NO enhances presynaptic currents during cerebellar mossy fiber-granule cell LTP

Nitric oxide (NO) is a candidate retrograde messenger in long-term potentiation (LTP). The NO metabolic pathway is expressed in the cerebellar granule cell layer but its physiological role remained unknown. In this paper we have investigated the role of NO in cerebellar mossy fiber-granule cell LTP, which has postsynaptic N-methyl-d-aspartate (NMDA) receptor-dependent induction. Pre- and postsynaptic current changes were simultaneously measured by using extracellular focal recordings, and NO release was monitored with an electrochemical probe in P21 rat cerebellar slices. High-frequency mossy fiber stimulation induced LTP and caused a significant NO release (6.2 +/- 2.8 nM; n = 5) in the granular layer that was dependent on NMDA receptor as well as on nitric oxide synthase (NOS) activation. Preventing NO production by perfusing the NOS inhibitor 100 microM NG-nitro-l-arginine (L-NNA), blocking extracellular NO diffusion by 10 microM MbO2, or inhibiting the NO target guanylyl cyclase (sGC) with 10 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-dione (ODQ) prevented LTP. Moreover, the NO donor 10 microM 2-(N,N-diethylamino)-diazenolate-2-oxide.Na (DEA-NO) induced LTP, which was mutually occlusive with LTP generated by high-frequency stimulation, prevented by ODQ, and insensitive to NMDA channel blockade (50 microM APV + 25 microM 7-Cl-kyn) or interruption of mossy fiber stimulation. Thus NO is critical for LTP induction at the cerebellar mossy fiber-granule cell relay. Interestingly, LTP manipulations were accompanied by consensual changes in the presynaptic current, suggesting that NO acts as a retrograde signal-enhancing presynaptic terminal excitability.     

NMDA receptor-dependent metaplasticity at hippocampal mossy fiber synapses

Hippocampal mossy fiber synapses have been reported to lack NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) of AMPA excitatory postsynaptic currents (EPSCs), unlike conventional glutamatergic synapses. An explanation for this difference may reside in the relatively low number of NMDARs at these synapses. Because mossy fiber synapses display LTP selective for NMDARs, we examined whether this would affect the plasticity rules at mossy fiber-CA3 synapses in mouse hippocampal slices. We found that LTP of NMDARs serves as a metaplastic switch making mossy fiber synapses competent for generating NMDAR-dependent LTP of AMPA EPSCs.     

Recurrent mossy fiber pathway in rat dentate gyrus: synaptic currents evoked in presence and absence of seizure-induced growth

A common feature of temporal lobe epilepsy and of animal models of epilepsy is the growth of hippocampal mossy fibers into the dentate molecular layer, where at least some of them innervate granule cells. Because the mossy fibers are axons of granule cells, the recurrent mossy fiber pathway provides monosynaptic excitatory feedback to these neurons that could facilitate seizure discharge. We used the pilocarpine model of temporal lobe epilepsy to study the synaptic responses evoked by activating this pathway. Whole cell patch-clamp recording demonstrated that antidromic stimulation of the mossy fibers evoked an excitatory postsynaptic current (EPSC) in approximately 74% of granule cells from rats that had survived >10 wk after pilocarpine-induced status epilepticus. Recurrent mossy fiber growth was demonstrated with the Timm stain in all instances. In contrast, antidromic stimulation of the mossy fibers evoked an EPSC in only 5% of granule cells studied 4-6 days after status epilepticus, before recurrent mossy fiber growth became detectable. Notably, antidromic mossy fiber stimulation also evoked an EPSC in many granule cells from control rats. Clusters of mossy fiber-like Timm staining normally were present in the inner third of the dentate molecular layer at the level of the hippocampal formation from which slices were prepared, and several considerations suggested that the recorded EPSCs depended mainly on activation of recurrent mossy fibers rather than associational fibers. In both status epilepticus and control groups, the antidromically evoked EPSC was glutamatergic and involved the activation of both AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors. EPSCs recorded in granule cells from rats with recurrent mossy fiber growth differed in three respects from those recorded in control granule cells: they were much more frequently evoked, a number of them were unusually large, and the NMDA component of the response was generally much more prominent. In contrast to the antidromically evoked EPSC, the EPSC evoked by stimulation of the perforant path appeared to be unaffected by a prior episode of status epilepticus. These results support the hypothesis that recurrent mossy fiber growth and synapse formation increases the excitatory drive to dentate granule cells and thus facilitates repetitive synchronous discharge. Activation of NMDA receptors in the recurrent pathway may contribute to seizure propagation under depolarizing conditions. Mossy fiber-granule cell synapses also are present in normal rats, where they may contribute to repetitive granule cell discharge in regions of the dentate gyrus where their numbers are significant.     

Silent synapses in developing cerebellar granule neurons

Silent synapses are excitatory synapses endowed exclusively with N-methyl-D-aspartate (NMDA) responses that have been proposed to acquire alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) responses during development and after long-term potentiation (LTP). These synapses are functionally silent because of the Mg(2+) block of NMDA receptors at resting potentials. Here we provide evidence for the presence of silent synapses in developing cerebellar granule cells. Using the patch-clamp technique in the whole-cell configuration, we recorded the spontaneous excitatory postsynaptic currents (sEPSCs) from rat cerebellar granule cells in culture and in slices at physiological concentration of Mg(2+) (1 mM). A holding potential of +60 mV removes Mg(2+) block of NMDA channels, allowing us to record NMDA-sEPSCs. We thus compared the frequency of AMPA-sEPSCs, recorded at -60 mV, with that of NMDA-sEPSCs, recorded at +60 mV. NMDA-sEPSCs occurred at higher frequency than the AMPA-sEPSCs in most cells recorded in slices from rats at postnatal day (P) <13 and in culture at 6-8 days after plating (DIV6-8). In a few cells from young rats (P6-9) and in most neurons in culture at DIV6 we recorded exclusively NMDA-sEPSCs, supporting the hypothesis of existence of functional synapses with NMDA and without AMPA receptors. Increasing glutamate release in the slice with cyclothiazide and temperature increased AMPA and NMDA-sEPSCs frequencies but failed to alter the relative ratio of frequency of occurrence. Frequency ratio of NMDA versus AMPA-sEPSCs in slices was correlated with the weighted time constant of decay (tau(w)) of NMDA-sEPSCs and decreased with development along the reported decrease of tau(w). We suggest that the prevalence of synaptic NR2A subunits that confer faster kinetics is paralleled by the disappearance of silent synapses early in cerebellar development.     

Corticostriatal paired-pulse potentiation produced by voltage-dependent activation of NMDA receptors and L-type Ca(2+) channels.

AMPA and N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses expressed differential paired-pulse plasticity when examined in the same cell using intracellular or whole cell voltage-clamp recordings. Electrical stimulation of corticostriatal afferents in brain slices bathed in artificial cerebrospinal fluid containing bicuculline produces excitatory postsynaptic potentials and excitatory postsynaptic currents (EPSCs) mediated primarily by AMPA receptors. Cell-to-cell variation existed in AMPA receptor paired-pulse plasticity, but within-cell plasticity was stable over a range of stimulation intensities. Addition of 6-cyano-7-nitroquinoxalene-2,3-dione blocked most of the synaptic response leaving behind a small AP-5-sensitive component. Increasing the stimulation intensity produced large, long-lasting NMDA receptor-mediated responses. In contrast to AMPA receptor-mediated responses, NMDA receptor responses consistently showed an increase in paired-pulse potentiation with increasing stimulation intensity. This relationship was restricted to interstimulus intervals shorter than 100 ms. Paired-pulse potentiation of NMDA receptor responses was voltage-dependent and reduced by removal of extracellular Mg(2+). Block of postsynaptic L-type Ca(2+) channels with nifedipine produced a voltage-dependent reduction of NMDA receptor excitatory postsynaptic currents (EPSCs) and a voltage-dependent reduction of NMDA receptor paired-pulse potentiation. These data indicate depolarization during the first NMDA receptor response causes facilitation of the second by removing voltage-dependent block of NMDA receptors by Mg(2+) and by activating voltage-dependent Ca(2+) channels.     

NMDA receptor-mediated currents in rat cerebellar granule and unipolar brush cells

The properties of N-methyl-D-aspartate (NMDA) receptor-mediated currents at the giant cerebellar mossy-fiber unipolar brush cell (UBC) synapse were compared with those of adjacent granule cells using patch-clamp recording methods in thin slices of rat cerebellar nodulus. In UBCs, NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) decayed as a single exponential whose time constant was independent of membrane potential. The EPSC was reduced in all cells by the NR1/NR2B-selective antagonist ifenprodil, and the Zn(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) produced a transient potentiation in 50% of cells. In contrast, the NMDA EPSC in granule cells decayed as a double exponential that dramatically switched to a slower rate at positive membrane potentials. The synaptic response in some granule cells also displayed a late second peak at positive potentials, and in others, activation of mossy fibers produced repetitive trains of EPSCs indicating they may be postsynaptic to the UBC network. Single-channel recordings of outside-out somatic patches from UBCs in magnesium-free solution revealed only high-conductance (50 pS) channels whose open time was increased with depolarization, but the opening frequency was decreased to yield a low (p(o) = 0.0298), voltage-independent opening probability. Lowering extracellular calcium (2.5-0.25 mM) had no effects on channel gating, although an increase of single-channel conductance was observed at lower calcium concentrations. Taken together, the data support the notion that the NMDA receptor in UBCs may comprise both NR1/NR2A and NR1/NR2B receptors. Furthermore, the properties of the EPSC in these two classes of feedforward glutamatergic interneurons display fundamental differences that may relate to their roles in synaptic integration.     

NMDA receptor-dependent plasticity of granule cell spiking in the dentate gyrus of normal and epileptic rats

Because granule cells in the dentate gyrus provide a major synaptic input to pyramidal neurons in the CA3 region of the hippocampus, spike generation by granule cells is likely to have a significant role in hippocampal information processing. Granule cells normally fire in a single-spike mode even when inhibition is blocked and provide single-spike output to CA3 when afferent activity converging into the entorhinal cortex from neocortex, brainstem, and other limbic regions increases. The effects of enhancement of N-methyl-D-aspartate (NMDA) receptor-dependent excitatory synaptic transmission and reduction in gamma-aminobutyric acid-A (GABA(A)) receptor-dependent inhibition on spike generation were examined in granule cells of the dentate gyrus. In contrast to the single-spike mode observed in normal bathing conditions, perforant path stimulation in Mg(2+)-free bathing conditions evoked graded burst discharges in granule cells which increased in duration, amplitude, and number of spikes as a function of stimulus intensity. After burst discharges were evoked during transient exposure to bathing conditions that relieve the Mg(2+) block of the NMDA receptor, there was a marked increase in the NMDA receptor-dependent component of the EPSP, but no significant increase in the non-NMDA receptor-dependent component of the EPSP in normal bathing medium. Supramaximal perforant path stimulation still evoked only a single spike, but granule cell spike generation was immediately converted from a single-spike firing mode to a graded burst discharge mode when inhibition was then reduced. The induction of graded burst discharges in Mg(2+)-free conditions and the expression of burst discharges evoked in normal bathing medium with subsequent disinhibition were both blocked by DL-2-amino-4-phosphonovaleric acid (APV) and were therefore NMDA receptor dependent, in contrast to long-term potentiation (LTP) in the perforant path, which is induced by NMDA receptors and is also expressed by alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptors. The graded burst discharge mode was also observed in granule cells when inhibition was reduced after a single epileptic afterdischarge, which enhances the NMDA receptor-dependent component of evoked synaptic response, and in the dentate gyrus reorganized by mossy fiber sprouting in kindled and kainic acid-treated rats. NMDA receptor-dependent plasticity of granule cell spike generation, which can be distinguished from LTP and induces long-term susceptibility to epileptic burst discharge under conditions of reduced inhibition, could modify information processing in the hippocampus and promote epileptic synchronization by increasing excitatory input into CA3.     

Genetic manipulation study of information processing in the cerebellum

The cerebellar circuitry consists of two main excitatory glutamatergic pathways. The inputs of mossy fibers and climbing fibers converge on Purkinje cells and deep cerebellar nuclei. In this circuitry, Golgi interneurons suppress granule cell excitability via the inhibitory GABA transmitter. A novel technique termed reversible neurotransmission blocking (RNB) was genetically established, in which granule cell transmission to Purkinje cells was selectively and reversibly blocked in the mouse cerebellar circuitry. This study revealed that Purkinje cells are essential for expression of conditioned eye-blink motor learning but that this memory is acquired and stored in deep cerebellar nuclei. A different technique termed immunotoxin-mediated cell targeting (IMCT) was developed to selectively ablate Golgi cells from the mouse cerebellar network. The study disclosed that excitatory glutamate receptors and inhibitory GABA receptors cooperatively act at Golgi cell–mossy fiber–granule cell synapses and are indispensable for motor coordination and adaptation. Finally, gene targeting of mGluR2 displayed that the metabotropic glutamate receptor acts collaboratively with the ionotropic AMPA receptors at granule cell–Golgi cell synapses and is crucial for the spatiotemporal regulation in the mouse cerebellar circuitry. The neural information is thus hierarchically regulated and integrated at different levels of the cerebellar network.     

Dendritic glutamate autoreceptors modulate signal processing in rat mitral cells

It has been shown recently that in mitral cells of the rat olfactory bulb, N-methyl-D-aspartate (NMDA) autoreceptors are activated during mitral cell firing. Here we consider in more details the mechanisms of mitral cell self-excitation and its physiological relevance. We show that both ionotropic NMDA and non-NMDA autoreceptors are activated by glutamate released from primary and secondary dendrites. In contrast to non-NMDA autoreceptors, NMDA autoreceptors are almost exclusively located on secondary dendrites and their activation generates a large and sustained self-excitation. Both intracellularly evoked and miniature NMDA-R mediated synaptic potentials are blocked by intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) and result from a calcium-dependent release of glutamate. Self-excitation can be produced by a single spike, and trains of spikes result in frequency facilitation. Thus activation of excitatory autoreceptors is a major function of action potentials backpropagating in mitral cell dendrites, which results in an immediate positive feedback counteracting recurrent inhibition and increasing the signal-to-noise ratio of olfactory inputs.     

Synaptic inputs to granule cells of the dorsal cochlear nucleus

The mammalian dorsal cochlear nucleus (DCN) integrates auditory nerve input with nonauditory signals via a cerebellar-like granule cell circuit. Although granule cells carry nonauditory information to the DCN, almost nothing is known about their physiology. Here we describe electrophysiological features of synaptic inputs to granule cells in the DCN by in vitro patch-clamp recordings from P12 to P22 rats. Granule cells ranged from 6 to 8 microm in cell body diameter and had high-input resistance. Excitatory postsynaptic currents consisted of both AMPA receptor-mediated and N-methyl-D-aspartate receptor-mediated currents. Synaptically evoked excitatory postsynaptic currents ranged from -25 to -140 pA with fast decay time constants. Synaptic stimulation evoked both short- and long-latency synaptic responses that summated to spike threshold, indicating the presence of a polysynaptic excitatory pathway in the granule cell circuit. Synaptically evoked inhibitory postsynaptic currents in Cl(-)-loaded cells ranged from -30 to -1,021 pA and were mediated by glycine and, to a lesser extent, GABA(A) receptors. Unlike cerebellar granule cells, DCN granule cells lacked tonic inhibition by GABA. The glycinergic synaptic conductance was mediated by heteromeric glycine receptors and was far stronger than the glutamatergic conductance, suggesting that glycinergic neurons may act to gate nonauditory signals in the DCN.     

Activity-dependent synaptic plasticity in the supraoptic nucleus of the rat hypothalamus

Activity-dependent long-term synaptic changes were investigated at glutamatergic synapses in the supraoptic nucleus (SON) of the rat hypothalamus. In acute hypothalamic slices, high frequency stimulation (HFS) of afferent fibres caused long-term potentiation (LTP) of the amplitude of AMPA receptor-mediated excitatory postsynaptic currents (EPSCs) recorded with the whole-cell patch-clamp technique. LTP was also obtained in response to membrane depolarization paired with mild afferent stimulation. On the other hand, stimulating the inputs at 5 Hz for 3 min at resting membrane potential caused long-term depression (LTD) of excitatory transmission in the SON. These forms of synaptic plasticity required the activation of NMDA receptors since they were abolished in the presence of d -AP5 or ifenprodil, two selective blockers of these receptors. Analysis of paired-pulse facilitation and trial-to-trial variability indicated that LTP and LTD were not associated with changes in the probability of transmitter release, thereby suggesting that the locus of expression of these phenomena was postsynaptic. Using sharp microelectrode recordings in a hypothalamic explant preparation, we found that HFS also generates LTP at functionally defined glutamatergic synapses formed between the organum vasculosum lamina terminalis and SON neurons. Taken together, our findings indicate that glutamatergic synapses in the SON exhibit activity-dependent long-term synaptic changes similar to those prevailing in other brain areas. Such forms of plasticity could play an important role in the context of physiological responses, like dehydration or lactation, where the activity of presynaptic glutamatergic neurons is strongly increased.     

Mossy fiber-granule cell synapses in the normal and epileptic rat dentate gyrus studied with minimal laser photostimulation.

Dentate granule cells become synaptically interconnected in the hippocampus of persons with temporal lobe epilepsy, forming a recurrent mossy fiber pathway. This pathway may contribute to the development and propagation of seizures. The physiology of mossy fiber-granule cell synapses is difficult to characterize unambiguously, because electrical stimulation may activate other pathways and because there is a low probability of granule cell interconnection. These problems were addressed by the use of scanning laser photostimulation in slices of the caudal hippocampal formation. Glutamate was released from a caged precursor with highly focused ultraviolet light to evoke action potentials in a small population of granule cells. Excitatory synaptic currents were recorded in the presence of bicuculline. Minimal laser photostimulation evoked an apparently unitary excitatory postsynaptic current (EPSC) in 61% of granule cells from rats that had experienced pilocarpine-induced status epilepticus followed by recurrent mossy fiber growth. An EPSC was also evoked in 13-16% of granule cells from the control groups. EPSCs from status epilepticus and control groups had similar peak amplitudes ( approximately 30 pA), 20-80% rise times (approximately 1.2 ms), decay time constants ( approximately 10 ms), and half-widths (approximately 8 ms). The mean failure rate was high (approximately 70%) in both groups, and in both groups activation of N-methyl-D-aspartate receptors contributed a small component to the EPSC. The strong similarity between responses from the status epilepticus and control groups suggests that they resulted from activation of a similar synaptic population. No EPSC was recorded when the laser beam was focused in the dentate hilus, suggesting that indirect activation of hilar mossy cells contributed little, if at all, to these results. Recurrent mossy fiber growth increases the density of mossy fiber-granule cell synapses in the caudal dentate gyrus by perhaps sixfold, but the new synapses appear to operate very similarly to preexisting mossy fiber-granule cell synapses.     

Simultaneous recording of presynaptic spikes and excitatory postsynaptic potentials from monosynaptically connected hippocampal neurons

A technique has been devised to activate single granule cells in the hippocampus, and to record simultaneously spikes from the particular granule cell and excitatory postsynaptic potentials from a monosynaptically connected CA3 neuron. The unitary excitatory postsynaptic potentials (EPSPs) sustained for long observation periods, and increased in size with increases in stimulus frequency and in external Ca2+ concentration. This technique may be useful for quantal analysis of transmission through the synapse between mossy fibers and CA3 neurons.     

Kindling induces transient NMDA receptor-mediated facilitation of high-frequency input in the rat dentate gyrus

To elucidate the gating mechanism of the epileptic dentate gyrus on seizure-like input, we investigated dentate gyrus field potentials and granule cell excitatory postsynaptic potentials (EPSPs) following high-frequency stimulation (10-100 Hz) of the lateral perforant path in an experimental model of temporal lobe epilepsy (i.e., kindled rats). Although control slices showed steady EPSP depression at frequencies greater than 20 Hz, slices taken from animals 48 h after the last seizure presented pronounced EPSP facilitation at 50 and 100 Hz, followed by steady depression. However, 28 days after kindling, the EPSP facilitation was no longer detectable. Using the specific N-methyl-D-aspartate (NMDA) and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproponic acid (AMPA) receptor antagonists 2-amino-5-phosphonovaleric acid and SYM 2206, we examined the time course of alterations in glutamate receptor-dependent synaptic currents that parallel transient EPSP facilitation. Forty-eight hours after kindling, the fractional AMPA and NMDA receptor-mediated excitatory postsynaptic current (EPSC) components shifted dramatically in favor of the NMDA receptor-mediated response. Four weeks after kindling, however, AMPA and NMDA receptor-mediated EPSCs reverted to control-like values. Although the granule cells of the dentate gyrus contain mRNA-encoding kainate receptors, neither single nor repetitive perforant path stimuli evoked kainate receptor-mediated EPSCs in control or in kindled rats. The enhanced excitability of the kindled dentate gyrus 48 h after the last seizure, as well as the breakdown of its gating function, appear to result from transiently enhanced NMDA receptor activation that provides significantly slower EPSC kinetics than those observed in control slices and in slices from kindled animals with a 28-day seizure-free interval. Therefore, NMDA receptors seem to play a critical role in the acute throughput of seizure activity and in the induction of the kindled state but not in the persistence of enhanced seizure susceptibility.     

Potentiation of spontaneous synaptic activity in rat mossy cells.

Recent studies have demonstrated the vulnerability of dentate mossy cells to seizure-induced damage. One source of potentially damaging synaptic input are spontaneously active granule cell terminals ('mossy terminals'.) We sought to test whether there were activity-dependent changes in the spontaneous excitatory input to mossy cells. Using the in vitro slice preparation, we examined the frequency and amplitude of spontaneous excitatory postsynaptic potentials (EPSPs) after intracellular current injection designed to mimic the extreme depolarization these neurons receive during repetitive afferent stimulation. In 4 of 7 neurons, depolarization with trains of current pulses resulted in a significant and persistent increase in frequency of spontaneous synaptic depolarizations (to an average of 178% of the initial baseline rate). In 3 of these affected neurons, an increased frequency of large amplitude, fast-rising EPSPs accounted for the majority of this change. Injection of hyperpolarizing current pulses failed to alter spontaneous activity in 3 other mossy cells. These results suggest spontaneous synaptic input to mossy cells in plastic and can be potentiated by depolarization of a single postsynaptic mossy cell. The ability of mossy cells to potentiate their excitatory input may be relevant to their vulnerability to excitotoxic injury during repetitive afferent stimulation.     

Transmembrane AMPA receptor regulatory proteins and AMPA receptor function in the cerebellum

Heterogeneity among AMPA receptor (AMPAR) subtypes is thought to be one of the key postsynaptic factors giving rise to diversity in excitatory synaptic signaling in the CNS. Recently, compelling evidence has emerged that ancillary AMPAR subunits—the so-called transmembrane AMPA receptor regulatory proteins (TARPs)—also play a vital role in influencing the variety of postsynaptic signaling. This TARP family of molecules controls both trafficking and functional properties of AMPARs at most, if not all, excitatory central synapses. Furthermore, individual TARPs differ in their effects on the biophysical and pharmacological properties of AMPARs. The critical importance of TARPs in synaptic transmission was first revealed in experiments on cerebellar granule cells from stargazer mice. These lack the prototypic TARP stargazin, present in granule cells from wild-type animals, and consequently lack synaptic transmission at the mossy fibre-to-granule cell synapse. Subsequent work has identified many other members of the stargazin family which act as functional TARPs. It has also provided valuable information about specific TARPs present in many central neurons. Because much of the initial work on TARPs was carried out on stargazer granule cells, the important functional properties of TARPs present throughout the cerebellum have received particular attention. Here we discuss some of these recent findings in relation to the main TARPs and the AMPAR subunits identified in cerebellar neurons and glia.     

Presynaptic and postsynaptic NMDA receptors mediate distinct effects of brain-derived neurotrophic factor on synaptic transmission

In addition to its effects on neuronal survival and differentiation, brain-derived neurotrophic factor (BDNF) plays an important role in modulating synaptic transmission and plasticity in many brain areas, most notably the neocortex and hippocampus. These effects may underlie a role for BDNF in learning and memory as well as developmental plasticity. Consistent with localization of the tropomyosin-related kinase B receptor to both sides of the synapse, BDNF appears to have pre- and postsynaptic effects, but the underlying cellular mechanisms are unclear and it is not known whether pre- and postsynaptic modulations by BDNF occur simultaneously. To address these issues, we recorded dual-component (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA] and N-methyl-D-aspartate [NMDA]) miniature excitatory postsynaptic currents (mEPSCs) from cortical and hippocampal pyramidal neurons and dentate gyrus granule cells from acute brain slices. BDNF had no effect on the fast component of mEPSC decay or on the peak amplitude, suggesting that BDNF did not modulate postsynaptic AMPA receptors, although BDNF rapidly modulated NMDA receptors, as seen by an enhancement of the slow component of mEPSC decay that was prevented by blocking postsynaptic NMDA receptors. At the same time, BDNF acted presynaptically to enhance mEPSC frequency. Surprisingly, the effect on frequency was also NMDA receptor dependent, but required activation of presynaptic, not postsynaptic, NMDA receptors. BDNF also enhanced action potential-dependent glutamate release via presynaptic NMDA receptors, an effect that was unmasked when voltage-gated calcium channels were partially inhibited. Our results indicate that BDNF acutely modulates presynaptic release and postsynaptic responsiveness through simultaneous effects on pre- and postsynaptic NMDA receptors.     

Long-term enhancement of excitatory synaptic inputs to layer V parahippocampal neurons by low frequency stimulation in rat brain slices

Excitatory inputs to layer V neurons of the parasubiculum and medial entorhinal cortex were examined in rat brain slices with intracellular and field potential recordings. Single extracellular stimuli to layer V evoked subthreshold excitatory postsynaptic potentials (EPSPs) or a long duration (>100 ms) depolarization that sustained high frequency firing. Repetitive stimulation at low frequencies (from 1/10 s to 1/min) induced stable long-lasting decreases in the threshold for firing in individual cells or population events, and also induced stable long-lasting increases in evoked intracellular or field response amplitudes. More stimuli were required to produce the equivalent changes in threshold and amplitude in the presence of MCPG (200 microM). Smaller changes in amplitude, but equivalent changes in threshold were elicited in the presence of CPP (10 microM), or CPPG (20 microM). No changes in threshold or amplitude were detected in the presence of CNQX (10 microM), even when used in combination with picrotoxin (100 microM). EPSP facilitation was enhanced greatly by firing in postsynaptic cells. It is suggested that stable changes in excitatory inputs to layer V parahippocampal neurons involve the activation of NMDA and metabotropic glutamate receptors, but requires AMPA receptor activation and postsynaptic cell firing.     

Astrocytic Ca2+ signaling evoked by sensory stimulation in vivo

Although astrocytes are the most abundant cell type in the brain, evidence for their activation during physiological sensory activity is lacking. Here we show that whisker stimulation evokes increases in astrocytic cytosolic calcium (Ca(2+)) within the barrel cortex of adult mice. Increases in astrocytic Ca(2+) were a function of the frequency of stimulation, occurred within several seconds and were inhibited by metabotropic glutamate receptor antagonists. To distinguish between synaptic input and output, local synaptic activity in cortical layer 2 was silenced by iontophoresis of AMPA and NMDA receptor antagonists. The antagonists did not reduce astrocytic Ca(2+) responses despite a marked reduction in excitatory postsynaptic currents in response to whisker stimulation. These findings indicate that astrocytes respond to synaptic input, by means of spillover or ectopic release of glutamate, and that increases in astrocytic Ca(2+) occur independently of postsynaptic excitatory activity.