Hsiu-Wen Yang

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.

Morphology of local axon collaterals of electrophysiologically characterised neurons in the rat medial septal/ diagonal band complex.

Neurons in the medial septal/diagonal band complex (MS/DB) in vivo exhibit rhythmic burst‐firing activity that is phase‐locked with the hippocampal theta rhythm. The aim was to assess the morphology of local axon collaterals of electrophysiologically identified MS/DB neurons using intracellular recording and biocytin injection in vitro. Cells were classified according to previous criteria into slow‐firing, fast‐spiking, regular‐spiking, and burst‐firing neurons; previous work has suggested that the slow‐firing neurons are cholinergic and that the other types are GABAergic. A novel finding was the existence of two types of burst‐firing neuron. Type I burst‐firing neurons had significantly longer duration after hyperpolarisation potentials when held at −60 mV, and at −75 mV, type I neurons exhibited a low‐threshold spike with more rapid activation and inactivation kinetics than those of type II neurons. We have, also for the first time, described the main features of the local axon collaterals of the five neuron types. All filled neurons possessed a main axon that gave forth 1–12 local primary axon collaterals. All electrophysiological types, except for the type I burst‐firing neuron, had a main axon that coursed toward the fornix. Myelination of the main axon was a prominent feature of all but the slow‐firing neurons. Branching of the primary axon collaterals of the fast‐spiking and type I burst‐firing neurons was more extensive than that of the other cell types, with those of the slow‐firing neurons exhibiting the least branching. All cell types possessed axon collaterals of the en passant type, and some in addition had twiglike or basketlike axon terminals. All cell types made synapses on distal dendrites; a proportion of the fast‐spiking and burst‐firing cells in addition had basketlike terminals that made synaptic contacts on proximal dendrites and on somata. Two morphological types of somata were postsynaptic to the basket cells: large (20–30‐μm) oval cells with dark cytoplasm, and large oval cells with paler cytoplasm, often with an apical dendrite. The presence of lamellar bodies in the large dark neurons suggests that they may be cholinergic neurons, because previous work has localised these structures in some neurons that stain for choline acetyltransferase. Our work suggests therefore that there may be GABAergic neurons in the MS/DB that form basket synaptic contacts on at least two types of target cell, possibly cholinergic and GABAergic neurons, which means that the basket cells could play a key role in the generation of rhythmic activity in the MS/DB. J. Comp. Neurol. 430:410–432, 2001.

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.

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.

The physiological and morphological characteristics of interneurons caudal to the trigeminal motor nucleus in rats

In this study we have characterized the membrane properties and morphology of interneurons which lie between the caudal pole of the trigeminal motor nucleus and the rostral border of the facial motor nucleus. Previous studies suggest that many of these interneurons may participate in the genesis of rhythmical jaw movements. Saggital brainstem slices were taken from rats aged 5–8 days. Interneurons lying caudal to the trigeminal motor nucleus were visualized using near‐infrared differential interference contrast (DIC) microscopy, and were recorded from using patch pipettes filled with a K‐gluconate‐ and biocytin‐based solution. The 127 neurons recorded could be categorized into three subtypes on the basis of their responses to injection of depolarizing current pulses, namely tonic firing (type I), burst firing (type II) and spike‐adaptive (type III) neurons. Type I interneurons had a higher input resistance and a lower rheobase than type II neurons. All three neuron subtypes showed ‘sag’ of the voltage response to injection of large‐amplitude hyperpolarizing current pulses, and, in addition, also showed rectification of the voltage response to injection of depolarizing current pulses, with type II neurons showing significantly greater rectification than type I neurons. The axonal arborizations were reconstructed for 44 of 63 neurons labelled with tracer. Neurons of each subtype were found to issue axon collaterals terminating in the brainstem nuclei, including the parvocellular reticular nucleus (PCRt), the trigeminal motor nucleus (Vmot), the supratrigeminal nucleus or the trigeminal mesencephalic nucleus. Twenty‐five of the 43 neurons issued collaterals which terminated in the Vmot and the other brainstem nuclei. When viewed under 100× magnification, the collaterals of some interneurons were seen to give off varicosities and end‐terminations which passed close to the somata of unidentified neurons in the trigeminal motor nucleus and in the area close to the interneuron soma itself. This suggests that the interneurons may make synaptic contacts both on motoneurons and also on nearby interneurons. These results provide data on the membrane properties of trigeminal interneurons and evidence for their synaptic connections both with nearby interneurons and also with motoneurons. Thus, the interneurons examined could play roles in the shaping, and possibly also in the generation, of rhythmical signals to trigeminal motoneurons.

Comparison of synaptic transmission and plasticity between sensory and cortical synapses on relay neurons in the ventrobasal nucleus of the rat thalamus.

Relay neurons in the ventrobasal nucleus of the thalamus transmit somatosensory information to the cerebral cortex and receive sensory and cortical (feedback) synaptic inputs via, respectively, medial lemniscal (ML) and corticothalamic (CT) fibres. Here, we report that calcium‐permeable AMPA receptors are expressed at CT synapses, but not ML synapses, and that the NMDA receptor (NMDAR)‐mediated/non‐NMDAR‐mediated synaptic current ratio is significantly larger at CT synapses than at ML synapses. Moreover, NMDAR‐dependent LTP and L‐type voltage‐gated calcium channel‐dependent LTD are readily induced at CT synapses, but not ML synapses. In particular, LTD of CT synaptic transmission is induced by spiking of postsynaptic relay neurons in continuous mode, but not burst mode, in current‐clamp recordings. These results show that the strength of the cortical input to thalamic relay neurons is selectively subjected to use‐dependent modification, which could be a mechanism for regulation of thalamocortical–corticothalamic interactions and the underlying sensory processing.

Physiological and morphological properties of, and effect of substance P on, neurons in the A7 catecholamine cell group in rats

The A7 catecholamine cell group consists of noradrenergic (NAergic) neurons that project to the dorsal horn of the spinal cord. Here, we characterized their morphology and physiology properties and tested the effect of substance P (Sub-P) on them, since the results of many morphological studies suggest that A7 neurons are densely innervated by Sub-P-releasing terminals from nuclei involved in the descending inhibitory system, such as the lateral hypothalamus and periaqueductal gray area. Whole cell recordings were made from neurons located approximately 200 microm rostral to the trigeminal motor nucleus (the presumed A7 area) in sagittal brainstem slices from rats aged 7-10 days. After recording, the neurons were injected with biocytin and immunostained with antibody against dopamine-beta-hydroxylase (DBH). DBH-immunoreactive (ir) cells were presumed to be NAergic neurons. They had a large somata diameter ( approximately 20 microm) and relatively simple dendritic branching patterns. They fired action potentials (AP) spontaneously with or without blockade of synaptic inputs, and had similar properties to those of NAergic neurons in other areas, including the existence of calcium channel-mediated APs and a voltage-dependent delay in initiation of the AP (an indicator of the existence of A-type potassium currents) and an ability to be hyperpolarized by norepinephrine. Furthermore, in all DBH-ir neurons tested, Sub-P caused depolarization of the membrane potential and an increase in neuronal firing rate by acting on neurokinin-1 receptors. Non-DBH-ir neurons with a smaller somata size were also found in the A7 area. These showed great diversity in firing patterns and about half were depolarized by Sub-P. Morphological examination suggested that the non-DBH-ir neurons form contacts with DBH-ir neurons. These results provide the first description of the intrinsic regulation of membrane properties of, and the excitatory effect of Sub-P on, A7 area neurons, which play an important role in pain regulation.

Neurokinin 1 receptor activates transient receptor potential-like currents in noradrenergic A7 neurons in rats.

Noradrenergic (NAergic) A7 neurons are involved in modulating nociception by releasing noradrenaline in the dorsal spinal cord. Since NAergic A7 neurons receive dense Substance P (Sub-P) releasing terminals from ventromedial medulla, here we tested the effect of Sub-P on them. Bath application of Sub-P induced an inward current (I(Sub-P)) in NAergic neurons, which was significantly blocked by Neurokinin 1 (NK1) receptor antagonist. The I(Sub-P) was reversed at approximately -20 mV, blocked by several TRP channel blockers, enhanced by OAG and negatively regulated by PKC. Immunohistochemistry staining showed that NAergic A7 neurons express high level of TRPC6 channel proteins, which is consistent with pharmacological properties of I(Sub-P) shown above, as TRPC6 channel is shown to be augmented by OAG and inhibited by PKC. In conclusion, the above results provide mechanism underlying postsynaptic action of Sub-P on NAergic A7 neurons and a role for TRPC6 channel in NAergic pain modulation.