Reduced salivation in rats following ventromedial hypothalamic lesions

Physiology & Behavior, Vol. 24, pp. 451--455.Pergamon Press and Brain Research Publ., 1980. Printed in the U.S.A.

Reduced Salivation in Rats Following Ventromedial Hypothalamic Lesions I FRANCIS W. FLYNN,

D A V I D L . S C H I R E R A N D J A M E S C. M I T C H E L L z

Department o f Psychology, Kansas State University, Manhattan, K S 66506 R e c e i v e d 19 S e p t e m b e r 1979 FLYNN, F. W., D. L. SCHIRER AND J. C. MITCHELL. Reduced salivation in rats following ventromedial hypothalamic lesions. PHYSIOL. BEHAV. 24(3) 451--455, 1980.--Salivation was assessed in normal rats and rats with bilateral lesions of the ventromedial hypothalamus (VMH). VMH lesioned rats demonstrated a reliable reduction of salivation. This hyposalivation occurred during a one month period that animals were maintained on dry rat chow and for a 16 day period that animals were maintained on a palatable liquid diet. VMH rats did not differ from control rats in the amount of saliva secreted in response to Prostigmin or wintergreen solution. Thus, while the VMH rat shows a reduced basal salivation level, such animals salivate normally in response to a strong taste substance. Ventromedial hypothalamus

Salivation

VMH syndrome

R E C E N T L Y , Powley [15] proposed a "Cephalic Phase Hypothesis" to account for the effects o f ventromedial hypothalamic (VMH) lesions. This hypothesis suggests that V M H lesions result in the modification of cranial nerve reflexes to appetitive stimuli. These cephalic reflexes, such as insulin secretion, gastric acid secretion, and salivation, occur in the intact animal but following VMH damage the amplitude o f these responses is exaggerated. Powley proposed that disinhibition of cephalic responses is responsible for the V M H syndrome. Following lesions of the V M H in dogs, there are changes in classical salivary conditioned reflexes [18]. Dogs showed differing salivary responses to a stimulus that was paired with food ( C S + ) and a stimulus that was paired with no food ( C S - ) . Lesions of the V M H did not change the salivary response to the C S + or unconditioned stimulus. However, salivation to the C S - and during the intertrial interval increased. In contrast, dogs with lateral hypothalamic (LH) lesions showed a reduction in salivation [19]. The L H rat displays prandial drinking similar to the totally desalivate animal suggesting that L H lesions interrupt salivary function [4, 10, 11, 12, 25]. However, direct measurement of saliva secretion in rats following L H lesions demonstrated a short period o f hypersalivation followed by a reduction in salivation to below normal levels [20]. The present studies were undertaken to determine whether V M H lesioned rats show any changes in salivation. Experiment 1 estimated basal salivation for one month to determine if fluctuation in salivary function occurs in rats following VMH destruction. Rats with lesions of the V M H and control rats were also tested for salivary responsiveness to oil o f wintergreen, a taste which normally elicits salivation, and to

Finickiness

injection of Prostigrnin, an anticholinesterase agent. Since Experiment 1 demonstrated a marked reduction in basal salivation in VMH lesioned rats, a second experiment was conducted to further examine this effect. Experiment 2 examined if basal salivation would be influenced by the type of diet the animal was maintained on.

EXPERIMENT 1 METHOD

Subjects Female hooded rats of the Long-Evans strain were matched by weight (230-260 g) and assigned to two groups of six control and six operate animals. The animals were housed in individual cages with ad lib food and water. The room temperature was maintained at 25-26°C. A constant 12 hr day/night cycle was kept with the lights on at 0700 hr and o f f a t 1900 hr.

Procedure Subjects were adapted to the method of saliva measurement for seven days prior to surgery. This method has been described in detail elsewhere [20]. Briefly, a 15 cm cotton tipped swab was weighed and then inserted into the mouth of the animal and held against the right cheek for one minute. The swab was then immediately reweighed to measure the weight of saliva collected. The measures were taken during the day portion of the day/night cycle (0900 hr). All weights were recorded to the nearest 0.1 mg. Evaporation during the

tThis research was supported in part by NIH Biomedical Research Support Grant RR07036. Portions of this paper were presented at the convention of the Society for Neuroscience, St. Louis, MO, 1978. ~Requests for reprints should be addressed to James C. Mitchell, Department of Psychology, Kansas State University, Manhattan, Kansas 66506.

C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/030451-05502.00/0

452

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brief period of time the swab was exposed to air did not influence the weight of saliva measured.

Surgery After seven days of measurement, lesions were produced with the aid of a stereotaxic device with the animals anesthetized with Chloropent (0.30 ml/100 g). With the skull level, bilateral lesions of the VMH were produced by passing a 2mA DC anodal current through the electrode for 15 sec (2.5 mm posterior to bregma, 0.5 mm lateral to the midline, and 0.8 mm dorsal from the base of the skull). The ear bars of the stereotaxic instrument served as the cathode. The sham operated animals were anesthetized but the integrity of the brain was not disrupted.

Postoperative Procedure Body weights and salivation measures were taken 8 hr and 18 hr following surgery to ascertain whether there was any immediate or short-lived alteration in salivation following VMH destruction. Salivation and body weights were then taken once a day for a period of nine days. Following this nine day period animals were placed on a six hr food deprivation schedule during the day portion (0900-1500 hr) of the day/night cycle. During Days 10--13, while animals were adapting to the food deprivation schedule, no saliva measurements were taken. Starting with Day 14, saliva measurements were taken twice daily until Day 33. The fwst daily saliva measurement was taken at the onset of the deprivation period and the second measurement just prior to the reintroduction of food. These saliva measurements continued for 20 days to determine whether saliva secretion differed in the morning and afternoon while on a food deprivation schedule and to determine whether salivation levels changed during this 20 day period. After this period of time, animals were returned to ad lib food access. Several days after the animals were returned to ad lib

food, animals were tested for salivary responsiveness to Prostigmin, a parasympathomemetic agent. Subcutaneous injections of Prostigmin (Roche Laboratories, neostigm;me methylsulfate, 0.10 mg/kg) were given to all animals con, secutively on three test days. A final salivation test was given to assess the animals' responsiveness to a natural taste stimulus. In a counterbalanced fashion, a s ~ m o u n t of U.S.P. oil of wintergreen or water was placed daily into the animal's mouth followed by saliva rneasurement. ~ s procedure involved six days of testing, allowing each animal to be tested three times with each substance.

Histology Following completion of the experiment, animals were deeply anesthetized and perfused with saline and 10% Formalin. Brains were extracted and embedded in e¢lloidin. Sections 30 tz thick were taken with every fifth section being saved and stained with cresyl violet. The extent o f thehesions was determined under microscopic examination and by projecting the sections onto representative photomicrograph plates of the Pellegrino and Cushman atlas [13].

RESULTS

Lesions of the VMH were total and generally symmetrical. Destruction included the arcuate and periventricular nuclei. There was no damage to the lateral hypothalamic area nor to the fornix. Histological examination verified sparing of the VMH in one subject and hence this subject's data were eliminated from analyses. Minimal and maximal lesion size is indicated in Fig. 1. Analyses using t-tests indicated that prior to operative procedures, the two groups to which animals were assigned did not differ significantly either in weight or salivation, p>0.05. VMH lesioned rats and control rats did not differ significantly in salivation 8 hr after surgery, p>0.05. How-

R E D U C E D S A L I V A T I O N IN VMH RATS

453

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FIG. 2. Salivation secretion of control and VMH rats during Experiment 1. For Days 14-33, the abscissa is blocked into 5 blocks of 4 days each. Vertical bars represent SEM.

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factor (treatmentxdays) analysis of variance. Rats with VMH lesions salivated significantly less than control rats, F(1,9)= 17.5, p<0.01. Figure 2 displays this reduction in salivation in VMH lesioned rats. Although days effect was significant, F(8,72)=12.6, p<0.01, no trend is apparent. The treatmentxdays interaction was not significant. On the same day, VMH rats always salivated less than control rats. The 20 day deprivation period during which salivation measures were taken in the morning and afternoon was collapsed into five blocks of four days each. A 2 (treatment)x 5 (blocks) × 2 (time of day) repeated measures analysis of variance revealed that VMH rats salivated significantly less than controls, F(1,9)=14.39, p<0.004. Main effect for blocks of days was not significant. Thus, salivation for the VMH rat remained suppressed for the additional 20 day period (see Fig. 2). The time of day that saliva measurements were taken (morning and afternoon) was not significant. However, the treatment x time of day interaction was significant, F(1,9)=6.5, p<0.03. Tukey's Test revealed that in comparing morning salivation, control rats salivated significantly more than VMH rats (o<0.05). Other comparisons between control, VMH rats, and time of measurement were not significant. No other interactions proved significant. Animals' responsiveness to Prostigmin and wintergreen solution is indicated in Fig. 3. As compared to baseline levels, control and VMH rats showed significant increases in salivation to Prostigmin and wintergreen solution, all p's<0.01. Control and VMH rats did not differ significantly in their salivary response to either substance, t ' s < l .

FIG. 3. Histogram of baseline salivation ( ___SEM) of control and VMH rats and salivary responses to wintergreen solution and Prostigmin during Experiment 1. The average saliva secretion of control and VMH rats for Days 1-33 is represented as baseline.

EXPERIMENT 2 METHOD

Subjects ever, 18 hr following surgery, VMH rats showed a significant reduction of salivation, t(9) = 3.13, p <0.02. For the initial nine days postoperatively, during which the animals had free access to food and water, the amount of saliva collected was analyzed by an unweighted means two

Fifteen female rats of the Long-Evans strain weighing 215--240 g served as subjects. Animals were individually housed with ad lib Purina Rat Chow and water. Animals were maintained on a 12 hr day/night cycle as in Experiment 1.

454

FLYNN, SCHIRER AND MITCHELL TABLE 1 OVERALLMEANSALIVASECRETIONACROSSDAYSAND BODYWEIGHTS AT THE TIMEOF SACRIFICEOF CONTROLAND VMH RATS ( -+ SEM INCLUDED) Experiment 1

Experiment 2

Group

Mg saliva

Body weight

Mg saliva

Body weight

Control VMH

11.7 + 2.9 5.0 _+ 1.6

299 - 8.3 418.4 _+ 18.8

17.2 _+ 3.1 8.5 _+ 1.8

313 -+ 4.6 400 +__25.0

Procedure Subjects were extensively handled and tamed for seven days prior to surgery. During this period no salivation measures were taken. Animals were assigned at random to the control (n=8) or VMH lesion group (n=7). Surgical procedures were the same as in Experiment 1. Animals were allowed a four day recovery period during which one VMH rat died. On day five, the animals' diet was changed to a liquid diet [14]. Animals were allowed undisturbed free access to the liquid diet and water for five days. Beginning on the 10th postoperative day, saliva measures were taken for each animal and thereafter every fourth day for a period of 16 days thus providing 4 measures for each rat. The technique for measuring saliva was the same as in Experiment 1. Following this 16 day period animals were sacrificed.

Histology Brains were extracted, fixed in sweetened Formalin and frozen in liquid nitrogen prior to being sectioned with the use of a cryostatic microtome. Every fifth section was stained and projected onto representative atlas plates [13] to verify lesion placement. RESULTS The minimal and maximal extent of VMH destruction is presented in Fig. 1. Lesions tended to be large and symmetrical destroying the majority of the VMH. The arcuate and periventricular nuclei were destroyed and in some rats, portions of the anterior hypothalamic nucleus, dorsomedial nucleus and ventral premammillary nucleus sustained damage. Despite the large size of the lesions, destruction extended laterally to the fornix in only one animal. A 2 (treatment)x4 (days) repeated measures analysis of variance indicated that lesions of the VMH produced a significant reduction in salivation as compared to control animals, F(1,12)=5.1, p<0.05. Neither the days effect nor the treatment x days interaction were significant. A summary of saliva secretion and body weights of control and VMH animals for Experiments 1 and 2 appear in Table 1. In Experiment 2 as in Experiment 1 VMH rats display a 50% reduction in saliva secretion. For Experiments 1 and 2, t-test comparisons indicated that VMH lesioned rats weighed significantly more than control rats at the time of sacrifice (p's<0.05). DISCUSSION The results of both experiments indicate that lesions of

the VMH reduce basal salivation. The VMH lesioned rat displayed the reduced salivation whether maintained on dry food or a palatable liquid diet. These results are consistent with the observed reduction in salivary gland weight following VMH lesions [9]. Thus, like rats recovered from LH aphagla, the rat with a VMH lesion shows a long-term reduction in salivation. However, unlike the LH rat [20], the VMH rat shows no short-term increase in salivation. When salivary flow is severely or totally blocked, animals adopt a feeding pattern where meals are frequently interrupted by bouts of drinking [10,23]. This prandial drinking has not been observed in the VMH rat but has been observed in the LH rat. While prandial drinking was interpreted to indicate impaired salivary function in LH rats [4, 5, 11], this dry mouth interpretation o f L H prandial drinking is inconsistent with several findings [ 16, 17, 20] which indicate that the lesion itself influences prandial drinking independently of the absence of saliva. Thus, prandiality need not be associated with a reduction in salivation. Despite the reduction in basal salivation observed in the VMH rat, both VMH and control rats displayed comparable elevations of saliva secretion in response to Prostigmin and wintergreen solution. The normal salivary response by VMH rats to wintergre, n is somewhat surprising if VMH lesions exaggerate cephalic responses to oropharyngeal stimulation. However, further study of salivation to food related stimuli is necessary to integrate these findings with Powley's cephalic phase hypothesis [15]. If the decreased basal salivation reflects also a decreased availability of saliva during a meal, this then may in part, contribute to the finicky behavior displayed by the VMH rat. For example, the VMH rat undereats when maintained on a bland powdered food yet gains weight rapidly when switched to a greasy food or a pellet food that is more easily ingested [3, 6, 24]. Thus, the ease or difficulty with which a food is swallowed may contribute to the finicky behavior displayed by the VMH animal. The VMH rat is identified as being hyperemotional and hyperreactive to handling and novel stimuli [7, 21, 22]. One could suggest that the reduced level of salivation was in response to the stress of the measuring procedure which involved handling the subjects. However, several factors lead us to doubt this. First, this possible confounding was anticipated initially. Special care in handling and adapting the animals to the measurement procedure was taken. Secondly, after the adaptation period, each animal received 60 or more of these measures and became relatively docile during the procedure. In spite of the variability in resistance to handling both within and between groups, there was no relationship between saliva secretion and the animals' response to the measuring procedure.

R E D U C E D SALIVATION IN VMH RATS

455 REFERENCES

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