Bath Perfusion Research Articles

Estradiol Drives the Anorexigenic Activity of Proopiomelanocortin Neurons in Female Mice.

Energy balance is regulated by anorexigenic proopiomelanocortin (POMC) and orexigenic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons of the hypothalamic arcuate nucleus. POMC neurons make extensive projections and are thought to release both amino acid and peptide neurotransmitters. However, whether they communicate directly with NPY/AgRP neurons is debated. Initially, using single-cell RT-PCR, we determined that mouse POMCeGFP neurons express Slc17a6 (Vglut2) and Slc18a2 (Vmat2), but not Slc31a1 (Vgat) mRNA, suggesting glutamate and non-canonical GABA release. Quantitative (q)RT-PCR of POMCeGFP cells revealed that Vglut2 and Vmat2 expression was significantly increased in E2- versus oil-treated, ovariectomized (OVX) female mice. Since 17β-estradiol (E2) is anorexigenic, we hypothesized that an underlying mechanism is enhancement of POMC signaling. Therefore, we optogenetically stimulated POMC neurons in hypothalamic slices to examine evoked release of neurotransmitters onto NPY/AgRP neurons. Using brief light pulses, we primarily observed glutamatergic currents and, based on the paired pulse ratio (PPR), determined that release probability was higher in E2- versus oil-treated, OVX female, congruent with increased Vlgut2 expression. Moreover, bath perfusion of the Gq-coupled membrane estrogen receptor (ER) agonist STX recapitulated the effects of E2 treatment. In addition, high-frequency (20 Hz) stimulation generated a slow outward current that reversed near Ek+ and was antagonized by naloxone, indicative of β-endorphin release. Furthermore, individual NPY/AgRP neurons were found to express Oprm1, the transcript for μ-opioid receptor, and DAMGO, a selective agonist, elicited an outward current. Therefore, POMC excitability and neurotransmission are enhanced by E2, which would facilitate decreased food consumption through marked inhibition of NPY/AgRP neurons.

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

Virally Mediated ClopHensorN Chloride Imaging in the Supraoptic Vasopressin Neurons

A number of physiological manipulations in rats, including 2% salt loading (SL), alter chloride homeostasis effecting GABAA mediated inhibition in vasopressin (VP) neurons in the supraoptic nucleus (SON). Brain Derived Neurotrophic Factor (BDNF) and tyrosine receptor kinase B (TrkB) contribute to changes in chloride homeostasis in SON associated with SL. However, it can be difficult to measure these changes using traditional patch clamp electrophysiology. In this study, we used ClopHensorN (Addgene Plasmid #50758 from Colin Akerman), a relatively new technique for ratiometric intracellular chloride [Cl]i imaging to measure changes in the responses of acutely dissociated VP SON neurons to the GABAA receptor agonist muscimol following a 7‐day 2% SL protocol. ClopHensorN was packaged in an AAV2 vector with a VP specific promotor (Addgene Plasmid #40868 from Harold Gainer). Adult male Sprague Dawley rats (250–300 g b w) were anesthetized with isoflurane and bilaterally injected in the SON (300 nl) with rAAV2‐0VP1‐ClopHensorN. After two weeks of recovery, 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 (100 mg/kg IP) and decapitated. The brains were removed and the SONs isolated for mechanical and chemical dissociation. The cells were plated on coverslips that were placed in a perfusion bath on an inverted microscope for ratiometric live cell imaging. Some brains (injected with a VP reporter, AAV2‐p2.OVPI‐eGFP) were sectioned in a vibratome and live 300 um sections containing the SONs were used for electrophysiological recording in order to confirm the ClopHensorN findings. ClopHensorN positive cells were tested for changes in 445/556nm fluorescence ratio indicating decreases or increase in [Cl]i after focal application of muscimol (100uM). To confirm the function of ClopHensorN, transfected cells were dialyzed with micropipettes containing either a high [Cl] (130 mM CsCl) or 0 [Cl] (140 mM KGlu). Dialysis with CsCl produced a decrease and KGlu produced an increase in ClopHensorN fluorescence consistent with Cl− influx and efflux, respectively. In experiments with muscimol on SON neurons from SL rats, 6 out of 10 cells showed significant increases in chloride efflux while 4 out of 10 had no change in chloride flux. TrKB antagonist incubation significantly blocked muscimol induced chloride efflux in SL rats. We also saw significant increase in [Cl]i in 5 of 9 cells from euhydrated rats with muscimol application and this was unaffected by TrkB antagonism. Using loose patch voltage clamp recordings, neurons from euhydrated rats (6/6) were inhibited by muscimol (100 uM). Neurons from SL rats were either unresponsive to muscimol (5/7) or responded with a rapid and robust increase in activity (2/7) followed by a period inactivity (characteristic of depolarization block). Results with ClopHensorN are consistent with electrophysiology recordings suggesting that it is a viable alternative to electrophysiological recordings. The ClopHensorN's ability measure chloride ion dynamics could make it an important tool.Support or Funding InformationSupported by R01 HL119458This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dendritic A-Current in Rhythmically Active PreBötzinger Complex Neurons in Organotypic Cultures from Newborn Mice.

The brainstem preBötzinger complex (preBötC) generates the inspiratory rhythm for breathing. The onset of neural activity that precipitates the inspiratory phase of the respiratory cycle may depend on the activity of type-1 preBötC neurons, which exhibit a transient outward K+ current, IA Inspiratory rhythm generation can be studied ex vivo because the preBötC remains rhythmically active in vitro, both in acute brainstem slices and organotypic cultures. Advantageous optical conditions in organotypic slice cultures from newborn mice of either sex allowed us to investigate how IA impacts Ca2+ transients occurring in the dendrites of rhythmically active type-1 preBötC neurons. The amplitude of dendritic Ca2+ transients evoked via voltage increases originating from the soma significantly increased after an IA antagonist, 4-aminopyridine (4-AP), was applied to the perfusion bath or to local dendritic regions. Similarly, glutamate-evoked postsynaptic depolarizations recorded at the soma increased in amplitude when 4-AP was coapplied with glutamate at distal dendritic locations. We conclude that IA is expressed on type-1 preBötC neuron dendrites. We propose that IA filters synaptic input, shunting sparse excitation, while enabling temporally summated events to pass more readily as a result of IA inactivation. Dendritic IA in rhythmically active preBötC neurons could thus ensure that inspiratory motor activity does not occur until excitatory synaptic drive is synchronized and well coordinated among cellular constituents of the preBötC during inspiratory rhythmogenesis. The biophysical properties of dendritic IA might thus promote robustness and regularity of breathing rhythms.SIGNIFICANCE STATEMENT Brainstem neurons in the preBötC generate the oscillatory activity that underlies breathing. PreBötC neurons express voltage-dependent currents that can influence inspiratory activity, among which is a transient potassium current (IA) previously identified in a rhythmogenic excitatory subset of type-1 preBötC neurons. We sought to determine whether IA is expressed in the dendrites of preBötC. We found that dendrites of type-1 preBötC neurons indeed express IA, which may aid in shunting sparse non-summating synaptic inputs, while enabling strong summating excitatory inputs to readily pass and thus influence somatic membrane potential trajectory. The subcellular distribution of IA in rhythmically active neurons of the preBötC may thus be critical for producing well coordinated ensemble activity during inspiratory burst formation.

The 2-aminoethoxydiphenyl borate analog, DPB161 blocks store-operated Ca2+ entry in acutely dissociated rat submandibular cells.

Cellular Ca2+ signals play a critical role in cell physiology and pathology. In most non-excitable cells, store-operated Ca2+ entry (SOCE) is an important mechanism by which intracellular Ca2+ signaling is regulated. However, few drugs can selectively modulate SOCE. 2-Aminoethoxydiphenyl borate (2APB) and its analogs (DPB162 and DPB163) have been reported to inhibit SOCE. Here, we examined the effects of another 2-APB analog, DPB161 on SOCE in acutely-isolated rat submandibular cells. Both patch-clamp recordings and Ca2+ imaging showed that upon removal of extracellular Ca2+ ([Ca2+]o=0), rat submandibular cells were unable to maintain ACh-induced Ca2+ oscillations, but restoration of [Ca2+]o to refill Ca2+ stores enable recovery of these Ca2+ oscillations. However, addition of 50 μM DPB161 with [Ca2+]o to extracellular solution prevented the refilling of Ca2+ store. Fura-2 Ca2+ imaging showed that DPB161 inhibited SOCE in a concentration-dependent manner. After depleting Ca2+ stores by thapsigargin treatment, bath perfusion of 1 mM Ca2+ induced [Ca2+]i elevation in a manner that was prevented by DPB161. Collectively, these results show that the 2-APB analog DPB161 blocks SOCE in rat submandibular cells, suggesting that this compound can be developed as a pharmacological tool for the study of SOCE function and as a new therapeutic agent for treating SOCE-associated disorders.

Optogenetic activation of VGLUT2-expressing excitatory neurons blocks epileptic seizure-like activity in the mouse entorhinal cortex

We investigated whether an anti-epileptic effect is obtained by selectively activating excitatory neurons expressing ChR2 under the promoter for the synaptic vesicular glutamate transporter 2 (VGLUT2). VGLUT2-expressing cells were optically stimulated while local field potential and whole-cell patch-clamp recordings were performed in mouse entorhinal cortical slices perfused with the proconvulsive compound 4-aminopyridine (4-AP). In control conditions, blue light flashes directly depolarized the majority of putative glutamatergic cells, which in turn synaptically excited GABAergic interneurons. During bath perfusion with 4-AP, photostimuli triggered a fast EPSP-IPSP sequence which was often followed by tonic-clonic seizure-like activity closely resembling spontaneous ictal discharges. The GABAA-receptor antagonist gabazine blocked the progression of both light-induced and spontaneous seizures. Surprisingly, prolonged photostimuli delivered during ongoing seizures caused a robust interruption of synchronous discharges. Such break was correlated with a membrane potential depolarization block in principal cells, while putative GABAergic interneurons changed their firing activity from a burst-like to an irregular single-spike pattern. These data suggest that photostimulation of glutamatergic neurons triggers seizure-like activity only in the presence of an intact GABAergic transmission and that selectively activating the same glutamatergic cells robustly interrupts ongoing seizures by inducing a strong depolarization block, resulting in the disruption of paroxysmal burst-like firing.

P135 Intermittent theta burst increases action potential threshold and spike firing in cortical layer 5 pyramidal neurons

Introduction Repetitive transcranial magnetic stimulation has become a popular method of inducing neural plasticity in humans. In particular, complex patterned stimulation such as intermittent theta burst stimulation (iTBS) is routinely used to increase corticospinal excitably. Despite its wide use, the mechanisms underlying iTBS-induced increases in corticospinal excitability remain unclear. Objectives Experimental (acute brain slices) models of repetitive magnetic stimulation (rMS) allow for direct investigation of iTBS-induced plasticity, such as changes at the single neuron level. Here we investigated whether iTBS rMS alters the active and passive electrophysiological properties of layer 5 pyramidal neurons in the juvenile mouse cortex. Materials & methods Acute brain slices were prepared from the motor and somatosensory cortex of P12-P15 C57Bl6J mice. rMS was delivered with a custom circular coil (8 mm diameter) placed underneath the perfusion bath and slice. Whole cell current clamp recordings were made from layer 5 pyramidal neurons to characterise (i) passive membrane properties (resting membrane potential, input resistance) (ii) active membrane properties (action potential threshold, rheobase) and (iii) spike firing. Baseline recordings were followed by 192s of control (n = 10/9 animals) or iTBS rMS (n = 9/7 animals). Recordings were made immediately post stimulation and again after 10 and 20 min. Results ITBS did not alter the resting membrane potential (p = 0.464) or input resistance (p = 0.418) of layer 5 pyramidal neurons. Interestingly, iTBS increased action potential threshold relative to baseline (control 1.855 mV ± 1.098 vs iTBS −1.537 mV ± 1.158, p = 0.048) but did not alter the rheobase current (p = 0.328). iTBS also increased the number of action potentials (spike firing) generated in response to suprathreshold currents (control 0.783 Hz ± 0.426 vs iTBS 3.167 Hz ± 0.449 p = 0.001). Conclusion Our data suggest that rTMS-induced plasticity may be in part due to electrophysiological changes at the single neuron level, such as altered action potential thresholds and spike firing. These results help elucidate the mechanisms of rTMS-induced plasticity which is essential for the optimisation of rTMS treatment in health and disease.

Propylparaben reduces the excitability of hippocampal neurons by blocking sodium channels

Propylparaben (PPB) is an antimicrobial preservative widely used in food, cosmetics, and pharmaceutics. Virtual screening methodologies predicted anticonvulsant activity of PPB that was confirmed in vivo. Thus, we explored the effects of PPB on the excitability of hippocampal neurons by using standard patch clamp techniques. Bath perfusion of PPB reduced the fast-inactivating sodium current (INa) amplitude, causing a hyperpolarizing shift in the inactivation curve of the INa, and markedly delayed the sodium channel recovery from the inactivation state. Also, PPB effectively suppressed the riluzole-sensitive, persistent sodium current (INaP). PPB perfusion also modified the action potential kinetics, and higher concentrations of PPB suppressed the spike activity. Nevertheless, the modulatory effects of PPB did not occur when PPB was internally applied by whole-cell dialysis. These results indicate that PPB reduces the excitability of CA1 pyramidal neurons by modulating voltage-dependent sodium channels. The mechanistic basis of this effect is a marked delay in the recovery from inactivation state of the voltage-sensitive sodium channels. Our results indicate that similar to local anesthetics and anticonvulsant drugs that act on sodium channels, PPB acts in a use-dependent manner.

Antagonist pharmacology of desensitizing and non-desensitizing nicotinic acetylcholine receptors in cockroach neurons

Two α-bungarotoxin-sensitive nicotinic acetylcholine (ACh) receptor subtypes in neurons of the American cockroach have been identified as desensitizing (nAChD) and selectively inhibitable with 100nM imidacloprid, and non-desensitizing (nAChN) and selectively inhibitable with 100pM methyllycaconitine. In this paper, the single-electrode voltage-clamp technique was used to measure concentration-response relations for the action of ACh and five antagonists on pharmacologically separated nAChD and nAChN receptors of acutely dissociated neurons from thoracic ganglia of the American cockroach. A dual bath and U-tube perfusion system was used to achieve rapid application of ACh in the continued presence of antagonists, which was essential to accurately measure inhibition by rapidly-reversible antagonists. ACh activated both receptors with an EC50 of 7μM and the antagonist potencies were (nAChD/nAChN in nM): dihydro-β-erythroidine: 1.0/5.6, d-tubocurarine: 1000/34, condelphine: 0.39/0.65, phencyclidine: 74/980 and mecamylamine 47/1150. While each of these antagonists displayed some subtype selectivity, none are selective enough to be used as subtype-selective tools. These results bring to a total of 16 the number of nicotinic compounds that have been measured on nAChD and nAChN currents. Characterization of these receptors is important for understanding the role of nAChRs in the insect nervous system and the mechanism of action of insecticides.

A High-Throughput Automated Microfluidic Platform for Calcium Imaging of Taste Sensing.

The human enteroendocrine L cell line NCI-H716, expressing taste receptors and taste signaling elements, constitutes a unique model for the studies of cellular responses to glucose, appetite regulation, gastrointestinal motility, and insulin secretion. Targeting these gut taste receptors may provide novel treatments for diabetes and obesity. However, NCI-H716 cells are cultured in suspension and tend to form multicellular aggregates, preventing high-throughput calcium imaging due to interferences caused by laborious immobilization and stimulus delivery procedures. Here, we have developed an automated microfluidic platform that is capable of trapping more than 500 single cells into microwells with a loading efficiency of 77% within two minutes, delivering multiple chemical stimuli and performing calcium imaging with enhanced spatial and temporal resolutions when compared to bath perfusion systems. Results revealed the presence of heterogeneity in cellular responses to the type, concentration, and order of applied sweet and bitter stimuli. Sucralose and denatonium benzoate elicited robust increases in the intracellular Ca2+ concentration. However, glucose evoked a rapid elevation of intracellular Ca2+ followed by reduced responses to subsequent glucose stimulation. Using Gymnema sylvestre as a blocking agent for the sweet taste receptor confirmed that different taste receptors were utilized for sweet and bitter tastes. This automated microfluidic platform is cost-effective, easy to fabricate and operate, and may be generally applicable for high-throughput and high-content single-cell analysis and drug screening.

Effects ofSaccharomyces cerevisiaeorboulardiiyeasts on acute stress induced intestinal dysmotility

AIMTo investigate the capacity of Saccharomyces cerevisiae (S. cerevisiae) and Saccharomyces boulardii (S. boulardii) yeasts to reverse or to treat acute stress-related intestinal dysmotility.METHODSAdult Swiss Webster mice were stressed for 1 h in a wire-mesh restraint to induce symptoms of intestinal dysmotility and were subsequently killed by cervical dislocation. Jejunal and colon tissue were excised and placed within a tissue perfusion bath in which S. cerevisiae, S. boulardii, or their supernatants were administered into the lumen. Video recordings of contractility and gut diameter changes were converted to spatiotemporal maps and the velocity, frequency, and amplitude of propagating contractile clusters (PCC) were measured. Motility pre- and post-treatment was compared between stressed animals and unstressed controls.RESULTSS. boulardii and S. cerevisiae helped to mediate the effects of stress on the small and large intestine. Restraint stress reduced jejunal transit velocity (mm/s) from 2.635 ± 0.316 to 1.644 ± 0.238, P < 0.001 and jejunal transit frequency (Hz) from 0.032 ± 0.008 to 0.016 ± 0.005, P < 0.001. Restraint stress increased colonic transit velocity (mm/s) from 0.864 ± 0.183 to 1.432 ± 0.329, P < 0.001 and frequency to a lesser degree. Luminal application of S. boulardii helped to restore jejunal and colonic velocity towards the unstressed controls; 1.833 ± 0.688 to 2.627 ± 0.664, P < 0.001 and 1.516 ± 0.263 to 1.036 ± 0.21, P < 0.001, respectively. S. cerevisiae also had therapeutic effects on the stressed gut, but was most apparent in the jejunum. S. cerevisiae increased PCC velocity in the stressed jejunum from 1.763 ± 0.397 to 2.017 ± 0.48, P = 0.0031 and PCC frequency from 0.016 ± 0.009 to 0.027 ± 0.007, P < 0.001. S. cerevisiae decreased colon PCC velocity from 1.647 ± 0.187 to 1.038 ± 0.222, P < 0.001. Addition of S. boulardii or S. cerevisiae supernatants also helped to restore motility to unstressed values in similar capacity.CONCLUSIONThere is a potential therapeutic role for S. cerevisiae and S. boulardii yeasts and their supernatants in the treatment of acute stress-related gut dysmotility.

A method for continuous and stable perfusion of tissue and single cell preparations with accurate concentrations of volatile anaesthetics

BackgroundIt is difficult to design a system to reliably deliver volatile anaesthetics such as halothane or isoflurane to in vitro preparations such as tissues or cells cultures: the very volatility of the drugs means that they can rapidly dissipate from even carefully-prepared solutions. Furthermore, many experiments require the control of other gases (such as oxygen or carbon dioxide) which requires constant perfusion. New methodWe describe a constant perfusion system that is air-tight (i.e., allows the accurate administration of hypoxic or hypercapnic gas mixtures), in which volatile anaesthetic is delivered via calibrated vaporisers by constant bubbling into the perfusing solution (and continuously monitored for stability by infrared spectroscopy in the headspace above the solution). ResultsWe have confirmed the accuracy (i.e., linear relationship of dissolved concentrations with vapour dial settings) and stability (i.e., over time) of the anaesthetic concentrations in solutions in samples taken from the bottles into which anaesthetic is bubbled, and from samples taken from the tissue perfusion bath, using gas chromatrography–mass spectrometry (GC–MS). ConclusionsIt is possible to deliver volatile anaesthetics in accurate concentrations to cell/tissue preparations whilst adjusting ambient air composition rapidly, stable over sustained time periods.

Development of theta rhythm in hippocampal formation slices perfused with 5-HT1A antagonist, (S)WAY 100135

Numerous studies have revealed that median raphe nuclei stimulation induces desynchronization of hippocampal field activity in vivo. Some findings provide evidence for tonic regulation of the theta oscillation of the septo-hippocampal system via the serotonergic system. To date the involvement of serotonergic transmission in theta rhythm generation in hippocampal slices has never been investigated. Thus the aim of the present study was to test whether HPC in vitro preparation is capable of producing theta in the presence of compounds modulating the activity of 5-HT1A receptors. To achieve this a series of experiments designed to determine the effect of different concentrations of the 5-HT1A receptor antagonist (S)WAY 100135 on HPC field activity was carried out. The dominant field potential pattern recorded within HPC slices was epileptiform activity, with a maximum frequency ranging from 0.19±0.06Hz to 0.69±0.10Hz. In addition, after the bath application of (S)WAY 100135 in concentrations 3 and 10µM rhythmic epochs in the theta frequency range were also noted. The highest probability of theta rhythm production was observed after the bath perfusion with a solution of (S) WAY 100135 at a concentration of 10μM. These theta rhythm epochs were characterized by a higher-than-average amplitude compared to carbachol-induced theta epochs and shorter time duration, with no apparent differences in the average frequency and duration of intervals between theta epochs. These results obtained herein of in vitro studies provide direct evidence for the involvement of serotonergic receptors in the depression of oscillatory activity in the HPC theta band.

Proarrhythmic effect of sustained EPAC activation on TRPC3/4 in rat ventricular cardiomyocytes

The Exchange Protein directly Activated by cAMP (EPAC) participates to the pathological signaling of cardiac hypertrophy and heart failure, in which the role of Ca2+ entry through the Transient Receptor Potential Canonical (TRPC) channels begin to be appreciated. Here we studied whether EPAC activation could influence the activity and/or expression of TRPC channels in cardiac myocytes. In adult rat ventricular myocytes treated for 4 to 6h with the selective EPAC activator, 8-pCPT (10μM), we observed by Fluo-3 confocal fluorescence a Store-Operated Ca2+ Entry (SOCE) like-activity, which was blunted by co-incubation with EPAC inhibitors (ESI-05 and CE3F4 at 10μM). This SOCE-like activity, which was very small in control incubated cells, was sensitive to 30-μM SKF-96365. Molecular screening showed a specific upregulation of TRPC3 and C4 protein isoforms after 8-pCPT treatment. Moreover, sustained EPAC activation favored proarrhythmic Ca2+ waves, which were reduced either by co-incubation with EPAC inhibitors or bath perfusion with TRPC inhibitors.Our study provides the first evidence that sustained selective EPAC activation leads to an increase in TRPC3 and C4 protein expression and induces a proarrhythmic SOCE-like activity in adult rat ventricular cardiomyocytes, which might be of importance during the development of cardiac diseases.

A Novel Isolated Glomerular Albumin Permeability Assay Based on Quantitative Fluorescence Microscopy

Vascular permeability is disrupted in several pathological conditions. We've taken advantage of the microscope densitometric technique to develop a new method to measure the solute permeability coefficient (Ps) of fluorescently labelled molecules ex vivo in glomeruli. Calibration experiments have been carried out to test the validity of this assay. Glomeruli were pre-perfused with Octadecyl-Rhodamine-B Chloride and Alexa Fluor 488 BSA (AF488-BSA) via whole-organ kidney perfusion in vivo. Following exsanguination and nephrectomy, glomeruli were sieved from the cortex of each kidney. Glomeruli were then secured in an imaging chamber and exposed to an albumin gradient, which drives efflux of fluorescent albumin from glomerular capillaries that can be quantified by time-lapse confocal imaging. Analysis and acquisition parameters were chosen using specific gain values. The fluorescence intensity values were calculated by selecting specific regions of interest inside different loops and in the perfusion bath. Using a similar protocol we also assessed the contribution of the z-stack position, the photobleaching effect, the free AF488 present in our solution, and differences between acellular and intact glomeruli on Ps values. There was no significant difference in photobleaching between AF488-BSA exposed to the laser for 90 min or 0min (n=4, Paired T Test = 0.20). Neither free dye present in our solution (Ps=0 n=4[1]), or z-stack position effected. Ps values were significantly higher in acellular glomeruli compared to non treated glomeruli. This technique is powerful to sensitively measure changes in albumin across a single glomerular capillary wall and has important implications, especially for isolated human glomeruli.

Tyrode Solution for Bath Perfusion

Tyrode Solution for Bath Perfusion

Rescue of homeostatic regulation of striatal excitability and locomotor activity in a mouse model of Huntington’s disease

We describe a fast activity-dependent homeostatic regulation of intrinsic excitability of identified neurons in mouse dorsal striatum, the striatal output neurons. It can be induced by brief bursts of activity, is expressed on a time scale of seconds, limits repetitive firing, and can convert regular firing patterns to irregular ones. We show it is due to progressive recruitment of the KCNQ2/3 channels that generate the M current. This homeostatic mechanism is significantly reduced in striatal output neurons of the R6/2 transgenic mouse model of Huntington's disease, at an age when the neurons are hyperactive in vivo and the mice begin to exhibit locomotor impairment. Furthermore, it can be rescued by bath perfusion with retigabine, a KCNQ channel activator, and chronic treatment improves locomotor performance. Thus, M-current dysfunction may contribute to the hyperactivity and network dysregulation characteristic of this neurodegenerative disease, and KCNQ2/3 channel regulation may be a target for therapeutic intervention.

Localized drug application and sub-second voltammetric dopamine release measurements in a brain slice perfusion device.

The use of fast scan cyclic voltammetry (FSCV) to measure the release and uptake of dopamine (DA) as well as other biogenic molecules in viable brain tissue slices has gained popularity over the last 2 decades. Brain slices have the advantage of maintaining the functional three-dimensional architecture of the neuronal network while also allowing researchers to obtain multiple sets of measurements from a single animal. In this work, we describe a simple, easy-to-fabricate perfusion device designed to focally deliver pharmacological agents to brain slices. The device incorporates a microfluidic channel that runs under the perfusion bath and a microcapillary that supplies fluid from this channel up to the slice. We measured electrically evoked DA release in brain slices before and after the administration of two dopaminergic stimulants, cocaine and GBR-12909. Measurements were collected at two locations, one directly over and the other 500 μm away from the capillary opening. Using this approach, the controlled delivery of drugs to a confined region of the brain slice and the application of this chamber to FSCV measurements, were demonstrated. Moreover, the consumption of drugs was reduced to tens of microliters, which is thousands of times less than traditional perfusion methods. We expect that this simply fabricated device will be useful in providing spatially resolved delivery of drugs with minimum consumption for voltammetric and electrophysiological studies of a variety of biological tissues both in vitro and ex vivo.

Patch clamp recordings of fast‐acting ion channels in rat dorsal root ganglion cells (654.3)

Manual patch clamp recordings offer direct insight into ion channel properties through the characterization of ion channel activity. Due to the high quality data it is considered the gold standard for ion channel research. Still, the limited throughput means that maximizing data from each cell, especially sensitive primary cells, is important.We have developed a patch clamp‐based assay for characterization fast‐acting ion channels, in this case exemplified by P2X, in primary dorsal root ganglion (DRG) neurons. This assay is based on the use of primary DRG neurons in culture as a cell model for chronic pain. These neurons retain their sensory functionality and remain responsive to thermal, mechanical and functional stimuli, and when supplemented with nerve growth factor (NGF) they can be used to mimic peripheral sensitization. The assay utilizes a microfluidic perfusion system, Dynaflow Resolve, to facilitate a stable recording situation and fast and programmable solution exchange. At the start of the experiment, compounds and buffer are loaded in the 16 wells of the Dynaflow Resolve chip. Micro‐channels connect each well to a recording chamber where the cells are added. To be able to provide fast solution exchange the DRG neuron is lifted using the patch pipette and positioned in front of the micro‐channel outlets. Then, the cell is scanned through the discrete flow zones formed outside the channels.A comparison between conventional bath perfusion and the automated Dynaflow Resolve system showed that both systems have similar capacity when it comes to obtaining successful high resistance seals. However, for collecting full 5‐8 point dose‐responses from single cells, the success rate for the Dynaflow Resolve superseded the conventional system. This enables a dramatically increased productivity when performing more complex pharmacological characterizations.

Local vasotocin modulation of the pacemaker nucleus resembles distinct electric behaviors in two species of weakly electric fish

The neural bases of social behavior diversity in vertebrates have evolved in close association with hypothalamic neuropeptides. In particular, arginine-vasotocin (AVT) is a key integrator underlying differences in behavior across vertebrate taxa. Behavioral displays in weakly electric fish are channeled through specific patterns in their electric organ discharges (EODs), whose rate is ultimately controlled by a medullary pacemaker nucleus (PN). We first explored interspecific differences in the role of AVT as modulator of electric behavior in terms of EOD rate between the solitary Gymnotus omarorum and the gregarious Brachyhypopomus gauderio. In both species, AVT IP injection (10μg/gbw) caused a progressive increase of EOD rate of about 30%, which was persistent in B. gauderio, and attenuated after 30min in G. omarorum. Secondly, we demonstrated by in vitro electrophysiological experiments that these behavioral differences can be accounted by dissimilar effects of AVT upon the PN in itself. AVT administration (1μM) to the perfusion bath of brainstem slices containing the PN produced a small and transient increase of PN activity rate in G. omarorum vs the larger and persistent increase previously reported in B. gauderio. We also identified AVT neurons, for the first time in electric fish, using immunohistochemistry techniques and confirmed the presence of hindbrain AVT projections close to the PN that might constitute the anatomical substrate for AVT influences on PN activity. Taken together, our data reinforce the view of the PN as an extremely plastic medullary central pattern generator that not only responds to higher influences to adapt its function to diverse contexts, but also is able to intrinsically shape its response to neuropeptide actions, thus adding a hindbrain target level to the complexity of the global integration of central neuromodulation of electric behavior.