Increase In Transmitter Release Research Articles

Augmentation Increases Vesicular Release Probability in the Presence of Masking Depression at the Frog Neuromuscular Junction

Synaptic augmentation is a short-term component of synaptic plasticity that increases transmitter release during repetitive stimulation and decays thereafter with a time constant of approximately 7 sec. Augmentation has typically been observed under conditions where there is little or no depression because of depletion of synaptic vesicles from the readily releasable pool (RRP) of transmitter. We now study augmentation under conditions of pronounced depression at the frog neuromuscular junction to gain additional insight into mechanism. If augmentation reflects an increase in the size of the RRP of transmitter, then augmentation should not be present with depression. Our findings using four different experimental approaches suggested that augmentation was still present in the presence of pronounced depression: mathematical extraction of augmentation from the changes in transmitter release after repetitive stimulation, identification of augmentation with Ba2+, correction of the data for the measured depletion of the RRP, and identification of an augmentation component of residual Ca2+. We conclude that the augmentation machinery still acts to increase transmitter release when depression reduces the RRP sufficiently to mask obvious augmentation. The masked augmentation was found to increase transmitter release by increasing the probability of releasing individual vesicles from the depressed RRP, countering the effects of depression. Because augmentation and depression have similar time courses, either process can mask the other, depending on their relative magnitudes. Consequently, the apparent absence of one of the processes does not exclude that it is still contributing to short-term synaptic plasticity.

New Type of Synaptically Mediated Epileptiform Activity Independent of Known Glutamate and GABA Receptors

It is well known that excitatory synaptic transmission at the hippocampal CA3-CA1 synapse depends on the binding of released glutamate to ionotropic receptors. Here we report that during long-term application of Cs+ (5 mM), stimulation of the Schaffer collateral-commisural pathway evokes an epileptic field potential (Cs-FP) in area CA1 of the rat hippocampal slice, which is resistant to antagonists of ionotropic glutamate and GABA(A) receptors. The Cs-FP was blocked by N-type but not L-type Ca2+ channel antagonists and was attenuated by adenosine (0.5 mM), as expected for a synaptically mediated response. These properties make the Cs-FP fundamentally different from other types of Cs(+)-induced epileptiform activity. Replacement of Cs+ with antagonists of the hyperpolarization-activated nonselective cation current I(h) and inwardly rectifying potassium channels (K(IR)) or partial inhibition of the Na(+)/K+ pump did not cause Cs-FP-like potentials, which indicates that such actions of Cs+ were not responsible for the Cs-FP. The effect of Cs+ was partly mimicked by 4-aminopyridine (4-AP; 2 mM), suggesting that an increase in transmitter release is involved. The group I metabotropic glutamate receptor (mGluR) agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) attenuated the Cs-FP. This effect was not, however, antagonized by group I mGluR antagonists. Selective and nonselective mGluR antagonists did not attenuate the Cs-FP. We conclude that long-term exposure to Cs+ induces a state where excitatory synaptic transmission can exist between area CA3 and CA1 in the hippocampus, independent of ionotropic and metabotropic glutamate receptors and GABA(A) receptors.

α-Synuclein produces a long-lasting increase in neurotransmitter release

Wild-type alpha-synuclein, a protein of unknown function, has received much attention because of its involvement in a series of diseases that are known as synucleinopathies. We find that long-lasting potentiation of synaptic transmission between cultured hippocampal neurons is accompanied by an increase in the number of alpha-synuclein clusters. Conversely, suppression of alpha-synuclein expression through antisense nucleotide and knockout techniques blocks the potentiation, as well as the glutamate-induced increase in presynaptic functional bouton number. Consistent with these findings, alpha-synuclein introduction into the presynaptic neuron of a pair of monosynaptically connected cells causes a rapid and long-lasting enhancement of synaptic transmission, and rescues the block of potentiation in alpha-synuclein null mouse cultures. Also, we report that the application of nitric oxide (NO) increases the number of alpha-synuclein clusters, and inhibitors of NO-synthase block this increase, supporting the hypothesis that NO is involved in the enhancement of the number of alpha-synuclein clusters. Thus, alpha-synuclein is involved in synaptic plasticity by augmenting transmitter release from the presynaptic terminal.

Measurement of presynaptic zinc changes in hippocampal mossy fibers

The hippocampal mossy fiber terminals of CA3 area contain high levels of vesicular zinc that is released in a calcium-dependent way, following high-frequency stimulation. However the properties of zinc release during normal synaptic transmission, paired-pulse facilitation and mossy fiber long-term potentiation are still unknown. Using the fluorescent zinc probe N-(6-methoxy-8-quinolyl)- para-toluenesulfonamide, we measured fast mossy fiber zinc changes indicating that zinc is released following single and low levels of electrical stimulation. The observed presynaptic zinc signals are maintained during the expression of mossy fiber long-term potentiation, assumed to be mediated by an increase in transmitter release, and are enhanced during paired-pulse facilitation. This zinc enhancement is, like paired-pulse facilitation, reduced during established long-term potentiation. The correlation between the paired-pulse evoked zinc and field potential responses supports the idea that zinc is co-released with glutamate.

Postsynaptic depolarisation enhances transmitter release and causes the appearance of responses at “silent” synapses in rat hippocampus

Recent data indicate that most “silent” synapses in the hippocampus are “presynaptically silent” due to low transmitter release rather than “postsynaptically silent” due to “latent” receptors of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type (AMPARs). That synapses bearing only N-methyl- d-aspartate (NMDAR) receptors do exist is suggested by the decreased number of transmission failures during postsynaptic depolarisation and by the presence of NMDA-mediated excitatory postsynaptic currents (EPSCs) in synapses silent at rest. We tested whether these effects could be due to potentiated transmitter release at depolarised postsynaptic potentials rather than removal of Mg 2+ block from NMDARs. Using whole-cell recordings of minimal EPSCs from CA1 and CA3 neurones of hippocampal slices we confirmed decreased incidence of failures at +40 mV as compared with −60 mV. This effect was associated with a gradual increase of EPSC amplitude after switching to +40 mV and with a decrease of paired-pulse facilitation. In initially silent synapses, potentiation of pharmacologically isolated AMPAR-mediated EPSCs was still observed at +40 mV and this persisted after stepping back to −60 mV. All above effects were blocked when the cell was dialysed with the Ca 2+ chelator BAPTA (20 mM). These observations are difficult to reconcile with the “latent AMPAR” hypothesis and suggest an alternative explanation, namely that the reduction in failure rates at positive potentials is due to potentiation of transmitter release following Ca 2+ influx through NMDARs. Our results suggest that silent synapses can be mainly “presynaptically” rather than “postsynaptically silent” and thus increased transmitter release rather than insertion of AMPARs is a major mechanism of early long-term potentiation maintenance.

Postsynaptic depolarisation enhances transmitter release and causes the appearance of responses at ?silent? synapses in rat hippocampus

Recent data indicate that most “silent” synapses in the hippocampus are “presynaptically silent” due to low transmitter release rather than “postsynaptically silent” due to “latent” receptors of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type (AMPARs). That synapses bearing only N -methyl- d -aspartate (NMDAR) receptors do exist is suggested by the decreased number of transmission failures during postsynaptic depolarisation and by the presence of NMDA-mediated excitatory postsynaptic currents (EPSCs) in synapses silent at rest. We tested whether these effects could be due to potentiated transmitter release at depolarised postsynaptic potentials rather than removal of Mg 2+ block from NMDARs. Using whole-cell recordings of minimal EPSCs from CA1 and CA3 neurones of hippocampal slices we confirmed decreased incidence of failures at +40 mV as compared with −60 mV. This effect was associated with a gradual increase of EPSC amplitude after switching to +40 mV and with a decrease of paired-pulse facilitation. In initially silent synapses, potentiation of pharmacologically isolated AMPAR-mediated EPSCs was still observed at +40 mV and this persisted after stepping back to −60 mV. All above effects were blocked when the cell was dialysed with the Ca 2+ chelator BAPTA (20 mM). These observations are difficult to reconcile with the “latent AMPAR” hypothesis and suggest an alternative explanation, namely that the reduction in failure rates at positive potentials is due to potentiation of transmitter release following Ca 2+ influx through NMDARs. Our results suggest that silent synapses can be mainly “presynaptically” rather than “postsynaptically silent” and thus increased transmitter release rather than insertion of AMPARs is a major mechanism of early long-term potentiation maintenance.

Impaired function of alpha2-adrenergic autoreceptors on sympathetic nerves associated with mesenteric arteries and veins in DOCA-salt hypertension.

The present study tested the hypothesis that there is impaired function of alpha(2)-adrenergic autoreceptors and increased transmitter release from sympathetic nerves associated with mesenteric arteries and veins from DOCA-salt rats. High-performance liquid chromatography was used to measure the overflow of ATP and norepinephrine (NE) from electrically stimulated mesenteric artery and vein preparations in vitro. In sham arteries, nerve stimulation evoked a 1.5-fold increase in NE release, whereas in DOCA-salt arteries there was a 3.9-fold increase in NE release over basal levels (P < 0.05). In contrast, stimulated ATP release was not different in DOCA-salt arteries compared with sham arteries. In sham veins, nerve stimulation evoked a 2.9-fold increase in NE release, whereas in DOCA-salt veins there was a 8.4-fold increase in NE release over basal levels (P < 0.05). In sham rats NE release, normalized to basal levels, was greater in veins than in arteries (P < 0.05). The alpha(2)-adrenergic receptor antagonist yohimbine (1 microM) increased ATP and NE release in sham but not DOCA-salt arteries. The alpha(2)-adrenergic receptor agonist UK-14304 (10 microM) decreased ATP release in sham but not DOCA-salt arteries. In sham veins, UK-14304 decreased, but yohimbine increased, NE release; effects that were not observed in DOCA-salt veins. These data show that nerve stimulation causes a greater increase in NE release from nerves associated with veins compared with arteries. In addition, impairment of alpha(2)-adrenergic autoreceptor function in sympathetic nerves associated with arteries and veins from DOCA-salt rats results in increased NE release.

Manipulating transmitter release at the neuromuscular junction of neonatal rats alters the expression of ChAT and GAP-43 in motoneurons

During early postnatal development, motoneurons innervating rat hindlimb muscles die following injury to the sciatic nerve. However, prematurely enhancing transmitter release from nerve terminals of neonatal rats renders motoneurons less vulnerable to nerve injury, whereas reducing transmitter release increases their susceptibility to injury. Thus, alterations in transmitter release may have an influence on motoneuron phenotype. Here we investigated the relationship between the vulnerability of motoneurons to injury, and the expression of proteins associated with axonal growth and neuromuscular transmission. We examined the effect of agents that affect transmitter release from nerve terminals and that have been shown to influence the expression of transmitter and growth related proteins in developing motoneurons in response to nerve injury. In newborn rats, implants containing either 4-aminopyridine (4-AP), to increase transmitter release, or magnesium sulphate (MgSO 4), to decrease release, were applied to the soleus muscle in one hindlimb. The effect of these treatments on the activity of choline acetyltransferase (ChAT) in nerve terminals in the soleus muscle was measured using a radiochemical assay. Levels of GAP-43 in sciatic nerve were also assessed, by Western blot analysis. The results showed that during normal development, there was a gradual increase in ChAT activity during the second week of postnatal development, whereas GAP-43 levels declined sharply between postnatal days 12–14. However, following 4-AP treatment, there was a dramatic increase in ChAT activity in nerve terminals contacting the treated soleus muscles and the levels of GAP-43 in the sciatic nerve declined at an earlier age than normal. Conversely, following treatment with MgSO 4 the normal increase in ChAT activity that occurs during the second postnatal week was delayed, and GAP-43 levels in the sciatic nerve were maintained for significantly longer than normal. Thus, manipulating transmitter release from nerve terminals in neonatal rats alters the normal pattern of expression of transmitter and growth related proteins in developing motoneurons. This alteration in protein expression may influence both the maturation of motoneurons and their ability to withstand nerve injury.

Signalling mechanism coupled to 5-hydroxytryptamine4 receptor-mediated facilitation of fast synaptic transmission in the guinea-pig ileum myenteric plexus.

5-hydroxytryptamine (HT)4 receptor agonists stimulate gastrointestinal motility partly by facilitating acetylcholine release from myenteric neurones. However, the signalling mechanisms that couple 5-HT4 receptor activation to increased transmitter release in the myenteric plexus are unknown. We used conventional intracellular electrophysiological methods to record fast excitatory postsynaptic potentials (fEPSPs) from neurones in the guinea-pig ileum myenteric plexus preparation. The substituted benzamide, renzapride, acted at 5-HT4 receptors to facilitate fEPSPs. This response was mimicked by forskolin, an activator of adenylate cyclase. Facilitation of fEPSPs by renzapride and forskolin was not blocked by treating tissues with pertussis toxin (PTX) (2 h, 2 microg mL-1). Facilitation of fEPSPs caused by renzapride was blocked by the non-selective protein kinase inhibitors, staurosporine (1 micromol L-1) and H-8 (30 micromol L-1) and by the selective protein kinase A (PKA) inhibitor, H-89 (10 micromol L-1). These data indicate that 5-HT4 receptors act via a PTX-resistant mechanism to activate PKA. Protein kinase A activation leads to an increase in transmitter release from myenteric nerve terminals and a facilitation of fast excitatory synaptic transmission.

Genetics of Childhood Disorders: LIV. Learning and Memory, Part 7: Maintenance of Long-Term Potentiation

Before moving on to the processes that contribute to the persistence of long-term potentiation (LTP), we need to address an apparently straightforward issue: which side of the synapse expresses LTP? We have seen that the main events of LTP induction occur in postsynaptic signaling pathways. Does this mean that the locus of LTP expression is also postsynaptic? At the CA3–CA1 synapse, the preponderance of electrophysiological evidence favors the postsynaptic side of the synapse, but this has been a contentious area of research and some data support the hypothesis of increased transmitter release in LTP. The problem of the site of LTP expression has been approached using several different strategies. One method is to determine the rate of glutamate release. The efflux of glutamate from a hippocampal slice can be directly measured, and early work showed an elevation during LTP. However, synaptic release represents only a fraction of total glutamate efflux, and metabolic changes could be largely responsible for the observed increases. A more sophisticated experimental approach exploits the active uptake of synaptic glutamate by glial cells, resulting in graded glial transporter currents that can be recorded. These currents are not increased during LTP, suggesting that the locus of the potentiation is postsynaptic. The hypothesis that LTP expression is restricted to the postsynaptic cell has been challenged by experiments in which vesicle recycling at the CA3–CA1 synapse was monitored using the presynaptic loading of a fluorescent dye. An increase in vesicle recycling would indicate that glutamate-containing vesicles were being turned over at a higher rate, presumably due to increased synaptic release. Such an effect occurred following LTP-inducing stimulation, supporting a presynaptic contribution to the expression of LTP. A second approach to identifying the locus of LTP exploits the voltage-dependent block of N-methyl-D-aspartate (NMDA) receptors described in the previous column, a property that is not shared by (AMPA)-type receptors. This difference can be used to separate the synaptic response into AMPA and NMDA components because the contribution of the NMDA-mediated current will depend upon the postsynaptic membrane voltage. If an increase in glutamate release is responsible for LTP, then the AMPA and NMDA receptor-mediated responses should increase equally. But most studies show that only the AMPA component is potentiated during LTP, consistent with a postsynaptic change that is restricted to AMPA receptors.

Solution Structure and Function of the “Tandem Inactivation Domain” of the Neuronal A-type Potassium Channel Kv1.4

Cumulative inactivation of voltage-gated (Kv) K(+) channels shapes the presynaptic action potential and determines timing and strength of synaptic transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type inactivation gating. Different from all other Kvalpha subunits, Kv1.4 harbors two inactivation domains at its N terminus. Here we report the solution structure and function of this "tandem inactivation domain" using NMR spectroscopy and patch clamp recordings. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, whereas ID2 (residues 40-50) is a 2.5-turn helix made up of small hydrophobic amino acids. Functional analysis suggests that only ID1 may work as a pore-occluding ball domain, whereas ID2 most likely acts as a "docking domain" that attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2 slows inactivation considerably and largely impairs cumulative inactivation. Together, the concerted action of ID1 and ID2 may promote rapid inactivation of Kv1.4 that is crucial for the channel function in short term plasticity.

L-type voltage-gated calcium channels are involved in the in vivo and in vitro expression of fear conditioning.

Fear conditioning, a behavioral model of fear learning and cue-related anxiety, causes enhanced neuronal transmission in the thalamic to lateral amygdala pathway.(1,2) In the expression phase of learned fear, this increased transmission recorded in vitro is revealed in increased amplitudes of excitatory postsynaptic currents (EPSCs) and occlusion of paired-pulse facilitation (PPF) implicating a presynaptic increase in transmitter release. Here we examined the contribution of L-type calcium channels in fear conditioning. We measured the effect of nimodipine (Nim, 1.5-20 mg/kg), an L-type calcium channel antagonist, on fear-potentiated startle in which startle was assessed in animals receiving paired or unpaired tone and foot shock. Nim administered intraperitoneally blocked fear-potentiated startle but not baseline startle in a dose-dependent manner. We also analyzed the effect of Nim (10 micro M) in vitro on synaptic facilitation of EPSCs and PPF in slices from naïve control, unpaired control, and fear-conditioned animals. In neurons from naïve control animals, Nim had no effect on EPSC amplitude or PPF, but in slices from fear-conditioned rats, Nim reduced EPSC amplitude, suggesting the recruitment of L-type calcium channels within the fear-conditioning pathway. Nim increased PPF in slices from fear-conditioned animals, suggesting that L-type calcium channels may contribute to increased probability of release in fear conditioning. In slices from unpaired animals, Nim decreased synaptic transmission but had little effect on PPF, suggesting that stress or contextual fear learning may induce L-type channel activity in fear-conditioned and unpaired control animal groups. We also analyzed protein expression of the alpha(1C) and alpha(1D) L-type calcium channel subunits isolated from the amygdala and found that alpha(1C) protein was significantly increased in fear-conditioned animals. These findings suggest that L-type calcium channels play a role in the amygdala in cued fear conditioning and have important implications in the treatment of anxiety and in emotional learning and plasticity.

Synaptic modulation by a neuropeptide depends on temperature and extracellular calcium.

The crayfish neuropeptide DRNFLRFamide increases transmitter release from synaptic terminals onto muscle cells. As temperature decreases from 20 to 8 degrees C, the size of excitatory junctional potentials (EJPs) decreases, and the peptide becomes more effective at increasing EJP amplitude. The goal of the present study was to determine whether the enhanced effectiveness of the peptide is strictly a temperature-related effect, or whether it is related to the fact that the EJPs are smaller at low temperature, allowing a greater range for EJP amplitude to increase. Decreasing temperature reduced the number of quanta of transmitter released per nerve impulse (assessed by recording synaptic currents) and increased input resistance in muscle fibers. As in earlier work, the ability of the peptide to increase EJP amplitude was enhanced by decreasing temperature. However, the peptide was also more effective at increasing EJP amplitude when transmitter output was lowered by reducing the ratio of calcium to magnesium ions in the bath. Thus the effectiveness of the peptide may be related to the level of output from the synaptic terminals.

5-hydroxyindole causes convulsions and increases transmitter release in the CA1 region of the rat hippocampus.

1 5-hydroxyindole (5-OHi) is a proposed tryptophan metabolite able to cause convulsions when systemically injected into rodents. We studied its effects using microdialysis in vivo and electrophysiological approaches in vitro. 2 Local administration of 5-OHi into the CA1 region of the rat hippocampus, via a microdialysis probe, significantly increased glutamate concentrations in the dialysates. 3 In rat hippocampal slices, using extracellular recordings in the CA1 region, 5-OHi (30-300 microM) increased the amplitude of population spikes and fEPSPs. 4 In the same preparation, using intracellular recordings in CA1 pyramidal neurons, 5-OHi reduced the latency of firing induced by direct depolarization and increased both evoked excitatory and slow inhibitory postsynaptic potential amplitudes, without affecting the resting membrane potential, the after-hyperpolarization or the neuronal input resistance. It also altered GABA(A)-mediated neurotransmission by increasing the frequency and the amplitude of pharmacologically isolated spontaneous inhibitory postsynaptic currents (sIPSC). 5 In separate experiments, performed by measuring AMPA or NMDA-induced depolarization in cortical wedges, 5-OHi did not modify glutamate receptor agonist responses. 6 Our results show that 5-OHi causes convulsions, modifies the properties and the function of the hippocampal circuitry, and facilitates the output of both excitatory and inhibitory transmitters.

Aβ(25–35) and Aβ(1–40) act on different calcium channels in CA1 hippocampal neurons

The acute effects of β-amyloid (25–35) and (1–40) on high voltage activated calcium channels were compared in CA1 pyramidal cells of adult mouse hippocampal slices using the whole-cell patch-clamp recording. Bath application of oligomeric β-amyloid (25–35) reversibly increased the barium current ( I Ba) to 1.61 (normalized amplitude), while oligomeric β-amyloid (1–40) reversibly enhanced the I Ba to 1.74. Reverse-sequence β-amyloid [(35–25) and (40–1)] had no effect. The effect of β-amyloid (25–35) was blocked by nifedipine, a selective antagonist of L-type calcium channels. In contrast, the effect of β-amyloid (1–40) was not blocked by nifedipine and I Ba was enhanced to 4.96. It is concluded that these oligomeric peptides may act through different types of calcium channels and/or receptors. The toxicity of Aβ(25–35) implicates a potentiation of L-type calcium channels while the one of Aβ(1–40) is related to an increase of non-L-type calcium channels, which may involve an increase in transmitter release.

Transmitter release from Rana pipiens vestibular hair cells via mGluRs: A role for intracellular Ca ++ release

The response of the semicircular canal (SCC) to the group I mGluR-selective agonist dihydroxyphenylglycine (DHPG; 300 μM) – facilitation of afferent discharge rate – was dose-dependently reduced by the phospholipase C inhibitor U-73122 (1–100 μM; IC 50: 22 μM), the smooth endoplasmic reticulum Ca ++ ATPase inhibitor thapsigargin (100 nM–3 μM; IC 50: 500 nM), and xestospongin C (100 pM–1 μM; IC 50: 11 nM), an inositol trisphosphate receptor (IP 3R) antagonist. Ryanodine, a modulator of Ca ++-induced Ca ++ release, biphasically facilitated, then suppressed this response (1 nM–1 mM; approximate IC 50: 50 μM). 5 mM caffeine increased the amplitude (34.6±13.4%) and duration (453±169.8%; n=4) of the response of the SCC to DHPG, while 50 mM caffeine eliminated this response ( n=2). The protein kinase C inhibitor bisindolylmaleimide I-HCl (10–100 μM; n=3) and the cyclic-ADP ribose antagonist 8-Br-cyclic-ADP ribose (1–10 μM; n=3) had no effect on the response of the SCC to DHPG. These data suggest that the increase in transmitter release following activation of group I mGluRs on vestibular hair cells is associated with intracellular Ca ++ release from both IP 3-sensitive and ryanodine/caffeine-sensitive intracellular Ca ++ stores. Such positive feedback on transmitter release may serve to enhance the contrast between the spontaneous and stimulus-evoked modes of hair cell transmitter release, thereby optimizing signal discrimination at the synapse between hair cells and vestibular afferent fibers.

Calpain inhibitors: a treatment for Alzheimer's disease.

Activation of the calpain system might contribute to the impairment of synaptic transmission in Alzheimer's disease (AD) (Liu et al., 1999; Rapoport, 1999; Selkoe, 1994). Calpains regulate the function of many proteins by limited proteolysis and initiate the complete degradation of other proteins. In particular, they modulate processes that govern the function and metabolism of proteins key to the pathogenesis of AD, including tau and amyloid precursor protein (APP). (Xie and Johnson, 1998; Wang, 2000). We have found that overexpression of APP(K670M:N671L) and PS1(M146L) proteins in hippocampal cultures derived from transgenic mice causes an increase in the frequency of spontaneous release of neurotransmitter. We have also found that calpain immunoreactive clusters are co-localized with immunoreactivity for the vesicle-associated presynaptic marker, synaptophysin. Moreover, application of calpain inhibitor reduces the frequency of spontaneous release of neurotransmitter. Therefore, we have hypothesized that calpains might contribute to the increase in transmitter release. Based on this hypothesis, we propose to test whether it is possible to restore normal synaptic transmission between cells derived from the transgenic model of AD by using calpain inhibitors. The transgenic mouse model also shows spatial learning impairment, a phenomenon that is thought to be associated with plastic changes at synaptic level. Therefore, we will also test whether we can rescue the learning impairment through a treatment with calpain inhibitors.

Chronic morphine treatment alters endogenous opioid control of hippocampal mossy fiber synaptic transmission.

Synaptic adaptations are thought to be an important component of the consequences of drug abuse. One such adaptation is an up-regulation of adenylyl cyclase that has been shown to increase transmitter release at several inhibitory synapses. In this study the effects of chronic morphine treatment were studied on mossy fiber synapses in the guinea pig hippocampus using extracellular field potential recordings. This opioid-sensitive synapse was chosen because of the known role of the adenylyl cyclase cascade in the regulation of glutamate release. Long-term potentiation (LTP) at the mossy fiber synapse was enhanced after chronic morphine treatment. In control animals, opioid antagonists increased LTP but had no effect in morphine-treated guinea pigs. In contrast, the long-lasting depression of transmission induced by a mGluR agonist and CA1 LTP were not altered. Chronic morphine treatment neither caused tolerance to mu- and kappa-receptor-mediated inhibition at the mossy fiber synapse nor modified total hippocampal dynorphin levels. The results suggest that the phasic inhibition of glutamate transmission mediated by endogenous opioids is reduced after chronic exposure to morphine.

Regulation of acetylcholine release by muscarinic receptors at the mouse neuromuscular junction depends on the activity of acetylcholinesterase.

Muscarinic acetylcholine receptors (mAChRs) play an important role in regulating the release of acetylcholine (ACh) in various tissues. We used subtype-specific antibodies and a fluorescent-labelled muscarinic toxin to demonstrate that mammalian neuromuscular junction expresses mAChR subtypes M1 to M4, and that localization of all subtypes is highly restricted to the innervated part of the muscle. To elucidate the roles of the mAChR subtypes regulating ACh release, we measured the mean quantal content of endplate potentials in isolated mouse phrenic--hemidiaphragm preparations in which release was reduced by a low Ca2+/high Mg2+ medium. Muscarine decreased evoked ACh release in normal junctions but, depending on the concentration, reduced or increased transmitter release in collagen Q-deficient junctions completely lacking acetylcholinesterase (AChE). Both effects were also seen in normal junctions when AChE was inhibited by various doses of fasciculin-2. Block of mAChRs by atropine had no effect on evoked release at normal junctions, but decreased release at junctions lacking AChE. The muscarine-elicited depression of ACh release in normal junctions was completely abolished by pertussis toxin or methoctramine pretreatment, but was not affected by muscarinic toxin MT-3, thus indicating the involvement of the M2 mAChR. The muscarine-induced increase of ACh release in AChE-deficient junctions was not affected by pertussis toxin, but was completely blocked by MT-7, a specific M1 mAChR antagonist. Our results show that the M1 and M2 mAChRs have opposite presynaptic functions in modulating quantal ACh release, and that regulation of release by the two receptor subtypes depends on the functional state of AChE at the neuromuscular junction.

Expression of fear-conditioning is accompanied by increased paired-pulse depression within the amygdala

Fear-conditioning is a model of fear learning and anxiety. The lateral nucleus of the amygdala (LA) provides a critical link for relaying thalamic and cortical auditory information to the rest of the amygdala during the fear conditioning process. Alterations in excitatory synaptic transmission in the thalamic to LA pyramidal cells was studied using whole-cell patch clamp recordings in brain slices from fear-conditioned animals. Following paired stimulation of the thalamic afferents, paired-pulse depression (PPD) could be recorded at 200-ms to 2-s intervals. Increasing transmitter release by decreasing the Mg 2+/Ca 2+ ratio enhanced PPD suggesting that PPD is reflective of changes in release probability. Analysis of the pairs of composite, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl- d-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs) showed that there was no correlation between EPSC pairs suggesting that PPD may be mediated through a release-independent mechanism of presynaptic origin. However, AMPA and NMDA receptor mediated PPD had a different time course and magnitude suggesting postsynaptic factors may be involved in PPD. After fear-conditioning PPD of the composite and AMPA receptor-mediated EPSCs was enhanced suggesting that neurotransmitter release may be increased in learned fear. The NMDA receptor-mediated PPD was however not altered in fear-conditioned animals. The differences in response of AMPA and NMDA receptor-mediated PPD suggest that postsynaptic mechanisms may also be involved in the expression of fear conditioning.