Metabotropic receptors and synaptic plasticity in the schaffar collateral pathway in Hippocampus

8/9/2019 Metabotropic receptors and synaptic plasticity in the schaffar collateral pathway in Hippocampus

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Metabotropic receptors

and synaptic plasticity in

the schaffar collateral

pathway in Hippocampus


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HEAD BIOTECH May 2010 Oslo, Norway. 2

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 transmitters

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, metabotropicreceptors 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 willaffect 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 ofSensitization 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 PKAs 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, mGluRsagonist 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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Metabotropic receptors and synaptic plasticity in the schaffar collateral pathway in Hippocampus

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