MOLECULAR BASIS OF LEARNING IN THE HIPPOCAMPUS AND THE AMYGDALA

Abstract

The hippocampus and the amygdala are structures of mammalian brain both involved in memorizing. However, they are responsible for different types of memory: the hippocampus is involved in creating and storing declarative engrams and the amygdala is engaged in some of non-declarative learning. During memorization, changes of synapses appear and it is believed that they encode information. Long-Term Potentiation (LTP) and Long-Term Depression (LTD) are two processes which provide to these changes which are called synaptic plasticity. LTP strengthens connections between neurons and because of that it is traditionally linked with learning. LTD as an opposite state is usually treated as forgetting. However, there are some evidences that it is true only for few types of non-declarative engrams. More sophisticated learning (like declarative learning) requires cooperation of these processes. Review is focused on functions and detailed signaling pathways of processes of synaptic plasticity.

Headnote

ABSTRACT

The hippocampus and the amygdala are structures of mammalian brain both involved in memorizing. However, they are responsible for different types of memory: the hippocampus is involved in creating and storing declarative engrams and the amygdala is engaged in some of non-declarative learning. During memorization, changes of synapses appear and it is believed that they encode information. Long-Term Potentiation (LTP) and Long-Term Depression (LTD) are two processes which provide to these changes which are called synaptic plasticity. LTP strengthens connections between neurons and because of that it is traditionally linked with learning. LTD as an opposite state is usually treated as forgetting. However, there are some evidences that it is true only for few types of non-declarative engrams. More sophisticated learning (like declarative learning) requires cooperation of these processes. Review is focused on functions and detailed signaling pathways of processes of synaptic plasticity.

KEY WORDS: synaptic plasticity, learning, Long-Term Potentiation, Long-Term Depression, Long-Term memory

There are three types of memory: sensory, short-term and long-term (Atkinsons et al., 1968). Sensory memory has big capacities and relates to sensory receptors. It lasts as long as information is transmitted further: to short-term memory. It can store engram consisted of 7±2 and stays few seconds (with a possibility to extend that time with active recalling) (Sperling et al., 1960, Miller et al,.1956, Squire et al., 2004). The last type is long-term memory with almost unlimited capacities and duration as long as whole life span. Long-term memory can also be divided further: by access of a consciousness to its engrams (stored information): declarative and nondeclarative memory. They consist of subtypes and detailed division is shown on fig. 1 (Squire et al., 2004).

There are many structures in brain involved in memorization but there are two especially well-studied: the hippocampus and the amygdala. The hippocampus is a symmetrical structure of medial temporal lobe, part of three-layered archicortex (MacLean et al., 1990). It consists of dentate gyrus (DG), Cornu Amonis (CA1, CA2 and CA3) and subiculum (Van Strien et al., 2009). Electrophysiological research and clinical observation of patients with selective lesions of temporal lobes have shown that the hippocampus is necessary in some parts of memory processes (Rugget al., 2012, Scoville et al., 1957). It has got a role not only in memorization but this is a structure where some of engrams are stored (O'Keefe et al., 1976).

The amygdala is placed near the hippocampus but it has different anatomy. It consists of two parts: basolateral complex and cortically medial complex (with the cortical nucleus, the medial nucleus and the central nucleus) (Amunts et al., 2005). The amygdala is responsible for emotional arousal which seems to enhance memorization (Kapp et al., 1992).

The amygdala is also necessary to many types of non-declarative learning like classical conditioning (Goosens et al., 2001). Because of the fact that medial part of the amygdala is a place where different brain pathways are crossing, some researchers claim that this is a structure of integration of conditioned (CS) and unconditioned stimulus (US) (Blair et al., 2001, 2005). In research on rats it was shown that that conditioning learning is impaired in both situations of blocking the amygdala: before and after an experiment (Campeau et al., 1995). So we can assume that this structure is involved not only in detection of stimuli coexistence but also in storage of some of engrams.

Synaptic plasticity is an adaptive value which is an ability of neural circuits to change its properties upon experiences (Citri et al., 2008). Changes on neural level can be observed also during learning and they are are viewed as molecular basis of memorization and they include enhancing and weakening of connections between neurons-synapses (Citri et al., 2008).

There are three types of synaptic plasticity: developmental, short-term and long-term plasticity but only second and third concerns to learning. Developmental synaptic plasticity is essential for brain development. Short-term synaptic plasticity relies on accumulation of calcium ions in pre-synapse and neurotransmitter release, leading to short-term memory. Long-term plasticity relates to long-term memory andis described further in detail (Citri et al., 2008).

Long-Term Potentiation (LTP) leads to temporary enhancement of excitatory postsynaptic potential amplitudes. It is the type of neural plasticity which provides long lasting strengths to synapses. These changes include increased number and size of dendritic spines, higher number of receptors on post-synapse and increased number of ribosomes. In experimental protocols, LTP appears after high-frequency (tetanic) stimuli of pre-synapse or pairing stimulation. Pairing stimulation is a stimulation of pre-synapse with simultaneous depolarization of post-synapse (Citri et al., 2008, Blair et al., 2001). Naturally, LTP appears as an effect of theta rhythm which isan oscillatory pattern of brain activity with frequency around 6-10Hz. This rhythm appears during exploration and exposure on novelity (Citri et al., 2008, Martin et al., 2000, Winson et al., 974).

LTP is not a homogenous process and it can be divided into four phases and each phase is essential to create long-lasting synaptic changes and depends on different molecular basis (Sweatt et al., 1999).

Post-tetanic potentiation (PTP) - It is the first phase of LTP and depends on Ca2+ entry into cytoplasm of post-synapse mediated by N-methyl-D-aspartate receptor (NMDAR). NMDAR open only in specific conditions: with co-activation of two ligands: glutamate and glycine (or D-serine). NMDAR is also blocked by magnesium ion which can be released by membrane. So opening NMDAR require co-activation of two pre-synapses (Nowak et al. 1984, Tsien et al., 2000, Malenka et al., 1993).In some parts of brain (for examples CA1, medial nuclei of the amygdala) Ca2+ can flow also by other channels like Voltage-Dependent Calcium Channels type L (VDCC) which can also mediate LTP: with or without NMDAR (Chapman et al., 1992).

Short-Term Potentiation (STP)-during this phase increased calcium level in cytoplasm is sustained by metabotropic glutamate receptors type 1 (mGluR1) (Erickson et al., 2010). Binding glutamate to mGluR leads to activation of proteinGq.Gqaffects phospholipase C (PLC) which provides to formation of Diglyceride (DAG) and inositol-1,4,5-trisphosphate (IP3). In turn, IP3 activate IP3 receptors on smooth endoplasmic reticulum which is a calcium store in a cell (Alberts et al. 2013).

Nitrous oxide (NO) laso plays a crucial role in STP (Bernabeu et al., 1995). NO acts retrogradely (on pre-synapse) and causes increased release of glutamate which expedites activation of glutamate receptors and thus increased Ca2+ entry (Alberts et al. 2013, Böhme et al., 1991).

Early-LTP (E-LTP)-Increased calcium level in cytoplasm causes a cascade of changes in cellleading to an LTP- phase dependent upon kinases. Higher level of Ca2+ causes activation of protein kinase A (PKA) and calcium dependent kinase II (CaMKII).CaMKII phosphorylates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) which can be activated by glutamate, thus being permeable for Ca2+ (Lisman et al., 2002, Derkach et al. 1999). CaMKII controls also RasGAP and provides increased activity of ras protein. In that way intracellular vessels with AMPARs heads to cell membrane which increase neuron excitability (Böhme et al., 1991). Insertion of AMPAR to cell membrane is also regulated by Phosphoinositide 3-kinase (PI3K) which acts through protein kinase B (PKB, also known as AKT) on t-SNARE proteins. (Lisman et al., 2002, Citri et al., 2008).

Another important consequences of E-LTP are cytoskeletal changes which are caused by increased calcium level. Integrinsand cadherins are activated bytyrosine kinases, protein Src, p190 RhoGAP and finally Rho GTPase which suppress actin depolimerization factor-cofilin. Actin can polymerize and create actin skeleton which can build new dendritic spines (Lamprecht et al. 2004). However, Rho GTPase phosphorylates also collapsin response mediator protein (CRMP2) which is responsible for microtubules' polymerization. Phosporylated CRMP2 loses that properties and with its inactivation of microtubule skeleton is suppressed (Arimura et al., 2005).

In E-LTP DAG has a role, by activating PKC and protein kinase G. Common place for all these kinases in LTP's pathway is mitogen-activated protein kinase (ERK, also known as MAPK). In one way, ERK phosphorylates potassium channels type A which increases potassium currents and excitability of neuron. In other, nearCaMKII, PKA, PKC and PKG, it is also an nuclear activator of cAMP response element-binding protein (CREB) (Silva et al., 1998). CREB is an element of next phase of LTP- Late LTP (L-LTP).

Late-LTP- this phase relies on protein synthesis which contributes to long-lasting changes in synapses. Phosphorylated by kinases CREB creates a complex with protein binding CREB (CBP) and in that complex it binds to specific region on DNA-CRE (Silva et al., 1998). CREB is an activator of immediate early genes (IEGs) like Arc, c-fos, Zif268 (Rosen et al., 1998). Activation of c-fos leads to expression of matrix metallopeptidase 9 (MMP-9) which is responsible for maturation of dendritic spines. MMP-9 posible is released to an extracellular matrix where it can process laminins. Once laminins are processed it acts asa substrate for b1 integrins which there by polymerises actin and lengthens dendritic spines. MMP-9 also influences VDCCs and NMDARs, however the mechanism of its regulation is not clear. Intracellular substrate for MMP-9 is CRMP2, which is activated by cleaving. In that way microtubule skeleton mounts up (Stawarski et al., 2014, Bajor et al. 2012).

Shortly after release of MMP-9 it is inactivated by tissue inhibitor of metalloproteinases 1 (TIMP1) and synapse gets mushroom-like shape (mature form of spine) (Stawarski et al., 2014).

Postsynaptic Long-Term Depression

The second process providing long-lasting changes inn synaptic transmission is long-term depression (LTD). LTD leads to weakening of synaptic efficency. This state can be experimentally induced by low-frequency stimuli or chemically (by activation of mGluR) (Kemp et al., 2007).

Induction of LTD. An enigmatic property of synaptic plasticity is that LTD just like LTP can be induced by activation of NMDAR or mGluR. What's more is this opposite state also depends on calcium ions entry to a cell (Kemp et al., 2007).One hypothesis claims that the difference between activation of these processes is in the level of Ca2+which enters to cytoplasm. In this hypothesis lower level of influent Ca2+ provides to LTD while higher provides to LTP. This hypothesis is consistent with different properties of some calcium detectors in LTP and LTD. For example activation of calmodulin (CaM) in LTD requires lower level of Ca2+than activation of CaMKII in LTP (Lisman et al., 1989).

Induction of LTD is based on NMDARs (especially containing subunit NR2B) or by mGluR (Kemp et al., 2007, Yashiro et al., 2008).These two ways of induction leads to different pathways so they will be described separately.

NMDAR-dependent LTD. In that type of LTD, Ca2+ is binded by CaM, which activates calcineurin and so on protein phosphatase 1 (PP1). PP1 suppress via dephosphorylation AMPARs and CaMKII (and thus LTP pathway). On the other hand, PP1 activates glycogen synthase kinase 3 beta (GSK3β) by its dephosphorylation (Collingridge et al., 2010).

GSK3β through phosphorylation of kinesin light chain 2 suppress that motor protein and microtubul transport at once (vessels with AMPAR are not able to internalize with membrane). GSK3β phosphorylates also β-catenin which provides to its degradation. β-catenin creates adherent junctions between pre- and post- synapsesothat degradation is therefore a possible cause of decreasing number of synapses after LTD (Bradley et al., 2012, Kaidanovich-Beilin et al., 2011).

Another effect of GSK3β is depolimerisation of microtubules. Increased cytoplasmic calcium level activates also protein interacting with PKC 1 (PICK1) which affects actin skeleton. Activated PICK1 binds to F-actin and Actin-Related Proteins 2/3-Arp2/3 which leads to actin (Collingridge et al., 2010).

mGluR-dependent LTD. Binding ligand to mGluR activates PLC and IP3 and DAG are created. IP3 provides to release of Ca2+ from smooth endoplasmic reticulum and this is direct source of increased cytoplasmic calcium level in that type of LTD (Collingridge et al., 2010, Alberts et al. 2011).

DAG activates PICK1 (through PKC) which phosphorylates subunit of AMPAR. That subunit splits off from AMPAR-binding protein and glutamate receptor interacting protein (ABP/GRIP). It provides to internalization of AMPAR and lesser excitability of neuron (Collingridgeet al., 2010).

Some parts of these two types pathways are probably common. However, there is a need of determination of theirs cascades, especially on their nuclear level. It is only known that eukaryotic elongation factor 2 (EEF2) can be nuclear activator. Some researchers claim that its effect is a translation of Arc, Protein tyrosine phosphatases (PTPs) and p38 MAPK (Collingridge et al., 2010).

Functions of LTP and LTD

LTP was a state observed during some types of learning and as described above- it was also observed as a consequence of theta rhythm in hippocampus. With its enhancing effect on synapsis it was a good candidate of molecular equivalent of learning (Rogan et al. 1997). LTD by contrast, as an opposite state matches perfectly as molecular basis of forgetting (Tsumoto et al., 1993).

However, with further reports, it was clear that way of thinking do not describe properly memorization. Firstly, theta rhythm in some parts of brain (for example in the amygdala) causes LTD, not LTP (Heinbockel et al. 2000). Secondly, it seems that some types of fear conditioning is based rather on weakening than strengthen of synapses (Paré et al., 2000). Finally, it was shown that blocking LTD (with LTP preserved) impairs contextual and spatial learning with no changes on non-contextual learning (Etkin et al., 2006). However selective blocking of LTP (by blocking NMDAR subunit- NR2A) also impaired spatial learning (Kemp et al., 2004).

On the other hand, it seems that in some types of associative learning LTP or LTD can be treated as an equality of memorization (with an opposite process as a forgetting). Associative learning is explained by theory of associative learning (also called Hebbian plasticity). Initially, Hebbian theory said that during repeatedly stimuli of two neurons, new synapses between these neurons are created. Later activation of one neuron would possess to activation alsopaired neuron (Citri et al., 2008). Now, this theory is linked with Konorski's theory which says that learning modify pre-existing connections between these neurons. (Konorski, 1948).

Upon new knowledge about molecular basis of learning it seems that there is a big need of wariness during interpretation of data about synaptic plasticity. Treating LTP as equality of memorization and LTD as equality of forgetting is too big simplifying. Probably some research about impact of various substances on LTP/LTD and learning should be reconsidered (Chen et al. 2014). It should be remembered that affecting on one type of synaptic plasticity often changes also an opposite. One solution to better understanding of LTP and LTD is using diversity of behavioral tests which can verify different types of memory.

Better understanding of LTD and LTP can also shed light onto some of unsolved questions. In research with mice lacking genes for MMP-9 have got impaired reward learning with intact aversive learning (Knapska et al., 2013). Upon a fact there is no activity of MMP-9 during LTD, it can be assumed that studied aversive learning (fear conditioning) was based rather on LTD than LTP.

It is still not clear how engrams are created and stored. It looks that proper learning requires balance between LTP and LTD. Especially, coding parts of declarative memory stays as guesses. One hypothesis claims, that during learning (with accompany of LTP) excess of active synapses is created. In that hypothesis, following LTD is necessary to stabilize neural network (Caroni et al., 2012). Some scientists claim that this rearrangement (at least in the hippocampus) is a process where engrams are encoded on a matrix, created during LTP (Kemp et al., 2007).

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