State-dependent Regulation Research Articles

Neuropeptide Y is crucial for nutritional state-dependent regulation of maternal behavior.

Lactation is indispensable for the survival of mammalian pups. However, any excess of energy expenditure for lactation over energy intake threatens the mother's survival. Here, we report that an orexigenic molecule, neuropeptide Y (NPY), mediates nutritional state-dependent regulation of maternal behavior. After 9h of fasting, dams showed a dramatic decrease in the expression of maternal behavior. Intracerebroventricular or direct dorsal raphe nucleus (DRN) injection of NPY inhibited the expression of maternal behavior in non-fasted dams. In contrast, injection of the NPY Y1 receptor antagonist BIBP-3226 into the DRN, in which the expression of the Y1 receptor was confirmed in serotonergic (5-HT) and GABAergic interneurons, recovered the expression of maternal behavior in fasted dams. When the pups were presented, the increase in the number of c-Fos-positive GABAergic, but not serotonergic, neurons was smaller in fasted than in non-fasted dams. These results suggest that NPY may inhibit pup-induced activation of GABAergic neurons via the Y1 receptor. Injection of a 5-HT1A agonist, GABAA receptor antagonist, or GABAB receptor antagonist into the DRN induced incomplete maternal behavior in non-fasted dams. In contrast, each of a 5-HT2A receptor agonist or a GABAB receptor agonist, but not a GABAA receptor agonist, recovered separate components of maternal behavior in fasted dams. These results suggest that NPY inhibits both 5-HT neuronal activity and its modulation via the GABA receptor in the DRN, resulting in the suppression of maternal behavior under food-restricted conditions.

Kiss1 neurons drastically change their firing activity in accordance with the reproductive state: insights from a seasonal breeder.

Kisspeptin (Kiss) neurons show drastic changes in kisspeptin expression in response to the serum sex steroid concentration in various vertebrate species. Thus, according to the reproductive states, kisspeptin neurons are suggested to modulate various neuronal activities, including the regulation of GnRH neurons in mammals. However, despite their reproductive state-dependent regulation, there is no physiological analysis of kisspeptin neurons in seasonal breeders. Here we generated the first kiss1-enhanced green fluorescent protein transgenic line of a seasonal breeder, medaka, for histological and electrophysiological analyses using a whole-brain in vitro preparation in which most synaptic connections are intact. We found histologically that Kiss1 neurons in the nucleus ventralis tuberis (NVT) projected to the preoptic area, hypothalamus, pituitary, and ventral telencephalon. Therefore, NVT Kiss1 neurons may regulate various homeostatic functions and innate behaviors. Electrophysiological analyses revealed that they show various firing patterns, including bursting. Furthermore, we found that their firings are regulated by the resting membrane potential. However, bursting was not induced from the other firing patterns with a current injection, suggesting that it requires some chronic modulations of intrinsic properties such as channel expression. Finally, we found that NVT Kiss1 neurons drastically change their neuronal activities according to the reproductive state and the estradiol levels. Taken together with the previous reports, we here conclude that the breeding condition drastically alters the Kiss1 neuron activities in both gene expression and firing activities, the latter of which is strongly related to Kiss1 release, and the Kiss1 peptides regulate the activities of various neural circuits through their axonal projections.

Get Over It! A Multilevel Threshold Autoregressive Model for State-Dependent Affect Regulation.

Intensive longitudinal data provide rich information, which is best captured when specialized models are used in the analysis. One of these models is the multilevel autoregressive model, which psychologists have applied successfully to study affect regulation as well as alcohol use. A limitation of this model is that the autoregressive parameter is treated as a fixed, trait-like property of a person. We argue that the autoregressive parameter may be state-dependent, for example, if the strength of affect regulation depends on the intensity of affect experienced. To allow such intra-individual variation, we propose a multilevel threshold autoregressive model. Using simulations, we show that this model can be used to detect state-dependent regulation with adequate power and Type I error. The potential of the new modeling approach is illustrated with two empirical applications that extend the basic model to address additional substantive research questions.Electronic supplementary materialThe online version of this article (doi:10.1007/s11336-014-9417-x) contains supplementary material, which is available to authorized users.

Pirin is an iron-dependent redox regulator of NF-κB

Pirin is a nuclear nonheme Fe protein of unknown function present in all human tissues. Here we describe that pirin may act as a redox sensor for the nuclear factor κB (NF-κB) transcription factor, a critical mediator of intracellular signaling that has been linked to cellular responses to proinflammatory signals and controls the expression of a vast array of genes involved in immune and stress responses. Pirin's regulatory effect was tested with several metals and at different oxidations states, and our spectroscopic results show that only the ferric form of pirin substantially facilitates binding of NF-κB proteins to target κB genes, a finding that suggests that pirin performs a redox-sensing role in NF-κB regulation. The molecular mechanism of such a metal identity- and redox state-dependent regulation is revealed by our structural studies of pirin. The ferrous and ferric pirin proteins differ only by one electron, yet they have distinct conformations. The Fe center is shown to play an allosteric role on an R-shaped surface area that has two distinct conformations based on the identity and the formal redox state of the metal. We show that the R-shaped area composes the interface for pirin-NF-κB binding that is responsible for modulation of NF-κB's DNA-binding properties. The nonheme Fe protein pirin is proposed to serve as a reversible functional switch that enables NF-κB to respond to changes in the redox levels of the cell nucleus.

Thalamic Control of Layer 1 Circuits in Prefrontal Cortex

Knowledge of thalamocortical (TC) processing comes mainly from studying core thalamic systems that project to middle layers of primary sensory cortices. However, most thalamic relay neurons comprise a matrix of cells that are densest in the "nonspecific" thalamic nuclei and usually target layer 1 (L1) of multiple cortical areas. A longstanding hypothesis is that matrix TC systems are crucial for regulating neocortical excitability during changing behavioral states, yet we know almost nothing about the mechanisms of such regulation. It is also unclear whether synaptic and circuit mechanisms that are well established for core sensory TC systems apply to matrix TC systems. Here we describe studies of thalamic matrix influences on mouse prefrontal cortex using optogenetic and in vitro electrophysiology techniques. Channelrhodopsin-2 was expressed in midline and paralaminar (matrix) thalamic neurons, and their L1-projecting TC axons were activated optically. Contrary to conventional views, we found that matrix TC projections to L1 could transmit relatively strong, fast, high-fidelity synaptic signals. L1 TC projections preferentially drove inhibitory interneurons of L1, especially those of the late-spiking subtype, and often triggered feedforward inhibition in both L1 interneurons and pyramidal cells of L2/L3. Responses during repetitive stimulation were far more sustained for matrix than for core sensory TC pathways. Thus, matrix TC circuits appear to be specialized for robust transmission over relatively extended periods, consistent with the sort of persistent activation observed during working memory and potentially applicable to state-dependent regulation of excitability.

Origin‐specific epigenetic program correlates with vascular bed‐specific differences inRgs5expression

Cells from multiple origins contribute to vascular smooth muscle cell (VSMC) development. Phenotypic heterogeneity of VSMCs is associated with their point of developmental origin; however, the mechanisms driving such differences are unknown. We here examined the mechanisms controlling vascular bed-specific differences in Rgs5 expression during development. Rgs5 levels were similar across different regions of the vasculature in neonatal animals but were >15-fold higher in descending aortas compared with carotid arteries of adult mice. Thus, vessel bed-specific changes in regulation of Rgs5 expression occurred during vessel maturation. Examination of adult Rgs5-LacZ reporter mice revealed lower Rgs5 expression in VSMCs originating from the third (carotid artery) branchial arch compared with those originating in the fourth and sixth (aortic B segment, right subclavian, and ductus arteriosus) branchial arches. Indeed, a mosaic Rgs5 expression pattern, with discreet LacZ boundaries between VSMCs derived from different developmental origins, was observed. Furthermore, Rgs5-LacZ expression was correlated with the site of VSMC origin (splanchic mesoderm ≈ local mesenchyme > somites > proepicardium > mesothelium). Surprisingly, Rgs5 reporter activity in cultured carotid artery- and descending aorta-derived cells did not recapitulate the differences observed in vivo. Consistent with a developmental origin-specific epigenetic mechanism driving the observed expression differences in vivo, the Rgs5 promoter showed increased methylation on CpG dinucleotides in carotid arteries compared with that in descending aortas in adult but not in neonatal mice. In vitro methylation of the Rgs5 promoter confirmed that its activity is sensitive to transcriptional down-regulation by CpG methylation. These data suggest that an origin-dependent epigenetic program regulates vascular bed- and maturation state-dependent regulation of VSMC-specific gene transcription.

State-dependent Regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gating by a High Affinity Fe3+ Bridge between the Regulatory Domain and Cytoplasmic Loop 3

The unique regulatory (R) domain differentiates the human CFTR channel from other ATP-binding cassette transporters and exerts multiple effects on channel function. However, the underlying mechanisms are unclear. Here, an intracellular high affinity (2.3 × 10(-19) M) Fe(3+) bridge is reported as a novel approach to regulating channel gating. It inhibited CFTR activity by primarily reducing an open probability and an opening rate, and inhibition was reversed by EDTA and phenanthroline. His-950, His-954, Cys-832, His-775, and Asp-836 were found essential for inhibition and phosphorylated Ser-768 may enhance Fe(3+) binding. More importantly, inhibition by Fe(3+) was state-dependent. Sensitivity to Fe(3+) was reduced when the channel was locked in an open state by AMP-PNP. Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe(3+) no matter whether NBD2 was present or not. Therefore, although ATP binding-induced dimerization of NBD1-NBD2 is required for channel gating, regulation of CFTR activity by Fe(3+) may involve an interaction between the R domain and CL3. These findings may support proximity of the R domain to the cytoplasmic loops. They also suggest that Fe(3+) homeostasis may play a critical role in regulating pathophysiological CFTR activity because dysregulation of this protein causes cystic fibrosis, secretary diarrhea, and infertility.

State-dependent vs. central motor effects of ethanol on breathing

Ethanol, one of the most widely used drugs in Western society, worsens obstructive sleep apnea in humans. No studies, however, have distinguished between two primary mechanisms that could mediate suppression of genioglossus (GG) activity with ethanol. We test the hypothesis that ethanol suppresses GG activity by effects at the hypoglossal motor pool and/or by state-dependent regulation of motor activity via independent influences on sleep/arousal processes. Intraperitoneal injections of ethanol (1.25 g/kg, n = 6 rats) resulted in maximum blood levels of 125.5 +/- 15.8 mg/dl, i.e., physiologically relevant levels for producing behavioral impairment in rats and humans. Ethanol decreased wakefulness, reduced sleep latency, and increased non-rapid eye movement sleep (P < 0.001, n = 10 rats) and significantly reduced postural muscle tone and electroencephalogram frequencies, consistent with sedation. Ethanol also caused a state-dependent (wakefulness only) decrease in respiratory-related GG activity (P = 0.018) but did not affect diaphragm amplitude or rate, with the magnitude of GG decrease related to baseline activity (P < 0.0002). Ethanol did not alter GG activity when applied to the hypoglossal motor pool (0.025-1 M, n = 16 isoflurane-anesthetized rats). In conclusion, ethanol promoted sleep and altered electroencephalogram and postural motor activities, indicative of sedation. The lack of effect on GG with ethanol at the hypoglossal motor pool indicates that the GG and postural motor suppression following systemic administration was mediated via effects on state-dependent/arousal-related processes. These data show that ethanol can suppress GG by primary influences on state-dependent aspects of central nervous system function independent of effects on the respiratory network per se, a distinction that has not previously been identified experimentally.

State-dependent regulation of cortical activity by cortisol: An EEG study

In the present study we investigated the effects of cortisol administration on EEG activity in eight healthy volunteers. We administered 35 mg of cortisol in a within-subjects double-blind placebo-controlled design. Cortisol administration caused a global decrease in cortical activity except for an increase frontally at the left, resulting in a significant change in frontal asymmetry. This pattern of results is almost the exact mirror image of one of our previous studies. Comparing subjective activation measures from the present and previous study showed that activation was substantially higher in the present study that was performed in formal testing conditions involving venipuncture, compared to the previous EEG study that was performed in non-formal testing conditions. However, the direction of the present cortisolinduced change in frontal activity asymmetry is consistent with the cortisolinduced change in asymmetric rotation behavior that we recently reported in similar testing conditions. We also found indications that, in contrast to the effects on the EEG measures, effects of cortisol administration on subjective anxiety and plasma oxytocin levels may be sex-dependent. These results are preliminary because of the post-hoc nature and the small number of subjects in the present study. However, they are in line with recent findings by others, suggesting that the effects of cortisol on cortical activity and subjective activation are state dependent and are influenced by testing conditions.

State‐dependent regulation of sensory‐motor transmission: role of muscarinic receptors in sensory‐motor integration in the crayfish walking system

The aim of this study was to investigate a potential mechanism for state-dependent regulation of sensory-motor transmission from sensory afferents of a proprioceptor to motoneurons (MNs) in the walking system of the crayfish. This study was performed using an in vitro preparation of thoracic ganglia including motor nerves and the proprioceptor that codes movements of the second joint (coxo-basal chordotonal organ - CBCO) of the leg. Application of movements to the CBCO elicits resistance reflex responses intracellularly recorded from Dep MNs. This reflex response is enhanced when Dep MNs are depolarized either spontaneously or by current injection. This enhancement is abolished in the presence of scopolamine (an antagonist of muscarinic acetylcholine receptors). Using pharmacology, we demonstrate that the monosynaptic connection from CBCO sensory neurons to the Dep MNs includes both nicotinic and muscarinic components. In addition, the shape of monosynaptic excitatory postsynaptic potentials (EPSPs) depends on the membrane potential: at a subthreshold depolarizing membrane potential, the time constant of the falling phase of the EPSPs is significantly increased compared with its value at resting potential. This change is suppressed in the presence of scopolamine, indicating that the muscarinic component may contribute to the activation of the Dep MN pool by sensory activity. This state-dependent amplification of the sensory input may be important for increasing the strength of sensory feedback at times when central activation of the Dep MNs is very strong (e.g. during walking).

Differential behavioral state-dependence in the burst properties of CA3 and CA1 neurons

Brief bursts of fast high-frequency action potentials are a signature characteristic of CA3 and CA1 pyramidal neurons. Understanding the factors determining burst and single spiking is potentially significant for sensory representation, synaptic plasticity and epileptogenesis. A variety of models suggest distinct functional roles for burst discharge, and for specific characteristics of the burst in neural coding. However, little in vivo data demonstrate how often and under what conditions CA3 and CA1 actually exhibit burst and single spike discharges.The present study examined burst discharge and single spiking of CA3 and CA1 neurons across distinct behavioral states (awake-immobility and maze-running) in rats. In both CA3 and CA1 spike bursts accounted for less than 20% of all spike events. CA3 neurons exhibited more spikes per burst, greater spike frequency, larger amplitude spikes and more spike amplitude attenuation than CA1 neurons.A major finding of the present study is that the propensity of CA1 neurons to burst was affected by behavioral state, while the propensity of CA3 to burst was not. CA1 neurons exhibited fewer bursts during maze running compared with awake-immobility. In contrast, there were no differences in burst discharge of CA3 neurons. Neurons in both subregions exhibited smaller spike amplitude, fewer spikes per burst, longer inter-spike intervals and greater spike amplitude attenuation within a burst during awake-immobility compared with maze running. These findings demonstrate that the CA1 network is under greater behavioral state-dependent regulation than CA3. The present findings should inform both theoretic and computational models of CA3 and CA1 function.

Dynamin-dependent NMDAR endocytosis during LTD and its dependence on synaptic state

BackgroundThe N-methyl-D-aspartate (NMDA)-type glutamate receptor expressed at excitatory glutamatergic synapses is required for learning and memory and is critical for normal brain function. At a cellular level, this receptor plays a pivotal role in triggering and controlling synaptic plasticity. While it has been long recognized that this receptor plays a regulatory role, it was considered by many to be itself immune to synaptic activity-induced plasticity. More recently, we and others have shown that NMDA receptor-mediated synaptic responses can be subject to activity-dependent depression.ResultsHere we show that depression of synaptic transmission mediated by NMDA receptors displays a state-dependence in its plasticity; NMDA receptors are resistant to activity-induced changes at silent and recently-silent synapses. Once synapses transition to the active state however, NMDA receptors become fully 'plastic'. This state-dependence is identical to that shown by the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor. Furthermore, the down-regulation of NMDAR-mediated responses during synaptic depression is prevented by disruption of dynamin-dependent endocytosis.ConclusionNMDA receptor-mediated synaptic responses are plastic in a state-dependent manner. Depending on the plasticity state in which a synapse currently resides, NMDA receptors will either be available or unavailable for down-regulation. The mechanism underlying the down-regulation of NMDA receptor-mediated synaptic responses is endocytosis of the NMDA receptor. Other potential mechanisms, such as receptor diffusion along the plane of the membrane, or changes in the activity of the channel are not supported. The mechanisms of AMPA receptor and NMDA receptor endocytosis appear to be tightly coupled, as both are either available or unavailable for endocytosis in the same synaptic states. Endocytosis of NMDA receptors would serve as a potent mechanism for metaplasticity. Such state-dependent regulation of NMDAR endocytosis will provide fundamental control over downstream NMDA receptor-dependent plasticity of neuronal circuitry.

Characteristics and thyroid state-dependent regulation of iodothyronine deiodinases in pigs.

Three iodothyronine deiodinases (D1, D2, and D3) regulate local and systemic availability of thyroid hormone. D1 and D2 activate the prohormone T4 to the thyromimetic T3, and D3 inactivates T4 and T3 to rT3 and 3,3'-diiodothyronine, respectively. The expression of the three deiodinases is tightly regulated with regard to developmental stage and cell type to provide fine tuning of T3 supply to target cells. Most studies regarding distribution and regulation of deiodinases have been carried out in rodents. However, in different respects, rodents do not seem to be the optimal experimental model for human thyroid hormone physiology. For instance, D2 expression has been observed in human thyroid and skeletal muscle but not in these tissues in rodents. In this study, we have explored the pig as an alternative model. Porcine D1, D2, and D3 were cloned by RT-PCR, and their catalytic properties were shown to be virtually identical to those reported for human and rodent deiodinases. The tissue distribution of deiodinases was studied in normal pigs and in pigs made hypothyroid by methimazole treatment or in pigs made hyperthyroid by T4 treatment. D1 activity in liver and kidney was increased in T4-treated pigs. D2 activities in cerebrum and pituitary were decreased after T4 treatment and strongly increased after methimazole treatment. Remarkably, D2 activity in thyroid and skeletal muscle was induced in hypothyroid pigs. Significant expression of D3 was observed in cerebrum and was positively regulated by thyroid state. In conclusion, the pig appears to be a valuable model for human thyroid hormone physiology. The expression of D2 activity in thyroid and skeletal muscle is of particular interest for studies on the importance of this enzyme in (hypothyroid) humans.

Different neuronal phenotypes in the lateral hypothalamus and their role in sleep and wakefulness.

The sleep disorder narcolepsy is now linked with a loss of neurons containing the neuropeptide hypocretin (also known as orexin). The hypocretin neurons are located exclusively in the lateral hypothalamus, a brain region that has been implicated in arousal based on observations made by von Economo during the viral encephalitic epidemic of 1916-1926. There are other neuronal phenotypes located in the lateral hypothalamus that are distinct and separate from the hypocretin neurons. Here the authors identify these neurons based on peptides and neurotransmitters that they express and review roles of these neurons in sleep. Given the heterogeneity of the neuronal phenotypes in the lateral hypothalamus, it is likely that hypocretin neurons, as well as other types of neurons in the lateral hypothalamus, influence sleep and provide state-dependent regulation of physiological functions.

State-dependent regulation of cortical blood flow and respiration in hamsters: response to hypercapnia during arousal from hibernation

State-dependent regulation of cortical blood flow and respiration in hamsters: response to hypercapnia during arousal from hibernation

State-dependent regulation of cortical blood flow and respiration in hamsters: response to hypercapnia during arousal from hibernation.

Hibernation is characterised by a global reduction of metabolism, body temperature and blood flow, while arousal from hibernation is achieved by the reversal of these processes. Our experiments were performed on Syrian hamsters that had been chronically implanted with a cortical thermocouple and an optical fibre over the contralateral cortex, and acutely implanted with thermocouples in the rectal, cheek pouch and interscapular brown adipose tissue (BAT). Measurements revealed large thermal gradients in the body of the arousing animals. Maximum whole-body metabolic rate, which was 2.4 times normal cenothermic resting metabolic rate, coincided not with rectal temperature but more closely with respiratory rate (RR) or BAT temperature. Regional cortical blood flow (rCBF), as measured by laser-Doppler flowmetry, changed in parallel with whole-body metabolic rate, peaking at 3.8 times the normal cenothermic resting levels, when rectal temperature was 15 degrees C. When BAT temperature was less than 25 degrees C, RR, rCBF and heart rate (HR) were decreased by breathing hypercapnic gas, but these parameters were unresponsive to hyperoxic gases. At cenothermia the RR and rCBF of anaesthetised hamsters was increased by exposure to hypercapnic gases. Exposure to hyperoxic gas decreased RR but had no effect on rCBF. The mechanisms regulating rCBF, HR and RR exhibit state-dependent sensitivities to hypercapnic and hyperoxic stimuli. The large increase in rCBF observed during arousal implies that cerebral autoregulation is temporarily suspended and suggests that hamsters effectively use endogenous mechanisms to minimise the pathology normally associated with dramatic increases in rCBF.

Influence of Prematurity and Extrauterine Development on the Sleep State Dependent Heart Rate Patterns. Einfluss von Fruhgeburtlichkeit und extrauteriner Entwicklung auf schlafstadienabhangige Herzfrequenzveranderungen

Question of the Study Premature birth and subsequent extrauterine development may lead to changes in brainstem functions. Central nervous cardiorespiratory regulation, especially the interaction between respiratory and cardiovascular systems, appears to be an essential component of brainstem maturation. The question arises whether premature birth and subsequent extrauterine development may influence sleep state dependent cardiorespiratory regulation.

Growth-dependent Regulation of Mammalian Pyrimidine Biosynthesis by the Protein Kinase A and MAPK Signaling Cascades

The carbamoyl phosphate synthetase domain of the multifunctional protein CAD catalyzes the initial, rate-limiting step in mammalian de novo pyrimidine biosynthesis. In addition to allosteric regulation by the inhibitor UTP and the activator PRPP, the carbamoyl phosphate synthetase activity is controlled by mitogen-activated protein kinase (MAPK)- and protein kinase A (PKA)-mediated phosphorylation. MAPK phosphorylation, both in vivo and in vitro, increases sensitivity to PRPP and decreases sensitivity to the inhibitor UTP, whereas PKA phosphorylation reduces the response to both allosteric effectors. To elucidate the factors responsible for growth state-dependent regulation of pyrimidine biosynthesis, the activity of the de novo pyrimidine pathway, the MAPK and PKA activities, the phosphorylation state, and the allosteric regulation of CAD were measured as a function of growth state. As cells entered the exponential growth phase, there was an 8-fold increase in pyrimidine biosynthesis that was accompanied by a 40-fold increase in MAPK activity and a 4-fold increase in CAD threonine phosphorylation. PRPP activation increased to 21-fold, and UTP became a modest activator. These changes were reversed when the cultures approach confluence and growth ceases. Moreover, CAD phosphoserine, a measure of PKA phosphorylation, increased 2-fold in confluent cells. These results are consistent with the activation of CAD by MAPK during periods of rapid growth and its down-regulation in confluent cells associated with decreased MAPK phosphorylation and a concomitant increase in PKA phosphorylation. A scheme is proposed that could account for growth-dependent regulation of pyrimidine biosynthesis based on the sequential action of MAPK and PKA on the carbamoyl phosphate synthetase activity of CAD.

Growth state-dependent regulation of plasminogen activator inhibitor type-1 gene expression during epithelial cell stimulation by serum and transforming growth factor-beta1.

Transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene appears to be growth state regulated in several cell types (e.g. , Ryan and Higgins, 1993, J Cell Physiol 155:376-384; Mu et al., 1998, J Cell Physiol 174:90-98). Transit of serum-stimulated normal rat kidney (NRK) epthelial cells through the first division cycle after release from quiescence (G(0)) provided a model system to assess the kinetics and mechanisms underlying PAI-1 expression in a growth "activated" phenotype. PAI-1 mRNA transcripts increased by more than 20-fold during the G(0)-->G(1) transition; induced expression had immediate-early response characteristics and abruptly declined prior to the onset of DNA synthesis. Transcriptional activity of the PAI-1 gene paralleled the steady-state mRNA abundance profile during this first synchronized growth cycle after release from quiescence. Although PAI-1 mRNA levels were up-regulated (approximately threefold) upon exposure to several different growth factors, neutralizing antibodies to transforming growth factor-beta1 (TGF-beta1) effectively attenuated the more than ninefold serum-associated PAI-1 inductive response by more than 70% (at both the mRNA transcript and protein levels). Similar to the metabolic requirements for serum-mediated PAI-1 transcription, PAI-1 induction upon addition of TGF-beta1 to quiescent NRK cell cultures was actinomycin D sensitive and resistant to cyclohexamide and puromycin, suggesting a primary mode of transcript control. The response to protein synthesis inhibitors, however, was complex. While cyclohexamide appeared to stabilize, or at least maintain, fetal bovine serum (FBS)- or TGF-beta1-stimulated PAI-1 mRNA levels, puromycin had no such affect. The amplitude and duration of induced PAI-1 expression were the same in either the presence or absence of puromycin. Cyclohexamide when used alone (i.e., in non-FBS- or TGF-beta1-treated cultures), moreover, effectively stimulated PAI-1 induction whereas puromycin was ineffective. Although TGF-beta1 was not a complete mitogen in the NRK cell system, incubation of quiescent renal cell cultures with TGF-beta1, prior to serum stimulation, resulted in a 10- to 12-fold increase in PAI-1 expression coincident with exit out of G(0). These data support a model in which PAI-1 gene expression is closely associated with creation of the growth-activated state and that cell cycle controls appear to be superimposed on the time course of the serum-induced expression of the PAI-1 gene.

S-8-1 - State-dependent regulation of neuronal excitability by dopamine

Since the discovery that the loss of the dopaminergic innervation of the striatum resulted in Parkinson's disease, physiologists have attempted to understand the role of dopamine on striatal activity. Hypotheses relying upon concepts derived from studies of fast synaptic transmission have consistently failed to explain the actions of dopamine or other receptors coupled to G-proteins which modulate the properties of voltage-dependent ionic conductances responsible for synaptic integration and spike activity. Recently, patch clamp studies have revealed that in medium spiny striatal neurons dopamine D1-class receptors modulate voltage-dependent Na+, K+ and Ca2+ channels. From a consideration of the biophysical properties of these channels and the state transitions that medium spiny neurons undergo while responding to cortical input, a novel picture of dopamine's actions is beginning to emerge. Our results and those of others suggest that D2-class receptors serve to make the transition to the depolarized 'upstate' from the hyperpolarized 'downstate' more probable in response to cortical input. But, once the transition has occurred, the alteration in excitability should be short-lived unless the neuron has recently been active. This state-dependent modulation provides a mechanism by which dopamine could shape global striatal activity governing the execution of motor behaviors.