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One small point of clarification, Iain: there won't be any myelin in
Sarah's neurons - these neurons are unmyelinated. However, more than
95% of their surface is closely apposed by sometimes several layers of
Schwann cell processes, so there is plenty of opportunity for the AM
ester to be soaked up before getting to the neurons...

IAN

On Wednesday, February 23, 2005, at 09:16  AM, Johnson, Iain wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Sarah:
>
> AM ester loading requires both penetration of the AM ester into the
> cytoplasm followed by activation by esterase cleavage. Unfortunately I
> think
> neurons are deficient on both counts - penetration gets impeded by the
> myelin sheath and the glial layer and there isn't much intracellular
> esterase activity compared to glia and astrocytes. Hence the results
> you
> observe and those reported in the Stosiek and Konnerth paper cited by
> George
> Macnamara.  For another example see Hirase et al in PLOS Biology 2:494
> (2004). Concurring with other respondents to your message, I think
> that to
> truly get axon specific staining, you have to put the dye in through a
> cut
> end or a patch clamp and use a dextran conjugate to keep it in situ.
> For a
> nice example, see Verbny Y, Zhang CL, Chiu SY. Coupling of calcium
> homeostasis to axonal sodium in axons of mouse optic nerve. J
> Neurophysiol.
> 88:802-816 (2002).
>
> As far as blocking dye efflux is concerned, the commonly used blockers
> such
> as probenecid primarily inhibit organic anion transporters expressed in
> epithelial cells (probenecid is what one takes to evade detection of
> "performance enhancing substances" in urine tests, or so I am
> told......).
> So its not clear to me that this technique is relevant to neurons.
>
> Iain
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On
> Behalf Of Locknar, Sarah A
> Sent: Friday, February 18, 2005 12:14 PM
> To: [log in to unmask]
> Subject: Neuronal loading advice?
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi everyone-
> Our group has done some calcium imaging in isolated cardiac neurons
> from
> guinea pig in the past using fluo-3 and fluo-4.  We are trying to
> duplicate some of those results in atrial whole mount preparations.  I
> loaded the tissue with 10 uM fluo-4 AM for 1 hour at RT in HEPES
> buffered Krebs and cleaved for another hour before imaging.  What we
> see
> is very strong fluorescence (either more dye or more Ca++) in small
> cells surrounding the neurons (presumably some sort of glia) and
> essentially no dye in the neurons themselves.  We applied high K to
> induce action potentials in the neurons and saw no fluorescence
> increases, making me believe that there's no dye in them.  It seems
> like
> the dye is either not making it through the glial layer, or perhaps it
> is being actively extruded from the neurons.  Does anyone have any
> ideas
> about getting the dye in an keeping it in?  Next, I was going to try
> loading in the refrigerator and cleaving at RT, as suggested on this
> list.
> Thanks in advance-
> Sarah
>
> -----------------------------------------------------------------------
> -
> ---------------
> Sarah Locknar, Ph.D.
> Director, Neuroscience COBRE Imaging / Physiology Core
> College of Medicine, University of Vermont
> E015 Given Building
> 89 Beaumont Ave.
> Burlington, VT 05405
> 802-656-0413
> 802-656-8704 (fax)
> -----------------------------------------------------------------------
> -
> ---------------
>
>

* * * * * * * * * * *
Prof Ian Gibbins
Anatomy & Histology
Flinders University
GPO Box 2100
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