Materials and methods off functions were generated, was initiated when stable

104 T [17,22] have been similarly exploited to examine sites ranged from 0.2 to 0.92 mm posterior to bregma, 0.6 to 2.0 activated by stimulating electrodes in LH reward sites. mm lateral to the mid-sagittal suture, and 7.6 to 8.2 mm Cytochrome oxidase activity is evident in the lateral septal ventral to the skull surface reading at bregma. The nucleus and nucleus accumbens when short pulse durations anesthetic consisted of a combination of sodium pen- are used to activate reward loci while a more widespread tobarbital Somnotol, 65 mg kg i.p. and xylazine Rom- activation is observed from the use of longer pulse pun, 0.05 ml i.m.; atropine sulfate 0.05 ml s.c. was durations [2]. In the case of glycogen phosphorylase, the administered just prior to surgery in order to minimize diagonal band of Broca and the caudate nucleus show respiratory distress. greater activation of reaction product on the stimulated side, and the habenula in the contralateral hemisphere in 2.2. Apparatus one study [22], and weak activation in the ipsilateral lateral septum in another study [17]. More recently, c-fos im- Electrical stimulation was delivered via an integrated munohistochemistry has been similarly exploited and circuit pulse generator, manufactured in house, and a generally supports the implication of basal forebrain constant-current amplifier [24]. The current was continu- structures such as the LPO as responsive to rewarding ously monitored on an oscilloscope by reading the voltage MFB stimulation [1,10,20,28]. drop across a 1 kV precision resistor in series with the rat. The purpose of this mapping study was to obtain In order to prevent polarization at the electrode tip in the information about the distribution of reward sites in the absence of a pulse, the output was shorted to ground via a region of the LPO and their spatial organization; the 1 kV resistor. location of self-stimulation sites within and around the LPO was therefore documented. At sites that reliably 2.3. Screening and training supported this behavior, the trade-off relationship between current and period was established in order to permit the Three days after surgery, the animals were trained to characterization of the neural density and the boundaries of lever-press on a continuous reinforcement schedule for 0.5 the behaviorally relevant bundle. Estimates of the relative s trains of square wave monophasic pulses, 0.1 ms in neural density of elements within the effective stimulation duration, using conventional operant shaping techniques. If field, at individual sites, can be gleaned from period after two screening sessions no self-stimulation behavior thresholds [23,45]. Due to the trade-off between current was evident, then the electrode was moved a ventral and period the reciprocal of frequency, any change in distance of 0.08 to 0.24 mm, aided by a calibrated driver; current must be accommodated by roughly the same the new site was evaluated the following day. This change in period in order to maintain equivalent levels of procedure continued until either bar-pressing was estab- behavior. The function that describes this relationship, lished or the full ventral travel of the electrode was used, when plotted in logarithmic space, ascends to level off at based on the estimated location of the electrode tip from high currents where the relationship between these two the Paxinos and Watson atlas [31]. parameters breaks down. Thus differences in slope across During the training phase of the study, the animals were sites can be used to infer the density of the relevant introduced to 60 s trials preceded by five priming stimula- substrate. The assumptions underlying this interpretation tions, separated by 1 s, set at the same values as the trial are explained in detail in Gallistel et al. [14]. This strategy parameters; the current was 200 mA. A descending se- has been profitably employed by others to map more quence of periods was presented, starting with a value at posterior reward sites from the stria medullaris [8,42] and which no response occurred. Each successive period was ventral pallidum regions [30] to the posterior mesence- 0.1 log 10 unit below the previous one. The series phalon [9,11], as well as rostrally to study the connections continued until the bar pressing rate exceeded the criterion between the caudate–putamen and the medial prefrontal of 35 responses min. A rate period curve was plotted for cortex in our own laboratory [7,41]. each current and the period threshold, i.e. the period required to support the criterion response rate was de- termined. The testing phase, during which complete trade-

2. Materials and methods off functions were generated, was initiated when stable

thresholds were obtained. The criterion for stability was 2.1. Subjects and surgery met when the standard error of the mean associated with the average period threshold across sessions did not exceed Twenty-two male Long–Evans rats Charles River 10 of the mean value. Generally, stable thresholds were Laboratories were housed separately in plastic cages under achieved within two sessions, during which repeated rate a 12-h light dark cycle with lights on at 07.00 h. Food and period functions at the baseline current were collected. water were available ad libitum. Moveable electrodes Kinetrode Inc. were implanted in all but one subject 2.4. Behavioral tests TH22 which received a fixed electrode. The electrodes were aimed at the LPO; the flat-skull coordinates [31] During this phase, collection of full trade-off functions T . Bushnik et al. Brain Research 881 2000 103 –111 105 was conducted; the currents used ranged from 80 mA to anterior to the LPO, the second, third, and fourth groups 1000 mA, incrementing in 0.2 log units. The period were found within the LPO nucleus at roughly anterior, 10 thresholds, as previously described, were then plotted in mid, and posterior levels, and the fifth group was situated order to generate the trade-off relationship between the posterior to the LPO, in the transition zone between the period threshold and the current. If the period thresholds LPO and the LH. associated with a baseline current administered at the The placements are shown in Fig. 1. Those of the first beginning and at the end of each session differed by more group n54 rats, 10 sites were found to be just rostral to than 0.1 log units, the data from the session were the midline convergence of the anterior commissure Fig. 10 discarded. When the trade-off functions were established 1, plate 1. This level is dominated by the caudate-putamen for a range of currents, the electrode was moved and the and the nuclei of the precommissural septum which site tested anew using the same procedure. An example of includes the horizontal and vertical limbs of the diagonal a rate period function generated at different currents has band, the medial septum, and compartments ‘a’ and ‘b’ of been published elsewhere [19]. the MFB [29]. The second group n57 rats, 24 sites is made up of electrodes located around or near the anterior 2.5. Histology part of the LPO Fig. 1, plate 2. At this level, the anterior commissure and the fornix, as yet not bifurcated, are still Following administration of a lethal dose of sodium evident. The LPO is bordered medially by the medial pentobarbital, animals were perfused intracardially with preoptic area, laterally by the ventral pallidum and sub- saline, followed by 10 formalin. The brains were re- stantia innominata, dorsally by the bed nuclei of the stria moved and stored in 10 formalin for at least 1 week. terminalis, and, ventrally by the nucleus of the horizontal Coronal sections 40 mm were placed on coated slides and limb of the diagonal band. Three subjects 20 sites stained with Cresyl Violet in order to better locate the contribute to the third grouping of electrode sites Fig. 1, electrode tips using the Paxinos and Watson atlas [31]. plate 3. This level is characterized by a large, well-defined third ventricle, the movement of the medial preoptic area from the midline to a more lateral position, and a more

3. Results dispersed optic chiasm. The LPO is bordered by the same