Expression of synaptic proteins and development of dendritic spines in fetal and postnatal neocortex of the pig, the European wild boar Sus scrofa

doi:10.21203/rs.3.rs-4943302/v1

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Expression of synaptic proteins and development of dendritic spines in fetal and postnatal neocortex of the pig, the European wild boar Sus scrofa

https://doi.org/10.21203/rs.3.rs-4943302/v1

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Synapse formation is a critical step in neuronal development. Current knowledge is largely based on altricial rodents where synapse formation and maturation proceed largely postnatally. In precocially born mammals such as guinea pig presynapse and spine formation start well before birth. Here, we analysed the developmental expression of proteins associated with synapse formation and maturation together with the development of basal dendritic spines of pyramidal neurons of visual and somatosensory cortex of the pig, an emerging translational model for human neurodegenerative disorders.

A total of 23 selected proteins was quantified with Western blots. Most were detectable from midgestation embryonal day (E) 65 onwards. About half reached the adult expression level seen in postnatal day (P) 90 pig cortex already two weeks before birth (gestation 114 days) in somatosensory, albeit not yet in visual cortex. For instance, major molecular components of synaptic plasticity such as GluN2B, CamKIIα, α-actinin-2, synaptopodin and T286 phosphorylated CamKIIα were expressed at E100 in somatosensory cortex. Dendritic spine type quantification with DiI-labeled material revealed an increase of total dendritic protrusion from E70 onwards. The increase was steepest in somatosensory cortex which had, at E110, a proportion of mushroom spines equal to the proportion present at P90. Together, matching the ungulate life history, a rapid development of functional synaptic connectivity in prenatal somatosensory cortex serves the somatomotor abilities essentially required by the newborn nest-fledgling. Results support the “cascading” model of a sequential maturation of cortical areas, and in precocial species the cascade starts well before birth.

NMDA receptor

GABA receptor

PSD95

GRIP1

GAD-65/67

fast-spiking interneurons

porcine

The development of the cerebral cortex of the pig, a precocial nest-fledgling, is mainly a prenatal process. Ungulates are born with their senses and motor system functional because they need to run with their mother quickly after birth. As expected, myelination commences early during pregnancy in precocial animals like pig (Sobierajski et al., 2023), guinea pig (Tolcos et al., 2011), cattle (Urban et al., 1997) and sheep (Barlow, 1969). When comparing temporal expression profiles, we found that myelin basic protein and myelin-associated glycoprotein are expressed around two weeks earlier in somatosensory cortex (SC; the representation of the rostrum has been analyzed) than in visual cortex (VC). Further, myelinated fibers are thicker in SC than in VC at the same age. Also, the axons of parvalbumin-expressing interneurons are more likely to be myelinated in SC than in VC at the same age (Sobierajski et al., 2023). This seems to be in line with the “cascading” hypothesis (Huttenlocher and Dabholkar, 1997) of neurodevelopment: somatosensory representations develop early and somehow trigger the formation of functional maps serving the other senses. Based on receptor binding assays, the opposing “integrative network” hypothesis claims that cortical fields follow a common developmental trajectory (Lidow et al., 1991), at least from birth onwards. Indeed, the blood vessel system of the pig cortex develops at the same time in SC and VC, and expression profiles of angiogenesis- and tight junction-related proteins reveals no difference between SC and VC (Sobierajski et al., 2024). Angiogenesis in postnatal cortex has an activity-dependent component (Fonta and Imbert, 2002; Tieman et al., 2004), and it appears as if very early forms of spontaneous activity (Powell et al., 2024) and sensory evoked activity also influences angiogenesis in fetal pig cortex. Overall, to our knowledge, time-dependent area differences e.g. of protein expression in early cortical development are understudied and literature evidence is scarce.

The two opposing findings (Sobierajski et al., 2023; Sobierajski et al., 2024) raised the question of whether expression of developmentally regulated neuronal proteins might also display time-dependent areal differences. If the SC matures more rapidly than the VC one would expect a higher level of proteins for neuronal function, e.g. for spines, presynapses, transmitter receptors, in SC and a delayed expression in occipital VC. Besides humans, knowledge about development and maturation of pre- and postsynaptic components have so far been obtained mainly from altricial rodents. Studies also described dendritic spine morphology, density, and dynamics in diverse neuronal populations and cortical areas (reviewed by Moyer and Zuo, 2018). For instance, spines from layer 5 pyramidal neurons in SC and auditory cortex (AC) of young mice are more dynamic than those in VC. Yet, the stability of mature mushroom spines and immature filopodia is comparable in the areas examined (Majewska et al., 2006). Further, in the same cortical area, layer 2/3 pyramidal cells have more spines along the apical dendrite than layer 5/6 pyramidal cells in both, motor and somatosensory cortex from P14 to P120 (Tjia et al., 2017). In pig, stellate neurons of layer 4 in SC and AC are more complex than those in VC at P3, and this reverses at 8 postnatal weeks (Jarvinen et al., 1998). Spine densities did not differ between the cortical areas at the two ages (Jarvinen et al., 1998).

Developmental studies in altricial species often have a focus on the critical period of sensory plasticity and malleability after birth. Precocial species such as guinea pig, ungulates and primates, and human with their perinatal brain development (possibly not as altricial as long believed; Gómez-Robles et al., 2024), have the period of rapid spine and synapse formation in the late fetal stage (Bourgeois, 1997; Huttenlocher and Dabholkar, 1997; Nacher et al., 2000). This explains why fetal growth restriction in these species substantially impairs cognitive abilities later in life. The fetal brain is constantly exposed to specific somatosensory input evoked by movements of the dam, of its siblings, or its own spontaneous muscle activity. In visual system, spontaneous retinal activity before eye opening in rodent and ferret drives the axonal wiring (Godement et al., 1984; Meister et al., 1991; Akerman et al., 2002; Adelsberger et al., 2005), and in primate and cat, eye-specific segregation is completed before birth (Rakic, 1976; Sretavan and Shatz, 1986). Functionally, in newborn sheep, the response properties of visual cortical cells such as orientation selectivity, receptive field orientation, ocular dominance distribution are indistinguishable from those in the adult; just that binocular cells are initially equally well responding to monocular stimuli suggesting that mature binocular interactions depend on visual experience (Clarke et al., 1979). Having so little information on prenatal development and having the two opposing findings on area differences of development of myelin-related proteins but not of angiogenesis-related proteins (Sobierajski et al., 2023; Sobierajski et al., 2024) prompted the present study on quantitative development of spines visualized with lipophilic dyes and synaptic proteins with Western blots in fetal and postnatal material of SC and VC of the pig.

Animal material The material is from our fetal pig brain collection (Ernst et al., 2018; Sobierajski et al., 2022). Our model of choice is a non-domesticated form, the European wild boar, Sus scrofa (Linné, 1758) which has an approximately 41% larger brain volume compared to domestic landrace pig (Böndel, 2017) without signs of domestication such as reduced sensory organs. Fetuses derived from mostly young (first pregnancy) sows and piglets individually hunted for population control in accordance with the German Game Law (Jagdrecht) or killed in road accidents. Law requests disposal of viscera including sexual organs. During evisceration, the uteri were examined for pregnancies. As described in detail (Sobierajski et al., 2022), most fetuses were dissected at the Forsthof Üfter Mark, embryonic membranes were removed, and fetuses immersed in cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer pH 7.4, or in cold Ringer solution for sampling raw material for blots. The P5 domestic “German Landrace” piglet was donated by Prof. Martin Schmelz and Prof. Dr. Maren Engelhardt, Institutes of Physiology and Anatomy, Medical Faculty, University Mannheim. Fine dissection of brain and body organs was done after transport to Ruhr University. Staging has been done using the crown-rump-length using the published formula for European wild boar (Henry, 1968) and by external features.

Table 1

Antibodies and reagents for staining and protein blotting.
Primary antibodies	Species, label, method	Source, order number, RRID	Dilution
α-Actinin-2	Rabbit, WB	Invitrogen, Carlsbad, Cat# PA5-27863 CA, USA, RRID: AB_254533	1:750
β-actin (housekeeping protein)	Mouse, WB	Sigma-Aldrich, St. Louis, MO, USA, Cat# A1978, RRID: AB_476692	1:6000
βIII-tubulin (housekeeping protein)	Mouse, WB	Sigma-Aldrich, St. Louis, MO, USA, Cat# T8660, RRID: AB_477590	1:3000
Caspase-3	Rabbit, WB	Cell Signaling, Cambridge, UK, Cat# 9662, RRID: AB_331439	1:1000
CaMKIIα	Mouse, WB	Abcam, Cambridge, UK, Cat# ab22609, RRID: AB_447192	1:1000
pCaMKIIα T286	Mouse, WB	Invitrogen, Carlsbad, CA, USA, Cat# MA1-047, RRID: AB_325402	1:1000
GABA_ARα1	Mouse, WB	NeuroMab, Davis, CA, USA, Cat# 75–136, RRID: AB_ 2877288	1:1000
GABA_ARβ3	Mouse, WB	NeuroMab, Davis, CA, USA, Cat# 75–149, RRID: AB_ 2109585	1:500
GAD-65/67	Mouse, WB	Enzo Life Sciences Inc., Farmingdale, NY, USA, Cat# ADI-MSA-225, RRID: AB_ 2039129	1:1000
GluA2	Mouse, WB	NeuroMab, Davis, CA, USA, Cat# 75 − 002, RRID: AB_ 2232661	1:500
GRIP1	Rabbit, WB	Synaptic Systems, Göttingen, DE, Cat# 151003, RRID: AB_10804287	1:1000
Homer-1	Rabbit, WB	Synaptic Systems, Göttingen, DE, Cat# 160033, RRID: AB_887730	1:1000
Kv3.2	Rabbit, WB	Alomone Labs Ltd, Jerusalem, IL, Cat# APC-011, RRID: AB_20401686	1:1000
Munc-18	Rabbit, WB	Synaptic Systems, Göttingen, DE, Cat# 116003, RRID: AB_2619783	1:1000
NMDAR subunit 1 (NR1/GluN1)	Mouse, WB	NeuroMab, Davis, CA, USA, Cat# 75–327, RRID: AB_2315839	1:500
NMDAR subunit 2B (NR2B/GluN2B)	Rabbit, WB	Millipore, Burlington, MA, USA, Cat# 06-600, RRID: AB_310193	1:1000
PSD95	Mouse, WB	Synaptic Systems, Göttingen, DE, Cat# 124011, RRID: AB_10804286	1:1500
p38/ Synapophysin	Mouse, WB	Synaptic Systems, Göttingen, DE, Cat# 101011, RRID: AB_887824	1:5000
p70/Synapsin1	Mouse, WB	Synaptic Systems, Göttingen, DE, Cat# 106001, RRID: AB_887805	1:2000
RIM1	Rabbit, WB	Synaptic Systems, Göttingen, DE, Cat# 140003, RRID: AB_AB_887774	1:1000
Synaptopodin	Rabbit, WB	Synaptic Systems, Göttingen, DE, Cat# 163002, RRID: AB_887825	1:1000
Synaptotagmin-2	Rabbit, WB	Synaptic Systems, Göttingen, DE, Cat# 105123, RRID: AB_2199465	1:1000
TOM70	Mouse, WB	Santa Cruz Technologies, Cat# Sc-3900545, RRID: AB_2714192	1:1000
Trim46	Rabbit, WB	Synaptic Systems, Göttingen, DE, Cat# 377008, RRID: AB_2905572	1:2500
Secondary antibodies
Anti-mouse	Rabbit, alkaline phosphatase, WB	Dako A/S, Glostrup, DEN, Cat# D0314	1:5000
Anti-rabbit	Goat, alkaline phosphatase, WB	Dako A/S, Glostrup, DEN, Cat# D0487, RRID: AB_2617144	1:3000
Reagents
DiIC18(3)	Fluorescent lipophilic dye	Molecular Probes via Invitrogen, Carlsbad, CA, USA,	crystalline
DAPI	Fluorescent counterstain	Sigma-Aldrich, St.Louis, MO, USA, Cat# D9542	1:3000

Tissue processing and DiI staining As described (Ernst et al., 2018; Sobierajski et al., 2022) dissected brains were immersion-fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) pH 7.4 with 5% (vol/vol) water-saturated picric acid for about 2 weeks at 8°C (refreshed once). For DiI staining small slabs of tissue containing one or two gyri were cut from the VC and SC and freed from remaining meninges. Using glas micropipettes, small crystals of DiI were implanted into gray matter (layers 3–5 at E110 and P90) to label nearby pyramidal cells via their horizontal axon collaterals. Tissue slabs were stored in fixative in the dark at 4°C for at least two weeks to allow dye diffusion. Slabs were cut with a vibratome (MA752, Campden Instruments) into 80–120 µm thick sections. The E70 and E85 tissue blocks were pre-embedded in 1% agarose to improve stability during slicing. The sections were stained with DAPI for 10 minutes, washed repeatedly, mounted on silanized slides, and coverslipped with ImmuMount.

Protein Blots Tissue blocks containing gray and upper white matter from VC and SC were taken during preparation of the fetuses (ages E65, E80, E95, E100, P90) as described (Sobierajski et al., 2023). VC and SC of P60 rat cortex was used as positive control. Tissue blocks were first pulverized on dry ice. A microspoon-sized powder sample was homogenized in standard RIPA lysis buffer. The amount of protein was determined with the Markwell assay. Proteins were separated on 8%, 10% or 14% polyacrylamide gels (30 µg protein per lane) with a current voltage of 7 mA per gel overnight. Proteins were transferred to nitrocellulose membranes (BA 85/Protran, 0.2 µm pore size, Schleicher and Schuell or Amersham) with a tank blotter (Hoefer, San Francisco, CA, USA). Smaller proteins were transferred with buffer containing 20% MeOH and no SDS, proteins above 70 kDa were transferred with buffer containing 15% MeOH and 0.05% SDS. The blots were stained with Ponceau-Red to visualize major protein bands. Membranes were then separated into horizontal stripes containing the desired protein and blocked with TBS (50 mM Tris, pH 7.4, 150 mM NaCl) containing 5% bovine serum albumin for 1 h. Primary antibodies (Table 1) were incubated overnight at 4°C, followed by washes for 10 min each in TBS, TBST (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween-20) and TBS. Matching alkaline phosphatase-conjugated secondaries (Table 1) were incubated for 90–120 min. Blots were stained with 0.18 mg/mL BCIP and 0.35 mg/mL NBT, washed with water and dried.

Several other antibodies have been tried. While all were staining the target band in rat, they did not deliver reliably measurable bands in pig. Nevertheless, we listed these antibodies in Supplementary Table 1 to inform future research.

Analysis DiI-stained sections were imaged with a Leica TSC SP5 confocal microscope (10x, 40× and 63x objective with 1.1 NA, 1024 x 1024 px). Raw confocal images were deconvolved with Huygens Essential (Scientific Volume Imaging B.V.) for improving image stack quality and removing background noise. Selected dendrites were optically isolated with the surface tool and a masking algorithm of Imaris 10.1. software (Oxford Instruments). The “filament” algorithm in Imaris allows 3D reconstruction. The initial semi-automatic reconstruction was strictly followed by manual work-over and classification of spine types in selected categories (Risher et al., 2014). This often involved two experienced observers equipped with the only reliable high-tech, the human retina and the human brain, to make concise decisions regarding the continuum of spine shapes and their intermediates known to exist during development and even in adulthood (Ofer et al., 2022). Spines along secondary and tertiary basal dendrites of pyramidal cells of infragranular layers were reconstructed. For documentation, global whole-picture contrast-, brightness-, color intensity- and saturation-settings were adjusted with Adobe Photoshop®. Scale bars were generated with Leica Acquisition Suite X and inserted with Adobe Photoshop® (CS6 Extended, Version 13.0 x64). Data management was done in Microsoft Excel 365. Graphs were made with SigmaPlot 12.3 (Systat Software GmbH). Plots and images were arranged with Adobe Photoshop®. Blots were photographed and relative band intensities were determined with ImageJ. All protein bands were normalized to the 42 kDa β-actin or the 55 kDa βIII-tubulin, which served as control for loading equivalency. Values were then expressed relative to the intensity at P90. P90 is shortly before puberty onset in pig and has been taken as equivalent to adult (Sobierajski et al., 2022). Statistical analysis of the spine density was done with a non-parametric Mann-Whitney rank sum test.

Developmental expression of neural and synaptic proteins

We selected proteins which, in rodent, are developmentally regulated and/or related to neuronal plasticity. Blot by blot we aimed to compare expression profiles in VC to SC beginning at midgestation E65, just after the begin of gyrification (Ernst et al., 2018), at late gestation E80, E95, E100, and at P90 (adult). Levels at P90 had been set to 1 because we expected proteins to increase towards adulthood.

Caspase-3 expression indicates cell death which is high early in development. As expected, expression was high early on in VC declining after E95. Decline in SC commenced already after E65. Lowest level was seen at P90 (Fig. 1A). Translocase of the outer membrane, TOM70, works in a complex to transport proteins into mitochondria. It is a general metabolic marker useful for assessing biogenesis of mitochondria (Kreimendahl and Rassow, 2020; Ma et al., 2023). Given the dramatic volume increase of the cortex from midgestation to postnatal and the rapid differentiation of energy-hungry neurons as well as the addition of microglia, myelin-forming oligodendrocytes and blood vessels (Ernst et al., 2018; Sobierajski et al., 2022; Sobierajski et al., 2023; Sobierajski et al., 2024) we expected an increase. Indeed, TOM70 expression increased substantially reaching the adult level at E100 in SC, albeit not yet in VC (Fig. 1B). Looking more to neurons, the neuron-specific RNA splice factor NeuN comes in two isoforms of 46 and 48 kDa. The larger isoform is faintly expressed at E65 (Fig. 1C). Both isoforms increased from E80 onwards, and the adult level is reached at E100 in SC, albeit not yet in VC. The E65 levels were higher than levels at E80 and E95 resulting, unexpectedly, in a U-shaped expression profile (Fig. 1C). The microtubule-associated Ring finger E3 ligase protein Trim46 is involved in neuronal polarity and axonal outgrowth (Curcio and Bradke, 2015). Expression was highest at E65 remaining at a constant level thereafter (Fig. 1D). The enrichment of Trim46 in the axon initial segment maintains neuronal polarity and it is in line with the presence of βIV-spectrin-positive axon initial segments in pig cortex as early at E70 (Ernst et al., 2018).

Growing axons secrete transmitters, and since synaptic vesicle proteins become selectively delivered to axons (Watson et al., 2023) their expression could be a proxy for development of axonal connectivity. The vesicle docking and fusion protein Munc-18 has a crucial role for exocytosis. It is at near-adult levels already from E65 onwards in VC and in SC (Fig. 1E). Munc-18 is involved in organizing the presynapse rather than being a marker for synaptogenesis, since it was already highly expressed when the vesicle-associated proteins p38 and p70 were still very low. Yet, transmitter release from nascent axons can occur without molecularly mature vesicles (Andreae and Burrone, 2014). P38/synaptophysin is a marker for presynapse development. It was present weakly in SC at E65 but not yet in VC at E65. Protein amounts increased slowly until E100 and then doubled to P90 (Fig. 1F). Similarly, p70/synapsin-1a/b was detectable at E65 with higher levels in SC than in VC and increasing in both areas (Fig. 1G). Another presynaptic active zone protein is RIM1a. The expression profile was U-shaped in VC and in SC. Levels at E100 were not yet near P90 levels in both areas (Fig. 1H).

We also compared P90 pig VC to rat VC (Supplemental Fig. 1A-D) by using blot images not included in Figs. 1–4. The aim was to confirm the quantification with independent blots and to explicitly focus on the delayed development of the VC. The comparison revealed that NeuN, Trim46, Munc-18, p70/synapsin-1 and RIM1 expression were fairly similar to rat in terms of postnatal band intensity and molecular weight. Pig caspase-3 ran lower than rat caspase-3 and the smaller isoform seen in pig up to E100 has not been reported in rodent. TOM70 had a doublet band in rat lysates but never in pig lysates. Rat p38/synaptophysin ran slightly lower than pig p38 (Supplemental Fig. 1A).

A. Cell-death-related caspase-3. B. Mitochondrial marker translocator of outer membrane, TOM70. C. Neuron-specific RNA splice factors, NeuN. D. Neuronal polarity and axon initial segment marker Trim46. E. Presynaptic organizer Munc-18. F. Presynaptic vesicle protein p38/synaptophysin. G. Presynaptic vesicle protein p70/synapsin-1a/b. H. Presynaptic organizer RIM1. β-actin at 42 kDa was used for normalization.

In Fig. 1, 2, 3, 4, VC is left, SC is right, each with 5 stages from embryonic day (E) 65, E80, E95, E100 to postnatal day P90 the average of which has been set to 1. Numbers in the bars indicate the number of independent lysates which delivered measurable bands. n.m., not measurable; the protein of interest was not detectable in these lysates. In case a lysate was run twice for a given marker, the average has been included to avoid technical repeats. Bars represent the mean with S.E.M.. Representative gel stripes are arranged together with the housekeeping protein from the same lanes. Molecular weight [kDa] is given.

Before continuing describing the other proteins, we shall briefly address the expression profiles. Seeing more intense bands with age, as with TOM70, p38/synaptophysin, p70/synapsin-1a/b, is commonly interpreted as an increase of protein amounts. This has been also the case for the expression of myelin-related proteins and GFAP detected with blots from the very same material (Sobierajski et al., 2023). Seeing less intense bands, as with caspase-3 or Trim46, is commonly interpreted as a decrease. This has been also the case PDGFRα, a marker for immature oligodendrocytes (Sobierajski et al., 2023). In contrast, U-shaped profiles as revealed for NeuN, RIM1 or PSD95 (Fig. 3) look peculiar. It might suggest a transient expression, or transient interaction with binding partners masking the antigenic epitope, up to harvest at a wrong circadian time point or simply postmortem protein degradation. Myelin proteins are particularly susceptible to the latter, however, within our postmortem time periods (Ernst et al., 2018) we could not detect any degradation (Sobierajski et al., 2023). Moreover, such interpretations make little sense in case of NeuN and PSD95 which are continuously increasing in neurons with ongoing differentiation, and both are not regulated by circadian mechanisms. Rather, we have to consider the dramatic volume increase of the brain from ~ 5 g at E45 to ~ 90 g at P90 (Ernst et al., 2018; Sobierajski et al., 2023). At E65, the future cortical gray matter consists mostly of densely packed immature neurons and the still migrating neurons of the cortical plate, whereas microglia (Sobierajski et al., 2022) and immature oligodendrocytes are still scarce, and astrocytic GFAP protein expression is barely detectable (Sobierajski et al., 2023). Therefore, at E65 neuronal proteins expressed by zillions of neurons will be the dominant proteins in a lysate of 30 µg total protein. With time, these early expressed neuronal proteins become diluted because proliferating, invading and/or maturating microglia, macroglia and endothelial cells contribute more and more non-neuronal proteins to the lysate. Accordingly, the band intensity drops transiently only to recover towards the postnatal stage with ongoing neuronal differentiation.

We therefore suggest an alternative interpretation. U-shaped profiles are seen with proteins which are expressed at substantial amounts early on and which then remain rather on a plateau. A constant protein level over the ages should be rather regarded as a continuous increase of expression with the “increase” being compensated for by the “dilution”, be it via neurons or glia which for instance also express many transmitter receptors. An increasing intensity of a band should be regarded as an even steeper increase of protein expression, again, in neurons or in glia cells. The caspase-3 expression then would initially increase before declining. Reports on U-shaped expression profiles are rare presumably because midgestation brain samples are rarely included. For instance, expression of protein tyrosine phosphatase 1D in fetal superior colliculus displays a U-shaped profile which has been interpreted as reflecting a switch in function (Reinhard et al., 2009). Increasing, decreasing and U-shaped profiles have also been shown for claudin-3, Jam-A and claudin-12, respectively, in fetal sheep from 0.60 gestational age onwards (Sadowska et al., 2015).

Developmental expression of plasticity-related proteins

PSD95 anchors NMDA receptors. The mRNA encoding the obligatory subunit GluN1 increases in rat cortex from birth to peak levels during the fourth postnatal week followed by a decline towards adulthood (Hofer et al., 1994) and the protein increases about 3-fold from birth to P40 (Luo et al., 1996). In pig, however, the strongest expression was at E65 in VC and less so also in SC with levels being 3-fold higher than at P90 (Fig. 2A) confirming the view that this subunit is not limiting the assembly of NMDA receptors. At the following fetal ages, the GluN1 band became extremely faint and was not always measurable by densitometry in all lysates tested. The band was again well detectable at P90. With all caution towards measuring such a faint expression, the bar graphs did suggest a small increase from E80 to E95. In contrast, the synaptic plasticity-related subunit GluN2B was well detectable from E65 onwards in VC and SC. A steep increase to the adult levels has occurred at E100 in SC, albeit not yet in VC (Fig. 2B). Of note, the expression of glutamate and GABA receptors may not solely mirror neuronal maturation since AMPA, NMDA and GABA_AR are also expressed in macroglia (Gundersen et al., 2015, for review).

The 55 kDa CamKIIα subunit is expressed in pyramidal cells and upregulates in rodent VC around eye opening at the end of the second postnatal week. CamKIIα autophosphorylation is essential for NMDAR-dependent plasticity (Gordon et al., 1996; Glazewski et al., 2000). Highly interesting, CamKIIα protein was expressed and steadily increasing in pig VC and SC from E65 onwards (Fig. 2C) suggesting that CamKIIα mirrored the functional differentiation of excitatory neurons. Also important, the T286-phosphorylated form was detectable at E100, a bit stronger already in SC than in VC. Substantially higher CamKIIα levels was seen at P90 (Fig. 2D) suggesting a massive increase after E100. The CamKIIα interaction with α-actinin-2 is required for structural long-term potentiation (Curtis et al., 2023) and spine growth and stability. Indeed, α-actinin-2 was well expressed in pig VC and SC and steadily increasing from E65 onwards (Fig. 2E). Also essential for spine maturation and maintenance is the spine apparatus with synaptopodin via its interaction with α-actinin-2. Synaptopodin comes as a long and a short transcript, and PCR detects both in brain and in kidney (Asanuma et al., 2005). At the protein level, only the smaller, ~ 100 kDa isoform is expressed in brain. In pig, expression was U-shaped with synaptopodin present at E65, not measurable at E85 and E95 and again detectable at near-adult levels at E100 in SC and less so in VC (Fig. 2F). Comparing pig VC to rat VC revealed the bands of GluN1, GluN2B, CamKIIα, α-actinin-2 and synaptopodin at the same molecular weight as in rat VC. The intensity of the P90 GluN1 and synaptopodin bands was lower in pig VC than in rat VC, which might reflect a difference in antibody binding (Supplemental Fig. 1B).

A. NMDAR subunit GluN1. B. NMDAR subunit GluN2B. C. Postsynaptic enzyme CamKIIα, total protein. D. T286 phosphorylated CamKIIα. E. Structural plasticity-related actin bundling protein α-actinin-2. F. Spine apparatus and cisternal organelle-associated synaptopodin. β-actin at 42 kDa was used for normalization. n.m., the protein was not measurable at this age in this cortex.

Developmental expression of proteins of the excitatory synapse

The postsynaptic scaffold PSD95 anchors NMDA receptors and interacts with AMPA receptors. PSD95 displayed a mild U-shaped expression profile with a strong increase in SC at E100 which was not yet seen in VC (Fig. 3A), and higher expression at P90. It resembles the human where PSD95 is strongly expressed in the neonate increasing two-fold towards adolescence (Webster et al., 2011). We tested two GluA2-specific antibodies, both yielding only faint bands in pig VC and SC. With all caution, GluA2 was present at the fetal stages and at higher levels at P90 (Fig. 3B). The glutamate receptor interacting protein GRIP1 is expressed postnatally in rodent cortex and parallels that of AMPA receptors. GRIP1 binds in particular GluA2 (Dong et al., 1999) and has an essential role in trafficking AMPA receptors into the postsynapse when cells undergo long-term potentiation. GRIP1 was strongly expressed from E65 onwards at near-adult levels in VC and SC in line with the presence of GluA2 (Fig. 3C). Homer-1 is a postsynaptic scaffold protein, interacts with Shank proteins and links metabotropic glutamate receptors with intracellular signaling partners such as the IP3 receptor. Expression from E65 onwards was steeply increasing between E100 to P90 in VC and earlier already in SC where near-adult levels were reached first (Fig. 3D). Comparing pig to rat VC revealed PSD95, GluA2, GRIP1 at the same molecular weight as in rat VC. GluN2A was at lower intensity in pig VC than in rat VC, possibly reflecting a difference in antibody binding. Homer-1 ran slightly lower in pig than in rat VC (Supplemental Fig. 1C).

A. Organizer of the excitatory postsynapse, postsynaptic density protein PSD95. B. AMPA-type glutamate receptor GluA2. C. AMPAR interacting protein GRIP1. D. Protein crosslinking metabotropic glutamate receptors to intracellular mediators, Homer-1. β-actin at 42 kDa was used for normalization.

Developmental expression of proteins of the inhibitory synapse

GABA_ARα1 and β3 are major components of GABA receptors on pyramidal neurons. The mRNAs encoding the two subunits have been also reported in oligodendrocyte precursors in fetal and postnatal human brain (Gutierrez et al., 2023). GABA_ARα1 was not yet detectable at E65 in VC and SC but increased thereafter reaching near-adult levels in SC before VC (Fig. 4A). This confirms protein blot data in fetal pig parietal cortex at E100 to P7 with immunoreactivity perinatally in pyramidal and nonpyramidal neurons (Kalanjati et al., 2017). GABA_ARβ3 was at near-adult level from E65 onwards in SC and very mildly U-shaped in VC (Fig. 4B). GAD-65/67 enzymes are expressed in inhibitory interneurons and synthesize GABA. Expression was not yet measurable at E65 in VC. In SC, the isoforms were detectable from E65 onwards and fetal expression levels were overall higher than in VC. Yet, the major increase towards the adult level seems to happen after E100 in both areas (Fig. 4C). Interneurons, in particular the fast-spiking types, express Kv3 channels for a fast repolarization of the action potential. Further, synaptotagmin-2/Syt-2 is the presynaptic calcium sensor specifically in fast-spiking basket and chandelier cells. Kv3.2 was expressed from E65 onwards increasing steeper in SC than in VC (Fig. 4D). Synaptotagmin-2 was not measurable at E65, but well present from E85 onwards. As with GAD-56/67 the major increase towards the adult level happened after E100 in both areas (Fig. 4E). Comparing pig to rat VC revealed the bands of GAD-65/67, synaptotagmin-2 and GABA_A receptors were at the same molecular weight and intensity as in rat VC. Kv3.2 expression levels tended to be higher in pig at P90. In terms of molecular weight, pig Syt-2 was running lower than rat Syt-2. (Supplemental Fig. 1D).

A. GABA_ARα1. B. GABA_ARβ3. C. Glutamate decarboxylase isoforms 65 and 67 kDa, GAD-65/67. D. Voltage-gated potassium channel Kv3.2. E. Calcium sensor in axon terminals of fast-spiking interneurons, synaptotagmin-2. β-actin at 42 kDa was used for normalization. n.m., the protein was not measurable at this age in this cortex.

Development of pyramidal cell dendritic spines and spine types

The density and morphology of dendritic spines are important indicators of the development of neuronal maturation. Here, we acquired confocal scans of DiI-stained neurons from the VC and SC from E70 to P90 and reconstructed the basal dendrites and classified the dendritic protrusions (addressed as ‘spines’ below). Only second and higher order dendritic segments were included. Dendritic end segments were excluded as the thin distal tips of dendrites often have fewer spines. Thick proximal dendritic shafts were excluded because short spines may be masked by the thick shaft (Xu et al., 2020). At E70, neurons had small somata (Fig. 5A). They form spines with immature morphologies which made classification difficult, and we did not determine spine types at this stage (Fig. 5A1). At E85, neurons had a much more mature morphology (Fig. 5B). Now, spine types could be determined (Fig. 5B1). A substantial increase in density proceeded between E85 and E110, and pyramidal cells developed larger somata with longer and more branched dendrites (Fig. 5C). Pyramidal neurons of the P5 domestic piglet did not obviously differ from E110 boar cells in morphology (Fig. 5D) but had a lower spine density (Fig. 5D1). Interestingly, volume, branching, and segment length of blood vessels of the P5 domestic piglet were somewhat lower than values obtained in E100/E110 pig marginal zone and gray matter suggesting that also vascular development tends to proceed slower in domestic pig than in wild boar (Sobierajski et al., 2024). Until P90, the complexity of pyramidal neurons (Fig. 5E) and the density of spines had increased further (Fig. 5E1).

The data base of the spine analysis is given in Fig. 6A. From E70 to E85, spine density moderately increased on average from 8 to 18 spines per 100 µm (Fig. 6B) followed by a roughly threefold increase until E110 to a median of 60 spines per 100 µm. As expected from the qualitative data, the P5 domestic piglet had a spine density of on average 43 spines per 100 µm which was in the lower range of what is present in boar at E110. Until P90, spine density increased to 79 spines per 100 µm (Fig. 6B). At E85, the majority of spines were classified as thin and filopodial, mushroom and stubby spines were rare (Fig. 6C). Thin spines were still the dominant fraction at E110, but the density of mushroom and stubby spines had substantially increased whereas filopodia became rare (Fig. 6D). This trend continued at P90 with stubby spines remaining at the level seen at E110, while thin and mushroom spines continued to increase (Fig. 6E).

When looking at Fig. 6B, the almost three-fold variability between individual cells at E110 and at P90 might indicate large cell-individual differences in spine densities. This is known to occur in vivo but could potentially suggest a sampling bias. The latter can be excluded. The variability decreased substantially when separating the cells by area. At E110 and at P90, SC neurons had significantly higher spine densities than VC neurons. Moreover, spine density increased significantly between E110 and P90 in VC neurons, whereas the spine density in SC neurons at E110 was statistically not different from the density at P90 (Fig. 6F). The developmental advance of SC pyramidal neurons and the much slower development of VC pyramidal cells, respectively, was mirrored by the proportion of mushroom spines. In SC, the proportion seen at P90 was reached already at E110, whereas in VC the proportion of mushroom spines was still low at E110, increasing to P90 albeit not yet to the level reached in P90 SC (Fig. 6G).

Our study delivered three major results. First, many activity-dependent and plasticity-related proteins tested here are expressed from midgestation onwards. Second, many reach the P90 postnatal levels already at E100 in SC, two weeks before birth, or display a steeper increase in SC than in VC. Third, basal dendrites of SC pyramidal cells display an adult number of spines and an adult proportion of spine types already at E110, while VC was still lagging. Protein expression and spine assessment support the “cascading” model which claims that cortical areas mature sequentially. In human, spine density increases more rapidly during second half or gestation and pruning of synapses during childhood proceeds earlier in auditory cortex than in prefrontal cortex (Huttenlocher and Dabholkar, 1997). Thus, the “cascade” starts well before birth and might have been missed by analyses beginning at birth. In line with this, the expression of myelin-related proteins commences 1–2 weeks earlier in prenatal SC than in VC (Sobierajski et al., 2023), suggestive of being driven by specific sensory input, in addition to spontaneous activity of the sensory periphery. Moreover, the myelin sheaths in SC were thicker on average at E100 than those in VC. An exception has been the blood vessel development which in terms of protein expression commences at about the same time in SC and VC (Sobierajski et al., 2024) presumably being influenced by very early activity patterns in networks extending over millimeters (Powell et al., 2024). In addition to spontaneous calcium oscillations, optic nerve stimulation at E55 in sheep (0.37 gestational age, equals ~ E45 in pig) can elicits visual cortical responses in form of a long-latency surface wave (Persson and Stenberg, 1972) which resembles the wave evoked by somatosensory stimulation at E55 (0.38 gestational age in sheep; equals E45 in pig). The latter wave presumably reflects postsynaptic potentials in basal dendrites of cortical pyramidal neurons of infragranular layers and subplate (Molliver, 1967). As reviewed recently (Sobierajski et al., 2024), spontaneous activity and activity evoked by peripheral stimulation is present very early in the ungulate fetal cortex. These activity pattern can regulate the protein expression as well as the spine development. In fact, many of the proteins assessed here as well as spine maturation are downstream of activity.

The spine assessment yielded highest numbers in the category „thin” spines which have a small difference between the width of the neck and the width of the head. Numbers might have been a bit overestimated because neck width has been reported to depend on fixation. After cryofixation spine neck width has been found to be 30% thinner (Tamada et al., 2020). Enhancing the difference between neck and head width might affect the classification, however, that would account to all ages equally. Of note, the P5 domestic piglet does not present with spine densities seen in wild boar. A reason could be that the piglet underwent a 1 h anaesthesia to demonstrate a nerve recording to medical students at University of Heidelberg before euthanasia. Perinatal exposure to anaesthetics rapidly decreases spine density, whereas exposure around the peak of synaptogenesis rapidly increases spine density without evoking cell death or dendritic reorganization (Briner et al., 2011). Given the advanced development of perinatal wild boar we expected the latter effect. However, the low level rather argues for a deficit evoked by domestication.

What innervates the early neurons in SC and VC to promote spinogenesis? First, spines offer docking space for local connections which innervate in particular basal dendrites. Second, specific afferents deliver sensory information to the SC and at least spontaneous activity generated in the retina will arrive in the VC. Spontaneous activity efficiently drives development of the functional architecture (Meister et al., 1991), and its spatial pattern rather than its frequency plays the instructive role for topographic map formation (Xu et al., 2011). Third, immature cortical areas are connected by long-range projections and the activity elicited in one area may easily travel to neighboring areas (Powell et al., 2024). Fourth, transient connections exist e.g. between auditory and visual areas (Clarke and Innocenti, 1990), and intraareal (Assal and Innocenti, 1993) and callosal projections exuberantly innervate deeper cortical layers. Even when not maintained into adulthood such connections will exert a modulatory role on functional and morphological maturation (Elberger, 1994; Innocenti and Price, 2005). The early presence of synaptic vesicle proteins in axons (Watson et al., 2023) and organizers of the pre- and postsynapse as well as receptors for glutamate and GABA suggest a robust increase of synaptic transmission from E65 onwards in pig cortex.

In pig VC and SC, CamKIIα, synaptopodin, and α-actinin-2 were detectable from E65 onwards. Synaptopodin is a marker of spine maturation (Deller et al., 2000) being enriched in mushroom spines. Synaptopodin is phosphorylated by CamKIIα, can then bind to a 14-3-3 protein which protects synaptopodin from proteolytic degradation (Faul et al., 2008). Synaptopodin mediates the long-term spine stability (Okubo-Suzuki et al., 2008; Fester et al., 2009; Yap et al., 2020) by stabilizing actin via interaction with RhoA and cdc42 (Asanuma et al., 2006) and by regulating local calcium signalling (Korkotian et al., 2014). Interaction with α-actinin-2 (Asanuma et al., 2005) is important for dendritic targeting and postsynaptic enrichment of synaptopodin (Kremerskothen et al., 2005).

Midgestation pyramidal neurons in pig cortex barely had basal dendrites and those present were not yet equipped with spine-like protrusions. Synaptopodin expression at E65 might serve another structure, the cisternal organelle of the axon initial segment (Peters et al., 1968). Pyramidal cells are equipped with axons descending into the future white matter from migration onwards. The βIV-spectrin-positive domain characterizing the axon initial segment is well present at E70 in pig cortex pyramidal cells as well as in NPY-positive subplate neurons. Further, the domain elongates substantially until P30 and continuously shifts closer to the soma (Ernst et al., 2018). In dissociated hippocampal neurons, the axon initial segment acquires a cisternal organelle within a few days after axonal outgrowth, and synaptopodin expression at this early stage is independent of the neuron’s action potential activity (Sánchez-Ponce et al., 2011).

In rodent, CamKIIα expression in rat cortex starts after P7 (Lin and Redmond, 2008). Also, synaptopodin upregulates postnatally reaching adult levels by the second postnatal week (Asanuma et al., 2005; Czarnecki et al., 2005). This increase is presumably driven by neuronal activity (Roth et al., 2001), because synaptopodin expression increases in rodent hippocampus after induction of LTP (Yamazaki et al., 2001). In pig, the activated T286 phospho-CamKIIα was detectable at E100 in SC and weakly in VC which argues for electrically active neurons. Moreover, CamKIIα has a unique function in structurally interacting with GluN2B via α-actinin-2 to regulate LTP (Wang et al., 2011; Curtis et al., 2023). GluN2B and GluN1 were expressed early in fetal pig VC and SC, and GluN2B reached the adult level at E100 in SC albeit not yet in VC. A functioning spine apparatus is important for LTP and spatial learning (Deller et al., 2003). Single unit recordings in late-fetal post-central (somatosensory) cortex of the Göttingen minipig reveal that the action potential activity at E110 is highly similar to spike patterns observed in prepubertal pig (Konda et al., 1979). Response properties of visual cortical cells in newborn sheep are indistinguishable from those in the adult; (Clarke et al., 1979). Together, this is suggestive of a presence of LTP-like mechanisms in E100/E110 pig sensory cortex.

The inhibitory system also developed from midgestation onwards. GAD expression was relatively low during the fetal period increasing to adult levels after E100. Quite surprisingly, markers for fast-spiking neurons such as Kv3.2 and synaptotagmin-2 (Sommeijer and Levelt, 2012) were expressed early which confirms the early maturation of interneurons (Ernst et al., 2018). In line, the calcium-binding protein parvalbumin, a marker for basket and chandelier cells, is well expressed at E100 and even more, their axons started to become myelinated weeks before birth (Sobierajski et al., 2023). Similarly, parvalbumin-positive neurons are well developed at birth in guinea pig (Nacher et al., 2000). In human, parvalbumin-positive cells are present at gestational week 36 (Honig et al., 1996). In contrast in rat cortex, parvalbumin comes up during the second postnatal week (Patz et al., 2004) and axons become myelinated earliest during the third week (Micheva et al., 2021).

GluN2B-containing NMDA receptors are important for basal dendritic development (Balu and Coyle, 2012; Gonda et al., 2020), and these dendrites grow heavily in late gestation pig cortex. Moreover, in SC at E110, the fraction of mushroom spines which preferentially harbor synaptopodin and which possibly had been stabilized by synaptopodin was at postnatal levels. Overall spine densities at E110 resemble numbers reported for rat P15 (Miller, 1988). Comparable results have been reported in guinea pig cortex where dendrites and spine densities are at near-adult dimensions well before birth (Schüz, 1981). Behaviorally, this makes sense for an ungulate which needs to orientate and run with the group minutes-to-hour after birth.

Funding Information

This work was supported by Deutsche Forschungsgemeinschaft WA 541/15 − 1 to P.W.

Author Contribution

Conceptualization and methodology: E.S, P.W.; Resources: C.B. and C.B.; Investigation and analysis: E.S., S.W., M.A.S., K.C., S.R.; Visualization: E.S.; Writing—original draft: E.S., P.W.; Writing—review and editing: E.S., P.W.; Supervision and project administration: P.W.; All authors reviewed and approved the manuscript.

Acknowledgement

We acknowledge the Regionalverband Ruhr, Essen, Germany, for the interest in our work. We thank Andrea Räk and Christian Riedel for technical support. We thank Prof. Schmelz and Prof. Engelhardt, University Mannheim, Germany, for donating the P5 piglet brain.

Data Availability Statement

We confirm that all data obtained are presented in this manuscript.

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Expression of synaptic proteins and development of dendritic spines in fetal and postnatal neocortex of the pig, the European wild boar Sus scrofa

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INTRODUCTION

MATERIAL AND METHODS

RESULTS

Developmental expression of neural and synaptic proteins

Developmental expression of plasticity-related proteins

Developmental expression of proteins of the excitatory synapse

Developmental expression of proteins of the inhibitory synapse

Development of pyramidal cell dendritic spines and spine types

DISCUSSION

Declarations

Funding Information

Author Contribution

Acknowledgement

Data Availability Statement

References

Additional Declarations

Supplementary Files

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