Studies on the molecular and cellular mechanisms underlying the process of learning and memory formation body

Investigations on Learning-Induced Remodeling of Hippocampal Mossy Fibers: The Role of Presynaptic Structural Plasticity in Long-Term Memory Chapter 1 Introduction 1.1 Introduction of

PART I Investigations on Learning-Induced Remodeling of

Hippocampal Mossy Fibers: The Role of Presynaptic Structural

Plasticity in Long-Term Memory

Chapter 1 Introduction

1.1 Introduction of hippocampal mossy fibers

1.1.1 Anatomy of the hippocampal mossy fibers

1.1.1.1 Introduction of the hippocampus

The hippocampus, a major component of the brain, has been considered to play an important role in the formation of new memories and in the consolidations of information from short-term memory to long-term memory Anatomically, the hippocampus proper has three subdivisions: CA3, CA2, and CA1 (CA: cornu ammonis) Together with other regions, including the dentate gyrus, subiculum, presubiculum, parasubiculum, and entorhinal cortex, constitute the hippocampal formation Figure 1.1 (Neves et al., 2008) shows the schematic diagram of the circuit in the hippocampal formation

The entorhinal cortex is the first step to receive much of the neocortical input in the intrinsic hippocampal circuit Neurons in the superficial layers (layer II) of the entorhinal cortex give rise to axons that are projecting to the dentate gyrus The projections from the entorhinal

the major hippocampal input pathways called the perforant path Perforant path axons form excitatory synaptic contacts to the dendrites of granule cells in dentate gyrus: axons from the medial and lateral entorhinal cortices innervate the middle and outer molecular layers of dentate gyrus, respectively Similarly, the granule cells of the dentate gyrus, project to the dendrites of the pyramidal cells in the CA3 through their axons which are called mossy fibers (MFs) In turn, the pyramidal cells of CA3 are the source of the major input to the CA1, comprising of the Schaffer collaterals These three sequential projections, traditionally called trisynaptic circuits, are the major pathways in the hippocampus In addition to this trisynaptic circuit, there are also other networks interconnecting two subfields For example, the distal apical dendrites of CA1 pyramidal neurons receive a direct input from layer III cells of the entorhinal cortex CA1, inversely, projects across subiculum to the inner deep layer of the entorhinal cortex Through these connections, CA1 and the subiculum enclose the hippocampal processing loop that begins in the superficial layers of the entorhinal cortex and ends in its deep layers Furthermore, there is also a direct input from the entorhinal cortex to CA3 pyramidal cells CA3 pyramidal neurons are extensively connected to each other via recurrent collateral synapses: associational/commissural synapse (Amaral & Lavenex, The Hippocampus Book, 2007)

Figure 1.1 Basic anatomy of the hippocampus formation (Neves et al., 2008) Transverse view of the hippocampus in the middle shows the trisynaptic loop interconnecting the hippocampal subfields, indicating the critical role of the hippocampus in the information processing.

1.1.1.2 Introduction of the mossy fibers

As mentioned in 1.1.1.1, the hippocampal MFs are the axons which arise from the granule cells of the dentate gyrus The MFs pathway, the only efferent projections from dentate gyrus

to neurons in the hilus and CA3 area, comprise the second synapse of the hippocampal circuit

In this trisynaptic model of the hippocampus, based on the observations of the MFs synapses and their locations, the basic functional role of MFs it is clear that the MFs pathway provides a strong excitatory input to the proximal apical dendrites of CA3 pyramidal cells (Blackstad and Kjaerheim, 1961; Andersen et al., 1966; Andersen et al., 1971)

The MFs form distinct synapses with excitatory and inhibitory cells of the hilus and area CA3 It has been shown that the MFs form synapses on large thorny excrescences of CA3 pyramidal neurons (Amaral and Dent, 1981) Besides, the MFs also make synaptic connecting to the MF-associated inhibitory interneurons (Maccaferri et al., 1988; Vida and Frotscher, 2000) In the rodents, each of the granule cells in the dentate gyrus gives rise to a single MF axon and the main MF axon gives rise to a great amount of fine collaterals to provide input to the polymorphic neurons of the hilus (Henze et al., 2000) Furthermore, the main MF axons leave the hilus and pass through CA3 area in a narrow band which is called

the stratum lucidum (SL), corresponding approximately to the proximal 100 μm of the apical

dendrites of CA3 pyramidal cells (Henze et al., 2000) Besides, there are present large numbers of active zones and their associated post-synaptic densities in the MFs synaptic complex (Acsady et al., 1998; Chicurel and Harris 1992) Therefore, because of its unique and basic structural property, the hippocampal MFs pathway is usually considered one of the pre-synaptic models in the investigations on the presynaptic morphological axonal readjustments

1.1.2 Histology of Mossy fiber pathways

It has been studied that the MFs terminals contain the highest concentration of zinc in the brain by Frederickson in 1983 This heavy metal is found in large dense core vesicles and is released from the MFs Timm’s staining method for heavy metals has the ability to reveal zinc, therefore it can be considered as an efficient histological tool to identify the distribution

of the MFs using the light microscope (Figure 1.2, reference image from Rekart et al., 2007a)

Figure 1.2 illustrates the organization of the MFs terminal regions in CA3 In CA3c, the proximal portion of CA3 that is close to the dentate gyrus, the MFs are distributed below, within, and above the pyramidal cell layer The fibers located below the layer are generally called the infrapyramidal MFs (INFRA; white arrow in Figure 1.2), while, the fibers located within the pyramidal cell layer are called the intrapyramidal MFs (INTRA; white arrowhead

in Figure 1.2) Often these two are treated as one pathway, the infra- and intrapyramidal MF (IIPMF) pathway The IIPMF pathway originates from hippocampal granule cells and terminates primarily upon the basal dendrites of superficial pyramidal cells in CA3b and CA3c

Figure 1.2 Hippocampal MFs pathways of a Wistar rat, distinguished by the Timm’s stain and cresyl violet counterstain Image is from reference Rekart et al., 2007a.

The fibers located above the pyramidal cell layer are called the suprapyramidal MFs, which terminate in a relatively narrow zone (SL in Figure 1.2) located within the proximal apical dendrites of CA3 pyramidal cell in CA3a Granule cell MFs

projections also terminate infrapyramidally in the stratum oriens (SO) and intrapyramidally in the stratum pyramidale (SP) in CA3a

In addition to the pathways above in a lamellar organization, there are descending MFs along the longitudinal axis of the hippocampus The descending pathway travels from the granule cell layer transversely through the SL, synapses on more temporally located CA3 cells (Lorente, 1934; Swanson et al., 1978; Amaral et al., 1981) The maximum distance of the descending MFs reaches as far as 2 mm in the temporal direction (Amaral and Witter, 1989)

The MF axons form three different types of synaptic contacts with the targets in the hilus and CA3 (Henze et al., 2000) Firstly, the characteristic large boutons, which are

up to 4-10 µm in diameter, synapse with the hilar mossy cells and proximal dendrites

of CA3 pyramidal cells These boutons appear as a cluster of large vesicles that containing zinc as one of the neurotransmitters in the MFs Their large size has attracted the attention of physiologists interested in using them for patch-clamp studies on transmitter release (Henze et al., 2000) Besides, the giant boutons together with the associated postsynaptic densities demonstrate the presence of multiple active

synaptic zones In this way, the CA3 pyramidal cells are associated with a number of the active zones associated with the MFs pathway Therefore, the MFs pathway is believed to have the potential to provide a strong excitatory input and trigger the action potential generation in CA3 pyramidal cells

The remaining two types of MFs synaptic contacts are small filopodial extensions emanated from the large MFs boutons They are associated with the GABA (γ-aminobutyric acid)-containing interneurons of the hilus and the CA3 (Acsady et al., 1998) The interneuron-associated boutons are smaller than the giant pyramidal cell associated boutons and also have active zones (Henze et al., 2000) Notably, the average size of the active zones at these synapses is larger than the actives zones observed at other excitatory synapses in CA3, CA1, and cortex (Acsady et al., 1998),suggesting that all synapses made by the MFs pathway are relatively strong

1.1.3 Anatomic plasticity of the mossy fibers

Besides the characterized structural properties summarized above, there is another unusual feature of the MFs pathway It has been revealed through anatomical techniques that the granule cells are continuously undergoing turnover throughout the life of the animal (Altman and Dascal, 1965; Angevine, 1965; Bayer, 1980; Kuhn et al., 1996; Gage, 2002) It was reported by Kaplan and Bell in 1984, that granule cells are being generated from stem cells located in the hilus continuously and the new

born cells are migrating outwards into the granule cell layer Notably, the total number of granule cells apparently does not change due to the increasing age of the animal, which is regulated by genetic factor (Kempermann et al., 1997, 2006) However, there is evidence indicating that the number of granule cells is regulated dynamically by the environmental factors For example, exposure to novel or enriched environments increases the proliferation (Kempermann et al., 1998) and enlarges the MFs boutons (Gogolla et al., 2009)

Strong activities such as epilepsy induced by kainite injection or kindling also affect the MFs pathway with the demonstration of the MFs sprouting (Represa and Ben-Ari, 1992a and 1992b; Wuarin and Dudek, 1996; Van-der-Zee et al., 1995) It has been reported that high-frequency stimulation induced LTP also induces MFs synaptogenesis (Adams et al., 1997; Escobar et al., 1997) Finally, the neurogenesis of the granule cell is decreasing and the appearance of the MFs synapses reduces under chronic stress (McEwen, 1999) All these findings on the neurogenesis of granule cells indicate the remodeling property of the MFs pathway

1.2 Synaptic plasticity of the mossy fibers

Investigations on the MFs synaptic plasticity are carried out through patch-clamp studies on transmitter release (Henze et al., 2000) The release of glutamate and the

neurotransmission at the MFs synapses Studies have showed that the mechanism of long-lasting synaptic plasticity at mossy fibre synapses differs significantly from that

at the Schaffer collateral synapse in CA1 and other typical cortical synapses (Nicoll and Schmitz, 2005)

1.2.1 Short-term plasticity

Short-term plasticity at the MFs synapses onto CA3 pyramidal neurons exhibit higher level in paired-pulse facilitation (PPF) than most other synapses in the central nervous system PPF, a presynaptic form of short-term plasticity, describes the ability

of synapses to increase neurotransmitter release on the second of two closely spaced afferent stimulations and depends on the residual Ca2+ concentrations in the presynaptic terminal (Nicoll and Schmitz, 2005) It has been shown that the MFs PPF

is approximately two times greater in amplitude than the other CA3 commissural/associational synapses (Salin et al., 1996)

Another unique property is the ability of the MFs synapses to undergo frequency facilitation, another form of short-term plasticity, in which increasing the frequency of stimulation from low to moderate (for example, from 0.05 Hz to 1 Hz) rates can cause manifold increases in synaptic strength In contrast to the MFs synapses, CA3 commissural/associational synapses and CA1 Schaffer collateral synapses show little facilitation For example, the MFs synapses showed frequency facilitation at inter-

stimulus intervals (ISIs) as long as 40 s, compared to less than 10 s of the synapses Moreover, the maximal frequency facilitation for the MFs synapses reached up to 600% of the control where as for CA3 commissural/associational synapses it was only 125% (Salin et al., 1996) The ability to induce frequency facilitation at the MFs synapses with low stimulation frequencies (>0.1 Hz) was also shown to be dependent

on calcium/calmodulin dependent kinase II and was also partially occluded by induction of long-term potentiation (LTP) (Salin et al., 1996) Notably, pronounced short-term facilitation is particularly prominent for the large MFs synapses, not in the synapses onto the interneurons (Toth et al., 2000) Therefore, the large MFs synapses onto CA3 pyramidal cells are considered to have a low probability of transmitter release under resting conditions (Jonas et al., 1993; Lawrence et al., 2004)

In addition, there is also a significant difference in the time-course of post-tetanic potentiation (PTP) between the MFs synapse and the Schaffer collateral to CA1 synapse PTP at the Schaffer collateral synapses decays with a time constant of less than 1 min, in contrast, PTP at the MFs synapses decays with a time constant of about

3 min (Langdon et al., 1995) The Ca2+ dynamics in the MFs boutons may be a reasonable factor that mediates the prolonged expression of the MFs PTP (Regehr et al., 1994)

1.2.2 Mechanisms of short-term plasticity

Multiple presynaptic glutamate receptor mediated control mechanisms may contribute to the specific properties of the MFs presynaptic plasticity Generally speaking, synaptically released glutamate can mediate both negative (mGluRs) and positive (KARs) feedback by acting on two distinct subtypes of presynaptic receptors, therefore facilitating the plasticity of the MFs synapses

1.2.2.1 A 1 adenosine receptors

It is reported that inhibition of Gi/o protein’s activity by the specific antagonist enhances transmission at the hippocampal MFs synapse, thereby reducing the short-term plasticity (Moore et al., 2003) Therefore, the MFs synapses onto CA3 pyramidal cells are strongly inhibited by tonic Gi/o protein activity in the presynaptic terminal Based on the fact that the tonic activity of the G protein is a consequence of the tonic activation of A1 adenosine receptors, removal of adenosine tone by A1 receptor antagonists, genetic deletion of A1 receptors or application of adenosine-degrading enzymes is proved to enhance synaptic transmission at this synapse and thereby reduces short-term potentiation (Moore et al., 2003) Adenosine acting on A1receptors therefore could be taken as a possible cause of the low initial release probability at the hippocampal MFs synapse onto CA3 pyramidal cells, which still needs further investigation

1.2.2.2 Metabotropic glutamate receptors

Metabotropic glutamate receptors (mGluRs) are the seven-transmembrane protein-coupled receptors expressed both pre- and postsynaptically mGluRs were discovered as a new type of glutamate receptor due to their unique coupling mechanisms and pharmacological characteristics

MFs express specific subtypes of mGluRs, e.g mGluR2, which are thought to strongly depress neurotransmitter release The type of mGluR expressed on the terminals of the MFs synapses has specificity in the target cell and the species It is generally accepted that mGluRs suppress neurotransmitter release via inhibiting Ca2+channels and Ca2+ entry is inhibited at the mossy fibre synapses, suggesting the role of these receptors in both short-term and long-term plasticity at the mossy fibre synapse (Nicoll and Schmitz, 2005)

The presynaptic mGluR2 also functions as the limitation on the magnitude of frequency facilitation, moreover, inhibiting surrounding synapses This is because they are reported not to be activated during low-frequency stimulation of about 0.05

Hz, but do become activated at higher frequencies, when release is facilitated and glutamate spreads from the release site (Toth et al., 2000; Scanziani et al., 1997)

1.2.2.3 Kainate receptors

Kainate receptors (KARs) are ionotropic glutamate receptors that constitute a separate group from the NMDA (N-methyld- aspartate) receptors (NMDARs) and AMPA (α-amino-3-hydroxy-5-methyl- 4-isoxazole propionic acid) receptors (AMPARs) Besides mGluRs, KARs is also present on both pre- and postsynaptical terminals (Nicoll and Schmitz, 2005).

Low concentrations of kainite at 0.1–1.0 μM could cause an increase in the excitability of the MFs by reducing the threshold for activating the mossy fibre axon (Kamiya et al., 2000; Schmitz et al., 2000), suggesting the pharmacological properties

of KARs Moreover, these concentrations can cause a strong reduction in presynaptic transmission, accompanied by an increase in PPF and a decrease the action potential-evoked Ca2+ transients in presynapse (Kamiya et al., 2000) These may be a result of the ionotropic depolarizing action of KARs (Nicoll and Schmitz, 2005) Furthermore, other studies have been completed indicating that enhanced synaptic transmission at hippocampal MFs synapses is observed with the application of much lower concentrations of kainate at 20–100 nM (Schmitz et al., 2001; Lauri et al., 2001) Repetitive stimulation on the MFs induces synaptically released glutamate, thereby activates KARs Activation of the presynaptic receptors facilitates a long-lasting evoked neurotransmitter release and contributes to frequency facilitation, which is the pronounced characteristic of the MFs synapses

1.2.2.4 Intrinsic conductances

There exist voltage-gated K+, Ca2+ channels and high density of Na+ channels in the MFs boutons (Geiger et al., 2000; Bischofberger et al., 2002; Engel and Jonas, 2005)

In the MFs presynapses, the inactivation of K+ channels dynamically regulates the

Ca2+ inflow during high frequency stimulation, which contributes to the control of synaptic efficacy at the MFs synapse onto CA3 pyramidal neurons Presynaptic Na+channels can amplify the action potential and thereby enhance Ca2+ inflow Therefore, the specific properties of presynaptic K+ and Na+ channels contribute to mossy fibre synaptic transmission and its dynamic regulation (Geiger et al., 2000; Bischofberger

et al., 2002; Engel and Jonas, 2005; Nicoll and Schmitz, 2005)

1.2.3 Long-term plasticity - Long-term potentiation

Long-term potentiation (LTP), a long-lasting enhancement in synaptic transmission resulted from repetitive stimulation, has been most studied at the Schaffer collateral synapse onto CA1 pyramidal cells and at most other synapses in the central nervous system Normally, LTP at these synapses requires the activation of NMDARs with an increase in AMPARs responses postsynaptically However, the mechanism of long-term plasticity at mossy fibre synapses onto CA3 pyramidal cells shows fundamental differences

1.2.3.1 NMDA receptor independent

There is a universal agreement that the induction of mossy fibre LTP is NMDAR independent The NMDAR dependent LTP is defined as the Hebbian LTP Hebbian forms of plasticity describe the associative memory formation, in which simultaneous activity at different synaptic inputs onto a given cell leads to pronounced increases in synaptic strength between those cells In contrast, non-Hebbian forms of LTP would not result in the associations of simultaneously active synaptic inputs A specific repeatedly activated input may induce the non-Hebbian LTP at the MFs synapses (Henze et al., 2000) The low number of NMDARs at the MFs synapses may explain the lack of NMDAR-dependent LTP; alternatively, synapses might lack the machinery downstream of NMDAR activation Elements of AMPAR trafficking found at synapses expressing NMDAR-dependent LTP are absent at the MFs synapses (Nicoll and Schmitz, 2005)

Actually it has been proved that LTP at the MFs synapse onto CA3 pyramidal cells can be either non-Hebbian or Hebbian, depending on the specific pattern of high-frequency stimulation (Urban and Barrionuevo, 1996) Interestingly, long lasting high-frequency stimulation (HFS) (three 1-s-duration 100-Hz trains presented at 0.1

Hz, L-HFS) induced non-Hebbian LTP, while, a brief tetanus (eight 0.1-s 100-Hz trains presented at 0.2 Hz, B-HFS) induces Hebbian form of LTP that requires

postsynaptic calcium influx and depolarization of the postsynaptic CA3 cell as well as the activity of the MFs

1.2.3.2 Postsynaptic calcium elevation

Both the L-HFS induced non-Hebbian LTP and B-HFS induced Hebbian LTP require an elevation in postsynaptic Ca2+ Different sources of postsynaptic Ca2+ are involved in two forms of LTP at the MFs synapses Voltage-dependent Ca2+ channels are activated during the B-HFS induced depolarization, thereby postsynaptic calcium

is elevated by influx via L-type calcium channels Alternately, Ca2+ could either be released from internal stores after mGluR activation or enter through Ca2+-permeable non-NMDARs, resulting in Ca2+ elevation that take place in the absence of postsynaptic depolarization (Henze et al., 2000; Nicoll and Schmitz, 2005)

1.2.3.3 Cyclic AMP

Cyclic adenosine monophosphate (cAMP)-dependent signaling cascades are confirmed to be involved in the induction of the MFs LTP through both pharmacological and genetic analysis (Huang et al., 1994; Weisskopf et al., 1994) Presynaptic calcium influx by the L-HFS triggers a cAMP cascade leading to neurotransmitter release In addition, the increased postsynaptic calcium also activates

a postsynaptic cAMP cascade, which then leads to the generation of a retrograde messenger In turn, the retrograde messenger activates a presynaptic cAMP cascade

1.2.3.4 Downstream targets of PKA and PKC

It is widely accepted that the expression of the MFs LTP is due to increasing release

of presynaptic glutamate, and requires the activation of PKA as well as the downstream targets of PKC and PKA Several substrates of PKA have been investigated for their possible role in the MFs LTP expression For example, one of the PKC substrate GAP-43 shows increased phosphorylation during the MFs LTP, suggesting the role in release process (Oestreicher et al., 1997) Similarly, the synaptic protein rabphilin and Rab3A are required for the MFs LTP expression (Lonart et al., 1998a and 1998b)

In summary, the biochemical mechanisms responsible for induction and expression

of presynaptic MFs LTP are mediated by the elevation of intracellular Ca2+concentration Presynaptic Ca2+ influx activates a calcium/calmodulin-regulated adenylyl cyclise, which increases cAMP levels and activates PKA, thereby the downstream substates However, how the cAMP cascades work remains a mystery

1.2.4 Long-term depression

Long-term depression (LTD) is another form of long-term plasticity, found in many excitatory synapses in the central nervous system Low-frequency stimulation (e.g 1

Hz for 15 min) induces LTD at the MFs synapses, with the reduction in neurotransmitter release Activation of presynaptic mGluR2 is involved, and associated with a rise in presynaptic Ca2+, leads to a decrease in adenylyl cyclise activity followed by a decrease in PKA activity (Tzounopoulo et al., 1998) So, the MFs LTD appears to be a reversal of the presynaptic process involved in the MFs LTP

1.3 Mossy fibers in learning and memory

1.3.1 Role of mossy fibers plasticity in learning and memory

Currently, most studies have been focused on the postsynaptic plasticity (Kasai et al., 2003) It has been reviewed that the postsynaptical dendritic spines are considered as the ideal substrates for information storage due to their motility in response to neural activity (Sarra and Harris, 2000) Enhanced dendritic spine density has been observed

as the results of LTP induced in CA1 pyramidal cells in hippocampal slice (Engert and Bonhoeffer, 1999), also as the results of spatial learning training in a water maze (Moser et al., 1994)

However, in contrast to the large examples of postsynaptic structural plasticity studies, there is a scarcity of examples of learning-related presynaptic structural plasticity Based on its physiological characteristics summarised above, the MFs shows strong potential as an important presynaptic model for the investigations on the learning-dependent morphological malleability, as well as the relations of synaptic plasticity with long-lasting memory

The correlations between the basal size of the IIPMF pathway and various forms of hippocampus-dependent learning were initially verified by Dr Lipp’s group (Lipp et al., 1988; Schwegler and Crusio, 1995) Since then, the theory that increased distribution of IIPMFs is associated with superior spatial learning has been established Morris water maze and radial arm maze are among the popular behavioural training tasks to test the hippocampal-dependent spatial learning and memory (Morris, 1981; Olton and Samuelson, 1976) Using the navigation water maze, the length of rat hippocampal IIPMF is observed and correlated with the performance in the training (Schopke, et al., 1991) In addition, there exist the differences in the distribution pattern of IIPMF pathways among various strains or species of rats and mice (Schopke, et al., 1991; Prior et al., 1997; Holahan et al., 2006; Rekart et al., 2007a) Pharmacological inhibition of the MFs terminals in CA3 during the acquisition phase

of the water maze tasks impairs the spatial memory storage as shown by failed recall

of the platform location in a hidden platform water maze task but not in the nonspatial water maze task Moreover, inactivation of the MFs synapses does not affect

acquisition of the task (Lassalle et al., 2000; Florian and Roullet, 2004; Stupien et al., 2003)

Non-pathological plasticity of the MFs pathways has been observed in water maze overtrained animals (Ramirez-Amaya et al., 1999, 2001; Holahan et al 2006, 2007; Rekart et al., 2007b) The overtraining in a hidden platform water maze task induces growth of rat’s hippocampal granule cell MFs terminal fields, 7 days after the last day

of training, from the SL of CA3 into the SO and SP using Timm’s staining In contrast,

no changes in the area of the MFs terminals were observed in animals that underwent

a cued visible platform and non-overtraining task Furthermore, the growth is of independence with any stress-related responses as confirmed in yoked control animals that just allowed swimming in the water maze with no platform present Taken together, the learning-induced presynaptic plasticity could be involved in the mechanisms underlying the hippocampal-recruited long-term memory

1.3.2 Species and strains difference in mossy fibers

As mentioned there exist differences in the distribution pattern of IIPMF pathways among various strains or species of rats and mice (Schopke, et al., 1991; Prior et al., 1997; Holahan et al., 2006; Rekart et al., 2007a), there are also clear distinctions in the learning-induced axonal remodelling of the hippocampal MFs system between rat

Wistar rat is the pioneer animals model to exam the MFs sprouting in CA3 area induced by overtraining in hidden platform water maze task (Ramirez-Amaya et al.,

1999, 2001; Holahan et al 2006) There is an easily recognized MFs sprouting from the SL to the SO in CA3 region in Wistar rats 7 days after being well trained In contrast, another strain, Long Evans (LE) rats, was used and showed faster acquisition

in the hidden platform water maze tasks than Wistar rats (Holahan et al., 2006) The sprouting of granule cell MFs terminals in LE rats takes place as soon as 2 days after the last day of overtraining process, suggesting that the rapidity of learning and recalled location of a hidden platform is reflected in the equally rapid expansion of the MFs to SO

Distinctions on the MFs morphology have been also found in mice In contrast to Wistar rats, mice do not demonstrate learning-induced MFs sprouting into SO in CA3 after water maze training (Rekart et al., 2007a) What is more, even no kainate induced MFs sprouting and no induction of GAP-43 mRNA in granule cells after these kainate-induced seizures are found in mice of three different strains (Routtenberg, 2010) These definite differences in behaviour and anatomy may due to the evolutionary divergence of two components of the MFs system in rat and mouse some 20 million years ago, suggesting the opportunity to achieve a more fundamental understanding of the evolutionary biology of memory even within the rodent order

1.4 Significance of studies

As the classic presynapse model, the system plays a critical role in the related learning and long-term memory procedures Investigations on the learning-induced presynaptic plasticity might light the shadows on the complicated mechanisms underlying the hippocampal-recruited long-term memory

hippocampal-1.4.1 Missing gaps

Although accumulating studies have been done on the non-pathological plasticity of

the MFs pathways induced by hippocampal-dependent behavioural learning tasks, histological staining techniques such as Timm’s staining and immunohistochemistic staining are the main methods used for detecting sprouting of the MFs terminals in brain sections As described above in 1.3, sprouting of the MFs in CA3 area is a time-

dependent process, thus, possible in vivo detection methods would be more valuable

for visualizing the dynamic remodelling of the MFs, meanwhile, providing the three dimension images on how the MFs pathway expand during information storage Magnetic resonance imaging (MRI) has been widely used to visualize the anatomical and functional characteristics of the brain in both animal and clinical studies (Budinger et al 1999; Farrall 2006) Recently, manganese-enhanced magnetic resonance imaging (MEMRI), at high spatial resolution, is being increasingly

employed for detection of the MFs plasticity induced by epilepsy in vivo (Nairismagi

et al., 2006; Immonen et al, 2008; Kuo et al., 2008) However, so far there are no

records on MEMRI in detecting learning-induced MFs plasticity Whether MEMRI can be competent for this job successfully needs to be tested

As mentioned that learning-induced expansion of the MFs terminals is dependent in rats, another rat strain e.g Long-Evens (LE) rats were used in comparison to Wistar rats that are well studied to represent the model with ability of growth of the MFs terminals in CA3 Enriched information on strain-different MFs systems may afford an opportunity for uncovering linkages between evolutionarily significant alterations in hippocampal circuitry in relation to learning and memory formation requirements

Furthermore, the molecular mechanisms underlying the MFs redistribution during learning and memory formation are still under investigation As introduced previously

in 1.2, enhancement of neurotransmitter release is always coupled with activation of the MFs In addition, the MFs terminals contain a cluster of large vesicles that contain

Zn2+, which is co-released with glutamate from the MFs terminals in the same manner

as neurotransmitters (Li et al., 2001; Huang et al., 2008) Therefore, the role of Zn2+ in the MFs is rising up in the investigations on the Zn-associated neurological dysfunctions and diseases Localization of Zn2+ and observation of cellular Zn2+ levels have been done using techniques such as histochemical staining (e.g Timm’s staining) methods, fluorescence labelling methods and stable-isotope dilution However, all of

these methods fail to provide accurate elemental concentrations in the required samples Nuclear microscopy, carried out using a focused 2-MeV proton beam (Watt

et al., 1994), has the ability to image the morphology of tissues and map the trace elements such as Fe, Ca, Zn and Cu in brain tissues as well as other tissues (Ong et al., 1999; Ren et al., 2003 and 2006; Rajendran et al., 2005 and 2009; Watt et al., 2006) Whether quantitative analysis of Zn in rat hippocampal MFs in the CA3 area could be done by nuclear microscopy is not yet any clear evidence

1.4.2 Objectives

There are three main objectives in the studies in Part I of this thesis:

1 To detect the in vivo progressing of the MFs remodelling during the spatial

memory formation using the MEMRI approach

2 In addition, the issue whether the remodelling of the MFs system is strain dependent was examined using two strains of rats: Wistar and Lister-Hooded (LH) rats

For objective 1 and 2, two strains of rats were employed for 5 days overtraining in a hidden platform water maze task and examined by MEMRI afterwards Systemic administration of MnCl2 intravenously was applied to label the MFs pathway In parallel, Timm’s stain was used for histological verification of sprouting of MFs

3 In order to accurately identify and quantify Zn in the brain, especially in the hippocampus, trace elemental analysis of Zn in the synapses from the MFs to CA3 was carried out without recourse to conventional staining, using nuclear microscopy based imaging techniques Timm’s staining onto serial tissue sections was conducted to provide the histological references for the hippocampus structures and Zn distribution in the MFs In this work we use nuclear microscopy to provide structural and elemental images of the hippocampus, and use these images to measure the concentrations of total Zn

in the SL, the layer containing the hippocampal MFs in CA3 region, and the two adjacent two layers: the SP and SO

Chapter 2 Materials and methods

2.1 Animal model

Rodents including rats and mice were used in the studies of the thesis Three species

of rats, i.e Wistar, LH and Sprague-Dawley (SD) were used in the studies in part I of the thesis All were adult male, aged between 6-8 weeks They were housed in pairs (Animal Hall, NUS) or groups of five (GlaxoSmithKline Research and Development China, Singapore Research Centre in Biological Resource Center Department One of A-Star, Singapore) per cage with free access to food and water ad libitum Animals were maintained with an average temperature of 22℃ and under an inverted 12 hr light/dark cycle (7:00 A.M to 7:00 P.M.) All the experiments using living animals were carried out in the light phase of a 12 hr light dark cycle All protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore

2.2 Neuroanatomical methods

The intricacy of the brain that is responsible for behavior is an emergent characteristic of the diverse neuronal cell types that make up the neural systems, the specificity of connections between these neurons, the neurochemical interactions that

occur through these connections, and the functional responses of neurons that result from such interactions Neuroanatomical procedures provide for the visualization of the structural organization of neural systems Most neuroanatomical procedures involve the following steps: (1) brain preparation, (2) sectioning the brain, (3) labeling and staining the brain sections, (4) microscopic analysis

All protocols using live animals and tissue collections were approved by the Institutional Animal Care and Use Committee (IACUC) of National University of Singapore

2.2.1 Brain tissue preparation

2.2.1.1 Preparation of unfixed fresh-frozen brain tissue

2.2.1.1.1 Materials

Isopentane, dry ice, and anesthetic are prepared before the procedure The dissection instruments include scissors, spatula and forceps In addition, a metal sieve-like basket and a metal container that is large enough to hold it are needed

2.2.1.1.2 Protocol

1) Place 300-500ml isopentane in the metal container Place the metal container with the isopentane in dry ice for 15 to 30 min, until the temperature of the isopentane reaches -70°C Isopentane may be reused, so it can be rebottled after the procedure is completed

2) Animals are scarified with an overdose of anesthesia

3) Remove the brain from the skull and place the brain on the mesh bottom of the sieve-like basket in a manner that preserves the normal shape of the brain 4) Immerse the brain in the cooled isopentane for 20-30 sec The time of immersion is absolutely critical; it must be long enough to result in complete freezing of the brain, but not so long that the brain cracks

5) Rapidly remove the frozen brain in the isopentane, detach it from the mesh, and place it briefly on absorbent paper to remove excess isopentane

6) Wrap the dried, frozen brain in foil and store at -20°C to -70°C until sectioning is performed Fresh-frozen brains can stored at -70°C for months and even years prior to being sectioned

2.2.1.2 Perfusion fixation

2.2.1.2.1 Materials

Saline (0.9% w/v NaCl) and sucrose-infiltration solution (30% sucrose in 0.1M PBS,

pH 7.4) are prepared and stored at 4°C; fixative solution (4% paraformaldehyde in 0.1M PBS, pH 7.4) for perfusion is made at room temperature Surgical instruments

include scalpel, scissors, clamps, hegenbarth clip-applying forceps, hemostats and bone rongeur Peristaltic perfusion pump with variable-speed standard drives is also needed

2.2.1.2.2 Protocol

1) Place saline (kept on ice to maintain cool) and fixative solution in separate flasks and set up the peristaltic pump in such a manner that the saline is first drawn through the pump into tubing that is to be connected to the animal Fixative solution is set to be drawn through the tubing at a later point Fill the system with saline and make sure that air is removed completely during the switch between the two fluids The end of tubing primed with saline is attached to a blunt 15-G hypodermic needle that will be used for perfusion 2) Prepare animal for infusion by administering a lethal dose of anesthesia Monitor it until the point when the animal fails to respond to pinching of the foot

3) Make an incision through the abdomen and ribs to expose the heart Clamp the sternum with a hemostat and fold the cut rib flap headward This procedure is most successful if the heart is still beating at the outset of perfusion

4) Make a small incision of the left ventricle and quickly insert a blunt 13-G hypodermic needle upward through the ventricle past the aortic valve so that it may be visualized about 5mm inside the ascending aorta Clamp the needle in place with a Hegenbarth clip-applying forceps or hemostat across the ventricle

5) Begin perfusion of saline slowly; meanwhile, cut the right atrium to allow an escape route for the blood and perfusion fluid Perfuse saline at a moderate to rapid rate (∼40 ml/min) until the effluent runs clear, which may require 200

to 500 ml of solution

6) After the effluent runs clear, stop the pump and introduce fixative into the peristaltic pump line running into the animal Perfuse fixative at a moderate to slow rate (∼20 ml/min) such that∼500 ml of fixative is perfused over 10 to 20 min

7) Stop perfusion until the whole body is stiff; remove the brain from the skull using a bone rongeur

8) Transfer the brain into a vessel containing 4°C sucrose-infiltration solution Incubate 24 to 48 hr at 4°C, until the brain sinks into the bottom of sucrose solution, indicating that the brain is ready to be sectioned

Certain procedures may be adjusted depends on the different histochemistry studies used in each project

2.2.2 Cryostat sectioning of frozen brain tissue

A cryostat is a microtome housed in a freezing chamber that allows the sectioning process to be performed at a temperature of -20° to -30°C A cryostat is required for sectioning fresh-frozen brains in order to maintain in a frozen state before they are

mounted onto a microscope slide Cryostat sectioning may also be used for fixed brains Sections are transferred to subbed microscope slides directly from the knife

perfusion-2.2.2.1 Materials

Embedding matrix, cryostat microtome, specimen holder for supporting specimen during sectioning are needed for cryostat section Poly-L-lysine coated slides at room temperature are used for collecting brain slices

2.2.2.2 Protocol

1) Chill the specimen holder using liquid nitrogen and apply embedding matrix

on the surface of specimen holder As the embedding matrix begins to freeze, place the frozen brain, base-down, into it so that the brain adheres to the specimen holder

2) Pour embedding matrix over the frozen brain to provide a thin coat that helps maintain the integrity of the brain sections during cutting Place the brain, mounted on the specimen holder, into the cryostat microtome for 10-15 min before sectioning the brain The temperature used for cutting brain slices is -16° to -20°C Thickness of brain slices can be adjusted depending on the further analysis

3) Collect sections from the specimen holder onto poly-L-lysine coated slides, and store at−20° to−80°C for further processing

2.2.3 Histological study

Histological stains have become invaluable tools for visualizing or differentially identifying anatomic structures of cells and tissues, with the examination under the microscope There are hundreds of various techniques that have been used to selectively stain cells and cellular components (Ross & Pawlina, 2006) Generally speaking, the Nissl method and Golgi's method are commonly useful in identifying neurons A counterstain may be employed in the aim of detecting immunohistologically unlabled cells or neuroanatomical landmarks Nissl stains, such

as crestyl violet, are commonly used for this purpose

2.2.3.1 Nissl stain

1) Prepare the 0.1% mg/ml cresyl violet solution

2) Dehydrate the tissues by immersing the tissue-containing slides for 30 sec in

H2O, then successively for 3 min each in ethanol at 70%, 95%, and 100% 3) Immerse slides 3 times in xylene, each tome for 5 min, then rehydrate the tissue by immersing slides successively for 3 min each in ethanol at 100%, 95%, then 70%, and finally in water

4) Immerse slides for 10 to 20 min in the cresyl violet solution, adjust the staining duration depending on the staining density of neurons

5) Rinse slides in distilled water and determine if a darker stain is desired by observing under a light microscope If the staining is acceptable, immerse

slides in 70% ethanol for several minutes until the desired level of differentiation of the stain is achieved

6) Immerse slides once for 2 min in 90% ethanol, then twice for 2 min each tome in 100% ethanol Immerse slides 3 times in xylene, each time for 3 min 7) Mount tissue with mounting medium and a coverslip Avoid the air bubbles

in between Leave the slides dry overnight at room temperature before observation under the light microscope

2.2.3.2 Timm’s stain

Timm’s sulfide/sliver staining technique is used to detect heavy metal i.e copper, ion and zinc etc in tissues It is a silver technique where heavy metal sulfide is converted to silver sulfide with the result that the heavy metal deposits are stained black It has been proved that this Timm’s sulfide/silver staining technique has the ability to detect free Zn in the mammalian brain, which is highly concentrated in the hippocampal MFs terminals (Danscher, 1981, 1996; Danscher et al., 1985)

2.2.3.2.1 Solution preparation

1) Sodium Sulfide:

Develop this solution in the hood Dissolve 400 mg sodium sulfide in 20 ml of 0.5

M PBS (pH 7.4) and bring solution to 100 ml with de-ionized water (diH2O) This solution is hygroscopic and is oxidized by the air, so it must be prepared fresh

2) 3% Glutaraldehyde:

Dissolve 7.5 ml of 50% glutaraldehyde in 75 ml of 4% paraformaldehyde and bring

to 300 ml with diH2O

3) Gum Arabic solution:

Dissolve 250 g gum Arabic powders in 500 ml of diH2O in a 37°C oven for at least

12 hours Filter the solution through cheesecloth and place in 120 ml aliquots to be frozen When removed from the freezer, the solution can be thawed and kept for one week in the refrigerator

4) 5% Hydroquinone solution:

Develop this solution in the hood Dissolve 3 g of hydroquinone in 40 ml of diH2O

by heating the water and bring final solution to 60 ml with diH2O This solution must

be used within a few hours of preparation

5) Citric Acid solution:

Dissolve 5.1 g of citric acid (anhydrate) in 10 ml of diH2O by heating the water slightly Once dissolved, add 4.7 g of sodium citrate This buffer may be kept at

room temperature for some days and is warmed partly before use to re-dissolve crystals

6) 7% Silver nitrate solution:

Dissolve 0.085 g of silver nitrate in diH2O until a total solution of 0.5 ml is obtained This solution may be kept for several days if refrigerated and protected from light It should be discarded if black grains of reduced silver are visible

7) Timm’s stain solution

The Timm’s stain solution is composed of 120 ml Arabic gum, 60ml 5% hydroquinone, 20ml citric acid and 500 l silver nitrate

The mixture of gum Arabic, hydroquinone and citric acid may be kept for several hours but the silver nitrate should only be added to the other three immediately before development The developer decays more quickly when illuminated but need not be protected from dimmed light during the 1-2 minutes it takes to stir in the silver nitrate and pour the solution into the staining jars Nor is the staining pattern qualitatively affected by occasional short inspections of sections during the staining During the staining, the developer decays spontaneously and becomes grayish-brown The speed of decay varies even if the developer is carefully prepared

2) Place brains in 30% sucrose-infiltration solution until they sink

3) Section the brain and mount sections on poly-L-lysine coated slides

4) Develop sections in fresh made Timm’s stain solution at 30°C for 90 minutes

in absolutely darkness

5) Rinse in diH2O for 10 minutes

6) Counterstain with cresyl violet

7) Clear and mount the slides for observation under light microscope

2.2.3.3 Immunohistochemistry

Immunohistological methods are used for visualization of proteins and neurochemicals in nervous system

Before immunostaining, brain tissues must be first fixed, infiltrated and sectioned

as described in 2.2.1 and 2.2.2 Fixation preserves the antigen in its native site and preserves tissue morphology Moreover, proper fixation allows the tissue to be sectioned more easily The 4% paraformaldehyde solution would adequately fix the tissue for the level of resolution provided by light microscope Infiltration using 30% sucrose can cryoprotect the tissue during sectioning procedure afterwards

Immunohistochemistry process includes blocking nonspecific sites, incubating tissue with primary and secondary antibodies in sequence, and forming tertiary complex that is to be visualized

2.2.3.3.1 Protocol for avidin-biotin complex (ABC) method

1) Rinse the tissue mounted onto the slides three times, each time by incubating

10 min in Tris-buffered saline (TBS) at 4°C The rinses will remove excess fixative or cryoprotectant

2) Incubate tissues 30 min in 4% normal animal serum (NAS)/avidin blocking buffer at 4°C

3) Rinse the sections three times in TBS as described in step 1

4) Dilute primary antibody against the antigen of interest to the desired concentration in antibody diluents Add the primary antibody solution onto the tissue sections and incubate overnight (~16 hr) at 4°C

5) On the second day, rinse the tissue sections three times in TBS as described in step 1

6) Prepare the biotinylated secondary antibody diluents; incubate the tissues with

it for 1 hr at 4°C

7) Rinse the tissue three times in TBS as described in step 1

8) Prepare the avidin-biotinylated horseradish peroxidase complex by mixing 1 drop each of reagents A and B per 10 ml TBS Incubates the tissue with the complex for 1 hr at room temperature

9) Rinse the tissue three times in TBS as described in step 1

10) Counterstain the tissue if necessary

11) Clear and mount the slides for observation under light microscope

2.3 Behavioral training tasks – Morris water maze

The Morris Water Maze, developed by Dr Richard G Morris in 1981, is one of the most popular behavioral procedures extensively used in behavioral neuroscience to study spatial learning and memory (Morris, 1981) Performance in the Morris water maze is acutely sensitive to manipulations of the hippocampus

In the typical paradigm, a subject is placed into a circular pool of warm (22oC, room temperature), opaque (by adding nontoxic whiter paint) water in a random start

Visual cues, such as colored shapes, are placed around the pool in plain sight of the animal During training trials, the subject swims around the pool in exploration of an exit, meanwhile various parameters are recorded, including the time spent in each quadrant of the pool, latency to find the platform location, and total distance traveled After several trials, a capable subject can swim directly from any release point to the platform, reflecting less latency comparing to the first trial This improvement in performance is because the subject has learned where the hidden platform is located relative to the conspicuous visual cues During probe trials, the platform is removed, and the percentage of time spent in the quadrant that normally contains the platform is compared to the time spent in other quadrants