Fig 1.
Fos-ir pattern of AAS nuclei elicited by appetitive behavior.
Left column in white (sex): Fos-ir after 30 min of exposure to receptive (proestrus n = 5) or to non-receptive (diestrus n = 5) females. Middle panel in light gray (drink): 48 hours of water deprivation followed by 30 minutes of enticing (Deprived enticed n = 14) or not (Deprived n = 6). Right column in dark gray (drug): 30 minutes of exposure to the place preference apparatus where the animals were conditioned to amphetamine (Amph. Conditioned), versus non-conditioned animals (Amph. non-Conditioned) and rats non-conditioned injected with saline, (saline). # p< 0.05 respect to the naïve circadian controls; * p< 0.05 between conditions. Kruskall Wallis one way ANOVA followed by all pairwise multiple comparisons (Dunn's Method).
Fig 2.
Photomicrograph of TMN neurons after re-exposure to the amphetamine place preferences apparatus.
(a) Double immunohistochemistry for Fos/ADA in the tuberomamillary nucleus of a naïve (circadian control) rat. (b) Double immunohistochemistry for Fos/ADA in the TMN of an amphetamine conditioned rat, after 30 minutes of re-exposure to the amphetamine place preference apparatus. Arrows in (a) depict ADA-ir neurons; arrows in (b) depict double-ir neurons Fos/ADA. Scale bar, 200 μm.
Fig 3.
Quantification of the OrxB-SAP ribotoxin lesion of the TMN and adjacent structures.
Photomicrographs of sections stained for ADA-ir (a, b) and Nissl substance (c, d). (a) and (c) show an intact TMN, (b) and (d) represent a large TMN lesion (77%) that involved the ventral (arrowhead) and the dorsal TMN (arrow). (e), the quantification of Orx-ir neurons in the LHA or TH-ir neurons in the ventral tegmental area (VTA), demonstrated that these adjacent regions that express Orx receptors were spared by the Orx-SAP injections. No statistical differences were detected (Mann Whitney test, p = 0.216 and 0.811 respectively). Scale bars = 200 μm. V3m mammillary recess of the third ventricule.
Fig 4.
Effect of TMN lesion on appetitive behavior.
(a) TMN lesion decreased the sniffing time of male rats challenged by a receptive female (proestrus) to a level similar to that elicited by non-receptive females (diestrus).(b) dorsal TMN and dorsal plus ventral TMN lesions were sufficient to reduce sexual appetitive behavior respect to the intact group. (c) TMN lesion decreased licking time of water deprived animals enticed by an empty drinking bottle. (d) The lesion had to involve both TMN divisions to be effective on drinking behavior. (e) TMN lesion decreased the time spent in the amphetamine-paired box in conditioned rats. (f), rats with a dorsal TMN lesion showed the largest drop in appetitive behavior directed to obtain amphetamine. ANOVA of Ranks (Kruskall Wallis) followed by a post-hoc of multiple comparison, Dunn's method. * indicates p<0.05 respect to intact rats, # p<0.05 respect to the two other conditions. n/d, not detected.
Table 1.
Linear regression between TMN lesion extension and behavior during the appetitive phase of sex, drink and drug motivated behaviors.