Behavioral comparison of the effectiveness of irritative and non-irritative lesions in producing hypothalamic hyperphagia

Behavioral comparison of the effectiveness of irritative and non-irritative lesions in producing hypothalamic hyperphagia

Physiology and Behavior. Vol. 3, pp. 417-420 Pergamon Press 1968. Printed in Great Britam Behavioral Comparison of the Effectiveness of Irritative an...

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Physiology and Behavior. Vol. 3, pp. 417-420 Pergamon Press 1968. Printed in Great Britam

Behavioral Comparison of the Effectiveness of Irritative and Non-lrritative Lesions in Producing Hypothalamic Hyperphagia' BERNARD

M. R A B I N 2 A N D C H A R L E S J. S M I T H

State University of New York, Buffalo, New York, U.S.A. (Received 31 August 1967)

RAaIN, B, M. ANDC. J. SMIxrt. Behavioralcomparison of the effectiveness of irritative and non-irritative lesions in producing hypothalamic hyperphagia. Privslot. BE,AV. 3(3) 417-420, 1968.--l_,¢sions were made in the ventromedial hypothalamus of rats using irritative (anodal electrolytic, stainless steel electrode) and non-irritative (cathodal electrolytic, stainless steel electrode; anodal electrolytic, platinum electrode; radio-frequency) techniques to assess the role of the deposition of metallic ions in the production of hypcrphagia. The results showed that while only 3 out of 35 rats in the non-irritative condition became hyperphagic, all animals in the irritative condition became hyperphagic. It was concluded that the deposition of metallic ions accompanying the production of a lesion with an irritative technique does play a significant role in the production of hyperphagia. Hypothalamus

Hyperphagia

Lesion techniques

REYNOLDS [7] reported that, unlike anodal d.c. stainless steel lesions, radio-frequency (r-f) lesions of the ventromedial hypothalamus (HVM) do not necessarily cause hyperphagia. These results directly contradict the earlier work which showed that hyperphagia consistently results from lesions of the HVM [13]. Reynolds [9] argued that the difference in results is due to the deposition of metallic ions that accompanies the destruction of tissue when anodal stainless steel or tungsten electrodes are used. In contrast, r-f power creates a lesion by the generation of heat in the tissue surrounding the electrode tip and does not, typically, involve the deposition of metallic ions at the electrode tip [1 ]. Also, there is a greater probability of hemorrhaging when a stainless steel anode is used, as compared to r-f power, with the resultant formation of scar tissue. Reynolds argued that the metallic ions and the scar tissue can serve as irritative foci which may act to stimulate the nearby lateral hypothalamic "feeding center" causing the observed increase in food intake, in much the same manner as does external stimulation of that center. Additional support for this interpretation comes from the finding that lesions of the ventrolateral hypothalamus consistently result in aphagia, whether anodal d.c. stainless steel or r-f lesion techniques are used [8]. This interpretation of the mechanisms producing hypothalamic hyperphagia has been questioned by Hoebel [4] who obtained hyperphagia in rats following r-f lesions of the HVM. Hoebel argued that these results demonstrate that increased

food intake consistently results from lesions of the HVM and, therefore, support the traditional view that this region is necessary for normal satiety. However, since the data that Hoebel [4] presented indicate that at least some of his experimental animals were not hyperphagic, the major question would seem to concern the efficacy of different lesion techniques in producing hyperphagia. If hyperphagia more consistently follows "irritative" lesions of the HVM, using anodal d.c. with a stainless steel electrode, this would support the view of Reynolds [9] that hyperphagia results from an irritative side effect of the lesion technique, rather than from the specific loss of tissue; and, therefore, that hyperphagia is not a necessary result of the removal of the HVM. If, however, there is no difference between "irritative" and "non-irritative" lesions with respect to. the frequency of hyperphagia, it might be that there is a wide range of overlap of weight and food intake increases between normal and operated animals. That is, the discrepant results between some studies may be due to the fact that a normal animal, at the high end of the normal distribution, may gain more weight than an animal with lesions of the HVM, at the low end of the experimental distribution. This would mean, therefore, that hyperphagia is not a discrete phenomenon but rather a continuous one; and, further, that the theories of hypothalamic regulation of food intake proposed by Stellar [12] and Teitlebaum [13] cannot be discounted on the basis of one discrepant study.

1This investigation was supported in part by Grant MH-6235 from the National Institute of Mental Health. sPresent Address: Istituto di Fisiologia, Universit/l di Pisa, 56100 Pisa (Italy). 417

418

RABIN AND SMIFH

The present study was designed to test the two alternatives by making lesions of the HVM using "irritative" and several "non-irritative" techniques and to compare the effectiveness of these techniques in producing hyperphagia. METHOD

The subjects were 69 male albino rats weighing 225-400 g at the time of surgery. These subjects were divided into six groups within three conditions. The first condition consisted of two control groups: an unoperated control and an operated control group that had an electrode passed through the HVM but with no current turned on. The "irritative" condition consisted of a single group in which anodal electrolytic lesions of the HVM were made with a stainless steel electrode. The "non-irritative" condition consisted of three groups in which the lesions were made using: (a) r-f power, (b) anodal d.c. with an electrode of 90 per cent platinum--10 per cent iridium, and (c) cathodal d.c. with a stainless steel electrode. All electrolytic lesions were made using a constant current d.c. lesion generator. The r-f lesions were made with a Grass LM-3 2 Mhz r-f lesion generator.

RESULTS

The results are summarized in Table I and Fig. 2. Only those animals in which histological examination showed a minimum of 75 per cent destruction of the HVM have been included. The results are presented in two forms: first, total postoperative weight gain, obtained by subtracting the preoperative weight from the animals' weight following surgery; and second, average weight gain per day (fl'om which the first three postoperative days have been excluded to make the data of all groups comparable). The data show that the total postoperative weight gain of 214 g for the anodal stainless steel group is considerably higher than for any other group. Also, there is basically no difference in total weight gain between the unoperated control (136 g), r-f (130 g) and platinum (135 g) groups. Figure 2 indicates the essential similarity of these groups with regard to average weight gain per day. The total postoperative weight gain for the operated control ~96 g) and cathodal stainless steel (68 g) groups were well below that of the other groups in their respective conditions. Table 1 indicates that there was no overlap in total postoperative weight gain between either of the control groups and

FIG. 1. Projection microscope drawings of coronal sections through the greatest extent of the lesions for representative animals. Lesions are shown in black. Abbreviations: HVM, ventromedial nucleus; HVL, ventrolateral hypothalamus; DMH, dorsomedial nucleus; FX, fornix; OT, optic tract. RT-20: anodal stainless steel lesions; lesion area of 5.9 sq. ram; weight gain of 186 g. RT-601 : cathodal stainless steel lesions; lesion area of 6.1 sq. ram; weight gain of 88 g. RT-235: platinum lesions; lesion area of 5.8 sq. ram; weight gain of 122 g. RT-271: platinum lesions; lesion area of 10.6 sq. ram; weight gain of 210 g. RT-446: r-f lesions; lesion area of 6.4 sq. mm ; weight gain of 200 g. RT-458 : r-f lesions; lesion area of 6.1 sq. ram; weight gain of 127 g.

The animals were weighed for 3 or 4 days prior to surgery to obtain their baseline weights. They were then anesthetized with Nembutal (40 mg/kg), placed in a stereotaxic instrument and the appropriate lesions made. Following surgery, the animals were weighed daily for 36 days and then sacrificed and their brains removed for histological examination. All animals were perfused with 10 per cent formalin saline. For those animals having lesions made with a stainless steel electrode, the perfusion solution also contained 1 per cent potassium ferrocyanide for Prussian blue staining of electrolytic iron deposits. The brains were fixed in formalin saline for several days and then frozen sections were cut at 40 I~ and stained with cresyl violet. The extent of each lesion was determined by using a polar planimeter.

the anodal stainless steel group. There was, however, some overlap between the r-f and platinum groups and the anodal stainless steel group. One platinum animal and two r-f animals gained more weight than the lowest animals in the anodal stainless steel group. The data on the extent of the lesions (defined as the area showing evacuation and/or giiosis) presented in Table 1 show that there is little difference between the anodal stainless steel, r-f, and platinum groups, while that of the cathodal stainless steel group was the smallest of all experimental conditions. With the exception of the platinum group, the rank order correlations between size of lesion and total postoperative weight gain were very low. While there was some variability in the anterior-posterior extent of the lesions, this did not

COMPARISON OF IRRITATIVE AND NON-1RRITATIVE LESIONS

l°° r

be attributed to differences in the extent of the lesions because this was similar for all groups and because the rank order correlations between size of lesion and total weight change were, for the most part, relatively small. While the extent of the lesions for the cathodal stainless steel group was somewhat smaller than for the other groups, it should be noted that a minimum of 75 per cent of the HVM was destroyed in each case and that there was only a small and negative correlation between total weight gain and size of lesion. These results also agree in part with those obtained by Hoebel [4] in showing that it is possible to obtain hyperphagia using non-irritative lesion techniques. However, since only 3 out of 35 animals in the non-irritative condition became hyperphagic, it would be difficult to argue that the specific loss of tissue following lesions of the HVM is the primary cause of hyperphagia. Pool [6] has also reported results which indicate that removal of the HVM is not a sufficient condition for the occurrence of hyperphagia. He found that while suction lesions of the HVM in female rats can cause hyperphagia, they do not reliably do so. These results do, however, indicate that the deposition of metallic ions is not a necessary condition for the production of hyperphagia and suggest that other factors may also be involved. There are several possible reasons for the occurrence of hyperphagia among the animals in the non-irritative condition in the present study. First, with r-f lesions, there may be

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TABLE 1 SUMMARY OF RESULTS

Group

n

Control Condition Unoperated Operated lrritative Condition Anodal Elect. Non-Irritative Condition Radio-Frequency Platinum Cathodal Elect.

Mean Weight Change/Day*

Range*

Total Weight Gain*

Range*

Mean Lesion Area (ram 2)

Range (ram 2)

Correlation**

9 15

3.4 3.8

3.1-4.7 1.7-4.6

136 96

112-172 36-152

10

7.1

5.7-8.8

214

186-268

8.2

5.6-10.1

0.26

16 11 8

3.7 4.3 2.7

2.0--5.6 2.1-6.2 1.8-3.6

130 135 68

64-201 69-210 39-98

8.6 7.2 5.9

4.5-15.4 5.0-10.6 3.8- 9.7

0.14 0.53 --0.10

*Grams **Rank order correlation between total weight gain and size of lesion.

appear to be related to weight gain. Further, it was not possible to distinguish between those animals which became hyperphagic and those which did not on the basis of the reconstructions of the lesions. DISCUSSION

The results tend to support the irritative hypothesis of the hypothalamic regulation of food intake proposed by Reynolds [9]. The fact that hyperphagia consistently followed irritative lesions and that it only rarely followed non-irritative lesions of the HVM supports the contention that the deposition of metallic ions with a stainless steel anode does play a significant role in the production of hyperphagia. This interpretation is further supported by the fact that the differences in weight gain between irritative and non-irritative conditions cannot

some rectification of current in the brain, changing the r-f power to a direct current. A second alternative is that irritative foci may be established by the operation of factors other than the deposition of metallic ions. Reynolds [9] has argued that the increased incidence of hemorrhage which occurs with the use of an electrolytic technique is an important factor. However, since cathodal electrolytic lesions cause a greater degree of hemorrhage than anodal lesions [5], the failure of any of the cathodal stainless steel group to become hyperphagic would seem to indicate that hemorrhage does not play a significant role in setting up irritative foci. Rather, the data seem to indicate that hemorrhage may be a purely disruptive factor since the cathodal stainless steel group required approximately 15 days to return to their preoperative base weight while the other operated groups reached this level within less than six days.

420

RABIN AND SMITH

While there is general agreement that tissue destruction with a cathodal electrolytic lesion occurs by the liberation of hydrogen gas at the electrode tip, the mechanism of tissue destruction with anodal d.c. lesions is not well understood [10]. Reynolds [9] has proposed that the deposition of ferric ions, when a stainless steel anode is used, is the primary cause of such destruction. However, this creates a problem in that if the ferric ions were responsible for the destruction of tissue, the cells suffering an invasion of ions could not serve as irritative foci since this latter function requires cells that are still firing, albeit abnormally. An alternative explanation is that the primary destruction of tissue from an anodal electrolytic lesion is due to the electrolysis of sodium chloride and therefore to the liberation of chlorine at the anode. For the purposes of electrochemical analysis the simplifying assumption may be made that the brain is composed primarily of an ultrafiltrate of blood of which the principle components are sodium and chlorine ions [3]. When current is passed through this electrolyte, several alternative reactions are possible at the anode. The reaction that will occur is the one that requires the least expenditure of energy [11]. If a stainless steel anode is used, the possible reactions are the release of ferric ions from the anode, the liberation of oxygen and the liberation of chlorine. Since the release of ferric ions requires the least expenditure of energy, this is the reaction that will occur.

However, it is possible for any number of reactions to occur simultaneously if the electrical potential for these reactions is reached [2]. While oxygen is usually evolved at the anode, when the electrolysis involves a chlorine solution, chlorine is also liberated at the anode. This reaction involving the liberation of chlorine results from the use of both stainless steel and platinum anodes [2]. A simple qualitative experiment is available to test these theoretical considerations. This involves passing current through an electrode immersed in a solution of isotonic saline at a voltage equivalent to that used in creating a lesion in the brain. At a constant current of 2 mA for making a lesion, the voltage varies between 5 and 13 V d.c. Passing 5 V through a stainless steel anode immersed in saline resulted in the liberation of enough gaseous chlorine for the smell to be noticeable, as well as in the release of some chlorine combined with ferric ions, probably as ferric chloride. When a platinum anode is used, there is also the liberation of chlorine at the electrode tip, but because of the extreme stability of the metal there is no release of metallic ions. Thus, it would seem that tissue destruction in anodal electrolytic lesions is caused by the liberation of chlorine at the electrode tip while the deposition of ferric ions, accompanying the use of a stainless steel electrode, serves to set up the irritative foci which are the primary cause of hyperphagia following lesions of the HVM.

REFERENCES

1. Aronow, S. The use of radio-frequency power in making lesions in the brain. J. Neurosurg. 17: 431--438, 1960. 2. Delahay, P. Instrumental Analysis. New York: Macmillan Co., 1957. 3. Eccles, J. C. The Neurophysiological Basis of Mind. London: Oxford University Press, 1953. 4. Hoebel, B. G. Hypothalamic lesions by electrocauterization: Disinhibition of feeding and self stimulation. Science 149: 452-453, 1965. 5. Mullan S., M. Mailis, J. Karasick, G. Vailati, and F. Beckman. A reappraisal of unipolar anodal electrolytic lesions. J. Neurosurg. 22: 531-538, 1965. 6. Pool, R. Suction lesions and hypothalamic hyperphagia. Am. J. Physiol. 213: 31-35, 1967. 7. Reynolds, R. W. Ventromedial hypothalamic lesions without hyperphagia. Am. J. Physiol. 204: 60-62, 1963. 8. Reynolds, R. W. Radio-frequency lesions in the ventrolateral bypothalamic "feeding center". J. comp. physiol. Psychol. 56: 965-967, 1963.

9. Reynolds, R. W. An irritative hypothesis concerning the hypothalamic regulation of food intake. PsychoL Rev. 72: 105-116, 1965. 10. Rowland, V., W. J. Maclntyre and T. G. Bidder. The production of brain lesions with electric currents: II. Bidirectional currents. J. Neurosurg. 17: 55-69, 1960. I 1. Sisler, H. H., C. A. Vander Werf and A. W. Davidson. General Chemistry. New York: Macmillan Co., 1959. 12. Stellar, E. Drive and motivation. In: Handbook of Physiology, Sec. 1, Neurophysiology, Vol. 3, edited by J. Field. Washington, D.C. : American Physiological Society, 1960, pp. 1501-1528. 13. Teitlebaum, P. Disturbances in feeding and drinking behavior after hypothalamic lesions. In: Nebraska Symposium onMotivation, edited by M. R. Jones. Lincoln, Nebraska: University of Nebraska Press, 1961, pp. 39-65.