Full Protocols for Morphological Interpretations

 

1–         Reporting axonal/dendritic locations based on a drawing or reconstruction of an actual neuron

2–         Reporting axonal/dendritic locations based on a schematic

3–         Reporting axonal/dendritic locations based on text where the locations are categorically stated

4–         Reporting axonal/dendritic locations based on text where the locations are equivocal

 

1–         Reporting axonal/dendritic locations based on a drawing or reconstruction of an actual neuron

A.  First, in order for a given layer to be considered, the axons or dendrites in that layer must be 15% or more of the arbor and must penetrate 15% or more of the layer.

 

B.  If the proportion of branches / boutons / synapses is reported (i.e. 25% in SO, 10% in SR, etc.), then the layer is recorded if the proportion is at least 15% of the entire tree.  If none of the proportions qualify (i.e. 12%, 10%, 8%, etc.), compare the relative sizes of the proportions to the largest proportion (i.e. 12/12, 10/12, 8/12, etc.) and report those layers where the proportions are at least half the size of the largest proportion.

 

C.  3-point Rule: Report an axonal/dendritic location if the given layer has at least 3 points worth of branches.  (e.g. 2 terminating branches, 1 terminating + 1 continuing branch, or 3 continuing branches), and the branches making up the three points are 15% or more of the total dendritic/axonal arbor.

 

Definitions:

1) A terminating branch ends in a given layer and satisfies the 15% rule (the length of the branch and the invasion depth into the layer are both at least 15% of the layer’s thickness). (also see note below)  (2 points for each encircled branch)

2) A continuing branch invades at least 15% of a layer but does not terminate in that layer. (also see note) (1 point for each encircled branch)

(note) A continuing branch that fails the definition of a terminating branch in the next layer is reported as a terminating branch in the continuing layer if the branch satisfies the 15% rule in the continuing layer, otherwise it is disregarded. (2 points for each encircled branch)

 

 

 

2–         Reporting axonal/dendritic locations based on a schematic

Report an axonal/dendritic location based on layers traversed by more than 15% in a schematic

 

e.g. In the figure to the right, the shaded box depicts axons, so report axons in stratum

pyramidale, but do not report axons in stratum oriens and stratum radiatum.

 

3–         Reporting axonal/dendritic locations based on text where the locations are categorically stated

e.g. Report CA1 O-LM neuron dendrites in CA1 stratum oriens: “In the CA1 area, O-LM cells are located in stratum oriens and have horizontally extending dendrites with hairy spines on distal segments.” (Klausberger T, Eur J Neurosci, 2009 Sep, 30 (6), pages: 947 – 957, PMID: 19735288).

 

4–         Reporting axonal/dendritic locations based on text where the locations are equivocal

e.g. Report dendrites in CA1 stratum radiatum (SR) and stratum lacunosum-moleculare (SLM), but omit dendrites in stratum pyramidale (SP) and stratum oriens (SO): “2. [CA1] Schaffer-associated interneurones. These five cells were multipolar, with most of their smooth or sparsely spiny dendritic arborisation contained within the SR and SLM (Figs. 10, 11) and only rarely entering the SP and SO.” (Pawelzik H, Hughes DI, and Thomson AM, J Comp Neurol, 2002 Feb 18, 443 (4), pages: 346 – 367, PMID: 11807843). 

 

1)   Trigger words or phrases:

 

Report “most” “majority” “superficial/deep layer X” “usually” “proximal/distal layer X” “septal/temporal layer X” “all layers”

Omit “a few/some” “minority” “rarely” “at the border of” “sometimes/occasional” “toward layer X” “a small fraction/number of”

 

2)   Interpreting ambivalent phrases. 

             i.        If not specifically defined otherwise by the author(s) …

…  entorhinal cortex deep layers are layers V-VI.

…  entorhinal cortex superficial layers are layers I-III.

…  the alveus is part of stratum oriens (SO).

…  hippocampus is CA1 and CA3.

…  apical dendrites are the portion of the dendritic tree located in both stratum radiatum (SR) and stratum lacunosum-moleculare (SLM) for CA1, CA2, and CA3.

…  the phrase “apical tuft” is interpreted as the portion of the apical dendritic tree located in stratum lacunosum-moleculare (SLM).

…  the phrase “oblique dendrites” is interpreted as a subset of the apical dendrites located in stratum radiatum (SR) for CA1/CA2.

…  the phrase “basal dendrites” is interpreted as the portion of the dendritic tree located in stratum oriens (SO).     

…  the stratum moleculare (SM) of the dentate gyrus (DG) is not specific enough for reporting locations of axons and dendrites.

…  the phrase “outer stratum moleculare” (SMo) of the dentate gyrus (DG) is interpreted as the two-thirds of the layer farthest from the stratum granulosum (SG).

…  the phrase “inner stratum moleculare” (SMi) of the dentate gyrus (DG) is interpreted as the one-third of the layer closest to the stratum granulosum (SG).

…  the phrase “perforant path termination zone” is interpreted as the outer stratum moleculare (SMo) for the dentate gyrus (DG), stratum lacunosum-moleculare (SLM) for CA1/CA2/CA3, and stratum moleculare (SM) for the subiculum.

…  the phrase “regio inferior” is interpreted as CA3 (see Blaabjerg M and Zimmer J, Prog Brain Res, 2007, pages: 85 – 107. PMID: 17765713).

…  the phrase “regio superior” is interpreted as CA1 (see Blaabjerg M and Zimmer J, Prog Brain Res, 2007, pages: 85 – 107. PMID: 17765713).

…  the phrase “CA4” is interpreted as the dentate gyrus (DG) hilus (see Blaabjerg M and Zimmer J, Prog Brain Res, 2007, pages: 85 – 107. PMID: 17765713).

…  the phrase “fascia dentata” is interpreted as the dentate gyrus (DG) stratum granulosum (SG) and stratum moleculare (SM) (see Blaabjerg M and Zimmer J, Prog Brain Res, 2007, pages: 85 – 107. PMID: 17765713).

…  the phrase “Brodmann Area 28” is interpreted as entorhinal cortex (EC) (see Witter M, 2011, Scholarpedia, 6 (10), page: 4380).

…  the phrase “lateral entorhinal area (LEA)” is interpreted as lateral entorhinal cortex (LEC).

…  the phrase “medial entorhinal area (MEA)” is interpreted as medial entorhinal cortex (MEC).

…  the phrase “Schaffer collaterals” refers specifically to CA3 pyramidal neuron axons.

…  the phrase “mossy fibers” refers specifically to dentate gyrus granule cell axons.

…  rats under 200 g and/or less than 3 weeks old are considered to be juveniles (Dumas TC, personal communication).

…  rats at least 200 g and/or more than 3 weeks old are considered to be adults (Dumas TC, personal communication).

 

            ii.        Despite what is stated by the author(s) …

…  the location of “apical proximal dendrites” is reported as stratum radiatum (SR) for CA1/CA2, stratum lucidum (SL) for CA3, and inner stratum moleculare (SMi) for the dentate gyrus (DG).

…  the location of “basal proximal dendrites” is reported as stratum oriens (SO) for CA1/CA2/CA3, and the hilus (H) for the dentate gyrus (DG).

…  perisomatic axonal targeting, such as somata, proximal dendrites, and axon initial segments, is reported as stratum pyramidale (SP) for CA1/CA2/CA3/subiculum (SUB) and stratum granulosum (SG) for the dentate gyrus (DG).  This rule applies specifically to basket and axo-axonic (a.k.a. chandelier) neurons.

…  depictions of axons or dendrites in a principal cell layer do not have their locations reported if they are within ~100 μm of the soma, as they are considered to be perisomatic, i.e. part of the soma, as defined by Freund and Katona (Neuron, 2007 Oct 4, 56 (1), pages: 33 – 42, PMID: 17920013).

…  CA2 stratum lucidum (SL) is interpreted as the incursion of mossy fibers, i.e. Granule cell axons, into CA2 stratum pyramidale (SP).

 

           iii.        Extending the principal cell layers:

The 15% of CA1/CA2/CA3 stratum oriens (SO) and CA1/CA2 stratum radiatum (SR) that are adjacent to CA1/CA2/CA3 stratum pyramidale (SP) are loosely interpreted as parts of SP, if not specifically defined otherwise by the author(s).

 

The 15% of the inner stratum moleculare (SMi) and the hilus (H) that are adjacent to stratum granulosum (SG) are loosely interpreted as parts of stratum granulosum, if not specifically defined otherwise by the author(s).

 

The 15% of the stratum moleculare (SM) and the polymorphic layer (PL) in the subiculum (SUB) that are adjacent to stratum pyramidale (SP) are loosely interpreted as parts of SP, if not specifically defined otherwise by the author(s).