Synaptic Blockade Research Articles

Genetically-targeted Recordings from Respiratory Chemoreceptor Neurons in the Retrotrapezoid Nucleus of Freely Behaving Mice by Fiber Photometry

Central respiratory chemoreceptors regulate breathing homeostasis in relation to arterial pH/PCO2. Neurons located in the retrotrapezoid nucleus (RTN) are considered to function as central respiratory chemoreceptors and/or to be part of the neural network that drives the central chemoreflex. RTN neurons are sensitive to changes in pH in vitro under synaptic blockade and in anesthetized preparations with artificial ventilation. However, there is limited information about their activity in vivo in non-anesthetized conditions. Recent studies have shown that Nmb-expressing neurons in the RTN have acid-sensitive properties and are necessary for the respiratory response to CO2. In this study, we hypothesized that RTN- Nmb neurons are activated during hypercapnia in freely behaving condition. To test this hypothesis, we measured the dynamic activity of RTN neurons using a genetically encoded calcium indicator (GCaMP7s) via fiber photometry in non-anesthetized mice. Expression of GCaMP7s was genetically targeted to RTN neurons by microinjection of a Cre-dependent viral vector (AAV1-Syn-Flex-jGCAMP7s) in transgenic Nmb-Cre mice. One month later, the animals were placed in a plethysmography chamber to simultaneously record breathing and GCaMP7s fluorescence from RTN- Nmb neurons in freely behaving conditions. During hypercapnia, RTN neurons were markedly activated, and the effect size was graded to FICO2 (DF/F0 from baseline: 1.9 ± 1.0 % at 0.03, 8.0 ± 4.0% at 0.06 and 12.0 ± 5.0% at 0.09, n=6), and significantly different for each level of CO2 (RM One-way ANOVA, p=0.0012). Importantly, the increase in RTN neuron activity correlates with changes in the FICO2 (p<0.0001, R2=0.97) and respiratory frequency (p<0.0001, R2=0.85). This study shows that RTN neurons are potently activated by hypercapnia in freely behaving conditions, and the response of RTN neurons to CO2 aligns with changes in breathing. These results are consistent with the concept that RTN neurons monitor arterial PCO2/pH and are essential for the hypercapnic ventilatory response. This study was supported by National Institutes of Health (HL148004 ) to Stephen BG Abbott. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Plasticity in Preganglionic and Postganglionic Neurons of the Sympathetic Nervous System during Embryonic Development.

Sympathetic preganglionic neurons (SPNs) are the final output neurons from the central arm of the autonomic nervous system. Therefore, SPNs represent a crucial component of the sympathetic nervous system for integrating several inputs before driving the postganglionic neurons (PGNs) in the periphery to control end organ function. The mechanisms which establish and regulate baseline sympathetic tone and overall excitability of SPNs and PGNs are poorly understood. The SPNs are also known as the autonomic motoneurons (MNs) as they arise from the same progenitor line as somatic MNs that innervate skeletal muscles. Previously our group has identified a rich repertoire of homeostatic plasticity (HP) mechanisms in somatic MNs of the embryonic chick following in vivo synaptic blockade. Here, using the same model system, we examined whether SPNs exhibit similar homeostatic capabilities to that of somatic MNs. Indeed, we found that after 2-d reduction of excitatory synaptic input, SPNs showed a significant increase in intracellular chloride levels, the mechanism underlying GABAergic synaptic scaling in this system. This form of HP could therefore play a role in the early establishment of a setpoint of excitability in this part of the sympathetic nervous system. Next, we asked whether homeostatic mechanisms are expressed in the synaptic targets of SPNs, the PGNs. In this case we blocked synaptic input to PGNs in vivo (48-h treatment), or acutely ex vivo, however neither treatment induced homeostatic adjustments in PGN excitability. We discuss differences in the homeostatic capacity between the central and peripheral component of the sympathetic nervous system.

Effects of aconitine on the respiratory activity of brainstem-spinal cord preparations isolated from newborn rats.

Aconitine is a sodium channel opener, but its effects on the respiratory center are not well understood. We investigated the dose-dependent effects of aconitine on central respiratory activity in brainstem-spinal cord preparations isolated from newborn rats. Bath application of 0.5-5μM aconitine caused an increase in respiratory rhythm and decrease in the inspiratory burst amplitude of the fourth cervical ventral root (C4). Separate application of aconitine revealed that medullary neurons were responsible for the respiratory rhythm increase, and neurons in both the medulla and spinal cord were involved in the decrease of C4 amplitude by aconitine. A local anesthetic, lidocaine (100μM), or a voltage-dependent sodium channel blocker, tetrodotoxin (0.1μM), partially antagonized the C4 amplitude decrease by aconitine. Tetrodotoxin treatment tentatively decreased the respiratory rhythm, but lidocaine tended to further increase the rhythm. Treatment with 100μM riluzole or 100μM flufenamic acid, which are known to inhibit respiratory pacemaker activity, did not reduce the respiratory rhythm enhanced by aconitine + lidocaine. The application of 1μM aconitine depolarized the preinspiratory, expiratory, and inspiratory motor neurons. The facilitated burst rhythm of inspiratory neurons after aconitine disappeared in a low Ca2+/high Mg2+ synaptic blockade solution. We showed the dose-dependent effects of aconitine on respiratory activity. The antagonists reversed the depressive effects of aconitine in different manners, possibly due to their actions on different sites of sodium channels. The burst-generating pacemaker properties of neurons may not be involved in the generation of the facilitated rhythm after aconitine treatment.

Distension evoked mucosal secretion in human and porcine colon in vitro.

It was suggested that intestinal mucosal secretion is enhanced during muscle relaxation and contraction. Mechanisms of mechanically induced secretion have been studied in rodent species. We used voltage clamp Ussing technique to investigate, in human and porcine colonic tissue, secretion evoked by serosal (Pser) or mucosal (Pmuc) pressure application (2-60 mmHg) to induce distension into the mucosal or serosal compartment, respectively. In both species, Pser or Pmuc caused secretion due to Cl- and, in human colon, also HCO3- fluxes. In the human colon, responses were larger in proximal than distal regions. In porcine colon, Pmuc evoked larger responses than Pser whereas the opposite was the case in human colon. In both species, piroxicam revealed a strong prostaglandin (PG) dependent component. Pser and Pmuc induced secretion was tetrodotoxin (TTX) sensitive in porcine colon. In human colon, a TTX sensitive component was only revealed after piroxicam. However, synaptic blockade by ω-conotoxin GVIA reduced the response to mechanical stimuli. Secretion was induced by tensile rather than compressive forces as preventing distension by a filter inhibited the secretion. In conclusion, in both species, distension induced secretion was predominantly mediated by PGs and a rather small nerve dependent response involving mechanosensitive somata and synapses.

Patch‐to‐Seq Reveals Unique Transcriptomic Profiles of Chemosensitive Serotonergic Neurons

Central respiratory chemoreceptors are specialized neurons with intrinsic sensitivity to hypercapnia and/or acidosis that couple breathing and pH/CO2 levels. Prior data indicate that a sub-population of brainstem serotonin (5-HT) neurons are likely central respiratory chemoreceptors. However, it remains unclear which 5-HT neurons develop chemosensitivity and what molecular markers may identify this unique sub-population of 5-HT neurons. Here we employed the “patch-to-seq” technique to measure action potential firing rate responses of 5-HT neuron to hypercapnic acidosis using cell-attached patch-clamp electrophysiology followed by isolation of that neurons’ intracellular RNA content for single cell RNA Sequencing. Acute brainstem slices (200 µm) from young (P18-23) transgenic rats expressing eGFP in all 5-HT neurons (SSeGFP) were recorded while superfused with artificial CSF (aCSF) containing inhibitors for synaptic blockade (10 mM CNQX, 50 mM D-AP5, 20 mM Gabazine) bubbled with either 5% CO2 (bal. O2; pH= 7.36; 5 min) or 15% CO2 (bal. O2; pH= 7.10). The Chemosensitivity Index (C.I.) determined cell phenotypes. pH-sensitive eGFP+ neurons (n=48) had an average C.I. of 169.8± 8.25 SEM whereas pH-insensitive eGFP+ neurons (n=44) had an average C.I. of 100.3 ± 1.4 (P < 0.0001; t-test). Cell extracts from 11 chemosensitive and 10 insensitive recorded cells were subjected to single cell RNA Sequencing (scRNA-Seq), from which an average of 48.3M reads were generated with a quality score of ~32.7 and ~75% mapping rate per sample with an average of ~8,000 genes detected. Purity of the isolated samples was confirmed by significantly more expression (Log2(FPKM+1)) of 6 serotonergic vs 7 neuronal and 8 glial gene markers, and more expression of 7 neuronal vs 8 known glial gene markers. There were 166differentially expressed genes (q < 0.05) between pH-sensitive and -insensitive 5-HT neuron populations none of which have a known contribution to pH regulation or sensing. Predicted upstream regulators of 352 DEGs (p<0.01) point to the importance of Egr2 activation, a developmental transcription factor previously shown to give rise to pH sensitive 5-HT neurons in mice. Machine learning algorithms identified unique markers of CO2/pH sensitive 5-HT neurons in rats, where supervised analyses (using Support Vector Machine learning and Recursive Feature Elimination (SVM-RFE)) yielded several genes in which expression levels significantly (P<0.001) and accurately (%) predict the predefined phenotype from the DEGs (CD46, 68.7%; Iba57, 71.2%; Chd5, 72.0%; and Maz, 74.0%) and among the 1,000s of detected genes (Hnmpk, 81.0%; Diablo, 87.9%) but the biological/function significance of these genes in CO2/pH sensing remains unclear. RNAScope confirmed 25% of Tph2+ neurons in the raphe midline co-express 4 or more RNA molecules of CD46 or Iba57 RNA. Together, these data suggest: 1) 5-HT pH sensitive neurons are transcriptionally distinct from insensitive 5-HT neurons, and 2) novel molecular markers of 5-HT pH sensitive rat neurons may be useful identifiers for this unique subpopulation.

Calcium Imaging Analysis of Cellular Responses to Hypercapnia and Hypoxia in the NTS of Newborn Rat Brainstem Preparation.

It is supposed that the nucleus of the solitary tract (NTS) in the dorsal medulla includes gas sensor cells responsive to hypercapnia or hypoxia in the central nervous system. In the present study, we analyzed cellular responses to hypercapnia and hypoxia in the NTS region of newborn rat in vitro preparation. The brainstem and spinal cord were isolated from newborn rat (P0-P4) and were transversely cut at the level of the rostral area postrema. To detect cellular responses, calcium indicator Oregon Green was pressure-injected into the NTS just beneath the cut surface of either the caudal or rostral block of the medulla, and the preparation was superfused with artificial cerebrospinal fluid (25–26°C). We examined cellular responses initially to hypercapnic stimulation (to 8% CO2 from 2% CO2) and then to hypoxic stimulation (to 0% O2 from 95% O2 at 5% CO2). We tested these responses in standard solution and in two different synapse blockade solutions: (1) cocktail blockers solution including bicuculline, strychnine, NBQX and MK-801 or (2) TTX solution. At the end of the experiments, the superfusate potassium concentration was lowered to 0.2 from 3 mM to classify recorded cells into neurons and astrocytes. Excitation of cells was detected as changes of fluorescence intensity with a confocal calcium imaging system. In the synaptic blockade solutions (cocktail or TTX solution), 7.6 and 8% of the NTS cells responded to hypercapnic and hypoxic stimulation, respectively, and approximately 2% of them responded to both stimulations. Some of these cells responded to low K+, and they were classified into astrocytes comprising 43% hypercapnia-sensitive cells, 56% hypoxia-sensitive cells and 54% of both stimulation-sensitive cells. Of note, 49% of the putative astrocytes identified by low K+ stimulation were sensitive to hypercapnia, hypoxia or both. In the presence of a glia preferential blocker, 5 mM fluoroacetate (plus 0.5 μM TTX), the percentage of hypoxia-sensitive cells was significantly reduced compared to those of all other conditions. This is the first study to reveal that the NTS includes hypercapnia and hypoxia dual-sensitive cells. These results suggest that astrocytes in the NTS region could act as a central gas sensor.

Coupling Electrophysiology with Single Cell RNA Sequencing to Uncover Transcriptional Markers of Chemosensitive Serotonergic Neurons

Central respiratory chemoreceptors are specialized neurons with intrinsic sensitivity to hypercapnia and/or acidosis that couple breathing and pH/CO2 levels. Previous data support the hypothesis that a sub‐population of brainstem serotonin (5‐HT) neurons serve as central respiratory chemoreceptors, where they become sensitive to pH/CO2 on or after post‐natal day 12 (P12) in rats. However, to date it remains unclear which 5‐HT neurons develop chemosensitivity and what molecular markers may identify this unique sub‐population of 5‐HT neurons. To address these questions, we employed a “patch‐to‐seq” technique to first distinguish 5‐HT neurons as chemosensitive (or insensitive) using electrophysiologic recording, and then to isolate each neurons’ RNA for single cell RNA Sequencing to identify potential cellular sensing mechanisms and/or unique transcriptional markers in each phenotypically‐identified 5‐HT neuron population. Acute brainstem slices (200 μm) were made from young (P18–23) transgenic rats which express eGFP in all 5‐HT neurons (SSeGFP), and action potential firing rates measured via a cell‐attached patch configuration in eGFP+ 5‐HT neurons during superfusion with artificial CSF (aCSF) containing inhibitors for synaptic blockade (10 mM CNQX, 50 mM D‐AP5, 20 mM Gabazine) bubbled with either 5% CO2 (bal. O2; pH= 7.36; 5 min) or 15% CO2 (bal. O2; pH= 7.10). Cellular phenotypes were assessed by calculating a Chemosensitivity Index (C.I.). pH‐sensitive eGFP+ neurons (n=7) had an average C.I. of 177.0±14.6 whereas pH‐insensitive eGFP+ neurons (n=9) had an average C.I. of 98.0 ± 2.9 (P &lt; 0.0001). Cellular contents of each recorded cell were collected and subjected to single cell RNA Sequencing (scRNA‐Seq), from which an average of 19.3M reads were generated with a quality score of ~33.7 and ~83% mapping rate per sample (32,883 known genes quantified). Preliminary analyses indicate very few differentially expressed genes (q &lt; 0.05) between pH‐sensitive and ‐ insensitive 5‐HT neuron populations, and of those differentially expressed no gene obviously contributes to pH regulation or sensing. In addition, unbiased hierarchical clustering and principal component analyses failed to clearly separate samples by phenotype. However, a supervised analysis using Support Vector Machine learning and Recursive Feature Elimination (SVM‐RFE) identified several genes in which expression levels significantly predict the predefined phenotype, but the biological/functional significance of these genes in CO2/pH sensing remains unclear and instead these genes may provide a molecular signature to distinguish pH/CO2 sensitive vs insensitive 5‐HT neurons. We conclude from these preliminary data that pH/CO2 sensitivity in 5‐HT neurons is not a transcriptionally regulated phenotype.Support or Funding InformationParker B. Francis FoundationNHLBI ‐ R01 HL122358

Functional strengthening through synaptic scaling upon connectivity disruption in neuronal cultures.

An elusive phenomenon in network neuroscience is the extent of neuronal activity remodeling upon damage. Here, we investigate the action of gradual synaptic blockade on the effective connectivity in cortical networks in vitro. We use two neuronal cultures configurations—one formed by about 130 neuronal aggregates and another one formed by about 600 individual neurons—and monitor their spontaneous activity upon progressive weakening of excitatory connectivity. We report that the effective connectivity in all cultures exhibits a first phase of transient strengthening followed by a second phase of steady deterioration. We quantify these phases by measuring GEFF, the global efficiency in processing network information. We term hyperefficiency the sudden strengthening of GEFF upon network deterioration, which increases by 20–50% depending on culture type. Relying on numerical simulations we reveal the role of synaptic scaling, an activity–dependent mechanism for synaptic plasticity, in counteracting the perturbative action, neatly reproducing the observed hyperefficiency. Our results demonstrate the importance of synaptic scaling as resilience mechanism.

Sleep-promoting effects of threonine link amino acid metabolism in Drosophila neuron to GABAergic control of sleep drive.

This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Experimental study of score direct muscle excitability after synaptic blockade by stimulating impedance myography by using volt-omics characteristics

Experimental study of score direct muscle excitability after synaptic blockade by stimulating impedance myography by using volt-omics characteristics

Pairing Electrophysiology and Single Cell RNA Sequencing to Identify Mechanisms of Cellular pH/CO2 Sensitivity in Respiratory Chemoreceptor Neurons

Respiratory chemoreceptors are specialized neurons that are excited by hypercapnic acidosis (HA) and encode brain pH and/or CO2 levels to drive the appropriate changes in breathing. A subset of serotonin‐producing (5‐HT) neurons develop an apparent intrinsic chemosensitivity to HA after ~P12, but the molecular mechanisms driving their cellular pH/CO2 sensitivity remains unknown. Given that Sudden Infant Death Syndrome (SIDS) in humans has been linked to developmental 5‐HT system defects and potential alterations in ventilatory chemoreflexes, we aimed to develop a new technique to identify transcriptional differences among pH‐sensitive and ‐insensitive 5‐HT neurons in order to ultimately define the genetic determinants of cellular pH/CO2 sensitivity. Acute brainstem slices (200 uM) were prepared from 18–23 day‐old transgenic Dahl Salt Sensitive rats that express eGFP in all 5‐HT neurons (SSeGFP). Firing rates were measured via cell‐attached patch in eGFP+ 5‐HT neurons superfused with an artificial CSF (aCSF) containing inhibitors for synaptic blockade (10 mM CNQX, 50 mM D‐AP5, 20 mM Gabazine) while cycling between control (5% CO2 bal. O2; pH= 7.36; 5 min) and HA (15% CO2 bal. O2; pH= 7.10; 10 min) conditions. Once the cellular phenotype was established for pHsensitive (n=6 cells) or ‐insensitive (n=5) cells, a second electrode filled with small amounts (&lt;1ul) of intracellular solution spiked with RNAse inhibitor was used to isolate the cellular contents for subsequent single cell RNA Sequencing (scRNA‐Seq). Specificity of cellular isolation was confirmed by high levels of expression of several serotonin and neuronal genes, and a lack of glial gene expression by sc‐qRT‐PCR (n=7) or sc‐RNAseq. The scRNA‐Seq read summary indicated an average of ~45M reads, a quality score of ~33.5, and ~80% mapping rate per sample, from which &gt;10,000 known genes were quantified. However, small sample sizes prevented obtaining statistical significance for differential expression (Q &gt; 0.05). Using R software packages, unsupervised analyses (hierarchical clustering and principal component analyses) failed to clearly separate samples by phenotype, and a power analysis based on FPKM values from the 11 samples suggested sample sizes of 20–60 cells/group are required. However, a supervised analysis (pre‐defining experimental groups) using Support Vector Machine (SVM) learning coupled with Recursive Feature Elimination (i.e. creates a model with all genes and recursively reduces the number of genes to identify the fewest genes to explain the predefined phenotype) revealed a candidate molecular signature for CO2 sensitive 5‐HT neurons. Overall, we demonstrated we can obtain transcriptomic data from electrophysiologically‐characterized 5‐HT neurons at the single cell‐level, and analyses of these pilot data suggest unsupervised and supervised machine learning approaches may identify unique genetic profiles in pH‐sensitive 5‐HT neurons. These data provide the field of respiratory physiology with preliminary markers of 5‐HT neuron CO2/pH sensitivity and validate that this approach may be used to address questions pertaining to genetic determinants of a given cellular phenotype.Support or Funding InformationNIH R01 HL12358 04 &amp; Parker B. Francis FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ca2+ Channels Involvement in Low-Frequency Stimulation-Mediated Suppression of Intrinsic Excitability of Hippocampal CA1 Pyramidal Cells in a Rat Amygdala Kindling Model

Low-frequency stimulation has demonstrated promising seizure suppression in animal models of epilepsy, while the mechanism of the effect is still debated. Changes in intrinsic properties have been recognized as a prominent pathophysiologically relevant feature of numerous neurological disorders including epilepsy. Here, it was evaluated whether LFS can preserve the intrinsic neuronal electrophysiological properties in a rat model of epilepsy, focusing on the possible involvement of voltage-gated Ca2+ channels. The amygdala kindling model was induced by 3 s monophasic square wave pulses (50 Hz, 1 ms duration, 12times/day at 5 min intervals). Both LFS alone and kindled plus LFS (KLFS) groups received four packages of LFS (each consisting of 200 monophasic square pulses, 0.1 ms pulse duration at 1 Hz with the after discharge threshold intensity), which in KLFS rats was applied immediately after kindling induction. Whole-cell patch-clamp recordings were made in the presence of fast synaptic blockers 24 h after the last kindling stimulations or following kindling stimulations plus LFS application. In the KLFS group, both the rebound excitation and kindling-induced intrinsic hyperexcitability were decreased, associated with a regular intrinsic firing as indicated by a lower coefficient of variation. The amplitude of afterdepolarization (ADP) and its area under the curve were both decreased in the KLFS group compared to the kindled group. LFS prevented the increasing effect of kindling on Ca2+ currents in the KLFS group. Findings provided evidence for a novel form of epileptiform activity suppression by LFS in the presence of synaptic blockade possibly by decreasing Ca2+ currents.

AgRP/NPY Neuron Excitability Is Modulated by Metabotropic Glutamate Receptor 1 During Fasting.

The potential to control feeding behavior via hypothalamic AgRP/NPY neurons has led to many approaches to modulate their excitability—particularly by glutamatergic input. In the present study using NPY-hrGFP reporter mice, we visualize AgRP/NPY neuronal metabotropic glutamate receptor 1 (mGluR1) expression and test the effect of fasting on mGluR1 function. Using the pharmacological agonist dihydroxyphenylglycine (DHPG), we demonstrate the enhanced capacity of mGluR1 to drive firing of AgRP/NPY neurons after overnight fasting, while antagonist 3-MATIDA reduces firing. Further, under synaptic blockade we demonstrate that DHPG acts directly on AgRP/NPY neurons to create a slow inward current. Using an in vitro approach, we show that emulation of intracellular signals associated with fasting by forskolin enhances DHPG induced phosphorylation of extracellularly regulated-signal kinase (1/2) in GT1-7 cell culture. We show in vivo that blocking mGluR1 by antagonist 3-MATIDA lowers fasting induced refeeding. In summary, this study identifies a novel layer of regulation on AgRP/NPY neurons integrated with whole body energy balance.

Differential electrical responses in retinal ganglion cell subtypes: effects of synaptic blockade and stimulating electrode location

Objective. Visual prostheses have shown promising results in restoring visual perception to blind patients. The ability to differentially activate retinal ganglion cell (RGC) subtypes could further improve the efficacy of these medical devices. Approach. Using whole-cell patch clamp, we investigated membrane potential differences between ON and OFF RGCs in the mouse retina when their synaptic inputs were blocked by synaptic blockers, and examined the differences in stimulation thresholds under such conditions. By injecting intracellular current, we further confirmed the relationship between RGC stimulation thresholds and resting membrane potentials (RMPs). In addition, we investigated the effects of stimulating electrode location on the differences in stimulation thresholds between ON and OFF RGCs. Main results. With synaptic blockade, ON RGCs became significantly more hyperpolarized (from −61.8 ± 1.4 mV to −70.8 ± 1.6 mV), while OFF RGCs depolarized slightly (from −60.5 ± 0.7 mV to −58.6 ± 0.9 mV). RGC stimulation thresholds were negatively correlated with their RMPs (Pearson r value: −0.5154; p-value: 0.0042). Thus, depriving ON RGCs of synaptic inputs significantly increased their thresholds (from 14.7 ± 1.3 µA to 22.3 ± 2.1 µA) over those of OFF RGCs (from 13.2 ± 0.7 µA to 13.1 ± 1.1 µA). However, with control solution, ON and OFF RGC stimulation thresholds were not significantly different. Finally, placement of the stimulating electrode away from the axon enhanced differences in stimulation thresholds between ON and OFF RGCs, facilitating preferential activation of OFF RGCs. Significance. Since ON and OFF RGCs have antagonistic responses to natural light, achieving differential RGC activation could convey more natural visual information, leading to better visual prosthesis outcomes.

α2 Receptors in the lateral parabrachial nucleus generates the pressor response of the cardiovascular chemoreflex, effects of GABAA receptor

The lateral parabrachial nucleus (LPBN) is a pontine area involved in cardiovascular chemoreflex. This study was performed to find the effects of reversible synaptic blockade of the LPBN on the chemoreflex responses, and to find the roles of GABAA receptor and α2-adenoreceptor (α2-AR) in chemoreflex. It also aimed to seek possible interaction between GABA and noradrenergic systems of the LPBN in urethane-anesthetized male rats. Cardiovascular chemoreflex was activated by intravenous injection of potassium cyanide (KCN, 80 μg/kg). The cardiovascular responses of chemoreflex were evaluated before (control), 5 and 15 min after microinjection of each drug (100 nl) into the LPBN.Microinjections of cobalt chloride (5 mM), a reversible synaptic blocker, into the LPBN greatly attenuated the chemoreflex pressor and bradycardic responses indicating that the LPBN plays a main role in chemoreflex. Local injection of yohimbine (10 nmol), an α2-AR antagonist, attenuated the pressor response with no effect on bradycardic response, suggesting that α2-adrenoreceptors are involved in producing the pressor response of the chemoreflex. Microinjection of bicuculline methiodide (BMI, 100 pmol), a GABAA antagonist, into the LPBN augmented the pressor response and attenuated the bradycardic response, indicating that GABA inhibits the sympathetic output to the heart and vasculature. Sequential injection of yohimbine and BMI had no significant effect on the pressor response but attenuated the bradycardia.In conclusion, the LPBN is essential for the chemoreflex responses. The pressor response of the chemoreflex, at least partly, is produced by α2- adenoreceptors. GABA in the LPBN inhibits the cardiovascular system. Finally, there is no interaction between GABAergic and adrenergic neurons of the LPBN in producing the cardiovascular chemoreflex.

In vitro and ex vivo screening of candidate therapeutics to restore neurotransmission in nerve terminals intoxicated by botulinum neurotoxin serotype A1

Botulinum neurotoxins (BoNTs) are exceedingly potent neurological poisons that block cholinergic release in the peripheral nervous system and cause death by asphyxiation. While post-exposure prophylaxis can effectively eliminate toxin in the bloodstream, there are no clinically effective treatments to prevent or reverse disease once BoNT has entered the neuron. To address the need for post-symptomatic countermeasures, we designed and developed an in vitro assay based on whole-cell, patch-clamp electrophysiological monitoring of miniature excitatory post-synaptic currents in synaptically active murine embryonic stem cell-derived neurons. This synaptic function-based assay was used to assess the efficacy of rationally selected drugs to restore neurotransmission in neurons comprehensively intoxicated by BoNT/A. Based on clinical reports suggesting that elevated Ca2+ signaling promotes symptomatic relief from botulism, we identified seven candidate drugs that modulate presynaptic Ca2+ signaling and assessed their ability to reverse BoNT/A-induced synaptic blockade. The most effective drugs from the screen were found to phasically agonize voltage-gated calcium channel (VGCC) activity. Lead candidates were then applied to ex vivo studies in BoNT/A-paralyzing mouse phrenic nerve-hemidiaphragm (PND) preparations. Treatment of PNDs with VGCC agonists after paralytic onset transiently potentiated nerve-elicited muscle contraction and delayed progression to neuromuscular failure. Collectively, this study suggests that Ca2+-modulating drugs represent a novel symptomatic treatment for neuromuscular paralysis following BoNT/A poisoning.

Synaptic activation of putative sensory neurons by hexamethonium-sensitive nerve pathways in mouse colon.

The gastrointestinal tract contains its own independent population of sensory neurons within the gut wall. These sensory neurons have been referred to as intrinsic primary afferent neurons (IPANs) and can be identified by immunoreactivity to calcitonin gene-related peptide (CGRP) in mice. A common feature of IPANs is a paucity of fast synaptic inputs observed during sharp microelectrode recordings. Whether this is observed using different recording techniques is of particular interest for understanding the physiology of these neurons and neural circuit modeling. Here, we imaged spontaneous and evoked activation of myenteric neurons in isolated whole preparations of mouse colon and correlated recordings with CGRP and nitric oxide synthase (NOS) immunoreactivity, post hoc. Calcium indicator fluo 4 was used for this purpose. Calcium responses were recorded in nerve cell bodies located 5-10 mm oral to transmural electrical nerve stimuli. A total of 618 recorded neurons were classified for CGRP or NOS immunoreactivity. Aboral electrical stimulation evoked short-latency calcium transients in the majority of myenteric neurons, including ~90% of CGRP-immunoreactive Dogiel type II neurons. Activation of Dogiel type II neurons had a time course consistent with fast synaptic transmission and was always abolished by hexamethonium (300 μM) and by low-calcium Krebs solution. The nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (during synaptic blockade) directly activated Dogiel type II neurons. The present study suggests that murine colonic Dogiel type II neurons receive prominent fast excitatory synaptic inputs from hexamethonium-sensitive neural pathways. NEW & NOTEWORTHY Myenteric neurons in isolated mouse colon were recorded using calcium imaging and then neurochemically defined. Short-latency calcium transients were detected in >90% of calcitonin gene-related peptide-immunoreactive neurons to electrical stimulation of hexamethonium-sensitive pathways. Putative sensory Dogiel type II calcitonin gene-related peptide-immunoreactive myenteric neurons may receive widespread fast synaptic inputs in mouse colon.

Firing properties of pre‐inspiratory neurons are altered in female rats exposed to chronic intermittent hypoxia

Female rats submitted to chronic intermittent hypoxia (CIH) presented a reduced time of inspiration, sympathetic overactivity and hypertension. The increase in sympathetic nerve discharge in CIH‐female rats is associated to an increase in the inspiratory modulation of sympathetic activity. In this context, we hypothesized that inspiratory neurons at the brainstem have change their activity in CIH‐female rats, which in turn, modulates the presympathetic neurons producing an increase in sympathetic activity during inspiration. To test this hypothesis, juvenile female Wistar rats (P20–21) underwent intermittent hypoxia for 10 days. One day after CIH, we performed the extracellular single unit recordings of respiratory neurons at the ventral surface of the medulla using the ventral approach of the working heart‐brainstem preparation. All the experimental protocols were approved by the institutional ethical committee (#091/2013). Pre‐inspiratory neurons (Pre‐I/I) of CIH‐female rats (n=16) have reduced their firing frequency (58.3 ± 9 vs 107.6 ± 12Hz, p=0.0027) compared to Pre‐I/I neurons of control (n=15). Synaptic blockade demonstrated that Pre‐I/I neurons (n=3) have auto‐depolarization property. Ramp‐I, Early‐I and Late‐I neurons presented no changes in their firing properties after CIH. Regarding the expiratory neurons, post‐inspiratory neurons (Post‐I) have increased the firing time relative to duration of expiration (92.2 ± 2 vs 81.3 ± 3 % of total duration of expiration, p=0.0127) in CIH‐female rats (n=15) compared to control (n=17). We conclude that CIH produces a reduction in the activity of pacemaker Pre‐I/I neurons that could explain the reduced time of inspiration and the increase in inspiratory modulation of sympathetic activity in CIH‐female rats. These findings contribute to a better understanding of the respiratory network activity associated with sympathetic overactivity and hypertension in female rats exposed to CIH.Support or Funding InformationFapesp, CAPES and CNPq

Role of the lateral preoptic area in cardiovascular and neuroendocrine responses to acute restraint stress in rats

The lateral preoptic area (LPO) is connected with limbic structures involved in physiological and behavioral responses to stress. Accordingly, exposure to stressors stimuli activates neurons within the LPO. In spite of these evidence, an involvement of the LPO on cardiovascular and neuroendocrine adjustments during aversive threats has not yet been investigated. Therefore, in the present study we tested the hypothesis that the LPO is involved in the control of cardiovascular and neuroendocrine responses to acute restraint stress in rats. Bilateral microinjection of the nonselective synaptic blocker CoCl2 (0.1nmol/100nl) into the LPO did not affect basal values of either arterial pressure, heart rate, tail skin temperature, or plasma corticosterone concentration. However, LPO treatment with CoCl2 enhanced the tachycardiac response and the increase in plasma corticosterone concentration caused by restraint stress. Conversely, LPO synaptic blockade decreased restraint-evoked pressor response. Sympathetic-mediated cutaneous vasoconstriction during restraint stress was not affected by LPO pharmacological treatment. These findings indicate an inhibitory influence of LPO on tachycardiac and plasma corticosterone responses evoked during aversive threats. Additionally, data suggest that LPO plays a facilitatory influence on stress-evoked pressor response.

A Cre Mouse Line for Probing Irradiance- and Direction-Encoding Retinal Networks.

Cell type-specific Cre driver lines have revolutionized the analysis of retinal cell types and circuits. We show that the transgenic mouse Rbp4-Cre selectively labels several retinal neuronal types relevant to the encoding of absolute light intensity (irradiance) and visual motion. In the ganglion cell layer (GCL), most marked cells are wide-field spiking polyaxonal amacrine cells (ACs) with sustained irradiance-encoding ON responses that persist during chemical synaptic blockade. Their arbors spread about 1 mm across the retina and are restricted to the inner half of the ON sublamina of the inner plexiform layer (IPL). There, they costratify with dendrites of M2 intrinsically photosensitive retinal ganglion cells (ipRGCs), to which they are tracer coupled. We propose that synaptically driven and intrinsic photocurrents of M2 cells pass through gap junctions to drive AC light responses. Also marked in this mouse are two types of RGCs. R-cells have a bistratified dendritic arbor, weak directional tuning, and irradiance-encoding ON responses. However, they also receive excitatory OFF input, revealed during ON-channel blockade. Serial blockface electron microscopic (SBEM) reconstruction confirms OFF bipolar input, and reveals that some OFF input derives from a novel type of OFF bipolar cell (BC). R-cells innervate specific layers of the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC). The other marked RGC type (RDS) is bistratified, transient, and ON-OFF direction selective (DS). It apparently innervates the nucleus of the optic tract (NOT). The Rbp4-Cre mouse will be valuable for targeting these cell types for further study and for selectively manipulating them for circuit analysis.