At least four morphologic classes of GFAP positive cells are present in the human cortex
At least four major morphologic subclasses of GFAP immunoreactive cells were identified in the adult human temporal lobe (). Layer 1 is characterized by densely packed interlaminar astrocytes, which extend millimeter long processes terminating in layers 2–4 (). Layers 2–6 are characterized by protoplasmic astrocytes of a larger size than found in rodents (). In layers 5–6, a small number of varicose projection astrocytes reside (). These cells extend long processes in a seemingly random manner. Finally, fibrous astrocytes populate the white matter (). Examination of primate cortex revealed that the cortex of higher order primates, specifically chimpanzee, also encompassed 4 subtypes of GFAP+ cells (Supplemental Figure 1
), although the cellular complexity did not compare to those of human brain. In contrast, cortex of lower order primates including the Rhesus Macaque and Squirrel monkeys contained 3 subtypes of astrocytes, whereas only protoplasmic and fibrous astrocytes were identified in rodents (Supplemental Figure 1
Figure 1 There are 4 classes of GFAP+ cells in the Human Cortex. Human brains were immunolabeled with GFAP and analyzed throughout all layers of the cortex to determine subclasses of human astrocytes. Layer 1 is composed of the cell bodies of interlaminar astrocytes, (more ...)
Varicose projection astrocytes – a novel GFAP positive cell in higher order primate cortex
One of the most striking features noted distinguishing humans and chimpanzee from other lower primate and rodent astrocytes was the presence of a previously undescribed pool of morphologically distinct GFAP+
cells residing in layers 5–6, characterized by long fibers with prominent varicosities (). The main GFAP+
processes of these varicose projection astrocytes are straighter than those of protoplasmic astrocytes, whose primary processes are typically tortuous and highly branched (compare ). Additionally, these cells also extend 1–5 long processes of up to a millimeter in length, which may terminate in the neuropil or on the vasculature (). The processes of these projection astrocytes are characterized by varicosities that are spaced approximately every 10 µm (, ). The processes frequently penetrate the process-delimited domains of neighboring astrocytes. These varicose projection astrocytes are relatively sparse but were present in all 30 specimens examined. In our analysis of primate tissue, we were able to locate a small number of varicose projection astrocytes within layers 5 of 6 of the chimpanzee cortex ( inset). These cells differed in comparison to those seen in human in that they were smaller and less complex, with fewer main GFAP+ processes. However, we believe that they are the same cell type in that they were located in the same area of cortex and also extended 1–3 long processes characterized by evenly spaced varicosities. We were unable to perform morphometric analysis to compare these cells directly to human due to the paucity of higher order primate tissue available. The other species of primates, including Rhesus Macaque and Squirrel monkeys, we were unable to identify this cell type, suggesting that varicose projection astrocytes are specific to higher order primates and humans (Supplemental Figure 1
). To our knowledge, astrocytes with this morphological phenotype have not been reported in any other species, and the function of their long processes is not known.
Figure 2 Varicose projection astrocytes in layers 5 and 6. A) Varicose projection astrocytes reside in layers 5–6 and extend long processes characterized by evenly spaced varicosites. Inset: Varicose projection astrocyte from chimpanzee cortex. GFAP; white, (more ...)
Figure 4 Protoplasmic astrocytes are larger and more complicated than the rodent counterpart. A) Typical mouse protoplasmic astrocyte. GFAP; white. SB=20µm. B) Typical human protoplasmic astrocyte in the same scale. SB=20µm. C–D) Human (more ...)
Figure 3 Primate specific interlaminar astrocytes populate layer 1. A) Pial surface and layers 1–2 of human cortex. GFAP; white, Dapi; blue. SB=100 µm. Yellow line indicates border between layer 1 and 2. B) High power image of layer 1 showing interlaminar (more ...)
GFAP immunolabeling reveals only the filamentous skeleton of an astrocyte. To visualize the full structure of varicose projection astrocytes, we therefore used DiI-mediated diolistic labeling – biolistic insertion of DiI into cells - of lightly fixed human tissue to better visualize the full extent of single astroglia (). DiI-tagged varicose projection astrocytes resemble protoplasmic astrocytes, with 1–5 long thin processes extending from a relatively symmetrical cell (). The fine processes of these cells are, however, spiny and sharp, unlike the bulbous processes previously described in rodent protoplasmic astrocytes ((Bushong et al., 2002
; Ogata and Kosaka, 2002
; Oberheim et al., 2008
), and see ). Many of the processes from varicose projection astrocytes contacted the vasculature, either through the direct extension of long processes to vascular walls, or by en passant contact of glial processes with the capillary microvasculature ().
Primate specific interlaminar fibers in the human cortex
The GFAP-labeled human cortex was also characterized by a preponderance of interlaminar astrocytes (). Interlaminar astrocytes are primate-specific cells that are densely packed and reside solely in layer 1. Interlaminar astrocytes present in primates have oblong cell bodies, and extend several processes both to the pial surface and also down through the cortical layers in a columnar fashion (Supplemental figure 1
). In addition to being more numerous, the morphology of interlaminar astrocytes is subtly different in humans. Human interlaminar astrocytes have small cell bodies and are round cells, not oblong, and have several short processes that extend in all directions that contribute to the pial glial limitains, creating a thick network of GFAP fibers not seen in the primate (, compare , Supplemental Figure 1
). These cells also extend 1–2 processes from layer 1, terminating in layers 2–4 of the cortex. This results in numerous millimeter long processes radiating though the outer cortical layers (). Interlaminar astrocyte processes are tortuous; they wind back and forth as they travel (). The diameter of their processes is constant without varicosities and can therefore easily be distinguished from those of polarized astrocytes (). The processes of interlaminar astrocytes do not respect the borders of protoplasmic astrocytes and frequently travel through their processes (). The processes terminate in the neuropil, and occasionally on the vasculature (data not shown and (Colombo and Reisin, 2004
)). GFAP immuno-EM showed that these processes frequently contained mitochondria ()(Colombo et al., 1997a
). The function of interlaminar astrocytes and their long processes remains unknown.
Human protoplasmic astrocytes are more complex than the rodent
Protoplasmic astrocytes are the most abundant cell population of the rodent, primate, and human cortex. However, human protoplasmic astrocytes are both more complex, and larger than their rodent and primate counterparts (, supplemental figure 1
). Protoplasmic astrocytes reside in layers 2–6, and vary in domain diameter from < 100 µm to >400 µm, with an average diameter of 142.6 ± 5.8µm based on GFAP immunolabeling (n=50 cells; 6 patients). This is 2.55-fold larger than rodent protoplasmic astrocytes, which have an average diameter of 56.0 µm ± 2.0 (n=65 cells from 7 animals, p
<.0001) ()(Oberheim et al., 2008
). Human protoplasmic astrocytes exhibited an approximately 2.6 fold larger length of their longest process, with an average of 97.9 ±5.2 µm in humans, and 37.2± 2.0 µm in rodents (human, n=50 cells from 6 patients, mouse n=65 cells from 7 animals, p
<0.0001)(Oberheim et al., 2006
). The diameter of the thickest GFAP positive process also differed (Human=2.9 ± 0.18 µm, mouse=2.2 ±0.13 µm, p<.005)(Oberheim et al., 2008
). Most significantly, human astrocytes extended 10-fold more GFAP positive processes from their cell body compared to the rodent with an average of 37.5 ± 5.17 processes radiating from the nucleus compared to 3.75 ± 0.115 in the rodent (human n=50 cells from 7 patients, rodent n=65 cells 7 animals, p<0.0001) ()(Oberheim et al., 2008
Protoplasmic astrocytes in primate have an intermediate phenotype between rodents and humans: they are smaller and less complex than the human, but larger and more complicated that those seen in rodent (Supplemental Figure 1
). For example, the average diameter of protoplasmic cortical astrocytes in the Chimpanzee brain was 81.7±1.9 µm (n = 36), which is significantly smaller than human astrocytes, but significantly larger than protoplasmic astrocytes in mouse brain (p< 0.001, ANOVA with Bonferroni post-hoc test).
We diolistically labeled both human and rodent tissue with DiI to examine the full structure of the protoplasmic astrocytes (). The human protoplasmic astrocytes were easily identified based on their characteristic bushy morphology. We were able to disclose the numerous delicate processes that are GFAP negative in both the rodent as well as in the human. The fine processes in human protoplasmic astrocytes are bulbous, similar to those previously described in rodent, and distinctly different the spiny and sharp processes of varicose projection astrocytes (). Overall, the complexity of the human protoplasmic astrocytes was apparent when their fine processes are visualized diolistically.
Human Protoplasmic Astrocytes are organized in domains
Rodent protoplasmic astrocytes are organized into domains (Bushong et al., 2002
; Ogata and Kosaka, 2002
; Halassa et al., 2007
). Each astrocyte occupies its own anatomical space with little overlap between adjacent cells. Thus all neuronal cell bodies, synapses, and vessel within an astrocytic domain will be in contact with processes from a single astrocyte only. It has been speculated that astrocytic domains constitute a unit for local integration and modulation of neural activity that is spatial and temporally distinct from synaptic transmission. To determine if domain organization has been preserved through evolution and exists in the human cortex, we used biolistic labeling to tag human and rodent cells in situ, with two spectrally-distinct lipophilic dyes, DiI and DiD (). When two adjacent astrocytes are labeled with different dyes, the borders between the two cells may be clearly visualized, and their domain organizations can be examined. We found that like rodent astrocytes, human protoplasmic astrocytes were also organized into domains, with limited overlap between adjacent cells (); the extent of this overlap was quantified in two ways. First, the border of the domain of the cell was outlined by connecting the tips of the processes of each cell. The area common between the two cells was then averaged from 10 optical sections, sampled 1 µm apart (). In additional, processes that crossed into the domain of its neighbor were also measured, and averaged over 10 optical sections (). We predicted that human domains would exhibit more overlap than rodent cells, due to their significantly greater size. The average area of overlap for each human astrocytic domain was 204.7 ± 44.1 µm2
, while the summated length of overlapping processes was 53.0 ± 10.7 µm (n=10 cells from 2 patients). As predicated, this was significantly greater than in corresponding rodent cortical samples, in which the average area of overlap was 11.8 ± 2.2 µm2
, comprised of processes with net overlapping length of 4.2 ± 0.7 µm (n=30 cells from 7 animals; area comparison significant to p
<0.0001; overlapping process lengths to p
<0.0001) (Oberheim et al., 2008
). The area of overlap in humans was larger than the predicted 6.5 fold increase, based on the 2.55 fold increase in diameter. Thus, human protoplasmic astrocytes were distributed evenly across cortex and were organized in domains, albeit with more extensive overlap of their processes than in rodents.
Figure 5 Human astrocytes are organized into domains. A) Cortical mouse astrocytes labeled with DiI (red) and DiD (green) demonstrating the presence of domains. Nuclei (sytox); blue. SB=10µm. B) Human protoplasmic astrocytes labeled with DiI (red) and (more ...)
Human protoplasmic astrocytes have increased GFAP in their endfeet
Endfeet are specialized structures of protoplasmic astrocytes, that contact the vasculature and contribute to the blood brain barrier (Simard and Nedergaard, 2004
). Astrocytic endfeet can be seen as distinct compartments with polarized expression of a number of proteins, such as aquaporin 4, Cx43 and Kir4.1 (Nagelhus et al., 2004
; Simard and Nedergaard, 2004
). To compare the endfeet of human and rodent astrocytes, we prepared acute slices identically from both species and immunolabeled with GFAP (). In both species, nearly every protoplasmic astrocyte contacted the vasculature, either through extension of long processes, or capillaries within its domain. Yet the microanatomy of their respective endfeet manifestly differed. In the human brain, GFAP completely encompassed the vessels, creating a cobblestone pattern along the vasculature (). In contrast, rodent astrocytes formed rosettes of GFAP around the vasculature (). Ultrastructurally, the astrocytic endfeet of humans and rats appeared similar; each contained an abundance of mitochondria ( arrows).
Figure 6 Astrocytic endfeet in rodents and humans. A) Cortical blood vessel in rat. GFAP; white, Nuclei (dapi); blue. SB=20µm. B) GFAP in endfeet forms rosettes on the vessel in the rat. SB=10µm. Yellow circles indicate individual endfeet. C) Human (more ...)
Human fibrous astrocytes are significantly larger than rodent
Fibrous astrocytes, found in the white matter of both rodents, primates and humans, are morphologically distinct from protoplasmic astrocytes. In rodents, they are larger than protoplasmic astrocytes, with an average GFAP-defined domain diameter of 85.6 ± 2.7 µm (n=50 cells from 3 mice, p<0.0001) (). Their processes are radially oriented in the direction of the axon bundles. When viewed after DiI-based biolistic labeling, they were noted to have fewer fine processes, resulting in a much smaller surface area-to-volume ratio than protoplasmic astrocytes (, compare ). Human fibrous astrocytes proved significantly larger than their rodent counterparts, with an average GFAP-defined domain diameter of 183.2 ± 6.1 µm (n=50 cells from 6 patients, p<.0001 rodent) (). Their relatively unbranched straight GFAP positive processes extended evenly from a small cell body. These processes interdigitate extensively with processes from neighboring fibrous astrocytes; therefore fibrous astrocytes are not organized within domains (). Their cell bodies, however, are equally spaced (). This regular spacing is most likely due to the structural support that the fibrous astrocytes provide for the axons tracts. Additionally, the majority of human fibrous astrocytes contact the vasculature, either close to their cell bodies, or through the extension of long processes. Diolistic labeling reveals that human fibrous astrocytes also have essentially none of the fine bulbous processes characteristic of the protoplasmic astrocytes found in the human cortex. ().
Figure 7 Human fibrous astrocytes are significantly larger than in rodent. A) Mouse fibrous astrocyte in white matter. GFAP; white, sytox; blue. SB=10µm. B) Human fibrous astrocytes in white matter. SB=10µm. C) Human fibrous astrocytes are approximately (more ...)
Functional properties of human astrocytes
To study Ca2+ signaling in human astrocytes, we prepared acute slices from human cortex immediately following surgical resection. The tissue was placed in ice-cold oxygenated ACSF and sectioned within 15 min after surgical resection, then loaded with the calcium indicator Fluo-4/am, and in some cases with caged Ca2+, NP-EGTA-AM. Ca2+ signals were then visualized using a 2-photon laser scanning microscope.
To determine if human astrocytes were able to support a calcium wave, we photoreleased caged calcium (NP-EGTA) in astrocytes in situ, in the presence or absence of TTX (). Photorelease of caged calcium led to an increase in intracellular calcium in the target cell, with subsequent calcium wave propagation to other cells in 25 of 28 experiments in the presence of TTX (6 slices from 2 patients), and in 7 of 7 experiments in the absence of TTX (4 slices from 1 patient). The speed of the calcium wave in the absence of TTX was 43.4 ± 4.7 µm/sec (n=7 waves); this was not significantly altered by TTX, added so as to block concurrent neuronal activity (36.3 ± 4.4 µm/sec; n=23 waves; p=0.41).
Figure 8 Human astrocytes can support faster calcium waves. A) Acute slice preparation of mouse cortex was loaded simultaneously with Fluo-4 AM as well as NP-EGTA AM caged calcium. A single astrocyte (indicated as x) was the target of photorelease of caged calcium (more ...)
To compare the rates of calcium wave transmission between human and rodent astrocytes, we next measured the speed of calcium wave transmission in TTX-treated rodent cortical slices prepared identically to their human counterparts. We found that the speed of the calcium wave in rodents was significantly slower than in humans: Mouse astrocytes propagated calcium waves at 8.6 ± 0.6 µm/s (n=8 waves from 3 animals), less than a fifth the speed of their human counterparts (43.4 ± 4.7 µm/sec, n=7; p<0.005) ().
Adult astrocytes in the rodent brain respond to both ATP and glutamate (Volterra and Meldolesi, 2005
). To assess the responses of human astrocytes to these stimuli, we next challenged human astrocytes with bath application of ATP (). The cells responded to concentrations of ATP ranging from 100 µM to 5 mM by increasing intracellular calcium, both in the presence and absence of TTX. In the absence of TTX, 93 ± 3% of loaded cells responded to ATP by increasing their florescence by >2 standard deviations from their baseline (n=161 cells; 9 slices from 3 patients) (). In the presence of TTX, 71 ± 15% of cells responded to ATP application (67 cells; 3 slices from 1 patient) (). In general, astrocytes returned to baseline calcium levels within 20–30 seconds. In some slices, astrocytes in layer 1 were imaged (). In addition to cell bodies, processes from these cells also loaded with the calcium indicator dye. Interestingly, in response to ATP stimulus, the processes of these cells also increased their intracellular calcium, and displayed propagation of the calcium signal along the interlaminar fibers (). Thus, human astrocytes respond to ATP through rises in intracellular calcium in both their cell bodies and processes.
Figure 9 Human astrocytes in situ have functional purinergic and glutamateric receptors and respond with increases in intracellular calcium. A) Acute slice preparation of human temporal lobe loaded with calcium indicator dye Fluo-4am. Astrocytes (white circles) (more ...)
Slices were also exposed to glutamate (1 – 15 mM) by bath application in the presence of TTX (). On average, 60 ± 8% of the loaded astrocytes responded with rises in intracellular calcium (54 cells, 4 slices from 3 patients). In order to determine if this response to glutamate was mediated by metabotropic glutamate receptors, slices were then exposed to (±)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), a group I and II metabotropic glutamate receptor agonist in the presence of TTX (). 79 ± 17% of loaded cells responded to 30 µm t-ACPD (64 cells, 2 slices from 1 patient). Astrocytes exhibited a rapid rise in intracellular calcium in response to both glutamate and t-ACPD, and return to baseline levels within seconds. Some cells maintained an oscillatory response, with serial elevations in intracellular calcium in response to the initial stimulus. Therefore, human astrocytes, like their rodent counterparts, respond to ATP and glutamate through rises in intracellular calcium. However, the signal propagation by human astrocytes is significantly and substantially more rapid than that exhibited by rodent astroglia.