The present study provides novel insight into the motivational state of the female rat by revealing that intact VTA function is required for the expression of CPP for a context paired with a natural (pup) stimulus but not CPP for a context paired with a pharmacological (cocaine) stimulus. Virgin females’ preference for a cocaine-paired context remained intact despite VTA inactivation (via bupivacaine), with preference remaining identical regardless of whether bupivacaine or saline was microinfused into the VTA. Postpartum females’ preference for a pup-paired context, however, was abolished by bupivacaine microinfusions into the VTA. Following saline microinfusions, females’ preference for the pup-paired context matched that of controls (groups 1–3). Postpartum females’ preference for the center and empty side chambers remained similar regardless of VTA function, confirming that disrupted CPP expression was restricted to the context paired with the salient pup stimulus. To our knowledge, this is the first demonstration that intact VTA function is critical for the expression of preference for a unique context paired with an appetitive natural stimulus but not one paired with an appetitive drug.
Chamber preferences following intra-VTA saline microinfusions matched those of control females (groups 1–3), confirming that neither VTA cannulation nor surgery itself altered CPP expression. As surgery occurred prior to the start of the CPP paradigm, any possible surgery-related confounds (e.g., anesthesia, stress, discomfort) or tissue damage caused by cannula implantation (e.g., Benveniste and Diemer, 1987
) did not affect CPP acquisition or expression, as reported by others (Herzig and Schmidt, 2007
As bupivacaine blocks Na+ channels to induce widespread neuronal inactivation, discrete bupivacaine microinfusions used in the present study presumably inactivated all neuronal types within the VTA. Neurons in the VTA vary widely in their connectivity and neurochemical content. While DAergic VTA neurons projecting to the NAc have received the most experimental attention regarding motivation and goal-directed behavior, VTA neurons containing gamma-amino butyric acid (GABA) (Van Bockstaele and Pickel, 1995
; Carr and Sesack, 2000
) or glutamate (Yamaguchi et al., 2007
) may also participate in motivated behavior and drug-related responsiveness. These non-DAergic subpopulations of VTA neurons send long-range projections to a variety of targets, including the NAc (Van Bockstaele and Pickel, 1995
), prefrontal cortex (PFC) (Carr and Sesack, 2000
), amygdala (Rosenkrantz and Grace, 2002), hypothalamus and MPOA (Swanson, 1982
; Miller and Lonstein, 2006
), ventral pallidum (Fields et al., 2007
) and lateral habenula (Swanson, 1982
), which may contribute importantly to different aspects of reward-related and goal-directed behavior, decision-making and affective processing (McBride et al., 1999
; Tzschentke, 2000
; Fields et al., 2007
; Matsumoto and Hikosaka, 2007
). Unlike blocking specific classes of receptors, bupivacaine depressed the activity of all neurochemical subpopulations of VTA neurons, effectively eliminating VTA input to a broadly distributed neural circuitry participating in motivated behavior.
As voltage-gated Na+ channels are also located at nodes of Ranvier on axonal fibers, bupivacaine also blocks axonal conductance in fibers of passage. One such fiber bundle, the fasciculus retroflexus (FR), projects from the habenula to the interpeduncular and raphé nuclei through the midbrain, just dorsal to the VTA. Though the habenula and its projections respond to the motivational value of stimuli and their predictive cues (Matsumoto and Hikosaka, 2007
), it is unlikely that bupivacaine-induced blockade of the FR within the VTA affected CPP, as our injection controls received bupivacaine into rostral and/or dorsal aspects of the FR yet retained normal CPP.
Given the injected volume and expected diffusion of bupivacaine, we posit that physiological inactivation induced by bupivacaine microinjection was limited to the VTA. The VTA extends approximately 1,000 microns (µm) mediolaterally, 800–1,000µm dorsoventrally, and 600–800µm rostrocaudally. Bupivacaine is structurally similar to the amide anesthetic lidocaine, which diffuses in an estimated sphere of 1000µm from the tip of the injector cannulae per 1µl microinfusion (Sandkühler et al., 1987
; Martin and Ghez, 1999
; Pereira de Vasconcelos et al., 2006
). This sphere of diffusion coincides with a spherical area of depressed physiological activity (Boehnke and Rasmusson, 2001
) and local glucose uptake (Martin and Ghez, 1999
), with little inactivation extending beyond that perimeter (Tehovnik and Sommer, 1997
). As expected for bupivacaine, lidocaine-induced neuronal inactivation is greatest at the center of the microinjection site, where drug concentration is greatest, and decreased gradually with increasing distance from injection site (Martin, 1991
; Martin and Ghez, 1999
). Given its smaller molecular mass (Tucker and Mather, 1998
), bupivacaine is presumed to spread to equal or slightly greater distances than an identical volume of lidocaine. Thus, our bupivacaine microinjections of 0.5µl/side likely diffused about 500–600µm from all directions of the injector cannula. Given the anatomical accuracy of cannula tip placement, we believe that bupivacaine inactivated the majority of the VTA without diffusing into neighboring structures.
The site specificity of microinfusions was also verified behaviorally in our locomotor analyses. In VTA females, locomotor habituation emerged consistently in each chamber, mean locomotor rates did not differ, females moved easily between CPP chambers and postpartum females carried and manipulated pups normally following either microinfusion. In contrast, unilateral inactivation of the adjacent substantia nigra (SN) produced rapid and profound locomotor impairments. Given the normal locomotor behavior observed in VTA females, we posit bupivacaine diffusion into SN was minimal.
Region-specific mesocorticolimbic lesions have been used frequently to alter the acquisition but not the expression of CPP (for review, see Tzschentke, 1998
). Interventions targeting the acquisition of CPP disrupt the ability of a subject to encode the valence of the primary stimulus (e.g., pups) and/or attribute incentive value to a unique context paired with that stimulus. In contrast, interventions targeting CPP expression alter a subjects’ motivation to seek out and spend time in a unique context attributed with a certain value. As mesolimbic manipulations may not affect consolidated learning (Dalley et al., 2005
; Berridge, 2007
) and thus subjects’ retrieval of learning about the conditioned value of each CPP context, impaired pup CPP following VTA inactivation likely reflects a selective disruption of goal-directed behavior toward unique contexts associated with pups. Only limited work has assessed expression of drug CPP after mesolimbic disruption (Sellings and Clarke, 2003
; Moaddab et al., 2008); this is the first study to assess CPP for cocaine- or pup-paired contexts by disrupting VTA function.
These findings extend growing evidence that the incentive value of pups depends upon connections between the maternally responsive medial preoptic area (MPOA) and the mesocorticolimbic system. In females, the MPOA mediates the expression of maternal behavior (Lee et al., 2000
; Numan et al., 2007
), motivation to reunite with offspring (Perrin et al., 2007
) and preference for a unique pup-paired context (Morrell et al., 2008
). The MPOA and ventral bed nucleus of the stria terminalis (vBNST) project directly to VTA (Numan and Smith, 1984
; Numan and Numan, 1997
) and these projections are activated during motivated pup-directed behavior (Numan and Numan, 1997
). Maternal behavior is disrupted following MPOA or VTA lesions (Gaffori and LeMoal, 1979
; Lee et al., 2000
; Perrin et al., 2007
) or by blocking select receptor subtypes within the VTA (Pedersen et al., 1994
; Thompson and Kristal, 1996
; Numan and Stolzenberg, 2009
). As DAergic projections from the VTA (Hansen et al., 1991
) and their upstream targets (PFC: Afonso et al., 2007
; NAc: Lee et al., 2000
) must also remain intact for females to express maternal behavior, the VTA may allow maternally relevant input from the MPOA/vBNST to access the mesocorticolimbic system (Numan, 2007
; Numan and Stolzenberg, 2008
) to facilitate preference for a pup-paired context.
Select impairments in motivated maternal behavior following VTA inactivation were also revealed in females tested for pup CPP. Transient VTA inactivation selectively disrupted pup retrieval and anogenital licking, though females could still carry/transport pups with their mouths, group and lick pups (see corporal licking data), and assume normal nursing postures (though retrieval impairments extended females’ latency to hover/crouch). As PFC lesions selectively disrupt pup retrieval and licking (Afonso et al., 2007
), the specific deficits revealed in the present study suggest that VTA inactivation disabled maternally relevant inputs to the PFC.
While the expression of preference for chambers paired with a natural stimulus required intact VTA function, preference for the cocaine-paired chamber remained intact despite widespread VTA inactivation. Extensive research suggests that the mesolimbic circuitry, including the VTA and NAc, contribute to an animal’s response to cocaine and cocaine-paired stimuli and its expression of cocaine-seeking behavior (Carelli et al., 2000
; Carelli and Ijames, 2001
; Carelli, 2002
; Phillips et al., 2003a
; Roitman et al., 2004
; Yun et al., 2004
; Sombers et al., 2009
). In the present study, however, the expression of cocaine CPP was unaffected by inactivation of the VTA and its projections.
Only one other study used a similar site-specific inactivation procedure, revealing that the expression of morphine CPP was reduced, but not abolished, following bilateral VTA inactivation and remained undisrupted following unilateral inactivation (Moaddab et al., 2008). Compared to the present study, Moaddab and colleagues (2008) used a different strain, sex and age of rats, number of conditioning sessions, and drug, dose and route of administration, each of which can influence CPP (reviewed in Bardo et al., 1995
; also, see Russo et al., 2003
; Seip et al., 2008
). A combination of these variables likely contributed to the difference in magnitude of CPP effects reported in these two studies.
A number of alternative, non-VTA dependent mechanisms may exist to maintain cocaine-seeking behavior during the expression of CPP. The basolateral amygdala (BLA) is strongly activated by preference for a cocaine-paired chamber (Mattson and Morrell, 2005
) and cocaine-paired cues (Carelli et al., 2003
) but is not activated as strongly by preference for a pup-paired chamber (Mattson and Morrell, 2005
). Given a sufficiently salient stimulus, such as cocaine, the BLA may be able to sustain CPP during VTA inactivation, possibly by directly modifying accumbal DA release (Floresco et al., 1998
; Phillips et al., 2003a
). The PFC also contributes to the reinforcing properties of cocaine (Isaac et al., 1989
) and participates in goal-directed behavior (Tzschentke, 2000
; Hitchcott et al., 2007
). As prefrontal disregulation can increase motivation to seek drugs and drug-related stimuli (Jentsch and Taylor, 1999
; Tzschentke, 2000
) but not necessarily natural stimuli like food (Levy et al., 2007
), inactivating the VTA and its projections to the PFC may facilitate responses to cocaine-paired cues to maintain cocaine CPP.
We conclude that the VTA may be differentially involved in motivated seeking behavior directed toward contexts associated with natural stimuli and contexts associated with drug stimuli. As virgin and postpartum females express identical CPP for intraperitoneal cocaine (Seip et al., 2008
) and, if sensitized to behave maternally, for a pup-paired chamber (Seip and Morrell, 2008
), we fully expect that, following VTA inactivation, cocaine CPP would remain intact in postpartum females and pup CPP would be abolished in maternal virgin females. Together, these findings emphasize the importance of characterizing females’ motivated behavior toward various salient stimuli in her environment. The choice to invest time with a specific stimulus over available alternatives can be evolutionarily advantageous, such as when a female seeks out stimuli that will directly benefit her progeny (e.g., food, maternal care) or maladaptive (e.g., drug-seeking). The present work now reveals that motivated seeking behavior directed toward contexts associated with natural, highly adaptive stimuli may be strikingly vulnerable to loss of VTA function compared to contexts paired with drugs of abuse.