Standardization of dissection protocols for INIA-West

Background

Historically, dissections have been done with relatively little quality control. INIA-West recommends utilizing markers that should be enriched (or depleted) in the regions of interest. For example, if one is dissecting the nucleus accumbens, there should be a substantial enrichment in mRNA for D1 and D2 dopamine receptors. In the central nucleus of the amygdale or the lateral posterior aspect of the bed nucleus of the stria terminalis, there should be a substantial enrichment in mRNA for CRF (see ppt examples). In cortical regions of interest, the calcium binding proteins are differentially expressed (see ppt examples). Overall, the approach is very similar to what many investigator used to do when purifying synaptic membranes – Na+,K+ ATPase needed to be enriched 10-fold, cytochrome oxidase activity needed to be negligible (a measure of mitochondrial contamination) and so on. Future developments will include identification of  2 or 3 markers per region and the details for the C57BL/6 mouse provided at the July 2007 meeting.

Key regions of interest at this point

Prefrontal Cortex (parvalbumin and calretinin)
Nucleus accumbens (D1 and D2 dopamine receptors, calretinin) – if we are comparing core and shell – calretinin will be higher in the shell whereas calbindin will be higher in the core)
Central nucleus (CRF, preproenkephalin, neurotensin)
Bed nucleus (CRF, preproenkephalin, neurotensin) (here the assumption is that most dissections will include both the lateral posterior and lateral dorsal)
Basolateral  (acetylcholine esterase, parvalbumin, choline acetyltransferase)
Ventral tegmental area (tyrosine hydroxylase, dopamine transporter)

Qrt-PCR (TaqMan) assays will be used to measure mRNA levels.

Frontal Cortex dissections in detail

A two-tier dissection of the frontal cortex is recommended. For the punch technique in rat and mouse, INIA-West recommends three punches: overall prefrontal cortex, orbitofrontal-insula, and cingulate punches. However, for more detailed analyses utilizing laser capture the individual components of the three global regions may be engaged. Listed below are the regions and Figure and steretotaxic coordinates from interaural Bregma for the rat from Paxinos and Watson The Rat Brain, 4th edition Academic Press (Elsevier).

Prefrontal cortex- interaural AP 11.70 (Figure 9),
Cg1 medial +0.5 mm, depth +8
PrL medial +0.5 mm, depth +6.5
IL medial +0.5 mm, depth  +5

Orbitofrontal-insula interaural AP 11.70 (figure 9)
GI, AID, AIV  medial +4, depth +5
LO, VO medial +3, depth +4.5

Cingulate interaural AP10.60- 7.60, (Figures 11 -24) medial +0.5, depth 7.5

Cg1
Cg2