There are three principle findings arising from these investigations. First, we demonstrate that a conditioned motor response with this behavioral paradigm was context-specific, results that are consistent with other studies (Ahmed et al., 1995
; Badiani et al., 1995
; Jodogne et al., 1994
; Shen et al., 2006
; Tirelli & Terry, 1998
). Secondly, non-contingent AMPH administration activated Fos in all brain regions examined except the VP, results that are consistent with other studies (Cadoni et al., 1995
; Dalia & Wallace, 1995
; Engber et al., 1998
; Niles et al., 1997
). Thirdly, AMPH administered in the context that reliably predicted AMPH (i.e., the AMPH-paired context) activated Fos in the hippocampus, BLA, NAcC, NAcS, and DLS. However, the administration of saline in the AMPH-paired context also activated Fos, but only in the BLA and NAcC. Finally, AMPH, but not saline, given in the AMPH-paired context was associated with increased immunoreactivity for synaptophysin in the hippocampus, BLA, and DLS, but not in the NAcC. Considered together, these data suggest thatcellular activation of the BLA and NAcC are involved in the context-elicited conditioned motor activity, but the change in the synaptic marker associated with CMS is restricted to the BLA, hippocampus, and the DLS.
We found, in an earlier investigation, in which we assayed the same brain regions, that the 1.0 mg/kg dose of AMPH and a similar conditioning procedure produced a significant increase in both Fos and synaptophysin immunoreactivity in the NAcC for rats that showed a significant CPP (Rademacher et al., 2006
). In the present study, we found an increase in Fos, but not synaptophysin, immunoreactivity in the NAcC and NAcS for rats that received an AMPH challenge injection in the AMPH-paired compartment. This important difference could be related to the two experimental paradigms. In the earlier study, all rats were tested for CPP, drug- and saline-free, 72 hours after the final conditioning session. During the test for CPP, animals had access to all three compartments of the place conditioning apparatus, and contextual cues presumably elicited approach behavior to the preferred compartment (see e.g., Bardo & Bevins, 2000
; Everitt et al., 1991
; Rademacher et al., 2000
). In contrast, for the current report, there was no test for CPP, and animals were confined to one compartment of the apparatus after the challenge injection. Thus, it is possible, that at least for the NAcC, synaptic change, as measured by an increase in synaptophysin immunoreactivity, only occurs when the memory trace is being reactivated and reconsolidated at the time of the test for CPP (Miller & Marshall, 2005b
). The strength of the initial context-drug memories and the nature of the reactivation events, important contributors to the degree of memory destabilization and reconsolidation (Bozon et al., 2003
; Milekic et al., 2006
; Suzuki et al., 2004
), could also have differed between the two studies. However, this possibility seems unlikely considering that memory reactivation and reconsolidation appear to be independent of the initial memory consolidation (Lee et al., 2004
). Finally, the expression of CPP could engage brain systems that include the NAcC, but these systems are not activated in the absence of a need to retrieve contextual cues, as at the time of the evaluation of CMS, a possibility that requires further investigation.
The finding that AMPH-administered in the AMPH-paired context increased cellular activation and synaptophysin expression in the DLS is consistent with the view that the DLS mediates the acquisition of stimulus-response (S-R) associations or habits (Everitt et al., 2001
; Packard & Knowlton, 2002
). Glutamatergic corticostriatal projections provide the DLS with sensory information underlying the formation of S-R associations (Alloway et al., 2006
). Dopaminergic input to the DLS provides a signal that effectively imprints S-R associations (White, 1989
). Glutamate and dopamine are involved in synaptogenesis (Petrak et al., 2005
; Spencer et al., 1998
) and activation of these two systems increase Fos expression in the DLS (Graybiel et al., 1990
; Liste et al., 1995
). The current findings indicate that the associative learning processes subsumed during AMPH-induced CMS may engage these, or similar systems, to alter synaptically mediated function in the DLS.
Augmented synaptophysin immunoreactivity could mean an increase in the number of vesicles, or, as been shown with electron microscopy, represent a true increase in the number of synapses (see e.g., Calhoun et al., 1996
; Liu & Ju, 2001
). Clearly one of the limitations of the present study was that it was conducted at the light microscopy level and so we are unable to distinguish synaptogenesis from enlarged boutons. Nevertheless, either outcome could result in synaptic enhancement. We have studies in progress at the electron microscopic level to establish the nature of this change.
A puzzling set of findings in the present study are those that demonstrate that the density of synaptophysin immunoreactivity is significantly greater in the CA1 field and DLS for animals that received a saline challenge injection in the AMPH- or saline-paired compartment compared to animals that received an AMPH challenge injection in the saline-paired compartment. One explanation is the density of synaptophysin immunoreactivity is dependent upon the pairing between the contextual CS and drug UCS. Certainly, the density of synaptophysin immunoreactivity generally increased whenever the AMPH challenge injections were administered in the “expected” compartment and decreased when given in the “unexpected” compartment. In addition, there was no difference in the density of synaptophysin immunoreactivity in any brain region for rats given saline in the AMPH-paired compartment compared to rats given saline in the saline-paired compartment. Further evaluation is required to determine whether or not the decrease in synaptophysin immunoreactivity for animals that received AMPH in the saline-paired compartment reflects a true decrease in the number of vesicles or synapses.
The AMPH-associated context significantly increased the expression of Fos in the BLA and NAcC, which is consistent with other reports of AMPH- or cocaine-paired environments activating this immediate early gene in these regions (Brown et al., 1992
; Ciccocioppo et al., 2001
; Franklin & Druhan, 2000
; Mead et al., 1999
; Miller & Marshall, 2004
; Neisewander et al., 2000
). However, as a context-elicited increase in synaptophysin density also occurs in the BLA but not in the NAcC, the formation of contextual CS-drug UCS associations may require adaptations in the BLA neurons for the appropriate motor output from the NAcC to be elicited (Rademacher et al., 2006
, Sellings & Clarke, 2006
; Sutton et al., 2000
). Supporting this concept, Di Ciano and Everitt (2004)
demonstrated that if the BLA-NAcC pathway is interrupted, cue-elicited cocaine seeking on a second order schedule of reinforcement is greatly attenuated.
Both Fos and synaptophysin are significantly elevated the BLA and hippocampus after AMPH-induced CMS. These two regions serve important roles in appetitive cue and contextual conditioning. Whereas the formation of associations between discrete, elemental CS occurs in the BLA (Everitt et al., 1991
; Ito et al., 2006
; McDonald & White, 1995
), the representation of spatial context probably resides in the hippocampus (Holland & Bouton, 1999
; Ito et al., 2006
). Thus, lesions of either the dorsal hippocampus (Ferbinteanu & McDonald, 2001
; Meyers et al., 2003
) or the BLA (Brown & Fibiger, 1993
; Everitt et al., 1991
; Hiroi & White, 1991
) block drug-induced CPP. We hypothesize that contextual CS-UCS associations, formed in the BLA and hippocampus, may gain control over the motor response through convergent input onto NAc neurons (see Pennartz et al., 1994
Finally, it is useful to describe our results in terms of an occasion-setting model. Critical to this consideration is our finding that saline given in the AMPH-paired and saline–paired context enhances and inhibits the expression of behavioral sensitization, respectively, by setting the occasion for the response. Whereas the AMPH-paired context reliably predicts drug administration and has taken-on occasion setting properties that act to enhance the motor response, the saline-paired context reliably predicts the absence of drug administration and has taken on occasion setting properties that act to diminish the motor response. Thus, the context-specific expression of AMPH-induced motor sensitization may, in part, be due to differential activation of the BLA-NAcC pathway by these occasion setters.