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Mechanism of nitric oxide action on inhibitory GABAergic signaling within the nucleus tractus solitarii

Sheng Wang^*, Anja G. Teschemacher, Julian F. R. Paton^* and Sergey Kasparov^*,1

Departments of

^* Physiology and

Pharmacology, School of Medical Sciences, University of Bristol, Bristol, UK

¹Correspondence: Department of Physiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK. E-mail: sergey.kasparov{at}bristol.ac.uk

SPECIFIC AIMS

Physiological functions of nitric oxide (NO) in the brain are usually linked to its ability to modulate neurotransmitter release. Although the effects of NO on glutamatergic synapses have been studied extensively, very little is known about its effects on -aminobutyric acid (GABA)-ergic transmission even though this could be equally important for various physiological and pathological processes. For example, in the nucleus tractus solitarii (NTS), NO-evoked GABA release may contribute to hypertension. NO could modulate GABAergic transmission by altering excitability of GABAergic interneurons or by direct modulation of transmitter release. At the presynaptic terminal, transmitter release is frequently regulated via changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) either by modulation of external Ca²⁺ influx or release from stores. NO-cGMP pathway has been demonstrated to facilitate Ca²⁺ release from ryanodine-sensitive stores in sea urchin eggs and glia. We hypothesized that NO directly regulates NTS GABAergic inhibitory interneurons via an evolutionary conserved cGMP-cyclic adenosine diphosphate ribose (cADPR)-Ca²⁺ pathway.

PRINCIPAL FINDINGS

1. Low nanomolar concentrations of NO increase [Ca²⁺]_i in GABAergic neurons, with the axons being the most responsive cellular compartment. Somatic depolarization is also observed in some cells
In cultured rat brainstem slices, NTS GABAergic neurons were targeted with an adenoviral vector expressing enhanced green fluorescent protein and studied using patch clamp and confocal Ca²⁺ imaging. The actual concentration of NO at the recording site was measured using an electrochemical NO sensor.

One and ten micromoles diethylamine NONOate (DEA/NO) generated 17 ± 7 and 121 ± 9 nM of NO (n=3 in both cases) in artificial cerebrospinal solution saturated with carbogen, respectively. DEA/NO increased [Ca²⁺]_i in somata of GABAergic neurons in a concentration-dependent manner. [Ca²⁺]_i responses in eight cells treated with 10 µM DEA/NO developed at a latency of 17 ± 1 s and increased over the next 0.3–5 min. Ten micromoles of DEA/NO increased [Ca²⁺]_i in somata (+20±4%) and dendrites (+30±4%), but the most dramatic increase occurred in putative axons (+40±10%, P<0.05 between somata and axons). [Ca²⁺]_i elevations induced by DEA/NO in almost all cases were reversible within 5–10 min.

2. NO action on [Ca²⁺]_i in GABAergic neurons is direct and not via facilitation of glutamatergic inputs or action potential dependent
To remove indirect effects of NO via glutamatergic synapses, 6-cyano-7-nitroxaline-2,3-dione (CNQX; 20 µM) was bath applied. CNQX did not prevent DEA/NO-induced rises in [Ca²⁺]_i and depolarization. One micromolar TTX also failed to prevent effects of DEA/NO. Thus, DEA/NO action is unlikely to result from glutamate release through polysynaptic connections and is action potential independent. Thus, NO has a direct effect on GABAergic interneurons.

3. NO-induced [Ca²⁺]_i elevations are not due to depolarization of GABAergic neurons
Ten micromoles of DEA/NO caused a moderate, reversible, and ¹H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)-sensitive depolarization in 66% of the neurons (+3.4±1 mV). However, depolarization occurred much later than the [Ca²⁺]_i rise (latency of 73±9 s in contrast to <20 s for Ca²⁺ responses). Depolarization can lead to transmembrane influx of extracellular Ca²⁺ and a rise in [Ca²⁺]_i. To control for that, membrane was depolarized using positive current injections by 5 mV (more than that induced by 10 µM DEA/NO). Within 3 min, F/F₀ in soma increased by 10 ± 2% and in dendrites by 14 ± 4%, which was significantly less than that after DEA/NO administration. In addition, DEA/NO increased [Ca²⁺]_i also in cells that did not depolarize. Therefore, NO-induced increases in [Ca²⁺]_i are not a result of membrane depolarization.

4. The pathway mediating NO-induced [Ca²⁺]_i elevations in GABAergic neurons involves cADPR
4.1. Effects of DEA/NO on GABAergic neurons are mediated by soluble guanylate cyclase
Pretreatment with a sGC inhibitor ODQ (10 µM) abolished DEA/NO-evoked [Ca²⁺]_i rises and depolarization.

4.2. IP₃-sensitive stores are not the main source of DEA/NO-evoked [Ca²⁺]_i responses
Preincubation with 100 µM 2-APB, a blocker of IP₃-sensitive stores, was without effect on DEA/NO-evoked [Ca²⁺]_i increases (Fig. 1 ).

View larger version (34K): [in this window] [in a new window]   Figure 1. Calcium stores responsible for DEA/NO-induced [Ca2+]i rises in GABAergic neurons. Average changes in relative fluorescence intensity (F/F0) in GABAergic NTS neurons after administration of DEA/NO and antagonists of different Ca2+ stores. Left bar) Effect of DEA/NO shown for comparison. Pretreatment with 2-APB (an inhibitor of the IP3-sensitive Ca2+ release) failed to prevent the DEA/NO effect. Nifedipine (a putative inhibitor of the NAADP-sensitive channel) did not block DEA/NO-elicited Ca2+ release, although there was a clear tendency to reduce it. Blockade of cADPR/ryanodine-sensitive stores with 8-Br-cADPR (a cADPR antagonist) abolished the effect of DEA/NO on Ca2+ release. Thus, cADPR/ryanodine-sensitive stores are likely to be the primary source of NO-released Ca2+. *P < 0.05, **P < 0.01, compared with control (paired t test). ##P < 0.01, compared with group treated with DEA/NO alone, unpaired t test.

4.3. A possible role for nicotinic acid adenine dinucleotide phosphate-sensitive store?
Nicotinic acid adenine dinucleotide phosphate (NAADP) might operate an independent Ca²⁺ release mechanism, and this pathway can be blocked by high concentrations (100 µM) of an L-type Ca²⁺ channel blocker nifedipine. Pretreatment with 100 µM nifedipine did not completely block DEA/NO action but reduced DEA/NO-induced [Ca²⁺]_i rises from 20 ± 4 to 12 ± 4% (n=4/5; P<0.05; Fig. 1 ).

4.4. 8-Bromo-cADPR (8-Br-cADPR) is a key link between NO and elevations in [Ca²⁺]_i
cADPR is thought to be an endogenous activator of ryanodine-sensitive Ca²⁺ stores. To test for the involvement of these stores 8-bromo-cADPR (8-Br-cADPR; 100 µM), a highly selective antagonist of cADPR that does not block IP₃-sensitive stores, was introduced from the patch pipette directly into the GABAergic neurons. In the presence of 8-Br-cADPR, DEA/NO failed to evoke [Ca²⁺]_i elevations (Fig. 1) . Membrane depolarization was also almost abolished. Therefore, NO acts via cADPR-sensitive Ca²⁺ stores to elevate [Ca²⁺]_i in GABAergic neurons.

5. cADPR mediates NO-induced potentiation of GABAergic inhibitory postsynaptic potentials
Having found that DEA/NO effects on [Ca²⁺]_i in GABAergic neurons are mediated by cADPR, we tested in acute brainstem slices whether cADPR also mediates the NO-induced potentiation of monosynaptic GABAergic inhibitory postsynaptic potentials (IPSPs). Experiments were performed in the presence of CNQX to cancel potential indirect effects related to release of glutamate. 1 µM DEA/NO (which released 55 nM of NO) potentiated IPSP by 32%. DEA/NO-induced IPSP potentiation was essentially abolished by pretreatment with 30 µM 8-Br-cADPR. Thus, NO potentiates monosynaptic GABAergic IPSP via a cADPR-dependent mechanism.

CONCLUSIONS AND SIGNIFICANCE

GABAergic inhibition is fundamental for brain function and is involved in all aspects of its activity. Our results lend support to the hypothesis of direct stimulation of GABA exocytosis by NO. We have found that potentiation of Ca²⁺ release from cADPR/ryanodine-sensitive stores is the principle mechanism by which NO enhances inhibitory transmission, at least in the NTS. This means that at least in some parts of the central nervous system NO may modulate not only excitatory but also inhibitory connections. We propose that the functional destiny of NO release may depend on both the threshold concentrations of NO required for affecting glutamatergic and GABAergic neurons and the spatial relationship between the sources of NO (i.e., the NO synthases) and its targets. Importantly, all effects reported in this study are mediated by sGC and therefore not related to other mechanisms characteristic of high NO concentrations, such as effects on cellular respiration, direct modification of proteins, or formation of the products of NO oxidation.

1. NO has a direct effect on GABAergic neurons
Several studies indicate that NO can elevate extracellular concentrations of glutamate and GABA, but to our knowledge this is the first study that shows that the effect on GABAergic transmission is direct, rather than a result of an increased glutamatergic drive. Our study directly demonstrates that at low nanomolar concentrations NO has two effects on GABAergic neurons (e.g., elevations in [Ca²⁺]_i and depolarization) both of which might contribute to an increase in GABAergic inhibition in the intact brain. These effects persist in the absence of glutamatergic transmission action potentials. Up to date this remained an unresolved issue, although a previous study strongly suggests that this could be the case. NO effect on [Ca²⁺]_i was most dramatic in putative axons, suggesting that axonal release Ca²⁺ may be strongly potentiated by NO, which would be most effective for modulating GABA release. It now becomes important to establish whether the threshold NO concentrations required for enhancing glutamate release is different to those for GABA, as it might be that the two transmitter systems are affected at different levels of activity of NO synthases. The other unresolved issue is the mechanism of NO-induced depolarization, which has been also seen in previous studies. Based on our data with 8-Br-cADPR, which also antagonized depolarization, one could speculate that it might be a downstream effect of cADPR production.

2. cADPR is the link between NO and elevations in [Ca²⁺]_i
Although previous studies suggested that in the paraventricular nucleus NO has direct effect on GABAergic interneurons, the mechanism of this effect remained unknown. We show that in the NTS GABAergic interneurons NO operates via an evolutionary conserved pathway. First described in sea urchin eggs by a previous study, this pathway involves cGMP mediated activation of cADPR production and Ca²⁺ release. cADPR is generated from NAD by ADP-ribosyl cyclase and activates the same stores as ryanodine. Importantly, cADPR/ryanodine-sensitive stores are also responsible for the Ca²⁺-induced Ca²⁺ release triggered by action potentials. Hence, the elevations in axonal [Ca²⁺]_i reported here may be expected to boost the action-potential evoked Ca²⁺ and transmitter release, as found in other cellular models (Fig. 2 ). Whether the NO-cGMP-cADPR-Ca²⁺ pathway is sufficiently powerful to lead to action potential-independent GABA release at present is unclear. In general, our results are consistent with earlier studies linking NO action on Ca²⁺ signaling with cADPR in invertebrates and with the proposed role of cADPR-operated stores in hippocampal LTD. However, to our knowledge, this is the first direct demonstration of the NO-cGMP-cADPR-Ca²⁺ pathway in GABAergic neurons. Importantly, cADPR does not only mediate [Ca²⁺]_i elevations in GABAergic neurons, but it is also crucial for NO potentiation of monosynaptic GABAergic IPSP.

View larger version (49K): [in this window] [in a new window]   Figure 2. Pathway mediating NO-evoked potentiation of GABA release. Background: a patched GABAergic NTS neuron transduced with an adenoviral vector. Plot on right is example of fluorescence response to NO in an axon of 1 such neuron.

Several questions remain unanswered. First, we have noticed that a putative antagonist of a hypothetical separate NAADP store nifedipine (100 µM) partially antagonized the effects of NO on [Ca²⁺]_i. Interestingly, NAADP is produced by the same enzyme that generates cADPR, the ADP-ribosyl cyclase. It would be very useful to find out the exact mechanism of this effect but more selective blockers are needed to convincingly prove a separate NAADP-dependent pathway. Second, it remains to be established whether activation of cADPR/ryanodine-sensitive stores also underlies NO effects on glutamatergic transmission in the NTS as well as other parts of the brain.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5547fje

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This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.05-5547fjev1 20/9/1537    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, S. Articles by Kasparov, S. Search for Related Content PubMed PubMed Citation Articles by Wang, S. Articles by Kasparov, S.