The mesopontine tegmentum, including the pedunculopontine and laterodorsal tegmental nuclei (PPN and LDT), provides major cholinergic inputs to midbrain and regulates locomotion and reward. To delineate the underlying projection-specific circuit mechanisms we employed optogenetics to control mesopontine cholinergic neurons at somata and at divergent projections within distinct midbrain areas. Bidirectional manipulation of PPN cholinergic cell bodies exerted opposing effects on locomotor behavior and reinforcement learning. These motor and reward effects were separable via limiting photostimulation to PPN cholinergic terminals in the ventral substantia nigra pars compacta (vSNc) or to the ventral tegmental area (VTA), respectively. LDT cholinergic neurons also form connections with vSNc and VTA neurons, however although photo-excitation of LDT cholinergic terminals in the VTA caused positive reinforcement, LDT-to-vSNc modulation did not alter locomotion or reward. Therefore, the selective targeting of projection-specific mesopontine cholinergic pathways may offer increased benefit in treating movement and addiction disorders.

Anandamide (AEA) and ⌬ 9 -tetrahydrocannabinol (THC) are endogenous and exogenous ligands, respectively, for cannabinoid receptors. Whereas most of the pharmacological actions of cannabinoids are mediated by CB1 receptors, there is also evidence that these compounds can produce effects that are not mediated by the activation of identified cannabinoid receptors. Here, we report that THC and AEA, in a CB1 receptorindependent manner, cause a significant potentiation of the amplitudes of glycine-activated currents (I Gly ) in acutely isolated neurons from rat ventral tegmental area (VTA) and in Xenopus laevis oocytes expressing human homomeric (␣1) and heteromeric (␣1␤1) subunits of glycine receptors (GlyRs). The potentiation of I Gly by THC and AEA is concentration-dependent, with respective EC 50 values of 86 Ϯ 9 and 319 Ϯ 31 nM for ␣1 homomeric receptors, 73 Ϯ 8 and 318 Ϯ 24 nM for ␣1␤1 heteromeric receptors, and 115 Ϯ 13 and 230 Ϯ 29 nM for native GlyRs in VTA neurons. The effects of THC and AEA are selective for I Gly , because GABA-activated current in VTA neurons or in X. laevis oocytes expressing ␣2␤3␥2 GABA A receptor subunits were unaffected by these compounds. The maximal potentiation by THC and AEA was observed at the lowest concentration of glycine; with increasing concentrations of glycine, the potentiation significantly decreased. The site for THC and AEA seems to be distinct from that of the alcohol and volatile anesthetics. The results indicate that THC and AEA, in pharmacologically relevant concentrations, directly potentiate the function of GlyRs through an allosteric mechanism.

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 a-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 D 1 receptor agonist, and by GBR 12935, a dopamine reuptake inhibitor, and they are blocked by SKF 83566, a D 1 antagonist, or by reserpine, which depletes dopamine stores. The enhancement of sEPSC frequency reaches a peak with 40 mM ethanol and declines with concentrations X80 mM ethanol, which is quite likely a result of D 2 receptor activation as raclopride, a D 2 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 D 1 receptors, ethanol at low concentrations increases glutamate release in the VTA, thus raising somatodendritic dopamine release, which further activates the presynaptic D 1 receptors. Enhancement of this positive feedback loop may significantly contribute to the development of alcohol addiction.

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Mesenchymal stem cells (MSCs) are mesoderm-derived cells, primarily resident in adult bone marrow (BM). MSCs exhibit lineage differentiation to generate cells such as BM stroma, fat, and cartilage. Recent studies have reported the transdifferentiation of MSCs to cells of ectodermal and endodermal origin. Previously, we have reported transdifferentiation of human (h) MSCs into neuronal cells using retinoic acid (RA) as a differentiating agent. This study presents a more efficient induction method and rigorously characterizes the development using molecular, cellular, and functional approaches. A cocktail of induction agents containing basic fibroblast growth factor (bFGF) and RA generated cells that expressed glial and neuronal progenitor markers (GFAP) at day 2 post-induction. By day 12, 90% of hMSCs differentiated into cells that expressed markers consistent for neurons, including transcription factors linked to the development of differentiated neurons. Furthermore, cell proliferation studies and western blots for cell cycle-specific proteins demonstrated day-12 induced cells to be post-mitotic cells with no evidence of cell death. The cells exhibited spontaneous post-synaptic currents and were capable of neurotransmitter synthesis, packaging, and release. Together, the improved induction protocol, combined with an interdisciplinary approach to verify that hMSCs can differentiate into neuronal cells, provides a step toward translational application with models of regenerative medicine.