Bath Perfusion Research Articles

The Paraventricular Thalamic Nucleus and Its Projections in Regulating Reward and Context Associations.

The paraventricular thalamic nucleus (PVT) is a brain region that mediates aversive and reward-related behaviors as shown in animals exposed to fear conditioning, natural rewards, or drugs of abuse. However, it is unknown whether manipulations of the PVT, in the absence of external factors or stimuli (e.g., fear, natural rewards, or drugs of abuse), are sufficient to drive reward-related behaviors. Additionally, it is unknown whether drugs of abuse administered directly into the PVT are sufficient to drive reward-related behaviors. Here, using behavioral as well as pathway and cell-type specific approaches, we manipulate PVT activity as well as the PVT-to-nucleus accumbens shell (NAcSh) neurocircuit to explore reward phenotypes. First, we show that bath perfusion of morphine (10 µM) caused hyperpolarization of the resting membrane potential, increased rheobase, and decreased intrinsic membrane excitability in PVT neurons that project to the NAcSh. Additionally, we found that direct injections of morphine (50 ng) in the PVT of mice were sufficient to generate conditioned place preference (CPP) for the morphine-paired chamber. Mimicking the inhibitory effect of morphine, we employed a chemogenetic approach to inhibit PVT neurons that projected to the NAcSh and found that pairing the inhibition of these PVT neurons with a specific context evoked the acquisition of CPP. Lastly, using brain slice electrophysiology, we found that bath-perfused morphine (10 µM) significantly reduced PVT excitatory synaptic transmission on both dopamine D1 and D2 receptor-expressing medium spiny neurons in the NAcSh, but that inhibiting PVT afferents in the NAcSh was not sufficient to evoke CPP.

PO-05-110 MULTI-CONTACT LEAD TO FACILITATE LEFT BUNDLE BRANCH PACING

Left bundle branch pacing (LBBP) has emerged as a preferred physiological pacing approach, however it is constrained by difficult implantation techniques and inconsistent LBB capture. Therefore, to facilitate LBBP and to improve implantation success, a novel pacing lead specifically for cardiac conduction system pacing that addresses these limitations is necessary. Develop a multi-contact lead (MCL) to facilitate pacing the conduction system at low threshold. A novel lead with five contacts at the distal end was designed and prototyped with fine silver wire wrapped around a central stainless-steel shaft. Insulation was removed from the silver wire with an Ultraviolet laser. The contacts were spaced apart from each other at distances ranging from 0.2 mm to 1.0 mm. The MCLs were tested on five isolated, perfused adult canine hearts. The right ventricular (RV) septum was exposed by opening the RV and leads were inserted from the RV septum to near the left ventricular (LV) septal surface. A standard clinical lead (Medtronic 3830) was also implanted from the RV septal surface. RV apex pacing was performed with a pair of pacing wires. Pacing thresholds for LBB capture were determined and QRS duration was measured from the pseudo-ECG recorded in perfusion bath. LBB capture was achieved for the standard lead and all combinations of MCL. LBB capture threshold was lower for the lowest combination of contacts on MCL compared to the standard 3830 lead values (0.68 ± 0.3 V vs. 1.63 ± 0.7 V, respectively, P < 0.01, Fig A). LBB capture was verified by comparing the shortest MCL QRS duration in each study with sinus rhythm and right ventricular pacing QRS durations (63.8 ± 9.6, 56.4 ± 5.9, and 106.8 ± 16.7 ms, respectively, Fig B). The novel MCL has the potential to facilitate the physiologic pacing process of LBBP. MCL enables a narrowly paced QRS complex and a low pacing capture threshold with a high success rate.

Pharmacological characterization and differential expression of NMDA receptor subunits in the chicken vestibular system during development.

Previous studies demonstrated that in vitro preparations of the isolated vestibular system of diverse animal species still exhibit stable resting electrical activity and mechanically evoked synaptic transmission between hair cells and primary afferent endings. However, there are no reports related to their neurodevelopment. Therefore, this research aimed to examine whether NMDA receptors mediate these electrical signals in an isolated preparation of the chicken vestibular system at three developmental stages, E15, E18, and E21. We found that the spontaneous and mechanically evoked discharges from primary afferents of the posterior semicircular canal were modulated by agonists NMDA and glycine, but not by the agonist d-serine applied near the synapses. Moreover, the individually applied by bath perfusion of three NMDA receptor antagonists (MK-801, ifenprodil, and 2-naphthoic acid) or high Mg2+ decreased the resting discharge rate, the NMDA response, and the discharge rate of mechanically evoked activity from these primary afferents. Furthermore, we found that the vestibular ganglion shows a stage-dependent increase in the expression of NMDA receptor subunits GluN1, GluN2 (A-C), and GluN3 (A-B), being greater at E21, except for GluN2D, which was inversely related to the developmental stage. However, in the crista ampullaris, the expression pattern remained constant throughout development. This could suggest the possible existence of presynaptic NMDA receptors. Our results highlight that although the NMDA receptors are functionally active at the early embryonic stages of the vestibular system, NMDA and glycine reach their mature functionality to increase NMDA responses close to hatching (E21).

Ex vivo capillary-parenchymal arteriole approach to study brain pericyte physiology.

.SignificanceVascular mural cells, defined as smooth muscle cells (SMCs) and pericytes, influence brain microcirculation, but how they contribute is not fully understood. Most approaches used to investigate pericyte and capillary interactions include ex vivo retinal/slice preparations or in vivo two-photon microscopy. However, neither method adequately captures mural cell behavior without interfering neuronal tissue. Thus, there is a need to isolate vessels with their respective mural cells to study functional and pathological changes.AimThe aim of our work was to implement an ex vivo method that recapitulates vessel dynamics in the brain.ApproachExpanding upon our established ex vivo capillary-parenchymal arteriole (CaPA) preparation, we isolated and pressurized arteriole-capillary branches. Using Alexa Fluor™ 633 Hydrazide, we distinguished arterioles (containing elastin) versus capillaries (lacking elastin). In addition, our transgenic SMMHC-GCaMP6f mice allowed for us to visualize mural cell morphology and signals. Lastly, isolated microvasculature was cultured in DMEM media (up to 72 h), mounted, and pressurized using our CaPA preparation.ResultsU46619 induced a decrease in capillary lumen diameter using both a bath perfusion and local application. In addition, U46619 increased signaling both globally and locally in contractile pericytes. In our SMMHC-GCaMP6f mice, we saw that thin strand pericytes had sparse processes while contractile pericytes had long, thick processes that wrapped around the lumen of the capillary. Fresh and cultured pericytes constricted in response to U46619 to the same level, and upstream arteriolar dilation induced by capillary stimulation with 10 mM remained unchanged by culture conditions adding another application of longer treatment to our approach.ConclusionOur ex vivo CaPA methodology facilitates observation of arteriolar SMC and pericyte dynamic changes in real-time without environmental factors. This method will help to better understand how mural cells differ based on microvasculature location.

Electrophysiological study of the effects of side products of RuBi-GABA uncaging on GABAA receptors in cerebellar granule cells.

The study of the GABAA receptor itself and its pharmacology is of paramount importance for shedding light on the role of this receptor in the central nervous system. Caged compounds have emerged as powerful tools to support research in this field, as they allow to control, in space and time, the release of neurotransmitters enabling, for example, to map receptors' distribution and dynamics. Here we focus on γ-aminobutyric acid (GABA)-caged compounds, particularly on a commercial complex called RuBi-GABA, which has high efficiency of uncaging upon irradiation at visible wavelengths. We characterized, by electrophysiological measurements, the effects of RuBi-GABA on GABAA receptors of rat cerebellar granule cells in vitro. In particular, we evaluated the effects of side products obtained after RuBi-GABA photolysis. For this purpose, we developed a procedure to separate the "RuBi-cage" from GABA after uncaging RuBi-GABA with a laser source; then, we compared electrophysiological measurements acquired with and without administering the RuBi-cage in the perfusing bath. In conclusion, to investigate the role of the "cage" molecules both near and far from the cell soma, we compared experiments performed changing the distance of the uncaging point from the cell.

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 &lt;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.

Hypertension Induces Contractile Dysfunction in Rat Mesenteric Lymph Vessels

Hypertension predisposes patients to a cluster of deadly cardiovascular and metabolic diseases involving inflammation, dyslipidemia, and fluid imbalance. Current medications fail to normalize blood pressure in 25% of hypertensive patients; therefore, we considered the possibility that the lymphatic circulation provides a new therapeutic target to alleviate hypertension and its associated co‐morbidities. The intrinsic driving force for the propulsion of lymph fluid is the robust, spontaneous rhythmic contractions of lymphatic muscle cells (LMCs) that facilitate the lymph flow which helps maintain fluid balance and the transport of lipids and immune cells. Thus, evaluation of lymphatic contractions and estimation of lymph flow during hypertension is important to understand the possibility of targeting lymphatic circulation in hypertension. Angiotensin II (Ang II) is a key regulator of blood pressure, and increased levels of Ang II are known to induce hypertension. However, the role of Ang II mediated hypertension on lymphatic contractile dysfunction is still a matter of debate. Here, we evaluated the rhythmic contractions of LVs isolated from Ang II infused hypertensive rats (AHT LVs) compared to normotensive controls (NT LVs). Ang II containing osmotic mini pumps (500 ng/kg/min) were implanted subcutaneously in adult (9‐13 weeks old) male Sprague Dawley (SD) rats for 14 days. Rat mesenteric LVs were dissected free from fat and then cannulated and pressurized (4‐5 mmHg) in a perfusion bath filled with physiological salt solution (PSS). Changes in diameter were monitored by real‐time edge detection software. Baseline rhythmic contractions were recorded for ~45 minutes and then a L‐type calcium channel antagonist, nifedipine (NIF; 1 µmol/L) was added to the bath for 10 mins followed by washout with calcium free PSS. AHT LVs had significantly increased calcium dependent tone to 14.3% ± 5.1% (n=8) compared to NT LVs, which remained at 5.2% ± 1.1% (n=8). AHT LVs also exhibited a larger response to nifedipine with 14.0% ± 6.5% (n=6) increase in dilation compared to NT LVs 3.9% ± 0.9% (n=8). Contractile traces also showed visible dysrhythmia, defined as an increased variation in contraction interval, in LVs isolated from AHT rats at 32.4% ± 14.5% (n=8) when compared with NT LVs at 2.3% ± 1.1% (n=8). An increased variance for the time to relaxation also was observed in AHT LVs (7.4% ± 4.6%; n=8) compared to NT LVs (0.6% ± 0.2%; n=8). These differences ultimately resulted in a significant decrease in calculated flow in AHT LVs (62.0 nL/s ± 8.4 nL/s; n=5) compared to NT LVs (92.4 nL/s ± 4.3 nL/s; n=5). Our data suggest that Ang II mediated hypertension can cause lymphatic contractile dysfunction leading to loss of lymph flow and may be a potential mechanism by which hypertension increases the risk of associated co‐morbidities. Studies are ongoing to assess the changes in lymph flow in vivo during Ang II mediated hypertensive versus normotensive rats.

Intraluminal Pressure, Multi‐Dimensional Field Imaging and Fluid Volume Recordings as a Useful Tool to Determine Peristaltic Propulsion

IntroductionPeristalsis is a collective process of intestinal muscle contractions for the passage of food through the gastrointestinal tract. Although, peristalsis has been extensively studied using spatiotemporal mapping and pressure transducers both in vivo and in vitro, the quantitative analysis of peristaltic propulsion is not fully understood. Lack of a definitive tool therefore, limited drug discovery in the field of constipation including IBSC. In this study, we used directional lighting, advanced field‐imaging techniques together with intraluminal pressure recordings to determine intestinal diameter, frequency of longitudinal muscle activity and pressure changes as well as velocity of anterograde and retrograde contractions and volume output measurements to help determine the net peristaltic activity.MethodsThe basal activity of intestinal peristalsis in terms of intraluminal pressure (ILP) changes, real‐time changes in longitudinal muscle activity and intestinal lumen diameter were measured in approximately 5 cm long segments from jejunum of mouse small intestine (male, NIH Swiss) in physiological Ringer solution. Intraluminal pressure was recorded using pressure transducers connected to the oral and aboral end of each segment mounted in Perfusion Bath for Tubular Organs, (Harvard apparatus, USA). Longitudinal muscle activity was studied using CVX400 series vision system, and LumiTrax™multi‐spectrum light (Keyence, USA) using multiple tracking points in each intestinal segment. The number, strength, and amplitude of oscillations in terms of intraluminal pressure changes and longitudinal muscle activity was analyzed using custom written programs. Luminal volume output was measured using a photo‐electric drop counter unit.ResultsThe number, amplitude, duration, and frequency of high and low amplitude changes in ILP and longitudinal muscle activity was determined using a lab‐written program. The frequency of longitudinal muscle activity correlated well with the frequency of low and high amplitude pressure changes. Frequency of both these oscillations were found to be 0.7 Hz. A reduction in the ILP was found to induce high amplitude contractions which subsided as the ILP increased. Changes in intestinal diameter with time correlated to ILP changes. A reduction in luminal diameter caused an increase in ILP and when not fully compensated caused increased fluid output. The mean velocity of the contractions over time in the regions tracked was determined to be 2.5 – 2.7mm/sec with peak anterograde contraction velocity of ‐4.06 mm/sec and retrograde contractions of 12 mm/sec. This mean velocity in presence of physiological Ringer solution was thought to determine the direction of contractions along the length of the GI tract under normal conditions.InferenceFrequency of longitudinal muscle activity and ILP oscillation showed similar frequency suggesting common pacemaker activity. The velocity of the contraction and volume output over time will help determine the overall peristaltic movement and could be used as a helpful index to gauge constipation.

Microglia-derived TNF-α inhibiting GABAergic neurons in the anterior lateral bed nucleus of the stria terminalis precipitates visceral hypersensitivity induced by colorectal distension in rats

Irritable bowel syndrome (IBS) is a common and debilitating gastrointestinal disorder that is exacerbated by stress and characterized by abdominal pain. Although microglia in the CNS have been implicated as an important mediator of the stress response, the role of microglia and microglia-GABAergic neuron interactions in the limbic area, most notably BNST, in the development of colorectal hypersensitivity has not been determined. We established a neonatal colorectal distension-induced chronic visceral hyperalgesia model in rats. The results showed that the frequency of spontaneous discharges of alBNST GABAergic neurons and the expression of GAD65/67 were significantly decreased in rats with chronic visceral pain. Moreover, ablation of BNST GABAergic neurons significantly reduced the visceral pain threshold in normal rats. Meanwhile, the number of M1 proinflammatory microglia and the expression of the M1 proinflammatory microglia-derived cytokines IL-6 and TNF-α were increased in the alBNST of rats with chronic visceral pain. Furthermore, alBNST infusion of the microglial inhibitor minocycline or IL-6 and TNF-α neutralizing antibodies significantly increased the visceral pain threshold. The decreased frequency of spontaneous discharges of alBNST GABAergic neurons in rats with chronic visceral pain was mimicked by a bath perfusion of TNF-α, but not IL-6, and was abolished by a perfusion of the microglial inhibitor minocycline. In addition, the alBNST infusion of the microglial inhibitor minocycline upregulated the expression of GAD65/67. Moreover, ablation of BNST GABAergic neurons significantly decreased the visceral pain threshold in normal rats, which was not reversed by a subsequent infusion of the microglial inhibitor minocycline. Our findings revealed this microglia-GABAergic neuron circuit in the alBNST, and this microglia-driven disinhibitory mechanism is essential for brain and gut dysfunction in stressful condition, providing a novel potential target for treating patients with IBS presenting visceral pain that is worsened during episodes of stress.

Differential and long-lasting changes in neurotransmission in the amygdala of male Wistar rats during extended amphetamine abstinence

Amphetamine addiction is associated with maladaptive actions that promotes continued use despite negative consequences, and a high risk of relapse even after protracted abstinence. Considering the role of the amygdala in regulating incentive motivation and reward-based behavior, the aim of this study was to assess neuroadaptations in subregions of the amygdala elicited by a brief period of discontinuous amphetamine exposure (2.0 mg/kg/day, 5 days) followed by abstinence (2 weeks, 1 month, 3 months) in male Wistar rats. Electrophysiological field potential recordings demonstrated that repeated amphetamine exposure significantly depressed evoked populations spikes in the basolateral amygdala (BLA). Evoked populations spikes were normalized after three months abstinence, but one challenge dose of amphetamine (0.5 mg/kg) was sufficient to reinstate synaptic depression in animals previously receiving amphetamine. In the central nucleus of the amygdala (CeA), amphetamine produced a long-lasting hyperexcitability that sustained even after three months abstinence. In the CeA, there were no significant differences between treatment groups following bath perfusion of the GABAA receptor antagonist bicuculline, indicating that amphetamine acts by reducing the inhibitory tone. Recordings performed in brain subregions interlinked with the amygdala, including medial prefrontal cortex, orbitofrontal cortex, and nucleus accumbens shell (nAc), revealed no significant neuroadaptations after two weeks abstinence. However, synaptic output was significantly depressed in the nAc after one- and three-month abstinence. In conclusion, the data presented here shows that five days of discontinuous exposure to amphetamine is sufficient to produce long-lasting neuroadaptations, which may contribute to compulsive drug taking and increase the risk for relapse.

An Immersible Microgripper for Pancreatic Islet and Organoid Research.

To improve the predictive value of in vitro experimentation, the use of 3D cell culture models, or organoids, is becoming increasingly popular. However, the current equipment of life science laboratories has been developed to deal with cell monolayers or cell suspensions. To handle 3D cell aggregates and organoids in a well-controlled manner, without causing structural damage or disturbing the function of interest, new instrumentation is needed. In particular, the precise and stable positioning in a cell bath with flow rates sufficient to characterize the kinetic responses to physiological or pharmacological stimuli can be a demanding task. Here, we present data that demonstrate that microgrippers are well suited to this task. The current version is able to work in aqueous solutions and was shown to position isolated pancreatic islets and 3D aggregates of insulin-secreting MIN6-cells. A stable hold required a gripping force of less than 30 μN and did not affect the cellular integrity. It was maintained even with high flow rates of the bath perfusion, and it was precise enough to permit the simultaneous microfluorimetric measurements and membrane potential measurements of the single cells within the islet through the use of patch-clamp electrodes.

Periaqueductal gray is required for controlling chronic stress-induced depression-like behavior

BackgroundEnduring exposure to psychological stress is associated with an elevated risk of major depressive disorder (MDD). There is an enormous need to investigate the unexplored mechanisms of MDD. We examined whether pain-free stress alters synaptic transmission, causing depression-like behaviors in the ventrolateral periaqueductal gray (vlPAG), a brain stem nucleus that controls stress-related depression-like behavior. MethodsIn the current study, we studied neuronal changes in the vlPAG and behavioral transforms using electrophysiological recordings, behavioral tests, and pharmacological approaches. ResultsWe found that chronic restraint stress (CRS) diminished glutamatergic transmission in the vlPAG, leading to maladaptive behavioral despair and anhedonia in mice demonstrated by the forced swimming test (FST), tail suspension test (TST) and female urine sniffing test (FUST). Moreover, CRS increased behavioral hypersensitivity shown by the von Frey test. Bath perfusion with the rapid-acting antidepressant (2R,6R)-hydroxynorketamine (HNK) increased both the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in vlPAG neurons in the CRS and control groups. Functionally, (2R,6R)-HNK directly enhanced the action potential firing rate in vlPAG neurons. Behaviorally, intravlPAG microinjection of (2R,6R)-HNK alleviated chronic restraint stress-induced depression-like behaviors and behavioral hypersensitivity. ConclusionsThese results demonstrate that psychological stress-elicited depression-like behavior is related to a remarkable decrease in glutamatergic transmission in the vlPAG. The maladaptive behaviors are attributed to hypoactivity of glutamatergic neurons in the vlPAG, and direct enhancement of glutamatergic neuronal activity in the vlPAG rescues depression-like behaviors. The present results prove that vlPAG is critical for controlling stress-induced depression-like behaviors through alteration of glutamatergic transmission.

Synedrella nodiflora Extract Depresses Excitatory Synaptic Transmission and Chemically-Induced In Vitro Seizures in the Rat Hippocampus.

Extracts of the tropical Cinderella plant Synedrella nodiflora are used traditionally to manage convulsive conditions in the West African sub-region. This study sought to determine the neuronal basis of the effectiveness of these plant extracts to suppress seizure activity. Using the hippocampal slice preparation from rats, the ability of the extract to depress excitatory synaptic transmission and in vitro seizure activity were investigated. Bath perfusion of the hydro-ethanolic extract of Synedrella nodiflora (SNE) caused a concentration-dependent depression of evoked field excitatory postsynaptic potentials (fEPSPs) recorded extracellularly in the CA1 region of the hippocampus with maximal depression of about 80% and an estimated IC50 of 0.06 mg/ml. The SNE-induced fEPSP depression was accompanied by an increase in paired pulse facilitation. The fEPSP depression only recovered partially after 20 min washing out. The effect of SNE was not stimulus dependent as it was present even in the absence of synaptic stimulation. Furthermore, it did not show desensitization as repeat application after 10 min washout produced the same level of fEPSP depression as the first application. The SNE effect on fEPSPs was not via adenosine release as it was neither blocked nor reversed by 8-CPT, an adenosine A1 receptor antagonist. In addition, SNE depressed in vitro seizures induced by zero Mg2+ and high K+ -containing artificial cerebrospinal fluid (aCSF) in a concentration-dependent manner. The results show that SNE depresses fEPSPs and spontaneous bursting activity in hippocampal neurons that may underlie its ability to abort convulsive activity in persons with epilepsy.

Topiramate inhibits offensive aggression through targeting ventrolateral periaqueductal gray

Topiramate is an approved antiepileptic drug clinically used to treat epilepsy and prevent migraines. Currently, topiramate has been found to be effective in treating aggressive symptoms in neuropsychiatric patients. In preclinical studies, however, the effects and mechanisms of topiramate on offensive aggression are still largely uninvestigated. Our previous studies indicated that glutamatergic transmission in the ventrolateral periaqueductal gray (vlPAG) plays a crucial role in regulating elements of offensive aggressive behaviors. In the present work, we investigated the actions of topiramate on vlPAG glutamatergic transmission and aggressive behaviors in group-housed (GH) and socially isolated (SI) rats. The results suggested that a single injection of topiramate systemically and dose-dependently inhibited elements of offensive aggressive behaviors of both GH and SI rats in the resident-intruder test (RIT), with long-lasting effective time profiles in SI rats. Moreover, systemic single administration of topiramate reduced the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in the vlPAG. Bath perfusion of topiramate directly decreased the frequency and amplitude of mEPSCs and shortened the amplitude of evoked excitatory postsynaptic currents (EPSCs) in the vlPAG. Furthermore, intra-vlPAG single microinjection of topiramate dose-dependently inhibited offensive aggressive behaviors in GH and SI rats in a time-dependent manner. Additionally, both systemic and local topiramate inhibited offensive aggressive behaviors in a (2R,6R)-hydroxynorketamine (HNK)-dependent rat model. In conclusion, the present results suggest that topiramate exerts anti-aggressive roles through its inhibitory actions on glutamatergic activities in the vlPAG. These preclinical results support topiramate as a candidate drug to treat patients with heightened offensive aggression.

A Computational Model to Investigate GABA-Activated Astrocyte Modulation of Neuronal Excitation.

Gamma-aminobutyric acid (GABA) is critical for proper neural network function and can activate astrocytes to induce neuronal excitability; however, the mechanism by which astrocytes transform inhibitory signaling to excitatory enhancement remains unclear. Computational modeling can be a powerful tool to provide further understanding of how GABA-activated astrocytes modulate neuronal excitation. In the present study, we implemented a biophysical neuronal network model to investigate the effects of astrocytes on excitatory pre- and postsynaptic terminals following exposure to increasing concentrations of external GABA. The model completely describes the effects of GABA on astrocytes and excitatory presynaptic terminals within the framework of glutamatergic gliotransmission according to neurophysiological findings. Utilizing this model, our results show that astrocytes can rapidly respond to incoming GABA by inducing Ca2+ oscillations and subsequent gliotransmitter glutamate release. Elevation in GABA concentrations not only naturally decreases neuronal spikes but also enhances astrocytic glutamate release, which leads to an increase in astrocyte-mediated presynaptic release and postsynaptic slow inward currents. Neuronal excitation induced by GABA-activated astrocytes partly counteracts the inhibitory effect of GABA. Overall, the model helps to increase knowledge regarding the involvement of astrocytes in neuronal regulation using simulated bath perfusion of GABA, which may be useful for exploring the effects of GABA-type antiepileptic drugs.

Activation of glycine receptors in the lateral habenula rescues anxiety- and depression-like behaviors associated with alcohol withdrawal and reduces alcohol intake in rats

The lateral habenula (LHb) is activated by a range of aversive states including those related to alcohol withdrawal and has glycine receptors (GlyRs), a sensitive target of alcohol. However, whether GlyRs in the LHb contribute to alcohol-related behaviors is unknown. Here, we report that rats experiencing withdrawal from chronic alcohol consumption showed higher anxiety and sensitivity to stress compared to their alcohol-naïve counterparts. Intra-LHb injection of glycine attenuated these aberrant behaviors and reduced alcohol intake upon alcohol re-access. Glycine's effect was blocked by strychnine, a GlyR antagonist, indicating that it was mediated by strychnine-sensitive GlyRs. Conversely, intra-LHb strychnine elicited anxiety- and depression-like behaviors in Naïve rats but not in withdrawal rats. Additionally, both the frequency and the amplitude of the spontaneous IPSCs were lower in LHb neurons in slices of withdrawal rats compared to naïve rats. Also, there were sporadic strychnine-sensitive synaptic events in some LHb neurons. Bath perfusion of strychnine induced a depolarizing inward current and increased action potential firings in LHb neurons. By contrast, bath perfusion of glycine or sarcosine, a glycine transporter subtype 1 inhibitor, inhibited LHb activity. Collectively, these data reveal that LHb neurons are under the tonic glycine inhibition both in physiological and pathological conditions. Activation of GlyRs reverses LHb hyperactivity, alleviates aberrant behaviors, and reduces alcohol intake, thus highlighting the GlyRs in the LHb as a potential therapeutic target for alcohol-use disorders.

A novel mechanism strengthens GABAergic inhibition in the hypothalamic PVN in proportion to the level of synaptic L‐glutamate spillover

Perhaps the most robust property of pre‐sympathetic neurons (PSNs) of the hypothalamic paraventricular nucleus (PVN) in unstressed animals is their state of near quiescence. Although discharge quiescence has been strongly linked to the dominance of GABAergic synaptic inhibition, mechanisms that govern the strength of local GABAergic inhibition remain to be fully elucidated. Here, whole cell patch‐clamp recordings were made from identified parvocellular PVN neurons in brain slices and GABAergic inhibitory postsynaptic currents (IPSCs) were evoked by electrical stimuli delivered to the surrounding peri‐nuclear zone. To investigate the impact of GABA synthesis and vesicular packaging on GABAergic IPSC amplitude, we compared eIPSC amplitude before and after bath application of the GABA synthetic substrate L‐glutamate (L‐Glu; 100 μM, 10 min). Within 3–5 minutes of L‐Glu washout, eIPSC amplitude suddenly and significantly increased (+23 ± 6%; P&lt;0.002). This response was unaffected by selective inhibition of glial L‐Glu uptake with dihydrokainate (DHK; 250 μM), but was nearly abolished (−77 ± 8.5%) by DL‐threo‐β‐benzyloxyaspartic acid (TBOA; 100 μM), a pan‐specific inhibitor of L‐glutamate transporters, suggesting that strengthening of GABAergic inhibition by L‐Glu depends on uptake by the neuronal L‐Glu transporter EAAT3. Similar results were obtained when local synaptic activity was increased by bath perfusion with high K+ (10 mM) aCSF. In current‐clamp recordings of spontaneous firing, high bath K+ caused a delayed reduction of discharge (1.58 ± 0.59Hz to 1.18 ± 0.51Hz; P&lt;0.03) that was not observed when depolarizing current was injected to mimic the spiking response to high K+. These findings suggest that GABAergic IPSC strengthening is sufficient to reduce PVN neuronal discharge. High frequency stimulation‐induced GABAergic synaptic depression/vesicular depletion revealed that exposure to bath L‐Glu did not increase quantal content (i.e., the number of GABA vesicles released during each IPSC), but enhance recovery of IPSC amplitude by +36 pA within 10 s. These data indicate that bath L‐Glu selectively increases the number of GABA molecules contained within each GABA vesicle. In anesthetized rats (n=6), acute blockade of GABA‐A receptors in the PVN with picrotoxin (PTX) increased renal sympathetic nerve activity (+23%). Prior PVN injection of L‐Glu greatly amplified the PTX response (+72%), which is consistent with increased functional inhibitory tonus. Collectively, our findings indicate that GABA synaptic vesicles are “over‐filled” when GABA is synthesized from L‐Glu under conditions of heightened L‐Glu availability. This process depends on EAAT3‐mediated L‐Glu uptake, likely into GABA terminals. As over‐filled vesicles transition to the readily releasable pool, the amplitude of IPSCs increases, allowing synaptic inhibitory tonus to increase in proportion to the level of synaptic L‐Glu spillover. This local mechanism is postulated to help prevent neuronal hyper‐activation under conditions of heightened glutamatergic excitation.Support or Funding InformationNIH HL088052 (GMT)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intracellular Chloride Regulation of Supraoptic Vasopressin Neurons during Salt Loading

Salt loading (SL) increases intracellular chloride concentration [Cl]i, impairing GABAA inhibition of arginine vasopressin (AVP) neurons in the supraoptic nucleus (SON) of hypothalamus. But the regulatory mechanisms leading to increased [Cl]i is not completely understood. Based on previous studies, we hypothesize that SL activates tyrosine receptor kinase B (TrkB) and downregulates K+/Cl− co‐transporter 2 (KCC2) membrane expression. Downregulation of KCC2 decreases the efflux of chloride ion causing increase in [Cl]i in SON AVP neurons. In this study, we have combined virally mediated ClopHensorN, a relatively new ratiometric chloride imaging technique with capillary based Simple Western technology to record changes in [Cl]i and specifically detect KCC2 protein expression in individual SON AVP neurons.Adult male Sprague Dawley rats were bilaterally injected in the SON with rAAV2‐0VP1‐ClopHensorN. The ClopHensorN (Addgene Plasmid #50758 from Colin Akerman) was packaged in an AAV2 vector with a AVP specific promotor (Addgene Plasmid #40868 from Harold Gainer). After 2 weeks, the rats were given either water or 2% NaCl to drink for 7 days. At the end of the protocol, the rats were anesthetized with inactin and their SONs were dissociated. The cells were plated on coverslips and placed in a perfusion bath on an inverted microscope for ratiometric live cell imaging. ClopHensorN positive neurons were tested for decrease or increase in [Cl]i to focal application of GABAA agonist muscimol (100uM). After imaging, individual neurons were collected by aspirating into a patch pipette to verify KCC2 and ß‐Actin protein expression. The standard matrix (12–230kDa) in Protein Simple Wes was used to identify and quantitate very low concentration of protein from single neuron. Data were analyzed by Chi‐squared test and one‐way ANOVA with Bonferroni comparisons.Muscimol application to SL SONs either significantly increased chloride efflux (p&lt;0.05;13/20) or did not change chloride flux (7/20). TrkB antagonist (AnA,50uM) significantly blocked the chloride effluxes. SONs from euhydrated rats showed muscimol induced chloride influx (p&lt;0.05;11/16). KCC2 antagonist (VU 0240551,10uM) significantly blocked the chloride influxes in euhydrated rats. SON AVP neurons that responded to pharmacological inhibitors during chloride imaging were viable and expressed KCC2 co‐transporter and ß‐Actin. Neurons that did not respond during chloride imaging did not had KCC2 and ß‐Actin protein expression. Salt loading increases [Cl]i in SON AVP neurons through TrKB‐KCC2 dependent mechanism.Support or Funding InformationSupported by R01 HL119458 and 18PRE34060035This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Kir2.1 Channel Regulation of Glycinergic Transmission Selectively Contributes to Dynamic Mechanical Allodynia in a Mouse Model of Spared Nerve Injury.

Neuropathic pain is a chronic debilitating symptom characterized by spontaneous pain and mechanical allodynia. It occurs in distinct forms, including brush-evoked dynamic and filament-evoked punctate mechanical allodynia. Potassium channel 2.1 (Kir2.1), which exhibits strong inward rectification, is and regulates the activity of lamina I projection neurons. However, the relationship between Kir2.1 channels and mechanical allodynia is still unclear. In this study, we first found that pretreatment with ML133, a selective Kir2.1 inhibitor, by intrathecal administration, preferentially inhibited dynamic, but not punctate, allodynia in mice with spared nerve injury (SNI). Intrathecal injection of low doses of strychnine, a glycine receptor inhibitor, selectively induced dynamic, but not punctate allodynia, not only in naïve but also in ML133-pretreated mice. In contrast, bicuculline, a GABAA receptor antagonist, induced only punctate, but not dynamic, allodynia. These results indicated the involvement of glycinergic transmission in the development of dynamic allodynia. We further found that SNI significantly suppressed the frequency, but not the amplitude, of the glycinergic spontaneous inhibitory postsynaptic currents (gly-sIPSCs) in neurons on the lamina II-III border of the spinal dorsal horn, and pretreatment with ML133 prevented the SNI-induced gly-sIPSC reduction. Furthermore, 5days after SNI, ML133, either by intrathecal administration or acute bath perfusion, and strychnine sensitively reversed the SNI-induced dynamic, but not punctate, allodynia and the gly-sIPSC reduction in lamina IIi neurons, respectively. In conclusion, our results suggest that blockade of Kir2.1 channels in the spinal dorsal horn selectively inhibits dynamic, but not punctate, mechanical allodynia by enhancing glycinergic inhibitory transmission.

Contrast-Enhanced Ultrasound to Quantifyc Perfusion in a Machine-Perfused Pig Liver.

This paper introduces a non-invasive, contrastenhanced ultrasound (CEUS) infusion method to quantify the health of viable donor livers. The method uses the infusion of microbubbles and their destruction and subsequent replenishment to measure the perfusion rate in the liver microvasculature. The proposed method improves on the previous parameter extraction approaches applied to the flashreplenishment technique by addressing the effects of the microbubble mixing within the perfusate bath and destruction rate. By doing so, the tissue perfusion rate can be extracted from the data even though the microbubble concentration is not constant throughout image acquisition. The measured changes in the tissue perfusion rate showed that CEUS infusion is a viable biomarker for assessing liver health.