Lecture notes:

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Way back when, we talked about localized, short-term, use-dependent manifestations of synaptic plasticity that have been extensively characterized at the frog nmj. These included synaptic depression, and several distinct phenomena that collectively lead to use-dependent increases in neurotransmitter release: Facilitation 1 and 2, augmentation and potentiation.

These sorts of changes allow a neuron to keep a sort of running summary of its history of use and to alter its output of neurotransmitter in response to changes in firing pattern -- they therefore contribute to the flexibility of neuronal function by allowing a particular neuronal pathway to be strengthened or weakened depending upon how rapidly the involved neurons are firing. However, because these changes in transmitter release are relatively transient, its unlikely that they’re directly involved in what we think of as learning and memory.

Now that we've covered a lot of material on neuronal cell biology and the regulation of gene expression, I'm going to return to the idea of plasticity in another context. I’m going to be speaking about other plastic changes in neuronal function that may underlie some forms of learning. I’m going to be concentrating on the cellular correlates of learning in the sea slug Aplysia.

I. Aplysia -- why study learning in a sea slug?

When Eric Kandel -- the researcher who’s done the most to popularize Aplysia as an experimental preparation in which to study learning -- and his colleagues first started studying Aplysia, a lot of neuroscientists said pretty much the same thing.

A. Simple ganglia and identified neurons

The great power of invertebrates like Aplysia is that their nervous systems are relatively simple. Unlike the mammalian CNS, which can contain billions of neurons, the CNS of Aplysia consists of 8 sets of linked ganglia, each of which contains 1-2000 neurons or so.

Moreover in Aplysia’s ganglia, there are certain specific large identifiable neurons. So you can recognize a given neuron from one preparation to the next as a certain, specific cell.

Once you’ve characterized a given neuron, you know whether it’s a sensory neuron or a motor neuron or an interneuron and you know what its characteristic firing pattern is like and what neurotransmitter it uses and so forth. And since these neurons are large, you can impale them with microelectrodes and see whether or not they respond to a given stimulus. So ultimately, you can map the neuronal circuits that appear to be involved in mediating certain specific behaviors.

That means that you can see whether the physiological properties of neurons in the circuit change along with changes in behavior. So you can identify a specific neuronal circuit that mediates a given behavior and see how physiological changes in these neurons correlate with changes in behavior. Many of the cells in Aplysia are so big, that you can also analyze what sorts of  biochemical changes are involved in mediating the physiological changes.

B. The siphon and gill withdrawal reflex

In response to a threatening stimulus -- like an experimenter squirting the siphon with a Water Pik -- the animal withdraws its gill and siphon reflexively. This gill withdrawal response can be quantified by placing a photocell underneath the gill.

C. Habituation

If you continue to squirt the siphon, you find that the response becomes progressively weaker. It will recover with time, if you stop stimulating the siphon, or if you give a strong mechanical stimulus. This behavior resembles habituation, a behavior observed in higher organisms that represents a progressive reduction in the behavioral response to to a repetitive non-noxious stimulus
 

clothes

subway trains

1.Underlying circuit

By selective stimulation and recording from different identified cells, Kandel and his colleagues were able to map a circuit of neurons involved in the gill withdrawal response. This is a simplified circuit of the neurons mediating this response. It's simplified because it only shows one sensory neuron synapsing with one motorneuron to the gill and one interneuron, when actually there are about 24 sensory neurons in parallel as well as a pool of motorneurons and interneurons.

Innervating the siphon skin are sensory neurons. These neurons make excitatory synapses directly on motorneurons to the gill -- this has been shown by electron microscopy -- as well as synapsing on interneurons which mediate various indirect effects. When you stimulate the siphon skin, this creates a receptor potential in the sensory neuron. If this receptor potential is superthreshold -- above threshold -- the sensory neuron will fire. The sensory neuron then makes an excitatory synapse on the motorneuron. Additionally, the motorneuron receives inputs from interneurons and other sensory neurons. If the inputs to the motorneuron are above threshold, then the motorneuron will fire. This, in turn will lead to an action potential in the muscle and ultimately, to contraction and withdrawal of the gill.

By simultaneously recording from the sensory and motor neurons while inducing habituation, they determined that habituation was associated with a depression of synaptic function -- that occurred simultaneously with the behavioral response. So both the EPSP and the gill withdrawal response were simultaneously reduced in amplitude.
 

(Caveat: Some researchers would argue that the circuit is really oversimplified, in that smaller, unidentified neurons are likely involved in the response. Optical mapping with membrane potential-sensitive dyes has suggested that many other neurons, in addition to those that have been identified in the response, likely play a role as well.)

Physiological correlate to a behavioral response.

b. short-term vs long-term habituation

You can train an organism to be habituated for either a short amount of time or a long amount of time. If you have repeated training sessions, you can get the habituation to last for a long time, perhaps a week or more, whereas otherwise it wears off fairly rapidly (ten minutes for ten stimuli). Whether they used training paradigms that induced habituation only briefly or training paradigms with which habituation lasted for a long time, synaptic depression was correlated with behavioral habituation. Suggests that the two processes are related to each other.

Kandel and his colleagues went on to show that the synaptic depression was presynaptic in origin -- less neurotransmitter being released by the presynaptic nerve terminal, rather than a reduction in sensitivity on the part of the post-synaptic receptors.

The depression of synaptic transmission that underlies short-term habituation appears to involve both inactivation of a class of calcium channels on the nerve terminal -- so less calcium would enter the cell in response to an action potential and therefore less transmitter will be released -- and reduction of the readily releasable pool, while long-term habituation correlates with stuctural changes in the sensory neurons: there's an actual loss in the number of sensory neuron nerve terminals synapsing on motor neurons.

D. Sensitization

If you expose an organism to some sort of strong, potentially dangerous NOXIOUS stimulus, you often find that it will then show a stronger response to some innocuous stimulus that would normally elicit only a mild response
 

Firecracker

This sort of enhanced response to a normally innocuous stimulus is called sensitization. Aplysia show sensitization as well. So if you give a shock to the tail and then stimulate the siphon, it will respond more strongly than otherwise. This sensitizing behavior is apparent both as a loss of habituation and with non-habituated responses as well.

As with habituation, you can get short-term sensitization that lasts for minutes after a single training session and long-term sensitization that lasts for days after repeated training sessions.

Like habituation, sensitization of the siphon response involves a change in the efficacy of synapses between the sensory neurons of the siphon and motor neurons to the gill:

So when you stimulate the sensory neurons from the siphon after a shock to the tail, they release more transmitter in the sensitized Aplysia than they would if it were not sensitized. And just as Aplysia shows short-term and long-term sensitization, it shows long term and short term facilitation as well.

While depression in Aplysia, is mediated homosynaptically, with the same synapses that mediate the response becoming depressed in response to repeated stimulation, facilitation in Aplysia requires activation of a separate neuronal pathway -- sensory neurons and interneurons from the tail (which is where the shock occurred) must be activated for this synapse here or this synapse here to be facilitated. So that's a heterosynaptic response.

a. Short-term facilitation

Both short-term facilitation and long-term facilitation in Aplysia involve an increase in the amount of neurotransmitter released every time the sensory neuron fires. And both of these are caused by activation of the second messenger cAMP in response to serotonin and peptide cotransmitters released from these "facilitating" interneurons.

Serotonin interacts with a G-protein coupled receptor linked to G_s as well as one linked to a G₀ associated with PLC

One of the targets for cAMP-dependent protein kinase is a K channel, involved in repolarizing the cell after it fires an action potential. This reduces the potassium conductance. This cAMP-dependent prolongation of action potential duration is responsible for most of the facilitation at synapses that haven’t been previously depressed by habituation.

Serotonin also leads to a direct increase in calcium entry into the cell through an L-channel and enhanced mobilization of vesicles into the readily releasable pool possibly involving phosphorylation of Synapsin I. The latter two effects, which are mediated through both cAMP and PLC pathways, are primarily responsible for "dishabituation" of previously depressed synapses.
 

b. Long-term facilitation
 

Unlike short-term facilitation, long-term facilitation can be eliminated by inhibition of new protein synthesis. (Hmmmmm)

With prolonged or repeated noxious stimulation (or repeated exposure to serotonin) the catalytic subunits of cAMP-dependent protein kinase translocate into the nucleus where they phosphorylate CREB

Phosphorylation of CREB leads to transcritional activation of at least two classes of genes:

A degradatory enzyme, Ubiquitin hydrolase (degrades reg units; leading to persistent activation of kinase and hence long-term phosphorylation of its target proteins) and is also involved in growth of the sensory neuron and establishment of new synaptic connections

Transcription factors

CREB IMPORTANT IN MEMORY PARADIGMS IN DROSOPHILA AND MICE AS WELL

When I first gave this lecture, that's where things stood; more recently people have realized that not only was CREB activation important for long-term sensitization, but that a CREB relative, CREB2, that functions as a transcriptional repressor, needs to be inactivated as well.

However, CREB2 has no consensus phosphorylation sequences for PKA, which presents a bit of a problem as far as using PKA activity to explain everything.

CREB2 does have a consensus sequence for another protein kinase, MAP kinase.

MAP kinase has traditionally been viewed as being involved in the response to growth factors; in this particular situation it isn't clear whether it's being turned on through cross-talk between the two pathways or, indirectly through cAMP-stimulated release of some trophic factor.
 

Cross-talk could occur through either (or both) of two known routes or an unknown one.  The known routes:

1) In Aplysia, PKA phosphorylation of K channel leading to enhanced calcium influx, as described above; calcium influx leads to Ras stimulation

2) In PC12 cells, an independent route to MAP Kinase Kinase has been described that bypasses Ras; possibly this pathway exists in Aplysia as well

In response to repeated stimulation (or repeated exposure to serotonin), MAP kinase also translocates to the nucleus, where it phosphorylates and inactivates CREB2 (and maybe CREB as well)

Cytoplasmically, it also phosphorylates the Aplysia version of N-CAM.

This phosphorylation may target N-CAM in active synapses for degradation by Ubiquitin Hydrolase; this appears to be required for the growth of new synaptic contacts.

The cytoplasmic phosphorylation may allow localized changes to take place in a given synapse in response to a global change in gene expression.

E. Associative learning

Involves associating one stimulus with another; classical conditioning: Pavlov’s dog

Researchers been able to classically condition Aplysia, by pairing a relatively innocuous stimulus (CS: lightly stimulating the siphon or even putting shrimp extract in the water) that elicits a small response with a powerful stimulus that elicits a big response (US: shocking the tail).

As in other forms of classical conditioning, the conditioned stimulus must slightly precede the unconditioned stimulus in order for it to be effective. So later, the innocuous stimulus causes a big response: it has become a conditioned stimulus.

The mechanism underlying classical conditioning in Aplysia appears to be similar to that underlying sensitization, except that is necessary for the neuron involved in the conditioned stimulus to fire prior to the delivery of the unconditioned stimulus.

Firing of the sensory neuron allows calcium to enter the cell, and calmodulin enhances the activity of AC to increase the levels of cAMP; thereby strengthening the response: but only if the neuron mediating the conditioned response is active (allowing calcium influx) prior to receiving input from the pathway mediating the unconditioned stimulus..
Calmodulin must be turned on when AC gets turned on.

Ca and cAMP also synergistically activate CREB; permitting long-term strengthening of the pathway mediating the conditioned response.

Finally, there appears to be some retrograde signal from the post-synaptic cell that leads to enhanced release from the presynaptic neuron.

LTP in the hippocampus

A. Hippocampal Circuitry
 

1. LTP in the CA3 region is non-associative and entirely presynaptic: it involves increased transmitter release from the mossy fiber neurons and appears to depend upon calcium entry.  Calcium activates calmodulin; this leads to calmodulin-dependent enhancement of adenylate cyclase, which leads to LTP.

2. LTP in the dentate gyrus and CA1 is associative, requiring activity of both pre and post-synaptoc cells, and appears to involve both pre and post-synaptic changes
 

induced post-synaptically: Calcium Influx through NMDA receptors is key. (Remember, the NMDA receptor is both glu and voltage dependent, so you need a big depolarization mediated by non-NMDA receptors to depolarize the cells enough to turn it on.)

3. Glu activation of a G-protein coupled receptor linked to PLC is also likely involved.

4. Post-synaptic changes involve increased sensitivity of non-NMDA glu receptors.

5. A retrograde signal (likely NO) seems to mediate presynaptic changes as well

6.  There's an early and a late phase of LTP.  Just like long-term sensitization in Aplysia, the late phase is dependent upon protein synthesis (hmmmm.....)

            CREB implicated in long term changes

            New synapses are actually formed (again, similar to Aplysia).
 

Slides

Kandel ER, Schwartz JH and Jessell TM (2000) "Cellular mechanisms of learning and and the Biological Basis of Individuality" in Principles of Neural Science, 4th ED., ER Kandel, JH Schwartz and TM Jessell (eds), McGraw-Hill, NY, p.1247.

Nicholls JG, Martin AR and Wallace BG (1992) "Leech and Aplysia: Two simple nervous systems" in From Neuron to Brain, JG Nicholls, AR Martin and BG Wallace (eds) Sinauer, Sunderland, MA, p 422-466.

Castellucci VF, Blumenfeld H, Goelet GP and Kandel ER (1989) "Inhibitor of protein synthesis blocks long-term behavior sensitization in the isolated gill-withdrawal reflex of Aplysia" Journal of Neurobiology 20:1-9.

Dash PK, Hochner B and Kandel ER (1990) "Injection of cAMP-responsive element into the nucleus of Aplysia sensory neurons blocks long-term facilitation" Nature 345:718-721.

Kornhauser JM and Greenberg ME (1997) "A kinase to remember: Dual roles for MAP kinase in long-term memory" Neuron 18:839-842.

Martin KC, Michael D, Rose JC, Barad M, Casadio A, Zhu H and Kandel ER (1997) "MAP kinase translocates into the nucleus of the presynaptic cell and is required for long-term facilitation in Aplysia" Neuron 18: 899-912.

Zecevic D, Wu JY, Cohen LB, London JA, Hopp HP and Falk CX (1989) "Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill withdrawal reflex" J Neuroscience 9:3681-3689.