Neuro Box 11

The mechanisms that mediate changes in synaptic strength operate in —

phases

Initially, local molecular mechanisms produce —— changes in synaptic strength.

short-term and rapid

Initially, local molecular mechanisms produce short-term and rapid changes in synaptic strength. Additional processes are then activated that result in——

lasting memories

Establishing a long-term memory depends on the activation of——

genes that manufacture new protein products that produce a lasting change in how a synapse works

Using drugs that block ——, early observations suggested that the manufacture of new proteins contributes to the formation of long-term memories

protein synthesis

Using drugs that block protein synthesis, early observations suggested that the manufacture of new proteins contributes to ——

the formation of long-term memories

molecular biology studies have revealed that a variety of learning mechanisms depend on: .

• the induction of common genetic codes

  • genes that encode some biological universals that have been well conserved through evolution.

molecular biology studies have revealed that a variety of learning mechanisms depend on:

• the induction of common genetic codes

• genes that encode some biological universals that have been well conserved through evolution.

Just as the mechanisms that underlie the generation of a neural signal (the action potential) are well conserved across species, so too may be—

the mechanisms that underlie synaptic plasticity.

Modern genetics has also given us new tools for studying the role of gene expression in learning. We can:

• read the genetic code

• identify the locus of the relevant genes

  • and experimentally manipulate how those genes operate.

If we believe that a particular protein plays an essential role in learning, we can test this by using—-

mice in which the relevant gene has been knocked out

If we believe that a particular protein plays an essential role in learning, we can test this by using mice in which the relevant gene has been knocked out

This provides a new and unique window into —-

the molecular mechanisms that underlie learning

Silva and his colleagues created genetically-engineered mice that exhibit specific deficits in the way they learn and remember

• Early studies addressed this issue by manipulating a protein known as ———

calmodulin-dependent protein kinase II (CaMKII)

One way a synapse can be strengthened is by—

 allowing calcium (Ca++) into the postsynaptic cell

— is an electrically charged particle that normally has a higher concentration outside of the neuron than inside.

Ca++

When Ca++ is allowed into the neuron by the NMDA receptor →

it engages CaMKII which enhances synaptic efficacy by activating the AMPA receptors that mediate the neural signal.

When Ca++ is allowed into the neuron by the NMDA receptor → it engages CaMKII which enhances synaptic efficacy by—-

activating the AMPA receptors that mediate the neural signal.

Silva created mice that lacked the gene that underlies the production of CaMKII within the ——

(the) hippocampus

Silva created mice that lacked the gene that underlies the production of CaMKII within the hippocampus.

• From other studies, Silva knew that the hippocampus plays a critical role in learning about spatial relations.

• He reasoned that if CaMKII is critical for learning, then knockout mice that lack this gene should have —-

difficulty remembering where a hidden platform is in a Morris water maze

Silva created mice that lacked the gene that underlies the production of CaMKII within the hippocampus.

• From other studies, Silva knew that the hippocampus plays a critical role in learning about spatial relations.

• He reasoned that if CaMKII is critical for learning, then knockout mice that lack this gene should have difficulty remembering where a hidden platform is in a Morris water maze.

That is precisely what occurred, providing—-

a link between learning and a particular protein product

A difficulty with studies of knockout mice is

that the mice may not develop normally.

A difficulty with studies of knockout mice is that the mice may not develop normally.

• When a gene is missing, other biochemical mechanisms can be —-

enlisted that help the organism compensate for its deficiency

A difficulty with studies of knockout mice is that the mice may not develop normally.

• When a gene is missing, other biochemical mechanisms can be enlisted that help the organism compensate for its deficiency.

  • This could yield—-

a brain that differs in a variety of ways from a normal brain

A difficulty with studies of knockout mice is that the mice may not develop normally.

• When a gene is missing, other biochemical mechanisms can be enlisted that help the organism compensate for its deficiency.

  • This could yield a brain that differs in a variety of ways from a normal brain.

• The abnormal neural environment can make it difficult to interpret the consequences of the genetic manipulation.

  • Neuroscientists are solving this problem by —

making mice in which the expression of a gene can be experimentally controlled.

A difficulty with studies of knockout mice is that the mice may not develop normally.

• When a gene is missing, other biochemical mechanisms can be enlisted that help the organism compensate for its deficiency.

  • This could yield a brain that differs in a variety of ways from a normal brain.

• The abnormal neural environment can make it difficult to interpret the consequences of the genetic manipulation.

  • Neuroscientists are solving this problem by making mice in which the expression of a gene can be experimentally controlled.

    • An interesting application of this technology involves the —-

creation of a transgenic mouse.

Instead of losing a gene (a knockout), the transgenic mouse—→

has an extra gene that makes a new protein product.

mice were engineered that made a mutant version of CaMKII that did not work properly.

• The expression of this gene was placed under the control of a ——- that is regulated by tetracycline transactivator (tTA)

bacterial promoter

mice were engineered that made a mutant version of CaMKII that did not work properly.

• The expression of this gene was placed under the control of a bacterial promoter that is regulated by ——

tetracycline transactivator (tTA)

mice were engineered that made a mutant version of CaMKII that did not work properly.

• The expression of this gene was placed under the control of a bacterial promoter that is regulated by tetracycline transactivator (tTA)

  • Because tTA is not normally present, its gene was also inserted, coupled to a promoter that was engaged within——

brain neurons late in development

mice were engineered that made a mutant version of CaMKII that did not work properly.

• The expression of this gene was placed under the control of a bacterial promoter that is regulated by tetracycline transactivator (tTA)

  • Because tTA is not normally present, its gene was also inserted, coupled to a promoter that was engaged within brain neurons late in development

    • Because the mutated CaMKII would only be expressed in cells that make tTA, limiting the expression of tTA assured —-

• that the mutated gene was only manufactured within the brain

What makes this transgenic system so powerful is that tTA can be inactivated by adding the chemical ——- to the animal’s food.

doxycycline (DOX)

What makes this transgenic system so powerful is that tTA can be inactivated by adding the chemical doxycycline (DOX) to the animal’s food.

• As long as DOX is present, tTA is——- and ——

inactivated and the mutated gene is silenced

What makes this transgenic system so powerful is that tTA can be inactivated by adding the chemical doxycycline (DOX) to the animal’s food.

• As long as DOX is present, tTA is inactivated and the mutated gene is silenced.

  • Under these conditions, the mutated mice exhibited ——- within a Morriz water maze

• normal LTP and learning

When DOX was removed, the mutated CaMKII was —— and ——-

expressed and, shortly thereafter, both LTP and spatial learning were disrupted.

The induction of LTP depends on—-

the NMDA receptor

The induction of LTP depends on the NMDA receptor.

• This receptor is formed from components (subunits), one of which changes with development.

  • Early in development, animals have a subunit called —— which appears to promote the induction of LTP.

NR2B

The induction of LTP depends on the NMDA receptor.

• This receptor is formed from components (subunits), one of which changes with development.

  • Early in development, animals have a subunit called NR2B which appears to promote the induction of LTP.

    • In adults, this subunit is replaced by an alternative form (——-) that down regulates LTP.

NR2A

The induction of LTP depends on the NMDA receptor.

• This receptor is formed from components (subunits), one of which changes with development.

  • Early in development, animals have a subunit called NR2B which appears to promote the induction of LTP.

  • In adults, this subunit is replaced by an alternative form (NR2A) that down regulates LTP.

    • The change from the juvenile form (NR2B) to the adult form (NR2A) could—-

• make it more difficult for an adult animal to learn about new environmental relations.

Tsein and his colleagues created mice that continued to make the juvenile form of the subunit (NR2B) into adulthood.

As expected, these mice showed —-

stronger LTP as adults. The mice also exhibited enhanced learning on an object recognition task and improved spatial memory in the Morris water Maze

lesioning the hippocampus disrupts context conditioning, which suggests that —-

this structure plays a necessary role

To strengthen the hypothesized link, we would also like to show that engaging these hippocampal neurons is—-

sufficient to activate the contextual memory

To strengthen the hypothesized link, we would also like to show that engaging these hippocampal neurons is sufficient to activate the contextual memory.

We cannot do this by simply activating the hippocampus with an excitatory chemical or electricity, because this would —-

engage hippocampal neurons in a non-selective manner.

To strengthen the hypothesized link, we would also like to show that engaging these hippocampal neurons is sufficient to activate the contextual memory.

We cannot do this by simply activating the hippocampus with an excitatory chemical or electricity, because this would engage hippocampal neurons in a non-selective manner.

Rather, what is needed is a trick that would allow us to ——

engage only those neurons activated by a particular context.

(needed something that would allow us to engage only those neurons activated by a particular context)

• The solution was provided by combining the transgenic approach with ——

a viral vector

(needed something that would allow us to engage only those neurons activated by a particular context)

• The solution was provided by combining the transgenic approach with a viral vector.

  • A virus infects cells by ———, causing it to produce a foreign l

binding to the cell membrane and depositing genes that the cell mistakes for its own

(needed something that would allow us to engage only those neurons activated by a particular context)

• The solution was provided by combining the transgenic approach with a viral vector.

  • A virus infects cells by binding to the cell membrane and depositing genes that the cell mistakes for its own, causing it to produce a foreign protein.

This provides researchers with another tool for ——-

inserting a gene into a cell

a virus was used that would deliver the gene for ——-, a light sensitive protein that is inserted into the neural membrane.

channelrhodopsin (ChR2)

a virus was used that would deliver the gene for channelrhodopsin (ChR2), a light sensitive protein that is inserted into the neural membrane.

Like the rhodopsin found in our retina, ChR2 is light sensitive; when illuminated by blue light it ———-, which ———

(it) allows positively charged ions (e.g., Na+) to enter the cell, (which) chemically activates the neuron

(a viral vector used to deliver the gene channelrhodopsin (ChR2) into the neural membrane)

• The expression of the ChR2 gene was placed under the control of the ——promoter.

tTA (promoter)

(a viral vector used to deliver the gene channelrhodopsin (ChR2) into the neural membrane)

• The expression of the ChR2 gene was placed under the control of the tTA promoter.

• As a result, ChR2 would only be made if tTA was present and here too, its expression could be silenced by——

feeding the animals DOX

(a viral vector used to deliver the gene channelrhodopsin (ChR2) into the neural membrane)

• The expression of the ChR2 gene was placed under the control of the tTA promoter.

• As a result, ChR2 would only be made if tTA was present and here too, its expression could be silenced by feeding the animals DOX.

Because this approach combines optical and genetic techniques, it is known as ——-

optogenetics

microinjected the ChR2 containing virus into a region of the hippocampus (the ———) using mice that contained the gene for tTA.

(dentate gyrus)

microinjected the ChR2 containing virus into a region of the hippocampus (the dentate gyrus) using mice that contained the gene for tTA.

• The expression of tTA was linked to another gene (——) that is engaged by neuronal activity.

c-fos)

(c-fos) is engaged by —-

neuronal activity.

(using mice that contained the gene for tTA) → Microinject ChR2 w/ virus into the hippocampal dentate gyrus) → The expression of tTA  is linked to (c-fos) that is engaged by neuronal activity.

• We now have an animal that———, and within the dentate gyrus, will ———

• expresses tTA in active neurons

• (will (engage the expression of ChR2.

(using mice that contained the gene for tTA) → Microinject ChR2 w/ virus into the hippocampal dentate gyrus) → The expression of tTA  is linked to (c-fos) that is engaged by neuronal activity.

• We now have an animal that expresses tTA in active neurons and, within the dentate gyrus, this will engage the expression of ChR2.

  • We can then activate these cells with ——

• blue light provided by a optical fiber inserted into the dentate gyrus

Make mouse that produce tTA gene → microinject ChR2 w/ viral vector into denate gyrus (hippocampus) → neural activity induced → cfos engaged → tTA expressed → ChR2 expression engaged in the denate gyrus

• feed mice DOX →

→ (silence the expression of ChR2)

Make mouse that produce tTA gene → microinject ChR2 w/ viral vector into denate gyrus (hippocampus) → neural activity induced → cfos engaged → tTA expressed → ChR2 expression engaged in the denate gyrus

• feed mice DOX → (silence the expression of ChR2) →

→ habituate mice to Context (A )

Make mouse that produce tTA gene → microinject ChR2 w/ viral vector into denate gyrus (hippocampus) → neural activity induced → cfos engaged → tTA expressed → ChR2 expression engaged in the denate gyrus

• feed mice DOX → (silence the expression of ChR2) → habituate mice to Context (A )→

→ remove DOX from diet

Make mouse that produce tTA gene → microinject ChR2 w/ viral vector into denate gyrus (hippocampus) → neural activity induced → cfos engaged → tTA expressed → ChR2 expression engaged in the denate gyrus

• feed mice DOX → (silence the expression of ChR2) → habituate mice to Context (A )→ remove DOX from diet →

→ present shock in a novel Context (B)

Make mouse that produce tTA gene → microinject ChR2 w/ viral vector into denate gyrus (hippocampus) → neural activity induced → cfos engaged → tTA expressed → ChR2 expression engaged in the denate gyrus

• feed mice DOX → (silence the expression of ChR2) → habituate mice to Context (A )→ remove DOX from diet → present shock in a novel Context (B) →

→ novel context (B) would engages cellular activity (c-fos expression) within the hippocampal cells that encode that context

Make mouse that produce tTA gene → microinject ChR2 w/ viral vector into denate gyrus (hippocampus) → neural activity induced → cfos engaged → tTA expressed → ChR2 expression engaged in the denate gyrus

• feed mice DOX → (silence the expression of ChR2) → habituate mice to Context (A )→ remove DOX from diet → present shock in a novel Context (B) → novel context (B) would engages cellular activity (c-fos expression) within the hippocampal cells that encode that context →

→ causes ChR2 to be expressed within those cells

Make mouse that produce tTA gene → microinject ChR2 w/ viral vector into denate gyrus (hippocampus) → neural activity induced → cfos engaged → tTA expressed → ChR2 expression engaged in the denate gyrus

• feed mice DOX → (silence the expression of ChR2) → habituate mice to Context (A )→ remove DOX from diet → present shock in a novel Context (B) → novel context (B) would engages cellular activity (c-fos expression) within the hippocampal cells that encode that context → causes ChR2 to be expressed within those cells

• Give mice DOX again →

→ (stop further ChR2 expression)

Make mouse that produce tTA gene → microinject ChR2 w/ viral vector into denate gyrus (hippocampus) → neural activity induced → cfos engaged → tTA expressed → ChR2 expression engaged in the denate gyrus

• feed mice DOX → (silence the expression of ChR2) → habituate mice to Context (A )→ remove DOX from diet → present shock in a novel Context (B) → novel context (B) would engages cellular activity (c-fos expression) within the hippocampal cells that encode that context → causes ChR2 to be expressed within those cells

• Give mice DOX again → (stop further ChR2 expression) →

→ mice returned to Context (A) and observed

Make mouse that produce tTA gene → microinject ChR2 w/ viral vector into denate gyrus (hippocampus) → neural activity induced → cfos engaged → tTA expressed → ChR2 expression engaged in the denate gyrus

• feed mice DOX → (silence the expression of ChR2) → habituate mice to Context (A )→ remove DOX from diet → present shock in a novel Context (B) → novel context (B) would engages cellular activity (c-fos expression) within the hippocampal cells that encode that context → causes ChR2 to be expressed within those cells

• Give mice DOX again → (stop further ChR2 expression) → mice returned to Context (A) and observed

In the absence of stimulation, the subjects exhibited:

no evidence of fear (freezing)

Make mouse that produce tTA gene → microinject ChR2 w/ viral vector into denate gyrus (hippocampus) → neural activity induced → cfos engaged → tTA expressed → ChR2 expression engaged in the denate gyrus

• feed mice DOX → (silence the expression of ChR2) → habituate mice to Context (A )→ remove DOX from diet → present shock in a novel Context (B) → novel context (B) would engages cellular activity (c-fos expression) within the hippocampal cells that encode that context → causes ChR2 to be expressed within those cells

• Give mice DOX again → (stop further ChR2 expression) → mice returned to Context (A) and observed

  • In the absence of stimulation, the subjects exhibited no evidence of fear (freezing)

  • When blue light was presented through the optical fiber, —-

it elicited freezing behavior—as if it had engaged the memory of the shocked context.

Make mouse that produce tTA gene → microinject ChR2 w/ viral vector into denate gyrus (hippocampus) → neural activity induced → cfos engaged → tTA expressed → ChR2 expression engaged in the denate gyrus

• feed mice DOX → (silence the expression of ChR2) → habituate mice to Context (A )→ remove DOX from diet → present shock in a novel Context (B) → novel context (B) would engages cellular activity (c-fos expression) within the hippocampal cells that encode that context → causes ChR2 to be expressed within those cells

• Give mice DOX again → (stop further ChR2 expression) → mice returned to Context (A) and observed

  • In the absence of stimulation, the subjects exhibited no evidence of fear (freezing)

  • When blue light was presented through the optical fiber, it elicited freezing behavior—as if it had engaged the memory of the shocked context.

• mice that were treated the same, but never received shock:

did not exhibit light- induced freezing.

Make mouse that produce tTA gene → microinject ChR2 w/ viral vector into denate gyrus (hippocampus) → neural activity induced → cfos engaged → tTA expressed → ChR2 expression engaged in the denate gyrus

• feed mice DOX → (silence the expression of ChR2) → habituate mice to Context (A )→ remove DOX from diet → present shock in a novel Context (B) → novel context (B) would engages cellular activity (c-fos expression) within the hippocampal cells that encode that context → causes ChR2 to be expressed within those cells

• Give mice DOX again → (stop further ChR2 expression) → mice returned to Context (A) and observed

  • In the absence of stimulation, the subjects exhibited no evidence of fear (freezing)

  • When blue light was presented through the optical fiber, it elicited freezing behavior—as if it had engaged the memory of the shocked context.

• mice that were treated the same, but never received shock: did not exhibit light- induced freezing.

  • Thus, optical activation of ———- elicited freezing behavior.

hippocampal cells that were active during context conditioning

Make mouse that produce tTA gene → microinject ChR2 w/ viral vector into denate gyrus (hippocampus) → neural activity induced → cfos engaged → tTA expressed → ChR2 expression engaged in the denate gyrus

• feed mice DOX → (silence the expression of ChR2) → habituate mice to Context (A )→ remove DOX from diet → present shock in a novel Context (B) → novel context (B) would engages cellular activity (c-fos expression) within the hippocampal cells that encode that context → causes ChR2 to be expressed within those cells

• Give mice DOX again → (stop further ChR2 expression) → mice returned to Context (A) and observed

  • In the absence of stimulation, the subjects exhibited no evidence of fear (freezing)

  • When blue light was presented through the optical fiber, it elicited freezing behavior—as if it had engaged the memory of the shocked context.

• mice that were treated the same, but never received shock: did not exhibit light- induced freezing.

  • Thus, optical activation of hippocampal cells that were active during context conditioning elicited freezing behavior.

This implies that engaging cells involved in the formation of a memory is ——

sufficient to induce the behavioral expression of that memory.