Lateral hypothalamic self-stimulation pathways in Rattus norvegicus

Physiology andSekavior. Voi. 7, pp. 419--432. Pergamon Press, 1971. Printed in Great Britain

Lateral Hypothalamic Self-Stimulation Pathways in Rattus Norvegicus' YUNG HWA HUANG ~ AND ARYEH ROUTTENBERG

Departments of Psychology and Biological Sciences, Northwestern University, Evanston, Illinois, U.S.A. (Received 5 November 1970)

HUANG, Y. H. AND A. RotrrrENnERG. Lateral hypothalamic self-stimulationpathways in Rattus Norvegicus. PHYSIOL. BEnAV. 7 (3) 419--432, 1971.---Self-stimulation in lateral hypothalamus involves the activation of several ascending and descending fiber systems. In an effort to define these fiber systems, self-stimulation loci were studied with a single low current level and bipolar nichrome electrodes made with wire 78.7 Iz dia. Prngrade degeneration revealed by the FinkHeimer technique and retrograde chromatolytic changes were described following lesions at self-stimulation sites. Neuronal activity following electrical stimulation of self-stimulation sites was also used to define the functional influence of these fiber systems. The relationship with the extrapyramidal systemwas emphasized, with convergent evidence suggesting an intimate relation between lateral hypothalamic neurons and those of substantia nigra, pars compacta. Lateral hypothalamus

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have been claimed to produce SS using larger electrodes and higher current levels [25, 27].

THE PAucn'v of knowledge concerning the neural pathways which subserve intraeranial self-stimulation (SS) has recently been emphasized [31]. We have boon studying such pathways directly by tracing degeneration following lesions at SS sites. After demonstration of SS loci in the brachium conjunetivum [27] and the medial, frontal cortex [24], fibers proceeding from these SS sites were traced using silver impregnation techniques [6, 14]. It has been suggested that the knowledge of these anatomical pathways might lead to a synthesis of SS points located along such fiber bundles [23]. The present study was concerned with pathways from lateral hypothalamus (LH) and medial forebrain bundle (MFB) self-stimulation sites. Since LH has reciprocal connections with other SS sites in the limbic system and since the highest SS rates are often obtained from LH loci, the assumption has been made that LH is the focus of SS [18]. In an effort to understand the pathways of SS fully, it was necessary to examine the pathways from LH self-stimulation sites and determine the relationship between the course of such pathways and SS sites which have been described previously.

Method Animals. One hundred and six male, adult, SpragueDawley rats weighing 230-450 g at the time of operation were used. Of these rats, 58 served only in Experiment 1, 21 also served in Experiment 2, 27 in Experiment 3. Apparatus. The bipolar stimulation electrode was formed by twisting together nichrome wires each 78.7 tL dia. The electrode was insulated with Epoxylite except at the cross section of the tip, and a Johnson-Kreig stereotaxic instrument was used for implantation. Self-stimulation was carried out in Skinner boxes manufactured by Scientific Prototype. They had 7.5 in. walls and a 9.25 in. by 8.0 in. grid floor. A bar 1.5 in. above the floor protruded from one wall. Depression of the bar produced 10 ttA, 60 Hz sine-wave brain stimulattion equal to bar press duration up to but not exceeding 0.5 see. Procedure. Electrode implantation was performed using sodium pentobarbital anesthesia (50 mg/kg, i.p.) administered to 24 hr food-deprived rats [22]. Each animal was implanted with only one bipolar electrode. The electrode was aimed at diencephalic and midbrain sites of 93 animals and in 13 rats at the dentate and interpositus subcortical nuclei. After a four-day postoperative recovery period the animals were allowed to bar press for brain stimulation in the Skinner boxes. This SS test continued for 12 consecutive days, one session/day, 15 rain/session. The greatest number of bar

EXPERIMENT 1 SELF-STIMULATION MAPPING

This experiment used small electrodes and a single low current level to map SS loci in the posterior hypothalarnus and the midbrain tegmeatum. Attention was also paid to placements in subthalamic structures such as Ha field of Forel (He) and the substantia nigra (SN), since these areas

tThis report is based upon a dissertation submitted by the first author under the direction of the second author in partial fulfillment for the doctoral degree at Northwestern University. This research was supported by MH17255 to A.R. to whom reprint requests at the Department of Psychology,Northwestern University, Evanston, Illinois 60201, should he sent. aPresent Address: Department of Physiology, University of Western Ontario, London, Ontario, Canada. 419

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presses produced in any one of the 12 sessions was taken as the score for a given animal. This score did not differ appreeiably from the mean of the last 3 days of testing and represented the best that the animal could perform with that placement. F o r subsequent histological work, the animal was sacririced with an overdose of sodium pentobarbital ( > 60 mg), perfused with physiological saline and then 10% formalin. Brain tissue was removed from the cranium and then stored in l0 % formalin for at least a week. Frozen coronal sections of 20-25 t~ thickness were taken and those near the electrode tips were stained either for cell bodies with cresyleeht violet or thionin, or for myelin sheaths [30]. Results

The SS scores in the 15 min test session ranged from 0-749. F o r convenience, these scores were classified as in the previous work [27] into the following rates: 0-49, neutral; 50-199, low; 200-499, moderate; and 500-999, high. The number of electrode placements falling into each rate category was: 57, neutral; 17, low; 22, moderate; and 9, high. The placements of electrodes and the SS rates associated with them are shown in Figs. 1-4. Certain features of the mapping data may be pointed out.

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Figures 2 B and F show three placements in H2 field of ForeI which resulted in a moderate or high rate of SS. Of 12 placements in SN (Figs. 2 H and 3A, C-E), 3 of 3 animals in the compact zone of SN (SNC) yielded SS with a mean SS of 468. In contrast, 6 of 9 in the reticular zone (SNR) produced SS, with a mean SS score of 153 for the 15 rain test session. One of four electrodes in BC (Figs. 3 F and G, 4 C) yielded SS. Eleven of 12 placements in MFB (Figs. 1 A and H, 2 A, C, D and F) yielded SS. The mean of the 12 SS scores associated with these MFB placements was 328. Dividing M F B into a dorsal, intermediate and ventral section, it was found that the mean SS score was 519 for dorsal MFB (n = 4), 318 for the intermediate MFB (n : : 2), and 180 for the ventral M F B (n = 5). Ten placements were outside the MFB, but within 0.3 mm of the circumference of M F B as delimited by K6nig and Klippel [11]. Of these 10 electrodes, 9 were effective in producing SS. The mean SS score for these 10 electrodes was 393. Figure 4 H shows 2 electrodes in the dentate nucleus, 3 in the interpositus nucleus, and 3 adjacent to these nuclei. None of the electrodes in or adjacent to these cerebeilar loci supported SS.

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FIG. 1. Drawings showing sites of stimulation and associated SS rates, The legend in Section A, indicating SS scores and symbols, applies to all sections of Figs. 1-4. The symbols are ~ t e d with the following c ~ o f SIS: half filled circles, neutral; filled squares, low; filled circles, moderate; filled stars, hi~l. S~tions A - H a ~ from KSnig and Klipp¢l ([11]; Figs. 17 b, 26 b, 30 b--34 b). A ~ . a t i o n s C A / , internal capsule; c,p, nucleus caudatus putamen; F, fornix; ha, anterior hypothalamus; MFB, medial forebrain bundle; tv, ventral thalamus,

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FIG. 2. Drawings ([11]; Figs. 37 b 4~ b) showing sites of stimulation and associated SS rates. Symbols as in Fig. 1. Abbreviations: CC, crus cerebri; H~,H~fieldofForcl; MFB, medial forcbrain bundle; SNR, substantianigra, parsrcticulata; sut, subthalamus; tv, ventralthalamus.

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FIG. 3. Drawings ([11]; Figs. 45 b-49 b, 51 b-52 b, 54 b) showing sites of stimulation and associated SS rates. Symbols as in FIG. 1. Abbreviations: El(2, brachium conjunctivum; MFB, medial forcbrain bundle; r, red nucleus; SNC, substantia nigra, pars compacta; SNR, substantia nigra, pars reticulata.

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Discussion 1. Methodological considerations. The electrodes used in the present study had an exposed surface area of 9.7 × 103 ~3, assuming a minimum effective elliptical area with the long axis twice the diameter of the wire, and the short axis equal to the wire diameter. This is considerably smaller than the electrodes usually used for SS. For instance, Olds and Olds [19], in mapping the brain for SS and for avoidance behavior, used wire 254 ~ dia, which yields an approximate effective surface area of 101.3 × 103 ~. Moreover, the l0 ~A current employed in the present study is low compared to that used by others. For instance, in the Olds and Olds study [19], 50 ~A current levels were used routinely. The use of low current stimulation and small diameter wire in the present study should restrict activation of nervous tissue, thus rendering more accurate the localization of SS loci. The placements obtained in the present study, therefore, probably provide a more precise identification of SS loci. Until some direct method is devised, however, to measure effective current spread using various electrode configurations and current levels, the field which is being activated cannot be specified. Since we consider negative SS points as important information it is necessary to consider the possibility that such points could yield SS were a different testing situation used. For example, would the map of Figs. 1-4 have been altered by using food deprived animals ? This question is important if and only if it can be demonstrated that such a manipulation

changes the qualitative pattern of SS points. If a modification is observed with all placements, then it is likely that the treatment has had a nonspecific effect. 2. Comparison with other studies. Self-stimulation was obtained from SN placements, confirming the observation of Routtenberg and Kramis [26] made in the gexbit, and Routtenberg and Malsbury [27] in the rat. That SNC is more effective in producing SS than is SNR suggests a functional localization within the divisions of substantia nigra. In agreement with the results obtained with the larger electrode by Routtenberg and Huang [25], the 3 H2 placements of the present study resulted in SS. Of the tegmental BC placernents in the present study, only one of four yielded SS. Self-stimulation from all BC placements was reported by Routtenbcrg and Malshury [27]. One possible reason for this discrepancy might be related to the presence of both SS and non-SS systems in BC which may have been revealed in the present study with the use of a lower current and a smaller electrode. The use of the low current and the small electrode may have also assisted in demonstrating that different parts of MFB were differentially effective in producing SS. Thus, the mean scores increased as the more dorsal parts of MFB were approached. It is interesting to note that SS was obtained from most (9 of I0) placements in the area immedia~ty outside MFB (0.3 mm within its circumference) just as most (10 of 11) MFB electrodes resulted in SS. These two regions, MFB and its

SELF-STIMULATION PATHWAYS

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immediate vicinity, also produced similar SS rates. This spatial proximity plus similar SS effectiveness suggests that the two regions may belong to the same SS-related system. That the mean SS scores for the two regions were similar suggests that SS obtained from the area adjacent to MFB was not due to current spread from that area to MFB. If current applied to the electrode adjacent to MFB were to spread to the latter simply by electrotonic flow, one would have expected the peripheral placements to yield a lower rate of SS than that obtained from MFB. In sum, stimulation of regions nearby MFB may perhaps involve direct activation of MFB.

EXPERIMENT

2

PROGRADE DEGENERATION FOLLOWING LESIONS AT SELF-STIMULATION SITES

This experiment studied the pathways and terminations of fibers arising from SS loci in MFB. For this purpose, lesions were made at SS sites in MFB, and the resulting prograde fiber degeneration was examined by means of the Fink and Heimer [6] staining technique.

Method Animals. Thirty-two rats were used. Their strain, sex and weight at the time of operation were as in Experiment 1. Apparatus. Electrodes for implantation, Skinner boxes for SS testing, and parameters of stimulation were the same as in Experiment 1. Procedure. Each rat was implanted with one electrode. The electrode was aimed at tn = 28) or 0.5 mm above (n = 4) LH using either a perpendicular approach or an angular (25°) approach from the contralateral side. Immediately following the SS test, electrolytic lesions were made by delivering either direct or alternating current to the electrode in those animals which showed a moderate (n = 10) or high (n = 5) rate of SS. To make the direct current lesions, a current of 1 mA was delivered for 10-30 sac, the anode to the electrode, the cathode to the ear. The alternating current lesions were made by connecting two leads from a Variac set at 40 or 80 V to the two wires of the implanted electrode for 20-30 sac. For 5 days after the lesion, animals were tested for self-stimulation; decrements in rate were noted in all animals indicating that the lesion was effective. The animals were sacrificed 5 days after the lesion and the brain was stored in formalin as in Experiment 1. Although multiple survival times are often desirable in visualizing terminal degeneration, it was ~ecessary to use a single survival time so that comparisons of degeneration patterns among animals could be made with less difficulty. Although other survival times could be used in future research, the 3-7 day survival period is, in fact, generally used. The 5 day period, then, is often used, and, in the present study, did permit the demonstration of terminal degeneration. For study of fiber degeneration, frozen sections were taken at 20 ~ and every third or fourth section was processed according to Procedure lI of Fink and Heimer [6]. The sections were taken in the same plane as the atlas [11] so that charting of degeneration could be performed on the line drawings of the atlas. Two brains, each implanted with a shorter electrode but with no lesion, were similarly processed as histological controls.

Results A total of nine brains were selected as representative of the group of 15 self-stimulation subjects and 4 controls. Seven had perpendicularly implanted electrodes and 2 had angled implants. 1. Degeneration from the electrode tract. It is important to describe the fiber degeneration from the electrode tract above LH, since such degeneration must be discounted in describing the degeneration following a lesion in the LH region. In control Rat 1734 (Fig. 5 A), the electrode tip was in HI field of Forel and did not produce SS. Prograde degeneration arising from the tract is schematically shown in Figs. 6 A-F. A few short fibers projected from the tip area to H,. Degeneration could not be observed in SN, nor in the midbrain tegmentum. 2. Descending projections from LH. Lesions at SS sites produced descending degeneration in MFB which, at the level of the ventral tegmental area of Tsai, distributed to the midbrain in three branches. This was demonstrated in case 1974 (Figs. 5 E; 7 F-H) but was most clearly shown in Case 1980 (Figs. 5 F; 8). The medial branch ran near the midhne of the midhrain to reach the dorsal rapbe. Some fibers terminated in the dorsal raphe while others turned laterally to end in the ventral part of the central gray laterocaudal to the trochlear nucleus. The intermediate branch coursed through the area between the red nucleus and the decussating BC, terminating in the ventral part of the central gray. The lateral branch turned laterodorsocaudally to the medial part of SNC where some fibers terminated (Figs. 9 A and B). Others passed through the medial SNC and, after running through the area dorsal to the lateral SNC, swept around the lateral edge of the medial lemniscus and spread into the nucleus mesencephalicus profundus, pars lateralis of Gillilan [7]. 3. Lateral projections from LH. The H2 field of Forel appeared to receive a few fibers from most lesioned MFB portions (Figs. 7 E, 10 D and 11 C) except from the medial part of MFB (Fig. 12 C-D). 4. Ascending pathways from LH. Rostral to the lesion, degeneration ascended in MFB and reached the medial septal nucleus (Fig. 7 A). Some collaterals diverged from the ascending MFB degeneration at the level of the anterior hypothalamus, entered the stria medullaris, and terminated in the lateral habenuclar nucleus (Figs. 7 B and C, 10 B and C). 5. Medial projections from LH. Degenerating fibers deviated from different MFB sites and distributed medially to ventromedial and dorsomedial hypothalamic nuclei, to supramamillary and ventral tegmental decussations, and to the periventricular system (Figs. 7 C-F; 10 E and 12 D). 6. Topographical organization in MFB. Although black spherules 0.5-2 ~ dia. indicating terminal degeneration were interspersed with MFB fiber degeneration at all levels of MFB, a topographical organization within MFB was still observed. As shown in Fig. 5 B-E, lesions in medial (B), dorsal (C), dorsolateral (D), and ventromedial (E) MFB destroyed tissue which was chiefly restricted to the region of the electrode tip. The resulting degeneration ran in both rostral and caudal directions primarily in that part of MFB lesioned at the electrode tip. This topographical organization extended from the level of the anterior hypothalamus caudally to the level of the supramamillary decussation. Beyond these two levels, the topographical differentiation was reduced. Rostrally, the blurring of the differentiation occurred because of MFB fibers running dorsomedially into the stria medullaris;

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caudally, MFB collaterals running medially to the supramamiUary decussation caused a reduction of the topographical character of this MFB projection. 7. Degeneration from H, field of Forel. In Cases 1730 (Fig. 5 (3) and 1732 (Fig. 5 H), the damaged tissue was restricted to the region of the electrode tip in H,. Since the resulting degeneration is similar for both cases, the result of only 1732 is presented (Figs. 11 E and F). From the tip locus some degenerating fibers spread dorsolaterally along the area immediately dorsal to the internal capsule. Their destination, however, could not be ascertained. Other fibers ran medially into the supramamillary decussation. The final termination of this latter projection could not be discerned. More caudally, fiber degeneration reached and appeared to terminate in SNC. A few longer fibers passed SNC towards the lateral portion of the medial lemniscus. Another system was seen to pass the dorsal part of the ventral tegmental area

of Tsai and, further along its course, to turn dorsally to run near the midline for a short distance. The termination site of this pathway could not be specified. In contrast to other, more medial self-stimulation sites in MFB, no deganerated terminals were seen in the ventral tegmental area of Tsai following the lesions at the lateral edge of MFB. Discussion 1. Comparison with brainstem self-stimulation aites. One purpose of Experiment 2 was to determine whether projections from lateral hypothalamic self-stimulation sites project to or through SS sites described in brainstem [28], present results indicated that pathways originating from LH do, indeed, pass through the interstitial nucleus of the ventral tegmental decussation, SNC, and parts of BC. I t cannot be concluded, however, that brainstcm SS is solely related to

FIG. 5. Microphotographs of tissue sections showing sites of stimulation or of lesions in brain 1734 (A), 1727 (B), 1728 (C), 1736 (D), 1974 (E), 1980 (F), 1930 (G) and 1932 (H). Prograde degeneration from these sites was studied. Fink and Heimer stain. × 24.

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FIG. 8. Microphotographs of degeneration that originated from lesion of both MFB and its neighboring areas in brain 1980. A-D show degenerated tegmental fibers that projected through three paths: medial path (m), intermediate path (i) and lateral path [1]. As shown in E-H, some of these tegmental fibers reached and terminated in the ventral part of the central~grey.Fink and Heimer stain. A, × 24; B-D, x 600; E, x 24; F, x 240; G and H, x 600.

FIG. 9. A and B show degeneration in SNC after MFB lesion in brain 1974. A, × 24, B, x 600. C D and E show degenerating fibers and terminals in the ventral tegmental area of Tsai after MFB lesion in brain 1974. C, x 2 4 ; D, x600; E, x960. F i n k a n d Heimer stain.

FIG. 13. Microphotographs of tissue sections showing sites of stimulation and of lesions in brains 2041 (A, H~ field of Forel: SS rate/15 min. was 578), 2055 (B, substantia nigra, zona compacta: SS rate---442), 1793 (C, dorsal substantia nigra, zona reticulata: SS rate--207), 2053 (D, ventral substantia nigra, zona reticutata: SS rate--103), 1986 (E, ventral to brachium conjunctivum: SS rate--31), 2047 (F, dorsal to brachium conjunctivum: SS rate~253), 2050 (G, in brachium conjunctivum: SS rate--335), and 1987 (H, ventrolateral to central grey: SS rate--403). Well stain, x 24.

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FIG. 14. Microphotographs of tissue sections showing retrograde degeneration after lesioning SS loci. A shows cell loss in the substantia nigra, pars compacta after ipsilateral FI2 lesion in brain 2041. B shows the substantia nigra, pars compacta at a level corresponding to that of A but on the contralateral side. C indicates cell reduction in the lateral hypothalamus after a lesion in the ipsilateral substantia nigra, pars compacta in brain 2055. D shows the corresponding lateral hypothalamus on the contralateral side. E indicates retrograde degeneration in the ipsilateral interpositus nucleus following a lesion ventrolateral to the central grey in brain 1987. F shows the corresponding interpositus nucleus on the contralateral side. G shows the complete disappearance of neurons in the trochlear nucleus on the ipsilateral side in brain 1987. Thionin stain. × 96.

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FIG. 7. Drawings ([11]; Figs. 15 b, 26 b, 35 b, 38 b, 42 b, 46 b, 49 b and 54 b) of degenerated fibers originating from the lesioned ventromedial part of MFB in brain 1974. Symbols for degenerated fibers and for degenerated terminal ramification are as in Fig. 6. Note that the fine dots in central grey denote terminals. Abbreviations: BC, brachium conjunctivum; CG, central gray; LM,m edial lenmiscus; SM, stria meduUaris; SNC, substantia nigra, pars compacta; SNR, substantia nigra, pars reticulata.

these pathways. Two examples should illustrate this difficulty. First, with regard to substantia nigra SS, it cannot be determined whether SS is derivative of the fibers which originate in LH or of fibers of passage which perforate through the LH region. Second, it is difficult to explain SS sites in BC that do not appear to have fiber pathways from LH passing through it. The present experiment indicates quite clearly, nonetheless, that inferences about the site of action of the electrode cannot be made with certainty solely on the basis of observation of the electrode tip locus. A similar conclusion was drawn in a study from our laboratory of forebrain SS pathways [24]. 2. Methodological considerations. Caution is required in interpreting degenerating fibers and disintegrated terminals because of the capricious character of silver staining techniques. In the present report, a statement of the presence of hypothalamofugal degeneration at any area is made, therefore, only ff the degeneration can be traced without interruption from LH to that area. A particularly difficult problem has been the interpretation of degeneration in SNC where axon fragnumts and terminals appear to be intermixed. It is possible that no terminals are, in fact, present in SNC, but on the basis of our fight microscopic data and electrophysiological results (Experiment 4), such a conclusion appears remote. Only electron microscopic data will unequivocally settle this issue.

With regard to inferring the presence of disintegrated terminals, only those degenerating fragments which meet the following criteria wore considered to be terminals: (a) degenerating fibers enter into an area which has perikarya; (b) elongated fibers indicating degenerating fibers change to spheroidal bodies; (c) linear patterns of degenerating fibers are replaced by a random orientation of scattered spheroids; (d) the size of fragments ranges between 0.5-2.0 t~ [6]. Another point of consideration concerns the size of lesions. In the present experiment, except for animals 1974 and 1980, lesion size was small compared to similar studies by other investigators [8, 32]. A small lesion is particularly valuable for the present experiment because only by a small lesion can it be certain that the resulting degeneration originates from the site of stimulation. Furthermore, different parts of MFB may not function exactly in the same way with regard to SS (Experiment 1) and hence a small lesion in MFB can describe pathways associated with individual parts of MFB. 3. Comparison with previous studi¢s. In reference to fibers distributing to SN, Nauta [15] mentioned a system in cat brain that originates from MFB and runs in the area dorsal to SN. Wolf and Sutin [32] reported a bundle in rat MFB that distributes to the dorsomedial part of SN. The present study found hypothalamofugal fibers that run both to the dorsomedial part of SN and then along the area dorsal to

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SN. These hypothalamofugal fibers traverse the medial part of SNC, and in our material, some fibers are seen to texminate there. On their further course, these fibers run to the dorsolateral part of SNC. The involvement of substantia nigra, pars reticulata (SNR), was not in evidence in any of our materiaL This may be of fuvctional importance or, perhaps, reflect the particular survival time used in the present study. In a study of deseneration following lesions at hypothalamic self-stimulation sites, Scott [28]; (personal communication) has noted terminals in SNC and on the most medial border of SNC and SNR. With regard to tegmental degeneration, all three branches mentioned in the present study have been variously reported by others using techniques other than the Fink and Heimer [6] method. The medial branch near the midJ!ne was recognized by Guillery [8], Nauta [5] and Wolf and Sutin [32]. The lateral system arching around and piercing through the lateral part of the medial lemniscus has been described by Nauta [5] and Wolf and Sutin [32]. Wolf and Sutin [32] also mentioned the intermediate system that runs caudodorsal to the red nucleus. Some fibers were seen to pass through Hi, althwagh this projection was not extensive. The presea~ of such fibers is supported by the Millhouse [13] study of Golsi impregnated

material in which a lateral group of fibers from MFB are seen to pass through H, field of Forel. The cells of origin of certain pathways dega'ibed above may be derived from frontal cortex. This lmssildlity must be given consideration in view of the denmnstrafion [23] that lesions at self-stimulation sites in frontal cortex give rise to degeneration in the most medial aspect of the capsule, intermixing thereby, with the lateral aspect of MFB~ Thus, terminals seen in SNC following lateral LH lesions may have been the result of the interruption of frontofugal fibers. EXPERIMENT 3 RETROGRADE DEGENERATION IN LATERAL HYPOTHALAMUS FOLLOWING LlglONS AT "EXTRAPYRAMIDAL" SELF-STIMULATION SITES

This experiment was performed to determine whether SS loci in the posterior dien~phalon and the midbrai~ s p e c i f r ~ y in H,, SN and BC, contain flbe~ whose cells of origin arise from LH. If such were the case, then one might demonstrate retrograde cellular chauses in LH after lesions made at such SS loci.

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Method Animals. A total of 35 rats were used of which 7 were selected for detailed cytological study. Strain, sex and weight at the time of operation were given in Experiment 1. Apparatus. Electrodes for implantation, Skinner boxes and stimulus parameters were described in Experiment 1. Procedure. Operative procedures were described in Experiment 1. Each animal was implanted with one electrode only. The electrode was aimed at H~, SN, BC, as well as other tegmental areas that were shown in Experiment 2 to contain LH fibers. To control the effect on chromatolysis of the damage along the electrode track, an electrode was aimed at the area 0.5 mm dorsal to each of these structures just mentioned. Following SS testing of these placements, electrolytic lesions using current parameters as in Experiment 2 were made at the electrode tip in those animals which showed SS at a moderate or high rate. A survival period of 1.5-2 months was allowed after the lesion was made. Animals were sacrificed with an overdose of barbiturate, the brains were removed and fixed in formalin and frozen sections of 25 ~t thickness were taken. For those brains which had been lesioned, every other section along MFB and every fourth section from the rest of the brain were stained with thionin. For those brains which had not been lesioned, histological work was performed only to locate the electrode placement. The number of brains studied for retrograde degeneration after lesioning of a particular SS locus was: 1 at H2, 3 at SN, and 3 at or near BC. For each of these structures the one brain implanted with the shorter electrode was also studied. Results Electrode placements, maximum extent of each lesion and SS scores are contained in Fig. 13. Selected retrograde cellular changes are shown in Fig. 14. The SNC lesion in Case 2055 (Fig. 13 B) resulted in some cell loss in LH on the ipsilateral side (Figs. 14 C and D). This conclusion was based on study of the rostral-caudal extent of L H on both the same and opposite side of the SN lesion. Discrete regions of cell loss were not observed, but a reduction in the number of cells was found. In this case (3055), SNR chromatolysis on the side of the SNC lesion was also observed. N o sign of retrograde degeneration could be detected in L H as a result of lesions at SS sites in H2, SNR or BC. The substantia nigra, pars compacta showed chromatolysis following a lesion at a self-stimulation site in H, field of Forel (Case 2041; Figs. 13 A, 14 A and B). Following a lesion at a self-stimulation site in BC, chromatolysis was observed in the interpositns and dentate nuclei on the homolateral side (Cases 1987, 2047, 2050; Figs. 13 F - H , 14 E and F). In Case 1987 (Figs. 13 H and 14 G) a complete disappearance of the homolateral trochlear nucleus was observed following destruction of part of BC and the area ventrolateral to the central gray. Discussion The results of this retrograde experiment demonstrated that SS from $NC might involve activation of the axons of the lateral hypothalanaic nucleus, since lesions at SNC caused retrograde changes in LH. It cannot be stated with certainty, however, wh~her the retrograde e f f ~ were the result of damage to terminals in SNC or to fibers of passage. It does seem reasonable to emphasize the relationship which

HUANG AND ROUTTENBERG

appears to exist between the lateral hypothalamus and the substantia nigra, in the compact zone. As in Experiment 2 no relation could be discerned between SNR and LH. With regard to BC SS, the retrograde changes observed in dentatus and interpositus indicated that these are the cells of origin of BC, and suggest that such cells may be related to BC SS. This result must be interpreted with caution, since no self-stimulation was observed following direct stimulation of these cells of origin (Experiment 1). If BC does support selfstimulation, it may be the case, then, that direct stimulation of cell bodies and associated axon terminals is not comparable to stimulation of heavily myelinated axons. If BC does not support self-stimulation, then some other fiber pathway, as yet not specified, must be sought. It is difficult to attribute the LH fibers going to tegmentum to BC SS seen in Fig. 13 (F and G), since even in the largest lesion (Fig. 5 F) degenerating fibers were not seen in this region of BC where SS was obtained. Although negative results in retrograde degeneration experiments may be viewed with some caution, no evidence was found that H~ or BC lesions at SS sites produced retrograde changes in LH neurons. Such negative data may be more acceptable in view of the presence of retrograde changes following these same lesions, but in other nuclear groups (Figs. 14 A, B, E and F). EXVEmME~rr4 MICROELECTRODE STUDY OF LATERAL HYPOTHALAMIC ACTIVATION OF "EXTRAPYRAMIDAL" NEURONS

This experiment determined the effect of stimulation at SS sites in LH upon extracellular neuronal activity in midbrain and pens. Since anatomical projections, as shown in Experiments 2 and 3, for example, do not indicate whether the influence is facilitating or suppressive, microelectrode recording was performed to gain such information. Method Animals. Thirty-six rats were used. Strain, sex and weight at time of operation have been given in Experiment 1. Apparatus. The electrode, Skinner boxes and the stimulus parameters used in the SS test were the same as those in Experiment 1. Recording procedures were similar to those described previously [25]. Briefly, the stimulus used during recording was a train of rectangular pulses obtained from a Tektronix 160 series, parameters of which were: 0-30 V, 0.01-0.03 msec pulse duration, 1-1.6 msec interpulse interval, and 1-10 msec train duration. Response to the stimulus was recorded using a tungsten microelectrode with a tip diameter of 1-3 ~ [9]. The shaft of the rm'croelectrode was insulated with Stoner-Mudge enamel (S-986-015) using the Bartlett [2] procedure. Neuronal spike potentials were amplified by a Tektronix 122 preamplifier with filters set to exclude frequencies below 80 Hz and above 10 KHz. Output from the preamplifier w a s displayed on a Tektronix 564 storage oseiUosc,ope, monitored over a speaker, and stored on magnetic tape using an audio recorder (Revox 77A). Procedure. Two bipolar electrodes were implanted into each of 36 rats. One electrode was aimed at LH, the other at the globus pallidus (GP), both on the same side of the brain. Both electrode plaeeznents were each tested for SS on 12 consecutive days, one trial per day, 15 rain per trial. The order in which the two placements were tested was alternated each day. The greatest number of bar presses per trial made

SELF-STIMULATION PATHWAYS

429

Results 1. Responses to hypothalamic stimulation. Of 146 neurons recorded, 28 were influenced by hypothalamic stimulation; 19 increased and 9 decreased their electrical activity. Excitation, defined here as increased spike discharge, had a latency of 5-25 mscc. Inhibition, defined here as decreased electrical activity, was immediate and lasted 70-500 see. The location of units recorded and the nature of their responses to L H stimulation are shown in Figs. 15 A - D . (Oscillographic examples of unit responses observed may be requested.) Of five neurons in H , or zona incerta (Fig. 15 A), only one responded, and this occurred with 5 msec latency to L H stimulation. Of 5 neurons in SNC, 3 were inhibited following L H stimulation (Fig. 15 B). Of 12 neurons in SNR, 2 were excited and 1 inl~ibited. Of 36 neurons recorded in the mesencephalic tegmentum, 7 were excited and 3 inhibited (Figs. 15 B-D). Two neurons in the BC region did not respond to hypothalamic stimulation. 2. Responses to corpus striatal stimulation. Thirty-three of 146 neurons responded to corpus striatal stimulation; 21 were excited and 12 inhibited. The latency for excitation ranged from 7-100 msec. Inhibition was immediate and lasted 70-270 msec. The nature of neuronal responses to the corpus striatal stimulation is shown in Figs. 15 E - H . 3. Comparison of neural activities following L H and corpus striatal stimulation. Of all cells recorded, 104 responded to neither stimulus, 9 responded only to LH stimulation, 14

in any one trial over the 12 days was taken as the score associated with that placement. Only those animals which self-stimulated through L H electrodes were employed for the electrophysiological recording study. The SS rates in L H of the 12 animals that met this criterion fell into three categories: low (n - - 1); m o d e r a t e (n = 10); and high (n = 1). SS rates on the other electrode were neutral (n = 7) or low (n = 3) in GP, and neutral (n = 1) or moderate (n = 1) in the striatum. F o r electrophysiological study, the rat was placed in the K o p f stereotaxic under Diabutal anesthesia (50 mg/kg, i.p.). Diabutal was injected about every 2 hr thereafter to maintain full anesthesia throughout the recording experiment. A microelectrode was lowered into the posterior diencephalon or the midbrain. Upon encountering neuronal activity, the rectangular pulse stimulus was delivered to one electrode, then to the other. Recordings were made from neurons encountered as the microelectrode was progressively lowered into the brain. Small lesions were made using a 6 V, 3 sec anodal current, with the ear attached to the cathode after recording from the first unit and the last unit of each penetration. Following the acute microelectrode study, the animal was killed with an overdose of Diabutal, and histological procedures that stained myelin sheath and cell bodies were used to estimate microelectrode loci following the general procedure of Hubel [10].

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430

HUANG AND ROUTTENBERG

responded only to corpus striatal stimulation and 19 responded to both stimuli. Comparison of parts A-D with parts E--H of Fig. 1[ suggested that the number of neurons which responded to both stimuli increased as the more caudal neurons were approached. Except for one neuron, all neurons that responded to the two stimuli reacted to both in the same way. Namely, those that increased or decreased their activity following one stimulus also increased or decreased their activity following the other stimulus. The exceptional case was in SNR. It discharged spikes 7 msec after LH stimulation, but was inhibited for 200 msec following corpus striatal stimulation. Discussion The results of the electrophysiological experiment indicate that neurons in SNC were inhibited by LH stimulation. This lends additional weight to the suggestion made in Experiments 2 and 3 that an intimate relation exists between substantia nigra and lateral hypothalamus. Thus, the terminals revealed in Experiment 2 might be inhibitory according to the results of this experiment. The significance of this inhibitory functional relation, however, is not known.

GENERAL DISCUSSION

The specific objective of the present paper was to determine whether there exist SS pathways from the LH focus of selfstimulation [18] to extrapyramidal structures which support SS [27]. The results of the present study suggest involvement of certain structures, and lack of involvement of others. The results further indicate the importance of applying several techniques when attempting to determine the fiber systems which subserve SS. The present discussion, then, will focus on the information gained from each technique in determining the involvement of certain extrapyramidal SS sites in mediating this behavior. 1. The substamia nigra. Several results pointed to a relation between LH and SNC. All placements in SNC resulted in SS (Experiment 1). Prograde degeneration from SS loci in MFB was traced to SNC (Experiment 2). Retrograde chromatolysis was seen in some LH neurons following a lesion of an SS locus in SNC (Experiment 3). Neurons in SNC decr~sed their activity following stimulation of LH (Experiment 4). Convergent evidence, then, indicated that LH projects to SN in the compact zone. This is not yet conclusive, however, since terminal degeneration in SNC (Experiment 2) and inhibitory effects of SNC neurons (Experiment 4) may arise following manipulation of descending frontal pathways mediating self-stimulation [24], while retrograde changes in LH following SNC lesions (Experiment 3) may be caused by destruction of SNC fibers of passage originating in LH. The poSSibility, then, that both the frontomesencephalic pathway and the lateral hypothalamo-tegnaental pathway originate from separate sources but converge at posterior and ventral diencephalon indicates an intimate, though still poorly understood, relation between the lateral hypothalamus and substantia nigra, pars compacta. The microelectrode data indicated that LH stimulation at SS sites suppressed SNC neuronal activity. This suggests the possibility of inhibitory terminals on SNC neurons originating from LH. When SNC is electrically stimulated the current may activate these inhibitory terminals causing a net decrease in neuronal activity of SNC cells. This result would be consistent with the possibility that lateral hypothalamic self-stimulation leads to depression of SNC neuronal

activity. The functional significance of this relation may perhaps be studied using the chronic multiunit recording technique developed by Olds [17]. It would be of considerable interest, for example, to observe the relation between these 2 systems during a variety of behavioral activities. 2. Brachium conjunctivum. The prograde degeneration studied in Experiment 2 demonstrated three descending fiber systems from LH which either pass through or were adjacent to the superior cerebellar peduncle. The three routes were: a medial path near the medial plane of the midbrain tegmentum, an intermediate path caudal to the red nucleus, and a lateral route around the lateral edge of the medial lemniscus, Following such LH lesions, however, all cases that were studied in Experiment 2, excepting Rat 1980, did not show degeneration at SS loci in BC demonstrated in Experiment 1 or Experiment 3. In the exceptional Case 1980, degenerating fibers of moderate density were observed in certain parts of BC, but not all, from which SS was obtained. It is also necessary to indicate that the exact origin of these degenerating fibers is unknown since the lesion of 1980 extended beyond MFB. The assumption that SS in BC may not be mediated by descending fibers originating from LH is supported by the fact that no unit activation or suppression was observed in BC following LH activation and by the fact that retrograde degeneration following lesions in or near SS sites in BC did not lead to retrograde changes in LH. Although these two lines of evidence are essentially negative, they do not provide positive evidence necessary for the alternative view that BC SS is mediated by descending LH fibers. The retrograde study also indicated that the cells of origin of BC SS were the dentate and interpositus cerebellar nuclei. SS was not obtained from electrodes in the dentate and interpositus nuclei, however, indicating the possibility that stimulation of the axons but not the cells of origin of the brachiurn support SS. It may be relevant to this apparent paradox that electrodes placed in these deep cerebellar nuclei may activate the inhibitory terminals from Purkinje cell axons [5]. This explanation was considered relevant to the relation between LH and SNC (see above), and may represent an important factor, as yet poorly understood, in the results obtained with brain stimulation experiments. This is particularly important for the approach to the study of the anatomical and physiological organization of SS as advocated here. 3. Red nucleus. Although it was suggested that the red nucleus was involved with SS [27], projections from LH in the present study curved around this extrapyramidal structure, but no fiber terminals wore soon to end there, The absence of terminals cannot be unequivocally convincing because of the presence of argentaffm spherules in the red nucleus [24]. In brief, the suggested importance of the red nucleus in SS [27] was not indicated by the results of the present report, but it would be premature to exclude a role in SS for this structure. 4. Globus pallidus. We could virtually exclude a role for GP in self-stimulation, based on observations made in Experiment 4. Only 3 of 10 placements in this structure yielded low rates of SS and this may have occurred as a result of activation of the descending SS pathway from frontal cortex [24], In addition, the SS that was observed may also have occurred because of the presence of a second electrode in LH. SS derived from the LH electrode may have promoted SS in the GP placements, because of cross-over effects [27].

SELF-STIMULATION PATHWAYS

431

There does not, in summary, appear to be compelling evidence in the present results to suggest an important involvement of GP in SS. 5. 1"I2 field o f Forel. All HI placements yielded SS in Experiment 1. The results of prograde degeneration in Experiment 2 following lesions at MFB SS sites indicated that HI does contain fibers from the MFB region. Chromatolysis in LH following an H2 lesion was not observed in Experiment 3. It is uncertain, therefore, whether SS obtained from Hm depends on the integrity of fibers running from MFB to H~. Future research will require evaluation of the involvement of LH dendrites and MFB axons [13], pallidofugal fibers [16], and internal capsule collaterals [3, 12, 21] in supporting self-stimulation in the H~ region. The present study demonstrated chromatolysis in SNC following lesions at SS sites in H2. This result may be of importance since it suggests the involvement of the projection of SNC axons in self-stimulation. This pathway, the nigrostriate bundle, has been shown to contain a major portion of brain dopamine, supplying dopamine terminals to the caudate nucleus [1]. 6. Ventral tegmentum. Experiment 2 showed a heavy projection through the ventral tegmental area with terminals in the area of Tsai and fibers of passage through the interstitial nucleus of the ventral tegmental decussation [11]. This nucleus and adjacent regions were found to support the highest rates of self-stimulation in our earlier work [27]. The present results indicate that electrodes in the region of the ventral tegmental decussation may be stimulating fibers descending from LH. The high rates of self-stimulation indicate a marked involvement of these LH fibers in ventral tegmental SS. The presence of other fiber systems traversing this region which support SS is not ruled out. In regard to this overlap between the sites of stimulation and this descending pathway from LH, it is worth noting that the effective points producing feeding in cat tegmentum [33] follow the lateral hypothalamo-tegrnental pathway. Interestingly, feeding was induced by stimulation in the substantia nigra, in the compact zone.

The relation of the present finding to the role attributed to norepinephrine is SS [29] deserves mention. One major nuclear group which is high in norepinephrine concentration and which projects to the hypothalamus is the ventral tegmental region, or A 10 according to Dahlstr6m and Fuxe [4]. Many of the lateral hypothalamic lesions of the present report did produce terminal degeneration in this region suggesting that stimulation in the LH activates or influences norepinephrine containing neurons in the ventral tegmentum. The present results, then, might provide the anatomical substrate for the recruitment of the norepinephrine system. Some caution is necessary because at least one placement (Figs. 11 E and F) did not send terminals to the ventral tegmental area of Tsai, although it did support self-stimulation. The present results indicate, therefore, that perhaps some, but not all, self-stimulation involves the direct influence of norepinephrine containing neurons. Finally, a comment concerning the general application of the present methods may be in order. It has been our intention to indicate the necessity for applying several techniques to the understanding of the site of action of localized brain manipulation. The same approach could be used in a lesion experiment where the immediate effects of the lesion on behavior are apparent. It would be appropriate, for example, to examine the pattern of degeneration from sites which lead to aphagia and sites which though nearby, do not produce decrements in food intake. Such an approach may be helpful in understanding the anatomical systems which are destroyed during aphagia. Whatever the application, the present series of experiments should suggest to the investigator interested in local brain manipulation that the area observed under the probe tip may not be the region of major importance in mediating the behavioral effect. Although this view will require further detailed investigation, such an effort will provide a more accurate assessment of the brain substrates of the behavior under study.

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9. Hubel, D. H. Tungsten microelectrodes for recording from single units. Science 125: 549-550, 1957. 10. Hubel, D. H. Single unit activityinstriatecortexofunrestrained cats. d. Physiol. 147: 226-238, 1959. 11. Kttnig, J. F. R. and R. A. Klippel. The Rat Brain. Baltimore: Williams and Wilkins, 1963. 12. Leonard, C. M. The prefrontal cortex of the rat. I. Cortical projections of the mediodorsal nucleus. II. Efferent connections. Brain Res. 12: 321-343, 1969. 13. Millhouse, O. E. A Golgl study of the descending media forebrain bundle. Brain Res. 15: 341-363, 1969. 14. Nauta, W. J. H. Silver impregnation of degenerating axons. In: New Research Techniques of Neuroanatomy, edited by W. F. Windle. Springlield, Ill.: Thomas, 1957. 15. Nauta, W. J. H. Hippocampal projections and related neural pathways to the midbrain in the cat. Brain 81: 319-340, 1958. 16. Nauta, W. J. H. and W. R. Mehler. Projections of the lentiform nucleus in the monkey. Brain Res. 1: 3-42, 1966. 17. Olds, J. The central nervous system and the reinforcement of behavior. Am. Psyehol. 24: 114--132, 1969. 18. Olds, J. and M. E. Olds. The mechanisms of voluntary behavior. In: The Role of Pleasure in Behavior, edited by R. G. Heath. New York: Hoeber, 1964.

432 19. Olds, M. E. and J. Olds. Approach-avoidance analysis of rat diencephalon. J. comp. NeuroL 120: 259-295, 1963. 20. Pellegrino, L. H. and A. J. Cushman. A Stereotaxic Atlas of the Rat Brain. New York: Appleton-Century Crofts, 1967 21 Ramon y Cajal, S. Histologie du Systeme Nerveux de l'Homme et des Vertebres. Paris: Maloine, 1911. 22. Routtenberg, A. Pentobarbital anesthesia of albino rats. J. exp. Analysis Behav. 11: 52, 1968. 23. Routtenberg, A. Self-stimulation pathways as substrate for stimulus-response integration. In: Efferent Organization and Integrative Behavior, edited by J. Maser. New York: Academic Press, 1972. 24. Routtenberg, A. Forebrain pathways of reward. J. comp. physiol. Psychol. 75: 269-276, 1971. 25. Routtenberg, A. and Y. H. Huang. Reticular formation and brainstem unitary activity: Effects of posterior hypothalamic and septal-limbic stimulation at reward loci. Physiol. Behav. 3: 611-617, 1968.

HUANG AND ROUTTENBERG 26. Routtenberg, A. and R. C. Kramis. Foot-stomping in the gerbil: Rewarding brain stimulation, sexual behavior, and foot shock. Nature 214: 173-174, 1967. 27. Routtenberg, A. and C. Malsbury. Brainstem pathways of reward, d. comp. phsyiol. Psychol. 68: 22-30, 1969. 28. Scott, J. W. Self-stimulation and diencephalic fiber pathways. APA Proceedings, 78th Annual Convention, 1970. 29. Stein, L. Self-stimulation of the brain and the central stimulant action of amphetamine. Fedn. Proe. 23: 836-850, 1964. 30. Weil, A. A rapid method for staining myelin sheaths. Arehs Neurol. Psychiat. 20: 392, 1928. 31. Wetzel, M. C. Self-stimulation's anatomy: Data needs. Brain Res. 10: 287-296, 1968. 32. Wolf, G. and J. Sutin. Fiber degeneration after lateral hypothalamic lesions in the rat. J. comp. Neurol. 127: 137-156, 1966. 33. Wyrwicka, W. and R. W. Dory. Feeding induced in cats by electrical stimulation of brain stem. Expl Brain Res. 1:152160, 1966.