2nd Postnatal Week Research Articles

Preproenkephalin gene expression and [Met 5]–enkephalin levels in the developing rat heart

[Met 5]–enkephalin, encoded by the preproenkephalin (PPE) gene, serves as a growth factor (opioid growth factor, OGF) during cardiac development in addition to its role as a neuroregulator. This study examined the ontogeny and relationship of gene and peptide expression in the mammalian heart during late embryonic, preweaning, and postweaning periods. Values for PPE mRNA of hearts in rats from embryonic day 16 (E16) to postnatal day 1 were 33 to 50% of levels found in adults. Adult values for the mature heart were comparable to those in the caudate, an area of the rat brain rich in PPE mRNA. Message gradually decreased during the first postnatal week to 10% of adult values and remained so until weaning. PPE mRNA on days 35 and 50 were three- and sevenfold, respectively, higher than at 21 days, and in adults was more than 50% greater than at day 50. Message for PPE in neonatal heart was regulated rapidly and in a sustained fashion by excess opioid agonist (OGF) or blockade of opioid-receptor interaction. [Met 5]–enkephalin levels increased sevenfold between E18 and E20, and another 1.6-fold until birth. Having reached a zenith in the neonate, values for enkephalin-like peptide decreased gradually through the 2nd postnatal week, and were extremely low in adulthood. Indeed, a 43-fold difference in peptide levels was detected between neonatal and adult rat heart. These data provide evidence for the expression of a tightly regulated and distinct growth factor (OGF) during the crucial periods of cell proliferation and differentiation in the mammalian heart, and reveal that the source of OGF is autocrine and/or paracrine.

Localization of the neuronal antigen recognized by anti-Tr antibodies from patients with paraneoplastic cerebellar degeneration and Hodgkin's disease in the rat nervous system.

Patients with paraneoplastic cerebellar degeneration and Hodgkin's disease develop autoantibodies (Tr-Ab) that immunoreact with the cytoplasm of the Purkinje cells and produce a characteristic punctate pattern in the molecular layer of the cerebellum. In the present study, we analyzed the structures of the adult rat cerebellar cortex identified by Tr-Ab and the expression of the antigen recognized by Tr-Ab in the developing rat brain. By laser confocal microscopy and immunoelectron microscopy, Tr-Ab immunoreactivity was found localized in the cytosol and outer surface of the endoplasmic reticulum of the perikarya of neurons of the molecular layer and the cell body and dendrites of Purkinje cells without a particular concentration in dendritic spines. Tr-Ab reactivity was more widespread in the developing rat brain. Tr-Ab labeling of Purkinje cells was already observed at P0 (day of birth). The staining of the molecular layer followed the development of the dendritic tree. The internal and inner level of the external granule cell layer were labeled with Tr-Ab with a dotted pattern that became almost negative by the 2nd postnatal week. The staining probably corresponded to granule cells as suggested by the positive Tr-Ab labeling of cultures of embryonic granule neurons. The present findings suggest that the antigen recognized by Tr-Ab appears early and is widely expressed in the developing rat brain. In the adult cerebellum, the antigen is localized in the cell body and dendrites of the Purkinje cells but is not concentrated in the dendritic spines.

Cyclooxygenase-2 in rat nephron development.

The inducible second isoform of cyclooxygenase (COX-2) that mediates inflammation also is expressed at low levels in normal adult rat kidneys and is upregulated in response to noninflammatory stimuli (R. C. Harris, J. A. McKanna, Y. Akai, H. R. Jacobson, R. N. DuBois, and M. D. Breyer, J. Clin. Invest. 94: 2504-2510, 1994). Roles in morphogenesis are indicated by reported teratogenicity of COX inhibitors and renal dysgenesis in COX-2 knockout mice (J. E. Dinchuk, B. D. Car, R. J. Focht, J. J. Johnston, B. D. Jaffee, M. B. Covington, N. R. Contel, V. M. Eng, R. J. Collins, P. M. Czerniak, A. G. Stewart, and J. M. Trzaskos, Nature 378: 406-409, 1995; S. G. Morham, R. Lagenbach, C. D. Loftin, H. F. Tiano, N. Vouloumanos, J. C. Jennette, J. F. Mahler, K. D. Kluckman, A. Ledford, C. A. Lee, and O. Smithies. Cell 83: 473-482, 1995). Blots from developing rat kidneys demonstrated that COX-2 mRNA and immunoreactive protein were present in neonates, peaked in the 2nd and 3rd postnatal weeks and declined to adult levels by the 3rd month. Immunolocalization and in situ hybridization detected intense COX-2 immunoreactivity and mRNA in a subset of thick ascending limb epithelial cells near the macula densa in each developing nephron; after 2 wk the COX-2 gradually waned. These data demonstrate that COX-2 expression is subject to normal developmental regulation and can be sustained over extended periods; they also support the conclusion that metabolites of COX-2 play important roles in the differentiation and early functions of mammalian nephrons.

Developmental fates and migratory pathways of dividing progenitors in the postnatal rat cerebellum.

To investigate the developmental fates and the migratory pathways of dividing progenitors in both the white matter (WM) and the external granule layer (EGL) in the early postnatal rat cerebellum, a replication-deficient retrovirus carrying the beta-galactosidase gene (BAG) was injected into the deep cerebellar tissue or the EGL of postnatal rats to label dividing progenitors. After 1-3 days post-injection (1-3 dpi) of BAG into the deep cerebellar tissue of postnatal day 4/5 (P4/5) rats, labeled immature, unipolar cells were found mainly in the WM. From 4 to 6 dpi, similar cells appeared in the internal granule (IGL), Purkinje cell, and molecular layers, although about half of the labeled cells still resided in the WM and appeared immature. The first morphologically definable Bergmann glia, astrocytes, and oligodendrocytes were also observed. From 14 to 20 dpi, most labeled cells had developed into Bergmann glia, astrocytes, oligodendrocytes, and interneurons in their appropriate layers. When BAG injections were performed at P14, unipolar cells were initially observed, but the majority of these differentiated into myelinating oligodendrocytes in the WM and IGL by 17 dpi. Few immature cells were labeled by injections administered at P20, and these did not develop into mature glia, but into cells with lacy, fine processes, possible representing immature oligodendrocytes. In contrast, BAG-labeled progenitors of EGL produced only granule neurons. Thus, within the first 2 postnatal weeks, dividing progenitors in the WM migrate as immature cells into the cortex before differentiating into a variety of glia and interneurons. The genesis of oligodendrocytes continues through the 2nd postnatal week and largely ceases by P20. EGL cells do not produce glia, but only granule cells.

Neuropathic pain in neonatal rats

The aim of this study was to determine if peripheral nerve injury in neonatal rats triggers neuropathic pain behaviors as it does in adults. The injury was produced in three groups of neonatal rats (1, 2, and 3 weeks old) by tightly ligating the left L5 and L6 spinal nerves. Behavioral tests for mechanical allodynia were conducted from the 15th day after birth for the 1- and 2-week groups, and 1 day after surgery for the 3-week group. Rats in the 3-week group developed behaviors representing mechanical allodynia as is seen in adults. For the younger animals, however, the signs of mechanical allodynia lasted for a shorter period of time. These data suggest that a factor which is critically important for the maintenance of neuropathic pain develops between the 2nd and 3rd postnatal weeks.

PHYSIOLOGICAL CONSEQUENCES OF OROGASTRIC TUBE FEEDING ON CEREBRAL HAEMODYNAMICS IN HEALTHY PRETERM INFANTS. † 1271

In a prospective study we investigated the influence of orogastric tube feeding on cerebral haemodynamics in 11 preterm infants (BW 1278±168 g and GA 30.5±2 wks). Data were collected at weekly intervals during the first 3 postnatal weeks. Heart rate, oxygen saturation and respiration were continuously recorded during the one hour procedure. Cerebral blood flow velocities, measured using pulsed-wave duplex-doppler, were assessed from the anterior cerebral artery 10 min before, during, and 10 and 20 min after tube feeding. For each step the doppler-data were recorded over a 2 min period. Milk was delivered via mechanical pump over a 5 min period in the first postnatal week, and a 10 min period in the 2nd and 3rd postnatal week, while the infants were placed in the prone position. The whole procedure was recorded on video for later analysis of the infants' behavioral state. To avoid the influence of tube insertion on measured parameters, milk delivery was started 2 min after the tube was inserted. Data were analyzed for all recordings (n=33) and separately for each postnatal week (n=11).

Development of the hypothalamic vasopressin system and nephrons in Meriones shawi during ontogenesis.

This study has evaluated the development of the hypothalamic vasopressin system and nephrons of the kidney in desert rodents, Meriones shawi, which effectively retain water by excretion of highly concentrated urine. The vasopressin system was studied immunocytochemically at the 18th fetal day, at the 2nd, 13th, 27th postnatal days and in adulthood. The kidneys were investigated at the 2nd, 13th postnatal days and in adulthood using microdissection technique. Occasional vasopressin-immunoreactive neurons were observed as early as the 18th fetal day, only in the paraventricular nucleus. From the 2nd postnatal day onwards, vasopressin neurons increased progressively in number, being mainly concentrated in the supraoptic and paraventricular nuclei, as well as in the ventral retrochiasmatic region. Transient neuronal populations were also observed at the 13th postnatal day in the lateral preoptic area and anterior hypothalamic nucleus. Apart from the neurons, the glandular cells of the tuberal lobe showed immunostaining from the 18th fetal day, the first age studied, until the 13th postnatal day. The fibers of differentiating vasopressin neurons grew towards the circumventricular/neurohemal organs, terminating in the organum vasculosum of the lamina terminalis and the lateral ventricles as early as the 18th fetal day, as well as the third ventricle, the posterior lobe and the external zone of the median eminence between the 2nd and 13th postnatal days. The kidney in 2-day-old Meriones comprised nephrons at different stages of development from an S-shaped body to well-differentiated nephrons. At the 13th postnatal day, as in adulthood, the nephrons were well differentiated and characterized by long, thin loops descending to different levels of papilla. Thus, according to our morphological data the hypothalamic vasopressin neurons and nephrons in the kidney of Meriones reach the definitive state by the end of the 2nd postnatal week.

NADPH-diaphorase-positive ganglion cells of the rat adrenal gland: age- and sex-related changes in their number, size, and distribution.

The rat adrenal gland contains ganglion cells able to synthesize nitric oxide (NO). This messenger molecule controls and modulates adrenal secretory activity and blood flow. The present study analyzed the number, size, and distribution of NO-producing adrenal neurons in adulthood and during postnatal development by means of beta-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. This method reliably visualizes the enzyme responsible for NO generation. The reactive neurons per adrenal gland were 350-400 in both male and female adult rats. The positive nerve cell bodies were mostly located in the medulla, few being detected within the cortex and the subcapsular region. Dual labeling with anti-microtubule-associated protein 2 antibody, specific for neuronal elements, confirmed this distribution. Anti-microtubule-associated protein 1b antibody identified a subset of NADPH-d-positive neurons, displaying different degrees of maturation according to their position within the adrenal gland. At birth, there were about 220 NADPH-d-labeled neurons per adrenal gland in both sexes. As confirmed by dual immunocytochemical labeling, their great majority was evenly distributed between the cortex and the subcapsular region, the medulla being practically devoid of stained neurons. After birth, the number of adrenal NADPH-d-positive ganglion cells displayed a strong postnatal increase and reached the adult-like distribution after 1-2 months. During the period of increase, there was a transient difference in the numbers of these cells in the two sexes. Thus we present here evidence of plasticity in the number, size, and distribution of NADPH-d-positive adrenal neurons between birth and adulthood; in addition, we describe transient sex-related differences in their number and distribution during the 2nd postnatal week, which are possibly related to the epigenetic action of gonadal hormones during this period.

NADPH‐diaphorase‐positive ganglion cells of the rat adrenal gland: Age‐ and sex‐related changes in their number, size, and distribution

The rat adrenal gland contains ganglion cells able to synthesize nitric oxide (NO). This messenger molecule controls and modulates adrenal secretory activity and blood flow. The present study analyzed the number, size, and distribution of NO-producing adrenal neurons in adulthood and during postnatal development by means of β-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. This method reliably visualizes the enzyme responsible for NO generation. The reactive neurons per adrenal gland were 350–400 in both male and female adult rats. The positive nerve cell bodies were mostly located in the medulla, few being detected within the cortex and the subcapsular region. Dual labeling with anti-microtubule-associated protein 2 antibody, specific for neuronal elements, confirmed this distribution. Anti-microtubule-associated protein 1b antibody identified a subset of NADPH-d-positive neurons, displaying different degrees of maturation according to their position within the adrenal gland. At birth, there were about 220 NADPH-d-labeled neurons per adrenal gland in both sexes. As confirmed by dual immunocytochemical labeling, their great majority was evenly distributed between the cortex and the subcapsular region, the medulla being practically devoid of stained neurons. After birth, the number of adrenal NADPH-d-positive ganglion cells displayed a strong postnatal increase and reached the adult-like distribution after 1–2 months. During the period of increase, there was a transient difference in the numbers of these cells in the two sexes. Thus we present here evidence of plasticity in the number, size, and distribution of NADPH-d-positive adrenal neurons between birth and adulthood; in addition, we describe transient sex-related differences in their number and distribution during the 2nd postnatal week, which are possibly related to the epigenetic action of gonadal hormones during this period. © 1996 Wiley-Liss, Inc.

Chronic prenatal ethanol exposure alters the normal ontogeny of choline acetyltransferase activity in the rat septohippocampal system.

In animal models of fetal alcohol syndrome (FAS), the hippocampus has been shown to be especially sensitive to the effects of prenatal ethanol exposure, exhibiting neuronal loss and alterations in neuritic process elaboration. We have characterized the influence of chronic prenatal ethanol treatment (CPET) on the postnatal expression of choline acetyltransferase (ChAT) in the hippocampus and the septal area that contains neurons that provide the primary cholinergic innervation to the hippocampus. On gestation days 1-22, pregnant rats were either fed an ethanol-containing liquid diet, pair-fed a calorically equivalent sucrose-containing diet, or given rat chow ad libitum. In Chow control animals, the ontogenetic progression of ChAT activity in the septal area and hippocampus was characterized by a significant period of upregulation during the 2nd and 3rd postnatal weeks, exhibiting and an approximate 5-fold increase (septal area) and 7-fold increase (hippocampus) by postnatal day 21 (P21). At P14, ethanol exposure reduced septal and hippocampal ChAT activity levels, compared with those of pair-fed offspring. ChAT activity reached control levels by P21 in ethanol-exposed pups, suggesting that the earlier decline in activity may reflect a delay in the ontogenetic upregulation. In addition, there was a trend toward increased septal and hippocampal ChAT activities at P1 and P7 in both liquid diet groups. This liquid diet-stimulated increase may mask the effects of ethanol on early postnatal ChAT expression in the septohippocampal system. The results suggest that prenatal ethanol exposure may influence factors that regulate the developmental expression of ChAT in the septohippocampal system.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplants Enhance Locomotion in Neonatal Kittens Whose Spinal Cords Are Transected: A Behavioral and Anatomical Study

We have studied the locomotor development of kittens that received complete low thoracic spinal cord transections and embryonic spinal cord transplants as newborns. Embryonic spinal cord (E21-E26) transplanted into the site of a transection integrated well with the host spinal cord and promoted the development of overground locomotion. Spinalized kittens with transplants were first distinguished from spinalized kittens during the 2nd and 3rd postnatal weeks when kittens with transplants positioned their hindlimbs underneath their bodies which promoted support of the hindquarters. By postnatal Week 6, kittens with transplants exhibited overground locomotion characterized by full weight support and moderate balance control. By 20 weeks of age, as many as 96% of the step cycles showed full weight support and as few as 2% of the step cycles were interrupted by a fall. Most kittens also showed coordination between the forelimbs and the hindlimbs. They differed from normal in the precocious onset of reflex stepping and in the less precise interlimb coordination and more precarious balance during overground locomotion. The overground locomotor performance of kittens with transplant greatly exceeded that of spinal kittens without transplants since few spinalized kittens showed any full-weight-supported step cycles and none showed coordination between the forelimbs and the hindlimbs. In the absence of a transplant, no fibers could grow across the lesion site. In the presence of a transplant, fibers grew across the lesion site and established anatomical connectivity with the host. Host segmental systems identified by the presence of calcitonin gene-related peptide- and substance P-immunoreactive fibers were found throughout the transplants. Descending host systems of supraspinal origin were identified by serotonin- and dopamine β-hydroxylase-immunoreactive fibers throughout the transplants. The growth of supraspinal axons into the transplant, and in one case into the caudal host spinal cord, provided a possible anatomical basis for the development of coordinated overground locomotion.

Distribution of Somatostatin Receptor Subtype 1 mRNA in the Developing Cerebral Hemispheres of the Rat (Part 2 of 2)

Somatostatin (SST) is one of the major peptide transmitters in the mammalian central nervous system and also seems to exert specific functions during brain development. In contrast to ligand binding experiments, by which two pharmacologically different binding sites were characterized, molecular cloning techniques have led to the identification of at least five different receptor subtypes (SSTR1–5), which according to RNA blot analyses seem to be differentially distributed and regulated in the developing brain. In order to provide more precise data on the distribution of SSTR1 during ontogenesis, we have performed an in situ hybridization analysis, using a 35S-labelled RNA probe, in the developing rat cortex between embryonic day (E)12 and adulthood. Within the cortical plate, expression of SSTR1 gene was first detected in parallel with the establishment of the deep laminae V/VI at E16, thereby following the characteristic morphogenetic gradients of cortical plate construction. Thus, with the subsequent addition of cells along the radial dimension, e.g. the deposition of the supragranular neurons beyond E18, the hybridization signal spreads as an uniform homogenous band through the entire cortical plate, whereby silver grains reach their peak density around birth. Similar developmental gradients were observed along the lateromedial and frontooccipital dimension, whereby SSTR1 transcripts were detected near the frontal pole and the lateral cortical areas roughly 2 days before they appeared in the occipital and medial cortical anlage, respectively. From the initially homogenous distribution, two distinct SSTR1 mRNA-positive bands coextensive with laminae V/VI and II/IH, respectively, and sparing lamina IV evolved during the first postnatal week, the grain density of which decreased during further postnatal development. Within the hippocampal formation, SSTR1 transcripts were initially observed at E18 in the subicular complex, and after birth also extending into the neighboring CA1 region. During the 1st and 2nd postnatal week, silver grains were observed over the pyramidal cell layer of CA2 and CA3 and as a faint supragranular band in the dentate gyrus. Similar to the isocortex, grain density decreased thereafter. Hypothetically, the pronounced temporospatial regulation of SSTR1 gene expression during brain development can be correlated with (1) the establishment and eventual reduction of transient cortical SSTergic neuron populations described for late pregnancy and early postnatal development and (2) a receptor subtype exchange during maturation as evidenced by the late (from postnatal day 7 onward) appearance of e.g. SSTR3.

Distribution of somatostatin receptor subtype 1 mRNA in the developing cerebral hemispheres of the rat.

Somatostatin (SST) is one of the major peptide transmitters in the mammalian central nervous system and also seems to exert specific functions during brain development. In contrast to ligand binding experiments, by which two pharmacologically different binding sites were characterized, molecular cloning techniques have led to the identification of at least five different receptor subtypes (SSTR1-5), which according to RNA blot analyses seem to be differentially distributed and regulated in the developing brain. In order to provide more precise data on the distribution of SSTR1 during ontogenesis, we have performed an in situ hybridization analysis, using a 35S-labelled RNA probe, in the developing rat cortex between embryonic day (E)12 and adulthood. Within the cortical plate, expression of SSTR1 gene was first detected in parallel with the establishment of the deep laminae V/VI at E16, thereby following the characteristic morphogenetic gradients of cortical plate construction. Thus, with the subsequent addition of cells along the radial dimension, e.g. the deposition of the supragranular neurons beyond E18, the hybridization signal spreads as an uniform homogenous band through the entire cortical plate, whereby silver grains reach their peak density around birth. Similar developmental gradients were observed along the lateromedial and frontooccipital dimension, whereby SSTR1 transcripts were detected near the frontal pole and the lateral cortical areas roughly 2 days before they appeared in the occipital and medial cortical anlage, respectively. From the initially homogenous distribution, two distinct SSTR1 mRNA-positive bands coextensive with laminae V/VI and II/III, respectively, and sparing lamina IV evolved during the first postnatal week, the grain density of which decreased during further postnatal development. Within the hippocampal formation, SSTR1 transcripts were initially observed at E18 in the subicular complex, and after birth also extending into the neighboring CA1 region. During the 1st and 2nd postnatal week, silver grains were observed over the pyramidal cell layer of CA2 and CA3 and as a faint supragranular band in the dentate gyrus. Similar to the isocortex, grain density decreased thereafter. Hypothetically, the pronounced temporospatial regulation of SSTR1 gene expression during brain development can be correlated with (1) the establishment and eventual reduction of transient cortical SSTergic neuron populations described for late pregnancy and early postnatal development and (2) a receptor subtype exchange during maturation as evidenced by the late (from postnatal day 7 onward) appearance of e.g. SSTR3.

Effects of maternal lead exposure on functional plasticity in the visual cortex and hippocampus of immature rats

We examined the amount of long-term potentiation (LTP) in slices from the visual cortex and hippocampus of pre- and postnatally lead-exposed rats and controls at postnatal days (PND) 12–20. A dietary lead intake of 750 ppm by the dams resulted in a mean blood lead concentration in the suckling offspring of about 17 μg/dl. While high-frequency stimulation (HFS) of the white matter induced LTP of the field potentials in layers II/III in cortical slices of ten out of the 14 control rats, only three of the twelve lead-exposed rats showed a small amount of LTP. However, in slices from seven of the twelve lead-exposed rats a long-term depression was found following HFS. Furthermore, paired-pulse inhibition was weaker in cortical slices from the lead-exposed as compared to the control rats. In the CA1 hippocampal region the amount of LTP was significantly reduced in the lead-exposed group only in slices taken from rats at PND 16–20, while no differences were seen in slices from younger animals. It is concluded that even low level lead exposure impairs functions of the visual cortex in the immature rat. We suggest that the developing hippocampus is able to compensate for lead-induced functional deficits in the 2nd postnatal week, being more vulnerable at older ages.

Transient expression of a slow-tonic MHC isoform by extrafusal fibers in the developing rat.

ALD 19, a monoclonal antibody that recognizes the slow-tonic myosin heavy chain (MHC) isoform, has been used extensively as a marker for nuclear bag intrafusal fibers of muscle spindles in developing and adult rats. Extrafusal fibers of adult rat hindlimb muscles do not express slow-tonic MHC. However, while using ALD 19 to trace the fate of intrafusal fibers following neonatal denervation, we noted that some extrafusal fibers of neonates also bound this antibody. The immunolabeled extrafusal fibers were a subset of slow fibers located in the deep axial regions of crural muscles. The same fiber subset transiently displayed a weak affinity for ALD 19 during the first postnatal week in normal muscles. Denervation at birth increased the intensity of ALD 19 immunolabelling by these extrafusal fibers and extended the duration of the slow-tonic immunoreactivity into the 2nd postnatal week, after which expression diminished or ceased. Demonstration that some developing extrafusal fibers have a nerve-independent capacity for transiently expressing slow-tonic MHC, an MHC previously though to be expressed only by intrafusal fibers, raises the possibility that both types of fiber originate from a subset of bipotential slow primary myotubes in rat hindlimbs.

Short-interval amygdala kindling in neonatal rats

The kindling paradigm provides a powerful tool for studying the generation, propagation and generalization of seizures. Such reproducible quantitative paradigms are a prerequisite for the experimental study of epilepsy in the developing brain. Kindling has been extensively utilized as a model of limbic seizures in the adult rat; amygdala short-interval kindling has been studied in ≧ 15- day-old rats . We applied the short-interval kindling method, i.e., stimulation at every 15 min, to 7-12-day-old rats. Stage-5 behavioral seizures were achieved even in 7-day-old rats; however, the progression of behavioral kindling differed somewhat from that of older rats. Correlation of electrographic discharges and behavioral phenomena was inversely related to age. Reliable progressive amygdala discharges were difficult to assess in most ≦ 10-day-old rats. Spontaneous seizures occurred relatively frequently in younger age groups. The amygdala short-interval kindling paradigm is reproducibly and reliably applicable to rats during the 2nd postnatal week. The presence of progressive focal to bilateral-generalized seizures suggests a significant functional maturity of the amygdala-limbic circuitry at this age.

Immunohistochemical localization of the S-100β protein in postnatal cat visual cortex: spatial and temporal patterns of expression in cortical and subcortical glia

The ontogenic expression of the glial-specific protein S100β was examined in postnatal cat visual cortex using immunocytochemical methods. Astrocytes in visual cortex and oligodendrocytes in the subcortical white matter exhibited distinct spatio-temporal gradients in their expression of the S100β protein. In the visual cortex, S100β-immunoreactivity was detected in astroglial cytoplasm, as well as in the extracellular interstitium, in a lamina-specific manner throughout postnatal development. Using double labeling procedures, the S100β protein was found to be strictly colocalized with GFAP-immunoreactive astroycytes when GFAP was present. The glial fibrillary acidic protein (GFAP), a marker of mature astrocytes, was not present at high levels until the 4th postnatal week. From the 2nd through 5th postnatal weeks, the expression of S100β was highest in the thalamocortical recipient, layer IV, of visual cortical areas 17 and 18. At ages beyond 6 postnatal weeks, S100β-immunoreactivity increased disproportionately in supra- and infragranular layers such that areas 17 and 18 were demarcated from adjacent cortices by lower levels in layer IV. The S100β protein was also highly expressed in oligodendroglial somata and processes in the subcortical white matter between the 2nd and 6th postnatal weeks. The levels of S100β in the subcortical white matter progressively diminished to adult levels, where it was localized only to a few remaining oligodendroglial somata. The differential laminar expression of the S100β protein in astrocytes during the period within which the visual cortex exhibits input- and experience-dependent synaptic modifications suggests that astrocytes, possibly via their release of S100β, may play a special role in mediating plasticity in visual cortical development. A consistent feature of the appearance of the S100β protein was its expression in immature astroglia and oligodendroglia, well before they are considered morphologically mature. This characteristic underscores the potential of S100β as a marker of distinct populations of glial cells and of their role in normal and abnormal development.

Ontogeny of GABA A/benzodiazepine receptor subunit mRNAs in the murine inferior olive: transient appearance of β3 subunit mRNA and [ 3H]muscimol binding sites

The GABA A/benzodiazepine receptor consists of at least four subunits, α, β, γ and δ, each comprised of several variants. The developmental expression of the α 1, β 1–3, γ 2 and δ subunits was studied in the murine inferior olivary nucleus by in situ hybridization with antisense cRNA probes. The postnatal appearance and distribution of [ 3H]flunitrazepam and [ 3H]muscimol binding sites, α and β subunit-specific ligands respectively, were also studied autoradiographically. The β 3 subunit was transiently expressed in each of the subnuclei of the inferior olive: The signal was strong at birth, increased throughout postnatal week 1 and rapidly declined thereafter to low adult levels. A similar pattern of labeling was observed with [ 3H]muscimol. Detectable levels of α 1 subunit mRNA hybridization signal and [ 3H]flunitrazepam binding sites were also present in the inferior olive at birth, decreasing thereafter. Low to moderate levels of β 1, β 2, and γ 2 subunit mRNAs were present in olivary neurons throughout postnatal development, while δ mRNAs were largely absent. It has been reported previously that, during the 2nd postnatal week, the ratio of climbing fiber terminals to Purkinje cells is reduced from 3 : 1, as observed in neonates, to the 1 : 1 relationship observed in the adult cerebellar cortex. Our results raise the possibility that the subunit composition of the GABA A/benzodiazepine receptor in inferior olivary neurons undergoes changes during development, and that this process may be related to the elimination of multiple climbing fiber innervation of cerebellar Purkinje cells.

Ontogeny of zeta (ζ), the opioid growth factor receptor, in the rat brain

Opioid growth factor (OGF), [Met 5]enkephalin, serves as an inhibitory influence on the developing nervous system and is especially targeted to cell proliferative events. OGF interacts with the zeta (ζ) opioid receptor to perform its function. Using [ 3H]-[Met 5]enkephalin, the ontogeny of the ζ receptor in the whole brain and cerebellum of rats was explored. Specific and saturable binding was recorded at the earliest time sampled, prenatal day 15 (E15). In the whole brain, binding capacity ( B max) was two-fold greater at E15 than at E18 adn E20. The quantity of ζ receptor appeared to increase in the first postnatal week, reaching a maximum on postnatal day 8. Binding decreased the remainder of the 2nd week and between postnatal days 15 and 25 binding was no longer recorded. In the cerebellum, binding capacity increased from E20 to the 2nd postnatal week, reaching a maximum on postnatal days 8–10. The B max receptor decreased precipitously on postnatal day 11, being 5.4-fold lower than on postnatal day 10. Between postnatal days 21 and 30, no binding was observed. The binding affinities of the whole brain and cerebellum were 2.3 and 2.7 nM, respectively, and no differences between ages could be observed. The binding affinities of the whole brain and cerebellum postnatal day 6 increased body weight, the B max of the ζ receptor in the whole brain and cerebellum (but not the K d), and increased the number of layers of germinal cells in the cerebellum. These results define the temporal expression of the ζ receptor in the rat brain, as well as some regulatory properties, and support the concept that the ζ opioid receptor is primarily related to the proliferation of cells in the nervous system.

The development of the somatosensory representation in the superior colliculus of visually deprived mice

Mice were enucleated bilaterally at birth in order to investigate whether early visual deprivation had any influence on the development of the somatosensory representation in the superior colliculus (SC). Single unit recordings were performed during the 2nd, 3rd, 5th and 8th postnatal week in both normal and enucleated mice. It was found that, in visually deprived mice, there was a dramatic increase of the SC somatosensory units starting from the 3rd postnatal week, as occurs in normal mice. During the 5th and 8th postnatal week, this increase was larger than in normal mice, mainly due to the invasion of the somatosensory input into the superficial layers. In visually deprived mice, both the orientation of the somatosensory map and the magnification of some body parts, such as the upper vibrassae, did not show any difference with respect to normal mice. These results indicate the involvement of both vision-dependent and independent mechanisms in the formation of the SC somatosensory representation. In fact, whereas the segregation of the somatosensory afferents in the deep layers depended on the presence of visual input, the postnatal emergence and the topographic arrangement of the somatosensory map resulted to be independent of vision.