Substance P: evidence for diverse roles in neuronal function from cultured mouse spinal neurons.

Mouse spinal neurons grown in tissue culture were used to examine the membrane mechanisms of action of the peptide substance P. Two functionally distinct actions were observed, one being a rapidly desensitizing excitation, and the other being a dose-dependent, reversible depression of excitatory responses to the putative amino acid neurotransmitter glutamate. These effects on excitability suggest that substance P may play more than one role in intercellular communication in the nervous system.

Firing patterns of hypothalamic supraoptic neurons during water deprivation in monkeys.

Water deprivation in monkeys caused an acceleration of action potential firing of supraoptic neurons, but not of neurons located 2 to 3 millimeters above the hypothalamic supraoptic nucleus. Whereas in the normally hydrated animal only 12 percent of the neuroendocrine cells discharged periodically, the proportion of these periodic bursters increased markedly with increasing plasma osmolarity. This finding suggests that such periodically firing supraoptic neurons are those engaged in active neurohypophyseal hormone secretion.

Immunocytochemically identified vasopressin neurons in culture show slow, calcium-dependent electrical responses.

From morphological characterization and intracellular recordings, monolayer cultures derived from fetal mouse hypothalami were found to include functionally differentiated peptide neurons, a number of which appear to contain vasopressin. These cells exhibited particular patterns of slow, calcium-dependent membrane depolarizations, resembling in their periodicity and duration the phasic activity of vasopressin neurons recorded extracellularly in vivo.

Membrane effects of thyrotropin-releasing hormone and estrogen shown by intracellular recording from pituitary cells.

The effects of thyrotropin-releasing hormone and 17 beta-estradiol on the electrical membrane properties of a prolactin-secretin pituitary cell line (GH3/B6) were studied with intracellular microelectrode recordings. Of the cells tested, 50 percent were excitable and displayed calcium-dependent action potentials when depolarized. When injected directly on the membrane of an excitable cell, thyrotropin-releasing hormone and 17 beta-estradiol induced action potentials within 1 minute. The spiking activity was preceded by a progressive increase of the input resistance without any detectable change in the resting membrane polarization. The results reveal a rapid effect of both substances on the membrane of GH3/B6 cells. In the case of thyrotropin-releasing hormone, which has both a short-term effect on release of prolactin and a long-term effect on its synthesis, the induced electrical activity may be associated with the stimulation of prolactin production. The physiological implication of 17 beta-estradiol-induced, calcium-dependent spiking activity remains to be elucidated.

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.

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.

Hormonal control of neural function in the adult brain.

Although the mechanisms by which peripheral hormones modulate complex behaviors are far from being well understood, recent advances in deciphering the mechanisms of hormone action in the brain are promising. Current areas of interest include the molecular mechanisms of steroid receptor action, the steroid modulation of synaptic function, and the mediation of steroid-regulated neuronal and glial plasticity by growth factors or proteins associated with brain development.

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.

Morphofunctional plasticity in the adult hypothalamus induces regulation of polysialic acid-neural cell adhesion molecule through changing activity and expression levels of polysialyltransferases.

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.

An evolutionary view of drug-receptor interaction: the bioamine receptor family.

The large molecular diversity of receptors and their subtypes means that the pharmacologist is faced with many puzzling characterization questions. First, the molecular diversity of the receptors is deciphered only in part by a pharmacological approach, which precludes a satisfactory receptor classification based solely on pharmacological characteristics. Second, the physiological counterpart of the numerous subtypes of receptors specifically activated by single endogenous ligands remains unclear. Here, Philippe Vernier and colleagues use the example of the bioamine G protein-coupled receptors to show that many of the apparent inconsistencies that emerge from pharmacological and molecular characterizations of receptors can be better understood if the evolutionary history of the receptors is taken into account.

Ion channels and the signal transduction pathways in the regulation of growth hormone secretion.

The secretion of GH from pituitary somatotrophs is mainly regulated by alterations in the levels of intracellular free Ca(2+) concentrations ([Ca(2+)](i)) that depend on the influx of Ca(2+) through voltage-gated Ca(2+) channels in the cell membrane. Hypothalamic stimulatory and inhibitory factors bind to specific receptors on the cell membrane to regulate membrane potential and activate second-messenger systems. The receptors are G-protein coupled, and activated G proteins directly influence membrane ion channels to regulate Ca(2+) influx. The function of cAMP-dependent protein kinase A is also modulated by receptor-coupled G proteins leading to the phosphorylation of Ca(2+) channel proteins and further alteration of Ca(2+) influx.

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)

Differential subcellular distribution and transcriptional activity of sigmaE3, sigmaE4, and sigmaE3-4 isoforms of the rat estrogen receptor-alpha.

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.

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.

Alternative splicing of the D2 dopamine receptor messenger ribonucleic acid is modulated by activated sex steroid receptors in the MMQ prolactin cell line.

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.

The electrical properties of isolated human prolactin-secreting adenoma cells and their modification by dopamine.

Human prolactinoma cells were maintained in culture for a period of at least 8 days and were able to secrete PRL in large amounts. This secretion was inhibited by bromocriptine, an agonist of dopaminergic receptors, in a dose-dependent manner. The cells showed electrical activity (action potentials) which was blocked by inhibitors of calcium current (cobalt, manganese), whereas it was insensitive to blockers of sodium current (tetrodotoxin). At the resting potential of the cell, dopamine induced a hyperpolarizing response such that action potentials no longer occurred. This effect was due to increase of the membrane conductance and depended on the cell potential. The reversal potential of this response was at -100 mV, which suggests the involvement of potassium ions. Bromocriptine and RU 24213, which are strong dopaminergic receptor agonists, both induced responses identical to the dopamine-induced response. The D2 receptor antagonists (haloperidol, domperidone, and spiperone) blocked the dopamine-induced response in a reversible manner. The D1 antagonist of dopaminergic receptors flupentixol had no effect on the dopamine response. It is concluded that the dopamine modulation of electrical activity involving calcium current may be an early important step in the mechanism by which dopamine inhibits PRL release.

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.

Stage- and region-specific expression of estrogen receptor alpha isoforms during ontogeny of the pituitary gland.

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.