Environmental enrichment results in cortical and subcortical changes in levels of synaptophysin and PSD-95 proteins

Abstract

Experience-dependent plasticity is thought to involve selective change in pre-existing brain circuits, involving synaptic plasticity. One model for looking at experience-dependent plasticity is environmental enrichment (EE), where animals are exposed to a complex novel environment. Previous studies using electron microscopy showed that EE resulted in synaptic plasticity in the visual cortex and hippocampus. However, the areas in the brain that have been examined following EE have been limited. The present study quantified potential synaptic plasticity throughout the brains of C57BL/6 mice using an enzyme-linked immunosorbent assay (ELISA) for two synaptic proteins, synaptophysin and PSD-95. EE resulted in increased synaptophysin and PSD-95 levels through major brain regions, including anterior and posterior areas of the forebrain, hippocampus, thalamus, and hypothalamus. However, no changes in synaptophysin were detected in the cerebellum. These results demonstrate that EE results in an increase in levels of both pre- and post-synaptic proteins in multiple regions of the brain, and it is possible that such changes represent the underlying synaptic plasticity occurring in EE.

Introduction

Our ability to interact, adapt, and respond to our environment highly depends upon the storage of past experiences through the process of learning and memory. Long-term storage of memories is known to be dependent on de novo protein synthesis, possibly involving changes in existing brain circuitry. Such changes are generally considered to involve plasticity, such as altered synaptic response or increased synaptic number or size. In a limited number of cases, experiences involving learning have resulted in an increase in synapse number (Black, Isaacs, Anderson, Alcantara, & Greenough, 1990; Federmeier, Kleim, & Greenough, 2002; Kleim, Lussnig, Schwarz, Comery, & Greenough, 1996; Wenzel, Kammerer, Kirsche, Matthies, & Wenzel, 1980) or change in dendritic spine density (Knafo, Grossman, Barkai, & Benshalom, 2001; Moser, Trommald, & Andersen, 1994; Moser, Trommald, Egeland, & Andersen, 1997; O’Malley, O’Connell, & Regan, 1998; O’Malley, O’Connell, Murphy, & Regan, 2000). Similarly, synaptogenesis has also been reported as a consequence of long-term potentiation (LTP), a leading mechanism for understanding learning and memory (Bolshakov, Golan, Kandel, & Siegelbaum, 1997; Engert & Bonhoeffer, 1999; Maletic-Savatic, Malinow, & Svoboda, 1999; Toni, Buchs, Nikonenko, Bron, & Muller, 1999).

Environmental enrichment (EE) is a model for both storage of new information and experience-dependent behavioral modification. EE was used to directly demonstrate experience-dependent synaptic plasticity, and results in increased synaptic size in visual cortex (Turner & Greenough, 1985) and increased synapse and spine density in the hippocampus (Moser et al., 1994; Rampon et al., 2000b). EE also has been shown to have a variety of other effects within the hippocampus, including altered synaptic transmission (Foster & Dumas, 2001; Green & Greenough, 1986), increased neurotrophin levels (Ickes et al., 2000), and enhanced neurogenesis (Kempermann, Kuhn, & Gage, 1997). EE has also delayed disease onset and progression in a mouse model of Huntington’s disease (Hockly et al., 2002). Furthermore, there are significant behavioral effects on animals. Exposing animals to EE improves learning and memory (Rampon et al., 2000b) and decreases stress (Nikolaev, Kaczmarek, Zhu, Winblad, & Mohammed, 2002).

The above studies thus show that EE is a good model for analysis of experience-dependent plasticity, however, analysis has mainly been restricted to the cortex and hippocampus. We were interested in developing a technique which would allow us quantitate the levels of synaptic markers rapidly and for many samples. This would allow us to do multiple comparisons across many different brain regions and determine if synaptic plasticity might have occurred. Therefore, we have exploited a simple and sensitive technique of enzyme-linked immunosorbent assays (ELISA) for synaptophysin, an integral membrane protein in synaptic vesicles (Jahn, Schiebler, Ouimet, & Greengard, 1985; Wiedenmann, Franke, Kuhn, Moll, & Gould, 1986). Synaptophysin generally co-localizes with axon terminals (Calhoun et al., 1996; Hiscock, Murphy, & Willoughby, 2000). Changes in levels of synaptophysin presumably reflect changes in synaptic vesicles and it thus follows that such changes are an indicator of synaptic plasticity.

We also developed an ELISA for the post-synaptic density protein, PSD-95. PSD-95 is thought to be a scaffolding protein for key signaling molecules at the synapse, including glutamate receptors (Cho, Hunt, & Kennedy, 1992; Kornau, Schenker, Kennedy, & Seeburg, 1995). Increases in PSD-95 gene expression or protein levels have been detected in cortex following EE and other experimental manipulations which induce synaptic plasticity (Rampon et al., 2000a; Skibinska, Lech, & Kossut, 2001). The current study aimed to examine the effect of EE, throughout the brain and to identify where changes in these markers might occur. We found enrichment induced changes in synaptophysin levels throughout major cortical regions. In addition, we found changes in major subcortical regions, in particular thalamus and hypothalamus. Parallel analysis of PSD-95 also revealed similar findings. Thus, enrichment results in changes in the levels of both pre- and post-synaptic proteins in multiple brain regions, including those involved in sensory processing, learning and memory.