Effects of prenatal hypoxia-ischemia on male rat periaqueductal gray matter: Hyperalgesia, astrogliosis and nitrergic system impairment.

Prenatal hypoxic-ischemic insult (HI) may lead to a variety of neurological consequences that may persist throughout adulthood. In the most severe cases, HI is known to increase pain sensitivity which profoundly impacts quality of life. Periaqueductal gray matter (PAG) is a relevant region of the descending pain pathway and its function may be modulated by a complex network that includes nitrergic neurons and glial response, among other factors. Astrocytes, central players in pain modulation, are known to respond to HI by inducing hyperplasia, hypertrophy and increasing the number of their processes and the staining of glial fibrillary acidic protein (GFAP). In this work we investigated the effects of prenatal HI on touch and pain sensitivity, besides the distribution of the neuronal isoform of Nitric Oxide Synthase (nNOS) and GFAP in the PAG of young and adult male rats. At 18 days of gestation, rats had their uterine arteries clamped for 45 min (HI group). SHAM-operated animals were also generated (SHAM group). At post-natal day 30 (P30) or 90 (P90), the offspring was submitted to the behavioral tests of Von Frey and formalin or histological processing to perform immunohistochemistry for nNOS and GFAP. Although there was no significant difference between the groups concerning touch sensitivity, we observed an increase in pain sensitivity in HI P30 and HI P90. The number of nNOS + cells was reduced in HI adult animals in dlPAG and vlPAG. GFAP immunostaining was increased in HI P90 in dlPAG and dmPAG. Our results demonstrated for the first time an increase in pain sensitivity as a consequence of prenatal HI in an animal model. It reinforces the relevance of this model to mimic the effects of prenatal HI, as hyperalgesia.

Serological Evidence of Rickettsia Exposure among Patients with Unknown Fever Origin in Angola, 2016-2017.

Spotted fever group Rickettsia (SFGR) is one among the aetiologies that cause fever of unknown origin in Angola. Despite their occurrence, there is little information about its magnitude in this country either because it is misdiagnosed or due to the lack of diagnostic resources. For this purpose, eighty-seven selected malaria- and yellow fever-negative serum specimens collected between February 2016 and March 2017 as part of the National Laboratory of Febrile Syndromes, from patients with fever (≥37.5°C) for at least 4 days and of unknown origin, were screened for Rickettsia antibodies through an immunofluorescence assay (IFA). Serological results were interpreted according to the 2017 guidelines for the detection of Rickettsia spp. Three seroreactive patients had detectable IgM antibodies to Rickettsia with an endpoint titre of 32 and IgG antibodies with endpoint titres of 128 and 256. These findings supported a diagnosis of Rickettsia exposure amongst these patients and highlight that rickettsioses may be among the cause of unknown febrile syndromes in Angola. Therefore, physicians must be aware of this reality and must include this vector-borne disease as part of aetiologies that should be considered and systematically tested in order to delineate appropriate strategies of diagnostic and control of Rickettsia in Angola.

Crenosoma Striatum in Lungs of European Hedgehogs (Erinaceus Europeus) from Portugal.

Crenosoma striatum is a host-specifi c metastrongiloid nematode causing respiratory tract disease in hedgehogs (Erinaceus europaeus). Since few studies have reported C. striatum in hedgehogs and little genetic data is available concerning this lungworm, this study aimed to determine the occurrence of C. striatum in a population sample of hedgehogs from Portugal, additionally providing morphological, histological and molecular data. From 2017 to 2018 a survey of infection was carried out in 11 necropsied hedgehogs. Worms were extracted from fresh lung tissues and microscopically evaluated. Molecular characterization of partial mitochondrial (12S rRNA) and nuclear (18S rRNA) genes was performed. The presence of lungworms in pulmonary tissues of five hedgehogs (45.5%) was detected. Morphological and histopathological analyses evidenced adult forms of nematodes consistent with C. striatum. Molecular characterization of 18S rRNA genes confirmed the classifi cation as C. striatum. Also, novel genetic data characterizing the mitochondrial (12S rRNA) gene of C. striatum is presented. This is the first report of C. striatum infection in hedgehogs of Portugal. The findings here reported provide new insights regarding the geographic distribution and the molecular identification of this lungworm species.

Neonatal Nicotine Exposure Leads to Hypothalamic Gliosis in Adult Overweight Rats.

Astrocytes and microglia, the immune competent cells of central nercous system, can be activated in response to metabolic signals such as obesity and hyperleptinaemia. In rats, maternal exposure to nicotine during lactation leads to central obesity, hyperleptinaemia, leptin resistance and alterations in hypothalamic neuropeptides in the offspring during adulthood. In the present study, we studied the activation of astrocytes and microglia, as well as the pattern of inflammatory mediators, in adult offspring of this experimental model. On postnatal day 2 (P2), osmotic minipumps releasing nicotine (NIC) (-6 mg/kg/day) or saline for 14 days were s.c. implanted in dams. Male offspring were killed on P180 and hypothalamic immunohistochemistry, retroperitoneal white adipose tissue (WAT) polymerase chain reaction analysis and multiplex analysis for plasma inflammatory mediators were carried out. At P180, NIC astrocyte cell number was higher in the arcuate nucleus (ARC) (medial: +82%; lateral: +110%), in the paraventricular nucleus (PVN) (+144%) and in the lateral hypothalamus (+121%). NIC glial fibrillary acidic protein fibre density was higher in the lateral ARC (+178%) and in the PVN (+183%). Interleukin-6 was not affected in the hypothalamus. NIC monocyte chemotactic protein 1 was only higher in the periventricular nucleus (+287%). NIC microglia (iba-1-positive) cell number was higher (+68%) only in the PVN, as was the chemokine (C-X3-C motif) receptor 1 density (+93%). NIC interleukin-10 was lower in the WAT (-58%) and plasma (-50%). Thus, offspring of mothers exposed to nicotine during lactation present hypothalamic astrogliosis at adulthood and microgliosis in the PVN.

Undernutrition during early life alters neuropeptide Y distribution along the arcuate/paraventricular pathway.

Perinatal nutrient restriction exerts profound influences on brain development. Animals that suffer undernutrition during lactation also display impaired weight gain. Feeding behavior is mainly modulated by neural and hormonal inputs to the hypothalamus. The arcuate-paraventricular neuropeptidergic Y pathway has a prominent role in appetite regulation. The aim of this work was to study the effects of protein undernutrition during lactation on this hypothalamic pathway. We used rats from 5 to 60 postnatal (P) days whose dams were fed a 0% protein diet (PFG) or a normoprotein diet (CG) from P1 to P10. To reproduce the same amount of calorie ingested by the PFG we used an underfed group (UFG). Immunohistochemistry was performed to assess neuropeptide Y (NPY) distribution in the arcuate, periventricular and paraventricular nuclei. Our results showed a NPY immunostaining peak at P10 in all nuclei in CG animals. In UFG animals this peak was observed by P15, while, in the PFG animals only by P20. Our results suggest that the neuropeptidergic arcuate-paraventricular pathway suffered a delay in NPY distribution in undernourished animals, particularly those fed a 0% protein diet, reflecting an effect on this pathway maturation that could explain previously reported alterations on feeding behavior in these animals.

Glycogen stores are impaired in hypothalamic nuclei of rats malnourished during early life.

Perinatal nutrition has persistent influences on neural development and cognition. In humans and other animals, protein malnutrition during the perinatal period causes permanent changes, inducing to adulthood metabolic syndrome. Feeding is mainly modulated by neural and hormonal inputs to the hypothalamus. Hypothalamic glycogen stores are a source of glucose in high energetic demands, as during development of neural circuits. As some hypothalamic circuits are formed during lactation, we studied the effects of malnutrition, during the first 10 days of lactation, on glycogen stores in hypothalamic nuclei involved in the control of energy metabolism. Female pregnant rats were fed ad libitum with a normal protein diet (22% protein). After delivery, each dam was kept with 6 male pups. During the first 10 days of lactation, dams from the experimental group received a protein-free diet and the control group a normoprotein diet. By post-natal day 10 (P10), glycogen stores were very high in the arcuate nucleus and median eminence of control group. Glycogen stores decreased during development. In P20 control animals, glycogen stores were lower when compared to P10 control animals. Animals submitted to malnutrition presented a staining even lower than control ones. After P45, it was difficult to determine differences between control and diet groups because glycogen stores were reduced. We also showed that tanycytes were the cells presenting glycogen stores. Our data reinforce the concept that maternal nutritional state during lactation may be critical for neurodevelopment since it resulted in a low hypothalamic glycogen store, which may be critical for establishment of neuronal circuitry.

A role for the MAPK/ERK pathway in oligodendroglial differentiation in vitro: stage specific effects on cell branching.

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway is important for both long-term survival and timing of the progression of oligodendrocyte differentiation. Oligodendroglial cells treated with MEK inhibitor were distinguished by using stage specific markers: NG2 proteoglycan, A2B5, 2'3'nucleotide-cyclic 3'phosphodiesterase (CNPase) and myelin basic protein (MBP), and classified according to their morphology into different developmental stages. Treatment significantly increased the number of cells with more immature morphologies and decreased the number of mature cells. Furthermore, it increased the number of rounded cells that could not be classified into any of the oligodendroglial developmental stages. The strongest effects were usually observed shortly after treatment. Rounded cells were CNPase/MBP positive and they were not stained by anti-NG2 or A2B5, indicating that they were mature cells unable either to extend and/or to maintain their processes. These data showed an effect of the MAPK/ERK pathway on oligodendroglial branching, with possible consequences for the formation of the myelin sheath.

Ultrastructural identification of oligodendrocyte/myelin proteins in corpus callosum of hypothyroid animals.

Thyroid hormone (T3) deficiency impairs the development of the CNS, particularly myelination. We have previously described an increase in the frequency of morphological abnormalities in the central myelin sheath in a hypothyroidism model, which reinforced the hypothesis of a role for T3 in myelin compaction. However, there are no data concerning the cellular distribution of myelin proteins in hypothyroid animals. In the present work, we describe the distribution of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), myelin basic protein (MBP) and proteolipid protein (PLP) throughout the central myelin sheath of a hypothyroidism model. We used euthyroid and hypothyroid adult rats at 90 days of age. In order to induce hypothyroid status, animals received 0.02% methimazol from the 19th gestation day onwards. After perfusion with a fixative mixture, small pieces of corpus callosum were obtained, dehydrated and embedded in LR White resin. Ultrathin sections were immunoreacted, using specific antibodies revealed by a secondary antibody coupled to colloidal gold particles of 10nm. Gold particle density per region of myelin sheath for each one of these proteins was obtained. In normal animals, CNPase, PLP and MBP were identified in sites that had already been described in previous studies. In hypothyroid animals, CNPase was identified in the region corresponding to compact lamellae, which normally does not contain this protein, while, in this same region, PLP and MBP immunolabeling were decreased. These results suggest that thyroid hormone deficiency impairs the distribution of the major oligodendrocyte/myelin markers. This effect may justify the reduction in myelin sheath compaction previously demonstrated in a similar model of hypothyroidism.

Thyroid hormone deficiency changes the distribution of oligodendrocyte/myelin markers during oligodendroglial differentiation in vitro.

Myelination depends on the proper differentiation of oligodendrocytes and several factors may influence this event. For instance, thyroid hormone (T3) affects the timing of differentiation and regulates the expression of several enzymes involved in the synthesis of complex lipids and in the expression of some myelin structural proteins. We investigated the effect of T3 deficiency on oligodendroglial differentiation and in the distribution of oligodendrocyte/myelin proteins 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and myelin basic protein (MBP). Oligodendroglial-enriched cultures were obtained from cerebra of neonate rats grown in a modified medium. The T3-deficient status was induced by using medium devoid of T3. We observed a delay, in T3-deficient cultures, in oligodendroglial maturation characterized by less extensive processes and membrane vellum than in controls. In control cultures, CNPase immunoreactivity was punctated, showing cell bodies and processes at earlier stages and redistribution to cytoskeleton vein-like structures in later stages. In T3-deficient cultures, CNPase remained in a punctated pattern and only at 10 days in vitro we observed CNPase redistribution to the presumptive cytoskeleton vein-like structures. MBP in control cultures was distributed through the whole cell body and processes whereas in T3-deficient cultures, MBP immunoreactivity was concentrated in the perinuclear region. These results reinforce the hypothesis that T3 is an important factor in oligodendrocyte differentiation, particularly regarding the distribution of myelin proteins.

Myelin basic protein accumulation is impaired in a model of protein deficiency during development.

During the development of the central nervous system (CNS) there is a great possibility of permanent effects in consequence of environmental disturbances. Nutritional deficiency is one of the factors that impair the normal CNS formation. In general, the protein deficiency evokes, beyond the damages in the maturation of nervous system, several consequences in body growth, biochemical maturation, motor function and the major cognitive functions. These effects were observed in undernourished children all over the world. Even in a restricted period, the malnutrition status may evoke permanent impairments in feeding behavior and in metabolism. Rats submitted to malnutrition during development, showed a marked decrease in the number of myelinated fibers. This condition may reflect a failure in the beginning of the wrapping of axons by oligodendroglial processes and/or a delay in the myelin synthesis. Myelin basic protein (MBP) is an intracellular oligodendrocyte protein that is directly related to the formation of the myelin sheath. In this study we verified the temporal pattern of MBP expression, by immunohistochemical and immunoblotting analyses, in a model of protein malnutrition induced during the first half of the lactation period. We showed that MBP expression was impaired in our malnutrition model and that some of the effects were maintained in adulthood, with possible consequences in the maturation of myelin sheath.

Hypothalamic nuclei nitric oxide synthase expression in rats malnourished during early lactation period.

In humans and other animals, it has been shown that protein malnutrition during the prenatal period leads to permanent changes, which in adulthood may cause chronic diseases. Molecules involved in the control of energy metabolism could be targets to alterations caused by nutritional status. Some hypothalamic nuclei as the paraventricular (PVN), ventro-medial and arcuate are related to energy metabolism regulation. Orexigenic and anorexigenic molecules are involved in this regulation. Some studies have showed that these nuclei present nitric oxide synthase (NOS) and that it is increased in obese rats. Recently it had been shown that rats malnourished during the lactation period presented metabolic alterations that persist in adulthood. The aim of this work was to study the expression of NOS in hypothalamic nuclei of rats submitted to malnutrition during the early lactation period. Rats from post-natal day (P10) to P90 were used. Control dams were fed with regular chow pellets and diet dams were fed with protein-free chow pellets during the first 10 days of lactation. NADPH-diaphorase or immunostaining techniques were used to access NOS expression in hypothalamic nuclei. Our results show a delay in NOS expression in the PVN and VMH of malnourished rats. It may affect the development of the hypothalamic circuitry, leading to a metabolic imprinting.

Selective effect of hypothyroidism on expression of myelin markers during development.

Thyroid hormones are critical for maturation of the central nervous system. In a previous study, we showed a change in the pattern of mature myelinated nerve fibers by 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in developing hypothyroid animals, which suggests a possible role for thyroid hormones in myelin compaction. The classical myelin markers myelin basic protein (MBP) and proteolipidic protein (PLP) are expressed later in oligodendroglial development, when myelin sheath formation is in progress. A myelin constituent designated myelin-associated/oligodendrocytic basic protein (MOBP) has been identified and related to myelin compaction. We assessed the developmental sequence of appearance of CNPase, MBP, MOPB, and PLP proteins in cerebellum (Cb) and corpus callosum (cc) in an experimental hypothyroidism model. The appearance of both MOBP isoforms occurred at postnatal day (P)25 and P30 in cc and Cb, respectively, followed by an increase with age in the control group. However, all the MOBP isoforms were weakly detectable in both regions at P30 from the hypothyroid (H) group, and the higher molecular weight isoform remains decreased in cc, even at P90. The developmental pattern of expression of CNPase, MBP, and PLP proteins was also delayed in the H group. CNPase and MBP expression was recovered in cc and Cb, whereas PLP remained below control levels at P90 in cc. Our data show that the experimental hypothyroidism affects the developmental pattern of the oligodendrocytic/myelin markers. Furthermore, thyroid hormone may modulate specific genes, as demonstrated by permanent down-regulation of MOBP and PLP expression in adulthood.

2'3'Cyclic nucleotide 3'phosphodiesterase immunohistochemistry shows an impairment on myelin compaction in hypothyroid rats.

The effects of hypothyroidism on oligodendroglial differentiation and myelination are for the first time studied by immunohistochemical localization of an early oligodendroglial marker, the 2'3'cyclic nucleotide 3'phosphodiesterase (E.C. 3.1.4.37-CNPase), in developing rats. Two groups received methimazol; one during gestation (H) and another postnatally (PN). One H sub-group received thyroxine after birth (T). We observed a delay in CNPase expression followed by a decrease in the number of CNPase immunoreactive fibers in both H and PN groups. The T sub-group was not different from controls. Furthermore, the immunoreactive fibers, in mature hypothyroid animals, showed a continuous pattern of staining in contrast with a discontinuous one in controls. Myelinogenesis is a highly regulated timed event. CNPase links myelin related proteins to the cytoskeleton also interacting with membrane lipids during extension and wrapping of the oligodendroglial process around the axon (ensheathment phase). In mature myelinated fiber the CNPase is absent from compact myelin sheath, being located only in the inner and outer loops and in paranodal loops. Thus, our data suggest a disorder in myelin compaction and point once more to the post-natal period as critical for the mechanisms that are thyroid hormone regulated in myelinogenesis.

Proliferation of differentiated glial cells in the brain stem.

Classical studies of macroglial proliferation in muride rodents have provided conflicting evidence concerning the proliferating capabilities of oligodendrocytes and microglia. Furthermore, little information has been obtained in other mammalian orders and very little is known about glial cell proliferation and differentiation in the subclass Metatheria although valuable knowledge may be obtained from the protracted period of central nervous system maturation in these forms. Thus, we have studied the proliferative capacity of phenotypically identified brain stem oligodendrocytes by tritiated thymidine radioautography and have compared it with known features of oligodendroglial differentiation as well as with proliferation of microglia in the opossum Didelphis marsupialis. We have detected a previously undescribed ephemeral, regionally heterogeneous proliferation of oligodendrocytes expressing the actin-binding, ensheathment-related protein 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), that is not necessarily related to the known regional and temporal heterogeneity of expression of CNPase in cell bodies. On the other hand, proliferation of microglia tagged by the binding of Griffonia simplicifolia B4 isolectin, which recognizes an alpha-D-galactosyl-bearing glycoprotein of the plasma membrane of macrophages/microglia, is known to be long lasting, showing no regional heterogeneity and being found amongst both ameboid and differentiated ramified cells, although at different rates. The functional significance of the proliferative behavior of these differentiated cells is unknown but may provide a low-grade cell renewal in the normal brain and may be augmented under pathological conditions.

Heterogeneous patterns of oligodendroglial differentiation in the forebrain of the opossum Didelphis marsupialis.

The differentiation of oligodendrocytes in the forebrain of the opossum (Didelphis marsupialis) has been studied by the immunohistochemical identification of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and by the autoradiographic detection of the uptake of 3H-thymidine. CNPase is expressed early in oligodendroglia somata and fibre sheaths (myelin) in the forebrain and its persistence in the cell bodies is regionally heterogeneous, being ephemeral in cells within the optic pathway, supraoptic decussation, and posterior commissure, of intermediate duration in the mamillo-thalamic fascicle, and stria medullaris, and long-lasting in other diencephalic and in telencephalic tracts. In the cerebral cortex, most CNPase+ cells have small somata and multiple processes (types I and II). CNPase-expressing oligodendrocytes are also regionally heterogeneous in terms of proliferative capability, which could not be detected in forebrain tracts or diencephalon, but has appeared in a small proportion of cells in the neocortical white matter and in the fimbria. Our findings provide additional evidence in favour of the heterogeneity of oligodendrocytes.

Proliferation of differentiated glial cells in the brain stem

Classical studies of macroglial proliferation in muride rodents have provided conflicting evidence concerning the proliferating capabilities of oligodendrocytes and microglia. Furthermore, little information has been obtained in other mammalian orders and very little is known about glial cell proliferation and differentiation in the subclass Metatheria although valuable knowledge may be obtained from the protracted period of central nervous system maturation in these forms. Thus, we have studied the proliferative capacity of phenotypically identified brain stem oligodendrocytes by tritiated thymidine radioautography and have compared it with known features of oligodentroglial differentation as well as with proliferation of microglia in the opossum Didelphis marsupialis. We have detected a previously undescribed ephemeral, regionally heterogenous proliferation of oligodendrocytes expressing the actin-binding, ensheathment-related protein 2' 3'- cyclic nucleotide 3' -phosphodiesterase (CNPase), that is not necessarily related to the known regional and temporal heterogeneity of expression of CNPase in cell bodies. On the other hand, proliferation of microglia tagged by the binding of Griffonia simplicifolia B4 isolectin, which recognizes an alpha-D-galactosyl-bearing glycoprotein of the plasma membrane of macrophages/microglia, is known to be long lasting, showing no regional heterogeneity and being found amongst both ameboid and differentiated ramified cells, although at different rates. The functional significance of the proliferative behavior of these differentiated cells is unknown but may provide a lowgrade cell renewal in the normal brain and may be augmented under pathological conditions.

Heterogeneity of median and lateral midbrain radial glia and astrocytes.

In the developing mammalian midbrain, radial glial cells are divided into median formations and lateral radial systems with differential properties including rate and timing of cell proliferation, expression of cytoskeletal and calcium-binding proteins, storage of glycogen and relations to afferent fiber systems. To test the hypothesis that radial glial cells of median and lateral midbrain sectors and/or their derivatives are heterogeneous in their relations with local neurons, an in vitro system has been developed and has also been characterized in terms of extracellular matrix (ECM) components. Confluent astrocyte cultures, derived from median (M) or lateral (L) embryonic mouse midbrain sectors, were used as substrates for culturing dissociated cells from median (m) or lateral (l) sectors of embryonic midbrains. In spite of the morphological invariance of glial substrates at confluency, cells that were plated onto these substrates and that were immunoreactive for neuronal markers (MAP2, polysialylated N-CAM or beta III tubulin) showed differences in the aggregation of somata and in the length, caliber and branching of neurites. These differences, which depend mostly on the sector of origin of astrocytes (L: permissive, M: non-permissive for neuronal growth), suggest that the substrates may differ in adhesiveness and/or their carrying of growth-promoting vs. growth-interfering molecules. Indeed, L and M cultures differ in laminin deposition patterns (L: fibrillar, M: punctate pattern). Furthermore, sulfated glycosaminoglycans (s-GAGs) isolated from the pericellular (P), intracellular (I) and extracellular (E) compartments of these sectoral cultures also showed correlations with the ability to support neurite growth. The total amount of s-GAGs in M cultures was twice that in L cultures and was particularly high in the P compartment, with about 3 times as much heparan sulfate (HS) and about 15 times as much chondroitin sulfate (CS) in this fraction of M than in the corresponding compartment of L glia. Our results indicate that cultured astrocytes have heterogeneous properties including different organization of their extracellular matrix that reflect the roles played by their parent radial glia in regions favorable to axonal growth or barrier regions of the developing brain.

The regional distribution of neuronal glycogen in the opossum brain, with special reference to hypothalamic systems.

Neurons that accumulate glycogen have been identified in the opossum brain stem and diencephalon by a modified histochemical method using alcoholic solutions and fuchsin proper (pararosanilin) rather than the Schiff reagent (leucosulphite derivative). Several of the glycogen-positive cell groups such as the mesencephalic trigeminal nucleus and the brainstem somatic and special visceral efferent nuclei have been previously detected in the developing brain of small, common laboratory mammals. Scattered glycogen-containing neurons also appear in the dorsal thalamus and basal forebrain. A conspicuous, often Golgi-like accumulation of glycogen has been found in neurons of the magnocellular and parvocellular hypothalamic systems. Together with available data on the metabolic rate of marsupials, our results suggest that the patterns of glycogen deposition may be common to several vertebrates and may be a constant although not exclusive property of cells with axonal endings outside the blood-brain barrier.

CNPase expression in the developing opossum brain stem and cerebellum.

Immunoreactivity to 2',3' cyclic nucleotide 3' phosphohydrolase (CNPase; EC 3.1.4.37) was studied in the developing opossum brain stem and cerebellum. Regional differences were found in oligodendrocytes concerning the time of appearance (early: medical longitudinal fascicle [mlf]; intermediate: inferior colliculus [IC], deep layers of the superior colliculus [SC] and white matter of cerebellar folia; late: optic layer of SC) and duration of immunoreactivity (short: optic layer of SC; intermediate: mlf; long: cerebellar folia, etc). The results suggest that regional heterogeneities in CNPase expression are linked to intrinsic properties of local and afferent axons.