to differ from that in rodents. Sheep could represent an alternative model to studying neurogenesis in primates because they possess a brain as large as a macaque monkey and have a similar life span. By using a marker of cell division, bromodeoxyuridine (BrdU), in combination with several markers, the maturation time of newborn cells in the dentate gyrus (DG) and the main olfactory bulb (MOB) was determined in sheep. In addition, to establish the origin of adult-born neurons in the MOB, an adeno-associated virus that infects neural cells in the ovine brain was injected into the subventricular zone (SVZ). A migratory stream was indicated from the SVZ up to the MOB, consisting of neuroblasts that formed chain-like structures. Results also showed a long neuronal maturation time in both the DG and the MOB, similar to that in primates. The first new neurons were observed at 1 month in the DG and at 3 months in the MOB after BrdU injections. Thus, maturation of adult-born cells in both the DG and the MOB is much longer than that in rodents and resembles that in nonhuman primates. This study points out the importance of studying the features of adult neurogenesis in models other than rodents, especially for translational research for human cellular therapy.

Adult neurogenesis in the olfactory system and neurodegenerative disease.

The olfactory system is unique in many respects-two of which include the process of adult neurogenesis which continually supplies it with newborn neurons, and the fact that neurodegenerative diseases are often accompanied by a loss of smell. A link between these two phenomena has been hypothesized, but recent evidence for the lack of robust adult neurogenesis in the human olfactory system calls into question this hypothesis. Nevertheless, model organisms continue to play a critical role in the exploration of neurodegenerative disease. In part one of this review we discuss the most promising recent technological advancements for studying adult neurogenesis in the murine olfactory system. Part two continues by looking at emerging evidence related to adult neurogenesis in neurodegenerative disease studied in model organisms and the differences between animal and human olfactory system adult neurogenesis. Hopefully, the careful application of advanced research methods to the study of neurodegenerative disease in model organisms, while taking into account the recently reported differences between the human and model organism olfactory system, will lead to a better understanding of the reasons for the susceptibility of olfaction to disease.