A Requirement of Nuclear Factor-κB Activation in Fear-potentiated Startle

Previous studies have shown that biochemical changes that occur in the amygdala during fear conditioning in vivo are similar to those occur during long term potentiation (LTP) in vitro. Electrophoretic mobility shift assay of nuclear extracts from startle-potentiated rats showed a selective increase in the amygdala of nuclear factor-κB (NF-κB) DNA binding activity. Supershift experiments further indicated that p65 and p50 subunits but not c-Rel were involved in DNA binding. The protein levels of IκB-α were reduced by treatments that reliably induced LTP in this area of the brain. This was accompanied by a decrease of NF-κB in the cytoplasm concomitant with an increase in the nucleus. Quantitative analysis of IκB kinase activity demonstrated that fear training led to an increase in kinase activity, and this effect was inhibited by thalidomide. Paralleled behavioral tests revealed that thalidomide inhibited fear-potentiated startle. Intra-amygdala administration of κB decoy DNA prior to training impaired fear-potentiated startle as well as LTP induction. Similarly, NF-κB inhibitors blocked IκB-α degradation and startle response. These results provide the first evidence of a requirement of NF-κB activation in the amygdala for consolidation of fear memory. Previous studies have shown that biochemical changes that occur in the amygdala during fear conditioning in vivo are similar to those occur during long term potentiation (LTP) in vitro. Electrophoretic mobility shift assay of nuclear extracts from startle-potentiated rats showed a selective increase in the amygdala of nuclear factor-κB (NF-κB) DNA binding activity. Supershift experiments further indicated that p65 and p50 subunits but not c-Rel were involved in DNA binding. The protein levels of IκB-α were reduced by treatments that reliably induced LTP in this area of the brain. This was accompanied by a decrease of NF-κB in the cytoplasm concomitant with an increase in the nucleus. Quantitative analysis of IκB kinase activity demonstrated that fear training led to an increase in kinase activity, and this effect was inhibited by thalidomide. Paralleled behavioral tests revealed that thalidomide inhibited fear-potentiated startle. Intra-amygdala administration of κB decoy DNA prior to training impaired fear-potentiated startle as well as LTP induction. Similarly, NF-κB inhibitors blocked IκB-α degradation and startle response. These results provide the first evidence of a requirement of NF-κB activation in the amygdala for consolidation of fear memory. It is generally believed that consolidation of long term memory in mammalian brain and long term facilitation in Aplysiarequire new protein synthesis (1Davis H.P. Squire L.R. Psychol. Bull. 1984; 96: 518-559Crossref PubMed Scopus (1249) Google Scholar, 2Castellucci V.F. Blumenfeld H. Goelet P. Kandel E.R. J. Neurobiol. 1989; 20: 1-9Crossref PubMed Scopus (162) Google Scholar, 3Bailey D.J. Kim J.J. Sun W. Thompson R.F. Helmstetter F.J. Behav. Neurosci. 1999; 113: 276-282Crossref PubMed Scopus (137) Google Scholar). Newly synthesized proteins are thought to deposit at the synapses that have been tagged by prior activity to encode enduring changes in synaptic strength (4Frey U. Morris R.G.M. Nature. 1997; 385: 533-536Crossref PubMed Scopus (1252) Google Scholar, 5Frey U. Morris R.G.M. Trends Neurosci. 1998; 21: 181-188Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). However, despite the importance of new protein synthesis for memory consolidation, little is known about signaling pathways leading to protein translation in neurons. NF-κB, originally identified as a regulator of immunoglobulin κ light chain gene expression, is a DNA-binding factor that functions as a dimer. Five mammalian members of the family have been identified; p50/NF-κB1, p65/RelA, c-Rel, RelB, and p52/NF-κB2 (6Siebenlist U. Franzoso G. Brown R. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2003) Google Scholar). NF-κB was localized mainly to the cytoplasm in an inactive form bound to an inhibitory protein termed IκB (7Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar). Upon stimulation by extracellular inducers, IκB was rapidly phosphorylated by the IκB kinase (IKK) complex on the serine residues 32 and 36. This phosphorylation led to the ubiquitination and subsequent degradation of IκB by the proteasome followed by nuclear translocation of NF-κB (8Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1159) Google Scholar, 9Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. 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Apart from its role in hematopoiesis, accumulated evidence indicates that NF-κB is involved in neuroprotection or neurodegeneration, depending on the particular system under investigation and the NF-κB subunits that became activated (16O'Neill L.A.J. Kaltschmidt C. Trends Neurosci. 1997; 20: 252-258Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar, 17Denk A. Wirth T. Baumann B. Cytokine Growth Factor Rev. 2000; 11: 303-320Crossref PubMed Scopus (113) Google Scholar, 18Castagne V. Lefevre K. Clarke P.G.H. Neuroscience. 2001; 108: 517-526Crossref PubMed Scopus (15) Google Scholar). Furthermore, a recent study implicated that NF-κB played an important role in the synaptic plasticity, because pretreatment of hippocampal slices with κB decoy DNA prevented induction of long term depression (LTD) 1The abbreviations used for: LTD, long term depression; CREB, cAMP-response element-binding protein; EMSA, electrophoretic mobility shift assay; IKK, IκB kinase; LTP, long term potentiation; D-APV, D-2-amino-phosphonovalerate; PI 3-kinase, phosphatidylinositol 3-kinase; TPCK, N-tosyl-l-phenylalanine chloromethyl ketone; TLCK, N α-p-tosyl-l-lysine chloromethyl ketone; TS, tetanic stimulation; LA, lateral; BLA, basolateral; ACSF, artificial cerebrospinal fluid; PMSF, phenylmethylsulfonyl fluoride. 1The abbreviations used for: LTD, long term depression; CREB, cAMP-response element-binding protein; EMSA, electrophoretic mobility shift assay; IKK, IκB kinase; LTP, long term potentiation; D-APV, D-2-amino-phosphonovalerate; PI 3-kinase, phosphatidylinositol 3-kinase; TPCK, N-tosyl-l-phenylalanine chloromethyl ketone; TLCK, N α-p-tosyl-l-lysine chloromethyl ketone; TS, tetanic stimulation; LA, lateral; BLA, basolateral; ACSF, artificial cerebrospinal fluid; PMSF, phenylmethylsulfonyl fluoride. and significantly reduced the magnitude of LTP (19Albensi B.C. Mattson M.P. Synapse. 2000; 35: 151-159Crossref PubMed Scopus (421) Google Scholar). We have recently demonstrated that acquisition of fear was associated with an activation of phosphatidylinositol 3-kinase (PI 3-kinase) and its downstream target Akt in the rat amygdala (20Lin C.H. Yeh H.W. Lin C.H. Lu K.T. Leu T.H. Chang W.C. Gean P.W. Neuron. 2001; 31: 841-851Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). PI 3-kinase and Akt were also activated in response to LTP-inducing tetanic stimulation (TS). In parallel, PI 3-kinase inhibitors interfered with TS-induced LTP as well as long term fear memory formation. Akt has been shown to induce the activation of NF-κB family of transcriptional factors (21Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-85Crossref PubMed Scopus (1866) Google Scholar, 22Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1654) Google Scholar). Therefore, it is of interest to investigate whether NF-κB plays any role in synaptic plasticity and memory formation. Here we show that NF-κB is activated in the amygdala following fear-potentiated startle, and disruption of this signaling pathway impairs fear memory. Rats (4–5 weeks old) were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally) and subsequently were mounted on a stereotaxic apparatus. Two cannula made of 22-gauge stainless steel tubing (C313G; Plastic Products) were implanted bilaterally into the lateral (LA) or basolateral (BLA) amygdala (anteroposterior, −2.8 mm; mediolateral, ±4.5 mm; dorsoventral, −7.0 mm) (23Paxinos G. Watson C. The Rat Brain in Stereotaxic Coordinates. Academic Press, Inc., New York1986Google Scholar). A 28-gauge dummy cannula was inserted into each cannula to prevent clogging. Three jewelry screws were implanted over the skull, serving as anchors and the whole assembly was affixed on the skull with dental cement. The rats were monitored and handled daily and were given 7 days to recover. NF-κB inhibitors were administered bilaterally in a volume of 0.5–0.8 μl at a rate of 0.5 μl/min. κB decoy and scrambled DNA were prepared and administered based on the method of Blondeau et al. (24Blondeau N. Widmann C. Lazdunski M. Heurteaux C. J. Neurosci. 2001; 21: 4668-4677Crossref PubMed Google Scholar). In brief, double-stranded κB decoy DNA was prepared by annealing complementary single strands with the sequences of 5′-GAGGGGACTTTCCCT-3′. Control DNA with a scrambled sequence was prepared by annealing oligonucleotides of the following sequence: 5′-GATGCGTCTGTCGCA-3′. Stocks of double-stranded DNA were prepared at a concentration of in κB decoy and scrambled DNA were into the amygdala at a rate of 0.5 at and conditioning was the startle in a startle as (20Lin C.H. Yeh H.W. Lin C.H. Lu K.T. Leu T.H. Chang W.C. Gean P.W. Neuron. 2001; 31: 841-851Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, M. Behav. PubMed Scopus Google Scholar). the was in a with an on the The whole was in a and The startle was a at an of The was a light by an to the of The which as the for fear was a with a of 0.5 was as the that during a the of the startle days each rat was to over In rats were in the startle and with startle at the of startle in the of rats were into with similar response the training rats were in the startle and the of light and but in a in which the occur at at following the rats were and were rapidly and in artificial cerebrospinal the brain was and slices of were the following and The was with and a of were made by stimulation of the which from the to the lateral with a in were at a of were to of the LTP was by of at at the stimulation used for κB decoy DNA amygdala slices from each rat were into in the were with κB decoy DNA for at were Control slices were for a similar of with scrambled DNA or in The lateral and basolateral of the amygdala were in (50 sodium sodium and In the LTP slices were given of or were with for the that reliably LTP in this area of the brain. slices were from the at as indicated the or the of the were at for and the was the by at for at concentration in the was a with as the of protein for each were in to and blocked in (50 of NF-κB and were with for or IκB-α Inc., was used for were in the used for The of the was by an analysis system the rats were the were rapidly and the and were a with a in PMSF, and sodium The was for at and was in PMSF, and sodium a were on for and with a The was at for The were in μl of of PMSF, sodium and were on for The nuclear was at for at and the was was on the amygdala from or at specific binding of protein was in binding and for at The double-stranded NF-κB was with kinase and has been K. M. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). was to the and for In the the nuclear extracts were with of the c-Rel proteins for the binding The of binding was demonstrated by the of or cAMP-response element-binding protein The were by in a with and 0.5 The were by an activity was an complex kinase assay as F. H. A. D.B. M. M. Manning A. A. 1997; PubMed Scopus Google Scholar). were prepared from or rats given thalidomide in μl of 0.5 μl of whole extracts were an the of activity was in kinase of PMSF, at at at and at for in the of the indicated The kinase was by the of to and by was and from with We whether conditioning was associated with activation of NF-κB by in which nuclear extracts from the amygdala of or rats were with NF-κB DNA A that fear training significantly the of NF-κB with NF-κB DNA binding activity was not in of NF-κB a the brain. whether it was specific for the experiments were to NF-κB DNA binding activity in the and in training of fear not to an increase in NF-κB binding activity in the and These results that of NF-κB activity in the of the brain involved in the of fear memory. Previous studies have shown that biochemical changes that occur in and during fear conditioning in vivo are similar to those occur during LTP in J. Neurosci. 2000; 20: PubMed Google Scholar, Kandel E.R. J. Neurosci. 2000; 20: PubMed Google Scholar, C. Jr., M. J. Neurosci. 2001; 21: PubMed Google Scholar). We whether or TS, the that reliably LTP in this area of the a similar effect as fear were with (50 for and nuclear extracts prepared from the and were for NF-κB activation A that NF-κB binding activity was in the of a of κB In effect the of binding. which NF-κB family members that were we A revealed that p65 and p50 the of proteins with the NF-κB experiments were following shown in assay indicated that the NF-κB complex induced by was also the the NF-κB and The induction of the of NF-κB phosphorylation and subsequent degradation of IκB-α (7Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar). We analysis to or have on the of A that IκB-α protein was by at The degradation was followed by an of IκB-α at degradation of IκB-α was at following the of We the PI 3-kinase and NF-κB We that degradation of IκB-α was by PI 3-kinase the of PI 3-kinase in NF-κB α-p-tosyl-l-lysine chloromethyl is a that was used as NF-κB T. T. M. P.A. Nature. PubMed Scopus Google Scholar, M. H. M. D. M. J. 1996; PubMed Scopus Google Scholar). in the degradation of IκB-α was blocked by We further the effect of on the NF-κB DNA binding activity. of training blocked binding of NF-κB However, the of to the nuclear extracts from the rats not the binding of NF-κB to the These that of NF-κB binding by is not to the transcription factor and the DNA but that to NF-κB activity. is the for the phosphorylation of we the of activity in the of was from whole extracts of rats that been were for kinase activity by with IκB-α protein in the of on a and by that fear training led to an increase in kinase activity by that the It has been shown that and of thalidomide were based on its to NF-κB activation of activity J.A. Jr., A.S. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). We whether thalidomide a similar effect on the by administration of this in μl of 0.5 μl Quantitative analysis of activity demonstrated that thalidomide inhibited the increase at and as with that thalidomide Paralleled behavioral tests training revealed that thalidomide inhibited fear-potentiated startle. were in the and in the slices were given of or were with for and extracts of were into and nuclear and by A that NF-κB p50 protein was reduced to of its in the In in the nuclear NF-κB p50 protein accumulated to of its Similarly, NF-κB p50 protein was reduced to of its in the of the in the nuclear NF-κB p50 protein accumulated to of its investigate whether the activation of NF-κB was not but also for we or 0.5 μl bilaterally into the or was A that impaired fear memory. The analysis of for startle revealed a effect for revealed the and the and mm) of was and mm; Similarly, of NF-κB chloromethyl 0.5 μl blocked the acquisition of fear memory The cannula are shown in B. rats with cannula at or the of and were in the We administered double-stranded κB decoy DNA or double-stranded DNA with a scrambled sequence bilaterally into the amygdala based on the method of et al. Z. D. Mattson M.P. J. Neurosci. 1999; 19: PubMed Google Scholar). the rats were and nuclear extracts from the amygdala were with NF-κB DNA shown in the increase in NF-κB DNA binding activity was not in rats that were given κB decoy Furthermore, of startle potentiation conditioning revealed a depression of the of potentiation of as with those scrambled DNA or rats the effect of κB decoy DNA on the amygdala rats that κB decoy DNA were for at days and subsequently were 7 that rats that impaired memory by κB decoy DNA a startle This that the effect of κB decoy DNA was and not to to the amygdala It has been shown that hippocampal slices were for in the of κB decoy of the κB decoy DNA into (19Albensi B.C. Mattson M.P. Synapse. 2000; 35: 151-159Crossref PubMed Scopus (421) Google Scholar). slices were with κB decoy DNA for at were Control slices were for a similar of with scrambled DNA in a that of of at an of a long of synaptic in and scrambled The of were and at and the the and of stimulation a potentiation (LTP) in κB decoy The at and the stimulation were and of The and κB decoy slices was at and We also experiments to whether the synaptic was by the and with κB decoy We that the was not by κB decoy DNA the of proteins with the NF-κB we and NF-κB with In the first of slices were given of TS, and nuclear extracts from the and were with and with NF-κB shown in in a increase in NF-κB of which bound to the effect was was in the of (50 with that LTP in this area is Kandel E.R. Neuron. 1998; 21: Full Text Full Text PDF PubMed Scopus Google Scholar). further the and NF-κB, nuclear extracts were first with NF-κB and with A similar increase of in the NF-κB and was TS, and this effect was blocked by (50 slices were with (50 for and nuclear extracts from the and were with and with NF-κB shown in activation of by a increase in NF-κB which bound to The effect of was blocked by the by protein kinase A. Similarly, nuclear extracts were first with NF-κB and with of which was to We have the role of NF-κB in amygdala synaptic Here we show for the first that fear-potentiated startle results in an activation of NF-κB and administration of NF-κB or κB decoy DNA memory consolidation and activated NF-κB to cognate DNA sequences to transcription of specific target genes, the results are with studies a requirement of new protein synthesis during fear conditioning and the of LTP in the amygdala Kandel E.R. J. Neurosci. 2000; 20: PubMed Google Scholar, J. Neurosci. 2000; 20: PubMed Google Scholar). The protein levels as well as the DNA binding activity of NF-κB were significantly in the the that and in an not any that NF-κB activation is specific to the of the In activation was to the amygdala but not the or the that amygdala but not or is involved in fear-potentiated startle. Previous also showed that fear conditioning was associated with an activation of J. J. Neurosci. 2001; PubMed Google and cAMP-response transcription S. K. R. C. Neurosci. 1998; PubMed Scopus Google in the Furthermore, of in the has been shown to long term memory of fear-potentiated startle C. Jr., M. J. Neurosci. 2001; 21: PubMed Google Scholar). training for conditioning or led to increase in cAMP-response gene in and of the S. K. R. C. Neurosci. 1998; PubMed Scopus Google Scholar). The of the which from and and in synapses on in the and is generally thought to and to and A. V. 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In the administration of κB decoy DNA or NF-κB inhibitors in an of LTP and memory formation. These results the that memory consolidation of fear and LTP in the a Supershift p65 or p50 subunits further indicated that the NF-κB complex induced by or was the This is with that and NF-κB in the system of (16O'Neill L.A.J. Kaltschmidt C. Trends Neurosci. 1997; 20: 252-258Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar, T. J. J. Neurosci. 1997; PubMed Scopus Google Scholar). we that protein levels of IκB-α were reduced by at or of followed by its at a This was accompanied by a decrease of NF-κB in the cytoplasm concomitant with an increase in the nucleus. The of IκB-α to the of NF-κB binding in the of IκB-α which was activated by NF-κB (7Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar, P.A. Ballard W.C. PubMed Scopus Google Scholar, Jr., A.S. 1994; PubMed Scopus Google Scholar, Miyamoto S. I.M. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). In the degradation of IκB-α was prevented by that PI 3-kinase is for activation of A role for PI 3-kinase in the of factor and factor activation of NF-κB has been in and (21Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-85Crossref PubMed Scopus (1866) Google Scholar, 22Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1654) Google Scholar). used as a and an for first to the leading to its from the It and and has to its for the of as and of 2000; PubMed Scopus Google Scholar). NF-κB is a regulator of genes, and it has been shown that thalidomide inhibited NF-κB DNA binding activity and transcriptional of in and a the of J.A. Jr., A.S. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In the we that fear training induced an increase in activity, in the activation of NF-κB in the with the in and thalidomide inhibited NF-κB activation an of behavioral study revealed that thalidomide blocked fear-potentiated startle, which the role of NF-κB in the acquisition of fear memory. The of new is thought to long changes in the strength of specific synaptic by specific in gene evidence indicates that is of the in the that term to long term synaptic plasticity A. Kandel E.R. 108: Full Text Full Text PDF PubMed Scopus Google Scholar, Neuron. 35: Full Text Full Text PDF PubMed Scopus Google the consolidation of fear memory (20Lin C.H. Yeh H.W. Lin C.H. Lu K.T. Leu T.H. Chang W.C. Gean P.W. Neuron. 2001; 31: 841-851Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, Kandel E.R. J. Neurosci. 2000; 20: PubMed Google C. Jr., M. J. Neurosci. 2001; 21: PubMed Google Scholar, S. K. R. C. Neurosci. 1998; PubMed Scopus Google Scholar). The experiments revealed that or the and NF-κB, and the effect was blocked by and It is that the NF-κB and transcriptional factors with for the cAMP-response and κB the role of a it is that TS, and fear training NF-κB the activation of PI 3-kinase, IKK, and subsequent degradation of IκB-α in the This NF-κB to into the nucleus, it to specific κB DNA sequences in the of to transcription and translation of new protein for memory consolidation In in an parallel, the results show that LTP in slices or fear-potentiated startle in NF-κB in the NF-κB on the IκB-α degradation as well as LTP and fear memory. These results provide the first evidence of a requirement of NF-κB activation in the amygdala for synaptic plasticity and memory Furthermore, the of NF-κB as a in the of fear-potentiated startle a new target for the of and in has to that a that IκB kinase and NF-κB activation are for memory in the has recently been in and A. We for of the