Perineuronal sulfated proteoglycans and dark neurons in the brain and spinal cord: A histochemical and electron microscopic study of newborn and adult mice

Neurons of intracerebellar nuclei in the mouse brain were demonstrated to possess a marked surface coat, formed 3–4 weeks after birth, which was stainable with cationic iron colloid or aldehyde fuchsin. Neurons with a similar surface coat were noted as relay or local interneurons in rather restricted areas such as the occipital cortex, retrosplenial cortex, zona incerta, hippocampal subiculum and spinal posterior horn. Dark neurons with condensed cytoplasm were also shown to be covered with the surface coat. The surface coat was stained doubly with cationic iron colloid and aldehyde fuchsin. Digestion with hyaluronidase eliminated the stainability of the surface coat to both agents. Combined digestion with chon-droitinase ABC, heparitinase and keratanase eliminated the cationic iron colloid staining of the surface coat, but did not interfere with the aldehyde fuchsin staining of the surface coat. Electron microscopy of ultrathin sections revealed that the iron particles indicating sulfated proteoglycans were preferentially deposited in the perineuronal tissue spaces. Many neurons in the hippocampal subiculum possessed cell surface glycoproteins which were labeled with lectin Vicia villosa or soybean agglutinin and formed 1–2 weeks after birth. Double staining revealed that these lectin-labeled neurons were identical in part with the neurons reactive to the cationic iron colloid. Dark neurons began to appear 3–4 weeks after birth. The formation of perineuronal sulfated proteoglycans and the appearance of dark neurons, both occurring during the weaning period, may reflect the morphological and physiological completion of the brain. Dark neurons are suggested to be exhausted cells that are restored to light or normal neurons after sleep.