Meredith Hay

Activation of glutamate metabotropic receptors (mGluRs) in nodose ganglia neurons has previously been shown to inhibit voltage-gated Ca++ currents and synaptic vesicle exocytosis. The present study describes the effects of mGluRs on depolarization-induced phosphorylation of the synaptic-vesicle-associated protein synapsin I. Depolarization of cultured nodose ganglia neurons with 60 mM KCl resulted in an increase in synapsin I phosphorylation. Application of mGluR agonists 1-aminocyclopentane-1s-3r-dicarboxylic acid (t-ACPD) and L(+)-2-Amino-4-phosphonobutyric acid (L-AP4) either in combination or independently inhibited the depolarization induced phosphorylation of synapsin I. Application of the mGluR antagonist (RS)-alpha-Methyl-4-carboxyphenylglycine (MCPG) blocked t-ACPD-induced inhibition of synapsin phosphorylation but not the effects of L-AP4. In addition, application of either t-ACPD or L-AP4 in the absence of KCl induced depolarization had no effect on resting synapsin I phosphorylation. RT-PCR analysis of mGluR subtypes in these nodose ganglia neurons revealed that these cells only express group III mGluR subtypes 7 and 8. These results suggest that activation of mGluRs modulates depolarization-induced synapsin I phosphorylation via activation of mGluR7 and/or mGluR8 and that this process may be involved in mGluR inhibition of synaptic vesicle exocytosis in visceral sensory neurons of the nodose ganglia.

The purpose of this chapter is to review some of the recent progress in the understanding of the cellular and biophysical mechanisms that are involved in the regulation of arterial baroreceptor neurotransmission. Synaptic depression or fatigue following repeated neuronal stimulation has been shown at central baroreceptor synapses in vivo and in vitro. As most of the central neurons have a limited number of vesicles, vesicle retrieval or endocytosis following exocytosis is thought to play a major role in preserving synaptic transmission. We have hypothesized that central baroreceptor terminals may inhibit their own synaptic transmission via feedback activation of presynaptic metabotropic glutamate receptors (mGluRs). We have analyzed the effects of mGluR autoreceptors (group III mGluRs) on voltage-gated calcium channels using standard patch-clamp techniques and on the process of exocytosis and endocytosis in aortic baroreceptor neurons using the quantitative imaging dye FM1-43 and FM2-10. Usng the whole-cell patch-clamp technique, we have found that activation of group III mGluRs with L-AP4 inhibits peak calcium channel current. Furthermore, activation of group III mGluRs with L-AP4 markedly decreases stimulation-induced exocytosis in aortic baroreceptor neurons, as measured with FM1-43, and inhibits synapsin I phosphorylation. These results suggest that activation of group III mGluRs may inhibit synaptic transmission by (1) inhibiting calcium influx, (2) decreasing synaptic vesicle exocytosis, and (3) modulating the mechanisms governing synaptic vesicle recovery and endocytosis. These effects of mGluRs on baroreceptor synaptic vesicles may contribute to the baroreceptor/nucleus tractus solitarius synaptic depression observed in vivo.

The effects of metabotropic glutamate receptor activation (mGluR) on voltage-gated potassium currents have been characterized in visceral sensory afferent neurons. L-Glutamate is known to be a primary neurotransmitter in visceral afferents which terminate at the level of the nucleus of the solitary tract (NTS). Synaptic communication between these afferents and the NTS has been shown to involve both postsynaptic ionotropic and presynaptic metabotropic glutamate receptor activation. The purpose of the present study was to determine the effects of mGluR activation on voltage-gated potassium currents in visceral sensory neurons. Application of mGluR agonist t-ACPD inhibited both the peak and the steady state voltage-gated potassium current in 39 out of 56 visceral afferent neurons tested (70%) by 22.0 +/- 3 and 22.8 +/- 2%, respectively. Voltage and pharmacological protocols were utilized to isolate the potassium current affected by mGluR activation. Increasing the holding potential from -100 mV to -30 mV only partially attenuated the inhibitory effects of t-ACPD (decreased effect by 11%), suggesting that t-ACPD modulates both a voltage insensitive and a voltage-sensitive potassium current. In addition, 4-aminopyridine (5 microM) was applied to eliminate the 4-AP sensitive transient current. Also, this protocol only partially attenuated the inhibitory effects of t-ACPD (decreased effect by 6.3%), suggesting that mGluR activation inhibits both a 4-AP-sensitive and 4-AP-insensitive potassium current in visceral afferent neurons. Results from this study suggest that mGluRs may regulate visceral sensory afferent neuronal activity through inhibition of voltage-gated potassium channels.

The present study tested the hypotheses that 1) nitric oxide (NO) is involved in attenuated responses to ANG II in female mice, and 2) there is differential expression of neuronal NO synthase (nNOS) in the subfornical organ (SFO) and paraventricular nucleus (PVN) in response to systemic infusions of ANG II in males vs. females. Aortic blood pressure (BP) was measured in conscious mice with telemetry implants. N(G)-nitro-l-arginine methyl ester (l-NAME; 100 microg x kg(.-1)day(-1)), an inhibitor of NOS, was administrated into the lateral cerebral ventricle for 14 days before and during ANG II pump implantation. Central infusion of l-NAME augmented the pressor effects of systemic ANG II in females (Delta21.5 + or - 2.2 vs. Delta9.2 + or - 1.5 mmHg) but not in males (Delta29.4 + or - 2.5 vs. Delta30.1 + or - 2.5 mmHg). Central administration of N(5)-(1-imino-3-butenyl)-l-ornithine (l-VNIO), a selective nNOS inhibitor, also significantly potentiated the increase in BP induced by ANG II in females (Delta17.5 + or - 3.2 vs. Delta9.2 + or - 1.5 mmHg). In gonadectomized mice, central l-NAME infusion did not affect the pressor response to ANG II in either males or females. Ganglionic blockade after ANG II infusion resulted in a greater reduction in BP in central l-NAME- or l-VNIO-treated females compared with control females. Western blot analysis of nNOS protein expression indicated that levels were approximately 12-fold higher in both the SFO and PVN of intact females compared with those in intact males. Seven days of ANG II treatment resulted in a further increase in nNOS protein expression only in intact females (PVN, to approximately 51-fold). Immunohistochemical studies revealed colocalization of nNOS and estrogen receptors in the SFO and PVN. These results suggest that NO attenuates the increase in BP induced by ANG II through reduced sympathetic outflow in females and that increased nNOS protein expression associated with the presence of female sex hormones plays a protective role against ANG II-induced hypertension in female mice.

1. Metabotropic glutamate receptors (mGluRs) have been suggested to modulate neurotransmission of glutamatergic pathways via autoreceptive action. Visceral sensory afferents and baroreceptor afferents in particular are thought to utilize L-glutamate (L-glu) as a primary neurotransmitter. The purpose of this study was to investigate whether visceral sensory afferents possess a mGluR and determine the effect of mGluR activation on voltage-gated calcium currents in these neurons. 2. Activation of mGluRs by the selective agonist trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD) reversibly suppressed the voltage-gated calcium currents in visceral sensory afferents of the nodose ganglion. Concentrations of t-ACPD ranging from 50 to 1,000 microM consistently decreased the evoked calcium current with a maximum suppression of the peak current of 25-30%. This response was repeatable and reversible within a given cell. 3. Metabotropic GluR activation selectively decreased the high-threshold calcium current evoked from step potentials greater than -30 mV and had no effect on the low-threshold calcium current. The inhibitory effects of t-ACPD on the high-threshold channel was partially blocked by omega-conotoxin (omega-CTx-GVIA) suggesting that at least part of the effects of mGluR inhibition of the voltage-gated calcium current is because of a modulation of the omega-CTx-GVIA sensitive high-threshold current. 4. Finally, the inhibitory effects of quisqualate (quis) on the high-threshold calcium current were blocked by pretreatment of the neurons with pertussis toxin (PTX). These results suggest that visceral sensory afferents do possess a PTX-sensitive mGluR and activation of this receptor results in the inhibition of a omega-CTx-GVIA sensitive high-threshold calcium channel.