methodological issues that make it difficult to draw firm conclusions about nuclear boundaries or patterns of connectivity. However one of us was able to demonstrate the feline pNr as consisting of a long cellular zone continuing from the ND to the rostral part of the mNr through the use of an HRP-tracing method (Onodera, 1984 [3]). After HRP deposition into the central core of the cat’s inferior olive, almost all the cells of both the ND and NB, located in the ventral central gray. A number of labelled small cells also were found in both the dorsomedial (dm)- and ventrolateral (vl)-pNr of the reticular formation. The cat possesses a continuous zonal neural sheet consisting of the ND, NB and the poorly- developed dm- and vl-pNr and it is readily seen that this mNr is well-developed for quadrupedalism (Onodera, 1984 [3]). mNr neurons in quadrupeds (e.g., cat: Drew et al., 1996 [4]) exhibit a significant increase in discharge frequency during of either fore- or hindlimbs movement when the animals cross obstacles, similar to what is seen in pyramidal tract neurons. These neurons are controlled by a network resembling a continuous zonal neural sheet, i.e., the mesodiencephalo-olivo-cerebellar system. Such animals execute voluntary gait modification of fore- and hindlimbs using visual and other sensory cues [5]. Pyramidal tract neurons contribute to fine, precise control of distal limb movements both by increases and decreases in the level of spinal interneuron activity. By contrast, mNr neurons may discharge more in relation to intralimb coordination as well as interlimb coordination [5]. mNr neurons of rats and monkeys, species that possess a manipulable hand, are provided with a ‘‘hand preshape’’ through their extension of digits via goal-directed limb move- ments, such as reaching to grasp [6,7,8,9]. Pyramidal tract neurons which are controlled by a continuous zonal neural sheet- olivo-cerebellar circuit, superimpose motor control for individual finger movements on grouped-finger extension, such action which is governed by rubrospinal tract neuron activity. In the bipedal human, the mNr is rudimentary. From an evolutionary perspective, human pyramidal tract neurons controlled by the rubro-olivo-cerebellar circuit would have subsumed the function of the framework governing limb movements that, until then, had been provided by the rubrospinal tract neurons, thereby introducing stability and accuracy of skilled movements of all muscle groups required for locomotion, enabling the advent of bipedalism. Although the human ND is identified as a separate body part from the well-developed red nucleus, demarcated by fiber bundles consisting of the superior cerebellar peduncle, CTT, MTT and the medial longitudinal fasciculus (MLF), the human NB could not be identified in the ventral central grey, as is seen for the cat’s NB. Materials and Methods Two adult female and male cats and three female macaques (Macaca fuscata) were employed as subjects in the present study. All experimental protocols were performed in accordance with the Animal Experimental Guidelines of Iwate Medical University and the Canadian Council of Animal Care, and the ethics protocol was approved by the Animal Users Subcommittee of Iwate Medical University. Procedures were designed to minimize animal suffering and reduce the number used. Human brains were obtained via informed donation for medical education and research of Iwate Medical University with the corresponding written consents given by donors and their families who agreed with policies relating to good–will cadaver donation. Human brain slice preparation approved by the Ethical Committee of Iwate Medical University is used annually in neuroanatomical courses at Iwate Medical University; cadaver identity data are anonymous. All experiments were performed between 1996 and 1998. New Model of Human Red Nucleus Animal Preparation Two cats in Case 1 (female, 2.8 kg) and Case 2 (male, 3.0 kg) and two macaques (Macaca fuscata) in Case 3 (female, 9.0 kg), and Case 4 (female,10.0 kg) were used for the demonstration of mesodiencephalic nuclei consisting of olivary projecting neurons. One macaque in Case 5 (female, 10.5 kg) was used for the demonstration of the cortico-mesodiencephalic projection. After induction of sedation by an intramuscular injection of ketamine hydrochloride (10 mg/kg body weight), animals were anesthetized deeply by intraperitoneal injections of sodium pentobarbital (30 mg/kg body weight). Then, atropine sulphate (60 mg/kg body weight) was injected intramuscularly. To gain sterotaxic access to the inferior olivary complex (Case 1–4), part of the occipital bone lying over the occipital cortex and cerebellum was removed. For demonstration of mesodiencephalic nuclei consisting of olivary projecting neurons, these animals received multiple stereotaxic injections (0.2 ml/one injection for the cat, 1.0 ml/one injection for the macaque) of 2% WGA-HRP (Toyobo, Tokyo, Japan) solution as retrograde tracer via a 1-ml Hamilton microsyringe into the entirety of the olivary complex (two cats, Cases 1 and 2: 0.2 ml62 and 0.2 ml64) or with the use of a 5-ml Hamilton microsyringe into its subnuclei (two macaques, Cases 3 and 4: 1.0 ml66). A large craniotomy was made to expose the appropriate region of frontal cortex of one macaque (Case 5). For demonstration of the cortico-mesodiencephalic projection, this animal received, under direct visual control, multiple injections (23 sites) of 10.6 ml 2% WGA-HRP solution as anterograde tracer using a 1-ml Hamilton microsyringe into the premotor area and frontal eye field (FEF). Animals were permitted to survive for 2 days after the injections, and were overdose-anesthetized prior to transcardial perfusion with 500 ml of warmed (37uC) physiological saline followed by 1,500 ml (for cats) or 2,500 ml (for macaques) of fixative containing 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4 and then by 1,000 ml PB, pH 7.4. Whole brains were stored overnight at 4uC in 0.1 M PB containing 30% sucrose. Coronal sections of 50 mm thickness were cut with a vibratome or a freezing microtome and collected in 0.1 M PB. Sections were processed for the histochemical demonstration of HRP with the tetramethylbenzidine method [10] and counterstained with neutral red. The distribution of labeled axons and somata were photographed and drawn through the use of a photomicroscope (Olympus) with a drawing tube attached. Human Brain Preparation After death and within 24 hrs postmortem, cadavers were perfused through the femoral artery with 10,000 ml of a 10% formalin solution. Brains were removed immediately from the skulls and immersed in the same fixative for several months. Well- fixed adult brain was selected and washed in running water. Macro- and microscopical examination of this brain did not reveal any lesions. The diencephalon and brainstem were cut into several blocks. These blocks were cut by a freezing microtome into 50 mm-thick serial coronal sections. Each section was picked up with a chrome alum gelatin-coated slide. Every twelfth section was stained with 1% cresyl violet. Other serial sections were stained with myelin stain using Luxol fast blue. The distributions of Nissl stained cells were plotted using a drawing tube. Drawing of 3D Image Frontal, dorsal and lateral views of the structures of the mesodiencephalic nuclei of the cat, macaque and human were PLoS ONE | www.plosone.org 2 August 2009 | Volume 4 | Issue 8 | e6623