Ventrolateral Nucleus Tractus Solitarius Research Articles

Characterization of Tyrosine Hydroxylase‐Positive Neurons Projections from the Paraventricular Nucleus of Hypothalamus to Hindbrain Autonomic Regulatory Centers

Hypothalamus is a key brain region for cardiovascular, behavioral, and metabolic regulation that contains complex type of excitatory or inhibitory neurons. Tyrosine hydroxylase (TH)-positive neurons in the hypothalamus play an important role in cardiometabolic regulation; however, the anatomical projections of TH neurons in the paraventricular nucleus hypothalamus (PVN) to key parasympathetic and sympathetic premotor nuclei in preautonomic regions of the brain and their connections to the liver remains undefined. This study aims to characterize the projections from THPVN neurons to ventral preautonomic regulating centers and their connection to liver neuronal projections. To achieve our goal, Cre recombinase-dependent TIT2L-eGFP reporter mice were microinjected with adeno-associated virus encoding the Cre recombinase driven by a rat TH promoter (AAV2-TH-Cre, 6.8 x 108 vg/site) bilaterally into the PVN using a stereotaxic apparatus. A retrograde pseudorabies virus with red fluorescence reporter (PRV-614-RFP) was injected into the liver (3 sites, 3ul/site) to identify liver-related neurons in the brainstem preautonotmic centers. One month after injection, mice were transcardially perfused and brain tissues were processed for confocal imaging. The AAV2-TH-Cre induced a robust expression of GFP mostly in the PVN neuronal cell bodies but not in other brain regions. In the brainstem preautonomic centers, we found dense projections in the nucleus tractus solitarius (NTS) and some projections to the dorsal motor nucleus of the vagus (DMV) and rostral ventrolateral medulla (RVLM). Interestingly, there was also dense projects into the median eminence (ME) that regulates neural hormones release. Importantly, the PRV-RFP retrograded from liver was found throughout the DMV and ventrolateral NTS . Particularly in the NTS, there was dense synaptic contact of the THPVN neuronal projections with the liver related neurons (PRV-RFP+ cell). In addition, we also observed that some PRV-RFP+ cells colocalized with TH-positive neurons in the PVN. In sum, the THPVN neurons project anatomically to the autonomic and neurosecretory regions of brain and may modulate the cardiometabolic activity and liver metabolism through these projections.

Midbrain control of breathing and blood pressure: The role of periaqueductal gray matter and mesencephalic collicular neuronal microcircuit oscillators.

Neural circuitry residing within the medullary ventral respiratory column nuclei and dorsal respiratory group interact with the Kölliker-Fuse and medial parabrachial nuclei to generate the corebreathing rhythm and patternduring resting conditions. Triphasic eupnea consists of inspiratory [I], post-inspiratory [post-I], and late-expiratory [E2] phases. Mesencephalic zones exert modulatory influences upon respiratory rhythm-generating circuitry, sympathetic oscillators, and parasympathetic nuclei. The earliest evidence supporting the existence of midbrain control of breathing derives from studies conducted by Martin and Booker in 1878. These authors demonstrated electrical stimulation of the deep layers of themesencephalic colliculi in the rabbitaugmented ventilation and sequentially elicited chest wall tremors and tetany. Investigations performed during the past several decadeswould demonstrate stimlation of distributed zones within the midbrain reticular formation elicits starkly disparate effects upon respiratory phase switching. Schmid, Böhmer, and Fallert demonstrated electrical stimulation of the nucleus rubreand emanating axon bundles alternately elicits or inhibits the activity of medullary expiratory- or inspiratory-related units and phrenic nerve discharge with differential latency. A series of studies would later indicate the red nucleus mediates hypoxic ventilatory depression. Periaqueductal gray matter neurons exhibit extensive afferent and efferent interconnectivity with suprabulbar, brainstem, and spinal cord zones aptly positioning these units to modulate breathing, autonomic outflow, nociception locomotion, micturtion, and sexual behavior.Experimental stimulatory activation of the tectal colliculi and periaqueductal gray matter via electrical current or glutamate, D,L-homocysteinic acid, or bicuculline microinjections coordinately modulates neuromotor inspiratory bursting frequency and amplitude and discharge of pre-Bötzinger complex, ventrolateral medullary late-I and post-I, and ventrolateral nucleus tractus solitarius decrementing early-I and augmenting and decrementing late-I neurons, elicits expiratory outflow and vocalization, and blunt the Hering-Breuer reflex in unanesthetzed decerebrate and anesthetized preprations of the cat and rat. Stimulation of the mesencephalic colliuli or dorsal divisions of the PAG potently amplifes renal sympathetic neural efferent activity, dynamic arterial pressure magnitude, and myocardial contraction frequency and elicits various behavioral defense responses. Elicited physiological effects exhibit extensive locoregional heterogeneity and variably enlist requisite contributions from the dorsomedial hypothalamus and/or lateral parabrachial nuclei. Stimulation of the dorsal mesencephalon occasionally elicits dynamic increases of arterial pressure magnitude exhibiting prominent oscillatory variability coherent with phrenic nerve discharge, perhaps by generating intra-neuraxial hysteresis, serving to intermittently deliver blood to organ vascular beds under high pressure in order to prevent organ edema, microcirculatory dysfunction, and downregulation of vascular smooth muscle alpha adrenergic receptors. Chemosensitive mesencephalic caudal raphéunits and projections of hypoxia-sensitive units in the caudal hypothalamus to the periaqueductal gray matter may imply the existence of a diencephalo-smesencephalic chemosensitive network modulating breathing and sympathetic discharge.

Identification of different types of respiratory neurones in the dorsal brainstem nucleus tractus solitarius of the rat

In Nembutal anaesthetised, spontaneously breathing rats, stereotaxic mapping of the nucleus tractus solitarius (NTS) for respiratory neuronal activity was undertaken. Eight different types of respiratory cells were found between 0.25 and 1.5 mm lateral to midline, extending 0.5 mm caudal to 1.5 mm rostral to obex, and 0.4–1.5 mm below the dorsal surface. A study of the respiratory motor (diaphragm EMG) and neuronal responses to excitatory amino acid (EAA) stimulation of the NTS areas was undertaken. Electrical stimulation of the vagus nerve was employed to study the NTS cellular responses to activation of pulmonary afferents. The effects of chemical activation of the midbrain periaqueductal grey (PAG) on NTS respiratory neuronal activity were investigated. EAA microinjections into the ventrolateral NTS rostral to the obex resulted in an increase in respiratory motor frequency along with increases to inspiratory cell discharge, whilst microinjections into the medial NTS caudal to the obex caused respiratory depression. EAA stimulation of calamus scriptorius produced apnea. NTS inspiratory neurones were inhibited following stimulation of ipsilateral vagus nerve, suggesting their involvement in the Hering–Breuer reflex pathway. PAG stimulation caused excitation of the NTS inspiratory cells indicating the presence of an excitatory respiratory pathway between the two nuclei. Following β-adrenergic antagonist pre-treatment of ventrolateral NTS, EAA microinjections into PAG did not evoke a cardiorespiratory effect. Based on the various findings the role of NTS in organising respiration in the rat is discussed.

Sonic hedgehog signalling in neurons of adult ventrolateral nucleus tractus solitarius

The transmembrane receptor Patched (Ptc) mediates the action of the diffusing factor Sonic hedgehog (Shh), which is implicated in establishing morphogenetic gradients during embryonic development. Whereas alteration of Ptc function is associated with developmental abnormalities and brain tumors, its functional activity and roles in the adult brain have yet to be elucidated. Here we describe the complementary pattern of Shh and Ptc expression in the rat dorsal vagal motor nucleus and the ventrolateral nucleus tractus solitarius (vNTS), respectively. Those two interconnected structures regulate the cardiorespiratory function during hypoxia. Bath application of a subnanomolar concentration of aminoterminal Shh protein (ShhN) to a slice preparation of the vNTS induces a rapid decrease in neuronal firing followed by a bursting activity that propagates in the neuronal network. Intracellular current injections show that bursts result from an action on the neuronal membrane electro-responsiveness. Both inhibiting and bursting effects are blocked by the monoclonal Shh antibody 5E1 and may require the Ptc binding site of ShhN. Thus, ShhN acting on specific neuronal sites controls electrophysiological properties of differentiated neurons of the vNTS. We speculate on a retrocontrol of cardiorespiratory signals in the vNTS, by Shh generated in dorsal vagal motoneurons.

Central neurocircuitry associated with emesis

Ingestion of toxin, traumatic events, adverse drug reactions, and motion can all result in nausea and emesis. In addition, cyclic vomiting syndrome is quite prevalent in the pediatric population. Coordination of the various autonomic changes associated with emesis occurs at the level of the medulla oblongata of the hindbrain. Chemosensitive receptors detect emetic agents in the blood and relay this information by means of neurons in the area postrema to the adjacent nucleus tractus solitarius (NTS). Abdominal vagal afferents that detect intestinal luminal contents and gastric tone also terminate in the NTS (gelatinosus, commissural, and medial subnuclei). The NTS is viscerotopically organized into subnuclei that subserve diverse functions related to swallowing (subnucleus centralis), gastric sensation (subnucleus gelatinosus), laryngeal and pharyngeal sensation (intermediate and interstitial NTS), baroreceptor function (medial NTS), and respiration (ventrolateral NTS). Neurons from the NTS project to a central pattern generator (CPG), which coordinates the sequence of behaviors during emesis, as well as directly to diverse populations of neurons in the ventral medulla and hypothalamus. Thus, it is critical to realize that there is not an isolated “vomiting center,” but rather groups of loosely organized neurons throughout the medulla that may be activated in sequence by a CPG. The newer antiemetic agents appear to block receptors in the peripheral endings of vagal afferents to reduce “perception” of emetic stimuli and/or act in the dorsal vagal complex. A primary site of action of 5-HT 3-receptor antagonists is by means of the vagal afferents. Neurokinin-1 receptor (NK 1R) antagonists are antiemetics, because they act at a site in the dorsal vagal complex. Part of their effectiveness may be the result of inhibition of the NK 1R on vagal motor neurons to prevent fundic relaxation, which is a prodromal event essential for emesis. Delta 9-tetrahydrocannabinol (Δ 9-THC), the major psychoactive component of marijuana, can be therapeutically useful as an antiemetic. The site of action of Δ 9-THC is on cannabinoid CB1 receptors in the dorsal vagal complex. However, it decreases fundic tone and antral motility. It is not easy to predict the potential antiemetic effects of drugs that alter motility. Although antiemetic drugs are available for management of acute chemotherapeutic-induced emesis, few treatments are effective for delayed emesis or cyclic vomiting syndrome.

Evaluation of pulmonary afferent fibers in the nucleus tractus solitarius: a horseradish peroxidase and c-fos like immunohistochemical study in the rat

Wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the rat lung parenchyma, just beneath the lateral surface of the left upper lobe, in order to demonstrate the pulmonary afferents. This injection resulted in heavy accumulation of labeled fibers in the medial nucleus tractus solitarius (NTS). The labeling in the medial NTS was divided into the ventral and dorsal parts at the level around the obex. Some labeling was found in the commissural and ventrolateral NTS. Further confirmation of the central distribution of these pulmonary afferent fibers was made by the expression of fos-like immunoreactivity (FOS-LI) induced by injection of formalin into the lung. It is concluded that afferents of lung parenchyma terminating predominantly in the medial NTS might come from alveoli and terminal bronchioles, because WGA-HRP and formalin injected into the lung are considered to be confined to the terminal areas of the respiratory tract.

Central connections of the motor and sensory vagal systems innervating the trachea

Cholera toxin β-subunit was used as both a transganglionic and retrograde cell body tracer to determine respectively the central sensory and motor systems innervating the trachea in three mammalian species, dog, ferret and rat. A basic pattern was found in all three animals. Sensory fibers terminated in three subnuclei of the nucleus tractus solitarius (NTS) with the densest concentration localized in a restricted part of the medial part of the rostral NTS. Weaker projections were identified in the ventrolateral NTS subnucleus and sparse labeling was seen in the commissural NTS subnucleus. No labeling was identified in the area postrema. The pattern of retrograde cell-body labeling was also similar in all three species. Two main sites were labeled: the rostralmost part of the dorsal vagal nucleus and the rostral nucleus ambiguus (NA). In the NA, cell labeling was found in mainly in the ventral (or external) portion of the nucleus, but some labeled neurons were consistently found in the compact NA as well. In addition, labeled neurons were also seen in the dorsomedial part of the C1–C2 ventral hom. In summary, the central sites of termination of the sensory fibers and cells of origin innervating the trachea were similar in all three species.

Opioid peptide gene expression in the nucleus trctus solitarius of rat brain and increases induced by unilateral cervical vagotomy: Implications for role of opioid neurons in respiratory control mechanisms

Neurons expressing messenger RNA encoding the opioid peptide precursors, preproenkephalin and preprodynorphin were localized in the medulla oblongata of the rat by in situ hybridization of specific DNA oligonucleotide probes. Neurons containing preproenkephalin messenger RNA were found throughout the medullary reticular formation in the gigantocellular and paragigantocellular reticular nuclei, the parvicellular and lateral reticular nuclei; commissural, medial and ventrolateral subnuclei in the nucleus tractus solitarius and the nucleus of the spinal trigeminal tract. Labelled cells were also concentrated in the more medial regions of the area postrema. In contrast, neurons containing preprodynorphin messenger RNA had a more restricted distribution and were detected in the commissural and ventrolateral nucleus tractus solitarius and nucleus of the spinal trigeminal tract, especially in the more dorsal regions. Expression of preproenkephalin and preprodynorphin messenger RNA was also examined in the dorsal vagal complex of rats that had undergone a unilateral nodose ganglionectomy or cervical vagotomy. Twenty-four hours after both cervical vagotomy and nodose ganglionectomy, there was a specific 1.5–2-fold elevation in preproenkephalin and preprodynorphin messenger RNA levels in the ventrolateral subnucleus of the contralateral nucleus tractus solitarius relative to levels in the ipsilateral nucleus tractus solitarius and in the nucleus tractus solitarius of sham-operated animals. Previous immunohistochemical studies demonstrating the co-localization of enkephalin and dynorphin in the ventrolateral nucleus tractus solitarius suggest that these changes occurred in the same population of neurons. In light of the suggested role of the ventrolateral nucleus tractus solitarius as a central respiratory centre and the activation of the intact pulmonary afferents that innervate this area following a unilateral vagotomy (which increases inspiration volume and expiratory time by affecting the Hering-Breuer reflex), our results suggest a specific involvement of enkephalin- and dynorphin-containing neurons in the ventrolateral nucleus tractus solitarius in central respiratory control mechanisms.

Dorsal medullary inspiratory neurons: effects of superior laryngeal afferent stimulation

In decerebrate paralyzed cats ventilated with a cycle-triggered pump, we examined the responses of inspiratory (I) neurons in the region of the ventrolateral nucleus tractus solitarius (NTS) to single electrical stimuli delivered to the ipsilateral superior laryngeal nerve (SLN). Sixty-five I neurons were classified as: I(−), I(0), I(+,early), I(+,late) or I(other) on the basis of responses to lung inflation, and as I(bulbophrenic) or I(non-bulbophrenic) on the basis of evidence of an excitatory projection to the contralateral phrenic motoneuron pool. The peristimulus histograms of contralateral phrenic activity showed an early peak of excitation with average latency of4.9 ± 0.1ms(mean±S.E.M.), followed by depression at7.3 ± 0.2ms, start of recovery from depression at22.7 ± 1.0ms, and recovery to control levels at28.4 ± 1.1.ms. The peristimulus histograms of ipsilateral I unit activity showed an initial excitation (latency2.9 ± 0.3ms), followed by spiking silence (latency6.0 ± 0.6ms) and recovery to control discharge frequency at38.8 ± 3.6ms. This time of inhibition was significantly longer than the time of phrenic depression, suggesting that other bulbophrenic excitatory projections are able to rapidly compensate for decreased NTS output. Subgroups of I neurons, as classified by lung inflation tests, did not differe significantly with respect to these timing variables. In contrast, latencies of excitation for I(bulbophrenic) neurons were significantly less than for I(non-bulbophrenic) neurons.

Excitation of upper cervical inspiratory neurons by inspiratory neurons of the nucleus retroambigualis in the cat

The experiments reported here examined inputs from inspiratory neurons of the nucleus retroambigualis to upper cervical inspiratory neurons. Antidromic mapping in contralateral C1 demonstrated the existence of axon collaterals for 9 of 19 nucleus retroambigualis axons tested (47%). Forty nucleus retroambigualis neurons were tested with antidromic mapping for a projection to ihe ipsilateral C1 segment; 9 40 (22%) had an ipsilateral axon (8 of these also projected contralaterally), and 2 40 (5%) had an axon collateral in ipsilateral C1. Cross-correlation histograms suggested monosynaptic excitation of an upper cervical inspiratory neuron by a contralateral nucleus retroambigualis neuron in 4 69 cases (6%) and common input to the pair in 2 69 cases (3%). Six of the 69 cross-correlograms were computed during antidromic activation of the nucleus retroambigualis neuron, and one of these six demonstrated direct excitation of the upper cervical inspiratory neuron by the retroambigualis neuron. We concluded that at least some upper cervical inspiratory neurons receive monosynaptic excitation from the contralateral, and possibly the ipsilateral, nucleus retroambigualis inspiratory neurons. These results, together with those published elsewhere for inputs from inspiratory neurons in the contralateral ventrolateral nucleus tractus solitarius, suggest that the rhythmic, inspiratory firing pattern of the upper cervical inspiratory neurons is due to excitatory fnputs from these two inspiratory bulbospinal neuron populations.

Monosynaptic excitation of thoracic motoneurones by inspiratory neurones of the nucleus tractus solitarius in the cat.

1. The connection between inspiratory neurones in the ventrolateral nucleus tractus solitarius (n.t.s.) and intercostal motoneurones was examined. 2. Descending axonal projections to contralateral T3-T5 spinal segments were found for 110 of 142 (77%) ventrolateral n.t.s. neurones examined. 3. Antidromic mapping was used to locate the axons of thirty-nine ventrolateral n.t.s. neurones in T4, and evidence for axon collaterals was found for thirty-two of forty-seven (68%) neurones examined. Axon collaterals were found in both T3 and T4 for four of nine neurones examined and in T3, T4 and T5 for two of three neurones examined. 4. Cross-correlation histograms were calculated for sixty-five ventrolateral n.t.s. neurones with the contralateral intercostal nerves. Peaks in the cross-correlograms were assessed for significance by calculating k, the ratio of the peak bin count to the mean bin count. Significant peaks (k ratios 1.07-1.24, mean 1.15) were found for twenty-eight (39%) cross-correlograms. Twelve of thirty-three (36%) were for the whole external intercostal nerve, ten of twenty-seven (37%) were for the whole internal intercostal nerve and six of eleven (54%) were for external intercostal nerve filaments. 5. Six of the cross-correlogram peaks were less than or equal to 1.2 ms in width at a level half-way between the peak and the mean bin count. The rest ranged from 2.0 to 4.6 ms (mean 3.0 ms). 6. Intracellular recordings from either internal or external intercostal motoneurones were made and averages of the intracellular potentials were computed using ventrolateral n.t.s. neurone spikes as triggers. 7. Thirty-two spike-triggered averages were computed for pairings between nineteen ventrolateral n.t.s. neurones and thirty-two intercostal motoneurones (twenty-five internal, seven external). Fast-rising, short-lasting depolarizations indicative of a monosynaptic e.p.s.p. were found for five ventrolateral n.t.s. neurones. 8. The characteristics of the cross-correlogram peaks were considered with respect to the e.p.s.p. shapes and it was concluded that the intercostal motoneurones receive a significant monosynaptic excitation from ventrolateral n.t.s. neurones.

Excitation of upper cervical inspiratory neurons by inspiratory neurons of the nucleus tractus solitarius in the cat

Experiments were conducted to investigate the connection from inspiratory neurons in the ventrolateral region of the nucleus tractus solitarius to the inspiratory neurons in the contralateral upper cervical (C1–C2) spinal cord. Microstimulation within the region usually produced long-latency (10 to 30 ms) synaptic activation of the upper cervical inspiratory neurons. Antidromic mapping of axons of the ventrolateral nucleus tractus solitarius in C1 demonstrated the existence of collateral arbors in the region of the upper cervical inspiratory neurons (7 of 15). Cross-correlation histograms showed the existence of short-term synchronization of firing for 12 of 74 pairs of ventrolateral nucleus tractus solitarius and upper cervical inspiratory neurons. Cross-correlation combined with antidromic activation showed that the shortterm synchronization was due to a direct connection in two cases and a common input in one case. It was concluded that at least some ventrolateral nucleus tractus solitarius inspiratory neurons may directly excite upper cervical inspiratory neurons, probably via monosynaptic connections. Both groups of neurons may also share common inputs.

The excitation by neurotensin of nucleus tractus solitarius neurons induces apneustic breathing

The possible involvement of neurotensin in the regulation of respiratory drive has been tested on single brainstem respiratory related neurons and on the global respiratory output. The neuropeptide was locally applied either by microiontopheresis or by pressure injection in the dorsal and ventral respiratory areas of the anesthetized bivagotomized cat. Effects of neurotensin applications were studied, on the one hand on the firing discharge of respiratory related neurons and on the other hand on the phrenic nerve activity and on arterial blood pressure. An increase of the firing frequency of respiratory related neurons was induced by neurotensin applied by iontophoresis or by pressure injection (0.005–33.5 fmol/s) on single neurons. In the latter case, neurotensin was active at concentration 10 3 times lower than glutamate. A bilateral apneustic pattern was induced on the phrenic nerve activities by microinjection of neurotensin (0.23–0.54 pmol/s) in one ventrolateral nucleus tractus solitarius without alteration of arterial blood pressure. These results suggest that the release of neurotensin in the nucleus tractus solitarius regulates respiratory rhythmogenesis by increasing the inspiratory duration.