Angelina Y. Fong

Immunohistochemical localization of GAD67‐expressing neurons and processes in the rat brainstem: Subregional distribution in the nucleus tractus solitarius

The role of γ‐aminobutyric acid (GABA) in homeostatic control in the brainstem, in particular, in the nucleus tractus solitarius (NTS), is well established. However, to date, there is no detailed description of the distribution of GABAergic neurons within the NTS. The goal of the current study was to reexamine the efficacy of immunohistochemical localization of glutamic acid decarboxylase (GAD) protein, specifically the 67‐kDa isoform (GAD67), as a marker for GABAergic neurons in the medulla and to provide a detailed map of GAD67‐immunoreactive (‐ir) cells within rat NTS by using a recently developed mouse monoclonal antibody. We describe a distribution of GAD67‐ir cells in the medulla similar to that reported previously from in situ hybridization study. GAD67‐ir cells were localized in regions known to contain high GABA content, including the ventrolateral medulla, raphe nuclei, and area postrema, but were absent from all motor nuclei, although dense terminal labeling was discerned in these regions. In the NTS, GAD67‐ir was localized in all subregions. Semiquantitative analysis of the GAD67‐ir distribution in the NTS revealed greater numbers of GAD67‐ir cells medial to the solitary tract. Finally, dense GAD67 terminal labeling was found in the medial, central, intermediate, commissural, and subpostremal subregions, whereas sparse labeling was observed in the ventral subregion. Our findings support the use of immunohistochemistry for GAD67 as a marker for the localization of GABAergic cells and terminal processes in the rat brainstem. Furthermore, the reported heterogeneous distribution of GAD67‐ir in the NTS suggests differential inhibitory modulation of sensory processing. J. Comp. Neurol. 493:274–290, 2005.

The conditional nature of the Central Rhythm Generator and the production of episodic breathing

Episodic breathing patterns have been observed in species of all vertebrate classes under certain conditions and/or at certain times in development. This breathing pattern can be considered part of a continuum between no breathing and continuous breathing. In birds and mammals it is also generally part of a developmental continuum in which episodic breathing occurs early in development and rarely in adults. Production of this pattern appears to be an intrinsic property of the medullary rhythm generating mechanism (possibly due to interactions between different rhythm generating sites) that is stabilized by pontine or midbrain inputs and, in intact animals, is primarily regulated by afferent inputs from chemoreceptors and pulmonary stretch receptors; i.e. there is a hierarchy of control. In all cases, episodes appear to be produced by quantal expression of a fundamental rhythm. At present NO, GABA(A) and glycine mediated processes, and possibly mu-opioid receptor mediated processes, are implicated in the clustering of breaths into episodes. The inter-breath interval, which may occur at either the end of the inspiratory or the expiratory phase in different species, is the primary regulated variable in this pattern. The biological significance of clustering breaths into episodes may relate to reducing the oxidative cost of breathing, enhancing gas exchange or minimizing oxidative damage to tissues.

EXPRESSION OF GROUP I METABOTROPIC GLUTAMATE RECEPTORS ON PHENOTYPICALLY DIFFERENT CELLS WITHIN THE NUCLEUS OF THE SOLITARY TRACT IN THE RAT

Group I metabotropic glutamate receptors (mGluRs) are G-coupled receptors that modulate synaptic activity. Previous studies have shown that Group I mGluRs are present in the nucleus of the solitary tract (NTS), in which many visceral afferents terminate. Microinjection of selective Group I mGluR agonists into the NTS results in a depressor response and decrease in sympathetic nerve activity. There is, however, little evidence detailing which phenotypes of neurons within the NTS express Group I mGluRs. In brainstem slices, we performed immunohistochemical localization of Group I mGluRs and either glutamic acid decarboxylase 67 kDa isoform (GAD67), neuronal nitric oxide synthase (nNOS) or tyrosine hydroxylase (TH). Fluoro-Gold (FG, 2%; 15 nl) was microinjected in the caudal ventrolateral medulla (CVLM) of the rat to retrogradely label NTS neurons that project to CVLM. Group I mGluRs were distributed throughout the rostral-caudal extent of the NTS and were found within most NTS subregions. The relative percentages of Group I mGluR expressing neurons colabeled with the different markers were FG (6.9+/-0.7) nNOS (5.6+/-0.9), TH (3.9+/-1.0), and GAD67 (3.1+/-1.4). The percentage of FG containing cells colabeled with Group I mGluR (13.6+/-2.0) was greater than the percent colabeled with GAD67 (3.1+/-0.5), nNOS (4.7+/-0.5), and TH (0.1+/-0.08). Cells triple labeled for FG, nNOS, and Group I mGluRs were identified in the NTS. Thus, these data provide an anatomical substrate by which Group I mGluRs could modulate activity of CVLM projecting neurons in the NTS.

TARGETED DELETION OF NEUROKININ-1 RECEPTOR EXPRESSING NUCLEUS TRACTUS SOLITARII NEURONS PRECLUDES SOMATOSENSORY DEPRESSION OF ARTERIAL BARORECEPTOR–HEART RATE REFLEX

Neurokinin-1 receptor (NK1-R) expressing neurons are densely distributed throughout the nucleus tractus solitarii (NTS). However, their fundamental role in arterial baroreflex function remains debated. Previously, our group has shown that activation of contraction-sensitive somatic afferents evoke substance P (SP) release in the NTS and resets the arterial baroreflex via activation of a GABAergic NTS circuit. Based on these findings, we hypothesized that modulation of arterial baroreflex function by somatic afferents is mediated by NK1-R dependent inhibition of barosensitive NTS circuits. In the present study, SP-conjugated saporin toxin (SP-SAP) was used to ablate NK1-R expressing NTS neurons. Contraction-sensitive somatic afferents were activated by electrically-evoked muscle contraction and the arterial baroreceptor-heart rate reflex was assessed by constructing reflex curves using a decerebrate, arterially-perfused preparation. Baseline baroreflex sensitivity was significantly attenuated in SP-SAP-treated rats compared with control rats receiving either unconjugated SAP or vehicle. Muscle contraction significantly attenuated baroslope in SAP and vehicle-treated animals and shifted the baroreflex curves to higher systemic pressure. In contrast, somatic afferent stimulation failed to alter baroslope or shift the baroreflex curves in SP-SAP-treated animals. Moreover, when reflex sensitivity was partially restored in SP-SAP animals, somatic stimulation failed to attenuate baroreflex bradycardia. In contrast, SP-SAP and somatic stimulation failed to blunt the reflex bradycardia evoked by the peripheral chemoreflex. Immunohistochemistry revealed that pretreatment with SP-SAP significantly reduced the number of NK1-R expressing neurons in the caudal NTS, while sparing NK1-R expressing neurons rostral to the injection site. This was accompanied by a significant reduction in the number of glutamic acid decarboxylase (GAD67) expressing neurons at equivalent levels of the NTS. These findings indicate that immunolesioning of NK1-R expressing NTS neurons selectively abolishes the depressive effect of somatosensory input on arterial baroreceptor-heart rate reflex function.

Neurokinin‐1 receptor activation in Bötzinger complex evokes bradypnoea

In the present study, we examined the role of the neurokinin‐1 receptor (NK1R) in the modulation of respiratory rhythm in a functionally identified bradypnoeic region of the ventral respiratory group (VRG) in the in situ arterially perfused juvenile rat preparation. In electrophysiologically and functionally identified bradypnoeic sites corresponding to the Bötzinger complex (BötC), microinjection of the selective NK1R agonist [Sar9‐Met(O2)11]‐substance P (SSP) produced a significant reduction in phrenic frequency mediated exclusively by an increase in expiratory duration (TE). The reduction was characterized by a significant increase in postinspiratory (post‐I) duration with no effect on either late‐expiratory duration (E2) or inspiratory duration (TI). In contrast, in a functionally identified tachypnoeic region, corresponding to the preBötzinger complex (Pre‐BötC), control microinjection of SSP elicited tachypnoea. Pretreatment with the NK1R antagonist CP99994 in the BötC significantly attenuated the bradypnoeic response to SSP injection and blunted the increase in TE duration. This effect of SSP mimicked the extension of TE produced by activation of the Hering‐Breuer reflex. Therefore, we hypothesized that activation of NK1Rs in the BötC is requisite for the expiratory‐lengthening effect of the Hering‐Breuer reflex. Unilateral electrical stimulation of the cervical vagus nerve produced bradypnoea by exclusively extending TE. Ipsilateral blockade of NK1Rs by CP99994 following blockade of the contralateral BötC by the GABAA receptor agonist muscimol significantly reduced the extension of TE produced by vagal stimulation. Results from the present study demonstrate that selective activation of NK1Rs in a functionally identified bradypnoeic region of the VRG can depress respiratory frequency by selectively lengthening post‐I duration and provide evidence that endogenous activation of NK1Rs in the BötC appears to be involved in the expiratory‐lengthening effect of the Hering‐Breuer reflex. In conclusion, our findings demonstrate that selective activation of NK1Rs in discrete regions of the VRG can exert functionally diverse effects on breathing.

Acute intermittent hypoxia induced neural plasticity in respiratory motor control

Respiratory neural networks can adapt to rapid environmental change or be altered over the long term by various inputs. The mechanisms that underlie the plasticity necessary for adaptive changes in breathing remain unclear. Acute intermittent hypoxia (AIH)‐induced respiratory long‐term facilitation (LTF) is one of the most extensively studied types of respiratory plasticity. Acute intermittent hypoxia‐induced LTF is present in several respiratory motor outputs, innervating both pump muscles (i.e. diaphragm) and valve muscles (i.e. tongue, pharynx and larynx). Long‐term facilitation is present in various species, including humans, and the expression of LTF is influenced by gender, age and genetics. Serotonin plays a key role in initiating and modulating plasticity at the level of respiratory motor neurons. Recently, multiple intracellular pathways have been elucidated that are capable of giving rise to respiratory LTF. These mainly activate the metabolic receptors coupled to Gq (‘Q’ pathway) and Gs (‘S’ pathway) proteins. Herein, we discuss AIH‐induced respiratory LTF in animals and humans, as well as recent advances in our understanding of the synaptic and intracellular pathways underlying this form of plasticity. We also discuss the potential to use intermittent hypoxia to induce functional recovery following cervical spinal injury.

Distribution of acetylcholine and catecholamines in fish gills and their potential roles in the hypoxic ventilatory response

Carotid body glomus cells in mammals contain a plethora of different neurochemicals. Several hypotheses exist to explain their roles in oxygen-chemosensing. In the present study we assessed the distribution of serotonin, acetylcholine and catecholamines in the gills of trout (Oncorhynchus mykiss) and goldfish (Carassius auratus) using immunohistochemistry, and an activity-dependent dye, Texas Red hydrazide (TXR). In fish the putative oxygen sensing cells are neuroepithelial cells (NECs) and the focus in recent studies has been on the role of serotonin in oxygen chemoreception. The NECs of trout and goldfish contain serotonin, but, in contrast to the glomus cells of mammals, not acetylcholine or catecholamines. Acetylcholine was expressed in chain and proximal neurons and in extrinsic nerve bundles in the filaments. The serotonergic NECs did not label with the HNK-1 antibody suggesting that if they are derived from the neural crest, they are no longer proliferative or migrating. Furthermore, we predicted that if serotonergic NECs were chemosensory, they would increase their activity during hypoxia (endocytose TXR), but following 30 min of hypoxic exposure (45 Torr), serotonergic NECs did not take up TXR. Based on these and previous findings we propose several possible models outlining the ways in which serotonin and acetylcholine could participate in oxygen chemoreception in completing the afferent sensory pathway.

Mechanism of Sympathetic Activation and Blood Pressure Elevation in Humans and Animals Following Acute Intermittent Hypoxia

Sleep apnea is associated with repeated episodes of hypoxemia, causing marked increase in sympathetic nerve activity and blood pressure. Considerable evidence suggests that intermittent hypoxia (IH) resulting from apnea is the primary stimulus for sympathetic overactivity in sleep apnea patients. Several IH protocols have been developed either in animals or in humans to investigate mechanisms underlying the altered autonomic regulation of the circulation. Most of these protocols involve several days (10-40 days) of IH exposure, that is, chronic intermittent hypoxia (CIH). Recent data suggest that a single session of IH exposure, that is, acute intermittent hypoxia (AIH), is already capable of increasing tonic sympathetic nerve output (sympathetic long-term facilitation, LTF) and altering chemo- and baroreflexes with or without elevation of blood pressure. This indicates that IH alters the autonomic neurocirculatory at a very early time point, although the mechanisms underlying this neuroplasticity have not been explored in detail. The purpose of this chapter is to briefly review the effects of AIH on sympathetic LTF and alteration of autonomic reflexes in comparison with the studies from CIH studies. We will also discuss the potential central and peripheral mechanism underlying sympathetic LTF.

Excessive Respiratory Modulation of Blood Pressure Triggers Hypertension

The etiology of hypertension, the worlds biggest killer, remains poorly understood, with treatments targeting the established symptom, not the cause. The development of hypertension involves increased sympathetic nerve activity that, in experimental hypertension, may be driven by excessive respiratory modulation. Using selective viral and cell lesion techniques, we identify adrenergic C1 neurons in the medulla oblongata as critical for respiratory-sympathetic entrainment and the development of experimental hypertension. We also show that a cohort of young, normotensive humans, selected for an exaggerated blood pressure response to exercise and thus increased hypertension risk, has enhanced respiratory-related blood pressure fluctuations. These studies pinpoint a specific neuronal target for ameliorating excessive sympathetic activity during the developmental phase of hypertension and identify a group of pre-hypertensive subjects that would benefit from targeting these cells.

Respiratory rhythm of brainstem-spinal cord preparations: Effects of maturation, age, mass and oxygenation

We examined the effect of age, mass and the presence of the pons on the longevity (length of time spontaneous respiratory-related activity is produced) of brainstem-spinal cord preparations of neonatal rodents (rats and hamsters) and the level of oxygenation in the medulla respiratory network in these preparations. We found the longevity of the preparations from both species decreased with increasing postnatal age. Physical removal of the pons increased respiratory frequency and the longevity of the preparation. However, tissue oxygenation at the level of the medullary respiratory network was not affected by removal of the pons or increasing postnatal age (up to postnatal day 4). Taken together, these data suggest that the effect of removing the pons on respiratory frequency and the longevity of brainstem-spinal cord preparations with increasing postnatal age are primarily due to postnatal development and appear to be unrelated to mass or changes in levels of tissue oxygenation.