Acid-sensing ion channels contribute to chemosensitivity of breathing-related neurons of the nucleus of the solitary tract.

Cellular mechanisms of central pH chemosensitivity remain largely unknown. The nucleus of the solitary tract (NTS) integrates peripheral afferents with central pathways controlling breathing; NTS neurons function as central chemosensors, but only limited information exists concerning the ionic mechanisms involved. Acid-sensing ion channels (ASICs) mediate chemosensitivity in nociceptive terminals, where pH values ∼6.5 are not uncommon in inflammation, but are also abundantly expressed throughout the brain where pHi s tightly regulated and their role is less clear. Here we test the hypothesis that ASICs are expressed in NTS neurons and contribute to intrinsic chemosensitivity and control of breathing. In electrophysiological recordings from acute rat NTS slices, ∼40% of NTS neurons responded to physiological acidification (pH 7.0) with a transient depolarization. This response was also present in dissociated neurons suggesting an intrinsic mechanism. In voltage clamp recordings in slices, a pH drop from 7.4 to 7.0 induced ASIC-like inward currents (blocked by 100 µM amiloride) in ∼40% of NTS neurons, while at pH ≤ 6.5 these currents were detected in all neurons tested; RT-PCR revealed expression of ASIC1 and, less abundantly, ASIC2 in the NTS. Anatomical analysis of dye-filled neurons showed that ASIC-dependent chemosensitive cells (cells responding to pH 7.0) cluster dorsally in the NTS. Using in vivo retrograde labelling from the ventral respiratory column, 90% (9/10) of the labelled neurons showed an ASIC-like response to pH 7.0, suggesting that ASIC currents contribute to control of breathing. Accordingly, amiloride injection into the NTS reduced phrenic nerve activity of anaesthetized rats with an elevated arterial P(CO(2)) .

Biocompatible nanoscale dispersion of single-walled carbon nanotubes minimizes in vivo pulmonary toxicity.

Excitement surrounding the attractive physical and chemical characteristics of single walled carbon nanotubes (SWCNTs) has been tempered by concerns regarding their potential health risks. Here we consider the lung toxicity of nanoscale dispersed SWCNTs (mean diameter approximately 1 nm). Because dispersion of the SWCNTs increases their aspect ratio relative to as-produced aggregates, we directly test the prevailing hypothesis that lung toxicity associated with SWCNTs compared with other carbon structures is attributable to the large aspect ratio of the individual particles. Thirty days after their intratracheal administration to mice, the granuloma-like structures with mild fibrosis in the large airways observed in mice treated with aggregated SWCNTs were absent in mice treated with nanoscale dispersed SWCNTs. Examination of lung sections from mice treated with nanoscale dispersed SWCNTs revealed uptake of the SWCNTs by macrophages and gradual clearance over time. We conclude that the toxicity of SWCNTs in vivo is attributable to aggregation of the nanomaterial rather than the large aspect ratio of the individual nanotubes. Biocompatible nanoscale dispersion provides a scalable method to generate purified preparations of SWCNTs with minimal toxicity, thus allowing them to be used safely in commercial and biomedical applications.

The chemical neuroanatomy of breathing.

The chemical neuroanatomy of breathing must ultimately encompass all the various neuronal elements physiologically identified in brainstem respiratory circuits and their apparent aggregation into "compartments" within the medulla and pons. These functionally defined respiratory compartments in the brainstem provide the major source of input to cranial motoneurons controlling the airways, and to spinal motoneurons activating inspiratory and expiratory pump muscles. This review provides an overview of the neuroanatomy of the major compartments comprising brainstem respiratory circuits, and a synopsis of the transmitters used by their constituent respiratory neurons.

Overview: the neurochemistry of respiratory control.

This special issue of Respiratory Physiology and Neurobiology surveys a broad range of topics focused on the neurochemical control of breathing. A variety of approaches have integrated the neurochemistry of breathing with the physiology of individual neurons, with the neuroanatomy of brainstem and forebrain respiratory circuits, and with the clinical pathology of respiratory disorders all of which has been fueled by the ongoing explosion of information in the molecular biology of the nervous system. Accordingly, substantial progress has identified neurotransmitters, neuromodulators, receptors, signaling cascades, trophic factors, hormones, and genes mediating normal and pathological breathing. Dynamic changes in the neurochemistry of breathing are addressed with respect to brainstem development, environmental challenges such as intermittent or chronic hypoxia, and as a function of the sleep-wake cycle. Respiratory disruption has also been identified in an increasing variety of genetic-based disorders and remarkable progress has been made in determining the affected genes and their mutations that negatively impact respiration.

Central pathways of pulmonary and lower airway vagal afferents.

Lung sensory receptors with afferent fibers coursing in the vagus nerves are broadly divided into three groups: slowly (SAR) and rapidly (RAR) adapting stretch receptors and bronchopulmonary C fibers. Central terminations of each group are found in largely nonoverlapping regions of the caudal half of the nucleus of the solitary tract (NTS). Second order neurons in the pathways from these receptors innervate neurons located in respiratory-related regions of the medulla, pons, and spinal cord. The relative ease of selective activation of SARs, and to a lesser extent RARs, has allowed for more complete physiological and morphological characterization of the second and higher order neurons in these pathways than for C fibers. A subset of NTS neurons receiving afferent input from SARs (termed pump or P-cells) mediates the Breuer-Hering reflex and inhibits neurons receiving afferent input from RARs. P-cells and second order neurons in the RAR pathway also provide inputs to regions of the ventrolateral medulla involved in control of respiratory motor pattern, i.e., regions containing a predominance of bulbospinal premotor neurons, as well as regions containing respiratory rhythm-generating neurons. Axon collaterals from both P-cells and RAR interneurons, and likely from NTS interneurons in the C-fiber pathway, project to the parabrachial pontine region where they may contribute to plasticity in respiratory control and integration of respiratory control with other systems, including those that provide for voluntary control of breathing, sleep-wake behavior, and emotions.

Sodium currents in medullary neurons isolated from the pre-Bötzinger complex region.

The pre-Bötzinger complex (preBötC) in the ventrolateral medulla contains interneurons important for respiratory rhythm generation. Voltage-dependent sodium channels mediate transient current (I(NaT)), underlying action potentials, and persistent current (I(NaP)), contributing to repetitive firing, pacemaker properties, and the amplification of synaptic inputs. Voltage-clamp studies of the biophysical properties of these sodium currents were conducted on acutely dissociated preBötC region neurons. Reverse transcription-PCR demonstrated the presence of mRNA for Nav1.1, Nav1.2, and Nav1.6 alpha-subunits in individual neurons. A TTX-sensitive I(NaP) was evoked in all tested neurons by ramp depolarization from -80 to 0 mV. Including a constant in the Boltzmann equation for inactivation by estimating the steady-state fraction of Na+ channels available for inactivation allowed prediction of a window current that did not decay to 0 at voltages positive to -20 mV and closely matched the measured I(NaP). Riluzole (3 microM), a putative I(NaP) antagonist, reduced both I(NaP) and I(NaT) and produced a hyperpolarizing shift in the voltage dependence of steady-state inactivation. The latter decreased the predicted window current by an amount equivalent to the decrease in I(NaP). Riluzole also decreased the inactivation time constant at potentials in which the peak window/persistent currents are generated. Together, these findings imply that I(NaP) and I(NaT) arise from the same channels and that a simple modification of the Hodgkin-Huxley model can satisfactorily account for both currents. In the rostral ventral respiratory group (immediately caudal to preBötC), I(NaP) was also detected, but peak conductance, current density, and input resistance were smaller than in preBötC region cells.

Pontine influences on breathing: an overview.

Historical and contemporary views of the functional organization of the lateral pontine regions influencing breathing are reviewed. In vertebrates, the rhombencephalon generates a breathing rhythm and detailed motor pattern that persist throughout life. Key to this process is an essentially continuous column of neurons extending from the spino-medullary border through the ventrolateral medulla, continuing through the ventral pons and arcing into the dorsolateral medulla. Comparative neuroanatomy and physiology indicate this is a richly interconnected network divided into serial, functionally distinct compartments. Serial compartmentalization of pontomedullary structures related to breathing also reflects the developmental segmentation of the rhombencephalon. However, with migration of cell groups such as the facial nucleus from the pons to the medulla during ontogeny, the boundaries of the adult pons are sometimes difficult to precisely define. Accordingly, a working definition of rostral and caudal pontine boundaries for adult mammals is depicted.

Parabrachial-lateral pontine neurons link nociception and breathing.

We investigated the role of the parabrachial complex in cutaneous nociceptor-induced respiratory stimulation in chloralose-urethane anesthetized, vagotomized rats. Noxious stimulation (mustard oil, MO) applied topically to a forelimb or hindlimb enhanced the peak amplitude of the integrated phrenic nerve discharge and, with forelimb application, increased phrenic nerve burst frequency. Bilateral inactivation of neural activity in the parabrachial complex with injection of the GABA agonist muscimol (3nl) markedly attenuated the response to MO application. Injection of the retrograde tracer FluoroGold within the medullary ventral respiratory column labeled neurons in dorsolateral pontine regions known to receive nociceptive inputs (i.e., Kolliker-Fuse, lateral crescent, and superior lateral subnuclei of the parabrachial complex). Extracellular recordings of 65 dorsolateral parabrachial neurons revealed about 15% responded to a noxious cutaneous pinch with either an increase or a decrease in discharge and approximately 40% of these exhibited a phasic respiratory-related component to their discharge. In conclusion, parabrachial pontine neurons contribute to cutaneous nociceptor-induced increases in breathing.

Extended amygdala and basal forebrain.

The basal forebrain is a confluence of systems that are crucial to understanding some of the most important functions of the brain, including reward and punishment, learning and cognition, and feeding and reproduction. Basic to understanding this broad spectrum of behavior is untangling the interwoven functional systems in basal forebrain. This has been grounded by the appreciation that the major nearby structures, that is, amygdala and basal ganglia, provide a context for interpreting basal forebrain areas that are best viewed as extensions of either of these larger regions. The components of basal forebrain, the ventral striatopallidal system and the medial and central divisions of extended amygdala, are subcortical relays for information garnered from brain stem, thalamus, and cortical areas. With respect to the classically defined amygdala of the temporal lobe, the lateral-basolateral complex, and the superficial amygdaloid nuclei may tentatively be viewed as specialized cortical regions. Their output targets both the striatopallidal complex and the extended amygdala, with some of the most massive basal forebrain efferents originating in the basolateral amygdaloid complex. The subcortical projections of the basolateral nucleus, at least in the rat, appear to be dichotomous, with anterior (or magnocellular) portions of the nucleus preferentially targeting striatum and ventral striatum (including the core of the nucleus accumbens), while the posterior (small-celled) portions of the basolateral nucleus target the extended amygdala as well as the shell of the nucleus accumbens. This divergence represents a particular opportunity for behavioral neuroscientists analyzing basal forebrain functions. Studies exploiting the dual subcortical projection of basolateral amygdala indicate distinct functional roles for striatum versus extended amygdala. These reinforce the identification of extended amygdala as a functional-anatomical entity distinct from the striatopallidal system.

Compartmentation of alpha 1 and alpha 2 GABA(A) receptor subunits within rat extended amygdala: implications for benzodiazepine action.

The extended amygdala, a morphological and functional entity within the basal forebrain, is a neuronal substrate for emotional states like fear and anxiety. Anxiety disorders are commonly treated by benzodiazepines that mediate their action via GABA(A) receptors. The binding properties and action of benzodiazepines depend on the alpha-subunit profile of the hetero-pentameric receptors: whereas the alpha1 subunit is associated with benzodiazepine type I pharmacology and reportedly mediates sedative as well as amnesic actions of benzodiazepines, the alpha2 subunit confers benzodiazepine type II pharmacology and mediates the anxiolytic actions of benzodiazepines. We determined the localization of alpha1 and alpha2 subunits within the extended amygdala, identified by secretoneurin immunostaining, to define the morphological substrates for the diverse benzodiazepine actions. A moderate expression of the alpha1 subunit could be detected in compartments of the medial subdivision and a strong expression of the alpha2 subunit throughout the central subdivision. It is concluded that the alpha1 and alpha2 subunits are differentially expressed within the extended amygdala, indicating that this structure is compartmentalized with respect to function and benzodiazepine action.

Respiratory rhythm generation: converging concepts from in vitro and in vivo approaches?

The timing and activation pattern of breathing movements are determined by the respiratory network. This network is amenable to a variety of in vivo and in vitro approaches, which offers a unique opportunity to investigate multiple organizational levels. It is only recently, however, that concepts obtained under in vivo and in vitro conditions are being integrated into a coherent model of breathing behavior. For example, the pre-Bötzinger complex as an essential site for rhythm generation was first identified in vitro, but has since been verified in vivo. Conversely, timing signals provided by other central and peripheral neuronal areas have so far been investigated in vivo, but it is now possible to address these issues with more complex in vitro preparations. Several key issues remain unresolved. For example, to what extent is the respiratory pattern controlled independently of the underlying rhythm? Answers to this and other questions require a dissection of mechanisms that is only possible through a complementary combination of experimental approaches.