RESUMO
Dorsal root ganglia (DRG) neurons transduce and convey somatosensory information from the periphery to the central nervous system. Adrenergic mediators are known to modulate nociceptive inputs in DRG neurons, acting as up- or down-regulators of neuronal excitability. They are also important in the development of sympathetic neuropathy. ATP-activated P2X channels and capsaicin-activated TRPV1 channels are directly involved in the transduction of nociceptive stimuli. In this work, we show that long-term (up to 3 days) in vitro stimulation of DRG neurons with selective α1-adrenergic agonist increased slow but not fast ATP-activated currents, with no effect on capsaicin currents. Selective agonists for α2, ß1 and ß3-adrenergic receptors decreased capsaicin activated currents and had no effect on ATP currents. Capsaicin currents were associated with increased neuronal excitability, while none of the adrenergic modulators produced change in rheobase. These results demonstrate that chronic adrenergic activation modulates two nociceptive transducer molecules, increasing or decreasing channel current depending on the adrenergic receptor subtype. These observations aid our understanding of nociceptive or antinociceptive effects of adrenergic agonists.
Assuntos
Agonistas Adrenérgicos , Capsaicina , Capsaicina/farmacologia , Agonistas Adrenérgicos/farmacologia , Nociceptividade , Canais Iônicos/farmacologia , Trifosfato de Adenosina/farmacologia , Gânglios Espinais , Canais de Cátion TRPVRESUMO
The dorsal cochlear nucleus (DCN) in the auditory brainstem integrates auditory and somatosensory information. Mature DCN fusiform neurons fall into two qualitatively distinct types: quiet, with no spontaneous regular action potential firing, or active, with regular spontaneous action potential firing. However, how these firing states and other electrophysiological properties of fusiform neurons develop during early postnatal days to adulthood is not known. Thus, we recorded fusiform neurons from mice from P4 to P21 and analyzed their electrophysiological properties. In the prehearing phase (P4-P13), we found that most fusiform neurons are quiet, with active neurons emerging after hearing onset at P14. Subthreshold properties underwent significant changes before hearing onset, whereas changes to the action potential waveform occurred mainly after P14, with the depolarization and repolarization phases becoming markedly faster and half-width significantly decreased. The activity threshold in posthearing neurons was more negative than in prehearing cells. Persistent sodium current (INaP) was increased after P14, coinciding with the emergence of spontaneous firing. Thus, we suggest that posthearing expression of INaP leads to hyperpolarization of the activity threshold and the active state of the fusiform neuron. At the same time, other changes refine the passive membrane properties and increase the speed of action potential firing of fusiform neurons.NEW & NOTEWORTHY Auditory brainstem neurons express unique electrophysiological properties adapted for their complex physiological functions that develop before hearing onset. Fusiform neurons of the DCN present two firing states, quiet and active, but the origin of these states is not known. Here, we showed that the quiet and active states develop after hearing onset at P14, along with changes in action potentials, suggesting an influence of auditory input on the refining of fusiform neuron's excitability.
Assuntos
Núcleo Coclear , Animais , Camundongos , Audição , Neurônios , Potenciais de Ação , Tronco EncefálicoRESUMO
This protocol provides instructions to acquire high-quality cellular contractility data from adult, neonatal, and human induced pluripotent stem cell-derived cardiomyocytes. Contractility parameters are key to unravel mechanisms underlying cardiac pathologies, yet difficulties in acquiring data can compromise measurement accuracy and reproducibility. We provide optimized steps for microscope and camera setup, as well as cellular selection criteria for different cardiomyocyte cell types, aiming to obtain robust and reliable data. Moreover, we use CONTRACTIONWAVE software to analyze and show the optimized results. For complete details on the use and execution of this profile, please refer to Scalzo et al. (2021).
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Recém-Nascido , Microscopia , Miócitos Cardíacos/metabolismo , Reprodutibilidade dos TestesRESUMO
Cell membrane deformation is an important feature that occurs during many physiological processes, and its study has been put to good use to investigate cardiomyocyte function. Several methods have been developed to extract information on cardiomyocyte contractility. However, no existing computational framework has provided, in a single platform, a straightforward approach to acquire, process, and quantify this type of cellular dynamics. For this reason, we develop CONTRACTIONWAVE, high-performance software written in Python programming language that allows the user to process large data image files and obtain contractility parameters by analyzing optical flow from images obtained with videomicroscopy. The software was validated by using neonatal, adult-, and human-induced pluripotent stem-cell-derived cardiomyocytes, treated or not with drugs known to affect contractility. Results presented indicate that CONTRACTIONWAVE is an excellent tool for examining changes to cardiac cellular contractility in animal models of disease and for pharmacological and toxicology screening during drug discovery.
Assuntos
Células-Tronco Pluripotentes Induzidas , Fluxo Óptico , Animais , Recém-Nascido , Humanos , Software , Miócitos Cardíacos , Células CultivadasRESUMO
Thiamine deficiency is associated with cerebellar dysfunction; however, the consequences of thiamine deficiency on the electrophysiological properties of cerebellar Purkinje cells are poorly understood. Here, we evaluated these parameters in brain slices containing cerebellar vermis. Adult mice were maintained for 12-13 days on a thiamine-free diet coupled with daily injections of pyrithiamine, an inhibitor of thiamine phosphorylation. Morphological analysis revealed a 20% reduction in Purkinje cell and nuclear volume in thiamine-deficient animals compared to feeding-matched controls, with no reduction in cell count. Under whole-cell current clamp, thiamine-deficient Purkinje cells required significantly less current injection to fire an action potential. This reduction in rheobase was not due to a change in voltage threshold. Rather, thiamine-deficient neurons presented significantly higher input resistance specifically in the voltage range just below threshold, which increases their sensitivity to current at these critical membrane potentials. In addition, thiamine deficiency caused a significant decrease in the amplitude of the action potential afterhyperpolarization, broadened the action potential, and decreased the current threshold for depolarization block. When thiamine-deficient animals were allowed to recover for 1 week on a normal diet, rheobase, threshold, action potential half-width, and depolarization block threshold were no longer different from controls. We conclude that thiamine deficiency causes significant but reversible changes to the electrophysiology properties of Purkinje cells prior to pathological morphological alterations or cell loss. Thus, the data obtained in the present study indicate that increased excitability of Purkinje cells may represent a leading indicator of cerebellar dysfunction caused by lack of thiamine.
Assuntos
Células de Purkinje/patologia , Deficiência de Tiamina/patologia , Deficiência de Tiamina/fisiopatologia , Potenciais de Ação/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Técnicas de Patch-ClampRESUMO
The basolateral amygdala (BLA) is critical in the control of the sympathetic output during stress. Studies demonstrated the involvement of the renin-angiotensin system components in the BLA. Angiotensin-(1-7) [Ang-(1-7)], acting through Mas receptors, reduces stress effects. Considering that angiotensin-converting enzyme 2 (ACE2) is the principal enzyme for the production of Ang-(1-7), here we evaluate the cardiovascular reactivity to acute stress after administration of the ACE2 activator, diminazene aceturate (DIZE) into the BLA. We also tested whether systemic treatment with DIZE could modify synaptic activity in the BLA and its effect directly on the expression of the N-methyl-d-aspartate receptors (NMDARs) in NG108 neurons in-vitro. Administration of DIZE into the BLA (200 pmol/100 nL) attenuated the tachycardia to stress (ΔHR, bpm: vehicle = 103 ± 17 vs DIZE = 49 ± 7 p = 0.018); this effect was inhibited by Ang-(1-7) antagonist, A-779 (ΔHR, bpm: DIZE = 49 ± 7 vs A-779 + DIZE = 100 ± 15 p = 0.04). Systemic treatment with DIZE attenuated the excitatory synaptic activity in the BLA (Frequency (Hz): vehicle = 2.9 ± 0.4 vs. DIZE =1.8 ± 0.3 p < 0.04). NG108 cells treated with DIZE demonstrated decreased expression of l subunit NMDAR-NR1 (NR1 expression (a.u): control = 0.534 ± 0.0593 vs. DIZE = 0.254 ± 0.0260) of NMDAR and increases of Mas receptors expression. These data demonstrate that DIZE attenuates the tachycardia evoked by acute stress. This effect results from a central action in the BLA involving activation of Mas receptors. The ACE2 activation via DIZE treatment attenuated the frequency of excitatory synaptic activity in the basolateral amygdala and this effect can be related with the decreases of the NMDAR-NR1 receptor expression.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Diminazena/análogos & derivados , Ácido Glutâmico/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Taquicardia/metabolismo , Angiotensina I/antagonistas & inibidores , Angiotensina II/análogos & derivados , Angiotensina II/farmacologia , Animais , Complexo Nuclear Basolateral da Amígdala/metabolismo , Diminazena/farmacologia , Neurônios/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Spider venoms, despite their toxicity, represent rich sources of pharmacologically active compounds with biotechnological potential. However, in view of the large diversity of the spider species, the full potential of their venom molecules is still far from being known. In this work, we report the purification and structural and functional characterization of GiTx1 (ß/κ-TRTX-Gi1a), the first toxin purified from the venom of the Brazilian tarantula spider Grammostola iheringi. GiTx1 was purified by chromatography, completely sequenced through automated Edman degradation and tandem mass spectrometry and its structure was predicted by molecular modeling. GiTx1 has a MW of 3.585 Da, with the following amino acid sequence: SCQKWMWTCDQKRPCCEDMVCKLWCKIIK. Pharmacological activity of GiTx1 was characterized by electrophysiology using whole-cell patch clamp on dorsal root ganglia neurons (DRG) and two-electrode voltage-clamp on voltage-gated sodium and potassium channels subtypes expressed in Xenopus laevis oocytes. GiTx1, at 2 µM, caused a partial block of inward (â¼40%) and outward (â¼20%) currents in DRG cells, blocked rNav1.2, rNav1.4 and mNav1.6 and had a significant effect on VdNav, an arachnid sodium channel isoform. IC50 values of 156.39 ± 14.90 nM for Nav1.6 and 124.05 ± 12.99 nM for VdNav, were obtained. In addition, this toxin was active on rKv4.3 and hERG potassium channels, but not Shaker IR or rKv2.1 potassium channels. In summary, GiTx1 is a promiscuous toxin with multiple effects on different types of ion channels.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana , Venenos de Aranha , Aranhas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Moscas Domésticas , Humanos , Camundongos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Domínios Proteicos , Ratos , Ratos Wistar , Venenos de Aranha/química , Venenos de Aranha/isolamento & purificação , Venenos de Aranha/toxicidade , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/isolamento & purificação , Bloqueadores do Canal de Sódio Disparado por Voltagem/toxicidade , Canais de Sódio Disparados por Voltagem/químicaRESUMO
Capsaicin, an agonist of TRPV1, evokes intracellular [Ca2+] transients and glutamate release from perfused trigeminal ganglion. The spider toxin PnTx3-5, native or recombinant is more potent than the selective TRPV1 blocker SB-366791 with IC50 of 47⯱â¯0.18â¯nM, 45⯱â¯1.18â¯nM and 390⯱â¯5.1â¯nM in the same experimental conditions. PnTx3-5 is thus more potent than the selective TRPV1 blocker SB-366791. PnTx3-5 (40â¯nM) and SB-366791 (3⯵M) also inhibited the capsaicin-induced increase in intracellular Ca2+ in HEK293â¯cells transfected with TRPV1 by 75⯱â¯16% and 84⯱â¯3.2%, respectively. In HEK293â¯cells transfected with TRPA1, cinnamaldehyde (30⯵M) generated an increase in intracellular Ca2+ that was blocked by the TRPA1 antagonist HC-030031 (10⯵M, 89% inhibition), but not by PnTx3-5 (40â¯nM), indicating selectivity of the toxin for TRPV1. In whole-cell patch-clamp experiments on HEK293â¯cells transfected with TRPV1, capsaicin (10⯵M) generated inward currents that were blocked by SB-366791 and by both native and recombinant PnTx3-5 by 47⯱â¯1.4%; 54⯱â¯7.8% and 56⯱â¯9.0%, respectively. Intradermal injection of capsaicin into the rat left vibrissa induced nociceptive behavior that was blocked by pre-injection with either SB-366791 (3 nmol/site i.d., 83.3⯱â¯7.2% inhibition) or PnTx3-5 (100 fmol/site, 89⯱â¯8.4% inhibition). We conclude that both native and recombinant PnTx3-5 are potent TRPV1 receptor antagonists with antinociceptive action on pain behavior evoked by capsaicin.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Capsaicina/farmacologia , Dor Facial/metabolismo , Neuropeptídeos/farmacologia , Nociceptividade/efeitos dos fármacos , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Gânglio Trigeminal/efeitos dos fármacos , Acroleína/análogos & derivados , Acroleína/farmacologia , Anilidas/farmacologia , Animais , Cálcio/metabolismo , Cinamatos/farmacologia , Modelos Animais de Doenças , Ácido Glutâmico/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Concentração Inibidora 50 , Masculino , Técnicas de Patch-Clamp , Ratos , Canal de Cátion TRPA1/efeitos dos fármacos , Canal de Cátion TRPA1/genética , Canais de Cátion TRPV/genética , Transfecção , Gânglio Trigeminal/metabolismoRESUMO
We examined effects of Group I metabotropic glutamate receptors on the excitability of mouse medial nucleus of the trapezoid body (MNTB) neurons. The selective agonist, S-3,5-dihydroxyphenylglycine (DHPG), evoked a dose-dependent depolarization of the resting potential, increased membrane resistance, increased sag depolarization, and promoted rebound action potential firing. Under voltage-clamp, DHPG evoked an inward current, referred to as IDHPG , which was developmentally stable through postnatal day P56. IDHPG had low temperature dependence in the range 25-34°C, consistent with a channel mechanism. However, the I-V relationship took the form of an inverted U that did not reverse at the calculated Nernst potential for K+ or Cl- . Thus, it is likely that more than one ion type contributes to IDHPG and the mix may be voltage dependent. IDHPG was resistant to the Na+ channel blockers tetrodotoxin and amiloride, and to inhibitors of iGluR (CNQX and MK801). IDHPG was inhibited 21% by Ba2+ (500 µM), 60% by ZD7288 (100 µM) and 73% when the two antagonists were applied together, suggesting that KIR channels and HCN channels contribute to the current. Voltage clamp measurements of IH indicated a small (6%) increase in Gmax by DHPG with no change in the voltage dependence. DHPG reduced action potential rheobase and reduced the number of post-synaptic AP failures during high frequency stimulation of the calyx of Held. Thus, activation of post-synaptic Group I mGlu receptors modifies the excitability of MNTB neurons and contributes to the reliability of high frequency firing in this auditory relay nucleus.