Cyclic adenosine 3'5'-monophosphate potentiates excitatory amino acid and synaptic responses of rat spinal dorsal horn neurons.

Intracellular recordings were made from rat dorsal horn neurons in the in vitro slice preparation to study the actions of cyclic adenosine 3',5'-monophosphate (cyclic AMP). In the presence of TTX, bath application of the membrane permeable analogue of cyclic AMP, 8-Br cyclic AMP (25-100 microM) caused a small depolarization of the resting membrane potential accompanied by a variable change in membrane input resistance. In addition, 8-Br cyclic AMP caused a long-lasting increase in the spontaneous synaptic activity and the amplitude of presumed monosynaptic excitatory postsynaptic potentials evoked in the substantia gelatinosa neurons by orthodromic stimulation of a lumbar dorsal root. When the fast voltage-sensitive Na conductance was blocked by TTX, 8-Br cyclic AMP enhanced in a reversible manner, the depolarizing responses of a proportion of dorsal horn neurons to N-methyl-D-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), quisqualic acid (QA) and kainic acid (KA). The effects of 8-Br cyclic AMP on the resting membrane potential and the NMDA response of dorsal horn neurons were mimicked by reducing phosphodiesterase activity with bath application of 3-isobutyl-1-methylxanthine, but not by cyclic AMP applied extracellularly. Moreover, we have found that intracellular application of a protein inhibitor of cyclic AMP-dependent protein kinase (PKI) into dorsal horn neurons prevents the 8-Br cyclic AMP-induced potentiation of the NMDA response of these cells. These results suggest that in the rat spinal dorsal horn the activation of the adenylate cyclase-cyclic AMP-dependent protein kinase system may be involved in the enhancement of the sensitivity of postsynaptic excitatory amino acid (NMDA, AMPA, KA) receptors and modulation of primary afferent neurotransmission, including nociception.     

Cyclic AMP accumulation alters calmodulin localization in SK-N-SH human neuroblastoma cells.

In SK-N-SH human neuroblastoma cells, the muscarinic agonist carbachol promotes polyphosphoinositide (PPI) hydrolysis via M3 receptors and increases cyclic AMP levels through an unidentified mechanism. Activation of PPI hydrolysis by carbachol elicits a robust translocation of CaM from membranes into cytosol which was previously shown to be mimicked by the addition of the calcium ionophore ionomycin and the phorbol ester TPA28. The effect of agonist-stimulated second messenger production on CaM localization was determined by activating receptors that increase and decrease adenylyl cyclase activity on SK-N-SH cells. VIP (10 microM), prostaglandin E1 (30 microM) and forskolin (10 microM) all increased adenylyl cyclase activity 8- to 10-fold above the activity with 1 microM GTP. Carbachol (100 microM) did not stimulate adenylyl cyclase activity. The alpha 2-adrenergic agonist UK 14,304 (0.1 microM) and the delta and mu opioid DPDPE (10 microM) and DAMGO (10 microM) inhibited forskolin-stimulated cyclic AMP formation by 27-32%. CaM did not stimulate adenylyl cyclase activity. Incubation of cells with vasoactive intestinal polypeptide (VIP), dibutyryl cyclic AMP and forskolin, resulted in 30% decrease in membrane CaM and an increase in cytosolic CaM of 40-50%. The CaM translocation with the combination of an agent that elevates cyclic AMP levels and a low dose of carbachol was not different from that observed with either agent alone. UK 14,304, DPDPE and DAMGO potentiated carbachol-stimulated increases in cytosolic CaM. Upon the addition of carbachol, a 5-fold increase in intracellular calcium concentration measured with fura-2 fluorescence was observed. VIP and UK 14,304 elevated intracellular calcium concentrations 2 to 3 fold, while forskolin (10 microM) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of Noradrenergic and Cholinergic Agonists with Ligands Increasing K-conductance of Guinea Pig Hippocampal Neurons,in vitro

Single electrode current clamp and voltage clamp recordings were employed to study the effects of noradrenergic agonists and a cholinergic agonist (carbachol, Cch) on the resting membrane potential of CA3 neurons in guinea pig hippocampal slices. Stimulation of muscarinic and beta-adrenergic receptors depolarized, and stimulation of alpha1-adrenergic receptor hyperpolarized, CA3 neurons but the membrane potential changes were small. Hyperpolarizations or outward currents induced by baclofen, adenosine or serotonin (5-HT) were strongly potentiated by alpha-noradrenergic agonists and suppressed by Cch at concentrations ten times lower than those having any direct effects on membrane potential. Both the enhancement of the baclofen-induced hyperpolarization by phenylephrine and its suppression by Cch were pronounced at low concentrations of baclofen, but diminished at higher concentrations. The modulatory effects persisted after blockade of sodium spikes by tetrodotoxin and after blockade of fast inhibitory and excitatory synaptic transmission by picrotoxin and 6-cyano-7-nitroquinoxaline-2,3-dione. Our data suggest that, through the postsynaptic interaction with ligands activating potassium conductance, noradrenergic and muscarinic receptor stimulation can exert a stronger inhibitory and excitatory effect on CA3 pyramidal neurons at their resting membrane potential than would be expected from the changes in membrane potential induced by these neuromodulators on their own.     

Dopamine action on hippocampal pyramidal cells

Dopamine (DA) was applied to CA1 region pyramidal cells in slices of guinea pig hippocampus maintained in vitro in order to examine its electrophysiological effect on CNS neurons. DA induced hyperpolarization of membrane potential and an increased conductance in 75% the 21 CA1 neurons to which it was applied. DA also augmented the afterhyperpolarizations and increased conductance which normally follow spike trains in these neurons. These effects were not altered by intracellular injections of Cl- but were blocked when slices were bathed in Mn2+ solutions. The Mn2+ blockade of DA-induced hyperpolarizations could be overcome when large amounts of agonists were applied. The DA effects were long lasting, were mimicked by the dopamine agonists apomorphine and Epinine, and were blocked by the dopamine antagonists flupenthixol and chlorpromazine. Extracellular or intracellular application of cyclic AMP mimicked the effects of DA. The results suggest that DA-induced hyperpolarization and conductance changes are mediated by a Ca2+-activated K+ conductance. DA may increase the intracellular Ca2+ concentration through effects on one of the Ca2+ buffering mechanisms. The long duration of these effects suggest that DA works though some intracellular intermediary, perhaps cyclic AMP, considering that the actions of cyclic AMP on membrane properties are similar to those of DA. The dopaminergic projection to the hippocampus should have a powerful inhibitory action, which would be most effective in modulating the activities of neurons exhibiting high levels of excitability, particularly cells involved in cyclical burst generation.     

The hyperpolarization‐activated non‐specific cation current (Ih) adjusts the membrane properties,excitability, and activity pattern of the giant cells in the rat dorsal cochlear nucleus

Giant cells of the cochlear nucleus are thought to integrate multimodal sensory inputs and participate in monaural sound source localization. Our aim was to explore the significance of a hyperpolarization‐activated current in determining the activity of giant neurones in slices prepared from 10 to 14‐day‐old rats. When subjected to hyperpolarizing stimuli, giant cells produced a 4‐(N‐ethyl‐N‐phenylamino)‐1,2‐dimethyl‐6‐(methylamino) pyridinium chloride (ZD7288)‐sensitive inward current with a reversal potential and half‐activation voltage of –36 and –88 mV, respectively. Consequently, the current was identified as the hyperpolarization‐activated non‐specific cationic current (I_h). At the resting membrane potential, 3.5% of the maximum I_h conductance was available. Immunohistochemistry experiments suggested that hyperpolarization‐activated, cyclic nucleotide‐gated, cation non‐selective (HCN)1, HCN2, and HCN4 subunits contribute to the assembly of the functional channels. Inhibition of I_h hyperpolarized the membrane by 6 mV and impeded spontaneous firing. The frequencies of spontaneous inhibitory and excitatory postsynaptic currents reaching the giant cell bodies were reduced but no significant change was observed when evoked postsynaptic currents were recorded. Giant cells are affected by biphasic postsynaptic currents consisting of an excitatory and a subsequent inhibitory component. Inhibition of I_h reduced the frequency of these biphasic events by 65% and increased the decay time constants of the inhibitory component. We conclude that I_h adjusts the resting membrane potential, contributes to spontaneous action potential firing, and may participate in the dendritic integration of the synaptic inputs of the giant neurones. Because its amplitude was higher in young than in adult rats, I_h of the giant cells may be especially important during the postnatal maturation of the auditory system.     

12-O-tetradecanoyl-phorbol-13-acetate-induced differentiation of human neuroblastoma cells is not accompanied by an increase in the intracellular concentration of cyclic AMP

Cultured human SH-SY5Y neuroblastoma cells could be induced to differentiate morphologically and biochemically followed by growth inhibition, by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The cells showed a limited differentiation when treated with substances known to increase the intracellular concentration of cyclic AMP. When these substances were combined with TPA, morphological differentiation and growth inhibition of the cells were potentiated. In contrast, these substances inhibited the TPA-induced increase in noradrenaline concentration and the relative activity of neuron-specific enolase. Both the intracellular concentration of cyclic AMP and the cytosolic level of cyclic AMP-binding components were similar in control and TPA-treated cells. It is suggested that cyclic AMP has a limited and non-regulatory role in the initiation of differentiation of SH-SY5Y cells. The effect of cyclic AMP is probably coupled mainly to the polymerization of microtubules, thus enhancing the morphological differentiation of the cells.     

Long-term potentiation and depression in layer III and V pyramidal neurons of the cat sensorimotor cortex in vitro

Synaptic plasticity of the cat sensorimotor cortex was examined intracellularly in vitro. After tetanic stimulation of the white matter, layer III and V pyramidal neurons showed long-term potentiation (LTP) of EPSPs in high incidence without GABA_A antagonist. The incidence and magnitude of LTP were very conspicuous in layer V cells. After an NMDA receptor antagonist application, the synaptic potentiation was blocked completely in layer III but not in layer V cells. Long-term depression (LTD) of the evoked EPSPs was also induced by the same stimulation in some layer III cells, where a transient hyperpolarization of the membrane potential was observed during tetanus.© 1997 Elsevier Science B.V. All rights reserved.     

A neuroglial cooperativity is required for the potentiation by 2-chloroadenosine of the muscarinic-sensitive phospholipase C in the striatum

In rat striatal slices, 2-chloroadenosine, which had no direct effect on inositol phosphate formation, potentiated in a dose-dependent manner the accumulation of inositol phosphates induced either by carbamylcholine (10(-3) M) or by noradrenaline (10(-4) M). Experiments made on pure populations of striatal neurons or striatal glial cells in primary culture from mouse embryos indicated that 2-chloroadenosine potentiated the noradrenaline-elicited phosphoinositide breakdown in striatal glial cultures but did not modify the responses evoked either by noradrenaline or by carbamylcholine in striatal neuronal cultures. However, 2-chloroadenosine enhanced both the carbamylcholine and the noradrenaline-induced accumulation of inositol phosphates in neuroglial cocultures just as it did in rat striatal slices. The potentiation by 2-chloroadenosine of the carbamylcholine response, which is neuron specific, involved a cooperative effect between neurons and glial cells and, as shown by additional experiments, required a brief contact only between the 2 types of cells. The potentiating effect of 2-chloroadenosine was blocked completely by a nonselective A1, A2 adenosine antagonist isobutylmethylxanthine either on rat striatal slices or on mouse embryonic cocultures (noradrenaline and carbamylcholine responses) or on mouse embryonic glial cultures (noradrenaline response). These data indicate the involvement of an extracellular membrane-bound adenosine receptor, possibly of the A1 subtype since N6-cyclohexyladenosine, an A1 adenosine receptor agonist, was more efficient than 5'-N-ethylcarboxamide-adenosine, a rather selective A2 adenosine receptor agonist. We propose that 2-chloroadenosine acts through an adenosine receptor located on glial cells and induces the synthesis of a substance that improves the coupling between carbamylcholine or noradrenaline and phospholipase C located in glial cells or neurons.     

Glial receptors and their intervention in astrocyto–astrocytic and astrocyto–neuronal interactions

As shown on cultured astrocytes from the mouse, in the presence of adenosine deaminase, 2-chloroadenosine by acting on A1-adenosine receptors potentiated the activation of phospholipase C induced by the α1-adrenergic agonist, methoxamine. This potentiation required the presence of external calcium and was blocked by pertussis toxin. Moreover, this potentiation resulted from a cascade of events: activation (by calcium and protein kinase C) of a phospholipase A2 coupled to A1-adenosine receptors, release of arachidonic acid, which inhibited the reuptake of glutamate into astrocytes and finally additional activation of phospholipase C by externally accumulated glutamate through metabotropic receptors. The effects of2-chloroadenosine and methoxamine were respectively mimicked by somatostatin and substance P while endothelins reproduced the combined effects of 2-chloroadenosine and methoxamine. Conditioned media from treated astrocytes enriched in glutamate stimulated phospholipase C in cultured striatal neurones. In addition, glutamate alone was also found to stimulate phospholipase A2 in astrocytes through receptors exhibiting a pharmacological profile distinct from metabotropic receptors coupled to phospholipase C and the glutamate response was potentiated by ATP. Moreover, the neuronal arachidonic acid production evoked by glutamate was potentiated by acetylcholine. Finally, the combined application of 2-chloroadenosine and methoxamine on striatal astrocytes reduced the permeability of gap junctions between astrocytes and this response was mimicked by arachidonic acid. Together, these results emphasized the contribution of astrocytes in the regulation of glutamatergic transmission. © 1994 Wiley-Liss, Inc.     

Muscarinic agonists depress calcium-dependent gK in bullfrog sympathetic neurons

Intracellular recordings were made from 'fast B' [9] neurons in bullfrog sympathetic ganglia. A single soma action potential was followed by a prolonged after-hyperpolarization lasting for several hundred milliseconds up to 2 s. The spike afterhyperpolarization, which is generated by calcium-dependent potassium conductance increase (gKCa) [3,20-24], was shortened by the muscarinic action of acetylcholine and oxotremorine (30-300 nM). These concentrations of muscarinic agonists were too low to cause any detectable changes in resting membrane potential, input resistance or action potential wave form. ACh released from presynaptic terminal under a physiological condition also caused the shortening of the calcium-dependent hyperpolarization. The results suggested that the shortening of calcium-dependent spike afterhyperpolarization may permit the neuron to pass the high frequency of discharge during the muscarinic excitation.     

Sensitivity of postsynaptic GABAB receptors on hippocampal CA1 and CA3 pyramidal neurons to ethanol

Baclofen-induced hyperpolarization of hippocampal CA1 and CA3 pyramidal neurons was examined to assess the impact of ethanol on postsynaptic GABA_B receptors. These receptors activate outward K⁺ currents via a pertussis toxin-sensitive G protein cascade to reduce membrane potential during the slow inhibitory postsynaptic potential. This inhibitory action may play a role in ethanol intoxication and withdrawal excitability. In both types of pyramidal neurons, baclofen applied consecutively in increasing concentrations caused concentration dependent hyperpolarization. There were no significant differences in resting membrane potential, input resistance, maximum baclofen-induced hyperpolarization or EC₅₀ between CA1 and CA3 neurons, although slope values were significantly smaller in the former neurons. These parameters were not significantly changed in the presence of ethanol 10–100 mM. Chronic ethanol treatment (12 days) sufficient to induce physical dependence also did not shift sensitivity or maximum response to baclofen in CA1 neurons. These results suggest that GABA_B receptors in this model are essentially insensitive to ethanol and do not confirm our earlier preliminary observation of a possible down-regulation of postsynaptic GABA_B receptor function by chronic ethanol treatment.     

Degeneration of CA1 neurons in hippocampus after ischemia in Mongolian gerbils: Cyclic AMP-systems

Bilateral ischemia induced by occlusion of the carotid arteries for 5 min causes a selective degeneration of CA 1 neurons of the hippocampus of Mongolian gerbils. The degeneration process is complete in 14 days as assessed by light microscopy. After one week, basal values for radioactive cyclic AMP in [3H]adenine-labeled tissue from the CA 1 region of hippocampus are greatly reduced as are the absolute magnitude of accumulations of cyclic AMP elicited by norepinephrine, 2-chloroadenosine and histamine. At 2 and 4 weeks, basal values for radioactive cyclic AMP have nearly attained control values and the response to 2-chloroadenosine is fully restored. The response of cyclic AMP-generating systems to norepinephrine is now significantly greater than in control, while the response to histamine remains reduced in magnitude. The ischemia has no effect on basal values for radioactive cyclic AMP or on responses in [3H]adenine-labeled slices from cerebral cortex. Histamine levels after ischemia are significantly increased above control in the CA 1 region. Basal and histamine-sensitive adenylate cyclase activity in the membrane preparations are slightly decreased during the first week after ischemia, followed by a recovery. There is an inverse correlation between histamine levels and adenylate cyclase activity in individual animals 4 days after ischemia.     

β-Adrenergic potentiation of E–C coupling increases force in rat skeletal muscle

We examined the mechanism(s) which allow terbutaline, a β₂-adrenergic agonist, to increase isometric force in bundles of normal and denervated rat soleus fibers. Terbutaline (10 μmol/L) potentiated tetanic contractions during exposure to 1 mmol/L ouabain, 10 μmol/L nifedipine, or 0.5 mmol/L iodoacetate. Terbutafine induced equivalent increases in submaximal potassium (K⁺) contracture and tetanic force: these effects were mimicked by 2 mmol/L dibutyrl-cyclic AMP. Therefore, terbutaline increased force by a cyclic AMP-dependent mechanism other than enhancement of sodium-pump activity, dihydropyridine sensitive Ca²⁺ currents, glycolysis, or action potentials. Pretreatment with 1 mmol/L caffeine induced submaximal potentiation of peak tetanic force but prevented further potentiation by terbutaline. This suggested that terbutaline did not influence the myofilaments, but acted on the sarcoplasmic reticulum (SR) to increase the myoplasmic Ca²⁺ concentration and hence force production. We speculate that force is potentiated following β-adrenoceptor activation by a cyclic AMP-dependent phosphorylation of Ca²⁺ release channels to facilitate SR calcium release during tetanic stimulation. © 1993 John Wiley & Sons, Inc.     

Differential intracellular regulation of cortical GABAA and spinal glycine receptors in cultured neurons

Using patch-clamp techniques we studied several aspects of intracellular GABA_A and glycine Cl⁻ current regulation in cortical and spinal cord neurons, respectively. Activation of PKA with a permeable analog of cyclic AMP (cAMP) produced a potentiation of the Cl⁻ current activated with glycine, but not of the current induced with GABA. The inactive analog was without effect. Activation of PKC with 1 μM PMA reduced the amplitude of the GABA_A and glycine currents. Internal application of 1 mM cGMP, on the other hand, had no effect on the amplitude of either current. The amplitude of these inhibitory currents changed slightly during 20 min of patch-clamp recording. Internal perfusion of the neurons with 1 μM okadaic acid, a phospatase inhibitor, induced potentiation in both currents. The amplitude of GABA_A and glycine currents recorded with 1 mM internal CaCl₂ and 10 mM EGTA (10 nM free Ca²⁺) decayed by less than 30% of control. Increasing the CaCl₂ concentration to 10 mM (34 μM free Ca²⁺) induced a transient potentiation of the GABA_A current. A strong depression of current amplitude was found with longer times of dialysis. The glycine current, on the contrary, was unchanged by increasing the intracellular Ca²⁺ concentration. Activation of G proteins with internal FAl⁻₄ induced an inhibition of the GABA_A current, but potentiated the amplitude of the strychnine-sensitive Cl⁻ current. These results indicate that GABA_A and glycine receptors are differentially regulated by activation of protein kinases, G proteins and Ca²⁺. This conclusion supports the existence of selectivity in the intracellular regulation of these two receptor types.     

Chrna2‐OLM interneurons display different membrane properties and h‐current magnitude depending on dorsoventral location

The hippocampus is an extended structure displaying heterogeneous anatomical cell layers along its dorsoventral axis. It is known that dorsal and ventral regions show different integrity when it comes to functionality, innervation, gene expression, and pyramidal cell properties. Still, whether hippocampal interneurons exhibit different properties along the dorsoventral axis is not known. Here, we report electrophysiological properties of dorsal and ventral oriens lacunosum moleculare (OLM) cells from coronal sections of the Chrna2‐cre mouse line. We found dorsal OLM cells to exhibit a significantly more depolarized resting membrane potential compared to ventral OLM cells, while action potential properties were similar between the two groups. We found ventral OLM cells to show a higher initial firing frequency in response to depolarizing current injections but also to exhibit a higher spike‐frequency adaptation than dorsal OLM cells. Additionally, dorsal OLM cells displayed large membrane sags in response to negative current injections correlating with our results showing that dorsal OLM cells have more hyperpolarization‐activated current (I_h) compared to ventral OLM cells. Immunohistochemical examination indicates the h‐current to correspond to hyperpolarization‐activated cyclic nucleotide‐gated subunit 2 (HCN2) channels. Computational studies suggest that I_h in OLM cells is essential for theta oscillations in hippocampal circuits, and here we found dorsal OLM cells to present a higher membrane resonance frequency than ventral OLM cells. Thus, our results highlight regional differences in membrane properties between dorsal and ventral OLM cells allowing this interneuron to differently participate in the generation of hippocampal theta rhythms depending on spatial location along the dorsoventral axis of the hippocampus.     

Characterization of Rebound Depolarization in Neurons of the Rat Medial Geniculate Body In Vitro

Rebound depolarization(RD) is a response to the offset from hyperpolarization of the neuronal membrane potential and is an important mechanism for the synaptic processing of inhibitory signals. In the present study, we characterized RD in neurons of the rat medial geniculate body(MGB), a nucleus of the auditory thalamus, using whole-cell patch-clamp and brain slices. RD was proportional in strength to the duration and magnitude of the hyperpolarization; was effectively blocked by Ni2or Mibefradil; and was depressed when the resting membrane potential was hyperpolarized by blocking hyperpolarization-activated cyclic nucleotide-gated(HCN)channels with ZD7288 or by activating G-protein-gated inwardly-rectifying K(GIRK) channels with baclofen.Our results demonstrated that RD in MGB neurons, which is carried by T-type Ca2channels, is critically regulated by HCN channels and likely by GIRK channels.     

Vasoactive intestinal peptide elevates cyclic AMP levels and potentiates secretion in bovine adrenal chromaffin cells

Vasoactive intestinal peptide (VIP) elevates the levels of cyclic AMP in primary cultures of highly purified chromaffin cells isolated from the bovine adrenal medulla, an effect that is potentiated by inhibitors of cyclic nucleotide phosphodiesterases. This elevation occurs within minutes and elevated levels of cyclic AMP are observed for as long as 24 hr in culture. The half-maximally effective concentration of VIP was approximately 1 microM. The VIP-induced elevation in chromaffin cell cyclic AMP levels was potentiated by simultaneous addition of secretagogues which alone also elevated cellular cyclic AMP levels. VIP alone failed to evoke catecholamine secretion from chromaffin cells, but potentiated potassium-, veratridine-, and nicotine-evoked secretion. Because VIP is found in nerve terminals innervating the adrenal medulla, this neuropeptide appears to be a modulator of chromaffin cell function, possibly via regulation of cyclic AMP levels.     

Neurite outgrowth of striatal neurons in vitro: involvement of purines in the growth-promoting effect of myenteric plexus explants

We have shown previously that a soluble factor(s) released by the myenteric plexus promotes neurite outgrowth from postnatal striatal neurons, and that this effect was abolished by tetrodotoxin. We have now investigated the possible involvement of purines in the mediation of this neuritogenic response, by examining their effect on neurite length of striatal neurons both in co-culture with myenteric plexus explants and cultured alone.Both ATP and 2-chloroadenosine partially reversed the inhibitory effect of tetrodotoxin in co-cultures with whole myenteric plexus, while the stable ATP analogue, α,β-methylene ATP, had no effect, suggesting that ATP was being broken down to adenosine before exerting its action. Further support for this view was that the ATP (P2) purinoceptor antagonist suramin did not reverse the effects of ATP, while the adenosine (P1) purinoceptor antagonist 8-(p-sulphophenyl)theophylline did antagonize the effects of ATP in tetrodotoxin-treated co-cultures. Further, both 8-(p-sulphophenyl)theophylline and adenosine deaminase reduced the effect of the myenteric plexus on striatal neurons in the absence of tetrodotoxin, and the adenylate cyclase activator forskolin completely reversed the effect of tetrodotoxin in our co-culture system.The neurite outgrowth-promoting effect of 2-chloroadenosine in tetrodotoxin-treated co-cultures was not further enhanced by a combination of neuropeptides. Serotonin and GTP were without effect on striatal neurons in the presence or absence of myenteric plexus explants. In experiments without myenteric plexus, both 2-chloroadenosine and forskolin caused a slight increase in striatal neurite length; ATP and GTP were ineffective. Basic fibroblast growth factor, nerve growth factor, neurotrophin-3 or neurotrophin-4/5 had no effect on neurite outgrowth in postnatal striatal cultures after two days in vitro. When these growth factors were added in combination with 2-chloroadenosine, the observed increase in mean neurite length did not exceed that induced by 2-chloroadenosine alone. Both 2-chloroadenosine and the ganglioside mix AGF1 increased neurite elongation of striatal neurons after two days in vitro, but an inhibition of enhanced neurite outgrowth was observed when both substances were added together. Both laminin and fibronectin were not neuritogenic for postnatal striatal neurons under our culture conditions. These observations suggest that a factor other than the growth factors tested here is involved in the promotion of striatal neurite outgrowth in co-culture with myenteric plexus explants.In summary, adenosine (probably acting through the A₂ subclass of the P1 purinoceptor) leads to increased striatal neurite outgrowth in co-culture with myenteric plexus and we propose that it does so either (1) by triggering the release of a neuritogenic factor, possibly from enteric glial cells, or (2) by acting synergistically with such a growth factor. Adenosine acts via PI purinoceptors, which leads to changes in cyclic AMP, and the response to forskolin suggests that cyclic AMP is probably involved in the events leading to increased striatal neurite outgrowth.     

Characterization of Rebound Depolarization in Neurons of the Rat Medial Geniculate Body <Emphasis Type="Italic">In Vitro</Emphasis>

Rebound depolarization (RD) is a response to the offset from hyperpolarization of the neuronal membrane potential and is an important mechanism for the synaptic processing of inhibitory signals. In the present study, we characterized RD in neurons of the rat medial geniculate body (MGB), a nucleus of the auditory thalamus, using whole-cell patch-clamp and brain slices. RD was proportional in strength to the duration and magnitude of the hyperpolarization; was effectively blocked by Ni²⁺ or Mibefradil; and was depressed when the resting membrane potential was hyperpolarized by blocking hyperpolarization-activated cyclic nucleotide-gated (HCN) channels with ZD7288 or by activating G-protein-gated inwardly-rectifying K⁺ (GIRK) channels with baclofen. Our results demonstrated that RD in MGB neurons, which is carried by T-type Ca²⁺ channels, is critically regulated by HCN channels and likely by GIRK channels.     

Bradykinin induces hyperpolarizations in rat glioma cells and in neuroblastoma × glioma hybrid cells

The effect of the nonapeptide bradykinin on the membrane potential of permanent cell lines from neural origin was studied. A hyperpolarizing response of 10–30 s duration was produced when bradykinin was iontophoretically applied onto polyploid rat glioma cells (clone C6-4-2). Starting from the resting membra potential the peak value of the hyperpolarizing response was reached within 0.5–1.5 s. Then the potential returned more slowly to the origin value. The hyperpolarization was associated with an approximately 50% decrease in membrane resistance. Neither Na⁺ nor Cl⁻ seemed to be important for the hyperpolarizing response, since bradykinin elicited similar hyperpolarizations in cells exposed to media in which Na⁺ or Cl⁻ were replaced by choline or isethionate, respectively. Ca²⁺ fluxes are unlikely to be involved, since the addition of D600 did not affect the hyperpolarizations induced by bradykinin. However, a 10-fold increase in the concentration of K⁺ in the medium reduced the amplitude of the hyperpolarization by 40 mV. Thus, the hyperpolarization induced by bradykinin is associated with a decrease in membrane resistance which is likely to be caused by an increased K⁺-conductance. The glioma cells showed a desensitization upon repeated application of bradykinin. However, the sensitivity of the cells to bradykinin was restored after 3–8 min of incubation in the absence of bradykinin. Since an antagonist of bradykinin is not known, the specificity of the action of bradykinin is difficult to assess. Nevertheless, the hyperpolarizing response to bradykinin appears to be specific insofar as other peptides, i.e. lutoliberin, thyroliberin, neurotensin, substance P and apamin, exerted no effect on the membrane potential of the glioma cells. Bradikynin-elicited hyperpolarizations with characteristics similar to those described above could also be demonstrated in neuroblastoma × glioma hybrid cells, but not in multinucleated fibroblast cells.