Frequency Of Spontaneous Action Potentials Research Articles

Differential effects of osmotic stress on synaptic vs. paracrine signaling mechanisms in oxytocinergic magnocellular neurons.

Acute hypernatremia promotes natriuresis in part by increasing activity of oxytocinergic magnocellular neurons in the hypothalamus that promote synaptic release of oxytocin (OT) from axon terminals in the neurohypophysis. Acute hypernatremia also blunts stress‐induced activation of the hypothalamic‐pituitary‐adrenal axis via a mechanism that we believe is likely to depend on central paracrine (rather than synaptic) release of OT from magnocellular dendrites. Interestingly, prior work from other groups indicates that acute hypernatremia produces an increase in the concentration of circulating OT significantly more rapidly than it creates an increase in the concentration of central OT. The current project was designed to develop a better understanding of the underlying cellular mechanisms through which osmotic stressors may plausibly promote both synaptic and paracrine release of OT from the same population of osmosensitive hypothalamic neurons, but with distinctly different temporal dynamics. Towards that end, we used the Cre‐LoxP system to generate OT‐reporter mice that express tdTomato in neurons that synthesize OT. Initial studies indicated a strong colocalization of tdTomato with OT/neurophysin1 throughout the hypothalamus. Whole‐cell patch clamp recordings from tdTomato positive magnocellular neurons in the paraventricular nucleus of the hypothalamus revealed expected intrinsic properties, including transient outward rectification produced by a prominent A‐type K+ current (IA). Initial imaging studies revealed that the dendrites of OT positive magnocellular neurons are passive conductors, with robust activity dependent dendritic calcium influx that diminishes substantially with increasing distance from the soma. Bath application of an external osmolyte (15 mM mannitol) produced a clear increase in the frequency of spontaneous action potentials observed in OT positive magnocellular neurons, and yet intriguingly, also produced a clear decrease in activity dependent dendritic calcium influx. This effect was also much more prominent in distal dendrites than in proximal dendrites, consistent with a mechanism that depends on decreased dendritic input resistance. Also consistent with that interpretation, we found that acute application of 15 mM mannitol significantly reduces the amplitude of evoked glutamatergic EPSCs recorded at the soma, as produced by a small theta glass stimulator placed near the distal dendrites. Interestingly, preliminary data suggest that parvocellular OT neurons lack similar osmosensitivty. Collectively, these results provide a partial mechanistic explanation for why acute osmotic stress is likely to initially favor synaptic over paracrine release of OT from osmosensitive hypothalamic magnocellular neurons.Support or Funding InformationSupported by NIMH R0104641This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

AT1aR Dependent GABAa Inhibition in the MnPO Following Chronic Intermittent Hypoxia

Chronic intermittent hypoxia (CIH) is an animal model that simulates the hypoxemia seen in obstructive sleep apnea (OSA). Rats exposed to CIH exhibit an increase in sympathetic tone and reactivity and a persistent elevation in blood pressure during periods of normoxia, similar to that observed in patients with OSA. The median preoptic nucleus (MnPO) exhibits increases in Angiotensin type 1a receptor (AT1aR) mRNA following CIH and blocking this increase in AT1aR mRNA prevents the sustained increase in blood pressure. Our lab has previously reported that AT1aR activation influences GABAa mediated inhibition within the MnPO. Here we investigate the role of Angiotensin II (Ang II) signaling via the AT1a receptor in the MnPO and its influence on excitatory/inhibitory balance in rats subjected to CIH. Under isoflurane (2–3%) anesthesia, male Sprague‐Dawley rats (250–350 g) received microinfusions (0.4 μL) of recombinant AAV construct containing GFP reporter and shRNA against AT1aR (AT1a KD) or an AAV construct containing the GFP reporter and a shRNA scramble (Scr) targeted to the MnPO. After recovery, rats were subjected to 7 days of CIH (0800–1600 hrs). The CIH protocol consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10×/hr for 8 consecutive hrs (during the normal inactive/sleep phase) on 7 consecutive days. After 7 days CIH, the rats were anesthetized with isoflurane (2–3%) and coronal slices (300 μm) containing the MnPO were cut using standard in vitro slice procedures. Loose patch recordings were obtained from GFP labeled neurons using borosilicate glass micropipettes containing aCSF as the internal solution (1–3 MΩ). Spontaneous action potential firing was recorded in response to focal application of Ang II (100 nM, 30s) or muscimol (100 uM, 30s). Brief focal application of Ang II produced a transient increase in spontaneous action potential frequency of MnPO neurons from both CIH/Scr (n = 11) and normoxic/Scr (n = 9) rats. The Ang II dependent increase in activity was blocked in neurons from AT1a KD rats from both CIH (n = 12) and normoxic (n = 10) groups. The GABAa agonist muscimol decreased action potential activity (n = 10) of neurons from normoxic/Scr rats. This GABAa mediated inhibition was blunted in neurons from CIH/Scr (n = 13) and normoxic/AT1a KD (n = 10) rats. However, GABAa activation from neurons in the CIH/AT1a KD (n = 15) group produced an increase in spontaneous activity. The current study shows AT1aR KD mediated reduction in GABAa inhibition is exacerbated to the point where GABAa activation is excitatory following CIH. Future studies will address the influence of reduced AT1a signaling combined with reduced GABAa mediated inhibition on downstream targets of the MnPO in AT1aKD from both normoxic and CIH rats.Support or Funding InformationSupported by P01 HL088052This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Trafficking patterns of NMDA and GABAA receptors in a Mg2+-free cultured hippocampal neuron model of status epilepticus

An altered pattern of receptor trafficking is one of the pathophysiologic mechanisms of status epilepticus (SE). The gradual internalization of GABAA receptors (GABARs) occurs in both in vitro and in vivo models of SE and is thought to be a cause of decreased GABAergic inhibition. Unlike GABARs, little is known about alterations in NMDA receptor (NMDAR) trafficking during SE, even though increased activity of NMDARs is indispensable for the induction and maintenance of SE. Therefore, we aimed to simultaneously investigate the changes in the trafficking patterns of GABARs and NMDARs in an in vitro cultured hippocampal neuron model of SE. For induction of epileptiform discharges, hippocampal neurons were exposed to external medium without Mg2+. Biotinylation assay and immunofluorescence staining for GABAR β2,3 and NMDAR NR1 subunits were performed to quantify and visualize surface GABARs and NMDARs, respectively. The frequency of spontaneous action potentials increased more than 4-fold after Mg2+-free induction. The level of surface GABARs decreased over time after Mg2+-free induction, dropping to approximately 50% of control levels an hour after Mg2+-free induction. By contrast, the trafficking of NMDARs to the surface was enhanced after a slight time lag, increasing by 30% of control levels an hour after Mg2+-free induction. Our data showed the changes of both NMDAR and GABAR trafficking during prolonged SE induced by a Mg2+-free extracellular environment and confirmed that this in vitro SE model is suitable for examining alterations in the receptor trafficking pattern by prolonged seizure activity. These results suggest that targeting of surface NMDAR could be a promising method in controlling benzodiazepine-resistant SE.

Electrophysiological properties and augmented catecholamine release from chromaffin cells of WKY and SHR rats contributing to the hypertension development elicited by chronic EtOH consumption

It is known that chronic ethanol (EtOH) consumption leads to hypertension development and has been associated with deleterious effects on the cardiovascular system. Whether this condition alters calcium (Ca2+) signaling and exocytosis in adrenal chromaffin cells (CCs) as the case is for genetic hypertension, is unknown. We explored this question in four randomized experimental groups, male Wistar Kyoto (WKY/EtOH) and Spontaneously Hypertensive (SHR/EtOH) rats were subjected to the intake of increasing EtOH concentrations (5–20%, for 30 days) and their respective controls (WKY/Control and SHR/Control) received water. WKY/EtOH developed hypertension and cardiac hypertrophy; blood aldehyde dehydrogenase (ALDH) and H2O2 were also augmented. In comparison with WKY/Control, CCs from WKY/EtOH had the following features: (i) depolarization and higher frequency of spontaneous action potentials; (ii) decreased Ca2+ currents with slower inactivation; (iii) decreased K+ currents; (iv) augmented K+-elicited cytosolic Ca2+ transients ([Ca2+]c); (v) enhanced K+-elicited catecholamine release. These cardiovascular, blood and CCs changes were qualitatively similar to those undergone by SHR/Control and SHR/EtOH. The results suggest that the hypertension elicited by chronic EtOH has pathogenic features common to genetic hypertension namely, augmented [Ca2+]c transients and catecholamine release from their CCs.

Atrial Spceific Pitx2 Insufficiencyincreases the Frequency of Calcium Sparks, Waves, and After-Depolarizations in Mouse Atrial Myocytes

The transcription factor Pitx2 has been proposed as a molecular link between single nucleotide polymorphisms on chromosome 4q25 and increased risk of atrial fibrillation in carriers of the risk variant. Since atrial fibrillation has been associated with calcium handling disturbances in isolated atrial myocytes, we here tested the hypothesis that Pitx2 insufficiency alters the calcium homeostasis in atrial myocytes.To test this hypothesis, we used right atrial myocytes from a transgenic mouse model with inducible atrial specific Pitx2 deletion. Spontaneous calcium release was detected with confocal calcium imaging and resulting ion currents or membrane depolarizations were measured with patch-clamp technique in myocytes from wild-type (Pitx2+/+) and heterozygous Pitx2+/- mice.Calcium imaging revealed that the frequency of calcium sparks (2.1±0.7 vs 0.2±0.1 events/cell/s, p<0.05) and waves (3.2±1.2 vs. 0 events/min, p<0.05) were significantly higher in Pitx2+/- mice. This was also true for the frequency of transient inward currants activated by calcium waves (2.8±0.5 vs 1.2±0.5 events/min, p<0.05). The higher frequency was of spontaneous calcium release was likely due to a higher caffeine releasable sarcoplasmic reticulum calcium load in the Pitx2+/- mice (23.8±5.8 vs 14.4±1.9 amol/pF, p<0.05). Moreover, only myocytes from Pitx2+/- had spontaneous action potentials at a resting potential of −80 mV (0.5±0.4/min). At −60 mV the frequency of spontaneous action potentials was 5.3±1.8/min for Pitx2+/- and 0.4±1.2/min for Pitx2+/+ mice. Importantly, spontaneous after-potentials were also recorded in myocytes subjected to field stimulation, but only in myocytes from Pitx2+/- mice. These results were not unique to right atrial myocytes as the calcium spark frequency was also 3.9 fold higher in left atrial myocytes from Pitx2+/- mice (p<0.001).Together, these results demonstrate that Pitx2 insufficiency promote both spontaneous calcium release and spontaneous action potentials. Both of these features are hallmarks of myocytes from patients with atrial fibrillation, suggesting that Pitx2-mediated modulation of intracellular calcium handling plays an important role in electrophysiological processes associated with atrial fibrillation.

Dual effects of eugenol on the neuronal excitability: An in vitro study

Besides its well-known actions on sensory afferents, eugenol also affects general excitability of the nervous system, but the mechanisms involved in the recent effect, especially through modulation of ion channels, have received much less attention. In this study, we studied the effects of eugenol on the excitability of central neurons of land snail Caucasotachea atrolabiata and tried to elucidate the underlying ionic mechanisms. The lower concentration of eugenol (0.5mM) reversibly reduced the frequency of spontaneous action potentials that was associated with elevation of threshold, reduction of maximum slope of rising phase and prolongation of actin potentials. These effects were mimicked by riluzole, suggesting that they might be mediated by inhibition of Na+ channels. Eugenol also prolonged the single-spike afterhyperpolarization and post stimulus inhibitory period, but these effects seemed to be consequent to action potential prolongation that indirectly augment Ca2+ inward currents and Ca2+-activated K+ currents. This concentration of eugenol was also able to prevent or abolish pentylenetetrazole-induced epileptiform activity. On the other hand, a higher concentration of eugenol (2mM) reversibly increased the frequency of action potentials and then induced epileptiform activity in majority of treated neurons. Several criteria suggest that the inhibition of K+ channels by higher concentration of eugenol and indirect augmentation of Ca2+ currents are central to the hyperexcitability and epileptiform activity induced by eugenol. Our findings indicate that while low concentration of eugenol could have antiepileptic properties, at higher concentration it induces epileptiform activity. It seems that does dependent inhibition of the ionic currents underlying rising and falling phases of action potential is relevant to the eugenol suppressant and excitatory actions, respectively.

Bothriurus bonariensis scorpion venom activates voltage-dependent sodium channels in insect and mammalian nervous systems

Animal venoms have been widely recognized as a major source of biologically active molecules. Bothriurus bonariensis, popularly known as black scorpion, is the arthropod responsible for the highest number of accidents involving scorpion sting in Southern Brazil. Here we reported the first attempt to investigate the neurobiology of B. bonariensis venom (BBV) in the insect and mammalian nervous system. BBV (32 μg/g) induced a slow neuromuscular blockade in the in vivo cockroach nerve-muscle preparations (70 ± 4%, n = 6, p < 0.001), provoking repetitive twitches and significantly decreasing the frequency of spontaneous leg action potentials (SNCAPs) from 82 ± 3 min−1 to 36 ± 1.3 min−1 (n = 6, p < 0.05), without affecting the amplitude. When tested in primary cultures of rat hippocampal cells, BBV induced a massive increase of Ca2+ influx (250 ± 1% peak increase, n = 3, p < 0.0001). The disturbance of calcium homeostasis induced by BBV on the mammalian central nervous system was not accompanied by cellular death and was prevented by the co-treatment of the hippocampal cells with tetrodotoxin, a selective sodium channel blocker. The results suggest that the biological activity of BBV is mostly related to a modulation of sodium channels function. Our biological activity survey suggests that BBV may have a promising insecticidal and therapeutic potential.

A comparison of the delayed outward potassium current between the nucleus ambiguus and hippocampus: sensitivity to paeonol

Whole-cell patch-clamp recordings investigated the electrophysiological effects of 2′-hydroxy-4′-methoxyacetophenone (paeonol), one of the major components of Moutan Cortex, in hippocampal CA1 neurons and nucleus ambiguus (NA) neurons from neonatal rats as well as in lung epithelial H1355 cells expressing Kv2.1 or Kv1.2. Extracellular application of paeonol at 100μM did not significantly affect the spontaneous action potential frequency, whereas paeonol at 300μM increased the frequency of spontaneous action potentials in hippocampal CA1 neurons. Paeonol (300μM) significantly decreased the tetraethylammonium-sensitive outward current in hippocampal CA1 neurons, but had no effect upon the fast-inactivating potassium current (IA). Extracellular application of paeonol at 300μM did not affect action potentials or the delayed outward currents in NA neurons. Paeonol (100μM) reduced the Kv2.1 current in H1355 cells, but not the Kv1.2 current. The inhibitor of Kv2, guangxitoxin-1E, reduced the delayed outward potassium currents in hippocampal neurons, but had only minimal effects in NA neurons. We demonstrated that paeonol decreased the delayed outward current and increased excitability in hippocampal CA1 neurons, whereas these effects were not observed in NA neurons. These effects may be associated with the inhibitory effects on Kv2.1 currents.

The Oxygen Sensor PHD2 Controls Dendritic Spines and Synapses via Modification of Filamin A.

SummaryNeuronal function is highly sensitive to changes in oxygen levels, but how hypoxia affects dendritic spine formation and synaptogenesis is unknown. Here we report that hypoxia, chemical inhibition of the oxygen-sensing prolyl hydroxylase domain proteins (PHDs), and silencing of Phd2 induce immature filopodium-like dendritic protrusions, promote spine regression, reduce synaptic density, and decrease the frequency of spontaneous action potentials independently of HIF signaling. We identified the actin cross-linker filamin A (FLNA) as a target of PHD2 mediating these effects. In normoxia, PHD2 hydroxylates the proline residues P2309 and P2316 in FLNA, leading to von Hippel-Lindau (VHL)-mediated ubiquitination and proteasomal degradation. In hypoxia, PHD2 inactivation rapidly upregulates FLNA protein levels because of blockage of its proteasomal degradation. FLNA upregulation induces more immature spines, whereas Flna silencing rescues the immature spine phenotype induced by PHD2 inhibition.

Alterations of the electrophysiological properties from cortical layer 5 pyramidal neurons in temporary rapamycin-treated rodent brain slices

The mammalian target of rapamycin (mTOR) signaling pathway is involved in neuro-developmental/degenerative and neuropsychiatric abnormalities. Rapamycin, a specific and potent inhibitor of mTOR signaling, could regulate synaptic plasticity and synaptic transmission of glutamatergic neurons following prolonged treatment. Its immediate effects on electrophysiological properties of cortical layer 5 (L5) pyramidal neurons where the information undergoes a sophisticated processing remain unknown. Here, we found that acute (within 2min) bath-application of rapamycin (0.5μgml−1) was able to depolarize the current-clamp baseline potentials significantly at postnatal day (P) 4, P10 in rats and P90 in mice (P<0.05), and altered the membrane current/voltage (I/V) curves in an age-dependent manner. Rapamycin not only increased the standard deviation or the peak amplitude of baseline membrane potential, but also increased the frequencies of spontaneous action potentials in more mature neurons (P10 and P90). In addition, rapamycin decreased the burst-firing frequencies of cortical L5 burst-spiking neurons from mature brains, and further switched their firing modes to regular-spiking ones. These findings suggest that acute inhibition of mTOR signaling by rapamycin induces an immediate impact on L5 pyramidal neurons’ electrophysiological properties, indicating that its effects might involve mechanisms of ion channel’s regulation.

Chronic intermittent hyperoxia alters the development of the hypoxic ventilatory response in neonatal rats

Chronic exposure to sustained hyperoxia alters the development of the respiratory control system, but the respiratory effects of chronic intermittent hyperoxia have rarely been investigated. We exposed newborn rats to short, repeated bouts of 30% O2 or 60% O2 (5 boutsh−1) for 4–15 days and then assessed their hypoxic ventilatory response (HVR; 10min at 12% O2) by plethysmography. The HVR tended to be enhanced by intermittent hyperoxia at P4 (early phase of the HVR), but it was significantly reduced at P14-15 (primarily late phase of the HVR) compared to age-matched controls; the HVR recovered when individuals were returned to room air and re-studied as adults. To investigate the role of carotid body function in this plasticity, single-unit carotid chemoafferent activity was recorded in vitro. Intermittent hyperoxia tended to decrease spontaneous action potential frequency under normoxic conditions but, contrary to expectations, hypoxic responses were only reduced at P4 (not at P14) and only in rats exposed to higher O2 levels (i.e., intermittent 60% O2). Rats exposed to intermittent hyperoxia had smaller carotid bodies, and this morphological change may contribute to the blunted HVR. In contrast to rats exposed to intermittent hyperoxia beginning at birth, two weeks of intermittent 60% O2 had no effect on the HVR or carotid body size of rats exposed beginning at P28; therefore, intermittent hyperoxia-induced respiratory plasticity appears to be unique to development. Although both intermittent and sustained hyperoxia alter carotid body development and the HVR of rats, the specific effects and time course of this plasticity differs.

Ghrelin alters neurite outgrowth and electrophysiological properties of mouse ventrolateral arcuate tyrosine hydroxylase neurons in culture

While the appetite-stimulating hormone ghrelin can act to acutely modulate electrical activity of neurons in the appetite regulating network, it also has a role in regulating neuronal outgrowth, synaptic connectivity and intrinsic electrophysiological properties. In this study, we investigated whether ghrelin may cause alteration in neurite outgrowth and electrophysiological properties of tyrosine hydroxylase (TH) neurons from the ventrolateral arcuate nucleus (VL-ARC), which are thought to contribute to regulation of energy balance. We prepared dissociated neuronal cultures from the VL-ARC of transgenic mice expressing EGFP under control of the tyrosine hydroxylase (TH) promoter, thus allowing visual identification of putative catecholaminergic (TH-EGFP) neurons. After five days of treatment with 100 nM ghrelin, TH-EGFP neurons exhibited significantly more and longer neurites than control treated neurons, and the effects of ghrelin were abolished by 100 μM ghrelin antagonist, D-Lys-GHRP-6. To investigate whether ghrelin altered electrophysiological properties of TH-EGFP neurons, we carried out patch clamp experiments measuring electrophysiological properties. No significant differences were identified for resting membrane potential or spontaneous action potential frequency, however we observed a hyperpolarization of threshold for action potentials and increased input resistance, indicating increased excitability. This increased excitability is consistent with an observed hyperpolarizing shift in the activation of voltage-gated Na+ currents. These data indicate that the hunger signal ghrelin induces plastic changes in TH-neurons from VL-ARC.

Functional G-protein-coupled receptor 35 is expressed by neurons in the CA1 field of the hippocampus

The G-protein-coupled receptor 35 (GPR35) was de-orphanized after the discovery that kynurenic acid (KYNA), an endogenous tryptophan metabolite, acts as an agonist of this receptor. Abundant evidence supports that GPR35 exists primarily in peripheral tissues. Here, we tested the hypothesis that GPR35 exists in the hippocampus and influences the neuronal activity. Fluorescence immunohistochemical staining using an antibody anti-NeuN (a neuronal marker), an antibody anti-GFAP (a glial marker), and an antibody anti-GPR35 revealed that neurons in the stratum oriens, stratum pyramidale, and stratum radiatum of the CA1 field of the hippocampus express GPR35. To determine the presence of functional GPR35 in the neurocircuitry, we tested the effects of various GPR35 agonists on the frequency of spontaneous action potentials recorded as fast current transients (CTs) from stratum radiatum interneurons (SRIs) under cell-attached configuration in rat hippocampal slices. Bath application of the GPR35 agonists zaprinast (1–10μM), dicumarol (50–100μM), pamoic acid (500–1000μM), and amlexanox (3μM) produced a concentration- and time-dependent reduction in the frequency of CTs. Superfusion of the hippocampal slices with the GPR35 antagonist ML145 (1μM) increased the frequency of CTs and reduced the inhibitory effect of zaprinast. Bath application of phosphodiesterase 5 inhibitor sildenafil (1 or 5μM) was ineffective, whereas a subsequent application of zaprinast was effective in reducing the CT frequency. The present results demonstrate for the first time that functional GPR35s are expressed by CA1 neurons and suggest that these receptors can be molecular targets for controlling neuronal activity in the hippocampus.

Neuroligin-1 Knockdown Suppresses Seizure Activity by Regulating Neuronal Hyperexcitability.

Abnormally synchronized synaptic transmission in the brain leads to epilepsy. Neuroligin-1 (NL1) is a synaptic cell adhesion molecule localized at excitatory synapses. NL1 modulates synaptic transmission and determines the properties of neuronal networks in the mammalian central nervous system. We showed that the expression of NL1 and its binding partner neurexin-1β was increased in temporal lobe epileptic foci in patients and lithium-pilocarpine-treated epileptic rats. We investigated electrophysiological and behavioral changes in epileptic rats after lentivirally mediated NL1 knockdown in the hippocampus to determine whether NL1 suppression prevented seizures and, if so, to explore the probable underlying mechanisms. Our behavioral studies revealed that NL1 knockdown in epileptic rats reduced seizure severity and increased seizure latency. Whole-cell patch-clamp recordings of CA1 pyramidal neurons in hippocampal slices from NL1 knockdown epileptic rats revealed a decrease in spontaneous action potential frequency and a decrease in miniature excitatory postsynaptic current (mEPSC) frequency but not amplitude. The amplitude of N-methyl-D-aspartate receptor (NMDAR)-dependent EPSCs was also selectively decreased. Notably, NL1 knockdown reduced total NMDAR1 expression and the surface/total ratio in the hippocampus of epileptic rats. Taken together, these data indicate that NL1 knockdown in epileptic rats may reduce the frequency and severity of seizures and suppress neuronal hyperexcitability via changes in postsynaptic NMDARs.

Androgens block outward potassium currents and decrease spontaneous action potentials in GH3 cells.

Androgens produce nongenomic effects in several cells by different mechanisms, including ion channel modulation. Adenohypophyseal cells express several K(+) channels, including voltage and Ca(2+)-dependent K(+) (BK) channels, which might be the target of androgens to modulate cellular action potentials and hormonal secretion. Androgen effects were studied in GH3 cells (from anterior pituitary rat tumor) by means of the patch-clamp technique. Cells were continuously perfused with saline solution, in the absence or presence of the androgens studied, while applying 40mV pulses of 400ms from a holding potential of -60mV in whole-cell configuration with nystatin-perforated patches. Androgens reversibly blocked noninactivating K(+) currents in a concentration-dependent manner without a latency period and with an order of efficacy of: 5β-dihydrotestosterone (DHT)>testosterone>5α-DHT. RT-PCR showed two isoforms of the α-pore forming subunits of BK channels. These channels are responsible for one third of the noninactivating current, according to the blockade of paxilline, a selective BK antagonist. Androgens seem to directly interact with BK channels since they were blocked in excised inside-out patches and independent of the whole-cell configuration and the NO-cGMP-dependent pathway. Testosterone, but not 5α- or 5β-DHT, increased BK currents in HEK-293 cells overexpressing the short isoform, suggesting a cellular selectivity based on the α-subunits. The effect on noninactivating currents may be responsible for the decrease of spontaneous action potential frequency. Long-term cellular incubation with testosterone did not modify noninactivating currents density in GH3 cells. It is remarkable that 5β-DHT, a reductase metabolite with weak androgenic activity, was the most efficient blocker.

Distinct Physiological Effects of Dopamine D4 Receptors on Prefrontal Cortical Pyramidal Neurons and Fast-Spiking Interneurons.

Dopamine D4 receptor (D4R), which is strongly linked to neuropsychiatric disorders, such as attention-deficit hyperactivity disorder and schizophrenia, is highly expressed in pyramidal neurons and GABAergic interneurons in prefrontal cortex (PFC). In this study, we examined the impact of D4R on the excitability of these 2 neuronal populations. We found that D4R activation decreased the frequency of spontaneous action potentials (sAPs) in PFC pyramidal neurons, whereas it induced a transient increase followed by a decrease of sAP frequency in PFC parvalbumin-positive (PV+) interneurons. D4R activation also induced distinct effects in both types of PFC neurons on spontaneous excitatory and inhibitory postsynaptic currents, which drive the generation of sAP. Moreover, dopamine substantially decreased sAP frequency in PFC pyramidal neurons, but markedly increased sAP frequency in PV+ interneurons, and both effects were partially mediated by D4R activation. In the phencyclidine model of schizophrenia, the decreasing effect of D4R on sAP frequency in both types of PFC neurons was attenuated, whereas the increasing effect of D4R on sAP in PV+ interneurons was intact. These results suggest that D4R activation elicits distinct effects on synaptically driven excitability in PFC projection neurons versus fast-spiking interneurons, which are differentially altered in neuropsychiatric disorder-related conditions.

Metabolic regulation of lateral hypothalamic glucose-inhibited orexin neurons may influence midbrain reward neurocircuitry.

Lateral hypothalamic area (LHA) orexin neurons modulate reward-based feeding by activating ventral tegmental area (VTA) dopamine (DA) neurons. We hypothesize that signals of peripheral energy status influence reward-based feeding by modulating the glucose sensitivity of LHA orexin glucose-inhibited (GI) neurons. This hypothesis was tested using electrophysiological recordings of LHA orexin-GI neurons in brain slices from 4 to 6week old male mice whose orexin neurons express green fluorescent protein (GFP) or putative VTA-DA neurons from C57Bl/6 mice. Low glucose directly activated ~60% of LHA orexin-GFP neurons in both whole cell and cell attached recordings. Leptin indirectly reduced and ghrelin directly enhanced the activation of LHA orexin-GI neurons by glucose decreases from 2.5 to 0.1mM by 53±12% (n=16, P<0.001) and 41±24% (n=8, P<0.05), respectively. GABA or neurotensin receptor blockade prevented leptin's effect on glucose sensitivity. Fasting increased activation of LHA orexin-GI neurons by decreased glucose, as would be predicted by these hormonal effects. We also evaluated putative VTA-DA neurons in a novel horizontal slice preparation containing the LHA and VTA. Decreased glucose increased the frequency of spontaneous excitatory post-synaptic currents (sEPSCs; 125 ± 40%, n=9, P<0.05) and action potentials (n=9; P<0.05) in 45% (9/20) of VTA DA neurons. sEPSCs were completely blocked by AMPA and NMDA glutamate receptor antagonists (CNQX 20 μM, n=4; APV 20μM, n=4; respectively), demonstrating that these sEPSCs were mediated by glutamatergic transmission onto VTA DA neurons. Orexin-1 but not 2 receptor antagonism with SB334867 (10μM; n=9) and TCS-OX2-29 (2μM; n=5), respectively, blocks the effects of decreased glucose on VTA DA neurons. Thus, decreased glucose increases orexin-dependent excitatory glutamate neurotransmission onto VTA DA neurons. These data suggest that the glucose sensitivity of LHA orexin-GI neurons links metabolic state and reward-based feeding.

Increased firing frequency of spontaneous action potentials in cerebellar Purkinje neurons of db/db mice results from altered auto-rhythmicity and diminished GABAergic tonic inhibition

The hormone leptin, by binding to hypothalamic receptors, suppresses food intake and decreases body adiposity. Leptin receptors are also widely expressed in extra-hypothalamic areas such as hippocampus, amygdala and cerebellum, where leptin modulates synaptic transmission. Here we show that a defective leptin receptor affects the electrophysiological properties of cerebellar Purkinje neurons (PNs). PNs from (db/db) mice recorded in cerebellar slices display a higher firing rate of spontaneous action potentials than PNs from wild type (WT) mice. Blockade of GABAergic tonic inhibition with bicuculline in WT mice changes the firing pattern from continuous, uninterrupted spiking into bursting firing, but bicuculline does not produce these alterations in db/db neurons, suggesting that they receive a weaker GABAergic inhibitory input. Our results also show that the intrinsic firing properties (auto-rhythmicity) of WT and db/db PNs are different. Tonic firing of PNs, the only efferent output from the cerebellar cortex, is a persistent signal to downstream cerebellar targets. The significance of leptin modulation of PNs spontaneous firing is not known. Also, it is not clear if the increased excitability of cerebellar PNs in db/db mice results from hyperglycemia or from the lack of leptin signaling, since both conditions coexist in the db/db strain.

Regional and genotypic differences in intrinsic electrophysiological properties of cerebellar Purkinje neurons from wild-type and dystrophin-deficient mdx mice

Cerebellar subregions are recognized as having specialized roles, with lateral cerebellum considered crucial for cognitive processing, whereas vermal cerebellum is more strongly associated with motor control. In human Duchenne muscular dystrophy, loss of the cytoskeletal protein dystrophin is thought to cause impairments in cognition, including learning and memory. Previous studies demonstrate that loss of dystrophin causes dysfunctional signaling at γ-aminobutyric acid (GABA) synapses on Purkinje neurons, presumably by destabilization of GABAA receptors. However, potential differences in the intrinsic electrophysiological properties of Purkinje neurons, including membrane potential and action potential firing rates, have not been investigated. Here, using a 2×2 analysis of variance (ANOVA) experimental design, we employed patch clamp analysis to compare membrane properties and action potentials generated by acutely dissociated Purkinje neurons from vermal and lateral cerebellum in wild-type (WT) mice and mdx dystrophin-deficient mice. Compared to Purkinje neurons from WT mice, neurons from mdx mice exhibited more irregular action potential firing and a hyperpolarization of the membrane potential. Firing frequency was also lower in Purkinje neurons from the lateral cerebellum of mdx mice relative to those from WT mice. Several action potential waveform parameters differed between vermal and lateral Purkinje neurons, irrespective of dystrophin status, including action potential amplitude, slope (both larger in the vermal region), and duration (shorter in the vermal region). Moreover, the membrane potential of Purkinje neurons from the vermal region of WT mice exhibited a significant hyperpolarization and concurrent reduction in the frequency of spontaneous action potentials compared to Purkinje neurons from the lateral region. This regional hyperpolarization and reduction in spontaneous action potential frequency was abolished in mdx mice. These results from mice demonstrate the presence of differential electrophysiological properties between Purkinje neurons from different regions of the WT mouse cerebellum and altered intrinsic membrane properties in the absence of dystrophin. These findings provide a possible mechanism for the observations that absence of cerebellar dystrophin contributes to deficits in mental function observed in humans and mouse models of muscular dystrophy. Moreover, these results highlight the importance of distinguishing functional zones of the cerebellum in future work characterizing Purkinje neuron electrophysiology and studies using the model of dissociated Purkinje neurons from mice.

Endogenous Subventricular Zone Neural Progenitors Contribute to the Formation and Hyperexcitability of Experimental Model of Focal Microgyria

Microgyria is associated with epilepsy and due to developmental disruption of neuronal migration. However, the role of endogenous subventricular zone-derived neural progenitors (SDNPs) in formation and hyperexcitability has not been fully elucidated. Here, we establish a neonatal cortex freeze-lesion (FL) model, which was considered as a model for focal microgyria, and simultaneously label SDNPs by CM-DiI. Morphological investigation showed that SDNPs migrated into FL and differentiated into neuronal and glia cell types, suggesting the involvement of endogenous SDNPs in the formation of FL-induced microgyria. Patch-clamp recordings in CM-DiI positive (CM-DiI(+)) pyramidal neurons within FL indicated an increase in frequency of spontaneous action potentials, while the resting membrane potential did not differ from the controls. We also found that spontaneous excitatory postsynaptic currents (EPSCs) increased in frequency but not in amplitude compared with controls. The evoked EPSCs showed a significant increase of 10-90% in rise time and decay time in the CM-DiI(+) neurons. Moreover, paired-pulse facilitation was dramatically larger in CM-DiI(+) pyramidal neurons. Western blotting data showed that AMPA and NMDA receptors were increased to some extent in the FL cortex compared with controls, and the NMDA/AMPA ratio of eEPSCs at CM-DiI(+) pyramidal neurons was significantly increased. Taken together, our findings provide novel evidence for the contribution of endogenous SDNPs in the formation and epileptogenicity of FL-induced focal microgyria.