Hominoid‐specific calretinin‐immunopositivity of the optic radiation (geniculocalcarine tract)

Abstract

It has been proposed that the optic radiation (OR) of primates is specifically revealed with parvalbumin immunohistochemistry. To test this proposition, the immunohistochemical expression of three calcium‐binding proteins (CaBPs, parvalbumin, calbindin, and calretinin), was investigated in the OR (also known as the geniculocalcarine tract) of five primate species, including a Strepsirrhini (Galago moholi—southern lesser galago), a Platyrrhini (Saimiri boliviensis—black‐capped squirrel monkey), a Cercopithecidae (Macaca nigra—crested macaque) and two Hominoidea (Hylobates lar—lar gibbon, Pan troglodytes—chimpanzee). The OR of the southern lesser galago did not reveal substantial immunostaining of any of the CaBPs investigated. The black‐capped squirrel monkey, and crested macaque evinced strong intensity parvalbumin‐immunostaining of the OR. In contrast, the OR of the lar gibbon and chimpanzee presented with strong intensity calretinin‐immunostaining, with weak to moderate parvalbumin‐immunostaining. These results indicate that the neurochemistry of the OR differs between the major primate lineages, although the trajectory of the OR through the white matter, including the temporal loop, is consistent across primates. While it is unclear precisely what effect this differing CaBP neurochemistry has on the processing of visual information, it is possible that these differences modulate axonal excitability or signal fidelity in the OR of hominoids when compared to other primates.

Calretinin‐immunostained coronal section through the primary (V1) and extrastriate (ExSt) cortex of the lar gibbon. Note that the optic radiation (OR) is strongly calretinin‐immunoreactive. This calretinin‐immunopositivity of the OR distinguishes the Hominoidea from other primates in terms of the neurochemistry of the OR.

1. INTRODUCTION

The primate visual system comprises subcortical nuclei and cortical areas that interact via a network of myelinated fiber pathways (Rokem et al., 2017) to hierarchically process visual input to shape perception (e.g., Oldenburg et al., 2024; Sondereker et al., 2020). The optic radiation (OR), also referred to as the geniculocalcarine tract, is the most prominent thalamocortical visual pathway, projecting from the dorsal lateral geniculate nucleus (LGN) of the thalamus to the primary visual cortex (V1) (Alvarez et al., 2015; Ebeling & Reulen, 1988). While often regarded as merely an input‐relay white matter tract, the OR transmits visual information crucial to sensory and perceptual processing within the cortex (Webb et al., 2022).

Although the OR is recognized as a distinct pathway within primate occipital white matter, as revealed by diffusion tensor imaging (DTI) tractography (Takemura et al., 2017; Bertani et al., 2018; Kaneko et al., 2020; Bryant et al., 2020, 2024), there is limited data on the neurochemistry of the axons forming this pathway in primates. Immunohistochemical staining of the OR for calcium (Ca²⁺)‐binding proteins (CaBPs) has been conducted in humans (Leuba & Saini, 1996, 1997) and common marmoset monkeys (Ma et al., 2023); however, to our knowledge, there are no reports available for other primate species with the purpose of enabling comparisons to infer evolutionary changes in the neurochemistry of this tract. In humans, the OR was observed to be strongly calretinin‐immunopositive and moderately parvalbumin‐immunopositive (Leuba & Saini, 1996, 1997), while in common marmoset monkeys the OR was found to be parvalbumin‐immunopositive (Ma et al., 2023). Expanding such neurochemical investigations across a broader phylogenetic sample of primates offers an avenue to identify lineage‐specific specializations and reconstruct the neurochemical evolutionary trajectory of visual pathway organization.

The CaBPs, such as parvalbumin, calbindin, and calretinin, play important and distinct roles in neuronal information processing, by acting as buffers that modulate calcium dynamics and signaling, and regulating neurotransmitter release and synaptic plasticity (e.g., Fairless et al., 2019). Given the observed difference in the presence of CaBPs in the OR when comparing humans (Leuba & Saini, 1996, 1997) and common marmoset monkeys (Ma et al., 2023), it is likely that the precise way neuronal information is transmitted by the OR in these species differs. The shift from parvalbumin to calretinin dominance in the human OR relative to common marmosets may reflect differences in axonal firing properties or calcium buffering capacity that support enhanced temporal precision or metabolic resilience in hominoid visual processing. Given the relative phylogenetic distance between humans and common marmosets—estimated at over 40 million years of divergence (e.g., Finstermeier et al., 2013)—understanding the neurochemistry of the OR, and how it varies across primate species, is important for interpreting experimental data in frequently studied models such as marmoset and macaque monkeys related to visual processing in humans. We examined aspects of the neurochemistry of the OR in five primate species, including representatives of the Strepsirrhini (prosimians), Platyrrhini (monkeys of the Americas), Cercopithecidae (monkeys from Africa and Asia), and the Hominoidea (apes), using immunohistochemical staining against the CaBPs parvalbumin, calbindin, and calretinin. The aim of this study was to determine whether parvalbumin is indeed a specific neurochemical “marker of the primate optic radiation” (our italics) as asserted in the title of the paper of Ma et al. (2023), or whether there is lineage‐specific neurochemical variance of the OR across primate species (Leuba & Saini, 1996, 1997).

2. MATERIALS AND METHODS

2.1. Species examined

In the current study, we examined immunohistochemically stained brain sections to detail the neurochemistry of the CaBPs in the OR in five species from across the primate radiation. The species examined include Galago moholi (southern lesser galago), Saimiri boliviensis (black‐capped squirrel monkey), Macaca nigra (crested macaque), Hylobates lar (lar gibbon), and Pan troglodytes (chimpanzee). To broaden the phylogenetic comparison, data detailing the presence of CaBPs within the LGN, OR, and V1 for common marmosets was obtained from Goodchild and Martin (1998), Yuasa et al. (2010), and Ma et al. (2023), while data for humans was derived from Leuba and Saini (1996, 1997). The data for southern lesser galago, black‐capped squirrel monkey, crested macaque, lar gibbon, and chimpanzee was derived from previously stained brain sections (curated by PRM) that were used for studies of white matter interstitial neurons (Swiegers et al., 2018; Swiegers, Bhagwandin, Maseko, et al., 2021; Swiegers, Bhagwandin, Williams, et al., 2021). All research was carried out in accordance with the guidelines of the University of Witwatersrand Animal Ethics Committee (Clearance number 2017/010/73/O), which correspond with those of the National Institutes of Health (NIH) for the care and use of animals in scientific experimentation.

2.2. Tissue acquisition, immunohistochemical staining, and antibody characterization

Full descriptions of the acquisition of brain specimens used in this study, the sectioning of the brains, the immunohistochemical staining protocol and the characterization of the antibodies used (see Table 1) have been provided previously (Swiegers et al., 2018; Swiegers, Bhagwandin, Maseko, et al., 2021; Swiegers, Bhagwandin, Williams, et al., 2021) and are thus not described in detail herein. Briefly, all animals used were overdosed with sodium pentobarbital (i.v.), in accordance with population management decisions, independent of the current study (Bertelsen, 2018). Immediately after euthanasia, the carotid arteries were cannulated and the heads were perfused with 0.9% saline solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB), both at 4°C. The brain was then extracted, preserved with the same fixative for 24 h at 4°C, cryoprotected, and stored in an anti‐freeze solution at −20°C until use (Manger et al., 2009). The brains were re‐immersed in a 30% sucrose solution in 0.1 M PB, frozen with dry ice, and sectioned at 50 μm thickness in the coronal plane using a sliding microtome. A 1 in 20 series of sections were taken with 4 series being used for immunohistochemical staining against antigens for a neuronal nuclear marker (NeuN), parvalbumin (PV), calbindin (CB), and calretinin (CR) (i.e., a section every 1 mm throughout the rostro‐caudal extent of the brain was immunostained for each antibody; see Swiegers et al., 2018; Swiegers, Bhagwandin, Williams, et al., 2021; Swiegers, Bhagwandin, Maseko, et al., 2021) for the complete immunostaining protocol (n.b., the calbindin immunostained sections for the black‐capped squirrel monkey were unfortunately broken and could not be used in this analysis). These stained sections allowed us to readily identify the architectonic boundaries and laminar pattern of subcortical and cortical regions, including the LGN, OR, and V1.

TABLE 1.

Sources and dilution of antibodies used in the current study.

Antibody	Host	Immunogen	Manufacturer	Catalogue No.	References	Dilution	RRID
NeuN	Rabbit	GST‐tagged recombinant protein corresponding to mouse NeuN	Merck‐Millipore	ABN78C3	Ngwenya et al. (2016)	1:500	AB_11204707
PV	Rabbit	Rat muscle parvalbumin	Swant	PV28	Celio (1990)	1:10,000	AB_10000343
CB	Rabbit	Rat recombinant calbindin D‐28k	Swant	CB38a	Celio (1990)	1:10,000	AB_10000340
CR	Rabbit	Recombinant human calretinin containing a 6‐his tag at the N‐terminal	Swant	7699/3H	Schwaller et al. (1997)	1:10,000	AB_10000321

Abbreviations: CB, calbindin; CR, calretinin; NeuN, neuronal nuclear marker; PV, parvalbumin.

The NeuN rabbit polyclonal antibody was raised against the GST‐tagged recombinant protein corresponding to mouse NeuN (ABN78C3; Merck‐Millipore; RRID: AB_11204707) (Ngwenya et al., 2016). The NeuN antibody revealed neurons throughout the brains of the primate studied, but as with other NeuN antibodies, it was absent from cerebellar Purkinje cells. The NeuN antibody was used at a dilution of 1:500. To reveal neuronal structures containing parvalbumin, we used the PV28 anti‐parvalbumin rabbit polyclonal antibody from Swant (PV28, Swant; RRID AB_10000343) at a dilution of 1:10,000. The pattern of immunoreactivity throughout the primate brains studied was like that seen in other mammals (Celio, 1990). To reveal neuronal structures containing calbindin, we used the CB38a anti‐calbindin rabbit polyclonal antibody from Swant (CB38a, Swant; RRID AB_10000340) at a dilution of 1:10,000. The pattern of immunoreactivity throughout the primate brains studied was like that seen in other mammals (Celio, 1990). To reveal neuronal structures containing calretinin, we used the 7699/3H anti‐calretinin rabbit polyclonal antibody from Swant (7699/3H, Swant; RRID: AB_10000321) at a dilution of 1:10,000. The pattern of immunoreactivity throughout the primate brains studied was like that seen in other mammals (Schwaller et al., 1997).

2.3. Analysis and image acquisition

A purely qualitative analysis was undertaken in the current study. All relevant sections for all species, apart from those where data was derived from published reports, were examined under both low and high magnification. A low‐power stereomicroscope was used to examine the NeuN‐immunostained sections and camera lucida drawings outlining architectural borders were made of the lar gibbon brain sections. The associated CR‐immunostained sections were matched to these drawings and the stained neuronal structures were marked. The drawings were then scanned and redrawn using the Canvas Draw 10 drawing program (Canvas GFX, Inc., FL, USA). Digital low and higher magnification photomicrographs were captured using a variety of microscopes. No pixelation adjustments, or manipulation of the captured images were undertaken, except for the adjustment of contrast, brightness, and levels using Adobe Photoshop software.

Abbreviations

3n: root of oculomotor nerve

4V: fourth ventricle

C: caudate nucleus

cc: corpus callosum

Cl: claustrum

CN: cerebellar nuclei

Cu: cuneate nucleus

cu: cuneate fasciculus

dh: dorsal horn of cervical spinal cord

DT: dorsal thalamus

ExSt: extrastriate cortex

f: fornix

fr: fasciculus retroflexus

Gr: gracile nucleus

gr: gracile fasciculus

HF: hippocampal formation

IC: inferior colliculus

IO: inferior olivary nuclear complex

LGN: lateral geniculate nucleus

LV: lateral ventricle

mcp: middle cerebellar peduncle

P: putamen nucleus

PAG: periaqueductal gray matter

PC: cerebral peduncle

pc: posterior commissure

Pn: pontine nucleus

py: pyramidal tract

pyx: decussation of the pyramidal tract

R: reticular nucleus of the thalamus

SC: superior colliculus

scp: superior cerebellar peduncle

STN: subthalamic nucleus

V1: primary visual, or striate, cortex

VC: ventral cochlear nuclear complex

vh: ventral horn of cervical spinal cord

3. RESULTS

The primate OR originates in the neurons forming the LGN, the axons forming a distinct pathway through the white matter prior to terminating specifically in the neuropil of primarily layers 3 and 4 of the primary visual cortex (V1) (Leuba & Saini, 1996, 1997; Ma et al., 2023). In this study we provide a qualitative description of the presence or absence of parvalbumin, calbindin, and calretinin, using immunohistochemical techniques, in the origin (soma of the neurons forming the LGN), pathway (the OR axons within the white matter), and termination (neuropil of layers 3 and 4 of V1) in five species representing all major branches of the primate radiation (Table 2).

TABLE 2.

Summary of qualitative staining intensity of the calcium binding proteins parvalbumin (PV), calbindin (CB), and calretinin (CR), in the lateral geniculate nucleus (LGN), optic radiation, and layer 4 of the primary visual cortex (V1) in the five species of primates investigated herein, with published data on the common marmoset monkey (Goodchild & Martin, 1998; Ma et al., 2023; Yuasa et al., 2010) and humans (Leuba & Saini, 1996, 1997).

Structure	Species	PV staining	CB staining	CR staining
LGN—M layer soma only	Galago moholi	−	++	−
	Callithrix jacchus	+++	+	+
	Saimiri boliviensis	+++	nd	+
	Macaca nigra	+++	−	−
	Hylobates lar	++	+	+++
	Pan troglodytes	+	++	+++
	Homo sapiens	+	+	++
LGN—P layer soma only	Galago moholi	−	++	+
	Callithrix jacchus	+++	+	++
	Saimiri boliviensis	+++	nd	+++
	Macaca nigra	+	−	−
	Hylobates lar	+	++	+++
	Pan troglodytes	+	+	+++
	Homo sapiens	+	+	++
LGN—K layer soma only	Galago moholi	−	++	+
	Callithrix jacchus	++	++	++
	Saimiri boliviensis	++	nd	+
	Macaca nigra	−	+	+
	Hylobates lar	−	+	+
	Pan troglodytes	−	+	++
	Homo sapiens	−	−	−
Optic radiation, specific to fibers of this pathway only	Galago moholi	−	−	−
	Callithrix jacchus	+++	−	−
	Saimiri boliviensis	++	nd	−
	Macaca nigra	++	−	−
	Hylobates lar	++	−	+++
	Pan troglodytes	+	−	+++
	Homo sapiens	+	−	+
V1 layers 3/4 neuropil only	Galago moholi	−	+	++
	Callithrix jacchus	++	++	+
	Saimiri boliviensis	++	nd	+
	Macaca nigra	+++	++	+
	Hylobates lar	++	+	+++
	Pan troglodytes	+	+	+++
	Homo sapiens	+	−	+

Abbreviations: Callithrix jacchus, common marmoset; Galago moholi, southern lesser galago; Hylobates lar, lar gibbon; Homo sapiens, human; K layers, koniocellular layers of LGN; M layers, magnocellular layers of LGN; Macaca nigra, crested macaque; P layers, parvocellular layers of LGN; Pan troglodytes, chimpanzee; Saimiri boliviensis, black‐capped squirrel monkey; −, absence of staining; +, weak staining intensity; ++, moderate staining intensity; +++, strong staining intensity; nd, no data available.

3.1. Strepsirrhini primates—southern lesser galago (G. moholi)

The soma of the LGN in our representative of the strepsirrhine primates, a southern lesser galago (Figures 1, 2), was all parvalbumin‐immunonegative; this includes soma from the M, P, and K layers of this nucleus (Figures 1b, 2d–f). A moderate staining intensity of calbindin‐immunoreactivity was observed in the soma of the M, P, and K layers of this nucleus (Figures 1c, 2g,h). Calretinin‐immunopositive soma were not observed in the M layer, while a weak staining intensity of calretinin‐immunopositivity was observed in the P and K layers (Figures 1d, 2j–l). The OR of the southern lesser galago exhibited no immunoreactivity to parvalbumin, calbindin, or calretinin (Figure 1e–h). Within the neuropil of layers 3 and 4 of V1, no immunoreactivity for parvalbumin was observed, while a weak staining intensity for calbindin, and a moderate staining intensity for calretinin were noted (Figure 1i–l).

FIGURE 1.

Photomicrographs of neuronal nuclear marker (a, e, i), parvalbumin (b, f, j), calbindin (c, g, k), and calretinin (d, h, l) immunostained sections in the lateral geniculate nucleus (LGN, a–d), optic radiation (OR, e–h) and primary visual cortex (V1, i–l) in the brain of the southern lesser galago (Galago moholi). Note the absence of immunohistochemical staining for any of the CaBPs in the OR of this strepsirrhine primate. Scale bar in (d) = 1 mm, and applies to (a)–(d). Scale bar in (h) = 1 mm, and applies to (e)–(h). Scale bar in (l) = 500 μm, and applies to (i)–(l).

FIGURE 2.

Photomicrographs of neuronal nuclear marker (a)–(c), parvalbumin (d)–(f), calbindin (g)–(i), and calretinin (j)–(l) immunostained sections in the P (a, d, g, j), K (b, e, h, k), and M (c, f, i, l) layers of the lateral geniculate nucleus in the brain of the southern lesser galago (Galago moholi). Scale bar in (l) = 50 μm and applies to all.

3.2. Platyrrhini primates—Black‐capped squirrel monkey (S. boliviensis)

In the black‐capped squirrel monkey (Figures 3, 4), strong staining intensity for parvalbumin was observed in the soma forming the M and P layers of the LGN, while the soma forming the K layer revealed moderate staining intensity for parvalbumin (Figures 3b, 4d–f). Moderate staining intensity for calbindin was noted in the K layer. The M and K layers revealed a weak staining intensity for calretinin, while in the P layer a strong staining intensity was observed (Figures 3c, 4g–i). The OR of the black‐capped squirrel monkey revealed a moderate staining intensity for parvalbumin (Figure 3e). No calretinin‐immunoreactivity was noted in the OR (Figure 3f). A moderate staining intensity for parvalbumin was noted in the neuropil of layers 3 and 4 of V1 (Figure 3h), while a weak staining intensity for calretinin was noted in the neuropil of layers 3 and 4 of V1 (Figure 3i).

FIGURE 3.

Photomicrographs of neuronal nuclear marker (a, d, g), parvalbumin (b, e, h), and calretinin (c, f, i) immunostained sections in the lateral geniculate nucleus (LGN, a–c), optic radiation (OR, d–f) and primary visual cortex (V1, g–i) in the brain of the black‐capped squirrel monkey (Saimiri boliviensis). Note the presence of parvalbumin‐immunopositive axons comprising the OR (e), but the absence of calretinin‐immunopositive axons in the OR. Scale bar in (c) = 1 mm, and applies to (a)–(c). Scale bar in (f) = 1 mm, and applies to (d)–(f). Scale bar in (i) = 500 μm, and applies to (g)–(i).

FIGURE 4.

Photomicrographs of neuronal nuclear marker (a)–(c), parvalbumin (d)–(f), and calretinin (g)–(i) immunostained sections in the P (a, d, g), K (b, e, h), and M (c, f, i) layers of the lateral geniculate nucleus in the brain of the black‐capped squirrel monkey (Saimiri boliviensis). Scale bar in (i) = 50 μm and applies to all.

3.3. Cercopithecidae primates—Crested macaque (M. nigra)

Within the LGN of the crested macaque (Figures 5a–d, 6), we noted strong staining intensity for parvalbumin in the soma forming the M layer, while in the P layer the soma exhibited a weaker staining intensity for parvalbumin, which was absent in the soma forming the K layer (Figures 5b, 6d–f). No staining for calbindin was noted in the soma forming the M and P layers, with only a weak staining intensity noted in the soma forming the K layer (Figures 5c, 6g–j). Calretinin‐immunoreactivity was absent in the soma of both the M and P layers, with a weak staining intensity for calretinin observed in the soma forming the K layer (Figures 5d, 6j–l). In the OR of the crested macaque, we noted a strong staining intensity for parvalbumin (Figure 5f), but no immunostaining was observed for either calbindin or calretinin (Figure 5g,h). A strong intensity staining for parvalbumin was noted in the neuropil of layers 3 and 4 of V1 in the crested macaque (Figure 5j), while a moderate staining intensity for calbindin, and a weak staining intensity for calretinin, were observed (Figure 5k,l).

FIGURE 5.

Photomicrographs of neuronal nuclear marker (a, e, i), parvalbumin (b, f, j), calbindin (c, g, k), and calretinin (d, h, l) immunostained sections in the lateral geniculate nucleus (LGN, a–d), optic radiation (OR, e–h), and primary visual cortex (V1, i–l) in the brain of the crested macaque (Macaca nigra). Note the presence of parvalbumin‐immunopositive axons comprising the OR (f), but the absence of calbindin‐ and calretinin‐immunopositive axons in the OR. Scale bar in (d) = 1 mm, and applies to (a)–(d). Scale bar in (h) = 3 mm, and applies to (e)–(h). Scale bar in (l) = 500 μm, and applies to (i)–(l).

FIGURE 6.

Photomicrographs of neuronal nuclear marker (a)–(c), parvalbumin (d)–(f), calbindin (g)–(i), and calretinin (j)–(l) immunostained sections in the P (a, d, g, j), K (b, e, h, k), and M (c, f, i, l) layers of the lateral geniculate nucleus in the brain of the crested macaque (Macaca nigra). Scale bar in (l) = 50 μm and applies to all.

3.4. Hominoidea primates—Lar gibbon (H. lar) and chimpanzee (P. troglodytes)

Within the soma of the M layer of the LGN a moderate staining intensity for parvalbumin was noted in the lar gibbon (Figures 7b, 8f), while these somas evinced a weak staining intensity for parvalbumin in the chimpanzee (Figures 7f, 9f). The soma forming the P layer of the LGN revealed a weak staining intensity for parvalbumin in both species, while no parvalbumin immunoreactivity was observed in the soma forming the K layer in both species (Figures 7b,f, 8d,e, 9d,e). Weak to moderate staining intensity for calbindin was observed in the soma of the M and P layers in both species, while weak intensity staining for calbindin was observed in the K layer (Figures 7c,g, 8g–i, 9g–i). Strong staining intensity for calretinin of the soma forming the M and P layers of both species was noted (Figures 7d,h, 8j,l, 9j,l). Weak intensity staining for calretinin was observed in the soma forming the K layer of the lar gibbon (Figures 7d, 8k), with moderate intensity staining in the K layer of the chimpanzee (Figures 7h, 9k).

FIGURE 7.

Photomicrographs of neuronal nuclear marker (a), (e), parvalbumin (b), (f,) calbindin (c), (g), and calretinin (d), (h) immunostained sections in the lateral geniculate nucleus (LGN), of the lar gibbon (a)–(d) and chimpanzee (e)–(h). Immunostaining of the soma forming the different layers exhibited weak to moderate staining intensities for parvalbumin and calbindin. In contrast, immunostaining for calretinin exhibited strong staining intensity in the M and P layers. Scale bar in (d) = 2 mm, and applies to (a)–(d). Scale bar in (h) = 3 mm, and applies to (e)–(h).

FIGURE 8.

Photomicrographs of neuronal nuclear marker (a)–(c), parvalbumin (d)–(f), calbindin (g)–(i), and calretinin (j)–(l) immunostained sections in the P (a, d, g, j), K (b, e, h, k), and M (c, f, i, l) layers of the lateral geniculate nucleus in the brain of the lar gibbon (Hylobates lar). Note the weak parvalbumin‐immunoreactivity, yet strong calretinin‐immunoreactivity in the soma of the P and M layers. Scale bar in (l) = 50 μm and applies to all.

FIGURE 9.

Photomicrographs of neuronal nuclear marker (a)–(c), parvalbumin (d)–(f), calbindin (g)–(i), and calretinin (j)–(l) immunostained sections in the P (a, d, g, j), K (b, e, h, k), and M (c, f, i, l) layers of the lateral geniculate nucleus in the brain of the chimpanzee (Pan troglodytes). Note the absent parvalbumin‐immunoreactivity, yet strong calretinin‐immunoreactivity in the soma of the P and M layers. Scale bar in (l) = 50 μm and applies to all.

The OR (see Section 3.5 for a description of the OR trajectory in the lar gibbon) was moderately to weakly intensely stained for parvalbumin in both species, while no calbindin‐immunopositivity was observed in the OR of both species. In both the lar gibbon and chimpanzee, a strong staining intensity for calretinin was observed in the OR (Figures 10, 11, 12, 13). In the neuropil of layers 3 and 4 of V1 (Figure 14) moderate to weak staining intensity for parvalbumin was noted in both species (Figure 14b,f). Weak to absent staining intensity was noted in the layer 3 and 4 V1 neuropil for calbindin in both species (Figure 14c,g). In the lar gibbon and chimpanzee strong staining intensity for calretinin was noted in the neuropil of layers 3 and 4 of V1 (Figures 13, 14). It should be noted that no calretinin‐immunopositive soma were observed in layer 4 of both the lar gibbon and chimpanzee (Figure 14d,h), indicating that the calretinin‐immunopositive neuropil staining likely represents the ramifying axons of the calretinin‐immunopositive OR axons.

FIGURE 10.

Photomicrographs of calretinin‐immunostained coronal sections through the lateral geniculate nucleus (LGN) and adjacent white matter in the lar gibbon (a)–(c) and chimpanzee (e), (f). Note the presence of a moderate to high density of calretinin‐immunopositive fibers, presumably emanating from the LGN, in the white matter adjacent to this nucleus. These fibers pass through the thalamic reticular nucleus (R), before forming a distinct fiber tract, the OR, (d) that terminates in the primary visual cortex. In all images dorsal is to the top and medial to the left. Scale bar in (f) = 500 μm and applies to (a)–(c), and (f). Scale bar in (d) = 50 μm and applies to (d) only. Scale bar in (e) = 250 μm and applies to (e) only.

FIGURE 11.

Low magnification photomicrographs of calretinin‐immunostained coronal sections through the brain of the lar gibbon from the level of the lateral genicular nucleus (LGN) (a), (b) through to the primary visual cortex (V1) (c), (d). The calretinin‐immunopositive OR emerges from the LGN, passes through the putamen (P) and thalamic reticular nucleus (R), before merging to form a very distinct pathway surrounding the lateral ventricle (LV). This pathway continues caudally and terminates exclusively in the primary visual cortex (V1). V1 is clearly demarcated by the presence of distinct calretinin immunostaining in layer 4. In all images dorsal is to the top and medial to the left. Scale bar in (d) = 5 mm and applies to all. See list for abbreviations.

FIGURE 12.

Low magnification photomicrographs of calretinin‐immunostained coronal sections through the brain of the chimpanzee from the rostral (a) and caudal (b) aspects of the occipital lobe. Note the presence of the calretinin‐immunopositive pathway surrounding the lateral ventricle and branching into the white matter deep to primary visual cortex (V1). In both images dorsal is to the top and medial to the left. Scale bar in (b) = 5 mm and applies to both images.

FIGURE 13.

Low magnification photomicrographs of calretinin‐immunostained sections through the visual cortex of the lar gibbon (a)–(d) and the chimpanzee (e), (f) at the border of primary visual, or striate, cortex (V1) and extrastriate cortex (ExSt). The border between V1 and ExSt is marked by the thicker line with short dashes, while the border of the cerebral cortex and the white matter is marked by the thinner line with longer dashes. Note the presence of calretinin‐immunopositive fibers exclusively below V1, while these fibers are not present below the ExSt. In all images dorsal is to the top and medial to the left. Scale bars in each image = 1 mm and only apply to the image in which they appear.

FIGURE 14.

Photomicrographs of neuronal nuclear marker (a), (e), parvalbumin (b), (f), calbindin (c), (g), and calretinin (d), (h) immunostained sections in the primary visual cortex of the lar gibbon (a)–(d) and chimpanzee (e)–(h). Note the strong staining intensity of the neuropil in layer 4 for calretinin, and the absence of calbindin‐immunopositive neurons in this layer. This indicates that this calretinin‐immunopositive neuropil staining represents the ramifying axons of the calretinin‐immunopositive optic radiation. Scale bar in (d) = 500 μm, and applies to (a)–(d). Scale bar in (h) = 500 μm, and applies to (e)–(h).

3.5. The trajectory of the OR in lar gibbon

The trajectory of the OR through the white matter, originating at the LGN and terminating in V1 was similar across the primates where clear immunolabeling by either parvalbumin or calretinin was observed. We describe and depict (Figures 11, 15), in the coronal plane, the trajectory of the OR in the lar gibbon as representative of all species studied, apart from the southern lesser galago as the OR in this species was not labeled by any of the antibodies used and we cannot be certain that the pathway follows the same trajectory. The calretinin‐immunopositive fibers labeling the OR emerged from the neurons forming the LGN, aggregating and exiting this nucleus on its lateral aspect (Figure 10). The OR then, after passing through the thalamic reticular nucleus (Figures 10, 11), spread mediolaterally and dorsoventrally to surround the caudal pole of the putamen deep to the claustrum (Figures 11, 15). Caudally, the OR continued to expand dorsolaterally, with a thin portion of the OR extending to surround the ventral aspect of the hippocampal formation, the temporal loop of the OR (Figures 11, 15). Further caudal, the ORwas observed to surround the medial, lateral, and ventral aspects of the hippocampal formation, as well as occupying the white matter lateral to the occipital horn of the lateral ventricle. As the OR progressed further caudal, it encircled the caudal pole of the occipital horn of the lateral ventricle (Figures 11, 15). At this coronal level, the OR began to invest into V1, showing distinct, but dorsoventrally wide, medial and lateral branches. The calretinin‐immunopositive axons of the OR were clearly associated with V1, and did not invest into any extrastriate cortical region (Figures 11, 13, 15). At the caudal aspect of the occipital lobe, most of the white matter was occupied by the calretinin‐immunopositive axons of the OR, reflecting the proportional occupation of the cortex by V1 (Figure 15).

FIGURE 15.

Serial drawings of coronal sections through one half of the lar gibbon brain from the level of the decussation of the lateral geniculate nucleus (LGN, dark gray shading) through to the caudal edge of the brain. (a) is the most rostral drawing, (l) the most caudal, with each drawing being spaced by approximately 3 mm. The architectural outlines were drawn using the neuronal nuclear marker stain, with the calretinin‐immunopositive optic radiation (OR) from the LGN to the primary visual or striate cortex (V1, dark gray shading) that traverses the white matter labeled in pale blue. The non‐striate cortex (ExSt, primarily extrastriate visual cortex, but with several non‐visual areas included) is outlined with light gray shading. See list for abbreviations.

4. DISCUSSION

In this study we examined the intensity of immunostaining for parvalbumin, calbindin, and calretinin (CaBPs), in the origin (soma of the neurons forming the LGN), pathway (the axons within the white matter comprising the OR), and termination (neuropil of layers 3 and 4 of V1) in the brains of five species of primates, including representatives from all major branches of the primate radiation. Distinct lineage‐specific differences in the expression of CaBPs were noted. These lineage‐specific differences suggest a phylogenetic shift in CaBP expression in the OR, with little to no immunostaining for CaBPs in the strepsirrhine examined, parvalbumin‐dominant labeling in platyrrhine and cercopithecid monkeys, and calretinin‐dominant labeling in hominoids (Figure 16), reflecting neurochemical divergence despite conserved OR trajectory in the white matter across primate species. We compare our results to those previously published for the common marmoset (Goodchild & Martin, 1998; Ma et al., 2023; Yuasa et al., 2010) and human (Leuba & Saini, 1996, 1997).

FIGURE 16.

A summary phylogeny showing the relationships of the primate species investigated in this study, and from previously published studies, and the primary results obtained regarding the neurochemistry of the optic radiation (OR). Note the presence of three distinct “lineages” when the neurochemistry is used as a cladistic trait.

4.1. Lineage‐specific variances of CaBPs in the primate optic radiation

While not an exhaustive study of primates, the current study has derived and compiled data on the presence (with qualitatively assessed staining intensity) or absence of three CaBPs (parvalbumin, calbindin, and calretinin) in the origin (LGN), pathway (OR), and termination (V1) in species representing all major branches of the primate radiation. The species analyzed include a Strepsirrhini (southern lesser galago), two Platyrrhini (the black‐capped squirrel monkey and published data from the common marmoset [Goodchild & Martin, 1998; Yuasa et al., 2010; Ma et al., 2023]), a Cercopithecidae (crested macaque), and three Hominoidea (lar gibbon, chimpanzee, and published data from human [Leuba & Saini, 1996, 1997]) (Figure 16).

In the representative Strepsirrhini studied herein (southern lesser galago), the LGN, OR, and V1 were not strongly immunoreactive for the CaBPs investigated. While calbindin‐immunoreactivity was observed, the staining intensity was considered moderate and did not clearly and specifically label the white matter axons of the OR. This lack of CaBP‐immunopositivity distinguished the Strepsirrhini primate from the Anthropoid (Platyrrhini, Cercopithecidae, Hominoidea) primates studied, indicating a potential Strepsirrhini lineage‐specific neurochemistry of the OR, although this needs confirmation through the investigation of additional Strepsirrhini primates.

The CaBP staining of the LGN, OR, and V1 in the Platyrrhini (black‐capped squirrel monkey and common marmoset) and Cercopithecidae (crested macaque) investigated showed many similarities. Of most salience was the distinct presence of parvalbumin in the axons forming the OR, supporting the observations of Ma et al. (2023). Other significant consistencies include strong staining intensity for parvalbumin in the soma of the M layer of the LGN (also seen in the tufted capuchin monkey, Soares et al., 2001), the neuropil of layers 3 and 4 of V1 (also seen in the rhesus macaque, Medalla et al., 2023), and the absence of calbindin and calretinin immunopositivity of the axons forming the OR. These consistencies indicate that the neurochemistry of the OR in the platyrrhine and cercopithecid monkeys can be considered lineage‐specific traits. Despite these similarities, there are some differences between the Platyrrhini and Cercopithecoidae (Table 2), but these are primarily based on qualitatively assessed staining intensity rather than distinct qualitative differences.

In the hominoid species investigated including published data from humans (Leuba & Saini, 1996, 1997), several clear similarities in CaBP immunostaining were noted. Of central relevance to the present study was the strong intensity calretinin immunostaining observed in the LGN, OR, and V1. This consistency in the pattern of calretinin immunostaining distinguishes the hominoids from the remaining primates studied. In addition, the weak to moderate staining intensity for parvalbumin within the LGN, OR, and V1 distinguishes the hominoids from the Platyrrhini and Cercopithecidae lineages (that show strong parvalbumin immunostaining). These results indicate a hominoid lineage‐specific neurochemistry of the OR, although studies in other phylogenetically relevant primates are needed to strengthen this conclusion.

This study has revealed that intense parvalbumin‐immunoreactivity of the LGN, OR, and V1 is not a feature common to all primates as proposed by Ma et al. (2023). Rather, the CaBP neurochemistry of the OR in primates varies between primate lineages (Figure 16). Notably, the hominoids were clearly distinguished from the remaining primate species due to the strong presence of calretinin in the OR.

4.2. Possible functional implications of different CaBPs in the OR

The three CaBPs investigated in the current study (parvalbumin, calbindin, and calretinin) have differential effects on the buffering of calcium that will affect information flow in neuronal pathways in different ways. While these CaBPs are most often found in GABAergic, or inhibitory, interneurons (e.g., Celio & Heizmann, 1981; Hof et al., 1993; Kemppainen & Pitkänen, 2000; McDonald & Mascagni, 2001), they are also present in excitatory, or projection, neurons (e.g., Baimbridge et al., 1992; Pór et al., 2005), as exemplified for the axons forming the OR in the current study and those of Leuba and Saini (1996, 1997) and Ma et al. (2023). It is known that parvalbumin acts as a slow buffer of calcium dynamics, with calbindin showing a similar but faster buffer of calcium dynamics, while calretinin appears to be a calcium modulator, showing dual kinetic properties rather than being a buffer (Barinka & Druga, 2010; Billing‐Marczak & Kuznicki, 1999; Schmidt et al., 2003). As the calcium transients produce short‐lived, but highly compartmentalized signals within the portions of the neurons where they are present, differing expression patterns of the various CaBPs may alter the action of the axons forming the OR. How the variation in the expression of CaBPs precisely affects visual information and perception processing remains uncertain, but speculatively, the strong presence of calretinin in the hominoid OR indicates that the input reaching and being processed in the hominoid primary visual cortex is likely to be modulated in a different manner to that observed in non‐hominoid primates. This may play a distinct role in the way visual perception is generated in the different primate lineages and how visual perception guides behavior, for example the global‐to‐local processing strategy used by chimpanzees compared to the local‐to‐global strategy observed in rhesus monkeys (Hopkins & Washburn, 2002), or with variations in the diversity of color vision capacities across primates (Kawamura, 2016). The current study adds to previous studies showing that while many aspects of the macaque and human visual system are comparable, differences in the macaque and human primary visual cortex are present (e.g., Preuss, 2003; Rapan et al., 2022; Tootell et al., 1996; Tootell et al., 2003). These differences are important to understand when extrapolating findings in model species, such as macaque and marmoset monkeys, to humans.

4.3. The gross anatomical path of the OR is similar across primates

The current study and those undertaken previously (Leuba & Saini, 1996, 1997; Ma et al., 2023) have outlined what might be considered an unusual appearance of the OR in primates when described with the precision allowed by chemical neuroanatomical studies, as the pathway (OR) between the LGN and V1 is not direct and includes the temporal loop that passes beneath the hippocampus proper. Despite this unusual trajectory of the OR across the primate species studied herein and previously, the broad organization of the trajectory of the OR appears very similar across primates, having been observed in all species investigated, including strepsirrhines (Ma et al., 2023; Takemura et al., 2017, 2024). Thus, in contrast to the neurochemical variation of the OR across primates, the trajectory of the primate OR is similar across species representing all major branches of the primate radiation. Thus, as concluded by Rapan et al. (2022), our study indicates that the OR in primates, while not being neurochemically identical in different lineages, has a trajectory in the white matter that is comparable across species. The effect on visual processing and perception based on neurochemical differences, and how this alters stimulus‐induced and self‐initiated behavior is of importance to understanding human vision.

AUTHOR CONTRIBUTIONS

Nelyane N. M. Santana: Investigation; writing – original draft; methodology; writing – review and editing. Jordan Swiegers: Methodology; writing – review and editing; investigation. Mads F. Bertelsen: Investigation; methodology; writing – review and editing. Chet C. Sherwood: Investigation; writing – review and editing. José R. L. P. Cavalcanti: Investigation; funding acquisition; methodology; writing – review and editing. Paul R. Manger: Conceptualization; investigation; funding acquisition; writing – original draft; methodology; validation; visualization; writing – review and editing; project administration; data curation; supervision; resources.

FUNDING INFORMATION

This work was supported by funding from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (NNMS, JRLPC & PRM), the Leakey Foundation (PRM & CCS), and the South African National Research Foundation (PRM).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT

Data have not been shared due to this study being based on histological sections.