Altered expression of neuropeptides in the primary somatosensory cortex of the Down syndrome model Ts65Dn
Introduction
Down syndrome, with an incidence of one in 800 live births (Roizen and Patterson, 2003), is one of the most common genetic disorders. The phenotype observed as a consequence of a trisomy on the chromosome 21 may include immune deficiencies, heart defects, increased risk of leukemia and early development of Alzheimer’s disease. The principal common feature among all DS individuals is the presence of mental retardation. The substrate for this retardation has not been fully understood and may include defects in the formation of neuronal networks and information processing. Alterations in synaptic plasticity have been related to impaired cognition in different murine models of this genetic alteration (Siarey et al., 2005, Siarey et al., 2006).
Among the murine models available to study this genetic alteration, the most widely used is the Ts65Dn mouse, which mimics most of the alterations observed in DS. Ts65Dn mice are segmentally trisomic for a portion of the murine chromosome 16, which 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). These mice display delays in the acquisition of a number of sensory and motor tasks (Holtzman et al., 1996, Costa et al., 1999), as well as defects in learning and in the execution of memory tasks mediated by the hippocampus (Reeves et al., 1995, Escorihuela et al., 1995, Escorihuela et al., 1998, Holtzman et al., 1996).
Many studies have shown deficits in the dendritic arborization of the principal cells of the neocortex and hippocampus of DS subjects and murine models for this disorder (Marín-Padilla, 1976, Becker et al., 1986, Vuksic et al., 2002, Takashima et al., 1981, Takashima et al., 1989, Kaufmann and Moser, 2000, Dierssen et al., 2003). This atrophy has been related to mental retardation and deficits in cognition (Dierssen and Ramakers, 2006).
Alterations at the synaptic level have been observed in both DS subjects and murine models. Studies analyzing the expression of synaptophysin (a reliable marker for synapses (Eastwood and Harrison, 2001, Masliah et al., 1990)) have observed that the area occupied by synaptophysin is higher in Ts65Dn mice when compared with euploid mice, suggesting an increase in the size of the synapse (Belichenko et al., 2004, Belichenko et al., 2007, Belichenko et al., 2009). The increased expression of synaptophysin was restricted to superficial layers of cortex (Pérez-Cremades et al., 2010). The detailed study of synapse subtypes reflects a reduction in the number of asymmetric (excitatory) synapses per neuron in the hippocampus and in the temporal cortex of Ts65Dn mice (Kurt et al., 2000). Moreover, the inhibitory contacts are redistributed on the dendrites of these principal neurons, resulting in an increased density of those contacting spines and in a decrease of those contacting dendritic shafts (Belichenko et al., 2004). There is also an increased density of inhibitory puncta (observed by immunohistochemistry for GAD-67) in every layer of the cortex (Pérez-Cremades et al., 2010). Altogether, these results suggest an unbalanced level of inhibitory and excitatory inputs in the neocortex and hippocampus in Down syndrome. Supporting this fact, an overactivation of the inhibitory system, causing a general inhibition in the brain, has been observed in Ts65Dn mice (Fernández et al., 2007). This increased inhibition may be responsible, at least to some extent, for the cognitive impairment observed in Down syndrome. In fact, some studies have attempted to reverse the cognitive impairments observed in Ts65Dn mice by using GABAA receptor antagonists (Fernández et al., 2007), obtaining promising results.
Changes in inhibitory puncta density and distribution could be related to changes in the number and types of interneurons in the affected regions. In fact, we have observed an increase in the total number of inhibitory neurons in the primary somatosensory cortex of Ts65Dn mice. The phenotypical characterization revealed that among the increased subpopulations of inhibitory neurons were the calretinin and the calbindin D-28k expressing neurons (Pérez-Cremades et al., 2010).
The aim of this study is to deepen into the changes in interneuron populations in a specific region of the neocortex of Ts65Dn mice, the primary somatosensory cortex. We have chosen this region because previous reports have shown atrophy in principal neurons (Dierssen et al., 2003) and increased density of inhibitory neurons (Pérez-Cremades et al., 2010) We analysed the distribution and density of interneurons expressing four different neuropeptides: cholecystokinin (CCK); somatostatin (SST), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) in this cortical region of Ts65Dn mice. The analysis of these neuropeptides allowed us to discriminate the different subtypes of interneurons that were not previously studied in this area. In this way to aim to complete our previous study where we studied calcium binding protein interneurons and observed an increased density of interneurons expressing calretinin (Pérez-Cremades et al., 2010).
Section snippets
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 purchased from the research colony of The Jackson Laboratory (Ben Harbor, Maine, USA) Euploid littermates of Ts65Dn mice served as controls. The genotypic characterization was established by qRT-PCR using SYBR Green PCR master mix (Applied Biosystems) from genomic DNA extracted of mice tails by mean of the phenol–chloroform method. The relative
Results
The analysis of volume per layer and total in the primary somatosensory cortex of Ts65Dn showed not significant changes when compared with their euploid littermates, in accordance with previous results (Belichenko et al., 2004).
Discussion
In this study we have analysed the changes in the subpopulations of interneurons expressing neuropeptides in the primary somatosensory cortex of Ts65Dn mice. Trisomic mice displayed a higher density of interneurons expressing somatostatin and VIP, and a lower density of interneurons expressing NPY than their euploid littermates. The differences were confined to specific layers, V and VI for somatostatin, IV for VIP, and VI for NPY. The study of the phenotype of the subpopulations that increased
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This study has been founded by Spanish Ministry of Education and Science (BFU2007-64130/BFI); Foundation Jerome Lejeune and The Spanish Ministry of Science and Innovation BFU2009-12284/BFI and PIM2010ERN-00577/NEUCONNECT in the frame of ERA-NET NEURON.
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