Research reportPiriform cortex alterations in the Ts65Dn model for down syndrome
Introduction
Down Syndrome (DS) is the most common chromosomal aneuploidy in humans, with an incidence of one in 1000 live births (Roizen and Patterson, 2003). Trisomy of the chromosome 21 induces a variable phenotype that may include immune deficiencies, heart defects, increased risk of leukaemia, and early development of Alzheimer’s disease (Ball and Nuttall, 1980, Folin et al., 2003, Hof et al., 1995, Holland et al., 2000, Nadel, 2003). The common feature among all DS subjects is the presence of intellectual disability reflected by impairment in learning and memory and a certain degree of olfactory dysfunction (Cecchini et al., 2016, Murphy and Jinich, 1996). Neural mechanisms underlying these alterations may include defects in the formation of neuronal networks, information processing and brain plasticity.
Several animal models that mimic the alterations in DS are available. Among them, one of the most studied is the Ts65Dn mouse. This model is segmentally trisomic for a portion of the mouse chromosome 16 that is orthologous to the long arm of the human chromosome 21. This segment contains approximately 140 genes, many of which are highly conserved between mice and humans (Gardiner et al., 2003, Rueda et al., 2012, Sturgeon and Gardiner, 2011). The phenotype of this model includes alterations in work memory and deficits in long-term memory, motor dysfunctions, reduced pain responsiveness and hyperactivity (Gardiner and Davisson, 2000, Holtzman et al., 1996, Rueda et al., 2012). Other remarkable features are the loss of cholinergic neurons related to age, a decrease in asymmetric synapses in temporal cortex, reduction in neuronal number in hippocampal regions, and deficiencies in beta-noradrenergic transmission in cerebral cortex and hippocampus (Granholm et al., 2000, Insausti et al., 1998, Kurt et al., 2000).
The piriform cortex is considered as primary olfactory cortex. This cortical region receives direct input from the olfactory bulb neurons through the lateral olfactory cortex. This region display a 3 layered lamination that is characteristic of paleocortical regions. The piriform cortex displays a strong association with other cortical regions, but also with the hippocampus or the hypothalamus. Moreover, the piriform cortex is a very interesting region to analyse due to the high level of adult structural plasticity (mainly in layer II) (Gómez-Climent et al., 2008). Structural plasticity can be defined as the ability to perform adaptive changes related to the structure and function of the central nervous system (Zilles, 1992). Structural plasticity is crucial during development, however, during adulthood it is restricted to some brain regions in the form of neurogenesis, neuritogenesis and synaptogenesis, necessary for learning and adaptability (Cotman et al., 1998, Gage, 2000). The piriform cortex is one of the regions displaying high levels of structural plasticity during adulthood under physiological conditions, including odour-input dependent spinogenesis, dendritic remodelling and synaptic reorganization (Knafo et al., 2001, Seki and Arai, 1991). One of the components of this structural plasticity is the presence of immature neurons in layer II expressing the Polysialylated form of the Neural Cell Adhesion Molecule (PSA-NCAM) and doublecortin (DCX) (Gómez-Climent et al., 2008, Nacher et al., 2002b, Seki and Arai, 1991). Previous studies from our group and others have shown alterations in structural plasticity in the hippocampus of the Ts65Dn model. Adult neurogenesis has been widely analysed and is clearly impaired in the Ts65Dn hippocampus (Chakrabarti et al., 2007, Clark et al., 2006, López-Hidalgo et al., 2016, Rueda et al., 2005). Moreover, we have observed alterations in the expression of proteins related to structural plasticity such as PSA-NCAM, BDNF or GAP-43 in the hippocampus of this model (Villarroya et al., 2018). Dendritic atrophy is one of the hallmarks of DS (Dierssen and Ramakers, 2006). Studies using Golgi techniques have shown dendritic atrophy in the neocortex of adult and young individuals with DS (Becker et al., 1986, Marin-Padilla, 1976, Takashima et al., 1981). Studies in the Ts65Dn model have demonstrated alterations in pyramidal neurons of the neocortex (Dierssen et al., 2003) and granule cells of hippocampus (Dang et al., 2014, Villarroya et al., 2018) among other areas. Also, we have observed a correlation between dendritic atrophy and reduced expression of markers for structural plasticity (Villarroya et al., 2018). Dendritic atrophy be related to the reduction of synaptic contacts and also to the unbalance between excitation and inhibition. Previous studies in this model have demonstrated over-inhibition in the hippocampus (Belichenko et al., 2004, Hernández-González et al., 2015) and in cortical regions (Hernández et al., 2012, Kurt et al., 2000, Pérez-Cremades et al., 2010).
Finally, piriform cortex is a region very sensitive to the development of epilepsy (Vaughan and Jackson, 2014, Young et al., 2019) and previous studies have observed a higher prevalence of epilepsy in DS individuals (Goldberg-Stern et al., 2001, Johannsen et al., 1996, Prasher, 1995, Pueschel et al., 1991, Stafstrom, 1993).
Thus, the piriform cortex displays a prominent position in information processing receiving directly the information from the olfactory bulb, which is altered in DS (Cecchini et al., 2016, Murphy and Jinich, 1996). Piriform cortex projects to other cortical regions and the hippocampus, which are also altered in DS (Belichenko et al., 2009b, Hernández-González et al., 2015). This cortical region also presents a high degree of structural plasticity, and plasticity is also impaired in DS. Finally, piriform cortex is a region involved in epilepsy generation (which is prevalent in DS individuals) and is particularly affected in Alzheimer’s Disease (disease present at early stages in DS individuals). All these reasons make relevant the study of piriform cortex in the Ts65Dn model of DS. With this aim in mind, we analysed the morphology (dendritic arborisation using Golgi-Cox method), synaptology (using immunohistochemistry against pre and postsynaptic markers, and electron microscopy) and structural plasticity (using immunohistochemistry against PSA-NCAM and DCX) in the piriform cortex of adult Ts65Dn mice.
Section snippets
Dendritic atrophy of neurons in the piriform cortex of the Ts65Dn mouse model.
We have analysed the dendritic complexity of neurons located in the piriform cortex using the Golgi-Cox method followed by Sholl analysis. We have analysed neurons located in layer II and layer III (Fig. 1). Neurons located in layer II (Fig. 1A, B) displayed a reduction in the total number of intersections: control 55.7 ± 3.8 intersections vs Ts65Dn 46.4 ± 1.8, p < 0.05 (Fig. 1C). The analysis of the number of intersections at different distances from the cell somata showed that the reduction
Discussion
In our study we have observed a moderate dendritic atrophy in the neurons located in the piriform cortex, an unbalance between excitation and inhibition, and an enlargement of the synaptic terminals under light and electron microscopy. The reduction in PSA-NCAM staining in the neuropil and in the number of immature neurons in piriform cortex layer II show that structural plasticity is impaired in TS65Dn mice. All these alterations could be related to the functional and pathological alterations
Experimental procedures
Experimental mice were generated by repeated backcrossing of Ts65Dn females to C57/6Ei 9 C3H/HeSnJ (B6EiC3) F1 hybrid males. The parental generation was obtained from the research colony of Jackson Laboratory. Euploid littermates of Ts65Dn mice served as controls. For this study, we used four- to five-month-old male mice. The genotypic characterization was established by qRT-PCR using SYBR Green PCR master mix (Applied Biosystems) from genomic DNA extracted from mice tails by mean of the
CRediT authorship contribution statement
Josep Carbonell: Investigation, Formal analysis, Visualization. José Miguel Blasco-Ibáñez: Investigation, Writing - review & editing. Carlos Crespo: Writing - review & editing. Juan Nácher: Writing - review & editing. Emilio Varea: Conceptualization, Methodology, Supervision, Writing - original draft.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study has been founded by Jerome Lejeune Foundation and the Ministry of Science, Innovation and Universities (RTI2018-098269-B-I00).
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