sympathetic activity following paraventricular or ventromedial hypothalamic lesions in rats

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Brain Research Bulletin, Vol. 20, pp. 461-465. 0 Pergamon Press plc, 1988. Printed in the U.S.A. 0361-9230188 $3.00 + .OO Sympathetic Activity Following Paraventricular or Ventromedial Hypothalamic Lesions in Rats’ T. SAKAGUCHI, G. A. BRAY’ AND G. EDDLESTONE Department of Medicine, University of Southern California Section of Diabetes and Clinical Nutrition, LA CountylUSC Medical Center, Los Angeles, CA Received 12 January 1987 SAKAGUCHI, T., G. A. BRAY AND G. EDDLESTONE. Sympathetic activity following paraventricular or ventrome- dial hypothalamic lesions in rats. BRAIN RES BULL 20(4) 461-465, 1988.-The present experiment was designed to measure the sympathetic firing rate of nerve filaments to interscapular brown adipose tissue in rats with acute and chronic lesions in the ventromedial hypothalamus or paraventricular nucleus. Female rats received injections of kainic acid into either the paraventricular nucleus or the ventromedial hypothalamus. The sympathetic activity was measured by recording the firing rate of nerve filaments to interscapular brown adipose tissue after mounting the filaments on silver wire electrodes connected to an amplifier and rate meter. In acute experiments performed 30 minutes after the lesion, the basal firing rate was reduced in both groups and was significantly lower in the VMH-lesioned rats than in those with PVN lesions. In the chronic experiment kainic acid was injected into the VMH or PVN and the sympathetic tiring rate was measured 7 to 9 days later. Both lesioned groups gained more weight than controls, but the VMH-lesioned rats gained more weight than the PVN-lesioned rats. The basal firing rate of sympathetic nerve filaments in the VMH-lesioned rats was lower than in the other groups. There was no significant difference in the sympathetic activity between PVN-lesioned and control rats. These experiments support the hypothesis that lesions of the ventromedial hypothalamus reduce the sympathetic activity but that lesions in the paraventricular nucleus do not. Kainic acid Obesity Brown adipose tissue OBESITY following damage to the ventromedial hypothal- amus (VMH) has been known for more than eighty years [2,7, lo]. The experimental syndrome is associated with hyper- phagia and a variety of metabolic disturbances which can be largely attributed to alterations in the function of the au- tonomic nervous system and the resulting hyperinsulinemia [7,13]. There is clear evidence for enhanced activity of the vagus nerve [3, 8, 141 which plays a major role in the hyperinsulinemia of this syndrome [5]. There is also evi- dence of reduced activity of the sympathetic nervous system following ventromedial hypothalamic lesions, but this data is less clear. The impaired mobilization of free fatty acids fol- lowing exposure to stressful conditions was among the first data to suggest that sympathetic activity was reduced in the hypothalamic-lesioned rats [ 181. The sympathetically inner- vated brown adipose tissue from VMH-lesioned rats is enlarged with triglyceride and shows an impaired response to electrical stimulation [21]. Norepinephrine turnover in brown adipose tissue is another index of activity of the sym- pathetic nervous system. In weanling rats norepinephrine turnover is decreased [24], but in adult animals norepineph- rine turnover is not slowed and may be increased [25,27,30]. Recently, direct measurements of sympathetic firing rate to brown adipose tissue [ 17,201 and to the splanchnic nerve [29] have shown a reduction in the firing rate of sympathetic nerves following ventromedial hypothalamic lesions. The possibility that there might be two syndromes of hy- pothalamic obesity was suggested in studies by Bray, Scla- fani and Novin [6]. They compared animals with parasagittal knife cuts and electrolytic hypothalamic lesions and found a dissociation between insulin levels and fatty acid mobiliza- tion. Studies by Leibowitz et al. [ 161, by Aravich and Scla- fani [ 11, by Weingarten [26], by Tokunaga et al. [22], and by Fukushima et al. [ 111 have shown that hypothalamic obesity can be produced by lesions in the paraventricular nucleus. Animals with PVN lesions do not show the enhanced activity of the vagus that was present in the VMH-lesioned rats [23]. Data on the effect of PVN lesions on sympathetic activity are contradictory. We have failed to demonstrate any change in norepinephrine turnover following PVN lesions which produce obesity [22,27], just as we had found no effect after VMN lesions. Yoshimatsu et al. [29], on the other hand, have found a decrease in sympathetic activity in the splanch- nit nerve. The present study was designed to test the ef- ‘This work was supported in part by grants DK 31988 and DK 32018 from the National Institutes of Health. Qequests for reprints should be addressed to George A. Bray, M.D., Section of Diabetes and Clinical Nutrition, University of Southern California, 2025 Zonal Avenue, Los Angeles, CA 90033. 461

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Page 1: Sympathetic activity following paraventricular or ventromedial hypothalamic lesions in rats

Brain Research Bulletin, Vol. 20, pp. 461-465. 0 Pergamon Press plc, 1988. Printed in the U.S.A. 0361-9230188 $3.00 + .OO

Sympathetic Activity Following Paraventricular or Ventromedial Hypothalamic Lesions in Rats’

T. SAKAGUCHI, G. A. BRAY’ AND G. EDDLESTONE

Department of Medicine, University of Southern California Section of Diabetes and Clinical Nutrition, LA CountylUSC Medical Center, Los Angeles, CA

Received 12 January 1987

SAKAGUCHI, T., G. A. BRAY AND G. EDDLESTONE. Sympathetic activity following paraventricular or ventrome- dial hypothalamic lesions in rats. BRAIN RES BULL 20(4) 461-465, 1988.-The present experiment was designed to measure the sympathetic firing rate of nerve filaments to interscapular brown adipose tissue in rats with acute and chronic lesions in the ventromedial hypothalamus or paraventricular nucleus. Female rats received injections of kainic acid into either the paraventricular nucleus or the ventromedial hypothalamus. The sympathetic activity was measured by recording the firing rate of nerve filaments to interscapular brown adipose tissue after mounting the filaments on silver wire electrodes connected to an amplifier and rate meter. In acute experiments performed 30 minutes after the lesion, the basal firing rate was reduced in both groups and was significantly lower in the VMH-lesioned rats than in those with PVN lesions. In the chronic experiment kainic acid was injected into the VMH or PVN and the sympathetic tiring rate was measured 7 to 9 days later. Both lesioned groups gained more weight than controls, but the VMH-lesioned rats gained more weight than the PVN-lesioned rats. The basal firing rate of sympathetic nerve filaments in the VMH-lesioned rats was lower than in the other groups. There was no significant difference in the sympathetic activity between PVN-lesioned and control rats. These experiments support the hypothesis that lesions of the ventromedial hypothalamus reduce the sympathetic activity but that lesions in the paraventricular nucleus do not.

Kainic acid Obesity Brown adipose tissue

OBESITY following damage to the ventromedial hypothal- amus (VMH) has been known for more than eighty years [2,7, lo]. The experimental syndrome is associated with hyper- phagia and a variety of metabolic disturbances which can be largely attributed to alterations in the function of the au- tonomic nervous system and the resulting hyperinsulinemia [7,13]. There is clear evidence for enhanced activity of the vagus nerve [3, 8, 141 which plays a major role in the hyperinsulinemia of this syndrome [5]. There is also evi- dence of reduced activity of the sympathetic nervous system following ventromedial hypothalamic lesions, but this data is less clear. The impaired mobilization of free fatty acids fol- lowing exposure to stressful conditions was among the first data to suggest that sympathetic activity was reduced in the hypothalamic-lesioned rats [ 181. The sympathetically inner- vated brown adipose tissue from VMH-lesioned rats is enlarged with triglyceride and shows an impaired response to electrical stimulation [21]. Norepinephrine turnover in brown adipose tissue is another index of activity of the sym- pathetic nervous system. In weanling rats norepinephrine turnover is decreased [24], but in adult animals norepineph- rine turnover is not slowed and may be increased [25,27,30].

Recently, direct measurements of sympathetic firing rate to brown adipose tissue [ 17,201 and to the splanchnic nerve [29] have shown a reduction in the firing rate of sympathetic nerves following ventromedial hypothalamic lesions.

The possibility that there might be two syndromes of hy- pothalamic obesity was suggested in studies by Bray, Scla- fani and Novin [6]. They compared animals with parasagittal knife cuts and electrolytic hypothalamic lesions and found a dissociation between insulin levels and fatty acid mobiliza- tion. Studies by Leibowitz et al. [ 161, by Aravich and Scla- fani [ 11, by Weingarten [26], by Tokunaga et al. [22], and by Fukushima et al. [ 111 have shown that hypothalamic obesity can be produced by lesions in the paraventricular nucleus. Animals with PVN lesions do not show the enhanced activity of the vagus that was present in the VMH-lesioned rats [23]. Data on the effect of PVN lesions on sympathetic activity are contradictory. We have failed to demonstrate any change in norepinephrine turnover following PVN lesions which produce obesity [22,27], just as we had found no effect after VMN lesions. Yoshimatsu et al. [29], on the other hand, have found a decrease in sympathetic activity in the splanch- nit nerve. The present study was designed to test the ef-

‘This work was supported in part by grants DK 31988 and DK 32018 from the National Institutes of Health. Qequests for reprints should be addressed to George A. Bray, M.D., Section of Diabetes and Clinical Nutrition, University of Southern

California, 2025 Zonal Avenue, Los Angeles, CA 90033.

461

Page 2: Sympathetic activity following paraventricular or ventromedial hypothalamic lesions in rats

462 SAKAGUCHI. BRAY AND F,DDLES’I-ONlf

60

ACUTE EFFECT OF KAINIC ACID

VMH

I I I I 7 I I I

-5 0 5 10 15 20 25 30 Time (min)

FIG. 1. Effect of acute injections of kainic acid on sympathetic firing rate. Animals were mounted in a stereotaxic apparatus and prepared for nerve recording. After obtaining basal readings kainic acid was injected at 0 time and the basal recording measured at 5 minute intervals for 30 minutes. The initial rise following the acute injec- tions and subsequent fall is visible for lesions in both areas. The saline injected animals (open symbols) showed no changes. The PVN-lesioned animals showed a small but not statistically signiti- cant reduction below baseline, while there was a significant reduc- tion in the firing rate in the VMH-lesioned animals. Data are mean+SEM (N=12 animals in each group).

feet of VMN and PVN lesions on the firing rate of the sympa- thetic nerves to brown adipose tissue following acute and chronic hypothalamic lesions.

METHOD

Animals

The 60 female rats used in these experiments were pur- chased from Harlan Industries (Madison, WI). The animals were received weighing approximately 200 to 240 g, and were maintained on Wayne Lablox (Chicago, IL) with tap water available ad lib. The vivarium in which they were housed was maintained at 22-c 1 degrees C with illumination from 0600 to 1800 hr.

Procedures

Hypothalamic lesions. Animals were anesthetized with pentobarbital (45 mg/kg) and mounted in a stereotaxic appa- ratus (Kopf Instrument Co., Tujunga, CA). Burr holes were placed in the cranium at sites which would allow introduc- tion of 28 gauge stainless steel cannulae into the paraven- tricular or ventromedial hypothalamic nucleus using coordi- nates from the atlas of Pellegrino et al. [ 191. Before lowering the cannula into position, the needle and attached No. 50 P.E. Tubing were filled with a 1.0 mM solution of kainic acid dissolved 0.15 M NaCl. One ~1 of the solution was infused over 2.5 min. The twenty-eight gauge cannula was allowed to remain in place for an additional two minutes. The control animals received 1 .O ~1 of 0.154 M NaCl.

Nerve recording. Animals were either studied im- mediately after injection of kainic acid (12 PVN-injected, 12 VMH-injected and 12 sham-injected controls) while under pentobarbital anesthesia, or 7 to 9 days following the injec-

PVN VMH

FIG. 2. Weight gain following injection of kainic acid. The control animals received saline injections and the lesioned animals received I nmole of kainic acid in the PVN (N=6) or VMH (N=6). Weight gain over the seven to nine days following lesion was significantly greater in both lesioned groups, and greater in the VMH-lesioned than in the PVN-lesioned rats. Data are mean?SEM for 6 rats per group.

tion of kainic acid (12 injected and 12 sham-injected con- trols). These latter rats were reanesthetized with pentobarbi- tal 45 mg/kg IP. The depth of anesthesia was maintained by injecting pentobarbital7.5 mgikg at 30 minute intervals. The interscapular brown adipose tissue was exposed through a mid-line incision, the right lobe gently retracted upward, and the five sympathetic nerve bundles were identified. To re- cord efferent nerve impulses one of the bundles was sec- tioned as close as possible to the interscapular brown adipose tissue (IBAT). The proximal segment of one bundle was microdissected longitudinally into several filaments, and a single filament was placed on a pair of silver wire elec- trodes and immersed in a mixture of liquid paraffin and petroleum jelly to prevent dehydration. Spontaneous effer- ent nerve activity from this single nerve filament was amplified through a differential amplifier and displayed on an oscilloscope. The spikes from the single nerve filament were converted to standard pulses through a window dis- criminator. A rate meter with a reset time of 5 seconds was used to observe the time course of nerve activity as dis- played on the oscilloscope. The output of the rate meter was also recorded on a 1 mV strip chart recorder. At the end of the experiment the animals were decapitated and blood col- lected in chilled tubes for measurement of glucose and insu- lin. The brains were removed and placed in buffered formalin for later identification of the lesioned areas.

Histology

The partially fixed brains were frozen on a freezing mi- crotome and coronal sections were cut at 25 PM distances. These sections were mounted individually on glass slides and stained with 1~x01 fast blue and cresyl violet. The extent of lesions in the kainic acid treated rats was established and a composite made using the sections from Pellegrino’s atlas.

Reagents and Analytical Methods

Glucose was determined with a glucose analyzer (Beckman Instruments, Fullerton, CA). Insulin was deter- mined in a double antibody assay using rat insulin standard (Nova Pharmaceutical, Copenhagen, Denmark).

Page 3: Sympathetic activity following paraventricular or ventromedial hypothalamic lesions in rats

SYMPATHETIC ACTIVITY AND HYPOTHALAMIC LESIONS 463

TABLE 1

EFFECT OF HYPOTHALAMIC LESIONS ON GLUCOSE, INSULIN AND ORGAN WEIGHTS

Sham-injection PVN-kainic acid

Sham-injection VMH-kainic acid

Glucose (mg/dl)

152.5 * 11.7 242.8 + 3.8”

150.2 2 3.2 205.1 ? 3.7a

Insulin (ng/mB

1.2 r 0.3 3.9 * 0.2s

1.3 f 0.1 4.8 f 0.5b

Naso-Anal Length (mm)

207.7 f 0.1 207.2 f 0.1

209.3 ? 0.2 208.6 ? 0.3

Liver (g)

8.9 * 0.2 9.4 2 0.3

8.6 2 0.2 9.9 2 0.3

Retroperitoneal Fat (mg)

2746 ? 99 3427 f 146”

2664 f lllb 4887 -+ 279’

Brown Fat (mg)

474 2 9.5 484 + 11.2

494 & 8.4 515 & 12.6

“p<O.OS PVN vs. Sham. bpcO.05 VMH Kainic acid vs. Sham. Cp<O.O5 VMH Kainic acid vs. PVH Kainic acid.

Statistics

Data were compared using unpaired t-tests between sham and experimental groups.

RESULTS

Acute Experiment

The reduction in firing rate after the acute injection of kainic acid into the VMH is significantly greater than when kainic acid was injected into the PVN (p<O.O5). The time course for these effects is shown in Fig. 1. The baseline integrated sympathetic activity was just above 40 spikes/5 seconds and was constant over the five minutes prior to injection of kainic acid. Following kainic acid, there was an initial acute increase in firing rate when injected into either the paraventricular nucleus or the ventromedial hypothala- mus. This initial rise in firing rate had dropped below baseline by 10 minutes and declined slowly over the ensuing 30 minutes of the study. The VMH-lesioned animals had significantly lower firing rates (p<O.O5), averaging 23 spikes/5 seconds 30 minutes after injecting kainic acid com- pared with a small but not statistically significant reduction in tiring rate of animals receiving kainic acid injections into the paraventricular nucleus.

Chronic Experiment

During the 7 to 9 days between kainic acid injections and the time of chronic study, weight gain in the control animals averaged 6* 1 g. In the PVN-lesioned animals, weight gain was 19+2.5 g, which was significantly greater than in the saline-injected controls (Fig. 2). For the VMH-lesioned animals weight gain was 42+3.5 g, which was more than twice that for the PVN-lesioned animals (p<O.OS). These differences in weight gain were reflected in the weight of several organs (Table 1). The white adipose tissue was signif- icantly heavier in both lesioned groups (p<O.O05), but the liver weight and weight of brown adipose tissue were not different. The concentrations of glucose and insulin were also significantly higher (p<O.OOS) in the two lesioned groups compared with the control animals.

The basal sympathetic f&g rate for the chronically treated animals is shown in Fig. 3. The firing rate for the two groups of sham-operated animals was similar and ranged be- tween 36 and 39 spikes/5 sec. The animals lesioned with kainic acid in the paraventricular nucleus showed no signifl- cant difference in basal tiring rate from that observed in

1 0 Sham

m Kainic Acid

i f’\IN

L

FIG. 3. Effect of kainic acid on basal fling rate of sympathetic nerves to brown adipose tissue. The basal fling rate of seven to nine days following the injection of saline or 1 nmole of kainic acid into the paraventricular or ventromedial hypothalamus is shown. The VMH-lesioned animals showed a significant reduction in firing rate compared to either saline treated or animals with kainic acid lesions in the PVN. Data are meanltSEM (N=6 animals per group). a=p<0.05 compared to Sham-VMH.

either of the saline treated groups. However, the animals lesioned with kainic acid in the ventromedial hypothalamus showed a significant reduction in firing rate (p<O.OOl).

Histological mapping of the composite lesions produced by kainic acid in the PVN and VMH is shown in Fig. 4a and b. The lesions are superimposed on top of drawings from the atlas of Pellegrino et al. 1191. It is clear that the lesions in the PVN (Fig. 4a) destroyed most of the nucleus, but did not damage the VMH. In the animals with injections into the VMH, this region was destroyed but other regions were largely spared (Fig. 4b).

DISCUSSION

The present study has demonstrated that kainic acid mjec- tions into the paraventricular nucleus (PVN) and ventrome- dial hypothalamus (VMH) can produce weight gain. These studies have also shown that, following an acute lesion in the ventromedial hypothalamus, the basal firing rate of the sym- pathetic nerves to interscapular brown adipose tissue is lower than in animals with lesions in the paraventricular nu- cleus. After recovery from the acute lesion the firing rate of

Page 4: Sympathetic activity following paraventricular or ventromedial hypothalamic lesions in rats

464 SAKAGUCHI. BRAY AND EDDLES’I-ONF.

a PVN - ILESION b VMH - LESION

FIG. 4. Histology of lesioned animais. Using the atlas of Pellegrino, a composite diagram of the areas destroyed have been blocked into the anatomic regions for PVN-lesioned animals (Fig. 4af on the left and VMH-lesioned animals in the right (Fig. 4b).

the sympathetic nervous system returns completely to nor- mal in the PVN-lesioned rats, but it remains signi~~antly reduced in the VMH-lesioned animals.

There is now abundant evidence that lesions in the paraventricular nucleus of rodents [ 11, 16,221 as well as dogs [ 121 will produce obesity. The magnitude of weight gain fol- lowing a PVN lesion is a function of the percentage of the paraventricular nucleus which is damaged, providing that regions outside the paraventricular nucleus are not simulta- neously injured [ 11,221. However, damage of 75% or more of the PVN does not produce as much weight gain as is ob- served in rats with the ventromedial hypothalamic lesions [11,22]. The present studies are consistent with these other studies in showing that animals with the PVN lesions gain signi~cantly less weight than animals with VMH lesions. The data of Weingarten et al. 1261 suggest that the syndrome of obesity following PVN lesions results from hyperphagia and that, if the hyperphagia is prevented, obesity does not occur.

Alterations in the function of the autonomic nervous sys- tem are a characteristic feature of the syndrome of ven- tromedial hypothalamic obesity [7]. These alterations are characterized by increased activity of the vagus nerve [3, 8, 151, which plays a major role in the hyperinsulinemia [5]. There is also a reduction in some parameters of sympathetic nervous system activity [14, 17, 18,2I, 25,291. The observa- tion of small salivary glands in animals with ventromedial hypothalamic lesions was the first to suggest reduced sympa- thetic activity [14]. It has been known for many years that the size of salivary glands is under sympathetic control and that they involute following sympathectomy [41. Subsequent studies on glucagon secretion [13], on the mobilization of

free fatty acids /18] and the impaired stimulation of ther- mogenesis in brown adipose tissue [21] are all consistent with the hypothesis of impaired sympathetic function follow- ing lesions in the ventromedial hypothalamus. Reduced nor- epinephrine turnover in weanling rats with VMH lesions which do not develop hype~hagia is also consistent with this hypothesis. However, norepinephrine turnover in IBAT of adult rats and mice with VMH lesions and hyperphagia is normal or increased [25,27, 301. The explanation of this later discrepancy is not at present clear. However, the prepon- derance of data currently support the reduction in sympa- thetic activity following VMH lesions. Direct measurements of reduced efferent firing rate to the sympathetic nerves in rats with VMH lesions has strongly buttressed this hypoth- esis [17, 20, 29). We have recently reported that VMH le- sions produced by kainic acid reduced firing rate of the sym- pathetic nervous system in rats [20]. The present studies are in harmony with these earlier reports, and document a re- duction in basal firing rate of the sympathetic nerves to brown adipose tissue in rats following acute injection of kainic acid into the ventromedial hypothalamus and in rats which have developed chronic lesions from this injection.

Kainic acid was used to produce hypothalamic lesions in this experiment because this drug has been reported to selectively damage neurons while sparing fibers of passage [9]. In the present studies we have failed to find a reduction in basal firing rate following kainic acid induced lesions in the paraventricular nucleus, even though obesity developed. This is consistent with a previous report from our laboratory showing that norepinephrine turnover in brown adipose tis- sue was not affected in animals with lesions in the paraven-

Page 5: Sympathetic activity following paraventricular or ventromedial hypothalamic lesions in rats

SYMPATHETIC ACTIVITY AND HYPOTHALAMIC LESIONS 465

tricular nucleus which became obese [27], although these studies failed to show a decrease in NE turnover after VMH lesions. The failure to modify basal sympathetic activity fol- lowing acute or chronic injection of kainic acid into the PVN is at variance with the data of Yoshimatsu et al. [29]. They found that electrolytic lesions in the PVN increased sympa- thetic firing in the splanchnic nerve in 4 of 6 rats and in 12 of 19 animals with VMH lesions. One possible explanation for this difference is the method of lesioning. The electrolytic lesions used by Yoshimatsu et al. [29] may have produced some damage to fibers to or from the VMN. Kainic acid lesions, used in our studies, will damage primarily cell bodies. In addition, the use of the splanchnic nerve versus sympathetic nerves to IBAT might help account for the difference. The present experiments provide further evi-

dence for two syndromes of hypothalamic obesity [6]. Dam- age to the ventromedial hypothalamus increases food intake, although this is not essential for the development of obestiy [8]. Hyperphagia, however, appears to be essential for de- velopment of obesity after PVN lesions. Hyperinsulinemia without hyperglycemia occurs after VMH lesions [3,23], but with PVN lesions, the hyperinsulinemia appears to be sec- ondary to the hyperglycemia [23]. Finally, sympathetic ac- tivity is reduced after VMH lesions [ 17, 20, 291 but not con- sistently after PVN lesions. We would thus tentatively con- clude that lesions in the ventromedial hypothalamus are more likely to be associated with disturbances in both com- ponents of the autonomic nervous system whereas lesions in the paraventricular nucleus may not disturb either the sym- pathetic or vagal systems.

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