Feeding related lateral hypothalamic neuron responses to odors depend on food deprivation in rats

Physiology & Behavior, Vol. 44, pp. 591-597. Copyright©Pergamon Press plc, 1988. Printed in the U.S.A.

0031-9384/88 $3.00 + .00

Feeding Related Lateral Hypothalamic Neuron Responses to Odors Depend on Food Deprivation in Rats TAKEMASA

SHIRAISHI

D e p a r t m e n t o f Physiology, Tokai University School o f Medicine, Bohseidai, lsehara 259-11 Japan

SHIRAISHI, T. Feeding related lateral hypothalamic neuron responses to odors depend on food deprivation in rats. PHYSIOL BEHAV 44(4/5) 591-597, 1988.--It has been investigated feeding related LHA neuronal activity and responses to odor stimulation in rats at various levels of satiation. Extracellular responses of 168 neurons to three odors, isoamylacetate (AA), cineole (CL), and isovaleric acid (VA), were recorded from 168 LHA neurons of Wistar-SPF male rats. Of 168 units, 107 (63.7%) respondod to from one to three odors, but not to light or phonic stimulation. Of the responding units, 94.4% (101/107) were excited, and 5.6% were inhibited. In response to a single electrical stimulation (0.5 msec, 1-10 V) of the OB, 61 units were excited with latencies of 6--43 msec (19.8+_12.0 reset, mean+_S.D.) indicating compound OB-LHA relations--mono- and polysynaptic through myelinated and nonmyelinated fibers. The results suggest predominantly excitatory effects of both electrical stimulation of the OB and odor stimulation on the LHA. Firing frequency in response to AA or VA was significantly (0<0.05) greater for the long fasting group (38 hr, LF, n=8) than for the NF (nonfasting, n= 12) group; differences between the LF and MF (24 hr, n=6) groups were not significant. Glucose-sensitive neurons (GSN, n= 19) responded more to odors than non-GSNs (n=86), and discharge frequency increase depended markedly on food deprivation. Food deprivation results suggest that responsiveness of feeding related LHA neurons to odors depends on the degree of satiation. In conclusion, it was confirmed that olfactory functions are important in the responses of hypothalamic feeding related neurons. Lateral hypothalamic neurons

Odor stimulation

Food deprivation

IT is established that the lateral hypothalamus (LHA) and the ventromedial hypothalamus (VMH) contribute to control of feeding behavior by their reciprocal activity (19, 20, 22, 26). It is also well known that L H A neurons respond to olfactory stimulation (16, 31, 37, 39). There is neuroanatomical evidence that L H A neurons receive olfactory inputs from the anterior olfactory nucleus, olfactory tubercle, amygdala, and pyriform cortex (1, 5--7, 11, 12, 18, 34, 38, 40, 48). Their pathways were also demonstrated by electrophysiological studies (3, 16, 31-33, 36, 39). Some L H A neurons responded to electrical stimulation of the olfactory bulb (OB), some responded to chemical stimulation of the olfactory mucosa and some responded to both (16,39). Neurophysiology o f olfaction has been studied systemically in unanesthetized animals in Dr. Takagi's laboratory (13-15, 45, 47). Many reports suggest that olfactory stimulation affects various behaviors, such as emotion and sex (3, 4, 12, 32, 37), but only a few electrophysiological studies can be found about the relationship between olfaction and food intake (9, 13, 30, 35). We recently reported that feeding related L H A neurons receive visceral information form both vagal and nonvagal afferents (44), while gastric vagal afferents reach the L H A either directly or via the OB (2,46). The experiments reported here were undertaken to clarify

Rats

how food deprivation influences single unit activity of olfactory related neurons in the L H A . Feeding related L H A neuronal activity and responses to odor stimulation in rats were investigated at various levels of satiation. Seventy-six male Wistar SPF-rats (Specific Pathogen Free), weighing between 280 and 460 g (average 320 g) were used. SPF-rats were used to ensure an open airway for extended artificial respiration. The rats were treated in compliance with the N I H Guide for the Care and Treatment of Laboratory Animals. An animal was fixed in stereotaxic apparatus (SN-20, Narishige, Tokyo) under ketamine anesthesia (10 mg/kg, IP, with booster applications every 20 to 30 min as required). Rectal temperature was maintained at 37--I°C. Neuronal activity was recorded from the L H A at posterior 3.0 mm, lateral 2.0 mm from the Bregma, and 8.0 to 8.5 mm vertical from the surface of the parietal bone. This region had previously been found to contain a population of feeding related L H A neurons (41,42). A multibarreled (3 to 5 barrels, tip o.d. about 1/zm) electrophoretic pipette for identification of feeding related glucose sensitive L H A neurons was cemented to a recording electrode wtih the electrode tip extending 20 to 30/~m beyond the pipette tip. Extracellular neuronal activity of feeding related and feeding unrelated L H A neurons was recorded through the glass microelectrodes filled with 3 M KCI or 4 M NaC1 (DC resistance, 50-80

591

592

SHIRAISHI

OB~LHA

Latency

histogram ( O B ~ L H A )

n=e

1 9 , 8 _+ 1 2 . 0 ( m s e c _+ M S D

P 20

A

15

A "5

10

E= Z

5 A

I A

I0

l~v

0

0

10

20

30

40

50

80 m ~c

Latency

ml

FIG. 1. LHA neuron responses to OB single stimulation. Left: Traces of complex wave form potentials evoked in LHA by single pulse (0.5 msec, 1-10 V) OB stimulation. Note the wide range of latency in responses of four different neurons despite almost equal stimuli. Arrow heads indicate stimulation artifacts. Calibrations: Ordinate, 1 inV. Abscissa, 20 msec. Right: Latency histograms of LHA evoked potentials in response to OB stimulation. Sixty-five units were excited with latencies of 0-5 to 55-60 msec (19.8_+12.0 mean_+S.D.) indicating compound OB-LHA relations, mono- and polysynaptic transmission through myelinated and nonmyelinated fibers. M~), with a silver indifferent electrode in the neck. Neuronal activity was amplified in a high input impedance preamplifier, observed on a cathode ray oscilloscope (VC10, Nihon Kohden, Tokyo), recorded by a continuous recording camera (PC-2B, Nihon Kohden), analyzed with a pulse rate meter for determination of neuronal discharge rate, and recorded on a four channel polygraph. Electrophoretic application of drugs to the feeding related and feeding unrelated L H A neurons was driven from a constant current source (25). Pipette barrels were filled with the following chemicals: 2 M 2-deoxy-D-glucose (2-DG, pH 5.2); 2 M glucose (pH 7.4); 0.5 M sodium L-glutamate (pH 5.6); 500 mM norpinephrine (pH 4.0); 250 mM tranylcypromine (pH 4.1); and 0.1% methyl blue; each in 154 mM NaCI (pH 6.2). Sodium L-glutamate was used to confirm viability of the assembly and proximity to the recorded neuron. Methyl blue was used for marking the tip location after completing an experiment. One barrel was filled 0.5 M NaCI to check osmosensitivity and current effects, and any neuron that responded to N a ÷ or C1- application was omitted from the data. A bipolar stimulating electrode, two 300 /~m diameter stainless steel wires, cashew varnish coated except for 0.2 mm at the tip, was implanted in a ventral part o f the OB according to the method of Chaput and Holly (9) with slight modification. The electrode was located in the ventral part of the mitral cell layer after confirming the second peak described by Chaput and Holly (9). Electrical stimulation in the OB was by a square pulse (1-10 V), with a duration of 0.5 msec from an electronic stimulator (SEN-1101, Nihon Kohden, Tokyo) with an isolator (SS-101J). After confirming OB stimulation in the preparation, and recording of neuronal activity from the L H A , the animal was immobilized with gallamine triethodide (5 mg/rat, IV) Teikoku Chemical Industry, Osaka) or tubocurarine chloride (1 mg/rat) (Amerizol, Takeda, Osaka) in addition

TABLE 1 SUMMARY O F R E S P O N S E S O F L H A N E U R O N S TO O D O R S

Response Items/Tested

+ 107/168 (63.7%)*

I'

~

-

101/107 (94.4%)t

6/107 (5.6%)

61/168 (36.3%)

*p<0.05, tp<0.01, (n=28). A total of 168 LHA neurons were examined. A significant number (63.7%, p<0.05) of LHA neurons responded to odors. These results suggest predominantly excitatory LHA responses to both electrical stimulation of the OB (olfactory bulb) and odor stimulation. +, response to odor; - , no response; ~', excitatory; ~, inhibitory.

to the ketamine anesthesia previously described, and a rodent respirator (681D, Harvard, Milhs, MA) was used to maintain stable respiration (15/min, VA=300-350 ml). Stimulation and recording were ipsilateral throughout the experiments. The following three odors were chosen for use in these experiments; isoamylacetate (AA) (Shuzui Chemical, Tokyo), cineole (CL) (Encalyptol, Sigma, St. Louis, MO), and isovaleric acid (VA) (Wako, Tokyo). The common sensations of these odors are: A A , vinegar; CL, eucalyptus; VA, sweat. Each of these substances was diluted to a concentration of 1 0 - ! 1 0 -8 M in an odorless mineral oil (Nujol, Plough Inc., Memphis, TN). A few drops of a diluted solution was stored in a 2.5 ml syringe. A 4.5 cm long odorless teflon tube, with an inner diameter of 1 mm, carried odors from the syringe to the animal. Each odorous vapor was applied with 0.5 ml of air for 5 see with intertrial intervals of 30 to 60 see. After each trial the air was deodorized by con-

ODOR RESPONDING L H A N E U R O N S A.

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i

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~ o -10-5m AA

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10-~ AA

10"~ AA

2O

0 i

i

30sec

FIG. 2. Various responding patterns of LHA neurons to odors. (A) Excitation type: Application of low concentration (10-s M) of VA (isovaleric acid) transiently increased discharge frequency. Subsequent application of higher concentration (10-e M) of VA increased neuronal activity with much longer duration than previously. (B) Persistent excitation type: Application of 10-* M CL (cineole) induced persistently increasing discharge frequency. (C) Inhibition type: AA (isoamyl acetate) at 10-s, 10-s and 10-4 M suppressed discharge rate of this neuron. Less than 10% of neurons tested were inhibited. Ordinates: neuronal discharge rate, pulses per sec. Abscissas time, 30 sec. calibration. Overbars, times of odor stimulation.

stantly flowing through activated charcoal and CaCOa at a rate o f 2 litersdmin. Different concentrations of odors were always applied from lower to higher to minimize adaptation. In addition, Nujol alone, light and sound stimuli were also applied as controls. The animals were divided into three food deprivation groups: a long fasting (LF) group (38 hr, n=8), a medium fasting (MF) group (24 hr, n=6), and a nonfasting (NF) group (n= 12). At the end o f all experiments, an animal was injected with an overdose of pentobarbital sodium (60 mg/kg, IP), then perfused with physiological slaine and 10% formalin. The brain was removed and fLxed, and 50/~m coronal sections were cut, mounted, and stained according to methods previously described (41). OB stimulation and L H A neuronal activity recording sites were checked against the rat brain atlas (17). All results are expressed as mean_S.D. Statistical analyses were by F-test, Student's t-test, Wilcoxon and/or chi-square test. LHA NEURON RESPONSESTO OB SINGLE STIMULATION The left part of Fig. 1 shows a complex wave form evoked in the feeding related region of the L H A by single pulse (0.5 msec, 1-10 V) OB stimulation. Note the wide range of latency in the responses of four different neurons despite almost equal stimuli. Latency histograms of L H A evoked potentials in response to OB stimulation are shown in the right part of Fig. 1. Response latencies of the 65 excited units ranged between 0 to 5 msec and 55 to 60 msec (19.8+ 12.0 msec, mean_+S.D.) indicating complex OB-LHA relations; mono- and polysynaptic connections through myelinated and nonmyelinated fibers. It was previously reported that responses of L H A neurons to single OB stimulation in anesthetized rats (16) had a median latency of 10 msec

(range, 4--16 msec). Another report stated that latencies to OB stimulation ranged from 3 to 90 msec (mean, 21.3 msec) in the L H A of anesthetized rats (39). Kogure and Onoda 05) reported that OB stimulation in unanesthetized rabbits elicited potentials in L H A neurons with latencies of 3.7 to 68.0 msec (mean, 19.4 msec). The broad nature of the latency distribution agrees with the existence of multiple routes between the OB and the L H A indicated by anatomical studies (1, 5-7, 11, 12, 18, 34, 38, 40, 48). LHA NEURON RESPONSESTO ODORSTIMULATION To classify responses to odor stimulation, the discharge rate immediately preceding stimulation (A) and the discharge rate during stimulation (B) were measured. From the ratio of B to A, responses were classified into the following groups, according to the criteria of Kogure and Onoda (15): 1) B/A > 1.5, facilitation; 2) B/A <0.5, inhibition; 3) increase or decrease less than 150 or 50%, respectively, no response. Of I68 L H A neurons examined in 28 rats, 107 (63.7%) a significant (p<0.05) number responded to odors. Of the 107 responding to neurons, a significant (p<0.01) number, 101 (94.4%), were facilitated, and 6 (5.6%) were inhibited. These results suggest predominantly excitatory effects of both electrical stimulation of the OB and odor stimulation on L H A neurons. Table 1 summarizes the odor responding L H A neurons. Fig. 2 A-C shows various responding patterns of L H A neurons to odors. These responses were divided into three types: Fig. 2A shows excitation, a low concentration (10 -8 M) of VA transiently increased the discharge frequency. Subsequent application of a higher concentration (10 -6 M) of VA increased neuronal activity for a much longer duration that the in'st one. Fig. 2B shows long lasting excita-

594

SHIRAISHI TABLE 2 EFFECTS OF FASTING ON LHA NEURONS TO ODOR (VA) Discharge Frequency (Hz)

Group

PreStimulation

PostStimulation

A%

L F (n=8) MF (n=6) N F (n=12)

18.6 + 4.3 21.3 - 11.4 16.8 --- 9.8

65.0 ___ 14.3 44.7 _+ 21.5 38.8 +_ 19.2

349.5 _+ 104.8" 209.9 _+ 77.7 231.0 -+ 112.4

*p<0.05 (LF vs. NF), n.s. (LF vs. MF, MF vs. NF). Firing frequency in response to VA was significantly (p<0.05) greater for the long fasting (LF) group than for the NF (nonfasting) group; differences between the LF and MF groups were not significant. Number of units tested were: LF=37, MF=20, and NF=50. n, number of animals.

tion induced by application of 10-4 M CL. Figure 2C shows the inhibition type; 10-5, 10-e and 10-4 M AA suppressed neuronal discharge. Although there were three different treatments used on these three different neurons, the differences were due to the neurons. In no tests did different treatment of one neuron produce different results for the odors described here. Inhibition was evident in less than 10% of the units tested as mentioned above. It was previously reported that hypothalamic neurons responded most strongly to the odor of AA, benzene or heptane (39), but responses to AA were relatively very few in the present study. The present results agree with other reports (15, 16, 39) in the predominance of excitatory responses. Excitatory responses to odors are found to be characteristic of LHA neurons, whereas in other regions of the brain, the OB (31), pyriform cortex (10), amygdala (8), and preoptic area (33), inhibitory and mixed responses are generally found more frequently. FOOD DEPRIVATION STUDY

A few previous studies have investigated relations between olfaction and food intake (9, 13, 30, 35). However their feeding relation was measured from stomach distention. The present experiment was undertaken to clarify how food deprivation influences neuronal activity of the olfactory related neurons in the feeding related region of the LHA. Discharge frequency for the 5 see interval from 7 sec before to 2 sec before stimulation was compared with the discharge rate in the 5 sec interval from 2 sec after to 7 sec after stimulation. The results using the most sensitive concentration of VA odor for each individual rat are summarized in Table 2. The firing frequency in response to VA was significantly (p<0.05) higher in the LF group than in the N F group; other differences were not significant. The average increase in discharge frequency of each group was 349.5% for the LF, 209.9% for the MF and 231.0% for the N F group. Thus, responses to odors, as well as to glucose, appeared to depend on food deprivation time. From this finding, it may be concluded that LHA neuronal responses to odor stimulation depend on level of satiation. Since only one odor was used in this analysis, it is not possible to decide whether the apparent selectivity of the modulation was connected with an essential property of the odor. Further studies including various control odors are necessary to fully determine which odor property affects responses. However, it is likely that the meaning of

p 2.0

p 2.5

p 3.0

p3.5

t

i

2 mm FIG. 3. Locations of odor sensitive neurons. Locations of 26 odor sensitive LHA neurons found in the food deprivation study. All units are within the LHA. Odor sensitive LHA neurons were distributed widely in the LHA, but most units were found in the region 3.0-3.5 mm posterior from the bregma. This indicates that odor sensitive LHA neurons are either feeding related or coexist with the feeding related neurons.

the olfactory signal with respect to the internal state of the animal is as much a determining factor as any intrinsic properties of the stimulus. This was demonstrated in experiments dealing with the control of olfactory input relative to the alimentary state (27,29), and was conf'Lrmed by a report that nonfood stimuli could elicit modulated responses if repeatedly associated with food intake (28). LOCATION OF ODOR SENSITIVE LHA NEURONS

Locations of 26 odor sensitive L H A units found in the food deprivation study are shown in Fig. 3. It appears that all odor-sensitive LHA neurons are distributed widely in the LHA, but most are in the region 3.0-3.5 mm posterior from the Bregma. Interestingly, this indicates that the odorsensitive LHA neurons either coexist with, or are feeding related neurons. We have previously reported the center of a population of feeding related LHA glucose-sensitive neurons (42,43). The next study was initiated to investigate how odor stimulation influences functionally specific glucose sensitive neurons, and how food deprivation might contribute to the effects.

ODOR R E S P O N D I N G L H A N E U R O N S

LHA

595

: GSN

A.

¢ 50hA i

2-DG 20

O

40 i

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ao

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30 i

t ,6 "~" 1 0

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0

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a.

-

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2-DG 30 ~

G 2-DG G 50nA 40 50 m =m,

C.

10"e= VA

Na +50 m

(t

lO'2mCL f

Cl -50 m

10"sin VA f

30 me

FIG. 4. Effects of various odors on LHA glucose-sensitive neurons (GSN). Glucose-sensitive neurons in the LHA were identified by their clear dose-related excitation in response to electrophoreticallyapplied 2-deoxy-D-glucose (2-13(3), and/or inhibition by glucose. (A) The discharge frequency increased by 2-DG electrophoretic applications at 20 and 40 hA, and decreased by glucose applications at 30 and 50 nA. This neuron, and the neurons in B and C were thus glucose-sensitive. Odor stimulation of 10-= M AA had no effect on this GSN in A. (B) Dose-dependent increase by 2-DG, and neuronal discharge rate increased by 10-8 M VA. Nonspecific osmosensitivity and current effects were not involved since no response was observed after Na ÷ or CI- applications. (C) Response ofa GSN in the LHA to two odors. Activity was increased by 2-DG, and decreased by glucose (G). Application of two different odors, 10-= M CL and 10-8 M VA, induced increase in discharge rate. Ordinates: neuronal discharge rates, pulses per 5 sec (A, C), and per sec (B). Abscissas: time 30 sec calibration. Overbars, electrophoretic applications, at currents indicated in nA. Arrows, times of odor stimulation.

GSN [

300

200 tO

ii!i

ilii

'5

"6

100 LF

M =

N

F

Groups FIG. 5. Effects of food deprivation on LHA GSN responses arid non-GSN responses to odor. LF, long; MF, medium; NF, nonfasted groups; GSN, glucose sensitive neuron (white columns); nonGSN, nonglucose-sensitive neuron (shaded columns). **p<0.01, F(7,28)=5.23, t(3)=0.93; *p<0.05, F(7,8)=2.57, t(15)=2.31.

E F F E C T S O F O D O R S ON L H A G L U C O S E - S E N S I T I V E N E U R O N S

Two types of glucose responding neurons in the hypothalamus have been demonstrated to be involved in feeding behavior and characterized (21-23, 26). Approximately 25% o f the neurons in the L H A and VMH are chemosensitive, their activity being influenced by glucose, F F A , insulin, and other endogenous metabolites and hormones related to feeding (24). Activity of glucose-sensitive neurons (GSN) in the L H A decreases and activity of glucoreceptor neurons (GRN) in the VMH increases when glucose is applied to electrophoresis, or by injection into the cerebral ventricles, or peripherally. G S N s have been previously shown to be feeding related (20,43). In the study reported here, G S N s in the L H A were identified by their clear dose-related excitation in response to electrophoretically applied 2-deoxy-D-glucose (2-DG), and/or inhibition by glucose. Specimen records of effects of various odors on G S N s in the L H A are shown in Fig. 4A-C. Figure 4A shows that the increase in discharge frequency after electrophoretic application of 2-DO at 20 and 40 nA, and decrease after glucose application at 30 and 50 nA. This neuron, and the neurons in B and C were GSNs. Odor stimulation of 10-2 M A A had no effect on the G S N in A. Figure 4B shows dose-dependent increases by 2-DG, and increase in neuronal discharge induced by 10-8 M VA. Nonspecific osmosensitivity and current effects were not involved since no response was observed after N a + or CI- applications. Fig. 4C shows the response o f a G S N in the L H A to two odors. The neuronal activity was increased by 2-DG, and decreased by glucose (G). Application o f the two different odors, 10 -2 M CL and 10-s M VA, induced increases in the discharge frequency. Figure 5 summarizes the effects of fasting on the

596

SHIRAISHI

responses of GSNs and non-GSNs in the LHA to all odors tested. Responses of GSNs were greater than responses of non-GSNs (p<0.01), although this difference was significant only in the LF group. Among the GSNs, responses to odors were significantly greater in the L F group than in the N F group (p<0.05). This indicates that feeding related LHA neurons, GSNs, respond more to odors than non-GSNs do, and their responses depend on the duration of fasting. SUMMARYAND CONCLUSIONS 1) Feeding related LHA neuronal activity and responses to odor stimulation in rats at various levels of satiation were investigated. 2) Extracellular responses of 168 neurons to three odors, isoamylacetate (AA), cineole (CL), and isovaleric acid (VA), were recorded from neurons in the feeding related region of the LHA. 3) Of 168 units (n=28 rats), 107 (63.7%) responded to one to three odors, but not to light or phonic stimulation. Of the responding units, 94.4% (101/107) were excited, and 5.6% were inhibited by odors. 4) In response to a single electrical stimulation (0.5 msec, 1-10 V) of the OB, 61 units were excited with latencies of 6--43 msec (19.8_+12.0 msec, mean_+S.D.) indicating com-

pound OB-LHA relations; mono-and polysynaptic transmission through myelinated and nonmyelinated fibers. The results suggest predominantly excitatory LHA responses to both electrical stimulation of the OB and odor stimulation. 5) Firing frequency in response to AA or VA was significantly (o<0.05) greater for the long fasting group (38 hr, LF, n=8) than for the N F (nonfasting, n = 12) group; differences between the LF and MF (24 hr, n=6) groups were not significant. 6) Glucose-sensitive neurons (GSN, n=21) responded more (p<0.01) to odors than non-GSNs (n=86), and discharge frequency increases depended markedly on food deprivation. Food deprivation results suggest that responsiveness of feeding related LHA neurons to odors depends on the degree of satiation. It is concluded that hypothalamic feeding related neurons respond to odors, and the degree of response depend on the extent of food deprivation.

ACKNOWLEDGEMENTS The author thanks Dr. A. D. Simpson, Ms. M. Kawashima and Ms. M. Kobayashi for their invaluable help in preparation of this manuscript.

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