Receptor-mediated Excitatory Postsynaptic Currents Research Articles

Differences in excitatory transmission between thalamic and cortical afferents to single spiny efferent neurons of rat dorsal striatum

The striatum is crucially involved in motor and cognitive function, and receives significant glutamate input from the cortex and thalamus. The corticostriatal pathway arises from diverse regions of the cortex and is thought to provide information to the basal ganglia from which motor actions are selected and modified. The thalamostriatal pathway arises from specific thalamic nuclei and is involved in attention and possibly strategy switching. Despite these fundamental functional differences, direct comparisons of the properties of these pathways are lacking. N-methyl-D-aspartate (NMDA) receptors at synapses powerfully affect postsynaptic processing, and incorporation of different NR2 subunits into NMDA receptors has profound effects on the pharmacological and biophysical properties of the receptor. Utilization of different NMDA receptors at thalamostriatal and corticostriatal synapses could allow for afferent-specific differences in information processing. We used a novel rat brain slice preparation preserving corticostriatal and thalamostriatal pathways to medium spiny neurons to examine the properties of NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) recorded using the whole-cell, patch-clamp technique. Within the same neuron, the NMDA/non-NMDA ratio is greater for excitatory responses evoked from the thalamostriatal pathway than for those evoked from the corticostriatal pathway. In addition, reversal potentials and decay kinetics of the NMDA receptor-mediated EPSCs suggest that the thalamostriatal synapse is more distant on the dendritic arbor. Finally, results obtained with antagonists specific for NR2B-containing NMDA receptors imply that NMDA receptors at corticostriatal synapses contain more NR2B subunits. These synapse-specific differences in NMDA receptor content and pharmacology provide potential differential sites of action for NMDA receptor subtype-specific antagonists proposed for the treatment of Parkinson's disease.

Postnatal development of excitatory postsynaptic currents in nucleus accumbens medium spiny neurons

We have recorded excitatory postsynaptic currents (EPSCs) evoked by local electrical stimulation in 243 nucleus accumbens (nAcb) neurons in vitro during postnatal development from the day of birth (postnatal day 0; P0) to P27 and in young adults rats (P59–P71). An EPSC sensitive to glutamatergic antagonists was found in all neurons. In the majority of cases (189/243), the EPSC had two distinct components: an early one sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and a late one that was sensitive to d-2-amino-5-phosphonovaleric acid (APV) showing that early and late components of the EPSC were mediated by AMPA/kainate (KA) and N-methyl-d-aspartate (NMDA) receptors respectively. During the first four postnatal days, the amplitudes of both the AMPA/KA and NMDA components of the EPSC were relatively small and then began to increase until the end of the second postnatal week. Whereas the amplitude of the early component appeared to stabilize from that point on, the late component began to decrease and became virtually undetectable in preparations from animals older than 3 weeks unless the AMPA/KA response was blocked with CNQX. In addition, the ratio between the amplitude of the NMDA and AMPA/KA receptor-mediated components of the EPSC followed a developmental pattern parallel to that of the NMDA receptor component showing an increase during the first two postnatal weeks followed by a decrease. Together, these results show that, during postnatal development, there is a period when NMDA receptor-mediated EPSC are preeminent and that time frame might represent a period during which the development of the nAcb might be sensitive to environmental manipulation.

N-methyl-D-aspartate-dependent long-term potentiation of excitatory transmission in trigeminal subnucleus oralis

This study for the first time demonstrates early developmental changes of passive/active membrane properties, and long-term potentiation (LTP) of excitatory synaptic transmission at spinal trigeminal subnucleus caudalis (Vc)-to-oralis (Vo) synapses. During postnatal development, the probability of Vo neurons with monosynaptic excitatory postsynaptic currents (EPSCs) upon Vc stimulation significantly increased, whereas the input resistances of Vo neurons and the latencies of monosynaptic EPSCs significantly decreased. Application of a 'pairing' protocol that comprises 2 Hz-conditioning stimulation of Vc with postsynaptic depolarization of Vo neuron to +30 mV generated LTP of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor-mediated monosynaptic EPSC amplitude in more than 70% of Vo neurons. The induction of LTP required the activation of N-methyl-D-aspartate receptor, but its magnitudes had correlation neither with postnatal ages nor with baseline EPSC amplitudes.

NMDA receptors regulate nicotine-enhanced brain reward function and intravenous nicotine self-administration: role of the ventral tegmental area and central nucleus of the amygdala.

Nicotine is considered an important component of tobacco responsible for the smoking habit in humans. Nicotine increases glutamate-mediated transmission throughout brain reward circuitries. This action of nicotine could potentially contribute to its intrinsic rewarding and reward-enhancing properties, which motivate consumption of the drug. Here we show that the competitive N-methyl-D-aspartate (NMDA) receptor antagonist LY235959 (0.5-2.5 mg per kg) abolished nicotine-enhanced brain reward function, reflected in blockade of the lowering of intracranial self-stimulation (ICSS) thresholds usually observed after experimenter-administered (0.25 mg per kg) or intravenously self-administered (0.03 mg per kg per infusion) nicotine injections. The highest LY235959 dose (5 mg per kg) tested reversed the hedonic valence of nicotine from positive to negative, reflected in nicotine-induced elevations of ICSS thresholds. LY235959 doses that reversed nicotine-induced lowering of ICSS thresholds also markedly decreased nicotine self-administration without altering responding for food reinforcement, whereas the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor antagonist NBQX had no effects on nicotine intake. In addition, nicotine self-administration upregulated NMDA receptor subunit expression in the central nucleus of the amygdala (CeA) and ventral tegmental area (VTA), suggesting important interactions between nicotine and the NMDA receptor. Furthermore, nicotine (1 microM) increased NMDA receptor-mediated excitatory postsynaptic currents in rat CeA slices, similar to its previously described effects in the VTA. Finally, infusion of LY235959 (0.1-10 ng per side) into the CeA or VTA decreased nicotine self-administration. Taken together, these data suggest that NMDA receptors, including those in the CeA and VTA, gate the magnitude and valence of the effects of nicotine on brain reward systems, thereby regulating motivation to consume the drug.

Genetic enhancement of trace fear memory and cingulate potentiation in mice overexpressing Ca2+/calmodulin‐dependent protein kinase IV

Long-term potentiation (LTP) is a key cellular model for studying mechanisms for learning and memory. Previous studies reported that the Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) is critical for gene regulation, and behavioral learning and memory. Less is known about the roles of CaMKIV in cortical plasticity and trace fear memory. Here we have found that LTP was significantly enhanced in the anterior cingulate cortex (ACC) of the mice overexpressing CaMKIV. By contrast, neither alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated basal excitatory synaptic transmission nor N-methyl-d-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents were affected. Furthermore, paired-pulse ratio in the transgenic mice is normal. In behavioral tests, we found that the CaMKIV transgenic mice exhibited significant enhancement in trace fear memory, while the acute sensory thresholds were not affected. Our results provide strong evidence that forebrain CaMKIV contributes to trace fear memory by enhancing synaptic potentiation in the ACC.

Lamotrigine inhibits postsynaptic AMPA receptor and glutamate release in the dentate gyrus

The dentate gyrus (DG) is a gateway that regulates seizure activity in the hippocampus. We investigated the site of action of lamotrigine (LTG), an effective anticonvulsant, in the regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) receptor-mediated excitatory synaptic transmission on DG. Evoked AMPA and NMDA receptor-mediated excitatory postsynaptic currents (eEPSCampa and eEPSCnmda) were recorded by whole-cell patch-clamp recording from the granule cells of DG in brain slice preparation of young Wistar rats (60-120 g). Exogenously applied AMPA and NMDA-induced currents and AMPA receptor-mediated miniature EPSC (mEPSCampa) were recorded in the presence of specific antagonists. LTG inhibited both eEPSCampa and eEPSCnmda, and had no effect on exogenously applied NMDA-induced current indicating LTG inhibited glutamate release. Previous studies demonstrated that alteration in glutamate concentration in synaptic cleft causes parallel changes of eEPSCampa and eEPSCnmda. Our results showed that LTG inhibited eEPSCampa significantly more than eEPSCnmda (p < 0.05), suggesting that LTG may also have blocked the postsynaptic AMPA receptor. The hypothesis is further supported by the facts that; (1) LTG (30-100 microM) inhibited direct exogenously applied AMPA-induced currents (to 90%), (2) LTG significantly reduced the amplitude, but not the frequency of mEPSCampa and asynchronous (EPSC), and (3) LTG-induced reduction of eEPSCampa was not associated with a modification of the paired-pulse ratio. To sum up, LTG exerts a postsynaptic inhibitory mechanism on the AMPA receptor. Our results demonstrate that LTG suppresses postsynaptic AMPA receptors and reduces glutamate release in granule cells of DG. The postsynaptic effect can be one of the underlying mechanisms of LTG's anticonvulsant action.

Tonic facilitation of glutamate release by glycine binding sites on presynaptic NR2B-containing NMDA autoreceptors in the rat visual cortex

We have previously shown that glycine binding sites on presynaptic NMDA receptors (NMDA-Rs) can tonically regulate glutamate release in the rat visual cortex. In the present study, we investigated the subunit composition of these presynaptic NMDA-Rs. We recorded miniature a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (mEPSCs) using whole-cell voltage clamp in layer II/III pyramidal neurons of the rat visual cortex with the open-channel NMDA receptor blocker, MK-801, in the recording pipette. We found that the frequency of mEPSCs is significantly reduced by 7-chloro-kynurenic acid (7-Cl KYNA) an NMDA-R glycine binding site antagonist, and glycine reverses this effect. Using a specific antagonist for NR2B-NMDA-Rs, Ro 25-6981 [(alphaR,betaS)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidinepropanol hydrochloride], instead of 7-Cl KYNA, we found that the frequency of mEPSCs is also significantly reduced but glycine cannot reverse this effect. Moreover, Zn(2+), an NR2A-NMDA-R antagonist, did not affect mEPSC frequency. These results suggest that presynaptic NR2B-containing NMDA-Rs are located in layer II/III pyramidal neurons of the rat visual cortex, and that the glycine binding site of these type NMDA-Rs tonically regulates glutamate release.

Morphology and physiology of lamina I neurons of the caudal part of the trigeminal nucleus

It has been suggested that in mammals, trigeminal lamina I neurons play a role in the processing and transmission of sensory information from the orofacial region. We investigated the physiological and morphological properties of trigeminal subnucleus caudalis (Sp5C) lamina I neurons in slices prepared from the medulla oblongata of 13- to 15-day-old postnatal rats using patch-clamp recordings and subsequent biocytin–streptavidin–Alexa labeling. Twenty-five neurons were recorded and immunohistochemically stained. The Sp5C lamina I consisted of several types of neurons which, on the basis of their responses to somatic current injection, can be classified into four groups: tonic neurons, which fired throughout the depolarizing pulse; phasic neurons, which expressed an initial burst of action potentials; delayed onset neurons, which showed a significant delay of the first action potential; and single spike neurons, characterized by only one to five action potentials at the very beginning of the depolarizing pulse even at high levels of stimulation intensity. Electrical stimulation of the spinal trigeminal tract evoked AMPA receptor-mediated excitatory postsynaptic currents (EPSC) exhibiting a strong polysynaptic component. AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSC) were characterized by a 10–90% rise time of 0.50±0.06 ms and a decay time constant of 2.5±0.5 ms. The kinetic properties of NMDA receptor-mediated EPSCs were measured at +40 mV. The 10–90% rise time was 8±2 ms and the deactivation time constants were 94±31 and 339±72 ms, respectively. Intracellular staining and morphological analysis revealed three groups of neurons: fusiform, pyramidal, and multipolar. Statistical analysis indicated that the electrophysiological properties and morphological characteristics are correlated. Tonic and phasic neurons were fusiform or pyramidal and delayed onset and single spike neurons were multipolar. Our results show that both the physiological and morphological properties of Sp5C lamina I neurons exhibit significant differences, indicating their specific integration in the processing and transmission of sensory information from the orofacial region.

The role of spontaneous activity in development of the endbulb of Held synapse

In the mouse brainstem cochlear nucleus, the auditory nerve to bushy cell synapse (endbulb of Held) is specialised for rapid, high-fidelity transmission. Development of this synapse is modulated by auditory nerve activity. Here we investigate the role of spontaneous auditory nerve activity in synaptic transmission using deafness ( dn/ dn) mutant mice that have abnormal hair cells and lack spontaneous auditory nerve activity. Evoked and miniature alpha amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor-mediated excitatory post-synaptic currents (eEPSCs, mEPSCs) were compared in deafness and normal mice before the age of hearing onset (postnatal day 7–11: P7–11) using variance-mean, miniature event and tetanic depression analyses. Amplitudes were significantly greater in deafness mice for eEPSCs (2.1-fold), mEPSCs (1.4-fold) and quantal amplitudes (1.5-fold). eEPSCs in deafness mice decayed more rapidly with increasing age, indicating an input-independent transition in post-synaptic AMPA receptor properties. A comparison of normal mice before and after the onset of hearing showed a change in synaptic parameters with an increase in eEPSC (1.7-fold), mEPSC (1.6-fold) and quantal amplitude (1.7-fold) after hearing onset while release probability remained constant (0.5). Overall, the results in deafness mice suggest that synaptic strength is altered in the absence of spontaneous auditory nerve activity.

NMDA receptor 2B subunit-mediated synaptic transmission in the superficial dorsal horn of peripheral nerve-injured neuropathic mice

Previous research has shown that peripheral inflammation and peripheral nerve injury alter the properties of NMDA receptors in the spinal dorsal horn. However, there is no direct evidence that demonstrates the influence of peripheral nerve injury on NMDA receptor-mediated synaptic transmission in the spinal dorsal horn. Using whole cell tight-seal methods, NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) were recorded from superficial dorsal horn neurons in adult mouse spinal cord slices. Peripheral nerve injury-induced changes in the pharmacological and electrophysiological properties of synaptic NMDA receptors were studied. The ratio of the amplitude of NMDA EPSCs to that of non-NMDA EPSCs was larger in nerve-ligated neuropathic mice than in sham-operated control mice. The decay phase of the NMDA EPSCs was slower in nerve-ligated neuropathic mice. The NR2B subunit-specific NMDA receptor antagonist ifenprodil (10 μM) reduced the amplitude of the NMDA EPSCs and shortened their decay phase. The sensitivity of NMDA EPSCs to ifenprodil was significantly larger in nerve-ligated neuropathic mice than in sham-operated control mice. Single-cell RT-PCR analysis performed on superficial dorsal horn neurons showed that the incidence of NR2A mRNA-expressing neurons was reduced in nerve-ligated neuropathic mice. This result, together with the electrophysiological findings, suggests that the subunit composition of the subsynaptic NMDA receptors in the superficial dorsal horn was altered by peripheral nerve injury. Pharmacological and electrophysiological changes observed in the present experiments might be the underlying causes of the hyperalgesia and allodynia induced by peripheral nerve injury and inflammation.

Human autoantibodies against early endosome antigen-1 enhance excitatory synaptic transmission

Early endosome antigen 1 (EEA1), a peripheral membrane protein associated with the cytoplasmic face of early endosomes, controls vesicle fusion during endocytosis, as extensively studied in non-neuronal cells. In neurons, early endosomes are involved in recycling of synaptic vesicles and neurotransmitter receptors. Since certain patients bearing autoantibodies that target EEA1 develop neurological disease, we studied the subcellular distribution of EEA1 in neurons and the effect on neurotransmission of purified immunoglobulins from the serum of a patient bearing EEA1 autoantibodies. EEA1 was localized in the soma and in the postsynaptic nerve terminals. Electrophysiological recordings in hippocampal slices including purified EEA1 antibodies in the patch pipette solution, revealed a run-up of AMPA, N-methyl- d-aspartate and kainate receptor-mediated excitatory post-synaptic currents recorded from CA3 pyramidal neurons, which was absent in the recordings obtained in the presence of control human immunoglobulin G. Inclusion of human EEA1 antibodies had no effect on inhibitory post-synaptic responses. Recordings in the presence of a dominant-negative C-terminal EEA1 deletion mutant produced a similar effect as observed with human anti-EEA1 antibodies. This specific effect on the excitatory synaptic transmission may be due to the impairment of internalization of specific glutamate receptors and their subsequent accumulation in the synapse. These results may account for the neurological deficits observed in some patients developing EEA1 autoantibodies.

Different composition of glutamate receptors in corticothalamic and lemniscal synaptic responses and their roles in the firing responses of ventrobasal thalamic neurons in juvenile mice

Thalamic ventrobasal (VB) relay neurons receive information via two major types of glutamatergic synapses, that is, from the medial lemniscus (lemniscal synapses) and primary somatosensory cortex (corticothalamic synapses). These two synapses influence and coordinate firing responses of VB neurons, but their precise operational mechanisms are not yet well understood. In this study, we compared the composition of glutamate receptors and synaptic properties of corticothalamic and lemniscal synapses. We found that the relative contribution of NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) to non-NMDA receptor-mediated EPSCs was significantly greater in corticothalamic synapses than in lemniscal synapses. Furthermore, NMDA receptor 2B-containing NMDA receptor- and kainate receptor-mediated currents were observed only in corticothalamic synapses, but not in lemniscal synapses. EPSCs in corticothalamic synapses displayed the postsynaptic summation in a frequency-dependent manner, in which the summation of the NMDA receptor-mediated component was largely involved. The summation of kainate receptor-mediated currents also partially contributed to the postsynaptic summation in corticothalamic synapses. In contrast, the contribution of NMDA receptor-mediated currents to the postsynaptic summation of lemniscal EPSCs was relatively minor. Furthermore, our results indicated that the prominent NMDA receptor-mediated component in corticothalamic synapses was the key determinant for the late-persistent firing of VB neurons in response to corticothalamic stimuli. In lemniscal synapses, in contrast, the onset-transient firing in response to lemniscal stimuli was regulated mainly by AMPA receptors.

NMDA Receptor Subtypes at Autaptic Synapses of Cerebellar Granule Neurons

We studied the action potential-evoked autaptic N-methyl-d-aspartate receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) using solitary cerebellar neurons cultured in microislands from wild-type (+/+), NR2A subunit knockout (NR2A-/-), and NR2C subunit knockout (NR2C-/-) mice. The peak amplitude of autaptic NMDA-EPSCs increased for all genotypes between days in vitro 8 (DIV8) and DIV13. Compared with +/+ cells at DIV13, NR2A-/- cells had smaller and NR2C-/- cells had larger NMDA-EPSCs. The decay time of these currents were all unexpectedly fast, except in NR2A-/- neurons, and showed small but significant shortening with development. Comparison of quantal parameters during development indicated an increase in quantal content in all genotypes. The synaptic portion of NMDA receptors measured using MK-801 blockade was roughly 50% in all genotypes at DIV8, and this percentage became slightly larger in NR2A-/- and NR2C-/- neurons at DIV12. The NR2B-selective antagonists Conantokin G and CP101,606 differed in their blocking actions with development, suggesting the presence of both heterodimeric NR1/NR2B and heterotrimeric NR1/NR2A/NR2B receptors. The most striking result we obtained was the significant increase of NMDA-EPSC peak amplitude and charge transfer in NR2C-/- mice. This was mainly the result of an increase in quantal size as estimated from miniature NMDA-EPSCs. The expression of NR2C subunit containing receptors was supported by the decreased Mg(2+) sensitivity of NMDA receptors at DIV13 in +/+ but not in NR2C-/- cells. Thus solitary cerebellar granule neurons provide a novel model to investigate the role of receptor subtypes in the developmental changes of synaptic NMDA receptors.

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.

Dopamine profoundly suppresses excitatory transmission in neonatal rat hippocampus via phosphatidylinositol-linked D1-like receptor

Dopamine modulates synaptic transmission in various brain regions. The disorder of dopamine system may be related to neurodevelopmental dysfunction. However, the action of dopamine on synaptic transmission during development is largely unknown. We studied the effect of dopamine on GABAergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from CA1 pyramidal cells. Dopamine (100 μM) profoundly decreased the amplitude of GABA A receptor-mediated postsynaptic currents (GABA A-PSCs) to 32.2±5.4% (mean±S.E.M., EC 50: 2.9 μM) in the first postnatal week, when GABA provides excitatory drive. Dopamine also decreased the amplitude of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (EPSCs) to 29.1±2.7% (EC 50: 18.7 μM) in the second postnatal week, when glutamate responses first appear. The dopamine-induced inhibition declined after these periods and became only partial after postnatal day 30. Further we identified the receptor subtype involved in the dopamine-induced inhibition as phosphatidylinositol-linked D1-like receptor, since 6-chloro-2,3,4,5-tetrahydro-3-methyl-1-(3-methylphenyl)-1H-3-benzazepine-7,8-diol hydrobromide (SKF 83959), a selective agonist for phosphatidylinositol-linked D1-like receptor, clearly mimicked the action of dopamine, and 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), an inhibitor of phospholipase C, significantly reduced the dopamine-induced inhibition. Dopamine did not change the response to puff-applied GABA or kainic acid, nor the amplitude of miniature GABA A-PSCs or miniature EPSCs. These results suggest that the activation of phosphatidylinositol-linked D1-like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus by presynaptic mechanisms. This study firstly demonstrates the effect of phosphatidylinositol-linked D1-like receptor on synaptic transmission.

Activation of Dopamine D4Receptors Induces Synaptic Translocation of Ca2+/Calmodulin-Dependent Protein Kinase II in Cultured Prefrontal Cortical Neurons

One of the important targets of dopamine D4 receptors in prefrontal cortex (PFC) is the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII). In the present study, we investigated the effect of D4 receptor activation on subcellular localization of CaMKII. We found that activation of D4 receptors, but not D2 receptors, induced a rapid translocation of alpha-CaMKII from cytosol to postsynaptic sites in cultured PFC neurons. Activated CaMKII (Thr286 phospho-CaMKII) was also redistributed to postsynaptic sites after D4 receptor stimulation. The translocation was blocked by inhibiting the phospholipase C/inositol 1,4,5-trisphosphate receptor/Ca2+ signaling. Point mutation of the calmodulin binding site (Ala302), but not the autophosphorylation site (Thr286), of alpha-CaMKII prevented the D4-induced CaMKII translocation. Moreover, D4 receptors failed to induce CaMKII translocation in the presence of an actin stabilizer, and D4 activation reduced the binding of CaMKII to F-actin. Concomitant with the synaptic accumulation of alpha-CaMKII in response to D4 receptor activation, a D4-induced increase in the CaMKII phosphorylation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor glutamate receptor 1 (GluR1) subunits and the amplitude of AMPA receptor-mediated excitatory postsynaptic currents was also observed. Thus, our results show that D4 receptor activation induces the synaptic translocation of CaMKII through a mechanism involving Ca2+/calmodulin and F-actin, which facilitates the regulation of synaptic targets of CaMKII, such as AMPA receptors.

Epileptogenesis Is Associated With Enhanced Glutamatergic Transmission in the Perforant Path

The perforant path provides the main excitatory input into the hippocampus and has been proposed to play a critical role in the generation of temporal lobe seizures. It has been hypothesized that changes in glutamatergic transmission in this pathway promote the epileptogenic process and seizure generation. We therefore asked whether epileptogenesis is associated with enhanced glutamatergic transmission from the perforant path to dentate granule cells. We used a rat model of temporal lobe epilepsy in which spontaneous seizures occur after an episode of pilocarpine-induced status epilepticus. Whole cell patch-clamp recordings were obtained from dentate granule cells in hippocampal slices from control and epileptic animals 3 wk after pilocarpine-induced status epilepticus. The paired pulse ratio of perforant path-evoked AMPA receptor-mediated excitatory postsynaptic currents (EPSCs) was reduced in tissue obtained from epileptic rats. This is consistent with an increase in release probability. N-methyl-d-aspartate (NMDA) receptor-mediated EPSCs were also prolonged. This prolongation could not be accounted for by decreased activity of glutamate transporters or by a change in NMDA receptor subunit composition in dentate granule cells, implying a change in NMDA receptor kinetics. This change in NMDA receptor kinetics was associated with the emergence of significant synaptic cross-talk, detected as a use-dependent block of receptors activated by medial perforant path synapses after lateral perforant path stimulation in MK-801. Enhanced glutamatergic transmission and the emergence of cross-talk among perforant path-dentate granule cell synapses may contribute to lowering seizure threshold.

Bidirectional regulations for glutamate and GABA release in the hippocampus by α7 and non-α7 ACh receptors

In the assay of glutamate and γ-aminobutyric acid (GABA) with a high-performance liquid chromatography, spontaneous release of glutamate and GABA from rat hippocampal slices was significantly enhanced by mecamylamine, an inhibitor of non-α7 ACh receptors, or α-bungarotoxin, an inhibitor of α7 ACh receptors in the absence of tetrodotoxin (TTX), but not in the presence of TTX. Nicotine significantly enhanced glutamate and GABA release in the absence of TTX, that is abolished by mecamylamine or α-bungarotoxin, while it had no effect on the release in the presence of TTX. In the recording of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (AMPA-EPSCs) and GABA A receptor-mediated inhibitory postsynaptic currents (GABA A-IPSCs) from CA1 pyramidal neurons of rat hippocampal slices, nicotine did not affect the rate and amplitude of AMPA-EPSCs and AMPA-miniature EPSCs. In contrast, nicotine significantly increased the rate of GABA A-IPSCs, without affecting the amplitude, but such effect was not obtained with GABA A-miniature IPSCs. The collective results suggest that α7 and non-α7 ACh receptors expressed in the hippocampus, activated under the basal conditions, inhibit release of glutamate and GABA controlled through multi-synaptic relays, but that otherwise, those receptors, highly activated by nicotine, stimulate both the release, with a part of GABA released from interneurons transmitting to CA1 pyramidal neurons. Furthermore, the results also suggest that α7 and non-α7 ACh receptors do not have potency sufficiently to modulate glutamate and GABA release controlled by single synapses.

Chronic BDNF deficiency permanently modifies excitatory synapses in the piriform cortex

Brain-derived neurotrophic factor (BDNF), aside from its classic neurotrophic role in development and survival of neurons, has been shown to be involved in modification and plasticity of central synapses. In mice with BDNF gene deletion (BDNF+/-), deficits in synaptic transmission are often observed but are reversed readily by administration of BDNF, suggesting its acute effect. In support, blockade of BDNF signaling in wild-type hippocampal slices by TrkB-IgG closely reproduces synaptic alterations observed in BDNF+/- mice. We demonstrate that in BDNF+/- mice, lateral olfactory tract (LOT) synapses exhibit decreased release probability of glutamate, suggested by increased paired-pulse facilitation (PPF) of field excitatory postsynaptic potentials (fEPSPs), as well as by slower blocking rate of N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs) by MK-801 in the pyramidal neurons of the piriform cortex. The changes in PPF were not mimicked in wild-type mice by acute blockade of BDNF signaling by TkrB-IgG. These data imply that BDNF deficit during development might lead to chronic changes of excitatory transmission in LOT synapses. Modification of the LOT synapses in BDNF+/- mice was associated with altered inhibitory drive onto the mitral cells from the granule and glomerular neurons, which in turn exhibited decreased renewal rate compared to that in wild-type mice. Taken together, these data suggest that BDNF deficiency can have both acute and more permanent effects on synaptic function, particularly when BDNF signaling is compromised during the early stages of brain development. In the latter case, altered synaptic properties in BDNF+/- mice could be secondary to other complex changes in the brain, e.g., cell survival/proliferation.

Long-term potentiation of silent synapses in substantia gelatinosa neurons

In this study, we observed quantal changes in single synapse excitatory postsynaptic currents to characterize N-methyl-D-aspartate receptor-mediated silent synapses between primary afferents and spinal substantia gelatinosa neurons. The failure rate of primary afferent quantal excitatory postsynaptic currents was lower at depolarized holding potentials than at hyperpolarized potentials. This lower failure rate at depolarized potentials was due to the activation of N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents. Pairing primary afferents with the postsynaptic depolarization induced a long-term decrease in the failure rate of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor-mediated excitatory postsynaptic currents, but did not alter the failure rate of N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents. Our findings suggest that pairing primary afferent with postsynaptic depolarization can convert silent substantia gelatinosa synapses to active synapses and the mechanism of this synaptic plasticity is associated with postsynaptic modification.