scholarly journals Organization and Properties of GABAergic Neurons in Solitary Tract Nucleus (NTS)

2008 ◽  
Vol 99 (4) ◽  
pp. 1712-1722 ◽  
Author(s):  
Timothy W. Bailey ◽  
Suzanne M. Appleyard ◽  
Young-Ho Jin ◽  
Michael C. Andresen
Keyword(s):  
Fluorescent Protein ◽  
Signal Propagation ◽  
Gabaergic Neurons ◽  
Visceral Afferents ◽  
Egfp Expression ◽  
Acid Decarboxylase ◽  
Solitary Tract Nucleus ◽  
Solitary Tract ◽  
Afferent Pathways ◽  
Postsynaptic Currents

Cranial visceral afferents enter the brain at the solitary tract nucleus (NTS). GABAergic neurons are scattered throughout the NTS, but their relation to solitary tract (ST) afferent pathways is imprecisely known. We hypothesized that most GABAergic NTS neurons would be connected only indirectly to the ST. We identified GABAergic neurons in brain stem horizontal slices using transgenic mice in which enhanced green fluorescent protein (EGFP) expression was linked to glutamic acid decarboxylase expression (GAD+). Finely graded electrical shocks to ST recruit ST-synchronized synaptic events with all-or-none thresholds and individual waveforms did not change with greater suprathreshold intensities—evidence consistent with initiation by single afferent axons. Most (∼70%) GAD+ neurons received ST-evoked excitatory postsynaptic currents (EPSCs) that had minimally variant latencies (jitter, SD of latency <200 μs) and waveforms consistent with single, direct ST connections (i.e., monosynaptic). Increasing stimulus intensity evoked additional ST-synchronized synaptic responses with jitters >200 μs including inhibitory postsynaptic currents (IPSCs), indicating indirect connections (polysynaptic). Shocks of suprathreshold intensity delivered adjacent (50–300 μm) to the ST failed to excite non-ST inputs to second-order neurons, suggesting a paucity of axons passing near to ST that connected to these neurons. Despite expectations, we found similar ST synaptic patterns in GAD+ and unlabeled neurons. Generally, ST information that arrived indirectly had small amplitudes (EPSCs and IPSCs) and frequency-dependent failures that reached >50% for IPSCs to bursts of stimuli. This ST afferent pathway organization is strongly use-dependent—a property that may tune signal propagation within and beyond NTS.

scholarly journals Substance P excites GABAergic neurons in the mouse central amygdala through neurokinin 1 receptor activation

2015 ◽  
Vol 114 (4) ◽  
pp. 2500-2508 ◽  
Author(s):  
L. Sosulina ◽  
C. Strippel ◽  
H. Romo-Parra ◽  
A. L. Walter ◽  
T. Kanyshkova ◽  
...  
Keyword(s):  
Substance P ◽  
Fluorescent Protein ◽  
Input Resistance ◽  
Receptor Activation ◽  
Gabaergic Neurons ◽  
Acid Decarboxylase ◽  
Central Amygdala ◽  
Cellular Mechanisms ◽  
Action Potential Firing

Substance P (SP) is implicated in stress regulation and affective and anxiety-related behavior. Particularly high expression has been found in the main output region of the amygdala complex, the central amygdala (CE). Here we investigated the cellular mechanisms of SP in CE in vitro, taking advantage of glutamic acid decarboxylase-green fluorescent protein (GAD67-GFP) knockin mice that yield a reliable labeling of GABAergic neurons, which comprise 95% of the neuronal population in the lateral section of CE (CEl). In GFP-positive neurons within CEl, SP caused a membrane depolarization and increase in input resistance, associated with an increase in action potential firing frequency. Under voltage-clamp conditions, the SP-specific membrane current reversed at −101.5 ± 2.8 mV and displayed inwardly rectifying properties indicative of a membrane K+ conductance. Moreover, SP responses were blocked by the neurokinin type 1 receptor (NK1R) antagonist L-822429 and mimicked by the NK1R agonist [Sar9,Met(O2)11]-SP. Immunofluorescence staining confirmed localization of NK1R in GFP-positive neurons in CEl, predominantly in PKCδ-negative neurons (80%) and in few PKCδ-positive neurons (17%). Differences in SP responses were not observed between the major types of CEl neurons (late firing, regular spiking, low-threshold bursting). In addition, SP increased the frequency and amplitude of GABAergic synaptic events in CEl neurons depending on upstream spike activity. These data indicate a NK1R-mediated increase in excitability and GABAergic activity in CEl neurons, which seems to mostly involve the PKCδ-negative subpopulation. This influence can be assumed to increase reciprocal interactions between CElon and CEloff pathways, thereby boosting the medial CE (CEm) output pathway and contributing to the anxiogenic-like action of SP in the amygdala.

scholarly journals Isoflurane Differentially Modulates Inhibitory and Excitatory Synaptic Transmission to the Solitary Tract Nucleus

2008 ◽  
Vol 108 (4) ◽  
pp. 675-683 ◽  
Author(s):  
James H. Peters ◽  
Stuart J. McDougall ◽  
David Mendelowitz ◽  
Dennis R. Koop ◽  
Michael C. Andresen
Keyword(s):  
Nucleus Tractus Solitarius ◽  
Second Order ◽  
Visceral Afferents ◽  
Gas Chromatography Mass Spectrometry ◽  
Gamma Aminobutyric Acid ◽  
Solitary Tract ◽  
Excitatory Postsynaptic Currents ◽  
Inhibitory Postsynaptic Currents ◽  
Postsynaptic Currents ◽  
Presynaptic Mechanisms

Background Isoflurane anesthesia produces cardiovascular and respiratory depression, although the specific mechanisms are not fully understood. Cranial visceral afferents, which innervate the heart and lungs, synapse centrally onto neurons within the medial portion of the nucleus tractus solitarius (NTS). Isoflurane modulation of afferent to NTS synaptic communication may underlie compromised cardiorespiratory reflex function. Methods Adult rat hindbrain slice preparations containing the solitary tract (ST) and NTS were used. Shocks to ST afferents evoked excitatory postsynaptic currents with low-variability (SEM &lt;200 mus) latencies identifying neurons as second order. ST-evoked and miniature excitatory postsynaptic currents as well as miniature inhibitory postsynaptic currents were measured during isoflurane exposure. Perfusion bath samples were taken in each experiment to measure isoflurane concentrations by gas chromatography-mass spectrometry. Results Isoflurane dose-dependently increased the decay-time constant of miniature inhibitory postsynaptic currents. At greater than 300 mum isoflurane, the amplitude of miniature inhibitory postsynaptic currents was decreased, but the frequency of events remained unaffected, whereas at equivalent isoflurane concentrations, the frequency of miniature excitatory postsynaptic currents was decreased. ST-evoked excitatory postsynaptic current amplitudes decreased without altering event kinetics. Isoflurane at greater than 300 mum increased the latency to onset and rate of synaptic failures of ST-evoked excitatory postsynaptic currents. Conclusions In second-order NTS neurons, isoflurane enhances phasic inhibitory transmission via postsynaptic gamma-aminobutyric acid type A receptors while suppressing excitatory transmission through presynaptic mechanisms. These results suggest that isoflurane acts through multiple distinct mechanisms to inhibit neurotransmission within the NTS, which would underlie suppression of homeostatic reflexes.

scholarly journals Convergence of Cranial Visceral Afferents within the Solitary Tract Nucleus

2009 ◽  
Vol 29 (41) ◽  
pp. 12886-12895 ◽  
Author(s):  
S. J. McDougall ◽  
J. H. Peters ◽  
M. C. Andresen
Keyword(s):  
Visceral Afferents ◽  
Solitary Tract Nucleus ◽  
Solitary Tract

scholarly journals Comparison of baroreceptive to other afferent synaptic transmission to the medial solitary tract nucleus

2008 ◽  
Vol 295 (5) ◽  
pp. H2032-H2042 ◽  
Author(s):  
Michael C. Andresen ◽  
James H. Peters
Keyword(s):  
Synaptic Transmission ◽  
Brain Stem ◽  
Glutamate Release ◽  
Second Order ◽  
Visceral Afferents ◽  
Solitary Tract Nucleus ◽  
Failure Rates ◽  
Solitary Tract ◽  
Frequency Dependent ◽  
Aortic Depressor Nerve

Cranial nerve visceral afferents enter the brain stem to synapse on neurons within the solitary tract nucleus (NTS). The broad heterogeneity of both visceral afferents and NTS neurons makes understanding afferent synaptic transmission particularly challenging. To study a specific subgroup of second-order neurons in medial NTS, we anterogradely labeled arterial baroreceptor afferents of the aortic depressor nerve (ADN) with lipophilic fluorescent tracer (i.e., ADN+) and measured synaptic responses to solitary tract (ST) activation recorded from dye-identified neurons in medial NTS in horizontal brain stem slices. Every ADN+ NTS neuron received constant-latency ST-evoked excitatory postsynaptic currents (EPSCs) (jitter <192 μs, SD of latency). Stimulus-recruitment profiles showed single thresholds and no suprathreshold recruitment, findings consistent with EPSCs arising from a single, branched afferent axon. Frequency-dependent depression of ADN+ EPSCs averaged ∼70% for five shocks at 50 Hz, but single-shock failure rates did not exceed 4%. Whether adjacent ADN− or those from unlabeled animals, other second-order NTS neurons (jitters <200 μs) had ST transmission properties indistinguishable from ADN+. Capsaicin (CAP; 100 nM) blocked ST transmission in some neurons. CAP-sensitive ST-EPSCs were smaller and failed over five times more frequently than CAP-resistant responses, whether ADN+ or from unlabeled animals. Variance-mean analysis of ST-EPSCs suggested uniformly high probabilities for quantal glutamate release across second-order neurons. While amplitude differences may reflect different numbers of contacts, higher frequency-dependent failure rates in CAP-sensitive ST-EPSCs may arise from subtype-specific differences in afferent axon properties. Thus afferent transmission within medial NTS differed by axon class (e.g., CAP sensitive) but was indistinguishable by source of axon (e.g., baroreceptor vs. nonbaroreceptor).

scholarly journals Visceral Afferents Directly Activate Catecholamine Neurons in the Solitary Tract Nucleus

2007 ◽  
Vol 27 (48) ◽  
pp. 13292-13302 ◽  
Author(s):  
S. M. Appleyard ◽  
D. Marks ◽  
K. Kobayashi ◽  
H. Okano ◽  
M. J. Low ◽  
...  
Keyword(s):  
Visceral Afferents ◽  
Solitary Tract Nucleus ◽  
Solitary Tract ◽  
Catecholamine Neurons

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

scholarly journals Mouse embryonic stem cell-derived cardiomyocytes cease to beat following exposure to monochromatic light: association with increased ROS and loss of calcium transients

2019 ◽  
Vol 317 (4) ◽  
pp. C725-C736
Author(s):  
Gurbind Singh ◽  
Divya Sridharan ◽  
Mahmood Khan ◽  
Polani B. Seshagiri
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Monochromatic Light ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Medical Diagnostics ◽  
Calcium Transients ◽  
Egfp Expression ◽  
Mitochondrial Activity

We earlier established the mouse embryonic stem (ES) cell “GS-2” line expressing enhanced green fluorescent protein (EGFP) and have been routinely using it to understand the molecular regulation of differentiation into cardiomyocytes. During such studies, we made a serendipitous discovery that functional cardiomyocytes derived from ES cells stopped beating when exposed to blue light. We observed a gradual cessation of contractility within a few minutes, regardless of wavelength (nm) ranges tested: blue (~420–495), green (~510–575), and red (~600–700), with green light manifesting the strongest impact. Following shifting of cultures back into the incubator (darkness), cardiac clusters regained beatings within a few hours. The observed light-induced contractility-inhibition effect was intrinsic to cardiomyocytes and not due to interference from other cell types. Also, this was not influenced by any physicochemical parameters or intracellular EGFP expression. Interestingly, the light-induced cardiomyocyte contractility inhibition was accompanied by increased intracellular reactive oxygen species (ROS), which could be abolished in the presence of N-acetylcysteine (ROS quencher). Besides, the increased intracardiomyocyte ROS levels were incidental to the inhibition of calcium transients and suppression of mitochondrial activity, both being essential for sarcomere function. To the best of our knowledge, ours is the first report to demonstrate the monochromatic light-mediated inhibition of contractions of cardiomyocytes with no apparent loss of cell viability and contractility. Our findings have implications in cardiac cell biology context in terms of 1) mechanistic insights into light impact on cardiomyocyte contraction, 2) potential use in laser beam-guided (cardiac) microsurgery, photo-optics-dependent medical diagnostics, 3) transient cessation of hearts during coronary artery bypass grafting, and 4) functional preservation of hearts for transplantation.

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