The ontogeny of [3H]5-hydroxytryptamine binding in the lamb: effects of in vivo thyroidectomy

The effects of congenital hypothyroidism in the late gestation ovine fetus include changes in serotonin concentrations in specific brain areas. To investigate possible ontogenic patterns of changes in 5-HT receptor function, we studied the binding characteristics of [3H]5-HT in the midbrain, hypothalamus and cerebral cortex in the late gestation ovine fetus and young lamb. We compared the binding characteristics of control fetuses to those of thyroidectomized fetuses, with or without thyroxine replacement therapy. In each of the areas examined, age-dependent changes in the receptor density (Bmax) for [3H]5-HT was observed. In cerebral cortex, Bmax was constant from 120 days gestation through the early neonatal period and increased significantly only at 25-30 days after birth. In hypothalamus, [3H]5-HT binding density decreased late in gestation (140-145 days) with a return after birth to values comparable to those at 120-125 and 130-135 days gestation. The midbrain also exhibited a significant age-dependent pattern of altered receptor density with a decrease in the 130-135 and 140-145 day gestational age groups compared to both younger and older lambs. In contrast, the affinity constant (Kd) for [3H]5-HT did not change over the ages evaluated in cerebral cortex or hypothalamus. In the midbrain, however, there was a significant increase in Kd at 1-5 days after birth compared to all other age groups. The ability of fetal thyroidectomy, with or without thyroxine replacement therapy, to alter patterns of [3H]5-HT binding was also tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of brain beta-adrenergic receptors after neonatal 6-Hydroxydopamine treatment

We used [125I]-cyanopindolol in vitro autoradiography and neonatal 6-hydroxydopamine treatment to study the development of beta-adrenergic receptor subtypes in rat brain. Brain regions receiving locus coeruleus innervation, such as cerebral cortex and cerebellum, displayed low receptor densities at birth and increased in density rapidly during the second and fourth weeks postnatally. By contrast, regions which receive little innervation from the locus coeruleus, such as substantia nigra, striatum, and globus pallidus, displayed relatively high beta-receptor densities even at birth. The striatum appeared to be an exception to these generalizations. 6-Hydroxydopamine administration was associated with an increase in the densities of beta-receptor subtypes and, unexpectedly, with a change in the proportions of the two subtypes. These data support the view that innervation determines the ontogenetic patterns of some receptors in brain.     

Ontogeny of substance P receptor binding sites in rat brain

The ontogeny of substance P (SP) receptor binding sites in rat brain has been studied using both membrane binding assays and in vitro receptor autoradiography. The density of SP binding sites is maximal 1 d before birth and decreases thereafter to reach adult values 14 d after birth. During the early postnatal period, the distribution of SP binding sites undergoes major modifications. For example, very high densities of SP binding sites are present in most brain stem nuclei from 1 to 14 d after birth, while it is not the case in adults. In the striatum, SP receptors are distributed in a "patchy" manner early after birth, while it is much more homogeneous in the adult. This demonstrates that SP receptors undergo major redistributions during postnatal development. The very high density of SP binding sites present in the brain at its early stages of development may indicate that SP could be an important factor involved in the early organization of the CNS.     

Blockade of NMDA receptors increases cell death and birth in the developing rat dentate gyrus

Excitatory input regulates cell birth and survival in many systems. The granule cell population of the rat dentate gyrus is formed primarily during the postnatal period. Excitatory afferents enter the dentate gyrus and begin to form synapses with granule cells during the first postnatal week, the time of maximal cell birth and death. In order to determine whether excitatory input plays a role in the regulation of cell birth and survival in the developing granule cell layers and their germinal regions, the subependymal layer and hilus, we treated rat pups with the N-methyl D-aspartate (NMDA) receptor antagonists MK-801, CGP 37849, or CGP 43487 during the first postnatal week and examined the numbers of ³H-thymidine-labeled cells, pyknotic cells, and healthy cells in these regions. In order to determine the cell type that was affected, sections from brains of MK-801-treated rats were processed for ³H-thymidine autoradiography combined with immunohistochemistry for the marker of radial glia, vimentin, and the marker of mature astrocytes, glial fibrillary acidic protein (GFAP). Within the dentate gyrus, NMDA receptor blockade resulted in the following changes: (1) the density of ³H-thymidine-labeled cells was increased, (2) the density of pyknotic cells was increased, (3) the density of ³H-thymidine-labeled pyknotic cells was increased, and (4) the density of healthy cells was decreased. The infrapyramidal blade/hilus showed changes throughout its extent, whereas the suprapyramidal blade showed changes only at the rostral level. No change in the numbers of ³H-thymidine-labeled vimentin-immunoreactive or GFAP-immunoreactive cells was observed in the dentate gyrus with MK-801 treatment, indicating that glia are not primarily affected by NMDA receptor blockade. Blockade of NMDA receptors resulted in gross morphologic changes in the dentate gyrus; in most cases, the infrapyramidal blade was indistinguishable from the hilus. Moreover, in several brains of animals treated with CGP 37849 or CGP 43487 on postnatal day (P)5, an abnormal aggregation of cells was observed ventral to the normal location of the infrapyramidal blade. This cellular cluster contained many pyknotic and ³H-thymidine-labeled cells and may represent cells that normally comprise the infrapyramidal blade. Dramatic changes to the subependymal layer were also seen following NMDA receptor blockade. The cross-sectional area of this region was significantly increased with MK-801, CGP 37849, or CGP 43487 treatment and contained a high density of ³H-thymidine-labeled cells and ³H-thymidine-labeled pyknotic cells. These results indicate that NMDA receptor activation is critical for the normal development of the rat dentate gyrus. The finding that blockade of NMDA receptors resulted in increased levels of cell death and birth supports the hypothesis that NMDA receptor activation is a natural signal for the inhibition of these processes in the developing dentate gyrus. © Wiley-Liss, Inc.     

Ontogeny of high-affinity GABA and benzodiazepine receptors in the rat cerebellum: an autoradiographic study

High-affinity GABA and benzodiazepine receptors were localized by light microscopic autoradiography in the developing rat cerebellum. [3H]muscimol was used for the labeling of GABA receptors and [3H]flunitrazepam for benzodiazepine receptors. Very low densities of GABA sites were found during the first postnatal week. GABA receptors start increasing linearly at the end of the second week up to adult levels around the fourth postnatal week. The increase in receptor density is concentrated in the developing granule cell layer. Benzodiazepine receptors are present at birth and increases in the density of receptors were observed already during the first postnatal week. Receptor concentrations reached adult values around the third to fourth weeks postnatally. The increase in benzodiazepine receptors in concentrated in the growing molecular layer with little change in the granule cell layer. The immature cell of the external granule layer were characterized by the absence of receptor sites. At least partial association of high-affinity GABA receptors with granule cells and benzodiazepine receptor with Purkinje cell dendrites is suggested by these developmental profiles.     

Spatiotemporal patterning of glutamate receptors in developing ferret striate cortex

We have studied glutamate receptor levels during very early phases of cortical formation by using quantitative in vitro autoradiography to map the expression of NMDA, AMPA and kainate receptors in the developing primary visual cortex of the ferret. NMDA and non-NMDA receptors exhibit very different developmental profiles in primary visual cortex. NMDA receptor density is low at birth and increases throughout the first 2 postnatal months, rising between threefold (layers II/III) and ninefold (layer VI). In contrast, AMPA receptors are abundant at birth and their density remains constant for the first postnatal month, before rising by a maximum of 1.7-fold (layer I) at around the time of eye-opening (postnatal day 32). Kainate receptors are also present in high levels at birth and their expression levels rise in the early postnatal period by between 1. 5-fold (layer I) and threefold (layers V/VI) to a peak just after eye-opening. The proportion of the total ionotropic glutamate receptor binding contributed by NMDA receptors thus rises from 5% at birth to a maximum of 22% at 2 months of age, while the AMPA receptor contribution falls from 87% to 72% over the same period. Below cortex, all three glutamate receptor subtypes are expressed in the subplate region for the first 3 postnatal weeks. These developmental patterns, combined with the fact that AMPA receptors are densely expressed in the proliferative zones underlying presumptive area 17, indicate that non-NMDA receptor expression levels in primary visual cortex are mostly specified much earlier than those of NMDA receptors.     

Mouse telencephalon exhibits an age-related decrease in glutamate (AMPA) receptors but no change in nerve terminal markers.

The central excitatory amino acid receptor selective for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) was examined in brain tissue from mice at 3 and 25 months after birth. Antibodies against the rat GluR-A glutamate receptor subunit (selective for kainate and AMPA) labeled a mouse brain component of about M(r) 100,000. Telencephalic tissue from the older group of mice exhibited 31% less immunoreactivity towards this component as compared with that from the young group. Binding of [3H]AMPA also decreased with age in the telencephalon to an extent which was similar to the loss of receptor immunoreactivity. Scatchard analysis revealed that this reduction is due to a decrease in receptor density and not to a change in binding affinity. In contrast, there were only small age-related changes in AMPA receptor immunoreactivity and binding levels in the brain stem and cerebellum. Binding to dopamine, serotonin, or GABA receptors was not significantly reduced in the older mice. Since the nerve terminal markers synaptophysin and the SV2 glycoprotein were not detectably different in the two groups of mice, the age-related reduction in AMPA receptors is not likely to be due to a general decrease in synaptic density. These data suggest that glutamatergic neurotransmission mediated by AMPA-type receptors is selectively impaired with aging in the telencephalon.     

Postnatal maturation of brain cholinergic systems in the precocial murid Acomys cahirinus: Comparison with the altricial rat

The spiny mouse (Acomys cahirinus) is the only precocial murid species. It has some neuroanatomical peculiarities such as a relatively thin cerebral cortex and a large hippocampus. The levels of choline acetyltransferase, membrane-bound acetylcholinesterase and muscarinic receptor sites (measured as [3H]quinuclidynil benzilate binding) were assessed in the whole brain on days 1, 7, 14, 21, 28 and 80 (adult), and compared with those of Wistar rats of the corresponding ages. At birth choline acetyltransferase was significantly higher in spiny mice than in rats but the adult levels were similar, with an overall increase of about 5.2- and 14-fold for the former and the latter species, respectively. Membrane-bound acetylcholinesterase level and maximal density of muscarinic receptor sites in spiny mice were considerably higher at birth, in contrast adult levels were significantly lower than in rats with a respective overall increase of about 1.5- and over 4.5-fold. The high degree of maturity attained at birth by spiny mice partially depends on the long gestation period. However, if we consider postconception age, the maturation of choline acetyltransferase appears to be delayed at birth in the spiny mice, probably in relation to the lack of external stimulation during intrauterine life. In the cerebral cortex, hippocampus and striatum of adult spiny mice, when compared with the rats, there were similar levels of choline acetyltransferase but lower levels of membrane-bound acetylcholinesterase and, in the cerebral cortex, lower density of muscarinic receptor sites.     

Brain and Pituitary Melatonin Receptors in Male Rat during Post-Natal and Pubertal Development and the Effect of Pinealectomy and Testosterone Manipulation

Using quantitative autoradiography, melatonin receptors have been studied during post-natal and pubertal development of the rat in 2 brain and 2 pituitary structures. In the pars distalis of anterior pituitary, melatonin receptors decrease gradually in density after birth and disappear in 30 day-old animals. In contrast melatonin binding is only expressed in the paraventricular nuclei of the thalamus at the age of 21–23 days and is always present in adult animals. In the suprachiasmatic nuclei and in the pars tuberalis of the pituitary, melatonin receptor density decreases after birth, remains stable for approximately 1 month and increases again at puberty to reach the birth values in the adult. This increase was absent in pinealectomized and in castrated animals but present in castrated animals receiving testosterone suggesting that it depends upon circulating testosterone and melatonin levels. These results show that melatonin receptors are differentially regulated during post-natal development in each of the 4 structures studied, and that melatonin and testosterone are 2 factors which could be involved in the regulation of melatonin receptor density in the suprachiasmatic nuclei and pars tuberalis.     

Postnatal development of striatal dopamine function. I. An examination of D1 and D2 receptors, adenylate cyclase regulation and presynaptic dopamine markers

We have characterized the postnatal development from 1 to 7 weeks after birth in rat striatal homogenates of D1 and D2 dopamine (DA) receptor sites, adenylate cyclase (AC) enzyme activity coupled to DA receptor function, guanine nucleotide binding sites and presynaptic markers of DA terminal function. D1 receptor density, expressed per unit of membrane protein, does not increase over this developmental interval, while maximum DA-stimulated AC activity per mg membrane protein increases 50-100%. D1 agonist affinity for D1 receptor sites doubles by 7 weeks of age but is consistently reduced by guanine nucleotide during development. Guanine nucleotide stimulation of AC develops a biphasic dose-response curve after 3 weeks of age. Between 2 and 4 weeks postnatal age there is a rapid increase in AC catalytic component activity as manifested by the capacity of forskolin or manganese ion to stimulate AC in presence of guanine nucleotide and DA. Reversible [3H]GppNHp (guanyldiphosphonateimidophosphate) binding to striatal homogenates is dependent on Mg2+, inhibited by Ca2+ and GppNHp analogues, and occurs in about a 300-fold excess over D1 sites. Presynaptic markers of dopaminergic function indicate a 7-fold increase in tissue DA levels, a 2-fold reduction in DA turnover and no apparent change in density of DA uptake sites, assayed by [3H]mazindol binding. Subcomponents of D1 and D2 DA receptors have distinct postnatal developmental profiles. Striatal D1 sites do not change significantly during development, but D2 receptors and GTP inhibition of AC increase and appear, respectively, at 3-4 weeks of age, at the same time as the massive matrix innervation of striatum by DA terminals.     

Evidence for developmental synaptic regression of cholinergic afferents to the rat main olfactory bulb

Choline acetyltransferase (ChAT) activity, acetylcholinesterase (AChE) activity and muscarinic cholinergic receptor binding were determined in homogenates of olfactory bulbs from rats killed at intervals from 4 days before through 60 days after birth. In addition, the localization of muscarinic receptors was determined using an in vitro autoradiographic technique in 6-millimicrons thick coronal sections of olfactory bulbs from rats killed at similar intervals after birth. All 3 cholinergic parameters were present in measurable quantities at birth and showed substantial increases between 1 and 20 days after birth. The most rapid increase in cholinergic parameters occurred between days 10 and 20 after birth. ChAT activity and muscarinic receptor binding decreased between days 20 and 35 and increased again between postnatal days 35 and 60. A similar developmental pattern was observed for autoradiographic grain density overlying the granule cell layer of the neonatal bulb. These data suggest that (1) centrifugal cholinergic afferents are present in the rat olfactory bulb at birth, (2) during the early postnatal period (between 10 and 20 days) synaptogenesis occurs resulting in an overproduction of cholinergic synapses and (3) between postnatal days 20 and 35, a period of synaptic reorganization occurs characterized by substantial regression.     

Pre- and postnatal development of GABA receptors in Macaca monkey visual cortex.

GABA is a putative inhibitory neurotransmitter in adult mammalian visual cortex but also has been implicated as playing a crucial role in cortical information processing during development. In order to understand better the role of GABA during primate visual cortex development, we have examined the time course of GABAA and GABAB receptor ontogenesis in 18 Macaca nemestrina monkeys ranging from fetal day 61 (F61d) to adulthood. The GABA and benzodiazepine binding sites of the GABAA receptor were detected by 3H-muscimol (3H-MS) and 3H-flunitrazepam (3H-FZ), respectively. GABAB receptors were detected by 3H-baclofen (3H-BA). All ligands were visualized by in vitro autoradiography. Quantitative analysis of film density was done to compare laminar changes during pre- and postnatal development. Saturation binding experiments were done for MS and FZ binding sites to determine receptor number (Bmax) and affinity (Kd) at selected pre- and postnatal ages. Both MS and FZ binding sites were present at F61d-72d throughout the cortical plate and marginal zone. FZ binding sites were more dense than MS binding sites over the cortical plate at young ages and were especially dense over the marginal zone. FZ binding sites also were present in lesser amounts over the subplate and intermediate zone, but not over the subventricular zone. By F119d-126d, layer 4 could be distinguished by its higher density for both ligands. The basic adult laminar pattern was established for both MS and BZ binding sites by birth (birth = F165d-170d). After birth, MS density increases dramatically in all layers, but layer 4C remains most dense to adulthood. FZ labeling is heavy in both layers 4 and 3 at birth but after 4 weeks after birth (P4 wk) it declines somewhat in the supragranular layers so that layer 4C now predominates. Labeling in layers 5/6 virtually disappears after birth. BA binding sites were present at F126d, at which time layer 4 was slightly lighter than the remainder of striate cortex; this laminar pattern remained basically the same throughout our series to adulthood. Competitive binding of agonist and antagonists for the GABAA receptor showed that MS binding characteristics were similar at F126d and P8.5 years (yr). MS binding site Bmax was about 8% of adult values at F72d, 24% by F126d, and 56% at F152d. Bmax then rose rapidly after birth to peak at P18wk at 169% of adult values, and then declined to P1yr. A second peak of 143% was found around P3.5yr, with adult values reached by P8.5yr.(ABSTRACT TRUNCATED AT 400 WORDS)     

Decreased density of benzodiazepine receptors in lymphocytes of anxious patients: reversal after chronic diazepam treatment

Peripheral-type benzodiazepine receptors were measured in human circulating lymphocytes using 3H-PK 11195 as specific ligand. In a group of outpatients with anxiety disorders a significant decrease of receptor density (-37%) was found compared with age-matched controls. In these patients long-term diazepam treatment restored binding density to normal levels: the effect persisted after drug withdrawal. Acute i.v. diazepam administration did not change receptor density. The observed receptor changes could reflect a down-regulation phenomenon and indicate that lymphocyte function reflect central nervous events.     

The effect of hypothyroidism on the development of the glycogen content of organ of Corti's hair cells

The density of glycogen particles in organ of Corti's sensory cells was measured to determine the effect of congenital hypothyroidism upon the normal development of this energy source. This density in both normal and hypothyroid inner hair cells remains in low values from birth to adulthood. On the other hand, that of normal outer hair cells undergoes a great increase between the 10th and the 20th postnatal days, coinciding with the maturation of both the efferent innervation of these cells and the tuning properties of the auditory receptor. The glycogen stores of the hypothyroid outer hair cells do not show any significant increase from birth to adulthood. This latter fact suggests that the congenital hypothyroidism restrains the development of an important energy source of outer hair cells, most surely disturbing the physiological processes relying on glycogen metabolism.     

Diameter and internodal length of axons of spinal interneurones excited by group I afferents in the cat

Examination of various biochemical characteristics of the GABAnergic nervous system in the rat brain was made between 15 days of gestation and adulthood. At birth, the concentration of GABA in whole brain and most regions is approximately 50% of adult levels, whereas the medulla-pons has achieved adult levels by this time. Compared to GABA levels, there is a marked lag in the development of the activity of glutamic acid decar?ylase in all areas studied; however, the activity of the enzyme increases in a linear fashion from birth to adulthood. The development of the uptake of GABA into particulate fractions prepared from whole brain and regions differs markedly from that of GABA and glutamic acid decar?ylase, with uptake near adult levels by birth, peaking considerably above that of the adult between one to two weeks after birth and then declining toward adult activity by 4 weeks after birth. Examination of the kinetics of GABA uptake into resuspended P₂ fractions demonstrates that the developmental changes in the uptake reflects differences in the V_max whereas theK_t remains constant. Studies on the development of the apparent postsynaptic receptor for GABA reveals that in all regions binding is approximately 25% of adult up to 8 days after birth, at which time it increases dramatically, approximating adult levels by 4 weeks after birth. The rise in the density of the apparent postsynaptic GABA receptor after 8 days postpartum correlates best with the increase in the activity of glutamic acid decar?ylase, a presynaptic marker.     

Development of laminar distributions of kainate receptors in the somatosensory cortex of mice

Kainate receptors were present at birth in the murine somatosensory cortex as revealed by quantitative in vitro autoradiography. During the first five postnatal days [³H]kainate binding rapidly increased and the maximum density in layer IV was reached at P12. The adult laminar pattern of receptor binding distribution was established by the third postnatal week with the heaviest labeling of infragranular layers. The sharp increase of kainate receptor during the first postnatal week coincides with the critical period for cytoarchitectonic plasticity of the barrels and establishment of functional thalamo-cortical connections in the barrel field.     

Autoradiographic evidence that intrastriatal administration of adenosine A(1) receptor antisense oligodeoxynucleotide decreases adenosine A(1) receptors in the rat striatum and cortex.

In this study, the effect of a phosphorothioated A(1) adenosine receptor antisense oligodeoxynucleotide on A(1) receptor density and mRNA in the striatum and cortex of rats was determined. Receptor autoradiography and in situ hybridization revealed a reduction in striatal and cortical A(1) receptor density and cortical A(1) receptor mRNA, respectively, in antisense-treated brains but not in those treated with a mismatch oligonucleotide. There was no change in A(2) receptor binding. These data imply that the corticostriatal pathway synthesizes A(1) receptors and transports them to its terminals.     

Effect of a simple visual pattern on the early postnatal development oF GABA Receptor sites in the chick optic lobe

It is known that manipulation of the visual environment results in changes in the developmental pattern of several neurotransmitter receptors and that the GABA receptor shows a high degree of plasticity in differential illumination experiments. In the present paper we investigated whether exposure to a visual pattern has a developmental effect on GABA receptor expression during early postnatal life. Two groups of newly hatched chicks were used: one was exposed to a simple and specific visual pattern and the other was deprived of any visual pattern. GABA receptors at each developmental stage were determined by binding experiments performed in a crude membrane fraction. Saturation studies were carried out in a fraction enriched in synaptic membranes. The developmental pattern of both high and low affinity GABA binding sites was affected by the visual pattern. This effect displays its maximal expression by the end of the first postnatal week. The modification in receptor expression was due to a change in the receptor density while the affinity was not affected. The change in receptor density induced by the presence of a visual pattern was highest at the end of the first postnatal week suggesting that at that time there is a brief period of higher plasticity for GABA receptor expression in the visual system than at other times. Our results also suggest that variations in GABA receptor density could be instrumental in adaptative changes in the visual system in response to variations in the environmental stimulation.     

Regulation by chronic treatment with cabergoline of dopamine D1 and D2 receptor levels and their expression in the striatum of Parkinsonian-monkeys

1. Chronic treatment for one month with the long-acting dopamine D2-like agonist cabergoline (0.25 mg/kg s.c. every 48 hours), had despite partial tolerance, sustained antiparkinsonian activity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Parkinsonian monkeys (Macaca fascicularis). 2. Cabergoline treatment decreased by half striatal D2 receptor binding density measured by [3H]spiperone autoradiography versus untreated MPTP monkeys. No change in D2 mRNA measured by in situ hybridization and D2 receptor immunostaining was observed. 3. No change in either D1 receptor binding density or D1 receptor mRNA levels was observed in cabergoline-treated MPTP-monkeys compared to untreated MPTP-monkeys, suggesting receptor subfamily specificity of cabergoline. 4. The present results suggest that the cabergoline-induced behavioral partial tolerance is accompanied by a decrease in D2 receptor binding but not due to alterations in the steady state of D2 mRNA levels.     

Influence of maternal melatonin on melatonin receptors in rat offspring

Summary Using quantitative autoradiography, we have studied the influence of maternal plasma melatonin on the expression and density of melatonin receptors in the brain and pituitary of rat offspring. At birth, the same structures displayed melatonin receptors whether the rats were born to and reared by intact or pinealectomized dams. The receptor density was, however, about 20% lower in the group born to pinealectomized dams. At postnatal day 9, when the pups of both groups synthetize rhythmically their own melatonin, this difference was suppressed. These results indicate that melatonin does not appear to be a requirement for the expression of its receptors, but seems to play a stimulatory role in their synthesis.