Loss of Piccolo Function in Rats Induces Cerebellar Network Dysfunction and Pontocerebellar Hypoplasia Type 3-like Phenotypes.

Piccolo, a presynaptic active zone protein, is best known for its role in the regulated assembly and function of vertebrate synapses. Genetic studies suggest a further link to several psychiatric disorders as well as Pontocerebellar Hypoplasia type 3 (PCH3). We have characterized recently generated Piccolo KO (Pclogt/gt ) rats. Analysis of rats of both sexes revealed a dramatic reduction in brain size compared with WT (Pclowt/wt ) animals, attributed to a decrease in the size of the cerebral cortical, cerebellar, and pontine regions. Analysis of the cerebellum and brainstem revealed a reduced granule cell layer and a reduction in size of pontine nuclei. Moreover, the maturation of mossy fiber afferents from pontine neurons and the expression of the α6 GABAA receptor subunit at the mossy fiber-granule cell synapse are perturbed, as well as the innervation of Purkinje cells by cerebellar climbing fibers. Ultrastructural and functional studies revealed a reduced size of mossy fiber boutons, with fewer synaptic vesicles and altered synaptic transmission. These data imply that Piccolo is required for the normal development, maturation, and function of neuronal networks formed between the brainstem and cerebellum. Consistently, behavioral studies demonstrated that adult Pclogt/gt rats display impaired motor coordination, despite adequate performance in tasks that reflect muscle strength and locomotion. Together, these data suggest that loss of Piccolo function in patients with PCH3 could be involved in many of the observed anatomical and behavioral symptoms, and that the further analysis of these animals could provide fundamental mechanistic insights into this devastating disorder.SIGNIFICANCE STATEMENT Pontocerebellar Hypoplasia Type 3 is a devastating developmental disorder associated with severe developmental delay, progressive microcephaly with brachycephaly, optic atrophy, seizures, and hypertonia with hyperreflexia. Recent genetic studies have identified non-sense mutations in the coding region of the PCLO gene, suggesting a functional link between this disorder and the presynaptic active zone. Our analysis of Piccolo KO rats supports this hypothesis, formally demonstrating that anatomical and behavioral phenotypes seen in patients with Pontocerebellar Hypoplasia Type 3 are also exhibited by these Piccolo deficient animals.

Correction: An Automated, Experimenter-Free Method for the Standardised, Operant Cognitive Testing of Rats.

[This corrects the article DOI: 10.1371/journal.pone.0169476.].

An Automated, Experimenter-Free Method for the Standardised, Operant Cognitive Testing of Rats.

Animal models of human pathology are essential for biomedical research. However, a recurring issue in the use of animal models is the poor reproducibility of behavioural and physiological findings within and between laboratories. The most critical factor influencing this issue remains the experimenter themselves. One solution is the use of procedures devoid of human intervention. We present a novel approach to experimenter-free testing cognitive abilities in rats, by combining undisturbed group housing with automated, standardized and individual operant testing. This experimenter-free system consisted of an automated-operant system (Bussey-Saksida rat touch screen) connected to a home cage containing group living rats via an automated animal sorter (PhenoSys). The automated animal sorter, which is based on radio-frequency identification (RFID) technology, functioned as a mechanical replacement of the experimenter. Rats learnt to regularly and individually enter the operant chamber and remained there for the duration of the experimental session only. Self-motivated rats acquired the complex touch screen task of trial-unique non-matching to location (TUNL) in half the time reported for animals that were manually placed into the operant chamber. Rat performance was similar between the two groups within our laboratory, and comparable to previously published results obtained elsewhere. This reproducibility, both within and between laboratories, confirms the validity of this approach. In addition, automation reduced daily experimental time by 80%, eliminated animal handling, and reduced equipment cost. This automated, experimenter-free setup is a promising tool of great potential for testing a large variety of functions with full automation in future studies.