The Architecture of the Human Fovea Webvision

By Helga Kolb, Ralph Nelson, Peter Ahnelt, Isabel Ortuo-Lizarn and Nicolas Cuenca

Abstract

We summarize the development, structure, different neural types and neural circuitry in the human fovea. The foveal pit is devoid of rod photoreceptors and of secondary and tertiary neurons, allowing light to directly stimulate cones and give us maximal visual acuity. The circuitry underlying the transmission to the brain occurs at the rim of the fovea. The predominant circuitry is concerned with the private cone to midget bipolar cell and midget ganglion cell pathways. Every cone drives two midget bipolar cells and two midget ganglion cells so that the message from a single cone is provided to the brain as a contrast between lighter signals (ON pathways) or darker signals (OFF pathways). The sharpening of this contrast message is provided by horizontal-cell feedback circuits and, in some pathways by amacrine circuitry. These midget pathways carry a concentric color and spatially opponent message from red and green cones.

Blue cones are sparse, even largely missing in the foveal center while occurring at somewhat higher density than elsewhere in the cone mosaic of the foveal slope. Signals from blue cones have different pathways to ganglion cells. The best understood is through an ON-type blue-cone-selecting bipolar cell to a non-midget, small bistratified ganglion cell. An OFF-center blue midget bipolar is known to be present in the fovea and connects to a blue OFF midget ganglion cell. Another OFF blue message is sent to a giant melanopsin ganglion cell that is present in the foveal rim area, but the circuitry driving that is less certain and possibly involves an intermediate amacrine cell. The H2 horizontal cells are thought to be feedback neurons primarily of the blue cone system.

Amacrine cells of the fovea are mostly small-field and glycinergic. The larger field GABAergic amacrines are present but more typically surround the fovea in a ring of processes, with little or no penetration into the foveal center. Thus, the small field glycinergic amacrines are important in some sort of interplay with the midget bipolarmidget ganglion cell channels. We have anatomical descriptions of their synaptology but only a few have been recorded from physiologically. Both OFF pathway and ON pathway amacrines are present in the fovea.

The central point of the visual field ahead of us is the image falling on the fovea in the human retina. This is the area of our visually sensitive retina where the cone photoreceptors are tightly packed, where rod photoreceptors are excluded and where all intervening layers of the retina are pushed aside concentrically to allow light to reach the densely packed sensory cones with minimum scatter from overlying tissues. The fovea is where focusing on fine detail in the image is perfected, allowing us to read, discriminate colors well and sense three-dimensional depth.

General features of the fovea

Figure 1. The normal human retina fundus photo shows the optic nerve (right), blood vessels and the position of the fovea (center).

Looking at the retina lining the back of the eyeball in a human, we can see the clear landmark of the optic nerve head (papilla) and radiating blood vessels (Figure 1). Temporal to the optic nerve head at a distance approximately 2.5 optic nerve (disk) diameters at roughly 3.4 mm distance lies a dark brown-yellowish area (Figure 1), in the center of which is the tiny circular fovea. The position of the fovea can be seen clearly in the retina illustrated in Figure 2A. This eye was treated with RNA-later for preservation, allowing for a clear view of a yellow macula lutea area and including the brown central point, (foveal pit) (Figure 2A).

Figure 2A.An isolated human retina shows the optic nerve (right), blood vessels and the fovea (center) with surrounding macula lutea (yellow). Cuenca et al, prepublication.

The area called the macula by ophthalmologists is a circular area around the foveal center of approximately 5.5 mm diameter (Figure 2B) The macula lutea with the yellow pigmentation extends across the fovea into the parafoveal region and a little beyond. This area is about 2.5 mm in diameter (Figure 2B). The actual fovea is about 1.5 mm in diameter and the central fovea consists of a foveal pit (umbo) that is a mere 0.15 mm across (Figure 2B). This foveal pit is almost devoid of all layers of the retina beneath the cone photoreceptors. On the edges of the foveal pit the foveal slope is still mainly devoid of other layers but some cell bodies of retinal interneurons, bipolar and horizontal cells and even some amacrine cell processes are becoming evident. By the 0.35 mm diameter circular area the first ganglion cell bodies, the retinal neurons sending signals to the brain, are beginning to appear. All the central fovea that measures 0.5 mm across is avascular (FAZ).

Figure 2B.A map of the whole macular area to show the dimensions of the foveal pit, foveal avascular zone, parafovea, perifovea, and the limits of the macula. Inset shows the dimensions of the foveal avascular zone, which is the fovea we are discussing here.

The avascular nature of the central fovea is depicted in Figure 3. A human retina wholemount has the blood vessels immunostained with antibodies against Collagen IV and is photographed by stacked images in a confocal microscope. It is absolutely clear that the smallest capillaries even, do not intrude into the foveal center (Figure 3, f) of 500 m diameter, thereby known as the avascular zone.

Figure 3.Wholemount of human retina with blood vessels immunostained with Collagen IV. The confocal microscopy of stacked images clearly shows the optic nerve head (ON) and all the blood vessels to the smallest capillaries. The capillaries surround the fovea (f), but do not enter it, thereby making the fovea avascular.

In vertical section of the human retina from the optic nerve head through the foveal pit and beyond (Figure 4), it is clear where the fovea is located relative to the nerve head (on). Figure 4 (a) is a confocal image after immunostaining with antibodies that are specific for cone photoreceptors [arrestin antibodies for cones, green; cytochrome C antibodies for mitochondria, blue; and for Mller glial cells and RPE, antibodies against cytoplasmic retinaldehyde binding protein (CRALBP), red]. In comparison is seen an optical coherence tomography (OCT) picture in Figure 4 (b) of exactly the same area of human retina. In both images it is clear that the second and third order neurons of the inner nuclear and ganglion cell layers respectively are not present in the foveal pit.

Figure 4.(a) An immunostained human retina section covering the optic nerve (ON) and the foveal pit. Cones, anti-arrestin (green); pigment epithelial and Mller cells, anti CRALPB (red) (109); mitochondria, anti-cytochrome C (blue). (b) An OCT image of the same retinal area in a normal human subject. The second and third order neurons of the retinal inner nuclear and ganglion cell layers respectively are not present in the foveal pit. Adapted from Cuenca, Ortuo-Lizarn and Pinilla 2018 (110).

In the foveal pit the only neurons are cone photoreceptors, all with slim inner segments, packed cell bodies, up to 6 layers deep reaching to the floor of the foveal pit (Figure 5, green cells). However, there are many expanded-looking Mller glia surrounding these cones (Figure 5, red profiles). A central bouquet of cones has their synaptic pedicles ending at the foveal pit floor (Figure 5, green spots, arrows), whereas the cones surrounding them stretch their axons (known as Henle fibers) and presynaptic pedicles away from the center of the foveal pit to the foveal slope area (Figure 5, green spots form a continuous line, arrows). The lack of blood vessels in the central pit can be seen by the absence of the blue circular profiles there (Figure 5, bv).

Figure 5.Vertical section of the human fovea immunostained with antibodies to cone arrestin (green), CRALBP (red) and Collagen IV (blue).

OBrien and colleagues (1) very elegantly illustrated the cone axons radiating out from the foveal pit forming the Henle fiber layer and terminating in distant pedicles in a whole mount monkey retina (Figure 6). The picture would be very similar in a human retina. The Henle fiber layer is a combination of outward radially directed axons of the cones, and where rods begin to appear, also rod axons, and Mller cell processes. It is interesting to note in Figure 5 that the pedicles of the very central bouquet of cones are widely spaced ending on the foveal pit floor. We know from Figure 5 that these central bouquet cone pedicles are separated by voluminous Mller cell elements.

Figure 6.A wholemount monkey fovea immunostained with cone arrestin. The axons of the cones radiate out to a ring of cone pedicles. Central bouquet cone axons stay in the foveal pit. From OBrien et al., 2012 (1).

Understanding how the primate fovea develops from fetal to adult stage of the retina has been a very difficult task in vision research. This has, of course been due to the difficulty of obtaining retinas from human pre-birth and baby eyes. Even fetal monkey material has been scarce to obtain. Dr. Anita Hendrickson (Figure 7) at the University of Washington, Seattle, spent most of her career pursuing this subject of retinal research, and has contributed almost all we know.

Figure 7.A young Anita Hendrickson at her microscope. From her obituary in 2017 (111).

The earliest fetal retinas examined (2) were from a week-22 eye. The fovea is not recognizable at this stage, because the central region of the retina, where the fovea will develop, consists primarily of several layers of ganglion cell bodies and inner nuclear layer cells (INL), presumably amacrine and bipolar cells (Figure 8, a). A single layer of developing cones stretches from outer plexiform layer (OPL) to pigment epithelium and choroid (Figure 8, a, right inset). A hint of a developing cone pedicle is seen (Figure 8, right red arrow) but there is no sign of outer segments of cones (Figure 8, right, apposing red arrowheads). By fetal week 28, an indentation of the retina at the thickest ganglion cell layer appears and can be considered the earliest sign of the foveal pit (Figure 8, b, P). The inner nuclear layer has become thinner and appears pushed out of the pit (P) but a kind of split is occurring in the middle of the INL known as the transient layer of Chievitz (TC, Figure 8, c) (3). By fetal week 37 (Figure 8, c) a pronounced foveal pit is evident (P), the ganglion cells are thinned to 2 or 3 deep and the TC area in the INL appears like a sheared, radially projecting area of probable Mller cell fibers. Through the latter two fetal stages, where the foveal pit is becoming obvious, the cones are still immature, arranged in a single layer and have no visible outer segments (Figure 8, b and c). However, there is the first suggestion of the cone axons being tilted away from their cell bodies to form the early Henle fiber layer.

Figure 8.Foetal human retina at (a) foetal week (Fwk) 22, (b) Fwk 28, and (c) Fwk 37. The foveal position is not noticed at week 22 but in later weeks becomes dimpled as ganglion cells become displaced out radially from the developing foveal pit. In the beginning the retina is thick, multilayered and cones are undeveloped with no outer segments or visual pigment (a: right enlarged photo, red arrow heads point to a cone nucleus, a stubby inner segment, and a developing cone pedicle). From Hendrickson et al., 2012 (26).

It is interesting to closely examine the cone photoreceptors in the fetal 35-to-37-week retinas as illustrated by Hendrickson and coauthors (2). Figure 9 shows how immature the cones of the foveal pit are compared with those of the cones at some distance from the fovea (Figure 9. 2 mm from fovea). At the foveal pit area, the cones are just stubby cells with a synaptic pedicle, little to no lengthened inner segment and zero outer segments (Figure 9, fovea). By 800 m to 2 mm from the developing foveal pit, the cones become elongated vertically and have definite cone pedicles. Most cell bodies descend away from the external limiting membrane and have elongating axons that are angled away from the foveal pit, forming the early Henle fiber layer. Inner segments are long, but the outer segments are still not formed. (Figure 9, 800 m and 2 mm).

Figure 9.Sections of the retina of a human foetus at 25 weeks gestation. The cones of the fovea are still undeveloped with no outer segments, and a synaptic area with no axon. From 800 m to 2 mm from the foveal center there are clear elongated inner segments but still no outer segments. The slanting of the cone axons out radially is beginning to be evidence of a developing Henle fiber layer. From Hendrickson et al., 2012 (26).

At birth of the human baby the retina in the eye is looking recognizably foveate (Figure 10, a). The foveal pit now contains a very thin, only one layer thick, ganglion cell layer, a thin inner plexiform layer (IPL) but a prominent inner nuclear layer (INL) (Figure 10, a). The cones are now evident as straight vertical cones with synaptic pedicles, cell bodies and inner segments. There are probably developing cone outer segments too (not easy to see at this magnification). But the pit is still several cell layers thick with only the cones on the foveal slope beginning to angle away from the pit. Further out on the foveal slope the cone Henle fiber layer is obvious now (Figure 10, a). By 15 months after birth, the baby retina has a definite fovea and even the central cones are angling out to the foveal slope. Inner and outer segments are well developed in the pit and no other layers of the retina are here anymore (Figure 10, b and c). By 13 years the fovea is completely developed (Figure 10, d) (2).

Figure 10.The foveal retina sections of a human from (a) postnatal 8 days (P8d), through (b) 15 months, to fully formed (d) 13 years. (c) At 15 months the cones are thin, have outer segments and squash together and, except for the central bouquet, send axons radially outwards as the Henle fiber layer. Second order neurons and ganglion cells are pushed along the foveal slope to form a pile of ganglion cell bodies at the foveal rim. From Hendrickson et al., 2012 (26).

What forces could cause this remarkable transformation of an evenly thick multi-cell, layered retina to become concavely dimpled, buckled up and stretched outwards to form a single layered pit at the fovea and a high sided sloping tissue with the highest concentration of cell layers at the foveal rim. The developmental effort is to ensure that a central area of the retina is concentrated with the slimmest packed cones with no obstruction of incoming light by secondary and tertiary cell layers.

The most recent investigations on this developmental phenomenon in the human (primate) retina provide evidence that the radial retinal glia the Mller cells and possibly the astrocytes of the ganglion cell layer are instrumental in this process (4). The Mller cells of the foveal pit are closely associated with the cone fibers and together they make up the Henle fibers layer (Figure 11A, red profiles). Bringmann and colleagues suggest that the Mller cells exert tractional forces onto cone axons fibers by a vertical contraction of the central most Mller cells and cones so they become elongated and very thin (Figure 11, B, blue arrows). After widening of the foveal pit by elimination of astrocytes in the pit and ganglion cell layers, the Henle fibers are forced, by horizontal contraction of their surrounding Mller cell processes in the outer plexiform layer, to pull the cone and then rod photoreceptor centrifugally away from the pit (Figure 11, B, orange arrows).

Figure 11.(A) A human fovea drawing to show that the Henle fiber layer consists of cone photoreceptor axons as well as envelopingMller cells and fibers (red). B) Drawing to show the central foveal cone bouquet of thin and closely packed cones in the foveal pit. The cone axons on the foveal slope move radially out with the Mller cells to form the Henle fiber layer and end in pedicles that make connection with bipolar cells at some distance from the foveal pit. Blue arrows show the vertical squeezing and packing of the cones in the foveal pit and orange arrows show the displacement horizontally of the foveal cone axons, during development of the adult fovea.

The term foveal cone mosaic generally refers to the strikingly regular patterns of condensed cone inner and outer segments with largely triangular crystalline organization, which nevertheless includes non-randomly distributed discontinuities (5, 6). The less familiar and less understood part of foveal cones is the further course towards their synaptic terminals. It includes a two-step transition. From a two-dimensional mosaic for image reception it is rearranged into to a three-dimensional somata tiling, which then again spreads out to establish the concentric monolayered pedicle meshwork (7-9).

The mature human fovea consists of 3 spectral types of cone: red or long wavelength sensitive cones, L-cones; green or medium wavelength cones, or M-cones; and blue or short wavelength cones, S-cones. These three types of cone are tightly packed and at their most concentrated (up to 200,000/mm2 in the fovea (8, 10) (see Webvision Facts and Figures). Rods are not present in the foveal pit, appearing first halfway into the foveal slope, beyond the 300 m diameter area (see Figure 2B).

It is extremely difficult to get a horizontal section through the central fovea particularly including the central bouquet of cones because of the concave nature of the fovea. Figure 12.1 manages to get such a view of a horizontal slice through the inner segments of the cones of a human fovea (7). The tiniest central cones in the center of the photograph (Figure 12.1) are very slim at 2.5-3 m in diameter and become progressively larger as they move along a radial gradient from the central bouquet. It is noticeable that the cones are not uniformly distributed in a hexagonal mosaic. Small patches of cones are hexagonal and then the patch is interrupted and shifts the surrounding patches slightly (Figure 12.1). Ahnelt and coauthors (11) noticed that these shifts in the mosaic usually were associated with the position of a slightly larger diameter cone. They proposed that these larger cones were the short wavelength cones, the S-cones, and described their morphological differences from the surrounding, more common L- and M-cones (11).

Figure 12.1.A horizontally sectioned and stained human retina at the foveal pit and rod free area. From Ahnelt et al, 1987 (11).

S-cones are relatively rare in the retina compared with the much more dominant L- and M- cones. The S-cones are, however, ubiquitous in all vertebrate retinas, with the exception of cetaceans (12). As far as other mammals are concerned S-cones are commonly paired with L-cones to give them a dichromatic color sense. These L-cones vary in spectral peak, and the more mid-spectral types are called M-cones. In old world monkeys and apes, and in man an L-opsin gene duplication and further mutation produced an extra mid-spectral L-cone opsin subtype, M-cone opsin. The combination of L-cones, M-cones and S-cones provides trichromacy. This trichromacy allows discrimination of green, yellow and blue/purple hues.

There are differences in the genetic structure and locus of the S-cone visual pigment compared with the M- and L-cone pigments (13), yet the S-cones always form a consistent 8-10% of the mammalian cone photoreceptor population (14, 15). In primates and humans of course, the S-cones are rather scarce in the foveal pit. Some authors suggest that there is a so-called blue cone blind spot (16). However, S-cones peak in number on the foveal slope of the human retina and here form about 12% of the population. Figure 12.2, (a) shows the peak S-cone distribution on the foveal slope in a human retina as identified by the larger size and arrangement in the mosaic breaking up the regular hexagonal pattern distribution of the other cone types. In Figure 12.2, (b) the S-cones have been colored in for clarity.

Figure 12.2.A whole-mount photograph of the foveal slope of a human retina. P (upper right corner) is the foveal pit. Larger cone profiles break up the mosaic of cones into disjointed groups of closely packed smaller profile cones [arrows in (a, b) and colored in as S-blue cones in (b)]. From Ahnelt et al., 1987 (11).

Since these earlier identifications of foveal S-cones on morphological criteria (11), antibodies against the S-cone pigments in the cone outer segments have been developed and are able to positively identify the S-cones in the overall population by immunocytochemical methods. In figure 13, the human foveal pit (FP) and foveal slope are immunostained with an S-cone antibody and illustrate the S-cones as black spots and angled black cone outer segments. In the foveal pit only a few S-cones appear interspersed in the mosaic of highest density (Figure 13). However, their proportion increases in surrounding areas and are at their highest density on the foveal slope (Figure 13 brown spots, top and right-hand side).

Figure 13.The foveal pit (FP) and part of the foveal slope are immunostained with an S-cone opsin in a human retina.

Figure 14 illustrates immunostaining in vertical section and the scarcity of S-cones in the foveal pit compared to the increase in number of this population of cones on the foveal slope, of a human retina. A map of the S- cone distribution in another human fovea is shown in Figure 15. The lighter to darker blue shading indicates less dense to denser S- cone presence. Note in both images (Figs. 14 and 15) there are very small numbers of S-cones in the foveal pit.

Figure 14.Vertical section of a human foveal pit immunostained with antibodies against cone arrestin for all cones (red), and JH455, which labels S-cones (green). Few S-cones are found in the foveal pit.

Figure 15.Every S-cone is labelled with S-cone opsin antibody in a human fovea. The more intense blue shading indicates greater densities of S-cones in the foveal slope where they reach 12% of the cone population.

It has been rather easy to identify S-cones in the human fovea and the rest of the retina by these immunocytochemical techniques where S- cones can be visualized and distinguished from the surrounding L- or M-cones. Figure 16 shows a spectacular confocal image of the cones in near peripheral human retina by immunolabeling with cone arrestin, and by the HJ455 antibody to S-cones, that shows up the S-cone opsin both in the outer and inner segments.

Figure 16.Near peripheral retinal human cones stained with HJ455 antibody that identifies the S-cones (green) amongst the arrestin (red) labeled cones.

Sadly, the L-cones and M-cones are not distinguishable on immunostaining techniques because their visual pigments are so close in structure. There is presently no antibody developed to separately mark them into L- or M- cone types. So, to identify L- and M-cones in the human fovea we must go to other more sophisticated techniques. Psychophysical measurements have suggested that L- cones usually outnumber M-cones by 2:1 in the human fovea (17). Microspectrophotometry of all cones in small patches of cones in the fovea of monkeys, has revealed that L- and M-cones occur in about equal proportion (18).

Newer techniques, introduced by Roorda and Williams (19), use adaptive optics to make direct measurements of spectral sensitivity of foveal cones in the living human eye (Figure 17). They found that humans varied greatly in the proportions of L-cones to M-cones: some individuals have almost equal proportions while others have a higher proportion of L-cones, even to the extreme of 16 L-cones to every M-cone (Figure17, BS). While the sparser S-cones are spaced regularly, the L- and M-cones lie randomly in the mosaic meaning that clusters of cones of the same spectral type will occur together as suggested from Mollon and Bowmakers paper (18). Roorda and coauthors (20) concluded that L- and M-cones are in a random distribution in the foveal center (21). Nevertheless, the human subjects HS and BS in Figure 17 would seem intuitively to have a different perception of color. But both subjects were reported to have normal color vision (19). A single cone is achromatic, and its stimulation doesnt result in color vision unless there is comparison to stimulation of a neighbouring cone with different opsin (22). This comparison is done by retinal and brain neural circuitry (see later section on horizontal cell roles in spectral antagonism). Some elegant recent human adaptive optics studies and psychophysical reporting found that 79% of targeted cones in the foveal center, tested for color perception, correctly identified the color (hue) (22). Interestingly, others, using similar techniques of adaptive optics and human reports of hue for single cone stimulation with colored light in the fovea, found a considerable proportion of cones produced only white sensations (21).

Figure 17.Method of adaptive optics shows mosaics of L (red), M (green) and S (blue) cones in four human subjects with normal color vision. The ratio of S to L and M cones is constant, but that of L to M cones varies from 2.7:1 (L:M) to 16.5:1 (L:M). Adapted from Roorda and Williams, 1999 (19).

The process of centrifugal displacement by the Henle layer affects cone pedicles in different ways, depending on their eccentricity (Figure 18).

Figure 18.Foveal pit in blue and the foveal slope to the foveal edge in grey. Cone pedicles lack telodendria in the foveal pit. Pedicles with increasing eccentricity along the slope have tadpole-like shape. More peripherally cone pedicles are round in shape and have telodendrial interconnections. The transition coincides with the appearance of capillaries (red) and microglia (green spots). The thin blue line denotes the elliptical course of the external limiting membrane sectioned at the foveal slope at 1 degree (300 m eccentricity).

In the central bouquet of cones in the foveal pit, the pedicles appear to stay in place (Figure 18). In serial semithin (Figure 19, a) and electron microscopic (Figure 19, b) sections, a few roundish pedicles can be found at the foveal floor (Figure 19, a-c, circles). They are isolated from each other, thus lacking any connections to other cones via telodendria. Still they are contacted by dendritic processes running horizontally from a few interneurons (presumably bipolar and horizontal cells) from the foveal slope or even those neurons lying embedded in voluminous Mller cell processes (Figure 19 b-c, red circles around pedicles).

Figure 19.LM and EM appearances of cone pedicles. (a), (b) and (c) are isolated pedicles of the foveal pit (red circles). There are large Mller-cell processes and neural processes running to the cone pedicles. (d) and (e) show tadpole-like cone pedicles on the foveal slope. (f) Pedicles at the first capillary zone are arranged in curved, bead-like series. (g) Higher magnification shows the telodendrial network between most cone pedicles in (f). (a) is from Ahnelt, 1998 (112), ganglion cell (gc), Mller cell (Mc), cone axon (ax), scale bar 50 m. (g) is from Ahnelt and Pflug 1986 (113).

From the outer central cones, Henle fibers of short length terminate in peculiar tadpole-like pedicles (Figure 18, Figure 19, d-e). They too are largely isolated from neighboring terminals and are characteristic of the cone pedicles until about 1 or 288 m out (23). Beyond this zone still almost entirely established by cone terminals only the pedicles make up a patchy mosaic (Figure 19, f-g). These terminals elaborate telodendrial networks that end on neighboring cone pedicles at gap junction connections (1, 24). This pedicle mosaic tends to establish radial arrays yet is locally influenced by interspersed glia (Figure 19, g).

The cones of the foveal pit project vertically downwards (Figure 20, a). As the concentrated central cones have to extend their axons radially out of the pit they, together with Mller cells, become the Henle fibers. The cone axons become longer and longer as they project onto the foveal slope and into the parafovea (Figure 20, b, 200-400 m long). From then on, further out into the perifovea, the axons begin to shorten and by 3 mm eccentricity from the foveal pit axons are essentially no length at all (Figure 20, c-d, 4000 m periphery). The Henle fiber layer is over as is the macula lutea (Figure 2A, Figure 2B).

Figure 20.Cone morphology in the foveal pit (a), foveal slope (b) and peripheral retina (c). Cones and ON bipolar cells are immunostained with GNB3 (green). Drawing (d) shows the cone morphologies in the different areas. An S-cone (blue-green) is shown in comparison with the M/L-cone types.

S-cones and M/L-cones differ in the time course of mitotic differentiation and expression of opsins. According to Xiao and Hendickson (25), S-opsin and various synaptic proteins are detectable at fetal week 11, while various synaptic and transduction proteins appear in M/L cone subclasses before their opsin visual pigments are detected at fetal week 13 (26). It is clear that S-cones develop in a different mosaic than M/L-cones. Ahnelt and coworkers (7) have noted that cones likely to be short wavelength sensitive tend to occur in irregular positions in both, foveal and peripheral areas. Figure 21A shows an opsin labeled S-cone (asterisk) positioned between seemingly linear series of unlabeled M/L-cone inner segments. Thus in the foveal all-cone mosaic, S-cones appear to interrupt the linear beads of L/M cone-cell inner segments and clearly do not belong to the mosaic of M- and L-cones (6).

Figure 21A.Human cone inner segment mosaic on the foveal slope. Note the first rod (r), and the bead-like arrangement (colored lines) of the M- and L-cones circumventing an S-cone labeled by an S-opsin antibody (asterisk).

The S-cones form a random mosaic like the M/L cones except at the foveal slope area where they are at highest concentration. Here they approach a non-random distribution (25).

Figure 21B shows a schematic summary (7) of cone arrangement in the mosaic of the foveal slope area where the S-cones develop first and reach the non-random mosaic arrangement (25, 27). Three L/M cone patches are exemplified with false colors (yellow, dark blue green and light green). These have migrated downward from an initial position near the external limiting membrane (ELM) to form bead-like arrangements of M/L cone cell bodies in the depths of the outer nuclear layer (ONL). Their axons (Henle fibers) emerge from the cone nuclear layer and radiate centrifugally towards their pedicles. At the intersection of the L/M patches sits an S-cone always with its cell body, unmigrated, up at the outer limiting membrane. Figure 21B left top, indicates the original position (transparent ovals) of M/L cell bodies before mosaic condensation and their presumed path (tapered rays) to their adult positions.

Figure 21B.The transformation of the foveal cone mosaic groups (yellow, dark green, light green) by condensation of their inner/outer segments to vertical sequences of beaded cell bodies and descending, radiating axons in the Henle fiber layer. At left, the original position of the yellow groups cell bodies (line of ovals) before mosaic condensation is indicated, as well as their eventual path (curved lines) to their adult positions. Apparently, S-cones (blue) do not participate in this process, as their cell bodies stay close to the ELM (external limiting membrane, large arrow). Adapted from Ahnelt et al, 2004 (7).

As we have illustrated in Figure 2B, the whole fovea is roughly 1.5 mm across and so any cell found within 750 m of the foveal center is considered a foveal associated cell. It has been hard to get good staining of horizontal cells (HC) of the fovea but some Golgi impregnated human retinas in our possession did allow us to see a few within the 750 m of eccentricity around the central foveal pit (Figure 22) (28).

Figure 22.The shape and size of horizontal cells in the human fovea (Golgi staining). The smallest HCs are in the avascular zone edge of the foveal slope (350 m). The closest HCs stained on the inner foveal slope (200 m) are stretched out, with dendrites following the circular foveal pit circumference and reaching into the central bouquet of cones. From Kolb et al., 1994 (28).

The closest to the foveal center, which is of course cell free except for cone photoreceptors and some dendrites running up to synapse with the central cones, would be the HC at 200 m from the foveal center (Figure 22, top cell). These horizontal cells are elongated and arranged concentrically in a circle around the foveal center and on the far edge of the foveal pit. The area could still be in the avascular zone. Note the dendrites are reaching quite far to contact central cones. The cells are axon bearing, but morphologically it is difficult to judge of which type. The cells at 350 m (Figure 22) are much smaller than the foveal edge HC but now recognizable as H1, H2 and H3 cell types (28). The smallest are the H1 cells that appear to contact about 4-5 cones, judging by their dendritic clusters. H2 cells are wirier and more irregular than H1 and H3 cells but have quite closely packed and profuse dendrites (Figure 22). These H2 cells would be reaching into the foveal slope area, where we know there is the highest density of S-cones, to contact the latter cone type. H3 cells may also be reaching into the foveal slope but we know from previous data they do not receive synapses from S-cones (29, 30). There are no evident axons on these Golgi stained horizontal cells (Figure 22, 350 m), which probably reflects understaining.

The three horizontal cells at 500 m from the foveal center (Figure 22) would also be foveal HCs but in an area where blood vessels occur and the first rod photoreceptors are present. As can be seen they are a little larger in dendritic field size (Figure 22). The H1 cell contacts 6 cones and the H3 about 8-9 cones (Figure 22). H1 and H2 types here have axons (small arrows in Figure 22), which will expand into axon terminals in contact with rods in the case of H1, and with S-cones in the case of H2 cells (31).

By confocal microscopy the central human fovea can be seen to contain parvalbumin immunoreactive horizontal cells (Figure 23, a-b; green cells under the cone pedicles). Parvalbumin identifies H1/H3 horizontal cell types and it is likely that the Golgi staining at the 200 m distance from the central foveal pit is therefore of these types. They are elongated and not closely packed. Their dendrites would be reaching to contact central foveal bouquet cones (Figure 23, b). In contrast, the H1s of the foveal slope are closely packed with vertically squashed cell bodies and small bushy dendrites reaching to the closely packed cone pedicles at the ends of the Henle-fiber-layer cone axons (Figure 23, c). These HCs are clearly the same as those in the Golgi preparations at 300-500 m (Figure 22).

Figure 23.Vertical section of the human fovea cut along the edge of the foveal pit. H1 horizontal cells are immunostained with anti-parvalbumin (green) and cone photoreceptors with recoverin (red). H1 cells are very crowded together in the foveal slope.

The H2 cells of the human retina are known to be particularly associated with the S-cone (blue) photoreceptors (see Webvision chapter on S-cone pathways). We know that H2 cells stain with antibodies to calbindin in the human retina as compared to parvalbumen staining for H1/H3 cells. Figure 24 (white arrows) shows a few calbindin positive HCs (red cells, arrows) on the foveal slope in human retina. In addition to the H2 cells with cell bodies close to the OPL, there are diffuse cone bipolar cells contacting several cones, and amacrine cells stained with calbindin. These red, diffuse bipolar cells have cell bodies lower in the inner nuclear layer and long slanted single apical dendrites as compared to the red H2 cells. Note in this section of human fovea the first rods are present on the foveal slope and the first rod bipolar cells are staining for the antibody to PKC (Figure 24, green cells).

Figure 24.Human foveal slope area immunolabeled with antibodies against calbindin (red) that marks H2 horizontal cells, some bipolar and some amacrine cell types. H2 cells are marked with arrows. The first rod bipolar cells on the foveal slope are labeled with PKC-alpha antibodies (green).

Horizontal cells of the vertebrate retina are known to have important roles in sharpening and scaling of responses from photoreceptors through the subsequent retinal pathways to influence the ganglion cell output (32). At the first level of the outer plexiform layer, horizontal cells are involved in feedback of signal from surrounding cones to each individual cones receptive field. This surround input is expanded well beyond the horizontal cells dendritic connectivity field by virtue of gap junctions that join the dendrites of many horizontal cells of the same type together. i.e. in human retina the H1-H1 cells would be joined in gap junctions and the H2 cells would likewise be joined to other H2 cells (See the Webvision chapter Myriad roles for gap junctions in retinal circuits). This large feedback effect provokes an expanded region of antagonistic signal compared with the central cone signal. In the case of M- or L-cones the antagonistic surround is a mixed M- and L-cone signal. In other words, individual M- and L-cones do not show classic spectral opponency just mixed M- / L-cone surround antagonism (33). The feedback in the case of an S-cone would come from H2 cells, whose contacts include surrounding M- and L-cones. Indeed S-cones have been recorded from in monkey retina and found to have blueyellow spectral opponency as well as center-surround organization (34, 35). Presumably spatial opponency would be transmitted from the M- and L-cones to their respective bipolar cell connections, and in the case of the S-cone, a true spectral opponency has been proven to be transmitted as well (34). No recordings have been made in foveal cones to really see if an M- or L-cone has a spectrally opponent surround like that of (albeit peripheral) S-cones (35).

A long time ago the great Spanish anatomist, Santiago Ramn y Cajal described the neurons of the different vertebrate retinas as seen by sectioned Golgi-stained material. He noted many different types of bipolar cells in the various species and that there were particularly tiny dendritic spreads for some bipolar cells in the bird retina (36). He suggested that these bipolar cells contacted single cones.

In 1941, Stephen Polyak (Figure 25) published books on the neural cell types revealed by Golgi and other silver methods in monkey and human retinas and brain. In central monkey and human retinas Polyak observed and illustrated several types of bipolar cells, but he was very concentrated on the remarkably small dendritic tops of some types that he construed as contacting single cones. He named these bipolar cells, midget bipolar cells (mbc).

Figure 25.Steven Polyak circa 1940.

Figure 26 shows Polyaks original drawing of these midget bipolar cells and larger dendritic field size bipolar cells that would appear to contact several cones (Figure 26, imb, fmb and dfb). Polyak also drew and commented briefly that the midget bipolar cells appeared to be of two varieties, one that had a long axon to the inner plexiform layer, and the other a much shorter axon ending higher in the inner plexiform layer. At the same time, there were midget ganglion cells that had small dendritic trees that came in the two varieties possibly reaching to the axon terminals of the two types of midget bipolar cells (Figure 26, mgcs).

Figure 26.Original drawings of Polyak (90). Bipolar cells and ganglion cells of the central retina. We now know that the invaginating midget bipolar cells (imb) and flat midget bipolar cells (fmb) are physiologically different. Polyak described midget ganglion cells (mgc) as of two types, which we now know are OFF mgc and ON mgc. These connect to fmbs and imbs respectively. Large field bipolar cells (dfb) and parasol ganglion cells were also described by Polyak. The cone spectral types have been colored in by the present authors.

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The Architecture of the Human Fovea Webvision

The Role of Reproductive Hormones in Postpartum Depression

CNS Spectr. Author manuscript; available in PMC 2016 Feb 1.

Published in final edited form as:

PMCID: PMC4363269

NIHMSID: NIHMS622897

Crystal Edler Schiller* has a Ph.D. from the University of Iowa. Dr. Schiller is an Assistant Professor in the Psychiatry Department at the University of North Carolina at Chapel Hill in Chapel Hill, NC. Samantha Meltzer-Brody has an M.D. from Northwestern University Medical School and an M.P.H. from the University of North Carolina at Chapel Hill. Dr. Meltzer-Brody is an Associate Professor in the Psychiatry Department at the University of North Carolina at Chapel Hill in Chapel Hill, NC. David R. Rubinow has an M.D. from the University of Connecticut School of Medicine. Dr. Rubinow is the Assad Meymandi Distinguished Professor and Chair of the Psychiatry Department at the University of North Carolina at Chapel Hill in Chapel Hill, NC

Despite decades of research aimed at identifying the causes of postpartum depression (PPD), PPD remains common, and the causes are poorly understood. Many have attributed the onset of PPD to the rapid perinatal change in reproductive hormones. Although a number of human and non-human animal studies support the role of reproductive hormones in PPD, several studies have failed to detect an association between hormone concentrations and PPD. The purpose of this review is to examine the hypothesis that fluctuations in reproductive hormone levels during pregnancy and the postpartum period trigger PPD in susceptible women. We discuss and integrate the literature on animal models of PPD and human studies of reproductive hormones and PPD. We also discuss alternative biological models of PPD to demonstrate the potential for multiple PPD phenotypes and to describe the complex interplay of changing reproductive hormones and alterations in thyroid function, immune function, HPA axis function, lactogenic hormones, and genetic expression that may contribute to affective dysfunction. There are three primary lines of inquiry that have addressed the role of reproductive hormones in PPD: non-human animal studies, correlational studies of postpartum hormone levels and mood symptoms, and hormone manipulation studies. Reproductive hormones influence virtually every biological system implicated in PPD, and a subgroup of women seem to be particularly sensitive to the effects of perinatal changes in hormone levels. We propose that these women constitute a hormone-sensitive PPD phenotype, which should be studied independent of other PPD phenotypes to identify underlying pathophysiology and develop novel treatment targets.

Despite decades of research aimed at identifying the causes of postpartum depression (PPD) and developing effective methods of screening, prevention, and treatment, PPD remains common, affecting between 7 and 20% of women following delivery1. PPD is one of the most important public health problems that we can address: it not only affects women at a highly vulnerable time, but it also has deleterious effects on children and families. Many have speculated that PPD is caused, at least in part, by the rapid change in the reproductive hormones estradiol and progesterone before and immediately after delivery2. Although a number of human and non-human animal studies suggest that changes in reproductive hormone levels contribute to PPD38, several studies have failed to detect an association between hormone concentrations and PPD symptoms911. For example, cross-sectional human studies examining between-group differences in ovarian hormones levels and depressive symptoms during the postpartum period have failed to demonstrate and association between absolute estrogen and progesterone concentrations and PPD911. In contrast, studies that have treated PPD with estradiol have successfully reduced depressive symptoms5,12, and animal studies have demonstrated that estradiol and progesterone withdrawal provoke depression-like behavior4,7,8.

The mixed results regarding the role of estradiol and progesterone in PPD is likely due to three factors. First, the PPD diagnosis contains enormous variability. A postpartum depressive episode can meet the diagnostic criteria in a number of different ways, which results in women with very different symptom presentations receiving the same diagnosis. Two women could share only one symptom of major depression, experience timing of onset of the episode during very different hormonal conditions (e.g., first trimester of pregnancy versus first week postpartum), and both receive a PPD diagnosis. Thus, PPD likely represents a number of depressive phenotypes, which may in large part account for the difficulty in identifying any biological or hormonal factor central to the disorder.

Second, based on epidemiologic studies of risk, social and psychological factors play a large role in the pathogenesis of PPD. For example, decreased social support, poor quality social support, and poor marital satisfaction increase the risk of PPD1315. The number of previous episodes of depression, a history of PPD, and depression during pregnancy are also significant risk factors for PPD1517. PPD, like any mood disorder, is therefore best seen as a clinical integration of risk and protective factors that culminate in the triggering of a mood episode in the context of a biological (or reproductive) state.

Third, the existing studies have used widely diverging methods to examine how reproductive hormones influence depressive symptoms: some have examined absolute hormone concentrations in those with and without the disorder911, some have examined the change in hormone levels during pregnancy and the immediate postpartum period and the attendant changes in depressive symptoms10,18, some have administered hormones to well individuals at high risk for PPD3, and some have used hormones as a treatment for PPD5,12. Any biological model of PPD has to account for all three of these problems.

The purpose of this review is to examine the evidence for a reproductive hormone model of PPD in which fluctuating reproductive hormone levels trigger affective dysregulation. We will define PPD and discuss the diagnostic issues that contribute to difficulties in identifying a single biomarker for the disorder. We will discuss alternative biological models of PPD to demonstrate the potential for multiple PPD phenotypes and to describe the complex interplay of changing reproductive hormones and alterations in thyroid function, immune function, HPA axis function, lactogenic hormones, and genetic expression that may contribute to affective dysfunction. We will present animal models and human studies of reproductive hormones and PPD and discuss methodological issues that have contributed to conflicting findings in the literature. We will provide evidence of a hormone-sensitive PPD phenotype, and discuss the potential neurobiological pathophysiology of PPD for this group of women. Finally, we will review human brain imaging and genetic studies as they pertain to the hormonal contribution to affective dysregulation during the perinatal period.

The DSM-5 expanded the definition of PPD to include major depressive episodes with a perinatal onset as those beginning in either pregnancy or within the first four weeks postpartum19. Although PPD and non-perinatal major depressive disorder have the same DSM diagnostic criteria (i.e., depressed mood, anhedonia, sleep and appetite disturbance, impaired concentration, psychomotor disturbance, lethargy, feelings of worthlessness or guilt, and suicidal ideation)19, the symptoms of psychomotor agitation and lethargy are more prominent in PPD than MDD20. Additional symptoms of PPD include mood lability and preoccupation with infant well-being. PPD also is frequently associated with symptoms of anxiety, ruminative thoughts, and panic attacks21. Indeed, most women with PPD have comorbid anxiety disorders21. Recent estimates suggest that 7% of women experience an episode of major depression in the first three months following delivery, and the prevalence increases to 20% when episodes of minor depression are also included1. The majority of existing studies suggest that PPD is no more common than non-postpartum depression22; however, the largest epidemiological study to date demonstrated an increased risk of depression during the postpartum period23.

PPD is distinguished from the postpartum blues, which are defined as normative mild dysphoria occurring in the first week after delivery22. Also distinct from PPD is postpartum psychosis, which has a rapid onset associated with hallucinations or bizarre delusions, mood swings, disorganized behavior, and cognitive impairment24,25. Many cases of postpartum psychosis are manifestations of bipolar disorder26,27, which may present as mania for the first time during the postpartum period. The perturbation in mood, limited reality testing, and gross functional impairment make postpartum psychosis particularly dangerous for mothers and babies24.

An important limitation of the DSM criteria for PPD is that it is not mechanistically based, which is why the National Institute of Mental Health (NIMH) Research Domain Criteria (RDoC) project may be an ideal framework for studying PPD. The RDoC project advocates study of basic dimensions of functioning (e.g., emotion processing) across multiple units of analysis (e.g., genetic risk and epigenetic modification, limbic system, self-reported affective state) in a specific context (e.g., reproductive hormonal state). The RDoC initiative, therefore, allows researchers to go beyond the DSM criteria to identify women who demonstrate patterns affective dysregulation related to reproductive states and examine the underlying neurobiological pathophysiology. For example, while some previous studies have strictly defined PPD according to the DSM criteria, most have used more inclusive criteria, including episodes of depression that began before or during pregnancy and carried over into the postpartum and episodes with an onset several months following delivery. A study by Forty and colleagues28 demonstrated that defining PPD onset within 8 weeks of delivery is optimal for studying the biological triggering of affective dysregulation. Using this definition, Deligiannidis et al.29 identified functional neural correlates of postpartum depressive symptoms that occur in the context of changing reproductive hormone and neurosteroid levels.

Many have hypothesized a role for reproductive hormones in PPD because of the temporal association between the substantial and rapid changes in hormone concentrations that occur at delivery and the onset of depressive symptoms11. However, there are several important reasons for hypothesizing that reproductive hormones play a role in PPD. First, reproductive hormones play a major role in basic emotion processing, arousal, cognition, and motivation, and thus, may contribute to PPD indirectly by influencing the psychological and social risk factors. However, reproductive hormones also regulate each of the biological systems implicated in major depression, which suggests that hormones may impact a womans risk for PPD directly. In the forebrain and hippocampus, ovariectomy decreases and estradiol increases brain-derived neurotrophic factor (BDNF) levels30, which are decreased by depression and stress and increased by antidepressants31. Estradiol also increases cAMP response element-binding (CREB) protein activity32 and the neurotrophin receptor protein trkA33, and it decreases GSK-3 beta activity34 in the rat brain similar to antidepressant medications. Progesterone also regulates neurotransmitter synthesis, release, and transport35. For example, progesterone up-regulates BDNF expression in the hippocampus and cerebral cortex36. The relevance of gonadal steroids to affective regulation is further suggested by modulatory effects on stress and the HPA axis, neuroplasticity, cellular energetics, immune activation, and cortical activity37, all processes that have been implicated as dysfunctional in depression.

Of particular note are the manifold effects of gonadal steroids on brain function as revealed by brain imaging studies. These studies, employing positron emission tomography (PET) or functional magnetic resonance imaging (fMRI) in asymptomatic women, have demonstrated that physiologic levels of gonadal steroids modulate the neurocircuitry involved in normal and pathological affective states. In a study of healthy women, regional cerebral blood flow (rCBF) was attenuated in the dorsolateral prefrontal cortex, inferior parietal lobule, and posterior inferior temporal cortex during GnRH agonist-induced hypogonadism, whereas the characteristic pattern of cortical activation reemerged during both estradiol and progesterone addback38. Studies of neural activity during the menstrual cycle have compared activation across menstrual phases within subjects. Goldstein and colleagues39 found increased amygdala activity during the late follicular phase (higher estradiol levels) compared to the early follicular phase (lower estradiol levels), and Protopopescu et al.40 demonstrated increased activity in the medial orbitofrontal cortex (a region that exerts inhibitory control over amygdalar function) during the luteal phase (higher estradiol levels) compared with the follicular phase (relatively lower estradiol levels). The opposite was true for the lateral orbitofrontal cortex, suggesting that sensory and evaluative neural functions are suppressed in the days prior to menstruation40. Ovarian hormones also modulate neural reward function in humans, with increased activation of the superior orbitofrontal cortex and amygdala during reward anticipation and of the midbrain, striatum, and left ventrolateral prefrontal cortex during reward delivery in the follicular phase (compared with the luteal phase)41. Thus, there is evidence that reproductive hormones influence the biological systems and neural circuits implicated in depression directly, suggesting that the hormone instability inherent in the perinatal period could contribute to mood dysregulation in PPD.

The hormonal changes of pregnancy and the postpartum period do not occur in isolation: several other biological systems are altered during pregnancy and have been implicated in PPD. Alterations in any of these systems may provoke PPD independent of the changing hormonal milieu, which would suggest that there are a number of PPD phenotypes, each with their own relevant biomarkers. Thus far, the search for one biomarker for the general category of PPD has been elusive, and further research is needed to determine whether there are multiple PPD phenotypes with distinct etiologies. It also stands to reason that perturbations of other biological systems act in concert with rapidly changing hormone levels to contribute to affective dysregulation. Indeed, reproductive hormones have been shown modulate all of the other biological systems implicated in PPD: thyroid function42, lactogenic function43, the hypothalamic-pituitary-adrenal (HPA) axis44,45, and the immune system46. As such, we will discuss the potential contribution of each of these systems to affective dysregulation during pregnancy and the postpartum period, and we will discuss the evidence of a genetic susceptibility to PPD.

Thyroid hormones have been proposed as a biomarker of PPD in large part because of the presumed relationship between thyroid dysfunction and major depression47: depression accompanies thyroid pathologies48,49, thyroid dysregulation accompanies depression50,51, and the administration of thyroid hormones is thought to augment and accelerate antidepressant treatment52,53. Estrogen increases thyroxine-binding globulin (TBG) and consequently increases circulating thyroxine (T4) levels54,55. Thyroid dysfunction is associated with pregnancy56 and may contribute to PPD in some women57,58. However, previous studies have failed to detect a clear association between thyroid hormone dysregulation and PPD in the majority of patients5961.

The lactogenic hormones oxytocin and prolactin have been implicated in PPD62. Failed lactation and PPD commonly co-occur, and lactogenic hormones regulate not only the synthesis and secretion of breast milk, but also maternal behavior and mood. Oxytocin, in particular, may account for the shared pathogenesis of unplanned early weaning and PPD63. Estrogen and progesterone modulate oxytocin mRNA expression in brain regions associated with maternal behavior and lactation64,65. Lower oxytocin levels during the third trimester are associated with increased depressive symptoms during pregnancy63 and the immediate postpartum period66. In a recent study by Stuebe and colleagues63, oxytocin secretion during breastfeeding was inversely associated with depression and anxiety symptoms at 8 weeks postpartum. Although depression and anxiety symptoms were not associated with breastfeeding success in this study, reduced oxytocin may predispose women to PPD and subsequently lead to unsuccessful breastfeeding. Moreover, low oxytocin levels in mothers with PPD are associated with low oxytocin levels in fathers and their children, suggesting a potential neuroendocrine mechanism for the increased risk of depression in children of depressed mothers67. Lastly, oxytocin has also been examined as a potential treatment for a wide range of psychiatric disorders, including PPD, but with inconsistent findings to date68,69.

Hypothalamic-pituitary-adrenal (HPA) axis dysfunction has also been implicated in the pathogenesis of PPD. HPA axis hyperactivity is one of the most consistent findings in the neuroendocrinology of depression70. Hypercortisolism is associated with depressive symptoms and corrected with antidepressant treatment70. Additionally, the HPA axis is dysregulated by stress and trauma71, both of which are known precipitants of PPD13,72,73. Levels of corticotropin-releasing hormone (CRH), ACTH, and cortisol increase substantially during pregnancy and drop four days following delivery74. HPA axis function normalizes at approximately 12 weeks postpartum74. The effects of pregnancy on HPA axis function may be at least partially attributable to the effects of estrogen on corticosteroid binding globulin75, CRH gene expression76, and circulating corticotropin concentrations44. Similar to the HPA axis dysregulation seen in nonpuerperal depression, basal concentrations of plasma cortisol are increased in women with PPD, and suppression of cortisol by dexamethasone is blunted59. In one study, for women with PPD there was no association between ACTH and cortisol levels in response to a stress test, whereas among non-depressed control women, there was a more regulated association with cortisol levels rising following the increase in ACTH77. Some evidence suggests that higher cortisol levels at the end of pregnancy are associated with increased blues symptoms78. However, it remains unclear whether HPA dysregulation contributes to the onset of PPD or occurs as an epiphenomenon.

Immune dysregulation has been hypothesized to contribute to the development of PPD79. During pregnancy, anti-inflammatory cytokines responsible for immunosuppression are elevated and promote pregnancy maintenance, whereas proinflammatory cytokines are downregulated. Delivery abruptly shifts the immune system into a proinflammatory state, which lasts for several weeks. Patients with depression tend to have higher levels of the proinflammatory cytokines tumor necrosis factor (TNF)- and interleukin (IL)-680, and administration of cytokines is associated with the onset of depression81. The immune axis is regulated by estradiol. Estradiol modulates cytokine production, cytokine receptor expression, activation of effector cells, both the number and function of dendritic cells and antigen presenting cells, and monocyte and macrophage immune function82. Differential patterns of gene expression that are functionally related to differences in immunity have been found to distinguish women with PPD from those without83. Although one recent study identified several prenatal immune markers of PPD84, other studies have failed to detect an association between immune dysfunction and postpartum depressive symptoms8587. Thus, the role of immune function in PPD remains unclear.

Evidence of a genetic vulnerability to PPD has emerged from family, candidate gene, genome-wide, and gene manipulation studies. Family and twin studies suggest that PPD aggregates in families28,88, is heritable89, and may be genetically distinct from nonpuerperal depression89. Although multiple genes likely contribute to PPD, the role of specific genetic variations remains unclear. Candidate gene studies of PPD have identified several of the same polymorphisms implicated in non-puerperal depression, including the Val66Met polymorphism of the BDNF gene90,91, the Val158Met polymorphism of the COMT gene92,93(p-), the BcII polymorphism of the glucocorticoid receptor and the rs242939 polymorphism of the CRH receptor 194, the short version of the serotonin-transporter linked polymorphic region (5-HTTLPR) genotype95,96, three polymorphisms in the serotonin 2A receptor (HTR2A) gene97, and three polymorphisms at protein kinase C, beta (PRKCB)98. There is also evidence of PPD biomarkers that are theoretically distinct from those of MDD and that implicate reproductive hormones. For example, polymorphisms in the estrogen receptor alpha gene (ESR1) have been found to be associated with PPD98,99. However, to date, the results of candidate gene studies of MDD and PPD have failed to replicate100, have not been statistically significant after correcting for multiple comparisons97,98, and there is little consistency in the specific polymorphisms tested and identified across studies, which means that any one genetic variant or set of genetic variants is of limited utility as a diagnostic indicator. Genomic studies aim to address some of these shortcomings, and there have been a few small genomic studies of PPD to date. In a genome-wide linkage study of 1,210 women, researchers identified genetic variations on chromosomes 1q21.3-q32.1 and 9p24.3-p22.3 that may increase susceptibility to PPD101. Of particular relevance here, the strongest implicated gene was Hemicentin 1 (HMCN1), which contains multiple estrogen binding sites. Although the results were no longer significant after accounting for multiple comparisons101, the association between the rs2891230 polymorphism of the HMCN1 gene and PPD was confirmed by a subsequent candidate gene study102. Similarly, a genome-wide association study yielded a third-trimester biomarker panel of 116 transcripts that predicted PPD onset with 88% accuracy in both the discovery sample of 62 women and the independent replication sample of 24 women103. Of these transcripts, ESR1 was the only enriched transcription factor binding site, again potentially implicating estrogen in the pathogenesis of PPD103. Estrogen-induced DNA methylation change has also been implicated in PPD, which suggests that women with PPD have an enhanced sensitivity to estrogen-based DNA methylation reprogramming104. In order to serve as reliable biomarkers of PPD, these genetic variants will require replication in larger, independent samples, which is currently an active area of investigation in the field.

Non-human animal studies largely support the role of reproductive hormones in PPD. Ovariectomized rats treated with 17-estradiol and progesterone followed by vehicle only, to induce a hormone withdrawal state similar to the rodent postpartum period, show increased immobility during the forced swim test4,7, a behavioral indicator of despair, and decreased sucrose consumption and preference105, a behavioral indicator of anhedonia. One recent study demonstrated that estradiol supplementation and withdrawal alone was sufficient to provoke immobility during the forced swim test and anhedonic behavior during lateral hypothalamic self-stimulation18. Increased depression-like behavior during the postpartum demonstrated in previous studies could therefore be attributed to estradiol withdrawal alone.

The effects of estradiol withdrawal on depressive behavior in non-human animals are well documented. Following bilateral ovariectomy, rats demonstrate increased immobility during the forced swim test, and these effects are reversed by treatment with estradiol alone106,107. In addition, reduced immobility following a single administration of estradiol lasts 23 days, and the behavioral effects are the same as those following fluoxetine treatment108. The antidepressant effects of estradiol during the forced swim test appear to involve selective actions at intracellular estrogen receptor- (ER) in the ventral tegmental area109 and, in fact, may require ER110. In addition, abrupt estradiol withdrawal following sustained high estradiol levels results in elevated brain cortical dehydroepiandrosterone sulfate (DHEA-S), a neuroactive steroid synthesized endogenously in the brain that attenuates GABA-ergic activity and may be relevant to postpartum depressive symptoms111. Chronic administration of estradiol leads to dopamine receptor up-regulation and increased presynaptic dopamine activity in the striatum112114, which, when followed by abrupt estradiol withdrawal, leads to dysregulation in brain dopaminergic pathways and depressive symptoms115.

Estradiol-withdrawal models of PPD have two weaknesses: 1) they have low face validity as models of PPD given that the human postpartum period is characterized by a drop in both estradiol and progesterone (whereas progesterone drops before delivery in rodents), and 2) they result in depression without the attendant anxiety often seen in women with PPD116. The addition of progesterone to hormone withdrawal models of PPD is relevant given that progesterone withdrawal provokes anxiety. As noted above, progesterone metabolites act on GABA receptors in the brain, producing sedative-like effects by enhancing GABA neurotransmission117. Abrupt decreases in progesterone are associated with anxiety118, and treatment with progesterone reduces anxiety119. The anxiolytic effects of progesterone appear to be mediated by the progesterone metabolite allopregnanolone (ALLO)120. Indeed, postpartum rats show increased depressive behavior (increased immobility, decreased struggling and swimming) compared with pregnant rats, and this affect appears to be mediated by low hippocampal ALLO levels during the postpartum period120.

To examine the effects of concurrent estradiol and progesterone withdrawal, Suda et al.8 created a novel rodent model of PPD by administering hormone levels more consistent with human pregnancy than rat pregnancy. The concurrent withdrawal of estradiol and progesterone resulted in decreased immobility during the forced swim test (i.e., less depression-like behavior); however, it also resulted in learned helplessness, which was indicated by a failure to avoid repeated foot shocks8. Animals in this study also showed increased anxiety. Taken together, the existing animal models suggest that the abrupt withdrawal of estradiol alone produces behavioral despair and anhedonia, whereas the concurrent withdrawal of progesterone and estradiol produces learned helplessness and anxiety. However, these studies do not explain how the same putative stimulus (i.e., hormone change) is capable of causing depression in some women and not others.

There is no consistent or convincing evidence that women who develop PPD experience more rapid postpartum hormone withdrawal, have lower reproductive hormone concentrations during the postpartum period, or experience greater reductions in hormone levels from pregnancy to the postpartum than women without PPD911,29,121. The onset of depressive symptoms, however, is temporally coincident with the rapid changes in estradiol and progesterone levels that occur at delivery, leading some researchers to view the change in reproductive hormones as a potent stressor in susceptible women11.

Evidence that a subgroup of women are vulnerable to perinatal changes in reproductive hormones comes from treatment studies examining the effects of administering exogenous estradiol to women at high risk for PPD or those with active PPD symptoms. In a pilot study of 11 women with a history of PPD and no other history of affective disorder, participants were prophylactically administered oral Premarin, a conjugated estrogen, immediately following delivery to prevent estrogen withdrawal and the onset of depressive symptoms6. Ten of the 11 women remained well during the postpartum and for the first year following delivery6. A later double-blind, placebo-controlled study of 61 women with PPD that began within three months following delivery, showed that women treated with estradiol (n=34) (delivered via a transdermal patch) improved significantly more than women who received placebo (n=27), although nearly half of the women in both groups were also taking antidepressant medication5. A subsequent study examined the effects of estradiol treatment on a group of 23 women with severe postpartum depression, many of whom had attempted treatment with antidepressant medication or psychotherapy without effect12. At baseline, 16 of the 23 patients had serum estradiol concentrations consistent with gonadal failure. All women in the study received sublingual estradiol treatment for 8 weeks. After the first week, depressive symptoms significantly decreased, and by the end of the eight weeks all patients had achieved depressive symptom scores consistent with clinical recovery. Although Ahokas et al.12 suggest that postpartum gonadal failure is a risk factor for PPD, they did not compare estradiol levels in women with and without PPD. Instead, their data support the notion that, in susceptible women, low or declining estradiol levels may trigger PPD, while stable or increasing estradiol levels may ameliorate depressive symptoms. Although these treatment studies suggest a role for estradiol in the pathogenesis of PPD, they are small, lacking control groups, and confounded by the effects of stress, lack of sleep, and homeostatic shifts attendant to childbirth.

In order to assess the role of reproductive hormones in PPD directly, Bloch et al.3 created a scaled-down hormonal model of the puerperium wherein euthymic women with or without a history of PPD were blindly administered high-dose estradiol and progesterone during ovarian suppression and then abruptly withdrawn. Women with a history of PPD showed increasing depressive symptoms during hormone addback and further exacerbation during hormone withdrawal, but women lacking a history of PPD experienced no perturbation of mood despite identical hormonal conditions. Increasing depressive symptoms during both hormone addback and withdrawal among those with a history of PPD is consistent with research demonstrating that one of the biggest risk factors for PPD is depression during pregnancy15. The advantage of this design is that the effects of reproductive hormones on mood were examined without the confounding biological and psychosocial stressors associated with childbirth. The results provide support for a hormone-sensitive PPD phenotype in which reproductive hormone change alone is sufficient to provoke mood dysregulation in otherwise euthymic women.

Some have hypothesized that the source of PPD vulnerability is in abnormal neural responses to the normal perinatal fluctuations in reproductive hormones. PPD is characterized by abnormal activation of the same brain regions implicated in non-puerperal major depression: the amygdala, insula, striatum, orbitofrontal cortex, and dorsomedial prefrontal cortex122124. PPD is also associated with reduced connectivity between the amygdala and prefrontal regions, which implicates dysregulation of the limbic system in the neural pathophysiology of PPD123. Despite similar levels of circulating progesterone and ALLO to controls, women with PPD also show reduced resting state functional connectivity between the anterior cingulate cortex, amygdala, hippocampus, and dorsolateral prefrontal cortex in the context of the postnatal decline progesterone and ALLO29. These neuroimaging studies suggest that the neural abnormalities associated with PPD are unique to the perinatal period and may be unmasked by changes in circulating reproductive hormone concentrations. Taken together, the results of the human studies are suggestive of a hormone-sensitive PPD phenotype characterized by neural abnormalities present during the puerperium when reproductive hormone concentrations change rapidly.

One potential mechanism by which changing reproductive hormone levels trigger PPD involves neurosteroid regulation of affect. Neurosteroids are metabolites of steroid hormones that are synthesized in the brain and nervous system and modulate -aminobutyric acid (GABA) and glutamate. Two neurosteroids in particular play a role in affective dysregulation: dehydroepiandrosterone (DHEA) and ALLO. Abnormal DHEA secretion has been implicated in major depression 126130, and DHEA is an effective antidepressant in both men and women131,132. The majority of research on neurosteroids in reproductive mood disorders, however, has focused on the progesterone metabolite ALLO. There are several reasons to speculate that ALLO plays a role in PPD. ALLO modulates the GABA receptor, which mediates anxiolysis133. ALLO supplementation has anxiolytic effects134136, whereas ALLO withdrawal produces anxiety and insensitivity to benzodiazepines118,137. ALLO levels are decreased in depression and increased with successful antidepressant treatment138143. ALLO also modulates the biological processes dysregulated in major depressive disorder, including HPA axis regulation144147, neuroprotection148,149, and immune function150. ALLO also regulates the neural circuits implicated in depression, including the limbic system151,152.

Cortical GABA and ALLO are reduced in postpartum women, regardless of the presence of PPD, compared with healthy women in the follicular phase153. Although there is no evidence of abnormalities in basal circulating ALLO levels in PPD, women with PPD show reduced resting state functional connectivity between the anterior cingulate cortex, amygdala, hippocampus, and dorsolateral prefrontal cortex in the context of the postnatal decline in ALLO29. In addition, we recently reported an association between changes in ALLO levels and depressive symptoms during GnRH agonist-induced ovarian suppression and ovarian steroid addback in women with a history of PPD but not in those without such a history154. These studies suggest that, even in the presence of normal absolute levels, perinatal fluctuations in reproductive hormones may precipitate symptoms in a vulnerable subpopulation of women as a result of changing ALLO levels.

The identification of biomarkers in humans is difficult because of a lack of experimental control over the patients environment and genetic background and inaccessibility of brain tissue required for analysis. Gene manipulation studies in non-human animals can help model how genetic variants and the environment interact to yield a distinct behavioral phenotypes155. Animal models that have demonstrated that the behavioral effects of maternal care are associated with gene expression changes that persist into adulthood and can be transmitted across generations provide a potent epigenetic model of PPD155. For example, estradiol withdrawal is clearly associated with estradiol-reversible anxiety in a strain-dependent fashion (Schoenrock et al., unpublished manuscript). One genetic knockout model potentially explains both the specificity of affective dysregulation during the perinatal period and also the variation in susceptibility to PPD among women 125. In this model, Maguire and Mody125 demonstrated a GABAA receptor subunit knockout that is behaviorally silent until an animal is exposed to pregnancy and the postpartum state, following which the dam displays depression-like behavior and cannibalizes its young. Thus, reproductive events may unmask the genetic susceptibility to affective dysregulation. Maguire and Mody125,156,157 observed that alterations in the GABAA receptor -subunit occur as ovarian hormone levels fluctuate during the menstrual cycle, pregnancy, and the postpartum period. During pregnancy, the expression of the GABAA receptor -subunit is downregulated as ALLO levels increase, and at parturition, the expression of the GABAA receptor -subunit recovers in response to rapidly declining neurosteroid levels157. The failure to regulate these receptors during pregnancy and the postpartum, consequent to the knockout of the GABAA receptor -subunit, appears to provoke behavioral abnormalities consistent with PPD. Thus, as noted above, GABAA receptor -subunit deficient mice exhibit normal behaviors prior to pregnancy, but they show insensitivity to ALLO during pregnancy, depression-like and anxiety-like behavior, and abnormal maternal behavior125. This model suggests that changes in reproductive hormone concentrations during pregnancy and the postpartum are capable of provoking affective dysregulation, particularly in those with a genetically determined susceptibility.

The cross-species role of reproductive hormones in depressive behavior suggests a neuroendocrine pathophysiology for PPD. PPD, as defined in contemporary research, includes depression that began during or before pregnancy; depression that occurred in the context of a childhood trauma history, traumatic labor or delivery, subthreshold thyroid dysfunction, psychosocial stress, or sleep deprivation; and depression that co-occurred with obsessive-compulsive disorder, PTSD, generalized anxiety disorder, or personality pathology. Logic would preclude consideration of all of these as the same disorder; consequently, when attempting to understand the contribution of hormonal signaling to postpartum affective dysregulation, it is therefore necessary to carefully define the study population and attempt to characterize and disentangle individual PPD phenotypes. The extant literature supports the existence of a hormone-sensitive PPD phenotype3. In order to study the clinical and neuroendocrine correlates of this phenotype, some researchers have selected women with a history of PPD and without a history of non-puerperal depressive episodes3,18. Although these studies are primarily relevant for understanding the risk of PPD recurrence, they represent the first step toward identifying factors that predict first onset PPD. There is sufficient evidence to suggest that reproductive hormone fluctuations trigger affective dysregulation in sensitive women. Even within the hormone-sensitive phenotype, alterations in multiple biological systems the immune system, HPA axis, and lactogenic hormones likely contribute to the pathophysiology of PPD. Studies are underway to disentangle the complex interplay of fluctuating reproductive hormones, neurosteroids, HPA axis reactivity, neural dysfunction, and genetics with a specific focus on identifying genomic, brain, and behavior relationships that contribute to affective dysfunction in the context of specific reproductive states. Consistent with the RDoC mission, this line of research represents not only an opportunity to identify novel treatment targets for PPD but alsocriticallythe potential to prevent PPD in susceptible women.

We thank Sarah Johnson and Erin Richardson for assisting with the literature review. This work was supported by the UNC Building Interdisciplinary Careers in Womens Health (BIRCWH) Career Development Program (K12 HD001441) and the National Institute of Mental Health of the National Institutes of Health under Award Number R21MH101409.

Disclosure of Commercial and Non-Commercial Interests

The authors do not have an affiliation with or financial interest in any organization that might pose a conflict of interest.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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The Role of Reproductive Hormones in Postpartum Depression

Century Therapeutics to Present at the 63rd American Society of Hematology Annual Meeting and Host Virtual Research & Development Update -…

PHILADELPHIA, Nov. 04, 2021 (GLOBE NEWSWIRE) -- Century Therapeutics (NASDAQ: IPSC), an innovative biotechnology company developing induced pluripotent stem cell (iPSC)-derived cell therapies in immuno-oncology, today announced that preclinical data from the Companys CNTY-101 program and CAR-iT platform will be presented in two posters at the 63rd American Society of Hematology (ASH) Annual Meeting & Exposition, on December 11-14, 2021 in Atlanta, Georgia and virtually.

The Company also announced today that it will host a virtual research & development update on Thursday, December 16, 2021 from 8:00 AM - 9:30 AM ESTto share progress on its iPSC technology platform and pipeline. Eduardo Sotomayor, M.D., director of the Cancer Institute at Tampa General Hospital,will discuss the current treatment paradigm for B-cell malignancies. For additional information on how to access the event, please visit the Events & Presentations section of Centurys website.

Details of the two poster presentations are as follows:

Abstract Number: 1729 Title: Development of Multi-Engineered iPSC-Derived CAR-NK Cells for the Treatment of B-Cell Malignancies Session Name: 703. Cellular Immunotherapies: Basic and Translational: Poster I Session Date: Saturday, December 11, 2021 Session Time: 5:30 PM - 7:30 PM Presenter: Luis Borges, Chief Scientific Officer, Century Therapeutics

Abstract Number: 2771 Title: Induced Pluripotent Stem Cell-Derived Gamma Delta CAR-T Cells for Cancer Immunotherapy Session Name: 703 Cell Therapies: Basic and Translational Session Date: Sunday, December 12, 2021 Session Time: 6:00 PM 8:00 PM Presenter: Mark Wallet, Vice President, Immuno-Oncology, Century Therapeutics

Full abstracts are currently available through the ASH conference website.

About Century Therapeutics

Century Therapeutics (NASDAQ: IPSC) is harnessing the power of adult stem cells to develop curative cell therapy products for cancer that we believe will allow us to overcome the limitations of first-generation cell therapies. Our genetically engineered, iPSC-derived iNK and iT cell product candidates are designed to specifically target hematologic and solid tumor cancers. We are leveraging our expertise in cellular reprogramming, genetic engineering, and manufacturing to develop therapies with the potential to overcome many of the challenges inherent to cell therapy and provide a significant advantage over existing cell therapy technologies. We believe our commitment to developing off-the-shelf cell therapies will expand patient access and provide an unparalleled opportunity to advance the course of cancer care. For more information on Century Therapeutics please visit http://www.centurytx.com.

Century Therapeutics Forward-Looking Statement

This press release contains forward-looking statements within the meaning of, and made pursuant to the safe harbor provisions of, The Private Securities Litigation Reform Act of 1995. All statements contained in this press release, other than statements of historical facts or statements that relate to present facts or current conditions, including but not limited to, statements regarding our clinical development plans, are forward-looking statements. These statements involve known and unknown risks, uncertainties and other important factors that may cause our actual results, performance, or achievements to be materially different from any future results, performance or achievements expressed or implied by the forward-looking statements. In some cases, you can identify forward-looking statements by terms such as may, might, will, should, expect, plan, aim, seek, anticipate, could, intend, target, project, contemplate, believe, estimate, predict, forecast, potential or continue or the negative of these terms or other similar expressions. The forward-looking statements in this presentation are only predictions. We have based these forward-looking statements largely on our current expectations and projections about future events and financial trends that we believe may affect our business, financial condition, and results of operations. These forward-looking statements speak only as of the date of this press release and are subject to a number of risks, uncertainties and assumptions, some of which cannot be predicted or quantified and some of which are beyond our control, including, among others: our ability to successfully advance our current and future product candidates through development activities, preclinical studies, and clinical trials; our reliance on the maintenance of certain key collaborative relationships for the manufacturing and development of our product candidates; the timing, scope and likelihood of regulatory filings and approvals, including final regulatory approval of our product candidates; the impact of the COVID-19 pandemic on our business and operations; the performance of third parties in connection with the development of our product candidates, including third parties conducting our future clinical trials as well as third-party suppliers and manufacturers; our ability to successfully commercialize our product candidates and develop sales and marketing capabilities, if our product candidates are approved; and our ability to maintain and successfully enforce adequate intellectual property protection. These and other risks and uncertainties are described more fully in the Risk Factors section of our most recent filings with the Securities and Exchange Commission and available at http://www.sec.gov. You should not rely on these forward-looking statements as predictions of future events. The events and circumstances reflected in our forward-looking statements may not be achieved or occur, and actual results could differ materially from those projected in the forward-looking statements. Moreover, we operate in a dynamic industry and economy. New risk factors and uncertainties may emerge from time to time, and it is not possible for management to predict all risk factors and uncertainties that we may face. Except as required by applicable law, we do not plan to publicly update or revise any forward-looking statements contained herein, whether as a result of any new information, future events, changed circumstances or otherwise.

For More Information: Company: Elizabeth Krutoholow investor.relations@centurytx.comInvestors: Melissa Forst/Maghan Meyers century@argotpartners.comMedia: Joshua R. Mansbach century@argotpartners.com

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Century Therapeutics to Present at the 63rd American Society of Hematology Annual Meeting and Host Virtual Research & Development Update -...