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Post-training lateral hypothalamus (LH) intracranial self stimulation (ICSS) has a reliable enhancing effect on explicit memory formation evaluated in hippocampus-dependent tasks such as the Morris water maze. In this study, the effects of ICSS on gene expression in the hippocampus are examined 4.5 h post treatment by using oligonucleotide microarray and real-time PCR, and by measuring Arc protein levels in the different layers of hippocampal subfields through immunofluorescence. The microarray data analysis resulted in 65 significantly regulated genes in rat ICSS hippocampi compared to sham, including cAMP-mediated signaling as one of the most significantly enriched Database for Annotation, Visualization and Integrated Discovery (DAVID) functional categories. In particular, expression of CREB-dependent synaptic plasticity related genes (c-Fos, Arc, Bdnf, Ptgs-2 and Crem and Icer) was regulated in a time-dependent manner following treatment administration. Immunofluorescence results showed that ICSS treatment induced a significant increase in Arc protein expression in CA1 and DG hippocampal subfields. This empirical evidence supports our hypothesis that the effect of ICSS on improved or restored memory functions might be mediated by increased hippocampal expression of activity-dependent synaptic plasticity related genes, including Arc protein expression, as neural mechanisms related to memory consolidation.
Intracranial self stimulation (ICSS), a form of deep-brain stimulation with electrodes implanted in specific rewarding areas such as the lateral hypothalamus (LH), has a strong enhancing effect on several kinds of learning and implicit memory in rats (Aldavert-Vera et al. 1997; Coulombe & White 1982; Huston & Mueller 1978; Ruiz Medina et al. 2008b). Over recent years, we have demonstrated that ICSS in the LH is also a very reliable way to improve explicit memory consolidation and behavioral flexibility evaluated in tasks such as delayed spatial alternation (Soriano Mas et al. 2005) and the Morris water maze (Ruiz Medina et al. 2008a). ICSS induces long-lasting structural changes such as increases in dendritic arborization, and spine and synaptic density in hippocampal neurons (Rao 1999). This kind of structural plasticity has been shown to be involved in the neural mechanisms related to memory consolidation (see Morgado Bernal 2011).
At a molecular level, activity-dependent immediate-early gene (IEG) expression is a critical component of the molecular cascades underlying synaptic plasticity and long-term memory formation. In a microarray study by our group (Huguet et al. 2009), performed in the hippocampus 90 min after ICSS treatment, we found numerous overexpressed gene encoding proteins related to signal-transduction machinery that have also been found to be expressed after hippocampal-dependent tasks. These include the c-Fos transcription factor (Fleischmann et al. 2003), prostagladin-endoperoxidase synthase-2 (Ptgs-2) (Teather et al. 2002) and particularly genes such as adenylate-cyclase activating polypeptide1 (Adcyap1) (Chen et al. 2006; Yaka 2003) related to cAMP-dependent signal-transduction machinery that might participate in the signaling of Arc or Bdnf neuroplasticity-associated proteins. However, ICSS induction of Bdnf and Arc expression was not detected after 90 min. An initial objective of this work was to investigate the effects of LH-ICSS on genomic mechanisms in the hippocampus after a greater delay post-treatment, by using oligonucleotide microarrays after 4.5 h post-ICSS. Moreover, since the cAMP/PKA/CREB pathway activation is a specific molecular mechanism involved in memory consolidation in the hippocampus (Bekinschtein et al. 2007), and given that this pathway represents one of the main targets for the development of cognitive enhancers for the treatment of patients with memory dysfunction (Arnsten et al. 2005), a second objective was to compare the c-Fos, Bdnf, Arc, Ptgs-2, Crem and Icer CREB-dependent synaptic plasticity-related IEG mRNA levels at 90 min and 4.5 h post-ICSS by real-time PCR. Finally, we attempted to examine the unexplored effects of ICSS on Arc protein expression, a marker of synaptic plasticity processes occurring during hippocampal memory consolidation (Tzingounis 2006), in CA1, CA3 and DG subfields.
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This report outlines three major observations regarding the molecular effects of ICSS memory-facilitative treatment in rat hippocampus. A single LH-ICSS session causes: (a) increases in the expression of IEGs related to synaptic plasticity and to learning and memory processes after 4.5 h; (b) the regulation of a subset of CREB family-dependent genes (including c-Fos, Arc, Bdnf, Ptgs-2, Crem and Icer) in a time-dependent manner following treatment administration; and (c) a significant induction, 4.5 h after administration, of Arc protein expression in CA1 and DG hippocampal subfields, revealing that ICSS specifically upregulates an effector protein related to morphological changes that underlie the synaptic modification involved in memory consolidation processes.
Although the nature of ICSS behavior makes it difficult to distinguish which components of ICSS are chiefly responsible for the observed results, differences between groups are attributable mainly to brain stimulation more than to the motor activity inherent to ICSS treatment, since no correlation was observed between ICSS-motor variables and Arc-immunofluorescence intensity or any of the genes analyzed by quantitative real-time PCR. In accordance with the idea that operant responding does not seem to be a critical variable, similar patterns of Fos immunoreactivity in self-stimulation and yoked-stimulation groups were observed (Hunt & McGregor 2002). Fos expression did not correlate with bar-pressing rate (Panagis et al. 1997), and both LH self-stimulation and LH experimenter-administration procedures have been shown to facilitate memory (White and Major 1978). All these considerations therefore lead us to believe that, in all probability, the observed effects are mainly due to the electrical brain stimulation of LH.
cAMP-dependent signal-transduction machinery, which included activation of CRE-dependent gene expression, has been seen to be widely involved in the neuronal plasticity underlying learning and memory across species (Wang et al. 2006). Interestingly, the ‘cAMP-mediated signaling’ was most highly enriched annotation cluster associated with ICSS obtained after Functional DAVID analysis of our microarray gene list. ICSS-upregulated genes in this functional category included Crem, a CREB family protein exhibiting transcriptional activator functions, and Adora2a, an adenosine receptor that exerts critical roles in neuronal plasticity (Rebola et al. 2008). In addition, DAVID analysis identified the ICSS-dependent expression of two ‘synaptic plasticity’- and ‘learning and memory’-related IEGs effectors (Bdnf and Ptgs-2) that are also downstream cAMP-inducible genes (Lonze & Ginty 2002). Specifically, further investigation into six synaptic plasticity CRE-related genes revealed that these were ICSS-induced in a time-dependent manner: In accordance with our results showing c-Fos and Arc mRNA induction only at 90 min, a transient early activation (since 30 min to 2 h) of c-Fos and Arc transcription in the hippocampus has also been described after different stimuli such as spatial water maze training (Guzowski 2002; Vazdarjanova et al. 2006). In contrast, Bdnf and Crem showed a slightly delayed mRNA expression that has also been observed following the acquisition of spatial tasks (Lu et al. 2008) or after visual conditioning (Konopka et al. 1998). Ptgs-2, a gene that have been involved in the consolidation of hippocampal-dependent memory (Teather et al. 2002), was induced at the at two-time point analyzed, as well as Icer. Icer is as a potent endogenous repressor of CRE-mediated gene transcription, and it might serve as a gene-controlling mechanism that allows only strong/persistent information to gain access to long-term storage (Borlikova & Endo 2009). Coordinated expression of the target genes in the CREB family-transcription factors may provide synaptic strengthening (Lonze & Ginty 2002). Thus, these genes may well play important roles as IEGs mediating the ICSS facilitation of memory effects in the hippocampus with specific temporal activation.
The ICSS enrichment of other DAVID functional categories such as ‘neuron development’ and ‘neuron differentiation’ also contribute to the idea that ICSS regulates neural plasticity that could either influence memory facilitation or else collaborate in the learning- and memory-restoring capacities of ICSS observed in aging and brain-damaged rats (Aldavert-Vera et al. 1997; Redolar Ripoll et al. 2003).
The critical role of Arc in the maintenance of changes in synaptic efficacy and in the consolidation of long-term memory after the spatial water task (Korb & Finkbeiner 2011) points to Arc as an excellent marker for analyzing the effects of ICSS in synaptic plasticity. Here, an overexpression of Arc protein in CA1 and DG, but not in the CA3 subfields, was observed after 4.5 h following ICSS treatment, indicating an anatomical distribution throughout the different hippocampal subfields after our experimental conditions. We observed ICSS induction of Arc protein later than the Arc mRNA early and transient induction. Other researchers have also found disparities between levels of Arc mRNA and protein (Kelly & Deadwyler 2003; Zalfa et al. 2003), and hippocampal expression of Arc protein has been observed at different time courses (between 30 min and up to 6 h) after different paradigms such as LTP (Messaoudi 2007); contextual fear conditioning (Lee 2004); and spatial exploration (Ramirez Amaya 2005). The region-specific effects of ICSS on Arc protein expression are especially interesting, since CA1, CA3 and DG hippocampal subfields have previously been described to differ in terms of synaptic plasticity mechanisms (Hussain & Carpenter 2005; McBain 2008). Indeed, the CA1 region has been shown to be the most sensitive hippocampal region in Alzheimer's disease (AD) resulting in dendritic spine loss and synaptic alterations (Knobloch 2008) and increasing CREB function in CA1 rescues spatial-memory deficits in a mouse model of AD (Yiu et al. 2011). Interestingly, ICSS specifically induced an increase of the CA1 radiatum/pyramidal Arc expression ratio, suggesting a mobilization of Arc from the soma to the neuronal projections in this hippocampal region. Different behavioral studies show different rules governing synaptic plasticity between layers of CA1, and highlight the importance of the immune-histochemical analysis of the hippocampus subregions (Kitanishi et al. 2009). Our immune-histochemical results are consistent with subregional ICSS-induced differences related to synaptic plasticity, and agree with our recent data (Chamorro López et al. 2012) showing that post-training ICSS facilitates the Morris water maze task and induces long-lasting structural changes, including an increase in dendritic arborization and synaptic density in CA1 measured three and 20 days post-ICSS. An increase in Arc activity may facilitate the dendritic remodeling underlying long-term memory and there is evidence that this specifically targets stimulated dendrite regions (McIntyre et al. 2012).
LH-ICSS induces a widespread increased expression of c-Fos, including LH and other brain areas such as the amygdala (Arvanitogiannis et al. 1997, 2000; Kadar et al. 2011a). ICSS could have activated the hippocampus through its LH direct projections, as the orexin projections, known to affect the reward-related behavior and plasticity underlying learning and memory (Akbari 2011; Peyron 1998). But we cannot rule out the involvement of other indirect projections in the regulation of hippocampal plasticity, such as that from the amygdala, as it has been suggested by McIntyre et al. (2012). Interestingly, different learning paradigms also increase c-Fos, Arc and/or Bdnf expression in the hippocampus and amygdala (Datta et al. 2008). Comparing the present results with our previous findings in the amygdala (Kadar et al. 2011a), ICSS induces a different temporal activation of c-Fos, Arc and Bdnf in these two memory-related regions. Thus, the possibility exists that ICSS could modulate memory through a time-dependent cooperative activation of different memory systems.
In summary, ICSS regulates CRE-dependent genes hippocampal expression and induces Arc protein expression changes between hippocampal layers. The hippocampal ICSS modulation of related learning IEGs may promote plasticity mechanisms that underlie memory consolidation. This hypothesis is also supported by previous structural and behavioral findings showing that ICSS induces CA1 dendritic branching and improves retention at 1, 3 and 10 days post-acquisition of spatial learning in the Morris water maze (Chamorro et al. 2012; Ruiz Medina et al. 2008a). This results also shed new light onto the initial aspects of Huston's Central Theory of Reinforcement (Huston et al. 1977), according to which the ICSS—by means of activating the brain's reward system—would ‘strengthen’ short-term memory trace and, thus, enhance memory consolidation.