Spinal cord sensory synapses are glutamatergic, but previous studies have found a great diversity in synaptic vesicle structure and have suggested additional neurotransmitters. The identification of several vesicular glutamate transporters (VGLUTs) similarly revealed an unexpected molecular diversity among glutamate-containing terminals. Therefore, we quantitatively investigated VGLUT1 and VGLUT2 content in the central synapses of spinal sensory afferents by using confocal and electron microscopy immunocytochemistry. VGLUT1 localization (most abundant in LIII/LIV and medial LV) is consistent with an origin from cutaneous and muscle mechanoreceptors. Accordingly, most VGLUT1 immunoreactivity disappeared after rhizotomy and colocalized with markers of cutaneous (SSEA4) and muscle (parvalbumin) mechanoreceptors. With postembedding colloidal gold, intense VGLUT1 immunoreactivity was found in 88-95% (depending on the antibody used) of C(II) dorsal horn glomerular terminals and in large ventral horn synapses receiving axoaxonic contacts. VGLUT1 partially colocalized with CGRP in some large dense-core vesicles (LDCVs). However, immunostaining in neuropeptidergic afferents was inconsistent between VGLUT1 antibodies and rather weak with light microscopy. VGLUT2 immunoreactivity was widespread in all spinal cord laminae, with higher intensities in LII and lateral LV, complementing VGLUT1 distribution. VGLUT2 immunoreactivity did not change after rhizotomy, suggesting a preferential intrinsic origin. However, weak VGLUT2 immunoreactivity was detectable in primary sensory nociceptors expressing lectin (GSA-IB4) binding and in 83-90% of C(I) glomerular terminals in LII. Additional weak VGLUT2 immunoreactivity was found over the small clear vesicles of LDCV-containing afferents and in 50-60% of C(II) terminals in LIII. These results indicate a diversity of VGLUT isoform combinations expressed in different spinal primary afferents.

Mammalian spinal motoneurons are considered to be output elements of the spinal cord that generate exclusively cholinergic actions on Renshaw cells, their intraspinal synaptic targets. Here, we show that antidromic stimulation of motor axons evokes depolarizing monosynaptic potentials in Renshaw cells that are depressed, but not abolished, by cholinergic antagonists. This residual potential was abolished by 2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione. In the presence of cholinergic antagonists, motor axon stimulation triggered locomotor-like activity that was blocked by 2-amino-5-phosphonovaleric acid. Some cholinergic motoneuronal terminals on both Renshaw cells and motoneurons were enriched in glutamate, but none expressed vesicular glutamate transporters. Our results raise the possibility that motoneurons release an excitatory amino acid in addition to acetylcholine and that they may be more directly involved in the genesis of mammalian locomotion than previously believed.

The study of hematopoietic colony-forming units using semisolid culture media has greatly advanced the knowledge of hematopoiesis. Here we report that similar methods can be used to study pancreatic colony-forming units. We have developed two pancreatic colony assays that enable quantitative and functional analyses of progenitor-like cells isolated from dissociated adult (2-4 mo old) murine pancreas. We find that a methylcellulose-based semisolid medium containing Matrigel allows growth of duct-like "Ring/ Dense" colonies from a rare (∼1%) population of total pancreatic single cells. With the addition of roof plate-specific spondin 1, a wingless-int agonist, Ring/Dense colony-forming cells can be expanded more than 100,000-fold when serially dissociated and replated in the presence of Matrigel. When cells grown in Matrigel are then transferred to a Matrigel-free semisolid medium with a unique laminin-based hydrogel, some cells grow and differentiate into another type of colony, which we name "Endocrine/Acinar." These Endocrine/Acinar colonies are comprised mostly of endocrine-and acinar-like cells, as ascertained by RNA expression analysis, immunohistochemistry, and electron microscopy. Most Endocrine/Acinar colonies contain beta-like cells that secrete insulin/C-peptide in response to D-glucose and theophylline. These results demonstrate robust self-renewal and differentiation of adult Ring/Dense colony-forming units in vitro and suggest an approach to producing beta-like cells for cell replacement of type 1 diabetes. The methods described, which include microfluidic expression analysis of single cells and colonies, should also advance study of pancreas development and pancreatic progenitor cells. extracellular matrix proteins | Sry-related HMG box (Sox) 9 | Promonin 1 (CD133) | neurogenin 3 | dickkopf1 (Dkk1)

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Prolonged benzodiazepine treatment leads to tolerance and increases the risk of dependence. Flurazepam (FZP) withdrawal is associated with increased anxiety correlated with increased AMPAR-mediated synaptic function and AMPAR binding in CA1 pyramidal neurons. Enhanced AMPAR synaptic strength is also associated with a shift toward inward rectification of synaptic currents and increased expression of GluR1, but not GluR2 subunits, suggesting augmented membrane incorporation of GluR1-containing, GluR2-lacking AMPARs. To test this hypothesis, the postsynaptic incorporation of GluR1 and GluR2 subunits in CA1 neurons after FZP withdrawal was examined using postembedding immunogold quantitative electron microscopy. The percentage of GluR1 positively-labeled stratum radiatum (SR) synapses was significantly increased in FZP-withdrawn rats (88.2 ± 2.2%) compared to controls (74.4 ± 1.9%). In addition, GluR1 immunogold density was significantly increased by 30% in SR synapses in CA1 neurons from FZP-withdrawn rats compared to control rats (FZP: 14.1 ± 0.3 gold particles/μm; CON: 10.8 ± 0.4 gold particles/μm). In contrast, GluR2 immunogold density was not significantly different between groups. Taken together with recent functional data from our laboratory, the current study suggests that the enhanced glutamatergic strength at CA1 neuron synapses during benzodiazepine withdrawal is mediated by increased incorporation of GluR1-containing AMPARs. Mechanisms underlying synaptic plasticity in this model of drug dependence are therefore fundamentally similar to those that operate during activity-dependent plasticity.

In a previous study we established colony assays suitable for studying murine adult (2-4 months) pancreatic progenitor cells plated in semisolid media containing methylcellulose and extracellular matrix proteins. Using these assays, we found robust in vitro progenitor cell activities (multilineage differentiation and self-renewal) from pancreatic cells of adult mice in the CD-1 outbred background. However, it was not clear whether progenitor cell activities can be detected from inbred mice, a preferred mouse model for various genetic studies. It was also not clear whether a single cell is sufficient to self-renew. Here, we show that fluorescent activated cell sorting pancreatic CD133+ but not CD133 -cells from adult C57Bl/6 inbred mice are enriched for progenitor cells that self-renew and give rise to multilineage colonies in vitro. The number of cells in a colony is in proportion to its diameter. Around 60% of single handpicked 3-week-old colonies express trilineage markers, indicating most progenitors are tripotent for ductal, acinar, and endocrine lineage differentiation. Approximately 80% of primary (freshly sorted) colony-forming progenitor cells are capable of giving rise to secondary progenitors in vitro, indicating that a majority of the primary progenitors self-renew. A single cell is sufficient for self-renewal and a Wnt agonist, R-Spondin1, enhances the number of secondary progenitors from the primary progenitors. Together, our pancreatic colony assays allow quantitative analyses of progenitors at a single-cell level from inbred mice. These assays will be useful for elucidating in vitro mechanisms of pancreatic progenitor cell biology.

Benzodiazepine withdrawal-anxiety is associated with enhanced AMPA receptor (AMPAR)-mediated glutamatergic transmission in rat hippocampal CA1 synapses due to enhanced synaptic insertion and phosphorylation of GluA1 homomers. Interestingly, attenuation of withdrawalanxiety is associated with a reduction in NMDA receptor (NMDAR)-mediated currents and subunit expression, secondary to AMPA receptor potentiation. Therefore, in this study ultrastructural evidence for possible reductions in NMDAR GluN1, GluN2A and GluN2B subunits was sought at CA1 stratum radiatum synapses in proximal dendrites using postembedding immunogold labeling of tissues from rats withdrawn for 2-days from 1-week daily oral administration of the benzodiazepine, flurazepam (FZP). GluN1-immunogold density and the percentage of immunopositive synapses were significantly decreased in tissues from FZPwithdrawn rats. Similar decreases were observed for GluN2B subunits, however the relative lateral distribution of GluN2B-immunolabeling within the postsynaptic density did not change after BZ withdrawal. In contrast to the GluN2B subunit, the percentage of synapses labeled with the GluN2A subunit antibody and the density of immunogold labeling for this subunit was unchanged. The spatial localization of immunogold particles asssociated with each NMDAR subunit was consistent with a predominantly postsynaptic localization. The data therefore provide direct evidence for reduced synaptic GluN1/GluN2B receptors and preservation of GluN1/GluN2A receptors in the CA1 stratum radiatum region during BZ withdrawal. Based on collective findings in this benzodiazepine withdrawal-anxiety model, we propose a functional model illustrating the changes in glutamate receptor populations at excitatory synapses during benzodiazepine withdrawal.

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