- Email: [email protected]
Long-term alterations induced by injury and by 5-HT in Aplysia sensory neurons: convergent pathways and common signals?
REVIEW Long-term alterations induced by injury and by 5=HT in Aplysia sensory neurons: convergent pathways and common signals? EdgarT. Walters and Richard T. Ambron Bodily neural
injury in Aplysia, as in mammals, loci. One consequence of injury,
hyperexcitability,
synaptic
facilitation,
produces long-lasting damage to peripheral
and growth
are induced in these cells by repeated exposure ing. An interesting question is to what extent axonal
injury
and 5-HT overlap.
initiate persistent memory of injury. Trends
Neurosci.
(1995)
sensory
One current
alterations
that
memory traces axons, produces
in Aplysio sensory
neurons.
at various long-term
Similar
effects
to 5-HT that is released during aversive leamcellular pathways that mediate the effects of focus is on identifying
contribute
to both
cytoplasmic
long-term
signals that
sensitization
and
18, 137-142
TRIKING ADVANCES have recently been made in analyzing cellular processes that are likely to be S important for inducing memory. Much of this work,
region. This site-specific sensitization can facilitate dramatically responses that protect the site of injury, and can persist for weeks or longer as the injury heals. however, has focused on forms of plasticity, such as In mammals and molluscs, long-term memory long-term potentiation (LTP) in hippocampal slice traces of bodily injury appear to be stored at multiple preparations, that are examined for l-10 h. Less is sites within the PNS and CNS (Refs 1 and 2). The known about the induction of memories that last days, most accessible long-term traces have been found in weeks, or longer. In the mammalian brain, the enor- centrally located sensory neurons whose peripheral mous complexity of systems that are specialized for receptive fields are injured’. In Aplysia, cutting, psychological memory makes it extremely difficult to pinching, or applying a brief sequence of strong eleclocalize neuronal memory traces and to identify tric shock to the tail causes a long-lasting enhancetheir induction signals conclusively. A complementary ment of the signaling effectiveness of nociceptive strategy is to search for potentially general memorysensory neurons innervating the tail: the peripheral induction mechanisms in simpler forms of long-term receptive fields show a decrease in mechanosensory plasticity that are more tractable for linking cellular threshold and an increase in size3, while synapses mechanism to behavioral function. Perhaps the most onto central motor neurons are facilitated, and the impressive demonstration of the power of this ap- excitability of sensory-neuron somata is enhanced4. proach has come from studies, in Aplysiu sensory neur- These neural effects, and the correlated site-specific ons, of cellular and molecular events that are associated sensitization of defensive behavior4, last for days or with long-term general sensitization. Here we review weeks. All, except the increase in receptive field size, similar long-term plasticity, in the same type of sensory are expressed within seconds. Sensory neurons with neurons, conducted under what has traditionally been receptive fields that are distant from the site of considered a quite diierent paradigm - biological reac- trauma (on the siphon) also show long-term synaptic tions to injury. A number of potential convergence facilitation after noxious tail stimulation, but only if points between cellular pathways that are known to the stimulation is applied repeatedly’. This neural be activated by 5-I-H (a primary signal for general correlate of general sensitization is associated with sensitization) and cellular signals that are likely to be growth of central branches and synaptic varicosities involved in long-term reactions to injury are discussed. in siphon sensory neuron?. Persistent correlates of general and site-specific sensitization in Aplysia have Memory of peripheral trauma is stored in Aplysia parallels in neural correlates of more traditional sensory neurons forms of memory reported in vertebrates, including The term ‘memory’ refers generally to storage by growth of new synapses’, and enhancement of the nervous system (or immune system) of acquired excitability’. Long-term traces of tail trauma stored information that is potentially useful to the organism. in the tail sensory neurons are triggered by a combiInformation about bodily injury is important biologination of intense spike activity and exposure to cally because it enables an animal to modify its behav- extracellular modulators3r4 that might be released ior to facilitate healing (for example, by avoiding from nearby neurons, damaged cells, and activated movements that exacerbate an injury) and to miniamebocytes’,‘. In the siphon sensory neurons, which mize the likelihood of attack by predators or parasites do not innervate the tail, persistent traces of tail that are attracted to a wound’. A common reaction to trauma are triggered by longer range neural or injury in Aplysia and humans (where it is called humoral signals, or both, that do not activate the hyperalgesia) is enhanced sensitivity of the injured sensory neurons. One of the extracellular signals that
Edgar T. Walters is at the Dept of Physiology and Cell Biology, University of Texas Medical School, 6431 Fannin Street, Houston, TX77030, USA, and Richard T. Ambron is at the Dept ofAnatomy and Cell Biology, Columbia University, New York, NY 10032, USA.
8 1995, Elsevier Science Ltd
137
Tlh’S Vol. 18, No. 3. 1995
REVIEW
Motor neuron
&
_
-
A
Fig. 1. Long-term alteration of sensory-neuron signaling effectiveness by retrograde/y transported signals fo//owing nerve crush in vivo. (A) Schematic diagram of Aplysia CNS showing locations of sensory neuron somata (dots) in each pleural ganglion and sensory neuron axons (black lines) in nerves leaving the ipsilateral pedal ganglion (only three of nine nerves are indicated). All pedal nerves on one side were crushed under anesthetic conditions that blocked synaptic transmission and minimized spike activity. (B) Examples of synaptic facilitation and hyperexcitability one week after nerve crush. Brief intracellular stimulation of the soma of a sensov neuron on the uncrushed, control side elicited a single spike and a small EPSP in the postsynaptic motor neuron. The same.stimulus on the crushed side elicited an immediate sensory-neuron spike followed by an atlerdischarge of ten additional spikes and large EPSPs in an ipsilateral motor neuron. Adapted from Ref. 12.
is likely to be involved in rapidly triggering both the local and long-range effects of trauma is S-HT (Refs 1, 10 and 11). As described below, a more delayed trigger for ‘cellular memory’ of local trauma is injury to a nociceptive sensory neuron’s own axon.
retrograde axonal transport13. Of particular interest was the finding that inhibitors of axonal transport do not themselves trigger long-term hyperexcitability in Aplysia sensory neurons; indeed, they block the hyperexcitability l3. This indicated that the signal carried by retrograde axonal transport is not a negaLong-term alterations are induced by axon injury tive one (for example, owing to interrupted transport Significant peripheral trauma is likely to damage of target-derived factors), but instead involves the sensory axons. Direct effects of axon injury on ApZysia activation of constitutive signal molecules within sensory neurons were first investigated in the otherthe axon. Studies of axotomized neurons in mamwise intact animal by crushing pedal nerves on one mals have implicated both negative17,18 and posiside of the body, thereby injuring axons from sensory tivel920 injury signals but the identities of such signeurons with somata in the ipsilateral pleural gan- nals have been elusive. glion” (Fig. 1A). The crushes were performed under To test more directly whether long-term alteranesthetic conditions that largely block both neural ations can be induced by molecules that are transactivation and release of neurotransmitters such as ported retrogradely from the site of injury, crushed 5-HT. After a latency of one to three days (depending and uncrushed pedal nerves were ligated close to the on the distance of the crush), axotomized sensory ganglion to accumulate retrogradely transported material’l. Axoplasm was collected immediately disneurons displayed the same persistent changes that had previously been demonstrated following receptive tal to the ligation, and injected into pleural sensoryfield trauma: synaptic facilitation; spike broadening; neuron somata from other animals. Electroand increased excitability (expressed as decreases in physiological tests performed 18-22 h after injection spike threshold, spike accommodation, and after- showed that axoplasm collected from crushed nerves hyperpolarization; Figs 1B and 2A). These effects can produced the same pattern of increased excitability last for over a month, and are specific to the sensory that was observed in these sensory neurons after neurons that receive axonal injury14. Using in vitro injury to either their own axons or their receptive preparations, it was shown that long-term reactions to fields. In contrast, injection of axoplasm from axon injury are not blocked by bathing the nerves in uncrushed nerves caused no significant changes in TTX (to block spike activity) or the CNS in Cd’+ (to excitability. Taken together, these results13,21demonblock synaptic transmission), and that the axonal- strate that axon injury activates molecular signals in injury signals move to the soma at a rate of about the axoplasm that are transported retrogradely and, 36mmd-’ (Ref. 13) (Fig. 2C). Once the signal reaches when they reach the soma, are sufficient to induce the soma there is a delay of about 10 h before the functional alterations lasting at least one day. excitability of the soma increases. Because injuryThe dependence of injury-induced plasticity on induced hyperexcitability is blocked by actinoretrograde axonal transport of positive injury signals, mycin D or anisomycin, this delay probably involves and on changes in gene expression and protein syntranscription of mRNA and synthesis of proteins”. thesis, suggested that axoplasmic proteins that conChanges in protein synthesis are likely to be importtain nuclear-localization signals (NLS) might play a ant in damaged sensory neurons not only for com- crucial role in the induction of memory traces in pensatory and protective enhancement of sensory these cells. It had been shown that when a viral sigfunction but also for growth associated with axon nal peptide (sp) containing a NLS was coupled to regeneration and central sprouting of new fibersl’j. mammalian proteins, and then injected into Aplysia axons, the protein constructs were transported efLong-term alterations might be triggered by ficiently to the soma (but not in the opposite direcunmasking nuclear localization signals in tion) and imported rapidly into the nucleus22~23. axoplasmic proteins Injecting large amounts of free sp into the axon The observation that axonal injury signals travel blocked retrograde transport of the constructs (Fig. 3). at a rate of 36mm d-’ suggested that they move by This was the first demonstration that retrograde 138
TINSvol.la,No.3,~~9s
E. Walters
axonal transport and nuclear import constitutes a tightly linked, effectively continuous, pathway. The use of an antibody to sp revealed several endogenous proteins in Aplysia axons that could be considered candidates for the hypothesized retrograde injury signaPz3. Of these, only one, sp97, displayed the predicted anterochange in grade-to-retrograde transport in response to nerve injuryzl. A second property expected of an NLS-containing retrograde injury signal is that the NLS, which enables the retrograde transport-nuclear import pathway to be accessed when exposed, should be masked until the protein is activated by axon injury. The finding that nerve injury increased the phosphorylation of a protein that corresponded to sp97 suggested that the NLS in sp97 is unmasked by one or more protein kinases that are activated by axon injury21. A related signaling system might exist in mammals, since the antibody to sp97 recognizes sp-containing proteins in rat sciatic nerve (C.C. Huang and R.T. Ambron, unpublished observations). Convergence of signaling pathways that are activated axon injury and 5-HT
by
and R. Ambron
- Memory
REVIEW
of injury
A Distal crush
Proximal crush 4OmV I IJ/Jf _I
,
IS
40mV I
2ms
C r
10 86-
It is of interest to compare ‘cellu4lar memory’ induced by axon injury and by 5-HT (a signal for 0 general sensitization) in ApZysia 2 sensory neurons for several reasons. First, there is likely to be some 1 I, I I I I I I I I I overlap between the underlying 01 mechanisms. If so, extensive 48 72 96 B 0 6 12 18 24 30 36 knowledge of induction signals Time after crush (h) for long-term, S-HT-induced alterations (provided largely by the Fig. 2. Sensory-neuron alterdons after nerve crush in vitro. Pleural-pedal gongfia, with pedal nerves left as long as investigations of Eric Kandel and possible, were maintained in culture medium for l-4 d. (A) Enhanced excitability (indicated by reduced spike accommodation during a 1 s depolarizing pulse injected into the soma) in a sensory neuron 30 h after its axon was crushed colleagues; see below) will greatly -JO mm (proximal crush) from the soma. At the same time, the sensory neuron whose axon was crushed -4Omm facilitate analysis of pathways that (distal crush) from the soma shows the marked accommodation typicul of uninjured sensory neurons from naive aniare activated by axon injury. ma/s. (B) Prolonged spike duration and reduced spike afterhyperpolarization in a sensory neuron that had received Second, release of 5-HT from proximal axon crush relative to a contralateral cell that had received distal crush. The same alterations (including interneuronsz4, and perhaps from decreased accommodation) were observed after injection of axoplasm from crushed nerves into sensory-neuron somata amebocytes, is likely to be particu(see text). (C) Difference in the development of hyperexcitability after proximal (-IOmm; closed circles) and distal larly pronounced during bodily (-40 mm; open circ/es) nerve crush. Excitability was monitored by the number of spikes elicited by a 7 s depolarizing injury. This raises the possibility pulse 2.5 xsoma spike threshold. Calculations based on the average temporal offset of the two curves and mean difthat some functions of 5-HT- ference in nerve length yielded a transport rate for the axonal injuv signal of -36 mm d-‘. Adapted from Ref. 13. induced plasticity normally involve interactions with axon-injury-induced plas- caused by 5-HT alone, but instead are a result of ticity. Third, most published studies of long-term, interactions between 5-HT and previously activated S-HT-induced effects have used preparations, either injury signals. Given the likelihood that release of dissociated pairs of sensory and motor neurons in S-HT, and sensory-neuron injury, normally co-occur under conditions that produce the largest sensory culture” or isolated ganglia in culture25~26,in which the sensory neurons have been axotomized. This alterations, their possible interactions are interesting poses no problems for the conclusions drawn about to consider. Figure 4 presents a very simplified view of cellular the actions of 5-HT in these preparations, but it raises the possibility that some of the results are not signaling pathways that are activated by S-HT and by TlNS Vol. 18, No. 3, 1995
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E. Walters
and R. Ambron
- Memory
of injury
Fig. 3. (Left.) Retrograde transport/nuclear import of a NLScontaining protein. A viral signal peptide (sp) containing a nuclearlocalization signal (MS) was coupled to fluorescent human-serum albumin, and microinjected into an oxonal voricosity of an Aplysia neuron in vitro. (A) Distribution of fluorescent protein 3h after microinjection (bottom). The top shows the same view in a brightfield micrograph. Nearly a// of the injected protein was transported to the soma and imported into the nucleus; no protein was transported in the onterograde direction. (6) inhibition of retrograde transport by co-injection of a 50-fold excess amount of sp (bottom). Most of the fluorescent protein remained in the varicosity that was injected. The top panel shows the same view in a bright-field micrograph. Scale bar, 700 pm. Adapted, with permission, from Ref. 23.
axonal injury in Aplysia sensory neurons. While detailed information is available about the S-HT pathway (indicated in blue), much less is known about the signals that are activated by injury in these cells. Seven levels of potential interaction are numbered in the diagram. Beginning at the termination of the pathways (levels 7 and 6), the persistent effects of axonal injury in ApZysia sensory neurons 140
TN? Vol. 18, No. 3, 1995
appear (thus far) to be the same as those previously shown to be produced by prolonged application of S-HT; that is, 5HT also produces long-term synaptic facilitation’0~26~27, soma hyperexcitabilityz8 and growth 29. It is likely, then, that many of the cellular effecters and their rorresponding genes overlap, and that at levels 7 and 6, the two pathways have largely converged. Indeed, some late genes show similar patterns of regulation after application of 5-HT, nerve stimulation (which probably releases S-HT), and axon injury3G3Z. More is known about the early steps in these pathways (levels 1 and 2). Initial intracellular mediators in the S-HT pathway include CAMP, which activates protein kinase A (PKA), and diacylglycerol (DAG), which activates protein kinase C (PKC). Both CAMP and DAG are involved in short-term synaptic facilitation33, whereas elevation of CAMP but not DAG is sufficient to induce long-term synaptic facilitation34 and perhaps soma hyperexcitability35. The initial intracellular mediators in the axon-injury pathway are not yet known, but rupture of membrane during injury should directly and indirectly (via the opening of voltage-gated Ca” channels in response to injury-induced depolarization and spike activity) produce a large influx of Ca’+, which can activate various protein kinases, proteases, and other enzymes. General roles for Ca” in signaling axon injury are supported by the finding that transient elimination of almost all extracellular Ca” during nerve crush blocks axon-injury-induced hyperexcitability in sensory neurons (J.D. Gunstream and E.T. Walters, unpublished observations). Under normal conditions, nerve injury would also be expected to release many extracellular modulators (including neurotransmitters, cytoklnes and growth factors) that might activate various kinases in affected sensory neurons. The strong possibility that influx of Ca2+ is an initial injury and memory signal points to several sites of convergence between the S-HT pathway and other cellular-signaling pathways that are activated during receptive-field trauma. Because local depolarization following axotomy might last for days36, and small depolarizations are sufficient to increase resting concentrations of Ca2+ in Aplysia sensory neurons3’, axotomy might result in prolonged entry of Ca2+at the injury site. In addition, under normal (unanesthetized) conditions, injury of the receptive field or axon causes high-frequency spike activity and a persistent afterdepolarization4,38, enabling voltagedependent entry of Ca2+into the soma and, presumably, the presynaptic terminals. Intracellular Ca2+ activates adenylate cyclase, enhancing the effects of
E. Walters
‘c ‘.__
w -----___
’
and R. Ambron
- Memoty
REVIEW
of injury
Retrograde transport
Axoplasm, cytoplasm
Unidentified transcription factors
Unidentified
Nucleus Cytoplasm, axoplasm
Fig. 4. Some potential interactions between introcelhdor pathways activated by 5-HT (blue) and by nerve injury in Aplysia sensory neurons. Many of the signals in the injury pathway remain hypothetical. Arrows do not necessarily indicate direct effects. Broken lines indicate potential effects of 54-U on events that are associated with cellular injury. Numbers refer to levels of possible interaction (see text). Roles in the induction of long-term alterations by other potential effects of 5-W and injury (for examp/e, effects on phosphatases, proteases and other enzymes) have received little or no study in these cells. Abbreviations: C/EN, CCAAT enhancer-binding protein; CRE, cAMP response element; CREB, CRE-binding protein; DAC, diacylglycerol; I, current; PKA, protein kinase A; PKC, protein kinase C; sp97, 97kDa protein recognized by antibody to o signal peptide containing o nuclear-localization signal (see text). TINS Vol. 18, No. 3, 1995
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E. Walters
Acknowledgements We are gra tefid to Eric Kandel for valuable comments, and to \im Pastore for preparing the figures. Work from the authors’ laboratories was supported by grants from the NlMH and NSF (ETW) and from the NIH (RW. 142
and R.Ambron
- Memoryofinjury
5-HT by amplifying SHT-induced synthesis of CAMP (Ref. 39). This mechanism in Aplysia sensory neurons was first proposed as a mechanism for classical conditioning4’,“, and later for site-specific sensitization of defensive behavior4. Conversely, 5-HT increases entry of Ca” by activating PKC, which directly increases a voltage-gated Ca” current4’ and PKA, which enhances voltagedependent entry of Ca” by closing K+ channels, thereby depolarizing the cell”‘, and increasing the duration and number of spikes elicited by subsequent stimuli”“f4’. Protein kinase A, PKC, and other protein kinases [perhaps Ca’+/calmodulin-dependent protein kinase (CaM kinase)] that are activated during application of 5-HT might participate in the activation of sp97 and other retrograde signals. An additional possibility is that CAMP enhances the rate of retrograde axonal transport (Fig. 4, level 3). The twoto threefold increase in the rate of retrograde transport following application of CAMP in other Aplysia neurons44 might cause a substantial (if temporary) increase in the amount of sp97 or other retrogradely transported signals reaching the soma. Calcium ions might also promote retrograde axonal transport, perhaps via a Ca’+-dependent protease4”. Protein kinase A is thought to be one retrograde signal that reaches the nucleus, since prolonged application of 5-HT can cause some translocation of free catalytic subunits of PKA into the nucleus 46. However, the efficiency of this translocation appears to be considerably less than that observed for the nuclear import of spcontaining proteins in Aplysia neurons22,23, suggesting that the catalytic subunit of PKA is not the only signal that enters the nucleus in response to 5-HT. Long-term synaptic facilitation after application of 5-HT requires binding of a nuclear transcription factor, CRE-binding protein (CREB), to specific DNA sequences [CAMP response element (CRE)] (Fig. 4, level 4)47. CRE-driven expression of microinjected reporter genes in response to 5-HT requires phosphorylation of a PKA-recognition site on CREB (Ref. 48), whereas phosphorylation of the same site by CaM kinase appears to be insufficient to activate CREdriven gene expression4’. These results point to an essential role for PKA in activating CREB but, as the authors of the study point out, the possibility remains that phosphorylation of other sites on CREB (or activation of other transcription factors) might also be required for inducing long-term facilitation. It will be interesting to see if sp97 or other axonal injury signals that access the retrograde transport-nuclear import pathway cause phosphorylation of nuclear-transcription factors. The possibility that additional kinases are involved in the nucleus is particularly intriguing, given evidence that phosphorylation of CREB by Ca’+dependent kinases is important for altering gene expression in some cells4’. At the genetic level (level 5), an elegant study has shown recently that an immediate-early gene product, CCAAT enhancerbinding protein (C/EBP), is necessary for 5-HT-induced long-term facilitation of Aplysia sensory-neuron synapsesSo. This study is particularly important for the question of overlap because, by showing that the gene is induced dramatically by tissue dissection alone, it provided strong evidence for a convergence of axonal-injury signals and 5-HT-induced signals at, or upstream from, C/EBP. TINS Vol. 18, No. 3, 1995
These preliminary indications of overlap between cellular pathways activated by 5-HT and by axon injury in AplysiLl raise interesting questions about molecular sites of convergence and the extent to which each pathway involves distinct signals and different functions. Further study of these cells might show how a variety of intrinsic and extrinsic signals can lead to adaptive, long-term alterations of neuronal and behavioral function. Selected
references
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13 Gunstream, (in press) 14 Clatworthy,
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M., Thompson, M.A. and Greenberg, Scimce252,1427-1430 50 Alberini, C.M. etal. (1994)CeZl76,1099-1114
M.E.
(1991)
injury in Aplysia, as in mammals, loci. One consequence of injury,
hyperexcitability,
synaptic
facilitation,
produces long-lasting damage to peripheral
and growth
are induced in these cells by repeated exposure ing. An interesting question is to what extent axonal
injury
and 5-HT overlap.
initiate persistent memory of injury. Trends
Neurosci.
(1995)
sensory
One current
alterations
that
memory traces axons, produces
in Aplysio sensory
neurons.
at various long-term
Similar
effects
to 5-HT that is released during aversive leamcellular pathways that mediate the effects of focus is on identifying
contribute
to both
cytoplasmic
long-term
signals that
sensitization
and
18, 137-142
TRIKING ADVANCES have recently been made in analyzing cellular processes that are likely to be S important for inducing memory. Much of this work,
region. This site-specific sensitization can facilitate dramatically responses that protect the site of injury, and can persist for weeks or longer as the injury heals. however, has focused on forms of plasticity, such as In mammals and molluscs, long-term memory long-term potentiation (LTP) in hippocampal slice traces of bodily injury appear to be stored at multiple preparations, that are examined for l-10 h. Less is sites within the PNS and CNS (Refs 1 and 2). The known about the induction of memories that last days, most accessible long-term traces have been found in weeks, or longer. In the mammalian brain, the enor- centrally located sensory neurons whose peripheral mous complexity of systems that are specialized for receptive fields are injured’. In Aplysia, cutting, psychological memory makes it extremely difficult to pinching, or applying a brief sequence of strong eleclocalize neuronal memory traces and to identify tric shock to the tail causes a long-lasting enhancetheir induction signals conclusively. A complementary ment of the signaling effectiveness of nociceptive strategy is to search for potentially general memorysensory neurons innervating the tail: the peripheral induction mechanisms in simpler forms of long-term receptive fields show a decrease in mechanosensory plasticity that are more tractable for linking cellular threshold and an increase in size3, while synapses mechanism to behavioral function. Perhaps the most onto central motor neurons are facilitated, and the impressive demonstration of the power of this ap- excitability of sensory-neuron somata is enhanced4. proach has come from studies, in Aplysiu sensory neur- These neural effects, and the correlated site-specific ons, of cellular and molecular events that are associated sensitization of defensive behavior4, last for days or with long-term general sensitization. Here we review weeks. All, except the increase in receptive field size, similar long-term plasticity, in the same type of sensory are expressed within seconds. Sensory neurons with neurons, conducted under what has traditionally been receptive fields that are distant from the site of considered a quite diierent paradigm - biological reac- trauma (on the siphon) also show long-term synaptic tions to injury. A number of potential convergence facilitation after noxious tail stimulation, but only if points between cellular pathways that are known to the stimulation is applied repeatedly’. This neural be activated by 5-I-H (a primary signal for general correlate of general sensitization is associated with sensitization) and cellular signals that are likely to be growth of central branches and synaptic varicosities involved in long-term reactions to injury are discussed. in siphon sensory neuron?. Persistent correlates of general and site-specific sensitization in Aplysia have Memory of peripheral trauma is stored in Aplysia parallels in neural correlates of more traditional sensory neurons forms of memory reported in vertebrates, including The term ‘memory’ refers generally to storage by growth of new synapses’, and enhancement of the nervous system (or immune system) of acquired excitability’. Long-term traces of tail trauma stored information that is potentially useful to the organism. in the tail sensory neurons are triggered by a combiInformation about bodily injury is important biologination of intense spike activity and exposure to cally because it enables an animal to modify its behav- extracellular modulators3r4 that might be released ior to facilitate healing (for example, by avoiding from nearby neurons, damaged cells, and activated movements that exacerbate an injury) and to miniamebocytes’,‘. In the siphon sensory neurons, which mize the likelihood of attack by predators or parasites do not innervate the tail, persistent traces of tail that are attracted to a wound’. A common reaction to trauma are triggered by longer range neural or injury in Aplysia and humans (where it is called humoral signals, or both, that do not activate the hyperalgesia) is enhanced sensitivity of the injured sensory neurons. One of the extracellular signals that
Edgar T. Walters is at the Dept of Physiology and Cell Biology, University of Texas Medical School, 6431 Fannin Street, Houston, TX77030, USA, and Richard T. Ambron is at the Dept ofAnatomy and Cell Biology, Columbia University, New York, NY 10032, USA.
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Fig. 1. Long-term alteration of sensory-neuron signaling effectiveness by retrograde/y transported signals fo//owing nerve crush in vivo. (A) Schematic diagram of Aplysia CNS showing locations of sensory neuron somata (dots) in each pleural ganglion and sensory neuron axons (black lines) in nerves leaving the ipsilateral pedal ganglion (only three of nine nerves are indicated). All pedal nerves on one side were crushed under anesthetic conditions that blocked synaptic transmission and minimized spike activity. (B) Examples of synaptic facilitation and hyperexcitability one week after nerve crush. Brief intracellular stimulation of the soma of a sensov neuron on the uncrushed, control side elicited a single spike and a small EPSP in the postsynaptic motor neuron. The same.stimulus on the crushed side elicited an immediate sensory-neuron spike followed by an atlerdischarge of ten additional spikes and large EPSPs in an ipsilateral motor neuron. Adapted from Ref. 12.
is likely to be involved in rapidly triggering both the local and long-range effects of trauma is S-HT (Refs 1, 10 and 11). As described below, a more delayed trigger for ‘cellular memory’ of local trauma is injury to a nociceptive sensory neuron’s own axon.
retrograde axonal transport13. Of particular interest was the finding that inhibitors of axonal transport do not themselves trigger long-term hyperexcitability in Aplysia sensory neurons; indeed, they block the hyperexcitability l3. This indicated that the signal carried by retrograde axonal transport is not a negaLong-term alterations are induced by axon injury tive one (for example, owing to interrupted transport Significant peripheral trauma is likely to damage of target-derived factors), but instead involves the sensory axons. Direct effects of axon injury on ApZysia activation of constitutive signal molecules within sensory neurons were first investigated in the otherthe axon. Studies of axotomized neurons in mamwise intact animal by crushing pedal nerves on one mals have implicated both negative17,18 and posiside of the body, thereby injuring axons from sensory tivel920 injury signals but the identities of such signeurons with somata in the ipsilateral pleural gan- nals have been elusive. glion” (Fig. 1A). The crushes were performed under To test more directly whether long-term alteranesthetic conditions that largely block both neural ations can be induced by molecules that are transactivation and release of neurotransmitters such as ported retrogradely from the site of injury, crushed 5-HT. After a latency of one to three days (depending and uncrushed pedal nerves were ligated close to the on the distance of the crush), axotomized sensory ganglion to accumulate retrogradely transported material’l. Axoplasm was collected immediately disneurons displayed the same persistent changes that had previously been demonstrated following receptive tal to the ligation, and injected into pleural sensoryfield trauma: synaptic facilitation; spike broadening; neuron somata from other animals. Electroand increased excitability (expressed as decreases in physiological tests performed 18-22 h after injection spike threshold, spike accommodation, and after- showed that axoplasm collected from crushed nerves hyperpolarization; Figs 1B and 2A). These effects can produced the same pattern of increased excitability last for over a month, and are specific to the sensory that was observed in these sensory neurons after neurons that receive axonal injury14. Using in vitro injury to either their own axons or their receptive preparations, it was shown that long-term reactions to fields. In contrast, injection of axoplasm from axon injury are not blocked by bathing the nerves in uncrushed nerves caused no significant changes in TTX (to block spike activity) or the CNS in Cd’+ (to excitability. Taken together, these results13,21demonblock synaptic transmission), and that the axonal- strate that axon injury activates molecular signals in injury signals move to the soma at a rate of about the axoplasm that are transported retrogradely and, 36mmd-’ (Ref. 13) (Fig. 2C). Once the signal reaches when they reach the soma, are sufficient to induce the soma there is a delay of about 10 h before the functional alterations lasting at least one day. excitability of the soma increases. Because injuryThe dependence of injury-induced plasticity on induced hyperexcitability is blocked by actinoretrograde axonal transport of positive injury signals, mycin D or anisomycin, this delay probably involves and on changes in gene expression and protein syntranscription of mRNA and synthesis of proteins”. thesis, suggested that axoplasmic proteins that conChanges in protein synthesis are likely to be importtain nuclear-localization signals (NLS) might play a ant in damaged sensory neurons not only for com- crucial role in the induction of memory traces in pensatory and protective enhancement of sensory these cells. It had been shown that when a viral sigfunction but also for growth associated with axon nal peptide (sp) containing a NLS was coupled to regeneration and central sprouting of new fibersl’j. mammalian proteins, and then injected into Aplysia axons, the protein constructs were transported efLong-term alterations might be triggered by ficiently to the soma (but not in the opposite direcunmasking nuclear localization signals in tion) and imported rapidly into the nucleus22~23. axoplasmic proteins Injecting large amounts of free sp into the axon The observation that axonal injury signals travel blocked retrograde transport of the constructs (Fig. 3). at a rate of 36mm d-’ suggested that they move by This was the first demonstration that retrograde 138
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axonal transport and nuclear import constitutes a tightly linked, effectively continuous, pathway. The use of an antibody to sp revealed several endogenous proteins in Aplysia axons that could be considered candidates for the hypothesized retrograde injury signaPz3. Of these, only one, sp97, displayed the predicted anterochange in grade-to-retrograde transport in response to nerve injuryzl. A second property expected of an NLS-containing retrograde injury signal is that the NLS, which enables the retrograde transport-nuclear import pathway to be accessed when exposed, should be masked until the protein is activated by axon injury. The finding that nerve injury increased the phosphorylation of a protein that corresponded to sp97 suggested that the NLS in sp97 is unmasked by one or more protein kinases that are activated by axon injury21. A related signaling system might exist in mammals, since the antibody to sp97 recognizes sp-containing proteins in rat sciatic nerve (C.C. Huang and R.T. Ambron, unpublished observations). Convergence of signaling pathways that are activated axon injury and 5-HT
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It is of interest to compare ‘cellu4lar memory’ induced by axon injury and by 5-HT (a signal for 0 general sensitization) in ApZysia 2 sensory neurons for several reasons. First, there is likely to be some 1 I, I I I I I I I I I overlap between the underlying 01 mechanisms. If so, extensive 48 72 96 B 0 6 12 18 24 30 36 knowledge of induction signals Time after crush (h) for long-term, S-HT-induced alterations (provided largely by the Fig. 2. Sensory-neuron alterdons after nerve crush in vitro. Pleural-pedal gongfia, with pedal nerves left as long as investigations of Eric Kandel and possible, were maintained in culture medium for l-4 d. (A) Enhanced excitability (indicated by reduced spike accommodation during a 1 s depolarizing pulse injected into the soma) in a sensory neuron 30 h after its axon was crushed colleagues; see below) will greatly -JO mm (proximal crush) from the soma. At the same time, the sensory neuron whose axon was crushed -4Omm facilitate analysis of pathways that (distal crush) from the soma shows the marked accommodation typicul of uninjured sensory neurons from naive aniare activated by axon injury. ma/s. (B) Prolonged spike duration and reduced spike afterhyperpolarization in a sensory neuron that had received Second, release of 5-HT from proximal axon crush relative to a contralateral cell that had received distal crush. The same alterations (including interneuronsz4, and perhaps from decreased accommodation) were observed after injection of axoplasm from crushed nerves into sensory-neuron somata amebocytes, is likely to be particu(see text). (C) Difference in the development of hyperexcitability after proximal (-IOmm; closed circles) and distal larly pronounced during bodily (-40 mm; open circ/es) nerve crush. Excitability was monitored by the number of spikes elicited by a 7 s depolarizing injury. This raises the possibility pulse 2.5 xsoma spike threshold. Calculations based on the average temporal offset of the two curves and mean difthat some functions of 5-HT- ference in nerve length yielded a transport rate for the axonal injuv signal of -36 mm d-‘. Adapted from Ref. 13. induced plasticity normally involve interactions with axon-injury-induced plas- caused by 5-HT alone, but instead are a result of ticity. Third, most published studies of long-term, interactions between 5-HT and previously activated S-HT-induced effects have used preparations, either injury signals. Given the likelihood that release of dissociated pairs of sensory and motor neurons in S-HT, and sensory-neuron injury, normally co-occur under conditions that produce the largest sensory culture” or isolated ganglia in culture25~26,in which the sensory neurons have been axotomized. This alterations, their possible interactions are interesting poses no problems for the conclusions drawn about to consider. Figure 4 presents a very simplified view of cellular the actions of 5-HT in these preparations, but it raises the possibility that some of the results are not signaling pathways that are activated by S-HT and by TlNS Vol. 18, No. 3, 1995
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Fig. 3. (Left.) Retrograde transport/nuclear import of a NLScontaining protein. A viral signal peptide (sp) containing a nuclearlocalization signal (MS) was coupled to fluorescent human-serum albumin, and microinjected into an oxonal voricosity of an Aplysia neuron in vitro. (A) Distribution of fluorescent protein 3h after microinjection (bottom). The top shows the same view in a brightfield micrograph. Nearly a// of the injected protein was transported to the soma and imported into the nucleus; no protein was transported in the onterograde direction. (6) inhibition of retrograde transport by co-injection of a 50-fold excess amount of sp (bottom). Most of the fluorescent protein remained in the varicosity that was injected. The top panel shows the same view in a bright-field micrograph. Scale bar, 700 pm. Adapted, with permission, from Ref. 23.
axonal injury in Aplysia sensory neurons. While detailed information is available about the S-HT pathway (indicated in blue), much less is known about the signals that are activated by injury in these cells. Seven levels of potential interaction are numbered in the diagram. Beginning at the termination of the pathways (levels 7 and 6), the persistent effects of axonal injury in ApZysia sensory neurons 140
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appear (thus far) to be the same as those previously shown to be produced by prolonged application of S-HT; that is, 5HT also produces long-term synaptic facilitation’0~26~27, soma hyperexcitabilityz8 and growth 29. It is likely, then, that many of the cellular effecters and their rorresponding genes overlap, and that at levels 7 and 6, the two pathways have largely converged. Indeed, some late genes show similar patterns of regulation after application of 5-HT, nerve stimulation (which probably releases S-HT), and axon injury3G3Z. More is known about the early steps in these pathways (levels 1 and 2). Initial intracellular mediators in the S-HT pathway include CAMP, which activates protein kinase A (PKA), and diacylglycerol (DAG), which activates protein kinase C (PKC). Both CAMP and DAG are involved in short-term synaptic facilitation33, whereas elevation of CAMP but not DAG is sufficient to induce long-term synaptic facilitation34 and perhaps soma hyperexcitability35. The initial intracellular mediators in the axon-injury pathway are not yet known, but rupture of membrane during injury should directly and indirectly (via the opening of voltage-gated Ca” channels in response to injury-induced depolarization and spike activity) produce a large influx of Ca’+, which can activate various protein kinases, proteases, and other enzymes. General roles for Ca” in signaling axon injury are supported by the finding that transient elimination of almost all extracellular Ca” during nerve crush blocks axon-injury-induced hyperexcitability in sensory neurons (J.D. Gunstream and E.T. Walters, unpublished observations). Under normal conditions, nerve injury would also be expected to release many extracellular modulators (including neurotransmitters, cytoklnes and growth factors) that might activate various kinases in affected sensory neurons. The strong possibility that influx of Ca2+ is an initial injury and memory signal points to several sites of convergence between the S-HT pathway and other cellular-signaling pathways that are activated during receptive-field trauma. Because local depolarization following axotomy might last for days36, and small depolarizations are sufficient to increase resting concentrations of Ca2+ in Aplysia sensory neurons3’, axotomy might result in prolonged entry of Ca2+at the injury site. In addition, under normal (unanesthetized) conditions, injury of the receptive field or axon causes high-frequency spike activity and a persistent afterdepolarization4,38, enabling voltagedependent entry of Ca2+into the soma and, presumably, the presynaptic terminals. Intracellular Ca2+ activates adenylate cyclase, enhancing the effects of
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Fig. 4. Some potential interactions between introcelhdor pathways activated by 5-HT (blue) and by nerve injury in Aplysia sensory neurons. Many of the signals in the injury pathway remain hypothetical. Arrows do not necessarily indicate direct effects. Broken lines indicate potential effects of 54-U on events that are associated with cellular injury. Numbers refer to levels of possible interaction (see text). Roles in the induction of long-term alterations by other potential effects of 5-W and injury (for examp/e, effects on phosphatases, proteases and other enzymes) have received little or no study in these cells. Abbreviations: C/EN, CCAAT enhancer-binding protein; CRE, cAMP response element; CREB, CRE-binding protein; DAC, diacylglycerol; I, current; PKA, protein kinase A; PKC, protein kinase C; sp97, 97kDa protein recognized by antibody to o signal peptide containing o nuclear-localization signal (see text). TINS Vol. 18, No. 3, 1995
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Acknowledgements We are gra tefid to Eric Kandel for valuable comments, and to \im Pastore for preparing the figures. Work from the authors’ laboratories was supported by grants from the NlMH and NSF (ETW) and from the NIH (RW. 142
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5-HT by amplifying SHT-induced synthesis of CAMP (Ref. 39). This mechanism in Aplysia sensory neurons was first proposed as a mechanism for classical conditioning4’,“, and later for site-specific sensitization of defensive behavior4. Conversely, 5-HT increases entry of Ca” by activating PKC, which directly increases a voltage-gated Ca” current4’ and PKA, which enhances voltagedependent entry of Ca” by closing K+ channels, thereby depolarizing the cell”‘, and increasing the duration and number of spikes elicited by subsequent stimuli”“f4’. Protein kinase A, PKC, and other protein kinases [perhaps Ca’+/calmodulin-dependent protein kinase (CaM kinase)] that are activated during application of 5-HT might participate in the activation of sp97 and other retrograde signals. An additional possibility is that CAMP enhances the rate of retrograde axonal transport (Fig. 4, level 3). The twoto threefold increase in the rate of retrograde transport following application of CAMP in other Aplysia neurons44 might cause a substantial (if temporary) increase in the amount of sp97 or other retrogradely transported signals reaching the soma. Calcium ions might also promote retrograde axonal transport, perhaps via a Ca’+-dependent protease4”. Protein kinase A is thought to be one retrograde signal that reaches the nucleus, since prolonged application of 5-HT can cause some translocation of free catalytic subunits of PKA into the nucleus 46. However, the efficiency of this translocation appears to be considerably less than that observed for the nuclear import of spcontaining proteins in Aplysia neurons22,23, suggesting that the catalytic subunit of PKA is not the only signal that enters the nucleus in response to 5-HT. Long-term synaptic facilitation after application of 5-HT requires binding of a nuclear transcription factor, CRE-binding protein (CREB), to specific DNA sequences [CAMP response element (CRE)] (Fig. 4, level 4)47. CRE-driven expression of microinjected reporter genes in response to 5-HT requires phosphorylation of a PKA-recognition site on CREB (Ref. 48), whereas phosphorylation of the same site by CaM kinase appears to be insufficient to activate CREdriven gene expression4’. These results point to an essential role for PKA in activating CREB but, as the authors of the study point out, the possibility remains that phosphorylation of other sites on CREB (or activation of other transcription factors) might also be required for inducing long-term facilitation. It will be interesting to see if sp97 or other axonal injury signals that access the retrograde transport-nuclear import pathway cause phosphorylation of nuclear-transcription factors. The possibility that additional kinases are involved in the nucleus is particularly intriguing, given evidence that phosphorylation of CREB by Ca’+dependent kinases is important for altering gene expression in some cells4’. At the genetic level (level 5), an elegant study has shown recently that an immediate-early gene product, CCAAT enhancerbinding protein (C/EBP), is necessary for 5-HT-induced long-term facilitation of Aplysia sensory-neuron synapsesSo. This study is particularly important for the question of overlap because, by showing that the gene is induced dramatically by tissue dissection alone, it provided strong evidence for a convergence of axonal-injury signals and 5-HT-induced signals at, or upstream from, C/EBP. TINS Vol. 18, No. 3, 1995
These preliminary indications of overlap between cellular pathways activated by 5-HT and by axon injury in AplysiLl raise interesting questions about molecular sites of convergence and the extent to which each pathway involves distinct signals and different functions. Further study of these cells might show how a variety of intrinsic and extrinsic signals can lead to adaptive, long-term alterations of neuronal and behavioral function. Selected
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