melanocyte - stimulating hormone release inhibiting factor-1 ( mif-1 ) , also known as plg based on its amino acid structure ( pro - leu - gly - nh2 ) , is an endogenous brain peptide that exerts a variety of pharmacological effects on the central nervous system .
clinical studies have shown that mif-1 can alleviate symptoms in parkinson 's disease ( pd ) and mental depression .
these have been summarized elsewhere , although the review overlooked a report in the chinese literature of favorable results of mif-1 in pd .
mif-1 was the first hypothalamic peptide shown to act   up  on the brain , not just   down  on the pituitary . in human plasma ,
a duration of 5 days is required for 50%   degradation of mif-1 at 37c .
this remarkable stability of mif-1 in human blood , coupled with its persisting biological activity , makes mif-1 a worthwhile candidate as a human therapeutic agent .  
the therapeutic potential of mif-1 indicates the importance of further determining how it acts on the brain .
there already is evidence that mif-1 activates cns pathways related to opiate and dopaminergic systems based on the following evidence : ( 1 ) mif-1 antagonizes opiate actions , and the first report of such activity correctly predicted the discovery of other endogenous antiopiate peptides blocking the analgesic effect of morphine and enkephalin in a radiant heat tail - flick assay .
mif-1 also significantly antagonized the effects of morphine in a double - blind study in humans .
( 2 ) mif-1 is effective in the dopa - potentiation , oxotremorine antagonism , and deserpidine antagonism tests . in a small number of patients with pd ,
barbeau   found its potentiation of the effects of levodopa to be remarkable ; ( 3 ) mif-1 can modulate dopaminergic transmission in - vitro as well as in - vivo , increasing the binding affinity of agonists to the high - affinity state of the dopamine receptor and shifting the ratio of high- and low - affinity states of the dopamine receptor in favor of the g - protein - coupled high - affinity state .
( 4 ) the actions of mif-1 appear rather selective toward dopamine receptors since it does not interact with other aminergic receptors such as adrenergic , gabaergic , or serotonergic receptors .
( 5 ) mif-1 also facilitates passive acquisition of brightness discrimination , passive avoidance retention , appetitive maze performance , and inhibits shock - suppressed water intake , findings interpreted as contributing to the processes of memory consolidation [ 1 , 12 ] .
however , mif-1 does not change camp in the brain in contrast to the increase induced by -msh , and its induction of cgmp is not robust in rat brain .
this led us to study alternative cellular signaling pathways . a number of exogenous stimuli , including an increase in neuronal activity , trigger the transcription of c - fos , which is an immediate early gene that in combination with the specific jun proteins forms the heterodimeric transcription regulator , ap-1 .
the extracellular signal - regulated kinase ( erk)-1/2 pathways are involved in the induction and regulation of c - fos . in this study , we performed a brain mapping of the neuronal expression of c - fos after intravenous ( iv ) or intracerebroventricular ( icv ) injection of mif-1 .
all studies were conducted following a protocol approved by the institutional animal care and use committee .
adult male c57bl/6j mice were purchased from jackson laboratories ( bar harbor , me ) and housed in the animal care facility for at least 2 weeks before study .
the mice were housed 4/cage and provided with regular rodent chow and water ad libitum .
the light span was 07:0019:00 hours in a light - dark ( ld ) 12:12 lighting regimen .   
mif-1 was purchased from phoenix pharmaceuticals ( burlingame , ca ) and reconstituted in phosphate - buffered saline ( pbs ) before use .
rabbit polyclonal anti - c - fos antibody , goat anti - pstat3-tyr antibody , and mouse anti - perk e-4 monoclonal antibody were purchased from santa cruz biotechnology ( santa cruz , ca ) .
louis , mo ) . in accordance with a protocol approved by the institutional animal care and use committee , c57bl/6j male mice ( 57 weeks old )
mif-1 was delivered either into the isolated left jugular vein ( iv ) at 10  g / mouse in a volume of 50  l ( n = 3 ) , or into the right lateral cerebral ventricle ( icv ) at 1  g / mouse in a volume of 1  l ( n = 3 ) .
the icv coordinates were 2  mm lateral and 0.2  mm posterior to the bregma , and 2.5  mm below the skull .
the mice were perfused intracardially with 30  ml of normal saline ( ns ) followed by 60  ml of 4% paraformaldehyde .
the brain was postfixed overnight in 4% paraformaldehyde and cryoprotected in 15% and then 30% sucrose .
coronal sections of 20  m thickness were obtained by the use of a cryostat .
the tissue sections were permeabilized with 0.3% triton x-100 and blocked with 10% normal donkey serum , and incubated with a primary antibody overnight at 4c .
the antibodies include rabbit polyclonal anti - c - fos antibody ( 1  :  200 , santa cruz biotechnology sc-52 ) and anti - rat neun antibody ( 1  :  100 , chemicon ) .
after thorough washing , they were incubated with respective alexa488-conjugated secondary antibodies at room temperature for 1 hour , washed , and mounted .
sh - sy5y human neuroblastoma cells ( american type culture collection , atcc , manassas , va ) were grown in dulbecco 's modified eagle 's medium ( dmem ) with 10% fetal bovine serum ( fbs ) .
the cells were differentiated by treatment with 10  m of all - trans retinoic acid ( sigma , st .
sixteen hours after serum starvation , the cells were treated with mif-1 ( 110  ng / ml for different time intervals ( 03 hours ) .
all cells were plated at the same time and treated according to the time intervals designed for individual experiments .
the cells were lysed in ice - cold ripa buffer ( 100  mm nacl , 10  mm tris , ph 7.2 , 0.1% sds , 1% triton x-100 , 1% deoxycholate , 5  mm edta ) in the presence of protease inhibitor cocktail ( pierce , rockford , il ) .
thirty to 50  g of protein was electrophoresed on 12% sds - polyacrylamide gel and transferred to a nitrocellulose membrane ( bio - rad , hercules , ca ) .
the membrane was blocked with 5% non - fat dry milk in tris - buffered saline ( ph 7.6 ) containing 0.1% tween-20 , and probed with rabbit anti - c - fos ( polyclonal , 1  :  200 , santa cruz biotechnology , sc-52 ) , goat anti - pstat3 tyr 705 ( polyclonal , 1  :  1,000 sc-7993 ) , mouse anti - perk e-4 ( monoclonal , 1  :  500 , sc-7383 and mouse anti--actin ( monoclonal , 1  :  10,000 , sigma , a2228 ) overnight at 4c . after thorough washing
, the membranes were incubated with horseradish peroxidase - conjugated secondary antibody for 1 hour at room temperature .
the signals were developed with enhanced chemiluminescence - plus western blotting detection reagents ( amersham biosciences , piscataway , nj ) .
we previously observed that mif-1 is saturably transported from blood to brain . in this study , we demonstrate that iv injection of mif-1 increased c - fos immunoreactivity in different brain regions .  
the regions that showed the highest c - fos activation include cingulate cortex ( figures 1(c3 ) and 2(a ) ) , infralimbic cortex ( figures 1(c1 ) and 2(e ) ) , nucleus accumbens ( figures 1(c1 ) and 2(i ) ) , paraventricular nucleus ( pvn ) in the hypothalamus ( figures 1(c5 ) and 2(f ) ) , medial basal amygdaloid nucleus ( figures 1(c6 ) and 2(h ) ) , fiber tract in piriform cortex ( figures 1(c6 ) and 2(g ) ) , paraventricular thalamic nucleus ( figures 1(c6 ) , 2(c ) , and 2(d ) ) , and other thalamic nuclei ( figures 1(c7 ) and 2(b ) ) .
double labeling with neun showed that many of the cells expressing c - fos were neurons .
the increase in c - fos expression was greater after iv injection than after icv injection in many brain regions .
this is consistent with the ability of stable substances in the cerebral circulation to reach all parts of the brain rapidly .
after icv , the increase of c - fos was mainly seen in the paraventricular nucleus of the hypothalamus ( figures 1(b5 ) and 2(f ) ) .
table 1 summarizes the number of c - fos immunopositive cells in different brain regions .
western blotting showed that the c - fos signal was increased 60 minutes after mif-1 treatment ( 10  ng / ml ) .
there was also a dose - dependent increase , highest at the maximal dose of 10  g / ml , but even the lowest concentration of mif-1 tested ( 6.25  ng / ml ) showed a robust induction of c - fos 1 hour later ( figure 3(b ) ) .    
western blotting analysis showed that perk expression was increased in sh - sy5y cells 10 minutes after mif-1 treatment ( 10  ng / ml ) ( figure 4 ) .
by contrast , the cells responded to mif-1 with an initial reduction of pstat3 at 10 and 60 minutes , followed by increased pstat3 expression at 2 and 3 hours ( figure 5 ) .
in this study , we showed that mif-1 induces c - fos activation in different regions of the brain and in cultured neurons .
this is the first report stating that mif-1 increases the phosphorylation of erk , indicative of activation of a mitogen - activated protein kinase ( mapk ) pathway .
our results show that mif-1 decreased pstat3 initially ( 10 and 60 minutes ) , but then increased pstat3 at later times ( 2 and 3 hours ) in neuronal culture , probably reflecting a secondary mechanism .
this also is the first report showing that mif-1 modulates pstat3 activation , although the functional consequences are not clear . these are not classical elements of the gpcr signaling pathway , and suggest a broader action of this orphan ligand .  
while a peptide like mif-1 may either show direct cns effects or activate secondary mediators , c - fos immunoreactivity reflects a final common pathway of brain activation as a result of mif-1 application .
the immediate early gene product c - fos is an easily identifiable and rather sensitive marker for cns activation .
the increase of c - fos has been demonstrated after various stimuli , including growth factors , ion channel activation , neurotransmitter release , and behavioral modifications .
the increase of its expression can be mediated by many intracellular signaling pathways , such as increases in camp , calcium influx , and activation of mapk .
our results show that the increase in c - fos expression is greater after iv injection than after icv injection in many brain regions .
after icv administration , the increase of c - fos immunoreactivity was mainly seen in the pvn of the hypothalamus . considering that less than 1% of mif-1 from blood permeates the blood - brain barrier to reach the brain , like most centrally active peptides and even l - dopa and morphine
, this indicates that blood - borne mif-1 is more effective than when administered into the slower moving ventricular system of the brain .
this is not surprising considering that no neuron is more than about 8  m from a capillary .
it is also possible that peripherally injected mif-1 activates additional mediators , thus triggering a cascade of signalling events .
the implications of mif-1-induced c - fos activation in different brain regions are discussed below .
( 1 ) cingulate cortex : the cingulate cortex is an anatomically and functionally heterogeneous region and it modulates emotion and mood . attention deficit in pd has been explained by dopamine depletion of the cingulate cortex .
fornix - induced lesions can reduce c - fos immunoreactivity in the cingulate cortex of rats .
earlier studies showed that lesions of the cingulate cortex affect behavior in the open - field and interspecies aggression behavior in rats .
melatonin ( 5-methoxy - n - acetyltryptamine ) was reported to be critically involved in the regulation of both mood and pain .
melatonin receptor type 1 ( mt1)-knockout mice display depression - like behavior with altered sensory responses and attention deficits .
these previous studies show that the central melatoninergic system might play an important role in the mechanism of interactions between pain and depression , and the anterior cingulate cortex could be a forebrain region of interest in this process .
mif-1 can potentiate the melanocyte - lightening effect of melatonin in rats , and its effects in patients with pd are associated with marked mood elevation .
therefore , the mif-1-induced c - fos activation in the cingulate cortex supports the speculation that the effectiveness of mif-1 in treating movement disorders may be associated with increased melatonin secretion .  
( 2 ) thalamus : the lateral geniculate nucleus of the thalamus is involved in fluctuations in both visual attention and visual awareness .
mif-1 can facilitate acquisition of brightness discrimination , probably by affecting processes of attention when rats are tested with a spatial extradimensional shift problem after acquisition of a visual task .
c - fos expression has been found in the medial geniculate body ( mgb ) of the thalamus after mice acquired a visually cued conditioned fear .
the activation of c - fos by mif-1 in the thalamus supports the direct effects of mif-1 there .  
( 3 ) infralimbic cortex : the infralimbic cortex ( il ) is a cortical region in the medial prefrontal cortex that is important in tonic inhibition of subcortical structures and emotional responses such as fear
this illustrates cortical control over extinction processes , which is one of the simplest forms of emotional regulation .
less c - fos immunoreactivity is present in the il of the extinction - resistant mouse compared with the control . in a 12-choice warden maze for a palatable food reward ,
rats receiving mif-1 have shorter latencies and make fewer errors than controls during learning , but not extinction , of the task .
( 4 ) nucleus accumbens : both typical and atypical antipsychotic drugs increase c - fos protein expression in the nucleus accumbens shell
. the ability of mif-1 to increase c - fos in the forebrain may have considerable predictive validity for antipsychotic drug actions .
administration of mif-1 to patients with depression showed substantial improvement within a few days after initiation of treatment [ 3335 ] .
previous studies showed that antipsychotic drugs like clozapine and amperozide preferentially increase dopamine release in the rat nucleus accumbens and prefrontal cortex .
hippocampal dopamine receptors modulate c - fos expression in the rat nucleus accumbens that is evoked by chemical stimulation of the ventral hippocampus .
thus , mif-1 may activate cells in the nucleus accumbens and modulate dopaminergic activity there to alleviate mood and schizoaffective disorder .
( 5 ) pvn : the pvn plays important roles in neuroendocrine and autonomic nervous system controls . for example
, many experimental challenges ( such as restraint stress , dehydration , and immune challenge ) induce c - fos expression in the pvn [ 38 , 39 ] . although mif-1 has no effects on a naloxone - sensitive peptide yy ( pyy ) model of hyperphagia after pyy injection into the pvn
, the induction of c - fos by mif-1 through both iv and icv injection indicates that pvn is a major site of action for mif-1 .   
( 6 ) medial amygdaloid nucleus : an avoidance task in the elevated t maze can increase fos protein expression in the medial amygdaloid nucleus .
the amygdala is a point of convergence for conditioned and unconditioned stimuli and seems to impart emotional value to sensory stimulation .
increased expression of immediate early genes in the amygdala has been reported in several paradigms of aversive conditioning .
these findings may provide a physiological basis for mif-1 induced c - fos activation in the amygdala .
( 7 ) piriform cortex : the presence of the c - fos protein has been shown in the piriform cortex at different stages during the acquisition of trace conditioning in rabbits .
mif-1 also facilitates passive avoidance retention and inhibits shock - suppressed water intake , findings interpreted as contributing to the processes of memory consolidation . in elevated - plus - maze tests , mice treated with tyr - mif-1
tend to spend more time in the open arms compared with the control group , suggesting the anxiolytic properties of this peptide that shows structural homology with mif-1 .
training induces c - fos mrna expression in the piriform cortex   and the neocortex of n-3-fatty acid depleted rats , who showed learning improvement in an olfactory discrimination task . mif-1 also improves the capacity of rats to store information received through olfactory cues in social investigatory behavior . in summary ,
the results show that mif-1 increased c - fos expression in brain regions involved in the regulation of mood , anxiety , depression , and memory .
blood - borne mif-1 induced a greater extent of activation than that after icv , suggesting a prominent role of blood - brain barrier permeation .
the activation status of perk and pstat3 may contribute to the overall expression level of c - fos .
thus , the results indicate the cellular mechanisms of actions of mif-1 that may underlie the therapeutic effects of mif-1 in the treatment of parkinson 's disease and depression .
 mif-1 ( pro - leu - gly - nh2 ) is a tripeptide for which the therapeutic potential in parkinson 's disease and depression has been indicated by many studies . 
 however , the cellular mechanisms of action of mif-1 are not yet clear . here 
 , we show the specific brain regions responsive to mif-1 treatment by c - fos mapping , and determine the kinetics of cellular signaling by western blotting of perk , pstat3 , and c - fos in cultured neurons . 
 the immunoreactivity of c - fos was increased 4 hours after mif-1 treatment in brain regions critically involved in the regulation of mood , anxiety , depression , and memory . 
 the number of cells activated was greater after peripheral treatment ( intravenous delivery ) than after intracerebroventricular injection . 
 in cultured sh - sy5y neuronal cells , c - fos was induced time- and dose - dependently . 
 the activation of cellular c - fos was preceded by a transient increase of mitogen - activated protein kinase perk but a reduction of phosphorylated signal transducer and activator of transcription ( pstat3 ) initially . 
 we conclude that mif-1 can modulate multiple cellular signals including perk , and pstat3 to activate c - fos . 
 the cellular activation in specific brain regions illustrates the biochemical and neuroanatomical basis underlying the therapeutic effect of mif-1 in parkinson 's disease and depression . 