Circulating Pro-Inflammatory Exosomes Worsen Stroke Outcomes in Aging.

[Figure: see text].

Expression and Function of Transient Receptor Potential Ankyrin 1 Ion Channels in the Caudal Nucleus of the Solitary Tract.

The nucleus of the solitary tract (NTS) receives visceral information via the solitary tract (ST) that comprises the sensory components of the cranial nerves VII, IX and X. The Transient Receptor Potential Ankyrin 1 (TRPA1) ion channels are non-selective cation channels that are expressed primarily in pain-related sensory neurons and nerve fibers. Thus, TRPA1 expressed in the primary sensory afferents may modulate the function of second order NTS neurons. This hypothesis was tested and confirmed in the present study using acute brainstem slices and caudal NTS neurons by RT-PCR, immunostaining and patch-clamp electrophysiology. The expression of TRPA1 was detected in presynaptic locations, but not the somata of caudal NTS neurons that did not express TRPA1 mRNA or proteins. Moreover, caudal NTS neurons did not show somatodendritic responsiveness to TRPA1 agonists, while TRPA1 immunostaining was detected only in the afferent fibers. Electrophysiological recordings detected activation of presynaptic TRPA1 in glutamatergic terminals synapsing on caudal NTS neurons evidenced by the enhanced glutamatergic synaptic neurotransmission in the presence of TRPA1 agonists. The requirement of TRPA1 for modulation of spontaneous synaptic activity was confirmed using TRPA1 knockout mice where TRPA1 agonists failed to alter synaptic efficacy. Thus, this study provides the first evidence of the TRPA1-dependent modulation of the primary afferent inputs to the caudal NTS. These results suggest that the second order caudal NTS neurons act as a TRPA1-dependent interface for visceral noxious-innocuous integration at the level of the caudal brainstem.

Treatment duration affects cytoprotective efficacy of positive allosteric modulation of α7 nAChRs after focal ischemia in rats.

To minimize irreversible brain injury after acute ischemic stroke (AIS), the time to treatment (i.e., treatment delay) should be minimized. However, thus far, all cytoprotective clinical trials have failed. Analysis of literature identified short treatment durations (≤72 h) as a common motif among completed cytoprotective clinical trials. Here, we argue that short cytoprotective regimens even if given early after AIS may only slow down the evolution of ischemic brain injury and fail to deliver sustained long-term solutions leading to relapses that may be misinterpreted for conceptual failure of cytoprotection. In this randomized blinded study, we used young adult male rats subjected to transient 90 min suture middle cerebral artery occlusion (MCAO) and treated with acute vs. sub-chronic regimens of PNU120596, a prototypical positive allosteric modulator of α7 nicotinic acetylcholine receptors with anti-inflammatory cytoprotective properties to test the hypothesis that insufficient treatment durations may reduce therapeutic benefits of otherwise efficacious cytoprotectants after AIS. A single acute treatment 90 min after MCAO significantly reduced brain injury and neurological deficits 24 h later, but these effects vanished 72 h after MCAO. These relapses were avoided by utilizing sub-chronic treatments. Thus, extending treatment duration augments therapeutic efficacy of PNU120596 after MCAO. Furthermore, sub-chronic treatments could offset the negative effects of prolonged treatment delays in cases where the acute treatment window after MCAO was left unexploited. We conclude that a combination of short treatment delays and prolonged treatment durations may be required to maximize therapeutic effects of PNU120596, reduce relapses and ensure sustained therapeutic efficacy after AIS. Similar concepts may hold for other cytoprotectants including those that failed in clinical trials.

Nicotine enhances inhibition of mouse vagal motor neurons by modulating excitability of premotor GABAergic neurons in the nucleus tractus solitarii.

The caudal nucleus of the solitary tract (NTS) serves as the site of the first synapse for visceral sensory inputs to the central nervous system. The NTS sends functional projections to multiple brain nuclei, with gastric-related projections primarily targeting the dorsal motor nucleus of the vagus (DMV). Previous studies have demonstrated that the majority of caudal NTS neurons that project to the DMV respond robustly to nicotine and express nicotinic acetylcholine receptors (nAChRs). However, the cytochemical identity and relationship with specific viscera of DMV-projecting, nicotine-responsive caudal NTS neurons have not been determined. The present study used transgenic mice that express enhanced green fluorescent protein (EGFP) under a GAD67 promoter in a subset of GABAergic neurons, in vivo retrograde pseudorabies viral labeling to identify gastric-related vagal complex neurons, and patch-clamp electrophysiology in acute brain stem slices to test the hypothesis that gastric-related and GABAergic inhibitory synaptic input to the DMV from the caudal NTS is under a robust modulatory control by nAChRs. Our results suggest that activation of nAChRs in the caudal NTS, but not DMV, potentiates GABAergic, but not glutamatergic, input to the DMV. Gastric-related caudal NTS and DMV neurons are directly involved in this nicotine-sensitive circuitry. Understanding the central patterns of nicotinic modulation of visceral sensory-motor circuitry may help develop therapeutic interventions to restore autonomic homeostasis in patients with autonomic impairments.

Reduced GABA(A) receptor-mediated tonic inhibition in aged rat auditory thalamus.

Age-related deficits in detecting and understanding speech, which can lead to social withdrawal and isolation, have been linked to changes in the central auditory system. Many of these central age-related changes involve altered mechanisms of inhibitory neurotransmission, essential for accurate and reliable auditory processing. In sensory thalamus, GABA mediates fast (phasic) inhibition via synaptic GABA(A) receptors (GABA(A)Rs) and long-lasting (tonic) inhibition via high-affinity (extrasynaptic) GABA(A)Rs, which provide a majority of the overall inhibitory tone in sensory thalamus. Due to a delicate balance between excitation and inhibition, alteration of normal thalamic inhibitory function with age and a reduction of tonic GABA(A)R-mediated inhibition may disrupt normal adult auditory processing, sensory gating, thalamocortical rhythmicity, and slow-wave sleep. The present study examines age-related homeostatic plasticity of GABA(A)R function in auditory thalamus or the medial geniculate body (MGB). Using thalamic slices from young adult (3-8 months) and aged (28-32 months) rats, these studies found a 45.5% reduction in GABA(A)R density and a 50.4% reduction in GABA(A)R-mediated tonic whole cell Cl(-) currents in the aged MGB. Synaptic GABA(A)R-mediated inhibition appeared differentially affected in aged lemniscal and nonlemniscal MGB. Except for resting membrane potential, basic properties were unaltered with age, including neuronal Cl(-) homeostasis determined using the gramicidin perforated patch-clamp method. Results demonstrate selective significant age-dependent deficits in the tonic inhibitory tone within the MGB. These data suggest that selective GABA(A)R subtype agonists or modulators might be used to augment MGB inhibitory neurotransmission, improving speech understanding, sensory gating, and slow-wave sleep for a subset of elderly individuals.

Projection target-specific action of nicotine in the caudal nucleus of the solitary tract.

The brainstem nucleus of the solitary tract (NTS) is the key integrating relay in the central processing of sensory information from the thoracic and from most subdiaphragmatic viscera. Modulation of neuronal excitability and synaptic activity in the NTS by nicotinic agents can have potent effects on vital physiological functions, such as feeding, digestion, respiration, and blood circulation. Caudal NTS neurons demonstrate considerable heterogeneity in projection targets, synaptic properties, and expression of nicotinic acetylcholine receptors (nAChRs). However, despite its heterogeneity, the caudal NTS may contain discrete subsets of neurons with unique projection target-specific properties. To test this hypothesis, we used in vivo fluorescent tracing and ex vivo patch-clamp electrophysiology to evaluate responsiveness to nicotine of anatomically identified caudal NTS neurons that project to the hypothalamic paraventricular nucleus (PVN) and the brainstem caudal ventrolateral medulla (CVLM). The results of this study demonstrate that responsiveness to nicotine correlates with where the neurons project. Specifically, PVN-projecting caudal NTS neurons respond to nicotine only presynaptically (i.e., via activation of presynaptic nAChRs and potentiation of synaptic release of glutamate), suggesting indirect, glutamate-dependent effects of nicotine on the PVN-projecting NTS circuitry. By contrast, CVLM-projecting caudal NTS neurons exhibit only limited presynaptic, but dominant somatodendritic, responsiveness to nicotine, suggesting that the effects of nicotine on the CVLM-projecting NTS circuitry are direct and largely glutamate independent. Understanding the relationships among function-specific brainstem/hypothalamic neuronal networks, nuclei, and individual neurons could help develop therapies targeting identifiable neuronal circuits to offset impaired autonomic homeostasis.

Responsiveness to nicotine of neurons of the caudal nucleus of the solitary tract correlates with the neuronal projection target.

The caudal nucleus of the solitary tract (NTS) is the key integrating center of visceral sensory-motor signaling supporting autonomic homeostasis. Two key projections of this nucleus are the parabrachial nucleus (PbN) and the dorsal motor nucleus of the vagus (DMV). The PbN integrates and relays viscerosensory information primarily to the forebrain, supporting behavioral, emotional, and endocrine responses to visceral events, while the DMV contains parasympathetic preganglionic cholinergic motoneurons that support primarily gastrointestinal reflexes. Subsets of caudal NTS neurons express presynaptic and somatodendritic nicotinic acetylcholine receptors (nAChRs). However, the anatomical identification of nicotine-responsive caudal NTS neurons has not been determined. This study used in vivo and ex vivo fluorescent tracing and slice patch-clamp electrophysiological recordings from anatomically identified caudal NTS neurons to test the hypothesis that the responsiveness of these cells to nicotine correlates with the target of their axonal projections. The results demonstrate that the majority of glutamatergic terminals that synapse on PbN-projecting caudal NTS neurons are unaffected by nicotine. Moreover, only a fraction of these cells express somatodendritic nAChRs. In contrast, the majority of DMV-projecting caudal NTS neurons exhibit robust presynaptic and somatodendritic responsiveness to nicotine. However, PbN-projecting neurons also exhibit significantly lower background frequencies of glutamatergic miniature postsynaptic currents than DMV-projecting neurons. Therefore, presynaptic unresponsiveness to nicotine may result from deficient glutamatergic innervation of PbN-projecting neurons. Nevertheless, the caudal NTS contains function-specific subsets of cells with target-specific responsiveness to nicotine. These results may support development of therapeutic strategies for selective targeting of specific autonomic pathways and impaired autonomic homeostasis.

α7 nicotinic ACh receptors as a ligand-gated source of Ca(2+) ions: the search for a Ca(2+) optimum.

The spatiotemporal distribution of cytosolic Ca(2+) ions is a key determinant of neuronal behavior and survival. Distinct sources of Ca(2+) ions including ligand- and voltage-gated Ca(2+) channels contribute to intracellular Ca(2+) homeostasis. Many normal physiological and therapeutic neuronal functions are Ca(2+)-dependent, however an excess of cytosolic Ca(2+) or a lack of the appropriate balance between Ca(2+) entry and clearance may destroy cellular integrity and cause cellular death. Therefore, the existence of optimal spatiotemporal patterns of cytosolic Ca(2+) elevations and thus, optimal activation of ligand- and voltage-gated Ca(2+) ion channels are postulated to benefit neuronal function and survival. Alpha7 nicotinic -acetylcholine receptors (nAChRs) are highly permeable to Ca(2+) ions and play an important role in modulation of neurotransmitter release, gene expression and neuroprotection in a variety of neuronal and non-neuronal cells. In this review, the focus is placed on α7 nAChR-mediated currents and Ca(2+) influx and how this source of Ca(2+) entry compares to NMDA receptors in supporting cytosolic Ca(2+) homeostasis, neuronal function and survival.

Mechanisms of facilitation of synaptic glutamate release by nicotinic agonists in the nucleus of the solitary tract.

The nucleus of the solitary tract (NTS) is the principal integrating relay in the processing of visceral sensory information. Functional nicotinic acetylcholine receptors (nAChRs) have been found on presynaptic glutamatergic terminals in subsets of caudal NTS neurons. Activation of these receptors has been shown to enhance synaptic release of glutamate and thus may modulate autonomic sensory-motor integration and visceral reflexes. However, the mechanisms of nAChR-mediated facilitation of synaptic glutamate release in the caudal NTS remain elusive. This study uses rat horizontal brainstem slices, patch-clamp electrophysiology, and fluorescent Ca(2+) imaging to test the hypothesis that a direct Ca(2+) entrance into glutamatergic terminals through active presynaptic non-α7- or α7-nAChR-mediated ion channels is sufficient to trigger synaptic glutamate release in subsets of caudal NTS neurons. The results of this study demonstrate that, in the continuous presence of 0.3 µM tetrodotoxin, a selective blocker of voltage-activated Na(+) ion channels, facilitation of synaptic glutamate release by activation of presynaptic nAChRs (detected as an increase in the frequency of miniature excitatory postsynaptic currents) requires external Ca(2+) but does not require activation of presynaptic Ca(2+) stores and presynaptic high- and low-threshold voltage-activated Ca(2+) ion channels. Expanding the knowledge of mechanisms and pharmacology of nAChRs in the caudal NTS should benefit therapeutic approaches aimed at restoring impaired autonomic homeostasis.

Spleen is not required for therapeutic effects of 4OH-GTS-21, a selective α7 nAChR agonist, in the sub-acute phase of ischemic stroke in rats.

Acute ischemic stroke (AIS) causes both central and peripheral inflammation, while activation of α7 nicotinic acetylcholine receptors (nAChRs) provides both central and peripheral anti-inflammatory and anti-apoptotic effects. Here, we provide evidence that 4OH-GTS-21, a selective α7 agonist, produces its therapeutic effects via primarily central sites of action because 4OH-GTS-21 was found equally effective in splenectomized and non-spenectomized rats in the sub-acute phase of ischemic stroke (≤1 week). However, the spleen may boost the therapeutic efficacy of 4OH-GTS-21 in certain behavioral tasks as our data also indicated. In our tests, AIS was modeled by transient middle cerebral artery occlusion (tMCAO). Splenectomy was done 2 weeks before tMCAO. We determined that: 1) Daily 4OH-GTS-21 treatments for 7 days after tMCAO significantly reduced neurological deficits and brain injury in both splenectomized and non-spelenectomized rats demonstrating that the spleen is not required for therapeutic benefits of 4OH-GTS-21; 2) The effects of 4OH-GTS-21 in the adhesive sticker removal test were significantly weaker in splenectomized animals suggesting that the spleen boosts the efficacy of 4OH-GTS-21 in the first week after tMCAO; and 3) Ischemic brain injury was not significantly affected by splenectomy in both vehicle-treated and 4OH-GTS-21-treated animals. These data support the hypothesis that the therapeutic efficacy of sub-chronic (≤1 week) 4OH-GTS-21 primarily originates from central sites of action. These results validate brain availability as a critical factor for developing novel α7 ligands for AIS.

Therapeutic efficacy of α7 ligands after acute ischaemic stroke is linked to conductive states of α7 nicotinic ACh receptors.

BACKGROUND AND PURPOSE: Targeting α7 nicotinic ACh receptors (nAChRs) in neuroinflammatory disorders including acute ischaemic stroke holds significant therapeutic promise. However, therapeutically relevant signalling mechanisms remain unidentified. Activation of neuronal α7 nAChRs triggers ionotropic signalling, but there is limited evidence for it in immunoglial tissues. The α7 ligands which are effective in reducing acute ischaemic stroke damage promote α7 ionotropic activity, suggesting a link between their therapeutic effects for treating acute ischaemic stroke and activation of α7 conductive states. EXPERIMENTAL APPROACH: This hypothesis was tested using a transient middle cerebral artery occlusion (MCAO) model of acute ischaemic stroke, NS6740, a known selective non-ionotropic agonist of α7 nAChRs and 4OH-GTS-21, a partial α7 agonist. NS6740-like ligands exhibiting low efficacy/potency for ionotropic activity will be referred to as non-ionotropic agonists or "metagonists". KEY RESULTS: 4OH-GTS-21, used as a positive control, significantly reduced neurological deficits and brain injury after MCAO as compared to vehicle and NS6740. By contrast, NS6740 was ineffective in identical assays and reversed the effects of 4OH-GTS-21 when these compounds were co-applied. Electrophysiological recordings from acute hippocampal slices obtained from NS6740-injected animals demonstrated its remarkable brain availability and protracted effects on α7 nAChRs as evidenced by sustained (>8 h) alterations in α7 ionotropic responsiveness. CONCLUSION AND IMPLICATIONS: These results suggest that α7 ionotropic activity may be obligatory for therapeutic efficacy of α7 ligands after acute ischaemic stroke yet, highlight the potential for selective application of α7 ligands to disease states based on their mode of receptor activation.

Systemic Administration of α7-Nicotinic Acetylcholine Receptor Ligands Does Not Improve Renal Injury or Behavior in Mice With Advanced Systemic Lupus Erythematosus.

There is a critical need for safe treatment options to control inflammation in patients with systemic lupus erythematosus (SLE) since the inflammation contributes to morbidity and mortality in advanced disease. Endogenous neuroimmune mechanisms like the cholinergic anti-inflammatory pathway can be targeted to modulate inflammation, but the ability to manipulate such pathways and reduce inflammation and end organ damage has not been fully explored in SLE. Positive allosteric modulators (PAM) are pharmacological agents that inhibit desensitization of the nicotinic acetylcholine receptor (α7-nAChR), the main anti-inflammatory feature within the cholinergic anti-inflammatory pathway, and may augment α7-dependent cholinergic tone to generate therapeutic benefits in SLE. In the current study, we hypothesize that activating the cholinergic anti-inflammatory pathway at the level of the α7-nAChR with systemic administration of a partial agonist, GTS-21, and a PAM, PNU-120596, would reduce inflammation, eliminating the associated end organ damage in a mouse model of SLE with advanced disease. Further, we hypothesize that systemic α7 ligands will have central effects and improve behavioral deficits in SLE mice. Female control (NZW) and SLE mice (NZBWF1) were administered GTS-21 or PNU-120596 subcutaneously via minipumps for 2 weeks. We found that the increased plasma dsDNA autoantibodies, splenic and renal inflammation, renal injury and hypertension usually observed in SLE mice with advanced disease at 35 weeks of age were not altered by GTS-21 or PNU-120596. The anxiety-like behavior presented in SLE mice was also not improved by GTS-21 or PNU-120596. Although no significant beneficial effects of α7 ligands were observed in SLE mice at this advanced stage, we predict that targeting this receptor earlier in the pathogenesis of the disease may prove to be efficacious and should be addressed in future studies.

Physiological concentrations of choline activate native alpha7-containing nicotinic acetylcholine receptors in the presence of PNU-120596 [1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea].

The use of PNU-120596 [1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea], a positive allosteric modulator of alpha7 nicotinic acetylcholine receptor (nAChR), may be beneficial for enhancing cholinergic therapies. However, the effects of PNU-120596 on activation of native alpha7-containing nAChRs by physiological concentrations of choline are not known and were investigated in this study using patch-clamp electrophysiology and histaminergic tuberomammillary neurons in hypothalamic slices. In the presence of PNU-120596, subthreshold (i.e., inactive) physiological concentrations of choline ( approximately 10 microM) elicited repetitive step-like whole-cell responses reminiscent of single ion channel openings that were reversibly blocked by 20 nM methyllycaconitine, a selective alpha7 nAChR antagonist. The effects of choline and PNU-120596 were synergistic as administration of 10 to 40 microM choline or 1 to 4 muM PNU-120596 alone did not elicit responses. In voltage clamp at -60 mV, the persistent activation of alpha7-containing nAChRs by 10 microM choline plus 1 microM PNU-120596 was estimated to produce a sustained influx of Ca(2+) ions at a rate of 8.4 pC/min ( approximately 0.14 pA). In the presence of PNU-120596 in current clamp, transient step-like depolarizations ( approximately 5 mV) enhanced neuronal excitability and triggered voltage-gated conductances; a single opening of an alpha7-containing nAChR channel appeared to transiently depolarize the entire neuron and facilitate spontaneous firing. Therefore, this study tested and confirmed the hypothesis that PNU-120596 enhances the effects of subthreshold concentrations of choline on native alpha7-containing nAChRs, allowing physiological levels of choline to activate these receptors and produce whole-cell responses in the absence of exogenous nicotinic agents. In certain neurological disorders, this activation may be therapeutically beneficial, more efficacious, and safer than treatments with nAChR agonists.

Evaluation of Ca2+ permeability of nicotinic acetylcholine receptors in hypothalamic histaminergic neurons.

Hypothalamic histaminergic tuberomammillary (TM) neurons express nicotinic acetylcholine receptors (nAChRs) with kinetic and pharmacological properties resembling those of highly Ca(2+) permeable alpha7 nAChRs. However, the Ca(2+) permeability of TM nAChR channels has not been determined. To directly evaluate the Ca(2+) permeability of TM nAChRs, patch-clamp recordings were conducted using non-cultured acutely dissociated TM neurons and external solutions containing low (2 mM) and high (20 mM) concentrations of Ca(2+). A shift in the reversal potentials was determined from the current-voltage relationships and the permeability ratio, P(Ca)/P(Na), was estimated within the Goldman-Hodgkin-Katz constant field approximation. TM nAChRs were found to be highly Ca(2+) permeable with the permeability ratio, P(Ca)/P(Na)(nAChR) being approximately 5.9 and the fractional Ca(2+) current, P(f)(nAChR) being approximately 10.1% at -60 mV. As a positive control for the applied methods and analysis, the permeability ratio, P(Ca)/P(Na)(NMDAR) being approximately 8.3 and the fractional Ca(2+) current, P(f)(NMDAR) being approximately 13.6% at -60 mV for NMDA receptors were determined using non-cultured acutely dissociated hippocampal pyramidal neurons and found similar to previously reported values. Therefore, these results demonstrate that native TM nAChRs are highly Ca(2+) permeable, but approximately 1.4 fold less permeable to Ca(2+) than native hippocampal pyramidal NMDA receptors.

Heterogeneity of nicotinic acetylcholine receptor expression in the caudal nucleus of the solitary tract.

The nucleus of the solitary tract (NTS) is the principal integrating relay in the processing of visceral sensory and gustatory information. In the present study, patch-clamp electrophysiological experiments were conducted using rat horizontal brainstem sections. Pre-synaptic and somatic/dendritic nicotinic acetylcholine receptors (nAChRs) expressed in neurons of the caudal NTS (cNTS) were found to be randomly distributed between pre-synaptic and somatic/dendritic sites (chi(2)=0.72, df=3, p>0.87, n=200). Pre-synaptic nAChRs were detected by their facilitating effects on glutamatergic neurotransmission of a sub-population of cNTS neurons (categorized as "effect-positive") upon brief picospritzer applications of 0.1-0.5mM nicotine. These effects were resistant to inhibition by 20nM methyllycaconitine (MLA) and 4muM dihydro-beta-erythroidine (DHbetaE), and were replicated by brief picospritzer applications of 0.2-1mM cytisine. Picospritzer applications of 0.2mM RJR-2403, a potent agonist of alpha4beta2 nAChRs, did not facilitate synaptic release of glutamate in effect-positive cNTS neurons. The population of somatic/dendritic nAChRs has been found to be heterogeneous and included nAChRs that were activated by RJR-2403 and/or cytisine, or insensitive to cytisine, or inhibited by MLA. The presented results are consistent with the expression of beta4-containing (i.e., beta4*) nAChRs, likely alpha3beta4*, in pre-synaptic terminals of effect-positive cNTS neurons. Somatic/dendritic nAChRs appear to involve both alpha7 and non-alpha7 subunits. Heterogeneity in the subunit composition of pre-synaptic and somatic/dendritic nAChRs may underlie diverse roles that these receptors play in regulation of behavioral and visceral reflexes, and may reflect specific targeting by endogenous nicotinic agents and nicotine.

Duration of isoflurane-based surgical anesthesia determines severity of brain injury and neurological deficits after a transient focal ischemia in young adult rats.

Tremendous efforts and funds invested in discovery of novel drug treatments for ischemic stroke have so far failed to deliver clinically efficacious therapies. The reasons for these failures are not fully understood. An indiscriminate use of isoflurane-based surgical anesthesia with or without nitrous oxide may act as an unconstrained, untraceable source of data variability, potentially causing false-positive or false-negative results. To test this hypothesis, a common transient suture middle cerebral artery occlusion (tMCAO) model of ischemic stroke in young adult male rats was used to determine the impact of a typical range of anesthesia durations required for this model on data variability (i.e., infarct volume and neurological deficits). The animals were maintained on spontaneous ventilation. The study results indicated that: (1) Variable duration of isoflurane anesthesia prior, during and after tMCAO is a significant source of data variability as evidenced by measurements of infarct volume and neurological deficits; and (2) Severity of brain injury and neurological deficits after tMCAO is inversely related to the duration of isoflurane anesthesia: e.g., in our study, a 90min isoflurane anesthesia nearly completely protected brain tissues from tMCAO-induced injury and thus, would be expected to obscure the effects of stroke treatments in pre-clinical trials. To elevate transparency, rigor and reproducibility of stroke research and minimize undesirable effects of isoflurane on the outcome of novel drug testing, we propose to monitor, minimize and standardize isoflurane anesthesia in experimental surgeries and make anesthesia duration a required reportable parameter in pre-clinical studies. Specifically, we propose to adopt 20-30min as an optimal anesthesia duration that both minimizes neuroprotective effects of isoflurane and permits a successful completion of surgical procedures in a suture tMCAO model of ischemic stroke in rodents. As the mechanisms and neuroprotective, metabolic and immune effects of general anesthesia are not fully understood, the results of this study cannot be blindly generalized to other anesthetics, animal species and experimental models.

Boosting Endogenous Resistance of Brain to Ischemia.

Most survivors of ischemic stroke remain physically disabled and require prolonged rehabilitation. However, some stroke victims achieve a full neurological recovery suggesting that the human brain can defend itself against ischemic injury, but the protective mechanisms are unknown. This study used selective pharmacological agents and a rat model of cerebral ischemic stroke to detect endogenous brain protective mechanisms that require activation of α7 nicotinic acetylcholine receptors (nAChRs). This endogenous protection was found to be (1) limited to less severe injuries; (2) significantly augmented by intranasal administration of a positive allosteric modulator of α7 nAChRs, significantly reducing brain injury and neurological deficits after more severe ischemic injuries; and (3) reduced by inhibition of calcium/calmodulin-dependent kinase-II. The physiological role of α7 nAChRs remains largely unknown. The therapeutic activation of α7 nAChRs after cerebral ischemia may serve as an important physiological responsibility of these ubiquitous receptors and holds a significant translational potential.

Cholinergic modulation of neurons in the gustatory region of the nucleus of the solitary tract.

The rostral portion of the nucleus of the solitary tract (rNST) is an obligatory relay for gustatory afferent input on its way to the forebrain. Previous studies have demonstrated excitation of rNTS neurons by glutamate and substance P and inhibition by gamma-aminobutyric acid (GABA) and met-enkephalin (ENK). Despite the existence of cholinergic neurons and putative terminals within the rNTS, there are no data on the effects of acetylcholine (ACh) on rNTS processing. Here, we use patch-clamp recording of rNTS neurons in vitro to examine ACh-mediated responses and voltage-gated conductances in these cells. Results revealed (1) intrinsic voltage-gated inhibition via activation of voltage-gated potassium A-channels (I(A)), found almost exclusively in the medial rNTS, and hyperpolarization-activated potassium/sodium channels (I(h)), found more frequently in the lateral rNST; and (2) ligand-gated inhibition via activation of muscarinic m2 ACh receptors (mAChRs) linked to inward rectifier potassium channels (K(ir)) evenly distributed throughout the rNTS, a mechanism dependent on cholinergic inputs. Muscarinic responses were blocked by AFDX-116, a selective m2 mAChR antagonist, and by BaCl2, an antagonist of K(ir) channels. In addition, many rNTS neurons exhibited excitation via alpha7 and non-alpha7 nicotinic AChRs. Non-alpha7 nAChRs, blocked by 10 microM mecamylamine, occurred more frequently in the lateral rNTS. In contrast, alpha7 nAChRs, blocked by 20 nM methyllcaconitine, were evenly distributed across the nucleus. As previously reported for voltage-activated conductances, none of these currents was related to neuronal morphology. These voltage- and ligand-dependent inhibitory mechanisms would be expected to contribute to the modulation of gustatory processing through the NST.

Histamine decreases myogenic tone in rat cerebral arteries by H2-receptor-mediated KV channel activation, independent of endothelium and cyclic AMP.

The effect of histamine on the pressure-induced constriction was characterized in rat cerebral arteries and mechanisms were investigated. Rat cerebral arteries were pressurized to 70 mm Hg in an arteriograph and the effect of histamine on myogenic tone was studied. Histamine and amthamine, a selective histamine H(2)-receptor agonist, concentration-dependently decreased myogenic tone, which was unchanged in the absence of endothelium. 2-(2-aminoethyl) pyridine, a selective histamine H(1)-receptor agonist, produced concentration-dependent constriction of arteries that was significantly increased in the absence of endothelium. Imetit, a selective histamine H(3)-receptor agonist, has no effect on myogenic tone. The dilation to histamine was antagonized by tiotidine, a selective antagonist of histamine H(2)-receptor subtype, giving a pK(B) of 7.86 that was not altered in the absence of endothelium. The histamine-mediated dilation was significantly antagonized by NF 449, a reversible inhibitor of Gs-protein activation but was not affected by ODQ and SQ 22536. Dilations to histamine and amthamine were accompanied by a decrease in arterial wall calcium measured by fura-2 ratios. The dilation to histamine was significantly reduced by partial depolarization of smooth muscle by 25 mM KCl (control 91+/-5%, 25 mM KCl 53+/-5%, P<0.002) and was not observed in the presence of strongly depolarizing 60 mM KCl. The histamine dilation was not affected by iberiotoxin, barium chloride and glibenclamide but was strongly antagonized by 4-aminopyridine (0.3 mM) and tetraethylammonium chloride (10 mM) (pEC(50): control: 5.6+/-0.1, 4-aminopyridine: 4.1+/-0.1 (P<0.001); tetraethylammonium chloride: 3.2+/-0.2 (P<0.0001)). These results suggest that histamine-mediated reversal of myogenic tone in rat cerebral arteries is endothelium-independent, mediated by histamine H(2)-receptor subtype with no involvement of guanylyl cyclase or adenylyl cyclase activation and most likely involves activation of K(V) potassium channels.

Allosteric Modulation of Nicotinic Acetylcholine Receptors: The Concept and Therapeutic Trends.

Expressing functional nicotinic acetylcholine receptors (nAChRs) may be beneficial to central neurons and neuronal networks because activation of nAChRs enhances neuronal resistance to injury, improves attention, cognitive performance, and produces robust anti-inflammatory and analgesic effects in mammals. Although exogenous orthosteric nAChR ligands present valuable tools in treatment of age- and trauma-related neurological deficits, therapeutic approaches that could amplify the brain's innate ability to maintain cholinergic homeostasis and resist injury may serve as intriguing and promising alternatives and have not been fully explored. One of these novel approaches utilizes positive allosteric modulators (PAMs) of nAChRs. Because of the ubiquitous expression of nAChRs in neuronal, glial and immune tissues, highly selective PAMs could amplify multiple endogenous neuroprotective, pro-cognitive, anti-inflammatory and anti-nociceptive cholinergic pathways to offset cholinergic hypofunction and generate therapeutic efficacy by targeting only a single player: i.e., nAChRs activated by endogenous cholinergic tone. In this article, I review the concept of allosteric modulation and current trends in therapeutic applications of nicotinic PAMs.