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

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 shortterm 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
cortex to the dentate gyrus, conveying the polymodal sensory information, make up one of
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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).

2


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.

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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 presynapses 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.

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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.

5


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.

6


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
7


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 (γaminobutyric 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
8

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
activation of postsynaptic glutamate receptors are involved in the excitatory
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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 inter10


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
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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 A1 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 shortterm 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 A1
receptors 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
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Metabotropic glutamate receptors (mGluRs) are the seven-transmembrane Gprotein-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

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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 potentialevoked 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.

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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 longterm plasticity at mossy fibre synapses onto CA3 pyramidal cells shows fundamental
differences.

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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 highfrequency 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

16


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

17


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

18

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).

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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
20


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
and mouse species and strains.
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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.

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1.4 Significance of studies

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

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 timedependent 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
23

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 straindependent 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
24


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.

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