ng2 cells ( neuron - glia chondroitin sulphate proteoglycan 2 ) , also referred to as synantocytes , polydendrocytes , -astrocytes , oligodendrocyte precursor cells ( opc ) have been recognized as the fourth type of glial cells in the mammalian central nervous system , representing about 58% of the glial cell population .
morphologically , at light microscopy they resemble astrocytes , though their cell body is less rounded and cytoplasmic processes slender . at electron microscopy ,
ng2 cells show flattened nucleus with decondensed chromatin , well - represented cytoplasmic organelles and , where opposed to neurons , focal membrane densities .
opposite to astrocytes , ng2 cells do not express gfap nor s100 protein , but express the ng2 chondroitin sulphate proteoglycan , a membrane protein with a large extracellular domain , whose function is still obscure . in the central nervous system , ng2 cells can be found in both gray and white matter and have been proposed as resident oligodendrocyte and astrocyte precursors . actually , their number increases in the vicinity of demyelization foci in some neuropathologies and after wound insult . on the other hand
, ng2 cells do not express glutamate transporters , but have calcium - permeable ampa receptors , that cluster in apposition to nerve terminals , forming conventional synapses .
this feature suggests that these cells must have a more complex function than to be a simple reservoir of oligodendrocytes or astrocytes .
the cerebellum , which has a pivotal role in motor control , timing and learning , has been relatively neglected in studies concerning aging . in elderly people ,
movement and stability control are less efficient , with increasing fall risk and serious consequences and social costs . here
, we describe that ng2 cells are not evenly distributed in the rat cerebellum and that their distribution and morphology undergo changes during aging .
sprague dawley rats , aged 4 ( 3 animals ) and 28 months ( 3 animals ) , purchased from harlan nossan ( correzzana , italy ) and maintained in standard conditions ( 12 h light / dark cycle , 23c temperature , food and water ad libitum ) for few days were used .
experiments were performed in accordance with the italian and european community law for the use of experimental animals and were approved by a local bioethical committee .
animals were deeply anesthetized with chloral hydrate ( 0.35 g / g body weight ) and perfused transcardially with 4% paraformaldehyde in pbs .
cerebella were excised , postfixed in the same fixative for 4 h and cryoprotected in 30% sucrose overnight .
two out of every 10 sections were processed for immunocytochemistry for ng2 chondroitin sulphate proteoglycan as follows . briefly , after treatment with 3% h2o2 in 10% methanol in pbs and with 10% normal serum in pbs containing 0.1% nan3 , the sections were incubated overnight with 1:200 rabbit polyclonal antibody against ng2 chondroitin sulphate proteoglycan ( chemicon , billera , ma , usa ) .
afterwards the sections were incubated for 90 min in anti - rabbit biotinylated igg , followed by streptavidin - hrp complex ( vectastain elite kit , vector , ca , usa ) .
. some adjacent sections from 28-month - old rats were processed for calbindin immunocytochemistry ( with 1:5000 rabbit polyclonal antibody against calbindin , swant , ch ) to check for the integrity of the cytoarchitecture . for the reaction control
, some sections were incubated with normal rabbit serum instead of the primary antibodies . to quantify the changes in the cerebellar area in which ng2 glia was densely scattered ,
sections were photographed at low magnification and a morphometric analysis was performed using nih image j software .
the total section area and the area in which ng2 cells were densely represented ( relative area ) were measured by outlining the contours .
since the total area of cerebellar sections can vary with age and among animals of the same group , the ratio between the relative and total areas was calculated to standardize data .
in the cerebellar vermis of 4-month - old rats , ng2 cells appear to be unevenly distributed . in lobules vi to viii according to larsell ng2 cells are uniformly and densely scattered in all cerebellar layers , including the white matter ( wm ) ; their long , slender cytoplasmic processes extend to form a spider web - like structure throughout all the cerebellar cortex ( figure 1a ) . on the contrary , in the more rostral and caudal lobules ( i  vi and ix  x ) ,
only sporadic cells may be observed in the wm and the granule cell layer ( igl ) , the majority of cells lying in the molecular layer ( ml ) .
in addition , they appear less intensely stained than in the more central lobules ( figure 2a ) . in control reaction sections
in fact , in mammals the central part of cerebellar vermis receives its main input from the cerebral cortex and is therefore called cerebrocerebellum . the anterior and posterior parts receive afferents primarily from the spinal cord and are therefore termed spinocerebellum . with regard to efferent connections ,
the cerebrocerebellum acts on the cerebral cortex and the spinocerebellum influences the spinal cord . in particular , the cerebrocerebellum receives information , via the corticopontine tract , about movements that are being planned and about commands that are sent out from the motor cortex . in response
, it can modulate the activity of the motor cortex so that movements are performed accurately and smoothly
. the high density of ng2 cells in the central lobules of the cerebellar vermis in the rat may therefore be due to a higher functional complexity of the cerebrocerebellum , which in turn requires a higher modulation from glial cells .
the functional significance of this rapid communication between neurons and ng2 cells is still obscure , but it has been suggested that through synapses ng2 cells can monitor neuronal activity and act consequently , maybe by releasing neurotrophic substances . 

figure 1a , b ) representative sections of the cerebellar vermis of 4- and 28-month - old rats showing the distribution of ng2 cells .
the cerebellar area in which ng2 cells are uniformly and densely scattered ( dotted line ) is enlarged in aging animals ; c ) a section from a 28-month - old animal incubated with normal rabbit serum instead of the primary antibody .
scale bar : 2 mm . 
  a , b ) representative sections of the cerebellar vermis of 4- and 28-month - old rats showing the distribution of ng2 cells .
the cerebellar area in which ng2 cells are uniformly and densely scattered ( dotted line ) is enlarged in aging animals ; c ) a section from a 28-month - old animal incubated with normal rabbit serum instead of the primary antibody .
figure 2 in both 4- and 28-old rats , in the more rostral and caudal lobules of the cerebellum , ng2 cells are only sporadically present , mainly in the molecular layer .
inset in b : in the molecular layer of both 4-and 28-month - old animals , immunostained cells show irregular soma and long , slender processes , typical of ng2 glia .
scale bar a , b : 400 m ; inset 20 m . 
  in both 4- and 28-old rats , in the more rostral and caudal lobules of the cerebellum , ng2 cells are only sporadically present , mainly in the molecular layer .
inset in b : in the molecular layer of both 4-and 28-month - old animals , immunostained cells show irregular soma and long , slender processes , typical of ng2 glia .
the distribution of ng2 cells in the cerebellar vermis of 28-month - old rats is similar to that observed in 4-month - old animals .
however , the area in which ng2 cells are more represented and uniformly scattered ( relative area ) appears to be enlarged , involving also the lobule v and part of the lobule ix ( figure 1b ) .
morphometric analysis revealed that the ratio between the relative and total areas of the sections is increased by 62% in aging animals ( 0.320.01 vs 0.520.02 , in 4- and 28-month - old rats , respectively ; p<0.0001 ) .
the expansion of the cerebellar area in which ng2 cells are densely represented in aged rats is an enigmatic finding .
the spinocerebellum is somatotopically organized as well as its afferent fibers , so that signals from different body parts are kept segregated .
the spinocerebellum is involved in the maintenance of the muscle tone and plays a role in the coordination of movements of walking .
taking into consideration that , if not exercised , muscle become hypotonic with aging , it is tempting to hypothesize that the increase in ng2 cells reflects a higher demand of neurotrophic factors or control from cerebellar neurons . obviously , this is a hypothesis that needs to be tested in animals subjected to daily exercise ( treadmill walking , for instance ) to maintain muscle tone , and that will have an unconfutable answer only when the function of ng2 cells will be fully elucidated . in aged rats , in lobules i  iv and ix  x , ng2 cells are no more present in the igl and wm and in the ml they appear paler ( figure 2b ) than in young - adult animal .
however , punctate immunoreaction product is present in correspondence with the apical pole and stem dendrite of purkinje cells ( figure 3 ) .
the punctate reaction in lobules belonging to the spinocerebellum may represent profiles of hypertrophic ng2 cell processes that ensheath the soma and stem dendrite of purkinje cells .
this is the cell region where climbing fibers from neurons of the inferior olivary complex form the majority of synapses with purkinje cells .
the cerebellum/ inferior olivary complex system plays a major role as a comparator of sensory information and motor output . climbing fibers inform about errors in the execution of a movement , so that the cerebellum can adjust it .
in this view , since climbing fibers establish synapses with ng2 cells , also ng2 cells could be informed and modulate purkinje cell activity accordingly . on the other hand , it can not be excluded that the punctate immunoreaction product represents extracellular product .
in fact , the large ng2 proteoglycan ectodomain may be cleaved and released in the extracellular matrix .
this would also explain the pale staining of ng2 cells residing in the ml in 28-month - old animals . 

figure 3a ) in the 4-month - old rat cerebellum , in the most rostral and caudal lobules , no immunoreaction product is present in the purkinje cell layer ; b , c ) representative photographs of portions of the most rostral and caudal cerebellar lobules in 28-month - old rats .
punctate immunoreaction product is visible at the apical pole of purkinje cells ; d ) particular of the most caudal cerebellar lobules in a section immunostained for calbindin from 28-month - old rat .
ml , molecular layer ; pc , purkinje cell layer ; igl , granule cell layer .
scale bar a , b , c : 20 m ; d : 50 m . 
 
a ) in the 4-month - old rat cerebellum , in the most rostral and caudal lobules , no immunoreaction product is present in the purkinje cell layer ; b , c ) representative photographs of portions of the most rostral and caudal cerebellar lobules in 28-month - old rats .
punctate immunoreaction product is visible at the apical pole of purkinje cells ; d ) particular of the most caudal cerebellar lobules in a section immunostained for calbindin from 28-month - old rat .
ml , molecular layer ; pc , purkinje cell layer ; igl , granule cell layer .
scale bar a , b , c : 20 m ; d : 50 m .
 we describe by ng2 ( neuron - glia chondroitin sulphate proteoglycan 2 ) immunocytochemistry an uneven distribution of ng2 glial cells in the rat cerebellum , being them more represented in the central lobules of the cerebellar vermis , belonging to the cerebrocerebellum . 
 the cerebellar distribution of ng2 cells changes in aging rats , in which the area where the cells appear to be densely scattered throughout all cerebellar layers involves also more rostral and caudal lobules . 
 in addition , in aging rats , in the most rostral and caudal lobules belonging to the spinocerebellum , punctate reaction product is present at the apical pole of purkinje cells , i.e. in the area where the majority of synapses between olivary climbing fibers and purkinje cells occur . 
 data suggest that the different distribution of ng2 cells is correlated to differences in physiology among cerebellar areas and reflects changes during aging . 