Learning & Memory

LEARNING & MEMORY

REAL MECHANISMS OF LONG-TERM MEMORY IN HABITUATION, SENSITIZATION & PRIMING

SKKU GBME GRAD CLASS KIM HF

Gluck2e_CH03.indd 74 28/11/12 2:03 PM Versatility dimension Execution dimension Time dimension

Flexible memory Working memory Short-term memory

Stable memory Long-term memory

Declarative memory Non-declarative memory (Explicit) (implicit)

Priming

Episodic memory Semantic memory Nonassociative learning: Associative learning: Procedural memory Sensitization Classical Skill & Habituation & Operant conditioning & Habit

Medial temporal lobe Neocortex Amygdala Cerebellum Striatum Diencephalon Reflex pathway SHORT-TERM & LONG-TERM MEMORY?

2 | CHAPTER 1 Introductory Module ■ THE PSYCHOLOGY OF LEARNING AND MEMORY

A few minutes later, he’d catch sight of her again and stage another passion- ate reunion. Clive now lived “in the moment,” caught in an endless loop of reawakening. His numerous jour- nals show his desperate efforts to make sense of what he was experiencing: “7:09 A.M.: Awake. 7:34 A.M.: Actually finally awake. 7:44 A.M.: Really perfectly awake . . . 10:08 A.M.: Now I am superla- tively awake. First time aware for years.

Jiri Rezac, 2006 10:13 A.M.: Now I am overwhelmingly Clive Wearing with his wife, awake. . . . 10:28 A.M.: Actually I am now ADeborah case of Clive Wearingfirst time awake for years. . . .” Each time he added a new entry, he might go back and scratch out the previous line, angry that a 1985: virus infection in temporal lobe including stranger had written misleading entries in his journal. Willem de Kooning hippocampus Yet even when Clive knew nothing else, he knew that he loved his wife. Emotional memory—love—survived when almost every-Alzheimer’s disease Anterograde amnesiathing else was gone. And he could still play the piano and con- duct an orchestra so competently that a nonmusician wouldn’t But memories whichsuspect were anything formed was wrong long with ago Clive’s are mind. fine. Those specialized skill memories survived, along with more mundane skills, such as making coffee or playing card games. And although Clive was unable to consciously learn any new facts, he could acquire some new habits through repeated practice. After moving to a nursing home, he eventually learned the route from the dining hall to his room, and when prompted to put on his coat for his daily walk past the local pond, he would ask if it was time to go feed the ducks (Wilson & Wearing, 1995). Clive’s memory was more like an imperfectly erased blackboard than a blank slate. Clive Wearing’s case is tragic but makes two important points. The first is the unrivaled importance of learning and memory to our lives. Most of the time, we take for granted our memories of who we are and what we know. When these are stripped away, life becomes a series of unrelated moments, isolated from past and future, like those fuzzy moments we all experience when we’ve just awakened and are disoriented. The second point is that speaking of memory as if it were a single, cohesive process is misleading. In fact, there are many different kinds of memory, and as happened in Clive’s case, some can be damaged while others are spared. Normally, these different kinds of memory function together seamlessly, and we aren’t aware of whether a given instance of learning has been preserved as a fact, habit, skill, or emotion. But this cohesion is in many ways an illusion. By confronting the limits of this illusion, we can

Gluck2e_CH01.indd 2 26/11/12 4:04 PM SHORT-TERM & LONG-TERM MEMORY?

2 | CHAPTER 1 Introductory Module ■ THE PSYCHOLOGY OF LEARNING AND MEMORY

Jiri Rezac, 2006 10:13 A.M.: Now I am overwhelmingly Clive Wearing with his wife, awake. . . . 10:28 A.M.: Actually I am now ADeborah case of Clive Wearingfirst time awake for years. . . .” Each time he added a new entry, he might go back and scratch out the previous line, angry that a 1985: virus infection in temporal lobe including stranger had written misleading entries in his journal. Willem de Kooning hippocampus Yet even when Clive knew nothing else, he knew that he loved his wife. Emotional memory—love—survived when almost every-Alzheimer’s disease Anterograde amnesiathing else was gone. And he could still play the piano and con- duct an orchestra so competently that a nonmusician wouldn’t But memories whichsuspect were anything formed was wrong long with ago Clive’s are mind. fine. Those specialized skill memories survived, along with more mundane skills, such as making coffee or playing card games. And although Clive was unable to consciously learn any new facts, he could acquire some new habits through repeatedLong practice.-term After memory moving to a nursing is stored somewhere by a certain mechanism. home, he eventually learned the route from the dining hall to his room, and when prompted to put on his coat for his daily walk past the local pond, he would ask if it was time to go feed the ducks (Wilson & Wearing, 1995). Clive’s memory was more like an imperfectly erased blackboard than a blank slate. Clive Wearing’s case is tragic but makes two important points. The first is the unrivaled importance of learning and memory to our lives. Most of the time, we take for granted our memories of who we are and what we know. When these are stripped away, life becomes a series of unrelated moments, isolated from past and future, like those fuzzy moments we all experience when we’ve just awakened and are disoriented. The second point is that speaking of memory as if it were a single, cohesive process is misleading. In fact, there are many different kinds of memory, and as happened in Clive’s case, some can be damaged while others are spared. Normally, these different kinds of memory function together seamlessly, and we aren’t aware of whether a given instance of learning has been preserved as a fact, habit, skill, or emotion. But this cohesion is in many ways an illusion. By confronting the limits of this illusion, we can

Gluck2e_CH01.indd 2 26/11/12 4:04 PM WHAT’S THE MECHANISMS OF MEMORY?

Imagine you are now living in 60’~70’. How can you make a hypothesis or an idea for where and how the brain encodes memory? WHAT’S THE MECHANISMS OF MEMORY?

Imagine you are now living in 60’~70’. How can you make a hypothesis or an idea for where and how the brain encodes memory? HOW TO STUDY?

Human brain is too complex! @_@ LET’S MAKE A MODEL SYSTEM!

Rodent model For example… WHEN CAN WE LEARN?

1. Repetitive learning

2. Associative learning

3. Intensive learning Tim Bliss

Long-term potentiation

Bliss and Lomo, 1973 Effect of gamma-amino butyric acid (GABA) on K42 LadislavTa u c (1926–1999) was born outflux from rabbit cortex. in Pardubice, Czechoslovakia. He was a French neuroscientist, BRINLEY FJ Jr, KANDEL ER, MARSHALL WH. and a pioneer in neuroethology and neuronal physiology, who J Neurophysiol. 1960 May;23:237-45 immigrated to France in 1949 to work at the Institut Marey in Paris. Tauc was the founder and former director of the Laboratoire de Neurobiologie Cellulaire et Moléculaire of Transient and long-lasting electrical responses to direct the French National Centre for Scientific Research (CNRS). hippocampal stimulation. He was one of the teachers of Eric R. Kandel. KANDEL ER, SPENCER WA, BRINLEY FJ Jr. Am J Physiol. 1960 Apr;198:687-92.

MECHANISM OF PROLONGED HETEROSYNAPTIC FACILITATION. KANDEL ER, TAUC L. Nature. 1964 Apr 11;202:145-7.

Eric R. Kandel Simple system is not so simple…

What’s the best animal? Aplysia! (Like Eureka!?) WHY APLYSIA? WHY APLYSIA?

1. Large cell body

2. Relatively simple circuit

3. Circuits in culture system

4. Various behavioral schemes BRAIN SUBSTRATES | 97

Dogs and cats are natural antagonists, as any dog or cat owner knows. Some of the earliest brain studies on habituation, using dogs and cats as subjects, seemed to bear out the view that these animals are fundamentally antitheti- cal. Ivan Pavlov, for example, found that when a dog’s cortex was removed, the dog no longer showed habituation to auditory stimuli: the dog would instead continue to show orienting responses to the sounds, even after many exposures (Pavlov, 1927). Such findings led researchers to suggest that the cortex was critical for habituation and that it actively suppressed reflexive orienting responses to stimuli perceived as familiar (Sokolov, 1963). The data from cats, however, seemed completely contradictory. Cats that had their brain disconnected from their spinal cord, called spinal cats, still showed habituation to tactile stimulation (Thompson & Spencer, 1966). This seemed to prove that the spinal cord by itself contained all the neural machinery necessary for habituation; the cortex— and indeed the rest of the brain—wasn’t needed. The cat data were consistent with the finding that many other organisms known to habituate, including roaches, protozoa, and numerous other invertebrates, don’t have any cortex. How to reconcile the dog data and the cat data? For one thing, the animals in these early studies were learning about different kinds of stimuli. Whether cortical processing is involved in habituation likely depends on the kinds of stimuli that are being repeated, where they are normally processed, and where memories of the stimuli are formed. One way to avoid these complications is to study habituation not with mammals, such as cats and dogs, but with smaller- brained animals such as everyone’s favorite—the sea slug. Invertebrate Model Systems Much work on the neural substrates of habituation has been conducted on a group of marine invertebrates called Aplysia, the sea slugs (or sea hares), such as the species Aplysia californica shown in Figure 3.9. Like many marine animals, Aplysia breathes through gills, which extend upward from between the two wings of the mantle, the animal’s outer covering. A structure called the siphon works like a tube to blow aerated water over the gills to assist respiration. The gills are delicate and easily damaged. When danger threatens, the sea hare tends to retract them under the safety of the mantle. This is called a gill-withdrawal reflex. ANATOMYOne advantage ofOF studying APLYSIA learning in Aplysia is that they have a relatively simple nervous system—only about 20,000 neurons, compared with the tens of billions in a cat or human. Plus, some of the neurons are very big. A few are

Mantle Figure 3.9 Aplysia Siphon californica, the sea hare This marine invertebrate, a shell-less mollusk, has a relatively simple nervous system, useful for studying the neural bases of learning. If the siphon is touched lightly, both siphon and gill are protectively withdrawn (the gill-withdrawal Tail reflex). With repeated light Head touches, the gill-withdrawal reflex habituates. In this photo, the gill is underneath the mantle. (Adapted from Squire and Kandel, 2000.) David Wrobel/Visuals David Unlimited Wrobel/Visuals

Gluck2e_CH03.indd 97 28/11/12 2:04 PM Gill Parapodia Siphon

Ten ta c l e

Eye

Aplysia nervous system Head

Ta i l NEURAL SYSTEM OF APLYSIA ANATOMY OF APLYSIA APLYSIA SYSTEM YOU CAN STUDY ANYTHING!

Behavior

In vivo recording

Semi in-vivo recording

Circuit recording

Culture recording

Molecular biology Aplysia Kurodai Gene expression Nervous system BEHAVIOR: SIPHON WITHDRAWAL REFLEX (SWR) (Kandel, Science, 1973)

Siphon CIRCUITS IN APLYSIA

Siphon-Gill Circuitry Ta i l -Siphon Circuitry Aplysia neural circuit for siphon sensitization

Siphon

Motor Inter- L7 neuron neuron

LE Sensory cluster neuron SWR Siphon withdrawal reflex

Motor neuron

Sensory neuron

Aplysia nerve system IN VIVO RECORDING SEMI IN-VIVO PREPARATION

SEMI IN-VIVO PREPARATION

!!!! Cellular model : easy to study molecular mechanism

Motor neuron

Sensory neuron MOLECULAR MECHANISM!

Kim et al., 2006 Computational Model of the Serotonergic Modulation of Sensory Realistic Simulation of the Aplysia Siphon-Withdrawal Reﬂex Circuit: Neurons in Aplysia Roles of Circuit Elements in Producing Motor Output

J. R. LIEB, JR. AND W. N. FROST 1 1 2 1 Department of Neurobiology and Anatomy, University of Texas, Houston Health Science Center, Houston, Texas 77225 DOUGLAS A. BAXTER, CARMEN C. CANAVIER, JOHN W. CLARK, JR., AND JOHN H. BYRNE 1250 J. R. LIEB, JR. AND W. N. FROST 1Laboratories of Origin, Department of Neurobiology and Anatomy, W. M. Keck Center for the Neurobiology of Learning and Memory, The University of Texas-Houston Medical School, Houston, 77225; and 2Department of Electrical and Lieb, J. R., Jr. and W. N. Frost. Realistic simulation of the ies have worked out the basic structurebranchial of a particular nerves circuit, to the gill, siphon, and other mantle organs. Ani- Aplysia siphon-withdrawal reﬂex circuit: roles of circuit elements the contribution made by each componentmals to (100–250 the processing g) were anesthetized by injecting one third to Computer Engineering, Rice University, Houston, Texas 77251 in producing motor output. J. Neurophysiol. 77: 1249–1268, 1997. of information as it ﬂows through the network is often, at The circuitry underlying the Aplysia siphon-elicited siphon-with- one half their body weight of isotonic (350 mM) MgCl2 into the 2916 D. A. BAXTER, C. C. CANAVIER, J. W. CLARK, AND J. H. BYRNE best, merely an educated guess. The Aplysia siphon-with-

hemocoel. After the ganglionDownloaded from and mantle organs were dissected drawal reflex has been widely used to study the cellular substrates drawal reflex circuit is an interesting case in point. Although of simple forms of learning and memory. Nonetheless, the func- the basic form of the behavior is quite simple,out, the theconnective underlying tissue surrounding the ganglion was lightly Baxter, Douglas A., Carmen C. Canavier, John W. Clark, Jr., and Acting via elevated levels of intracellular adenosine cyclictional roles of the different neurons and synaptic connections modi- fixed by a 35-s exposure to 0.5% glutaraldehyde in artificial sea John H. Byrne. Computational model of the serotonergic modulation fied with learning have yet to be firmly established. In this study we circuitry is rather complex, consisting of monosynaptic and monophosphate (cAMP) and the subsequent activation of pro-constructed a realistic computer simulation of the best-understood polysynaptic pathways, excitatory andwater inhibitory (ASW) interneu- (Instant Ocean, Aquarium Systems). The entire of sensory neurons in Aplysia. J. Neurophysiol. 82: 2914–2935, 1999. Downloaded from tein kinase A (PKA), application of 5-HT decreased the mag-component of this network to better understand how the siphon- rons, chemical and electrical synapses,preparation and a layer was of in- next transferred to a two-chamber recording dish Serotonergic modulation of the sensory neurons that mediate the gill- nitude of I (Fig. 1A) (Baxter and Byrne 1990a; Bernier et al.withdrawal circuit works. We used an integrate-and-fire scheme to terneuronal processing that includes bothwhose recurrent bottom and surfacelat- was covered with a transparent Sylgard and tail-withdrawal reflexes of Aplysia is a useful model system for K,S simulate four neuron types (LFS, L29, L30, L34) and 10 synaptic eral inhibition (Frost and Kandel 1995). What is the func- 1982; Jarrard et al. 1993; Ocorr and Byrne 1985; Pollock andconnections. Each of these circuit components was individually (Dow Corning) pinninghttp://jn.physiology.org/ surface. The abdominal ganglion was studies of neuronal plasticity that contributes to learning and memory. tional significance of all this complexityplaced given in the the apparent smaller of the two chambers, in a solution composed Camardo 1987; Pollock et al. 1985; Shuster and Siegelbaumconstructed to match the mean or typical example of its biological simplicity of the behavior? Physiological studies have led The effects of serotonin (5-HT) are mediated, in part, via two protein counterpart on the basis of group measurements of each circuit of 50% isotonic MgCl and 50% ASW (maintained at 14–157C), kinases (protein kinase A, PKA, and protein kinase C, PKC), which in 1987; Shuster et al. 1985; Siegelbaum et al. 1982; Sugita et al. to several speculations regarding the roles of the different 2 element. Once each cell and synapse was modeled, its free parame- components of this circuit (Cleary et al.and 1995; the mantle Fischerorgans and were placed directly into ASW in the larger ters were fixed and not subject to further manipulation. The LFS turn, modulate at least four membrane currents, including a S (“sero- 1997a; Walsh and Byrne 1989). Because 5-HT produced a Carew 1993, 1995; Frost 1987; Frostchamber and Kandel (maintained 1995; at room temperature), with the siphon and tonin-sensitive”) K1 current (I ), a steeply voltage-dependent K1 motor neurons respond to sensory input with a brief phasic burst K,S broadening of the action potential and enhanced the excitabilityfollowedhttp://jn.physiology.org/ by a long-lasting period of tonic firing. We found that Hawkins and Schacher 1989; Hawkinsbranchial et al. 1981; nerves Trudeau passing through two narrow Vaseline-covered slits current (I ), a slow component of the Ca21-activated K1 current K-V of sensory neurons, both of these changes originally werethe assembled model network responded to sensory input in a and Castellucci 1992, 1993a,b; Wrightin et a al. dividing 1991), wall yet a between the two chambers. After the ganglion 21 means of testing these ideas has remained elusive. (IK,Ca-S), and a L-type Ca current (ICa-L). The present study inves- qualitatively similar fashion, suggesting that many of the interneu- was pinned out and its left ventral side was desheathed, the CNS attributed to the reduction of IK,S (e.g., Klein et al. 1986). It hasrons important for producing the LFS firing response have now One straightforward approach to determining the func- by 10.220.33.2 on October 9, 2016 tigated how the modulation of these currents altered the spike duration was revived by perfusing the smaller chamber with ASW. The become clear, however, that the mechanisms underlying thebeen identified. By selectively removing different circuit elements, tional roles of the different circuit elements would be to Downloaded from and excitability of sensory neurons and examined the relative contri- we determined the contribution of each to the LFS firing pattern. siphon was revived by first injecting it directly with ASW, and butions of PKA- and PKC-mediated effects to the actions of 5-HT. A 5-HT-induced changes in the biophysical properties of sensory study the effect on motor neuron firing of removing specific Our first finding was that the monosynaptic sensory neuron to interneurons or pathways. Although thisthen has perfusing been attempted it throughout the experiment either through a poly- Hodgkin-Huxley type model was developed that described the ionic neurons are more complicated than the activation of a singlemotor neuron pathway contributed only to the initial brief burst of in a few instances (Fischer and Carewethylene 1993, 1995; tube Wright inserted into a sinus located in its base or through a conductances in the somata of sensory neurons. The descriptions of the LFS firing response, whereas the polysynaptic pathway deter- and Carew 1995), the fact that most interneurons in the second-messenger/protein kinase system and the modulation ofmined the overall duration of LFS firing. By making more selective needle inserted directly into its base. Both chambers were perfused 1 these currents and their modulation were based largely on voltage- a single K current. deletions, we found that the circuit elements responsible for trans- circuit are multiply represented hasseparately made this approachthroughout the experiment. clamp data from sensory neurons. Simulations were preformed with Downloaded from forming brief sensory neuron discharges into long-lasting LFS fir- rather difficult in practice. An alternativeCells approach were is toidentified use on the basis of their size, location, pigmen- Several additional components contribute to 5-HT-induced by 10.220.33.6 on October 9, 2016 the program SNNAP (Simulator for Neural Networks and Action ing were the slow components of the L29-LFS fast/slow excitatory physiological data to construct a realistic computer simula- modulation of action potentials and excitability of sensorypostsynaptic potentials. The inhibitory L30 neurons exerted a sig- tion of the circuit, which can then betation, used synaptic to study theconnections with other identified neurons, and re-

Potentials). The model was sufficient to replicate empirical data that http://jn.physiology.org/ neurons (Fig. 1B). First, elevated levels of cAMP modulate atnificant braking action on the flow of excitatory information detailed contribution of any neuron orsponse synaptic to connection tactile stimulation of the siphon (Frost and Kandel 1995; describes the membrane currents, action potential waveform and through the circuit. Interestingly, L30 lost its ability to reduce the to network function. Here we have usedHawkins this latter et approach al. 1981). The LFS motor neurons were further identi- excitability as well as their modulation by application of 5-HT, least three currents in addition to IK,S. Walsh and Byrne (1989)duration of LFS firing at high stimulus intensities. This was found to study the operation of the Aplysia siphon-elicited siphon- 21 1 fied on the basis of the characteristic siphon movements they pro- increased levels of adenosine cyclic monophosphate or application of described a slow component of the Ca -activated K currentto be due to the intrinsic nature of L30’s current-frequency relation- withdrawal reflex circuit. We employedduce a single-compart- when driven with intracellular current injection (Frost and active phorbol esters. In the model, modulation of I by PKC played ship. Some circuit elements, including interneuron L34, and the ment, integrate-and-fire simulation scheme (Getting 1989a) K-V (IK,Ca-S) that was active near the resting potential of the cell andelectrical coupling between L29 and L30 were found to have little Kandel 1995; Hickie and Walters 1995). Recordings were made a dominate role in 5-HT-induced spike broadening, whereas the to model all known circuitry conveying excitatory siphon-

http://jn.physiology.org/ impact when subtracted from the network. These results represent that was decreased by intracellular injection of cAMP (or elicited input to one class of siphon motorwith neurons, microelectrodes the LFS (10–20 MV) filled either with 3 M KCl or, concurrent modulation of IK,S and IK,Ca-S by PKA primarily ac- a detailed dissection of the functional roles of the different elements when recording inhibitory postsynaptic potentials (IPSPs), with 4 application of 5-HT). Braha et al. (1993; see also Edmonds etof the siphon-elicited siphon-withdrawal circuit in Aplysia. Be- cells. Our model network includes four neuron types (L29, counted for 5-HT-induced increases in excitability. Finally, simula- L30, L34, and LFS), nine chemical synapses,M potassium and one acetate. elec- tions indicated that a PKC-induced increase in excitability resulted al. 1990; Eliot et al. 1993) reported that intracellular injectioncause many vertebrate andFIG invertebrate. 1. A: circuit circuits diagram perform of similar the modeled portion of the Aplysia siphon- tasks and contain similarelicited information siphon-withdrawal processing elements, reflex aspects network.trical The synapse model (Fig. circuit 1A represents). 5 from decreases of I and I , which was likely the indirect result of cAMP (or application of 5-HT) enhanced a dihydropyridine-of these results may be of general significance for understanding K,S K,Ca-S L29s, 2 L34s, 3 L30s, and an LFS siphon motorIn this neuron. paper This we circuit describe contains first the construction of our by 10.220.33.2 on October 9, 2016 21 the function of motor networks. In addition, because several sites Modeling cells of cross-talk between the PKC and PKA systems. The results provide sensitive and slowly inactivating component of the Ca cur- recurrent and lateral inhibition, electricalmodel, coupling, and and then both how monosynaptic the model was used to address several several predictions that warrant additional experimental investigation 21 in this network store learning-related information, these results are specific issues regarding the organization of the circuit. FIG rent similar to the L-type Ca current (ICa-L). Baxter andrelevant for elucidatingand the functional polysynaptic significance sensory of neuron the distrib- to motor neuron pathways. B: physiologi- Because our aim was to model the typical rather than an arbi- . 2.and Model illustrate of a sensory the importance neuron. A: equivalent of considering electrical indirect circuitas of thewell cell as membrane. direct Linear conductances are indicated by These issues include the following. What are the relative resistors and nonlinear (i.e., voltage-gated) conductances are indicated by variable resistors.Byrne Each conductance (1989; see is associated also White with et al. 1994) reported that appli-uted storage of learned informationcal recording in Aplysia. of an LFS siphon motorcontributions neuron response of the to a monosynaptic brief tactile andtrarily polysynaptic selected path- example of each neuron, we characterized the elec- effects of modulatory agents on the modulation of membrane currents. siphon stimulus. These cells characteristically respond with a brief, high- a equilibrium potential (E). In parallel with the membrane capacitance (CM) are 10 ionic conductances:cation of a leakage 5-HT conductance decreased (gL the); conductance and slowed the ways to the characteristic phasic/tonictrophysiological motor neuron firing properties of each of the four cell types (L29, 1 21 21 frequency burst of action potentials (phasic response), followed by a long- a fast Na conductance (gNa); a slowly inactivating L-type Ca conductance (gCa-L); a rapidly inactivating N-type Ca 1 response to sensory input? What specific role does interneur- by 10.220.33.6 on October 9, 2016 L30, L34, and LFS) in several different preparations. In each case, 1 21kinetics of1 a large, steeply voltage-dependent K currentINTRODUCTION lasting low-frequency spike train (tonic response). conductance (gCa-N); a fast transient A-type K conductance (gK-A); a fast voltage- and Ca -activated K conductance (gK,Ca-F); onal processing play in shaping motor neuron firing? For 21 1 (I ). Goldsmith and Abrams (1992) reported that application measurements were made of resting potential (Vr , the cell’s mem- a slow voltage-independent Ca -activated K conductance (gK,Ca-S); an instantaneous, modestlyK-V voltage-dependent component of Determining the exact functional roles played by different example, do each of the identified interneuron types (L29, INTRODUCTION1 1 neurons and synaptic connections in neural circuits is not a brane potential when at rest), input resistance (Rinput , the asymp- the S (“serotonin”)-type K conductance (gK,S-I); a voltage- and time-dependent component ofof the analogues S-type K conductance of cAMP ( partiallygK,S-V); mimicked the 5-HT-induced the connections among the interneuronsL30, L34) (electrical make unique synapses, contributions to the motor neuron and a delayed, steeply voltage-dependent K1 conductance (g ). The application of extrinsic stimulating and/or bias currents is simple matter. Even after detailed electrophysiological stud- firing response, or are their contributionstotic redundant? voltage How change do produced by a hyperpolarizing constant cur- By changing the waveform of actionK-V potentials and excit- slowing of the activation kinetics of IK-V. Similarly, Hochner recurrent inhibition, and lateral inhibition) contribute to net- rent pulse divided by the current applied), input capacitance (C, represented by IStim. B: intracellular regulatory pathways of the model. Model also incorporated a description of an intracellular 21 21 and Kandel21 (1992) reported that specific blockers of PKA work function? obtained by dividing the time constant of the input resistance charg- pool of Caability,. Calcium serotonin influx via (5-HT)-inducedgCa-L and gCa-N contributed modulation to the Ca of membranepool. Dynamics of the Ca pool were modeled as a 0022-3077/97 $5.00 Copyright q 1997 The American Physiological Society 1249 21 partially blocked the 5-HT-induced slowing of the activation first-ordercurrents process. The in intracellular the sensory pool of neurons Ca , in that turn, regulated mediate several the gill- membrane and conductances. Both gCa-L and gCa-N were / 9k0e$$mr18ThisJ390-6 study represents 09-02-97 a detailed 13:48:49 dissection neupal of LP-Neurophys the func- ing curve by the input resistance itself), and steady-state action 21 21 kinetics of I . These results indicate that the 5-HT-induced regulated negativelytail-withdrawal by [Ca reflexes] (i.e., Ca of-dependentAplysia inactivation),is believed and to both be gK,Ca-S a keyand gK,Ca-F were activatedK-V (i.e., positively tional roles of some of the different elements of the siphon- potential threshold (uss , the membrane potential at which action regulated) by [Ca21]. Although not explicitly modeled as biochemical components, the regulatory relationships of PKC and PKA mechanism underlying short-term heterosynaptic facilitation modulation of IK-V is mediated, at least in part, by the cAMP/ elicited siphon-withdrawal circuit in Aplysia (see also Blazis potentials were initiated by just-threshold depolarizing constant are illustrated. See text for details. 1, enhancement or positive regulation; 2, decrease or negative regulation. current pulses). The spontaneously active LFS neurons were si- (for recent review, see Byrne and Kandel 1996). The first- PKA system. Moreover, studies of Goldsmith and Abrams et al. 1993). These issues are of interest in part because this 1 (1992; see also Shuster et al. 1991) suggested that the origi- circuit has been used for some time as a model preparation lenced by hyperpolarization before applying the depolarizing cur- et al. 1994).discovered Version 5 “serotonin-sensitive” of SNNAP was used and current the software was a was novel run K wascur- composed of a membrane capacitance (CM), which was assumed rent pulses to determine uss . The intracellular electrodes used in mnally2 described IK,S consisted of two components, a moderately for studies of learning-related cellular and synaptic plasticity under therent Windows that was 95/NT termed operating the systems S current on (PC-typeIK,S) (Klein microcom- et al. 1982).to be 1 F/cm (Almers 1978), in parallel with three inward currents these recordings were carefully balanced before making measure- puters (Baxter and Byrne 1999). The Euler method with a fixed time (1 Na1 andvoltage-dependent 2 Ca21 currents), six and K1 slowly-activatingcurrents, and a leak component current. The (IK,S-V), (Byrne and Kandel 1996; Castellucci et al. 1970; Frost et and an instantaneous (i.e., time-independent) “steady-state” ments of Rinput and uss . All examples of each neuron type were step of 30-mThes was costs used of publication for numerical of this integration. article were defrayed When simulations in part by the paymentionic currents in the model were described by Hodgkin-Huxley-type al. 1988; Hawkins et al. 1993; Kandel and Schwartz 1982). from different preparations. After these measurements, all neurons were begun,of page there charges. typically The article was amust small therefore (,500 be herebymV), brief marked (; “advertisement1 s) equations” component in which generalized that was Boltzman-type activated at the equations resting defined potential the (IK,S-I). In addition, because many features of the circuit, such as were injected with a series of depolarizing constant current pulses transientin before accordance the resting with 18 membrane U.S.C. Section potential 1734 settledsolely to to indicate its steady- this fact.voltage- andThus time-dependent 5-HT-induced activation increases and in the inactivation levels of of cAMP conduc- can lead to 21 recurrent and lateral inhibition, are common to vertebrate to assess their repetitive firing properties (see RESULTS for details). state value (250 mV). To avoid analysis during this or any other tances. In addition, the descriptions of four conductances (the 2 Ca motor circuits (Dowling 1992; Eccles 1964; Pompeiano Except for L29, all repetitive firing measurements were made in transient, $10 s of simulated time was allowed to elapse before data conductances and 2 of the K1 conductances) were expanded to 2914 0022-3077/99 $5.00 Copyright © 1999 The21 American Physiological Society 21 1984; Renshaw 1941), our results may have some relevance ASW. L29 posed the difficulty that it recruits recurrent IPSPs back were taken. include Ca -dependent regulation (e.g., Ca -dependent inactiva- onto itself from the L30 neurons, causing it to fire in a characteristic The soma of a sensory neuron was modeled as a sphere 20 mm in tion and activation, respectively; Fig. 2B). The details of the equations to motor circuits in general. 4 2 stuttering fashion (Frost et al. 1988). To eliminate this feedback diameter that had a surface area of 1.2 3 10 mm . This surface area and parameters are given in the APPENDIX and Table 1. Some aspects of this work have been published previously in abstract form (Frost et al. 1991). and thus allow us to measure L29’s intrinsic current-frequency included a factor of 10 to account for membrane invagination (Gor- Whenever possible, previously published voltage-clamp data from relationship, we measured L29 excitability in three preparations in man and Mirolli 1972; Mirolli and Talbott 1972). In the present study, sensory neurons were used to formulate the descriptions of the cur- the presence of 100 mM d-tubocurarine, which blocks the L30 we assumed the axon did not make a significantly contribution to the rents. These data have been collected from sensory neurons in both METHODS IPSP (Trudeau and Castellucci 1993a), thereby allowing L29 to biophysical properties of the sensory neuron or to their responses to abdominal and pleural ganglia. In the present study, these two sets of Experimental preparation fire smoothly to injected depolarizing current pulses. 5-HT. This assumption was based, in part, on previously published sensory neurons were assumed to have identical biophysical proper- Each model cell was constructed with the use of a four-step studies of action potentials, excitability, and their modulation by 5-HT ties and responses to 5-HT. This assumption was based, in part, on the A semi-intact preparation was used for all experiments, con- procedure (see APPENDIX for an explanation of the equations and in isolated somata and in ganglia preparations (cf. Sugita et al. 1992, work of Wright and Kirschman (1995), who made direct comparisons sisting of the abdominal ganglion attached by the siphon and parameters used in our simulation scheme). First, the experimen- 1997). Apparently identical results have obtained in both types of of the firing properties and effects of 5-HT on siphon versus tail preparation. The model consisted of a membrane model represented sensory neurons. They concluded that the properties and responses to by an equivalent circuit (Fig. 2A) coupled to equations that describe 5-HT of these two classes of sensory neurons were indistinguishable. the regulation of intracellular Ca21 (Fig. 2B). The membrane model Thus we (and others) generally have considered these two sets of / 9k0e$$mr18J390-6 09-02-97 13:48:49 neupal LP-Neurophys Approach using this system Memory Memory enhancement regulation/prediction New learning paradigm Memory paradigm Spatial & associative

Neural circuit Sensory-Motor neural circuit

Principle of synaptic plasticity New LTF induction protocol

Molecules A novel factor Bottom-up Reductionism "The whole is the sum of its parts" Memory regulation/prediction

Memory paradigm Holism Neural circuit "The whole is more than the sum of its parts"

Principle of synaptic plasticity

Molecules Bottom-up HABITUATION HABITUATION -BEHAVIOR

1. What’s the habituation? 2. Make your own model for habituation using Aplysia 3. Make your own hypothesis for mechanism of habituation WHAT’S THE HABITUATION? NEURAL CIRCUITS

IN APLYSIA98 | CHAPTER 3 LearningGILL Module- WITHDRAWAL■ HABITUATION, SENSITIZATION, AND FAMILIARIZATIONREFLEX

Figure 3.10 Neural Siphon Sensory neuron S circuits in Aplysia gill- withdrawal reflex (a) Sensory neurons S, T, and U respond to a touch on the siphon, tail, and Tail Sensory neuron T Motor neuron M Gill muscles upper mantle, respectively. These neurons converge on motor neurons such as M, which contract the gill muscles. (b) When sensory Mantle Sensory neuron U neuron S fires, it releases the neurotransmitter glutamate into (a) the synapse between S and M. The glutamate molecules (shown in yellow) may dock at receptors on neuron M. If enough receptors S S are activated, neuron M will fire, retracting the gill. (c) If neuron S is activated repeatedly, it gradually Fewer molecules releases less glutamate each time, released, so less decreasing the response of M. transmission This synaptic depression underlies Glutamate habituation of the gill-withdrawal response in sea hares. M M Glutamate receptor (c) (b)

large enough to be seen with the naked eye. Best of all, the pattern of neurons in Aplysia seems to be “hardwired,” meaning that researchers can identify a particular neuron in one sea hare (say, motor neuron L7G) and find the same neuron in the same place in another member of the species. This type of nervous system makes things much easier for a neuroscientist trying to understand how the brain encodes new memories. Neuroscientists have documented each of the neurons involved in Aplysia’s gill-withdrawal reflex. The siphon contains 24 sensory neurons that are directly connected to 6 motor neurons that innervate the gill. Figure 3.10a shows a simplified scheme of this system of neurons, consisting of three sensory neurons S, T, and U and one motor neuron M. When the siphon is touched, sensory neuron S fires, releasing a neurotransmitter, glutamate, into the synapse (Figure 3.10b). Molecules of glutamate diffuse across the synapse to activate receptors in motor neuron M. If enough receptors are activated, neuron M fires, causing the muscles to retract the gill for a few seconds. As simple as sea hares are, they are still capable of adapting their behavior in response to experience. Aplysia show habituation, sensitization, and several other forms of learning, just as rats and humans do. In Aplysia, however, scientists can actually watch the nervous system in action as these learning processes occur. Habituation in Sea Hares An initial, light touch on a sea hare’s siphon will activate the gill-withdrawal response, but if the touch is repeated, the gill-withdrawal reflex gradually becomes weaker, or habituates. The amount of habituation is proportional to the intensity of the stimulus and the repetition rate. If a sufficiently light touch is delivered every minute, the withdrawal response habituates after 10 or 12 touches and can last for 10 to 15 minutes (Pinsker, Kupfermann, Castellucci, & Kandel, 1970). In sea hares, we can see exactly what is causing this habituation. Refer back to the schematic diagram in Figure 3.10a. Recall that touching the siphon

Gluck2e_CH03.indd 98 28/11/12 2:04 PM HABITUATION -BEHAVIOR HABITUATION -SYNAPTIC MECHANISM HABITUATION -NEURAL MECHANISM SENSITIZATION SENSITIZATION -BEHAVIOR

1. What’s the sensitization? 2. How can you make a model for sensitization? 3. What’s your hypothesis for sensitization mechanism? WHAT’S THE MECHANISM OF SENSITIZATION?

Hint: Opposite to habituation => what’s the final goal in cell level? SENSITIZATION -BEHAVIOR SENSITIZATION -BEHAVIOR What does the tail shock induce in this circuit? SIPHON WITHDRAWAL REFLEX CIRCUIT & NEURAL ACTIVITY SENSITIZATION CIRCUIT

100 | CHAPTER 3 Learning Module ■ HABITUATION, SENSITIZATION, AND FAMILIARIZATION

Siphon Sensory neuron S

Tail Sensory neuron T Sensory neuron M Gill muscles

I N

Mantle Sensory neuron U (a)

Figure 3.11 Sensitization in Aplysia (a) A tail shock activates sensory neuron T, which activates motor neuron M, causing the motor S

response. T also activates an interneuron IN, which delivers a neuromodulator (such as serotonin) to the axons of neurons S and U. (b) Subsequent More vesicles, activation of neuron S will cause a larger release of so more neurotransmitter (glutamate), leading to greater transmission activation of neuron M than was previously evoked by S.

(b)

interneuron , as its name suggests, is a neuron that neither directly receives sensory inputs nor produces motor outputs but instead carries a message between two other neurons. A modulatory interneuron is an interneuron that alters the strength of the message being transmitted. You’ll recall from Chapter 2 that neuromodulators are neurotransmitters that can affect activity in entire brain areas rather than just at a single synapse. In Aplysia, interneuron IN connects neuron T to both S and U, communicating with them by releasing a neuromodulator such as serotonin. Serotonin increases the number of glutamate vesicles available to release glutamate from neuron S each time it fires. In effect, the interneuron does not tell S whether to fire; instead, it tells S, “When you do fire, fire strongly.” Suppose the sea hare now experiences a mild touch to the siphon. Before the tail shock, this mild touch would have caused neuron S to release neurotransmitter from a small number of vesicles, leading to a weak gill withdrawal (as in Figure 3.10b). But now, this mild touch causes neuron S to release neurotransmitter from a larger number of vesicles (Figure 3.11b), so that M is more likely to fire, leading to a stronger gill-withdrawal response (Brunelli, Castellucci, & Kandel, 1976; Castellucci & Kandel, 1976). In effect, the prior tail shock at T has put the slug on alert, making it sensitive to a subsequent light touch. The key to sensitization is that it is heterosynaptic, meaning that it involves changes across several synapses, including synapses that were not activated by the sensitizing event. Because of this feature, a tail shock increases responses to any future stimulus. For example, if the experimenter touched the upper mantle (activating sensory neuron U) instead of the siphon (neuron S), the same over- reaction would occur. In effect, the tail shock has increased the sea hare’s level of arousal, making it more likely to respond to any stimulus that follows. As with habituation, the degree of sensitization depends on the strength of the initial stimulus: a single mild shock can produce sensitization that lasts for minutes; four or five shocks together can produce sensitization that lasts two or more days (Marcus et al.,

Gluck2e_CH03.indd 100 28/11/12 2:04 PM Heterosynaptic facilitation 5-HT works! & Molecular pathway: PKA & PKC pathway

Neuromodulator

How about the lasting time? SENSITIZATION -NEURAL MECHANISM

Imagine what’re changed in synapse!

Short-term memory 1. Channel 2. Vesicle release Molecular mechanism of STM Cellular model : easy to study molecular mechanism

1x5-HT

Motor neuron

Sensory neuron Short-term sensitization Molecular mechanism Art by HFK LONG-TERM MEMORY FORMATION SENSITIZATION -NEURAL MECHANISM

Imagine what’re changed in synapse!

Short-term memory

1. Channel 2. Vesicle

How can you make long-term change in synapse? SENSITIZATION -NEURAL MECHANISM

Imagine what’re changed in synapse!

Short-term memory Long-term memory

1. Channel 1. Change of synaptic structure 2. Vesicle

How can you make long-term change in synapse? SENSITIZATION -STRUCTURAL CHANGE

What’s necessary for structural change? WHAT’S NECESSARY FOR THE SYNAPTIC CHANGE? Cellular model : easy to study molecular mechanism

5x5-HT

Motor neuron

Sensory neuron SENSITIZATION -MOLECULAR MECHANISM SENSITIZATION -TRANSCRIPTION & TRANSLATION Long-term memory Sensory neuron CREB1

AF CREB (cAMP response element- C/EBP binding protein) is a cellular transcription factor. It binds to certain DNA sequences called cAMP response elements (CRE), thereby increasing or decreasing the transcription of the downstream genes. Interneuron PKA

Late genes

G 5x5-HT Synaptic 5-HTR change Long-term memory Motor neuron SENSITIZATION -NEURAL MECHANISM

Imagine what’re changed in synapse!

Short-term memory Long-term memory

1. Channel 1. Change of synaptic structure 2. Vesicle 2. Biosynthesis Nu

FURTHER QUESTION

One neuron has lots of synapses. How does it discriminate a specific information? SENSITIZATION -SYNAPTIC : IT’S SPECIFICITY SENSITIZATION -SYNAPTIC : IT’S SPECIFICITY SENSITIZATION -SYNAPTIC TAGGING & CAPTURE SENSITIZATION -SYNAPTIC TAGGING & CAPTURE

Synaptic capture

Synaptic tagging

When is it necessary? SENSITIZATION -SYNAPTIC CAPTURE &TAGGING : MOLECULAR MECHANISMS SENSITIZATION -LOCAL PROTEIN SYNTHESIS

CPEB is one of the tagging molecules.

CPEB, or cytoplasmic polyadenylation element binding protein, is a highly conserved RNA- binding protein that promotes the elongation of the polyadenine tail of messenger RNA. PRIMING

Practical application! Let’s make a model of priming! What’s the priming? Priming is an implicit memory effect in which exposure to one stimulus (i.e., perceptual pattern) influences the response to another stimulus.

What’s difference in the molecular mechanism? Let’s extract the important points of concepts!

Weak stimulus STM 1. Learning Strong stimulus LTM

2. Learning more Strong stimulus LTM Enhancement quickly

Weak stimulus

3. Priming? Stimulus No effect Effect

Weak stimulus What’s difference in the molecular mechanism?

Siphon pinching (SP) Tail shock Siphon withdrawal reflex

t 30 min 24 hr 5 min 60 mA, 1 sec Siphon pinching (SP) Tail shock Siphon withdrawal reflex

t 30 min 24 hr 5 min 60 mA, 1 sec PRIMING! APLYSIA REMEMBERS A PREVIOUS EVENT!

Siphon pinching

Abdominal ganglion

Siphon The Role of ApLLP in Synaptic Plasticity 715

previous reports showing that coincident pairing with tetanus and 5-HT induces long-lasting facilitation (Eliot PRIMING!et al., 1994; Sun and Schacher, 1998). In the previous study, in order to mimic classical conditioning in Aplysia, APLYSIA5-HT was appliedREMEMBERS to a cocultured Aplysia neuron 0.5 s after the onset of tetanus (paired) or 3 min after the A endPREVIOUS of tetanus (unpaired). Long-lastingEVENT! facilitation is induced only in paired neurons immediately or 24 hr after pairing of the two stimuli. This facilitation is believed to result from gene expression by calcium/calmodulin- sensitive cyclase-mediated signaling, which is only activated by temporally coincident stimulation of tetanus and 5-HT (Abrams et al., 1991). However, in our experiments, depolarization itself increases the ApC/EBP mRNA level through ApLLP expression, 15 min after high potassium treatment. There- fore, the long-term increase in synaptic strength can be produced by 5-HT treatment in presynaptic sensory neurons that have high levels of ApC/EBP mRNA, and this increase is mediated by the ApLLP induction. More- over, because a noxious stimulus produces the ApLLP and ApC/EBP mRNA induction (Figure 7A), our model suggests that if sufficient neural stimulus is given to the Aplysia neurons prior to 5-HT treatment for inducing activity-dependent transcription and translation, it is possible to induce a long-term increase in synaptic strength and long-term memory in Aplysia. However, because noxious stimulus could induce not only the depolarization of sensory neurons but also the secretion of (Kim et al., 2006) neuromodulators including 5-HT, nitric oxide, and hista- mine in the abdominal ganglion of freely moving Aplysia, it is possible that SNS-induced neuromodulators contribute to the ApLLP induction (Jacklet et al., 2004). Figure 8. Schematic Model of ApLLP Functions in Synaptic Plastic- Noxious Stimulus-Elicited ApLLP Induction Could ity and Siphon Noxious Stimulus-Facilitated Sensitization Facilitate Long-Term Memory Formation in The siphon sensory neuron (siphon SN), siphon motor neuron Siphon-Elicited Siphon Withdrawal Reflex of (siphon MN), and serotonergic neuron (5-HT) are represented sche- Aplysia: Its Implication for a Learning Paradigm matically. SNS-induced neural activity activates calcium signaling and induces ApLLP although the molecular mechanism of ApLLP in Aplysia and Mammalian Models induction is not clear yet (represented as question marks). The In this study, we showed that the long-term sensitization induced-ApLLP directly binds to the ApC/EBP promoter and acti- can be produced by only a single tail shock after 30 min vates the transcription of ApC/EBP mRNA. This ApLLP-dependent after a noxious stimulus that induces both ApLLP and ApC/EBP induction can lower the threshold for the 5-HT-elicited ApC/EBP expression. The high potassium treatment LTF. Thus, treatment with a single 5-HT pulse, which produces has been used to mimic the axotomy-induced strong only short-term synaptic plasticity by transient modification of proteins, switches a short-term to a long-term increase in synaptic neural activity in the Aplysia neurons (Weragoda et al., strength. As parallel, a single tail shock, which produces only 2004). Therefore, ApLLP was induced by strong neural short-term sensitization, induces the long-term sensitization in activity that was generated by a stressful event such Aplysia behavior model. as by pinching the Aplysia siphon for 5 min. It is reported that some stressful experiences before the learning event facilitate learning and memory forma- and long-term memory. Upon treatment of one pulse tion in the mammalian system. Shors et al. (1992) have 5-HT or a single tail shock (learning), which produces shown that exposure to an acute stressor of restraint only short-term plasticity by transient modification of and intermittent tail shocks enhances classical eyeblink proteins (Bartsch et al., 2000; Lee et al., 2001), ApLLP-in- conditioning after a day in the male rat. However, the duced ApC/EBP switches short-term into long-term fa- cellular and molecular mechanisms underlying these be- cilitation or sensitization (Figure 8). These suggest that haviors have not been well studied. ApLLP-elicited ApC/EBP induction by noxious stimulus In the cellular model of ApLLP-induced LTF, the before the learning could be a molecular trace of stress- strong neural activity elicited by SNS (stressful experi- ful event to enhance the long-term memory formation. ence) induces ApLLP expression through calcium-acti- The homologs of ApLLP have been searched in the vated signaling cascade although the molecular mecha- EST databases of various organisms (Kim et al., 2003). nism of ApLLP induction remains to be investigated Recently, yeast and human homologs of ApLLP have (Finkbeiner and Greenberg, 1998; Lin et al., 2003). been identified in the yeastgfp database and nucleolar Then, ApLLP induces ApC/EBP expression and subse- proteome database (http://yeastgfp.ucsf.edu/ and quently lowers the threshold for the induction of LTF http://lamondlab.com/nopdb/). The sequence analysis P

Unexpressed cellular memory Cellular unconsicousness memory (?) MODELING Dual processINTERACTIONS theory OF DEPRESSION AND FACILITATION

Figure 1: The combination of habituation and sensitization to produce dual-process learning. (A1–A4) Decomposition of dual- process learning curve (solid line) into component habituation learning curve (broken line labeled H) and habituating sensitization learning curve (broken line labeled S) at four different stimulus intensities increasing between A1 and A4. Empirical data are shown as dots. (B) The contribution of op- posing processes at different stimulus intensities. Stimulus intensities used in A are illustrated as open arrows pointing along the stimulus axis. Below each of these arrows are shown the learning processes recruited at that stimulus intensity. The arrow marked H indicates habituation (response decrement by depression); the arrow marked S indicates sensitization (response increment by facilitation). Stimulus 1 elicits strong habituation; stimulus 4 elicits only weak habituation but also elicits strong sensitization; stimuli 2 and 3 are intermediate. An important addition to this interaction is that the lower limit for eliciting sensitization increases (indicated by arrow marked with star) consequent to depression early in the neural circuit (see text), meaning that the balance between habituation and sensitization shifts in favor of habituation over time, explaining the bumpy shape of the curves seen in A. A is reprinted, with permission, from Groves and Thompson (1970). Copyright 1970 by the American Psychological Association. balance between learning processes is dynamic, 1996). Data also suggest that dual-process learning shifting as the stimulus is repeated.Homosynaptic Extending be- maydepression occur in the siphon-elicited GSW reflex (see yond Groves and Thompson’s discussion, is there a below). The occurrence of dual-process learning is physiological basis in the neuralHeterosynpatic network for this notfacilitation explicitly recognized in all of these publica- changing balance? The data presented herein sug- tions, but in each case, the reflex exhibits plasticity gest that the kinetics of dual-process learning can like that described by the learning curves in Figure be explained by the relative positioning of learning 1, A1–A4, namely transient sensitization followed processes within the neural network. by habituation or, at least, a delayed onset of net Groves and Thompson’s ideas were based on habituation. investigations of the hindlimb flexion reflex of the acute spinal cat and the startle response of the intact rat, both of which exhibit dual-process learn- Review of Physiological Data ing (Groves and Thompson 1970). More complex HINDLIMB FLEXION REFLEX IN THE CAT behaviors such as the mobbing response of chaffinches (Hinde 1970) and aggression in three- The dual-process theory of plasticity stemmed spined sticklebacks (Peeke 1969, 1983; Peeke and largely from work on the spinal cat. In these ex- Veno 1973) also exhibit plasticity consistent with periments, electrical stimulation is applied to the the dual-process theory, as do certain human be- skin or to a cutaneous nerve, and the reflex con- haviors such as infant visual attention (Bashinski et traction of a flexor muscle is measured isometri- al. 1985; Kaplan and Werner 1986). Dual-process cally. Habituation of the flexion reflex occurs in learning is also common, though not ubiquitous, the acute spinal cat with all nine parametric char- amongst invertebrate withdrawal responses. These acteristics described by Thompson and Spencer responses include the withdrawal response in the (1966). The reflex also exhibits extrinsic sensitiza- earthworm (Roberts 1966), whole body with- tion (Spencer et al. 1966a; Thompson and Spencer drawal in the snail Lymnaea (Cook 1975), tentacle 1966) and intrinsic sensitization (Groves et al. withdrawal in the snail Helix (Christoffersen et al. 1969a,b). Interestingly, both forms of sensitization 1981; Zakharov and Balaban 1987; Balaban 1993; habituate if the sensitizing stimulus is repeatedly Prescott and Chase 1996; S.A. Prescott and R. applied (Spencer et al. 1966a; Thompson and Spen- Chase, in prep.), local bending in the leech (Lock- cer 1966; Groves and Thompson 1970). ery and Kristan 1991), escape response in the crab To date, the polysynaptic circuit mediating the (Rakitin et al. 1991), swim response in Tritonia reflex remains incompletely understood (see Mos- (Brown et al. 1996), and tail-induced siphon and chovakis et al. 1992; Burke 1998), making it impos- tail withdrawal in Aplysia (Stopfer and Carew sible to localize precisely the plastic loci. Data are

LEARNING& MEMORY 449 SUMMARY Mechanism of long-term memory 1. New transcription factor 2. New scheme for synaptic plasticity

3. New memory paradigm sensitization (6). Both short-term sensitiza- As a first step in exploring the molecular before or after 5-HT application did not tion, lasting minutes to hours, and long- mechanisms underlying long-term plasticity, block long-term facilitation. These results term sensitization, lasting days, involve we have further simplified the analysis of the suggest that long-term synaptic facilitation changes in the strength of the connections neuronal circuit by reconstituting the mono- requires the synthesis of gene products not that the sensory neurons make on their synaptic sensorimotor component of the essential for the expression of short-term central target cells, the interneurons and the gill-withdrawal reflex in dissociated cell cul- facilitation. Protein and RNA synthesis is motor neurons. Short-term sensitization re- ture. We simulated the behavioral trainingS CIENCErequired’ S C OMPASSduring a period of 1 to 3 hours, sults from the heterosynaptic facilitation28. F.of Desdouits,necessary J. C. Siciliano,to P.produce Greengard, J.long-term A. Girault, 40.sensitization A. A. Fienberg et al.,duringScience 281and, 838immediately (1998). afterhavethe workedapplication in our laboratory. I would particularly Proc. Natl. Acad. Sci. U.S.A. 92, 2682 (1995). 41. P. B. Allen, C. C. Ouimet, P. Greengard, Proc. Natl. like to mention A. C. Nairn, who has been a close transmitter release from the terminals of29.the A. Nishi,in G. L.the Snyder,intact P. Greengard,animalJ. Neurosci.(repeated17, trainsAcad.of Sci.head U.S.A. 94,of 99565-HT. (1997). colleague and friend for more than 20 years. This 8147 (1997). 42. Z. Yan et al., Nature Neurosci. 2, 13 (1999). work has also benefited enormously from collabora- sensory neurons produced by the actions30. G.of L. Snyder,or A.tail A. Fienberg,shocks R. L. Huganir,every P.30 Green-minutes43. J.–A.over Girault, H.2 C. HemmingsA single Jr., K. R. Williams,brief A.application C. tions withof excellent5-HT scientistsin at several other uni- serotonergic and other modulatory neuronsgard, J. Neurosci.hours),18,by 10297using (1998) five repeated applicationsNairn, P. Greengard, J. Biol. Chem. 264, 21748 versities. Our work on regulation of ion pumps was 31. P. Svenningsson et al., Neurosci. 84, 223 (1998). (1989). dissociated cell culture producedcarried outshort-term in collaboration with A. Aperia at the activated by tail or head shock (7). Long-32. A. Nishi,of G. L.serotonin Snyder, A. C.(5-HT). Nairn, P. Greengard,These repeated44. F. Desdouits,appli- D. Cohen,facilitation A. C. Nairn, P. Greengard,of the J.-A.sensorimotorKarolinskaconnection Institute. We continue to collaborate with J. Neurochem. 72, 2015 (1999). Girault, J. Biol. Chem. 270, 8772 (1995). R. L. Huganir, who was at The Rockefeller University, termn sensitization also leads to an enhance-33. P. Svenningssoncationset al.,producedJ. Neurochem. 75long-term, 248 (2000). facilitation45. J. A. Bibb etof al., Naturecomparable402(6762), 669in (1999).amplitude andand istime now atcourse The Johnsto Hopkins University School of 34. G. L. Snyder et al., J. Neurosci. 20, 4480 (2000). 46. E. F. da Cruz e Silva et al., J. Neurosci. 15(5), 3375 Medicine and with E. J. Nestler, who was at the Yale ment in the strength of the connections35. A. A. Fienberg,the P.connection Greengard, Brain Res.between Rev. 31, 313the sensory(1995). and the facilitation evoked in theUniversityintact Schoolnervous of Medicine and is now at the (2000). 47. P. Greengard, J. Jen, A. C. Nairn, C. F. Stevens, Science University of Texas Southwestern Medical Center. between sensory and motor neurons (6).36. P.In Svenningssonmotoret al.neurons, Proc. Natl.detected Acad. Sci. U.S.A.1 day later.253, 1135This (1991). system with natural stimulationMuch of our electrophysiologicalor with work has been done addition, long-term sensitization results in97a, 1856long-term (2000). facilitation was blocked,48. L.-Yselective- Wang, M. W. Salter,exogenous J. F. MacDonald,5-HITScience(9). Wein collaborationtherefore with D. J. Surmeier at Northwestern 37. A. Nishi et al., Proc. Natl. Acad. Sci. U.S.A. 97, 12840 253, 1132 (1991). University. The workfirst of our research group has been growth process leading to an increase (2000).in ly, without interfering with short-term49. L. Hsieh-Wilsonfa- et al.examined, unpublished data.whether these veryconnections generously supportedcan for over 30 years by the 38. J. A. Bibb et al., Nature 410, 376 (2001). 50. C. Rosenmund et al., Nature 368, 853 (1994). National Institutes of Health, including the National both the number of varicosities of the senso-39. H. C. Hemmingscilitation Jr., P.when Greengard,a H.specific Y. L. Tung,inhibitor P. 51. Theof workeither summarizedundergo here reflectslong-term outstanding con-facilitationInstitutein ofvitro Mentalwhen Health, the National Institute on iy neurons and in the incidence of activeCohen, Natureprotein310, 503or (1984).RNA synthesis was appliedtributions fromto- manyexposed highly giftedto associatesrepeated who applicationsDrug Abuse,of and5-HT. the National Institute on Aging. zones for transmitter release (8). REVIEW:getherNEUROSCIENCEwith 5-HT. Inhibitors applied either We co-cultured the gill motor cell L7 to- The Molecular Biology of Memory Storage: Fig. 1. Long-term facilitation of sensorimotor A i 5X 5-HT - 5-Ht connections with repeated applications of 5-HT is A DialogueIv BetweenV V V Genes and SynapsesT i blocked in the presence of inhibitors of protein or -4 -1 0 I 2 Hours on October 9, 2016 RNA synthesis. (A) Experimental protocol. After Eric R. Kandel* 24 I - Anisomycin or Emetine - 4 5 days in culture, sensory and motor cells were * 5-minute,form. Thiss-iM was5-HT the approach that Alden Spen- impaled and the motor cell was hyperpolarizedOne50 of the most remarkableI- Actinom,-D aspects of-I an animal’s behavior is the ability to modify a Recordingcer and Isessions took when we joined forces on October 3, 2016 at NIH in mV below the resting potential to permit accuratethat behavior by learning, an ability that reaches its highest form in human beings. For 1958 to study the cellular properties of the measurement of the amplitude of the EPSP. Theme, learning and memory have proven to be endlessly fascinating mental processes hippocampus, the part of the mammalian brain because they address one of the fundamental features of human activity: our ability thought to be most directly involved in aspects first recording session (0 hour, dark bar), eitherto in acquire new ideas from experience and to retain these ideas over time in memory. of111zu 2 complex memory (1). We initially asked, the absence or presence of the inhibitors, consist-Moreover,B unlike other mental processes such asC thought, language, and conscious-+ + _ ,- ratherI F naı¨vely:i Are the electrophysiological ed of the following. Every 20 seconds, a singleness, learning seemed from the outset to be readily accessibleM2I to cellular andIF , properties.. of the pyramidal cells of the hip- spike was evoked in the sensory neurons molecularby analysis. I,u therefore, have been curious to know: What changes in the brain pocampus, which were thought to be the key when we learn? And, once something is learned, how is that information0 x retainedxLa into1xe xin x10I tox injecting a depolarizing pulse. After the fifth 2- hippocampal cells involved in memory storage, the brain? I have tried to address these questions through a reductionist approach that http://science.sciencemag.org/ Control 100 fundamentally different from other neurons in action potential, 5-HT was applied in the bathwouldto allow me to investigate elementary forms of learning and memory at a cellular the brain? With study, it became9. clearW. toA. usBerggren, that D. V. Kent, J. J. Flynn, J. A. Van a final concentration of 1 pM and the extentmolecularof level—asSomeL.E JL-authorities specific molecularjL-have suggested activities withinthat, identifiedbe- tions, nervea cells.chronology that accords well with facilitation assessed with three additional evoked -.84 morphological analysesallthat nerveplace cells,some includingspe- the pyramidalCouvering, cells ofGeol. Soc. Am. BuU. 96, 1407 (1985). 2cause of their many primitive anthropoidc the hippocampus, have similar10. signalingR. Said, prop-The Geology ofEgypt (Elsevier, Amsterdam, EPSP's (see Fig. 2). In cultures with two sensory characteristics, one group of Fayum.c 60pri- cies at the base of the highererties. Therefore,primate theradia- intrinsic signaling1962). proper- neurons, short-term facilitation was assessed dur-first became interested in the study of mem-i the National0 Institutes of Health (NIH) in Be- ties of neurons would themselves11. The nothigher give usprimate status of the still poorly known mates, the parapithecids, may be relatedat 40-to tion. Burmese primates Amphipithecus and ing the recording from the second cell. Fourory in 1950IX 5-HT as a result of my readings in thesda from 1957 to 1960, I focused on learning key insights into memory storage (2). The Pondaungia http://science.sciencemag.org/ psychoanalysisthe New whileWorld still an undergraduateplatyrrhines (14).more aboutA majorS theCIENCE biology’ ofS theC OMPASS brain and became unique functions of the hippocampus[R. L. hadCiochon, to D. E.Downloaded from Savage, T. Tint, B. Maw, minutes later, the 5-HT was washed outI by Science 229, 756 (1985)] is unsettled. at Harvard College.LE Later, during medical train- interesteda in20 knowing how learning produces arise not so much from the intrinsic properties perfusion with 50% L15 and 50% artificial sea- line28. F.of Desdouits,evidence J. C. Siciliano,against P. Greengard,an J.African A. Girault, origin40. A. A. Fienbergfor et al., Science 281, 838 (1998). have worked in our laboratory. I would particularly12. P. Andrews, in Ancestors: The Hard Evidence, E. ing, I began to findProc. the Natl. psychoanalytic Acad. Sci. U.S.A. 92 approach, 2682 (1995). changes41. P. in B. the Allen, neural C. C. Ouimet, networks P. Greengard, ofREFERENCES theProc. brain. Natl. ANDoflikepyramidal to mentionNOTES A. C.neurons Nairn, who but has from been a the close pattern of water,pH 7.6, at a rate of 1.5 ml per minutelimiting(0.5- becauseplatyrrhines29. A. it Nishi, tended G. L. to Snyder,has treat P. theGreengard,been brain,J.the Neurosci. the apparent17, My purposeAcad.differ- Sci. U.S.A. in translating94, 9956T (1997). questions about functionalcolleague and friendinterconnections for more than 20 of years. these ThisDelson, cells,Ed. and (Liss, New York, 1985), pp. 14-22. 8147 (1997). 42. Z. 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Jen, A.combine C. Nairn, C. theF. Stevens, mentalisticScience esUniversity behavioral of Texas output. Southwestern This Medical was Center. a formidable thropol.Downloaded from Paris 4, 927 (1977). ter each 5-HT application, 5-HT was washedalysts,out couldmillion36. be P. investigated Svenningssonyearsetolder with al., Proc. thethan Natl. methods Acad.the Sci.supposed U.S.A.psychology253age,of 1135 memoryof (1991). storageCorruccini, with theEds. biology(Plenum,challenge,MuchNew of our electrophysiologicalYork, since1983), the hippocampus workpp. has been done has a large 97, 1856 (2000). 48. L.-Y Wang, M. W. Salter, J. F. MacDonald, Science B. J. B. at a rate of 1.5 ml minute. Control cultures The cultures were 15 in 181-210. in collaboration with D. J. Surmeier at Northwestern15. MacFadden etal.,J. Geol. 93, 223 (1985); J. per of modern biology.37. A. Nishi Aset al.a, result,Proc. Natl.then my Acad.perfused interest Sci. U.S.A. in97for, 12840of neuronalminutes253, 1132 signaling. (1991).Summary I hoped furtherof long-term that the facilitationnumberUniversity. Theofinduced neuronswork of our andby research5- an group immense has beenMacFadden,J. number of Paleontol. 169 the (2000).Fayum primates. However, more49. L.recent Hsieh-Wilson et al.4., unpublishedV. Maglio data. and H. B. S. Cooke, Eds., Evolution of Vet. 5, (1985). received the same number of evoked EPSP'smemorybut shifteddrug-free frommedium. a psychoanalyticActinomycin to a Dbiological(50 ,Lg/ml) analysis ofHT memory might carry with interconnections.very generouslyRNA supported It for seemed over 30 years unlikely by the that we 38. J. A. Bibb et al., Nature 410, 376 (2001). 50. C. Rosenmund et al., NatureandAfrican368blockade, 853Mammals (1994). by(HarvardproteinNationalUniv.and InstitutesPress, ofCambridge,synthesis Health, including the16. NationalWe thank the Geological Survey of Egypt for assist- either no 5-HT or a single of biologicalwas approach.studies39.added H. C. AsHemmingsof a1 postdoctoralthehour Jr.,geology P. Greengard,before fellow H.of Y.the at L.the Tung,firstitSalla P. anrecording51. extrabasin The work bonus,in summarized that here the reflects study outstanding of memory con- wouldInstitute of be Mental able Health, to work the National out Institutein anyance on reasonableand cooperation, especially B. Issawi, R. Eissa, application 5-HT, Cohen, Nature 310, 503 (1984). tributions from manyinhibitors. highly1978); giftedA,T. associatesanisomycin;M. whoBown andDrugE, E.emetine; Abuse,L. andSimons, theAD, NationalNatureacti- Institute on Aging. and were continuously perfused for 2 hours. After session,Boliviaafter(15)whichhavetheshowncells werethatperfusedstoragethe mightearliestwith revealnomycin new(London) aspectsD; a-A, of308, neuronala-amanitin.447 (1984).periodThe ofheight time howof each the neural networks,B. el-Khashab, in A. A. el Awady Kandil, A. el Ghany REVIEW: NEUROSCIENCE signaling. Indeed, this has proven true. which the hippocampus was embedded,Ibrihim partic-Shellaby,KandelandMolecularM. Askalany; Brain 2012, 5and:14 M. Kraus, 2 hours, both experimental and control culturesHoward Hughesdrug-freeplatyrrhines Medicalmedium. Institute,are CenterActinomycinprobably for Neuro- fromD wasaboutnot per-25 bar5.isT.theM.meanBown±et SEMal., J. Hum.of theEvol.percent11, 603change(1982).in 6. J. G. Fleagle, T. M. Bown,ipateJ. D. inObradovich, behavior andE. howL. those networksP. Chatrath, are D.http://www.molecularbrain.com/content/5/1/14Domning, T. Rasmussen, P. Gin- were retumed to culture medium and replacedbiologyin andfused Behavior,continuously College of Physiciansbecause and Sur-a 1-hourA Radicalexposure Reductionistamplitude Strategyof the tofirst evokedaffectedEPSP byrecorded learning. at 0 geons of Columbiamillion University,years Newago, York Statesubstantially Psy- younger Simons, in Primate J. G. Else and P. C. gerich, R. Kay, S. Wing, and D. Krause for informa- the incubator at 18°C. At 24 both irreversibly blocked total RNA Learningin and Memory Evolution, hours, experi-chiatric Institute,than 1051theThe RiversideFayum Drive, Molecularanthropoids. New York, NYsynthesisThus, the BiologyAply-new hoursLee,compared ofEds. Memory(Cambridgeto the firstUniv.evokedTo bringPress, Storage:EPSP theCambridge, powerof the of moderntion biologyand suggestions. to Supported in part by BNS mental and control cultures were reexamined.10032,The USA.sia E-mail:neurons [email protected] more than 90%. (B) AtSensorimotor first thought, someonesame interestedconnection1986), pp. in3-17. learningreexaminedbearat 24 on thehours study(see ofFig. learning, it seemed8310913, nec-BNS 8546024, and BNS 8521655, from same cells were impaled, and the strength of*Thisthe essayconnections isminimum adapted fromAshowage the Dialogue author’sestimatelong-term addressoffacilitation tothe andJebel Between memoryafterQatranire- might1B). be7. temptedT. GenesEachM. Bownculture to tackleand(oneM. andthe J.L7Kraus,essarywith SynapsesU.S. toone takeGeol.or aSurv.two veryProfLE different approach—aNSF and 70869600REVIEWand 809479 from the Smith- Open Access the Nobel Foundation, December 2000. problem in its most complex and interesting radically reductionist approach.sonian We neededForeign Currency Program Duke Primate in on October 9, 2016 connections and short-term facilitation reexam- peatedFormation5-HT applications.indicates thatThe firsttheevokedFayumEPSPpri- Ericsensory R. Kandel*Pap.,neuronpress.synaptic connections) was treated ined (24 hour, dark bar) as during the 0-hour inmatesthe motorare cellsubstantiallyL7 [top tracesolderin (B,),than(B2),the andfirst as 8.a E.singleL. Simons,sample.Sci. TheAm. 217,number28 (Decemberof samples1967);is Center publication 415. 1030 2 NOVEMBER 2001 VOL 294 SCIENCEand www.sciencemag.orgA. E. Wood,form.Bull. This wasPeabody the approachMus. Nat. that Alden Spen- The molecular biology of memory: cAMP, PKA, recording session. Cultures treated with drugs (B3),appearancesOneduring of thethe mostof0-hourall remarkablemodemrecording aspectsanthropoid of ansession animal’sradia-(left behaviorindicated is theHist. ability28,in to1 modifyparentheses.(1968). cer andIn I tookcases whenwhere we joinedtwo forces at NIH in14 July 1986; accepted 10 October 1986 alone Table also received identical stimula- that behavior by learning, anthe ability that reaches its highest form in human beings. For 1958 to study the cellular properties of the CRE, CREB-1, CREB-2, and CPEB (see 1) column)]me, learningis compared and memoryto have provenfirst evoked to be endlesslyEPSP fascinatingsensory mentalneurons processesformedhippocampus,synaptic theconnections part of the mammalian brain tion regimens but no 5-HT (23). Anisomycin (10 for thebecausesame theyconnection address oneat ofthe the24-hour fundamentalrecording features of humanwith activity:L7 in oura single ability culture,thoughtthe to bepercent most directlychange involved in aspects Eric R Kandel p.M) or emetine (100 pM) was added to the sessionto acquire(right newcolumn). ideas fromWhen experienceno 5-HT and to retainapplica- these ideaswas overcalculated time in memory.as the meanof complexchange memoryfor (the1). Wetwo initially asked, Moreover, unlike other mental processes such as thought, language, and conscious- rather naı¨vely: Are the electrophysiological cultures 1 hour before the initial recording and tionsness,were learninggiven seemed[control from(B,)] thethe outsetamplitude to be readilyof accessiblesynapses. toAn cellularoverall and analysispropertiesof variance of the pyramidalindicated cells of the hip- molecular analysis. I, therefore, have been curious to know: What changes in the brain pocampus, which were thought to be the key Abstract was present continuously until 30 minutes after the EPSPwhen weshows learn? And,a slight once somethingdecline. isTwenty-four learned, how is thata informationdifference retainedwith intreatment (F = 3.02; df = 6, The interpretation of earlier experiments, A Critical Period for Macromolecular Synthesishippocampal cells involvedin in memory storage, Cell,The Vol. analysis 95, 211–223, of the October contributions 16, 1998, toCopyright synaptic1998 plasticity by Cell and Press memory of cAMP, PKA, CRE, CREB-1, CREB-2, and CPEB the last 5-HT application. The transcriptional hourstheafter brain?a Isingle have tried5-HT to addressapplication these questions[1x 5-HT through a reductionistP < approachA that means http://science.sciencemag.org/ 41; 0.05). comparisonfundamentallyof the different from(mul- other neurons in on October 3, 2016 would allow me to investigate elementary forms of learning and memory at a cellular based on the systemichas recruitedapplication the efforts ofof manyprotein laboratories all over the world. These are six key steps in the molecular biological the brain? With study, it became clear to us that inhibitor a-amanitin (2 ,ug/ml), which penetrates (B2)]molecularthe EPSP level—asis unchanged. specific molecularA significant activities withinin- identifiedtiple nervecomparison cells. t test) indicated that 5 x 5-HT delineation of short-term memory and its conversion to long-term memory for both implicit (procedural) and Long-Term Heterosynaptic Facilitation inall nerveAplysia cells, including the pyramidal cellssynthesis of inhibitors, was limited, however, cells slowly, was added to cultures 4 hours before crease in the amplitude of the EPSP was obtained CREB1explicit (declarative)Encodes memory. I here a first Nuclear trace the background Activator, for the clinical and a behavioral Repressor, studies of implicit significantly increased EPSPthe hippocampus,amplitude havecompared similar signaling prop- in two ways. memoryFirst, thatthe madeprotein a molecularsynthesis biology of memory storage possible, and then detail the discovery and early the initial recording and was present in the perfu- after five applications of 5-HT [5x 5-HT (B3)]. to the other treatmentserties.(CR Therefore,= 57, theP intrinsic< 0.05), signaling proper- and a Cytoplasmic Modulator that Form a Regulatory sion medium throughout the 5-HT applications ForP.detailsfirstG. becameMONTAROLO,*on interestedrecording in thesessions study ofP.and mem-GOELET,protocol,the Nationalsee V. InstituteswhereasF. ofCASTELLUCCI, Healththe (NIH)other intreatments Be- tiesJ. ofMORGAN, neuronsdid not woulddiffer themselvesfrom not giveinhibitors us oftenhistoryled ofto theseserious six molecularside stepseffects and their roles in explicit memory. ory in 1950 as a result of my readings in thesda from 1957 to 1960, I focused on learning key insights into memory storage (2). The Unit Critical for Long-Term Facilitation until 30 minutes after the last 5-HT application. Fig.E.psychoanalysisLA.R.CalibrationKANDEL, while stillbars:S. an undergraduate10SCHACHERtmV, 20 msec.more about(C) the biologyeach ofother the brainor andcontrol. became unique functions of the hippocampussuch had to as seizureDownloaded from and sickness (3). Second, the I Background: Simple systems in the study of memory that had been advanced up to that time: the at Harvard College. Later, during medical train- interested in knowing how learning produces arise not so much from the intrinsic properties of implicit learning†‡ and memory† † aggregate field approach advocated by Karl Lashley in ing, I began to find the psychoanalytic approach changes in the neural networks of the brain. of pyramidal neurons but from the patternstudies of were Dusanbased Bartsch,on theAndreaexamination Casadio, Kevinof A. Karl,* neurotrophins, CRE sequences are often found in the I250 SCIENCE, VOL. 234 PauloBy 1969, Serodio, we had† and already Eric R. learned Kandel* from† the pioneering theupstream 1950sregulatory and by Ross regions Adey of genesin the transcriptionally 1960s, which Bothlimitinglong-term because it tendedand to treatshort-term the brain, the sensitizationMy purpose in translatingofthe gill questionsand aboutsiphonfunctionalwithdrawal interconnectionsreflex of thesein cells,behavioral and performance rather than on the organ that generates behavior, as a black box. In the psychology of learning into the empirical how those interconnections are affected by *Howardwork of Hughes Brenda MilnerMedical that Institute certain forms of memory assumedresponsive that to information these signaling is stored pathways in the bioelectric (for review, field see † Aplysiathe mid-1950s,involve while stillfacilitation in medical school,of Ithelanguagemonosynaptic of biology was notconnections to replace the logicbetweenlearning. Tothe tacklesensory that problemand we neededanalysis to of thewereCentercellular stored for Neurobiologycorrelates in the hippocampus andresponsible Behavior and the medial temporal generatedHabener etby al., the 1995). aggregate activity of many neurons; began to appreciate that during my lifetime the of psychology or psychoanalysis with the logic know how sensory information about a learning Collegelobe. In of addition, Physicians the workand Surgeons of Larry Squire of Columbia revealed that andThe the initialcellular molecular connectionist characterization approach, which of the derived role of motorblack box ofneurons. the brain wouldTo be openedanalyze and thattheofrelationship cellular molecular biology,between but to trythese to join twotask reachesforms theof hippocampussynaptic and howfor infor-the neuronalthereUniversity areplasticity two and major memoryunderlying systems inthe the brain: explicit from cAMP, Santiago PKA,and Ramon CREB y Cajal's in synaptic idea [5] plasticity which postulated came from the problems of memory storage, once the ex- these two disciplines and to contribute to a new mation processed by the hippocampus influenc- Newor declarative; York State implicit Psychiatric or procedural. Institute Explicit memory, a thatstudies learning of Aplysia resultswhere from changes the synapse in the between strength the of sen- the at altered New York,Inhibitors New Yorkof 10032 sory and motor neurons of the gill-withdrawal reflex can facilitationclusive domain of psychologiststhe cellular and psychoan-andsynthesismolecular that wouldlevel, combinethis the mentalisticmonosynaptices behavioralsensorimotor output. This was a formidable behavior.memory for facts andprotein events—forsyn- people, places, and synapse. The cellular-connection idea was later renamed be reconstituted in primary cell culture. In response to alysts, could be investigated with the methods psychology of memory storage with the biology challenge, since the hippocampus has a large objects—requires, as Milner has pointed out, the medial synaptic plasticity by Konorski in his 1948 book: Condi- component of the gill-withdrawal reflex ofAplysia can be reconstituted in dissociated thesis can affect behavioral performance not one pulse of serotonin (5-HT), a modulatory transmitter of modern biology. As a result, my interest in of neuronal signaling. I hoped further that the number of neurons and an immense number of temporal lobe and the hippocampus [1-3]. By contrast, tioned Reflexes and Neuronal Organization [6]. Konors- released by sensitization stimuli, this synapse under- cellmemoryculture. shifted fromWhereas a psychoanalyticone brief to a biologicalapplication analysis ofof memory1 ,uM mightserotonin carry with interconnections.produced short-term It seemed unlikely thatonly we through Summarytheir action on memory pro- http://science.sciencemag.org/ we knew less about the localization of implicit memory, a kigoes’s idea cAMP-mediated was incorporated short-term into a facilitation, more specific whereas model in biological approach. As a postdoctoral fellow at it an extra bonus, that the study of memory would be able to work out in any reasonable memory for perceptual and motor skills and other forms of certain types of learning by Hebb in 1949. Spencer facilitation in the sensorimotor connection that lasted minutes, five applications over cesses, but alsoAlthoughthrough CREB seemstheir toaction be importanton for memory response to five pulses of 5-HT this synapse undergoes storage might reveal new aspects of neuronal period of time how the neural networks, in of procedural memory which proved to involve not one and I concluded our review by emphasizing the need to formation, it is not known which of the isoforms of long-term facilitation. The long-term facilitation requires signaling. Indeed, this has proven true. which the hippocampus was embedded, partic- but a number of different brain systems: the cerebellum, develop tractable behavioral systems in which one could 1.5Howardhours Hughes Medicalresulted Institute,in Centerlong-term for Neuro- facilitation that lasted more than 24 hours. Inhibitors other systemsCREB,that CREM,influence or ATF1performance, are expressed in the neurons transcription and translation and is selectively blocked ipate in behavior and how those networks are the striatum, the amygdala, and in the most elementary distinguish between these alternatives by relating, in a biology and Behavior, College of Physicians and Sur- A Radical Reductionist Strategy to affected by learning. that undergo long-term synaptic changes and what by injection of CRE oligonucleotides into the nucleus of ofgeonsprotein of Columbiasynthesis University, Newor YorkRNA State Psy- synthesis selectively blocked long-term facilitation, but such as attentioninstances,or motivation. simple reflex pathwaysAs a result, themselves. Moreover, causal way, specific changes in the neuronal components Learning and Memory To bring the power of modern biology to roles they have in memory formation. We have found the sensory neurons (Dash et al., 1990). Furthermore, chiatric Institute, 1051 Riverside Drive, New York, NY we knew even less about the mechanisms of any form of ofrepeated a behavior pulses to modification of 5-HT will activate of that behaviorin these sensory during not10032,short-term USA. E-mail: [email protected], indicatingAt first thought,that someonelong-term interestedfacilitation in learning bearrequires on the studythe of learning,expres- it seemedit nec-has not beenapossible single Aplysiato determine CREB1 genewheth- homologous to both and memory might be tempted to tackle the essary to take a very different approach—a mammalianmemory storage. CREB Indeed, and CREM we did and not have even characterized know whether learningneurons and a CREB memory reporter storage gene [4]. (Kaang et al., 1993). The sion*This essayofgene is adapted from the author’snot addressessential to for short-term Moreover, the inhibitors er new protein synthesis is important for central role of CREB in long-term memory was further the Nobel Foundation,products December 2000. problem in its most complexfacilitation. and interesting radically reductionist approach. We needed inthe the storage sensory mechanisms neurons were that synaptic mediate or gill-withdrawal non-synaptic. The first behavioral systems to be analyzed in this only blocked long-term facilitation when given during the serotonin applications; the memory and whetherreflexIn 1968, the expressionthe Aldencharacteristic Spencer and and function I weretime of invited the three to write pro- a mannerdemonstrated were simple in studies forms of learningolfactory in memory the context in Dro- of teinsperspective that it encodes: of learning CREB1a, for Physiological CREB1b, Reviewsand CREB1c., which implicitsophila. memory. In transgenic From flies, 1964 the to induced 1979, several expression useful of 1030 2 NOVEMBER 2001 VOL 294 SCIENCE www.sciencemag.org inhibitors did not block the facilitation when given either before or after serotonin window for CREB1along-termwe entitled is a“ transcriptionalCellularmemory Neurophysiological activatorreflects thata Approaches is both nec-Downloaded from in modela dCREB2b systems repressor emerged: selectively the flexion blocked reflex of translation- cats, the essarythe Study and, of upon Learning. phosphorylation,” [4] In it we sufficient pointed for out long- that eye-blinkdependent response long-term of rabbits, memory and a (Yin variety et al.,of simple 1994). forms Con- application. These results parallel those for behavioral performance in vertebrates and specific requirementtermthere facilitation. was nofor frame CREB1bmacromolecular of reference is a repressor for studying of long-term memory ofversely, reflex learning overexpression in inverteb of therates: dCREB2a the gill-withdrawal activator facilitated memory so that a task that normally requires facilitation.because one Cytoplasmic could not yet CREB1c distinguish, modulates experimentally, both the reflex of Aplysia,olfactorylearninginthefly,thees- indicate that the critical time window characteristic of the requirement for macromo- synthesis by particular neurons involved in multiple spaced training sessions was acquired in one short-between and the long-term two conflicting facilitation. approaches Thus, in to the the sensory biology cape reflex of Tritonia, and various behavioral modifi- neurons, CREB1 encodes a critical regulatory unit training trial (Yin et al., 1995a). Finally, mice with dis- lecular synthesis in long-term heterosynaptic facilitation is not a property of complex the storage of long-term information. cations in Hermissenda, Pleurobranchaea, and Limax, converting short- to long-term synaptic changes. rupted expression of a and d isoforms of CREB (Humm- crayfish, and honeybees. The studies were aimed at defi- circuitry, but an intrinsic characteristic of specific nerve cells and synaptic connections To overcomeCorrespondence:these limitations,[email protected] we ana- ler et al., 1994) also show impairment in both LTP and IntroductionDepartment of Neuroscience, Columbia University, 1051 Riverside Drive, ning,long-term then pinpointing, memory (Bourtchuladze the neural circuits et al., 1994). that mediate involved in the long-term storage of information. lyzed memory#664,processes New York, NY 10032,at the USA cellular and theseDespite behaviors evidence and the that critical CRE-binding synaptic sites proteins within are these important components of a general switch that converts molecular level,Bothusing invertebratethe andgill-withdrawal vertebrate© 2012 nervous Kandel; licensee systems BioMed store Central Ltd. This is an Open Access article distributed under the terms of the Creative information for short- and long-termCommons memory Attribution License by chang- (http://creativecommons.org/licenses/by/2.0),short-term to long-term which permits synaptic unrestricted and use, behavioral distribution, and plastic- ME EMORY, THE RETENTION OF ately after training (3). There is no deficit in reflex ofAplysia.ing theThe strengthneural of theircircuitry synapticreproduction connectionsof this in any medium, (Bliss provided and the originality, work it is is properly not cited. known which of the many CREB/ATF or Collingridge, 1993; for review, see Bailey et al., 1996). CREM protein isoforms are involved in the specific cells learned information over time, is the retention of the learned behavior when reflex behaviorStudieshas been in Aplysiadelineated, Drosophila,(5), andand mice suggest that that store particular forms of long-term memory and short-term memory storage is accompanied by transient what role each of these isoforms has in the storage thought to have at least two com- the inhibitors are applied even 1 to 2 hours one importantchangescellular in thelocus strengthhas ofbeen synapticidenti- connections by co- process. This lack of information not only reflects the ponents-short-term memory lasting min- after training. This time window is remark- fied in mediatingvalentshort-term modifications ofand preexistinglong-term proteins. By contrast, difficulty in localizing and studying the critical cells that long-term memory storage is accompanied by enduring participate in memory storage, but also the complexity utes to hours, and long-term memory last- ably characteristic and has been demonstrat- changes in synaptic strength that require both transcrip- of CREB/ATF and CREM genes in higher eukaryotes (for tion and translation of genes (Montarolo et al., 1986; review, see Habener et al., 1995). In both mammals and ing for days to weeks or even years (1). ed for memory produced by associative as Nguyen et al., 1994). These persistent changes are, in Drosophila, CREB mRNAs undergo extensive splicing Behavioral studies in vertebrates suggest well as nonassociative forms of learning and Howard Hughes someMedical cases,Institute accompaniedand Center by growthfor Ncu- of new synaptic (Waeber et al., 1991; Ruppert et al., 1992; Yin et al., robiology and Behavior,connectionsColumbia (Bailey andUniversity, Chen, 1983).College 1995b). In addition to the multiple isoforms of CREB, that long-term memory requires the synthe- for various vertebrate species (3). Although In Aplysia, Drosophila, and rodents, the conversion there is at least an equal number of CREM isoforms of Physicians andofSurgeons short- toand long-termNew York synapticState plasticityPsychi- and memory (Foulkes et al., 1991; Molina et al., 1993). Given the sis of new protein, whereas short-term comparable data are lacking in humans, atric Institute, 722formationWest requires168 Street, an increaseNew inYork, intracellularNY cAMP and variety of CREB/ATF and CREM isoforms and the possi- convulsions and 10032. recruitment of the cAMP-dependent protein kinase A bility of functional compensation between them, it be- memory does not (2). Moreover, long-term analogous clinical studies of (PKA). In eukaryotic cells, transcriptional regulation in comes particularly important to know which specific memory is most sensitive to disruption by head trauma indicate that human long-term response to cAMP is primarily mediated by transcrip- isoforms are expressed and to determine what role, if *Present address: tionalDipartimento activatorsdi andAnatomie repressorse Fisiologia of the CREB/ATF and any, each of these isoforms plays in the specific cells inhibitors of protein synthesis when the memory is particularly susceptible to disrup- Umana, C. so RaffaelloCREM30, families.10125 ConsistentTorino, withItaly. a role for the CREB/ that participate in memory storage. ATF and CREM families of transcription factors in the To examine the role of CREB protein isoforms in syn- inhibitors are applied during and immedi- tion during and soon after acquisition (4). tTo whom correspondenceregulation ofshould genesbe responsivesent. to cAMP, calcium, or aptic plasticity in mature Aplysia sensory neurons, we cloned an Aplysia CREB1 gene that appears to be the 5 DECEMBER ‡ To whom correspondenceREPORTS should be addressed1249 (e-mail: db57@ only member of the CREB, CREM, and ATF1 family of I986 columbia.edu). genes expressed in Aplysia neurons. We find that three NEXT CLASS…

Classical conditioning