there currently is considerable interest in developing therapeutic strategies to enhance plasticity of the adult central nervous system ( cns ) .
physical exercise , diet , and various forms of environmental / cognitive enrichment have all been proposed to facilitate plasticity [ 14 ] .
for example , the maturation and strength of gabaergic signaling act as a critical regulator of plasticity in cortical networks , with increasing inhibitory tone during postnatal development generally limiting plasticity [ 59 ] .
consequently , manipulations aimed at reducing the strength of gabaergic transmission are thought to constitute promising candidate strategies to enhance plasticity of mature , less plastic cortical circuits [ 7 , 8 ] .
interestingly , recent reports have provided evidence for the notion that chronic treatment with the antidepressant and selective serotonin reuptake inhibitor ( ssri ) fluoxetine can reduce the strength of gabaergic inhibition and promote plasticity of forebrain synapses .
for example , chronic ( 4 weeks ) fluoxetine administration restored ocular dominance shifts in the primary visual cortex ( v1 ) of adult rats , a form of developmentally regulated plasticity that is significantly reduced in the mature brain .
in addition , fluoxetine treatment allowed v1 synapses to express greater long - term potentiation ( ltp ) , an electrophysiological index of the ability of synapses to undergo an upregulation of synaptic strength .
these plasticity - promoting effects of chronic fluoxetine administration appeared to be mediated by a decrease in intracortical inhibition and translated into significant behavioral effects , as assessed by the restoration of visual functions in a rat model of adult amblyopia .
thus , chronic ssri treatment may offer significant , therapeutic potential for the restoration of plasticity to levels normally present only during the earlier stages of postnatal brain development . the notion that chronic ssri treatment can exert a prominent ,
facilitating effect on plasticity has also been supported by investigations employing structural and neuroanatomical measures .
noted that , in rats , 14-day treatment with fluoxetine resulted in an increase in immediate early gene ( c - fos ) expression in the somatosensory cortex , together with an increased spine density of cortical pyramidal cells ; similar results have also been obtained in hippocampal pyramidal cells . finally , it is now well established that fluoxetine administration enhances neurogenesis in the hippocampal formation of adult animals [ 1416 ] , an effect that appears to be a critical mediator of some of the behavioral effects seen with ssri treatment .
the evidence summarized above indicates the potential of fluoxetine to affect plasticity of forebrain synapses .
it is important to note , however , that some investigations have failed to detect beneficial effects ( or noted adverse outcomes ) of chronic fluoxetine treatment on plasticity or in animal models of several neurological diseases , some of which clearly involve deficient plasticity mechanisms ( down syndrome , fetal alcohol syndrome , and neurotoxic brain damage ) [ 1720 ] .
consequently , there is a need for further , detailed investigations of the effects of ssri treatment on plasticity mechanisms across various forebrain networks . in the present study
, we assessed the effect of chronic fluoxetine treatment on ltp in the thalamocortical auditory system between the medial geniculate nucleus ( mgn ) and primary auditory cortex ( a1 ) of adult rats .
ltp in this projection system shows a sharp , age - dependent decline over postnatal life , with high levels of ltp present during the first 5 - 6 weeks of postnatal life , but only modest levels after postnatal day ( pd ) 100 .
here , we tested whether chronic fluoxetine treatment of adult rats would restore ltp to levels normally seen only in juveniles , similar to the effects reported for plasticity in v1 of adult rodents .
experiments were conducted on adult , male long - evans rats ( obtained from charles river laboratories inc . , saint - constant , qubec , canada ; 200250  g body weight or about 5055 days old at the arrival in the animal colony ; at least 9095 days old at the time of the electrophysiological procedures ) .
rats were individually housed ( cage dimensions 40  20  20  cm ) with ad libitum access to food and water .
the colony room was maintained under a reversed 12  /12 hour dark / light cycle ( lights on at 7 pm ) .
experimental procedures were performed in accordance with the published guidelines of the canadian council on animal care and approved by the queen 's university animal care committee .
all efforts were made in order to minimize animal suffering and the number of animals employed for these experiments .
all animals were allowed at least 1 week of acclimatization to the animal colony prior to the onset of fluoxetine treatment .
the fluoxetine treatment regimen was the same as that described by vetencourt et al . .
fluoxetine ( capsules containing 10  mg fluoxetine hydrochloride , obtained from the kingston general hospital pharmacy , kingston , on , canada ) was dissolved in the drinking water at 0.2  mg / ml and was available ad libitum ; control animals received drinking water without drug .
drinking bottles were covered with cardboard or tinfoil to prevent photodecomposition of fluoxetine and were refilled every 48 hours .
treatment continued for 4 - 5 weeks ( mperiod = 4.5 and 4.7 weeks for fluoxetine and water rats , resp . ) , with fluid intake ( every 48 hours ) and body weight ( every 7 days ) recorded throughout the treatment period .
electrophysiological assessments were carried out at the end of the treatment period ( rat age of about 9095 days ) and followed previously established procedures [ 21 , 22 ] .
rats were anesthetized using urethane ( sigma - aldrich , oakville , on , canada ; 1.5  g / kg , given as three intraperitoneal ( i.p . )
injections of 0.5  g / kg each , every 20  min , further supplements as required to reach deep , surgical anesthesia ) .
in addition , the local anesthetic marcaine ( 0.2 - 0.3  ml ) was administered subcutaneously under the skin covering the skull . throughout the experiment
after anesthesia induction , rats were placed in a stereotaxic apparatus , the skull was exposed , and small holes were drilled over the mgn ( ap 5.5 , l + 4.0 , v 5.4 to 6.4 ) and the ipsilateral a1 ( ap 4.5 , l + 7.0 , v 3.2 to 5.4 , all measurements from bregma ) .
additional holes over the cerebellum and frontal cortex were used to secure reference and ground connections ( jewelry screws ) , respectively . a stimulation electrode ( concentric bipolar electrode , sne-100 ,
rhodes medical instruments , david kopf , tujunga , ca ) was lowered into the mgn , and a recoding electrode ( 125  m diameter teflon - insulated stainless steel wire ) was placed in a1 .
the final , ventral placement of both electrodes was optimized to yield the maximum amplitude of field postsynaptic potentials ( fpsps ) in a1 in response to single pulse stimulation of the mgn .
signals were amplified ( model 1800 , a - m systems inc . , carlsborg , wa , half - amplitude filter settings at 0.3  hz to 1  khz ) , digitized ( at 10  khz using a powerlab/4s system , running scope software v.4.0.2 , ad instruments , toronto , on , canada ) , and stored for subsequent offline analysis . stimulation ( 0.2  ms pulse duration ) of the mgn was delivered by means of a stimulus isolation unit ( ml 180 stimulus isolator , ad instruments ) . for each rat , a 30  min period following the final electrode adjustments was given to allow for stabilization prior to the onset of data collection .
following stabilization , an input - output curve was generated by stimulating the mgn at successively increasing intensities ( 0.11.0  ma , in 0.1  ma increments ) .
the intensity that elicited fpsps of 5060% of the maximal fpsp amplitude was chosen for the formal data collection .
baseline fpsps were recorded every 30  s until a 30  min period of stable baseline recordings was established .
subsequently , theta - burst stimulation ( tbs ) of the mgn was applied as trains of 10 bursts ( delivered at 5  hz ) , with each burst consisting of five pulses at 100  hz . stimulation trains were repeated every 10  s for a total of four trains .
this induction protocol has previously been shown to elicit reliable ltp in the thalamocortical auditory system in vivo [ 21 , 22 ] .
after the tbs delivery , fpsps were recorded for 60  min ( every 30  s ) , followed by a second tbs episode ( same as above ) and a final 60  min of fpsp recordings . immediately after the experiment
, rats were perfused through the heart with 0.9% saline , followed by 10% formalin .
brains were extracted and immersed in formalin prior to sectioning ( 40  m ) using a cryostat
. the locations of all electrodes were examined using standard histological techniques and only animals with accurate placements were included in the analysis of the electrophysiology data .
the fpsp amplitude was computed offline by scope software ( v.4.1.1 , ad instruments ) .
values for each rat were averaged over 10  min intervals and these averages were normalized by dividing them by the averaged baseline ( pre - tbs ) amplitude of that animal .
data were statistically evaluated by repeated measures analysis of variance ( anova ) and , if statistically appropriate , simple effects tests using the clr anova software package ( v.1.1 , clear lake research inc . , houston , tx ) .
note that the results of all statistical analyses are reported in the appropriate figure captions .
for a 4-week period , rats were given access to drinking water ( n = 18 ) or drinking water containing fluoxetine ( 0.2  mg / ml ; n = 20 ) . during this period ,
weight gain was significantly reduced in rats given access to fluoxetine relative to control ( water ) animals ( figure 1(a ) ) .
the total weight gain from week 0 ( before treatment onset ) to week 4 was 37  3% and 54 + 3% for fluoxetine and water animals , respectively , findings that are consistent with the substantial literature demonstrating appetite - suppressant effects of fluoxetine treatment [ 19 , 20 , 23 ] .
fluoxetine also reduced water intake ( figure 1(b ) ) , with fluid consumption over 48  h averaging 31  1  ml and 51  3  ml in fluoxetine and water animals , respectively .
after 4 - 5 treatment weeks ( 4.5 and 4.7 weeks for fluoxetine and water animals , resp . ) , each rat was anesthetized with urethane to allow for the placement of a stimulating and recording electrode in the mgn and a1 , respectively ( figure 2(a ) ) .
consistent with previous wok [ 21 , 22 ] , extracellular recordings in the middle layers ( iii / iv ) of a1 revealed that single pulse stimulation of the mgn elicited fpsps consisting of two negative - going components with peak latencies of about 68 and 1416  ms , respectively ( figure 2(b ) ) .
previous work using current - source density analysis and pharmacological approaches has revealed that these two negative peaks correspond to current sinks associated with the sequential activation of direct , thalamocortical synapses ( layer iv ; first fpsp peak ) and subsequent , intracortical synapses ( layers ii / iii ; second fpsp peak ) [ 22 , 24 ] .
it is important to note that the urethane dose required for deep , surgical anesthesia did not differ significantly between the two groups of animals , with water and fluoxetine rats receiving a final dose of 2.12  0.09 and 2.07  0.06  g / kg of urethane , respectively ( figure 2(c ) ) .
thus , chronic fluoxetine treatment did not appear to alter the response to urethane anesthesia .
initially , fpsps elicited by single - pulse mgn stimulation were recorded for 30  min in order to establish a measure of baseline synaptic strength prior to ltp induction .
stimulation intensities used for the two groups of animals did not differ significantly , with mgn stimulation pulses of 0.48  0.04  ma and 0.5  0.02  ma for water ( n = 11 ) and fluoxetine ( n = 14 ) animals , respectively ( figure 2(d ) ) .
further , the amplitude of baseline fpsps did not differ between the treatment groups , with the amplitude of the first peak at 1.05  0.15  mv and 1.0  0.15  mv in water and fluoxetine animals , respectively ( figure 2(e ) ) .
the amplitude of the second fpsp peak was 0.48  0.07  mv and 0.59  0.08  mv in the water and fluoxetine group , respectively ( figure 2(f ) ) .
it is noteworthy that the amplitude difference between the two groups amounts to 23% , even though the statistical analysis did not indicate this effect to approach significance ( p = 0.3 ) .
following the completion of baseline recordings ( 30  min ) , two episodes of tbs were delivered to the mgn , each followed by 60  min of fpsp recordings . in water animals , tbs resulted in successful ltp induction , with the first tbs episode resulting in a potentiation of the first and second fpsp peak to 115% and 123% of baseline , respectively ( figure 3 ; all values reported here are mean values for recordings taken between 3160  min after tbs delivery ) .
the second tbs episodes resulted in further potentiation in water animals , with the two peaks reaching 120% and 135% of baseline ( figure 3 ) . in fluoxetine animals ,
tbs also resulted in ltp , but the amplitude of the first and second fpsp peak reached only 111% and 110% of baseline , respectively , following the first tbs episode ( figure 3 ) .
further , after the second tbs , both peaks reached only 113% of baseline ( figure 3 ) .
thus , fluoxetine animals showed less potentiation than that seen in the water group , an effect that was significant for the second fpsp peak representing intracortical synapses ( see caption for figure 3 ) .
as mentioned , the second fpsp peak in fluoxetine rats exhibits a baseline amplitude 23% higher than that seen in water animals . even though this effect did not reach statistical significance
, it might nevertheless indicate a minor enhancement of intracortical synaptic strength following chronic fluoxetine exposure .
such an enhancement may act to limit ( occlude ) further ltp induction by tbs delivery . in order to assess this possibility
, we performed additional analysis by plotting and correlating baseline fpsp amplitude against levels of ltp for the second fpsp peak in all fluoxetine animals ( figure 4 ) . however , correlations using either raw or rank - ordered data ( figure 4 ) both failed to indicate a significant relation between baseline fpsp amplitude and subsequent potentiation induced by tbs in fluoxetine rats .
with the present experiments , we examined whether chronic fluoxetine treatment alters ltp of synapses in the mature a1 of adult rats .
several recent reports have provided support for the notion that chronic fluoxetine treatment leads to an enhancement of plasticity in the adult cns [ 7 , 10 , 1214 , 25 ] .
in contrast to these findings , we found no evidence of an upregulation of plasticity at a1 synapses .
in fact , there was a clear suppression of ltp in fluoxetine - treated rats , in particular for the second peak of the cortical fpsp , thought to reflect synaptic currents originating at intracortical synapses in a1 ( see below ) .
previous work has shown that , in rats , fpsps elicited by mgn stimulation in vivo typically consist of two distinct , negative peaks that correspond to current sinks associated with the successive activation of thalamocortical and intracortical a1 synapses , respectively [ 22 , 24 , 26 ] . for both sets of synapses ,
ltp induced by thalamic stimulation shows a clear , age - related decline , with significant potentiation present up to pd 50 , modest ltp around pd 100 ( about the time ltp was assessed in the present study ) , and very little or no ltp after pd 200 [ 21 , 24 ] . as such , ltp in the rat thalamocortical auditory system provides an appropriate model to study the developmental decline of plasticity in a central sensory system .
the present data confirm that adult rats show modest levels of plasticity under the present experimental conditions , with thalamocortical and intracortical synapses expressing ltp of about 120% and 135% of baseline , respectively .
surprisingly , rats given chronic fluoxetine showed less ltp , with both thalamocortical and intracortical synapses expressing potentiation of only 113% of baseline , respectively , observations that are indicative of an inhibition of ltp induction mechanisms , especially for intracortical synapses in a1 .
the results summarized above regarding ltp in control animals are consistent with previous work , which has also shown that intracortical synapses in the adult a1 show higher ltp levels relative to thalamocortical synapses [ 21 , 22 , 24 ] .
interestingly , receptive field plasticity of a1 neurons ( i.e. , shifts in the optimal response to different sound frequencies ) also occurs by a potentiation of intracortical but not thalamocortical synapses , suggesting that the ltp measured here plays a direct role in receptive field plasticity and associated changes of the tonotopic map present in a1 . consequently , it is possible that chronic fluoxetine exposure may also impair a1 receptive field shifts in rodents , a hypothesis that clearly requires examination .
we employed the same fluoxetine dosing regimen ( 0.2  mg of fluoxetine in 1  ml of drinking water ) as that used in previous work showing a restoration of ocular dominance plasticity and enhancement of ltp in v1 slice preparations obtained from adult rats . in the present investigation , rats in the fluoxetine condition consumed an average of about 15.5  ml of fluid every 24 hours , equaling an intake of about 3.1  mg of fluoxetine .
this drug amount corresponds to a daily dosage of about 10.7  mg / kg and 7.9  mg / kg of body weight at the beginning ( body weight of about 290  g ) and the end ( 390  g ) of the treatment period , respectively .
these dosages are very similar to those used in previous chronic administration studies demonstrating behavioral and/or neurochemical effects following fluoxetine treatment [ 10 , 17 , 19 , 28 ] . in our experiments ,
fluoxetine reduced body weight gain during the treatment period , a classic effect of ssri treatment [ 17 , 19 , 20 , 23 ] .
in addition , we also noted a suppression of water intake , also consistent with the results of previous work .
together , these results confirm the bioavailability and bioactivity of fluoxetine , as administered in the present investigation . while fluoxetine has been suggested to enhance plasticity of the mature cns , empirical evidence assessing this contention has been inconsistent .
stewart and reid   noted that 15-day treatment with fluoxetine reduced levels of ltp in the dentate gyrus of anesthetized rats , and inhibitory effects on hippocampal ( area ca1 ) ltp in rats have also been reported for acute ( single - dose ) fluoxetine treatment .
an elegant , recent investigation revealed that a chronic ( 4 week ) fluoxetine regimen resulted in deficits in the induction of ltp in the hippocampal ca1 field of adult rats , while dentate gyrus ltp was intact .
interestingly , the same authors also noted a disruption of ltd , which again was specific for the ca1 field .
the ltd impairment is of significance since it suggests that any observed reduction in ltp is unlikely to be related exclusively to an upregulation of synaptic strength following long - term fluoxetine exposure ( see ) .
typically , such an effect reduces ltp but yields greater ltd , due to the fact that enhanced synaptic connectivity represents a potentiated state , which limits further potentiation but leaves greater room for synaptic weakening [ 3133 ]
. it will be important for future work to assess whether chronic fluoxetine impairs or facilitates ltd induction in areas other than the hippocampal formation .
the present experiments did not provide reliable evidence for an upregulation of a1 synaptic strength following chronic fluoxetine , with baseline fpsp amplitudes showing no significant differences between the two treatment groups .
previous work has found evidence for an enhancement of field potential strength in rats after fluoxetine administration in both the dentate gyrus and ca1 field [ 17 , 30 ] , even though a lack of fpsp facilitation in the rat dentate gyrus has also been reported .
it is noteworthy , however , that the second fpsp peak was 23% larger in fluoxetine rats relative to water animals , in particular since this value is very similar to the reduction of ltp for the second fpsp peak in the fluoxetine group ( 135% and 113% potentiation in water and fluoxetine rats , resp .
, we carried out additional analyses to assess whether greater baseline fpsp amplitude was related to lower levels of tbs - induced ltp , suggestive of an occlusion - like effect of chronic fluoxetine in ltp .
however , these analyses did not provide any suggestion of an association between baseline fpsp amplitude and subsequent ltp magnitude .
we can not rule out that such a relation , or a significant difference in baseline synaptic strength , may emerge with larger sample sizes or alternative methodologies to study synaptic connectivity and plasticity in a1 ( e.g. , optical imaging or in vitro approaches ) .
it is also possible that some cns regions ( e.g. , hippocampal formation ) exhibit greater sensitivity to the potential , neurotrophic effects elicited by fluoxetine than areas such as the primary sensory fields of the neocortical mantle .
previous work has shown that local application of an nmda receptor antagonist directly in a1 of rats blocks the induction of ltp elicited by mgn stimulation in vivo [ 22 , 34 ] .
it is well documented that the precise subunit composition of the nmda receptor exerts profound effects on ltp induction by altering the level of calcium influx across the postsynaptic membrane .
receptors containing the nr2b subunit exhibit prolonged channel opening duration and greater calcium influx relative to nr2a - expressing receptors , effects that lead to enhanced ltp induction [ 21 , 22 , 3537 ] .
interestingly , in rats , chronic fluoxetine exposure can alter nmda subunit composition by increasing the relative expression of nr2a subunits , raising the possibility that this effect contributes to the reduction of ltp noted here and in previous work [ 17 , 30 ] .
future work is required to examine this hypothesis and delineate the precise mechanisms that mediate the effects ( facilitating and inhibitory ) of long - term fluoxetine exposure on the induction of different forms ( e.g. , structural and physiological ) of cns plasticity . in recent years
, there has been considerable excitement regarding the potential of various ssris to enhance cns plasticity [ 7 , 8 , 10 , 14 , 39 ] .
not only have the plasticity - promoting actions been suggested to mediate mood - enhancing effects [ 1416 , 39 ] , but ssris may also facilitate plasticity and functional recovery in neurological conditions unrelated to mood disorders ( e.g. , [ 7 , 10 , 1820 ] ) . while some evidence is clearly supportive of this notion
[ 10 , 12 , 13 , 25 ] , the present study confirms and extends previous investigations that have failed to detect an enhancement of plasticity following chronic fluoxetine treatment [ 17 , 30 ] .
in fact , both electrophysiological ( ltp ) and structural investigations are compatible with the view that fluoxetine exposure can result in a strengthening and stabilization of synaptic connectivity , which reduces the ability of neurons to express further plasticity [ 12 , 13 , 30 , 38 ] .
thus , the effects of fluoxetine on plasticity appear to be complex and bidirectional , findings that clearly require consideration when discussing the use of fluoxetine and other ssris for the modulation of plasticity of the mammalian forebrain .
 several recent studies have provided evidence that chronic treatment with the selective serotonin reuptake inhibitor ( ssri ) fluoxetine can facilitate synaptic plasticity ( e.g. , ocular dominance shifts ) in the adult central nervous system . here , we assessed whether fluoxetine enhances long - term potentiation ( ltp ) in the thalamocortical auditory system of mature rats , a developmentally regulated form of plasticity that shows a characteristic decline during postnatal life . 
 adult rats were chronically treated with fluoxetine ( administered in the drinking water , 0.2  mg / ml , four weeks of treatment ) . 
 electrophysiological assessments were conducted using an anesthetized ( urethane ) in vivo preparation , with ltp of field potentials in the primary auditory cortex ( a1 ) induced by theta - burst stimulation of the medial geniculate nucleus . 
 we find that , compared to water - treated control animals , fluoxetine - treated rats did not express higher levels of ltp and , in fact , exhibited reduced levels of potentiation at presumed intracortical a1 synapses . 
 bioactivity of fluoxetine was confirmed by a reduction of weight gain and fluid intake during the four - week treatment period . 
 we conclude that chronic fluoxetine treatment fails to enhance ltp in the mature rodent thalamocortical auditory system , results that bring into question the notion that ssris act as general facilitators of synaptic plasticity in the mammalian forebrain . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion