9% saline followed by 4% paraformaldehyde in 0.15 M sodium phosphate buffer solution (NaPBS, pH 7.4, 21 °C). The brainstem was removed and fixed in 4% paraformaldehyde at 4 °C and refrigerated overnight. The brainstem was sectioned coronally at 100-μm thickness using a Vibratome. Sections were placed in buffer solution (KPBS, pH 7.4, 21 °C), reacted with cytochrome oxidase (CO), and mounted on gelatin-coated glass slides, air dried overnight, and coverslipped. Sections were digitized and reconstructed in Photoshop. The borders of CN and Selumetinib order neighboring gracilis and spinal trigeminal nuclei were identified from CO-stained sections and used to generate a morphological map. A physiological map was
produced from the receptive field data collected
from each electrode penetration, and this map was superimposed on the morphological map by aligning the locations of lesions in the 2 maps that served as fiducials. The mismatch between morphological and physiological maps never exceeded 25 μm at any of the lesion sites. Electrode penetrations and receptive field(s) recorded along these penetrations were then extrapolated from the lesion data and plotted in relationship to the underlying morphological map. Electrode tracks could often be seen where blood had coagulated, and these tracks were also used for receptive field reconstruction. Data collected for this study were obtained at approximately 300 μm anterior to the tip of the obex where a complete map of CO-stained clusters representing the forelimb Selleck TGF beta inhibitor was present. Animals were grouped according to the time of amputation and mapping. The 1-week deafferent group (1-WD) had 4 rats that were mapped 1 week after amputation. The 2-week deafferent group (2-WD) had 4 rats that were mapped 2 weeks after amputation, and the 3-week deafferent group (3-WD) had 5 rats that were mapped 3 weeks after amputation. The 4-week deafferent group (4-WD) had 3 rats that were mapped 4 weeks after amputation, and the 5-week deafferent group (5-WD) had 4 rats that were mapped 5 weeks after amputation. The 6-through 8-week deafferent
group Etomidate (6–8-WD) had 6 rats – 2 rats that were mapped 6 weeks after amputation, 2 rats that were mapped 7 weeks after amputation, and 2 rats that were mapped 8 weeks after amputation. The 9- through 12-week deafferent group (9–12-WD) had 6 rats – 1 rat that was mapped 9 weeks after amputation, 1 rat mapped at 10 weeks after amputation, 3 rats that were mapped 11 weeks after amputation, and 1 rat that was mapped 12 weeks after amputation. The 26-week deafferent group (26-WD) and 30-week deafferent group (30-WD) each had 1 rat. All rats were amputated between 6 and 8 weeks of age. Areal measurements of physiological maps and total areas of CN and total areas of medial, central, and lateral zones were made using Image J (NIH).