Rats in the SAC 1 day, SAC 6 day, and IAC 6 day groups received about the same number of cocaine infusions in all sessions (), indicating that the rats in each of these groups on average had similar amounts of cocaine on-board on day of killing and that the self-administration history for the SAC 6 day and IAC 6 day rats was on average similar. However, as indicated in , responding (numbers of nose pokes) and locomotor activity, respectively, declined over several daily sessions in the SAC 6 day group to values significantly below the mean of the SAC 1 day group, likely reflecting the tendency of SAC 6 day rats to make fewer ineffective approaches and nose pokes as they accumulate experience in self-administering. SAS rats were about half as active and delivered about 35% of the infusions recorded for SAC rats. However, responding and locomotor activity in the SAS 6 day group were comparable with that of SAS 1 day rats throughout the course of six consecutive self-administration sessions.
Diagrams illustrating the behavior of the rats used in the study in terms of numbers of cocaine and saline infusions (a), numbers of responses (b) and (c) locomotor activity. Means and SEMs are shown.
Some rats in the group that self-administered cocaine for 5 days and was anesthetized in the home cage on day 6 (SAC 5 days ‘basal Fos' group) delivered lesser amounts of cocaine than did rats in the SAC 1 and 6 day groups (). Two such rats actually stopped self-administering on days 4 and 5. Despite these drug intake anomalies, uniformly low Fos expression was detected in all of the ‘basal Fos' rats, whether SAS or SAC and whether they discontinued self-administering or persisted through the full 5 days (see below).
Fos labeling, expressed as mean density of Fos-ir neuronal nuclei in ROIs after 1 session and 6 consecutive daily sessions of SAC or SAS, is shown in , , and , where saline controls (SAS) are paired with the directly comparable cocaine-receiving (SAC and IAC) groups. Each ROI () is represented by a cluster of five vertical bars and a horizontal reference for ‘basal Fos' expression. Acute groups are represented by a pair of bars: SAS (1 day, n=6, white bars with hatches) and SAC (1 day, n=6, white bars). Repeated administration groups are represented by 3 bars: SAS (6 day, n=8, gray bars with hatches), SAC (6 day, n=9, gray bars), and IAC (6 day, n=8, black bars). Widths of the horizontal bars reflect the magnitude of the SEM above and below the mean for ‘basal Fos' expression. As noted above, because significant differences were not detected between the basal SAC and basal SAS groups, the data from those groups were pooled. The results of the ANOVA and LSD post hoc tests applied to the data are given in the cross-reference tables beneath the graphs (, , and ) and in . shows, in addition, the actual magnitudes of significant responses, reflected in the mean SAC and 1 day values after the respective SAS and 6 day means were subtracted out.
Comparisons of Means Graphed in Figures (6, 7, 8 and 9) Reflecting Fos Densities (Fos-ir nuclei/mm2) for (A) Cocaine (SAC) vs Saline (SAS) Self-Administration On Days 1 and 6 and (B) After 6 vs 1 Day of SAC or SAS
SAC vs SAS
Cocaine, whether self-administered over 1 or 6 sessions or administered by the investigator over 6 sessions, mainly increased Fos expression as compared with the relevant SAS group (, , and ; , column A). Two different kinds of response could be observed. In one, Fos expression was low in the SAS group () and much greater in the corresponding SAC group () after both 1 and 6 self-administration sessions, resulting in marked increases in both the intensity of immunostaining of Fos-ir/nucleus and density of Fos-ir neurons. Evaluated structures exhibiting this type of response included the ventral pallidum (), caudate-putamen ( and ), globus pallidus (), subthalamic nucleus (), substantia nigra reticulata (), intermediate division of the lateral septum (), lateral division of the lateral habenula (), zona incerta (), VTA (), and rostromedial tegmental nucleus (). The other response type was reflected in moderate amounts of SAS-elicited Fos labeling that could be moderately greater in the corresponding SAC groups, after either 1 or 6 self-administration session(s), but generally not both. Structures exhibiting this kind of response after 1, but not 6, session(s) included the accumbens rostral pole (), dorsomedial division of the bed nucleus of stria terminalis (), medial division of the lateral habenula (), supraoptic nucleus (), and prelimbic and anterior cingulate cortex (). Structures that exhibited this response after 6, but not 1, sessions included the dorsolateral division of the bed nucleus of stria terminalis () and the pedunculopontine and laterodorsal tegmental nuclei ().
Figure 10 Photomicrographs illustrating Fos immunoreactive neurons in the caudate-putamen (CPu) in the 1 (1d) and 6 (6d) day saline self-administration (SAS, panels a, b, e, and f) and cocaine self-administration (SAC, panels c, d, g, and h) groups. b, d, f, and (more ...)
Three (of 32) quantitatively evaluated structures—the lateral division of the central amygdaloid nucleus (), ventral division of the lateral septum (), and supraoptic nucleus ()—exhibited a significantly lesser density of Fos-ir nuclei following cocaine administration as compared with the corresponding SAS group after 6, but not 1, self-administration sessions. Interestingly, of these, the lateral division of the central amygdaloid nucleus and supraoptic nucleus exhibited greater than control Fos expression after the first cocaine self-administration session. Note, however, that the apparent decreases in SAC Fos-expressing neurons in the lateral division of the central amygdaloid nucleus and ventral division of the lateral septum on day 6 were due not to fewer SAC-elicited Fos-expressing neurons, but rather to greatly increased SAS-elicited Fos expression (; ). In contrast, the change in density of Fos-expressing neurons between day 1 and day 6 in the supraoptic nucleus reflected a true decrease in the numbers of Fos-expressing neurons ( and ). No significant differences distinguishing SAS and SAC groups at 1 or 6 days were observed in the accumbens shell or core (), piriform cortex, forelimb/hindlimb somatosensory cortex, or SCN.
Figure 11 Photomicrographs illustrating Fos immunoreactive neurons in the accumbens (Acb) in the 1 (1d) and 6 (6d) day cocaine self-administration (SAC) groups. b and d are enlargements of the boxed areas in a and c, respectively. ac, anterior commissure; co,Acb (more ...) Day 6 vs Day 1
, column B shows that the density of Fos-ir neurons after 6 daily sessions of cocaine self-administration differed significantly in a number of brain structures from that observed in the same structures following only 1 self-administration session. The same column, in addition, provides the actual magnitudes of significant responses, reflected in the mean day 6 values after the respective day 1 means had been subtracted out. Structures in which Fos-ir neuron density was greater after 6 sessions as compared with 1 included the ventral pallidum (), globus pallidus (), substantia nigra pars reticulata (), dorsolateral and ventral divisions of the bed nucleus of stria terminalis (), lateral division of the central amygdaloid nucleus (), intermediate division of the lateral septum (), zona incerta (), and VTA (). Structures in which Fos-ir neuronal density decreased after 6 sessions as compared with 1 included the accumbens rostral pole and shell (), central and ventrolateral parts of caudate-putamen (), medial and lateral divisions of the lateral habenula (), rostral and caudal parts of the rostromedial tegmental nucleus (), hypothalamic supraoptic and paraventricular nuclei (), and anterior cingulate cortex (). Fos-ir neuron density in the SAS group was increased in a number of structures after 6 as compared with 1 sessions. These included the dorsomedial and ventral divisions of the bed nucleus of stria terminalis (), medial and lateral divisions of the central amygdaloid nucleus (), ventral division of the lateral septum (), zona incerta (), and piriform cortex (). Fos-ir neuron density decreased after 6 as compared with 1 saline self-administration sessions in the central part of the caudate nucleus (), laterodorsal tegmental nucleus (), supraoptic nucleus (), and the somatosensory cortical barrel field ().
Patterns of Fos Response
One subset of evaluated structures exhibited negligible Fos expression following SAS, but robust Fos expression after both 1 session and 6 consecutive daily sessions of cocaine administration. Another group of evaluated structures exhibited moderate to robust expression of Fos following SAS and many of these showed a further, generally modest, augmentation of Fos expression, after either 1 or 6 sessions of cocaine self-administration. The different Fos responses were not randomly distributed among basal forebrain and brainstem structures, but instead clustered in patterns reflecting the identities of various components of basal forebrain macrosystems ().
Thus, evaluated basal ganglia input nuclei, which include subterritories of the accumbens and caudate-putamen, adhered to a general pattern in which cocaine mainly enhances Fos expression in a robust manner that desensitizes after repeated administrations. Fos expression in these structures was greater or statistically similar in the SAC as compared with SAS condition on days 1 and 6, while SAC-elicited Fos expression after 6 sessions as compared with 1 session was mainly decreased. SAS-elicited Fos expression after 6 sessions as compared with 1 session was mainly unchanged in these structures. This pattern, ie, acute cocaine-elicited robust enhancement of Fos expression that desensitizes on repeated administrations, was also observed in the lateral habenula, rostromedial tegmental nuclei, pedunculopontine and laterodorsal tegmental nucleus, hypothalamic paraventricular and supraoptic nuclei, and anterior cingulate and barrel field cortex.
In evaluated basal ganglia output and intrinsic nuclei, including the ventral pallidum, globus pallidus, subthalamic nucleus, substantia nigra reticulata, and VTA, and also in the intermediate division of the lateral septum, lateral division of the central amygdaloid nucleus, and zona incerta, SAC as compared with SAS on days 1 and 6 was accompanied by robustly increased Fos expression. In all but the subthalamic nucleus, SAC-elicited Fos expression was further increased after 6 sessions as compared with 1 session. SAS-elicited Fos expression after 6 sessions as compared with 1 session was uniformly unchanged in all of these structures, except the lateral division of the central amygdala and zona incerta, where it was increased. Thus, in this pattern, cocaine robustly enhances Fos expression on days 1 and 6 in a manner that sensitizes with repeated administrations.
Evaluated extended amygdala structures and the ventral division of the lateral septum exhibited SAC-elicited Fos expression that was statistically similar or marginally greater than the relevant SAS control on days 1 or 6. In nearly all of these structures, however, robustly sensitized Fos responses were observed after repeated saline administrations.
SAC vs IAC
Means for Fos-ir neuron density were significantly greater in the IAC 6 day group than in the SAC 6 day group in the accumbens rostral pole () and core (), central part of the caudate-putamen (), intermediate division of the lateral septum (), medial division of the lateral habenula (), zona incerta (), VTA (), rostral division of the rostromedial tegmental nucleus (), laterodorsal tegmental nucleus (), piriform cortex (), prelimbic cortex (), anterior cingulate cortex (), and the forelimb/hindlimb part of the S1 somatosensory area (). In contrast, the IAC 6 day value was significantly below that of the SAC 6 day group in the medial division of the central amygdaloid nucleus () and it trended toward lesser in the dorsolateral division of the bed nucleus of stria terminalis () and paraventricular nucleus of the hypothalamus (). In the remaining structures the IAC 6 day and SAC 6 day values were not different.