Regulation of neuronal plasticity and fear by a dynamic change in PAR1-G protein coupling in the amygdala.

Fear memories are acquired through neuronal plasticity, an orchestrated sequence of events regulated at circuit and cellular levels. The conventional model of fear acquisition assumes unimodal (for example, excitatory or inhibitory) roles of modulatory receptors in controlling neuronal activity and learning. Contrary to this view, we show that protease-activated receptor-1 (PAR1) promotes contrasting neuronal responses depending on the emotional status of an animal by a dynamic shift between distinct G protein-coupling partners. In the basolateral amygdala of fear-naive mice PAR1 couples to Gαq/11 and Gαo proteins, while after fear conditioning coupling to Gαo increases. Concurrently, stimulation of PAR1 before conditioning enhanced, but afterwards it inhibited firing of basal amygdala neurons. An initial impairment of the long-term potentiation (LTP) in PAR1-deficient mice was transformed into an increase in LTP and enhancement of fear after conditioning. These effects correlated with more frequent 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) receptor-mediated miniature post synaptic events and increased neuronal excitability. Our findings point to experience-specific shifts in PAR1-G protein coupling in the amygdala as a novel mechanism regulating neuronal excitability and fear.

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.

Synaptic transmission: well-placed modulators.

Metabotropic glutamate receptors are involved in the modulation of synaptic transmission; their localization in perisynaptic areas would appear to limit their activation by endogenous glutamate, but recent reports suggest that this strategic placement allows use-dependent activation of these synaptic modulators.

The binaural auditory pathway: membrane currents limiting multiple action potential generation in the rat medial nucleus of the trapezoid body.

In this paper we describe the membrane currents of neurons in the medial nucleus of the trapezoid body (MNTB), which serves as an inverting relay in the binaural auditory pathway. In the following paper (Forsythe & Barnes-Davies (Proc. R. Soc. Lond. B 251, 151 (1993))) we describe the synaptic inputs to the MNTB and discuss the significance of these results for transmission through this nucleus, where the fidelity of information transfer will depend on the integration of synaptic responses with the intrinsic postsynaptic membrane properties. Whole-cell patch clamp recordings were made from MNTB neurons using a thin-slice preparation of the rat brain stem. Resting potentials were -70 mV with a neuronal input resistance of 250 M omega and a membrane time constant of 14 ms. Voltage-clamp studies showed that MNTB neurons possess an inward sodium current, an outward current similar to a delayed rectifier and an inward rectifier. In addition, a novel transient outward current exhibiting rapid kinetics and a sustained current are present, which are both blocked by micromolar concentrations of 4-aminopyridine (4AP). Current-clamp recording showed that MNTB neurons respond to depolarization with a single overshooting action potential (AP); 4AP blocked a fast after-hyperpolarization, increased AP duration, and converted the single AP response on depolarization to a train of action potentials.

The binaural auditory pathway: excitatory amino acid receptors mediate dual timecourse excitatory postsynaptic currents in the rat medial nucleus of the trapezoid body.

We show here that synaptic transmission to the medial nucleus of the trapezoid body (MNTB) is mediated principally by excitatory amino acid receptors and has two components. A fast excitatory postsynaptic current (EPSC) is mediated by non-NMDA receptors and a slow EPSC is mediated by NMDA receptors. Each neuron receives a large synaptic input (calyx of Held) which produces an EPSC with a mean peak conductance of 37 nS. The somatic location of this synapse gives good resolution of the EPSC timecourse with the fast EPSC decaying with a time constant of 1.1 ms (at 25 degrees C). The slow EPSC exhibits a double exponential decay with time constants of 41 ms and 106 ms and is voltage dependent in the presence of extracellular magnesium. Other smaller EPSCS mediated by NMDA and non-NMDA receptors, and a strychnine-sensitive synaptic current, are also present. Although the intrinsic membrane properties of MNTB neurons (Forsythe & Barnes-Davies (Proc. R. Soc. Lond. B 251, 143 (1993)), preceding paper) promote high-fidelity transmission, we show that voltage-dependent modulation of synaptic transmission can occur. Given the specialization of the calyx of Held, it seems that the NMDA-receptor ion channel complex is not primarily serving to potentiate a subthreshold input, but may be involved in the development and maintenance of this exuberant somatic synapse.

Elevation of cytosolic calcium by cholinoceptor agonists in SH-SY5Y human neuroblastoma cells: estimation of the contribution of voltage-dependent currents.

1. Muscarinic but not nicotinic receptor stimulation in SH-SY5Y human neuroblastoma cells induces a concentration-dependent increase in [3H]-inositol phosphate formation and a biphasic increase in [Ca2+]i. The latter involves release from both an intracellular store and Ca2+ entry across the plasma membrane. Here we examine the possibility that this agonist-stimulated Ca2+ entry occurs indirectly, as a consequence of depolarization. 2. Electrophysiological characterization, by whole cell patch-clamp techniques revealed that SH-SY5Y cells possess a tetrodotoxin-sensitive inward sodium current, a dihydropyridine-insensitive calcium current and an outward potassium current which was blocked by tetraethylammonium, 4-aminopyridine and intracellular caesium ions. The outward potassium current showed voltage-dependent activation and inactivation, similar to that seen for A-currents. 3. Application of nicotinic agonists evoked an inward current in cells voltage-clamped at negative holding potentials, but this current rectified, resulting in little or no outward current flow at positive potentials. The mean amplitude at a holding potential of -60 mV was -1.14 nA. Extrapolation of the current-voltage relation gave a reversal potential of +8 mV, indicative of a non-specific cationic permeability. 4. Application of muscarinic agonists had no detectable effect in most of the cells tested. However, in one third of cells studied, a small slowly activating inward current was observed. The mean amplitude of this current at a holding potential of -60 mV was -8.3 pA.5. This study confirms that SH-SY5Y cells possess voltage-dependent sodium, potassium and calcium currents. In addition, these cells are strongly depolarized by nicotinic agonists, which produce little change in [Ca2t]1. On the other hand, muscarinic agonists produce profound changes in [Ca2+1J with only a small inward current (depolarization). The contrasting effects of these two cholinoceptor agonists strongly implies that the Ca2+ entry after muscarinic receptor activation is not primarily due to activation of voltage-dependent calcium channels.

Intracellular recording of identified dorsal spinocerebellar tract neurones from guinea pig spinal cord in vitro.

Recent studies on the monosynaptic connection between primary afferent fibres and dorsal spinocerebellar tract (DSCT) neurones in the spinal cord of anaesthetized cats have been undertaken to investigate the mechanisms of excitatory synaptic transmission in the mammalian central nervous system. The need to extend these observations to a study of the effects of changes in the extracellular environment of DSCT neurones prompted us to develop the in vitro preparation described in this paper. We have developed an isolated spinal cord preparation from young guinea pigs, in which DSCT neurones can be identified and recorded from intracellularly. The spinal cord can be maintained in vitro for at least 7 hours. This preparation is sufficiently stable to allow intracellular penetration of DSCT cells in excess of 2 h, with resting membrane potentials of -65 mV obtainable. Reconstruction of neurones stained with horseradish peroxidase confirmed their location in Clarke's column. The dendritic morphology of the reconstructed DSCT neurones was found to be similar to DSCT neurones described in the cat spinal cord. Since the motor system of the guinea pig is quite advanced at birth, this preparation should provide a valid extension to in vivo results on neuronal membrane properties and synaptic potentials in DSCT neurones.

A chamber for electrophysiological recording from cultured neurones allowing perfusion and temperature control.

With the increasing use of cell culture in electrophysiology there has arisen a need for more rigorous control of the extracellular fluid composition and temperature. We describe here an environmental chamber designed to permit perfusion and temperature control of the extracellular medium during electrophysiological studies of cultured or acutely dissociated neurones in 35 mm Petri dishes. The chamber consists of a Peltier driven heat exchange, which allows temperature control above and below ambient. The Petri dish temperature is controlled in 3 ways: (1) through direct conduction from the controlled surface; (2) by flow of gas around the dish; and (3) by flow of extracellular medium to the Petri dish. We demonstrate the temperature control achieved with this equipment and the ability to exchange the extracellular medium in the whole dish. Using the whole-cell-patch recording technique to record from hippocampal neurones, Q10 parameters were estimated for the membrane time constant, input resistance and action potential halfwidth, over a range of 15-36 degrees C. The mean Q10 values (+/- S.E.M.) were 0.71 +/- 0.05, 0.58 +/- 0.03 and 0.35 +/- 0.1, respectively. Because of rapid diffusion and mixing of drugs when using puffer pipettes, quantitative pharmacological studies or manipulations of the extracellular recording medium are not possible. By perfusing the whole recording chamber the final concentration can be accurately known. Using this method we obtained an estimate of the relative potency of the non-specific excitatory amino acid antagonist, kynurenate, for synaptically activated N-methyl-d-aspartate (NMDA) receptors of 1:0.62.

Demonstration of the synaptic origin of primary afferent depolarisation (PAD) in the isolated spinal cord of the hamster.

Intracellular recordings have been made from 31 primary afferent fibres within the dorsal horn of an isolated mammalian spinal cord. In 17 fibres stimulation of an adjacent dorsal root evoked primary afferent depolarization (PAD); these fibres also showed spontaneous depolarizations. Replacement of the calcium in the perfusing medium by manganese blocked both evoked and spontaneous activity showing them to be of synaptic origin. Observations on the effects of current injection and of bicuculline support an involvement of GABA in the generation of PAD.

An investigation of the dorsal root reflex using an in vitro preparation of the hamster spinal cord.

A detailed description is given of an hemisected spinal cord preparation from adult golden hamsters and this preparation has been used to investigate the physiology of the dorsal root reflex. In addition to antidromic reflex discharges which could be recorded from lumbar dorsal roots following stimulation of adjacent dorsal roots or the dorsal columns, spontaneous firing was also recorded from the dorsal roots. This activity reached a peak at 27 degrees C and was abolished at temperatures above 35 degrees C. Both the evoked and the spontaneous dorsal root activity were demonstrated to be travelling antidromically along the dorsal roots out of the cord, and replacement of the calcium in the bathing medium by manganese showed them to be of synaptic origin. Stimulation of a lumbar dorsal root was found to evoke a reflex in up to 4 adjacent spinal segments in both rostral and caudal directions, and a period of depressed activity was demonstrated following both evoked and spontaneous discharges. A time-locked relationship was found between the dorsal root reflex and the slow dorsal horn potential recorded from within the spinal cord.

Spinocerebellar neurones in the guinea pig--a morphological study.

The morphology and distribution of spinocerebellar neurones were examined in the guinea pig. Horseradish peroxidase was injected into the cerebellum, and after a survival time of 72 h retrogradely labelled cells were examined in the spinal cord. The distribution of spinocerebellar cells was similar to that previously demonstrated in the rat. Three major groups of neurones were distinguished: the central cervical nucleus (C1-C2), Clarke's column (T2-L3) and spinal border cells (L3-L6). Neurones in the central cervical nucleus were multipolar, had mean equivalent diameters of about 24 microns, and their axons ascended on the contralateral side of the spinal cord. Neurones in Clarke's column were spindle-shaped, approximately 25 X 35 microns, and their axons ascended on the ipsilateral side of the spinal cord. Spinal border cells were multipolar, with mean equivalent diameters of about 35 microns; their axons were predominantly crossed.

Synaptic and non-synaptic components of the dorsal horn potential in isolated hamster spinal cord.

Slow negative potentials, evoked by stimulation of the lumbar dorsal roots, have been demonstrated in the dorsal horn of an isolated, hemisected spinal cord preparation from golden hamsters. Paired stimuli revealed a period of partial suppression of this slow potential persisting for up to 2 s following the conditioning stimulus, but with high stimulation frequencies this effect was masked and above 20 Hz a tetanic train of stimuli produced a smoothly rising potential. The response evoked by tetanic stimulation was shown to consist of two components, a manganese-sensitive, synaptically generated component, and a manganese-resistant, frequency-dependent element. Treatment with 10(-4) M 4-aminopyridine blocked the manganese-resistant tetanic response but did not reduce the manganese-sensitive component. Bicuculline, picrotoxin and tubocurare had little effect upon the tetanic response, but 10(-3) M procaine blocked it completely. The possibility that the manganese-resistant response was due to the release of potassium ions is considered.

Possible modulatory role of voltage-activated Ca(2+) currents determining the membrane properties of isolated pyramidal neurones of the rat dorsal cochlear nucleus.

Voltage-activated Ca(2+) currents have been studied in pyramidal cells isolated enzymatically from the dorsal cochlear nuclei of 6-11-day-old Wistar rats, using whole-cell voltage-clamp. From hyperpolarized membrane potentials, the neurones exhibited a T-type Ca(2+) current on depolarizations positive to -90 mV (the maximum occurred at about -40 mV). The magnitude of the T-current varied considerably from cell to cell (-56 to -852 pA) while its steady-state inactivation was consistent (E(50)=-88.2+/-1.7 mV, s=-6. 0+/-0.4 mV). The maximum of high-voltage activated (HVA) Ca(2+) currents was observed at about -15 mV. At a membrane potential of -10 mV the L-type Ca(2+) channel blocker nifedipine (10 microM) inhibited approximately 60% of the HVA current, the N-type channel inhibitor omega-Conotoxin GVIA (2 microM) reduced the current by 25% while the P/Q-type channel blocker omega-Agatoxin IVA (200 nM) blocked a further 10%. The presence of the N- and P/Q-type Ca(2+) channels was confirmed by immunochemical methods. The metabotropic glutamate receptor agonist (+/-)-1-aminocyclopentane-trans-1, 3-dicarboxylic acid (200 microM) depressed the HVA current in every cell studied (a block of approximately 7% on an average). The GABA(B) receptor agonist baclofen (100 microM) reversibly inhibited 25% of the HVA current. Simultaneous application of omega-Conotoxin GVIA and baclofen suggested that this inhibition could be attributed to the nearly complete blockade of the N-type channels. Possible physiological functions of the voltage-activated Ca(2+) currents reported in this work are discussed.

N-methyl-D-aspartate receptors influence neuronal survival in developing spinal cord cultures.

Neuronal cell death, which exhibits precise spatial and temporal regulation, serves to remodel and optimize function in the developing nervous system. The mechanisms underlying neuronal cell death are poorly understood, but electrical activity and trophic substances appear to be among the important determinants of survival. We find that N-methyl-D-aspartate (NMDA) receptor antagonists induce neuronal cell death in developing spinal cord cultures. The magnitude of cell death is similar in amount to that produced by blocking action potentials with tetrodotoxin (TTX). The NMDA antagonists and TTX accelerate neuronal death in 2-week-old cultures but not in those that are 1 month old. Low concentrations of NMDA increased neuronal survival under conditions of electrical blockade with TTX. In addition, treatment with low levels of a calcium ionophore also decreased cell death associated with TTX. These results suggest that the NMDA receptor is an important determinant of neuronal survival and that this influence is stage-dependent and likely to be calcium-mediated.

Calcium channels triggering transmitter release in the rat medial superior olive.

We used whole cell voltage clamp recordings from neurones in rat auditory brainstem slices to study the Ca(2+) channel types involved in triggering synaptic glutamate and glycine release in the medial superior olivary nucleus. Glutamate release from the anterior ventral cochlear (aVCN) bushy neurone synapse did not involve L-type Ca(2+) channels (alpha(1C-D); Ca(V)1.2-1.3), but was mediated with similar efficacies by both N-type (alpha(1B); Ca(V)2.2) and the P/Q-type Ca(2+) channels (alpha(1A); Ca(V)2.1). Glycine release from the medial nucleus of the trapezoid body (MNTB) synapse was mediated predominantly by P/Q-type Ca(2+) channels, but with a significant contribution from N-type Ca(2+) channels. Combined application of the P/Q- and N-type Ca(2+) channel toxins, omega-agatoxin IVA and omega-conotoxin GVIA, left a very small remnant of both the inhibitory and excitatory postsynaptic currents, probably reflecting a minimal contribution of R-type Ca(2+) channels (alpha(1E); Ca(V)2.3) to transmitter release. In contrast with aVCN bushy neurones, MNTB somata lacked both T- (alpha(1G-I); Ca(V)3.1-3.3) and L-type channels, but expressed a higher proportion of P/Q-type Ca(2+) channels.

P/Q-type calcium channel ablation in a mice glycinergic synapse mediated by multiple types of Ca²+ channels alters transmitter release and short term plasticity.

Ca(v)2.1 channels (P/Q-type) play a prominent role in controlling neurotransmitter release. Transgenic mice in which the α1A pore-forming subunit of Ca(v)2.1 channels is ablated (KO) provide a powerful tool to study Ca(v)2.1 function in synaptic transmission in vivo. Whole-cell patch clamp was used to measure inhibitory glycinergic postsynaptic currents (IPSCs) from the lateral superior olive (LSO). Comparing wild-type (WT) and KO mice, we investigated the relevance of P/Q-type calcium channels at a glycinergic synapse mediated by multiple types of Ca(2+) channels, in opposition to synapses where only this type of Ca(2+) channels are in charge of transmitter release. We found that in KO mice, N-type and L-type Ca(2+) channels control synaptic transmission, resulting in a functional but reduced glycinergic transmitter release. Pair pulse facilitation of synaptic currents is retained in KO mice, even when synaptic transmission is driven by either N or L-type calcium channels alone, in contrast with lack of this phenomenon in other synapses which are exclusively mediated by P/Q-type channels. Thus, pointing a difference between P/Q- and N-type channels present in single or multiple types of calcium channels driven synapses. Significant alterations in short-term synaptic plasticity were observed. KO mice exhibited a stronger short term depression (STD) of IPSCs during repetitive stimulation at high frequency and recovered with a larger time constant compared to WT mice. Finally, transmitter release at the LSO synapse from KO mice was strongly modulated by presynaptic GTP-binding protein-coupled receptor γ-aminobutyric acid type B (GABA(B)).

Acceleration of AMPA receptor kinetics underlies temperature-dependent changes in synaptic strength at the rat calyx of Held.

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.