Thalamic and tegmental mechanisms for sodium intake: Anatomical and functional relations to lateral hypothalamus

Physiology and Behavior. Vol. 3, pp. 997-1002. Pergamon Press, 1968. Printed in Great Britain

Thalamic and Tegmental Mechanisms for Sodium Intake: Anatomical and Functional Relations to Lateral Hypothalamus' GEORGE WOLF'

Department of Anatomy, Mount Sinai School of Medicine, New York, N.Y. 10029 U.S.A. (Received 14 June 1968) WOLF,G. Thalami•andtegmenta•mechanismsf•rs•diumintake:anat•micalandfuncti•nalrelati•nst•lateralhyp•thalamus. PHYSIOL.BEHAV.3 (6) 997-1002, 1968.--Lesions were placed in the thalamic gustatory relay and surrounding regions in the rat. Approximately 2 months after lesioning, the rats were tested for sodium intake after sodium depletion and after injection of desoxycorticosterone. Lesions involving the gustatory nucleus or the tegmental region immediately caudal to this nucleus resulted in significant impairments of sodium intake regulation. Lesions rostral, dorsal, medial, or lateral to the gustatory nucleus had no effect. Pathways which might connect the effective mesencephalic area with the thalamic gustatory nucleus and the lateral hypothalamic feeding area are described. Reticular formation

Lateral hypothalamus

Sodium appetite

As A RESULT of ingenious behavioral research over the past decade, we are gaining insight into some of the functions of the hypothalamus in the control of alimentary behavior. The findings suggest that the lateral hypothalamic area plays a role in the motivational aspects of feeding and drinking, while the associated sensory and motor functions are organized in anothor part of the b r a i n - - p r o b a b l y outside the hypothalamus. F o r example, during the initial stages of the lateral hypothalamic syndrome rats will perform the acts of locating and ingesting water if they are motivated by fear but not by dehydration [23]. Conversely, such rats will make no attempt to obtain nourishment when they are food-deprived even if they are not required to perform normal ingestive acts (e.g. when food is delivered directly to the stomach via a cannula when a lever is depressed [19]). It is also known that lateral rats can discriminate between different tastes because they are very sensitive to the palatability of the foods they are offered [21]. While the hypothesis of a simple motivational function of the lateral area does not account for all the data [12, 27] and considerable experimental and conceptual work remains to be done, it seems that there is sufficient information to warrant forming further hypotheses, especially with regard to how the proposed motivational function of the hypothalamic feeding area is integrated with sensory and motor functions relevant to the regulation of food intake. It seems likely that the lateral area performs a similar function in feeding, drinking, and the ingestion of specific

Gustatory nucleus

Desoxycorticosterone

nutrients such as sodium salts. A close behavioral relationship between motivation and gustation is seen in the regulation of sodium intake: the sodium-deficient rat without previous experience ingests sodium salts immediately upon tasting them but rejects non-sodium salts within a few seconds after the initial taste [11]. Rapid repetitive ingestive behavior is triggered when the two conditions of drive and sodium taste are present simultaneously but not when either one is absent (this statement does not hold for weak saline solutions, which appear palatable to normal rats). This close innate behavioral relationship suggests close neuroanatomical relationships between motivational, gustatory, and motor systems involved in sodium intake. Previous experiments have shown that destruction of the presumed drive mechanism in the lateral area results in complete impairment of sodium intake regulation [27] but have not yet elucidated the pathways by which relevant information is conveyed to extrahypothalamic systems [24, 26]. Since behavioral experiments show that taste plays a critical role in sodium ingestion [20], it seems important to determine which structures of the gustatory system are involved and how they may be anatomically related to the hypothalamic feeding area. The present experiments show that lesions in the region of the thalamic gustatory nucleus and in the reticular formation immediately caudal to this nucleus impair sodium intake regulation. (The term "gustatory nucleus" is used for convenience; the nucleus actually receives tactile as well as gustatory input from the tongue [4].

aPresented at the E.P.A. Symposium on Hypothalamic Mechanisms in the Regulation of Ingestive Behavior. April, 1968 in Washington, D.C. sWork done during tenure of an Established Investigatorship of the American Heart Association and supported by USPHS Grant MH 13189. The author is thankful to Dr. Christiana Leonard for assistance in the application and analysis of the Fink-Heimer stain. 997 o

998

W()I.[

We refer to the area between the posterior thalamic nucleus and the central grey as "reticular formation", assuming that this ill-defined region corresponds to the anterior extent of the nucleus mesencephalicus profundus of Gillilan [8], or the nucleus cuneiformis as defined by Valverde [22]). Pathways which might provide connections between these regions and the hypothalamic feeding area are discussed.

METHODS

Lesioning and Postoperative Procedures Adult male Sprague-Dawley rats were used. Lesions were placed in and around the thalamic gustatory nucleus by passing anodal d.c. through stereotaxically guided stainless steel electrodes insulated except for the tip. Rats which did not eat or drink after the operation were fed by stomach tube and given palatable wet foods until recovery of normal food intake. At least 2 months' postoperative recovery was allowed before commencement of the experiment, and all rats appeared behaviorally and physiologically normal at the time of testing.

After completion of the experiment, brains were prepared for histology as described elsewhere [30]. Cases with bilaterally asymmetrical lesions were discarded or included in the control group (when they were small and bilaterally heterotopic).

Behavioral Tests Rats were given 0.50 M NaCI solution, 0.30 M KCI solution, and distilled water in standard drinking tubes attached to the fronts of the individual cages. Condensed milk was given as the sole food, in a beaker also attached to the front of the cage. The positions of the solutions were fixed during each of the two phases of the experiment but were changed between the phases. Intake and body weight were measured daily. The two salt solutions appeared about equally aversive to the rats, and within a few days intake of these solutions dropped to less than 1 ml per day. After intake stabilized, the rats were injected with 2.5 ml of 1.5 % formalin to induce sodium depletion [28], and intake during the next 24 hr was observed. On the day after formalin injection, the positions of the solutions were changed and,

Gustatory Nucleus

o NZo

Caudal

Dorsal

Rostral ,

Medial

Lateral

/

FIG. 1. Projection drawings of sections near the centers of the lesions in individual rats of the Gustatory Nucleus and Caudal groups and in representative rats of the Rostra], Dorsal, Medial, and Lateral groups. In the Gustatory Nucleus and Caudal groups, the number to the bottom left of each section identifies the rat and the number to the bottom right gives the total increase in sodium intake of the rat on both tests (to the nearest mEq). The gustatory nucleus is not specifically outlined in the sections because it is generally not distinguishable from surrounding structures throughout its extent, and it was beyond our ability to precisely identify the remnants of the nucleus after distortion of surrounding structures resulting from prolonged postoperative cicatrization.

THALAMIC AND TEGMENTAL MECHANISMS FOR Na INTAKE

999

the central grey matter. The lesions in the lateral group involved the lateral part of the ventral complex and the reticular and ventral lateral geniculate nuclei. A few were placed somewhat more caudally and included the medial geniculate nuclei. Note that dorsal, medial, and lateral lesions were centered some-what caudal to the level of the gustatory nucleus. Table 1 gives median increases in sodium chloride intake after formalin and D O C A treatments, and in water intake after formalin treatment for a mixed control group of 42 rats with no brain lesions, unilateral lesions, or small grossly asymmetrical lesions and for each of the 6 experimental groups with bilaterally symmetrical lesions. The increase scores represent the difference in intake from the day before to the day after treatments. Also shown are the number of rats in each group and the percentage which did not respond to the treatments by a significant increase in sodium intake (total increase in sodium not exceeding 1.0 mEq on both tests). KCI intake was not significantly affected by either treatment and water intake was not significantly affected by D O C A treatment in any of the groups and these scores are therefore omitted from the Table. Because of the presence of occasional extreme scores in some of the groups, a nonparametric median test was used to determine the significance of the differences between each experimental group and the control group. All groups learned to avoid the salt solutions within a few days, indicating that they could discriminate between the strong salt solutions and water. Those groups which manifested increases in sodium intake after the treatments demonstrated that they could also discriminate between the NaC1 and the KCI solutions since the increase was always specific to the NaCI solution. In the control group the median increase in sodium intake after formalin was 4.5 mEq and after D O C A was 6.0 mEq. Only about 10 per cent of the control rats failed to respond to both tests. Significant decrements in sodium intake in comparison to the control group occurred in the gustatory nucleus and caudal groups (see Table 1 for p values on various tests). In the gustatory nucleus group, 9 of the 13 rats did not respond to the treatments by a total

about 1 week later when fluid intake was again stable, the rats were injected with 5.0 mg of desoxycorticosterone acetate (DOCA) on 3 successive days to induce need-free sodium appetite [18]. Intake during the 24 hr period after the final D O C A injection was analyzed. RESULTS

Lesioned rats were divided into 6 groups depending on lesion placement. The center of the lesion sites was the region of the thalamic relay for taste (gustatory nucleus group) and the remaining lesions were either caudal, rostral, dorsal, medial, or lateral to this area. Lesions ventral to the gustatory area were not attempted because of the proximity of the lateral hypothalamus. Figure 1 shows projection drawings of sections near the centers of the lesions for individual rats in the gustatory nucleus group and in the caudal group, both of which manifested significant decrements on the sodium intake tests. Representative lesions are shown for the rostral, dorsal, medial, and lateral groups, which did not manifest significant decrements. In the gustatory nucleus group, the lesions damaged the medial portion of the ventral nuclear complex and portions of adjacent structures such as the centromedian and parafascicular nuclei. Some of the lesions were centered in the posterior part of the gustatory nucleus and included the anterior tip of the caudally adjacent reticular formation. The lesions in the caudal group varied somewhat in placement but always included the anterior part of the mesencephalic reticular formation. Other structures, such as the nucleus of the posterior commissure and the posterior thalamic nucleus, were also damaged by most of these lesions. Although there was some overlap in the areas damaged in this group and in the gustatory nucleus group, in no case did the caudal lesions cause bilateral damage to the gustatory relay. In the rostral group the lesions damaged the medioventral and mediodorsal nuclei and the anteromedial part of the ventral complex. The dorsal lesions involved the parafascicular and pretectal nuclei, and the medial protions of the lateral thalamic nuclei. The medial lesions were restricted almost entirely to

TABLE 1 CHANGESIN SODIUMCHLORIDEAND WATERINTAKEAFTERTREATMENTS

% Group

N

Median Increase in Na (mEq) After Formalin After DOCA

Control

42

4.5

6.5

Gustatory Nucleus

13

0 ( p < 0.05)*

0

( p < 0.01)

69.2 (p < 0.001)

Caudal

10

0 (p < 0.10)

0

(p < 0.01)

60.0 (p .< 0.01)

Rostral

10

5.5

5.0

Dorsal

6

4.0

Medial

8

3.5

Lateral

10

6.0

Non-responders Median Increase (total increase in H,O (ml) in Na < 1.1 mEq) After Formalin 9.5

14.0 15.0 8.5 (p < 0.02)

10.0

22.0

2.0

0

16.0

4.5

12.5

17.0

5.5

0

18.0

*p values represent significance of difference between lesioned group and control group by median and chi-square tests.

1000

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increase of more than 1.0 mEq in sodium intake, and in the caudal group 6 of the 10 rats did not respond. The remaining rats in these groups responded normally or somewhat sub-normally but, as can be seen in Fig. 1. which gives the total increase in sodium intake of the individual rats, there are no consistent differences in the lesions of responders and non-responders. The other lesioned groups did not differ from the control group on any of the measures, although there was some variability in the average magnitudes of the responses among these 4 groups. Only the group with caudal lesions manifested a significant decrement in water intake after formalin (p < 0.02 in comparison to controls). The group with rostral lesions appeared to ingest considerably more water, on the average, than the controls after formalin but variability was great and the difference was not statistically significant. DISCUSSION

There are great limitations to the amount and kind of anatomical information which can be gained by the lesion method. The method is useful primarily for initial orientation in the early stages of research when there is little information on the anatomical systems subserving a given function. Thus, the results of the present study serve to delineate brain regions which organize or transmit information which is important for normal regulation of sodium intake but they do not clarify the nature of that function, the degree to which it is specifically and directly related to sodium ingestion, or the manner in which it is related to other relevant brain functions. F o r example, although lesions which included the thalamic gustatory relay resulted in significant impairments on the sodium intake tests, it cannot be concluded that the impairment was due specifically to disruption of gustatory functions. First, it is possible that the decrement was due to destruction of nongustatory fibers passing through this area rather than to destruction of the cell bodies of the gustatory relay. Second, the lesions were not restricted to the disc-shaped gustatory nucleus but also involved surrounding structures. It is important to be aware that brain lesions contract greatly during prolonged postoperative periods. The rats of the present study were not sacrificed until about 3 months after lesioning, and it is certain that the initial area of destruction was considerably larger than it appears from the histological sections. Decrements in sodium intake also occurred when lesions were placed caudal to the gustatory relay. One difference between the effects of the tegmental and the thalamic lesions was that the tegmental lesions, like lateral hypothalamic lesions [24] resulted in decrements in water intake after formalin (hypovolemic thirst) while the thalamic lesions did not. There was also a tendency for tegmental lesions to produce a transient postoperative aphagia and adipsia while the thalamic lesions did not usually produce such an effect. F o u r of the 10 rats in the tegmental group (22T, 353, 390, and 402) suffered complete aphagia and adipsia of more than 1 week duration, while only 1 of the t 3 rats in the thalamic group (350) manifested aphagia and adipsia of more than 1 week duration. Unlike lateral hypothalamic lesions, which consistently produce decrements in sodium appetite, those in the thalamus and tegmentum were not always effective. There was no apparent relationship between the size and placement of these lesions and the occurrence of decrements. F o r example, in rat #546 the lesions were perfectly placed in the gustatory nucleus, but this animal had no deficit on the

sodium intake tests and, in filct, ingested excessive amounts after the treatments. Other rats with almost identical lesions manifested complete impairments. There was also no relationship between sodium intake and the duration of postoperative decrements in food and water intake in either of the groups. While we cannot yet account for the inconsistency of the effects of the thalamic and tegmental lesions, this fact must be taken into account in the formulation of hypotheses about the function of these areas. It seems likely that the thalamic and tegmental lesions involved the same, or closely related, functional systems. Lesions of the gustatory nucleus result in degeneration of axons which pass caudally into the anterior part of the reticular formation [25]. (However, there is no evidence that the cells of origin are actually in the gustatory nucleus3 We have been studying the ascending pathways from the mesencephalic tegmentum using the methods of Nauta [15] and of Fink and Heimer [5]. Lesions of the mesencephalic reticular formation produce widespread thalamic degeneration some of which passes through the region of the gustatory nucleus (Fig. 2). Sections stained by the Fink-Heimer method suggested some sparse termination of fibers in the gustatory nucleus among much more dense terminal fields in adjacent structures such as the parafasicular nucleus. These pathways provide a potentially relevant connection between the reticular formation and the ventromedial thalamus. Since lesions of the lateral hypothalamus, ventromedial thalamus, and mesencephalic reticular formation cause impairments of sodium intake regulation, a question arises as to how these regions might be related. There are apparently no direct connections between the lateral hypothalamus and the thalamic gustatory nucleus [10, 25, 29]. The lateral hypothalamus is known to be connected with the mesencephalic reticular formation via the medial forebrain bundle [10, 29], but when this connection is severed, sodium appetite as measured by the present tests is not disrupted [24]. There is another possible route through which the lateral area and the reticular formation may interact. In a previous study [29], we observed a pathway which entered the lateral hypothalamus through the sub-thalamus and which degenerated after large caudal diencephalic lesions which included the effective sites of the present experiment. We have now observed this pathway in 6 of 7 rats with lesions placed more caudally in the mesencephalic tegmentum. It runs rostrally through the prerubral fields and then turns ventrally into the lateral hypothalamus and entopeduncular nucleus {Fig. 2). At least some of the fibers continue rostrally to enter the globus pallidus. Sections stained by the Fink-Heimer method indicate termination in the lateral area, but this is somewhat confounded by the fact that some degenerating fibers of the medial forebrain bundle are intermingled with those entering from the subthalamus. However, further evidence for termination of this fiber system in the lateral hypothalamus is a progressive diminution in the density of the fibers as they course rostrally. With the possible exception of its hypothalamic component, the pathway follows closely the trajectory of the ansa lenticularis and may be considered, at least in part, as an ascending component of that fiber system [2]. Evidence for a hypothalamic trajectory of pallidofugal fibers in the rat is contradictory [t?f.. 14, 29]. In our previous studies [29], we did not observe degeneration in this system after lateral hypothalamic lesions, but the lesions were probably too medially or caudally placed to interrupt these fibers. It is important to note that our observations in the rat indicate that fibers in the trajectory

THALAMIC AND TEGMENTAL MECHANISMS FOR Na INTAKE

1001

00

J

G

H

I

FIG. 2. Ascending degeneration (depicted by dots) from a large tegmental lesion. Degeneration in the tectum and ventral supraoptic commissure is omitted. Thalamic degeneration is widespread but appears mainly in two areas. A dorsal group of fibers passes through the region of the internal medullary lamina entering the parafascicular and centromedian nuclei and the lateral part of the mediodorsal nucleus. A ventral group of fibers from the H-I field enters the medioventral nucleus and the anterior part of the ventral complex. Less dense degeneration is found scattered through the ventral nuclear complex including the region of the gustatory relay. Fibers cross to contralateral thalamic nuclei via the commissure above the rhomboid nucleus. Subthalamic degeneration passes through Forel's fields separating somewhat indistinctly into two groups. The dorsal group appears to disperse through the zona incerta. The ventral group enters the internal capsule and lateral hypothalamus. Capsular fibers continue rostratly into the entopeduncular nucleus and globus pallidus. In some cases, fibers were seen entering the striatum but this was not consistent. Degeneration passing rostrally from the midbrain in the medial forebrain bundle was relatively sparse. Elucidation of the origins of the above pathways in the rat awaits further study, but see Nauta and Kuypers [16] for a detailed study in the cat. of the ansa originate and terminate [see also, 3] over a widespread area of the tegmentum and are not all restricted to the region of the red nucleus. As suggested by Morgane [14], it is likely that the impairments in feeding behavior observed after far lateral hypothalamic lesions are at least partially attributable to damage to this fiber system. Further evidence that this system is important in feeding is that lesions along its trajectory in the globus pallidus [14], the entopeduncular nucleus [9], and the reticular formation [17] result in disruptions of feeding behavior. The mesencephalic reticular formation is in a position to integrate the various functions involved in alimentary behavior. In addition to projections from the hypothalamus, inflow from motor and sensory systems to the reticular system is well documented [e.g. 3, 6, 22]. Although the evidence

is indirect or incomplete, it seems highly probable that second order gustatory fibers from the solitary nucleus project heavily into the reticular formation at some level [1, 7, 13]. The only finding incompatible with the hypothesis that the reticular formation integrates the various functions involved in alimentary behavior is that the decrements in food, water, and salt intake [17 and present results] do not appear as severe and consistent as one might expect if the reticular formation plays such an important role. It might be that the area involved in the integrative process is quite extensive and that the lesions have only partially destroyed it. In any case, the behavioral and anatomical data thus far implicate the mesencephalic reticular formation in the control of alimentary functions and suggest further experiments exploring its relation to hypothalamic functions.

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