Rapamycin inhibits mTOR/p70S6K activation in CA3 region of the hippocampus of the rat and impairs long term memory
Introduction
The hippocampus is critical for learning and memory. Age related decline in hippocampal function may underlie impaired memory abilities in about half of the population over 60 years of age (Hedden and Gabrieli, 2004, Small et al., 2002). The hippocampal regions CA1, CA3 and dentate gyrus, although interconnected via the trisynaptic pathway, display striking anatomical differences (Amaral & Witter, 1989) and show distinct functions, contributing to specific types of information processing such as novelty detection, encoding, short-term memory, intermediate-term memory and retrieval. In particular, CA3 and CA1 pyramidal neurons perform distinct, yet complementary, functions in the processing of spatial and contextual information (Vazdarjanova & Guzowski, 2004). The CA3 hippocampus supports processes associated with the rapid formation of spatial or contextual memory (Kesner et al., 2004, Lee and Kesner, 2002, Lee and Kesner, 2003, Nakazawa et al., 2003). However, CA3 lesions, or experimentally-induced dysfunctions of CA3, also impair spatial memory (Lee and Kesner, 2004, Nakazawa et al., 2003) and object-place associations (Hunsaker & Kesner, 2008; Langston, Stevenson, Wilson, Saunders, & Wood, 2010). Intrahippocampal CA3 information processing is also important for memory-based behavior and can modulate activity in the CA1 (O’Reilly, Alarcon, & Ferbinteanu, 2014). The relative contribution of CA3 and CA1 regions to memory is not completely understood. A recent review of the learning and memory literature suggested (Stokes, Kyle, & Ekstrom, 2015) that CA3/DG and CA1 have distinct and separate roles in the representation of a spatial context during the formation of memories.
Long term memory requires protein synthesis; mTOR signaling is of crucial importance in this process, especially at the level of neuronal synaptodendritic compartment (Giovannini & Lana, 2016). Activation of mTOR/p70S6K pathway in CA1 hippocampal pyramidal neurons is instrumental to the process of formation of a long term inhibitory avoidance (IA) memory, and the cholinergic input through muscarinic and nicotinic receptor blockade impairs short term, but not long term IA memory (Lana et al., 2013).
IA is an emotionally-arousing paradigm (Giovannini et al., 2005, Giovannini et al., 2008, Izquierdo et al., 1997a, Lana et al., 2013, Maren, 2001) that involves a spatial memory component as the animal remembers the location where the noxious stimulus was given during acquisition (Cammarota, Bevilaqua, Medina, & Izquierdo, 2007), an explicit, associative component to the context, and an operant-like conditioning component to the shock as the animal may avoid the aversive stimulus (Wilensky, Schafe, & LeDoux, 2000). The IA response is a learning task that depends upon the activation of the hippocampal cholinergic system (Giovannini et al., 2005), as shown by the impairment by pre-training (Giovannini et al., 1999, Izquierdo et al., 1998a) or post-training administration of muscarinic receptor antagonists (Giovannini et al., 1999, Izquierdo et al., 1998b, McGaugh and Izquierdo, 2000). The recall test, performed at different times after acquisition, offers insight into the mechanisms involved in short term (Izquierdo et al., 1998a, Izquierdo et al., 1998b) and long term memory (Izquierdo et al., 2002). The step-down IA is acquired in one trial by activation of different brain structures by sensorial stimuli, including spatial and visual perceptions, pain, and fear (Izquierdo, 1989, Izquierdo and Medina, 1997b).
In this paper we will extend our analysis on mTOR pathway dynamics in the CA3 region of the hippocampus during the formation of an IA memory. By comparing the similarities and differences between CA1 and CA3 we will be better able to define the relative contribution of these two hippocampal regions in the encoding of an IA memory. Furthermore, we will define whether in CA3 pyramidal neurons the mTOR pathway is modulated by the cholinergic input and whether activation of this pathway triggers the encoding of long term memories in a similar manner to what we had demonstrated in CA1 hippocampus (Lana et al., 2013).
Section snippets
Animals
Male adult (3 months old) Wistar rats, weighing 200–225 g, were (Harlan Nossan, Milano, Italy) housed in macrolon cages until experiment with ad libitum food and water and maintained on a 16 h light – 8 h dark cycle with light at 7:00 am. The room temperature was 23 ± 1 °C. All rats were kept for at least 1 week in the animal house facility of the University of Florence before initiating the experiment and were frequently handled. All animal manipulations were carried out according to the European
Results
This investigation determined whether the mTOR/p70S6K cascade was activated in the CA3 region of the hippocampus by the acquisition of short and long term IA memory.
Discussion
The current study compared the dynamics of mTOR/p70S6K activation in the CA1 and CA3 regions of the hippocampus during the formation of IA memory with the goal of determining the differential contribution of these two hippocampal regions in the encoding of an IA memory. We discovered that the mTOR/p70S6K pathway in CA3 pyramidal neurons is activated by cholinergic input in order to trigger the formation of long term memory. Taken together with our previous study (Lana et al., 2013) this report
Conclusions
In this paper we have demonstrated that in the CA3 region of the hippocampus, activation of the mTOR pathway is necessary for the formation of a long term inhibitory avoidance memory. A serendipitous, possibly important finding was that after administration of rapamycin the activation of mTORC2 increases in microglial cells, poses the basis for a reappraisal of the beneficial/adverse effects of rapamycin administration.
Acknowledgements
We thank Dr. A. Melani for help in animal care and Miss. M.R. Malatacca, for help in immunohistochemistry and quantification analysis. This work was partly supported by Fondazione Ente Cassa di Risparmio Grant N. 2014/0663 and Grant 2015 from Università di Firenze. D.L. was supported by “Prize from Italian Society of Pharmacology”, with the contribution of MSD Italia, Prize Edition 2014. GLW was supported by U.S. Public Health Service, RO1 AG037320.
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