cannabinoid   receptors are a 
 family of g - protein coupled , presynaptic receptors [ 1 , 2 ] . autoradiography 
 studies using the cannabinoid receptor ligand cp55,940 [ 35 ] show that cb1rs are 
 distributed throughout neuronal tissue . these studies report a dense binding of 
 cp55,940 in the basal ganglia , specifically the substantia nigra pars reticulata the globus pallidus ( gp ) 
 and also in cerebellum . in the cerebrum , the hippocampal formation and the entorhinal 
 cortex ( ec ) show the highest density of staining for cb1r . cannabinoids 
 are known to exert powerful control over gabaergic inhibitory signalling in the 
 cns [ 68 ] , and it is reported that 
 cb1r inhibition of gabaa receptor mediated synaptic transmission 
 occurs through the inhibition of voltage - dependent calcium channels ( vgccs ; ) . in the hippocampus , the activation of 
 presynaptic cb1r depresses gaba release onto postsynaptic target cells [ 9 , 10 ] , and in these studies , endogenous 
 and exogenous cb1r agonists have been shown to reduce the amplitude and 
 frequency of gabaergic spontaneous inhibitory postsynaptic currents ( sipscs ) , 
 but not to affect action - potential - independent miniature ( m)ipscs . other 
 studies have shown that cannabinoid receptor activation enhances network 
 oscillatory activity . however , in the parahippocampal region ( phr ) , the effects of cannabinoid 
 receptors are less well described . here , we have investigated the functional 
 effects of cbrs on neuronal network activity modelled in vitro by kainate ( ka- ) induced persistent oscillations [ 8 , 12 ] . persistent oscillatory 
 activity in the gamma frequency band ( 3080 hz ) has been the most commonly 
 reported and studied form of network activity in the in vitro slice preparation , and can be elicited by metabotropic 
 glutamate receptors   or application of kainic acid 
 [ 8 , 12 ] and/or the muscarinic 
 agonist carbachol   neuronal network oscillatory activity reflects the phasic inhibition of principal cells by gabaergic interneurones , which act to entrain and 
 synchronize principal cell activity ( cobb et al . , ) . the mec has been reported to express gamma oscillations ( 30100 hz ) in response to application of nanomolar concentrations of kainate 
 [ 15 , 16 ] , and oscillatory 
 power was greatest in superficial layers ii / iii . combined ec - hippocampal slices were prepared from young male 
 wistar rats ( 50110 g ) as previously described . all experiments were performed in accordance with the uk animals 
 ( scientific procedures ) act 1986 and european communities council directive 
 1986 ( 86/609/eec ) . rats were anaesthetised with isoflurane and n2/o2 , 
 until cardiorespiratory arrest , and decapitated . the brain was rapidly removed 
 and immersed in oxygenated artificial cerebrospinal fluid ( acsf ) chilled to 4c . slices ( 450 m ) were cut using a vibrating microtome ( microm , 
 germany ) , and stored in acsf continuously bubbled with 95% o2/5% co2 , 
 at room temperature . following a recovery period of at least 1 hour , individual 
 slices were transferred to a recording chamber mounted on the stage of an 
 olympus ( bx50wi ) upright microscope . the chamber was continuously perfused with 
 oxygenated acsf at 3032c , at a flow rate of approximately 2 ml / min . the acsf 
 contained the following ( in mm ) : nacl ( 126 ) , kcl ( 3.25 ) , nah2po4 ( 1.25 ) , nahco3 ( 24 ) , mgso4 ( 2 ) , cacl2 ( 2.5 ) , 
 and d - glucose ( 10 ) . the solution was continuously bubbled with 95% o2/5% 
 co2 to maintain a ph of 7.4 . patch - clamp electrodes 
 were pulled from borosilicate glass ( 1.2 mm od , 0.69 i d ; harvard apparatus ) and 
 had open tip resistances of 4 - 5 m. they were filled with a solution containing 
 the following ( in mm ) : cscl ( 90 ) , hepes ( 33 ) , qx-314 ( 5 ) , egta ( 0.6 ) , mgcl2 ( 5.0 ) , 
 tea - cl ( 10 ) , phosophocreatine ( 7 ) atp ( 4 ) , gtp ( 0.4 ) . the solution was adjusted 
 to 290 mosmol with sucrose and to ph 7.4 with csoh . whole - cell voltage clamp recordings were made from neurones in layers ii and v 
 of the medial division of the ec , using an axopatch 700 series amplifier 
 ( molecular devices , usa ) . under 
 these experimental conditions , layer ii / v neurones exhibited sipscs , mediated 
 by gaba acting primarily at gabaa receptors . data were recorded 
 directly to computer hard disk using axoscope software ( molecular devices , usa ) . mini analysis ( synaptosoft , usa ) was used 
 for analysis of sipscs offline . sipscs were detected automatically using a 
 threshold - crossing algorithm , and their frequency and amplitude are analysed . 200 sipscs were sampled during a continuous recording period for each neurone 
 under each condition . the nonparametric kolmogorov - smirnoff ( ks ) test was used 
 to assess the significance of shifts in cumulative probability distributions of 
 interevent interval ( iei ) . differences between drug and control situations in 
 studies of sipscs were assessed by means of a one - way anova . all salts 
 used in preparation of acsf were analar grade and purchased from merck / bdh ( uk ) . ly320135 and acpa were obtained from tocris cookson 
 ( uk ) . for field 
 recordings of oscillatory activity , slices were placed into an interface chamber 
 ( brsc-1 , digitimer , uk ) and the chamber was 
 continuously perfused with oxygenated acsf at 3032c , at a flow rate of 
 approximately 1 - 2 ml / min . extracellular 
 population recordings were made with glass microelectrodes filled with acsf , of 
 resistance 13 m.   signals were amplified 1000-fold and recorded 
 unfiltered . low amplitude 50 hz interference was removed using a humbug ( quest scientific , canada ) . signals were digitized and recorded at 10 khz using an npi ext-02f amplifier ( npi , germany ) 
 and pclamp 10 software ( molecular devices , usa ) . following 3090 minutes control period of stable 
 oscillatory activity , pharmacological oscillatory activity was 
 analysed using the fast fourier transform ( fft , clampfit 10 ) , cross - correlation 
 analysis and morlet - wavelet time - frequency spectrogram analysis ( matlab 2007r , 
 mathworks ) . we analysed oscillations at beta 
 ( 1529 hz ) and gamma ( 3090 hz ) bands , using bandpass filters ( clampfit 10.1 ) 
 and measurement of the area under the power spectrum curve in sigmaplot 8.0 . whilst 
 recording from layers ii and v of the mec , we applied the cbr agonist 
 arachidonylcyclopropylamide ( acpa ) at 10 m , onto slices from which stable gamma 
 activity had been induced by 300400 nm kainate . as figure 1(a ) shows , ka - induced gamma oscillations in layer ii were 
 broadly similar to those reported by cunningham et al . . hence , mean area power in the -band was 561      179 v and mean control 
 gamma frequency was 40.7  2.4 hz . following 4060 minutes bath application of acpa , we applied the 
 cb1r - specific inverse agonist ly320135 ( 500 nm ; ) . figure 1(b ) shows the power spectral density of activity bandpass filtered between 2100 hz , and figure 1(c ) shows similar data 
 filtered at gamma frequency ( 3090 hz ) . as figure 1(c ) shows , there was a tendency towards an increase in gamma power in acpa in some 
 recordings , but this was not significant overall ( p      .19 , n   =   9 ) . in pooled data , acpa did 
 significantly reduce mean peak gamma frequency to 35.6  1.8 hz ( p      .04 , n   =   9 ) , although this 
 effect was variable , and some recordings showed multiple peaks . following perfusion of the cb1r - specific 
 inverse agonist ly320135 , there was a marked reduction in normalised gamma 
 power to 39.4  10.1% of control , and this was highly significant ( p      .0006 , n   =   9 ) . in addition , mean peak 
 frequency returned to 41.2  1.8 hz ( p      .04 , 
 n   =   9 ) . when 
 beta power in layer ii was measured , we noted a similar pattern of drug 
 responses to that observed for gamma activity . mean area power 
 in the beta band was lower than that of gamma activity at 26      6 v and mean peak beta 
 frequency in control conditions was 25.6  1.4 hz . figure 2(a ) shows the power spectral density of activity bandpass 
 filtered between 2100 hz , and figure 2(c ) shows similar data filtered at beta frequency ( 1529 hz ) . as figure 2(c ) shows , there was no significant 
 change in beta power in acpa ( 81.4      15% of control , p .14 , n   =   9 ) , 
 and acpa had no significant effect on mean peak beta frequency ( 27.6  1.43 hz , p      .25 , n   =   9 ) . however , when we added the cb1r - specific inverse 
 agonist ly320135 , there was a reduction in normalised beta power to 57      13% 
 of control , and this was highly significant ( p      .008 , n   =   9 ) . ly320135 had no effect on mean peak frequency ( 27.9  0.52 hz , p      .4 , n   =   9 ) . during the above experiments , we 
 simultaneously recorded oscillatory activity in deep entorhinal cortex ( layer 
 v ) . oscillatory activity in layer v was lower in power in layer v compared to 
 layer ii , with mean area gamma power just 60      10 v and mean peak frequency 
 was similar to layer ii at 39.19  3.1 hz . when we applied acpa whilst recording in layer v we observed a 
 significant increase in mean   gamma power ( figure 3(a ) ) , 
 by 38.1  13.4% of control ( p      .03 , n   =   9 ) , however , baseline gamma power was very 
 low in this layer , and the absolute change in gamma power was difficult to 
 discern . peak frequency was again slightly reduced to 36.0  2.4 hz , but 
 this was not significant ( p      .31 , n   =   9 ) . on subsequent addition 
 of the cb1r - specific inverse 
 agonist ly320135 , there was a strong increase in normalised gamma power to 
 108.4      58% of control , and this reached significance ( p   =   .049 , 
 n   =   9 ) . again , ly320135 did not significantly alter mean 
 peak gamma frequency ( 35.7  2.41 hz in ly320135 , p      .45 , n   =   9 ) . when beta power in layer v was analysed , 
 we noted a similar pattern of drug responses to that observed for gamma 
 activity . mean area power in the beta band was lower than that of 
 gamma activity at 9.6  0.6 v and mean control beta frequency was 27.9  0.52 hz . figure 4(a ) shows field oscillations recorded in layer v before drug 
 application and during acpa and ly320135 periods . figure 4(b ) shows the power spectral density of activity bandpass 
 filtered between 2100 hz , and figure 4(c ) shows similar data filtered at beta frequency ( 1529 hz ) . as figure 4(c ) shows , there was a slight 
 tendency towards an increase in beta power ( by 27      14% ) in acpa in some 
 recordings , but this was not significant overall ( p      .06 , 
 n   =   9 ) . acpa had no significant effect on mean peak beta frequency ( 28.4  0.7 hz , p      .5 , n   =   9 ) . when we next 
 applied the inverse agonist ly320135 , we noted an increase in normalised beta 
 power by 142.4      88% of control , and this just failed to reach significance ( p      .07 , n   =   9 ) . ly320135 ( 26.3  1.5 hz , p      .3 , n   =   9 ) had no effect on peak frequency . we 
 hypothesised that the lack of effects of acpa in layer ii might reflect 
 constitutive or tonic activation of cbr , perhaps due to persistent 
 kainate - induced activation of pyramidal neurones . to test this hypothesis , we applied 
 ly320135 in the absence of acpa . application of ly320135 suppressed gamma band activity to 19.5      11% of control , and this was highly significant ( p      .01 , n   =   5 ) . when beta activity was measured , it 
 was apparent that in ly320135 , there was a significant reduction in mean 
 normalised beta power ( 58.4      12% of control ; p      .04 , n   =   5 ) . the data presented up to this point 
 indicated that , in general , gamma and beta power decreased in layer ii in 
 response to blockade or inverse agonism of cb1r , and that in layer v , the 
 opposite was seen , with an increase in gamma and beta power . figures   5(a)-5(b ) shows summary bar charts indicating the effects of acpa and ly320135 on normalised mean area power in 
 the gamma and beta bands in layers ii and v of the mec . we hypothesised 
 that the alterations in oscillatory power seen during drug application would 
 relate to the effects of acpa and ly320135 on sipscs impinging on neurones in 
 deep and superficial entorhinal cortex . to measure these effects , we performed 
 whole - cell voltage clamp recordings of sipscs , whilst bath applying acpa and 
 ly320135 at concentrations similar to those used above . figure 6(a ) shows typical 
 recordings of inward sipscs made from a layer ii pyramidal neurone . as figure 6(b ) shows , the application of acpa 
 ( 10 m ) had subtle 
 effects on sipscs in layer ii , decreasing their frequency without affecting mean 
 amplitude . cumulative probability plots for sipsc amplitude in the presence of 
 acpa ( figure 6(c ) ) indicated a shift in 
 amplitude distribution , and mean amplitude showed a slight increase from 101.7  3.2 pa in 
 control to 108.3  3.4 pa in acpa , but this was nonsignificant ( p      .168 , anova ) . when we analysed amplitude distribution using the 
 nonparametric kolmogorov - smirnov test , the shift towards higher amplitude 
 sipscs was just significant ( p      .021 ks test ) . in the case of interevent interval 
 ( iei ; the reciprocal of frequency ) , we noted a shift to the right in the 
 cumulative probability plot ( figure 6(d ) ) , 
 indicating an increase in the likelihood of greater iei values ( reduced 
 frequency ) . mean median iei 
 increased from 34.6  5.5 milliseconds in control to 47.6  9.7 milliseconds in 
 acpa ( p      .0001 , anova , n   =   5 ) , and the 
 increase in iei time distribution towards higher values was highly significant ( p      .006 , ks test ) . 
 when we performed similar experiments using ly320135 ( 500 nm ) , we noted effects 
 which tended towards the opposite of those seen with acpa , that is , increased 
 sipsc amplitude and frequency . figure 7(a ) shows a typical recording of inward sipscs made from a layer - ii pyramidal 
 neurone . as figure 7(b ) shows , the 
 application of ly320135 increased sipsc frequency and amplitude . mean amplitude 
 increased from 51.7  3.6 pa in control to    69.0  5.9 pa in ly320135 , and this was 
 significant ( p      .013 , anova , n   =   6 ) . similarly , the 
 shift in distribution towards larger amplitudes was highly significant ( p      .002 , ks test ) . the mean median iei 
 showed a slight decrease from 86.6  15.8 milliseconds in control to 80.6  16.1 milliseconds ly320135 , but this decrease in iei time was not significant ( p      .116 , anova , n   =   6 ) . the 
 cumulative probability plots for sipsc amplitude ( figure 7(c ) ) and iei ( figure 7(d ) ) 
 in the presence of ly320135 indicate the shifts in distribution of these 
 parameters in the presence of ly320135 . in contrast to the effects observed 
 in layer ii and layer v we noted a significant reduction in sipsc frequency in 
 response to acpa application .   as figure 8(a ) shows , sipscs in layer v are 
 considerably less frequent than those in layer ii ( see woodhall et al . , ) . when acpa was applied , sipsc 
 frequency was greatly attenuated ( figure 8(b ) ) , 
 but there was no overall shift 
 in amplitude distribution ( confirmed by a nonsignificant ks test ( p      .23 ) ) . mean amplitude rose slightly from 
 59.41  7.33 pa in control to 70.49  8.89 pa in acpa , but this increase was not 
 significant ( p      .33 , anova , n   =   6 ) . when 
 we analysed iei , the change in distribution towards larger iei values was significant 
 ( p      .0001 ks test ) , and mean median iei was found 
 to increase very significantly from 792      41 milliseconds in control to 1317      75 milliseconds in acpa ( p      .0001 , anova , n   =   6 ) . this effect of acpa on iei 
 in layer v was consistent in all recordings . cumulative 
 probability plots for sipsc amplitude ( figure 8(c ) ) 
 and iei ( figure 8(d ) ) illustrate the 
 effects of acpa on sipsc amplitude and iei . when we performed similar 
 experiments using ly320135 ( 500 nm ) , we again noted robust effects , which opposed 
 those seen with acpa , that is , increased sipsc amplitude and frequency . figure 9(a ) shows a typical recording of 
 inward sipscs made from a layer - ii pyramidal neurone . as figure 9(b ) shows , the application of ly320135 had marked effects on 
 sipscs in layer v , increasing their frequency and amplitude . mean sipsc 
 amplitude in layer v increased from 47.0 pa      3.0 to 87.9 pa      7.4 pa in 
 ly320135 , and this increase was significant ( p      .001 , anova 
 n   =   7 ) . cumulative probability plots for sipsc amplitude in the presence of ly320135 
 ( figure 9(c ) ) show the shift in amplitude 
 distribution , and this was confirmed statistically ( p      .0004 , ks test ) . in the case of iei , we noted 
 a shift to the left in the cumulative probability plot ( figure 9(d ) ) , indicating an increase in the likelihood of lower iei values 
 ( increased frequency ) . during ly320135 application the mean median iei 
 decreased from 477.1  108.0 milliseconds in control to 300.0  71.5 milliseconds 
 in ly320135 showing that an overall increase in sipsc frequency has occurred . the decrease in mean median iei between control and ly320135 periods was 
 significant ( p      .014 
 anova , n   =   7 ) , as was the change in distribution ( p      .016 , ks test ) . we 
 found that the cannabinoid receptor agonist acpa had little effect on either 
 oscillatory activity or synaptic inhibition in superficial layers of the mec , 
 and that effects in deep layers were more robust , especially in the case of 
 sipsc frequency . however , the inverse agonist , ly320135 , strongly suppressed 
 oscillatory activity in superficial mec even while its effects on sipsc 
 frequency and amplitude were not great . we also observed that suppression of 
 oscillatory activity in layer ii by ly3201235 was accompanied by augmentation 
 of oscillatory power in layer v , and that the suppression of sipscs by acpa and 
 subsequent enhancement by ly320135 in this layer were marked . we 
 have reported previously   that spontaneous inhibition is much greater in superficial layer 
 ii of mec than in deep layer v. in addition , more than 90% of ipscs in layer ii 
 are action - potential ( ap ) independent , whereas in layer v , ap - dependent events 
 comprise a much greater ( > 50% ) proportion of sipscs . given that cbrs act 
 only on ca dependent release of gaba and have no effect on mipscs 
 (;   it seems likely that cannabinoid ligands would show greater 
 effects in the deep layers , where activity is low at baseline , and probably 
 more sensitive to modulation since it is more likely to be ap - dependent . since , 
 compared to layer ii , both acpa and 
 ly320135 had more profound effects on synaptic inhibition in layer v , it seems 
 likely that the relative dominance of mipscs in layer ii may mask cb1r effects 
 on the minority of ap - dependent sipscs to some degree . the lack of a robust 
 effect of acpa on oscillatory activity in layer v suggests , however , that cb1r 
 may already be activated by ongoing network activity , and that further attempts 
 at activation using an agonist did not increase any effect that cb1r might have 
 on oscillatory power . this appears to be supported by the effects of the 
 inverse agonist ly320135 in layer ii . here , we observed a robust reduction in 
 both beta and gamma power in layer ii , suggesting that cb1r do contribute to 
 maintaining oscillatory activity in this layer . the apparently contradictory 
 result of enhanced oscillatory activity in layer v in response to ly320125 may 
 relate to effects that are secondary to activity in layer ii , for example , bragin , working in vivo , noted that ablation of superficial ec causes augmented 
 oscillatory activity in ca3-ca1 , and it may be that a similar mechanism allows 
 suppression of oscillations in layer ii to unmask activity in layer v , which 
 receives inputs from ca1 . similarly , previous reports   indicate that superficial 
 layers ( especially layer iii ) show the strongest gamma power , perhaps 
 suggesting a role in driving oscillatory activity in other layers . however , 
 layer v is not driven directly by layers ii or iii , and hence any effect in layer v may well be 
 indirect . cannabinoid 
 receptors exert powerful control over gaba release from presynaptic terminals , 
 with cb1 receptors having been shown to suppress both ipsps and ipscs in 
 pyramidal neurones ( ipsps , ; ipscs , ) . endocannabinoids , such as 2-arachidonyl glycerol and 
 anandamide also suppress inhibition in cns ( see , for review ) . cannabinoids are also believed to 
 mediate the phenomenon of depolarisation - induced suppression of inhibition 
 ( dsi ; [ 2224 ] ) . recently , studies have suggested 
 that cb1r are present at terminals from specific subsets of inhibitory 
 interneurones . for example , fast spiking ( fs ) inhibitory neurones in neocortex 
 express parvalbumin ( pv ) but not cb1r , and by contrast , irregular spiking ( is ) 
 neurones express cb1r but not pv [ 25 , 26 ] . recently , galaretta et al . 
 have demonstrated that synapses between is neurones and pyramidal cells express 
 cb1r and show dsi , whereas synapses between fs neurones and 
 pyramidal cells show neither cb1r nor dsi . fs cells are thought to 
 pace fast oscillatory network rhythms such as gamma activity (; and is cells are thought to possess properties that 
 predispose towards nonrhythmic activity [ 25 , 29 ] . a subset of neurones that express cb1r but not pv expresses cholecystokinin 
 ( cck ) , and these neurones have been suggested to act , through dsi , to 
 differentiate subgroups of pyramidal cells into neuronal assemblies which are 
 then entrained by fs cells (  sparse coding  in place cell assemblies , 
 ) . in this 
 scenario , pyramidal cell activation leads to endocannabinoid synthesis and 
 release , which inhibits is - cell inputs to the somata and proximal dendrites of 
 active cells , but allows is - cell - mediated inhibition to remain intact ( and 
 ongoing ) at less active pyramids . this effect , in turn , allows fs - cells to 
 entrain oscillatory activity only at the disinhibited population of pyramidal 
 cells , effectively selecting that subset for rhythmic activity . it 
 seems possible that pv/cck+/cb1r+ inhibitory interneurones might similarly select 
 populations of pyramidal cells involved in rhythmogenesis in the mec , which 
 contains both pv+ and pv neurones [ 31 , 32 ] and cck+ interneurones , which also express cb1r . we used a selective cannabinoid 
 receptor inverse agonist to globally inhibit cb1rs during persistent gamma and 
 beta band oscillations in brain slices from the mec . under conditions in which 
 cb1 were subject to blockade or inverse agonist effects , we observed a decrease 
 in oscillatory power in gamma and beta bands in layer ii . this is consistent 
 with the literature described above [ 27 , 30 ] and we propose that , in layer ii , blockade or inverse agonism of 
 cbrs results in increased irregular phasic inhibition from is - cells onto 
 pyramidal cells , decreasing the population available to participate in network 
 oscillations and hence reducing field oscillatory power . this appears to be 
 supported by our voltage - clamp recordings showing that ly320135 increased 
 phasic gabaergic inhibition at principal cells in layer ii . when 
 we measured oscillatory activity in layer v , inverse agonists at cbr increased gamma and beta power and this 
 appeared to be correlated with decreased superficial beta and gamma power . at 
 first , this appears paradoxical , however , oscillatory activity in specific 
 laminae does not exist in isolation , and we might expect interactions between , 
 as well as within , networks of neurones . bragin et al .   have demonstrated that , in vivo , bilateral ablation of the ec suppresses gamma activity 
 in the dentate gyrus ( dg ) , but augments gamma oscillations in ca3-ca1 . as 
 previously discussed , superficial mec projects to dg , and ca1 projects to deep mec 
 layers . given that in our experiments , oscillatory activity in superficial mec 
 was suppressed , it is reasonable to suggest that this may depress gamma and/or 
 beta activity in dg and enhance such activity in ca3-ca1 . this , in turn , would 
 feed through to layer v , where increased gamma and beta power is seen . hence , 
 although phasic inhibition in layer v appeared to increase in ly320135 , it may 
 be that this effect is not involved in selection of neuronal assemblies for 
 oscillatory activity in layer v ; rather , excitatory inputs to this area from hippocampus 
 may be the dominant influence on pyramidal cell activity .
cannabinoids modulate inhibitory gabaergic neurotransmission in many brain regions . within the temporal lobe , cannabinoid receptors 
 are highly expressed , and are located presynaptically at inhibitory terminals . here , we have explored the role of type-1 cannabinoid receptors ( cb1rs ) at the level of inhibitory synaptic currents and field - recorded network oscillations . 
 we report that arachidonylcyclopropylamide ( acpa ; 10 m ) , an agonist at cb1r , inhibits gabaergic synaptic transmission onto both superficial and deep medial entorhinal ( mec ) neurones , but this has little effect on network oscillations in beta / gamma frequency bands . 
 by contrast , the cb1r antagonist / inverse agonist ly320135 ( 500 nm ) , increased gabaergic synaptic activity and beta / gamma oscillatory activity in superficial mec , was suppressed , whilst that in deep mec was enhanced . 
 these data indicate that cannabinoid - mediated effects on inhibitory synaptic activity may be constitutively active in vitro , and that modulation of cb1r activation using inverse agonists unmasks complex effects of cbr function on network activity .