Thursday, March 31, 2011

GFAP and Mouse Myenteric Plexus

Our Neuronal-Glial Markers are important tools for our customers investigating expression in the CNS and PNS. I have posted images showing staining of mouse retinal astrocytes and in the ventral horn, funiculus of adult rat spinal cord and mouse medulloblastoma stem cells using our GFAP antibodies.

I wanted to share an excellent image generated by Dr. Kate Ellacott's lab at Vanderbilt University.

GFAP (Chicken-Cat#: CH22102) staining in of enteric glia in the myenteric plexus of the mouse gastrointestinal tract. Staining was performed in methanol/acetone fixed frozen sections using 1:1000 dilution of the antibody followed by 1:500 anti-chicken Alexa 594.

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Wednesday, March 23, 2011

STEMEZ hNP1 Neural Progenitors and Ion Channels

In my conversation with neuro-drug discover researchers, I am frequently being asked about the potential of using our STEMEZ(TM) hNP1 Human Neural Progenitors Expansion Kits for studying ion channels. How effective are these cells as a source for studying neurodegenerative diseases and for drug screening assays? There is good news.

When differentiated, these  neural progenitors express subunits of glutamatergic,  GABAergic, nicotinic, purinergic and transient receptor potential receptors. In addition, sodium  and calcium channel subunits were also expressed. Functionally, virtually all the NP cells exhibited delayed rectifier potassium channel currents and some differentiated cells exhibited  tetrodotoxin sensitive, voltage-dependent sodium channel current under whole-cell voltage clamp and action potentials could be elicited by current injection under whole-cell current clamp.  These results indicate that removing basic fibroblast growth factor from the neural progenitor cell cultures leads to a post-mitotic state, and also results in the capability to produce excitable cells that can generate action potentials. This is the first data demonstrating capabilitiesof these cells for ionotrophic receptor assays and ultimately for electrically active human neural cell assays for drug discovery.

Images: Glutamate receptor expression in hNP cells and differentiated hNP cells The expression of ionotropic glutamate receptors might also be an indicator of neuronal maturation. These receptors are composed of three distinct families: NMDA, kainate and AMPA receptors. The hNP cells and differentiated hNP cells cultured in the absence of bFGF for 2 weeks were analyzed for mRNA expression of subunits of each glutamate receptor subtype relative to hESCs. Significant increases (p<0.05) in Grin2b were seen in hNP cells (20 fold) and differentiated hNP cells (25 fold) relative to hESCs (Figure 3A). Additionally, Grin1 and Grin2d were significantly increased (p<0.05) only in differentiated hNP cells relative to hESCs, but not in undifferentiated hNP cells (Figure 3A). Of the kainate receptors, Grik4 and Grik5 were significantly (p<0.05) increased only in undifferentiated hNP cells relative to hESCs (Figure 3B); whereas, Grik2 was significantly (p<0.05) increased only in hNP cells where bFGF had been removed (Figure 3B). AMPA receptor subunits were also examined. Gria1 and Gria4 were up regulated in hNP cells relative to hESCs (Figure 3C). Two week differentiated hNP cells showed significant (p<0.05) up regulation of Gria2 and Gira4 relative to hESCs (Figure 3C). To determine if functional glutamate channels exist in differentiated hNP cells, calcium influx in response to AMPA, kainic acid or NMDA application was measured on hNP cells, 14 days after the removal of bFGF. Figure 3G indicates that NMDA could not depolarize differentiated or undifferentiated hNP cells enough to cause significant calcium influx above background. In contrast, AMPA and kainic acid can cause calcium influx which can be potentiated by AMPA receptor specific modulator, cyclothiazide (50 μM, Figure 3G).Calcium influx was detected in the presence of cyclothiazide in calcium activity as measured (Figure 3H).

Images: Sodium channel activity in differentiated hNP cells was measured using whole cell voltage clamp. 81 total hNP cells cultured in the absence of bFGF from 4 to 27 days were analyzed. Of these, 34 exhibited no fast inward currents in response to a step depolarization indicating the 348 absence of functional voltage gated sodium channels (Figure 4G). The remaining cells yielded between 0.04 - 1.5 nA of inward current in response to the step depolarization (Figures 4B and 4G). These currents inactivated rapidly in all cases (Figures 4B and 4C) and could be abolished with the addition of 1 μM TTX (n = 3 cells; Figure 4C). Voltage-dependent steady state inactivation (n = 11 cells; Figure 4D) and recovery from fast inactivation (n = 5 cells; Figure 4E) were also observed on several positive cells. A subset of these cells was subjected to current clamp and action potentials were elicited by current injection (n = 8 cells, Figure 4F). In support of this, increasing concentrations of a sodium channel activator veratridine in a FLIPR assay on differentiated hNP cells show an increasing calcium response (Figure 4H). This probably resulted from voltage-gated sodium channel depolarization of cells that subsequently allowed calcium influx through calcium channels. These data indicate that differentiation of hNP cells by removal of bFGF can lead to a neuronal cell that can generate action potentials and depolarize the cell. The 58% hit rate for voltage-gated sodium channel function (Figure 4G), does not reflect the true proportion of sodium channel positive cells in our differentiated hNP cells, but rather our ability to morphologically distinguish these cells from negative cells by eye. An example of the morphology of a sodium channel positive cell is shown in Figure 4A. The positive cells were phase bright with a few long processes.

Tuesday, March 15, 2011

pERK1/2, TRPV1 and Scalding Burn Pain

I can only imaging the intense pain suffered by people with scalding pain injuries.

Understanding the bio-processes behind this form of nociceptive pain is a step towards finding better analgesics. In  this study, the authors study several key markers for pain...phosphoERK1/2 and TRPV1 (surprisingly not a major piece of the this pain signaling process).

John P.M. Whitea, Chin Wing Koa, Antonio Rei Fidalgoa, Mario Cibellia, Cleoper C. Paulea, Peter J. Andersona, Celia Cruzb, Szabolcs Gombad, Klara Matesze, Gabor Veressd, Antonio Avelinob and Istvan Nagya. Severe burn injury induces a characteristic activation of extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons. European Journal of Pain.doi:10.1016/j.ejpain.2010.12.006...pERK1/2 (1:1000, Neuromics; RA15002)..
Abstract: We have studied scalding-type burn injury-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the spinal dorsal horn, which is a recognised marker for spinal nociceptive processing. At 5 min after severe scalding injury to mouse hind-paw, a substantial number of phosphorylated ERK1/2 (pERK1/2) immunopositive neurons were found in the ipsilateral dorsal horn. At 1 h post-injury, the number of pERK1/2-labelled neurons remained substantially the same. However, at 3 h post-injury, a further increase in the number of labelled neurons was found on the ipsilateral side, while a remarkable increase in the number of labelled neurons on the contralateral side resulted in there being no significant difference between the extent of the labelling on both sides. By 6 h post-injury, the number of labelled neurons was reduced on both sides without there being significant difference between the two sides. A similar pattern of severe scalding injury-induced activation of ERK1/2 in spinal dorsal horn neurons over the same time-course was found in mice which lacked the transient receptor potential type 1 receptor (TRPV1) except that the extent to which ERK1/2 was activated in the ipsilateral dorsal horn at 5 min post-injury was significantly greater in wild-type animals when compared to TRPV1 null animals. This difference in activation of ERK1/2 in spinal dorsal horn neurons was abolished within 1 h after injury, demonstrating that TRPV1 is not essential for the maintenance of ongoing spinal nociceptive processing in inflammatory pain conditions in mouse resulting from at least certain types of severe burn injury.

Wednesday, March 09, 2011

TrkB and Assembly/Maintenance of GABAergic Neurons

Publications that provide unique insight into neuro-and synptogenesis catch my attention.

This publication focuses on the mechanisms that direct inhibitory circuits. Specifically, the authors study assembly and maintenance of GABAergic inhibitory synapses between Golgi and granule cells in the mouse cerebellar cortex.

Albert I. Chen, Cindy N. Nguyen, David R. Copenhagen, Sylvia Badurek, Liliana Minichiello, Barbara Ranscht, and Louis F. Reichardt. TrkB (Tropomyosin-Related Kinase B) Controls the Assembly and Maintenance of GABAergic Synapses in the Cerebellar Cortex . The Journal of Neuroscience, 23 February 2011, 31(8): 2769-2780; doi: 10.1523/​JNEUROSCI.4991-10.2011.

Our cell adhesion marker-Contactin-1 was referenced: Immunohistochemistry 1:1,000; western blot: 1:1,000

Conclusion Highlights:  Kinase activity of TrkB is required not simply to initiate GABAergic synapse formation, but also to maintain these synapses in adulthood. We also show that the localization of Contactin-1 at the synaptic contacts between Golgi and granule cells requires TrkB suggesting that TrkB promotes synapse formation and maintenance, in part, by controlling the localization of cell adhesion molecules.

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