Adult Rat Sympathetic Neurons Research Articles

Target validation: Weak selectivity of LY341495 for mGluR2 over mGluR4 makes glutamate a less selective agonist.

Metabotropic glutamate receptors (mGluRs) are class C G protein coupled receptors with widespread expression in the central nervous system. There are eight mGluRs in the mammalian genome. Research on mGluRs relies on the availability of selective compounds. While many selective allosteric compounds have been described, selectivity of orthosteric agonists and antagonists has been more difficult due to the similarity of the glutamate binding pocket across the mGluR family. LY341495 has been used for decades as a potent and selective group II mGluR antagonist. The selectivity of LY341495 was investigated here between mGluR2, a group II mGluR, and mGluR4, a group III receptor, heterologously expressed in adult rat sympathetic neurons from the superior cervical ganglion (SCG), which provides a null‐mGluR background upon which mGluRs were examined in isolation. The compound does in fact selectively inhibit mGluR2 over mGluR4, but in such a way that it makes signaling of the two receptors more difficult to distinguish. The glutamate potency of mGluR2 is about 10‐fold higher than mGluR4. 50 nmol L−1 LY341495 did not alter mGluR4 signaling but shifted the mGluR2 glutamate dose‐response about 10‐fold, such that it overlapped more closely with that of mGluR4. Increasing the LY341494 dose to 500 nmol L−1 further shifted the glutamate dose‐response of mGluR2 by another ~10‐fold, but also shifted that of mGluR4 similarly. Thus, while glutamate is a moderately selective agonist of mGluR2 over mGluR4 when applied alone, in the presence of increasing concentrations of LY341495, this selectivity of glutamate is lost.

Expression of G-protein signaling components in adult mammalian neurons by microinjection.

Although methods for expressing foreign proteins in clonal cell lines are well established, mature neurons remain a difficult preparation for the introduction of foreign genes. Microinjection is a reliable method for producing robust targeted expression in neurons that has advantages over conventional transfection/infection methodologies. Here, I describe procedures for expressing signaling proteins in adult rat sympathetic neurons by direct microinjection of cRNA and cDNA into the cytoplasm and nucleus, respectively. The methods are applicable to a wide variety of peripheral and central neuron preparations, as well as clonal cell lines.

P75 and TrkA receptors are both required for uptake of NGF in adult sympathetic neurons: use of a novel fluorescent NGF conjugate

We have developed and tested the biological activity and specificity of a novel fluorescent dextran-Texas Red–nerve growth factor (DTR–NGF) conjugate. DTR–NGF was found to promote survival and neurite outgrowth in cultured dissociated sympathetic neurons similarly to native NGF. The conjugate was taken up and transported retrogradely by terminal sympathetic nerves innervating the iris to neurons in the ipsilateral superior cervical ganglion (SCG) of young adult rats. Uptake and transport was assessed by counting numbers of labelled neurons and by measuring intensity of neuronal labelling using confocal microscopy and image analysis. DTR–NGF labelling in SCG neurons was shown to be dose-dependent with an EC 50 of 75 ng. Similar concentrations of unconjugated DTR resulted in no neuronal labelling. DTR–NGF uptake was competed off using a 50-fold excess of native NGF, resulting in a 73% reduction in numbers of labelled neurons. Pretreatment of nerve terminals with function-blocking antibodies against the low (p75) and high (TrkA) affinity NGF receptors resulted in a large (85–93%) reduction in numbers of DTR–NGF labelled neurons. Anti-p75 and anti-TrkA antibodies had comparable effects which were concentration-dependent. These findings indicate that both receptors are required for uptake of NGF in adult rat sympathetic neurons. In particular, the results provide strong evidence that the p75 receptor plays a more active role in transducing the NGF signal than has been proposed.

Heterologous expression and coupling of G protein-gated inwardly rectifying K+ channels in adult rat sympathetic neurons.

1. G protein-gated inwardly rectifying K+ (GIRK) channels were heterologously expressed in rat superior cervical ganglion (SCG) neurons by intranuclear microinjection. The properties of GIRK channels and their coupling to native receptors were characterized using the whole-cell patch-clamp technique. 2. Following coinjection of either GIRK1-2 or GIRK1-4 cDNA, application of noradrenaline (NA) produced large inwardly rectifying K+ currents. Injection of cDNA encoding individual GIRK subunits produced only small and inconsistent NA-activated inward currents. Current arising from the native expression of GIRK channels in SCG neurons was not observed. 3. NA-mediated activation of GIRK channels was abolished by pertussis toxin (PTX) pretreatment, indicating coupling via G proteins of the Gi/Go subfamily. Conversely, vasoactive intestinal peptide (VIP) activated GIRK channel currents via a cholera toxin-sensitive pathway suggesting coupling through Galphas. Pretreatment of neurons with PTX caused a significant increase in amplitude of the VIP-mediated GIRK channel currents when compared with untreated cells. 4. Application of adenosine, prostaglandin E2 and somatostatin resulted in activation of GIRK channel currents. Activation of m1 muscarinic acetylcholine receptors (i.e. application of oxotremorine M to PTX-treated neurons) failed to elicit overt GIRK channel currents. 5. GIRK channel overexpression decreased basal Ca2+ channel facilitation significantly when compared with uninjected neurons. Furthermore, the NA-mediated inhibition of Ca2+ channels was significantly attenuated. 6. In summary, the ability to heterologously express GIRK channels in adult sympathetic neurons allows the experimental alteration of receptor-G protein-effector stoichiometry. Such studies may increase our understanding of the mechanisms underlying ion channel modulation by G proteins in a neuronal environment.

Heterologous expression of metabotropic glutamate receptors in adult rat sympathetic neurons: Subtype-specific coupling to ion channels

A novel heterologous expression system was used to examine the coupling of metabotropic glutamate receptors (mGluRs) to neuronal voltage-gated ion channels. Cytoplasmic injection of mGluR2 cRNA into adult rat sympathetic neurons resulted in the expression of receptors that negatively coupled to N-type Ca2+ channels through a pertussis toxin-sensitive pathway. Injection of mGluR1 alpha cRNA resulted in the expression of receptors that inhibited M-type K+ channels via a pertussis toxin-insensitive pathway. Coupling was restricted to specific transduction elements and effectors, since mGluR2 did not inhibit M channels and mGluR1 alpha had minimal effects on Ca2+ channels. These findings demonstrate that heterologously expressed, and thus unambiguously identified, mGluR subtypes modulate specific neuronal ion channels through discrete signal transduction pathways.

Increase in [Ca2+]i by CCh in adult rat sympathetic neurons are not dependent on intracellular Ca2+ pools.

We have examined the effects of the muscarinic agonists, carbachol (CCh) and oxotremorine (Oxo), on the intracellular free Ca2+ concentration ([Ca2+]i) in acutely dissociated sympathetic neurons from adult rats using fura 2-based microfluorometry. The drugs increased [Ca2+]i by 86 +/- 7 and 38 +/- 10 nM for CCh and Oxo, respectively (both 10 microM). Basal [Ca2+]i was 52 +/- 3 nM. Depletion of the caffeine-sensitive Ca2+ store or blockade of the Ca(2+)-adenosinetriphosphatase with thapsigargin did not alter the effect of either agonist on the rise in [Ca2+]i. On the other hand, the omission of Ca2+ from the perfusion solution or the use of TA-3090, a Ca2+ channel antagonist, blocked the effects of CCh and Oxo. In whole cell current-clamp recordings, the muscarinic agonists elicited a depolarization and action potential firing, which probably explained the rise in [Ca2+]i observed with microfluorimetric recording. In addition to their direct effects on the [Ca2+]i, muscarinic agonists also reduced the rise in [Ca2+]i induced by a nicotinic agonist. This inhibitory effect, observed in 68% of cells that responded to the nicotinic agonist, was blocked by atropine and pertussis toxin, whereas the muscarinic agonist-induced increase in [Ca2+]i was blocked by atropine but was pertussis toxin insensitive. These results suggest that at least two muscarinic receptors are present on sympathetic neurons and that they mediate opposite effect on the fluctuation of [Ca2+]i.

Modulation of Ca(2+)-channel currents by protein kinase C in adult rat sympathetic neurons

1. Modulation of Ca(2+)-channel currents by phorbol-12-myristate-13-acetate (PMA) was investigated in acutely dissociated adult rat superior cervical ganglion neurons using the whole cell variant of the patch-clamp technique. 2. PMA (500 nM) increased the current amplitudes, accelerated the inactivation of step currents, retarded the deactivation of tail currents, and shifted the tail current activation to more negative potentials. 3. The effects of PMA were concentration and voltage dependent and mediated through activation of protein kinase C (PKC). PMA also increased Ca2+ currents recorded with the perforated patch technique. 4. PMA affected the N-type Ca2+ channels and an omega-conotoxin GVIA-resistant current component. Ca2+ currents affected by PMA were not sensitive to omega-agatoxin IVA or nimodipine. 5. PMA not only attenuated Ca(2+)-channel inhibition induced by alpha 2-adrenoceptor agonist, which modulates Ca2+ channels via a pertussis toxin (PTX)-sensitive pathway, but also attenuated current inhibition by vasoactive intestinal polypeptide, which modulates Ca2+ channels via a PTX-insensitive but cholera toxin-sensitive pathway. 6. PMA reversed Ca(2+)-channel inhibition induced by tonic activation of G-protein in the absence of neurotransmitter (even in neurons pretreated with PTX) or induced by activation of G-proteins with guanosine 5'-O-(3-thiotriphosphate) (GTP)-gamma-S. 7. Inhibition of phosphatase by okadaic acid or substitution of Ba2+ for Ca2+ in the external solutions accelerated the PMA effect. 8. Our results suggest that activation of PKC antagonizes G-protein mediated inhibition of Ca2+ channels by shifting Ca2+ channels from the "reluctant" state to the "willing" state. The G-proteins and, more likely, the N-type Ca2+ channels may be the target of PKC phosphorylation. Protein phosphatases may be involved in counteracting the PKC phosphorylation in rat sympathetic neurons.

Galanin expression increases in adult rat sympathetic neurons after axotomy

Changes in neuropeptide expression occur in sensory, motor, and sympathetic neurons following axotomy. The particular pattern of peptide changes that occurs varies among the three cell types. We have studied the regulation in the rat superior cervical ganglion of the expression of galanin, a peptide previously shown to increase in axotomized sensory and motor neurons. While normally only an occasional neuron exhibiting galanin-like immunoreactivity is found in this ganglion, at two days after transection of the postganglionic internal and external carotid nerves, immunostaining can be observed in many neurons throughout the ganglion. Similar changes are found when ganglia are placed in organ culture for two days. The distribution of immunostained neurons after section of only one of the postganglionic trunks suggests that changes in galanin-like immunoreactivity occur only within neurons whose axons are transected. None the less, even when both nerve trunks are transected, only about half of the neurons in the ganglion exhibit galanin-like immunoreactivity, indicating that only a proportion of the axotomized neurons exhibit a detectable response. The few immunostained neurons seen after section of the cervical sympathetic trunk may also represent axotomized neurons. Galanin-like immunoreactivity extracted from the ganglion co-chromatographs with authentic galanin, and the level of this immunoreactivity increases dramatically after axotomy and explantation, and modestly after decentralization. These same manipulations produce parallel increases in the level of galanin messenger RNA. Together, the findings indicate that the expression of galanin increases in sympathetic neurons after axotomy. Galanin is thus the first neuropeptide whose expression has been shown to increase after transection of all three types of peripheral axons that have been studied.

Modulation of the enkephalinergic phenotype of rat sympathetic neurons by hormonal and transsynaptic mechanisms.

Most sympathetic neurons contain one or more neuropeptides in addition to catecholamines. Although the regulation of catecholamines has been studied extensively, comparatively little is known about the regulation of neuropeptides. Since glucocorticoids and preganglionic innervation regulate catecholaminergic properties in chromaffin cells, we examined the effects of these factors on a co-localized neuropeptide, leucine enkephalin (L-Enk), in adult rat sympathetic neurons in vivo. Lowered serum glucocorticoid levels as a consequence of bilateral adrenalectomy resulted in a reduction of ganglionic L-Enk content that was reversed by exposure of adrenalectomized animals to the synthetic glucocorticoid, dexamethasone. Surgical denervation of the SCG eliminated L-Enk-IR preganglionic fibers and caused a dramatic increase in the number of L-Enk-IR neurons. Inhibition of the enkephalinergic component of the preganglionic innervation by chronic exposure to the opiate receptor antagonist naloxone increases the number of L-Enk-IR cell bodies and total ganglionic L-Enk content. None of the experimental manipulations noticeably altered the number or distribution of NPY-IR neurons, suggesting that the effects of glucocorticoids and the innervation on ganglionic L-Enk levels were specific and not simply an alteration of the biosynthetic state of the cells. These results demonstrate that circulating glucocorticoids and the preganglionic innervation regulate L-Enk levels in sympathetic neurons. Since both glucocorticoid levels and preganglionic activity are known to be altered by stressful stimuli, acute regulation of sympathetic L-Enk levels may constitute an important component of the autonomic response to stress.

Adenosine modulates voltage-gated Ca2+ channels in adult rat sympathetic neurons

1. Ca(2+)-channel modulation by adenosine was investigated in enzymatically dispersed adult rat superior cervical ganglion (SCG) neurons using the whole-cell variant of the patch-clamp technique. 2. Adenosine produced a concentration-dependent decrease in the Ca(2+)-current amplitude with an EC50 of 174 nM and maximum inhibition of 36%. The effects of adenosine on the Ca2+ current were both time and voltage dependent. The inhibition was maximal at +10 mV and decreased at either hyperpolarizing or depolarizing potentials. 3. The inhibitory response desensitized after prolonged (> 1 min) exposure to 10 microM adenosine, whereas multiple brief (< 30 s) applications slightly decreased the subsequent response. 4. Adenosine-induced Ca2+ current inhibition was mediated by an A1-type adenosine receptor, because the half-maximal inhibition value for an A1 receptor selective agonist, chloro-N-cyclopentyladenosine, was 1,000-fold lower than that for an A2 receptor selective agonist, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarbozamido adenosine hydrochloride (33 nM vs. 40 microM, respectively). 5. A guanine nucleotide binding protein (G protein) appeared to be involved in the action of adenosine, because: 1) the adenosine-induced current inhibition could be largely relieved by depolarizing voltage prepulses; 2) tail current analysis revealed that adenosine shifted Ca(2+)-channel activation to more depolarized potentials; and 3) adenosine inhibition was abolished by 2 mM intracellular guanosine 5'-O-(2-thiodiphosphate) or 500 ng/ml pertussis toxin pretreatment. 6. Adenosine did not appear to inhibit L-type Ca2+ channels, because the prolonged tail current component induced by the dihydropyridine "agonist" 2,6-dimethy-3-carbomethoxy-5-nitro-4-(2-trifluoromethyl-phenyl)- 1,4-dihydropyridine (2 microM) was not affected by adenosine. 7. Adenosine-induced inhibition was reduced to approximately 15% after application of 10 microM omega-conotoxin GVIA, suggesting that adenosine primarily inhibits N-type Ca2+ channels. The Ca(2+)-current component resistant to omega-conotoxin GVIA was also resistant to omega-agatoxin IVA (200 nM), suggesting a lack of P-type of Ca2+ channels in SCG neurons. 8. In conclusion, adenosine produces a dose-, time-, and voltage-dependent inhibition of Ca2+ currents in SCG neurons. Adenosine acts on an A1 adenosine receptor subtype in SCG neurons via a pertussis toxin-sensitive G protein to inhibit N-type Ca2+ channels and an unidentified Ca(2+)-current component. Modulation of Ca2+ currents by adenosine may be an important mechanism for its inhibitory effect on neurotransmitter release in sympathetic neurons.

2,3-Butanedione monoxime blockade of Ca 2+ currents in adult rat sympathetic neurons does not involve ‘chemical phosphatase’ activity

Effects of the ‘chemical phosphatase’ 2,3-butanedione monoxide (BDM) on voltage-activated Ca 2+ currents of adult rat superior cervical ganglion neurons were investigated using the whole-cell patch-clamp technique. BDM produced a rapid (<10 s), reversible and dose-dependent ( IC 50 = 18.3 mM) inhibition of Ca 2+ currents. The action of BDM was not prevented by 1 mM 8-(4-chlorophenylthio)-cAMP or 50 μM isoproterenol. H-7, a non-specific protein kinase inhibitor at 200 μM, did not prevent the rapid recovery from BDM-induced inhibition. Our results suggest that BDM inhibition of Ca 2+ currents in rat sympathetic neurons does not require a ‘chemical phosphatase’ activity.

Norepinephrine-induced Ca 2+ current inhibition in adult rat sympathetic neurons does not require protein kinase C activation

Experiments were performed to investigate if protein kinase C is involved in the norepinephrine-induced α 2-adrenoceptor-mediated inhibition of the Ca 2+ current in adult rat superior cervical ganglion neurons. Ca 2+ currents were recorded from dispersed superior cervical ganglion neurons, acutely isolated from adult rats, using the whole-cell patch-clamp technique. Both norepinephrine and the protein kinase C activator, 1,2-dioctanoylglycerol (diC8) decreased the Ca 2+ current induced by step depolarizations to +10 mV from a holding potential of −80 mV. In the presence of norepinephrine, the Ca 2+ current rising phase was adequately fit by a double exponential with a second time constant much larger than control, whereas in the presence of diC8 the rising phase became mono-exponential and the current displayed a prominent decay. Control tail current activation curves were described by the sum of two Boltzmann functions. Both norepinephrine and diC8 reduced peak tail current amplitude. Norepinephrine preferentially reduced the component activated at more hyperpolarized potentials, while diC8 preferentially reduced the component activated at more depolarized potentials. Intracellular application of three protein kinase C inhibitors: protein kinase C pseudosubstrate (PKC-19-36) (2 μM), staurosporine (1 μM) and 1-(5-isoquinolinylsulonyl)-2-sulfonylpiperazine (H-7) (50 μM), failed to affect norepinephrine-induced Ca 2+ current inhibition. In addition, these protein kinase C inhibitors did not decrease the Ca 2+ current inhibition induced by diC8. Thus, it appears that norepinephrine and diC8: (a) inhibit Ca 2+ currents by different mechanisms, (b) the α 2-adrenoceptor-mediated inhibition of the Ca 2+ current of superior cervical ganglion neurons from adult rats is not transducted by protein kinase C activation, and (c) diC8 can inhibit Ca 2+ currents by a protein kinase C-independent mechanism.