both central and peripheral administration of leptin reduce food intake and body weight [ 1 , 2 ] . leptin receptors are localized to the hypothalamus [ 3 , 4 ] and choroid plexus . direct application of leptin has been shown to increase the activity of glucoreceptor neurons in the ventromedial hypothalamus ( vmh ) and inhibit the activity of glucose - sensitive and nonglucose - sensitive neurons in the lateral hypothalamus ( lha ) . this finding suggests that leptin is liberated from wat into the bloodstream , transported into the hypothalamus through the choroid plexus , and bound to receptors in the hypothalamic neurons to modulate their activities . in addition to the direct action of blood - borne leptin in the hypothalamus , the existence of afferent signaling from the leptin sensors in the wat of the epididymis and the sensors ' role in reflex modulation of sympathetic nerve activity have been reported [ 7 , 8 ] . it is reasonable to assume that leptin sensors exist in the hepatoportal region because the portal vein is a main pathway for leptin secreted from the visceral wat to arrive in the portal venous blood . further , it has been reported that several types of chemosensors , such as glucose sensors , amino acid sensors , and interleukin-1 sensors , exist in the hepatoportal region and send their information through the hepatic branch of the vagus nerve to the brain . the aim of the present study was to investigate the effect of leptin on afferent signals from leptin sensors in the hepatoportal region and the sensors ' reflex effect on autonomic outflow . all animals were housed in a room maintained at approximately 24c and illuminated for 12  h ( 07:0019:00 ) . food ( type mf ; oriental yeast co. , tokyo ) and water were freely available . all procedures were performed in accordance with the japanese physiological society 's guidelines for animal care . experiments were conducted after the animals had been allowed to adapt to their housing conditions for one week . after the laparotomy , a catheter was inserted into the portal vein or the inferior vena cava for administration of leptin . with a dissection microscope , an isolated nerve filament from the peripheral cut end of the hepatic branch of the vagus nerve was placed on a pair of silver wire recording electrodes to record the nerve activity . afferent signals were sometimes recorded from the gastric or celiac branches of the vagus nerve . to record the efferent nerve activity of the sympathetic or vagal nerve branch , a nerve filament dissected from its central cut end was used . the recording electrodes were immersed in a mixture of liquid paraffin and petroleum jelly to prevent dehydration . the nerve activity was amplified in a condenser - coupled differential amplifier , monitored by an oscilloscope , and stored on magnetic tape . all nerve activity were analyzed after conversion of raw data to standard pulses by a window discriminator , which separated discharges from background noise . the discharge rate was displayed on a pen recorder by means of a rate - meter with a reset time of 5 seconds . the effects of leptin injection on nerve activity were investigated by comparing the mean number of impulses per 5 seconds over 50 seconds ( i.e. , the mean value of 10 successive measured samples ) before and after the injection . statistical significance was determined by analysis of variance ( anova ) ( p < .05 ) . recombinant leptin ( sigma ) was kept in a freezer ( 20c ) and dissolved in physiological saline before use . the leptin solution was injected through a catheter inserted into the inferior vena cava or portal vein . figure 1 illustrates typical examples of the effects of two successive iv injections of leptin on the afferent nerve activities of the hepatic , gastric , and celiac branches of the vagus nerve , as well as of the splanchnic nerve . all recordings were made from the nerve filament dissected from the peripheral cut end of the nerve . the second iv injection ( 10  ng , 0.1  ml ) was made 60 minutes after the first injection ( 1  ng , 0.1  ml ) . afferent discharge rates of vagal hepatic afferents just before , 30 minutes after , and 60 minutes after the first injection were 77.6  1.9 , 79.7  36.1 , and 84.7  2.3 * impulses per 5 seconds , respectively . those just before , 30 minutes after , and 60 minutes after the second injection were 84.7  2.3 , 100.2  1.9 * , and 105.7  2.8 * impulses per 5 seconds , respectively ( * represents significant increase , p < .05 ) . these results indicate that iv injections of leptin at doses of 1  ng and 10  ng significantly increased the afferent discharge rate , and the responses were dose related . however , no significant changes in afferent discharge rates were observed in the gastric or celiac branches of the vagus nerve , nor in the splanchnic nerve , after iv injections of leptin ( 1  ng and 10  ng ) . the results of these experiments clearly suggest the existence of leptin sensors at the terminals of vagal hepatic afferents . to confirm the existence of leptin sensors in the hepatoportal region , the effect of leptin injection into the portal vein ( ipv ) on the afferent activity of the hepatic branch of the vagus nerve figure 2(a ) shows an example of the effect of an ipv injection of leptin ( 100  pg , 0.1  ml ) on the vagal hepatic afferents . as shown in the trace , an injection of leptin caused a gradual and long - lasting increase in afferent activity , which reached a peak value approximately 60 min after the injection . the discharge rates just before and 30 , 60 , 90 , and 120 minutes after ipv injection were 65.2  2.2 , 113.6  4.1 * , 128.5  2.9 * , 149.8  5.8 * , and 83.1  2.5 * impulses per 5 seconds , respectively ( * indicates a significant increase , p < .05 ) ( figure 2(b ) ) .   figure 2(c ) presents the afferent action potentials at times a , b , and c in figure 2(a ) . figure 2(d ) presents the mean discharge rates of five different preparations before and 30 , 60 , and 90 minutes after ipv injection of leptin ( 100  pg , 0.1  ml ) . the mean discharge rates were 71.0  5.7 , 95.7  10.0 * , 100.7  9.6 * , and 115.3  11.7 * impulses per 5 seconds , respectively ( * indicates a significant increase , n = 5 , p < .05 ) . further , each of five preparations demonstrates a significant increase in afferent discharge rates 60 and 90 minutes after ipv injection ( table 1 ) . it was further observed that an ipv injection of saline ( 0.1  ml ) as a control resulted in no significant change in discharge rates ; ipv injection of 10  pg leptin resulted in no significant increase . reflex effects of ipv infusion of leptin on the efferent activity of the sympathetic branch to the adrenal medulla , pancreas , liver , epididymal wat , interscapular brown adipose tissue ( bat ) , as well as that of the celiac and pancreatic branches of vagus nerve , were investigated . figure 3 presents typical examples of the effects of ipv infusion of leptin 1  ng ( 0.1  ml ) on sympathetic and vagal outflows . the injection activated sympathetic nerve activity and inhibited vagal nerve activity to the organs mentioned above . figure 4 and table 2 show the mean discharge rates of sympathetic or vagal nerve activities before and after iv injection of leptin ( 1  ng , 0.1  ml ) . these observations demonstrated that ipv or iv administration of leptin at a dose of 1  ng activates sympathetic and inhibits vagal outflow . in subsequent experiments , the effects of ipv injection of leptin ( 1  ng , 0.1  ml ) on the efferent nerve activity of the adrenal sympathetic and vagal celiac nerves were compared in normal and hepatic vagotomized rats . as shown in the upper part of the left panel of figure 5 , leptin injection ( 1  ng , 0.1  ml ) caused a significant increase in adrenal nerve activity in normal rats ; however , no significant change in nerve activity occurred following ipv injection of leptin at the same dose in hepatic vagotomized rats . the lower part of the left panel of the figure 5 presents the effects of leptin on vagal celiac efferents . in the normal rat , leptin injection suppressed vagal nerve activity , although no significant change in nerve activity was observed following injection of leptin in hepatic vagotomized rats . the right panel of figure 5 demonstrates that iv administration of leptin ( 1  ng , 0.1  ml ) caused an increase in efferent activity of the sympathetic adrenal efferents ; further , in bilateral or hepatic vagotomized rats , no remarkable changes in efferent activity following iv injection of leptin at doses of 1  ng , 10  ng , 100  ng , or 1  g were observed . however , iv administration of a large amount of leptin ( 10  g ) resulted in a remarkable increase in efferent activity . this response might be the direct effect of blood - borne leptin on the hypothalamus . these observations indicate that ipv injection of leptin results in reflex activation of sympathetic outflow and reflex suppression of vagal outflow to visceral organs that regulate metabolic functions . it was stated in the introduction that both central and peripheral administration of leptin reduces food intake and body weight , and that the site of action of leptin is the hypothalamus [ 1 , 2 , 12 ] . it was also reported that leptin receptors are distributed within the hypothalamus and choroid plexus [ 35 ] , and that leptin directly stimulates activity of vmh ( satiety enter ) neurons and inhibits activity of lha ( feeding center ) neurons , and stimulates / inhibits activity of arc ( dual center ) neurons . interestingly , several lines of evidence support that leptin signaling in the central nervous system regulates autonomic outflow to white adipose tissue , brown adipose tissue , muscle and liver . in relation to this , the importance of the role played by the hepatic sympathetic nerves has been reported . the most widely accepted hypothesis is that blood - borne leptin is transported into the brain by the choroid plexus and acts on the leptin receptors in the hypothalamus ; chemical signals may thereby be translated into neural signals . it was recently reported that leptin sensors exist in the wat of the epididymis and send signals through afferent nerve fibers innervating wat to the central nervous system ; those afferent signals then evoke reflex activation of sympathetic nerve activity to the visceral organs , such as the adrenal medulla , pancreas , liver , wat , and bat , and evoke reflex inhibition of vagal nerve activity to the pancreas and liver [ 13 , 14 ] . further , it is possible to assume that peripheral leptin sensors also exist in the hepatoportal region , are sensitive to leptin liberated from the visceral wat into the portal venous blood , and send signals to the brain via vagal hepatic afferent fibers . the results of the present study demonstrated that an ipv or iv injection of leptin ( 100  pg to 1  ng , 0.1  ml ) activated afferent nerve activity of the hepatic branch of the vagus nerve ; an iv or ipv injection of leptin ( 1  ng , 0.1  ml ) caused facilitation of sympathetic nerve activity to visceral organs such as the adrenal medulla , wat , and bat , and inhibition of vagal celiac and pancreatic nerve activity ; the same amount of leptin injection was without effect in totally vagotomized or hepatic vagotomized rats ; the doses of leptin ( 100  pg to 10  ng ) used for iv or ipv injections were in the physiological range of plasma leptin concentrations ( 6  1  ng / ml ) ; iv or ipv injection of physiological saline was without effect on the afferent activity of the hepatic branch of the vagus nerve as well as on the reflex change in autonomic outflow ; iv or ipv injection of a larger amount of leptin ( 1  g or 10  g ) increased adrenal sympathetic nerve activity in totally or hepatic vagotomized rats . further , the author observed that injections of leptin at doses of 0.51.0  g ( 1.5  l ) into the lateral ventricle activated efferent activity of the adrenal sympathetic nerve ( unpublished data ) . in addition to the blood - borne leptin pathway and afferent signaling system from leptin sensors in the wat , the present study demonstrated a third pathway of the leptin signaling system from hepatoportal leptin sensors to the brain . leptin secreted from the abdominal visceral white adipose tissue into the portal vein may play an important role in reflex regulation of metabolic function . the existence of three different leptin signaling systems ( the blood - borne system , afferent signaling system from wat , and that from hepatoportal leptin sensors ) , is likely to represent a fail - safe alarm system . the results of the present experiments demonstrate the reflex modulation of metabolic functions , including acceleration of lipolysis in wat , increase in catecholamine secretion from the adrenal medulla , facilitation of glycolysis in the liver , and reduction in insulin release from the pancreas . the reflex center is in the hypothalamus or brainstem , and the afferent limb is made up of afferent fibers in the hepatic branch of the vagus nerve . the exact sites of hepatoportal leptin sensors , as well as morphological and immunohistochemical properties of these sensors , remain to be studied . the results of the present experiments indicate that afferent signals from leptin sensors in the hepatoportal region play a role in reflex regulation of metabolic functions through modulation of autonomic outflow .
afferent nerve signals were recorded from a peripheral cut end of the small nerve bundle of the hepatic branch of the vagus nerve in anesthetized rats . 
 an injection of leptin ( 100  pg , 0.1  ml ) into the portal vein facilitated the afferent activity . 
 the response was dose dependent . 
 further , an intravenous ( iv ) injection of leptin ( 1  ng , 0.1  ml ) facilitated the efferent nerve activity of the sympathetic nerve to the adrenal gland and suppressed that of the celiac branch of the vagus nerve . 
 in hepatic vagotomized rats , no change in efferent activity of the adrenal sympathetic nerve nor celiac branch of the vagus nerve was observed following iv administration of leptin . 
 these observations suggest that leptin sensors in the hepatoportal region play a role in reflex modulation of autonomic outflow in relation to metabolic functions .