Jiang Hong Ye

Neurons Derived From Human Mesenchymal Stem Cells Show Synaptic Transmission and Can Be Induced to Produce the Neurotransmitter Substance P by Interleukin‐1α

Mesenchymal stem cells (MSCs) exhibit immune‐suppressive properties, follow a pattern of multilineage differentiation, and exhibit transdifferentiation potential. Ease in expansion from adult bone marrow, as well as its separation from ethical issues, makes MSCs appealing for clinical application. MSCs treated with retinoic acid resulted in synaptic transmission, based on immunostaining of synaptophysin and electrophysiological studies. In situ hybridization indicated that the neurotransmitter gene preprotachykinin‐I was expressed in these cells. However, translation of this gene only occurred after stimulation with interleukin (IL)‐1α. This effect was blunted by costimulation with IL‐1 receptor antagonist. This study reports on the ability of MSCs to be transdifferentiated into neurons with functional synapses with the potential to become polarized towards producing specific neurotransmitters.

Patch-clamp studies in the CNS illustrate a simple new method for obtaining viable neurons in rat brain slices: glycerol replacement of NaCl protects CNS neurons.

Viable neurons in brain slices are crucial for electrophysiological studies. The present study describes a new method for obtaining viable cells in several regions of the central nervous system including the ventral tegmental area, the hypothalamus, the periaqueductal grey matter and the spinal cord. The essence of the method was to use a modified artificial cerebrospinal fluid (ACSF) in which all NaCl was replaced initially by equi-osmotic glycerol. This modified glycerol-based ACSF was used during slice preparation. The underlying principle for the modification is to prevent the possible acute neurotoxic effects of passive chloride entry, subsequent cell swelling and lysis. This method significantly increased the live/dead ratio in morphology compared to the normal ACSF or sucrose-base ACSF, in which NaCl was replaced by sucrose. An examination of some electrophysiological and pharmacological properties of the neurons in these preparations, by means of current-clamp and voltage-clamp recordings, revealed similar properties of those neurons obtained with the traditional ACSF method. Due to the increase in the number of viable neurons, the new ACSF increases the productivity of experiments. Based on our data, we propose that this glycerol-based solution may protect CNS neurons.

Ethanol Facilitates Glutamatergic Transmission to Dopamine Neurons in the Ventral Tegmental Area

The cellular mechanisms underlying alcohol addiction are poorly understood. In several brain areas, ethanol depresses glutamatergic excitatory transmission, but how it affects excitatory synapses on dopamine neurons of the ventral tegmental area (VTA), a crucial site for the development of drug addiction, is not known. We report here that in midbrain slices from rats, clinically relevant concentrations of ethanol (10–80 mM) increase the amplitude of evoked EPSCs and reduce their paired-pulse ratio in dopamine neurons in the VTA. The EPSCs were mediated by glutamate α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. In addition, ethanol increases the frequency but not the amplitude of spontaneous EPSCs. Furthermore, ethanol increases extracellular glutamate levels in the VTA of midbrain slices. The effects of ethanol are mimicked by SKF 38393, a dopamine D1 receptor agonist, and by GBR 12935, a dopamine reuptake inhibitor, and they are blocked by SKF 83566, a D1 antagonist, or by reserpine, which depletes dopamine stores. The enhancement of sEPSC frequency reaches a peak with 40 mM ethanol and declines with concentrations ⩾80 mM ethanol, which is quite likely a result of D2 receptor activation as raclopride, a D2 receptor blocker, significantly enhanced 80 mM ethanol-induced enhancement of sEPSCs. Finally, 6, 7-dinitroquinoxaline-2, 3-dione (DNQX), an AMPA receptor antagonist, attenuates ethanol-induced excitation of VTA DA neurons. We therefore conclude that, acting via presynaptic D1 receptors, ethanol at low concentrations increases glutamate release in the VTA, thus raising somatodendritic dopamine release, which further activates the presynaptic D1 receptors. Enhancement of this positive feedback loop may significantly contribute to the development of alcohol addiction.

Presynaptic Glycine Receptors on GABAergic Terminals Facilitate Discharge of Dopaminergic Neurons in Ventral Tegmental Area

GABA-mediated postsynaptic currents (IPSCs) were recorded from dopaminergic (DA) neurons of the ventral tegmental area (VTA) of rats, in acute brain slices, and from enzymatically or mechanically dissociated neurons. In young rats (3-10 d of age), where GABA is excitatory, glycine (1-3 μm) and taurine (10-30 μm) increased the amplitude of evoked IPSCs (eIPSCs) and the frequency of spontaneous IPSCs (sIPSCs) but had minimal postsynaptic effects. Strychnine (1 μm) blocked the action of glycine; when applied alone, it reduced the amplitude of eIPSCs and the frequency of sIPSCs, indicating a tonic facilitation of GABAergic excitation by some endogenous glycine agonist(s). In medium containing no Ca2+, or with Cd2+ or tetrodotoxin added, the amplitude and especially the frequency of sIPSCs greatly diminished. In many cells, glycine had no effect on remaining miniature IPSCs, suggesting a preterminal site of glycine receptors (GlyRs). Fura-2 fluorescent imaging showed a glycine-induced increase of [Ca2+] in nerve terminals (on DA neurons), which was suppressed by strychnine or 3 μm ω-conotoxin MVIIA. Therefore, the presynaptic GlyR-mediated facilitation of GABAergic transmission seems to be mediated by N- and/or P/Q-type Ca2+ channels. In older rats (22-30 d of age), where GABA causes inhibition, the effect of strychnine on GABAergic IPSCs was reversed to facilitation, indicating a tonic glycinergic inhibition of GABA release. Furthermore, glycine (1-3 μm) reduced the amplitude of eIPSCs and the frequency of sIPSCs. Hence, the overall effect of the presynaptic action of glycine is to enhance the firing of DA cells, both in very young and older rats.

Brain‐derived neurotrophic factor facilitates maturation of mesenchymal stem cell‐derived dopamine progenitors to functional neurons

The generation of dopamine (DA) neurons from stem cells holds great promise in the treatment of Parkinson’s disease and other neural disease associated with dysfunction of DA neurons. Mesenchymal stem cells (MSCs) derived from the adult bone marrow show plasticity with regards to generating cells of other germ layers. In addition to reduced ethical concerns, MSCs could be transplanted across allogeneic barriers, making them desirable stem cells for clinical applications. We have reported on the generation of DA cells from human MSCs using sonic hedgehog (SHH), fibroblast growth factor 8 and basic fibroblast growth factor. Despite the secretion of DA, the cells did not show evidence of functional neurons, and were therefore designated DA progenitors. Here, we report on the role of brain‐derived neurotrophic factor (BDNF) in the maturation of the MSC‐derived DA progenitors. 9‐day induced MSCs show significant tropomyosin‐receptor‐kinase B expression, which correlate with its ligand, BDNF, being able to induce functional maturation. The latter was based on Ca2+ imaging analyses and electrophysiology. BDNF‐treated cells showed the following: increases in intracellular Ca2+ upon depolarization and after stimulation with the neurotransmitters acetylcholine and GABA and, post‐synaptic currents by electrophysiological analyses. In addition, BDNF induced increased DA release upon depolarization. Taken together, these results demonstrate the crucial role for BDNF in the functional maturation of MSC‐derived DA progenitors.

Ethanol Enhances Glutamate Transmission by Retrograde Dopamine Signaling in a Postsynaptic Neuron/Synaptic Bouton Preparation From the Ventral Tegmental Area

It is well documented that somatodendritically released dopamine is important in the excitability and synaptic transmission of midbrain dopaminergic neurons. Recently we showed that in midbrain slices, acute ethanol exposure facilitates glutamatergic transmission onto dopaminergic neurons in the ventral tegmental area (VTA). The VTA is a brain region critical to the rewarding effects of abused drugs, including ethanol. We hypothesized that ethanol facilitation might result from an increase in somatodendritically released dopamine, which acts retrogradely on dopamine D1 receptors on glutamate-releasing axons and consequently leads to an increase in glutamate release onto dopaminergic neurons. To further test this hypothesis and to examine whether ethanol facilitation can occur at the single-cell level, VTA neurons were freshly isolated from rat brains using an enzyme-free procedure. These isolated neurons retain functional synaptic terminals, including those that release glutamate. Spontaneous excitatory postsynaptic currents (sEPSCs) mediated by glutamate α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors were recorded from these freshly isolated putative dopaminergic neurons. We found that acute application of clinically relevant concentrations of ethanol (10–80 mM) significantly facilitated the frequency of sEPSCs but not their mean amplitude. Ethanol facilitation was mimicked by the D1 agonist SKF 38393 and by the dopamine uptake blocker GBR 12935 but was blocked by the D1 antagonist SKF 83566, and by depleting dopamine stores with reserpine, as well as by chelating postsynaptic calcium with BAPTA. Furthermore, the sodium channel blocker tetrodotoxin eliminated the facilitation of sEPSCs induced by ethanol but not by SKF 38393. These results constitute the first evidence from single isolated cells of ethanol facilitation of glutamate transmission to dopaminergic neurons in the VTA. In addition, we show that ethanol facilitation has a postsynaptic origin and a presynaptic locus. Furthermore, ethanol stimulation of a single dopaminergic neuron is capable of eliciting the release of somatodendritic dopamine, which is sufficient to influence glutamatergic transmission at individual synapses.

Physiology and pharmacology of native glycine receptors in developing rat ventral tegmental area neurons.

Properties of whole-cell glycine-activated currents (I(Gly)) of neurons freshly isolated from ventral tegmental area of rats between postnatal day (P) 0 and 40 (P0-P40) were examined using the gramicidin-perforated patch technique. I(Gly) was present throughout the period of investigation and displayed age-related alternations in its kinetics, reversal potentials (E(Gly)) and sensitivity to antagonists. During development, the time constant for desensitization of I(Gly) decreased about two-fold, and the E(Gly) shifted to more negative potentials. Specifically, E(Gly) was -29 mV in neonatal (P0-P7) and decreased to -50 mV in adult (P24-P40) neurons. While the neonatal glycine receptors (GlyRs) are resistant to strychnine, the adult GlyRs are sensitive to STR. The values for 50% inhibition (IC(50)) of I(Gly) was 110 and 12 nM in P0-P3 and P24-P40 neurons, respectively. Picrotoxin, on the other hand, was more effective in blocking neonatal neurons. The IC(50) values were 17 and 231 microM for the neonatal and adult neurons, respectively. These observations are discussed in terms of developmental changes of subunit composition of GlyRs and of intracellular Cl(-) concentrations.

Histamine regulates activities of neurons in the ventrolateral preoptic nucleus

The neurons responsible for the onset of sleep are thought to be located in the ventrolateral preoptic nucleus (VLPO), which receives a dense histaminergic innervation from the tuberomammillary nucleus (TMN). Yet, the role of histamine in the VLPO remains unclear. Here we report that microinjection of histamine into the VLPO increases the motor activity of rats. Moreover, a bath application of histamine to acute brain slices inhibits the majority of VLPO neurons, which are also inhibited by noradrenaline. Histamine hyperpolarizes the membrane potential and lowers the firing rate. These effects are associated with an increase in the frequency but not in the amplitude of spontaneous GABAA receptor‐mediated inhibitory postsynaptic currents, and are blocked by gabazine or tetrodotoxin, indicating an indirect action. Conversely, on the noradrenaline‐excited VLPO neurons, histamine depolarizes the membrane potential and increases the firing rate via activation of H1 and H2 subtype histamine receptors. Moreover, histamine‐induced depolarization persists in the presence of gabazine or tetrodotoxin, indicating a direct action. Based on these findings, we propose that in the VLPO, noradrenaline‐inhibited neurons may normally be under the inhibitory control of noradrenaline‐excited neurons. By facilitating the inhibitory control of the noradrenaline‐excited neurons, histamine may inhibit the noradrenaline‐inhibited neurons, resulting in excitation of histamine‐releasing neurons in the TMN through disinhibition. This effect of histamine in the VLPO may contribute to the maintenance of wakefulness.

Taurine activates excitatory non-synaptic glycine receptors on dopamine neurones in ventral tegmental area of young rats

The physiological and pharmacological properties of taurine‐induced responses were investigated in dopaminergic (DA) neurones from the ventral tegmental area (VTA) of young rats aged 1–13 postnatal days, either in acute brain slices or acutely dissociated neurones. When whole‐cell responses were recorded from current‐clamped neurones using the gramicidin‐perforated technique, the application of taurine (0.01–30 mm) accelerated firings and induced membrane depolarization. In voltage‐clamped neurones, taurine induced a current which was antagonized by strychnine and by picrotoxin, but not by bicuculline. In addition, taurine‐induced current showed complete cross‐desensitization with glycine‐activated currents but not with γ‐aminobutyric acid (GABA)‐activated currents. Thus, taurine is a full agonist of the glycine receptors (GlyRs) in the VTA. Further studies found that taurine acted mainly on non‐synaptic GlyRs. The application of 20 μm bicuculline abolished the spontaneous inhibitory post‐synaptic currents (IPSCs) in 40/45 neurones, and 93% of the evoked IPSCs. The addition of 1 μm strychnine completely eliminated the remaining IPSCs. These results suggest that GABAergic IPSCs predominate, and that functional glycinergic synapses are present in a subset of the VTA neurones. The application of 1 μm strychnine alone induced an outward current, suggesting that these neurones were exposed to tonically released taurine/glycine. In conclusion, by activating non‐synaptic GlyRs, taurine may act as an excitatory extra‐synaptic neurotransmitter in the VTA during early development.

MicroRNA expression profile and functional analysis reveal that miR-382 is a critical novel gene of alcohol addiction

Alcohol addiction is a major social and health concern. Here, we determined the expression profile of microRNAs (miRNAs) in the nucleus accumbens (NAc) of rats treated with alcohol. The results suggest that multiple miRNAs were aberrantly expressed in rat NAc after alcohol injection. Among them, miR‐382 was down‐regulated in alcohol‐treated rats. In both cultured neuronal cells in vitro and in the NAc in vivo, we identified that the dopamine receptor D1 (Drd1) is a direct target gene of miR‐382. Via this target gene, miR‐382 strongly modulated the expression of DeltaFosB. Moreover, overexpression of miR‐382 significantly attenuated alcohol‐induced up‐regulation of DRD1 and DeltaFosB, decreased voluntary intake of and preference for alcohol and inhibited the DRD1‐induced action potential responses. The results indicated that miRNAs are involved in and may represent novel therapeutic targets for alcoholism.