Metabotropic receptors

and synaptic plasticity in

the schaffar collateral
pathway in Hippocampus
Background

Transfer of electric current (In the form of ions) from one neuron cell (pre synaptic cell) to the
next (post synaptic cell) can take place by two broad mechanisms. Electric transmission can either
be transferred by electrical or chemical synaptic transmission.

In electrical transmission the current is able to flow directly from the presynaptic cell to the
post synaptic cell via open gap-junction channels that connects the two neurons.
Depolarization of the membrane potential in the synaptic region of the presynaptic cell opens
the gap-junction channels that connect the pre- and post synaptic cells. Electric current in the
form of ions can then flow into the postsynaptic cell and affect the membrane potential
directly and contribute to opening of voltage gated channels that again can trigger a new
action potential.

In chemical synapses there is no such direct transmission of current flow. Transmission of the
electric signal over chemical synapses is mediated through several steps. This process makes
the transmission of electric signal become more time consuming compared to the usage of gap
junction channels in direct electric transmission (1).

The usage of electric and chemical synaptic transmission in a neural network is adapted to
physiological processes in the organism which the neural connection in question is
responsible for. For example, electric transmission is usually involved in physiological
processes that are very dependent on signalling speed. For example, fast reflexes in muscle
groups that are responsible for fast flight responses from dangerous situations in animals. On
the other side, such fast electric transmission signals lose complexity in their signal
processing compared to chemical transmission.

Chemical synaptic transmission is a multistep process that are triggered by the influx of Ca2+
ions as a response to the incoming electric current down the neuron axon to the synapse
terminal. Voltage gated Ca2+ channels are located as parallel rows in proximity to the active
zone in the synaptic terminal area (figure1). Vesicle storing neurotransmitters are located in
the same area in the presynaptic terminal. On influx of Ca2+ the ions trigger the discharge of
neurotransmitters that are stored inside the vesicles by the means of exocytosis into the
synaptic cleft. Ca2+ ions are believed to discharge quantitative pockets of transmitter’s
depending on the concentration of available Ca2+. A specific number of Ca2+ ions are

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needed to trigger the release of each individual vesicle, thus the concentration of available
Ca2+ ions will decide the extent of available vesicles that will go through exocytosis.

Diffusion of the released neurotransmitters into the synaptic cleft binds to postsynaptic
neurotransmitter receptors which usually are ion channels that control the influx of synaptic
ion content into the post synaptic cell. This will usually result in a membrane potential
depression that will directly, or indirectly, trigger an action potential that will open voltage
gated ion channels in the same area for further voltage depression. This will result in a new
flow of ionic current that spread down the new cell against the synaptic terminals of the cell.
This multistep chemical transmission process can involve different types of membrane
receptors and regulatory pathways that make complex processing of the initial pre synaptic
incoming signal possible (2). A consequence is that chemical transmission processes are set to
control complex behaviours such as learning and memory processes in neural networks.

In general, receptors that control ion permeability can be of two types, either ionotropic or
metabotropic. Ionotropic receptors respond to signal molecule binding (such as a
neurotransmitter) by a conformational change within the same protein as the transmitter binds.
This conformational change leads to channel opening which cause the biochemical change on
the cytoplasmic side of the cell the receptors are set to regulate.

Metabotropic receptors respond in an indirect manner. That is, the connection between the
binding events to give the biochemical response on the cytoplasmic side of the cell they are
set to regulate goes through several regulated steps. Metabotropic receptors merely trigger a
molecular signalling event that spread from the extracellular binding domain to the
cytoplasmic side. Then this signal can diffuse over long or short distances to affect
intracellular target molecules (for example ion channels) to cause a biochemical cytoplasmic
change. Multiple signalling pathways exists that can reach different downstream targets.
Downstream targets can be many different kinds of cellular components. For example
inactivated transcription factors can be the downstream target of one type of signalling
pathway (for example the PKA-MAPK-CREB pathway) which can lead to a change in the
cells gene expression profile. This pathway can lead to long term changes in the cell working
for days. Other pathways can simply have ion channels as downstream targets which can last
for milliseconds.

The ionotropic receptor type are only able to open channels as a response, metabotropic
receptors can open as well as close. They can also adjust the permeability of ion channels

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without either close or open them. Therefore metabotropic receptors are often referred to as
achieving modulatory synaptic actions, rather than causing an on/off response. Metabotropic
receptors are divided into two families based on the types of transmembrane protein families
they represent (G-protein coupled receptors and the Receptor Tyrosine Kinase) (3).

Figure 1. Synaptic transmission can be mediated by two major mechanisms. Figure on left
show direct electric transmission via the opening of gap junction channels. Figure on right
show a chemical transmission mechanism that involves several stages. Neurotransmitters that
are stored in vesicles are released into the synaptic cleft by exocytosis which binds to
postsynaptic neurotransmitter receptors and open them for ion influx (4).

Usually, diffusible molecules (called Secondary messengers) triggers a cascade of signalling

events that eventually ends up modifying the downstream target. Secondary messengers can
be of various types but are usually categorized as being of gaseous or non-gaseous origins.
Gaseous types are typically Nitric oxide (NO) or carbon monooxide (CO). The most common
non-gaseous types are either Ca2+ or cyclic adenosine monophosphate (cAMP) (3). The
signalling cascades that secondary messengers activate are carried out by protein kinases
which phosphorylate other kinases as well as other molecular substrates within the cell. These
protein kinases transfer one or several high energy phosphate ions to their substrates. This will
affect the energy level within the substrate molecule and cause conformational changes that

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will change their properties. Usually such a phosphate energy transfer typically functions as
an on/off binary switch to trigger or shut down the specific function the substrate in question
has in the cell (5). The main signalling pathway types typically use different secondary
messengers.

Memory can be defined as storage of experience-based learning that is important for the
survival of the organism. Generation of memory in mammals is believed to be mediated by
the modification of the strength in synaptic connections between neuronal cells within the
nervous system. However, not all neurones in the nervous system are capable of modify their
synaptic strengths to store memory. There are also different types of memory types, and
molecular mechanisms to how they are generated.

There are memory types that are of voluntary nature (explicit memory) and memories that are
automatically generated (implicit memory). A classical example of implicit memory is muscle
withdrawal reflexes that are developed to avoid dangerous experiences. Painful experiences
can strengthen synaptic connections between sensory, inter- and motor neuron networks in the
nervous system that give automated withdrawal reflexes in response to painful sensations.
Such learning will save time to the animal since an already pre-programmed locally reflex
circuit can make a motor decision without involving the conscious mind.

Enhancement in such reflex responses after experiences is refereed to as Sensitization.

However, there are also possible to “learn how to forget” a non-important sensation.
Habituation is the opposite of Sensitization in that an experience that is learned not to be
meaningful to the organism will be seen as neutral and therefore not be learned. This process
can develop after numerous exposures to the stimuli, which again and again will lead to
neither negative nor positive consequences to the organism.

A learning process can either be storage as a short term memory or a long term memory. A
long term memory must first be stored as a short term memory before conversion to long term
memory. This process is known as consolidation. The best studied brain region in vertebrates
that are occupied with learning and memory is the hippocampus. Storage of short and long
term memories in hippocampus are referred to as short term depression (LTD) and long term
potentiation (LTP). One type of synapse connection in hippocampus between CA3 and CA1
pyramidal cells called the Schaffer collateral fibres is in particular well studied.

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LTP can be induced by bursts of electrical signals between connected neuronal cells, or burst
of spike activity in the presynaptic cell. Generation of LTP is based on three principal
properties; cooperativity, associativity and input specificity. These properties are usually
simplified from a principal rule (the Hebbian rule) to be expressed into one sentence “Cells
that fire together, wire together”. Usually the Hippocampus creates a complex memory of a
situation that is based on many different incoming sensation inputs from different brain
regions. This gives the Hebbian rule an important aspect in LTP in that these different
sensation inputs into the hippocampus must share a time window to be able to be associated
together (6).

The molecular generation of LTP is known to be heavily based on Ca2+ ion levels both in the
presynaptic terminals and postsynaptic receiving compartments. However, in hippocampus
Ca2+ role in LTP is believed to have its functional role exclusively in the postsynaptic cell.
Ca2+ influx is believed to enter the postsynaptic cell via three pathways. These are input
through ionotropic glutamate receptors (GluRs) in particular N-methyl-D-aspartate receptors
(NMDARs), voltage gated channels (VGCCs) and Ca2+ release from intracellular stores.
NMDARs are in particular important because they need a certain input intensity to be opened,
because most of these channels are blocked by an Mg2+ ion that need sufficient
depolarization to be removed.

A NMDAR isoform called AMPAR is not voltage dependent by Mg2+ blockage and open
directly on glutamate binding. Thus, whenever glutamate is released into the synaptic cleft the
AMPAR channels will respond but NMDARs is dependent on signal intensity.

High intensity signals (or burst of spike activity) will increase the post synaptic Ca2+
concentration significantly. This increased concentration triggers Ca2+ dependent enzymes in
which Calmoduline-dependent kinase II plays a major role in LTP induction. This kinase can
trigger signalling pathways that result in cellular changes that can last for days or even weeks.
This is achieved by activation of intracellular signalling pathways that activate expression of
gene profile that codes for proteins important in synapse formation and strengthening. The
best known transcription factor involved in LTP is CREB. CREB is a transcription factor that
mediates the expression of a variety of genes in the cell and can be activated by
phosphorylation by protein kinases. CREB is involved in a great variety of biological
processes other than long term storage processes in the hippocampal region.

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Two important events caused by CREB in synapse strengthening are;

1) Expression of genes that codes for protein that are involved in the physical building of
synapse connection and 2) expression of ubiquitin mediated proteases that specifically
degrade regulatory subunits of PKA (a protein kinase). By removing the inhibiting regulatory
subunits a PKA will not be inactivated to the same degree and are then said to be autonomous.
As a result PKA’s will continue to phosphorylate molecular components in the postsynaptic
region that are important for the strengthening of synaptic connection.

These trials have showed that inhabitation of protein synthesis, CREB or PKA all have
affected long term memory function without affecting short term memories (6).

Result

Metabotropic receptors are involved in all kind of learning and memory processes and are
conserved throughout the animal kingdom.

It has been shown that metabotropic glutamate receptors (mGluRs) can cause long term
depression (LTD) in rat brains. More specifically in the CA1 region of the Hippocampus.
Memory storage in this region is mediated by consolidation based modification of synaptic
strengths between afferent presynaptic cells from CA3 to postsynaptic pyramidal cells in CA1
(figure 2), which is referred to as the collateral pathway.

mGluRs working in the collateral pathway are believed to cause depression of excitatory
transmission in the synaptic cleft between CA3 and CA1 pyramidal cells. This depression is
caused by mGluRs reduction of CA2+ in scaffer collateral presynaptic region. Ca2+ is known
to trigger release of neurotransmitter from vesicles located at the active zones into the
synaptic cleft by exocytosis (Figure 1). In addition they are found to cause LTD that is
maintained after the effect of mGluRs on Ca2+ levels in the presynaptic region. Thus this
repression is believed to be caused by downstream effects of the acute reduction in Ca2+
levels.

By simultaneous measuring of Ca2+ levels in CA3 cells and recording of membrane

potentials in the CA1 postsynaptic cells after electrical stimulation of CA3 cells with
electrodes both effects could be measured. To trigger activation of the mGluRs, mGluRs
agonist DHPG was applied.

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As seen in figure 2 B washout of the agonist only partly recover the membrane potential. This
is seen as a LTD effect that was maintained for the entire observation which lasted up to one
hour.

It is reason to believe that the acute effect in Ca2+ levels caused by mGluRs is mediated by
repressive modification of Ca2+ channel opening. However the full mechanism of these
behaviours is not fully understood especially the induction of the LTD effect. An attempt to
solve this was done by investigating effects of different metabotropic isoforms that was
believed to be active in the process. Two of these are mGluR1 and mGluR5. The DHPG
agonist has the same effect on both isoforms (mGluR1 and mGluR5) thus it is difficult to
separate the effects caused by the two if there is such a pattern. This was solved by using
mGluR1 and 5 specific antagonists together with the application of DHPG. LY36738 was
used to antagonize mGluR1 and MPEP was used to antagonize mGluR5.

Figure 2. A) Set up of the experimental design to measure Ca2+ levels in response to

application of electrical stimuli to the Scaffar collateral pathway in Hippocampus. B) After
wash out of mGlurs agonist DHPG depression of membrane potential is still seen which
indicated an LTP effect by the mGluRs (7).

Figure 3 shows the effect of combining the general agonist DHPG with agonist for the two
respective isoform in separated trials. The result of measuring Ca2+ levels in the presynaptic
terminal region after applying electrical stimuli, DHPG and MPEP are illustrated in figure 3A.
The level of Ca2+ is unaffected in the immediately acute effect of DHPG which indicate that

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mGluR is not responsible for the acute effect of signal depression. But the LTD affect does
not take place when compared to the control in which the mGluR agonist MPEP is not
present.

Figure 3B show the effect inhibition of the mGlur1 has in the same experimental experiment
as over. The antagonist LY367385 hinders mGlur1 in decreasing Ca2+ influx which is
illustrated by the higher acute levels of Ca2+ in response to DHPG treatment. At the same
time the level of presynaptic Ca2+ later in the trial shows lower concentration probably due to
the effect of non-inhibited mGlur5 receptors (7).

Figure 3. Plots show Ca2+ levels in presynaptic terminal in CA3 cells after applying electrical
stimuli and adding the mGlurs isoform 1 and 5 agonist DHPG. Ca2+ levels are measured by
the fluorescent fura-2. A) Isoform mGluR5 is inhibited by MPEP. B) Isoform mGluR1 is
inhibited with LY367385 (7).

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 Conclusion

Synaptic connections between neural cells in the neuron system of biological organisms share
many common principals for storage of information. Modulation of synaptic strength between
two neuron cells is a mechanism used to learn and remember environmental contexts that are
important to the organism.

Storage and retrial of memory can either by implicit (unconscious) or explicit (conscious).
Many withdrawal reflexes that link sensory information with motor responses are implicitly
controlled. In higher vertebrates explicit memory strategies are best known to take place in a
brain region called Hippocampus. Storage of information in this brain region use chemical
transmission of electrical stimulation signalling between presynaptic and postsynaptic cells.
This transmission of electric signalling between cells is more time consuming compared to
electric transmission but open up for more complex processing of signalling information that
are needed to handle explicit memory strategies. The storage of memory in the hippocampus
is mediated by principals that can be described by the Hebbian rule that states “Cells that fire
together, wire together”. Storage of memories that can last for days and weeks are created
through molecular processes called Long time Potentiation (LTP) while decreasing them
Long time Depression (LTD).

Many receptor types are involved in these processes such as NMDAR, AMPAR and VGCC
channels.

Ione channels in general can be of two major types called ionotropic and metabotropic
receptors. Ionotropic receptors trigger an immediate response to transmitter binding in the
postsynaptic membrane. Binding results in ion channel opening due to conformational
changes caused by transmitter binding.

Metabotropic receptors are not directly coupled to channel opening mechanisms after
transmitter binding; they are rather signalling mediators that transfer the binding of
transmitter on the extracellular side to a variety of cytoplasmic signalling pathways. Each
pathway can be linked to different downstream target that mediate different functions in the
cell. Two examples of important downstream targets that are important to the generation of
memory in Hippocampus are ion channels and transcription factors that are affectors of gene
expression profiles. Both types of metabotropic downstream targets can mediate synaptic
modulation by physically strengthening them. Activation of gene expression profiles is in

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particularly important for generation of LTP in expressing genes that code for proteins
important in the generation of new synaptic connections.

The Scaffar collateral nerve pathway in hippocampus which connects the CA3 region with the
Ca1 region has been extensively studied. Metabotropic recepetors are believed to be
responsible for regulation of both long time potentiation as well as long time depression.

Metabotropic receptors that are involved in long time depression in the scaffar collateral
pathway have experimentally shown to exsist. To show this in a wetlab experiment influx of
Ca2+ in the presynaptic terminal in CA3 cells was measured with a fluorescent dye as a
response to electric stimuli and the mGluR agonist DHPG. By usage of different mGluRs
isoform-antagonist a pattern emerged which showed that isoform mGluR1 was responsible for
acute Ca2+ effects while isoform mGluR5 was responsible for long time effects.

This emerging pattern suggests that mGluR isoform 5 is responsible for the triggering of long
time depression (LTD) in the Scaffar collateral pathways in hippocampus.

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4. G.J. Augistine, D. Fitzpatrick, L.C. Katz, A.S. LaMantia, J.O. McNamara, S.M. Williams.
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5. G. Zasshi N. Ganka. Physiology and pathology of visual information processing. 2007

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6. E.R. Kandel. Principles of neural science. Fourth edition. Chapter 63.

7. H. Adwanikar, G.C. Faas, R.W. Gereau, P. Saggau. Modulation of Presynaptic Calcium Transients by
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