ProNGF\NGF imbalance triggers learning and memory deficits, neurodegeneration and spontaneous epileptic-like discharges in transgenic mice.

ProNGF, the precursor of mature nerve growth factor (NGF), is the most abundant form of NGF in the brain. ProNGF and mature NGF differ significantly in their receptor interaction properties and in their bioactivity. ProNGF increases markedly in the cortex of Alzheimer's disease (AD) brains and proNGF\NGF imbalance has been postulated to play a role in neurodegeneration. However, a direct proof for a causal link between increased proNGF and AD neurodegeneration is lacking. In order to evaluate the consequences of increased levels of proNGF in the postnatal brain, transgenic mice expressing a furin cleavage-resistant form of proNGF, under the control of the neuron-specific mouse Thy1.2 promoter, were derived and characterized. Different transgenic lines displayed a phenotypic gradient of neurodegenerative severity features. We focused the analysis on the two lines TgproNGF#3 and TgproNGF#72, which shared learning and memory impairments in behavioral tests, cholinergic deficit and increased Aß-peptide immunoreactivity. In addition, TgproNGF#3 mice developed Aß oligomer immunoreactivity, as well as late diffuse astrocytosis. Both TgproNGF lines also display electrophysiological alterations related to spontaneous epileptic-like events. The results provide direct evidence that alterations in the proNGF/NGF balance in the adult brain can be an upstream driver of neurodegeneration, contributing to a circular loop linking alterations of proNGF/NGF equilibrium to excitatory/inhibitory synaptic imbalance and amyloid precursor protein (APP) dysmetabolism.

Postsynaptic alteration of NR2A subunit and defective autophosphorylation of alphaCaMKII at threonine-286 contribute to abnormal plasticity and morphology of upper motor neurons in presymptomatic SOD1G93A mice, a murine model for amyotrophic lateral sclerosis.

Although amyotrophic lateral sclerosis (ALS) has long been considered as a lower motor neuron (MN) disease, degeneration of upper MNs arising from a combination of mechanisms including insufficient growth factor signaling and enhanced extracellular glutamate levels is now well documented. The observation that these mechanisms are altered in presymptomatic superoxide dismutase (SOD1) mice, an ALS mouse model, suggests that defective primary motor cortex (M1) synaptic activity might precede the onset of motor disturbances. To examine this point, we assessed the composition of AMPAR and NMDAR subunits and of the alphaCa²(+)/calmodulin-dependent kinase autophosphorylation at threonine-286 in the triton insoluble fraction from the M1 in postnatal P80-P85 SOD1(G93A) and wild-type mice. We show that presymptomatic SOD1(G93A) exhibit a selective decrease of NR2A subunit expression and of the alphaCa²(+)/calmodulin-dependent kinase autophosphorylation at threonine-286 in the triton insoluble fraction of upper MNs synapses. These molecular alterations are associated with synaptic plasticity defects, and a reduction in upper MN dendritic outgrowth revealing that abnormal neuronal connectivity in the M1 region precedes the onset of motor symptoms. We suggest that the progressive disruption of M1 corticocortical connections resulting from the SOD1(G93A) mutation might extend to adjacent regions and promote development of cognitive/dementia alterations frequently associated with ALS.

Depression of mGluR-mediated IPSCs by 5-HT in dopamine neurons of the rat substantia nigra pars compacta.

Dopamine neurons of the substantia nigra pars compacta receive a prominent serotonin (5-HT) projection from the dorsal raphe nucleus and important functional interactions between the serotonergic and the dopaminergic system have been postulated. In the present report we examined the role of 5-HT in the modulation of the metabotropic glutamate receptor-mediated inhibitory postsynaptic current (mGluR-IPSC) in midbrain dopamine neurons, and we found a reversible depression of this synaptic response at concentrations of 5-HT ranging from 100 nm to 30 microm (EC50 1.06 microm). This resulted in a shift towards excitation of the overall dopamine neuron response to glutamatergic synaptic input. This effect was not because of a direct modulation of the Ca2+-sensitive K+ conductances underlying the mGluR-IPSC, but was associated with a decrease in the intracellular calcium signal triggered by mGluR stimulation. Similar results were obtained with alpha-methyl-5-hydroxytryptamine and 5-methoxytryptamine, but not with 5-carboxamidotryptamine or 1-(3-chlorophenyl) piperazine. No significant depression of the mGluR-IPSC by 5-HT was observed in the presence of the 5-HT2 antagonist cinanserin or the 5-HT4 receptor antagonist RS 23597-190, whereas the 5-HT2C antagonist RS 102221 was ineffective. Our results demonstrate a powerful inhibition of the mGluR-IPSC by 5-HT in midbrain dopamine neurons, most probably through stimulation of 5-HT2A and 5-HT4 receptors.

Altered long-term corticostriatal synaptic plasticity in transgenic mice overexpressing human CU/ZN superoxide dismutase (GLY(93)-->ALA) mutation.

Apart from the extensive loss of motor neurons, degeneration of midbrain dopaminergic cells has been described in both familial and sporadic forms of amyotrophic lateral sclerosis (ALS). Mice overexpressing the mutant human Cu/Zn superoxide dismutase (SOD1) show an ALS-like phenotype in that they show a progressive death of motor neurons accompanied by degeneration of dopaminergic cells. To describe the functional alterations specifically associated with this dopaminergic dysfunction, we have investigated the corticostriatal synaptic plasticity in mice overexpressing the human SOD1 (SOD1+) and the mutated (Gly(93)-->Ala) form (G93A+) of the same enzyme. We show that repetitive stimulation of the corticostriatal pathway generates long-term depression (LTD) in SOD1+ mice and in control (G93A-/SOD1-) animals, whereas in G93A+ mice the same stimulation generates an N-methyl-D-aspartic acid receptor-dependent long-term potentiation. No significant alterations were found in the intrinsic membrane properties of striatal medium spiny neurons and basal corticostriatal synaptic transmission of G93A+ mice. Bath perfusion of dopamine or the D(2) dopamine receptor agonist quinpirole restored LTD in G93A+ mice. Consistent with these in vitro results, habituation of locomotor activity and striatal-dependent active avoidance learning were impaired in G93A+ mice. Thus, degeneration of dopaminergic neurons in the substantia nigra of G93A+ mice causes substantial modifications in striatal synaptic plasticity and related behaviors, and may be a cellular substrate of the extrapyramidal motor and cognitive disorders observed in familial and sporadic ALS.

Glutamate receptor stimulation induces a persistent rhythmicity of the GABAergic inputs to rat midbrain dopaminergic neurons.

The substantia nigra pars compacta and the ventral tegmental area are part of a complex network in the basal ganglia involved in behaviours as diverse as motor planning, generation of pleasure and drug addiction. Here we report that in the dopaminergic neurons of the rat ventral midbrain a brief coactivation of group I metabotropic and NMDA glutamate receptors may transform a temporally dispersed synaptic GABAergic input into a rhythmic pattern (range 4.5-22.5 Hz), probably through a mechanism involving electrotonic couplings. The plastic and long-lasting modification in the temporal code of the inhibitory synaptic activity induced by glutamate may be a key element in determining the function of midbrain dopaminergic neurons in both normal and pathological behaviour.

Differences in amplitude-voltage relations between minimal and composite mossy fibre responses of rat CA3 hippocampal neurons support the existence of intrasynaptic ephaptic feedback in large synapses.

Computer simulations and electrophysiological experiments have been performed to test the hypothesis on the existence of an ephaptic interaction in purely chemical synapses. According to this hypothesis, the excitatory postsynaptic current would depolarize the presynaptic release site and further increase transmitter release, thus creating an intrasynaptic positive feedback. For synapses with the ephaptic feedback, computer simulations predicted non-linear amplitude-voltage relations and voltage dependence of paired-pulse facilitation. The deviation from linearity depended on the strength of the feedback determined by the value of the synaptic cleft resistance. The simulations showed that, in the presence of the intrasynaptic feedback, recruitment of imperfectly clamped synapses and synapses with linear amplitude-voltage relations tended to reduce the non-linearity and voltage dependence of paired-pulse facilitation. Therefore, the simulations predicted that the intrasynaptic feedback would particularly affect small excitatory postsynaptic currents induced by activation of electrotonically close synapses with long synaptic clefts. In electrophysiological experiments performed on hippocampal slices, the whole-cell configuration of the patch-clamp technique was used to record excitatory postsynaptic currents evoked in CA3 pyramidal cells by activation of large mossy fibre synapses. In accordance with the simulation results, minimal excitatory postsynaptic currents exhibited "supralinear" amplitude-voltage relations at hyperpolarized membrane potentials, decreases in the failure rate and voltage-dependent paired-pulse facilitation. Composite excitatory postsynaptic currents evoked by activation of a large amount of presynaptic fibres typically bear linear amplitude-voltage relationships and voltage-independent paired-pulse facilitation. These data are consistent with the hypothesis on a strong ephaptic feedback in large mossy fibre synapses. The feedback would provide a mechanism whereby signals from large synapses would be amplified. The ephaptic feedback would be more effective on synapses activated in isolation or together with electrotonically remote inputs. During synchronous activation of a large number of neighbouring inputs, suppression of the positive intrasynaptic feedback would prevent abnormal boosting of potent signals.

Postsynaptic hyperpolarization increases the strength of AMPA-mediated synaptic transmission at large synapses between mossy fibers and CA3 pyramidal cells.

In chemical synapses information flow is polarized. However, the postsynaptic cells can affect transmitter release via retrograde chemical signaling. Here we explored the hypothesis that, in large synapses, having large synaptic cleft resistance, transmitter release can be enhanced by electrical (ephaptic) signaling due to depolarization of the presynaptic release site induced by the excitatory postsynaptic current itself. The hypothesis predicts that, in such synapses, postsynaptic hyperpolarization would increase response amplitudes "supralinearly", i.e. stronger than predicted from the driving force shift. We found supralinear increases in the amplitude of minimal excitatory postsynaptic potential (EPSP) during hyperpolarization of CA3 pyramidal neurons. Failure rate, paired-pulse facilitation, coefficient of variation of the EPSP amplitude and EPSP quantal content were also modified. The effects were especially strong on mossy fiber EPSPs (MF-EPSPs) mediated by the activation of large synapses and identified pharmacologically or by their kinetics. The effects were weaker on commissural fiber EPSPs mediated by smaller and more remote synapses. Even spontaneous membrane potential fluctuations were associated with supralinear MF-EPSP increases and failure rate reduction. The results suggest the existence of a novel mechanism for retrograde control of synaptic efficacy from postsynaptic membrane potential and are consistent with the ephaptic feedback hypothesis.

Alpha(1)-adrenoceptor-mediated excitation of substantia nigra pars reticulata neurons.

The effect of noradrenaline was studied in principal neurons of the substantia nigra pars reticulata in rat brain slices using patch clamp recordings. Perfusion of noradrenaline or the alpha(1)-adrenoceptor agonist phenylephrine increased the spontaneous firing activity of reticulata cells. The alpha(1)-adrenoceptor antagonist prazosin counteracted the effects of noradrenaline. In contrast, the beta-adrenoceptor agonist isoproterenol did not affect the activity of reticulata cells and the beta-adrenoceptor antagonist pindolol did not prevent noradrenaline's effect. In whole-cell recordings, at -60 mV holding potential, noradrenaline caused a tetrodotoxin-resistant inward current with a time-course similar to the increase in firing activity. Analysis of the reversal potential of this current did not give homogeneous results. The net noradrenaline current could be associated with a conductance decrease or increase, or in some cases it did not reverse over a range from -120 to -30 mV. It is suggested that noradrenaline increases the excitability of substantia nigra reticulata cells through alpha(1)-adrenoceptors. Both a reduction and an increase in membrane conductance may mediate this effect. The increase in the tonic firing of principal reticulata cells caused by noradrenaline may have significant consequences in regulating the final output of the basal ganglia and consequently in motor-related behaviours.

Tissue plasminogen activator controls multiple forms of synaptic plasticity and memory.

Induction of long-term depression (LTD) in rat striatal slices revealed that this form of synaptic plasticity is coupled to an increased expression of tissue-plasminogen activator (t-PA) mRNA, as detected by the mRNA differential display technique. To further investigate the involvement of this gene in synaptic remodelling following striatal LTD, we recorded electrical activity from mice lacking the gene encoding t-PA (t-PA-KO) and from wild-type (WT) mice. Tetanic stimulation induced LTD in the large majority of striatal neurons recorded from WT mice. Conversely, LTD was absent in a significant proportion of striatal neurons obtained from mice lacking t-PA. Electrophysiological recordings obtained from hippocampal slices in the CA1 area showed that mainly the late phase of long-term potentiation (LTP) was reduced in t-PA-KO mice. Learning and memory-related behavioural abnormalities were also found in these transgenic mice. Disruption of the t-PA gene, in fact, altered both the context conditioning test, a hippocampus-related behavioural task, and the two-way active avoidance, a striatum-dependent task. In an open field object exploration task, t-PA-KO mice expressed deficits in habituation and reactivity to spatial change that are consistent with an altered hippocampal function. Nevertheless, decreased rearing and poor initial object exploration were also observed, further suggesting an altered striatal function. These data indicate that t-PA plays a critical role in the formation of various forms of synaptic plasticity and memory.

Muscarinic receptors depress GABAergic synaptic transmission in rat midbrain dopamine neurons.

The effects of muscarine and nicotine on evoked and spontaneous release of GABA were studied using intracellular and whole-cell patch-clamp recordings from rat midbrain dopamine neurons in an in vitro slice preparation. Muscarine (30 microM) reversibly depressed the pharmacologically isolated inhibitory postsynaptic potential evoked by local electrical stimulation. The maximal inhibition of the inhibitory postsynaptic potential amplitude was 39.6+/-5%. This depressant effect of muscarine was blocked by the M3/M1 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (100 nM), but was slightly affected by the M1/M3 receptor antagonist pirenzepine (1 microM). In addition, muscarine decreased the frequency of the miniature synaptic currents without any effect on their amplitude. Moreover, muscarine did not change the GABA-induced hyperpolarization, indicating that its effect on the inhibitory postsynaptic potential is mediated by presynaptic receptors. On the contrary, the cholinergic agonist nicotine did not change the frequency or the amplitude of the spontaneous glutamatergic and GABAergic synaptic currents. Our data indicate that a prevalent activation of presynaptic M3 muscarinic receptors inhibits the GABA-mediated synaptic events, while the activation of nicotinic receptors does not affect the release of glutamate and GABA on midbrain dopamine neurons.

Long-term synaptic changes induced by intracellular tetanization of CA3 pyramidal neurons in hippocampal slices from juvenile rats.

Minimal excitatory postsynaptic potentials were evoked in CA3 pyramidal neurons by activation of the mossy fibres in hippocampal slices from seven- to 16-day-old rats. Conditioning intracellular depolarizing pulses were delivered as 50- or 100-Hz bursts. A statistically significant depression and potentiation was induced in four and five of 13 cases, respectively. The initial state of the synapses influenced the effect: the amplitude changes correlated with the pretetanic paired-pulse facilitation ratio. Afferent (mossy fibre) tetanization produced a significant depression in four of six inputs, and no significant changes in two inputs. Quantal content decreased or increased following induction of the depression or potentiation, respectively, whereas no significant changes in quantal size were observed. Compatible with presynaptic maintenance mechanisms of both depression and potentiation, changes in the mean quantal content were associated with modifications in the paired-pulse facilitation ratios, coefficient of variation of response amplitudes and number of response failures. Cases were encountered when apparently "presynaptically silent" synapses were converted into functional synapses during potentiation or when effective synapses became "presynaptically silent" when depression was induced, suggesting respective changes in the probability of transmitter release. It is concluded that, in juvenile rats, it is possible to induce lasting potentiation at the mossy fibre-CA3 synapses by purely postsynaptic stimulation, while afferent tetanization is accompanied by long-lasting depression. The data support the existence not only of a presynaptically induced, but also a postsynaptically induced form of long-term potentiation in the mossy fibre-CA3 synapse. Despite a postsynaptic induction mechanism, maintenance of both potentiation and depression is likely to occur presynaptically.

Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices.

Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices. Giant depolarizing potentials (GDPs) are generated by the interplay of the depolarizing action of GABA and glutamate. In this study, single and dual whole cell recordings (in current-clamp configuration) were performed from CA3 pyramidal cells in hippocampal slices obtained from postnatal (P) days P1- to P6-old rats to evaluate the role of ionotropic glutamate receptors in GDP generation. Superfusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10-40 microM) completely blocked GDPs. However, in the presence of CNQX, it was still possible to re-induce the appearance of GDPs with GABA (20 microM) or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxadepropionate (AMPA) (5 microM). This effect was prevented by the more potent and selective AMPA receptor antagonist GYKI 53655 (50-100 microM). In the presence of GYKI 53655, both kainic or domoic acid (0.1-1 microM) were unable to induce GDPs. In contrast, bath application of D-(-)-2-amino-5-phosphonopentanoic acid (50 microM) or (+)-3-(2carboxy-piperazin-4-yl)-propyl-L-phosphonic acid (20 microM) produced only a 37 +/- 9% (SE) and 36 +/- 11% reduction in GDPs frequency, respectively. Cyclothiazide, a selective blocker of AMPA receptor desensitization, increased GDP frequency by 76 +/- 14%. Experiments were also performed with an intracellular solution containing KF to block GABAA receptor-mediated responses. In these conditions, a glutamatergic component of GDP was revealed. GDPs could still be recorded synchronous with those detected simultaneously with KCl-filled electrodes, although their amplitude was smaller. Similar results were found in pair recordings obtained from minislices containing only a small portion of the CA3 area. These data suggest that GDP generation requires activation of AMPA receptors by local release of glutamate from recurrent collaterals.

A novel form of long-term depression in the CA1 area of the adult rat hippocampus independent of glutamate receptors activation.

In young rats, low frequency (1-2 Hz) stimulation of the Schaffer collaterals for 15 min induces in the CA1 area of the hippocampus a homosynaptic and N-methyl-D-aspartate receptor-dependent form of long-term depression (LTD) of synaptic efficacy. In the adults, while a similar stimulation paradigm is able to depress previously potentiated synapses, it leads to conflicting results when applied to naive synapses. In the present experiments, different stimulation paradigms have been used to induce LTD in the CA1 area of the adult rat hippocampus in vitro. Thus, stimulation of the afferent pathway at frequencies higher than those used to produce LTD in young animals (5-10 Hz, for 15 min) reliably induced a homosynaptic form of LTD. This form of LTD was associated with a significant increase in paired-pulse facilitation ratio and was insensitive to ionotropic (CNQX, 10 microM and CPP, 20 microM) and metabotropic (S-MCPG, 1 mM) glutamate receptors antagonists, suggesting a presynaptic mechanism for both LTD induction and expression. In conclusion, our experiments clearly show that LTD is not a purely developmental phenomenon but is present also in mature rats, which possess the whole machinery for LTD induction and this will greatly enhance the flexibility and the storing capacity of neuronal circuits.

Nitric oxide sensitive depolarization-induced hyperpolarization: a possible role for gap junctions during development.

Electrical coupling is a widespread feature of developing neuronal circuits and it contributes to the generation of patterned activity. In the developing rat hippocampus, release of GABA by coactive hilar interneurones generates widespread synchronized activity. Here it is shown that hilar interneurones strongly rectify in the outward direction when depolarized. This depolarization-induced hyperpolarization, abolished by gap junction uncouplers, is modulated by nitric oxide. This phenomenon might represent a current-shunting mechanism of the excess current by providing functional inhibition at a developmental stage when GABA is excitatory. Spatial buffering of the current might represent an osmotic mechanism for growth and differentiation.

Cholinergic function in the hippocampus of juvenile rats chronically deprived of NGF.

Intracellular and extracellular recordings were used to assess the cholinergic function in hippocampal slices from juvenile rats chronically deprived of NGF. NGF was neutralised by implanting into the lateral ventricle of postnatal (P) day 2 rats, alphaD11 hybridoma cells (secreting monoclonal antibodies specific for NGF). Parental myeloma cells (P3U) were used as controls. At P15-P18, slow cholinergic EPSPs could be elicited in cells from both alphaD11- and P3U-treated rats. However, slices from alphaD11-implanted rats exhibited a 50% reduction in acetylcholine release following stimulation of cholinergic fibres. This effect was associated to a significant increase in the sensitivity of pyramidal cells to carbachol, as suggested by the shift to the left of the dose/response curve. This may reflect a compensatory mechanism for the reduced efficacy of cholinergic innervation in NGF-deprived rats. In both alphaD11- and P3U-treated rats, carbachol was able to induce a similar concentration-dependent depression of the field EPSPs, evoked by Schaffer collateral stimulation, suggesting that presynaptic muscarinic receptors were not altered. In rats implanted with alphaD11 cells at P15 and sacrificed at P21-P24, no changes in the sensitivity to carbachol were found. At this developmental stage, no differences in acetylcholine release were observed between P3U- and alphaD11-treated animals. These results provide physiological evidence for a regulatory role of NGF in the cholinergic function of the hippocampus during postnatal development.

Two distinct forms of long-term depression coexist at the mossy fiber-CA3 synapse in the hippocampus during development.

During a critical period of postnatal development, between postnatal days 6 and 14, a high-frequency stimulation train (100 Hz for 1 s) to the mossy fibers induces a long-term depression (LTD) of synaptic efficacy of 29 +/- 5.2%. This form of LTD is homosynaptic. It is independent of the activation of N-methyl-D-aspartate or metabotropic glutamate receptors but needs an increase in calcium into the postsynaptic cell for its induction. At the same synapse LTD also could be induced by low-frequency stimulation of the mossy fibers (1 Hz for 15 min). In this case the magnitude of the depression is 37 +/- 4.2%. This form of LTD is N-methyl-D-aspartate independent but requires the activation of metabotropic glutamate receptors because it is prevented by (S)-alpha-methyl-4-carboxyphenylglycine (1 mM). Moreover its induction appears to be presynaptic, because, in contrast with the high-frequency one, it is not blocked by loading the postsynaptic cell with the calcium chelator EGTA or bis-(-o-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid (BAPTA). Saturation of one form of LTD does not occlude the other, suggesting that high and low frequency-induced LTD depend on distinct mechanisms of induction and expression. Quantal (noise deconvolution) analysis of minimal excitatory postsynaptic potentials shows, similarly to high-frequency LTD, a decrease in quantal content without any change in quantal size after low-frequency LTD, suggesting that in both forms of LTD the site where maintenance mechanisms are located is presynaptic.

NGF antibodies impair long-term depression at the mossy fibre-CA3 synapse in the developing hippocampus.

Nerve growth factor (NGF) and other neurotrophins are proteins involved in neuronal survival and differentiation. Much experimental evidence is now drawing attention into a role of neurotrophins in activity-dependent synaptic plasticity processes. We now show that slices from rats chronically deprived of NGF, by intraventricular injection of alpha D11 hybridoma cells, which produce monoclonal antibodies against NGF, display a reduced probability of induction of long-term depression at the mossy fibre-CA3 synapse.

Tonic facilitation of glutamate release by presynaptic N-methyl-D-aspartate autoreceptors in the entorhinal cortex.

N-Methyl-D-aspartate receptors are fundamental for neuronal plasticity and development in the CNS. Most studies have examined postsynaptic roles of this receptor, but there are also indications for a presynaptic location and function. Here, we provide electrophysiological evidence for the existence of presynaptic N-methyl-D-aspartate receptors which can tonically facilitate glutamate release in the CNS. The N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonopentanoate reduced the frequency, but not amplitude, of glutamate-mediated spontaneous excitatory postsynaptic currents in layer II neurons of the rat entorhinal cortex. This effect was also observed in the presence of tetrodotoxin and when postsynaptic N-methyl-D-aspartate receptors were blocked by dialysis with dizocilpine maleate. When extracellular calcium was replaced with strontium, 2-amino-5-phosphonopentanoate reduced the "tail" of spontaneous excitatory postsynaptic currents that followed an evoked excitatory postsynaptic current. Finally, there was a tendency for paired-pulse facilitation of excitatory postsynaptic currents evoked at short (50 ms) intervals with postsynaptic N-methyl-D-aspartate receptors blocked) to be reduced by 2-amino-5-phosphonopentanoate, although this did not reach significance. These data strongly support the presence of presynaptic N-methyl-D-aspartate autoreceptors which may facilitate glutamate release in layer II of the entorhinal cortex.

A comparison of spontaneous EPSCs in layer II and layer IV-V neurons of the rat entorhinal cortex in vitro.

1. We have compared the characteristics of spontaneous excitatory postsynaptic currents (sEPSCs) in neurons of layer IV-V and layer II of the rat entorhinal cortex (EC) using whole cell voltage-clamp recordings in a slice preparation. 2. The frequency of sEPSCs was similar in the two layers, but the events in layer IV-V had a larger mean amplitude, faster rise time, and were faster to decay. The difference in amplitude could be attributed to the presence of a population of larger events in the layer IV-V neurons that were not present in layer II. 3. Electrotonic length was greater in layer II neurons, suggesting that the difference in kinetics of the sEPSCs may be explained partly by electrotonic attenuation. 4. The frequency of sEPSCs in both layers was reduced by tetrodotoxin (TTX) to a similar extent (15-20%). However, the amplitude distribution was unchanged in layer II, whereas in layer IV-V TTX abolished most of the larger amplitude sEPSCs. 5. 6-cyano-7-nitroquinoxaline-2,3-dione or 6-nitro-7-sulphamoylbenzo (f)-quinoxaline-2,3-dione, abolished most of the sEPSCs in neurons of both layers. However, even at negative holding potentials, a population of slower time-course sEPSCs remained in the presence of these antagonists. 6. The slow sEPSCs were more frequent in layer IV-V but had similar characteristics in both layers, being increased in amplitude at more positive holding potentials or in Mg2+-free medium, and blocked by 2-amino-5-phosphonovalerate (AP5). 7. AP5 alone (i.e., without addition of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid antagonists) reduced the peak amplitude and decay phase of sEPSCs in layer IV-V neurons but appeared to have little effect on amplitude and only a weak effect on decay phase in layer II. 8. Thus both layer IV-V and layer II neurons of the EC suffer continuous spontaneous excitation. However, layer IV-V neurons exhibit larger amplitude sEPSCs, probably mediated by release of multiple quanta of neurotransmitter. In addition, although both types of neurons display spontaneous excitation mediated by N-methyl-D-aspartate receptors, this component appears more pronounced in the deeper layers.

The beta-carboline derivative DMCM decreases gamma-aminobutyric acid responses and Ca(2+)-mediated K(+)-conductance in rat neocortical neurons in vitro.

Electrophysiological recordings from neurons of rat frontal neocortical slices have been used to investigate the action of the beta-carboline methyl-6,7-dimethoxy-4-ethyl-beta- carboline-3-carboxylate (DMCM), on responses to gamma-aminobutyric acid (GABA) and on the excitability of the neurons. Iontophoretic application of GABA close to the intracellularly recorded cells (resting membrane potential -74 +/- 0.9 mV) elicited a depolarization associated with a decrease of input resistance, mediated by GABAA receptors. Bath application of DMCM (0.1-1 microM) reduced these GABA responses decreasing the affinity of the receptors for GABA. This effect was blocked by the benzodiazepine receptor (BZR) antagonist ZK 93426 (1 microM). DMCM (0.1 microM) also decreased the hyperpolarization that followed a train of action potentials (AHP), mediated by Ca(2+)-dependent K+ conductance, and increased the duration of Ca(2+)-dependent action potentials recorded after blockade of Na+ and K+ conductances. Neither effect was blocked by BZR antagonists. These results indicate that DMCM increases the excitability of neurons not only by reducing the gain of the GABAA/BZR complex, but also by modulating intrinsic membrane mechanisms.