Stimulation Of Tractus Solitarius Research Articles

Astrocytic glutamate transporters reduce the neuronal and physiological influence of metabotropic glutamate receptors in nucleus tractus solitarii.

Astrocytic excitatory amino acid transporters (EAATs) are critical to restraining synaptic and neuronal activity in the nucleus tractus solitarii (nTS). Relief of nTS EAAT restraint generates two opposing effects, an increase in neuronal excitability that reduces blood pressure and breathing and an attenuation in afferent [tractus solitarius (TS)]-driven excitatory postsynaptic current (EPSC) amplitude. Although the former is due, in part, to activation of ionotropic glutamate receptors, there remains a substantial contribution from another unidentified glutamate receptor. In addition, the mechanism(s) by which EAAT inhibition reduced TS-EPSC amplitude is unknown. Metabotropic glutamate receptors (mGluRs) differentially modulate nTS excitability. Activation of group I mGluRs on nTS neuron somas leads to depolarization, whereas group II/III mGluRs on sensory afferents decrease TS-EPSC amplitude. Thus we hypothesize that EAATs control postsynaptic excitability and TS-EPSC amplitude via restraint of mGluR activation. To test this hypothesis, we used in vivo recording, brain slice electrophysiology, and imaging of glutamate release and TS-afferent Ca2+. Results show that EAAT blockade in the nTS with (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartic acid (TFB-TBOA) induced group I mGluR-mediated depressor, bradycardic, and apneic responses that were accompanied by neuronal depolarization, elevated discharge, and increased spontaneous synaptic activity. Conversely, upon TS stimulation TFB-TBOA elevated extracellular glutamate to decrease presynaptic Ca2+ and TS-EPSC amplitude via activation of group II/III mGluRs. Together, these data suggest an important role of EAATs in restraining mGluR activation and overall cardiorespiratory function.

Enhancement of excitatory transmission in NTS neurons projecting to ventral medulla of rats exposed to sustained hypoxia is blunted by minocycline.

Rats subjected to sustained hypoxia (SH) present increases in arterial pressure (AP) and in glutamatergic transmission in the nucleus tractus solitarius (NTS) neurons sending projections to ventrolateral medulla (VLM). Treatment with minocycline, a microglial inhibitor, attenuated the increase in AP in response to SH. The increase in the amplitude of glutamatergic postsynaptic currents in the NTS-VLM neurons, induced by postsynaptic mechanisms, was blunted by minocycline treatment. The number of microglial cells was increased in the NTS of vehicle-treated SH rats but not in the NTS of minocycline-treated rats. The data show that microglial recruitment/proliferation induced by SH is associated with the enhancement of excitatory neurotransmission in NTS-VLM neurons, which may contribute to the observed increase in AP. Short-term sustained hypoxia (SH) produces significant autonomic and respiratory adjustments and triggers activation of microglia, the resident immune cells in the brain. SH also enhances glutamatergic neurotransmission in the NTS. Here we evaluated the role of microglial activation induced by SH on the cardiovascular changes and mainly on glutamatergic neurotransmission in NTS neurons sending projections to the ventrolateral medulla (NTS-VLM), using a microglia inhibitor (minocycline). Direct measurement of arterial pressure (AP) in freely moving rats showed that SH (24h, fraction of inspired oxygen ( ) 0.1) in vehicle and minocycline (30mg/kg i.p. for 3days)-treated groups produced a significant increase in AP in relation to control groups under normoxic conditions, but this increase was significantly lower in minocycline-treated rats. Whole-cell patch-clamp recordings revealed that the active properties of the membrane were comparable among the groups. Nevertheless, the amplitudes of glutamatergic postsynaptic currents, evoked by tractus solitarius stimulation, were increased in NTS-VLM neurons of SH rats. Changes in asynchronous glutamatergic currents indicated that the observed increase in amplitude was due to postsynaptic mechanisms. These changes were blunted in the SH group previously treated with minocycline. Using immunofluorescence, we found that the number of microglial cells was increased in the NTS of vehicle-treated SH rats but not in the NTS neurons of minocycline-treated rats. Our data support the concept that microglial activation induced by SH is associated with the enhancement of excitatory neurotransmission in NTS-VLM neurons, which may contribute to the increase in AP observed in this experimental model.

Characterization of synapses in the rat subnucleus centralis of the nucleus tractus solitarius.

The nucleus tractus solitarius (NTS) receives subdiaphragmatic visceral sensory information via vagal A- or C-fibers. We have recently shown that, in contrast to cardiovascular NTS medialis neurons, which respond to either purinergic or vanilloid agonists, the majority of esophageal NTS centralis (cNTS) neurons respond to vanilloid agonists, whereas a smaller subset responds to both vanilloid and purinerigic agonists. The present study aimed to further investigate the neurochemical and synaptic characteristics of cNTS neurons using whole cell patch-clamp, single cell RT-PCR and immunohistochemistry. Excitatory postsynaptic currents (EPSCs) were evoked in cNTS by tractus solitarius stimulation, and in 19 of 64 neurons perfusion with the purinergic agonist αβ-methylene ATP (αβMeATP) increased the evoked EPSC amplitude significantly. Furthermore, neurons with αβMeATP-responsive synaptic inputs had different probabilities of release compared with nonresponsive neurons. Single cell RT-PCR revealed that 8 of 13 αβMeATP-responsive neurons expressed metabotropic glutamate receptor 8 (mGluR8) mRNA, which our previous studies have suggested is a marker of glutamatergic neurons, whereas only 3 of 13 expressed glutamic acid dehydroxylase, a marker of GABAergic neurons. A significantly lower proportion of αβMeATP-nonresponsive neurons expressed mGluR8 (2 of 30 neurons), whereas a greater proportion expressed glutamic acid dehydroxylase (12 of 30 neurons). Esophageal distension significantly increased the number of colocalized mGluR8- and c-Fos-immunoreactive neurons in the cNTS from 8.0 ± 4% to 20 ± 2.5%. These data indicate that cNTS comprises distinct neuronal subpopulations that can be distinguished based on their responses to purinergic agonists and that these subpopulations have distinct neurochemical and synaptic characteristics, suggesting that integration of sensory inputs from the esophagus relies on a discrete organization of synapses between vagal afferent fibers and cNTS neurons.

Glial cells modulate the synaptic transmission of NTS neurons sending projections to ventral medulla of Wistar rats.

There is evidence that sympathoexcitatory and respiratory responses to chemoreflex activation involve ventrolateral medulla-projecting nucleus tractus solitarius (NTS) neurons (NTS-VLM neurons) and also that ATP modulates this neurotransmission. Here, we evaluated whether or not astrocytes is the source of endogenous ATP modulating the synaptic transmission in NTS-VLM neurons. Synaptic activities of putative astrocytes or NTS-VLM neurons were recorded using whole cell patch clamp. Tractus solitarius (TS) stimulation induced TS-evoked excitatory postsynaptic currents (TS-eEPSCs) in NTS-VLM neurons as well in NTS putative astrocytes, which were also identified by previous labeling. Fluoracetate (FAC), an inhibitor of glial metabolism, reduced TS-eEPSCs amplitude (−85.6 ± 16 vs. −39 ± 7.1 pA, n = 12) and sEPSCs frequency (2.8 ± 0.5 vs. 1.8 ± 0.46 Hz, n = 10) in recorded NTS-VLM neurons, indicating a gliomodulation of glutamatergic currents. To verify the involvement of endogenous ATP a purinergic antagonist was used, which reduced the TS-eEPSCs amplitude (−207 ± 50 vs. −149 ± 50 pA, n = 6), the sEPSCs frequency (1.19 ± 0.2 vs. 0.62 ± 0.11 Hz, n = 6), and increased the paired-pulse ratio (PPR) values (∼20%) in NTS-VLM neurons. Simultaneous perfusion of Pyridoxalphosphate-6-azophenyl-2′,5′-disulfonic acid (iso-PPADS) and FAC produced reduction in TS-eEPSCs similar to that observed with iso-PPADS or FAC alone, indicating that glial cells are the source of ATP released after TS stimulation. Extracellular ATP measurement showed that FAC reduced evoked and spontaneous ATP release. All together these data show that putative astrocytes are the source of endogenous ATP, which via activation of presynaptic P2X receptors, facilitates the evoked glutamate release and increases the synaptic transmission efficacy in the NTS-VLM neurons probably involved with the peripheral chemoreflex pathways.

Of apples and oranges: GABA and glutamate transmission in neurones of the nucleus tractus solitarii could not be more different

The nucleus tractus solitarii (NTS) is the principal visceral sensory nucleus in the brain and comprises neurochemically and biophysically distinct neurons located in the dorsomedial medulla oblongata. The NTS conveys information from the gustatory, cardiorespiratory, oesophageal and subdiaphragmatic gastrointestinal visceral systems that is subsequently assimilated with homeostatic signals arriving from an impressive network of connections from other integrative centres of the pons, diencephalon and forebrain. Myelinated A-type and unmyelinated C-type sensory afferent fibres from the trigeminal, facial, glossopharyngeal and vagus nerve form the tractus solitarius (TS) and their terminals overlap along the rostro-caudal extent of the NTS. Regardless of their origin or type, though, afferent fibres entering the brainstem activate NTS neurones via the release of excitatory neurotransmitters, mainly glutamate (Andresen & Kunze, 1994; Baptista et al. 2005). Perhaps the most prominent neurochemical phenotypes encountered in the NTS are GABAergic and glutamatergic, which provide postsynaptic inhibitory and excitatory influences to adjacent motor nuclei such as the dorsal motor nucleus of the vagus, the nucleus ambiguus and the ventrolateral medulla. An impressive amount of work has been conducted in recent times by Andresen's group to characterize the glutamatergic transmission from the tractus solitarius onto NTS neurones. This group has used sophisticated electrophysiological analyses to demonstrate that glutamatergic excitatory transmission is highly effective, has an almost unfailing unitary transmission from primary sensory fibres, and can be associated with forms of synaptic plasticity that are unique to autonomic regulation (Peters et al. 2010). Earlier work by Mifflin (Mifflin, 1996; Mifflin & Felder, 1988) and Smith (Smith et al. 1998), however, showed that TS stimulation can evoke a sequence of excitatory responses that were followed by inhibitory currents, indicating the possible presence of a local GABAergic interneurone. This inhibitory interneurone, however, has not yet been demonstrated with anatomical tools. (Note that the presence of GABAergic neurons within the NTS is not in doubt. What remains to be confirmed, however, is the anatomical proof of their interneurone status, i.e. a neurone that does not project beyond the NTS itself.) The publication by McDougall and Andresen (2012) in this issue of The Journal of Physiology utilises an isolated horizontal brainstem slice in which TS fibres can be stimulated to evoke unitary currents in second order NTS neurones; these currents can then be identified as either mono- or polysynaptic. The authors showed that, in contrast to glutamatergic currents, monosynaptic GABAergic currents are evoked by focal stimulation disproportionately rarely and only following a stimulus that was located in close proximity to the recorded neurone. In addition to the high failure rate, large amplitude variability, low calcium sensitivity and modest frequency-dependent depression, the monosynaptic nature of these GABAergic currents was confirmed by a low jitter (albeit greater than that of glutamatergic synapses) and lack of sensitivity to glutamatergic receptor blockade. Overall, these data suggest rather weak GABAergic transmission whose efficacy must rely on converging activation of inhibitory fibres. These characteristics suggest strongly that the GABA release properties are fundamentally different from the powerful and synaptically reliable glutamatergic inputs impinging onto NTS neurones. When taken together with previous observations reporting that each TS-driven glutamatergic input targets individual NTS neurons selectively, and that the physiological and pharmacological responses of NTS neurones are target-coded (Bailey et al. 2006; Browning et al. 2011; Jin et al. 2004), the overall picture of the NTS strengthens the image of a hyperorganized nucleus with ‘task matching’ capabilities conferring an ability to cope with widely variable demands, both in terms of timing as well as duration and modulation of a response. Furthermore, this type of cellular organization within the NTS should force us, once again, to be extremely cautious in the often simplistic physiological interpretation of observations, often driven by high intensity stimuli, that analyse a single outcome such as cFos expression or calcium oscillations, which provide limited information regarding the excitability of neurones and provide no information at all regarding inhibitory transmission. Several questions remain to be answered before one can elucidate the role of these synaptic connections. For example, what are the inputs that activate these second order GABAergic neurones? What is their origin? Are they anatomically defined interneurones, or do they project beyond the NTS? Are they related to a particular pathway? Do they have a specific physiological role or are they ‘simply’ a low pass filter for excitatory TS-driven inputs?

Synaptic profile of nucleus tractus solitarius neurons involved with the peripheral chemoreflex pathways

The glomus cells in the carotid bodies (CB) detect alterations in pH and pCO₂ and low pO₂ level in arterial blood. The carotid sinus nerve conveys the information related to the oxygen level to 2nd-order neurons in the nucleus tractus solitarius (NTS) via tractus solitarius (TS), which is part of the chemoreflex pathways. It has been demonstrated that in 2nd-order NTS neurons receiving inputs from the aortic depressor nerve (ADN), the TS stimulation presents high temporal fidelity. However, the temporal properties of synaptic activity in NTS neurons receiving inputs from CB were not yet fully investigated. Herein using patch-clamp recordings in NTS brainstem slices, we studied TS-evoked excitatory postsynaptic currents (TS-eEPSCs) on morphologically identified 2nd-order NTS neurons that receive afferent inputs from the CB and compared with 2nd-order ADN-NTS neurons recorded in the same experimental conditions. The amplitudes of TS-eEPSCs were similar in both groups, but the latencies and standard deviation (SD) of latency were significantly higher in the CB-NTS neurons (latency: 4±0.2 ms, SD: 0.49±0.03 ms) than in ADN-NTS neurons (latency: 3.3±0.3 ms, SD: 0.19±0.02 ms; P=0.049 for latency and P<0.001 for SD of latency). In a series of double-labeling experiments, we confirmed that some CB-NTS 2nd-order neurons send direct projections to the rostral ventrolateral medulla (RVLM). We conclude that: (a) CB-NTS 2nd-order neurons present temporally distinct postsynaptic currents when compared with ADN-NTS 2nd-order neurons; (b) low SD of latency of TS-eEPSCs is not necessarily a characteristic of all 2nd-order neurons in the NTS; and (c) the presence of direct connections between these 2nd-order neurons in the NTS and RVLM is indicative that these synaptic properties of CB-NTS neurons are relevant for the processing of respiratory and autonomic responses to chemoreflex activation.

Synaptic properties of peripheral chemoreflex second-order neurons in the NTS of rats

Background/aims: The glomus cells in the carotid bodies (CB) detect low pO2 level in arterial blood and the tractus solitarius (TS) conveys this information to 2nd-order neurons in the nucleus tractus solitarius (NTS), which is part of the chemoreflex pathways. It has been demonstrated that 2nd-order NTS neurons receiving inputs from the aortic depressor nerve (ADN), related to the baroreflex, present low temporal variability in response to TS stimulation. However, the temporal variability of synaptic activity in CB–NTS neurons is not yet completely understood.

A novel postsynaptic group II metabotropic glutamate receptor role in modulating baroreceptor signal transmission.

The nucleus tractus solitarius (NTS) is essential for orchestrating baroreflex control of blood pressure. When a change in blood pressure occurs, the information is transmitted by baroreceptor afferent fibers to the central network by glutamate binding to ionotropic glutamate receptors on second-order baroreceptor neurons. Glutamate also activates presynaptic group II and III metabotropic glutamate receptors (mGluRs), depressing both glutamate and GABA release to modulate baroreceptor signal transmission. Here we present a novel role for postsynaptic group II mGluRs to further fine-tune baroreceptor signal transmission at the first central synapses. In a brainstem slice with ionotropic glutamate and GABA receptors blocked, whole-cell patch-clamp recordings of second-order baroreceptor neurons revealed that two group II mGluR agonists evoked concentration-dependent membrane hyperpolarizations. The hyperpolarization remained when a presynaptic contribution was prevented with Cd(2+), was blocked by a postsynaptic intervention of intracellular dialysis of the G-protein signaling inhibitor, was mimicked by endogenous release of glutamate by tractus solitarius stimulation, and was prevented by a group II mGluR antagonist. Postsynaptic localization of group II mGluRs was confirmed by fluorescent confocal immunohistochemistry and light microscopy. Group II mGluR induced-currents consisted of voltage-dependent outward and inward components, prevented by tetraethylammonium chloride and tetrodotoxin, respectively. In contrast to group II mGluR-induced hyperpolarization, there was no effect on intrinsic excitability as determined by action potential shape or firing in response to depolarizing current injections. The data suggest a novel mechanism for postsynaptic group II mGluRs to fine-tune baroreceptor signal transmission in the NTS.

Electrophysiological and morphological characteristics of nucleus tractus solitarii neurons projecting to the ventrolateral medulla

Electrophysiological and morphological properties of a direct projection from the nucleus of the tractus solitarius (NTS) to the ventrolateral medulla (VLM) were investigated. NTS neurons projecting to the VLM exhibit a monosynaptic excitatory response followed by an inhibitory one after the tractus solitarius stimulation. These neurons show spontaneous inhibitory postsynaptic currents, and have medium to large soma (14–26 μm in diameter). It is concluded that the projection from the NTS to the VLM is mediated mostly by medium to large neurons that are inhibited locally by GABAergic interneurons within the NTS.

Bursting discharges evoked in vitro, by solitary tract stimulation or application of , in neurons of the rat nucleus tractus solitarii

Extracellular recordings of the activity of neurons in the isolated nucleus tractus solitarii (NTS) showed that repetitive stimulation of the tractus solitarius (TS) elicited either long-lasting discharges at low frequency (type A neurons) or short-duration bursting discharges at low or high frequency (type B and C neurons, respectively). Bath application of N-methyl-D-aspartate (NMDA; 60-120 microM; 4-5 min duration) elicited a pattern of rhythmic bursting in most type B (21/24) and in all type C neurons and only a repetitive firing in type A neurons, even with applications of longer duration (6-10 min). The rhythmic bursting pattern was characterized by trains of action potentials occurring at a regular rate with each neuron (mean = 0.9 +/- 0.45 Hz). The present findings suggest that local mechanisms within the NTS (synaptic interactions or neuronal intrinsic properties) are involved in the burst firing elicited either by TS stimulation or NMDA application. The data are discussed in terms of the generation of swallowing taking into account the key role of NTS neurons in the organization of this motor pattern.

Neurons of the nucleus tractus solitarius, in vitro, generate bursting activities by solitary tract stimulation

Extracellular recordings of the activity of nucleus tractus solitarius (NTS) neurons were performed on rat brainstem slice preparations. Neurons localized in the medial part of the lateral NTS, which displayed a synaptic response to single pulse stimulation of the tractus solitarius (TS), generated bursting activity following repetitive TS stimulation (20-50 Hz frequency, 100-600 ms duration). According to their patterns of discharge and to the duration and frequency of their bursting activities, these neurons were classified in three groups called type A, B and C. We suggest that different cellular intrinsic properties, rather than local synaptic interactions, might be involved in the generation of these three types of bursting activities. These results are discussed in terms of the role of NTS neurons in the generation of the swallowing motor pattern.

Synaptic responses of neurons of the nucleus tractus solitarius in vitro

Postsynaptic response sof neurons in the nucleus tractus solitarius (NTS) have been studied in an in vitro slice preparation using extra- and intracellular recording. Single or paired pulse stimulations were delivered to afferent fibers within the tractus solitarius (TS) to activate orthodromic responses in these neurons. Most NTS neurons displayed an initial synaptic excitation followed by inhibition of spontaneous or evoked firing lasting up to 150–200 ms after stimulation. Excitatory postsynaptic potentials (EPSPs), recorded intracellularly, were increased in amplitude by membrane hyperpolarization. Large afterhyperpolarizations followed action potentials triggered by the EPSPs or evoked by intracellular current injections. Intracellular evidence for synaptic inhibition within the NTS included: (1) the presence, after Cl −injection, of flurries of spontaneous PSPs likely to be inverted inhibitory postsynaptic potentials; (2) reduction of the size of a test EPSP by a previous subthreshold TS conditioning volley; and (3) hyperpolarizing PSPs recorded in some neurons. Other NTS neurons exhibited prolonged excitatory responses to TS stimulation and could be local inhibitory interneurons. These results may help specify synaptic mechanisms in the NTS that could play an integrative role in the relay of visceral sensory inputs to higher order effectors.