mRNAs for ApKLC2 and ApKHC1 are induced by 5HT
We cloned three KLC isoforms and two KHC isoforms by degenerate PCR and by mining an Aplysia
neuronal EST database (Supplementary results and Figure S1
). We focused on ApKHC1 and ApKLC2 because they are highly expressed in neurons, and cloned their full-length cDNAs by screening Aplysia
To explore the possibility that these neuronal isoforms might have specific functions in experience-dependent plasticity and synaptic growth of neurons, we examined whether the levels of ApKLC2 and ApKHC1 are regulated by 5HT. We reasoned that for the long-term changes of synaptic strength, communication between the nucleus and the synapses might require an enhancement of axonal transport. If this were so, the requirement for transport might be accompanied either by an increase in the total number of motor molecules or by a modification of pre-existing motor molecules so that they could carry more cargo or carry it more efficiently. Alternatively, there might be an increase in the total number of cargos in response to 5HT or a modification of pre-existing cargos such that they now bind kinesin. The latter possibility has the advantage of providing specificity to the anterograde transport process. To test these possibilities, we first examined whether the mRNA levels of ApKLC2 and ApKHC1 change with application of 5HT.
We first examined the mRNA levels of ApKLC2 and ApKHC1 by semi-quantitative RTPCR. Each of the two pleural ganglia of the central nervous system (CNS) contains a large cluster of sensory neurons. We therefore isolated total RNA from pleural ganglia dissected from the CNS of Aplysia that were exposed to five pulses of 10 μM 5HT (0 minutes and 30 minutes after 5HT treatment). We found an upregulation of (~2 fold, p< 0.01, Student’s t test) ApKLC2 and ApKHC1 mRNA at thirty minutes after treatment with 5HT (). To further confirm the upregulation of kinesins by 5HT, we performed real-time RTPCR to quantify transcript level changes. Using Aplysia GAPDH levels to normalize the data, we found a ~4 fold increase in transcript levels [ApKHC1 (4.5 ± 0.8) and ApKLC2 (4.8 ± 0.7), p< 0.01, Student’s t test] in 30 minutes after application of 5HT ().
ApKLC2 and ApKHC1 are induced by 5HT
Is the increase in mRNAs induced by repeated pulses of 5HT specific to the presynaptic or the postsynaptic cell of the gill-withdrawal reflex neural circuit? To examine induction of mRNA at single cell level, we studied mRNA levels of ApKHC1 in sensory (SN) and motor (MN) neuron cocultures. RNA in situ hybridization experiments revealed that at 30 minutes following 5HT stimulation, there was an upregulation of ApKHC1 in both the sensory and motor neurons (fold increase in mean fluorescence intensity: SN 3.8 ± 0.4, MN 4.2 ± 0.5; p<0.01, Student’s t test; ). These results suggest that long-term synaptic facilitation is associated with enhanced kinesin mediated transport in both pre- and postsynaptic cells.
We next asked: Are these increases in the mRNA levels of ApKLC2 and ApKHC1 induced by 5HT reflected in their protein levels? We isolated total proteins from the pleural ganglia treated with five pulses of 5HT (0 minutes and 30 minutes after treatment) and immunoblotted them using affinity-purified anti- ApKLC and ApKHC antibodies (Supplementary Figure S3
). Following application of five pulses of 5HT, there was also an increase in the protein levels of ApKLC and ApKHC (fold increase: ApKHC 1.8 ± 0.2; ApKLC 1.6 ± 0.2; p< 0.01, Student’s t
test; ). We verified the increase in the protein levels induced by 5HT in sensory neurons using immunohistochemical analysis of KHC and KLC in sensory neurons (fold increase in mean fluorescence intensity: ApKHC 1.2 ± 0.02; ApKLC 1.6 ± 0.3; p< 0.05, Student’s t
The rapid increase in their mRNA levels suggested to us that ApKLC2 and ApKHC1 might function as primary response or immediate early genes. The induction of immediate early genes is independent of new protein synthesis and only dependent on transcriptional upregulation by constitutively present transcriptional regulators such as CREB in Aplysia
sensory neurons. To characterize the transcriptional induction of kinesins by 5HT, we focused on the expression of ApKHC1. We found that the induction of ApKHC1 is not affected by the treatment of pleural ganglia with protein synthesis inhibitors emetine or anisomycin but the induction was blocked by the exposure to transcriptional inhibitor Actinomycin D (Supplementary results, Figure S2
The failure of protein synthesis inhibitors to block 5HT mediated upregulation of the kinesin mRNA suggests that a protein synthesis independent signal is responsible for this transcriptional upregulation. Since 5HT upregulates cAMP level (Bacskai et al., 1993
), we asked whether an artificial increase in cAMP could induce ApKHC1 expression. We found that application of forskolin, a stimulator of cAMP levels, which in turn can activate CREB, resulted in increased levels of ApKHC1 mRNA in isolated sensory clusters, and this increase was blocked by the treatment with14-22 amide, a myristoylated catalytic site-specific inhibitor of PKA (Supplementary results, Figure S2
). These results suggest that ApKHC1 is an immediate early gene and that its transcriptional induction depends on cAMP and PKA.
Kinesins are thought to be relatively abundant proteins. Hence, one might wonder why kinesins would be upregulated to meet the transport requirements of nerve cells during learning. One possibility is that kinesins have a short half-life and become limiting for anterograde transport of cargos. The other possibility is that more kinesin is required irrespective of its relative abundance in response to the remodeling and growth of new synapses that accompany LTF. To test these possibilities, we investigated the rate of synthesis of new ApKHC protein in unstimulated neurons by pulse-chase experiment using 35
S-methionine labeling followed by coimmunoprecipitation of ApKHC and ApKLC. Contrary to the possibility that kinesins have short half-life, we found that both ApKHC and ApKLC showed two different half-lives, presumably relating to two different protein pools. One pool, which we could not measure precisely, is short-lived with half-lives of less than three hours (Table S2 and S3, and Supplementary results
). The other pool, which we measured reliably, is long-lived (ApKLC: 16.5 hours, r2
=0.98 and ApKHC: 20 hours, r2
=0.98; Supplementary Figure S4, Table S1
5HT increases vesicle trafficking in Aplysia sensory neurons
Does the increase in kinesin level induced by 5HT actually affect the anterograde trafficking of cargos? As a first step in understanding the effect of 5HT on cargo trafficking, we focused on vesicle trafficking in sensory neurons using video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy (Schnapp et al., 1985
). We recorded movement of vesicles for 10 minutes before and 30 minutes after the application of five pulses of 5HT. We observed in the sensory neurons, vesicles of varying sizes and mobility. For reliable quantitation of vesicle trafficking, we focused only on vesicles of ~300 nm size. These ~300 nm size vesicles move to the distal processes at a velocity of about 200-325 nm per second. In response to five pulses of 5HT, these vesicles showed an increase in their number, moving in the anterograde direction in sensory neurons (; % change: 5HT, 43 ± 4.5; untreated control, 10 ± 3.5; n=7, p<0.05, Student’s t
test). Application for 60 minutes of 5 μM nocodozole (Grigoriev et al., 1999
), an inhibitor of tubulin polymerization, blocked movement of these vesicles (data not shown) suggesting that transport of these vesicles depends on microtubules. Since only a fraction of the transport can be visualized with VEC-DIC microscopy, the ~40% increase in trafficking we observed may only represent a fraction of the net effect on vesicular transport.
5HT upregulates vesicle trafficking in sensory neurons
Antisense inhibition of ApKHC1 or ApKLC2 in presynaptic sensory neurons inhibits LTF
Is kinesin-mediated transport necessary for long-term synaptic facilitation? To test this idea, we first examined the kinesin heavy chain. We used the sensory neuron-motor neuron coculture in which a single motor neuron is innervated by two sensory neurons (Montorolo et al., 1986
). To specifically degrade ApKHC1 mRNAs, we used antisense oligonucleotides to deplete ApKHC1 (Supplementary Figure S5 and Supplementary results
). We microinjected antisense oligonucleotides (50 μg/ml) into one of the sensory neurons in coculture using the other sensory neuron as an internal control. First, we examined whether ApKHC1 antisense microinjection has any effect on short-term facilitation (STF) of sensory to motor neuron synapses. Cells were treated with one pulse of 10 μM 5HT for five minutes 20 hours after oligo microinjection and excitatory postsynaptic potentials (EPSPs) were measured 10 minutes after 5HT application (; % change in EPSP amplitudes: 5HT alone 99.3 ± 15.2, n=8; basal −4.5 ± 4.9, n=9; 5HT + antisense oligo 100 ± 13.1, n= 7; antisense oligo alone −3.4 ± 3.2, n=8; sense oligo + 5HT 93 ± 10.7, n=8). The antisense ApKHC1 did not affect 5HT induced STF (n=7, p>0.05, Student’s t
Kinesins are necessary for the induction of long-term memory storage in Aplysia
We then explored ApKHC1 function in LTF. After 3 hours of oligo injection into sensory neurons, cultures treated with five pulses of 10 μM 5HT and EPSPs were measured 24 hours after 5HT application (; % change in EPSP amplitudes: 5HT alone 113.9 ± 27.9, n=8; basal −6.7 ± 7.4, n=9; 5HT + anti sense oligo −2.1± 6.8, n= 7; antisense oligo alone −5.9 ± 8.8, n=8; sense oligo + 5HT 99.8 ± 30.6, n=8). There was a significant reduction in mean EPSP amplitude in the sensory neuron that received the antisense ApKHC1 oligo and five pulses of 5HT exposure (p < 0.01, n= 7, Student’s t test). Microinjection of sense oligos (control for the antisense oligo injections) to the sensory neurons did not have any significant effect on the 5HTinduced increase in mean EPSP amplitudes measured at 24 hours. Taken together these results suggest that presynaptic ApKHC1 levels are necessary for the induction of LTF.
To determine that the kinesin light chain was also necessary for LTF, we used antisense oligos designed specifically to degrade ApKLC2 mRNAs (Supplementary Figure S5 and Supplementary results
), and microinjected (50 μg/ml) into one of the sensory neurons in coculture using the other sensory neuron as an internal control. We first examined whether ApKLC2 antisense or sense (as control) oligo microinjection into sensory neuron has any effect on STF. The antisense or sense ApKLC2 oligo did not affect 5HT induced STF (n=7, p>0.05, Student’s t
test, , % change in EPSP amplitudes: 5HT alone 95.3 ± 15.8, n=8; basal −9.0 ± 7.1, n=5; 5HT + antisense oligo 104.3 ± 12.8, n= 7; antisense oligo alone −10 ± 7.3, n=8; sense oligo + 5HT 93 ± 13.3, n=7).
We then studied ApKLC2 function in LTF by injecting antisense or sense oligos into sensory neurons (; % change in EPSP amplitudes: 5HT alone 49.0 ± 10.3, n=8; basal −6.1 ± 6.8, n=9; 5HT + antisense oligo −10.2 ± 13.3, n= 8; antisense oligo alone −7.0 ± 11.9, n=8; sense oligo + 5HT 57.7 ± 12.3, n=8). The significant reduction in mean EPSP amplitude in the sensory neuron that received antisense ApKLC2 oligo and five pulses of 5HT (p < 0.01, n= 8, Student’s t test) suggests that kinesin light chain levels in the presynaptic neuron are also necessary for the induction of LTF.
Postsynaptic antisense inhibition of ApKHC1 in the motor neurons also inhibit LTF
We then examined the significance of kinesin-mediated transport in postsynaptic neurons by depleting ApKHC1 in the motor neuron. We used Aplysia
bifurcated neuronal cultures in which a single bifurcated sensory neuron is innervated by two spatially separated motor neurons (Martin et al., 1997
). We injected antisense or sense ApKHC1 oligo into one motor neuron and used the other as an internal control.
At 24 hours after bath application of five pulses of 10 μM 5HT, we observed a significant reduction in the mean EPSP amplitudes (p < 0.05, n=5, Student’s t test) at the motor neuron–sensory neuron synapses that received antisense oligo injection (; % change in EPSP amplitudes: 5HT alone 81.6 ± 12.2, n=7; basal −5.8 ± 3.5, n=6; 5HT + anti sense oligo 10.4 ± 18.8, n= 5; antisense oligo alone −28.3 ± 9.1, n=5; sense oligo + 5HT 74.3 ± 12.5, n=6). These results suggest that postsynaptic kinesin levels are also critical for the induction of LTF.
Persistence of synaptic facilitation does not depend on Kinesin mediated transport
Long-term memory storage has at least two phases, an induction phase and a persistence phase. We have recently found that these phases have distinct molecular requirements (Martin et al., 1997
; Si et al., 2003
). Local protein synthesis mediated by the cytoplasmic polyadenylation element binding protein (CPEB) is not required for the induction of LTF in Aplysia
, but is critical for the persistence of LTF at 72 hours after 5HT stimulation. This would suggest that the initial steps in new synapse formation use proteins synthesized in the cell body and transported to the synapses. Consistent with this idea, we find that blocking of ApKHC1 either in the presynaptic or the postsynaptic neuron blocks induction of LTF. Once induced, does LTF at 72 hours require enhanced levels of kinesin? Dependence of the persistence phase of LTF on kinesin levels would suggest the requirement for a continuous, enhanced delivery of cargos from the cell body to the synapses, whereas independence on kinesin levels would suggest that once LTF is established, synapses become autonomous with respect to transport from cell body and hence elevated kinesin mediated transport is not necessary during persistence.
To discriminate between these possibilities we injected ApKHC1 antisense oligos into sensory neurons 24 hours after induction of LTF and measured EPSPs at 72 hours (). We found no significant difference (n=6, p>0.05, paired t test.) of mean EPSP amplitudes measured at 72 hours due to microinjection of ApKHC1 antisense oligos at 24 hours after 5HT application compared to controls that did not receive antisense oligonucleotides (% change in EPSP amplitudes: 5HT alone: @ 24 hours 91.9 ± 13.8, n=6; @ 72 hours 65.5 ± 17.2, n= 6; 5HT alone @ 24 hours before antisense oligo injection 101.8 ± 22.8, n=6; 5HT + ApKHC1 oligo injection at 24 hours: EPSP @ 72 hours 55.7 ± 18.2, n=6; untreated control : @ 24 hr 1.7 ± 9.5, n=6; @ 72 hours −4.4 ± 10.6, n=6). These results suggest that once LTF is induced at the synapses and the requisite gene products are delivered for immediate growth of new synaptic connections, elevated kinesin levels are not required for the maintenance phase of LTF.
Overexpression of ApKHC1 in presynaptic neurons causes an increase in long-term synaptic strength
If the kinesin level is a limiting factor for the induction of LTF, would an increase in kinesin level be sufficient to induce an increase in synaptic strength? To address this question, we examined the effect of an ectopic increase in the level of ApKHC1 on the strength of the synaptic connections between the sensory and motor neuron by measuring EPSPs. To overexpress ApKHC1 in sensory neurons, we cloned full-length ApKHC1 in frame with EGFP into a plasmid construct while overexpression of EGFP alone in cocultures was used as control. Because of the variability due to differences in efficiency of injection of plasmid constructs into cells and differences in time taken to first detect the fluorescence and varying expression levels, we arbitrarily took the time of injection as time zero for our analysis. Overexpression of ApKHC1-EGFP resulted in increase in EPSPs () at 12 hours (n=12, p<0.0001, Student’s t test) after injecting the construct. This increase then decayed gradually over 72 hours (% change in EPSP amplitudes: ApKHC1-EGFP injections @12 hours 115.4 ± 23.2, n=12; @ 24 hours 67.9 ± 17.1 n=9; @48 hours 15.5 ± 13.5, n= 5; @ 72 hours 3.7 ± 17.4, n=4; EGFP injections: @12 hours 0.1 ± 3.6, n=7; @ 24 hours 4.0 ± 2.1, n=6; @48 hours −4.0 ± 3.4, n= 7; @ 72 hours −3.3 ± 7.5, n=7). Overexpression of EGFP alone did not enhance synaptic strength. Thus, an increase in kinesin levels alone is sufficient to cause an induction of long-term facilitation but it does not enhance maintenance as shown by a decay of EPSPs over the period of 72 hours. This supports the idea that kinesin mediated fast axonal transport is limiting and that this increase in kinesin level is sufficient for the induction of LTF, presumably by carrying cargos that are required for induction.
Overexpression of full length ApKHC1 in sensory neurons causes an increase in EPSPs
ApKHC carries synaptic proteins as cargo
How does the enhanced transport result in these distinct forms of synaptic facilitation? To begin to address this question, we decided to analyze what molecules are transported by kinesin in these neurons and we examined the protein components of the kinesin cargo. Protein cargos are synthesized in the cell body and transported to the distal processes as multi-protein complexes or as vesicular components. Using biochemical and genetic methods, proteins such as tubulin (Terada et al., 2000
), glutamate receptors (Setou et al., 2000
), APP (Kamal et al., 2000
), and JIP scaffolding proteins (Verhey, et al., 2001
) have been found to be associated with kinesin. Recently several RNA binding proteins associated with kinesin have been identified using proteomics (Kanai et al., 2004
To identify proteins in Aplysia
neurons that are associated with the kinesin heavy chain, we used both proteomic and candidate approaches ( and Table S4
). With the proteomics approach
, we tried to identify systematically the protein components of the kinesin complex by coimmunoprecipitation followed by mass spectrometric sequencing of protein bands. The lack of Aplysia
genome sequence information limited our efforts to achieve a comprehensive list of protein cargos using the proteomics approach. However, mass spectrometry allowed us to reliably identify several proteins (Table S4 and Supplementary Figure S6
The presence of the kinesin heavy chain and light chain in the immunoprecipitated complex indicated that we had successfully maintained the integrity of the kinesin transport complex isolated from Aplysia
neurons (). Interestingly, we identified a Ca2+
channel as part of the cargo complex, probably transported as a vesicular component. Recently a voltage gated K+
channel transported by Kif7 has been described (Chu et al., 2006
). In the kinesin complex we also found the Ca2+
/ calmodulin dependent protein kinase II, which has been described as important in various forms of synaptic plasticity (Frankland et al., 2001
; Mayford et al., 1996
), and the molecular motor, myosin heavy chain (MHC), that is involved in actin dependent dendritic transport of molecules. MHC has been previously identified to interact with kinesin (Ohashi et al., 2002
). Finally we found that the cargo of the kinesin complex (Supplementary Table S4
) also contains cytoskeletal components such as actin, tubulin and intermediate filament protein. Previously it had been shown that disruption of kinesin (Kif5a) caused abnormal neurofilament transport (Xia et al., 2003
). Taken together these results suggest that ApKHC cargo contain molecules that are required for cytoskeletal remodeling and synaptic plasticity.
In the Candidate approach
, we assumed that molecules that are important for synapse formation might be transported by kinesin. We therefore used specific antibodies to probe the ApKHC complex for the presence of such proteins. By western blotting we detected the presence of proteins required for de novo
synapse formation during development, such as neurexin (Dean et al., 2003
) and neuroligin (Ichtchenko et al., 1995
; Scheiffele et al., 2000
), and presynaptic active zone proteins piccolo (Fenster et al., 2000
) and bassoon (tomDieck et al., 1998
) associated with the kinesin complex (). However, proteins such as the transcriptional regulator Aplysia
CREB2 were not found (Supplementary Figure S3b
) in the complex. The presence of these synaptic proteins in the complex suggested that their sub-cellular distribution could be regulated by kinesin-mediated transport.
Is the upregulation of kinesin in response to 5HT accompanied by an increase in the synthesis of cargos for synapse formation? Indeed, the increase in the ApKHC level would not necessarily be accompanied by an increase in cargo. To explore this possibility, we examined whether the protein levels of neuroligin, neurexin, piccolo and bassoon are regulated independently by 5HT. We found, using western blotting, that the protein levels of neurexin, neuroligin, piccolo and bassoon are indeed upregulated in the pleural ganglia by five pulses of 5HT (; n=4, p<0.05, Student’s t test), suggesting not only that levels of kinesin motor but also levels of some of its cargos are upregulated in a coordinate fashion by 5HT.
Antisense inhibition in the presynaptic sensory neurons of Piccolo–an ApKHC cargo blocks induction of LTF
We now focused on piccolo to investigate the physiological significance of cargo upregulation by 5HT. We cloned a fragment of the Aplysia
homolog of piccolo based on the available sequence information in the Aplysia
Neuronal EST database and designed antisense oligos to degrade piccolo mRNAs (Supplementary Figure S8 and Supplementary results
). These oligos were microinjected into sensory neurons of the coculture as described above.
Piccolo antisense microinjection into sensory neurons did not affect STF (). EPSPs were measured following one pulse application of 5HT (% change in EPSP amplitudes: 5HT alone 85.17 ± 14.8, n=8; basal −2.5 ± 5.8, n=9; 5HT + anti sense oligo 101.4 ± 24.6, n= 7; antisense oligo alone 1.3 ± 6, n=8; sense oligo + 5HT 84.2 ± 14.5, n=8; sense oligo alone −4.2 ± 5.9, n=6) and did not show any significant difference (n=7, p>0.05, Student’s t
test) between antisense or sense oligo injection in the presence of 5HT and 5HT application alone. Measurements of EPSPs at 24 hours show that microinjection of antisense oligo against piccolo into sensory neurons blocked the induction of LTF (p < 0.05, n = 6, Student’s t
test) induced by application of five pulses of 10 μM 5HT (; % change in EPSP amplitudes: 5HT alone 73.4 ± 11.7, n=10; basal −9.8 ± 11.4, n=5; 5HT + anti sense oligo 26.1 ± 9.1, n= 14; antisense oligo alone −2.8 ± 13.4, n=14; sense oligo + 5HT 68.5 ± 18.3, n=6). We then injected piccolo antisense oligos into motor neurons to examine whether piccolo has any postsynaptic function. EPSP measurements after 24 hours of microinjection and 5HT application show that piccolo antisense oligo injection in motor neurons does not block induction of LTF (n=6, p>0.05, Student’s t
test). These data suggest that piccolo levels in sensory neurons are critical for induction of LTF. Piccolo is thought to perform scaffolding functions at active zones along with bassoon, RIMs, CASK, CAST, Velis and Mints (Sudhof, 2004
). Because long-term memory storage requires formation of new synapses, availability of the molecules involved in synaptic architecture such as the piccolo-bassoon complex at the presynaptic active zones is important for induction of LTF.
Necessity and sufficiency of ApKHC1 function in memory storage
Wong et al. (2002)
have suggested that the overexpression of kinesin in mouse brain leads to activation of a positive feedback loop which turns on CREB mediated transcription of some cargos. To test for this possibility in Aplysia
, we examined whether overexpression of kinesin by itself could induce an increase in piccolo mRNA levels and whether long-term increase in EPSPs due to kinesin overexpression required CREB activity. We found that piccolo is a target of CREB and its mRNA levels in sensory neurons showed no increase at 12 hours but a modest increase of 1.4 ± 0.04 fold at 24 hours after ApKHC1 overexpression (Supplementary results, Figure S8
We next asked whether CREB activity is required for the observed increase in EPSPs due to ApKHC1 overexpression. To specifically block CREB activity, we injected the CRE element to which phosphoCREB binds (Dash et al., 1990
) into sensory neuron and also overexpressed ApKHC1-GFP. We used a mutant CRE (mCRE) element to which phosphoCREB cannot bind as a control. We first studied the effect of CRE injection on LTF at 12 hours and 24 hours. We found that CRE injection blocked the 5HT induced increase in EPSPs () at both 12 hours (n=6; p<0.001, Student’s t
test) and at 24 hours (n=8; p <0.001, Student’s t
test). By contrast the control mCRE injection did not affect 5HT induced increase in EPSPs at 12 or 24 hours (% change in EPSP amplitudes: basal −5.2 ± 7.2, n=9; 5HT alone @ 12 hours 72 ± 11.6, n=7; 5HT + CRE @ 12 hours 9.4 ± 12, n= 7; 5HT + mCRE @ 12 hours 71.9 ± 11.2, n= 4; 5HT alone @ 24 hours + 66.3 ± 13.2, n=8; 5HT + CRE @ 24 hours 16 ± 13, n= 5; 5HT + mCRE @ 24 hours 68.2 ± 11.9, n= 4; CRE alone −5.4 ± 10.3, n=5; mCRE alone −2.8 ± 7.7, n=5). These results are consistent with the findings (Dash et al., 1990
) that CREB activity is required for the LTF induced by 5HT.
Necessity and sufficiency of ApKHC1 on LTF
We then blocked CREB activity in sensory neurons that overexpress ApKHC1 to test whether CREB is required for the long-term increase in EPSPs induced by overexpression of ApKHC1. CRE or mCRE was injected two hours after injection of ApKHC1-GFP construct and EPSPs were measured at 12 hours and 24 hours. Our results show that CRE injection into sensory neurons did not block the overexpression of ApKHC1 induced increase in EPSPs at 12 hours (n=5; p<0.0001, Tukey-Kramer multiple comparison test) (). By contrast CRE injection blocked the increase at 24 hours (n=5; p<0.001, Tukey-Kramer multiple comparison test; % change in EPSP amplitudes: basal @ 12 hours 5.7 ± 2.4, n=11; basal @ 24 hours 1.5 ± 2.7, n=11; KHC-GFP alone @ 12 hours 34.8 ± 2.8, n=8; KHC-GFP alone @ 24 hours 18.1 ± 1.3, n=8; KHC-GFP + CRE @ 12 hours 30 ± 2.1, n= 5; KHC-GFP + CRE @ 24 hours −4.7 ± 2.1, n= 8; KHC-GFP + mCRE @ 12 hours 31.2 ± 3.2, n= 6; KHC-GFP + mCRE @ 24 hours 22.8 ± 1.8, n= 5). These results suggest that the overexpression of ApKHC1 is sufficient to induce an increase in EPSPs at 12 hours without requiring CREB by increasing axonal transport of pre-existing cargos in the cell body. However, the EPSP increase at 24 hours is dependent on CREB at least at the higher levels of ApKHC overexpression achieved in our experiments and supports the idea proposed by Wong et al. that overexpression of KHC could lead also to the activation of CREB through a positive feed back loop. This is consistent with the results with piccolo. Our data further show that this positive feedback loop is a late event in the establishment of LTF induced by overexpression.
To further investigate the necessity and sufficiency of ApKHC1, we examined the effects of down regulation of piccolo mRNA levels on the increase in EPSPs induced by ApKHC overexpression. We injected sense or antisense piccolo oligonucleotides into sensory neurons two hours after injection of ApKHC1-GFP construct and measured EPSPs at 12 hours and 24 hours. We found that piccolo antisense or sense injection into sensory neurons does not block ApKHC1 overexpression induced increase in EPSPs at 12 hours (; n=8; p<0.0001, Tukey-Kramer multiple comparison test) but antisense piccolo oligonucleotides blocked the increase at 24 hours (n=7; p<0.001, Tukey-Kramer multiple comparison test; % change in EPSP amplitudes: basal @ 12 hours 7.6 ± 1.3, n=5; basal @ 24 hours 6.7 ± 1, n=5; KHC-GFP + sense Piccolo @ 12 hours 60.2 ± 29, n= 6; KHC-GFP + antisense Piccolo @ 12 hours 68.2 ± 5.7, n= 8; KHC-GFP + sense Piccolo @ 24 hours 24.3 ± 3.6, n= 6; KHC-GFP + antisense Piccolo @ 24 hours 6.3 ± 3, n= 7). These results are in agreement with the data that CREB mediated activation of transcription is not required for the increase in EPSP at 12 hours, but CREB is required for the EPSP increase at 24 hours. Consistent with these results, we found that piccolo knockdown block the EPSP increase at 24 hours in ApKHC1 overexpressing neurons or in neurons that are exposed to five pulses 5HT.
We then examined whether ApKHC1 is required for the EPSP increase at 12 hours by 5HT. We injected ApKHC1 antisense oligos into sensory neurons and exposed the cultures to five pulses of 5HT. The consequent down regulation of ApKHC1 by antisense ApKHC1 blocked the 5HT induced increase (p<0.001, Tukey-Kramer multiple comparison test; ) in EPSPs at 12 hours suggesting that ApKHC1 function is critical for the EPSP increase at 12 hours (% change in EPSP amplitudes: 5HT alone 82.73 ± 11.6, n=8; basal −2.89 ± 3.61, n=7; 5HT + anti sense oligo 24.7 ± 4.9, n= 7; sense oligo + 5HT 73.9 ± 4.4, n=7).
In summary, these results suggest that the electrophysiological phenotype that we described for overexpression of ApKHC1 has at least two phases, a 12-hour and 24-hour phase. ApKHC1 is both necessary and sufficient to induce an increase in EPSPs at 12 hours. By contrast CREB activity is also required at 24 hours.