Interlaminar Glia and Other Glial Themes Revisited: Pending Answers Following Three Decades of Glial Research

neuroglia

Perspective Interlaminar Glia and Other Glial Themes Revisited: Pending Answers Following Three Decades of Glial Research

Jorge A. Colombo

Unit of Applied Neurobiology (UNA, CEMIC-CONICET), Buenos Aires 1053, Argentina; [email protected]

Received: 23 January 2018; Accepted: 22 February 2018; Published: 1 March 2018

Abstract: This review aims to highlight the various significant matters in glial research stemming from personal work by the author and associates at the Unit of Applied Neurobiology (UNA, CEMIC-CONICET), and some of the pending questions. A reassessment and further comments on interlaminar astrocytes—an astroglial cell type that is specific to humans and other non-human primates, and is not found in rodents, is presented. Tentative hypothesis regarding their function and future possible research lines that could contribute to further the analysis of their development and possible role(s), are suggested. The possibility that they function as a separate entity from the “territorial” astrocytes, is also considered. In addition, the potential significance of our observations on interspecies differences in in vitro glial cell dye coupling, on glial diffusible factors affecting the induction of this glial phenotype, and on their interference with the cellular toxic effects of cerebrospinal fluid obtained from L-DOPA treated patients with Parkinson´s disease, is also considered. The major differences oberved in the cerebral cortex glial layout between human and rodents—the main model for studying glial function and pathology—calls for a careful assessment of known and potential species differences in all aspects of glial cell biology. This is essential to provide a better understanding of the organization and function of human and non-human primate brain, and of the neurobiological basis of their behavior.

Keywords: interlaminar astrocytes; role(s) of interlaminar astrocytes; control of interlaminar glia development; thalamic regulation of interlaminar glia; comparative dye coupling; glial diffusible factors

1. Introduction Following retirement from active laboratory work, a change in the experimental line of research at the Unit of Applied Neurobiology (UNA, CEMIC-CONICET) laboratories—at present aimed at studying more decisively neurocognitive issues—has provided the opportunity to propose this sort of brief account and reassessment of unresolved and pending questions on “glial issues” that were dealt with in our neurobiological laboratory in recent decades. This personal viewpoint is intended to stress and revisit several aspects of our own observations on glial physiology and comparative studies, which are aimed at encouraging further research in the ﬁeld. Thorough updates, comprehensive reviews, and inspirational thoughts on other related aspects of glial physiology can be found in Kettenmann and Ransom (2005), Verkhratsky and Butt (2013), and Verkhratsky and Nedergaard (2018) [1–3], as well as in numerous individual articles, for it, seems evident that experimental research on neuroglia has entered an era of further fertile analysis. Yet, as it will be considered later, caution and weighed decisions should be exerted in attempting interspecies extrapolations—in the present context referred to brain glia—and building our understanding of human and non-human primate brain organization and phyisology based on what can be considered

Neuroglia 2018, 1, 7–20; doi:10.3390/neuroglia1010003 www.mdpi.com/journal/neuroglia Neuroglia 20182018,, 11, 3 2 of 148

can be considered “general mammalian” characteristics, stemming from non‐comparative Rodentia “generalapproaches. mammalian” This may characteristics,limit our views stemming on the neurobiological from non-comparative basis of primate Rodentia brain approaches. evolution Thisand maybehavior. limit our views on the neurobiological basis of primate brain evolution and behavior.

2. Interlaminar Astrocytes and Primate Brain Evolution InterlaminarInterlaminar gliaglia or interlaminaror interlaminar astrocytes astrocytes (Figure (Figure1) essentially 1) essentially represent arepresent primate evolutionary a primate developmentevolutionary development [4–8] linked to [4–8] the split linked between to the split prosimians between and prosimians Anthropoidea. and Anthropoidea. They are characterized They are bycharacterized a cell soma by placed a cell in soma lamina placed I—in in general lamina next I—in to thegeneral glia limitans—ofnext to the glia the limitans—of cerebral cortex, the and cerebral long, descending,cortex, and long, cell processes descending, that cell could processes extend that for aboutcould 1extend mm, thus for about traversing 1 mm, more thus than traversing one cortical more laminae.than one Remainingcortical laminae. characteristics Remaining are characteristics mentioned below. are mentioned below.

Figure 1. Coronal section from the striate cerebral cortex obtained from an adult Saimiri boliviensis. Figure 1. Coronal section from the striate cerebral cortex obtained from an adult Saimiri boliviensis. Note dense packing of glial fibrillary acidic protein‐immunoreactivity (GFAP‐IR) astroglial Note dense packing of glial ﬁbrillary acidic protein-immunoreactivity (GFAP-IR) astroglial processes, processes, the relative height of the band of processes and the frequent appearance of slender the relative height of the band of processes and the frequent appearance of slender bulbous endings. bulbous endings. Broken line indicates the limit of lamina I. Scale bar; 100 μm (Adapted from Figure Broken line indicates the limit of lamina I. Scale bar: 100 µm. Adapted from Figure 1 in [9]. 1 in [9]).

The expression of interlaminar glia among orders and species, according to our screening of available samples and histological identificationidentification procedures, is incipient—theyincipient—they take form of isolated events—in the prosimian lemur (no brain samples were available at that time from galago and tarsier),tarsier), it isis absentabsent inin CallithricidaeCallithricidae (marmosets(marmosets and the tamarins),tamarins), constant and yet variable in itsits palisadepalisade expressionexpression in in Ceboidea Ceboidea (New (New World World monkeys) monkeys), and, and fully fully expressed expressed in Cercopithecidae in Cercopithecidae (Old World(Old World monkeys) monkeys) and Hominoideaand Hominoidea (great (great apes apes and and humans) humans) [4–8 [4–8].]. Their Their original original development development in primatesin primates possibly possibly relates relates to to point point mutation mutation or or epigenesis—predating epigenesis—predating some some of of the the factorsfactors thatthat are hypothetically linkedlinked to to the the later later increase increase in in brain brain size size (e.g., (e.g., expensive expensive tissue tissue hypothesis hypothesis of Aiello of Aiello and Wheelerand Wheeler [10]; socio-ecological[10]; socio‐ecological hypothesis hypothesis of Dunbar of Dunbar [11] and [11] other and other convergent convergent hypotheses hypotheses on brain on sizebrain evolution size evolution in humans). in humans). The author The author considers considers that its that emergence its emergence is probably is probably associated associated with the with less promoted—inthe less promoted—in terms of evolutionaryterms of evolutionary impact on brainimpact function—development on brain function—development of columnar organizationof columnar oforganization the cerebral of cortex, the cerebral which cortex, remains which to be remains further to comparatively be further comparatively explored in termsexplored of distributedin terms of neuraldistributed circuits neural (or modules) circuits (or and modules) of its impact and on of cerebral its impact cortex on informationcerebral cortex efficiency information (response efficiency speed, unit(response assembly speed, synchrony, unit assembly cognitive synchrony, fluidity). cognitive In this respect, fluidity). the possibilityIn this respect, that theythe possibility contribute that to a “non-territorial”they contribute managementto a “non‐territorial” of the cerebral management cortex intercellular of the cerebral space cortex and intercellular intercellular interactions space and intercellular interactions should be analyzed. Consequently, whether they are coupled or not to

Neuroglia 2018, 1 9 should be analyzed. Consequently, whether they are coupled or not to “local–territorial–astrocytes” could add to the characterization of their physiological role(s) and integration into the cerebral cortex processing. It seems apparent that brain evolution among anthropoid primates has proceeded in a series of continuous structural and functional—neurotransmitter and receptor dynamics for once—deletions and aggregates, which were not exclusively confined to neurons (see e.g., [12]). Among them, appearance of interlaminar astrocytes would represent an evolutionary—“primate-specific”—brain cell trait added to the “general mammalian” brain glial cell family, as suggested by Colombo and colleagues [4,7,8]. Although functional insertion of interlaminar astrocytes into cerebral cortex organization remains highly speculative (see below), their ontogenetic development and some interactions and responses following experimental procedures and pathologies, provide grounds for further research inquiries and analysis. One important obstacle to experiments aimed at advancing into the general understanding of human brain evolution and organization, resides in the limited access to extant anthropoid species for comparative research purposes—for interlaminar astrocytes are not a “general mammalian” event. In order to minimize the need for more general invasive protocols, perhaps the use of biopsic material would help to bypass such limitation, besides the possibility of novel methodological imaging approaches. Overcoming these problems is imperative, because the positive selection of interlaminar glia in the primate order calls for a full characterization and understanding of its role in cerebral cortex function. It may be opportune to state upfront that although other laboratory species provide valuable insights into “general mammalian” brain organization and evolution, the above-mentioned limitation generates an unavoidable and objective conceptual “gap” when extrapolating results from non-primate mammals to primates. Such consideration arises since the subtle, bioelectric, ionic/molecular dynamic interactions with cellular receptors operating in a particular brain organization is what provides the finely tuned scaffolding—besides structural or hardware connectivity—for the generation and expression of the characteristic complex and fluid human cognitive and emotional (manifest or introspective) behaviors. In this regard, further advances in the comparative—neuronal and glial—analysis within the primate order—specifically with genetically close extant species—seems critically needed. Perhaps, following the historically theoretical preeminence of the “neuronal doctrine”, most studies have been performed on these cells in anthropoid species, generating a clear gap with respect to advances made on glial cells. In such respect, although not intended to be reviewed here, numerous studies regarding associated neuronal and behavioral issues have been performed in primates by several authors. In particular, for example, some recent studies on the comparative distribution of neurotransmitter receptors in the brains of humans and extant primate species were reported by Zilles and colleagues [13,14], as well as comparative genetic studies by Pu et al. [15], Muntané et al. [16] and Mitchell and Silver [17]. Yet, the ethical limitations on the experimental use of primate species, and the needed minimization of invasive actions to be taken on them, as well as added limitations imposed on the inclusion in experimental protocols of extant species of great apes—mostly those genetically closer as Pan troglodytes and Pan paniscus (chimpanzee and bonobo, respectively)—calls for developing imaginative and minimally invasive research tools and procedures. The “handicap” of glial cells in terms of lacking readily in situ detectable bioelectric signals adds to the limitations on this field of the neurosciences. At any rate, as pointed out by several authors (e.g., [4,12,18]), it has become unavoidable to include the spectrum of glial cells into theoretical constructions of brain evolution and organization. Certainly, this stage has been built following studies based on the access to laboratory rodent species. But time has come to work on new approaches and theoretical models to avoid known limitations to expand glial research into primates. Developmental studies tracing the ontogenetic cellular origin of interlaminar glia remain missing. In the human brain, the developmental expression of interlaminar glia takes place during early Neuroglia 2018, 1 10 Neuroglia 2018, 1, 3 4 of 14 postnataland attaining life, the following adult‐like a periodconfiguration of “physiological of interlaminar astrogliosis” astrocytes by by 20–40 the second days month of postnatal of life [19]. life, andGenetic attaining analysis the adult-likefollowing configuration the isolation of of interlaminar these cells astrocytescould instigate by the detailed second month studies of lifeof their [19]. Geneticevolutionary analysis origin following and cell lineage. the isolation of these cells could instigate detailed studies of their evolutionaryThe soma origin of interlaminar and cell lineage. glial cells is closely apposed to the glia limitans and its short superficialThe soma processes of interlaminar are probably glial cells functionally is closely apposedassociated to thewith glia the limitans subarachnoid and its short space superficial [9]. This processeslayout suggests are probably that their functionally most superficial associated withaspect the could subarachnoid be linked space to [exchange9]. This layout with suggeststhe pial thatvasculature/subarachnoid their most superficial aspect space, could while be linked their to exchangedistal ending—usually with the pial vasculature/subarachnoid a slender bulbous space,formation—has while their been distal shown ending—usually [20] to be connected a slender to bulbous a blood formation—has vessel (Figure been2) or shown“floating” [20] in to the be connectedintercellular to aspace. blood Hence, vessel (Figurea role 2in) or“fast “floating” sink” operations in the intercellular appears space.as a possibility, Hence, a role besides in “fast ion sink”exchange operations through appears the membrane as a possibility, of its long besides interlaminar ion exchange processes. through This the glial membrane morphotype of its rather long interlaminarthan engulfing processes. synaptic This terminals glial morphotype and intimately rather than interacting engulfing with synaptic them terminals as the type and intimatelyof layout interactingreported by with Grosche them et as al. the [21] type for of parenchymal layout reported astroglia, by Grosche interlaminar et al. [21 ]processes for parenchymal would appear astroglia, to interlaminar“navigate” in processes the extracellular would appear space, to “navigate” perhaps inmonitoring the extracellular and regulating space, perhaps ionic/molecular monitoring andimbalances. regulating In ionic/molecularthis regard, their imbalances. membrane In dynamic this regard, characteristics their membrane remain dynamic to be characteristicsdetermined. It remainwould tobe be of determined. significant interest It would to be establish of significant whether interest this totype establish of glia whether is independent this type from of glia the is independentastroglial syncytia, from the and astroglial whether syncytia, they form and a parallel whether network they form that a parallelis perhaps network interconnected that is perhaps at the interconnectedsubpial level. However, at the subpial our attempts level. However, to analyze our possible attempts dye to analyzecoupling possible of interlaminar dye coupling glia—in of interlaminarsamples from glia—in a non samples‐human fromprimate—failed a non-human due primate—failed to technical reasons due to technical (previous reasons exposure (previous to an exposureantifreeze to medium an antifreeze for transport medium of fresh for transport sections affected of fresh membrane sections affected permeable membrane characteristics), permeable and characteristics),the limited number and of the sections limited to number work with. of sections to work with.

FigureFigure 2.2. InterlaminarInterlaminar bulbous endingending inin anan agedaged humanhuman cerebralcerebral cortexcortex sample.sample. Note inin ((AA)) aa mitochondrion,mitochondrion, andand bridgebridge (arrow)(arrow) connectingconnecting withwith bloodblood vesselvessel (asterisk)(asterisk) (also(also inin ((BB)).)). InIn ((BB),), notenote thethe multilamellarmultilamellar structure.structure. Scale bar: 50 50 μµm in (A) and 200 nm inin ((BB).). AdaptedAdapted fromfrom FigureFigure 44 inin ColomboColombo etet al.al. [[20].20].

TheThe possibilitypossibility of of vesicular vesicular transport transport as as reported reported by by Potokar Potokar et al. et [al.22 ][22] for rodentfor rodent glia shouldglia should also bealso considered. be considered. Attainment of ﬁnal length, density and palisade display of interlaminar processes (Figure1) dependsAttainment on species of final and subspecieslength, density characteristics: and palisade for display example, of some interlaminar New World processes monkeys (Figure could 1) presentdepends a non-systematic,on species and subspecies patchy and characteristics: more scattered for or unpredictableexample, some palisade, New World such asmonkeys is the case could of Cebuspresent paella a non(tufted‐systematic, capuchin), patchy while and a moremore typicalscattered palisade or unpredictable occurs in Saimiri palisade, boliviensis such as(black-capped is the case of squirrelCebus paella monkey). (tufted According capuchin), to experimental while a more data, thetypical presence palisade and length occurs of interlaminarin Saimiri processesboliviensis are(black signiﬁcantly‐capped squirrel affected bymonkey). interaction According with thalamic to experimental cortical afferents, data, at the least presence in areas thatand arelength related of tointerlaminar the visual system processes [23] are and significantly spinal cord somatosensoryaffected by interaction input [24 ].with thalamic cortical afferents, at leastWhat in areas are that the signalsare related involved to the invisual determining system [23] the and characteristics spinal cord ofsomatosensory the palisade? input Physiological [24]. signalsWhat driving are morphologicalthe signals involved changes in ofdetermining interlaminar the processes characteristics under the of the reported palisade? conditions Physiological remain undetermined.signals driving Whether morphological effects on changes interlaminar of interlaminar glia (Figures processes3 and4) represent under the a trophic reported or regulatory conditions roleremain of thalamic undetermined. afferents, Whether or an indirecteffects on one interlaminar through their glia cerebral (Figures cortex 3 and projections4) represent on a trophic neuronal or regulatory role of thalamic afferents, or an indirect one through their cerebral cortex projections on neuronal activity [25,26], remains an open question. The disruption of the interlaminar cortical

Neuroglia 2018, 1, 3 5 of 14 palisade after 11–13 months following spinal cord transection—with a lack of evidence of additional astrogliosis—and a somewhat “wavy” individual process display [24], suggests a rather long‐term impact on the rearrangement of the local neuropil, or that it acquired a new steady state condition followingNeuroglia 2018 lesioning,, 1 with loss or perturbation of the original columnar arrangement, and perhaps11 sharing an expanded spatial monitoring due to local disruption of the columnar modules. Neuroglia 2018, 1, 3 5 of 14 activity [25,26], remains an open question. The disruption of the interlaminar cortical palisade after 11–13palisade months after following11–13 months spinal following cord transection—with spinal cord transection—with a lack of evidence a oflack additional of evidence astrogliosis—and of additional aastrogliosis—and somewhat “wavy” a somewhat individual “wavy” process individual display [process24], suggests display a [24], rather suggests long-term a rather impact long on‐term the rearrangementimpact on the rearrangement of the local neuropil, of the or local that itneuropil, acquired or a newthat steadyit acquired state conditiona new steady following state condition lesioning, withfollowing loss or lesioning, perturbation with ofloss the or original perturbation columnar of the arrangement, original columnar and perhaps arrangement, sharing anand expanded perhaps spatialsharing monitoring an expanded due spatial to local monitoring disruption due of theto local columnar disruption modules. of the columnar modules.

Figure 3. Interlaminar GFAP‐IR events observed in coronal (A,B) and tangential (flattened) (C,D) sections of the striate cortex from control, intact (C) and three months visually deprived (D) adult Cebus apella monkey. Asterisk and broken line in (A) indicate the limit of lamina I. Vascular elements in (C,D) are marked by an asterisk. Note paucity of events in sections on the right side. Scale bar: 100 μm. Adapted from Colombo et al. [23].

Quite interestingly, following cortical lesioning or, most clearly, under cerebral cortex Figure 3. Interlaminar GFAP-IR events observed in coronal (A,B) and tangential (ﬂattened) (C,D) pathologicalFigure 3. conditionsInterlaminar (such GFAP as‐IR Alzheimer’s events observed disease in coronal or advanced (A,B) and Down’s tangential syndrome), (flattened) (orC,D aging,) sections of the striate cortex from control, intact (C) and three months visually deprived (D) adult interlaminarsections ofglia the do striate not cortexshow fromreactive control, forms intact (in (C contrast) and three to monthsparenchymal visually astrocytes),deprived (D )but adult rather Cebus apella monkey. Asterisk and broken line in (A) indicate the limit of lamina I. Vascular elements in disappearCebus apella[19,27], monkey. tending Asterisk to lose and their broken characteristic, line in (A) indicateordered, the lay limit out of of lamina long interlaminarI. Vascular elements processes, (inC ,D)(C,D are) are marked marked by anby asterisk.an asterisk. Note Note paucity paucity of events of events in sections in sections on the on rightthe right side. side. Scale Scale bar: bar: 100 µ100m. or to acquire increased bulbous endings (Figures 3 and 4, and [28]). Adaptedμm. Adapted from from Colombo Colombo et al. et [23 al.]. [23].

Quite interestingly, following cortical lesioning or, most clearly, under cerebral cortex pathological conditions (such as Alzheimer’s disease or advanced Down’s syndrome), or aging, interlaminar glia do not show reactive forms (in contrast to parenchymal astrocytes), but rather disappear [19,27], tending to lose their characteristic, ordered, lay out of long interlaminar processes, or to acquire increased bulbous endings (Figures 3 and 4, and [28]).

Figure 4.4.Morphology Morphology and and spatial spatial arrangement arrangement of GFAP-IR of GFAP interlaminar‐IR interlaminar processes in theprocesses somatosensory in the somatosensorycortex of control cortex and long-term of control spinal and cord-transected long‐term spinalMacaca cordindividuals.‐transected (AMacaca) Control; individuals. (B) Operated. (A) Control;Note extreme (B) Operated. departure Note from extreme the rectilinear departure trajectory from the and rectilinear several “terminal trajectory masses”and several (arrowheads). ‘‘terminal µ masses’’Large arrow (arrowheads). indicates direction Large arrow of the indicates cortical surface.direction Scale of the bar: cortical 40 m. surface. Adapted Scale from bar: Reisin 40 μ andm. AdaptedColombo from [24]. Reisin and Colombo [24].

Quite interestingly, following cortical lesioning or, most clearly, under cerebral cortex pathological conditionsFigure (such 4. Morphology as Alzheimer’s and disease spatial orarrangement advanced Down’s of GFAP syndrome),‐IR interlaminar or aging, processes interlaminar in the glia do not showsomatosensory reactive forms cortex (in of contrast control toand parenchymal long‐term spinal astrocytes), cord‐transected but rather Macaca disappear individuals. [19,27], tending(A) Control; (B) Operated. Note extreme departure from the rectilinear trajectory and several ‘‘terminal masses’’ (arrowheads). Large arrow indicates direction of the cortical surface. Scale bar: 40 μm. Adapted from Reisin and Colombo [24].

Neuroglia 2018, 1 12

Neurogliato lose their2018, 1 characteristic,, 3 ordered, lay out of long interlaminar processes, or to acquire increased6 of 14 bulbous endings (Figures3 and4, and [28]). TheThe description description ofof thethe “wavy”“wavy” terminalterminal (15–30(15–30 μµmm inin length)length) segmentsegment [[5,19]5,19] in interlaminar glia (Figures 55–7)–7) usuallyusually shows a tortuous “corkscrew” shape shape that that was was tentatively tentatively interpreted interpreted as as a locala local increase increase in in membrane membrane surface, surface, as as it it also also was was considered considered to to be be its its normally slender bulbous ending (10–15 μµmm inin diameter),diameter), in in some some cases cases being being associated associated to to blood blood vessels vessels [20 [20].]. The The presence presence of of a amitochondrion mitochondrion embedded embedded in in its its terminal terminal implies implies a speciﬁc a specific local local energy energy requirement requirement associated associated with withionic/molecular ionic/molecular exchange exchange mechanisms mechanisms or possibly or possibly general general terminal terminal structural structural maintenance. maintenance. These Thesebulbous bulbous terminals terminals acquire largeracquire and larger sometimes and sometimes even “massive” even proportions “massive” (30proportionsµm in diameter) (30 μm with in diameter)ageing and with in Alzheimer ageing and´s disease,in Alzheimer´s thus possibly disease, representing thus possibly a maladaption representing to a suchmaladaption conditions, to such also conditions,observed, for also example, observed, in Albert for example, Einstein´s in Albert brain samplesEinstein´s [6 brain,29]. samples [6,29].

Figure 5. Striate cerebral cortex sample from 2.5-months-old2.5‐months‐old Saimiri boliviensis. Glial fibrillaryfibrillary acidic protein immunostaining with Nissl stain. Scale Scale bar: bar: 100 100 μµm.m. Adapted Adapted from from Figure Figure 2 2 in in [19]. [9]. Note marked wavy configurationconfiguration of the long (interlaminar) cellular processes. Attempts to immunohistochemically label interlaminar processes with other cytoskeletal or Attempts to immunohistochemically label interlaminar processes with other cytoskeletal or neurotrasmitter markers, besides glial fibrillary acidic protein (GFAP), failed systematically in our neurotrasmitter markers, besides glial fibrillary acidic protein (GFAP), failed systematically in our hands, except at early postnatal ages, as illustrated (Figure6), in which they could be labelled hands, except at early postnatal ages, as illustrated (Figure 6), in which they could be labelled with with a-vimentin. a‐vimentin.

Neuroglia 2018, 1 13 Neuroglia 2018, 1, 3 7 of 14

Figure 6.6. Intrasurgical sample. Human Human frontal frontal cerebral cerebral cortex from aa seven-year-old.seven‐year‐old. Vimentin immunohistochemistry. LongLong processes processes with with “corkscrew” “corkscrew” appearance. appearance. Pia Pia mater mater is is towards towards the the top top of ofthe the ﬁgure figure asterisk asterisk indicates indicates cortical cortical surface. surface. Scale Scale bar: bar: 20 20µm. μm. Adapted Adapted from from Figure Figure 5 in 5 [in9]. [19]. Neuroglia 2018, 1, 3 8 of 14 The role of interlaminar glia in the regulation of the extracellular space and intercellular interactions is also unknown, although some speculative hypotheses were proposed by Reisin and Colombo [30], which were linked to the columnar organization of the neocortex “as proposed by Jakob and Onelli (1913), and later formalized by Lorente de No (1938) and electrophysiologically characterized by Powell and Mountcastle (1959) and Hubel and Wiesel (1965)” cf. [30]. To take this work forward requires studies on fresh tissue sections and/or using procedures, such as “tissue printing”. We have successfully applied tissue printing procedures aimed at developing a new approach to analyze the ionic/molecular dynamics of single interlaminar processes [31], based on previous procedures developed in rats [32]. Unfortunately, these studies could not be continued, but this less invasive procedure—if based on surgical biopsies—could partially overcome the limited access to human and non‐human primate brain samples.

Figure 7. Tangential section of striate cortex from an adult Saimiri boliviensis monkey, reacted for Figure 7. Tangential section of striate cortex from an adult Saimiri boliviensis monkey, reacted for GFAP GFAP with Nissl counterstain, note wavy terminal segments and slender bulbous terminals with Nissl counterstain, note wavy terminal segments and slender bulbous terminals decorating them. decorating them. Scale bar: 20 μm. Adapted from Figure 5 in [5]. Scale bar: 20 µm. Adapted from Figure 5 in [5].

3. Analysis of Astroglia in the In Vitro Conditions Applying lucifer yellow dye coupling procedures comparative studies were performed in human, non‐human primate, and rat cerebral cortex astroglial cell cultures. Their hypothetical role in cerebral cortex modularity was analyzed following initial observations on cell coupling in the cerebral cortical and striatal cells from Cebus apella (tufted capuchin) monkeys, demonstrating its functional preservation following freezing and thawing procedures [33]. Later, a comparison between rat, monkey, and human cell cultures from regional brain samples that were obtained at early ages and stored deep frozen, was undertaken by Lanosa et al. [34]. Rat striatum and cerebral cortex showed different coupling characteristics. Significant differences were observed in dye coupling between rodent and human brain cortical cells in in vitro conditions (Figure 8), which were suggestive of a comparatively reduced diffusion of dye coupling in human samples. It remains to be proven that these observations hold true in situ using brain slices. Should it be confirmed, it could be speculated that in Homo there is less tendency to spread cell coupling, which would aid in limiting spatial compromise of ionic/molecular perturbations and hence contribute more precise spatial (modular) characteristics, through reduction of glial territories.

Figure 8. Interspecies analysis of astroglial coupling levels following lucifer yellow pressure injections in astroglia‐enriched cultures from rat, monkey and human cerebral cortex, expressed as the percentage of cells coupled out of the total cells essayed (percentage of cell coupling). Numbers in parentheses indicate sample size. Significant differences were detected using Pearson’s chi‐square test (* p = 0.006). Adapted from Figure 3 in [34].

For the aforementioned experiments, the ages that were examined were developmentally early, as determined by the availability of samples from human and non‐human primates, using cerebral

Neuroglia 2018, 1, 3 8 of 14

Neuroglia 2018, 1 14

The role of interlaminar glia in the regulation of the extracellular space and intercellular interactions is also unknown, although some speculative hypotheses were proposed by Reisin and Colombo [30], which were linked to the columnar organization of the neocortex “as proposed by Jakob and Onelli (1913), and later formalized by Lorente de No (1938) and electrophysiologically characterized by Powell and Mountcastle (1959) and Hubel and Wiesel (1965)” cf. [30]. To take this work forward requires studies on fresh tissue sections and/or using procedures, such as “tissue printing”. We have successfully applied tissue printing procedures aimed at developing a new approach to analyze the ionic/molecular dynamics of single interlaminar processes [31 ], based on previousFigure procedures 7. Tangential developed section of in striate rats [cortex32]. Unfortunately,from an adult Saimiri these boliviensis studies could monkey, not reacted be continued, for but thisGFAP less with invasive Nissl procedure—if counterstain, basednote wavy on surgical terminal biopsies—could segments and partiallyslender bulbous overcome terminals the limited accessdecorating to human them. and Scale non-human bar: 20 μ primatem. Adapted brain from samples. Figure 5 in [5].

3. Analysis of Astroglia in the In Vitro ConditionsConditions Applying luciferlucifer yellow yellow dye dye coupling coupling procedures procedures comparative comparative studies studies were performedwere performed in human, in non-humanhuman, non primate,‐human andprimate, rat cerebral and rat cortex cerebral astroglial cortex cellastroglial cultures. cell Their cultures. hypothetical Their hypothetical role in cerebral role cortexin cerebral modularity cortex wasmodularity analyzed was following analyzed initial following observations initial on observations cell coupling on in cell the cerebralcoupling cortical in the andcerebral striatal cortical cells from and Cebusstriatal apella cells(tufted from capuchin) Cebus apella monkeys, (tufted demonstrating capuchin) monkeys, its functional demonstrating preservation its followingfunctional freezing preservation and thawing following procedures freezing [33 ].and Later, thawing a comparison procedures between [33]. rat, Later, monkey, a comparison and human cellbetween cultures rat, frommonkey, regional and brainhuman samples cell cultures that were from obtained regional at brain early samples ages and that stored were deep obtained frozen, at wasearly undertaken ages and stored by Lanosa deep frozen, et al. [34 was]. Rat undertaken striatum andby Lanosa cerebral et cortexal. [34]. showed Rat striatum different and coupling cerebral characteristics.cortex showed Signiﬁcantdifferent coupling differences characteristics. were observed Significant in dye coupling differences between were rodent observed and humanin dye braincoupling cortical between cells inrodentin vitro andconditions human brain (Figure cortical8), which cells werein in vitro suggestive conditions of a comparatively (Figure 8), which reduced were diffusionsuggestive of of dye a comparatively coupling in human reduced samples. diffusion It remainsof dye coupling to be proven in human that samples. these observations It remains holdto be trueproven in situ that using these brain observations slices. Should hold true it be in conﬁrmed, situ using itbrain could slices. be speculated Should it be that confirmed, in Homo there it could is less be tendencyspeculated to that spread in Homo cell coupling, there is less which tendency would aidto spread in limiting cell spatialcoupling, compromise which would of ionic/molecular aid in limiting perturbationsspatial compromise and hence of ionic/molecular contribute more preciseperturbations spatial (modular)and hence characteristics, contribute more through precise reduction spatial of(modular) glial territories. characteristics, through reduction of glial territories.

Figure 8.8.Interspecies Interspecies analysis analysis of astroglialof astroglial coupling coupling levels followinglevels following lucifer yellowlucifer pressure yellow injectionspressure ininjections astroglia-enriched in astroglia cultures‐enriched from cultures rat, monkey from rat, and monkey human cerebral and human cortex, cerebral expressed cortex, as the expressed percentage as ofthe cells percentage coupled of out cells of coupled the total out cells of essayed the total (percentage cells essayed of (percentage cell coupling). of cell Numbers coupling). in parenthesesNumbers in indicateparentheses sample indicate size. Signiﬁcantsample size. differences Significant were differences detected usingwere Pearson’sdetected using chi-square Pearson’s test (* chip =‐square 0.006). Adaptedtest (* p = from 0.006). Figure Adapted 3 in [from34]. Figure 3 in [34].

For the aforementioned experiments, the ages that were examined were developmentally early, early, as determineddetermined by thethe availabilityavailability ofof samplessamples fromfrom humanhuman andand non-humannon‐human primates,primates, usingusing cerebralcerebral cortex astroglial culture stocks of rat (ﬁve-day-old male), monkey (three-month-old male) and human (six-month-old female; intrasurgical sample) from postnatal origin, kept frozen under N2-atmosphere until they were used [34]. Hence, an effect of age cannot be overlooked. Yet, the results open Neuroglia 2018, 1 15 up an intriguing additional potential difference in glial functional (coupling) characteristics among mammalian species.

4. Glial Cells in Subcortical White Matter: Elements of a Subcortical, Distributed Information Neural Control Circuit? An additional issue regarding the roles of glial cells in the regulation of information transfer is whether the so called subcortical “interstitial” (neuronal and glial) cells are solely residual (neurons) and maintenance (glia) of projection and association ﬁbers, or they are part of regionally distributed, subcortical, neuron–glial networks, with a role in the control of information transfer probability through such axonal ﬁbers. This matter has been further discussed in [35,36], and the regulation of timing and signal transfer probability in the subcortical white matter alternatively considered. This possibility calls for adequate experimental testing.

5. Diffusible Factors Released by Astroglia According to observations by Colombo and Napp [37,38], and Hunter and Hatten [39], conditioned medium by conﬂuent embryonic cell cultures of astroglia showed evidence of releasing diffusible inducers of radial glia and neuritogenesis in the in vitro settings. Our contribution further stressed the compatibility and potential usefulness of this in vitro model for the ex vivo analysis of factors affecting the molecular dynamics that are involved in neuronal migration. Figure9 illustrates characteristic neuronal adhesion to radial glia and their leading and trailing processes. These radial glial cells expressed laminin and 401-R antigens. Neuroglia 2018, 1 16 Neuroglia 2018, 1, 3 10 of 14

Figure 9. ((AA)) Adhesion Adhesion of of cerebral cerebral cortex cortex primary primary fetal fetal (embryonic (embryonic day 17 17 (E17)) (E17)) cells cells onto elongated elongated (“radial(“radial-like”)‐like”) processes processes of of subcultured cerebral cerebral cortex cortex glia exposed to to cerebral cortex astroglial conditionedconditioned medium during 24 h, 3 h after seeding primary cells. Note Note in in (B) (B) evidence evidence of leading leading and trailingtrailing processesprocesses of primaryof primary cells, cells, adherent adherent to a cell process.to a cell Induced process. processes Induced were processes laminin-positive were lamininand Rat-401‐positive antisera-positive. and Rat‐401 antisera Adapted‐positive. from Figure Adapted 1 in from [37]. ScaleFigure bar: 1 in 10 [37].µm Scale (A,B ).bar: 10 μm (A,B).

InIn pathological conditions, such such as Parkinson’s disease, when considering that cerebrospinal fluidfluid (CSF) from L‐-DOPADOPA treated Parkinson’s disease disease patients patients is is dystrophic dystrophic to to neuronal neuronal cultures, cultures, possible diffusible messengers released released into into the the CSF affecting astroglial cells [40] [40] may intervene in in thethe progression of associated brain pathology. Figure Figure 10 illustratesillustrates the the effect of CSF preincubation with cultured controlcontrol glia glia on on the the otherwise otherwise deleterious deleterious effects effects of cerebrospinalof cerebrospinal fluid fluid from from patients patients with withParkinson’s Parkinson’s disease disease treated treated with withL-DOPA. L‐DOPA. The reported reported trophic trophic influences influences of of glia glia [37,38,40,41] [37–41] prompted prompted its its implementation implementation inin a cell transplantationtransplantation chymera chymera in in a a non non-human‐human primate primate in in the the inin vivo system, based on the intracarotid administration of the neurotoxin1‐methyl‐1‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced parkinsonism in Cebus apella monkeys [42]. Following bilateral astroglial transplantation, significant

Neuroglia 2018, 1 17

Neurogliaadministration 2018, 1, 3 of the neurotoxin1-methyl-1-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced11 of 14 parkinsonism in Cebus apella monkeys [42]. Following bilateral astroglial transplantation, signiﬁcant performance improvement in in a spatial delayed response task was observed, although it failed to modify perseveration in in an object retrieval detour task, or to improve motormotor clinicalclinical rating.rating. These observations suggestsuggest the the possibility possibility of of dissociating dissociating brain brain circuits circuits that that are are subserving subserving various various motor motor and andcognitive cognitive performances. performances. It should It should be added be that added the experimental that the experimental design should design take intoshould consideration take into considerationcognitive premorbid cognitive training premorbid effects, training to avoid effects, any interference to avoid any with interference the interpretation with the of interpretation changes due ofto changes transplantation due to transplantation proper [43]. proper [43].

Figure 10. ((AA––CC)) Effect Effect of of astroglial astroglial conditioning conditioning of of three three different sources of cerebrospinal fluid ﬂuid (CSF) from Parkinson’s disease patients on neuronal processes. Percentage Percentage of of emitting emitting cells cells in in rat primary cultures of striatum (dotted bars) or ventral mesencephalon (horizontally dashed bars) after 24 h in culture with CSF before (pre) glial conditioning or after (post) 24 h conditioning with fetal mesencephalic astroglia. astroglia. Pearson’s Pearson’s chi chi-square‐square test; test; ** ** p p< <0.01; 0.01; *** *** p

6. Environmental Environmental Effects on Glial Response to Injury and Dye Coupling The reported effects of environmental enrichment (EE) on neuronal events [44–46] [44–46] suggested the need to analyze glial cell responses to such contingencies. We We have studied this this in two different protocols. One, in terms of recovery after cortical mechanical lesioning [47], [47], with results suggesting that thethe exposure exposure to EEto conditions EE conditions prior to prior injury to attenuates injury theattenuates post-lesional the astroglialpost‐lesional GFAP-response astroglial GFAP‐response in the perilesional cortex of rats, and, hence, could modulate post‐lesional reactive components. This requires further studies to characterize the mechanisms that are involved. In the second protocol, we analyzed glial dye coupling in fresh tissue sections from rat motor cortex, after varying periods of EE. These studies reported several observations, but in terms of EE

Neuroglia 2018, 1 18 in the perilesional cortex of rats, and, hence, could modulate post-lesional reactive components. This requires further studies to characterize the mechanisms that are involved. In the second protocol, we analyzed glial dye coupling in fresh tissue sections from rat motor cortex, after varying periods of EE. These studies reported several observations, but in terms of EE on cell coupling in cortical laminae II–III, 30 days of EE resulted in a signiﬁcant increase in the area of cell coupling, but not in the number of coupled cells, with a concomitant decrease in cell density, suggesting a volume increase in intercellular—gliopil—space [48]. These results can be interpreted as a spatial expansion of the glial net, perhaps involving an increase in glial connectivity. Further studies should clarify the mechanisms underlying such gliopil expansion (e.g., astrocyte hypertrophy), and whether changes in neuronal and vascular elements also take part in the process following EE.

7. Summary and Conclusions Significant recent advances have been made in our understanding of general mammalian characteristics of glial cells and their roles in brain functional organization. However, care must be taken when extrapolating data to primate species. The absence of interlaminar glia in rodent species is an excellent case in point of such limitations. Critical functional knowledge must be based on primate species if we are to understand the role of glia in the complex organization of human and primate brains. In particular, “general mammalian” glial functional characteristics need to be critically assessed in light of interspecies differences in order to appreciate their significance in the cognitive and emotional processes that underlie primate and human behavior. For this purpose, continuously improved experimental procedures in primate species, such as in vitro cell culture, ex vivo organotypic brain slice preparations, and in vivo functional brain imaging and limited brain sampling procedures, would provide efficient means to accomplish such critical aims in highly protected species.

Acknowledgments: Contribution by Fundación Conectar to the development of the present review is gratefully acknowledged. Dedicated research involvement of colleagues and technical support personnel at the Unit of Applied Neurobiology (UNA, CEMIC-CONICET) during several demanding years, and sustained financial suport to the UNA research projects by various local and foreign granting agencies, is also gratefully acknowledged. Conflicts of Interest: The author declares no conflict of interest.

References

1. Kettenmann, H.; Ransom, B.R. Neuroglia; Oxford University Press: New York, NY, USA, 2005; ISBN 0-19-515222-0. 2. Verkhratsky, A.; Butt, A. Glial Physiology and Pathophysiology; Wiley-Blackwell: Oxford, UK, 2013; ISBN 978-0-470-97852-8. 3. Verkhratsky, A.; Nedergaard, M. Physiology of Astroglia. Physiol. Rev. 2018, 98, 239–389. [CrossRef] [PubMed] 4. Colombo, J.A. Interlaminar astroglial processes in the cerebral cortex of adult monkeys but not of adult rats. Acta Anat. 1996, 155, 57–62. [CrossRef][PubMed] 5. Colombo, J.A. A columnar-supporting mode of astroglial architecture in the cerebral cortex of adult primates? Neurobiology 2001, 9, 1–16. [CrossRef][PubMed] 6. Colombo, J.A. The interlaminar glia: From serendipity to hypothesis. Brain Struct. Funct. 2017, 222, 1109–1129. [CrossRef][PubMed] 7. Colombo, J.A.; Fuchs, E.; Härtig, W.; Marotte, L.R.; Puissant, V. “Rodent-like” and “primate-like” types of astroglial architecture in the adult cerebral cortex of mammals: A comparative study. Anat. Embryol. 2000, 201, 111–120. [CrossRef][PubMed] 8. Colombo, J.A.; Sherwood, C.; Hof, P. Interlaminar astroglial processes in the cerebral cortex of great apes. Anat. Embryol. 2004, 429, 391–394. [CrossRef][PubMed] 9. Colombo, J.A.; Lipina, S.; Yáñez, A.; Puissant, V. Postnatal development of interlaminar astroglial processes in the cerebral cortex of primates. Int. J. Dev. Neurosci. 1997, 15, 823–833. [CrossRef] 10. Aiello, L.C.; Wheeler, P.T. The Expensive-Tissue Hypotesis: The brain and the digestive system in human and primate evolutiom. Curr. Anthropol. 1995, 36, 199–221. [CrossRef] Neuroglia 2018, 1 19

11. Dunbar, R.I.M. The social brain hypothesis and its implications for social evolution. Ann. Hum. Biol. 2009, 36, 562–572. [CrossRef][PubMed] 12. Robertson, J.M. Astrocytes and the evolution of the human brain. Med. Hypotheses 2014, 82, 236–239. [CrossRef][PubMed] 13. Zilles, K.; Schlaug, G.; Matelli, M.; Luppino, G.; Schleicher, A.; Qü, M.; Dabringhaus, A.; Seitz, R.; Roland, P.E. Mapping of human and macaque sensorimotor areas by integrating architectonic, transmitter receptor, MRI and PET data. J. Anat. 1995, 187, 515–537. [PubMed] 14. Zilles, K.; Palomero-Gallagher, N. Multiple transmitter receptors in regions and layers of the human cerebral cortex. Front. Neuroanat. 2017, 11, 1–26. [CrossRef][PubMed] 15. Pu, M.M.; Yao, J.; Cao, X. Genomics: Disclose the influence of human specific genetic variation on the evolution and development of cerebral cortex. Hereditas 2016, 38, 957–970. [PubMed] 16. Muntané, G.; Santpere, G.; Verendeev, A.; Sherwood, C. Interhemispheric gene expression differences in the cerebral cortex of humans and macaque monkeys. Brain Struct. Funct. 2017, 222, 3241–3254. [CrossRef] [PubMed] 17. Mitchell, C.; Silver, D.L. Enhancing our brains: Genomic mechanisms underlying cortical evolution. Semin. Cell Dev. Biol. 2017.[CrossRef][PubMed] 18. Oberheim, N.A.; Takano, T.; Han, X.; He, W.; Lin, J.H.; Wang, F.; Xu, Q.; Wyatt, J.D.; Pilcher, W.; Ojemann, J.G.; et al. Uniquely hominid features of adult human astrocytes. J. Neurosci. 2009, 29, 3276–3287. [CrossRef] [PubMed] 19. Colombo, J.A.; Reisin, H.D.; Jones, M.; Bentham, C. Development of interlaminar astroglial processes in the cerebral cortex of control and Down’s syndrome human cases. Exp. Neurol. 2005, 193, 207–217. [CrossRef] [PubMed] 20. Colombo, J.A.; Gayol, S.; Yáñez, A.; Marco, P. Immunocytochemical and electron microscope observations on astroglial interlaminar processes in the primate neocortex. J. Neurosci. Res. 1997, 48, 352–357. [CrossRef] 21. Grosche, J.; Matyash, V.; Moller, T.; Verkhratsky, A.; Reichenbach, A.; Kettenmann, H. Microdomains for neuron–glia interaction: Parallel fiber signaling to Bergmann glial cells. Nat. Neurosci. 1999, 2, 139–143. [CrossRef][PubMed] 22. Potokar, M.; Kreft, M.; Andersson, J.D.; Pangrsic, T.; Chowdhury, H.H.; Pekny, M.; Zorec, R. Cytoskeleton and vesicle mobility in astrocytes. Traffic 2007, 8, 12–20. [CrossRef][PubMed] 23. Colombo, J.A.; Yáñez, A.; Lipina, S. Disruption of immunoreactive glial fibrillary acidic protein patterns in the Cebus apella striate cortex following loss of visual input. J. Brain Res. 1999, 39, 447–451. 24. Reisin, H.; Colombo, J.A. Glial changes in primate cerebral cortex following long-term sensory deprivation. Brain Res. 2004, 1000, 179–182. [CrossRef][PubMed] 25. Peters, A.; Feldman, M.L. The projection of the lateral geniculate nucleus to area 17 of the rat cerebral cortex. IV Terminations upon spiny dendrites. J. Neurocytol. 1977, 6, 669–689. [CrossRef][PubMed] 26. Biane, J.S.; Takashima, Y.; Scanziani, M.; Conner, J.M.; Tuszynski, M.H. Thalamocortical projections onto behaviorally relevant neurons exhibit plasticity during adult motor learning. Neuron 2016, 89, 1173–1179. [CrossRef][PubMed] 27. Colombo, J.A.; Quinn, B.; Puissant, V. Disruption of astroglial interlaminar processes in Alzheimer’s disease. Brain Res. Bull. 2002, 58, 235–242. [CrossRef] 28. Colombo, J.A.; Yañez, A.; Lipina, S. Interlaminar astroglial processes in the cerebral cortex of non-human primates: Response to injury. J. Brain Res. 1997, 38, 503–512. 29. Colombo, J.A.; Reisin, H.D.; Miguel-Hidalgo, J.J.; Rajkowska, G. Cerebral cortex astroglia and the brain of a genius: A propos of A. Einstein’s. Brain Res. Rev. 2006, 52, 257–263. [CrossRef][PubMed] 30. Reisin, H.D.; Colombo, J.A. Considerations on the astroglial architecture and the columnar organization of the cerebral cortex. Cell. Mol. Neurobiol. 2002, 22, 633–644. [CrossRef][PubMed] 31. Colombo, J.A.; Napp, M.I.; Yañez, A.; Reisin, H. Tissue printing of astroglial interlaminar processes from human and non-human primate cerebral cortex. Brain Res. Rev. 2001, 55, 561–565. [CrossRef] 32. Barres, B.A.; Koroshetz, W.J.; Chun, L.L.Y.; Corey, D.P. Ion channel expression by white matter glia: The type 1 astrocyte. Neuron 1990, 5, 527–544. [CrossRef] 33. Gayol, S.; Pannicke, T.; Reichenbach, E.; Colombo, J.A. Cell–cell coupling in cultures of striatal and cortical astrocytes of the monkey Cebus apella. J. Brain Res. 1999, 4, 473–478. Neuroglia 2018, 1 20

34. Lanosa, X.A.; Reisin, H.D.; Santacroce, I.; Colombo, J.A. Astroglial dye-coupling: An in vitro analysis of regional and interspecies differences in rodents and primates. Brain Res. 2008, 1240, 82–86. [CrossRef] [PubMed] 35. Colombo, J.A.; Bentham, C. Immunohistochemical analysis of subcortical white matter astroglia of infant and adult primates, with a note on resident neurons. Brain Res. 2006, 1100, 93–103. [CrossRef][PubMed] 36. Colombo, J.A. Cellular complexity in subcortical white matter: A distributed control circuit? Brain Struct. Funct. 2018, 223, 981–985. [CrossRef][PubMed] 37. Colombo, J.A.; Napp, M.I. Ex vivo astroglial-induced radial glia express in vivo markers. J. Neurosci. Res. 1996, 46, 674–677. [CrossRef] 38. Colombo, J.A.; Napp, M.I. Forebrain and midbrain astrocytes promotes neuritogenesis in cultured chromaffin cells. Restor. Neurol. Neurosci. 1994, 7, 111–117. [PubMed] 39. Hunter, K.E.; Hatten, M.E. Radial glial cell transformation to astrocytes in bidirectional regulation by a diffusible factor in embryonic forebrain. Proc. Natl. Acad. Sci. USA 1995, 92, 2061–2065. [CrossRef][PubMed] 40. Colombo, J.A.; Napp, M.I. Cerebrospinal fluid from L-dopa-treated Parkinson’s disease patients is dystrophic for various neural cell types ex vivo: Effects of astroglia. Exp. Neurol. 1998, 154, 452–463. [CrossRef] [PubMed] 41. Uceda, G.; Colombo, J.A.; Michelena, P.; López, M.G.; García, A.G. Rat striatal astroglia induce morphological and neurochemical changes in adult bovine, adrenergic-enriched adrenal chromaffin cells in vitro. Restor. Neurol. Neurosci. 1995, 8, 129–136. [PubMed] 42. Lipina, S.J.; Colombo, J.A. Dissociated functional recovery in parkinsonian monkeys following transplantation of astroglial cells. Brain Res. 2001, 911, 176–180. [CrossRef] 43. Lipina, S.J.; Colombo, J.A. Premorbid exercising in specific cognitive tasks prevents impairment of performance in parkinsonian monkeys. Brain Res. 2007, 1134, 180–186. [CrossRef][PubMed] 44. Diamond, M.C.; Krech, D.; Rosenzweig, M.R. The effects of an enriched environment on the histology of the rat cerebral cortex. J. Comp. Neurol. 1964, 123, 111–120. [CrossRef][PubMed] 45. Globus, A.; Rosenzweig, M.R.; Bennett, E.L.; Diamond, M.C. Effects of differential experience on dendritic spine counts in rat cerebral cortex. J. Comp. Physiol. Psychol. 1973, 82, 175–181. [CrossRef][PubMed] 46. Sirevaag, A.M.; Greenough, W.T. Differential rearing effects on rat visual cortex synapses. III. Neuronal and glial nuclei, boutons, dendrites, and capillaries. Brain Res. 1987, 424, 320–332. [CrossRef] 47. Lanosa, X.A.; Santacroce, I.; Colombo, J.A. Exposure to environmental enrichment prior to a cerebral cortex stab wound attenuates the postlesional astroglia response in rats. Neuron Glia Biol. 2011, 7, 1–13. [CrossRef] [PubMed] 48. Santacroce, I. Plasticity of Astroglial Networks in the Cerebral Cortex of the Rat: Response to Environmental Enrichment and Physicochemical Variables. Ph.D. Thesis, University of Buenos Aires, Buenos Aires, Argentina, 2017.

© 2018 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).