Rimonabant decreases breakpoints
During baseline and vehicle conditions rats reached breakpoints following an average of 14 (S.E.M =0.45) and 14 (S.E.M = 0.87) pellets respectively. These correspond to an average breakpoint or last response ratio completed during baseline of 78.2 (S.E.M =7.39) and during vehicle of 89.8 (S.E.M =16.85). Rimonabant (150 µg/kg, i.v.) robustly reduced the breakpoint in all animals tested, average breakpoint of 18.4 (S.E.M =6.30) with the corresponding reduction in the number of pellets earned [6.8 (S.E.M =1.62)]. Repeated measures ANOVA showed a significant effect of rimonabant across conditions (F(2,10) = 12.47, p < 0.001). Post-hoc comparisons showed that breakpoints obtained under rimonabant were significantly lower than those obtained during baseline and vehicle ().
Rimonabant significantly reduces breakpoints obtained in a progressive ratio schedule (N=6).
Nucleus accumbens cell classification according to waveform characteristics
Neurons in the NAc are compromised mostly by medium spiny neurons (MSNs), which constitute ~95% of the total neurons in this structure (Kemp and Powell 1971
), with the rest of neuronal population consisting mainly of cholinergic interneurons (Armstrong et al.,1983
), persistent low-threshold spiking interneurons (Bevan et al., 1998
), and fast-spiking interneurons (FSI) (Berke et al., 2004
; Lansink et al., 2010
; Morra et al., 2010
). In behaving animals most of the neurons that show a correlation with operant tasks are believed to be MSNs; whereas FSIs do not show a precise spike synchronization with different events in operant tasks (Berke, 2008
), but they may control LFP oscillatory power (Berke et al., 2004
; Berke, 2005
; van der Meer et al., 2010
). Given these differential characteristics between MSNs and FSIs it was necessary to distinguish these populations of neurons. A total of 133 (mean number of neurons per rat = 26.6; SEM=4.03) neurons were recorded in the NAc during the progressive ratio task; of those neurons 7 (5.26%) were classified as FSI and 125 (93.98%) as MSN and 1 as other (0.75%). The Characterization of neuronal types was done based on waveform shape (
); putative FSIs were identified as having a valley full width at half maximal (FWHM) equal or inferior than 150 µs, and a firing rate of more than 7.5 Hz. The rest were treated as putative medium spiny neurons. The neuron with a valley width of more than 500 µs was classified as other. Representative FSI and MSN waveform shapes are presented in respectively.
Figure 2 Sorting criteria for identifying putative medium spiny neurons (MSNs) and fast-spiking interneurons (FSIs). The total of number neurons recorded was 133 (a) Criteria used for identifying putative neuron types were firing rate (z-axis), amplitude (x-axis) (more ...)
Average and SEM of the criteria used to identify medium spiny neurons (MSNs) and fast-spiking interneurons (FSIs).
Nucleus accumbens cell firing patterns during progressive ratio responding
Nicola and Deadwyler (2000)
showed that the firing rate of neurons in the NAc can be broadly classified into lever press-excited (LPE) and lever press-inhibited (LPI) when subjects are working for cocaine under a progressive ratio schedule. We aimed to verify if this classification applied for rats working for food pellets and whether these distinctive firing rates encompassed a CB1R-mediated component. Analysis of integrated firing rate histograms identified 21 neurons (16%) as LPE and 14 neurons (11%) as LPI whereas the remaining neurons did not show any recognizable firing patterns during the task, consistent with previous studies (Carelli and Deadwyler, 1994
; Carelli et al, 2000
). None of the neurons showing LPE or LPI firing patterns were identified as FSI. The firing rates of LPE cells increased gradually across trials with increases in firing before the onset of each bout of lever pressing (). The highest overall firing rate was observed at breakpoint. During vehicle, peak firing rates for LPE neurons averaged 1.99 Hz (S.E.M=0.59); following rimonabant this peak did not significantly change 2.16 Hz (S.E.M= 0.78; t(20)
= −0.61; p=0.54). In contrast, the firing rates of LPI cells decreased across trials. Specifically, these neurons showed a decrease in activity at the beginning of the session which was sustained for the duration of responding (). Rimonabant significantly reduced activity prior to the beginning of the session compared to vehicle (t(13)
=−3.42; p= 0.004) from 1.43 (S.E.M.=0.29) to 1.08 Hz (S.E.M.=0.24).
Figure 3 Rate histograms showing a LPE neuron. Black tick marks show lever presses and red ticks indicate reward delivery. Vehicle and rimonabant were injected 1 minute before the start of the schedule and neural recording (represented by the vertical gray lines). (more ...)
Figure 4 Rate histograms showing a typical LPI neuron. Black tick marks show lever presses and red ticks indicate reward delivery. Vehicle and rimonabant were injected 1 minute before the start of the schedule and neural recording (represented by the vertical (more ...)
Event-specific characteristics of NAc neuron activity
In order to analyze time-locked firing patterns within the task, we examined activity at reward delivery and its associated cues, and cue onset at the beginning of each trial. shows the percentage of neurons showing different responses types to these events. Of the 133 neurons recorded during the task, 78 (58.64%) exhibited no patterned activity. The remaining 55 cells (41.35 %), all MSNs, exhibited excitatory or inhibitory firing patterns relative to cue presentation or reward delivery. Specifically, 36 (27%) neurons exhibited patterned activity relative to reward delivery; of these 27 (20%) showed a transient increase in firing rate, 5 (3.75%) showed a sustained increase, and 4 (3%) showed a sustained inhibition. 19 (14.3%) neurons exhibited patterned activity relative to the cue presentation; of these 10 (7.5%) showed a transient increase in firing rate, 4 (3%) showed a sustained increase, and 5 (3.75%) showed a sustained inhibition.
Number and percentage of NAc neurons exhibiting different types of patterned discharges during reward delivery and cue onset.
CB1 receptor blockade modifies neural encoding associated with reward delivery
A three dimensional representation of the perievent rasters of representative neurons responsive to reward delivery and its associated cues following vehicle and rimonabant administration, along with the normalized population activity graph are shown in . Neural activity is depicted 8 seconds before and after reward delivery, which is set at 0 across all trials. The translucent rectangle marks the injection of rimonabant. depicts a representative neuron that showed a transient increase in firing during vehicle at reward delivery and a profound decrease in this transient activity was observed following rimonabant. The decrease in transient activity is evident when the mean normalized firing rate from the 27 neurons that displayed phasic excitation upon reward delivery is plotted (). Rimonabant significantly attenuated the peak observed when contrasted against vehicle (t(26) =3.510; p= 0.0008).
Figure 5 3D rasters and normalized average from neurons responsive to reward delivery. The 3D perievent raster are aligned to reward delivery at time 0; rimonabant administration is denoted by the translucent rectangle. (a) Shows a representative neuron that presented (more ...)
shows a representative neuron that showed a sustained increase in firing rate after reward delivery and its associated cues, and the effect of rimonabant in this particular type of patterned discharge (). Paired t-tests carried out for the collapsed data before and after the reward delivery shows that there is a significant difference before and after the reward when the firing rate is recorded under vehicle (t(4) = −9.460; p= 0.0006) which remains unchanged following rimonabant (t(4) = −2.262; p= 0.086). However, when firing rates after reward delivery are compared, a statistical difference (t(4)= 10.441; p= 0.0004) is observed, confirming that rimonabant significantly reduced the post-reinforcement excitation.
Finally, a subset of neurons showed a time-locked decrease in firing at reward delivery and its associated cues. In these neurons rimonabant failed to significantly alter the observed decrease ( shows a representative neuron). Firing rate after reward delivery for this population of cell was not significantly different between vehicle and rimonabant (; t(3)= −2.177; p= 0.117).
CB1 receptor blockade modifies neural encoding associated with cue that indicates trial start
The three dimensional representation of the perievent rasters of representative neurons responsive to the cue that indicates the beginning of the trial during vehicle and rimonabant treatment, along with a normalized Z-score of population neural activity graph are shown in . As before, the translucent rectangle in the 3D graphs marks the injection of rimonabant. shows a representative neuron displaying a brief cue-evoked increase in firing at the cue. CB1R blockade significantly attenuated this pattern compared to vehicle (; t(9) =6.010; p= 0.0002).
Figure 6 3D rasters and normalized average from neurons responsive to cue onset. The 3D perievent raster are aligned to cue onset at time 0; as before, rimonabant administration is denoted by the translucent rectangle on the 3D rasters. (a) Shows a representative (more ...)
CB1R blockade also disrupted neurons showing a sustained increase in firing rate observed after cue onset (; t(3)= 3.365; p= 0.0435). This significant effect indicates that the sustained firing rate obtained under rimonabant is lower than that observed following vehicle administration. Lastly, rimonabant treatment altered the firing rate decrease observed after cue onset compared to vehicle; such that there was no differentiation in firing activity before or after cue onset (t(4)= −1.310; p = 0.130). Such effect is evident in the representative example () as well as in the mean normalized firing rate graph for the pooled population data (t(4)= −13.4737; p = 0.0001; ).
Collective synchrony in NAc neurons is affected by rimonabant
Functional connectivity among simultaneously recorded neurons was analyzed via pair-wise cross-correlations (). For each subject, each of the recorded neurons was used as a reference and cross-correlated with the rest of the neurons (all cell-pair combinations were analyzed). During vehicle there were 29 cross-correlograms with a peak (mean Z score peak= 4.85; SEM= 0.57) and 6 with a trough at time 0 (mean Z score trough = −4.38; SEM= 0.42). Following rimonabant administration there was a significant reduction in the peak of the population cross-correlogram (mean Z score peak = 2.54 SEM= 0.37; ; t(28)= 4.55 p= 0.0001). A reduction in the population trough was also observed following rimonabant (mean Z score trough = −2.43 SEM= 1.031; ); but was not statistically reliable (t(5)= 0.73; p = 0.49). None of the cross-correlograms analyzed that utilized FSI neurons as a reference yielded significant cross-correlations with either MSNs or FSIs.
average (a) peak (n=29) and (b) trough (n=6) cross-correlations. Rimonabant significantly reduces positive functional cross-correlations observed among neighboring neurons.
Accumbal LFP activity in the gamma range is disrupted following CB1 receptor blockade
LFP oscillations are believed to reflect the organization and synchronized activity between different brain areas related to different cognitive and motor processes (Buzsáki, 2006
; Sanes and Donoghue, 1993
). Here we measured LFPs with the same electrodes used to record action potentials. We constructed session-wide spectrograms to analyze changes in LFPs before and after the administration of rimonabant (). Rimonabant changed the frequency at which gamma power peaked and attenuated overall power in the gamma band. Specifically, the peak of the gamma band shifted from 64.11 Hz (S.E.M.= 2.38) during vehicle to 55.84 Hz (S.E.M.= 1.49) following rimonabant (t(4)
= 2.99 p=0.0402). shows the distribution of power across all spectral bandwidths during the task obtained after vehicle (blue) and rimonabant (red) administration. The only significant changes were observed in the gamma bandwidth (55–100 Hz; t(4)
= 2.93 p=0.0424).
Figure 8 (a) Power spectral densities obtained from recording electrodes during the progressive ratio task; after vehicle and rimonabant administration (n=6). Under the influence of rimonabant there is a change in the frequency at which the power peaks and a change (more ...)
Gamma oscillations are hypothesized to couple to FSIs in the striatum (Berke, 2005
; Kalenscher et al., 2008; van der Meer, 2009
). We analyzed these neurons to calculate spike-field coherence to determine whether LFPs were partially affected by the CB1R antagonist. We found, as previously reported (Berke, 2009
; van der Meer and Redish, 2009
), that there were two groups of FSI neurons one of which changed their firing rate differentially with “gamma-50” (45–55 Hz) and the other with “gamma-80” (70–85 Hz) powers (). Following rimonabant administration, FSIs lost their synchronous firing to these frequency bands and became coherent to multiple frequencies.
Gamma oscillations are differentially modulated by rimonabant during cue presentation and reward delivery
Perievent spectrograms showing the frequencies of the low and high gamma spectral bands were constructed around cue presentation () and reward delivery (). The different bands were studied at cue onset for either vehicle (), or rimonabant (). Baseline-subtracted spectrograms depicts time-locked changes in power at the different spectral bands during vehicle (), or rimonabant (). Mean “gamma-50” (45–55 Hz) power Z-scores increased slightly following cue onset during vehicle. Under rimonabant this increase disappears and instead a through in “gamma-50” power is observed (). This decrease is confirmed when the Z values of the troughs observed after cue onset, from the baseline subtracted powers, are contrasted (t(4)=3.34 p=0.002).
Figure 9 Perievent spectrogram for cue onset. The spectrogram is aligned to the cue that indicates the beginning of the trial at time 0. (a) Shows that after vehicle administration there is not an evident time-locked change in the power of the different spectral (more ...)
Figure 10 Perievent spectrogram for reward delivery and its associated cues. The spectrogram is aligned to reward delivery at time 0. (a) Shows that after vehicle administration there is an increase in “gamma-80” power immediately after reward delivery; (more ...)
depicts spectrograms obtained during vehicle administration at reward delivery and its associated cues. A significant increase in power is observed around high gamma immediately following reward delivery (t(4)=3.20 p=0.03; ). Rimonabant administration (), significantly reduced high gamma after reward delivery and its associated cues (t(4)=2.31 p=0.02, ). Mean “gamma-80” (70–85 Hz) power Z-scores observed are transiently elevated following vehicle and this increase is completely abolished by rimonabant administration (). Analysis of gamma power peaks, obtained from the baseline subtracted powers, confirms a highly significant statistical difference between vehicle and rimonabant (t(4)= 3.80; p=0.01).