Patent Application: US-84343204-A

Abstract:
several genes encoding subunits of the neuronal nicotinic acetylcholine receptors have been cloned and regulatory elements involved in the transcription of the ∝: 2 and ∝: 7 - subunit genes have been described . yet , the detailed mechanisms governing the neuron - specific transcription and the spatio - temporal expression pattern of these genes remain largely uninvestigated . the β2 - subunit is the most widely expressed neuronal nicotinic receptors subunit in the nervous system . we have studied the structural and regulatory properties of the 5 ′ sequence of this gene . a fragment of 1163 bp of upstream sequence is sufficient to drive the cell - specific transcription of a reporter gene in both transient transfection assays and in transgenic mice . deletion analysis and site - directed mutagenesis of this promoter reveal two negative and one positive element . the positively acting sequence includes one functional e - box . one of the repressor elements is located in the transcribed region and is the nrse / re1 sequence already described in promoters of neuronal genes .

Description:
the descriptions and examples below are exemplary of the embodiments and scope of this invention . the invention is not limited to the scope of this description . furthermore , this description together with the accompanying sections of this specification and the material incorporated by reference enables the practice of all of the claims which follow . the examples and embodiments that follow of course can be modified by techniques known in the art . variations in the nucleic acid sequences described or claimed can be produced by known methods without altering the effects or advantages the inventors have shown . such variations are therefore included within the scope of this description and invention . the pcx49 plasmid ( deneris et al ., 1988 ) containing the entire rat cdna ( kindly provided by drs . j . boulter and s . heinemann , the salk institute , san diego , calif .) was cut with ecori , the ˜. 2 . 2 kb fragment was isolated and used as a probe to screen an embl3 bacteriophage library of mouse dba2 genomic dna . one unique clone was obtained spanning ˜ 15 kb of dna upstream and ˜ 5 kb downstream from the first exon . fig1 shows the nucleotide sequence of 1 . 2 kb upstream from the initiator atg . hybridization conditions can be modified by known techniques 29 to determine stringent conditions for this probe . changes in the hybridization conditions such as temperature ( from about 45 ° c . to about 65 ° c .) and ssc buffer concentration ( from about 0 . 1 × ssc to about 6 × ssc ), as well as changes in the temperature of and the buffer for the washing condition can be made to develop sufficiently stringent conditions that allow hybridization to the β2 - subunit sequences . other related sequences can thus be isolated from other libraries based on this hybridization procedure . human sequences will be isolated by using hybridization conditions such as 45 ° c . and 6 × ssc . three deposits were made on dec . 13 , 1994 at the collection nationale de cultures de microorganismes ( cncm ), institut pasteur , 25 rue du docteur roux , 75724 paris cedex 15 , france . a phage , λβ2 nachr , is deposited under the accession number i - 1503 . this phage contains 15 - 20 kb of genomic dna including the promoter sequences and the coding sequences for all of the exons of the murine β2 - subunit of neuronal nachr . two e . coli cultures bearing plasmids have also been deposited . plasmid psa9 in e . coli dh5α has accession number i - 1501 and contains 9 kb of murine genomic dna including the regulatory sequences and regions coding for exons 1 , 2 and 3 of the β2 - subunit . plasmid pea5 in e . coli dh5α has accession number i - 1502 and contains 5 kb of murine genomic dna including a region of about 1 . 2 kb upstream of the eco47 - iii site and a region coding for exons 1 to 5 of the β2 - subunit . the inventors intend to deposit the nucleotide sequence data reported here in the embl , genbank and ddbj nucleotide sequence databases under the accession number : x82655 . for the mrna mapping , we used different batches of total rna extracted from dba2 embryos at stage e13 or e15 . the rna samples were first digested with dnase i to avoid dna contamination . rnase protection . an xbai / psti fragment containing part of intron 1 was inserted into bluescript sk ( stratagene ). the plasmid was then linearized by bglii , and an rna probe was synthesized using the t7 promoter . the protection experiments were then performed as described in ausubel et al . ( 1994 ). race - pcr ( frohman et al ., 1988 ). the mrna was hybridized 5 minutes at 80 ° c . with 10 pmol of primer . the synthesis of the cdna was performed using 400 u mmlv ( gibco ) for 45 minutes at 37 ° c . in the buffer recommended by the supplier . after a phenol / chloroform extraction , the cdna was ethanol precipitated . the terminal transferase reaction was performed in 0 . 2 m potassium cacodylate ; 25 mm tris - hcl ph 6 . 6 ; 25 mg / ml bsa ; 1 . 5 mmcocl 2 ; 50 nm datp and 50 u terminal transferase ( boehringer ) for 30 minutes at 37 ° c . after phenol / chloroform extraction and ethanol precipitation , one tenth of the terminal transferase reaction was amplified using promega &# 39 ; s taq dna polymerase ( 30 cycles , 1 minute at : 94 ° c . ; 55 ° c . ; 72 ° c .). the amplified fragment was then loaded on an agarose gel . the gel was blotted and hybridized to oligonucleotide p2 . we used pex2 as a primer for cdna synthesis , and p0 / bept for pcr to map mrna from brain . oluci3 ( synthesis of cdna ) and oluci2 / bept ( pcr ) were used to map mrna from transfected cells . slic ( dumas milnes edwards et al ., 1991 ). the cdna was first synthesized from 5 μg total rna using pex3 ( 6 pmol ) as a primer in 50 mm tris - hcl ph 8 . 3 ; 8 mm kcl ; 1 . 6 mm mgcl 2 ; 5 mm spermidine ; 0 . 5 mm dntp ; 1 u / μl rnasin ; 0 . 1 mg / ml bsa ; 70 mm β - mercaptoethanol ; 80 u amv reverse transcriptase ( promega ) at 420 for 45 minutes . the rna was subsequently degraded in naoh . the first strand of the cdna was then ligated with the oligonucleotide a5 ′. the resulting single stranded cdna was then submitted to two rounds of pcr amplification with oligonucleotides a5 ′- 1 / p0 and a5 ′- 2 / pl ( 35 cycles 94 ° c . 1 minute ; 60 ° c . 30 seconds ; 72 ° c . 45 seconds ). a5 ′: 5 ′- ctgcatctatctaatgctcctctcgctacctgctcactctgc gtgacatc a5 ′- 1 : 5 ′- gatgtcacgcagagtgagcaggtag a5 ′- 2 : 5 ′- agagtgagcaggtagcgagaggag p0 : 5 ′- ccaaagctgaacagcagcgccatag p1 : 5 ′- agcagcgccatagagttggagcacc p2 : 5 ′- aggcggctgcgcggcttcagcaccacggac pex2 : 5 ′- gccgctcctctgtgtcagtacgcaaaaccc pex3 : 5 ′- acattggtggtcatgatctg bept : 5 - gcgggatccgaatc ( t ) 21 a / c / g oluci3 : 5 ′- cgaagtattccgcgtacgtgatg oluci2 : 5 ′- accagggcgtatctcttcatagc ks - luci : the hindiii / kpni restriction fragment of the psvoal plasmid ( de wet et al ., 1987 ) was subcloned in the corresponding site of bluescript ks . the most 5 ′ ecori / bsmi ( 45 bp ) fragment of the luciferase gene was then deleted according to ( de wet et al ., 1987 ) and replaced by a sal i site . the 342 bp pvuii / hindiii restriction fragment of sv40 containing the polyadenylation sites was subsequently subcloned into the eagi sites using adaptors . ee1 . 2 - luci : the 1 . 2 kbp ecori / eco47ii fragment of the λβ2 phage was inserted in the eagi / sali sites of ks - luci using adaptors . the 5 ′ end deletions of the promoter were obtained using bai3 . 1 exonuclease as in current protocols in molecular biology ( ausubel , et al ., 1994 ). the mutations were introduced using the sculptor kit ( amersham ). in the nrse / re1 sequence , the mutated sequence was : + 24 accacttaca instead of accacggaca , as this mutation was shown to reduce the activity of the nrse element ( mori et al ., 1992 ). in the e - box sequence , the mutated sequence was : − 120 tcctcagg instead of tccacttg . fig7 shows that a nuclear protein is able to bind to the wild type sequence , but not to the mutated sequence . neuroblastomas n1e115 , human sk — n — be , hela and 3t6 fibroblasts , 293 human kidney cells and svlt striatal cells ( evrard et al ., 1990 ) were grown in dmem + 10 % fcs supplemented with 1 % glutamine and 1 % streptomycin . pc 2 cells were grown in dmem + 10 % hs + 5 % fcs supplemented with 1 % glutamine and 1 % streptomycin . cells were plated at 10 5 to 4 × 10 5 cells / 60 mm 2 plates . the next day cells were transfected in 750 μl of dmem + 2 % penicillin / streptomycin for 5 to 12 hours with 1 μg dna mixed with 2 . 5 pl of transfectam ( ibf / sepracor ) in 150 mm nacl . the luciferase activity was measured 48 hours later . dna was prepared using qiagen or wizard prep ( promega ) kits . when plasmid activities were compared , all plasmids were prepared the same day . at least two different dna preparations were tested for each plasmid . all transfections were done in duplicate and repeated at least three times . the luciferase gene from ee1 . 2 - luci was excised and replaced by the nlslacz gene ( kalderon et al ., 1984 ). the β2 - promoter / nlslacz fragment was electroeluted from a tae agarose gel then further purified by ethanol precipitation , and finally resuspended in tris - hcl 10 mm ph 7 . 5 ; edta 0 . 1 mm . the dna solution ( 3 ng / ml ) was injected into fertilized oocytes of c57bl6xsjl hybrids . staining of tissues was performed as described in mercer et al ., 1991 . oligonucleotides were labeled either with y [ 32 p ] atp and t4 polynucleotide kinase , or with ∝[ 32 p ] ctp and klenow enzyme as in current protocols in molecular biology . nuclear extracts were prepared from ≅ 10 7 cells as described ( bessis et al ., 1993 ). for binding , 1 nmol of labeled oligonucleotide was mixed with 0 . 5 μg of protein extract in 10 mm hepes ph 8 , 10 % glycerol ], 0 . 1 mm edta , 0 . 1 m nacl , 2 mm dtt , 0 . 1 mg / ml bsa , 4 mm mgcl 2 , 4 mm spermidine , 1 mm pmsf , 1 μg polydldc in 20 μl . the reaction was incubated for 10 minutes on ice . the dna - protein complexes were then analyzed on a 7 % polyacrylamide gel . the oligonucleotides used in this experiments were double stranded with the following sequences ( the underlined nucleotides are changed between the mutated and the wild type oligonucleotides ): e - d : 5 ′- tcctcccctagtagttcc a c tt gtgttccctag mut - e : 5 ′- cctcccctagtagttcc t c ag gtgttccctaga s - e : 5 ′- ctagctccggggcggagactcctcccctagtagttccagttg tgttccctag a λ phage containing the gene encoding the β2 - subunit was cloned and a region surrounding the initiator atg was sequenced ( fig1 ). the transcription initiation site was first mapped by rnase protection ( fig2 a ). this method allowed us to detect at least three initiation sites . however , minor additional start sites might not have been detected in these experiments . the size of the main protected band was estimated at about 150 nucleotides . to confirm and locate the initiation sites more precisely , we performed both race - pcr ( rapid amplification of cdna ends ; frohman et al ., 1988 ) and slic ( single strand ligation of cdna ; dumas milnes edwards et al ., 1991 ) which consist in the amplification of the primer extension product ( fig2 b ). both techniques allowed us to subclone and sequence the same fragments corresponding to the four initiation sites described in fig1 . it is probable that the − 13 start site is very rare and was not detected by rnase mapping . analysis of the sequence of the flanking region ( fig1 ) revealed several consensus dna binding elements : an sp1 site (− 146 ), a camp responsive element binding ( creb ) site (− 287 ; sassone - corsi , 1988 ), a nuclear receptor response element (− 344 to − 356 ; parker , 1993 ), a gata - 3 site (− 1073 ; ko and engel , 1993 ), and a weakly degenerate octamer motif (− 522 ). moreover , an e - box (− 118 ) contained in a dyad symmetrical element could be recognized . the proximal region (− 245 to + 82 ) also has an unusually high gc content ( 67 %) and a high number of dinucleotide cpg that may have some regulatory significance ( antequera and bird , 1993 ). finally , a 20 bp sequence identical to the nrse ( neural restrictive silencer element ; mori et al ., 1992 ) or re1 ( restrictive element ; kraner et al ., 1992 ) sequence was found in the 3 ′ end of the 1 . 2 kbp fragment (+ 18 to + 38 ). a 1 . 2 kbp fragment of flanking sequence of the β2 - subunit gene promotes a construct was generated containing the 1163 bp ecori / eco47iii fragment ( from − 1125 to + 38 ) of the β2 - subunit 5 ′ flanking region fused to the luciferase gene ( de wet et al ., 1987 ) ( plasmid ee1 . 2 - luci ). the polyadenylation sites of sv40 were inserted upstream from the β2 - subunit sequences to avoid readthrough . the transcriptional activity of the plasmid ee1 . 2 - luci was then tested by transient transfection into pheochromocytoma ( pc12 ) cells , neuroblastoma cell lines nie 115 and sk — n — be , svlt , a striatal cell line ( evrard et al ., 1990 ), nih3t6 or hela fibroblasts and human kidney cell line 293 . using rt - pcr , we verified that the neuroblastomas and the pc12 cells normally express the β2 - subunit mrna but not the striatal svlt cell lines or the 3t6 fibroblasts . fig3 shows that in pc12 cells and neuroblastomas , the 1 . 2 kbp fragment is 20 to 180 - fold more active in mediating transcription of the reporter gene than in the other cell lines . in fibroblasts , 293 cells and svlt cells , the transcriptional activity of the 1 . 2 kbp fragment is not significantly higher than that of the promoterless vector ( fig3 ). therefore , the β2 - subunit promoter is not active in these cell lines . these in vitro transfection experiments demonstrate that the 1163 bp fragment mimics the expression pattern of the endogenous β2 - subunit gene , and thus contains a cell - specific promoter . to test the 1163 bp promoter in vivo , the ecori / eco47iii fragment was linked upstream from the nis - β - galactosidase reporter gene ( kalderon et al ., 1984 ). the polyadenylation signals from sv40 were ligated downstream of the coding sequences . the resulting 4 . 7 kb fragment was subsequently microinjected into the male pronuclei of fertilized eggs from f1 hybrid mice ( c57b16xsjl ). dna extracted from the tails of the offspring was analyzed for the presence of the β - galactosidase gene by the polymerase chain reaction ( pcr ). three independent founders were obtained and analyzed for expression . two lines ( 13 and 26 ) had expression in neurons and the third line did not express at all . this shows that the 1163 bp promoter contains regulatory elements sufficient to drive neuron - specific expression in vivo . in the peripheral nervous system pns , both lines expressed in the same structure . in contrast , in the cns the labelling pattern of line 26 is a subset of that of line 13 . we will only describe line 13 in detail . as expected , most peripheral β2 - expressing ganglia expressed β - galactosidase ( β - gal ), whereas in the cns only a subset of β2 - positive regions expressed the β - gal . for instance , fig4 c shows that the vast majority of the neurons of the lumbo - sacral spinal cord express the β2 - subunit transcripts , whereas only a subset of neurons in the ventral and dorsal horns display β - gal activity . the expression of the transgene could be detected in the peripheral ganglia in e10 . 5 and e11 embryos . the labelling was examined in e13 total embryos ( fig4 a ) and in brains at later ages ( e17 , po and adulthood ). at e13 , labelling was prominent in pns : strong labelling was observed in the dorsal root ganglia ( drg , fig4 and 5 c , d ); some ganglia associated with the cranial nerves ( the trigeminal see fig5 a , geniculate , glossopharyngeal and vagal ganglia ); the ganglia of the sympathetic chain ( fig5 c , d ); the ganglionic cells of the retina ( fig5 a ); and putative parasympathetic ganglia in the cardiac wall ( fig5 b ). at e13 , clusters of positive cells were also present at several levels of the neuraxis , in both the brainstem and the proencephalon . clusters of stained neurons were also observed in the ventral and lateral spinal cord . later in development ( e17 ), positive neurons were found clustered in several basal telencephalic nuclei whereas dispersed cells were stained in the caudate - putamen . at the diencephalic level , positive clusters were present in the zona incerta and reticular thalamic nucleus , and in many hypothalamic nuclei . in the brainstem , most motor nuclei of cranial nerves ( with the exception of the dorsal motor nucleus of the vagus nerve ) showed some to high labelling . in addition , the dispersed cells of the v mesencephalic nucleus appeared strongly stained , as well as the pontine nuclei , the prepositus hypoglossal nucleus and a few dispersed cells in the pontine tegmentum . at po in line 13 , the distribution of positive cells already appeared more restricted than at previous ages ( for example labelling in basal telencephalon and oculomotor nuclei was clearly diminished ). in the cns of adult animals labelled cells were detected only in the hypothalamus . in line 13 , some clusters of cells were stained in the mucosa of the gastrointestinal tract ( stomach and duodenum ) and in the pancreas . ectopic labelling was detected in the genital tubercle and in several superficial muscles of line 13 , but none of these tissues were stained in the line 26 . to investigate in more detail the regulatory elements involved in the promoter activity , we generated a series of plasmids containing 5 ′ deletions of the 1163 bp promoter . these plasmids were tested by transient transfection into fibroblasts and sk — n — be cells . these two cell lines were chosen as they were the most easily transfected cell lines . moreover , the neuroblastoma line was initially isolated from peripheral structures ( biedler et al ., 1978 ) and is a convenient tool to study the regulatory elements carried by the 1163 bp promoter . when 157 bp were deleted from the 5 ′ end of the 1163 bp promoter ( plasmid 1006e - luci , described in fig1 ), the luciferase activity did not significantly change in neuroblastomas but increased in fibroblasts ( fig6 ). when 301 bp were further deleted , the activity of the remaining promoter continued to increase in the fibroblasts but not in neuroblastomas ( see plasmid 862e - luci , fig6 ). thus , the 157 and 301 bp deleted plasmids carry repressor elements which are only active in fibroblasts . however , the truncated 862 bp promoter still displayed a neuron - specific activity ( fig6 , compare activity of 862e - luci in both cell lines ), showing that additional regulatory elements are carried by the 1 . 2 kbp promoter . moreover , a repressor could be present between − 824 and − 245 ( compare the activities of 862e and 283e - luci in the neuroblastomas ). this putative regulatory element was not further analyzed . indeed , a 283 bp promoter ( plasmid 283e - luci ) is still ≅ 160 times more active in neuroblastomas than in fibroblasts , confirming the presence of another neuron - specific regulatory elements in this proximal portion of the promoter . when 150 bp were deleted from the 5 ′ end of the proximal 283 bp promoter , a very strong decrease of the transcriptional activity was detected in both fibroblasts and neuroblastomas ( see activity of plasmid 133e - luci ). this shows that crucial positive regulatory elements have been deleted . these positive and negative elements were further investigated by deletion and mutation studies of the proximal portion of the promoter . the 3 ′ end of the β2 - subunit promoter contains putative protein factor binding sites . to analyze the role of these elements in β2 - subunit gene regulation , we generated plasmids containing mutations in these binding sites . using deletion experiments , an activator was detected between − 95 and − 245 ( see fig3 , the difference between 283e and 133e - luci ). as the e - box located at nt - 1l8 was a good candidate , we analyzed the effect of mutations in this element on transcriptional activity . table 1a shows a 40 % reduction of the transcriptional activity of the mutated promoter compared to that of the wild type promoter . the role of the e - box in non - neuronal tissues was more difficult to assess as the basal level of transcription was already low in fibroblasts . to further understand the role of the e - box in the regulation of the promoter , we investigated the protein complexes able to interact with this sequence . gel shift assays were performed using the 33 bp sequence ( nt - 135 to - 103 , oligonucleotide e - d ) as a probe . when the 32 - p labelled oligonucleotide was mixed with nuclear extracts from neuroblastomas or fibroblasts , three complexes were observed ( fig7 ). all of them were fully displaced by an excess of the unlabelled oligonucleotide e - d . in contrast , no competition was observed when the competitor oligonucleotide was mutated within the e - box / dyad ( oligonucleotide mut . e , see fig7 lane “ mut - eu ”). this shows that the e - box / dyad is the only element contained within the − 135 / 103 sequence able to bind nuclear protein . this sequence is likely to be involved in the activity of the β - subunit promoter . an nrse / re1 sequence is also present in the proximal region and has been shown to act as a silencer in fibroblasts but not in pc12 cells or neuroblastomas ( kraner et al ., 1992 ; li et al ., 1993 ; mori et al ., 1992 ). point mutation of this sequence in the context of the 1163 bp promoter resulted in a 105 - fold increase in the transcriptional activity in fibroblasts , and only a 3 - fold increase in neuroblastomas ( table 1a ). this sequence is thus responsible for at least part of the cell - specific expression of the β2 subunit gene . elimination of high affinity nicotine receptor in transgenic mice results in alteration of avoidance learning the β2 - subunit of the nachr was disrupted in embryonic stem ( es ) cells , and mice deficient in this subunit were subsequently generated ( fig8 ). β2 −/− mice were viable , mated normally and showed no obvious physical deficits . overall brain size and organization were normal ( see for example fig9 , a and b ). western blot analysis of total brain homogenates using anti - β2 monoclonal 270 11 ( fig8 d ) and immunocytochemistry throughout the brain using a polyclonal anti - β2 antibody 9 demonstrated that the immunoreactivity detected in control mice was absent in β2 −/− mice and was diminished in β +/− mice . p2 - encoding mrna was undetectable in β2 −/− mice by in situ hybridization using β2 - antisense oligonucleotides ( fig9 a ). the distributions of the α4 - and β2 - subunits largely overlap in the brain , and these subunits are thought to combine to form the predominant nachr isoform in the cns 12 . based on oocyte expression experiments 6 , β4 - is the only subunit identified thus far that might also be able to form functional heteropentamers with the α4 - subunit . the β4 - subunit was expressed normally in the brains of β2 +/− mice or β2 −/− mice , with expression in the medial habenula ( mhb ) and the interpeduncular nucleus ( ipn ) 10 , and no upregulation elsewhere in the brain to replace the β2 - subunit ( fig9 a ). nor was the expression of the α4 - ( fig9 a ), α5 - or β3 - subunit mrnas significantly altered in mutant mice . equilibrium binding experiments have shown that nicotine binds to a population of high affinity sites ( kd near 10 nm 13 , 14 ), whose distribution tallies well with that of the α4 - and β2 - subunits 13 - 15 . quantitative receptor autoradiography was performed using 3 h - nicotine ( 4 nm ) to visualize high affinity nachr in brain sections from β2 +/+, +/− and −/− mice ( fig9 b ). nicotine binding in situ was completely abolished in β2 −/− animals , and was reduced by approximately 50 % in all brain areas in β2 +/− animals implicating the β2 - subunit in mediating this high affinity binding . neurons of the anterior thalamus , which express very high levels of β2 ( and α4 ) subunit mrnas ( fig9 a ), were studied for an electrophysiological response to nicotine . this area , easily accessible in a slice preparation , responded consistently to 10 μm nicotine in wild type animals with an average inward current of 155 +/− 73 pa which was blocked by 1 μm dihydro - β - erythroidine . the agonist order of the response was compatible with that seen for α4 / β2 - containing nicotinic receptors in vitro 6 ( nicotine & gt ; dmpp & gt ; cytisine ) ( fig1 a ). anterior thalamic neurons required several minutes to an hour for complete recovery of the agonist response , suggesting that receptor response is prone to desensitization . moreover , a relatively high dose of 1 μm was required for a reproducible response , implying that nicotine does not bind to its high affinity site to activate . high affinity nicotine binding sites may therefore be nachrs in a desensitized conformation . in β2 −/− mice the response of anterior thalamic neurons to nicotine was completely abolished in 100 % of neurons tested ( fig1 b ). as a control , neurons in the mhb , where both α3 and β4 are strongly expressed , were also tested . nicotine caused an average inward current of 505 +/− 132 pa in wild type mice , and the agonist potency of this response followed the rank order for the α3 / β4 containing receptor ( cytisine = nicotine & gt ; dmpp ) ( fig1 a ). as expected , the response of cells in the mhb to nicotine was maintained in mutant mice . the β2 subunit is expressed in the ganglia of wild type animals 8 - 10 , but there was no apparent difference in heart rate or basal body temperature . spontaneous locomotor activity , which is sensitive to high doses of nicotine and is not modified by drugs selective for the β2 / α4 isoform of the nachr 16 , was not significantly different in β2 −/−, b +/ 1 and β +/+ mice . learning and memory were examined in mutant and wild type mice using two procedures . the morris water maze 17 , 18 evaluates spatial orientation learning . the performance of mutant mice on this test did not differ from that of wild type mice when tested on the visible platform task , or on the hidden platform task ( minimum swim - time reached after 5 days of training : mutants ( n = 8 ): 7 . 4 +/− 1 . 4 sec ; wild type ( n = 8 ): 8 . 2 +/− 2 . 0 sec ). in the transfer test both groups of animals spent approximately 35 % of the time in the platform quadrant , with the same number of platform crossings ( mutants : 4 +/− 0 . 4 ; wild type : 3 . 9 +/− 0 . 6 ). retention of an inhibitory avoidance response was assessed using the passive avoidance test , which was also chosen for its pharmacological sensitivity to nicotine administration 19 , 20 . this test consisted of a training trial in which the mouse was placed in a well - lighted chamber of a shuttle box , and the latency to enter the adjacent dark chamber was measured . upon entry to the dark chamber , a mild , inescapable foot shock was delivered , and vehicle or nicotine ( 10 μg / kg ) was injected into the mouse . twenty - four ( 24 ) hours later , retention was assessed by measuring the latency to enter the dark chamber . time spent in the light chamber ( retention latency ) increased proportionally to the applied foot shock in both mutant and wild type mice . however , treatment with nicotine consistently facilitated retention ( p & lt ; 0 . 01 ) by shifting the curve upward by approximately 80 sec only in wild type mice ( fig1 a ). nicotine administration was completely ineffective in mutant mice . interestingly , retention latency was significantly higher for mutant mice than for their non - mutant , vehicle - injected siblings ( p & lt ; 0 . 05 ) ( fig1 b ). increased retention in the passive avoidance test can be observed in animals with a decreased pain threshold or increased emotionality . therefore , further behavioral testing was performed on all mice included in this experiment . mutant mice did not differ from their non - mutant siblings for flinch , vocalization or jump response to foot shock . emotionality was tested by measuring exploratory activity in a two compartment apparatus for 15 min 21 , 22 . the average time spent in the dark compartment , the locomotor activity in the dark compartment and the transitions between compartments did not differ between the mutant and wild type mice . therefore , neither changes in pain sensitivity nor changes in emotionality can account for the difference in retention latency observed in passive avoidance testing . studies using low doses of nicotine 23 or specific nicotinic agonists 16 suggest that high affinity nachrs in the brain mediate the effects of nicotine on passive avoidance . accordingly , nicotine cannot change the performance of β2 −/− mice on this test , as they lack high affinity binding sites . the enhanced performance of mutant mice versus wild type mice is quite surprising , however . several explanations for the paradoxical effect of the β2 - subunit mutation can be proposed . one hypothesis is that nicotine injection improves performance of wild type mice on passive avoidance as a result of desensitization , and thus inactivation of aachrs , leading to enhanced performance on the test . therefore , the behavior of mice lacking the receptor might mimic that of mice whose receptors have been desensitized 24 . another possibility is that nachrs may be present in at least two pathways that interact with opposite effects to generate the behavior measured in passive avoidance . if one pathway is physiologically more active than the other , the inactive pathway will be preferentially stimulated by injection of nicotine in wild type animals , while the more active pathway will be preferentially influenced by β2 - gene inactivation . the experiments described above demonstrate that nachrs containing the β - subunit mediate the effects of nicotine on passive avoidance , a specific learning task . these mice provide a model system for studying the pharmacological effects of nicotine in the cns , and are useful in elucidating the role of high affinity nachrs in cognitive processes , nicotine addiction , and dementias involving deficits of the nicotinic system . each reference below is hereby specifically incorporated into this specification by reference . anand , r . and lindstrom , j . 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