Excitation of locus coeruleus neurons by vasoactive intestinal peptide: evidence for a G-protein-mediated inward current

Vasoactive intestinal polypeptide (VIP) caused a reversible increase in the firing rate of locus coeruleus (LC) neurons. Voltage-clamp at −60 mV revealed that VIP induced an inward current, associated with a small increase in conductance. The inward current persisted in the presence of Co²⁺ (to block Ca²⁺ channels) or tetrodotoxin (to block fast voltage-dependent Na⁺ channels). Substitution (80%) of Na⁺ with choline or Tris reduced the VIP-elicited inward current by approximately 75%. Changing external K⁺ concentrations did not alter the effect of VIP. The inward current induced by VIP became irreversible after the intracellular administration of GTPγS, a hydrolysis-resistant analog of GTP which can cause a prolonged activation of G-proteins. The intracellular application of GDPβS, which can interfere with G-protein activation, attenuated the effect of VIP. Pertussis toxin, an inactivator of certain G-proteins, did not block the effect of VIP. We conclude that VIP directly excites LC neurons by inducing a largely Na-dependent inward current. As this effect became irreversible in the presence of intracellular GTPγS, was attenuated by GDPβS, and was not eliminated by pertussis toxin, mediation through a pertussis toxin-insensitive G-protein is suggested.     

Reduction by sevoflurane of adenosine 5′-triphosphate-activated inward current of locus coeruleus neurons in pontine slices of rats

Increasing evidence indicates that volatile general anesthetics exert their effects by affecting various types of membrane conductance expressed in the central nervous system (CNS), such as ligand-gated receptor-channels. The most recently identified family of the receptor-channels in the CNS are the extracellular ATP-gated channels (P2X purinoceptors). In the present study, we tested whether volatile anesthetics can affect P2X receptor function in the CNS network. We recorded whole-cell currents of locus coeruleus (LC) neurons in pontine slices from young rats. Adenosine 5′-triphosphate (ATP) sodium (0.03–3 mM) evoked a rapidly rising and moderately desensitizing inward current (50–200 pA) in a dose-dependent manner in LC neurons at a holding potential of −80 mV. Perfusion with clinically relevant concentration of sevoflurane (0.1–0.5 mM) reduced the ATP-induced inward current in a dose-dependent manner (to 56.8±5.9% of control with 0.5 mM sevoflurane; mean±S.E.M., n=13). Estimated IC₅₀ of sevoflurane was 0.59 mM. We conclude that the attenuation of extracellular ATP-mediated signaling in the central nervous system might be one of the multiple actions of volatile anesthetics.     

Adenosine inhibits locus coeruleus neurons: An intracellular study in a rat brain slice preparation

Intracellular recording was used to study the effect of adenosine (3-100 microM) on rat locus coeruleus (LC) neurons in a brain slice preparation. Bath application of adenosine (100 microM) reduced the rate of spontaneous firing in 88% of LC neurons. In some LC neurons adenosine also caused membrane hyperpolarization (2-10 mV) and reductions in input resistance of 9-24%. Adenosine effects were dose-dependent and antagonized by the adenosine receptor antagonist theophylline.     

Reduction by sevoflurane of adenosine 5'-triphosphate-activated inward current of locus coeruleus neurons in pontine slices of rats.

Increasing evidence indicates that volatile general anesthetics exert their effects by affecting various types of membrane conductance expressed in the central nervous system (CNS), such as ligand-gated receptor-channels. The most recently identified family of the receptor-channels in the CNS are the extracellular ATP-gated channels (P2X purinoceptors). In the present study, we tested whether volatile anesthetics can affect P2X receptor function in the CNS network. We recorded whole-cell currents of locus coeruleus (LC) neurons in pontine slices from young rats. Adenosine 5'-triphosphate (ATP) sodium (0.03-3 mM) evoked a rapidly rising and moderately desensitizing inward current (50-200 pA) in a dose-dependent manner in LC neurons at a holding potential of -80 mV. Perfusion with clinically relevant concentration of sevoflurane (0.1-0.5 mM) reduced the ATP-induced inward current in a dose-dependent manner (to 56.8+/-5.9% of control with 0.5 mM sevoflurane; mean+/-S.E.M., n=13). Estimated IC(50) of sevoflurane was 0.59 mM. We conclude that the attenuation of extracellular ATP-mediated signaling in the central nervous system might be one of the multiple actions of volatile anesthetics.     

Excitatory action of N-methyl- -aspartate on the rat locus coeruleus is mediated by nitric oxide: an in vivo voltammetric study

Biochemical, electrophysiological and behavioural studies have provided evidence that activation of N-methyl- -aspartate (NMDA) receptors contributes to the hyperactivity of noradrenergic neurons of the locus coeruleus (LC) in precipitated opioid withdrawal. Recently, it was demonstrated that central administration of nitric oxide (NO) synthase inhibitors suppresses this hyperactivity suggesting that NO mediates the NMDA receptor activation of LC in opioid withdrawal. Using a combination of microdialysis and in vivo voltammetry, this study examined whether local application of NMDA to the LC in opioid naive animals mimics the NO-dependent LC response seen in opioid withdrawal. In the urethane anaesthetized rat, perfusion of the LC (2 μl min⁻¹) with a solution of NMDA (5 mmol) via a microdialysis probe for 9 min resulted in a rapid and robust increase (290.1±32.2% above baseline) in the catechol oxidation current (CA·OC) recorded from the LC using differential normal pulse voltammetry (DNPV). The NMDA microdialysis also produced a large increase in the blood pressure (150.4±6.9% above baseline). An injection of the non-competitive NMDA receptor antagonist (+)MK-801 (0.5 mg kg⁻¹ i.v.), given 45 min after the start of NMDA application, rapidly returned both the CA·OC signal and the blood pressure response to baseline levels. Pretreatment of animals with intraventricular nitric oxide synthase (NOS) inhibitor, N^ω-nitro- -arginine methyl ester ( -NAME) (100 μg) significantly inhibited NOS activity in the LC, PAG-PVG and cerebellum. This dose of -NAME, administered prior to application of NMDA by microdialysis abolished the NMDA-induced rise in the CAOC recorded in the LC and the increase in systolic blood pressure. The results show that in voltammetry experiments, NMDA produces hyperactivity of LC and hypertension, responses that are dependent upon the synthesis of NO. Thus, in opioid naive rats, regional NMDA application via microdialysis mimics characteristics of the LC response that occur during the antagonist-precipitated opioid withdrawal.     

Opiate- and alpha 2-adrenoceptor-induced hyperpolarizations of locus ceruleus neurons in brain slices: reversal by cyclic adenosine 3':5'-monophosphate analogues

The purpose of this study was to investigate the ionic and second messenger mechanisms underlying the hyperpolarizations induced by the selective alpha 2-adrenoceptor agonist clonidine and the opiate agonist morphine in the locus ceruleus. Intracellular recordings were carried out in rat brain slices, and drugs at known concentrations were administered in the perfusate. The cyclic adenosine 3':5'-monophosphate (cAMP) analogues 8-bromo-cAMP and dibutyryl cAMP, while not altering basal activity, reversed the hyperpolarizations induced by clonidine or morphine. In contrast, administration of the parent compound adenosine failed to affect these responses. These results are consistent with previous biochemical studies suggesting that alpha 2-adrenergic and opiate agonists might signal their actions by reducing intracellular cAMP levels. Under manual voltage clamp, both clonidine and morphine elicited outward currents. The algebraic sum of the individual currents elicited by morphine and clonidine significantly exceeded the actual current elicited by their co-administration. This nonadditivity, as well as the observation that cAMP analogues reverse the morphine- and clonidine-induced hyperpolarizations, suggests that these compounds hyperpolarize locus ceruleus neurons through a shared ionic mechanism the activation of which might be signaled by a decrease in intracellular cAMP.     

Pacemaker activity of locus coeruleus neurons: whole-cell recordings in brain slices show dependence on cAMP and protein kinase A

Noradrenergic neurons of the rat locus coeruleus (LC) are endogenous pacemakers that exhibit slow, tonic firing even in the complete absence of synaptic inputs. In the present study a time-dependent decline in LC spontaneous firing activity was found on intracellular dialysis during whole-cell recording with low-resistance patch electrodes; this decline was accentuated by a specific inhibitor of cAMP-dependent protein kinase (PKI5-24). Conversely, the inclusion of cAMP, 8-Br-cAMP, or the catalytic subunit of cAMP-dependent protein kinase (PKAcat) in the patch pipettes dose-dependently increased firing rate; intracellular PKI5-24 blocked both 8-Br-cAMP and PKAcat-induced firing in LC neurons. These results indicate that endogenous cAMP, via a phosphorylation-dependent route, drives tonic pacemaker activity in LC neurons.     

Peptidergic and adrenergic regulation of the intracellular 3',5'-cyclic adenosine monophosphate content in cultured rat Schwann cells

We investigated the role of neuropeptides and adrenergic agonists in the regulation of intracellular 3',5'-cyclic adenosine monophosphate (cyclic AMP) contents in cultured Schwann cells from sciatic nerve of neonatal Sprague-Dawley rats. Of the neuropeptides examined, vasoactive intestinal polypeptide (VIP) and secretin markedly stimulated the accumulation of intracellular cyclic AMP in a time- and dose-dependent manner with half maximum at 3 and 12 min, and 2.8 X 10(-5) and 5.0 X 10(-5) M, respectively. While somatostatin, substance P, adrenocorticotropin (ACTH), beta-endorphin, and nerve growth factor (NGF) did not show any effect on cyclic AMP metabolism, isoproterenol (IP), norepinephrine (NE) and epinephrine (E) also markedly elevated the Schwann cell cyclic AMP concentration. The rank-order of potency of these adrenergic catecholamines on cyclic AMP accumulation was isoproterenol greater than norepinephrine greater than epinephrine. Simultaneous addition of VIP or secretin to the Schwann cell culture synergistically enhanced the norepinephrine-induced elevation of intracellular cyclic AMP. The effect of norepinephrine was antagonized by a selective beta 1-adrenergic antagonist but not by beta 2- nor alpha-adrenergic antagonists. These results suggest that VIP, secretin, and beta 1-adrenergic agonists alone or synergistically may play a part in the regulation of metabolism of Schwann cells mediated through a cyclic AMP-dependent mechanism.     

Norepinephrine and cyclic adenosine 3':5'-cyclic monophosphate enhance a nifedipine-sensitive calcium current in cultured rat astrocytes

We employed two microelectrode current-clamp and voltage-clamp methods to examine the modulation of Ca++ channels by norepinephrine and cyclic AMP (cAMP) in cultured astrocytes from the rat cerebral cortex. Currents owing to Ca++ channels were maximized by replacing Ca++ with Ba++ in the extracellular solution and pharmacologically blocking K+ and Na+ currents. In current-clamp experiments, we observed that norepinephrine, isoproterenol (an agonist of beta-receptors for norepinephrine), or dibutyryl cAMP (dbcAMP, a membrane permeant analogue of cAMP) induced or enhanced slow Ba++-dependent action potentials in the cells. In voltage-clamp experiments, we confirmed that the slow action potentials were generated by a voltage-activated and Ba++-dependent inward current. This current was mediated by channels that resembled L-type calcium channels (cf. McCleskey et al., Journal of Experimental Biology 124:177-190, 1986) in their voltage-activation range, slow inactivation, and sensitivity to blockage by Co++, Cd++, and nifedipine. DbcAMP, or isoproterenol, enhanced the Ba++ current. Modulation of Ca++ channel function in glial cells could have functional implications.     

Exogenousl-5-hydroxytryptophan is decarboxylated in neurons of the substantia nigra pars compacta and locus coeruleus of the rat

The aim of the present study is to examine by immunohistochemistry whether exogenousl-5-hydroxytryptophan (l-5HTP) is decarboxylated in neurons of the substantia nigra pars compacta (SNC) and locus coeruleus (LC) of the rat. In normal rats, neurons of the SNC and LC stained intensely for aromaticl-amino acid decarboxylase (AADC). No serotonin (5HT)-positive cells were found in the two regions of the normal rats. In rats that were intraperitoneally injected withl-5HTP alone, the SNC neurons stained deeply for 5HT, but the LC neurons showed only a faint staining for 5HT. In rats that intraperitoneally received both a monoamine oxidase (MAO) inhibitor andl-5HTP, when compared with thel-5HTP-injected rats, the LC neurons became much darker in 5HT staining, but the SNC neurons showed only a slight increase in 5HT staining. The present findings suggest that (i) AADC in dopaminergic neurons of the SNC and in noradrenergic neurons of the LC can catalyze the in vivo decarboxylation of exogenousl-5HTP to produce 5HT, and (ii) most of the newly produced 5HT in the LC neurons is rapidly degraded by endogenous MAO.     

Functional characterization of the H-current in SCN neurons in subjective day and night: a whole-cell patch-clamp study in acutely prepared brain slices

Neurons of the rat suprachiasmatic nucleus (SCN) exhibit a circadian rhythm in spontaneous firing rate. In this whole-cell patch-clamp study in slices, we examined the possibility that H-current (I_H) contributes to the spontaneous firing rate of SCN neurons. Most of our experiments were performed during the subjective day, because this is the time epoch during which one would expect the largest excitatory effect of I_H if it were to fluctuate in a circadian rhythm. Current-clamp experiments showed that blockade of I_H by Cs⁺ (1 mM) did not influence the spontaneous firing rate and resting membrane potential. Voltage-clamp experiments revealed that I_H, when activated at the resting membrane potential, is probably too small in magnitude and too slow in activation to make a significant contribution to the spontaneous firing rate. Both results suggest that I_H does not significantly contribute to the spontaneous firing of SCN neurons. In addition, we investigated whether the kinetics and voltage dependence of I_H were modulated in a circadian manner. However, no substantial day–night differences in I_H were found. We conclude that I_H, as recorded in whole-cell mode, does not contribute significantly to spontaneous firing in most SCN neurons and that this current, is more likely to be involved in `rescuing' SCN neurons from large and long-lasting hyperpolarizations by depolarizing the membrane.     

Effect of hallucinogens on spontaneous and sensory-evoked locus coeruleus unit activity in the rat: reversal by selective 5-HT2antagonists

As previously reported, systemic administration of the hallucinogens D-lysergic acid diethylamide (LSD) (5-10 micrograms/kg) and mescaline (2 mg/kg) in the anesthetized rat produced a decrease in spontaneous activity but, paradoxically, facilitated activation of locus coeruleus (LC) neurons by sciatic nerve stimulation. In the present study, the hallucinogen 2,5-dimethoxy-4-methylamphetamine (DOM) (20-80 micrograms/kg) was found to have similar effects. Systemic administration of the selective 5-HT2 antagonists LY 53857 (0.02-0.8 mg/kg) and ritanserin (0.1-0.3 mg/kg) completely reversed both actions of the hallucinogens on the LC. In contrast, LY 53857 did not reverse the effects of (+)-amphetamine (0.5 mg/kg) on the spontaneous or sensory-evoked activity of the LC. These results suggest that the common actions of indoleamine and phenethylamine hallucinogens displayed in the LC are mediated via 5-HT2 receptors; however, these receptors appear to be located outside the LC itself.     

Exogenousl-5-hydroxytryptophan is decarboxylated in neurons of the substantia nigra pars compacta and locus coeruleus of the rat

The aim of the present study is to examine by immunohistochemistry whether exogenousl-5-hydroxytryptophan (l-5HTP) is decarboxylated in neurons of the substantia nigra pars compacta (SNC) and locus coeruleus (LC) of the rat. In normal rats, neurons of the SNC and LC stained intensely for aromaticl-amino acid decarboxylase (AADC). No serotonin (5HT)-positive cells were found in the two regions of the normal rats. In rats that were intraperitoneally injected withl-5HTP alone, the SNC neurons stained deeply for 5HT, but the LC neurons showed only a faint staining for 5HT. In rats that intraperitoneally received both a monoamine oxidase (MAO) inhibitor andl-5HTP, when compared with thel-5HTP-injected rats, the LC neurons became much darker in 5HT staining, but the SNC neurons showed only a slight increase in 5HT staining. The present findings suggest that (i) AADC in dopaminergic neurons of the SNC and in noradrenergic neurons of the LC can catalyze the in vivo decarboxylation of exogenousl-5HTP to produce 5HT, and (ii) most of the newly produced 5HT in the LC neurons is rapidly degraded by endogenous MAO.     

Pyramidal neurons in rat prefrontal cortex show a complex synaptic response to single electrical stimulation of the locus coeruleus region: Evidence for antidromic activation and GABAergic inhibition using in vivo intracellular recording and electron microscopy

Cognition and acquisition of novel motor skills and responses to emotional stimuli are thought to involve complex networking between pyramidal and local GABAergic neurons in the prefrontal cortex. There is increasing evidence for the involvement of cortical norepinephrine (NE) deriving from the nucleus locus coeruleus (LC) in these processes, with possible reciprocal influence via descending projections from the prefrontal cortex to the region of the LC. We used in vivo intracellular recording in rat prefrontal cortex to determine the synaptic responses of individual neurons to single electrical stimulation of the mesencephalic region including the nucleus LC. The most common response consisted of a late-IPSP alone or preceded by an EPSP. The presence of an early-IPSP following the EPSP was sometimes detected. Analysis of the voltage dependence revealed that the late-IPSP and early-IPSP were putative K⁺- and Cl⁻ dependent, respectively. Synaptic events occurred following short delays and were inconsistent with the previously reported time for electrical activation of unmyelinated LC fibers. Moreover, systemic injection of the adrenergic antagonists propranolol (β receptors), or prazosin (α1 receptors), did not block synaptic responses to stimulation of the LC region. Finally, certain neurons were antidromically activated following electrical stimulation of this region of the dorsal pontine tegmentum. Taken together, these results suggest that the complex synaptic events in pyramidal neurons of the prefrontal cortex that are elicited by single electrical stimulation of the LC area are mainly due to antidromic activation of cortical efferents. Further insight into the chemical circuitry underlying these complex synaptic responses was provided by electron microscopic immunocytochemical analysis of the relations between the physiologically characterized neurons and either 1) GABA or 2) dopamine-β-hydroxylase (DBH), a marker for noradrenergic terminals. GABA-immunoreactive terminals formed numerous direct symmetric synapses on somata and dendrites of pyramidal cells recorded and filled with lucifer yellow (LY). In contrast, in single sections, noradrenergic terminals immunoreactive for DBH rarely contacted LY-filled somata and dendrites. These results support the conclusion that IPSPs observed following single electrical stimulation of the LC region are mediated bu GABA, with little involvement of NE. These IPSPs, arising from antidromic invasion of mPFC cells innervating the LC, may improve the signal-to-noise ratio and favor a better responsiveness of neighboring neurons to NE released in the mPFC. © 1996 Wiley-Liss, Inc.     

Transient increase in intracellular concentration of adenosine 3':5'-cyclic monophosphate results in morphological and biochemical differentiation of C6 glioma cells in culture

Incubation of C6-BU1 glioma cells in the presence of isoproterenol and Ro20-1724--a potent cAMP phosphodiesterase inhibitor--results in a transient increase in intracellular cAMP levels, followed by a rapid efflux of cyclic AMP from the cells into the media. Two distinct types of morphological changes could be seen: rounded cell bodies with multipolar processes and beadings after 30 minutes of incubation--this period coincides with a 70-80-fold increase in intracellular cAMP levels, and elongated cell bodies with extended bipolar processes after 24-48 hours. By this time the intracellular cAMP concentration dropped to a low level, which was only three- to four fold higher than that in control. The transient increase in intracellular cAMP concentration results in retardation of cell growth, diminished uptake of 3H-2-deoxyglucose, and abolition of enhanced synthesis of cyclic AMP by concanavalin A.     

Effect of denervation of the locus coeruleus projections by DSP-4 treatment on [3H]-raclopride binding to dopamine D(2) receptors and D(2) receptor-G protein interaction in the rat striatum

Changes in the control of dopaminergic neurotransmission by noradrenergic locus coeruleus (LC) projections has been implicated in such disorders as depression, drug addiction, and Parkinson's disease. In the present study, the effect of DSP-4, a neurotoxin highly selective for LC projections, on D(2) receptor abundance as assessed by [3H]-raclopride binding in the striatum was studied in rats after administration in doses of 10 and 50 mg/kg either 3 days or 1 month before decapitation. Three days after DSP-4 the levels of noradrenaline in the frontal cortex were dose-dependently reduced; after 1 month, noradrenaline levels were lowered only by the higher dose. DOPAC levels were dose-dependently reduced in the frontal cortex and striatum 3 days but not 1 month after DSP-4 treatment. Cortical 5-HIAA levels were reduced 3 days but not 1 month after DSP-4. The apparent number of D(2) receptor binding sites in the striatum was higher 1 month after either dose of DSP-4. DSP-4 treatment had no effect on [3H]-raclopride binding affinity, the ability of dopamine (DA) to compete with [3H]-raclopride binding and to activate [35S]GTPgammaS binding or on the binding affinities of GDP and [35S]GTPgammaS for corresponding G proteins 1 month after administration of the neurotoxin. These data suggest that after administration of DSP-4, short-term reduction in DA and 5-HT metabolism occurs. Subsequently, an upregulation of D(2) receptor binding sites develops in the striatum even after a minor denervation of the LC projections. Thus, alterations in the LC projection systems elicit lasting adaptive changes in DA-ergic neurotransmission that can serve as a substrate for psychiatric disorders.     

Lewis and Fischer rat strains display differences in biochemical, electrophysiological and behavioral parameters: studies in the nucleus accumbens and locus coeruleus of drug naive and morphine-treated animals

In previous studies, we demonstrated that tyrosine hydroxylase and neurofilament proteins are regulated by chronic morphine and chronic cocaine treatments in the ventral tegmental area in Sprague-Dawley rats and that the imbred Lewis and Fischer 344 rat strains, under drug-naive conditions, show different levels of these proteins specifically in this brain region. In the current study, we compared Lewis and Fischer rats with respect to levels of adenylate cyclase, cyclic AMP-dependent protein kinase and G-proteins in the nucleus accumbens (NAc) and locus coeruleus (LC), brain regions in Sprague-Dawley rats where these proteins are regulated by chronic exposure to morphine or to cocaine. We found that levels of adenylate cyclase and cyclic AMP-dependent protein kinase activity are higher in the NAc and LC of Lewis rats compared to Fischer rats, whereas levels of G_iα and G_β were lower. These strain differences were not seen in several other brain regions analyzed and no strain differences were detected in levels of other G-protein subunits. Lewis and Fischer rats also differed in the ability of chronic morphine to regulate adenylate cyclase and cyclic AMP-dependent protein kinase in the NAc and LC. In the NAc, chronic morphine increased levels of the two enzymes in the Fischer strain only, whereas in the LC chronic morphine increased levels of the enzymes in both strains, with more robust effects seen in the Lewis rat. To understand possible physiological consequences of these strain differences in the cyclic AMP pathway, we studied LC neuronal activity under basal and chronic morphine-treated conditions. LC neurons of Lewis rats showed higher spontaneous firing rates in brain slices in vitro than those of Fischer rats and also showed greater morphine-induced increases in responsiveness to bath-applied 8-bromo-cyclic AMP. These electrophysiological findings are generally consistent with the biochemical observations. Moreover, Lewis and Fischer rats displayed very different opiate withdrawal syndromes, with different types of behaviors elicited upon precipitation of opiate withdrawal with the opiate receptor antagonist, naltrexone. The possible relationship between these behavioral findings and the biochemical and electrophysiological data is discussed. These studies provide further support for the possibility that Lewis and Fischer rat strains provide a useful model system in which some of the genetic factors that contribute to drug-related behaviors can be investigated.     

Regulation of the concentration of adenosine 3',5'-cyclic monophosphate and the activity of tyrosine hydroxylase in the rat superior cervical ganglion by three neuropeptides of the secretin family

Preganglionic nerve stimulation leads to an acute elevation of tyrosine hydroxylase (TH) activity in the rat superior cervical ganglion. This effect is mediated in part by acetylcholine, acting via nicotinic receptors, and in part by a noncholinergic neurotransmitter. As a first step in an attempt to identify this noncholinergic transmitter, we have examined a number of biogenic amines, purine nucleotides, neuropeptides, and other compounds for their ability to increase TH activity. Secretin, vasoactive intestinal peptide (VIP), and PHI (a 27-amino acid peptide with an NH2-terminal histidine and a COOH-terminal isoleucine amide), all members of the secretin family of peptides, increased TH activity acutely. Human pancreatic growth hormone-releasing factor, glucagon, and gastric inhibitory peptide (three other members of this peptide family) and all other transmitter candidates tested had no effect on this enzyme activity. We have examined the possibility that this peptidergic regulation of TH activity is mediated via changes in adenosine 3',5'-cyclic monophosphate (cAMP) levels. When the six members of the secretin family were tested for their ability to increase cAMP levels in the ganglion, secretin, VIP, and PHI significantly increased this cyclic nucleotide, whereas growth hormone-releasing factor, glucagon, and gastric inhibitory peptide produced no significant effects. The rank orders of potency and of efficacy of secretin, VIP, and PHI in altering TH activity and cAMP levels were identical. Furthermore, a strong correlation was found between the cAMP level and the TH activity in individual ganglia exposed to these peptides. Finally, 8-bromoadenosine 3',5'-cyclic monophosphate and forskolin also increased TH activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Real-time monitoring of endogenous noradrenaline release in rat brain slices using fast cyclic voltammetry: 1. Characterisation of evoked noradrenaline efflux and uptake from nerve terminals in the bed nucleus of stria terminalis, pars ventralis

Fast cyclic voltammetry (FCV) at carbon fibre microelectrodes was used to monitor endogenous noradrenaline (NA) efflux in superfused slices of bed nucleus of stria terminalis pars ventralis (BSTV) in 'real time'. NA efflux was evoked by local electrical stimulation at bipolar tungsten stimulating electrodes. Confirmation of the identity of the released species as NA was made on the basis of anatomical, electrochemical and pharmacological proofs. Firstly, the signal matched the NA innervation density; efflux of monoamine was greater in BSTV than in the pars dorsalis of the nucleus. Secondly, the voltammogram of the released species was indistinguishable from those of the catecholamines NA and dopamine (DA) but dissimilar to that of the indoleamine serotonin (5-hydroxytryptamine, 5-HT). Thirdly, amine efflux was influenced in a predictable fashion by the drugs tested. Tetrodotoxin (10(-6) M) or omission of Ca2+ from the superfusate reversibly reduced amine efflux by 90.2 and 88.0% respectively. Ro 4-1284 (10(-6) M) decreased amine efflux by 75.8%. Desipramine (5 x 10(-8) M), the selective NA uptake blocker, significantly increased amine efflux and uptake half-life (to 214.3 and 389.5% of control respectively). Fluvoxamine (5 x 10(-7) M) and GBR 12909 (3 x 10(-7) M), blockers of 5-HT and DA uptake respectively, had no effect on amine efflux, although fluvoxamine caused a modest (91.0%) increase in the uptake half-life. Pargyline (2 x 10(-6) M) affected neither efflux nor uptake. The combined anatomical, electrochemical and pharmacological data confirm that the monoamine detected in BSTV by local electrical stimulation was NA. Stimulated NA efflux was stable and reproducible over at least 2.5 h (longest period tested). This study demonstrates the ability of FCV to selectively monitor endogenous NA efflux and uptake in 'real time' and with high spatial resolution.     

Behavioral depression produced by an uncontrollable stressor: Relationship to norepinephrine, dopamine, and serotonin levels in various regions of rat brain

This paper presents two experiments that continue efforts to determine the neurochemical changes responsible for stress-induced behavioral depression. These expriments measured active motor behavior in a swim tank as well as levels of norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in various brain regions of rats after the animals had (a) been exposed to electric shocks they could control (Avoidance-escape condition), or (b) received the same shocks with no control over them (Yoked condition), or (c) received no shock (No-shock condition). In the first experiment, measures were taken 90 min after the shock session ended. In the swim test, Yoked animals showed a depression of active behavior relative to the other groups. From measures of monoamine levels, the change found to be most closely related to this post-stress behavioral depression was in NE in the locus coeruleus (LC), where Yoked animals showed a considerable depletion of NE. In the second study, the same measures were taken 48 h and 72–96 h after the stress session. Yoked animals tested at 48 h post-stress showed motor depression, but those tested after 72–96 h did not. NE in the LC was significantly depleted in Yoked animals tested at 48 h post-stress but showed only slight (and non-significant) depletion in those tested 72–96 h post-stress. These results, together with others, suggest that large stress-induced depletion of NE in the LC is involved in mediating behavioral depression brought about by severe stress. It is further suggested that the time course for behavioral recovery and for the disappearance of NE depletion in the LC that was seen in Yoked animals after stress parallels the time course previously reported by other investigators for induction of catecholamine-synthesizing enzymes — tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH) — in the LC, so that induction of TH and DBH activity may be a neurochemical mechanism to bring about recovery from poststress behavioral depression.