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![A–D Detection of active synapses with calcium imaging. A A fluorescence image (380 nm excitation) over the MNTB nucleus from a slice loaded with fura-2 AM. Scale bar is 50 m. B The change of [Ca 2+ ] i following electrical stimulation (200 Hz for 200 ms) of the same field of cells as A. Scale bar is 50 m. C Graph of the calcium concentration of the cells in A during the electrical stimulation (at arrow). Only 1 of the 17 cells (indicated by the arrow head in A and B) responds to the electrical stimulation . D [Ca 2+ ] i recorded from two further cells during repeated stimulation (at arrows), in the presence and absence of 4 mM kynurenate and 1 M tetrodotoxin (TTX). The response in the top cell represents antidromic stimulation, whereas the lower cell demonstrates orthodromic , synaptic activation](profile/Ian-Forsythe-3/publication/11194254/figure/fig1/AS:394612586893331@1471094380816/A-D-Detection-of-active-synapses-with-calcium-imaging-A-A-fluorescence-image-380-nm.png)
A–D Detection of active synapses with calcium imaging. A A fluorescence image (380 nm excitation) over the MNTB nucleus from a slice loaded with fura-2 AM. Scale bar is 50 m. B The change of [Ca 2+ ] i following electrical stimulation (200 Hz for 200 ms) of the same field of cells as A. Scale bar is 50 m. C Graph of the calcium concentration of the cells in A during the electrical stimulation (at arrow). Only 1 of the 17 cells (indicated by the arrow head in A and B) responds to the electrical stimulation . D [Ca 2+ ] i recorded from two further cells during repeated stimulation (at arrows), in the presence and absence of 4 mM kynurenate and 1 M tetrodotoxin (TTX). The response in the top cell represents antidromic stimulation, whereas the lower cell demonstrates orthodromic , synaptic activation
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The study of synaptic transmission in brain slices generally entails the patch-clamping of postsynaptic neurones and stimulation of identified presynaptic axons using a remote electrical stimulating electrode. Although patch recording from postsynaptic neurones is routine, many presynaptic axons take tortuous turns and are severed in the slicing pr...
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The effects of substance P (SP) on whole cell currents were studied in neurons of the medial olivocochlear efferent system (MOCS) in the ventral nucleus of the trapezoid body (VNTB) of brain stem slices from neonatal rats. Each neuron was identified by retrograde labeling with Fast Blue injected into the cochlea. Bath application of SP (0.1-10 micr...
Electroporation creates transient pores in the plasma membrane to introduce macromolecules within a cell or cell population. Generally, electrical pulses are delivered between two electrodes separated from each other, making electroporation less likely to be localised. We have developed a new device combining local pressure ejection with local elec...
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... Astrocytes are known to sequester synaptically released glutamate via EAATs and a direct activation of glial glutamate transporters following synaptic stimulation has been observed in the cerebellum (Clark & Barbour, 1997) and hippocampus (Bergles & Jahr, 1997), but not in the MNTB (Reyes-Haro et al. 2010). This difficulty in observing synaptically induced astrocytic transporter currents could be a result of the high conductance of the astrocyte membrane or as a consequence of the small number (<10%) of MNTB cells that retain active synaptic connections following the brainstem slice procedure (Billups et al. 2002). Moreover, inhibiting astrocytic glutamate transport influences the levels of glutamate in and around the synapse and alters the properties of synaptic transmission at the calyx of Held synapse (Renden et al. 2005), as well as at other calyceal synapses (Otis et al. 1996), in the cerebellum (Barbour et al. 1994;Overstreet et al. 1999;Carter & Regehr, 2000;Marcaggi et al. 2003) and in the hippocampus (Scanziani et al. 1997;Huang et al. 2004). ...
Key points Following release of glutamate from excitatory synapses, excitatory amino acid transporters (EAATs) sequester this glutamate into neighbouring astrocytes.
The signalling effects on the astrocyte and the mechanisms by which this glutamate is recycled back to the synapse are currently unclear.
In this study we use electrophysiological recording from neurones and astrocytes to show that a surge of the neurotransmitter glutamate, as usually occurs during neuronal activity, activates astrocytes and causes them to rapidly release the amino acid glutamine.
This glutamine mediates a fast signal back to the neurones, where it is sequestered and is available for the biosynthesis of further neurotransmitters.
Our data demonstrate a novel feedback mechanism by which astrocytes can potentially modulate neuronal function, and pave the way for development of new therapeutic approaches to treat neurological disorders.
... Before recording, slices were kept in aCSF for 30 min to allow de-esterification of the AM dye. Fura 2 fluorescence was detected using imaging technique described previously (Billups et al., 2002) and viewed using a PentaMax intensified CCD camera (Princeton Instruments, Inc). The fluorescent image (emission >505nm) was displayed using Metafluor imaging software (version 7, Molecular Devices), the light source was a Polychrome II Monochromator (TILL Photonics, Martinsried, Germany). ...
... Hence, dynamic imaging has been commonly used to assess neuronal functionality in differentiated cultures, primarily using Ca 2+ -sensitive dyes [3,19] Ca 2+ indicators can be delivered to neurons either by single cell injection (electroporation: [20]; whole cell patch-clamp: [21]) or bulk loading [22]. Except when the Ca 2+ indicator is delivered locally using pressure ejection [23], the loading process of cultures takes longer than one hour [22] and can alter the physiological properties of the tissue [24]. The labeling of cells with JPW3027 on the other hand side takes between 5 and 10 minutes, does not require cell-toxic detergents (e.g. ...
Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca(2+)-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate.
In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters.
Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture.
... Cultured slices, containing dextran retrograde labelled MNTB neurons, were loaded with 5 μM Fura-2 AM (Molecular Probes, Eugene, OR, USA, dissolved in dimethyl sulphoxide (DMSO) containing 5% pluronic acid) for 10 min in aCSF. Before recording, slices were kept in aCSF for 30 min to allow de-esterification of the AM dye, using techniques described previously (Billups et al. 2002). In order to avoid glial cell debris and dead neurons, fura-2 fluorescence (excitation at 340 and 380 nm) was recorded only in confirmed MNTB neurons containing the retrograde dextran signal (543 nm excitation wavelength). ...
Principal neurons of the medial nucleus of the trapezoid body (MNTB) express a spectrum of voltage-dependent K(+) conductances mediated by Kv1-Kv4 channels, which shape action potential (AP) firing and regulate intrinsic excitability. Postsynaptic factors influencing expression of Kv channels were explored using organotypic cultures of brainstem prepared from P9-P12 rats and maintained in either low (5 mm, low-K) or high (25 mm, high-K) [K(+)](o) medium. Whole cell patch-clamp recordings were made after 7-28 days in vitro. MNTB neurons cultured in high-K medium maintained a single AP firing phenotype, while low-K cultures had smaller K(+) currents, enhanced excitability and fired multiple APs. The calyx of Held inputs degenerated within 3 days in culture, having lost their major afferent input; this preparation of calyx-free MNTB neurons allowed the effects of postsynaptic depolarisation to be studied with minimal synaptic activity. The depolarization caused by the high-K aCSF only transiently increased spontaneous AP firing (<2 min) and did not measurably increase synaptic activity. Chronic depolarization in high-K cultures raised basal levels of [Ca(2+)](i), increased Kv3 currents and shortened AP half-widths. These events relied on raised [Ca(2+)](i), mediated by influx through voltage-gated calcium channels (VGCCs) and release from intracellular stores, causing an increase in cAMP-response element binding protein (CREB) phosphorylation. Block of VGCCs or of CREB function suppressed Kv3 currents, increased AP duration, and reduced Kv3.3 and c-fos expression. Real-time PCR revealed higher Kv3.3 and Kv1.1 mRNA in high-K compared to low-K cultures, although the increased Kv1.1 mRNA was mediated by a CREB-independent mechanism. We conclude that Kv channel expression and hence the intrinsic membrane properties of MNTB neurons are homeostatically regulated by [Ca(2+)](i)-dependent mechanisms and influenced by sustained depolarization of the resting membrane potential.
... In order to identify neurones with intact synaptic connections, an imaging technique was used as previously described (Billups et al. 2002). Briefly, cells were loaded with 7 μM Fura 2 acetoxymethyl ester (Fura 2-AM; Molecular Probes, Eugene, OR, USA) for ∼4-7 min and then viewed with a Photometrics Cool SNAP-fx camera after a single 100 ms exposure to light at 380 nm wavelength (provided by a xenon arc lamp controlled by a Cairn Optoscan monochromater; Cairn Instruments, Faversham, UK). ...
... Before recording, slices were kept in aCSF for 30 min to allow de-esterification of the AM dye. Fura 2 fluorescence was detected as described previously (Billups et al. 2002) and viewed using a PentaMax intensified CCD camera (Princeton Instruments, Inc., Trenton, NJ, USA). The fluorescence image (emission >505 nm) was displayed using Metafluor imaging software (v. 7, Molecular Devices), and the light source was a Polychrome II Monochromator (TILL Photonics, Martinsried, Germany). ...
NMDA receptors (NMDARs) mediate a slow EPSC at excitatory glutamatergic synapses throughout the brain. In many areas the magnitude of the NMDAR-mediated EPSC declines with development and is associated with changes in subunit composition, but the mature channel composition is often unknown. We have employed the calyx of Held terminal with its target, the principal neuron of the medial nucleus of the trapezoid body (MNTB), to examine the NMDAR-mediated EPSC during synapse maturation from P10 to P40. Our data show that the calyx has reached a mature state by around P18. The NMDAR-mediated EPSC amplitude (and dominant decay ) fell from around 5 nA (: 40-50 ms) at P10/11 to 0.3-0.5 nA (: 10-15 ms) by P18. The mature NMDAR-EPSC showed no sensitivity to ifenprodil, indicating lack of NR2B subunits, and no block by submicromolar concentrations of zinc, consistent with NR1-1b subunit expression. Additionally, from P11 to P18 there was a reduction in voltage-dependent block and the apparent dissociation constant for [Mg(2+)](o) (K(o)) changed from 7.5 to 14 mm. Quantitative PCR showed that the relative expression of NR2A and NR2C increased, while immunohistochemistry confirmed the presence of NR2A, NR2B and NR2C protein. Although the mature NMDAR-EPSC is small, it is well coupled to NO signalling, as indicated by DAR-4M imaging. We conclude that native mature NMDAR channels at the calyx of Held have a fast time course and reduced block by [Mg(2+)](o), consistent with dominance of NR2C subunits and functional exclusion of NR2B subunits. The pharmacology suggests a single channel type and we postulate that the mature NMDARs consist of heterotrimers of NR1-1b-NR2A-NR2C.
... The input resistances measured over the range −70 to −50 mV was 106 ± 14 M (n = 18). A bipolar electrode for stimulating trapezoid axons was placed at the midline and a DS2A isolated stimulator (Digitimer, Welwyn garden city, UK) delivered 0.2 ms pulses of 4–10 V. Synaptic connections were detected using an imaging technique described previously (Billups et al., 2002). Briefly, slices were bathed in 7 M Fura2-AM (Molecular Probes, Invitrogen) for ∼4 min after which excess Fura2-AM was washed off. ...
Most current clamp studies trigger action potentials (APs) by step current injection through the recording electrode and assume that the resulting APs are essentially identical to those triggered by orthodromic synaptic inputs. However this assumption is not always valid, particularly when the synaptic conductance is of large magnitude and of close proximity to the axon initial segment. We addressed this question of similarity using the Calyx of Held/MNTB synapse; we compared APs evoked by long duration step current injections, short step current injections and orthodromic synaptic stimuli. Neither injected current protocol evoked APs that matched the evoked orthodromic AP waveform, showing differences in AP height, half-width and after-hyperpolarization. We postulated that this ‘error’ could arise from changes in the instantaneous conductance during the combined synaptic and AP waveforms, since the driving forces for the respective ionic currents are integrating and continually evolving over this time-course. We demonstrate that a simple Ohm's law manipulation of the EPSC waveform, which accounts for the evolving driving force on the synaptic conductance during the AP, produces waveforms that closely mimic those generated by physiological synaptic stimulation. This stimulation paradigm allows supra-threshold physiological stimulation (single stimuli or trains) without the variability caused by quantal fluctuation in transmitter release, and can be implemented without a specialised dynamic clamp system. Combined with pharmacological tools this method provides a reliable means to assess the physiological roles of postsynaptic ion channels without confounding affects from the presynaptic input.
... To investigate the role of NO in auditory brainstem signaling we used the fluorescent probe, DAR-4M (Kojima et al., 2001) to monitor NO production in brainstem slice preparations of the mouse SOC. Presynaptic calyceal APs were evoked using midline bipolar stimulation at 36-37 o C (Billups et al., 2002) and stimulation rates of up to 100Hz. These rates of synaptic stimulation were well within the auditory pathway physiological range for both spontaneous and sound-driven inputs. ...
... EPSCs were evoked with a bipolar platinum electrode placed at the midline and using a DS2A isolated stimulator (~3-8 V, 0.2ms; Digitimer, Welwyn Garden City, UK). Synaptic connections were detected using a Fura2 AM (Molecular Probes, Eugene, OR, USA) imaging technique described previously (Billups et al., 2002). ...
... Synaptic stimulation: EPSCs were evoked with a bipolar platinum electrode placed across the slice on the trapezoid body axons at the midline and using a DS2A isolated stimulator (~3-8 V, 0.2ms; Digitimer, Welwyn Garden City, UK). Calyceal synaptic connections were detected using a Fura2 AM (Molecular Probes, Eugene, OR, USA) imaging technique described previously (Billups et al., 2002). Stimulation of non-calyceal excitatory inputs to MNTB neurones was minimised, since they require higher stimulus intensities (Hamann et al., 2003). ...
Neuronal nitric oxide synthase (nNOS) is broadly expressed in the brain and associated with synaptic plasticity through NMDAR-mediated calcium influx. However, its physiological activation and the mechanisms by which nitric oxide (NO) influences synaptic transmission have proved elusive. Here, we exploit the unique input-specificity of the calyx of Held to characterize NO modulation at this glutamatergic synapse in the auditory pathway. NO is generated in an activity-dependent manner by MNTB principal neurons receiving a calyceal synaptic input. It acts in the target neuron and adjacent inactive neurons to modulate excitability and synaptic efficacy, inhibiting postsynaptic Kv3 potassium currents (via phosphorylation), reducing EPSCs and so increasing action potential duration and reducing transmission fidelity. We conclude that NO serves as a volume transmitter and slow dynamic modulator, integrating spontaneous and evoked neuronal firing, thereby providing an index of global activity and regulating information transmission across a population of active and inactive neurons.
... When whole-cell current recording was accomplished, the large glutamatergic postsynaptic current was clearly visible following stimulation (Fig. 9D). This current was blocked by 20 μM NBQX in both cellattached and whole-cell recording ( Fig. 9E and D respectively) and was therefore mediated by the activation of glutamatergic AMPA receptors as classically reported (Billups et al., 2002). ...
... However the process of cutting brain slices unavoidably damages many longer axons, making it very difficult to identify functional synaptic connections in certain brain areas. Within the medial nucleus of the trapezoid body, less than 10% of the cells retain viable synaptic inputs following the slicing procedure (Billups et al., 2002). We showed that presynaptic axons can be easily traced using local ejection of dextran amine combined to local electroporation of the presynaptic pathway. ...
... This allowed electrophysiological recording from pre-selected postsynaptic cells that were innervated by functional synaptic connections. Combining pressure with local electroporation provides an alternative possibility to tracing functional synaptic connections using calcium indicators (Billups et al., 2002). In this study, brainstem slices were loaded with fura-2AM and stimulation of the synaptic inputs caused intracellular calcium concentration to rise in postsynaptic neurones with active synaptic connections. ...
Electroporation creates transient pores in the plasma membrane to introduce macromolecules within a cell or cell population. Generally, electrical pulses are delivered between two electrodes separated from each other, making electroporation less likely to be localised. We have developed a new device combining local pressure ejection with local electroporation through a double-barrelled glass micropipette to transfer impermeable macromolecules in brain slices or in cultured HEK293 cells. The design achieves better targeting of the site of pressure ejection with that of electroporation. With this technique, we have been able to limit the delivery of propidium iodide or dextran amine within areas of 100-200 micrometer. We confirm that local electroporation is transient and show that when combined with pressure ejection, it allows local transfection of EGFP plasmids within HEK293 cells or within cerebellar and hippocampal slice cultures. We further show that local electroporation is less damaging when compared to global electroporation using two separate electrodes. Focal delivery of dextran amine dyes within trapezoid body fibres allowed tracing axonal tracts within brainstem slices, enabling the study of identified calyx of Held presynaptic terminals in living brain tissue. This labelling method can be used to target small nuclei in neuronal tissue and is generally applicable to the study of functional synaptic connectivity, or live axonal tracing in a variety of brain areas.
... Imaging and electrophysiology. Imaging was used as previously described (Billups et al. 2002) to identify neurones with intact calyceal synaptic connections. Briefly, MNTB neurons were loaded with 7 μm Fura2 acetoxymethyl ester (Fura2-AM; Molecular Probes, Eugene, OR, USA) for ∼4 min and then viewed with a Photometrics Cool SNAP-fx camera after a single 100 ms exposure to light at 380 nm wavelength (provided by a xenon arc lamp controlled by a Cairn Optoscan monochromater; Cairn Instruments, Faversham, UK). ...
Sustained activity at most central synapses is accompanied by a number of short-term changes in synaptic strength which act over a range of time scales. Here we examine experimental data and develop a model of synaptic depression at the calyx of Held synaptic terminal that combines many of these mechanisms (acting at differing sites and across a range of time scales). This new model incorporates vesicle recycling, facilitation, activity-dependent vesicle retrieval and multiple mechanisms affecting calcium channel activity and release probability. It can accurately reproduce the time course of experimentally measured short-term depression across different stimulus frequencies and exhibits a slow decay in EPSC amplitude during sustained stimulation. We show that the slow decay is a consequence of vesicle release inhibition by multiple mechanisms and is accompanied by a partial recovery of the releasable vesicle pool. This prediction is supported by patch-clamp data, using long duration repetitive EPSC stimulation at up to 400 Hz. The model also explains the recovery from depression in terms of interaction between these multiple processes, which together generate a stimulus-history-dependent recovery after repetitive stimulation. Given the high rates of spontaneous activity in the auditory pathway, the model also demonstrates how these multiple interactions cause chronic synaptic depression under in vivo conditions. While the magnitude of the depression converges to the same steady state for a given frequency, the time courses of onset and recovery are faster in the presence of spontaneous activity. We conclude that interactions between multiple sources of short-term plasticity can account for the complex kinetics during high frequency stimulation and cause stimulus-history-dependent recovery at this relay synapse.
... Synaptic connections were detected using an imaging technique previously described (Billups et al. 2002). Briefly, cells were loaded with 7 µm acetoxymethyl ester form of Fura2 (Fura2-AM; Molecular Probes, Eugene, OR, USA) for ∼4 min and then viewed using a Photometrics CoolSNAP-fx camera after a single 100 ms exposure to 380 nm (provided by a xenon arc lamp controlled by a Cairn Optoscan monochromater; Cairn Instruments, Faversham, UK). ...
It is well established that synaptic transmission declines at temperatures below physiological, but many in vitro studies are conducted at lower temperatures. Recent evidence suggests that temperature-dependent changes in presynaptic mechanisms remain in overall equilibrium and have little effect on transmitter release at low transmission frequencies. Our objective was to examine the postsynaptic effects of temperature. Whole-cell patch-clamp recordings from principal neurons in the medial nucleus of the trapezoid body showed that a rise from 25 degrees C to 35 degrees C increased miniature EPSC (mEPSC) amplitude from -33 +/- 2.3 to -46 +/- 5.7 pA (n=6) and accelerated mEPSC kinetics. Evoked EPSC amplitude increased from -3.14 +/- 0.59 to -4.15 +/- 0.73 nA with the fast decay time constant accelerating from 0.75 +/- 0.09 ms at 25 degrees C to 0.56 +/- 0.08 ms at 35 degrees C. Direct application of glutamate produced currents which similarly increased in amplitude from -0.76 +/- 0.10 nA at 25 degrees C to -1.11 +/- 0.19 nA 35 degrees C. Kinetic modelling of fast AMPA receptors showed that a temperature-dependent scaling of all reaction rate constants by a single multiplicative factor (Q10=2.4) drives AMPA channels with multiple subconductances into the higher-conducting states at higher temperature. Furthermore, Monte Carlo simulation and deconvolution analysis of transmission at the calyx of Held showed that this acceleration of the receptor kinetics explained the temperature dependence of both the mEPSC and evoked EPSC. We propose that acceleration in postsynaptic AMPA receptor kinetics, rather than altered presynaptic release, is the primary mechanism by which temperature changes alter synaptic responses at low frequencies.
