446 In order to gain more knowledge about the course of the pallido- and cerebellothalamic tracts and their stereotacti- cal positions in the subthalamic region, we conducted an anatomical study in human post-mortem brain using mye- lin staining techniques, immunohistochemical procedures and the Morel’s atlas of the human thalamus and basal ganglia (Morel et al. 1997; Morel 2007). The histological demonstration of the ﬁbre tracts was also correlated with high-resolution post-mortem MRI and the variability of their trajectory and position in different brains was evaluated. Materials and methods This study is based on post-mortem examination of ﬁve brains from normal subjects with no history of neurological diseases or pathological signs at autopsy. The characteris- tics of the subjects (age, cause of death, post-mortem delays) are given in Table 1, except for case Hb5, for which these informations were lacking. This case, how- ever, showed no pathological signs by post-mortem examination and was included for correlations with high- resolution MRI obtained prior to histological processing. The procedures for ﬁxation, stereotactic blocking, and sectioning follow those described previously (Morel et al. 1997, 2002; Morel 2007). In brief, whole brains were ﬁxed for 2–3 weeks in paraformaldehyde (PAF) 4% in 0.1 M phosphate buffer (PB) or in formalin 10% for few weeks to several months. After interhemispheric section, a special guillotine was used to make blocks of the thalamus and basal ganglia in planes parallel or orthogonal to the Table 1 List of brains and histological procedures Brain Struct Funct (2008) 212:443–463 reference stereotactic plane (i.e. passing through the cen- ters of the anterior and posterior commissures). The blocks were then postﬁxed 2–3 days (for PAF ﬁxed brains) and cryoprotected over 2–3 weeks in increasing (10, 20, and 30%) sucrose concentrations in PB or in formalin. Except for two blocks that were frozen in cryostat, all other were frozen by immersion in isopentane at *-30 C and then stored at –75 C. Sections were cut at 40 or 50 lm and from six to nine series (300–500 lm intervals between series) were collected in PB. Adjacent series were stained for Nissl with cresyl violet and for myelin with a modiﬁed He- idenhain procedure applied to free-ﬂoating or mounted sections (Hutchins and Weber 1983; Burgel et al. 1997). The two myelin staining methods were applied in case Hb5, the ﬁrst for its lower background and ﬁner ﬁbre staining (see Fig. 2), the second to reduce section shrink- age and deformation due to successive alcohol treatments. The shrinkage factor due to free-ﬂoating myelin process- ing, which was evaluated by measuring distances on adjacent free-ﬂoating and mounted myelin sections (in case Hb5) amounted to 7%. Mounted myelin sections were similar to Nissl stained sections. The overall deformation due to histological processing was evaluated by comparing distances measured in MRI and in corresponding Nissl stained sections. This was minimal (1.02) within the plane of section but slightly higher for distances measured across sections (as also reported in Morel 2007). Other series were processed for acetylcholinesterase (AChE) with a modiﬁed Koelle–Friedenwald method (Mesulam 1982), or immu- nocytochemically with antibodies against calcium-binding proteins (CaBP) and the non-phosphorylated neuroﬁlament protein (with SMI-32) (see Table 1). Case No. Hb1 Hb2 Hb3 Hb4c Hb5d 123 Age (years)/ gender 70/female 57/female 74/female 59/female – PM delay (h) \24 \24 4 16 – Cause of death Coronary infarct Lung carcinoma Lung carcinoma Anal carcinoma – ac-pc (mm)a 25 30 24 26 24 Fixation Formalin Formalin PAF PAF Formalin Hemisphere/plane of section Le/SAG Re/HOR R/FRO L/HOR R/HOR L/SAG Re/SAG Le/FRO Re/HOR Le/FRO Histologyb CaBP CaBP CaBP/SMI/AChE CaBP/SMI/AChE CaBP/SMI a Intercommissural (ac-pc) distances are indicated in millimeters ‘‘in vivo’’ b Staining procedures additional to standard Nissl and myelin c Post-mortem 3 T 3D T1-weighted MRI (see protocol in ‘‘Materials and methods’’) d Post-mortem 3 T proton-density MRI (see protocol in ‘‘Materials and methods’’) e Hemispheres used for stereotactic mapping