Metabotropic Responses Research Articles

Primary mouse myoblast metabotropic purinoceptor profiles and calcium signalling differ with their muscle origin and are altered in mdx dystrophinopathy

Mortality of Duchenne Muscular Dystrophy (DMD) is a consequence of progressive wasting of skeletal and cardiac muscle, where dystrophinopathy affects not only muscle fibres but also myogenic cells. Elevated activity of P2X7 receptors and increased store-operated calcium entry have been identified in myoblasts from the mdx mouse model of DMD. Moreover, in immortalized mdx myoblasts, increased metabotropic purinergic receptor response was found. Here, to exclude any potential effects of cell immortalization, we investigated the metabotropic response in primary mdx and wild-type myoblasts. Overall, analyses of receptor transcript and protein levels, antagonist sensitivity, and cellular localization in these primary myoblasts confirmed the previous data from immortalised cells. However, we identified significant differences in the pattern of expression and activity of P2Y receptors and the levels of the “calcium signalling toolkit” proteins between mdx and wild-type myoblasts isolated from different muscles. These results not only extend the earlier findings on the phenotypic effects of dystrophinopathy in undifferentiated muscle but, importantly, also reveal that these changes are muscle type-dependent and endure in isolated cells. This muscle-specific cellular impact of DMD may not be limited to the purinergic abnormality in mice and needs to be taken into consideration in human studies.

Tyrosine phosphorylation differentially fine-tunes ionotropic and metabotropic responses of human α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor is involved in neurological, neurodegenerative, and inflammatory disorders. It operates both as a ligand-gated cationic channel and as a metabotropic receptor in neuronal and non-neuronal cells. As protein phosphorylation is an important cell function regulatory mechanism, deciphering how tyrosine phosphorylation modulates α7 dual ionotropic/metabotropic molecular function is required for understanding its integral role in physiological and pathological processes. α7 single-channel activity elicited by ACh appears as brief isolated openings and less often as episodes of few openings in quick succession. The reduction of phosphorylation by tyrosine kinase inhibition increases the duration and frequency of activation episodes, whereas the inhibition of phosphatases has the opposite effect. Removal of two tyrosine residues at the α7 intracellular domain recapitulates the effects mediated by tyrosine kinase inhibition. The tyrosine-free mutant receptor shows longer duration-activation episodes, reduced desensitization rate and significantly faster recovery from desensitization, indicating that phosphorylation decreases α7 channel activity by favoring the desensitized state. However, the mutant receptor is incapable of triggering ERK1/2 phosphorylation in response to the α7-agonist. Thus, while tyrosine phosphorylation is absolutely required for α7-triggered ERK pathway, it negatively modulates α7 ionotropic activity. Overall, phosphorylation/dephosphorylation events fine-tune the integrated cell response mediated by α7 activation, thus having a broad impact on α7 cholinergic signaling.

Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons.

Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.

Vasopressin-induced intracellular Ca2+ concentration responses in non-neuronal cells of the rat dorsal root ganglion

Arginine-vasopressin (AVP) is a nonapeptide of hypothalamic origin that has been shown to exert many important cognitive and physiological functions in neurons and terminals of both the central and peripheral nervous system (CNS and PNS). Here we report for the first time that AVP induced an increase in intracellular Ca²⁺ concentration ([Ca²⁺](i)) in non-neuronal cells isolated from the rat dorsal root ganglion (DRG) and cultured in vitro. The ratiometric [Ca²⁺](i) measurements showed that AVP evoked [Ca²⁺](i) responses in the non-neuronal cells and these concentration-dependent (100 pM to 1 μM) responses increased with days in vitro in culture, reaching a maximum amplitude after 4-5 day. Immunostaining by anti-S-100 antibody revealed that more than 70% of S-100 positive cells were AVP-responsive, indicating that glial cells responded to AVP and increased their [Ca²⁺](i). The responses were inhibited by depletion of the intracellular Ca²⁺ stores or in the presence of inhibitors of phospholipase C, indicating a metabotropic response involving inositol trisphosphate, and were mediated by the V₁ subclass of AVP receptors, as evidenced by the use of the specific blockers for V₁ and OT receptors, (d(CH₂)₅¹,Tyr(Me)²,Arg⁸)-Vasopressin and (d(CH₂)₅¹,Tyr(Me)²,Thr⁴,Orn⁸,des-Gly-NH₂⁹)-Vasotocin, respectively. V(1a) but not V(1b) receptor mRNA was expressed sustainably through the culture period in cultured DRG cells. These results suggest that AVP modulates the activity of DRG glial cells via activation of V(1a) receptor.

Pharmacological evidence of functional inhibitory metabotrophic glutamate receptors on mouse arousal-related cholinergic laterodorsal tegmental neurons

Cholinergic neurons of the pontine laterodorsal tegmentum (LDT) are importantly involved in neurobiological mechanisms governing states of arousal such as sleep and wakefulness as well as other appetitive behaviors, such as drug-seeking. Accordingly, mechanisms controlling their excitability are important to elucidate if we are to understand how these LDT neurons generate arousal states. Glutamate mediates the vast majority of excitatory synaptic transmission in the vertebrate CNS and while presence of glutamate input in the LDT has been shown and ionotropic responses to glutamate have been reported in the LDT, characterization of metabotropic responses is lacking. Therefore, electrophysiological responses and changes in levels of intracellular Ca2+ in mouse cholinergic LDT neurons following application of specific mGluR agonists and antagonists were examined. Unexpectedly, both the mGluR5specific agonist, CHPG, and the group II mGluR (mGlu2/3) agonist, LY379268 (LY), induced a TTX-insensitive outward current/hyperpolarization. Both outward currents were significantly reduced by the mGluR antagonist MCPG and the CHPG-induced current was blocked by the specific mGluR5 antagonist MTEP. Concurrent Ca2+imaging revealed that while CHPG actions did include release of Ca2+ from CPA/thapsigargin-sensitive intracellular stores, actions of LY did not. Both CHPG- and LY-induced outward currents were mediated by a TEA-sensitive potassium conductance. The large-conductance, Ca2+-dependent potassium (BK) channel blocker, iberiotoxin, attenuated CHPG actions. Consistent with actions on the BK conductance, CHPG enhanced the amplitude of the fast component of the after hyperpolarizing potential, concurrent with a reduction in the firing rate. We conclude that stimulation of mGluR5 and group II (mGluR2/3) elicits postsynaptically-mediated outward currents/hyperpolarizations in cholinergic LDT neurons. Effects of glutamatergic input would be, thus, expected not only to be excitation via stimulation of ionotropic glutamate receptors and mGluR1, but also inhibition via actions at mGluR5 and mGluR2/3 on these neurons. As these two processes counteract each other, these surprising findings necessitate revision of predictions regarding the net level of excitation generated by glutamate input to cholinergic LDT cells and, by extension, the functional outcome of glutamate transmission on processes which these neurons regulate.This article is part of a Special Issue entitled ‘Metabotropic Glutamate Receptors’.

Ionotropic and metabotropic mechanisms in chemoreception: 'chance or design'?

Chemosensory receptors convert an enormous diversity of chemical signals from the external world into a common language of electrical activity in the brain. Mammals and insects use several families of transmembrane receptor proteins to recognize distinct classes of volatile and non-volatile chemicals that are produced by conspecifics or other environmental sources. A comparison of the signalling mechanisms of mammalian and insect receptors has revealed an unexpected functional distinction: mammals rely almost exclusively on metabotropic ligand-binding receptors, which use second messenger signalling cascades to indirectly activate ion channels, whereas insects use ionotropic receptors, which are gated directly by chemical stimuli, thereby leading to neuronal depolarization. In this review, we consider possible reasons for this dichotomy, taking into account biophysical, cell biological, ecological and evolutionary influences on how information is extracted from chemosensory cues by these animal classes.

Neurotransmitter responsiveness during early maturation of neural progenitor cells

Neurotransmitters are potential regulators of proliferation and differentiation of neural progenitor cells (NPC). To gain insight into the dynamics of neurotransmitter responsiveness, neurospheres were prepared from the lateral ventricles of postnatal day 6/7 mice. Individual NPCs migrating out from spheres were simultaneously monitored using Ca 2+ imaging, during the initial 8 days of differentiation, at an area between the inner edge of the sphere and outer periphery of the area of migration. At the first day of differentiation most cells showed metabotropic responses (Ca 2+ discharge from stores) to glutamate (pharmacologically identified as metabotropic glutamate receptor 5, mGluR 5), norepinephrine (NE), acetylcholine (Ach) and ATP, and a smaller proportion of cells also responded to substance P (SP). When outside the neurosphere, many of mGluR5 responding cells gained immunostaining for markers of neuronal lineage (Tuj-1 and NeuN). The number of cells responding through mGluR5 (and responses to Ach, NE and SP) showed during subsequent days of differentiation (day 2–3 onwards) a decline with time and progressively disappeared at the outer periphery of the area of migration. Conversely the number ionotropic glutamate responses as well as responses to depolarization increased in this area. After 5–8 days of differentiation mGluR5 responses could only be observed at the very inner edge of the neurosphere. At 8 days the migrated cells showed very robust ionotropic responses to glutamate, NMDA and depolarization comparable to mature neurons. Taken together, the data presented here suggest that differentiation of NPCs is a dynamic process triggered by cell migration, which leads to a loss of regulatory influences imposed by the inner milieu of the neurosphere. The subsequent switch or loss of metabotropic responses to glutamate, SP, NE, Ach and ATP with the gain of excitable characteristics such as ionotropic responses appears to be a key event in the final differentiation process.

Synaptic inhibition by glycine acting at a metabotropic receptor in tiger salamander retina

Glycine is the lone fast neurotransmitter for which a metabotropic pathway has not been identified. In retina, we found a strychnine-insensitive glycine response in bipolar and ganglion cells. This glycine response reduced high voltage-activated calcium current. It was G-protein mediated and protein kinase A dependent. The EC(50) of the metabotropic glycine response is 3 mum, an order of magnitude lower than the ionotropic glycine receptor in the same retina. The bipolar cell glutamatergic input to ganglion cells was suppressed by metabotropic glycine action. The synaptic output of about two-thirds of bipolar cells and calcium current in two-thirds of ganglion cells are sensitive to the action of glycine at metabotropic receptors, suggesting this signal regulates specific synaptic pathways in proximal retina. This study resolves the curious absence of a metabotropic glycine pathway in the nervous system and reveals that the major fast inhibitory neurotransmitters, GABA and glycine, both activate G-protein-coupled pathways as well.

Involvement of ryanodine receptors in IP 3-mediated calcium signalling in neurons : A modelling approach

Phospholipase C (PLC)-coupled metabotropic receptors trigger the release of intracellular Ca 2+ through activation of IP 3 receptors (IP 3Rs). Increasing evidence suggests that they can also and perhaps more efficiently mobilize Ca 2+ through ryanodine receptors (RyRs). We constructed a model allowing a variable PLC stimulation level (via the parameter γ) as well as a variable involvement of RyRs (via the parameter A). The sole presence of RyRs ( A ≠ 0) affected the basal Ca 2+ concentration [Ca 2+] i. To keep Ca 2+ below 160 nM, we fixed the upper limit of A to 0.2, a value that is compatible with the numerical ratio between RyRs and IP 3Rs in cerebellar Purkinje neurons. Metabotropic responses were simulated by abruptly raising the value of γ to various levels. In the absence of RyRs, the model starts to oscillate with γ = 0.4. For lower levels of PLC stimulation ( γ ≤ 0.3), the presence of RyR is capable of triggering an oscillatory signal. When A ≤ 0.18, the frequency of the Ca 2+ oscillations augments from 0.1 to 1.3 Hz as a function of γ. Conversely, as the frequency increases, the amplitude of the oscillations is reduced from 1 μM to 50 nM. With higher values of A, the oscillating pattern is definitively inhibited. It is concluded that RyRs have the potentiality to strikingly affect the temporal pattern of the Ca 2+ signalling triggered by IP 3-related metabotropic responses.

GABA Provides a Swell Route to Calcium

In addition to its role as neurotransmitter, γ-aminobutyric acid (GABA) mediates trophic effects during neuronal development. These long-term actions have been hypothesized to depend on Ca 2+ influx through voltage-dependent Ca 2+ channels, a consequence of activation of GABA A receptors (ligand-gated ion channels that are permeable to Cl − and HCO 3 − ), which can lead to depolarization in developing neurons. Chavas et al. loaded interneurons in cerebellar slices from juvenile rats with a calcium indicator and observed that application of the GABA agonist muscimol elicited a rise in [Ca 2+ ] i , which was greater in dendrites than in the cell body. The authors used cell-attached recordings in which they measured currents through single BK channels to simultaneously evaluate membrane potential and [Ca 2+ ] i , and they observed that a late phase of increased [Ca 2+ ] i did not correlate with membrane depolarization. Moreover, the [Ca 2+ ] i increase was not blocked by cadmium, and the threshold for calcium channel activation was higher than the peak depolarization in response to muscimol. Both changes in membrane potential and changes in [Ca 2+ ] i required HCO 3 − ; the late phase of increased [Ca 2+ ] i was inhibited by ryanodine. Dendrites expanded in size during muscimol application, and hypoosmotic solutions mimicked, whereas hyperosmotic solutions inhibited, the [Ca 2+ ] i response to muscimol. Thus, the authors proposed that the rise in [Ca 2+ ] i that occurs following GABA A receptor activation depends on increased osmotic tension following bicarbonate-dependent influx of Cl − . The sensitivity to ryanodine suggested that this might involve release of Ca 2+ from intracellular stores, linking ion flux through a ligand-gated channel to a metabotropic receptor-like response. J. Chavas, M. E. Forero, T. Collin, I. Llano, A. Marty, Osmotic tension as a possible link between GABA A receptor activation and intracellular calcium elevation. Neuron 44 , 701-713 (2004). [Online Journal]

The sleep inducing brain lipid cis-oleamide (cOA) does not modulate serotonergic transmission in the CA1 pyramidal neurons of the hippocampus in vitro

cis-Oleamide (cOA) is a novel sleep inducing brain lipid with an unknown mechanism of action. High affinity interactions with metabotropic 5-HT receptors (2A/C and 1A subtypes) in frog oocytes and expression systems have been reported, but functional in vitro evidence for the modulatory effect is still lacking. Here, we addressed the ability of cOA to modulate 5-HT-induced cellular actions in the CA1 neurons of the rat hippocampal slice. 5-HT (0.1–100 μM) concentration dependently reduced the amplitude of the evoked field population spike (fPS), and produced a hyperpolarising shift in the resting membrane potential ( V r) and a drop in input resistance ( R in). The effects of a low dose of 5-HT (3.2 μM) on fPS, V r and R in were reversed by the specific 5-HT 1A-receptor antagonist WAY 100135 (10 μM). cOA (1 μM) failed to potentiate 5-HT 1A receptor mediated effects on fPS, V r or R in. High doses of 5-HT also recruited both 5-HT 2 and 5-HT 3 receptors, causing an increase in the rate and amplitude of sIPSCs. cOA (1 μM), in the presence of Y 25130, failed to potentiate the 5-HT 2 receptor induced enhancement of sIPSCs. In summary, cis-oleamide failed to modulate metabotropic responses to exogenous 5-HT in this microelectrode study at concentrations well in excess of those reported to modulate 5-HT 1A and 5-HT 2A/C systems in earlier studies.

Coexpression of functional P2X and P2Y nucleotide receptors in single cerebellar granule cells

The present study describes the presence and expression of functional nucleotide receptors, both ionotropic and metabotropic, in highly purified cultures of cerebellar granule neurons. Microfluorimetric experiments have been carried out to record specific [Ca(2+)](i) transients in individual granule neurons after challenge with diverse nucleotides. Although great heterogeneity was found in nucleotide responses in single cells, these responses all became modified during the course of granule cell differentiation, not only at the level of the number of responding cells, but also in the magnitude of the response to nucleotides. These in vitro developmental changes were more significant in metabotropic responses to pyrimidine nucleotides, UTP and UDP, which were down- and upregulated, respectively, during the time in culture. At least two types of ADP-specific receptors seem expressed in different granule cell subpopulations responding to 2MeSADP, as the specific P2Y(1) antagonist MRS-2179 inhibited Ca(2+) responses in only one of these populations. The great diversity of metabotropic responses observed was confirmed by the RT-PCR expression of different types of P2Y receptors in granule cell cultures: P2Y(1), P2Y(4), P2Y(6), and P2Y(12). Similarly, ionotropic nucleotide responses were confirmed by the presence of specific messengers for different P2X subunits, and by immunolabeling studies (P2X(1), P2X(2), P2X(3), P2X(4) and P2X(7)). Immunolabeling reflected great variety in the P2X subunit distribution along the granule neuron cytoarchitecture, with P2X(2), P2X(3) and P2X(4) present at somatodendritic locations, and P2X(1), P2X(7), and P2X(3), located at the axodendritic prolongations. The punctuated labeling pattern obtained for P2X(3) and P2X(7) subunits is particularly notable, as it presents a high degree of colocalization with synaptophysin, a specific marker of synaptic vesicles, suggesting specialized localization and function in granule neurons.

Group I metabotropic glutamate receptors activate a calcium-sensitive transient receptor potential-like conductance in rat hippocampus.

In CA3 pyramidal neurons from organotypic slice cultures, activation of G(q)-coupled group I metabotropic glutamate receptors (mGluRs) induces a non-selective cationic conductance that enhances excitability. We have found that this response shares several properties with conductances that are mediated by the transient receptor potential (TRP) family of ion channels, including inhibition by La(3+), 2-aminoethoxydiphenylborane (2APB), cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL 12,330A) and a doubly rectifying current-voltage relationship. Stimulation of mGluR1 and mGluR5 converged to activate the TRP-like conductance in a synergistic manner, and activation of either subtype alone produced only a fraction of the normal response. Activation of the cationic current required elevated intracellular Ca(2+). Chelating intracellular Ca(2+) or blocking Ca(2+) entry through voltage-gated Ca(2+) channels attenuated responses to the activation of mGluRs. Conversely, raising intracellular Ca(2+) potentiated mGluR activation of the TRP-like conductance. Under control conditions, blocking G protein activation using intracellular GDPbetaS with or without N-(2, 6-dimethylphenylcarbamoylmethyl) triethylammonium chloride (QX-314) prevented mGluR-mediated activation of the TRP-like conductance. Following G protein blockade, however, the coupling between mGluRs 1 and/or 5 and the TRP-like conductance was rescued by increasing intracellular Ca(2+). This suggests that a G protein-independent signalling pathway is also activated by group I mGluRs. Such a pathway may represent an alternative transduction mechanism to maintain metabotropic responses under conditions where G proteins are functionally uncoupled from their cognate receptors.

Glutamate receptors and transporters in genetic and acquired models of epilepsy

Glutamate, the principal excitatory neurotransmitter in the brain, acts on three families of ionotropic receptor—AMPA (α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid), kainate and NMDA ( N-methyl- d-aspartate) receptors—and three families of metabotropic receptor (Group I: mGlu1 and mGlu5; Group II: mGlu2 and mGlu3; Group III: mGlu4, mGlu6, mGlu7 and mGlu8). Glutamate is removed from the synaptic cleft and the extracellular space by Na +-dependent transporters (GLAST/EAAT1, GLT/EAAT2, EAAC/EAAT3, EAAT4, EAAT5). In rodents, genetic manipulations relating to the expression or function of glutamate receptor proteins can induce epilepsy syndromes or raise seizure threshold. Decreased expression of glutamate transporters (EAAC knockdown, GLT knockout) can lead to seizures. In acquired epilepsy syndromes, a wide variety of changes in receptors and transporters have been described. Electrically-induced kindling in the rat is associated with functional potentiation of NMDA receptor-mediated responses at various limbic sites. Group I metabotropic responses are enhanced in the amygdala. To date, no genetic epilepsy in man has been identified in which the primary genetic defect involves glutamate receptors or transporters. Changes are found in some acquired syndromes, including enhanced NMDA receptor responses in dentate granule cells in patients with hippocampal sclerosis.

Intracellular sigma1 receptor modulates phospholipase C and protein kinase C activities in the brainstem.

Most physiological effects of sigma1 receptor ligands are sensitive to pertussis toxin, suggesting a coupling with cell membrane-bound G proteins. However, the cloning of the sigma1 receptor has allowed the identification of an intracellular protein anchored on the endoplasmic reticulum. Here, we show, using the isolated adult guinea pig brainstem preparation, that activation of the sigma1 receptor results in its translocation from the cytosol to the vicinity of the cell membrane and induces a robust and rapid decrease in hypoglossal activity, which is mediated by phospholipase C. The subsequent activation of protein kinase C beta1 and beta2 isoforms and the phosphorylation of a protein of the same molecular weight as the cloned sigma1 receptor lead to a desensitization of the sigma1 motor response. Our results indicate that the intracellular sigma1 receptor regulates several components implicated in plasma membrane-bound signal transduction. This might be an example of a mechanism by which an intracellular receptor modulates metabotropic responses.

Depletion of caffeine-sensitive calcium store results in diminution of ATP-induced metabotropic calcium responses in rat neocortical neurons

ATP receptor-mediated changes in the Ca2+ concentration were recorded from neurons of the sensorimotor cortex in brain slices from 3-week-old rats. To measure the cytoplasmic concentration of Ca2+, slices were incubated with Fura-2/AM, and a microfluorimetry system was focused on an individual cell. Possible glutamatergic signals resulting from ATP-evoked glutamate release were excluded. After elimination of calcium from the extracellular solution, the first ATP-induced [Ca2+] i transient decreased to 62±9% of a similar response in the normal solution, suggesting the participation of metabotropic purinoreceptor-triggered Ca release in transient generation. Depletion of the caffeine-sensitive calcium store results in diminution of ATP-induced [Ca2+] i transient in the Ca2+-free solution by 31.4±7.0% (P<0.01). This may indicate that in pyramidal neurons of the sensorimotor cortex InsP3- and Ca-induced Ca-releases demonstrate noticeable functional interaction. Nevertheless, there is no single compartment in the endoplasmic reticulum bearing both IICR and CICR channels.

G‐protein‐mediated desensitization of metabotropic glutamatergic and muscarinic responses in CA3 cells in rat hippocampus.

1. Desensitization of a metabotropic response was investigated in CA3 pyramidal neurons in hippocampal slice cultures using the patch-clamp technique. 2. 1S,3R-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD), an agonist at metabotropic glutamate receptors (mGluRs), and metacholine (MCh), an agonist at muscarinic receptors, induced a cationic current that appears to be activated through a G-protein-independent transduction process, as previously shown. Prolonged or repetitive bath application of agonists led to rapid desensitization of the cationic current with a time constant of approximately 20 s. 3. Complete recovery from desensitization was observed within 6 min. 4. These responses mediated by mGluRs and muscarinic receptors cross-desensitized. 5. Preventing the activation of G-proteins by loading cells with GDP beta S strongly reduced or suppressed desensitization, and resulted in a sustained inward cationic current. When cells were filled with GTP gamma S to irreversibly activate G-proteins, the desensitization process was enhanced such that a first application of agonist caused a markedly reduced response. 6. These results show that a cationic current induced by metabotropic agonists in hippocampal pyramidal cells undergoes apparent desensitization and suggests that this process occurs through a G-protein-mediated inhibition of the underlying membrane conductance.

Metabotropic Glutamate Receptor Activation in Cerebellar Purkinje Cells as Substrate for Adaptive Timing of the Classically Conditioned Eye-Blink Response

To understand how the cerebellum adaptively times the classically conditioned nictitating membrane response (NMR), a model of the metabotropic glutamate receptor (mGluR) second messenger system in cerebellar Purkinje cells is constructed. In the model, slow responses, generated postsynaptically by mGluR-mediated phosphoinositide hydrolysis and calcium release from intracellular stores, bridge the interstimulus interval (ISI) between the onset of parallel fiber activity associated with the conditioned stimulus (CS) and climbing fiber activity associated with unconditioned stimulus (US) onset. Temporal correlation of metabotropic responses and climbing fiber signals produces persistent phosphorylation of both AMPA receptors and Ca(2+)-dependent K+ channels. This is responsible for long-term depression (LTD) of AMPA receptors. The phosphorylation of Ca(2+)-dependent K+ channels leads to a reduction in baseline membrane potential and a reduction of Purkinje cell population firing during the CS-US interval. The Purkinje cell firing decrease disinhibits cerebellar nuclear cells, which then produce an excitatory response corresponding to the learned movement. Purkinje cell learning times the response, whereas nuclear cell learning can calibrate it. The model reproduces key features of the conditioned rabbit NMR: Purkinje cell population response is timed properly; delay conditioning occurs for ISIs of up to 4 sec, whereas trace conditioning occurs only at shorter ISIs; mixed training at two different ISIs produces a double-peaked response; and ISIs of 200-400 msec produce maximal responding. Biochemical similarities between timed cerebellar learning and photoreceptor transduction, and circuit similarities between the timed cerebellar circuit and a timed dentate-CA3 hippocampal circuit, are noted.

Excitation of sympathetic preganglionic neurons via metabotropic excitatory amino acid receptors.

The role of excitatory amino acid metabotropic receptors in the regulation of excitability of sympathetic preganglionic neurons was investigated. This study used both conventional intracellular and whole-cell patch clamp techniques to record from sympathetic preganglionic neurons in transverse spinal cord slices of the rat (9-21 days old). The metabotropic receptor agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) (10-200 microM, superfused for 2-60 s) and quisqualate (1-50 microM, superfused for 2-60 s) induced concentration-dependent depolarizing responses which did not desensitize. These responses were unaffected by the glutamate ionotropic receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-50 microM), 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 microM), dizocilpine (MK-801, 10-40 microM), 3-[(R)-2-carboxy-piperazin-4-yl]-propyl-1-phosphonic acid (D-CPP, 10-50 microM) and DL-2-amino-5-phosphonovaleric acid (DL-AP5, 20-100 microM). Depolarizing responses to 1S,3R-ACPD and quisqualate were unaffected by L-2-amino-3-phosphonopropionic acid (L-AP3, 30 microM-1mM) and L-2-amino-4-phosphonobutanoic acid (L-AP4, 100 microM-1 mM)). The responses to 1S,3R-ACPD and quisqualate were reduced by including the G-protein blocker GDP-beta-S (400 microM) in the patch pipette solution by 77 +/- 2% (mean +/- S.E) of control (n = 3), suggesting that these agonists activate a G-protein-coupled receptor. Metabotropic receptor-mediated responses were maintained in the presence of tetrodotoxin (500 nM), progressively reduced with increased membrane hyperpolarization to around -95 mV and associated with either an increase of 16.5 +/- 2.8% (data from four neurons) in the majority of neurons (n = 22 of 34) or no measurable change (n = 12) in neuronal input resistance. These data suggest that the agonists exert a direct action on 1S,3R-ACPD and quisqualate had several effects on sympathetic preganglionic neuron membrane properties including: inhibition of a slow apamin-insensitive component of the afterhyperpolarization; a reduction in spike frequency adaptation leading to increases in firing frequency from 6.4 +/- 2.8 Hz in control experiments up to 14.7 +/- 3.0 Hz (n = 6 neurons) in the presence of a metabotropic receptor agonist: a broadening of the action potential by 37.5 +/- 6.4% (n = 6 neurons) of control. These observations suggest that the metabotropic receptor-mediated depolarization is due, at least in part, to the reduction of potassium conductances involved in the spike afterhyperpolarisation potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulatory effects of NMDA on phosphoinositide responses evoked by the metabotropic glutamate receptor agonist 1S,3R‐ACPD in neonatal rat cerebral cortex

1. The effect of NMDA-receptor stimulation on phosphoinositide signalling in response to the metabotropic glutamate receptor agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) has been examined in neonatal rat cerebral cortex slices. 2. Total [3H]-inositol phosphate ([3H]-InsPx) accumulation, in the presence of 5 mM LiCl, in [3H]-inositol pre-labelled slices was concentration-dependently increased by 1S,3R-ACPD (EC50 16.6 microM) and, at a maximally effective concentration, 1S,3R-ACPD (300 microM) increased [3H]-InsPx accumulation by 12.8 fold over basal values. 3. [3H]-InsPx accumulation stimulated by 1S,1R-ACPD was enhanced by low concentrations of NMDA (3-30 microM), but not by higher concentrations (> 30 microM). [3H]-InsPx accumulations stimulated by 1S,3R-ACPD in the absence or presence of 10 microM NMDA were linear with time, at least over the 15 min period examined; however, in the presence of 100 microM NMDA the initial enhancement of 1S,3R-ACPD-stimulated phosphoinositide hydrolysis progressively decreased with time. 4. In the presence of a maximal enhancing concentration of NMDA (10 microM), the response to 1S,3R-ACPD (300 microM) was increased 1.9 fold and the EC50 for agonist-stimulated [3H]-InsPx accumulation decreased about 4 fold. The enhanced response to the metabotropic agonist was concentration-dependently inhibited by competitive and uncompetitive antagonists of NMDA-receptor activation. 5. 1S,3R-ACPD also stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation with an initial peak response (5-6 fold over basal) at 15 s decaying to a smaller (2 fold), but persistent elevated accumulation (1-10 min). 6. Co-addition of 10 or 100 MicroM NMDA enhanced the initial peak Ins(1,4,5)P3 response to 1S,3RACPD.However, the enhancing effect was only maintained over 10 min in the presence of 1O Micro MNMDA, whilst in contrast, 100 MicroM NMDA ceased to cause a significant enhancement of the metabotropic response by 5 min and completely suppressed lS,3R-ACPD-stimulated Ins(1,4,5)P3 accumulation at 10 min.7. Both basal and 1S,3R-ACPD-stimulated Ins(1,4,5)P3 accumulations were reduced when slices were incubated in nominally Ca2"-free medium. Under these conditions only a concentration-dependent enhancement of the response was observed (EC50 for NMDA facilitation of lS,3R-ACPD-stimulated Ins(1,4,5)P3 accumulation of 32 MicroM).8. These experiments have revealed that at low concentrations, NMDA can dramatically potentiate1S,3R-ACPD-stimulated phosphoinositide hydrolysis, probably by a Ca2"-dependent facilitation of agonist-stimulated phosphoinositide-specific phospholipase C activity. Higher concentrations of NMDA result in time-dependent inhibition of the metabotropic agonist-stimulated response. We believe the former effect could be fundamental in glutamate receptor 'cross-talk', whereas the latter may reflect a Ca2+-dependent neurotoxic effect of NMDA on the neonatal cerebral cortex slices.