g-Yuan Min

Activation of AMPA, Kainate, and Metabotropic Receptors at Hippocampal Mossy Fiber Synapses: Role of Glutamate Diffusion

Glutamatergic transmission at mossy fiber (MF) synapses on CA3 pyramidal neurons in the hippocampus is mediated by AMPA, kainate, and NMDA receptors and undergoes presynaptic modulation by metabotropic glutamate receptors. The recruitment of different receptors has thus far been studied by altering presynaptic stimulation to modulate glutamate release and interfering pharmacologically with receptors and transporters. Here, we introduce two novel experimental manipulations that alter the fate of glutamate molecules following release. First, an enzymatic glutamate scavenger reduces the postsynaptic response as well as presynaptic modulation by metabotropic receptors. At physiological temperature, however, the scavenger is effective only when glutamate uptake is blocked, revealing a role of active transport in both synaptic and extrasynaptic communication. Second, AMPA and kainate receptor-mediated postsynaptic signals are enhanced when extracellular diffusion is retarded by adding dextran to the perfusion solution, as is feedback modulation by metabotropic receptors, suggesting that the receptors are not saturated under baseline conditions. These results show that manipulating the spatiotemporal profile of glutamate following exocytosis can alter the involvement of different receptors in synaptic transmission.

Transformation of Human Umbilical Mesenchymal Cells into Neurons in vitro

Neuronal transplantation has provided a promising approach for treating neurodegenerative diseases. Recently, efforts have been directed at in vitro induction of various stem cells to transform into neurons. We report the first successful quantities in an in vitro attempt at directing the transformation into neurons of human umbilical mesenchymal cells, which are capable of rapid proliferation in vitro and are easily available. When cultured in neuronal conditioned medium, human umbilical mesenchymal cells started to express neuron-specific proteins such as NeuN and neurofilament (NF) on the 3rd day and exhibited retraction of the cell body, elaboration of processes, clustering of cells and expression of functional mRNA responsible for the synthesis of subunits of the kainate receptor and glutamate decarboxylase on the 6th day. Between the 9th and 12th days, the percentage of human umbilical mesenchymal cells expressing NF was as high as 87%, while functionality was demonstrated by glutamate invoking an inward current. At this stage, cells were differentiated into mature neurons in the postmitosis phase.

Change in bi-directional plasticity at CA1 synapses in hippocampal slices taken from 6-hydroxydopamine-treated rats: the role of endogenous norepinephrine

The object of the present study is to investigate the role of endogenous adrenergic innervation in regulating bi‐directional synaptic plasticity in rat hippocampal CA1 synapses. The endogenous adrenergic system was eliminated by giving subcutaneous injection of 6‐hydroxydopamine (6‐OHDA) to rats immediately after birth, and the animals were killed for experiments at postnatal ages of 25–35 days. In hippocampal slices taken from 6‐OHDA‐treated animals, theta‐burst stimulation at 100 Hz failed to induce long‐term potentiation (LTP) at CA1 synapses. However, the induction of long‐term depression (LTD) by prolonged low frequency stimulation at 1 Hz was unaffected in slices from 6‐OHDA‐treated animals. Bath application of norepinephrine (NE) restored LTP to control levels and blocked LTD. This effect was mimicked by β‐ but not α‐adrenergic receptor agonists, i.e. by isoproterenol but not phenylephrine. The activators of adenylyl cyclase and protein kinase A (PKA), i.e. forskolin and 8‐bromoadenosine‐3′, 5′‐cyclic monophosphate, respectively, restored LTP in slices from 6‐OHDA‐treated animals. In addition, application of the D1/D5 receptor agonist, dihydrexidine, also restored LTP in slices from 6‐OHDA‐treated animals. These results suggest that physiologically the recruitment of catecholamine innervation may be important for induction of LTP at hippocampal CA1 synapses during tetanic stimulation, while it may not be essential for LTD induction by prolonged 1 Hz stimulation. The released NE and dopamine exert their role in modulating synaptic plasticity via activation of β‐adrenergic and D1/D5 receptors, respectively, which in turn increase the levels of cytoplasm adenosine‐3′,5′‐cyclic monophosphate and PKA.

Glycine-immunoreactive terminals in the rat trigeminal motor nucleus: light- and electron-microscopic analysis of their relationships with motoneurones and with GABA-immunoreactive terminals.

Post-embedding immunolabelling methods were applied to semi-thin and ultrathin resin sections to examine the relationships between glycine- and gamma-aminobutyric acid (GABA)-immunoreactive terminals on trigeminal motoneurones, which were identified by the retrograde transport of horseradish peroxidase injected into the jaw-closer muscles. Serial sections were cut through boutons and alternate sections were incubated with antibodies to glycine and GABA. Light-microscopic analysis of semi-thin sections revealed a similar pattern of glycine and GABA-immunoreactive boutons along the motoneurone soma and proximal dendrites, and of immunoreactive cell bodies in the parvocellular reticular and peritrigeminal areas surrounding the motor nucleus. Immunoreactive synaptic terminals on motoneurones were identified on serial ultrathin sections at electron-microscopic level using a quantitative immunogold method. Three populations of immunolabelled boutons were recognized: boutons immunoreactive for glycine alone (32%), boutons immunoreactive for GABA alone (22%), and boutons showing co-existence of glycine and GABA immunoreactivities (46%). Terminals which were immunoreactive for glycine only contained a higher proportion of flattened synaptic vesicles than those which were immunoreactive for GABA only, which contained predominantly spherical vesicles. Terminals which exhibited both immunoreactivities contained a mixture of vesicle types. All three classes of terminal formed axo-dendritic and axo-somatic contacts onto retrogradely labelled motoneurones. A relatively high proportion (25%) of boutons that were immunoreactive for both transmitters formed synapses on somatic spines. However, only GABA-immunoreactive boutons formed the presynaptic elements at axo-axonic contacts: none of these were found to contain glycine immunoreactivity. These data provide ultrastructural evidence for the role of glycine and GABA as inhibitory neurotransmitters at synapses onto jaw-closer motoneurones, but suggest that presynaptic control of transmission at excitatory (glutamatergic) synapses on motoneurones involves GABAergic, but not glycinergic inhibition.

Role of Extracellular Signal-Regulated Kinase in Synaptic Transmission and Plasticity of a Nociceptive Input on Capsular Central Amygdaloid Neurons in Normal and Acid-Induced Muscle Pain Mice

Application of phorbol 12,13-diacetate (PDA) caused marked enhancement of synaptic transmission of nociceptive parabrachio-amygdaloid (PBA) input onto neurons of the capsular central amygdaloid (CeAC) nucleus. The potentiation of PBA–CeAC EPSCs by PDA involved a presynaptic protein kinase C (PKC)-dependent component and a postsynaptic PKC–extracellular-regulated kinase (ERK)-dependent component. NMDA glutamatergic receptor (NMDAR)-dependent long-term potentiation (LTP) of PBA–CeAC EPSCs, which was also dependent on the PKC–ERK signaling pathway, was induced by tetanus stimulation at 100 Hz. In slices from mice subjected to acid-induced muscle pain (AIMP), phosphorylated ERK levels in the CeAC increased, and PBA–CeAC synaptic transmission was postsynaptically enhanced. The enhanced PBA–CeAC synaptic transmission in AIMP mice shared common mechanisms with the postsynaptic potentiation effect of PDA and induction of NMDAR-dependent LTP by high-frequency stimulation in normal slices, both of which required ERK activation. Since the CeAC plays an important role in the emotionality of pain, enhanced synaptic function of nociceptive (PBA) inputs onto CeAC neurons might partially account for the supraspinal mechanisms underlying central sensitization.

Mice lacking Asic3 show reduced anxiety‐like behavior on the elevated plus maze and reduced aggression

Sensing external stimulation is crucial for central processing in the brain and subsequent behavioral expression. Although sensory alteration or deprivation may result in behavioral changes, most studies related to the control of behavior have focused on central mechanisms. Here we created a sensory deficit model of mice lacking acid‐sensing ion channel 3 (Asic3−/−) to probe behavioral alterations. ASIC3 is predominately distributed in the peripheral nervous system. RT‐PCR and immunohistochemistry used to examine the expression of Asic3 in the mouse brain showed near‐background mRNA and protein levels of ASIC3 throughout the whole brain, except for the sensory mesencephalic trigeminal nucleus. Consistent with the expression results, Asic3 knockout had no effect on synaptic plasticity of the hippocampus and the behavioral tasks of motor function, learning and memory. In anxiety behavior tasks, Asic3−/− mice spent more time in the open arms of an elevated plus maze than did their wild‐type littermates. Asic3−/− mice also displayed less aggressiveness toward intruders but more stereotypic repetitive behaviors during resident–intruder testing than did wild‐type littermates. Therefore, loss of ASIC3 produced behavioral changes in anxiety and aggression in mice, which suggests that ASIC3‐dependent sensory activities might relate to the central process of emotion modulation.

Identification and characterization of a subset of mouse sensory neurons that express acid-sensing ion channel 3

Acid-sensing ion channel 3 (ASIC3) is the most sensitive acid sensor in sensory neurons that innervate into skin, muscle, heart, and visceral tissues. ASIC3 is involved in ischemia sensing, nociception, mechanosensation, and hearing, but how ASIC3-expressing neurons differ in their firing properties is still unknown. We hypothesized that ASIC3-expressing neurons have specialized firing properties, which, coupled with the heterogeneity of acid-sensing properties, accounts for various physiological roles. Here, we successfully identified ASIC3-expressing lumbar dorsal root ganglion (DRG) neurons whose transient proton-gated currents were blocked by salicylic acid (SA). The salicylic acid-sensitive (SAS) neurons did not exist in DRG neurons of mice lacking ASIC3. SAS neurons expressed distinct electrophysiological properties as compared with other DRG neurons. Especially, SAS neurons fired action potentials (APs) with large overshoot and long afterhyperpolarization duration, which suggests that they belong to nociceptors. SAS neurons also exhibited multiple nociceptor markers such as capsaicin response (38%), action potential (AP) with inflection (35%), or tetrodotoxin resistance (31%). Only in SAS neurons but not other DRG neurons was afterhyperpolarization duration correlated with resting membrane potential and AP duration. Our studies reveal a unique feature of ASIC3-expressing DRG neurons and a basis for their heterogeneous functions.

Spike‐timing‐dependent plasticity at resting and conditioned lateral perforant path synapses on granule cells in the dentate gyrus: different roles of N‐methyl‐d‐aspartate and group I metabotropic glutamate receptors

We examined the mechanisms underlying spike‐timing‐dependent plasticity induction at resting and conditioned lateral perforant pathway (LPP) synapses in the rat dentate gyrus. Two stimulating electrodes were placed in the outer third of the molecular layer and in the granule cell layer in hippocampal slices to evoke field excitatory postsynaptic potentials (fEPSPs) and antidromic field somatic spikes (afSSs), respectively. Long‐term potentiation (LTP) of LPP synapses was induced by paired stimulation with fEPSP preceding afSS. Reversal of the temporal order of fEPSP and afSS stimulation resulted in long‐term depression (LTD). Induction of LTP or LTD was blocked by d,l‐2‐amino‐5‐phosphonopentanoic acid (AP5), showing that both effects were N‐methyl‐d‐aspartate receptor (NMDAR)‐dependent. Induction of LTP was also blocked by inhibitors of calcium–calmodulin kinase II, protein kinase C or mitogen‐activated/extracellular‐signal regulated kinase, suggesting that these are downstream effectors of NMDAR activation, whereas induction of LTD was blocked by inhibitors of protein kinase C and protein phosphatase 2B. At LPP synapses previously potentiated by high‐frequency stimulation or depressed by low‐frequency stimulation, paired fEPSP–afSS stimulation resulted in ‘de‐depression’ at depressed LPP synapses but had no effect on potentiated synapses, whereas reversal of the temporal order of fEPSP–afSS stimulation resulted in ‘de‐potentiation’ at potentiated synapses but had no effect on depressed synapses. Induction of de‐depression and de‐potentiation was unaffected by ap5 but was blocked by 2‐methyl‐6‐(phenylethynyl) pyridine hydrochloride, a group I metabotropic glutamate receptor blocker, showing that both were NMDAR‐independent but group I metabotropic glutamate receptor‐dependent. In conclusion, our results show that spike‐timing‐dependent plasticity can occur at both resting and conditioned LPP synapses, its induction in the former case being NMDAR‐dependent and, in the latter, group I metabotropic glutamate receptor‐dependent.

Retrieval of Context-Associated Memory is Dependent on the Cav3.2 T-Type Calcium Channel

Among all voltage-gated calcium channels, the T-type Ca2+ channels encoded by the Cav3.2 genes are highly expressed in the hippocampus, which is associated with contextual, temporal and spatial learning and memory. However, the specific involvement of the Cav3.2 T-type Ca2+ channel in these hippocampus-dependent types of learning and memory remains unclear. To investigate the functional role of this channel in learning and memory, we subjected Cav3.2 homozygous and heterozygous knockout mice and their wild-type littermates to hippocampus-dependent behavioral tasks, including trace fear conditioning, the Morris water-maze and passive avoidance. The Cav3.2 −/− mice performed normally in the Morris water-maze and auditory trace fear conditioning tasks but were impaired in the context-cued trace fear conditioning, step-down and step-through passive avoidance tasks. Furthermore, long-term potentiation (LTP) could be induced for 180 minutes in hippocampal slices of WTs and Cav3.2 +/− mice, whereas LTP persisted for only 120 minutes in Cav3.2 −/− mice. To determine whether the hippocampal formation is responsible for the impaired behavioral phenotypes, we next performed experiments to knock down local function of the Cav3.2 T-type Ca2+ channel in the hippocampus. Wild-type mice infused with mibefradil, a T-type channel blocker, exhibited similar behaviors as homozygous knockouts. Taken together, our results demonstrate that retrieval of context-associated memory is dependent on the Cav3.2 T-type Ca2+ channel.

Heat-shock pretreatment prevents suppression of long-term potentiation induced by scopolamine in rat hippocampal CA1 synapses.

We examined the effect of heat-shock pretreatment on long-term potentiation (LTP) in the CA1 hippocampal slices of the rat using the muscarinic blocker scopolamine as the LTP (memory) suppressor. Time course study using immunohistochemical techniques indicated peak expression of HSP70 16 h after heat-shock treatment. Focusing on that time point we found tetanic stimulation (at 100 Hz) induced LTP of 191.1+/-12.2% in control slices (n=7), which was suppressed by scopolamine to 114.5+/-2.8 %. Heat-shock pretreatment successfully prevented such suppression (216.6+/-38.2% and 190.2+/-10.6% with and without scopolamine, respectively, n=7). Both HSP expression and LTP responses were relatively small taken either 2 or 48 h after heat-shock or sham pretreatment. These results suggest that the induction of HSPs is time-dependent and can prevent scopolamine-mediated LTP suppression.