The neural circuitry of the cerebellar cortical sheet is in many respects uniform throughout, implying that functional differences between different regions mainly reflect differences in the origins of the mossy and climbing fibre afferents and in the destinations of the axons of the Purkinje cells that form the output path from the cortex. Evidence also exists (e.g. Voogd & Glickstein, 1998) that these connections are so organized as to create a population of discrete ‘longitudinal’ cortical zones, each comprising a population of smaller microzones. These features imply that adequate interpretation of cortical neuronal activity recorded during behaviours is unlikely to be achieved without detailed knowledge of the patterns of spatial localisation present in the pathways to and from the different regions.
We are therefore using axonal transport tracers to study, in the cat, the connectivity of two regions (the paravermal part of the anterior lobe and Crus I of the cerebellar hemisphere) in which microelectrodes are being used to study the discharge patterns of individual neurones active in relation to the execution of visually guided reaching movements.
Small volumes (▓le│ 200 nl) of tracer are pressure-injected at a depth of 0.5 mm into the tips of selected folia in animals surgically anaesthetised with propofol (Rapinovet; Schering-Plough; 0.1 mg kg-1 min-1, continuous I.V. infusion) following initial deep sedation with medetomidine hydrochloride (Domitor; Pfizer; 150 µg kg-1, subcutaneous). Operative and post-operative analgesia was obtained with carprofen (Rimadyl; Pfizer; 4.0 mg kg-1 I.V. for 24 h cover). The experiments were carried out in accordance with UK legislative requirements and at the end the animals were humanely killed.
By making one injection of rhodamine-labelled latex microspheres (‘beads’; a retrograde tracer; Lumafluor Inc.) mixed with 20 % Fluororuby (an orthograde tracer; Molecular Probes), another of fluorescein-labelled beads (Lumafluor) mixed with Fluoro-emerald (Molecular Probes) and another of cholera toxin subunit B (a bi-directional tracer; List Biologicals) it is possible in each animal to investigate the afferent and efferent connections of three small (▓le│ 1 mm2 ) areas of the cortex in known spatial relationship to each other and to the folial pattern. Use of two kinds of beads allows detection (via double labelling) of neurones with axons branching to provide climbing or mossy fibres to both fluorescent-tracer injection sites. Moreover, because the injection sites are small it is sometimes possible to place two side by side in the same zone in the same folium and therefore investigate the extent to which different parts across the width of a zone differ in their connections.
The demonstration will provide further details of the operative, histological and microscopical techniques employed and example patterns of retrograde labelling (in pontine nuclei, nucleus reticularis tegmenti pontis, lateral reticular nucleus and inferior olive) and orthograde labelling (in the deep cerebellar nuclei) will be illustrated.
We thank Rachel Bissett and Clare Everard for their excellent technical assistance. This work was supported by The Wellcome Trust.
- Voogd, J. & Glickstein, M. (1998). Trends Neurosci. 21, 370-375.