obesity increases the risk for hypertension and is a major driver of morbidity and mortality due to cardiovascular diseases ( dustan , 1983 ; poirier et  al . , 2006 ) . studies in rodents with diet - induced obesity ( dio ) suggest that increased sympathetic nerve activity ( sna ) is an important mediator of obesity - induced hypertension as  and  adrenergic receptor antagonists and renal denervation significantly blunt the rise in blood pressure ( bp ) associated with weight gain ( carlyle et  al . , 2002 ; however , the precise molecular and neural mechanisms that link changes in weight with changes in bp have not been fully elucidated . circulating concentrations of the adipocyte - derived hormone leptin increase in proportion to adipose tissue mass and fall with weight loss ( considine et  al . , we hypothesized that leptin may be involved in coupling changes in body weight ( bw ) to changes in bp . leptin regulates energy homeostasis by acting on hypothalamic neuronal circuits expressing the signaling isoform of the leptin receptor ( lepr ) to reduce calorie intake and increase energy expenditure ( halaas et  al . , 1997 ; leptin can increase sna , leading to increases in  bp and heart rate ( hr ) ( haynes , 2000 ; mark et  al . , 1999 ) . in the arcuate nucleus of the hypothalamus ( arh ) , leptin stimulates the expression of pro - opiomelanocortin ( pomc ) and increases the activity of pomc neurons , which release the pomc peptides (  ,  , and  melanocyte - stimulating hormones [ mshs ] ) that act on melanocortin 4-receptor ( mc4r)-expressing neurons in the paraventricular nucleus of the hypothalamus ( pvh ) and other brain regions to increase sna ( cone , 2005 ; cowley et  al . , 1999 , 2001 ; however , pomc neurons become unresponsive to  leptin in obesity , and leptin can act independently of mc4r signaling ( enriori et  al . , 2011 ; patterson et  al . , 2011 ; scott et  al . , 2009 ) . therefore , leptin s effects beyond the melanocortin circuits need to be investigated . the dorsomedial hypothalamus ( dmh ) is critical for leptin s ability to regulate brown adipose tissue ( bat ) temperature and the cardiovascular system ( enriori et  al . , 2011 ; ; rezai - zadeh et  al . , 2014 ) . here , we investigated the development of obesity - induced hypertension . we demonstrate that , in dio mice , increasing levels of leptin directly lead to an increase in hr and bp and that blocking the actions of leptin reverses these effects via neural circuits originating in the dmh . furthermore , humans with loss - of - function mutations in leptin and its receptor have normal bp despite severe obesity , suggesting that these mechanisms are likely to be preserved in humans . the temporal association between weight gain , changes in circulating leptin levels , hr , and bp were first examined . four - week - old c57bl/6j mice on a chow diet were implanted with radiotelemetric bp probes ; baseline measurements were recorded at 6  weeks . as bw increased ( figure  1a ) , plasma leptin levels also progressively increased ( figure  1b ) . after 4  weeks of hfd , hr became significantly elevated ( figure  1c ) and remained elevated throughout the 20  week recording period compared to chow fed mice . systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were significantly elevated after 12  weeks of hfd ( figures 1d and 1e ) , subsequent to the rise in plasma leptin concentration . when hfd - fed mice were returned to a chow diet following 20  weeks of hfd feeding , mice lost 3.9  g in the first week , 3.9  g in the second week , 3.4  g in the third week , and 0.4  g in the fourth week ( figure  1a ) . dbp reduced after 1  week ( figure  1e ) , and sbp ( figure  1d ) and hr ( figure  1c ) reduced after 2  weeks of chow feeding . these findings suggest that leptin appears to increase before hr and bp increases in dio , effects that are reversed with weight loss . we examined whether the effects seen in dio mice are dependent upon leptin signaling , using mice lacking leptin ( ob / ob ) or the lepr ( db / db ) . hfd - fed ob / ob and db / db mice were significantly ( p  < 0.001 ) heavier than hfd - fed dio and chow - fed lean , ob / ob , and db / db mice ( figure  2a ) . despite the increased severity of obesity in the ob / ob and db / db mice , only dio mice exhibited elevated hr , sbp , and dbp ( figures 2b2 g ) , demonstrating that increased bw alone is not the cause of the increase in bp in obesity . there was a greater difference in hr and bp between dio mice to other mice during the dark period compared to the light period ( figures s1a  s1c available online ) . the increase in cardiovascular parameters in dio mice was not due to increased activity ( figures s1d and s1e ) . strong correlations were found in c57bl/6j mice between plasma leptin concentration and hr ( figure  2h ) , sbp ( figure  2i ) , and dbp ( figure  2j ) . no correlation was found between plasma insulin concentration and hr , sbp , or dbp ( figures s1g  s1i ) . to examine the direct effect of leptin on bp , leptin - deficient ob / ob mice were treated for 11  days with either leptin or vehicle . after 11  days of treatment , the average plasma leptin concentration of ob / ob mice treated with leptin was 27.7  2.9  ng / ml compared to 36.3  4.9  ng / ml in dio mice and 6.6  1.1  ng / ml in lean mice ( figure  s1f ) . exogenous leptin treatment in ob / ob mice progressively decreased bw ( figure  s1 m ) and food intake ( fi ) ( figure  s1n ) compared to leptin - treated db / db mice and vehicle - treated ob / ob mice . despite this decrease in bw , leptin treatment increased hr ( figures 2k and 2l ) and sbp ( figures 2 m and 2n ) in leptin - treated ob / ob mice compared to leptin - treated db / db mice and vehicle - treated ob / ob mice . dbp also increased in ob / ob leptin - treated mice , however , not to the same extent as sbp ( figures 2o and 2p ) . the magnitude of the increase in hr and bp in leptin - treated ob / ob mice decreased with time , coincident with their substantial reduction in bw . the 24  hr circadian rhythm of hr ( figure  s2j ) , sbp ( figure  s2k ) , and dbp ( figure  s1l ) of leptin - treated ob / ob mice was increased toward the baseline recordings of dio mice . peripheral administration of a leptin antibody into dio mice for 5  days caused a significant reduction in hr ( figure  3a ) , sbp ( figure  3b ) , and dbp ( figure  3c ) , although no change in bw was observed ( figure  3d ) . central administration of a leptin receptor antagonist ( lra ) into the lateral ventricle ( lv ) of hypertensive dio mice over a 7  day period significantly reduced the elevated hr ( p  < 0.001 ; figure  3e ) . by the seventh day of lra treatment , hr was comparable to the average hr of lean animals ( figures 3f and s2a ) . systolic bp also progressively decreased when dio mice were treated with the lra , and by day 5 of lra treatment , sbp was significantly ( p  0.05 ) decreased compared to vehicle - treated dio mice ( figure  3 g ) . by the seventh day of lra treatment , sbp was significantly ( p  < 0.05 ) lower compared to the sbp at baseline in dio mice and was comparable to the baseline sbp of lean mice ( figures 3h and s2b ) . dbp also decreased throughout the lra treatment period ( figures 3i , 3j , and s2c ) . these changes were seen despite the absence of a significant change in fi ( figure  3k ) , indicating that these changes are mediated by blocking the effects of leptin signaling rather than through changes in fi and bw . to determine whether leptin s effects on bp and hr were mediated by neurons in the dmh , the lra was injected directly into the dmh of hypertensive dio mice daily over 7  days . lra treatment in the dmh decreased hr ( figures 4a , 4b , and s3a ) and sbp ( figures 4c , 4d , and s3b ) by day 7 of treatment . no significant change in dbp ( figures 4e , 4f , and s3c ) was observed . furthermore , injection of an aav expressing a short hairpin rna directed against the lepr ( hommel et  al . , 2006 ) in the dmh of dio mice led to a sustained decrease in sbp ( figures 4 g , 4h , and s3d ) after 4  weeks . utilizing mice in which the lepr is flanked by loxp sites and can be deleted by an aav cre , hfd for 20  weeks induced increased sbp at baseline , whereas aav cre administration into the dmh ( hence deleting the lepr in only the dmh regions ) decreased sbp ( figure  4i ) . additionally , we examined the effects of reactivation of the lepr by injection of an aav - expressing cre recombinase into the dmh of normotensive morbidly obese lepr null mice ( expressing a loxp flanked stop codon in front of the lepr , called lepr transcriptional blocker or both hr ( figure  4j ) and sbp ( figure  4k ) increased rapidly after lepr expression was reactivated in just the dmh of obese lepr - deficient mice . to determine the electrophysiological effect of leptin on dmh neurons , we recorded the electrical activity of neurons expressing lepr in lepr - cre - yfp mice ( leshan et  al . , 2012 ) . application of 100  nm leptin induced membrane depolarization and/or an increase in spontaneous action potential firing rate in 38.2% of recorded neurons ( figure  5a ) . leptin induced a mean peak amplitude depolarization of 4.9  1.0  mv from a mean resting potential of 49.9  3.1  mv to a new steady - state membrane potential of 45.0  2.7  mv ( p  = 0.0004 ) . leptin - induced excitation was associated with an increase in firing rate from a mean control basal level of 0.48  0.28  hz to 0.64  0.34  hz in the presence of leptin ( n  = 5 ) , effects that were at least in part reversible after washout of leptin ( p  = 0.59 ) . in two neurons , voltage - current relations , generated in response to a range of depolarizing and hyperpolarizing rectangular - wave current pulses ( 150 to  + 100  pa , 1,000  ms duration ) revealed that leptin - induced excitation was principally associated with a trend for a decrease ( p  = 0.86 ) in neuronal input resistance from 841  111 m in the absence to 743  138 m in the presence of leptin . plots of the voltage - current relations revealed extrapolated reversal potentials for leptin - induced excitation around 35  mv ( figure  5b ) . taken together , these data suggest that leptin - induced excitation is mediated via activation of one or more nonselective cation conductances . in addition to these effects on membrane input conductance , leptin also induced modulation of intrinsic subthreshold active conductance in a subpopulation of dmh neurons . in dmh neurons , membrane depolarization from negative holding potentials ( < 65  mv ) or depolarization at the offset of the membrane response to hyperpolarization from potentials close to resting potential ( 45 to 50  mv ) evoked a transient depolarizing potential consistent with activation of a low - threshold t - type calcium conductance . in the presence of leptin , this potential was prolonged ( figure  5b ) , the half - time to  decay increasing from 132  59  ms in the absence to 179  61  ms in the presence of leptin . these data are consistent with leptin modulating intrinsic active conductances in a subpopulation of dmh neurons . in addition to these postsynaptic effects , leptin induced an increase in spontaneous excitatory postsynaptic potentials ( epsps ) in a subpopulation ( n  = 2 ) of dmh neurons ( figure  s4b ) . the mean frequency of spontaneous epsps increased from 0.07  0.02  hz to 0.31  0.11  hz in the presence of leptin . these epsps could be of sufficient magnitude to reach threshold for firing , suggesting that indirect presynaptic effects of  leptin on dmh neurons can contribute to leptin - induced increases in neuronal excitability . in  vivo , the effects of directly altering the neuronal activity of  lepr - expressing dmh neurons were assessed using engineered pharmacologically selective chimeric ion channels for activating and silencing neuron activity ( magnus et  al . , 2011 ) . briefly , this technology requires the injection of a virus , which only infects and replicates in a cre - dependent manner , into cre - expressing mice . the virus drives the cre - dependent expression of a modified ion channel , containing a psam element . after injection ( intraperitoneal injection ) , the otherwise inert molecule psem binds to an ion channel that contains a psam element , which opens the ion pore and allows ion flux across the cell membrane , which depolarizes or hyperpolarizes virus - infected , cre - expressing neurons . using male 20-week - old chow - fed lean lepr - cre - yfp transgenic mice ( leshan et  al . , 2012 ) , we investigated whether activation of dmh lepr - expressing  neurons could increase hr and bp . lean lepr - cre - yfp mice were injected bilaterally with activator virus , an engineered ionotropic serotonin receptor packaged in an aav2 ( psyn::flex - rev - psam y115f:5ht3 hc - ires - gfp ) into the dmh . in lean mice 21  days after virus injection , twice daily intraperitoneal ( i.p . ) administration of the receptor ligand psem ( i.p . 2x daily [ 5  mg / kg ] ) for 3  days significantly increased hr and bp ( p  0.05 ; figures s4d lepr - cre - yfp mice , an inhibitory virus , an engineered ionotropic glycine receptor packaged into an aav2 ( aav2 : psyn::flex - rev - psam l141f : glyr - ires - gfp ) was administered bilaterally into the dmh . compared to vehicle treated mice , psem acutely decreased hr ( figures 5c and 5d ) and sbp ( figures 5e and 5f ) , but no significant change in dbp was observed ( figures 5 g and 5h ) . sub - chronic daily treatment with psem reduced hr ( by 72.6  5.6  bpm ) , sbp ( by 6.1  1.7  mmhg ) and dbp over 3  days ( figures 5i , 5j , and s4c ) , effects which were reversed once psem treatment ceased . treatment of mice with psem , prior to virus administration had no effect on hr or bp ( figures s4 g and s4h ) . cumulatively , these findings indicate that leptin signaling is required for the changes in bp seen in dio and that lepr expressing neurons in the dmh are necessary and sufficient to , cause these effects . these data also support the premise that the hypertension induced by leptin in the dmh is due to leptin - induced depolarization of dmh neurons . homozygous complete loss - of - function mutations in the gene encoding leptin ( lep ) are associated with undetectable leptin levels and severe early - onset obesity in humans ( montague et  al . , 1997 ) . we measured bp in the fasted rested state in eight leptin - deficient children and 42 equally obese controls of the same age with normal leptin levels for the degree of obesity studied in the same conditions ( table s1 ) . sbp was significantly lower in leptin - deficient children compared to age- and bmi - matched controls ( figure  6a ; p  < 0.05 ) ; there was no difference in dbp . a statistically significant attenuation of sbp was also seen in severely obese children who were homozygous for complete loss - of - function mutations in the lepr gene ( n  = 12 ) compared to 48 age- and bmi - matched controls ( figure  6b ; p  < there were no significant differences in resting hr between the groups ( data not shown ) . administration of recombinant methionyl human leptin to individuals with congenital leptin deficiency leads to significant weight loss ( farooqi et  al . , 1999 ; ozata et  al . , 1999 ) . in a previously reported experimental paradigm ( galgani et  al . , 2010 ) , three leptin - deficient adults were studied before and after treatment with recombinant leptin for 19  weeks , which was sufficient to cause 15.5  0.5  kg weight loss . in parallel , three age- and bmi - matched controls were studied before and after a diet and exercise intervention , which achieved a comparable degree of weight loss ( 14.8  1.76  kg ) . the three adult leptin - deficient individuals were found to have normal bp despite severe obesity ( figure  6c ) . whereas the map of the obese control group decreased as expected ( figure  6d ) , the bp of the leptin - deficient adults did not change . similarly , there was no statistically significant change in bp after recombinant leptin administration to leptin - deficient children ( data not shown ) . these studies in rare individuals completely lacking leptin or functioning lepr support the assertion that leptin is necessary for the increased bp associated with obesity in humans . cumulatively , these studies demonstrate that leptin signaling is  necessary for obesity - induced increased bp . we have used multiple convergent methods , including making lean mice obese , by adding leptin systemically and by restoring lepr to the  dmh of lepr - deficient mice . in all these studies , bp increased . similarly , we have studied reduced leptin signaling in leptin- and lepr - deficient mice , neutralized circulating leptin with a systemic antibody , infused lepr antagonist icv and intra - dmh ; used shrna to knock down lepr expression ; selectively deleted lepr ; and inhibited lepr - expressing neurons in  the dmh of obese hypertensive mice . in all of these experiments , reducing leptin signaling reduced bp , even in the presence of  obesity . clinical studies in severely obese humans with two  different forms of defective leptin signaling show that  these  observations are relevant to human physiology and pathophysiology . leptin can acutely increase hr , bp , and bat , even in anesthetized animals , presumably through activation of the sns ( enriori et  al . intracerebroventricular ( icv ) leptin increases sna to numerous organs , including the lumbar , kidney , and bat regions ( haynes et  al . , 1999 ) . central antagonism of lepr caused a reduction in bp and hr in dio hypertensive mice . although early lesion studies confirm the importance of the dmh for the control of energy homeostasis , little is known about the neurochemical phenotype of the neurons present in the dmh ( elmquist et  al . , 1999 ; the dmh appears to have a critical ability to control thermogenesis and is  involved in elevated bat - mediated thermogenesis found in obese mice ( enriori et  al . , 2011 ; fan et  al . , 2005 ; morrison et  al . , 2008 ) . in the studies conducted with antagonism or knocking down of the lepr in dio mice these results suggest that the dmh lepr - expressing neurons are not essential for leptin s effects on bw because the mice lost no additional weight when leprs were inactivated . although lesion studies have previously demonstrated a critical role of the dmh in the control of bw , we have shown that long - term knockdown of the lepr does not significantly affect bw , despite the significant reduction in bp and previously reported increase in bat temperature in obesity ( elmquist et  al . , 1999 ; enriori et  al . , 2011 ) . additionally , acute activation or inhibition of lepr - expressing neurons did not change fi or bw . contrary to our expectations , we did not observe an increase in bp with the administration of leptin to leptin - deficient patients . first , the magnitude of leptin s effect on bp may be too small to be detected given the sensitivity of the tools for measurement of bp in humans and the small number of individuals studied . notably , despite weight loss with continued leptin administration , we did not see a significant decrease in bp in these patients as would be expected in more common forms of obesity . these effects are comparable to the effects of leptin on total energy expenditure ( tee ) , another phenotype mediated by sympathetic tone . we have previously shown that , in contrast to weight loss in obese controls where tee decreases , tee does not change with leptin administration in leptin deficiency ( galgani et  al . , 2010 ) . whether leptin increases bp ( to a small degree ) or leptin - deficient individuals respond differently to weight loss can not be deduced from our data . however , our results are in line with the suggestion that the effect of leptin administration may be to prevent the reduction in bp expected in congenitally leptin - deficient individuals as they lose weight . in previous trials of leptin in common obesity , no effects on bp were observed ( heymsfield et  al . , 1999 ) . rosenbaum and leibel have shown that controlled 10% weight loss in an experimental setting is sufficient to reduce leptin levels and is associated with decreased sna ( rosenbaum et  al . , 2005 ) . it is possible that leptin could be producing some of its cardiovascular effects by acting peripherally . lepr are expressed throughout the brain , including the brainstem ; additionally , lepr are expressed on cardiac myocytes , the kidney , and arteries , including the coronary arteries ( knudson et  al . we treated dio mice peripherally with a leptin antibody and then in a separate experiment , dio mice were treated centrally with the lepr antagonist , and in both experiments , similar responses were observed . in mice lacking the lepr , re - expression of the lepr in only the dmh caused a dramatic increase in bp and hr . the opposite effects occurred when the leptin receptors were deleted from only the dmh region in hypertensive mice . thus , although lepr expression in peripheral regions may play a role , the results presented here clearly demonstrate a key role for the dmh lepr - expressing neurons in mediating the changes in bp in obesity . experiments in rodents and humans support a role for melanocortin signaling in the regulation of bp . central administration of -msh increases bp and hr in wild - type mice , but not in mc4r mice , which maintain a normal bp despite severe obesity ( kuo et  al . , 2003 heterozygous loss - of - function mutations in  mc4r are associated with a reduced prevalence of hypertension , low sbp , lower urinary noradrenaline excretion , and reduced peripheral nerve sna ( greenfield et  al . , 2009 ; sayk et  al . , 2010 ) . moreover , systemic administration of a centrally active melanocortin receptor agonist acutely increased bp in obese volunteers ( greenfield et  al . , 2009 ) . as such , some of leptin s effects on bp may be mediated by the melanocortin system . however , there are some indications that , in obesity , the pomc neurons in the arh become nonresponsive to leptin ( this has been termed leptin resistance ) , and this may limit how much the actions of leptin can be transduced by the pomc neurons in the arh ( enriori et  al . , 2007 ; knight et  al . , 2010 ; previous research has implicated the pomc neurons of the arh in the regulation of obesity - induced hypertension via activation of the ikk/nf-b pathway ( purkayastha et  al . , 2011 ; zhang et  al . , how pomc neurons interact with the dmh lepr - expressing neurons has not been addressed in these studies , but the dmh lepr - expressing neurons do send direct efferent projections to arh neurons ( gautron et  al . it is possible that the lepr - expressing neurons in the dmh express mc4r or that the dmh lepr - expressing neurons act through other neurons that express mc4r ( liu et  al . , 2003 ) . the degree to which leptin s effects on bp in obesity are dependent upon intact melanocortin signaling remains to be determined ( do carmo et  al . , 2011 ; harlan et  al . dmh lepr - expressing neurons are known to project to numerous brain regions , including the pvh ( elmquist et  al . , 1998 ; gautron et  al . dmh neurons also project to other nuclei , including the raphe pallidus nucleus ( rpa ) and rostral ventrolateral medualla ( rvlm ) ( cao and morrison , 2003 ; horiuchi et  al . microinjection of leptin into the dmh of anesthetized rats can induce an acute increase in hr and bp ( marsh et  al . , 2003 ) . the dmh - rpa connection has already been recognized to be important in the regulation of bat thermogenesis in response to leptin ( zhang et  al . , 2011 ) . the rvlm , however , appears to have a greater control over the regulation of bp , compared to regulation of thermogenesis and hr ( horiuchi et  al . the neurochemical phenotype of the lepr - expressing neuron populations in the dmh is still debated ( lee et  al . , 2012 ) . further studies will be necessary to characterize the dmh circuits that contribute to leptin s effects on blood pressure and to characterize the mechanisms by which these neurons modulate sympathetic outputs to the heart and peripheral blood vessels . although results here strongly demonstrate the role of leptin in contributing to elevated bp in obesity , a number of other hormones that change in response to weight gain could contribute . as expected , we found that , compared to lean mice , plasma insulin was elevated in all obese models . despite this increase in plasma insulin , it was only dio mice that exhibited significantly elevated bp , and insulin levels were not correlated with hr or  bp , suggesting that insulin contributes little to the chronic elevation of bp seen in obesity . also , insulin sensitivity as measured by hyperinsulinemic euglycemic clamps is comparable in human mc4r deficiency versus obese controls despite a  lower bp and reduced urinary catecholamine excretion in mc4r - deficient subjects ( greenfield et  al . , 2009 ) . therefore , our data do not support a role for insulin in mediating obesity - induced increases in bp . in conclusion , these observations suggest that pharmacological approaches based on the modulation of leptin s effects on specific subpopulations of neurons could represent a potentially useful therapeutic strategy for the treatment of obesity - associated hypertension and for the prevention of some obesity - associated cardiovascular disease . all mice were housed in a controlled environment in which lights were on a 12  hr light/12  hr dark cycle ; temperature and humidity remained constant . in experiments to examine the development of hypertension , 4-week - old male c57bl/6j mice were implanted with radiotelemetry probes ( dsi , usa , model ta11pa - c10 ) . these mice were allowed 2  weeks recovery postsurgery and , following baseline recordings , mice were spilt onto either chow ( 4.8% of fat , mouse and rat rodent chow diet , specialty feeds , glen forrest , australia ) or hfd ( 43% of fat , sf04 - 001 , specialty feeds , western australia , australia ) for 20  weeks ( 140  days ) in which recordings were taken every 13 to 15  days . after 20  weeks ( 140  days ) , the hfd fed mice were swapped onto a chow diet ( 4.8% of fat , sf04 - 001 , specialty feeds , western australia , australia ) for 4  weeks . in all other experiments , male c57bl/6j , leptin - deficient ( ob / ob ) , lepr - deficient ( db / db ) , lepr - deficient ( leprtb ( berglund et  al . , 2012 ) , lepr flox , and lepr - cre - yfp mice ( leshan et  al . , 2012 ) were placed on either a chow diet or hfd diet at 4  weeks of age and continuing for 20 consecutive weeks , after which mice were used for experiments . specific experimental procedures for radiotelemetry , pharmacological studies , genetic manipulations , and electrophysiological studies are detailed in the supplemental information . all human studies were conducted according to the principles outlined in the declaration of helsinki and after approval by local ethical committees . systolic and diastolic bp were measured in the rested , fasted state using wrist bp monitors ( omron healthcare , hamburg , germany ) in leptin - deficient ( n  = 8) and leptin - receptor - deficient children ( n  = 12 ) ( farooqi et  al . , 2002 , 2007 ) . these control subjects had been tested for mutations in leptin , leptin receptor , and mc4r . control subjects were age and bmi matched to leptin - deficient and leptin - receptor - deficient subjects ( n  = 42 and 48 , respectively ) ( wheeler et  al . , 2013 ) . leptin - deficient adult patients received pretreatment testing at the pennington center , along with weight- and bp - matched control subjects . the leptin - deficient patients then received a 3  month treatment of recombinant leptin ( leptin [ amgen inc . , thousand oaks ; galgani et  al . , 2010 ] ) . leptin levels were administered a low - calorie diet ( galgani et  al . , 2010 ) for 920  weeks to cause the same weight loss and also returned to the pennington center for posttesting . the study design was approved by the ethics committee of the pennington biomedical research center . approval for leptin replacement included ucla irb approval ( april 9 , 2001 ) and fda approval ( ind application number 61690 ) . p  values were calculated by either unpaired or paired two - tailed student s t  test , one - way anova with bonferroni post hoc test or two - way anova with bonferroni post hoc test . p  < 0.05 , p  < 0.01 , and p  < 0.001 . extended experimental proceduresradiotelemetryradiotelemetry probes were implanted into the left carotid artery ( ta11pa - c10 data science international ) under isoflurane anesthesia . all mice received 2  weeks recovery before any baseline recordings were measured.chronic leptin infusionchow fed , ob / ob or db / db mice were implanted with radiotelemetry probes , and baseline recordings were made . alzet 14  day osmotic minipumps ( model 1002 , cupertino , ca ) to administer either vehicle or leptin ( peprotech , rocky usa ) ( 30  g / per day in 6  l of solution [ 1.25  g/0.25  l per hour ] ) were implanted into a subcutaneous pocket 3hrs prior to the dark period . pumps had been prepared and incubated at 37c , 8 - 9hrs prior to implantation . pumps where removed after 11  days ( prior to the onset of the dark period ) and the mice recovered from treatment while radiotelemetry measurements continued to be recorded ( db / db mice were killed at day 11).immunohistochemistry and radioimmunoassayimmunohistochemistry ( ihc ) for pstat3 and radioimmunoassay ( ria ) measurement to determine plasma leptin concentration were performed as previously described ( enriori et  al . , 2007).peripheral neutralization of endogenous leptinto neutralize leptin s actions a goat igg against recombinant mouse leptin ( af498 from r and d systems , minneapolis ) was peripherally ( i.p . ) administered into dio mice for 5 consecutive days , following a sham injection period . injections of 24  g in 90  l of antibody solution or 90  l of igg control ( antibodies australia , victoria , australia ) . the dose of the antibody was determined from previous research ( konstantinides et  al . , 2004 ) . all mice where randomly treated.central blockade of endogenous leptinto reduce the activation of lepr expressing neurons in the brain , a leptin receptor antagonist ( lra ) ( mouse leptin antagonist mutant l39a / d40a / f41a , 0.5  l of 5g/l , protein laboratories , rehovot , israel ) was used as previously reported ( enriori et  al . , 2011 ) . in dio mice that where implanted with radiotelemetry probes , they were additionally equipped with either a lateral ventricle ( lv ) ( plastics one , 2  mm long 22 gauge , implanted 0.5  mm posterior and 1  mm lateral to bregma , 2  mm below the surface of the skull ) or dmh cannula ( plastics one , 4  mm long , 26 gauge , implanted 1.9  mm posterior , 0.4  mm lateral to bregma , 4  mm below the surface of the skull ) as described previously ( enriori et  al . , 2011 ) . following a 7  day recovery period , mice where all injected for 5  days twice daily with sham acsf injection ( 0.5  l ) into cannulas . mice then received acsf or the lra , injected directly into cannulas , twice daily for 7  days , immediately following the dark period and immediately prior to the dark period . following the 7  day injection period mice where monitored daily for a 7  day recovery period.chronic knockdown of leptin receptors in the dorsomedial hypothalamusdio mice were implanted with radiotelemetry probes and after 14  days of recovery baseline recordings mice were injected bilaterally into the dmh region ( coordinated 1.8  mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2  mm below the surface of the skull ) 0.5  l of either an aav scrambled shrna or aav leprshrna over a 20  min period each side ( hommel et  al . , 2006 ) . bw and fi were measurements daily and radiotelemetry recordings were measured periodically throughout the treatment period.reactivation and deletion of leptin receptor in the dmhlepr loxtb mice and lepr flox mice ( berglund et  al . , 2012 ) after which mice were implanted with radiotelemetry probes . following the surgery ( 2  weeks ) , baseline recordings where measured . mice where then injected into the dmh ( coordinates 1.8  mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2  mm below the surface of the skull ) with an aav cre virus . mice where then monitored and cardiovascular parameters measured throughout.neuron activation and silencing with psam / psem systemsto examine the effect of activation or inhibition of dmh lepr expressing neurons , we injected a virus that only infects and replicates in a cre dependent manner , into lepr - cre - yfp mice ( leshan et  al . , 2012 ) . after injection , the otherwise inert molecule psem binds to an ion channel that contains a psam element which causes opening of ion pore in the channel and allows ion flux across the cell membrane , which causes depolarization or hyperpolarization of cre - expressing neurons . the psam element only binds psem , this system is similar to dreadds , but uses direct ligand gated ion channels rather than gpcr s to drive neuron specific effects ( magnus et  al . , 2011 ) . these mice where placed on either the chow or hfd for 20  weeks then implanted with radiotelemtry probes and 2  weeks later bp radiotelemetry baseline recordings were made . following this the lepr - cre - yfp dio mice were injected in the dmh ( coordinates 1.8  mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2  mm below the surface of the skull ) with the inhibitor virus , psyn::flex - rev - psam l141f : glyr - ires - gfp ( aav2 , unc gene therapy center vector core , nc , usa ) , lean chow fed lepr - cre - yfp mice were injected in the dmh ( coordinated 1.8  mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2  mm below the surface of the skull ) with the activator virus , psyn::flex - rev - psam y115f:5ht3 hc - ires - gfp ( aav2 , unc gene therapy center vector core , nc , usa ) . injection of either of these virus s did not change baseline cardiovascular function , until the peripheral injection of the inert psem ligand , that caused opening of ion pores and activation or inhibition of neurons . after a 21  day recovery period cardiovascular parameters were re - examined and then mice where sham injected followed by acute or 3 consecutive days of treatment with psem ( 5  mg / kg , i.p . , twice daily ; apex scientific inc . stony brook , ny ) or vehicle , randomized , crossover design ( magnus et  al . , 2011).electrophysiologyexperiments were undertaken on lepr - yfp - cre mice ( leshan et  al . , 2012 ) following 20  weeks of chow diet feeding , ad lib . on the day of electrophysiological experiment the brain was rapidly removed , submerged in ice cold artificial cerebrospinal fluid ( acsf ) and coronal slices ( 200 - 250  m thick ) cut using a vibratome ( leica , vt1000s , uk ) . slices were maintained at room temperature in oxygenated acsf ( gassed with 95% 02 , 5% co2 ) for at least an hour prior to recording . for recording , slices were continuously perfused at room temperature with acsf of the following composition ( in mm ) : 127.0 nacl , 1.9 kcl,1.2 kh2po4 , 26.0 nahco3 , 2.0 d - glucose , 8.0 d - mannitol , 1.3 mgcl2 , 2.4 cacl2 , equilibrated with 95% o2 , 5% co2 , ph 7.3  7.4 . lepr - yfp expressing neurons in the dmh were visualized using fluorescence , fitted with appropriate filters and infrared video microscopy with dic optics and recordings obtained from these neurons with an axopatch 700a amplifier . patch pipettes were pulled using a horizontal puller ( sutter instrument co. , novato , ca , usa ) from thin - walled , filamented borosilicate glass ( gc150tf-10 ; harvard apparatus ltd ) with resistances between 4 and 8 m when filled with intracellular solution . the pipette solution comprised ( mm ) : 140 k - gluconate , the ph was adjusted to 7.3 with koh and the osmolarity to around 310 mosm with sucrose . data were filtered at 2  5 khz , digitized at 2  10 khz ( digidata 1322 , mds analytical technologies ) and stored on pc running pclamp 9 data acquisition software . analysis of electrophysiological data was carried out using clampfit 9.2 software ( mds analytical technologies ) . was determined using unpaired , non - parametric student s t test with p  < 0.05 considered as significant . leptin was made up as a concentrated stock solution and diluted to the concentration required ( 100 nm ) in acsf immediately before commencement of electrophysiological recording . radiotelemetry probes were implanted into the left carotid artery ( ta11pa - c10 data science international ) under isoflurane anesthesia . chow fed , ob / ob or db / db mice were implanted with radiotelemetry probes , and baseline recordings were made . alzet 14  day osmotic minipumps ( model 1002 , cupertino , ca ) to administer either vehicle or leptin ( peprotech , rocky usa ) ( 30  g / per day in 6  l of solution [ 1.25  g/0.25  l per hour ] ) were implanted into a subcutaneous pocket 3hrs prior to the dark period . pumps had been prepared and incubated at 37c , 8 - 9hrs prior to implantation . pumps where removed after 11  days ( prior to the onset of the dark period ) and the mice recovered from treatment while radiotelemetry measurements continued to be recorded ( db / db mice were killed at day 11 ) . immunohistochemistry ( ihc ) for pstat3 and radioimmunoassay ( ria ) measurement to determine plasma leptin concentration were performed as previously described ( enriori et  al . , 2007 ) . to neutralize leptin s actions a goat igg against recombinant mouse leptin ( af498 from r and d systems , minneapolis ) was peripherally ( i.p . ) administered into dio mice for 5 consecutive days , following a sham injection period . injections of 24  g in 90  l of antibody solution or 90  l of igg control ( antibodies australia , victoria , australia ) . the dose of the antibody was determined from previous research ( konstantinides et  al . , 2004 ) . all mice where randomly treated . to reduce the activation of lepr expressing neurons in the brain , a leptin receptor antagonist ( lra ) ( mouse leptin antagonist mutant l39a / d40a / f41a , 0.5  l of 5g/l , protein laboratories , rehovot , israel ) was used as previously reported ( enriori et  al . , 2011 ) . in dio mice that where implanted with radiotelemetry probes , they were additionally equipped with either a lateral ventricle ( lv ) ( plastics one , 2  mm long 22 gauge , implanted 0.5  mm posterior and 1  mm lateral to bregma , 2  mm below the surface of the skull ) or dmh cannula ( plastics one , 4  mm long , 26 gauge , implanted 1.9  mm posterior , 0.4  mm lateral to bregma , 4  mm below the surface of the skull ) as described previously ( enriori et  al . , 2011 ) . following a 7  day recovery period , mice where all injected for 5  days twice daily with sham acsf injection ( 0.5  l ) into cannulas . mice then received acsf or the lra , injected directly into cannulas , twice daily for 7  days , immediately following the dark period and immediately prior to the dark period . following the 7  day injection period mice where monitored daily for a 7  day recovery period . dio mice were implanted with radiotelemetry probes and after 14  days of recovery baseline recordings were measured . in a stereotaxic apparatus ( stoelting , usa ) mice were injected bilaterally into the dmh region ( coordinated 1.8  mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2  mm below the surface of the skull ) 0.5  l of either an aav scrambled shrna or aav leprshrna over a 20  min period each side ( hommel et  al . , 2006 ) . bw and fi were measurements daily and radiotelemetry recordings were measured periodically throughout the treatment period . lepr loxtb mice and lepr flox mice ( berglund et  al . , 2012 ) after which mice were implanted with radiotelemetry probes . following the surgery ( 2  weeks ) , baseline recordings where measured . mice where then injected into the dmh ( coordinates 1.8  mm posterior , 0.3/-0.3  mm lateral from bregma and 5.2  mm below the surface of the skull ) with an aav cre virus . mice where then monitored and cardiovascular parameters measured throughout . to examine the effect of activation or inhibition of dmh lepr expressing neurons , we injected a virus that only infects and replicates in a cre dependent manner , into lepr - cre - yfp mice ( leshan et  al . , 2012 ) . after injection , the otherwise inert molecule psem binds to an ion channel that contains a psam element which causes opening of ion pore in the channel and allows ion flux across the cell membrane , which causes depolarization or hyperpolarization of cre - expressing neurons . the psam element only binds psem , this system is similar to dreadds , but uses direct ligand gated ion channels rather than gpcr s to drive neuron specific effects ( magnus et  al . , 2011 ) . these mice where placed on either the chow or hfd for 20  weeks then implanted with radiotelemtry probes and 2  weeks later bp radiotelemetry baseline recordings were made . following this the lepr - cre - yfp dio mice were injected in the dmh ( coordinates 1.8  mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2  mm below the surface of the skull ) with the inhibitor virus , psyn::flex - rev - psam l141f : glyr - ires - gfp ( aav2 , unc gene therapy center vector core , nc , usa ) , lean chow fed lepr - cre - yfp mice were injected in the dmh ( coordinated 1.8  mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2  mm below the surface of the skull ) with the activator virus , psyn::flex - rev - psam y115f:5ht3 hc - ires - gfp ( aav2 , unc gene therapy center vector core , nc , usa ) . injection of either of these virus s did not change baseline cardiovascular function , until the peripheral injection of the inert psem ligand , that caused opening of ion pores and activation or inhibition of neurons . after a 21  day recovery period cardiovascular parameters were re - examined and then mice where sham injected followed by acute or 3 consecutive days of treatment with psem ( 5  mg / kg , i.p . , twice daily ; apex scientific inc . stony brook , ny ) or vehicle , randomized , crossover design ( magnus et  al . , 2011 ) . experiments were undertaken on lepr - yfp - cre mice ( leshan et  al . , 2012 ) following 20  weeks of chow diet feeding , ad lib . on the day of electrophysiological experiment the brain was rapidly removed , submerged in ice cold artificial cerebrospinal fluid ( acsf ) and coronal slices ( 200 - 250  m thick ) cut using a vibratome ( leica , vt1000s , uk ) . slices were maintained at room temperature in oxygenated acsf ( gassed with 95% 02 , 5% co2 ) for at least an hour prior to recording . for recording , slices were continuously perfused at room temperature with acsf of the following composition ( in mm ) : 127.0 nacl , 1.9 kcl,1.2 kh2po4 , 26.0 nahco3 , 2.0 d - glucose , 8.0 d - mannitol , 1.3 mgcl2 , 2.4 cacl2 , equilibrated with 95% o2 , 5% co2 , ph 7.3  7.4 . lepr - yfp expressing neurons in the dmh were visualized using fluorescence , fitted with appropriate filters and infrared video microscopy with dic optics and recordings obtained from these neurons with an axopatch 700a amplifier . patch pipettes were pulled using a horizontal puller ( sutter instrument co. , novato , ca , usa ) from thin - walled , filamented borosilicate glass ( gc150tf-10 ; harvard apparatus ltd ) with resistances between 4 and 8 m when filled with intracellular solution . the pipette solution comprised ( mm ) : 140 k - gluconate , the ph was adjusted to 7.3 with koh and the osmolarity to around 310 mosm with sucrose . data were filtered at 2  5 khz , digitized at 2  10 khz ( digidata 1322 , mds analytical technologies ) and stored on pc running pclamp 9 data acquisition software . analysis of electrophysiological data was carried out using clampfit 9.2 software ( mds analytical technologies ) . was determined using unpaired , non - parametric student s t test with p  < 0.05 considered as significant . leptin was made up as a concentrated stock solution and diluted to the concentration required ( 100 nm ) in acsf immediately before commencement of electrophysiological recording .