Frequency Of Spontaneous Currents Research Articles

Changes in PVN Neurons after Low‐Frequency Acute Optogenetic Stimulation

The paraventricular nucleus of the hypothalamus (PVN) is an important autonomic control center. It receives afferent inputs from many brain regions, including the median preoptic nucleus (MnPO). The connection between the MnPO and the PVN is particularly important in generating chronic intermittent hypoxia phenotypes such as increased sympathetic activity leading to the development of hypertension. PVN‐projecting MnPO neurons discharge frequency is <5Hz. To gain more insight into the acute change in PVN neuron properties after low‐frequency stimulation, whole‐cell patch‐clamp recordings were performed on PVN neurons. Adult 250‐350g male rats were injected bilaterally in the PVN with 100 nL retrogradely transported adeno‐associated virus encoding Channelrhodopsin (AAVrg‐CaMKIIa‐hChR2(H134R)‐mCherry) or with AAVrg‐CAG‐tdTomato as control. Three weeks after the injections, the rats were anesthetized with isoflurane (2‐3%) and sacrificed to prepare horizontal brain slices containing the PVN and MnPO. Postsynaptic currents were recorded from PVN neurons in whole‐cell voltage clamp configuration (Vhold=‐60 mV) under constant bath perfusion of normal aCSF (2‐3 ml/min). Axon terminals in the PVN were stimulated with 5Hz LED‐generated blue light (470nM) pulses of 50‐ms duration, 5 seconds off/on for a total of 1 min, delivered using optical fiber directed to the MnPO. Photo‐evoked currents were measured during 5 min before and after the photo‐stimulation train. Amplitude and frequency of spontaneous currents were compared before and after stimulation as well. At the end of each recording, cells were characterized as type I (magnocellular) or type II (parvocellular) PVN neurons based on the presence of transient outward rectification. Data were analyzed offline using Easy Electrophysiology v2.3.3 software. For both cell types, 5 Hz optogenetic stimulation caused a significant reduction in the optogenetic‐evoked current amplitude during the first 15 minutes after stimulation (before stimulation 94.88±2.85 %, n=9 cells vs. 15 minutes after stimulation 67.52±6.15 %, n=9. P=0.0027) before returning to baseline values. Post‐stimulation spontaneous postsynaptic current amplitude and frequency decreased in type II PVN neurons from retrograde ChR2 animals (pre‐stimulation amplitude 100±0.62 % vs. post‐stimulation amplitude 86.50±0.54 %, P=0.0371; pre‐stimulation frequency 3.82±1.7 Hz, post‐stimulation frequency 2.396±1.2 Hz, P= 0.0385) but not in type I PVN neurons from retrograde ChR2 animals or cells from control animals. Kinetic profiles of spontaneous current in type I and II PVN neurons did not change after 5 Hz optogenetic stimulation. The results suggest that low‐frequency stimulation discriminately alters spontaneous presynaptic neurotransmitter release and possibly postsynaptic responsiveness in PVN neurons. Additional experiments will be needed to specifically stimulate PVN afferents from the MnPO.

LRRK2 modulates neuronal vesicles cycle through protein interactions

Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson’s disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity and a C-terminal WD40 domain devoted to protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. We have investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Interestingly, a sequence variant (G2385R) within the WD40 domain has been implicated as a risk factor in PD, but its physiological and pathological function has not been systematically addressed yet. We analyzed molecular features of the WD40 domain and we addressed the functional implication of the G2385R variant. Our results suggest that LRRK2 WD40 domain serves as a hub for protein interactions setting LRRK2 as part of a protein network involved in synaptic vesicle trafficking. Furthermore we showed that the G2385R mutation influences WD40 domain features in terms of domain folding and binding properties and has an impact on synaptic vesicle dynamic. Our results suggest that different PD mutation might influence synaptic vesicle release via modulation of LRRK2 macro-molecular complex.

Short‐term sustained hypoxia affects synaptic transmission in the nucleus tractus solitarius neurons of juvenile rats

In the present study we evaluated the effects of a short‐term sustained hypoxia [(SH) 24 hours ‐ 10% O2] on intrinsic properties and synaptic transmission of nucleus tractus solitary (NTS) neurons of juvenile Wistar rats (P21). After 24 hours inside hypoxic or normoxic chambers rats were anesthetized, the brains removed and brainstems sliced. Intrinsic properties and excitatory currents of NTS neurons were recorded using whole‐cell patch clamp. SH did not change the resting membrane potential (control: −78.7 ± 2.4 mV n=15; SH: −72.3 ± 3.7 mV n=15), input resistance (control: 1.4 ± 0.4 G Ohm n=8; SH: 1.4 ± 0.4 G Ohm n=9) or capacitance of neurons (control: 14.2 ± 0.9 pF n=17; SH: 12.4 ± 0.8 pF n=15), indicating that SH does not change the intrinsic properties of NTS neurons. However, it was observed an increase in frequency of spontaneous currents (control: 3.6 ± 0.4 Hz n=18; SH: 7.1 ± 1.5 Hz n=15) and in peak amplitude of evoked currents (control: −289 ± 51 pA n=15; SH: −472 ± 64 pA n=15). Moreover, after a train of 5 stimuli, NTS neurons from SH group presented higher depression in glutamatergic events (relative amplitude of evoked currents; control: 100, 44, 34, 32 and 28%; SH: 100, 40, 28, 22 and 17%, *p<0.001, n=15), suggesting that SH affects the vesicular release probability. The data show that short‐term SH increases the spontaneous and evoked neurotransmitter release in the NTS neurons, which may explain, at least in part, the autonomic and respiratory changes observed in this experimental model.Financial support: FAPESP and CNPq.

Nickel modulates the electrical activity of cultured cortical neurons through a specific effect on N-methyl-d-aspartate receptor channels

Nickel (Ni2+) is a toxic metal that affects the function of several ionic channels. In the N-methyl-d-aspartate (NMDA) subtype of glutamate receptor (NR), it causes activity enhancement of the channels containing the NR2B subunit and voltage-independent inhibition of those containing NR2A. Thus, it may represent a functional marker for the identification of NR native channel subunits. We investigated the effect of Ni2+ on spontaneous NR currents in cortical neurons, dissociated from 18-day rat embryos and maintained in culture for up to ∼40 days. In whole-cell voltage-clamp at −60 mV, in a Mg2+-free bath containing the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) (10 μM), spontaneous currents were blocked by 10 μM D(–)-2-Amino-5-phosphonopentanoic acid (APV) (10 μM), and by NR2B antagonists, ifenprodil (10 μM) or Ro25-6981 (Ro25, 1 μM), indicating that they are due to NRs containing predominantly the NR2B subunit. In the presence of Ni2+ (30 μM) the amplitude and the frequency of spontaneous currents were increased and the decay time decreased. A higher dose (300 μM) blocked all electrical activity. In current-clamp, Ni2+ (30 μM) caused a ∼5 mV reversible depolarization. The effect of Ni2+, as well as that of NR2B antagonists, was almost independent of days in vitro (DIV) in the range from 18 to 33 DIV. The electrical activity of the neuronal networks measured by microelectrode arrays (MEAs) was also affected by Ni2+, which caused a decrease in firing rate, but an increase in burst duration, while Ro25 (1–10 μM) caused a decrease in both firing rate and burst duration. Finally, reverse transcription polymerase chain reaction (RT-PCR) revealed a predominant expression of NR2B, with no modification during DIV. These results demonstrate that, in these cultured cells, the NR spontaneous current is almost entirely due by NR2B-containing receptors and that Ni2+ affects the electrical activity through a specific effect on NR channels.

Spontaneous autocrine release of protons activates ASIC‐mediated currents in HEK293 cells

When examining HEK293 cells by whole-cell patch-clamp electrophysiology we found spontaneous currents, present in almost all cells. These currents were carried by Na(+) ions, were inhibited by amiloride and by cells exposure to acidic (pH 6.3) extracellular solutions. These properties (ion carrier, amiloride-sensitivity, and inactivation by constant lowering of extracellular pH) were similar to the properties of proton-activated currents measured from the same cells. Spontaneous currents required intracellular ATP, were completely inhibited by intracellular Ca(2+) buffering with BAPTA and were suppressed by intracellular administration of vesicular H(+)ATPase inhibitor bafilomycin. ATP-induced Ca(2+) influx through P2X receptors in HEK293 cells stably transfected with P2X(2), P2X(2/3) or P2X(4) purinoreceptor subunits transiently potentiated amplitude and frequency of spontaneous currents; this effect was antagonized by bafilomycin. We concluded that spontaneous currents represent activation of acid-sensitive ion channels (ASICs) by autocrine vesicular release of protons from HEK cells.

Modulatory role of adenosine receptors in insect motor nerve terminals.

The effects of adenosine and ATP were studied on blowfly larvae Calliphora vicina neuromuscular preparation. Adenosine diminished (IC50 = 40 +/- 3 microM) the amplitude of nerve-evoked postsynaptic currents (EPSCs) and slightly decreased the frequency of spontaneous currents without affecting their amplitude. EPSCs were slightly reduced by ATP, and this effect was prevented by concanavalin A. Presynaptic inhibition by adenosine was temperature-dependent and insensitive to pertussis toxin. A1 agonists of vertebrate adenosine receptor CPA and NECA failed to reproduce the effect of adenosine, and 2-CADO enhanced the EPSCs. A1 antagonist DPCPX competitively inhibited adenosine action. A2 agonist DPMA potentiated EPSCs, and its effect was abolished by A2 antagonist DMPX. Adenosine and ATP failed to affect the nonquantal release of glutamate. The results show for the first time the presence of presynaptic adenosine receptors regulating transmitter release at insect motor nerve terminals and point to differences in pharmacological properties of adenosine receptor subtypes in insects and vertebrates.