Targeting Astrocyte Signaling Alleviates Cerebrovascular and Synaptic Function Deficits in a Diet-Based Mouse Model of Small Cerebral Vessel Disease.

Despite the indispensable role that astrocytes play in the neurovascular unit, few studies have investigated the functional impact of astrocyte signaling in cognitive decline and dementia related to vascular pathology. Diet-mediated induction of hyperhomocysteinemia (HHcy) recapitulates numerous features of vascular contributions to cognitive impairment and dementia (VCID). Here, we used astrocyte targeting approaches to evaluate astrocyte Ca2+ dysregulation and the impact of aberrant astrocyte signaling on cerebrovascular dysfunction and synapse impairment in male and female HHcy diet mice. Two-photon imaging conducted in fully awake mice revealed activity-dependent Ca2+ dysregulation in barrel cortex astrocytes under HHcy. Stimulation of contralateral whiskers elicited larger Ca2+ transients in individual astrocytes of HHcy diet mice compared with control diet mice. However, evoked Ca2+ signaling across astrocyte networks was impaired in HHcy mice. HHcy also was associated with increased activation of the Ca2+/calcineurin-dependent transcription factor NFAT4, which has been linked previously to the reactive astrocyte phenotype and synapse dysfunction in amyloid and brain injury models. Targeting the NFAT inhibitor VIVIT to astrocytes, using adeno-associated virus vectors, led to reduced GFAP promoter activity in HHcy diet mice and improved functional hyperemia in arterioles and capillaries. VIVIT expression in astrocytes also preserved CA1 synaptic function and improved spontaneous alternation performance on the Y maze. Together, the results demonstrate that aberrant astrocyte signaling can impair the major functional properties of the neurovascular unit (i.e., cerebral vessel regulation and synaptic regulation) and may therefore represent a promising drug target for treating VCID and possibly Alzheimer's disease and other related dementias.SIGNIFICANCE STATEMENT The impact of reactive astrocytes in Alzheimer's disease and related dementias is poorly understood. Here, we evaluated Ca2+ responses and signaling in barrel cortex astrocytes of mice fed with a B-vitamin deficient diet that induces hyperhomocysteinemia (HHcy), cerebral vessel disease, and cognitive decline. Multiphoton imaging in awake mice with HHcy revealed augmented Ca2+ responses in individual astrocytes, but impaired signaling across astrocyte networks. Stimulation-evoked arteriole dilation and elevated red blood cell velocity in capillaries were also impaired in cortex of awake HHcy mice. Astrocyte-specific inhibition of the Ca2+-dependent transcription factor, NFAT, normalized cerebrovascular function in HHcy mice, improved synaptic properties in brain slices, and stabilized cognition. Results suggest that astrocytes are a mechanism and possible therapeutic target for vascular-related dementia.

Identifying vagal bronchopulmonary afferents mediating cough response to inhaled sulfur dioxide in mice.

Sulfur dioxide (SO2), a common environmental and industrial air pollutant, possesses a potent effect in eliciting cough reflex, but the primary type of airway sensory receptors involved in its tussive action has not been clearly identified. This study was carried out to determine the relative roles of three major types of vagal bronchopulmonary afferents [slowly adapting receptors (SARs), rapidly adapting receptors (RARs), and C-fibers] in regulating the cough response to inhaled SO2. Our results showed that inhalation of SO2 (300 or 600 ppm for 8 min) evoked an abrupt and intense stimulatory effect on bronchopulmonary C-fibers, which continued for the entire duration of inhalation challenge and returned toward the baseline in 1-2 min after resuming room air-breathing in anesthetized and mechanically ventilated mice. In stark contrast, the same SO2 inhalation challenge generated a distinct and consistent inhibitory effect on both SARs and phasic RARs; their phasic discharges synchronized with respiratory cycles during the baseline (breathing room air) began to decline progressively within 1-3 min after the onset of SO2 inhalation, ceased completely before termination of the 8-min inhalation challenge, and then slowly returned toward the baseline after >40 min. In a parallel study in awake mice, inhalation of SO2 at the same concentration and duration as that in the nerve recording experiments evoked cough responses in a pattern and time course similar to that observed in the C-fiber responses. Based on these results, we concluded that stimulation of vagal bronchopulmonary C-fibers is primarily responsible for triggering the cough response to inhaled SO2.NEW & NOTEWORTHY This study demonstrated that inhalation of a high concentration of sulfur dioxide, an irritant gas and common air pollutant, completely and reversibly inhibited the neural activities of both slowly adapting receptor and rapidly adapting receptor, two major types of mechanoreceptors in the lungs with their activities conducted by myelinated fibers. Furthermore, the results of this study suggested that stimulation of vagal bronchopulmonary C-fibers is primarily responsible for triggering the cough reflex responses to inhaled sulfur dioxide.

Mechanisms underlying the stimulatory effect of inhaled sulfur dioxide on vagal bronchopulmonary C-fibres.

KEY POINTS: Brief inhalation of SO2 of concentration >500 p.p.m. triggered a pronounced stimulatory effect on vagal bronchopulmonary C-fibres in anaesthetized rats. This stimulatory effect was drastically diminished by a pretreatment with NaHCO3 that raised the baseline arterial pH, suggesting a possible involvement of acidification of airway fluid and/or tissue generated by inhaled SO2 . The stimulation was completely abolished by pretreatment with antagonists of both acid-sensing ion channels and transient receptor potential vanilloid type-1 receptors, indicating that this effect was caused by acid activation of these cation channels expressed in airway sensory nerves. This conclusion was further supported by the results obtained from studies in isolated rat vagal bronchopulmonary sensory neurones and also in the cough response to SO2 inhalation challenge in awake mice. These results provide new insight into the underlying mechanism of harmful irritant effects in the respiratory tract caused by accidental exposure to a high concentration of SO2 . ABSTRACT: Inhalation of sulfur dioxide (SO2 ) triggers coughs and reflex bronchoconstriction, and stimulation of vagal bronchopulmonary C-fibres is primarily responsible. However, the mechanism underlying this stimulatory effect is not yet fully understood. In this study, we tested the hypothesis that the C-fibre stimulation was caused by SO2 -induced local tissue acidosis in the lung and airways. Single-unit activities of bronchopulmonary C-fibres in response to inhalation challenges of SO2 (500-1500 p.p.m., 10 breaths) were measured in anaesthetized rats. Inhalation of SO2 reproducibly induced a pronounced and sustained stimulation (lasting for 15-60 s) of pulmonary C-fibres in a concentration-dependent manner. This stimulatory effect was significantly attenuated by an increase in arterial pH generated by infusion of sodium bicarbonate (NaHCO3 ), and completely abrogated by a combined pretreatment with amiloride (an antagonist of acid-sensing ion channels, ASICs) and AMG8910 (a selective antagonist of the transient receptor potential vanilloid type-1 receptor, TRPV1). Furthermore, in isolated rat vagal pulmonary sensory neurones, perfusion of an aqueous solution of SO2 evoked a transient increase in the intracellular Ca2+ concentration; this response was also markedly diminished by a pretreatment with amiloride and AMG8910. In addition, inhalation of SO2 consistently evoked coughs in awake mice; responses were significantly smaller in TRPV1-/- mice than in wild-type mice, and almost completely abolished after a pretreatment with amiloride in TRPV1-/- mice. These results suggested that the stimulatory effect of inhaled SO2 on bronchopulmonary C-fibres was generated by acidification of fluid and/or tissue in the lung and airways, which activated both ASICs and TRPV1 expressed in these sensory nerves.

Immediate and delayed potentiating effects of tumor necrosis factor-α on TRPV1 sensitivity of rat vagal pulmonary sensory neurons.

We studied acute effects of tumor necrosis factor-α (TNFα) on the sensitivity of isolated rat vagal pulmonary sensory neurons. Our results showed the following. First, a brief pretreatment with a low dose of TNFα (1.44 nM, 9 min) enhanced the sensitivity of transient receptor potential vanilloid type 1 (TRPV1) receptors in these neurons in two distinct phases: the inward current evoked by capsaicin was amplified (Δ = 247%) immediately following the TNFα pretreatment, which gradually declined toward control and then increased again reaching another peak (Δ = 384%) after 60-90 min. Second, the immediate phase of this potentiating effect of TNFα was completely abolished by a pretreatment with a selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, whereas the delayed potentiation was only partially attenuated. Third, in sharp contrast, TNFα did not generate any potentiating effect on the responses to non-TRPV1 chemical activators of these neurons. Fourth, the selectivity of the TNFα action on TRPV1 was further illustrated by the responses to acid (pH 6.0); TNFα did not affect the rapid transient current mediated by acid-sensing ion channels but significantly augmented the slow sustained current mediated by TRPV1 in the same neurons. Fifth, in anesthetized rats, a similar pattern of acute sensitizing effects of TNFα on pulmonary C-fiber afferents and the involvement of COX-2 were also clearly shown. In conclusion, a brief pretreatment with TNFα induced both immediate and delayed potentiating effects on the TRPV1 sensitivity in pulmonary sensory neurons, and the production of COX-2 arachidonic acid metabolites plays a major role in the immediate sensitizing effect of TNFα.

Cough and expiration reflexes elicited by inhaled irritant gases are intensified in ovalbumin-sensitized mice.

This study was designed to determine the effect of active sensitization with ovalbumin (Ova) on cough responses to inhaled irritant gases in mice. Conscious mice moved freely in a recording chamber, while the pressure change in the chamber and audio and video signals of the mouse movements were recorded simultaneously to measure the frequencies of cough reflex (CR) and expiration reflex (ER). To further verify the accuracy of cough analysis, the intrapleural pressure was also recorded by a telemetry sensor surgically implanted in the intrapleural space in a subgroup of mice. During the irritant gas inhalation challenge, sulfur dioxide (SO2; 200 and 400 ppm) or ammonia (NH3; 0.1% and 0.2%) was drawn into the chamber at a constant flow rate for 8 min. Ova sensitization and sham sensitization with vehicle (Veh) were performed over a 25-day period in separate groups of mice. Our results showed that 1) both SO2 and NH3 inhalation challenges increased CR and ER frequencies in a concentration-dependent manner before Ova sensitization; 2) the baseline CR frequency was significantly elevated after Ova sensitization, accompanied by pronounced airway inflammation; and 3) Ova sensitization also markedly augmented the responses of CR and ER to both SO2 and NH3 inhalation challenges; in sharp contrast, the cough responses did not change after sham sensitization in the Veh group. In conclusion, Ova sensitization caused distinct and lingering increases in baseline cough frequency, and also intensified both CR and ER responses to inhaled irritant gases, which probably resulted from an allergic inflammation-induced hypersensitivity of airway sensory nerves.

Sustained sensitizing effects of tumor necrosis factor alpha on sensory nerves in lung and airways.

Tumor necrosis factor alpha (TNFα) plays a significant role in the pathogenesis of airway inflammatory diseases. Inhalation of aerosolized TNFα induced airway hyperresponsiveness accompanied by airway inflammation in healthy human subjects, but the underlying mechanism is not fully understood. We recently reported a series of studies aimed to investigate if TNFα elevates the sensitivity of vagal bronchopulmonary sensory nerves in a mouse model; these studies are summarized in this mini-review. Our results showed that intratracheal instillation of TNFα induced pronounced airway inflammation 24 h later, as illustrated by infiltration of eosinophils and neutrophils and the release of inflammatory mediators and cytokines in the lung and airways. Accompanying these inflammatory reactions, the sensitivity of vagal pulmonary C-fibers and silent rapidly adapting receptors to capsaicin, a selective agonist of transient receptor potential vanilloid type 1 receptor, was markedly elevated after the TNFα treatment. A distinct increase in the sensitivity to capsaicin induced by TNFα was also observed in isolated pulmonary sensory neurons, suggesting that the sensitizing effect is mediated primarily through a direct action of TNFα on these neurons. Furthermore, the same TNFα treatment also induced a lingering (>7days) cough hyperresponsiveness to inhalation challenge of NH3 in awake mice. Both the airway inflammation and the sensitizing effect on pulmonary sensory neurons caused by the TNFα treatment were abolished in the TNF-receptor double homozygous mutant mice, indicating the involvement of TNF-receptor activation. These findings suggest that the TNFα-induced hypersensitivity of vagal bronchopulmonary afferents may be responsible for, at least in part, the airway hyperresponsiveness caused by inhaled TNFα in healthy individuals.

Hypersensitivity of vagal pulmonary C-fibers induced by increasing airway temperature in ovalbumin-sensitized rats.

Our recent study has shown that hyperventilation of humidified warm air (HWA) triggered cough and reflex bronchoconstriction in patients with mild asthma. We suggested that a sensitizing effect on bronchopulmonary C-fibers by increasing airway temperature was involved, but direct evidence was lacking. This study was carried out to test the hypothesis that HWA enhances the pulmonary C-fiber sensitivity in Brown-Norway rats sensitized with ovalbumin (Ova). In anesthetized rats, isocapnic hyperventilation of HWA for 3 min rapidly elevated airway temperature to a steady state of 41.7°C. Immediately after the HWA challenge, the baseline fiber activity (FA) of pulmonary C-fibers was markedly elevated in sensitized rats, but not in control rats. Furthermore, the response of pulmonary C-fibers to right atrial injection of capsaicin in sensitized rats was significantly higher than control rats before the HWA challenge, and the response to capsaicin was further amplified after HWA in sensitized rats (ΔFA = 4.51 ± 1.02 imp/s before, and 9.26 ± 1.74 imp/s after the HWA challenge). A similar pattern of the HWA-induced potentiation of the FA response to phenylbiguanide, another chemical stimulant of C-fibers, was also found in sensitized rats. These results clearly demonstrated that increasing airway temperature significantly elevated both the baseline activity and responses to chemical stimuli of pulmonary C-fibers in Ova-sensitized rats. In conclusion, this study supports the hypothesis that the increased excitability of these afferents may have contributed to the cough and reflex bronchoconstriction evoked by hyperventilation of HWA in patients with asthma.

Hemorrhagic hypotension-induced hypersensitivity of vagal pulmonary C-fibers to chemical stimulation and lung inflation in anesthetized rats.

This study was carried out to investigate whether hemorrhagic hypotension (HH) altered the sensitivity of vagal pulmonary C-fibers. The fiber activity (FA) of single vagal pulmonary C-fiber was continuously recorded in anesthetized rats before, during, and after HH was induced by bleeding from the femoral arterial catheter into a blood reservoir and lowering the mean systemic arterial pressure (MSAP) to ∼40 mmHg for 20 min. Our results showed the following. First, after MSAP reached a steady state of HH, the peak FA response to intravenous injection of capsaicin was elevated by approximately fivefold. The enhanced C-fiber sensitivity continued to increase during HH and sustained even after MSAP returned to baseline during the recovery, but slowly returned to control ∼20 min later. Second, responses of FA to intravenous injections of other chemical stimulants of pulmonary C-fibers (phenylbiguanide, lactic acid, and adenosine) and a constant-pressure lung hyperinflation were all significantly potentiated by HH. Third, infusion of sodium bicarbonate alleviated the systemic acidosis during HH, and it also attenuated, but did not completely prevent, the HH-induced C-fiber hypersensitivity. In conclusion, the pulmonary C-fiber sensitivity was elevated during HH, probably caused by the endogenous release of chemical substances (e.g., lactic acid) that were produced by tissue ischemia during HH. This enhanced C-fiber sensitivity may heighten the pulmonary protective reflexes mediated through these afferents (e.g., cough, J reflex) during hemorrhage when the body is more susceptible to other hazardous insults and pathophysiological stresses.

Interaction between TRPA1 and TRPV1: Synergy on pulmonary sensory nerves.

Transient receptor potential ankyrin type 1 (TRPA1) and vanilloid type 1 (TRPV1) receptors are co-expressed in vagal pulmonary C-fiber sensory nerves. Because both these ligand-gated non-selective cation channels are sensitive to a number of endogenous inflammatory mediators, it is highly probable that they can be activated simultaneously during airway inflammation. Studies were carried out to investigate whether there is an interaction between these two polymodal transducers upon simultaneous activation, and how it modulates the activity of vagal pulmonary C-fiber sensory nerves. Our studies showed a distinct potentiating effect induced abruptly by simultaneous activations of TRPA1 and TRPV1 by their respective selective agonists, allyl isothiocyanate (AITC) and capsaicin (Cap), at near-threshold concentrations. This synergistic effect was demonstrated in the studies of single-unit recording of vagal bronchopulmonary C-fiber afferents and the reflex responses elicited by activation of these afferents in intact animals, as well as in the isolated nodose and jugular bronchopulmonary sensory neurons. This potentiating effect was absent when either AITC or Cap was replaced by non-TRPA1 and non-TRPV1 chemical activators of these neurons, demonstrating the selectivity of the interaction between these two TRP channels. Furthermore, the synergism was dependent upon the extracellular Ca(2+), and the rapid onset of the action further suggests that the interaction probably occurred locally at the sites of these channels. These findings suggest that the TRPA1-TRPV1 interaction may play an important role in regulating the function and excitability of pulmonary sensory neurons during airway inflammation, but the mechanism underlying this positive interaction is not yet fully understood.

Hydrogen sulfide induces hypersensitivity of rat capsaicin-sensitive lung vagal neurons: role of TRPA1 receptors.

The sensitization of capsaicin-sensitive lung vagal (CSLV) afferents by inflammatory mediators is important in the development of airway hypersensitivity. Hydrogen sulfide (H2S) is an endogenous mediator inducing hyperalgesia through transient receptor potential ankyrin 1 (TRPA1) receptors located on nociceptors. We conducted this study to determine whether H2S elevates the sensitivity of rat CSLV afferents. In anesthetized, artificially ventilated rats, the inhalation of aerosolized sodium hydrosulfide (NaHS, a H2S donor) caused no significant changes in the baseline activity of CSLV afferents. However, the afferent responses to right atrial injection of capsaicin or phenylbiguanide and to lung inflation were all markedly potentiated after NaHS inhalation. By contrast, the inhalation of its vehicle or NaOH (with a similar pH to NaHS) failed to enhance the afferent responses. Additionally, the potentiating effect on the afferent responses was found in rats inhaling L-cysteine (a substrate of H2S synthase) that slowly releases H2S. The potentiating effect of NaHS on the sensitivity of CSLV afferents was completely blocked by pretreatment of HC-030031 (a TRPA1 receptor antagonist) but was unaffected by its vehicle. In isolated rat CSLV neurons, the perfusion of NaHS alone did not influence the intracellular Ca(2+) concentration but markedly potentiated the Ca(2+) transients evoked by capsaicin. The NaHS-caused effect was totally abolished by HC-030031 pretreatment. These results suggest that H2S induces a nonspecific sensitizing effect on CSLV fibers to both chemical and mechanical stimulation in rat lungs, which appears mediated through an action on the TRPA1 receptors expressed on the nerve endings of CSLV afferents.

Bronchoconstriction triggered by breathing hot humid air in patients with asthma: role of cholinergic reflex.

RATIONALE: Hyperventilation of hot humid air induces transient bronchoconstriction in patients with asthma; the underlying mechanism is not known. Recent studies showed that an increase in temperature activates vagal bronchopulmonary C-fiber sensory nerves, which upon activation can elicit reflex bronchoconstriction. OBJECTIVES: This study was designed to test the hypothesis that the bronchoconstriction induced by increasing airway temperature in patients with asthma is mediated through cholinergic reflex resulting from activation of these airway sensory nerves. METHODS: Specific airway resistance (SR(aw)) and pulmonary function were measured to determine the airway responses to isocapnic hyperventilation of humidified air at hot (49°C; HA) and room temperature (20-22°C; RA) for 4 minutes in six patients with mild asthma and six healthy subjects. A double-blind design was used to compare the effects between pretreatments with ipratropium bromide and placebo aerosols on the airway responses to HA challenge in these patients. MEASUREMENTS AND MAIN RESULTS: SR(aw) increased by 112% immediately after hyperventilation of HA and by only 38% after RA in patients with asthma. Breathing HA, but not RA, triggered coughs in these patients. In contrast, hyperventilation of HA did not cause cough and increased SR(aw) by only 22% in healthy subjects; there was no difference between their SR(aw) responses to HA and RA challenges. More importantly, pretreatment with ipratropium completely prevented the HA-induced bronchoconstriction in patients with asthma. CONCLUSIONS: Bronchoconstriction induced by increasing airway temperature in patients with asthma is mediated through the cholinergic reflex pathway. The concomitant increase in cough response further indicates an involvement of airway sensory nerves, presumably the thermosensitive C-fiber afferents.

Age- and sex-dependent alterations in primary somatosensory cortex neuronal calcium network dynamics during locomotion.

Over the past 30 years, the calcium (Ca2+ ) hypothesis of brain aging has provided clear evidence that hippocampal neuronal Ca2+ dysregulation is a key biomarker of aging. Age-dependent Ca2+ -mediated changes in intrinsic excitability, synaptic plasticity, and activity have helped identify some of the mechanisms engaged in memory and cognitive decline based on work done mostly at the single-cell level and in the slice preparation. Recently, our lab identified age- and Ca2+ -related neuronal network dysregulation in the cortex of the anesthetized animal. Still, investigations in the awake animal are needed to test the generalizability of the Ca2+ hypothesis of brain aging. Here, we used in vigilo two-photon imaging in ambulating mice, to image GCaMP8f in the primary somatosensory cortex (S1), during ambulation and at rest. We investigated aging- and sex-related changes in neuronal networks in the C56BL/6J mouse. Following imaging, gait behavior was characterized to test for changes in locomotor stability. During ambulation, in both young adult and aged mice, an increase in network connectivity and synchronicity was noted. An age-dependent increase in synchronicity was seen in ambulating aged males only. Additionally, females displayed increases in the number of active neurons, Ca2+ transients, and neuronal activity compared to males, particularly during ambulation. These results suggest S1 Ca2+ dynamics and network synchronicity are likely contributors of locomotor stability. We believe this work raises awareness of age- and sex-dependent alterations in S1 neuronal networks, perhaps underlying the increase in falls with age.

Inhibitory effect of sulfur dioxide inhalation on Hering-Breuer inflation reflex in mice: role of voltage-gated potassium channels.

Slowly adapting receptors (SARs), vagal mechanosensitive receptors located in the lung, play an important role in regulating the breathing pattern and Hering-Breuer inflation reflex (HBIR). Inhalation of high concentration of sulfur dioxide (SO2), a common environmental and occupational air pollutant, has been shown to selectively block the SAR activity in rabbits, but the mechanism underlying this inhibitory effect remained a mystery. We carried out this study to determine if inhalation of SO2 can inhibit the HBIR and change the eupneic breathing pattern, and to investigate further a possible involvement of voltage-gated K+ channels in the inhibitory effect of SO2 on these vagal reflex-mediated responses. Our results showed 1) inhalation of SO2 (600 ppm; 8 min) consistently abolished both the phasic activity of SARs and their response to lung inflation in anesthetized, artificially ventilated mice, 2) inhalation of SO2 generated a distinct inhibitory effect on the HBIR and induced slow deep breathing in anesthetized, spontaneously breathing mice, and these effects were reversible and reproducible in the same animals, 3) This inhibitory effect of SO2 was blocked by pretreatment with 4-aminopyridine (4-AP), a nonselective blocker of voltage-gated K+ channel, and unaffected by pretreatment with its vehicle. In conclusion, this study suggests that this inhibitory effect on the baseline breathing pattern and the HBIR response was primarily mediated through the SO2-induced activation of voltage-gated K+ channels located in the vagal bronchopulmonary SAR neurons.NEW & NOTEWORTHY This study demonstrated that inhaled sulfur dioxide completely and reversibly abolished the activity of vagal bronchopulmonary slowly adapting receptors, significantly inhibited the apneic response to lung inflation, and induced slow deep breathing in anesthetized mice. More importantly, our results further suggested that this inhibitory effect was mediated through an action of sulfur dioxide and its derivatives on the voltage-gated potassium channels expressed in the slowly adapting receptor sensory neurons innervating the lung.

Falling Short: The Contribution of Central Insulin Receptors to Gait Dysregulation in Brain Aging.

Insulin resistance, which manifests as a reduction of insulin receptor signaling, is known to correlate with pathological changes in peripheral tissues as well as in the brain. Central insulin resistance has been associated with impaired cognitive performance, decreased neuronal health, and reduced brain metabolism; however, the mechanisms underlying central insulin resistance and its impact on brain regions outside of those associated with cognition remain unclear. Falls are a leading cause of both fatal and non-fatal injuries in the older population. Despite this, there is a paucity of work focused on age-dependent alterations in brain regions associated with ambulatory control or potential therapeutic approaches to target these processes. Here, we discuss age-dependent alterations in central modalities that may contribute to gait dysregulation, summarize current data supporting the role of insulin signaling in the brain, and highlight key findings that suggest insulin receptor sensitivity may be preserved in the aged brain. Finally, we present novel results showing that administration of insulin to the somatosensory cortex of aged animals can alter neuronal communication, cerebral blood flow, and the motivation to ambulate, emphasizing the need for further investigations of intranasal insulin as a clinical management strategy in the older population.

Sensitivity of the S1 neuronal calcium network to insulin and Bay-K 8644 in vivo: Relationship to gait, motivation, and aging processes.

Neuronal hippocampal Ca2+ dysregulation is a critical component of cognitive decline in brain aging and Alzheimer's disease and is suggested to impact communication and excitability through the activation of a larger after hyperpolarization. However, few studies have tested for the presence of Ca2+ dysregulation in vivo, how it manifests, and whether it impacts network function across hundreds of neurons. Here, we tested for neuronal Ca2+ network dysregulation in vivo in the primary somatosensory cortex (S1) of anesthetized young and aged male Fisher 344 rats using single-cell resolution techniques. Because S1 is involved in sensory discrimination and proprioception, we tested for alterations in ambulatory performance in the aged animal and investigated two potential pathways underlying these central aging- and Ca2+ -dependent changes. Compared to young, aged animals displayed increased overall activity and connectivity of the network as well as decreased ambulatory speed. In aged animals, intranasal insulin (INI) increased network synchronicity and ambulatory speed. Importantly, in young animals, delivery of the L-type voltage-gated Ca2+ channel modifier Bay-K 8644 altered network properties, replicating some of the changes seen in the older animal. These results suggest that hippocampal Ca2+ dysregulation may be generalizable to other areas, such as S1, and might engage modalities that are associated with locomotor stability and motivation to ambulate. Further, given the safety profile of INI in the clinic and the evidence presented here showing that this central dysregulation is sensitive to insulin, we suggest that these processes can be targeted to potentially increase motivation and coordination while also reducing fall frequency with age.

TRPV1 as a cough sensor and its temperature-sensitive properties.

In the respiratory tract, TRPV1, a non-selective cation channel and a polymodal transducer, is expressed primarily in non-myelinated sensory nerves. A significant role of TRPV1 in eliciting the cough reflex has been extensively documented. Inhalation of capsaicin aerosol, a selective agonist of TRPV1, consistently and reproducibly evoked coughs in a dose-dependent manner in both healthy humans and in patients with airway inflammatory diseases. A number of endogenous inflammatory mediators known to upregulate the TRPV1 sensitivity, such as prostaglandin E(2) and bradykinin, also enhanced the cough sensitivity. Furthermore, a substantial increase of TRPV1-immunoreactive nerve profiles was found in the bronchial tissue of patients with chronic cough. In addition to the cough reflex, activation of TRPV1-expressing sensory nerves in the airways is also known to elicit reflex bronchoconstriction and mucus secretion mediated through cholinergic pathways. One of the physiological stimuli known to activate TRPV1 receptor directly is high temperature. Recent studies have demonstrated that increasing temperature within the normal physiological range significantly elevated the baseline activity and sensitivity of isolated rat vagal pulmonary sensory neurons, and the sensitizing effect of hyperthermia appeared to be mediated selectively through the TRPV1 channel. This temperature-sensitive property of TRPV1 may play an important role in regulating the physiological function of the TRPV1-expressing airway sensory nerves and the sensitivity of their reflex responses, such as cough and reflex bronchoconstriction.

A Distinct Difference Between Air and Mucosal Temperatures in Human Respiratory Tract.

Extensive evidence indicates that several types of temperature-sensitive ion channels are abundantly expressed in the sensory nerves innervating airway mucosa. Indeed, airway temperature is known to play an important role in regulating respiratory functions. However, the actual airway mucosal temperature and its dynamic changes during the respiratory cycle have not been directly measured. In previous studies, airway tissue temperature was often estimated by indirect measurement of the peak exhaled breath temperature (PEBT). In view of the poor thermal conductivity of air, we believe that the airway tissue temperature cannot be accurately determined by the exhaled air temperature, and this study aimed to test this hypothesis. We applied a miniature rapid-response temperature probe to measure directly the mucosal temperatures of trachea, major, lobar, and segmental bronchi in eight human subjects during a bronchoscopy procedure. Unlike the air temperature in the airway lumen, the mucosal temperature in these airway segments remained relatively stable and did not exhibit the phasic changes synchronous with respiratory cycles. The airway mucosal temperature increased progressively from the extra-thoracic trachea (35.7 ± 0.2°C) toward the segmental bronchus (36.9 ± 0.2°C). Most importantly, the temperatures measured directly at the mucosa of all these airway segments were substantially higher than the PEBT (31.7 ± 0.8°C). The recent findings of a close association between an increased PEBT and airway tissue inflammation have revealed the implication and potential of incorporating the PEBT measurement in the future clinical diagnosis of airway inflammation. Therefore, it is imperative to recognize this distinct difference in temperature between airway mucosa and exhaled air.

Calcium transient evoked by TRPV1 activators is enhanced by tumor necrosis factor-{alpha} in rat pulmonary sensory neurons.

TNFα, a proinflammatory cytokine known to be involved in the pathogenesis of allergic asthma, has been shown to induce hyperalgesia in somatic tissue via a sensitizing effect on dorsal root ganglion neurons expressing transient receptor potential vanilloid type 1 receptor (TRPV1). Because TRPV1-expressing pulmonary sensory neurons play an important role in regulating airway function, this study was carried out to determine whether TNFα alters the sensitivity of these neurons to chemical activators. Responses of isolated nodose and jugular ganglion neurons innervating the rat lungs were determined by measuring the transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). Our results showed the following. 1) A pretreatment with TNFα (50 ng/ml) for ∼24 h increased significantly the peak Δ[Ca(2+)](i) evoked by capsaicin (Cap) in these neurons. A pretreatment with the same concentration of TNFα for a longer duration (∼48 h) did not further increase the response, but pretreatment for a shorter duration (1 h) or with a lower concentration (25 ng/ml, 24 h) failed to enhance the Cap sensitivity. 2) The same TNFα pretreatment also induced similar but less pronounced and less uniform increases in the responses to acid (pH 6.5-5.5), 2-aminoethoxydiphenyl borate (2-APB), a common activator of TRPV1, V2, and V3 channels, and allyl isothiocyanate (AITC), a selective activator of TRPA1 channel. 3) In sharp contrast, the responses to ATP, ACh, and KCl were not affected by TNFα. 4) The TNFα-induced hypersensitivity to Cap was not prevented by pretreatment with indomethacin (30 µM). 5) The immunoreactivity to both TNF receptor types 1 and 2 were detected in rat vagal pulmonary sensory neurons. In conclusion, prolonged treatment with TNFα induces a pronounced potentiating effect on the responses of isolated pulmonary sensory neurons to TRPV1 activators. This action of TNFα may contribute in part to the airway hyperresponsiveness induced by this cytokine.

Elevating Insulin Signaling Using a Constitutively Active Insulin Receptor Increases Glucose Metabolism and Expression of GLUT3 in Hippocampal Neurons.

Insulin signaling is an integral component of healthy brain function, with evidence of positive insulin-mediated alterations in synaptic integrity, cerebral blood flow, inflammation, and memory. However, the specific pathways targeted by this peptide remain unclear. Previously, our lab used a molecular approach to characterize the impact of insulin signaling on voltage-gated calcium channels and has also shown that acute insulin administration reduces calcium-induced calcium release in hippocampal neurons. Here, we explore the relationship between insulin receptor signaling and glucose metabolism using similar methods. Mixed, primary hippocampal cultures were infected with either a control lentivirus or one containing a constitutively active human insulin receptor (IRß). 2-NBDG imaging was used to obtain indirect measures of glucose uptake and utilization. Other outcome measures include Western immunoblots of GLUT3 and GLUT4 on total membrane and cytosolic subcellular fractions. Glucose imaging data indicate that neurons expressing IRß show significant elevations in uptake and rates of utilization compared to controls. As expected, astrocytes did not respond to the IRß treatment. Quantification of Western immunoblots show that IRß is associated with significant elevations in GLUT3 expression, particularly in the total membrane subcellular fraction, but did not alter GLUT4 expression in either fraction. Our work suggests that insulin plays a significant role in mediating neuronal glucose metabolism, potentially through an upregulation in the expression of GLUT3. This provides further evidence for a potential therapeutic mechanism underlying the beneficial impact of intranasal insulin in the clinic.

Electrophysiological and Imaging Calcium Biomarkers of Aging in Male and Female 5×FAD Mice.

BACKGROUND: In animal models and tissue preparations, calcium dyshomeostasis is a biomarker of aging and Alzheimer's disease that is associated with synaptic dysfunction, neuritic pruning, and dysregulated cellular processes. It is unclear, however, whether the onset of calcium dysregulation precedes, is concurrent with, or is the product of pathological cellular events (e.g., oxidation, amyloid-ß production, and neuroinflammation). Further, neuronal calcium dysregulation is not always present in animal models of amyloidogenesis, questioning its reliability as a disease biomarker. OBJECTIVE: Here, we directly tested for the presence of calcium dysregulation in dorsal hippocampal neurons in male and female 5×FAD mice on a C57BL/6 genetic background using sharp electrodes coupled with Oregon-green Bapta-1 imaging. We focused on three ages that coincide with the course of amyloid deposition: 1.5, 4, and 10 months old. METHODS: Outcome variables included measures of the afterhyperpolarization, short-term synaptic plasticity, and calcium kinetics during synaptic activation. Quantitative analyses of spatial learning and memory were also conducted using the Morris water maze. Main effects of sex, age, and genotype were identified on measures of electrophysiology and calcium imaging. RESULTS: Measures of resting Oregon-green Bapta-1 fluorescence showed significant reductions in the 5×FAD group compared to controls. Deficits in spatial memory, along with increases in Aß load, were detectable at older ages, allowing us to test for temporal associations with the onset of calcium dysregulation. CONCLUSION: Our results provide evidence that reduced, rather than elevated, neuronal calcium is identified in this 5×FAD model and suggests that this surprising result may be a novel biomarker of AD.