Hypothalamic circuitry of neuropeptide Y regulation of neuroendocrine function and food intake via the Y5 receptor subtype.

Neuropeptide Y (NPY) displays diverse modes of action in the CNS including the modulation of feeding behavior, gonadotropin releasing hormone release, and stress responses. Many of the above physiological actions have been at least partially attributed to actions of NPY on the NPY Y5 receptor subtype. We utilized an antibody directed against the NPY Y5 receptor to characterize the distribution of this receptor in the rat brain. Using Western blot analysis, this antibody recognized a single major band at approximately 57 kD. To further verify the specificity of the antibody, animals were treated for 5 days with antisense oligonucleotides for the Y5 receptor. The antisense treatment significantly reduced food intake and body weight. Furthermore, the Y5 antibody detected a significant decrease in Y5 receptor protein. Y5-like immunoreactivity (-ir) was observed throughout the hypothalamus, thalamus, hippocampus and cortex. Double-label immunofluorescence demonstrated that Y5-ir was colocalized with the following neuronal phenotypes in the hypothalamus, gonadotropin-releasing hormone, neurophysins, corticotropin-releasing hormone, and gamma-amino butyric acid. In addition, functional interactions were demonstrated by the presence of close appositions of NPY fibers with Y5-ir expressing cells. The wide distribution of the Y5 receptor-ir, as well as the colocalization within specific neuronal populations, agrees with the distribution of the Y5 receptor mRNA and the known physiological roles of the NPY/Y5 system. The role of the NPY/Y5 receptor system as a mediator between signals of peripheral energy availability and reproductive neuroendocrine function is discussed.

Neuropeptide Y Y5 receptor protein in the cortical/limbic system and brainstem of the rat: expression on gamma-aminobutyric acid and corticotropin-releasing hormone neurons.

Neuropeptide Y displays diverse modes of action in the CNS including the modulation of cortical/limbic function. Some of these physiological actions have been at least partially attributed to actions of neuropeptide Y on the Y5 receptor subtype. We utilized an antibody raised against the Y5 receptor to characterize the distribution of this receptor subtype in the rat cortical/limbic system and brainstem. Y5-like immunoreactivity was located primarily in neuronal cell bodies and proximal dendritic processes throughout the brain. In the cortex, Y5 immunoreactivity was limited to a subpopulation of small gamma-aminobutyric-acid interneurons (approximately 15 microm diameter) scattered throughout all cortical levels. Double label immunofluorescence was also used to demonstrate that all of the Y5 immunoreactive neurons in the cortex displayed intense corticotropin releasing hormone immunoreactivity. The most intense Y5 immunoreactive staining in the hippocampus was located in the pyramidal cell layer of the small CA2 subregion and the fasciola cinerea, with lower levels of staining in the hilar region of the dentate gyrus and CA3 subregion of the pyramidal cell layer. Nearly all of the Y5 immunoreactive neurons in the hilar region of the hippocampus displayed gamma-aminobutyric-acid immunoreactivity. In the brainstem, Y5 immunoreactivity was most intense in the Edinger-Westphal nucleus, locus coeruleus and the mesencephalic trigeminal nucleus. The present study provides neuroanatomical evidence for the possible sites of action of the neuropeptide Y/Y5 receptor system in the control of cortical/limbic function. The presence of Y5 immunoreactivity on cell bodies and proximal dendritic processes in specific regions of the hippocampus suggests that this receptor functions to modulate postsynaptic activity. These data also suggest that the neuropeptide Y/Y5 system may play a role in the modulation of a specific population of GABAergic neurons in the cortex, namely those that contain corticotropin-releasing hormone. The location of the Y5 receptor immunoreactivity fits with the known physiological actions of neuropeptide Y and this receptor.

Neuronal and glial properties coexist in a novel mouse CNS immortalized cell line.

A mes-c-myc A1 (A1) cell line was generated by retroviral infection of cultured embryonic mesencephalic cells and selected by neomycin resistance. A1 cells cease to divide and undergo morphological differentiation after serum withdrawal or addition of c-AMP. Proliferating or morphologically differentiated A1 cells are all positive for vimentin and nestin, a marker of neural precursor, and show neuronal markers such as microtubule-associated protein 1, neuron-specific enolase and peripherin, and the glial marker glial fibrillary acidic protein. Neuronal and glial markers coexist in single cells. Furthermore, A1 cells show presence of glutamic acid decarboxylase 67 mRNA and its embryonic form EP10 and accumulate the neurotransmitter GABA. Electrophysiological studies demonstrate that morphologically differentiated A1 cells display voltage-gated sodium and potassium channels in response to depolarizing stimuli. A1 cells thus represent a novel, bipotent neural cell line useful for studying CNS differentiation and plasticity, as well as the molecular mechanisms underlying development of GABAergic neurotransmission.

Feeding response to neuropeptide Y-related compounds in rats treated with Y5 receptor antisense or sense phosphothio-oligodeoxynucleotide.

Neuropeptide Y (NPY), NPY 3-36 and pancreatic polypeptide (PP) increase short-term (2-h) food intake to varying degrees when given intracerebroventricularly (i.c.v.). Various Y receptor subtypes are proposed to participate in Y receptor ligand-induced stimulation of food intake. Here, we used an antisense phosphothio-oligodeoxynucleotide sequence (-5 relative to the initiating ATG) to the Y5 receptor subtype, which has been suggested to mediate NPY-induced feeding. Rats were treated with i.c.v. antisense or sense phosphothio-oligodeoxynucleotide for 3.5 days before NPY, NPY 3-36, or PP i.c.v. administration. The results show that antisense to the Y5 receptor had no effect on either spontaneous 2-h or NPY-, NPY 3-36-, or PP-stimulated 2-h food intake. However, there was a significant decrease relative to the sense control group in 10-h food intake following the initial 2-h feeding response to NPY (n = 10, p < 0.0001) or NPY 3-36 (n = 10, p < 0.05). The data suggest that the Y5 receptor has a modulatory role in the maintenance of feeding, but not as the critical receptor to confer for NPY and NPY 3-36 action on food intake.

Interaction of 3beta, 5alpha-tetrahydrodeoxycorticosterone in rat and guinea-pig neurons: a comparison of Ca2+ - and GABA(A)-CI- -channel current modulation.

A comparison of the interaction of 3beta, 5alpha-tetrahydrodeoxycorticosterone (TDOC) on voltage-gated Ca2+ -and the gamma-aminobutyric receptor (GABA(A)) gated-Cl- -channels was examined in freshly dissociated guinea-pig (GP) and rat hippocampal CA1 neurons and rat hypothalamic ventromedial nucleus (VMN) neurons. The steady-state inhibition of the peak Ca2+ channel current evoked by depolarized steps from -80 to -10 mV by TDOC increased in concentration-dependent manner with IC50 values of 1 and 6 pM for rat and GP CA1 neurons, respectively and 3 nM for rat VMN neurons. TDOC rapidly and reversibly inhibited a fraction (up to 26%) of the total Ca2+ channel current in all neurons. Intracellular dialysis with GDP-beta-S (500 microM) significantly diminished the TDOC inhibition of the Ca2+ channel current, suggesting a G-protein involvement. In neurons isolated from pertussis-toxin-treated animals by chronic intracerebroventricular (1000 ng/24/48 h) infusion, the TDOC inhibition was also significantly diminished, suggesting modulation by the Galphai and/or Galphao G-protein subunits. The peak GABA-gated inward Cl- current was enhanced in both species from 0.1 to 10 microM with the greatest increase (48% at 10 microM) seen in the VMN. There was no difference in the enhancement of the GABA current in the CA1 region of both species. The results show that in contrast to the 3a-series, the 3beta-series weakly enhance the GABA-evoked Cl- current but potently inhibit the Ca2+ channel current. In addition, these results also suggest a common mode of action and a lack of interspecies difference for this steroid.

Neuropeptide Y-related compounds and feeding.

Neuropeptide Y (NPY) and related compounds increase short-term feeding. Previous studies have used different animal models, feeding schedules, sources of the compounds, and time and routes of administration. These differences in methodology are important in the variability reported on the potency of NPY-related compounds. To obtain reliable data on the relative efficacy, we tested NPY, NPY 3-36, and pancreatic polypeptide (PP) using an identical protocol and the same commercial source. These three NPY-related compounds were tested using the intracerebroventricular (i.c.v., into the third ventricle) administration, and the profile of the feeding enhancement including the dose response and potency was determined. Compounds were tested in parallel on at least 2 successive days. NPY, NPY 3-36, and PP exhibited different potencies in enhancing 2-h food intake. Comparison of their dose responses (using 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 microg/rat) demonstrated an overall potency of NPY 3-36 > NPY > PP for the high doses. To study ligand interactions, we examined the effects of various combinations of NPY-related compounds administered concomitantly. These combinations were justified based on the data obtained from the individual dose responses. The data show that the effects of NPY plus NPY 3-36 or NPY 3-36 plus PP were less than additive. When compared to the individual responses, the effects of NPY 3-36 were almost identical to those induced by the combinations using low doses of NPY plus NPY 3-36, or low and high doses of PP plus NPY 3-36. The results support the notion that NPY and its analogues induce a short-term feeding response by activating multiple receptor subtypes.

In vivo IL-1beta-induced modulation of G-protein alphaO subunit subclass in the hypothalamic ventromedial nucleus: implications to IL-1beta-associated anorexia.

The intracerebroventricular (i.c.v.) administration of interleukin-1beta (IL-1beta) induces anorexia in rats at doses that yield estimated pathophysiological concentrations in the cerebrospinal fluid. IL-1beta also induces anorexia when administered into the hypothalamic ventromedial nucleus (VMN), an important brain site for the control of feeding. A variety of guanine nucleotide binding protein (G-protein) coupled receptors (e. g., for neurotransmitters and neuropeptides) participate in the integrative regulation of feeding. Our previous studies reported that the VMN G-protein alphaO common subunit subclass is involved in the control of normal feeding, and that IL-1beta modulates calcium channel currents via a pertussis toxin (PTX)-sensitive G-protein (GalphaO/Galphai). Here, we examined the profile of GalphaO protein expression in the hypothalamic VMN during IL-1beta-induced anorexia. Intracerebroventricular microinfusion of IL-1beta (0.5 to 8.0 ng/24 h for 72 h) into the third cerebral ventricle dose-dependently induced anorexia (p<0.001) and decreased the VMN GalphaO common protein levels (p<0.001). Heat-inactivated IL-1beta and IL-1beta plus IL-1 receptor antagonist (a competitive inhibitor of IL-1beta action) had no effect on food intake or on VMN GalphaO common protein content. RT-PCR analysis of VMN RNA from IL-1beta-treated rats generated an expression profile for GalphaO common subunit; however, no modulation at the mRNA level was observed. The results suggest that anorexia induced by the central administration of IL-1beta involves modification of G-protein alphaO common subunit profile in the central nervous system.

GABA(A) receptor alpha4 subunit suppression prevents withdrawal properties of an endogenous steroid.

The hormone progesterone is readily converted to 3alpha-OH-5alpha-pregnan-20-one (3alpha,5alpha-THP) in the brains of males and females. In the brain, 3alpha,5alpha-THP acts like a sedative, decreasing anxiety and reducing seizure activity, by enhancing the function of GABA (gamma-aminobutyric acid), the brain's major inhibitory neurotransmitter. Symptoms of premenstrual syndrome (PMS), such as anxiety and seizure susceptibility, are associated with sharp declines in circulating levels of progesterone and, consequently, of levels of 3alpha,5alpha-THP in the brain. Abrupt discontinuation of use of sedatives such as benzodiazepines and ethanol can also produce PMS-like withdrawal symptoms. Here we report a progesterone-withdrawal paradigm, designed to mimic PMS and post-partum syndrome in a rat model. In this model, withdrawal of progesterone leads to increased seizure susceptibility and insensitivity to benzodiazepine sedatives through an effect on gene transcription. Specifically, this effect was due to reduced levels of 3alpha,5alpha-THP which enhance transcription of the gene encoding the alpha4 subunit of the GABA(A) receptor. We also find that increased susceptibility to seizure after progesferone withdrawal is due to a sixfold decrease in the decay time for GABA currents and consequent decreased inhibitory function. Blockade of the alpha4 gene transcript prevents these withdrawal properties. PMS symptoms may therefore be attributable, in part, to alterations in expression of GABA(A) receptor subunits as a result of progesterone withdrawal.

Antisense oligodeoxynucleotides to G-protein alpha-subunit subclasses identify a transductional requirement for the modulation of normal feeding dependent on G alpha OA subunit.

A variety of G-protein-coupled receptors are proposed to participate in the modulation of ingestive behavior and in the mode of action of antiobesity drugs. In the present study, we investigated the involvement of G-protein alpha-subunit subclasses (molecular transducers of multiple chemical signals) in the control of ingestive behavior. We report here that the chronic intracerebroventricular (i.c.v.) microinfusion for 72 h (via osmotic minipumps) with antisense phosphothio-oligodeoxynucleotides corresponding to G-protein alpha-subunitO common (to OA and OB) and OA subclasses decrease the nighttime food intake without affecting water intake in rats. Computerized analyses of the microstructure of feeding indicate that the G alpha OA antisense depresses feeding by reducing meal frequency, while meal size and meal duration increased slightly, but not significantly. The effects of G alpha O common and G alpha OA antisense on feeding are specific since the chronic i.c.v. microinfusion of sense to G alpha O common or G alpha OA, antisense to the related subclass G alpha OB, and antisense to other G-protein alpha-subunits (G alpha S, G alpha Q, G alpha 11 and G alpha i common) had no effect on food or water intake. The observed effects by G alpha O common and G alpha OA antisense imply a direct action in the central nervous system since the chronic subcutaneous microinfusion of G alpha O common and G alpha OA antisense in doses equivalent to two-fold higher relative to those administered centrally had no effect on food intake. The chronic microinfusion of G alpha O common antisense drastically decreased the levels of G alpha O protein detected in immunoblots of hypothalamic ventromedial nuclei. The results suggest that the G-protein alpha-subunit subclass G alpha OA is critical for the integrative modulation of normal feeding behavior, and that changes in its activity may be associated with modifications of feeding. These studies also show a novel approach to study the molecular basis of specific behaviors by manipulating elements of the transductional systems.

Residual Ca2+ channel current modulation by megestrol acetate via a G-protein alpha s-subunit in rat hypothalamic neurones.

1. The inhibition of voltage-activated Ca2+ channel currents by the orally active progesterone derivative, megestrol acetate (MA), was examined in freshly dissociated rat ventromedial hypothalamic nucleus (VMN) neurones using the whole-cell voltage-clamp technique with 10 mM Ba2+ as the charge carrier. 2. The steady-state inhibition of the peak high-threshold Ca2+ channel current evoked by depolarization from -80 to -10 mV by MA increased in a concentration-dependent fashion. MA inhibited a fraction of the whole-cell Ca2+ channel current while progesterone had no effect on the peak Ca2+ channel current (7% at 10 microM). The low-threshold Ca2+ (T-type) current, evoked from -100 to -30 mV, was unaffected by MA. 3. Intracellular dialysis with MA had no effect on the Ca2+ channel current. Concomitant extracellular perfusion of MA showed normal inhibitory activity, suggesting that the MA binding site can only be accessed extracellularly. 4. The high-threshold Ca2+ channel current in VMN neurones was found to consist of four pharmacologically distinguishable components: an N-type current, an L-type current, a P-type current, and a residual current. MA had no effect on the N-, L- and P-type Ca2+ channel currents, but inhibited the residual current. 5. In neurones isolated from cholera toxin-treated animals, the MA-induced inhibition of the Ca2+ channel current was significantly diminished, suggesting a G-protein alpha S-subunit involvement. 6. Treatment with antisense phosphothio-oligodeoxynucleotides to the G alpha S-subunit (antisense-G alpha S) significantly reduced the MA-induced inhibition of the Ca2+ channel current. Treatment with either sense-G alpha S or antisense-G alpha 11 had no effect, confirming a G alpha S-subunit involvement. 7. These results suggest that appetite enhancement induced by MA in cachectic patients may in part be due to a novel central nervous system action, that is, inhibition of a fraction of the whole-cell Ca2+ channel current to attenuate the firing of VMN neurones that may be involved in satiety mechanisms.

Withdrawal from the endogenous steroid progesterone results in GABAA currents insensitive to benzodiazepine modulation in rat CA1 hippocampus.

1. The withdrawal properties of the endogenous steroid progesterone (P) were tested in female rats as a function of benzodiazepine modulation of gamma-aminobutyric acid-A (GABAA)-gated current with the use of the whole cell patch-clamp technique on acutely dissociated CA1 hippocampal neurons. In a previous study, this steroid was shown to exhibit withdrawal properties, behaviorally. 2. One day withdrawal from in vivo administration of physiological doses of P (5 mg ip, 5 days/wk for 3 withdrawal cycles) or its metabolite, the GABAA modulator 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-THP or allopregnanolone, 20 mg/kg ip) prevented the normally potentiating effect of lorazepam (LZM; 10(-7)-10(-4) M) on GABAA-gated current. Withdrawal from 500 micrograms P administered concomitantly with 2 micrograms 17 beta-estradiol also markedly diminished LZM potentiation of GABAA current. This effect was seen only after three withdrawal cycles. 3. P withdrawal produced no inhibitory effect on either basal levels of GABAA-evoked current, the GABAA EC50, or barbiturate (+/-Pentobarbital, 10(-7)-10(-4) M) modulation of this parameter. 4. The effect of steroid withdrawal on LZM modulation of GABAA-evoked current was blocked by picrotoxin as well as by indomethacin, a drug that prevents conversion of P to its metabolite, the GABAA modulator 3 alpha,5 alpha-THP. These results suggest that the withdrawal properties of P may be due to changes in GABAA receptor function produced by 3 alpha,5 alpha-THP.

Cortisol inhibition of calcium currents in guinea pig hippocampal CA1 neurons via G-protein-coupled activation of protein kinase C.

The inhibition of voltage-activated Ca2+ channel currents by cortisol (hydrocortisone), the principal glucocorticoid in man and guinea pig, was examined in freshly dissociated pyramidal neurons from the adult guinea pig hippocampal CA1 region using whole-cell voltage-clamp recordings. Steady-state inhibition by cortisol of the peak Ca2+ channel current evoked by depolarization from -80 to -10 mV increased in a concentration-dependent fashion, with a maximal inhibition of 63 +/- 4% of the total current at 100 microM. Cortisone had a maximal 17 +/- 2% inhibition at 10 microM. Corticosterone and the metabolite allotetrahydrodeoxycorticosterone exhibited a plateau of inhibition of around 15% and 25%, respectively, between 10 pM and 100 nM; both compounds continued to inhibit at concentrations > 10(-7) M. Analysis of tail currents at -80 mV showed that cortisol and corticosterone had no effect on the voltage-dependent activation or deactivation of the Ca2+ channel current. However, cortisol slowed the activation of the current. Cortisol inhibited both the N-type or omega-conotoxin (CgTX)-sensitive, and the L-type or nifedipine (NIF)-sensitive Ca2+ channel current but had no effect on the CgTX/NIF-insensitive Ca2+ channel current. In neurons isolated from pertussis toxin (PTX)-treated animals, the cortisol inhibition was significantly diminished. Intracellular dialysis with GDP-beta-S (500 microM) or with the specific inhibitors of protein kinase C (PKC), the pseudosubstrate PKC inhibitor (PKCI 19-31) (2 microM) and bisindolylmaleimide (BIS) (1 microM) significantly diminished the cortisol inhibition of the Ca2+ channel current. The specific inhibitor of cAMP-dependent protein kinase (PKA) inhibitor, Rp-cAMPS (100 microM) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium current characterization in dissociated adult guinea-pig jugular ganglion neurons.

Voltage-dependent calcium (Ca2+) channel currents in freshly dissociated adult guinea-pig jugular ganglion neurons were examined and characterized using the whole-cell patch-clamp technique. Electrophysiological analysis demonstrated a high-threshold current, but no low-threshold or T-type current. A fraction of the total Ca2+ current was inhibited by omega (omega)-conotoxin GVIA (Cg (inTX; 10 microM); the dihydropyridine antagonist nifedipine (NIF; 10 microM), inhibited a large fraction of the CgTX-insensitive current. The remaining CgTX/NIF-insensitive current was completely inhibited by omega-agatoxin IVA (AgIVA; 100 nM). These results demonstrated that the whole-cell Ca2+ channel current consisted only of N-, L- P-type components. Of these currents, only the L-type was partially inhibited by both histamine and carbachol (0.01-100 microM).

Neurosteroids modulate calcium currents in hippocampal CA1 neurons via a pertussis toxin-sensitive G-protein-coupled mechanism.

The inhibition of Ca2+ channel currents by endogenous brain steroids was examined in freshly dissociated pyramidal neurons from the adult guinea pig hippocampal CA1 region. The steady-state inhibition of the peak Ca2+ channel current evoked by depolarizing steps from -80 to -10 mV occurred in a concentration-dependent manner with the following IC50 values: pregnenolone sulfate (PES), 11 nM; pregnenolone (PE), 130 nM; and allotetrahydrocorticosterone (THCC), 298 nM. THCC, PE, and PES depressed a fraction of the Ca2+ channel current with a maximal inhibition of 60% of the total current. However, substitution of an acetate group for the sulfate group on PES resulted in a complete loss of activity. Progesterone had no effect (4% inhibition at 100 microM). Intracellular dialysis of PES had no effect on the Ca2+ current; concomitant extracellular perfusion of PES showed normal inhibitory activity, suggesting that the steroid binding site can only be accessed extracellularly. Analysis of tail currents at -80 mV demonstrated that THCC and PES slowed the rate of Ca2+ current activation and deactivation with no change in the voltage dependence of activation. Inhibition of the Ca2+ channel current by THCC and PES was voltage dependent. THCC primarily inhibits the omega-conotoxin (CgTX)-sensitive or N-type Ca2+ channel current. PE was nonselective in inhibiting both the CgTX- and the nifedipine (NIF)-sensitive Ca2+ channel current. These neurosteroids had no effect on the CgTX/NIF-insensitive current. In neurons isolated from pertussis toxin (PTX)-treated animals by chronic intracerebroventricular infusion (1000 ng/24 hr for 48 hr), the Ca2+ channel current inhibition by PES, PE, and THCC was significantly diminished. Intracellular dialysis with GDP-beta-S (500 microM) also significantly diminished the neurosteroid inhibition of the Ca2+ channel current. Intracellular dialysis with the general kinase inhibitors H-7 (100 microM), staurosporine (400 nM), and a 20 amino acid protein kinase inhibitor (1 microM) also significantly prevented the THCC and PES inhibition of the Ca2+ channel current. Intracellular dialysis with the more specific inhibitors of protein kinase C (PKC), the pseudosubstrate inhibitor (PKCI 19-36) (1-2 microM) and bisindolylmaleimide (1 microM) significantly diminished the THCC and PE inhibition of the Ca2+ channel current. Rp- cAMP (100 microM), a specific inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the THCC and PE inhibition of the Ca2+ current.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscarine modulation by a G-protein alpha-subunit of delayed rectifier K+ current in rat ventromedial hypothalamic neurones.

1. Rat cultured ventromedial hypothalamic (VMH) neurones obtained from embryonic hypothalamus were used to study the muscarinic (carbachol) modulation of voltage-gated K+ currents with the whole-cell patch-clamp technique. 2. Carbachol produced a potent and concentration-dependent (100 fM to 100 microM) decrease of the outward delayed rectifier K+ current (IK) with an IC50 of 44 pM and a Hill coefficient of 0.4. The carbachol-induced depression of IK was reduced by pirenzepine (1-10 microM) and atropine (1 microM). Carbachol had no effect on the transient outward K+ current (IA). 3. Intracellular dialysis with guanosine 5'-O-(2-thiodiophosphate) (GDP-beta-S, 500 microM) significantly diminished the carbachol-induced depression of IK, suggesting GTP-binding protein (G-protein) involvement. Pre-incubation of VMH neurones with pertussis toxin (200-400 ng ml-1) or cholera toxin (1 microgram ml-1) for 24-48 h had no effect on the carbachol-induced depression of IK. This suggested that the G alpha o, G alpha i, and G alpha s G-protein alpha-subunits were not involved in mediating the carbachol-induced depression of IK in VMH neurones. 4. Treatment (24-48 h) of VMH neurones with antisense phosphothio-oligodeoxynucleotides to the G alpha 11 G-protein subunit (10 microM) significantly diminished the carbachol-induced depression of IK. Treatment with 10 microM of either G alpha 11 sense or antisense to G alpha q had no effect. 5. These results demonstrate a novel and potent muscarinic depression of IK in VMN neurones, and that this depression is specifically mediated by the G alpha 11 G-protein subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 beta inhibits Ca2+ channel currents in hippocampal neurons through protein kinase C.

Interleukin-1 beta depresses the voltage-gated Ca2+ channel currents in acutely dissociated guinea-pig hippocampal CA1 neurons. This depression is observed with pathophysiological concentrations found in the cerebrospinal fluid (> or = 1.0 pg interluekin-1 beta/10 microliters). Interleukin-1 receptor antagonist (in concentrations 25-fold higher than interleukin-1 beta) completely blocked the interleukin-1 beta-induced depression of the Ca2+ channel current. This suggests that interleukin-1 beta action is through a specific interaction with an interleukin-1 membrane receptor site. The application of other cytokines and growth factors (interleukin-6, epidermal growth factor, and basic fibroblast growth factor), or bacterial lipopolysaccharide (endotoxin) had no effect, indicating specificity of action of interleukin-1 beta. The depression of the Ca2+ channel current by interleukin-1 beta was prevented by the extracellular application of pertussis toxin, and by the intracellular application of GDP[beta S], H-7, staurosporine or bisindolylmaleimide. Application of phorbol 12-myristate 13-acetate also depressed the Ca2+ channel current, but this phorbol ester-induced depression was not additive to that induced by interleukin-1 beta. These results suggest mediation of interleukin-1 beta action through a pertussis toxin-sensitive G-protein coupled interleukin-1 receptor associated with the activation of protein kinase C. The depression of the Ca2+ channel current by interleukin-1 beta may be involved in the regulation of neuronal excitability during pathological conditions and in the induction and/or progression of neurodegenerative processes.

Calcium current block by (-)-pentobarbital, phenobarbital, and CHEB but not (+)-pentobarbital in acutely isolated hippocampal CA1 neurons: comparison with effects on GABA-activated Cl- current.

Block of a voltage-activated Ca2+ channel current by phenobarbital (PHB), 5-(2-cyclohexylideneethyl)-5-ethyl barbituric acid (CHEB), and the optical R(-)- and S(+)-enantiomers of pentobarbital (PB) was examined in freshly dissociated adult guinea pig hippocampal CA1 neurons; the effects of the barbiturates on GABA-activated Cl- current were also characterized in the same preparation. (-)-PB, PHB, and CHEB produced a reversible, concentration-dependent block of the peak Ca2+ channel current (3 mM Ba2+ as the charge carrier) evoked by depolarization from -80 to -10 mV (IC50 values, 3.5, 72, and 118 microM, respectively). In contrast, (+)-PB was nearly inactive at concentrations up to 1 mM. The inhibitory action of PHB was decreased at acid pH, indicating that the dissociated (anionic) form of the molecule is the active species. Block by (-)-PB was voltage dependent with the fractional block increasing at positive membrane potentials; calculations according to the method of Woodhull indicated that the (-)-PB blocking site senses approximately 40% of the transmembrane electric field. The time course and voltage dependence of activation of the Ca2+ channel current were unaffected by (-)-PB, PHB, and CHEB. The rate of inactivation was enhanced by (-)-PB and CHEB, with the major effect being acceleration of the slow phase of the biexponential decay of the current. GABA-activated Cl- current was potently enhanced by (-)-PB and PHB (EC50 values, 3.4 and 12 microM), whereas (+)-PB was only weakly active. At concentrations of (-)-PB > 100 microM and PHB > 200-300 microM, Cl- current responses were activated even in the absence of GABA. These results demonstrate that in CA1 hippocampal neurons, PB causes a stereoselective block of a voltage-activated Ca2+ current; PHB is also effective, but at higher concentrations. For (-)-PB, the effect on Ca2+ channel current occurred at similar concentrations as potentiation of GABA responses. In contrast, PHB was more potent as a GABA enhancer than as blocker of Ca2+ current, but the maximal potentiation of GABA responses was 40% of that obtained with (-)-PB. Consequently, the anticonvulsant action of PHB at clinically relevant concentrations may relate to modest enhancement of GABA responses and partial blockade of Ca2+ current, whereas the sedative effects that occur at higher concentrations could reflect stronger Ca2+ current blockade. The powerful sedative-hypnotic action of (-)-PB may reflect greater maximal enhancement of GABA responses in conjunction with strong inhibition of Ca2+ current.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin-2 modulates calcium currents in dissociated hippocampal CA1 neurons.

Interleukin-2 and its receptors are present in hippocampal structures, particularly the pyramidal cell layer. In the present study, we examined the interaction between recombinant human interleukin-2 (rhIL-2) and the voltage-dependent calcium (Ca2+) current using the whole-cell patch-clamp technique. RhIL-2 depressed a fraction of the inward Ca2+ current in acutely dissociated guinea-pig hippocampal CA1 neurons. This depression is rapid, dose-dependent with 0.001-1.0 ng rhIL-2/10 microliters (6.8 x 10(-12) to 6.8 x 10(-9) M) and reversible in > 80% of the neurons. Heat-inactivated rhIL-2 had no effect. The application of anti-rhIL-2 monoclonal antibody inhibited the rhIL-2-induced depression of the Ca2+ current. The depression of the Ca2+ current by IL-2 may play a role in the induction and/or progression of neuropathological processes of immunological origin, and in the induction of neurological manifestations during IL-2 immunotherapy.

Zinc modulates transient outward current gating in hippocampal neurons.

The actions of zinc ions (Zn2+) on transient outward current (TOC) were studied in cultured embryonic rat hippocampal neurons and adult guinea pig CA1 neurons acutely isolated from hippocampal slices. Zn2+ (1-1000 microM) shifted both the activation and inactivation curves for the TOC in the depolarizing direction. These effects of Zn2+ were modeled, assuming a single class of binding sites for Zn2+, yielding apparent dissociation constants for Zn2+ between 10 and 35 microM. In addition, the activation kinetics of the TOC were significantly slowed by Zn2+; deactivation was slowed at the highest concentrations of Zn2. These complex actions of Zn2+ on the TOC are likely to delay repolarization and promote spontaneous activity, resulting in action potential prolongation and hyperexcitability of hippocampal neurons.

Phencyclidine block of calcium current in isolated guinea-pig hippocampal neurones.

1. Phencyclidine (PCP) block of Ca2+ channel current in enzymatically dissociated neurones from the CA1 region of the adult guinea-pig hippocampus was studied using whole-cell voltage clamp techniques. Ca2+ channel current was recorded with 3 mM-Ba2+ as the charge carrier. Na+ currents were blocked with tetrodotoxin and K+ currents were eliminated by using tetraethylammonium and N-methyl-D-glucamine as the predominant extracellular and intracellular cations, respectively. 2. Peak Ca2+ channel current evoked by depolarization from -80 to -10 mV was reduced in a use-dependent fashion by PCP. The apparent forward and reverse rate constants for block at the depolarized voltage were 10(6) s-1 M-1 and 11-14 s-1, respectively. These values were at least 60 times faster than the corresponding rates at the resting voltage. The steady-state block produced by PCP increased in a concentration-dependent fashion with an IC50 of 7 microM. Other dissociative anaesthetic drugs were substantially weaker inhibitors of the current (tiletamine > dizocilpine (MK-801) > ketamine). 3. The Ca2+ channel current recorded under identical conditions in rat dorsal root ganglion neurones was less sensitive to blockade by PCP (IC50, 90 microM). 4. PCP block of the hippocampal Ca2+ channel current occurred in a voltage-dependent fashion with the fractional block decreasing at positive membrane potentials. Analysis indicated that the PCP blocking site senses 56% of the transmembrane electric field. 5. Analysis of tail currents recorded at -80 mV demonstrated that PCP does not affect the voltage-dependent or time-dependent activation or deactivation of the Ca2+ channel current. 6. The rate and extent of inactivation of the Ca2+ channel current was maximal at -10 mV and diminished at more positive potentials. Experiments with Ba(2+)-free external solution demonstrated that inactivation of the Ca2+ channels is largely voltage-dependent and is not affected by Ba2+ influx. 7. PCP markedly increased the apparent extent of inactivation of the Ca2+ channel current during prolonged voltage steps. This increase in apparent inactivation was more pronounced at depolarized potentials. Inactivation at -10 mV proceeded in two exponential phases; PCP had little effect on the fast decay phase and caused a moderate speeding of the slow decay phase. Although block of the activated state evolved on the same time scale as inactivation, the apparent rate of inactivation was not increased in a concentration-dependent fashion by PCP indicating that the block does not occur by a conventional open channel mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)