Hypothalamic corticotrophin-releasing factor and norepinephrine mediate sympathetic and cardiovascular responses to acute intracarotid injection of tumour necrosis factor-alpha in the rat

Systemic administration of tumour necrosis factor (TNF)-α induces the release of norepinephrine in the paraventricular nucleus (PVN) of hypothalamus and an increase in expression of corticotrophin-releasing factor (CRF) and CRF type 1 receptors. We explored the hypothesis that CRF and norepinephrine in PVN mediate the cardiovascular and sympathetic responses to acute systemic administration of TNF-α. In anaesthetised rats, the increases in arterial pressure and heart rate induced by intracarotid artery injection of TNF-α were attenuated by intracerebroventricular (ICV) injection of either the α₁-adrenergic antagonist prazosin or the CRF antagonist α-helical CRF. Prazosin blocked the TNF-α-induced increase in renal sympathetic nerve activity (RSNA), whereas α-helical CRF substantially reduced the RSNA response. Conversely, CRF and the α₁-adrenergic agonist phenylephrine, administered ICV, both elicited increases in PVN neuronal activity, RSNA, arterial pressure and heart rate. Microinjection of CRF and phenylephrine directly into PVN evoked smaller responses. These results are consistent with the hypothesis that norepinephrine and CRF in the PVN mediate the cardiovascular and sympathetic responses to acute systemic administration of TNF-α.     

p21ras Oncogene Protein Selectively Increases Low-voltage-activated Ca2+ Current Density in Embryonic Chick Dorsal Root Ganglion Neurons

p21^ras protein resembles the α subunit of trimeric G-proteins, which regulate ion channel function. We now report a modulation of Ca²⁺ channels in vertebrate sensory neurons by p21^ras in addition to its role in cell growth and differentiation. Quantitative microinjection of oncogenic p21-H-ras into embryonic chick dorsal root ganglion neurons was performed. After 4 h the current density of the low-voltage-activated (LVA; T-type) Ca²⁺ channels was increased. However, in contrast to trimeric G-proteins, which inhibit high-voltage-activated (HVA) Ca²⁺ channels in chick dorsal root ganglion neurons, p21^ras did not significantly affect HVA Ca²⁺ currents. To study the time course of p21^ras action, guanosine triphosphate-preloaded p21^ras was added to the patch pipette. Full-length ras was effective only after a delay of 20 -30 min. C-terminal modification by cellular enzymes is required to activate full-length ras, and can account for the observed delay. Unexpectedly, C-terminal-truncated p2l^ras, which was found to be inactive in biological assays, enhanced LVA Ca²⁺ currents within minutes. This suggests a G-protein-like modulation of the LVA Ca²⁺ channel by p21^ras. In an early phase of neuronal differentiation, dorsal root ganglion neurons express only LVA Ca²⁺ currents. The regulatory role of p21^ras on LVA channels may therefore be particularly important during differentiation.     

Central α2-Adrenergic Control of Colonic Transit in Rats

The effects of central and peripheral administration of α₂-adrenoceptor agonists on colonic propulsion were examined in conscious rats chronically fitted with a catheter inserted in the lumen of the proximal colon and a cannula placed in a cerebral lateral ventricle. The mean retention time of a marker in the colon was determined by administration of a bolus of [⁵¹Cr]sodium chromate into the proximal colon and collection of the feces at hourly intervals. In control studies the colonic mean retention time was 7.5 ± 1.6 hours. Clonidine administered intraperitonedy or intracerebroventricularly produced a dose-dependent increase in mean retention time but was more potent when given intracerebroventricularly. St-91, a quaternary derivative of clonidine which poorly crosses the blood-brain barrier, delayed colonic transit after intracerebroventricular, but not after intraperitoneal, administration at the same dose. The increase in colonic mean retention time induced by intraperitonedy administered clonidine was antagonized by intracerebroventricular administration of yohimhine but not of prazosin, an α₁-adrenoceptor antagonist. Yohimbine intracerebroventricularly or intraperitonedy administered alone at the same dose accelerated the colonic transit. Such data suggest a possible role of central α₂-adrenoceptors in the regulation of colonic motility in rats.     

Functional Properties of Neuronal Nicotinic Acetylcholine Receptor Channels Expressed in Transfected Human Cells

To study how subunit composition affects the functional properties of neuronal nicotinic acetylcholine receptors (nAChRs), we examined the behaviour of acetylcholine (ACh)-induced single-channel currents in human BOSC 23 cells transiently transfected with various subunit cDNA combinations. For all nAChRs examined (chick and rat α₃β₄, chick α_<3/β₂, α₄β₂, α₄β₄, α₇and α₈, expression levels were high enough to allow measurements of acetylcholine-evoked whole-cell currents and nicotine-elicited Ca²⁺ transients as well as the functional characterization of nAChR channels. Unitary acetylcholine-evoked events of α₈ nAChR had a slope conductance of 23 pS, whereas two conductance classes (19–23 and 32–45 pS) were identified for all other nAChR channels. The mean channel open times were significantly longer for homomeric α₇ and α₈ nAChRs (6–7 ms) than for heteromeric nAChRs (1–3 ms), with the exception of α₃α₄nAChRs (8.4 ms for rat, 7 ms for chick). At least two species of heterologously expressed nAChRs (α₃α₄and α₃α₂) exhibited single-channel characteristics similar to those reported for native receptors. The variety of nAChRs channel conductance and kinetic properties encountered in human cells transfected with nAChR subunits contributes to the functional diversity of nAChRs in nerve cells.     

Differential expression of voltage-activated calcium channels in III and XII motoneurones during development in the rat

To further our understanding of the role that voltage-activated Ca2+ channels play in the development, physiology and pathophysiology of motoneurones (MNs), we used whole-cell patch-clamp recording to compare voltage-activated Ca2+ currents in oculomotor (III) and hypoglossal (XII) MNs of neonatal [postnatal day (P)1-5] and juvenile (P14-19) rats. In contrast to III MNs that innervate extraocular muscles, XII MNs that innervate tongue muscles mature more rapidly, fire bursts of low frequency action potentials and are vulnerable to degeneration in amyotrophic lateral sclerosis. In neonates, low voltage-activated (LVA) Ca2+ current densities are similar in XII and III MNs but high voltage-activated (HVA) Ca2+ current densities are twofold higher in XII MNs. The HVA Ca2+ channel antagonists (nimodipine and nifedipine for L-type, omega-agatoxin-TK for P/Q-type and omega-conotoxin-GVIA for N-type) revealed that, while N- and P/Q-type HVA Ca2+ channels are present in both MN pools, a 3.5-fold greater P/Q-type Ca2+ current in XII MNs accounts for their greater HVA Ca2+ currents. Developmentally, LVA and HVA Ca2+ current densities decrease in III MNs but remain unchanged in XII MNs. Thus, the differences between these MN pools increase developmentally so that, in juveniles, the LVA Ca2+ current density is twofold greater and the HVA Ca2+ current density is threefold greater in XII compared with III MNs. We propose that this differential expression of LVA and HVA Ca2+ channels in XII and III MNs during development contributes to their distinct physiology and may also be a factor contributing to the greater susceptibility of XII MNs to degeneration as seen in amyotrophic lateral sclerosis.     

Stimulation of Alpha-Adrenoceptors Facilitates the Release of Growth Hormone-Releasing Hormone from Rat Hypothalamus in vitro

While extensive evidence suggests that adrenoceptors play an important role in the control of growth hormone in the rat, there are few studies involving the direct measurement of growth hormone-releasing hormone (GHRH). We have therefore developed a radioimmunoassay for rat GHRH, and used it to investigate the modulation of GHRH release by noradrenaline from incubated rat hypothalamus in vitro. The GHRH radioimmunoassay had no significant cross-reactivity with other hypothalamic or GHRH-related peptides, and was sensitive to 4 pg/tube; intra- and interassay coefficients of variation were 6% and 12% respectively. Single incubated rat hypothalami produced a stable and readily measurable output of GHRH in successive 20 min incubations after an initial 60 min preincubation; the release of GHRH was increased in the presence of 56 mM KCI, but did not respond to KCI-depolarization when calcium was excluded from the medium. Stimulated GHRH release was identical to synthetic rat GHRH(1–43) on high-performance liquid chromatography and Sephadex G-75 chromatography.
Noradrenaline stimulated GHRH secretion in a dose-dependent manner in the concentration range 10⁻¹⁰— 10⁻⁶M, with a plateau in response at 10⁻⁷M. Stimulation with noradrenaline 10⁻⁷M was blocked by idazoxan 10⁻⁵M and attenuated by thymoxamine 10⁻⁵M, but was unaffected by timolol 10⁻⁵M. Both the α₂-adrenoceptor agonist guanfacine, and the α₁-adrenoceptor agonist methoxamine, specifically stimulated GHRH secretion.
It is concluded that noradrenaline stimulates the release of GHRH at both α₁ and α₂-adrenoceptors.     

Noradrenalin Enhances the Activity of Cochlear Nucleus Neurons in the Rat

The cochlear nucleus of rats is heavily innervated by noradrenergic fibres from the locus coeruleus. The physiological meaning of this innervation is poorly understood. Therefore, iontophoretically applied noradrenalin was tested on single neurons of the cochlear nucleus in urethane-anaesthetized rats. Iontophoresis of noradrenalin had a dual effect. During application noradrenalin led to moderate inhibition of tone-evoked activity in 37% of the tested neurons. In contrast, ∼20-30 s after the onset of iontophoresis a long-lasting increase in discharge activity was found in most neurons. Data from iontophoresis of the α₁-receptor agonist phenylephrine and the α₂-receptor agonist clonidine suggest that the fast moderate inhibition is mediated by α₂-receptors while the pronounced long-lasting elevated neuronal firing is mediated by α₁-receptors. However, these data do not exclude the possibility that part of the response to noradrenalin is also mediated by β-receptors. Electrical stimulation of the locus coeruleus resulted in an increase in discharge activity comparable with iontophoresis of noradrenalin or phenylephrine. Thus, activation of the locus coeruleus predominantly increases spontaneous and tone-evoked neuronal firing in the cochlear nucleus of the rat. This α₁-receptor-mediated enhanced discharge activity may serve to increase the sensitivity of acoustic processing mechanisms or to lower the threshold for short-latency acoustic reflexes.     

Dopamine inhibits voltage-activated calcium channel currents in rat pars intermedia pituitary cells

Several lines of evidence suggest that dopamine acts as a neurotransmitter that inhibits both hormone secretion and electrical activity in pituitary intermediate cells (melanotrophs). In this study we examined the effects of exogenously applied dopamine on voltage activated calcium currents recorded with the whole-cell mode of the patch-clamp technique from short-term primary cultures of melanotrophs. Two types of calcium currents were distinguished by their voltage dependence and kinetics of inactivation similar to the low voltage-activated currents (LVA; or T-type) and high voltage-activated currents (HVA; N&L-types) of calcium currents. Exogenously applied dopamine (2-20 microM) reversibly reduced both LVA and HVA types of calcium currents. Evidence for these results came from experiments in which LVA and HVA calcium currents were separated by stepping to different membrane potentials from a fixed holding potential (Vh) or by changing Vh. These results suggest that dopamine can regulate the entry of calcium into melanotrophs by acting on at least two different populations of calcium channels thereby affecting hormone secretion and electrical activity.     

Differential down-regulation of voltage-gated calcium channel currents by glutamate and BDNF in embryonic cortical neurons

In the embryonic brain, post-mitotic cortical neurons migrate from their place of origin to their final location. Various external factors such as hormones, neurotransmitters or peptides regulate their migration. To date, however, only a few studies have investigated the effects of these external factors on the electrical properties of the newly formed embryonic cortical neurons. The aim of the present study was to determine whether glutamate and brain-derived neurotrophic factor (BDNF), known to regulate neuronal cell migration, could modulate currents through voltage-gated calcium channels (ICa) in cortical neurons isolated from embryonic day 13 (E13) mouse foetuses. Whole cell recordings of ICa showed that E13 cortical cells kept 1 day in vitro expressed functional low- and high-voltage activated (LVA and HVA) Ca2+ channels of T-, L- and N-types. A 1-day glutamate treatment non-specifically inhibited LVA and HVA ICa whereas BDNF down-regulated HVA with N-type ICa being more depressed than L-type ICa. The glutamate-induced ICa inhibition was mimicked by NMDA. BDNF exerted its action by recruiting trkB receptors and SKF-96365-sensitive channels. BAPTA prevented the glutamate- and the BDNF-dependent inhibition of Ica, indicating a Ca2+-dependent mechanism of action. It is proposed that an influx of Ca2+ through NMDA receptors depresses the expression of LVA and HVA Ca2+ channels whereas a Ca2+ influx through SKF-96365-sensitive TRPC (transient receptor potential protein of C subtype) channels preferentially inhibits the expression of HVA Ca2+ channels. Glutamate and BDNF appear as potent modulators of the electrical properties of early post-mitotic neurons. By down-regulating ICa they could exert a neuroprotective action on embryonic cortical neurons.     

Postnatal development of GABAB receptor-mediated modulation of voltage-activated Ca2+ currents in mouse brain-stem neurons.

GABA_B receptors modulate respiratory rhythm generation in adult mammals. However, little is currently known of their functional significance during postnatal development. In the present investigation, the effects of GABA_B receptor activation on voltage-activated Ca²⁺ currents were examined in rhythmically active neurons of the pre-Bötzinger complex (PBC). Both low- (LVA) and high-voltage-activated (HVA) Ca²⁺ currents were present from the first postnatal day (P1). The density of LVA Ca²⁺ currents increased during the first week, whilst the density of HVA Ca²⁺ currents increased after the first week. In the second postnatal week, the HVA Ca²⁺ currents were composed of L- (47 ± 10%) and N-type (21 ± 8%) currents plus a ‘residual’ current, whilst there were no N-type currents detectable in the first few days. The GABA_B receptor agonist baclofen (30 μm ) increased LVA Ca²⁺ currents (30 ± 11%) at P1–P3, but it decreased the currents (35 ± 11%) at P7–P15 without changing its time course. At all ages, baclofen (30 μm ) decreased the HVA Ca²⁺ currents by ≈ 54%. Threshold of baclofen effects on both LVA and HVA Ca²⁺ currents was 5 μm at P1–P3 and lower than 1 μm at P7–P15. The effect of baclofen was abolished in the presence of the GABA_B receptor antagonist CGP 55845A (50 n m ). We conclude that both LVA and HVA Ca²⁺ currents increased postnatally. The GABA_B receptor-mediated modulation of these currents undergo marked developmental changes during the first two postnatal weeks, which may contribute essentially to modulation of respiratory rhythm generation.     

Modulation of Ca2+ currents in rat thalamocortical relay neurons by activity and phosphorylation

Rhythmic low and high frequency activity in thalamocortical networks depend critically on activation of low- and high-voltage-activated (LVA, HVA) Ca2+ currents. In order to test whether Ca2+ currents are modified during repetitive activation, acutely isolated thalamocortical relay neurons of rats, at postnatal days 12 (P12) to P20, were investigated using patch-clamp, Ca2+ imaging and Western blot techniques. High-voltage-activated, but not LVA Ca2+ currents were reduced significantly during 2 Hz stimulation. Ca2+ imaging experiments demonstrated a close correlation between the increase in intracellular Ca2+ levels and the decrease in HVA Ca2+ current amplitudes. Further examination of HVA Ca2+ currents revealed a 'U-shaped' inactivation curve and a time-dependent inactivation process that could be described by a two-exponential function. The 'U-shape' was significantly reduced, current amplitude was increased significantly and time-dependent inactivation revealed a one-exponential decline with Ba2+ as the charge carrier, following activation of the cAMP/PKA pathway, and following application of phosphatase inhibitors (ascomycin, calyculin A). Western blot analysis and the effect of ascomycin indicated an involvement of calcineurin in the inactivation process. Isolation of HVA Ca2+ current components by subtype-specific blockers revealed that changes in time-dependent inactivation, inactivation curve and current amplitude were carried mainly by L-type and N-type Ca2+ currents. Furthermore, Ca2+-dependent inactivation was operative during stimulation protocols mimicking tonic action potential firing. These data indicate a modulation of L- and N-type Ca2+ channels by phosphorylation, resulting jointly in an increased intracellular Ca2+ influx during activity of the ascending brainstem system, the latter occurring during states of wakefulness.     

Partial compensation for N-type Ca2+ channel loss by P/Q-type Ca2+ channels underlines the differential release properties supported by these channels at cerebrocortical nerve terminals

N-type and P/Q-type Ca²⁺ channels support glutamate release at central synapses. To determine whether the glutamate release mediated by these channels exhibits distinct properties, we have isolated each release component in cerebrocortical nerve terminals from wild-type mice by specifically blocking N-type Ca²⁺ channels with ω-conotoxin-GVIA and P/Q-type Ca²⁺ channels with ω-agatoxin-IVA. In addition, we have determined the release properties at terminals from mice lacking the α_1B subunit of N-type channels (Ca_v 2.2) to test the possibility that P/Q-type channels can compensate for the loss of N-type Ca²⁺ channels. We recently demonstrated that, while evoked glutamate release depends on P/Q- and N-type channels in wild-type nerve terminals, only P/Q-type channels participate in these knockout mice. Moreover, in nerve terminals expressing solely P/Q-type channels, metabotropic glutamate receptor 7 (mGluR7) fails to inhibit the evoked Ca²⁺ influx and glutamate release. Here, we show that the failure of mGluR7 to modulate evoked glutamate release is not due to a lack of receptors, as nerve terminals from mice lacking N-type Ca²⁺ channels express mGluR7. Indeed, we show that other receptor responses, such as the inhibition of forskolin-induced release, are preserved in these knockout mice. N-type channels are more loosely coupled to release than P/Q-type channels in nerve terminals from wild-type mice, as reflected by the tighter coupling of release in knockout nerve terminals. We conclude that the glutamate release supported by N- and P/Q-type channels exhibits distinct properties, and that P/Q-type channels cannot fully compensate for the loss of N-type channels.     

Developmental regulation of T‐, N‐ and L‐type calcium currents in mouse embryonic sensory neurones

We investigated the development of a low (T-type) and two high voltage-activated (N- and L-type) calcium channel currents in large diameter dorsal root ganglion neurones acutely isolated from embryonic mice using the whole-cell patch-clamp technique. The low and high voltage-activated barium currents (LVA and HVA) were identified by their distinct threshold of activation and their sensitivity to pharmacological agents, dihydropyridines and ω-conotoxin-GVIA, at embryonic day 13 (E13), E15 and E17–18, respectively, before, during and after synaptogenesis. The amplitude and density of LVA currents, measured during a –40 mV pulse from a holding potential of –100 mV, increased significantly between E13 and E15, and remained constant between E15 and E17–18. The density of global HVA current, elicited by 0 mV pulse, increased between E13 and E15/E17–18. The density of the N-type current studied by the application of ω-conotoxin-GVIA (1 μm ) increased significantly between E13 and E15/E17–18. The use of the dihydropyridine nitrendipine (1 μm ) revealed that the density of L-type current remained constant at each stage of development. Nevertheless, application of dihydropyridine Bay K 8644 (3 μm ) demonstrated a significant slowing of the deactivation tail current between embryonic days 13 and 15, which may reflect a qualitative maturation of this class of calcium channel current. The temporal relationship between the changes in calcium channel pattern and the period of target innervation suggests possible roles of T-, N- and L-type currents during developmental key events such as natural neurone death and onset of synapse formation.     

Pharmacological characterization of calcium currents and synaptic transmission between thalamic neurons in vitro.

We recorded from pairs of cultured, synaptically connected thalamic neurons. Evoked excitatory postsynaptic currents (EPSCs) reversed at +17 mV and were blocked reversibly by 1 mM kynurenic acid, a glutamate receptor antagonist. NMDA and non-NMDA receptors mediated excitatory post-synaptic responses, as shown by selective block of EPSC components with 50 microM (+/-)-2-amino-5-phosphonopentanoic acid and 10 microM 6,7-dinitroquinoxaline-2,3-dione, respectively. Inhibitory postsynaptic responses were evoked less frequently and were blocked by the GABAA receptor antagonist (-)-bicuculline methochloride. The pharmacological profiles of whole-cell calcium currents and evoked EPSCs were compared. With 50 microM cadmium chloride (Cd), whole-cell low voltage-activated (LVA) calcium currents were reduced in amplitude and high voltage-activated (HVA) calcium currents and excitatory synaptic transmission were completely blocked. This suggests that the residual calcium influx through LVA channels into the presynaptic terminal does not suffice to trigger transmitter release. A saturating concentration of omega-conotoxin GVIA (omega-CgTx) (2.5 microM) blocked one-third of whole-cell HVA calcium currents and evoked EPSCs. The dihydropyridine nifedipine (50 microM) reversibly reduced whole-cell HVA calcium currents in a voltage-dependent manner but not excitatory synaptic transmission. Cd and omega-CgTx did not alter amplitude distributions of miniature EPSCs, demonstrating that the inhibition of synaptic transmission was due to block of presynaptic calcium channels. We conclude that excitatory glutamatergic transmission in thalamic neurons in vitro was mediated mainly by HVA calcium currents, which were insensitive to omega-CgTx and nifedipine.     

Protein tyrosine kinase inhibitors reduce high-voltage activating calcium currents in CA1 pyramidal neurones from rat hippocampal slices

We have investigated the effects of protein tyrosine kinases (PTKs) inhibitors on high-threshold voltage activating (HVA) calcium currents in CA1 pyramidal neurones, whole-cell patch-clamp recorded from rat hippocampal slices. Genistein (100 μM) and tyrphostin B42 (100 μM), two PTKs inhibitors, reduced the steady-state barium current (I_Ba). On the other hand, daidzein and genistin (100 μM), two inactive analogues of genistein, had no effect on I_Ba amplitude. The inhibition induced by genistein was more pronounced at negative potentials. In order to characterize the calcium channels subtypes inhibited by PTKs inhibitors, we examined the effect of genistein in the presence of different calcium channel blockers. When L-type calcium channels were blocked by nifedipine, genistein induced a strong inhibition of the nifedipine-resistant I_Ba, suggesting an effect on non-L-type channels. Genistein did not antagonize the depressant effect of ω-Conotoxin-GVIA, a selective N-type calcium channel blocker, suggesting that N-type channels were not blocked by genistein. ω-Conotoxin-MVIIC (3–10 μM), a selective P/Q-type calcium channel blocker, greatly antagonized the depressant effect of genistein. Our results suggest that PTKs inhibitors reduce P-/Q-type, but not L- or N-types calcium currents in neurones of the CNS. The possible modulation of calcium channels by endogenous PTKs is discussed.     

Adenosine Inhibits L- and N-Type Calcium Channels in Pituitary Melanotrophs. Evidence for the Involvement of a G Protein in Calcium Channel Gating

It has been previously demonstrated that activation of A₁ adenosine receptors in frog melanotrophs causes inhibition of spontaneous action potential discharges and alpha-melanocyte-stimulating hormone secretion. In the present study, we have investigated the effect of adenosine on high-voltage-activated (HVA) calcium currents in cultured melanotrophs, using the whole-cell variant of the patch-clamp technique with barium as a charge carrier. Adenosine and the specific A₁ adenosine receptor agonist R-PIA (50 μM each) produced a decrease of the amplitude of the barium current, while the selective A₂ adenosine receptor agonist CGS 21680 did not affect the current. The inhibitory effect of R-PIA was observed throughout the activation range of the current, with stronger responses at more positive potentials. R-PIA inhibited both the L- and N-type components of the current, the effect on the N-component being two-fold higher than on the L-component. The inhibitory effect of R-PIA was rendered irreversible by addition of GTPyS (100 μM) to the intracellular solution. Pre-treatment of the cells with pertussis toxin (1 μg/ml; 12 h) totally abolished the effect of R-PIA on the HVA calcium channels. Conversely, addition of a high concentration of cAMP (100 μM) together with the phosphodiesterase inhibitor IBMX (100 μM) to the intracellular solution did not modify the effect of R-PIA on the current.
It is concluded that, in frog melanotrophs, adenosine induces inhibition of L- and N-calcium currents and that this effect is mediated by a pertussis toxin-sensitive G protein. Our data also indicate that the inhibitory effect of adenosine on the calcium currents is not mediated by inhibition of adenylyl cyclase.     

Neurotensin Excitation of Serotonergic Neurons in the Dorsal Raphe Nucleus of the Rat In Vitro

Neurotensin-containing terminals and radioligand binding sites are present in the dorsal raphe nucleus. The purpose of this study was to test, in brain slices containing this nucleus, the effect of neurotensin on the electrical activity of serotonergic neurons. In extracellular recordings, the cells were identified by the ability of the α₁-adrenoceptor agonist phenylephrine to induce firing, and serotonin to reduce this effect. After washout of phenylephrine, neurotensin (10 nM to 10 μM) induced a concentration-dependent increase in the firing rate of serotonergic neurons (EC₅₀= 142 nM; maximum effect ˜1 μM). The neurotensin excitation, which was mimicked by neurotensin fragments 8–13 but not neurotensin peptide fragment 1–8 and selectively blocked by SR 48692 (100 nM), was observed mainly in the ventral part of the nucleus. Most serotonergic neurons showed marked desensitization to neurotensin, even at low concentrations. The neurotensin response was occluded by supramaximal concentrations of phenylephrine. In intracellular recordings using KCl-containing electrodes, neurotensin induced an inward current associated in some cases with a decrease in apparent input conductance. In conclusion, neurotensin was found to have an excitatory action on serotonergic neurons in the ventral part of the dorsal raphe nucleus, an effect which could be subject to desensitization and was occluded by phenylephrine. This occlusion phenomenon may be important for the physiological role of neurotensin in the dorsal raphe nucleus.     

Possible modulatory role of voltage-activated Ca(2+) currents determining the membrane properties of isolated pyramidal neurones of the rat dorsal cochlear nucleus.

Voltage-activated Ca(2+) currents have been studied in pyramidal cells isolated enzymatically from the dorsal cochlear nuclei of 6-11-day-old Wistar rats, using whole-cell voltage-clamp. From hyperpolarized membrane potentials, the neurones exhibited a T-type Ca(2+) current on depolarizations positive to -90 mV (the maximum occurred at about -40 mV). The magnitude of the T-current varied considerably from cell to cell (-56 to -852 pA) while its steady-state inactivation was consistent (E(50)=-88.2+/-1.7 mV, s=-6. 0+/-0.4 mV). The maximum of high-voltage activated (HVA) Ca(2+) currents was observed at about -15 mV. At a membrane potential of -10 mV the L-type Ca(2+) channel blocker nifedipine (10 microM) inhibited approximately 60% of the HVA current, the N-type channel inhibitor omega-Conotoxin GVIA (2 microM) reduced the current by 25% while the P/Q-type channel blocker omega-Agatoxin IVA (200 nM) blocked a further 10%. The presence of the N- and P/Q-type Ca(2+) channels was confirmed by immunochemical methods. The metabotropic glutamate receptor agonist (+/-)-1-aminocyclopentane-trans-1, 3-dicarboxylic acid (200 microM) depressed the HVA current in every cell studied (a block of approximately 7% on an average). The GABA(B) receptor agonist baclofen (100 microM) reversibly inhibited 25% of the HVA current. Simultaneous application of omega-Conotoxin GVIA and baclofen suggested that this inhibition could be attributed to the nearly complete blockade of the N-type channels. Possible physiological functions of the voltage-activated Ca(2+) currents reported in this work are discussed.