Aggrecan-based extracellular matrix shows unique cortical features and conserved subcortical principles of mammalian brain organization in the Madagascan lesser hedgehog tenrec (Echinops telfairi Martin, 1838)

doi:10.1016/j.neuroscience.2009.08.018

Neuroscience

Volume 165, Issue 3, 3 February 2010, Pages 831-849

https://doi.org/10.1016/j.neuroscience.2009.08.018 Get rights and content

Abstract

The Madagascan tenrecs (Afrotheria), an ancient mammalian clade, are characterized by unique brain anatomy. Striking features are an expanded paleocortex but a small and poorly differentiated neocortex devoid of a distinct granular layer IV. To investigate the organization of cortical areas we analyzed extracellular matrix components in perineuronal nets (PNs) using antibodies to aggrecan, lectin staining and hyaluronan-binding protein. Selected subcortical regions were studied to correlate the cortical patterns with features in evolutionary conserved systems. In the neocortex, paleocortex and hippocampus PNs were associated with nonpyramidal neurons. Quantitative analysis in the cerebral cortex revealed area-specific proportions and laminar distribution patterns of neurons ensheathed by PNs. Cortical PNs showed divergent structural phenotypes. Diffuse PNs forming a cotton wool-like perisomatic rim were characteristic of the paleocortex. These PNs were associated with a dense pericellular plexus of calretinin-immunoreactive fibres. Clearly contoured PNs were devoid of a calretinin-positive plexus and predominated in the neocortex and hippocampus. The organization of the extracellular matrix in subcortical nuclei followed the widely distributed mammalian type. We conclude that molecular properties of the aggrecan-based extracellular matrix are conserved during evolution of mammals; however, the matrix scaffold is adapted to specific wiring patterns of cortical and subcortical neuronal networks.

Section snippets

Animals

Four adult (3–8 years) lesser hedgehog tenrecs (Echinops telfairi) of both sexes, weighing 110–157 g, from the breeding colony of Dr. Künzle (Künzle et al., 2007), were investigated. All animals used in this study were treated in agreement with the German law on the use of laboratory animals and following the ethical guidelines of the laboratory animal care and use committee at the University of Munich. All efforts were made to minimize the number of animals and their suffering.

Cytochemistry

For the

Results

The aggrecan-based extracellular matrix in the Echinops brain showed a characteristic organization at the regional and cellular level especially in the cerebral cortex. The regional patterns were related to the number and structural phenotype of PNs but also to the differential expression of matrix components in neuropil zones. The patterns in subcortical regions appeared evolutionary conserved resembling in many aspects the patterns shown in rats (Seeger et al., 1994) and mice (Brückner et

Discussion

The results of the present study show that the extracellular matrix is an integral part of the unique brain architecture of the Madagascan lesser hedgehog tenrec. Different structural phenotypes of PNs are associated with neurons in region-specific patterns. This is clearly evident in the cerebral cortex. In addition, basic molecular properties of the extracellular matrix are evolutionary conserved. Aggrecan is a major chondroitin sulfate proteoglycan specifically related to the

Acknowledgement

This work was supported by the German Research Foundation GRK 1097 “INTERNEURO”, the grant Ku 624/3-3, the EU-Project “Neuropro” (Grant Agreement No. 223077) and the Interdisciplinary Center of Clinical Research (IZKF) at the Faculty of Medicine of the Universität Leipzig (project C01) in the course of the MD/PhD program at the Universität Leipzig.

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The human auditory brainstem, especially the cochlear nucleus (CN) and the superior olivary complex (SOC) are characterized by a high density of neurons associated with perineuronal nets (PNs). PNs build a specific form of extracellular matrix surrounding the neuronal somata, proximal dendrites and axon initial segments. They restrict synaptic plasticity and control high-frequency synaptic activity, a prominent characteristic of neurons of the auditory brainstem. The distribution of PNs within the auditory brainstem has been investigated in a number of mammalian species. However, much less is known regarding PNs in the human auditory brainstem. The present study aimed at the immunohistochemical identification of PNs in the cochlear nucleus (CN) and superior olivary complex (SOC) in the human brainstem. We focused on the complex nature and molecular variability of PNs in the CN and SOC by using specific antibodies against the main PN components (aggrecan, brevican, neurocan and hyaluronan and proteoglycan link protein 1). Virtually all subnuclei within the ventral CN and SOC were found to be associated with PNs. Direct comparison between gerbil and human yielded similar fine structure of PNs and confirmed the typical tight interdigitation of PNs with synaptic terminals in both species. Noticeably, an elaborate combination of immunohistochemical labelings clearly supports the still debated existence of the medial nucleus of trapezoid body (MNTB) in the human brain. In conclusion, the present study demonstrates that PNs form a prominent extracellular structure on CN and SOC neurons in the human brain, potentially stabilizing synaptic contacts, which is in agreement with many other mammalian species.
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The IC is also rich in multipolar cells classified as both excitatory and inhibitory interneurons. Studies in rat, mouse, hedgehog, tenrec, guinea pig, and rhesus monkey show PN-labeling covering subsets of IC neurons to the most part in its central nucleus (Costa et al., 2007; Foster et al., 2014; Friauf, 2000; Hilbig et al., 2007; Morawski et al., 2010b). These cells were identified as multipolar cells (Friauf, 2000), most of which were GABAergic (Table 1, Fig. 4) (Foster et al., 2014).
Perineuronal nets (PNs) are a unique and complex meshwork of specific extracellular matrix molecules that ensheath a subset of neurons in many regions of the central nervous system (CNS). PNs appear late in development and are supposed to restrict synaptic plasticity and to stabilize functional neuronal connections. PNs were further hypothesized to create a charged milieu around the neurons and thus, might directly modulate synaptic activity. Although PNs were first described more than 120 years ago, their exact functions still remain elusive. The purpose of the present review is to propose the nuclei of the auditory system, which are highly enriched in PN-wearing neurons, as particularly suitable structures to study the functional significance of PNs. We provide a detailed description of the distribution of PNs from the cochlear nucleus to the auditory cortex considering distinct markers for detection of PNs. We further point to the suitability of specific auditory neurons to serve as promising model systems to study in detail the contribution of PNs to synaptic physiology and also more generally to the functionality of the brain.
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A common finding of the experiments performed on rat, mouse and Madagascan tenrecs (Afrotheria), an ancient mammalian clade, is that CSPGs are detected in PNNs of vestibular neurons (Bertolotto et al., 1996; Hagihara et al., 1999; Deepa et al., 2006; Costa et al., 2007; Morawski et al., 2010; Deak et al., 2012; Rácz et al., 2013). In addition, HA was found in the PNNs of rat, mouse and human vestibular nuclei (Yasuhara et al., 1994; Deepa et al., 2006; Morawski et al., 2010; Deak et al., 2012; Rácz et al., 2013). Furthermore, rat vestibular neurons were found to have TN-R and HAPLN1 link protein-positive PNNs (Hagihara et al., 1999; Rácz et al., 2013).
The axons of transected and re-apposed vestibulocochlear nerve of the frog, in contrast to mammalian species, regenerate and establish functional contacts within their original termination areas of the vestibular nuclear complex and the cerebellum. The lack of regenerative capability of the mammalian central nervous system (CNS) is partially attributed to various extracellular matrix (ECM) molecules, such as chondroitin sulfate proteoglycans (CSPG) and tenascin-R (TN-R), which exert inhibition on axon regeneration. In contrast to these molecules, hyaluronan (HA) was reported to be permissive for CNS regeneration. Using histochemical and immunohistochemical methods, we investigated the distribution pattern of these molecules in the medial (MVN), lateral (LVN), superior and descending vestibular nuclei and the cerebellum of the frog and detected regional differences in the organization of the ECM. In the vestibular nuclear complex, pericellular condensation of the ECM, the perineuronal nets (PNNs) were recognizable in the LVN and MVN and were positive only for HA. The neuropil of the vestibular nuclei showed either a diffuse appearance with varying intensity of reactions, or dots and ring-like structures, which may represent the perinodal ECM of the vestibular fibers. In the cerebellum, indistinct PNNs that were only labeled for HA were present in the granular layer. Our findings suggest that the HA-rich, but CSPG and TN-R-free PNNs may be associated with the high degree of plasticity and regenerative potential of the amphibian vestibular system.
Comparison of sensory neuron growth cone and filopodial responses to structurally diverse aggrecan variants, in vitro
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Following spinal cord injury, a regenerating neurite encounters a glial scar enriched in chondroitin sulfate proteoglycans (CSPGs), which presents a major barrier. There are two points at which a neurite makes contact with glial scar CSPGs: initially, filopodia surrounding the growth cone extend and make contact with CSPGs, then the peripheral domain of the entire growth cone makes CSPG contact. Aggrecan is a CSPG commonly used to model the effect CSPGs have on elongating or regenerating neurites. In this study, we investigated filopodia and growth cone responses to contact with structurally diverse aggrecan variants using the common stripe assay. Using time-lapse imaging with 15-s intervals, we measured growth cone area, growth cone width, growth cone length, filopodia number, total filopodia length, and the length of the longest filopodia following contact with aggrecan. Responses were measured after both filopodia and growth cone contact with five different preparations of aggrecan: two forms of aggrecan derived from bovine articular cartilage (purified and prepared using different techniques), recombinant aggrecan lacking chondroitin sulfate side chains (produced in CHO-745 cells) and two additional recombinant aggrecan preparations with varying lengths of chondroitin sulfate side chains (produced in CHO-K1 and COS-7 cells). Responses in filopodia and growth cone behavior differed between the structurally diverse aggrecan variants. Mutant CHO-745 aggrecan (lacking chondroitin sulfate chains) permitted extensive growth across the PG stripe. Filopodia contact with the CHO-745 aggrecan caused a significant increase in growth cone width and filopodia length (112.7% ± 4.9 and 150.9% ± 7.2 respectively, p < 0.05), and subsequently upon growth cone contact, growth cone width remained elevated along with a reduction in filopodia number (121.9% ± 4.2; 72.39% ± 6.4, p < 0.05). COS-7 derived aggrecan inhibited neurite outgrowth following growth cone contact. Filopodia contact produced an increase in growth cone area and width (126.5% ± 8.1; 150.3% ± 13.31, p < 0.001), and while these parameters returned to baseline upon growth cone contact, a reduction in filopodia number and length was observed (73.94% ± 5.8, 75.3% ± 6.2, p < 0.05). CHO-K1 derived aggrecan inhibited neurite outgrowth following filopodia contact, and caused an increase in growth cone area and length (157.6% ± 6.2; 117.0% ± 2.8, p < 0.001). Interestingly, the two bovine articular cartilage aggrecan preparations differed in their effects on neurite outgrowth. The proprietary aggrecan (BA I, Sigma-Aldrich) inhibited neurites at the point of growth cone contact, while our chemically purified aggrecan (BA II) inhibited neurite outgrowth at the point of filopodia contact. BA I caused a reduction in growth cone width following filopodia contact (91.7% ± 2.5, p < 0.05). Upon growth cone contact, there was a further reduction in growth cone width and area (66.4% ± 2.2; 75.6% ± 2.9; p < 0.05), as well as reductions in filopodia number, total length, and max length (75.9% ± 5.7, p < 0.05; 68.8% ± 6.0; 69.6% ± 3.5, p < 0.001). Upon filopodia contact, BA II caused a significant increase in growth cone area, and reductions in filopodia number and total filopodia length (115.9% ± 5.4, p < 0.05; 72.5% ± 2.7; 77.7% ± 3.2, p < 0.001). In addition, filopodia contact with BA I caused a significant reduction in growth cone velocity (38.6 nm/s ± 1.3 before contact, 17.1 nm/s ± 3.6 after contact). These data showed that neuron morphology and behavior are differentially dependent upon aggrecan structure. Furthermore, the behavioral changes associated with the approaching growth cone may be predictive of inhibition or growth.
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NeuroanatomyResearch PaperAggrecan-based extracellular matrix shows unique cortical features and conserved subcortical principles of mammalian brain organization in the Madagascan lesser hedgehog tenrec (Echinops telfairi Martin, 1838)

Abstract

Section snippets

Animals

Cytochemistry

Results

Discussion

Acknowledgement

Neuroscience

Brain Res

Neuroscience

Brain Res

J Chem Neuroanat

Neuroscience

Neuroscience

J Chem Neuroanat

Brain Res

Neuroscience

Trends Neurosci

Brain Res

Brain Res

Brain Res

J Chem Neuroanat

Brain Res

Neuron

Curr Opin Neurobiol

Anim Reprod Sci

Neurosci Res

Brain Res

Exp Neurol

Trends Neurosci

Neuroscience

Brain Res Rev

Exp Neurol

Neurosci Res

Brain Res

Characterization of cochlear nucleus principal cells of Meriones unguiculatus and Monodelphis domestica by use of calcium-binding protein immunolabeling

J Chem Neuroanat

Perineuronal nets provide a polyanionic, glia-associated form of microenvironment around certain neurons in many parts of the rat brain

Glia

Postnatal development of perineuronal nets in wild-type mice and in a mutant deficient in tenascin-R

J Comp Neurol

Extracellular matrix organization in various regions of rat brain grey matter

J Neurocytol

Cortical perineuronal nets in the gray short-tailed opossum (Monodelphis domestica): a distribution pattern contrasting with that shown in placental mammals

Anat Embryol

Central nervous system

Upregulation of aggrecan, link protein 1, and hyaluronan synthases during formation of perineuronal nets in the rat cerebellum

J Comp Neurol

Perineuronal nets of extracellular matrix around parvalbumin-containing neurons of the hippocampus

Hippocampus

Calcium-binding protein parvalbumin as a neuronal marker

Nature

Disruption of the retinoid signalling pathway causes a deposition of amyloid beta in the adult rat brain

Eur J Neurosci

Microstructure of the neocortex: comparative aspects

J Neurocytol

Neuroanatomy
Research Paper
Aggrecan-based extracellular matrix shows unique cortical features and conserved subcortical principles of mammalian brain organization in the Madagascan lesser hedgehog tenrec (Echinops telfairi Martin, 1838)