We reported the colocalization of two proteins at LM and EM level, and mRNAs by in situ hybridization.

The below is extracted from "Abstract".

"The colocalization was more frequent in the primary somatosensory (S1) area than in both the primary visual (V1) and the motor areas; of area S1 cortical layers, colocalization was most evident in layer IV barrels at postnatal day (P) 7 and in adulthood."

Ref

Nakamura Ko, Watakabe A, Hioki H, Fujiyama F, Tanaka Yasuyo, Yamamori T, *Kaneko T. (2007) Transiently increased colocalization of vesicular glutamate transporters 1 and 2 at single axon terminals during postnatal development of mouse neocortex: a quantitative analysis with correlation coefficient. Eur J Neurosci 26(11):3054-3067.

PMID: 18028110

Dear Nicholas,

in our experience, we have detected the mRNA coding vGlut2 in pyramidal neurons troughout layers III and V of the cortex. Please have a look to panel C in Figure 2 of the enclosed manuscript.

Best, Jose L.

For some unkonwn reasons, I cannot attach the related manuscript. The complete reference is Lanciego et al, J. Psychopharm 25(2011):97-104. If you provide me with your email, I will send it to you immediately

Hi Nicholas,

Sorry for the former misunderstanding. I was considering that you were asking about endocannabinoid receptors type 2 instead of vGLUTs. With regards to vGluts, we have been working for a while on the thalamic expression of vGlut1 and vGlut2. At the mRNA level, we have found that thalamic neurons located in associative and motor-related thalamic nuclei co-express both vGluts isoforms. This is relevant to your questions since most of these neurons are the main contributors to thalamocortical projections. You can have a look to Barroso-Chinea et al., J Comp Neurol 501(2007):703-715 and Barroso-Chinea et al., J Chem Neuroanat 35(2008):101-107 (the latter related to Vglut coexpression within neurons giving rise to thalamostriatal projections).

As far as I know, vGluT2 itself is expressed in cortical cells, mainly in layer IV moderately and in layer V highly.

You can take a look at the following website:

http://mouse.brain-map.org/experiment/show/73818754

Somatosensory cortex , thalamus , hippocampus

Just to clarify, I am specifically asking about expression of vGluT2 and vGluT1/2 in neocortical cells and synapses. I would like to find out if there is any evidence that A) cells in cortical layer 4 express vGluT2, and B) whether these cells might express vGluT1 as well, possibly leading to the vGluT1/2 coexpression I observe at synaptic terminals. Thanks for all the replies!

Dear Nicholas,

My feeling is that antibodies against vGlut1 and vGlut2 will probably fail to properly identify vGlut-positive cortical neurons. My best advice is to conduct single- or double-in sity hybridization looking for expression of the mRNA coding vGlut1 and vGlut2. If needed, I can provide you with the required riboprobes. Please take a look to a number of technical papers we have published in the past dealing with this topic. Among others, please see Perez-Manso et al., Brain res 1072(2006):91-98; Barroso-Chinea et al., J. Chem. Neuroanat. 35(2008):101-107; Conte-Perales et al., J. Neurosci. Meth 194(2010):28-33; and Barroso-Chinea et al., J. Neurosci. Meth. 162(2007):119-128.

All the best and have a nice day, Jose L.

in my preliminary experiments in cerebral cortex of rats VGLUT1 and VGLUT2 colocalization was about 5% so two population are segregated it is likely that vglut2 is expressed by thalamocortical synapses.

Hi Nicholas,

I'm working on somatosensory system in mouse and I've used pretty often Vglut2 as a very specific marker for thalamocortical axonal terminals in layer IV of S1, allowing to clearly visualize the barrel hollows. I have never seen any labeling of spiny stellate neurons or any other cortical neurons in S1 with Vglut2. On the other side I've never used Vglut1, but if you check the ISH on the Allen Brain Atlas you can see that Vglut1 is spread throughout the whole cortex, while Vglut2 is not there and mainly expressed in the thalamus.

Hope I've been helpfull!

Ilaria, thanks for the comment. I don't see vGluT2 staining in S1 L4 cells either, but then again we're looking for protein, rather than mRNA, and many synaptic proteins are synthesized (or at least concentrated) only at the synaptic terminal. If you look at the allen brain atlas images that Ming Shi provided earlier (http://mouse.brain-map.org/experiment/show/73818754) it does actually look like L4/L5 cells (hard to tell) express some level of vGluT2.

I see what you mean, there are very few cells stained both for mRNA and protein but I would definetly say that they're in layer V. I don't have in mind any paper describing the expression of vGlut2 in the cortical neurons though. I see it hard by the way to check for colocalization of vGlut1/2 in synaptic terminals of cortical neurons. I guess that the fact you can easily stained vGlut2-positive thalamic terminals in layer IV depends on the fact that these axons are clustered together, while it won't be the case for cortical neurons, that would be your goal if I understood correctly. If that's the case I would at least try some amplifying staining, rather than using normal immunofluorescence staining.

We reported the colocalization of two proteins at LM and EM level, and mRNAs by in situ hybridization.

The below is extracted from "Abstract".

"The colocalization was more frequent in the primary somatosensory (S1) area than in both the primary visual (V1) and the motor areas; of area S1 cortical layers, colocalization was most evident in layer IV barrels at postnatal day (P) 7 and in adulthood."

Ref

Nakamura Ko, Watakabe A, Hioki H, Fujiyama F, Tanaka Yasuyo, Yamamori T, *Kaneko T. (2007) Transiently increased colocalization of vesicular glutamate transporters 1 and 2 at single axon terminals during postnatal development of mouse neocortex: a quantitative analysis with correlation coefficient. Eur J Neurosci 26(11):3054-3067.

PMID: 18028110

Hiroyuki – great! Thanks for the reference.

Ilaria, actually using our technique (array tomography - see http://goo.gl/nTlG4) we can visualize individual synaptic terminals. Unfortunately we don't think the vGluT2 antibody we're using is ideal (what antibody is), and it probably doesn't label all vGluT2 positive terminals. Still, we see a fair amount of colocalization.

My pleasure~ Good Luck!

Hi Nicholas,

Your question is a very interesting one and I believe I have an article to share that answers it (see links and PDF attachment below). As Jose Lanciego has already noted, immunohistochemical data will not answer the question of whether there are cortical neurons expressing VGLUT2 because VGLUT1/2 are generally undetectable in the cell body. Instead, in situ hybridization for VGLUT mRNA is needed is identify VGLUT1/2 neurons. In my own studies of VGLUT mRNA distribution focusing on the diencephalon, I have also noticed weak labeling for VGLUT2 in some cortical lamina, which seems specific because this labeling does not appear in my sense probe control sections from the same brains. More recently, a Swedish group has closely examined not only the cortical expression of VGLUT2 mRNA but also the functional significance of these VGLUT2 neurons in mice with a cortical and amygdala specific knockout of VGLUT2 using CaMKIIa-driven CRE. So, here are links to that paper as well as an attached PDF version. This paper includes a nice series of photos comparing VGLUT2 expression in wild-type vs KO mice.

Hope this helps!

http://www.ncbi.nlm.nih.gov/pubmed/19228977

http://www.jneurosci.org/content/29/7/2238.long

Hi,

VGLUT1 and VGLUT2 are both expressed by glutamatergic neurons but they are found in different subtypes of neurons. Indeed, VGLUT1 is expressed by most cortical neurons, and VGLUT2 by the thalamocortical neurons. You can obtain a VGluT2 staining in the cortex L4 and L6 because there is thalamic projections. You should check the materials and methods of the following article if you want to use an immunohistochemical approach: "Lack of Evidence for Vesicular Glutamate Transporter Expression in Mouse Astrocytes" Li D., et al Journal of neuroscience march 2013.