Indocyanine green (ICG) is an FDA approved dye widely used for fluorescence imaging in research, surgical navigation, and medical diagnostics. However, ICG has a few drawbacks, such as concentration-dependent aggregation and absorbance, nonspecific cellular targeting, and rapid photobleaching. Here, we report a novel DNA-based nanosensor platform that utilizes monomers of ICG and cholesterol. Using DNA origami, we can attach ICG to a DNA structure, maintaining its concentration, preserving its near-infrared (NIR) absorbance, and allowing attachment of targeting moieties. We characterized the nanosensors\' absorbance, stability in blood, and voltage sensing in vitro. This study presents a novel DNA-based ICG nanosensor platform for cellular voltage sensing for future in vivo applications.

The ventral pallidum (VP) is an integral locus in the reward circuitry and a major target of GABAergic innervation of both D1-medium spiny neurons (MSNs) and D2-MSNs from the nucleus accumbens. The VP contains populations of GABAergic [VPGABA, GAD2(+), or VGluT(-)] and glutamatergic [VPGlutamate, GAD2(-), or VGluT(+)] cells that facilitate positive reinforcement and behavioral avoidance, respectively. MSN efferents to the VP exert opponent control over behavioral reinforcement with activation of D1-MSN afferents promoting and D2-MSN afferents inhibiting reward seeking. How this afferent-specific and cell type-specific control of reward seeking is integrated remains largely unknown. In addition to GABA, D1-MSNs corelease substance P to stimulate neurokinin 1 receptors (NK1Rs) and D2-MSNs corelease enkephalin to activate μ-opioid receptors (MORs) and δ-opioid receptors. These neuropeptides act in the VP to alter appetitive behavior and reward seeking. Using a combination of optogenetics and patch-clamp electrophysiology in mice, we found that GAD2(-) cells receive weaker GABA input from D1-MSN, but GAD2(+) cells receive comparable GABAergic input from both afferent types. Pharmacological activation of MORs induced an equally strong presynaptic inhibition of GABA and glutamate transmission on both cell types. Interestingly, MOR activation hyperpolarized VPGABA but not VGluT(+). NK1R activation inhibited glutamatergic transmission only on VGluT(+) cells. Our results indicate that the afferent-specific release of GABA and neuropeptides from D1-MSNs and D2-MSNs can differentially influence VP neuronal subtypes.

OBJECTIVE: To investigate the electrophysiological properties of pyramidal neurons in mouse motor cortex during the early postnatal development.
METHODS: Thirty-six mice were randomly divided into postnatal 1-, 2-, 3-Week and 1-, 2-,3-Month groups (n=6). Membrane properties, action potentials (AP) and spontaneous excitatory postsynaptic currents (sEPSCs) of motor cortex pyramidal neurons were recorded to evaluate the changes in the intrinsic electrophysilogical characteristics by using whole cell patch clamp. Pyramidal neurons and interneurons were distinguished according to the AP firing patterns.
RESULTS: Comparing with interneurons, pyramidal neurons exhibited regular spiking (RS) with smaller frequency. During the period of postnatal 1 Week-3 Months, some of the intrinsic membrane properties of motor cortex pyramidal neurons changed. Compared to the 1-Week mice, the resting membrane potential (RMP) of 2-Week decreased significantly (P＜0.01), and the membrane input resistance (Rin) of 1-Month got a hyperpolarization (P＜0.01), and they showed no significant change in the next period, while the membrane capacitance (Cm) showed no significant changes during the whole postnatal development. The AP dynamic properties changed significantly during this period. Compared to the 1-Week mice, the absolute value of the AP threshold and the AP amplitude of the 3-Week increased significantly (P＜0.01), while the spike half width of the 2-Week decreased substantially (P＜0.05), and they showed no significant change in the next period. The sEPSCs frequency and amplitude of 1- Month increased significantly compared to the 1-Week mice(P＜0.01), while during the period of next 1 Month-3 Months, the amplitude and frequency showed no significant change.
CONCLUSIONS: These results suggest that the motor cortex pyramidal neurons have time-specific eletrophysilogical properties during the postnatal development. The electrophysiological properties can be used as a functional index to detect the degree of neurons maturity, and as a marker to distinguish the pyramidal neurons and interneurons.
目的: 研究小鼠生后发育过程中运动皮层锥体神经元电生理特性的变化。方法: 选取出生后不同发育阶段的小鼠共计36只,随机分为1、2、3周龄组(1-, 2-, 3-Week)、1、2、3月龄组(1-, 2-, 3-Month ) (n=6)。应用全细胞膜片钳及生物胞素细胞内标记技术区分锥体神经元与中间神经元,同时记录各组小鼠脑片运动皮层锥体神经元的被动膜特性、动作电位(AP)及兴奋性突触后电流(sEPSCs)。结果: 与中间神经元相比,小鼠运动皮层锥体神经元的AP放电特征表现为规则放电(RS),放电频率较为缓慢。小鼠运动皮层锥体神经元的被动膜特性在出生后发育期间表现为:与1周龄组小鼠相比,2周龄组的静息膜电位(RMP)表现为显著超极化(P＜0.01),2周后再无明显改变;1月龄组的膜输入阻抗(Rin)呈现显著下降的趋势(P＜0.01),在1月龄后无明显变化;膜电容(Cm)无明显变化。 AP在发育早期的变化表现为:与1周龄组小鼠相比,3周龄组AP阈电位绝对值和幅值显著增加(P＜0.01),2周龄组AP半波宽显著降低(P＜0.05),在此之后无显著变化。 sEPSCs随发育过程的变化表现为:1月龄小鼠sEPSCs的频率和幅值相较于1周龄组均显著增加(P＜0.01),在1月后趋于稳定,无显著差异。结论: 在小鼠出生后发育过程中,运动皮层锥体神经元电生理特性的变化存在时间特异性,电生理特性的成熟程度可以作为检测神经元是否成熟的功能学指标,同时电生理特性可以作为皮质中间神经元与锥体神经元区分的标志。.

T-type Ca channels are strongly expressed and important in the developing heart. In the adult heart, these channels play a significant role in pacemaker tissues, but there is uncertainty about their presence and physiological relevance in the working myocardium. Here, we show that the T-type Ca channel isoforms Cav3.1 and Cav3.2 are expressed at a protein level in ventricular cardiomyocytes from healthy adult C57/BL6 mice. Myocytes isolated from adult wild-type and Cav3.2 KO mice showed considerable whole cell T-type Ca currents under beta-adrenergic stimulation with isoprenaline. We further show that the detectability of basal T-type Ca currents in murine wild-type cardiomyocytes depends on the applied experimental conditions. Together, these findings reveal the presence of functional T-type Ca channels in the membrane of ventricular myocytes. In addition, electrically evoked Ca release from the sarcoplasmic reticulum was significantly impaired in Cav3.2 KO compared to wild-type cardiomyocytes. Our work implies a physiological role of T-type Ca channels in the healthy adult murine ventricular working myocardium.

Carotid body (CB) chemoreceptors are hyperactive in hypertension, and their acute activation produces bronchoconstriction. We show that the respiratory-modulated bronchiolar tone, pulmonary parasympathetic efferent activity, and the firing frequency and synaptic excitation of bronchoconstrictor motoneurones in the nucleus ambiguus were all enhanced in spontaneous hypertensive (SH) rats. In SH rats, CB denervation reduced the respiratory-related parasympathetic-mediated bronchoconstrictor tone to levels seen in normotensive rats. Chemoreflex evoked bronchoconstrictor tone was heightened in SH versus normotensive rats. The intrinsic electrophysiological properties and morphology of bronchoconstrictor motoneurones were similar across rat strains. The heightened respiratory modulation of parasympathetic-mediated bronchoconstrictor tone to the airways in SH rats is caused by afferent drive from the CBs.
Much research has described heightened sympathetic activity in hypertension and diminished parasympathetic tone, especially to the heart. The carotid body (CB) chemoreceptors exhibit hyperreflexia and are hyperactive, providing excitatory drive to sympathetic networks in hypertension. Given that acute CB activation produces reflex evoked bronchoconstriction via activation of parasympathetic vagal efferents, we hypothesised that the parasympathetic bronchoconstrictor activity is enhanced in spontaneously hypertensive (SH) rats and that this is dependent on CB inputs. In situ preparations of Wistar and SH rats were used in which bronchiolar tone, the pulmonary branch of the vagus (pVN) and phrenic nerves were recorded simultaneously; whole cell patch clamp recordings of bronchoconstrictor vagal motoneurones were also made from the nucleus ambiguus. Bronchiolar tone, pVN and bronchoconstrictor motoneurones were respiratory modulated and this modulation was enhanced in SH rats. These differences were all eliminated after CB denervation. Stimulation of the CBs increased the phrenic frequency that caused a summation of the respiratory-related increases in pVN, resulting in the development of bronchoconstrictor tone. This tone was exaggerated in SH rats. The enhanced respiratory-parasympathetic coupling to airways in SH rats was not due to differences in the intrinsic electrophysiological properties of bronchoconstrictor motoneurones but reflected heightened pre-inspiratory- and inspiratory-related synaptic drive. In summary, in SH rats the phasic respiratory modulation of parasympathetic tone to the airways is elevated and the greater development of this bronchoconstrictor tone is caused by the heightened afferent drive originating from the CBs. Thus, targeting the CBs may prove effective for increasing lower airway patency.

Long-term consequences of stroke significantly impair the quality of life in a growing population of stroke survivors. Hippocampal adult neurogenesis has been hypothesized to play a role in the pathophysiology of cognitive and neuropsychiatric long-term sequelae of stroke. Reliable animal models of stroke are paramount to understanding their biomechanisms and to advancing therapeutic strategies. We present a detailed protocol of a transient cerebral ischemia model which does not cause direct ischemic damage in the hippocampus, allowing investigations into the pathophysiology of long-term neurocognitive deficits of stroke. Furthermore, we describe a protocol for obtaining acute hippocampal slices for the purpose of electrophysiological and morphological characterization of adult-borne granule cells. Particularities relating to performing electrophysiological recordings from small cells, such as immature adult-borne granule cells, are also discussed. The present protocol may be complemented by multi-modal investigations (behavioral, morpho-structural, biochemical), to hopefully facilitate research and advances into the long-term sequelae of stroke and the discovery of new therapeutic opportunities.

The carotid body\'s glomus cells are the primary sensors of hypoxia in mammals. Previous studies suggested that the glomus cells\' hypoxia sensitivity is mediated by lactate in mice. This molecule increases the intracellular [Ca2+] and induces exocytosis in glomus cells, activating the carotid sinus nerve (the axons of chemoreceptive petrosal neurons). On the other hand, how lactate affects the activity of carotid body of rats is still unknown. We hypothesized that lactate activates the carotid body of rats. In Wistar rats, we measured the changes in the electrical properties of isolated glomus cells and petrosal chemoreceptive neurons in in situ preparations in response to different concentrations of lactate. Superfusion of both physiological and supraphysiological concentrations of lactate did not affect the membrane conductance and potential of glomus cells. Moreover, lactate injected into the carotid body did not activate the anatomically and physiologically identified chemoreceptive petrosal neurons. We conclude that the carotid body of Wistar rats is not sensitive to lactate.

Neuronal nitric oxide synthase (nNOS) is considered a regulator of Cav1.2 L-type Ca2+ channels and downstream Ca2+ cycling in the heart. The commonest view is that nitric oxide (NO), generated by nNOS activity in cardiomyocytes, reduces the currents through Cav1.2 channels. This gives rise to a diminished Ca2+ release from the sarcoplasmic reticulum, and finally reduced contractility. Here, we report that nNOS inhibitor substances significantly increase intracellular Ca2+ transients in ventricular cardiomyocytes derived from adult mouse and rat hearts. This is consistent with an inhibitory effect of nNOS/NO activity on Ca2+ cycling and contractility. Whole cell currents through L-type Ca2+ channels in rodent myocytes, on the other hand, were not substantially affected by the application of various NOS inhibitors, or application of a NO donor substance. Moreover, the presence of NO donors had no effect on the single-channel open probability of purified human Cav1.2 channel protein reconstituted in artificial liposomes. These results indicate that nNOS/NO activity does not directly modify Cav1.2 channel function. We conclude that-against the currently prevailing view-basal Cav1.2 channel activity in ventricular cardiomyocytes is not substantially regulated by nNOS activity and NO. Hence, nNOS/NO inhibition of Ca2+ cycling and contractility occurs independently of direct regulation of Cav1.2 channels by NO.

Parkinson\'s disease (PD) is predominantly idiopathic in origin, and a large body of evidence indicates that gastrointestinal (GI) dysfunctions are a significant comorbid clinical feature; these dysfunctions include dysphagia, nausea, delayed gastric emptying, and severe constipation, all of which occur commonly before the onset of the well-known motor symptoms of PD. Based on a distinct distribution pattern of Lewy bodies (LB) in the enteric nervous system (ENS) and in the preganglionic neurons of the dorsal motor nucleus of the vagus (DMV), and together with the early onset of GI symptoms, it was suggested that idiopathic PD begins in the ENS and spreads to the central nervous system (CNS), reaching the DMV and the substantia nigra pars compacta (SNpc). These two areas are connected by a recently discovered monosynaptic nigro-vagal pathway, which is dysfunctional in rodent models of PD. An alternative hypothesis downplays the role of LB transport through the vagus nerve and proposes that PD pathology is governed by regional or cell-restricted factors as the leading cause of nigral neuronal degeneration. The purpose of this brief review is to summarize the neuronal electrophysiological findings in the SNpc and DMV in PD.

Nifedipine and isradipine are prominent examples of calcium channel blockers with a 1,4-dihydropyridine (DHP) scaffold. Although successfully used in clinics since decades for the treatment of hypertension, the binding mechanism to their target, the L-type voltage-gated calcium channel Cav1.2, is still incompletely understood. Recently, novel DHP derivatives with a condensed ring system have been discovered that show distinct selectivity profiles to different calcium channel subtypes. This property renders this DHP class as a promising tool to achieve selectivity towards distinct calcium channel subtypes. In this study, we identified a common binding mode for prominent DHPs nifedipine and isradipine using docking and pharmacophore analysis that is also able to explain the structure-activity relationship of a small subseries of DHP derivatives with a condensed ring system. These findings were used to guide the synthesis of twenty-two novel DHPs. An extensive characterization using 1H NMR, 13C NMR, mass spectra and elemental analysis was followed by whole cell patch clamp assays for analyzing activity at Cav1.2 and Cav3.2. Two compounds were identified with significant activity against Cav1.2. Additionally, we identified four compounds active against Cav3.2 of which three were selective over Cav1.2. Novel binding modes were analyzed using docking and pharmacophore analysis as well as molecular dynamics simulations.