Department of Morphology, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil Projections From the Anterior Interposed Nucleus to the Red Nucleus Diminish With Age in the Mouse H. H. V. Olyntho-Tokunaga 1 , M. L. Pinto 1 , C. Souccar 2 , G. H. M. Schoorlemmer 3 and R. C. R. S. Lapa 1 * Departments of 1 Morphology, 2 Pharmacology and 3 Physiology, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil; *Corresponding author: Tel.: 55-11-5576-4271; fax: 55-11-5576-4268; e-mail: rcrslapa.morf@epm.br With 2 figures Received September 2007; accepted for publication June 2008 Summary To analyse the effect of ageing on the projection of the anterior interposed nucleus to the red nucleus, we injected the retro- grade tracer fluorogold in the red nucleus of 3-, 6- and 12- month-old mice. The number of labelled neurones in the anterior interposed nucleus fell by 9% between 3 and 6 months and by another 9% between 6 and 12 months (all P < 0.001). This suggests that loss of neurones from the cerebellar nuclei starts well before old age. Introduction Ageing is accompanied by functional and structural changes in the brain, including changes in metabolic rate, local reductions in the number of synapses, reductions in neuron size and loss of neurones (Jucker and Ingram, 1997). Changes in the hippocampus during ageing, including synaptic alterations (Kelly et al., 2006) and the loss of neurones from some of the hippocampal regions (West, 1993) may be related to changes in memory. Ageing is also accompanied by changes in motor control, which may be related to cerebellar shrinkage (Raz et al., 2005), neuron loss in the cerebellum (Khutoryan, 2005; Sturrock, 1989, 1990a; Zhang et al., 2006) and neuron loss in the red nucleus (Sturrock, 1990b). However, it is noted that while changes in the processing of motor information occur long before old age, reductions in the number of neurones are typically seen only late in life. We investigated neurones in the cerebellar anterior inter- posed nucleus that project to the red nucleus. The red nucleus is a pre-motor structure whose main function is in the control of the limb muscles (Canedo, 1997; Massion, 1967). This nucleus projects to the lateral part of the intermediate zone of the spinal grey matter, in lamina V through VII (Kostyuk and Skibo, 1975), where inter-neurones influencing the motor neurones innervating proximal and distal musculature of the limbs are concentrated (Kuypers, 1964). The red nucleus receives a large projection from the interposed nucleus, which in turn receives much of its input from cerebellar Purkinje neurones. Lesions in the interposed nucleus reduce the activity of rubrospinal neurones, and therefore reduce the excitability of motor neurones, reducing muscular tone (Kandel et al., 1991). In addition, such lesions reduce the accuracy of reach movements (Kandel et al., 1991). In this study, the projection from the anterior interposed nucleus to the red nucleus was analysed in mice. Retrograde tracers were injected into the red nucleus and the number of marked neurones in the interposed nucleus was quantified. We focused on changes that occur before old age. We used age groups of 3 months (young adults), 6 months and 12 months (middle aged). The results suggest that loss of neurones in the cerebellum can occur well before old age. Materials and Methods We used 13 C57BL/10 male mice of 3 months, 8 of 6 months and 15 of 12 months. Mice were housed in groups of six in plastic cages on a floor of wood chips, in a room with controlled temperature (22–25°C) and light-dark cycle ( lights on between 06:00 and 18:00 hours). They had free access to water and chow. All experimental procedures conformed to the guidelines of the Brazilian College for Animal Experimen- tation (COBEA) and were approved by the Ethics in Research Committee of the Federal University of Sa˜o Paulo (Protocol 0478/06). Mice were anaesthetized by i.p. injection of 1 mg ketamine and 0.2 mg xylazine. They were placed in a stereotaxic instrument and small holes were drilled in the skull above the left and right red nucleus. Injections were made with a 0.5 ll Hamilton syringe, connected with polyethylene tubing to a stainless steel injection needle with an outer diameter of 0.2 mm. The injection coordinates were 3.35 mm caudal, 0.55 mm lateral and 4.3 mm ventral of the bregma (Franklin and Paxinos, 1997). On one side, 200 nl of 2% fluorogold dissolved in sterile saline was injected. On the contralateral side, 400 nl of a suspension of latex microspheres (20–200 nm diameter) impregnated with a tracer was injected. In half of the cases, the label was rhodamine, in the rest, fluorescein (red and green Retrobeads, respectively; Lumafluor Inc., Naples, Italy). Five to seven days after injection of the tracers, the mice were anaesthetized again and perfused through the heart with 100 ml 0.9% NaCl, followed by 200 ml 4% formaldehyde in 0.1 mol/l phosphate buffer, pH 7.4. The brains were removed, incubated for 24 h in the fixative and incubated in phosphate buffer with 30% sucrose for 48 h. The brain stems were cut on a cryostat in 35 lm coronal slices and mounted on microscope slides with Jung Tissue freezing medium (Leica, Nussloch, Germany). The spread of the tracers was analysed on an epifluorescence microscope (BX60; Olympus, Tokyo, Japan) with appropriate filters (fluorogold: excitation 340–360 nm, absorbance 420 nm; rhodamine: excitation 510–560 nm, absorbance 590 nm; fluo- rescein: excitation 450–490 nm, absorbance 520 nm). The Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag Anat. Histol. Embryol. 37, 438–441 (2008) doi: 10.1111/j.1439-0264.2008.00877.x ISSN 0340–2096