Ian D. Forsythe

Presynaptic Calcium Current Modulation by a Metabotropic Glutamate Receptor

Metabotropic glutamate receptors (mGluRs) regulate transmitter release at mammalian central synapses. However, because of the difficulty of recording from mammalian presynaptic terminals, the mechanism underlying mGluR-mediated presynaptic inhibition is not known. Here, simultaneous recordings from a giant presynaptic terminal, the calyx of Held, and its postsynaptic target in the medial nucleus of the trapezoid body were obtained in rat brainstem slices. Agonists of mGluRs suppressed a high voltage-activated P/Q-type calcium conductance in the presynaptic terminal, thereby inhibiting transmitter release at this glutamatergic synapse. Because several forms of presynaptic modulation and plasticity are mediated by mGluRs, this identification of a target ion channel is a first step toward elucidation of their molecular mechanism.

INACTIVATION OF PRESYNAPTIC CALCIUM CURRENT CONTRIBUTES TO SYNAPTIC DEPRESSION AT A FAST CENTRAL SYNAPSE

Voltage-gated calcium channels are well characterized at neuronal somata but less thoroughly understood at the presynaptic terminal where they trigger transmitter release. In order to elucidate how the intrinsic properties of presynaptic calcium channels influence synaptic function, we have made direct recordings of the presynaptic calcium current (I(pCa)) in a brainstem giant synapse called the calyx of Held. The current was pharmacologically classified as P-type and exhibited marked inactivation. The inactivation was largely dependent upon the inward calcium current magnitude rather than the membrane potential, displayed little selectivity between divalent charge carriers (Ca2+, Ba2+ and Sr+), and exhibited slow recovery. Simultaneous pre- and postsynaptic whole-cell recording revealed that I(pCa) inactivation predominantly contributes to posttetanic depression of EPSCs. Thus, because of its slow recovery, I(pCa) inactivation underlies this short-term synaptic plasticity.

Contribution of the Kv3.1 potassium channel to high-frequency firing in mouse auditory neurones.

1 Using a combination of patch‐clamp, in situ hybridization and computer simulation techniques, we have analysed the contribution of potassium channels to the ability of a subset of mouse auditory neurones to fire at high frequencies. 2 Voltage‐clamp recordings from the principal neurones of the medial nucleus of the trapezoid body (MNTB) revealed a low‐threshold dendrotoxin (DTX)‐sensitive current (ILT) and a high‐threshold DTX‐insensitive current (IHT). 3 I HT displayed rapid activation and deactivation kinetics, and was selectively blocked by a low concentration of tetraethylammonium (TEA; 1 mm). 4 The physiological and pharmacological properties of IHT very closely matched those of the Shaw family potassium channel Kv3.1 stably expressed in a CHO cell line. 5 An mRNA probe corresponding to the C‐terminus of the Kv3.1 channel strongly labelled MNTB neurones, suggesting that this channel is expressed in these neurones. 6 TEA did not alter the ability of MNTB neurones to follow stimulation up to 200 Hz, but specifically reduced their ability to follow higher frequency impulses. 7 A computer simulation, using a model cell in which an outward current with the kinetics and voltage dependence of the Kv3.1 channel was incorporated, also confirmed that the Kv3.1‐ like current is essential for cells to respond to a sustained train of high‐frequency stimuli. 8 We conclude that in mouse MNTB neurones the Kv3.1 channel contributes to the ability of these cells to lock their firing to high‐frequency inputs.

Pre- and postsynaptic glutamate receptors at a giant excitatory synapse in rat auditory brainstem slices.

1. Whole‐cell patch recordings were used to examine the EPSC generated by the calyx of Held in neurones of the medial nucleus of the trapezoid body (MNTB). Each neurone receives a somatic input from a single calyx (giant synapse). 2. A slow NMDA receptor‐mediated EPSC peaked in 10 ms and decayed as a double exponential with time constants of 44 and 147 ms. A fast EPSC had a mean rise time of 356 microseconds (at 25 degrees C), while the decay was described by a double exponential with time constants of 0.70 and 3.43 ms. 3. Cyclothiazide slowed the decay of the fast EPSC, indicating that it is mediated by AMPA receptors. The slower time constant was slowed to a greater extent than the faster time constant. Cyclothiazide potentiated EPSC amplitude, partly by a presynaptic mechanism. 4. The metabotropic glutamate receptor (mGluR) agonists, 1S,3S‐ACPD, 1S,3R‐ACPD and L‐2‐amino‐4‐phosphonobutyrate (L‐AP4) reversibly depressed EPSC amplitude. A dose‐response curve for 1S,3S‐ACPD gave an EC50 of 7 microM and a Hill coefficient of 1.2. 5. Analysis of the coefficient of variation ratio showed that the above mGluR agonists acted presynaptically to reduce the probability of transmitter release. Adenosine and baclofen also depressed transmission by a presynaptic mechanism. 6. alpha‐Methyl‐4‐carboxyphenylglycine (MCPG; 0.5‐1 mM) did not antagonize the effects of 1S,3S‐ACPD, while high concentrations of L‐2‐amino‐3‐phosphonopropionic acid (L‐AP3; 1 mM) and 4‐carboxy‐3‐hydroxyphenyglycine (4C3HPG; 500 microM) depressed transmission. 7. There was a power relationship between [Ca2+]o and EPSC amplitude with co‐operativity values ranging from 1.5 to 3.4. 8. The mechanism by which mGluRs modulate transmitter release appeared to be independent of presynaptic Ca2+ or K+ currents, since ACPD caused no change in the level of paired‐pulse facilitation or the duration of the presynaptic action potential (observed by direct recording from the terminal), indicating that the presynaptic mGluR transduction mechanism may be coupled to part of the exocytotic machinery. 9. Our data are not consistent with the presence at the calyx of Held of any one known mGluR subtype. Comparison of the time course and pharmacology of the fast EPSC with data from cloned AMPA receptors is consistent with the idea that GluR‐Do subunits dominate the postsynaptic channels.

Nitric Oxide Signaling in Brain Function, Dysfunction, and Dementia

Nitric oxide (NO) is an important signaling molecule that is widely used in the nervous system. With recognition of its roles in synaptic plasticity (long-term potentiation, LTP; long-term depression, LTD) and elucidation of calcium-dependent, NMDAR-mediated activation of neuronal nitric oxide synthase (nNOS), numerous molecular and pharmacological tools have been used to explore the physiology and pathological consequences for nitrergic signaling. In this review, the authors summarize the current understanding of this subtle signaling pathway, discuss the evidence for nitrergic modulation of ion channels and homeostatic modulation of intrinsic excitability, and speculate about the pathological consequences of spillover between different nitrergic compartments in contributing to aberrant signaling in neurodegenerative disorders. Accumulating evidence points to various ion channels and particularly voltage-gated potassium channels as signaling targets, whereby NO mediates activity-dependent control of intrinsic neuronal excitability; such changes could underlie broader mechanisms of synaptic plasticity across neuronal networks. In addition, the inability to constrain NO diffusion suggests that spillover from endothelium (eNOS) and/or immune compartments (iNOS) into the nervous system provides potential pathological sources of NO and where control failure in these other systems could have broader neurological implications. Abnormal NO signaling could therefore contribute to a variety of neurodegenerative pathologies such as stroke/excitotoxicity, Alzheimer’s disease, multiple sclerosis, and Parkinson’s disease.

The calyx of Held

The calyx of Held is a large glutamatergic synapse in the mammalian auditory brainstem. By using brain slice preparations, direct patch-clamp recordings can be made from the nerve terminal and its postsynaptic target (principal neurons of the medial nucleus of the trapezoid body). Over the last decade, this preparation has been increasingly employed to investigate basic presynaptic mechanisms of transmission in the central nervous system. We review here the background to this preparation and summarise key findings concerning voltage-gated ion channels of the nerve terminal and the ionic mechanisms involved in exocytosis and modulation of transmitter release. The accessibility of this giant terminal has also permitted Ca2+-imaging and -uncaging studies combined with electrophysiological recording and capacitance measurements of exocytosis. Together, these studies convey the panopoly of presynaptic regulatory processes underlying the regulation of transmitter release, its modulatory control and short-term plasticity within one identified synaptic terminal.

Two Heteromeric Kv1 Potassium Channels Differentially Regulate Action Potential Firing

Low-threshold voltage-gated potassium currents (ILT) activating close to resting membrane potentials play an important role in shaping action potential (AP) firing patterns. In the medial nucleus of the trapezoid body (MNTB), ILT ensures generation of single APs during each EPSP, so that the timing and pattern of AP firing is preserved on transmission across this relay synapse (calyx of Held). This temporal information is critical for computation of sound location using interaural timing and level differences.ILT currents are generated by dendrotoxin-I-sensitive, Shaker-related K+ channels; our immunohistochemistry confirms that MNTB neurons express Kv1.1, Kv1.2, and Kv1.6 subunits. We used subunit-specific toxins to separate ILT into two components, each contributing approximately one-half of the total low-threshold current: (1) ILTS, a tityustoxin-Kα-sensitive current (TsTX) (known to block Kv1.2 containing channels), and (2) ILTR, an TsTX-resistant current. Both components were sensitive to the Kv1.1-specific toxin dendrotoxin-K and were insensitive to tetraethylammonium (1 mm). This pharmacological profile excludes homomeric Kv1.1 or Kv1.2 channels and is consistent withILTS channels being Kv1.1/Kv1.2 heteromers, whereas ILTR channels are probably Kv1.1/Kv1.6 heteromers. Although they have similar kinetic properties,ILTS is critical for generating the phenotypic single AP response of MNTB neurons. Immunohistochemistry confirms that Kv1.1 and Kv1.2 (ILTSchannels), but not Kv1.6, are concentrated in the first 20 μm of MNTB axons. Our results show that heteromeric channels containing Kv1.2 subunits govern AP firing and suggest that their localization at the initial segment of MNTB axons can explain their dominance of AP firing behavior.

Facilitation of the presynaptic calcium current at an auditory synapse in rat brainstem

1 The presynaptic calcium current (IpCa) was recorded from the calyx of Held in rat brainstem slices using the whole‐cell patch clamp technique. 2 Tetanic activation of IpCa by 1 ms depolarizing voltage steps markedly enhanced the amplitude of IpCa. Using a paired pulse protocol, the second (test) response was facilitated with inter‐pulse intervals of less than 100 ms. The facilitation was greater at shorter intervals and was maximal (about 20 %) at intervals of 5–10 ms. 3 When the test pulse duration was extended, the facilitation was revealed as an increased rate of IpCa activation. From the current‐voltage relationship measured at 1 ms from onset, facilitation could be described by a shift in the half‐activation voltage of about −4 mV. 4 I pCa facilitation was not attenuated when guanosine‐5′‐O‐(3‐thiotriphosphate) (GTPγS) or guanosine‐5′‐O‐(2‐thiodiphosphate) (GDPβS) was included in the patch pipette, suggesting that G‐proteins are not involved in this phenomenon. 5 On reducing [Ca2+]o, the magnitude of facilitation diminished proportionally to the amplitude of IpCa. Replacement of [Ca2+]o by Ba2+ or Na+, or buffering of [Ca2+]i with EGTA or BAPTA attenuated IpCa facilitation. 6 We conclude that repetitive presynaptic activity can facilitate the presynaptic Ca2+ current through a Ca2+‐dependent mechanism. This mechanism would be complementary to the action of residual Ca2+ on the exocytotic machinery in producing activity‐dependent facilitation of synaptic responses.

Characterisation of inhibitory and excitatory postsynaptic currents of the rat medial superior olive

1 The medial superior olive (MSO) is part of the binaural auditory pathway, receiving excitatory projections from both cochlear nuclei and an inhibitory input from the ipsilateral medial nucleus of the trapezoid body (MNTB). We characterised the excitatory and inhibitory synaptic currents of MSO neurones in 3‐ to 14‐day‐old rats using whole‐cell patch‐clamp methods in a brain slice preparation. 2 A dual component EPSC was mediated by AMPA and NMDA receptors. The AMPA receptor‐mediated EPSC decayed with a time constant of 1.99 ± 0.16 ms (n= 8). 3 Following blockade of glutamate receptors, a monosynaptic strychnine‐sensitive response was evoked on stimulation of the MNTB, indicative of a glycine receptor‐mediated IPSC. GABAA receptors contributed to IPSCs in rats under 6 days old (bicuculline blocked 30% of the IPSC). In older rats little or no bicuculline‐sensitive component was detectable, except in the presence of flunitrazepam. These glycinergic IPSCs showed a reversal potential that varied with changes in [Cl−]i, as predicted by the Nernst equation. 4 The IPSC exhibited two developmentally relevant changes. (i) At around postnatal day 6, the GABAA receptor‐mediated component declined, leaving a predominant glycine‐mediated IPSC. The isolated glycinergic IPSC decayed with time constants of 7.8 ± 0.3 and 38.3 ± 1.7 ms, with the slower component contributing 7.8 ± 0.6% of the peak amplitude (n= 121, 3‐11 days old, ‐70 mV, 25°C). (ii) After day 11 the IPSC fast decay accelerated to 3.9 ± 0.3 ms (n= 12) and the magnitude of the slow component declined to less than 1%. 5 Spontaneous miniature glycinergic IPSCs (mIPSCs) were variable in amplitude and were of large conductance (1.83 ± 0.19 nS, n= 8). The amplitude was unchanged on lowering [Ca2+]o. 6 The time course of evoked and spontaneous miniature glycinergic IPSCs were compared. The 10‐90% rise times were 0.7 and 0.6 ms, respectively. The evoked IPSC decayed with a fast time constant of 7.2 ± 0.7 ms, while the mIPSC decayed with a fast time constant of 5.3 ± 0.4 ms in the same seven cells. 7 The glycinergic IPSC decay was voltage dependent with an e‐fold change over 118 mV. The temperature dependence of the IPSC decay indicated a Q10 value of 2.1. Picrotoxin and cyanotriphenylborate had little or no effect on IPSCs from 6‐ to 14‐day‐old animals, implying homomeric channels are rare. 8 We conclude that the MSO receives excitatory inputs mediated by AMPA and NMDA receptors and a strong glycinergic IPSC which has a significant contribution from GABAA receptors in neonatal rats. Functionally, the IPSC could increase membrane conductance during the decay of binaural glutamatergic EPSCs, thus refining coincidence detection and interaural timing differences.

Presynaptic K+ channels: electrifying regulators of synaptic terminal excitability

Potassium channels are crucial regulators of neuronal excitability, setting resting membrane potentials and firing thresholds, repolarizing action potentials and limiting excitability. Although most of our understanding of K+ channels is based on somatic recordings, there is good evidence that these channels are present in synaptic terminals. In recent years the improved access to presynaptic compartments afforded by direct recording techniques has indicated diverse roles for native K+ channels, from suppression of aberrant firing to action potential repolarization and activity-dependent modulation of synaptic activity. This article reviews the growing evidence for multiple roles and discrete localization of distinct K+ channels at presynaptic terminals.