Differential effects of developmental hypo- and hyperthyroidism on acetylcholinesterase and butyrylcholinesterase activity in the spinal cord of developing postnatal rat pups.

The plasticity and vulnerability of the rat spinal cord (SC) during postnatal development has been less investigated compared to other CNS structures. In this study, we determined the effects of thyroid hormonal (TH) deficiency and excess on postnatal growth and neurochemical development of the rat SC. The growth as well as the specific and total activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes of the SC were determined in hypo- and hyperthyroid rat pups at postnatal (P) days P1, P5, P10 and P21 (weaning), and were compared to age-matched untreated normal controls. AChE is a cholinergic synaptic enzyme while BuChE is a metabolic enzyme mainly found in glial cells and neurovascular cells. The SC is rich in somatic motor, autonomic cholinergic neurons and associated interneurons. Daily subcutaneous injection of pups with thyroxine (T4) and administration of antithyroid goitrogen propylthiouracil (PTU) in the litter's drinking water were used to induce hyper- and hypothyroidism, respectively. Enzyme assays were carried out spectrophotometrically at the above-mentioned ages, using SC homogenates with acetylthiocholine-chloride as the substrate, together with specific cholinesterase inhibitors, which specifically target AChE and BuChE. SC weights were significantly lower at P10 and P21 in hypothyroid pups but unchanged in the hyperthyroid ones. Hypothyroidism significantly reduced both specific and total AChE activity in SC of P10 and P21 rat pups, while having no effects on the BuChE activity, although total BuChE activity was decreased due to reduced total tissue weight. In contrast both specific and total AChE activities were markedly and significantly increased (>100%) in the P10 and P21 hyperthyroid pups. However, BuChE specific activity was unaffected by this treatment. The results indicate that hypothyroid condition significantly reduces, while hyperthyroidism increases, the postnatal development of cholinergic synapses, thereby influencing the functional development of this major sensory and motor structure. However, the neurochemical development of glia and other non-neuronal cells, where BuChE is mainly localized, is comparatively unaffected in these abnormal developmental conditions.

Postnatal growth hormone deficiency in growing rats causes marked decline in the activity of spinal cord acetylcholinesterase but not butyrylcholinesterase.

The effects of growth hormone (GH) deficiency on the developmental changes in the abundance and activity of cholinesterase enzymes were studied in the developing spinal cord (SC) of postnatal rats by measuring the specific activity of acetylcholinesterase (AChE), a marker for cholinergic neurons and their synaptic compartments, and butyrylcholinesterase (BuChE), a marker for glial cells and neurovascular cells. Specific activities of these two enzymes were measured in SC tissue of 21- and 90 day-old (P21, weaning age; P90, young adulthood) GH deficient spontaneous dwarf (SpDwf) mutant rats which lack anterior pituitary and circulating plasma GH, and were compared with SC tissue of normal age-matched control animals. Assays were carried out for AChE and BuChE activity in the presence of their specific chemical inhibitors, BW284C51 and iso-OMPA, respectively. Results revealed that mean AChE activity was markedly and significantly reduced [28% at P21, 49% at P90, (p<0.01)] in the SC of GH deficient rats compared to age-matched controls. GH deficiency had a higher and more significant effect on AChE activity of the older (P90) rats than the younger ones (P21) ones. In contrast, BuChE activity in SC showed no significant changes in GH deficient rats at either of the two ages studied. Results imply that, in the absence of pituitary GH, the postnatal proliferation of cholinergic synapses in the rat SC, a CNS structure, where AChE activity is abundant, is markedly reduced during both the pre- and postweaning periods; more so in the postweaning than preweaning ages. In contrast, the absence of any effects on BuChE activity implies that GH does not affect the development of non-neuronal elements, e.g., glia, as much as the neuronal and synaptic compartments of the developing rat SC.

Effects of increasing durations of immobilization stress on plasma corticosterone level, learning and memory and hippocampal BDNF gene expression in rats.

Stress effects on learning and memory are widely recognized, but less agreement exists on whether they are positive or negative as well as on their neuronal and neuromolecular correlates. Stress involves expression of certain genes such as neurotrophin BDNF (brain derived neurotrophic factor), which is also involved in learning, but results are not consistent. Here effects of stress on memory and BDNF expression were studied using on adult male rats exposed to "immobilization stress" for various "short" durations, i.e., 1-h, 3-h, 5-h and "long-term" ones (2-h/day for 1 week). Learning and memory was measured using passive avoidance testing (STL=step-through-latency scores) as well as plasma corticosterone (CSt) levels and hippocampal BDNF gene expression. CSt increased in the 3-h and longer stressed groups but differences were significant in the 5-h and 1-week stressed subgroups. Three and 5-h of stress markedly and significantly (60-69%, p<0.01) decreased memory retention in the stressed animals, while 1-h of stress had no effect; prolonged stress (2-h daily for 1-week) increased memory significantly (33%, p<0.05). Hippocampal BDNF gene expression increased in the 1-h and 3-h stressed groups (44%, p<0.05 and 71%, p<0.01); but this parameter steadily declined in the 5-h stressed group (26%, p<0.05) and weeklong stressed group (6%, not significant). Statistical analysis revealed an apparent but significant negative correlation between changes in memory and those of BDNF gene expression, indicating that BDNF may possibly play a compensatory role, reversing deleterious effects of stress on hippocampal memory functions.

Marked recovery of functional metabolic activity and laminar volumes in the rat hippocampus and dentate gyrus following postnatal hypothyroid growth retardation: a quantitative cytochrome oxidase study.

Similar to cretinism in human children, absence or deficiency of thyroid hormones in rats and mice during early postnatal development results in marked retardation of brain development along with behavioral and cognitive deficits. Less is known about brain recovery from postnatal hypothyroidism. [Farahvar, A., Meisami, E., 2007. Novel two-dimensional morphometric maps and quantitative analysis reveal marked growth and structural recovery of the rat hippocampal regions from early hypothyroid retardation. Experimental Neurology.] found, by means of morphometric maps, that surface areas of hippocampal cortex and its CA1-CA4 regions which were significantly reduced in developing hypothyroid rats, show nearly complete growth recovery upon restoration of thyroid function. Here we explore the ability of hippocampal synapse-rich neuronal fiber layers to show recovery from early hypothyroid growth retardation. Rat pups were made hypothyroid from birth to day 25 (weaning) or up to young adulthood (day 90) by a treatment with the reversible goitrogen, PTU (n-propylthiouracil), in the drinking water. Recovery was induced by withdrawal of PTU at weaning and analysis of cytochrome oxidase (CytOx)-stained serial sections of the hippocampus and dentate gyrus at the ages of 25 and 90 days. CytOx stains the synapse-rich fiber layers of the hippocampal formation (HCF). Volumetric growth of molecular layer, stratum oriens and radiatum and dentate hilar region showed complete or nearly complete recovery from marked and significant growth retardation induced by early postnatal hypothyroidism. Also the reduced CytOx staining intensity in the hypothyroid rat HCF layers showed marked recovery following hormonal restoration. Results indicate remarkable growth plasticity of the HCF and ability of the synapse-rich fiber layers to show complete recovery of metabolic and functional neural activity from deleterious effects of early hypothyroidism. Mitochondrial CytOx is highly localized to the synapse-rich fiber layers of the HCF and its activity and histochemical staining intensity correlates positively with functional metabolic activity of neural tissue. Thus hippocampus and dentate gyrus neuronal fiber layers and their oxidative activity show remarkable ability to recover from the postnatal hypothyroid growth retardation. The results indicate that some brain regions are less vulnerable to early developmental insults and can recover.

Novel two-dimensional morphometric maps and quantitative analysis reveal marked growth and structural recovery of the rat hippocampal regions from early hypothyroid retardation.

Effects of postnatal hypothyroidism and recovery from this condition on regional growth of the rat hippocampus (HC) were studied using two-dimensional (2D) foldout, morphometric maps of HC and its constituent CA1-CA4 regions. The maps were derived from unfolding serial coronal sections of the rat forebrain, consisting of the entire rostrocaudal extent of HC pyramidal cell layer in the normal control and hypothyroid weanling (P25, postnatal day 25) and young adult (P90) male rats, as well as animals allowed to recover from hypothyroid-induced growth retardation at weaning. The maps revealed novel views of HC regions for assessment of topological relationships and measurement of surface areas of the HC cortical sheet (pyramidal cell layer). In normal control P90 rats, the unfolded HC on each side extended 4 times more laterally than rostrocaudally; total HC surface area was about 40 mm(2), compared to 30 mm(2) in the weanling, indicating 35% growth from P25 to P90; CA1 took up 52% of the total HC surface area, followed by CA3 (31%) and CA2 and CA4, 8% each. Hypothyroidism resulted in significant (p<0.01) 11% and 20% reductions in the HC surface area in P25 and P90 rats, respectively; CA1 and CA4 regions suffered the most reductions while CA3 and CA2 regions the least. Recovering rats examined at P90 exhibited remarkable growth plasticity and recovery in HC regions, as evident by their near normal HC cortical surface area values, compared to age-matched controls. The 2D maps also revealed growth deficits in all HC regions of the hypothyroid rats; recovery in these parameters occurred across all dimensions, although the anterior-posterior growth was more severely affected than the mediolateral one. These results are confirmed and extended by volumetric analysis of laminar volumes of HC regions presented in a companion paper [Farahvar, A., Darwish, N., Sladek, S., Meisami, E., in press. Marked recovery of functional metabolic activity and laminar volumes in the rat hippocampus and dentate gyrus following postnatal hypothyroid growth retardation: a quantitative cytochrome oxidase study. Exp. Neurol.]. These results imply that HC regions, in contrast to whole brain, possess exceptional growth plasticity, as shown by ability to dramatically recover from early hypothyroid retardation; also 2D morphometric maps are useful tools to visualize complex and convoluted regional sheet of HC cortex and depict quantitative aspects of growth in normal and experimental conditions.

Cytochrome oxidase staining reveals functionally important activity bands in the olfactory epithelium of newborn rat.

We used cytochrome oxidase (CytOx) staining intensity, which is correlated with neuronal functional activity, to evaluate maturity and functionality of newborn rat olfactory epithelium (OE) and olfactory receptor neurons (ORNs). Nasal olfactory tissue of neonatal rats was stained with CytOx and analyzed qualitatively and quantitatively. Results revealed that newborn OE shows six differentially stained horizontal bands. Bands run parallel to the OE surface and were categorized as very light, medium or darkly stained. A narrow and pale Band 1 overlapped with horizontal basal cells. Next, a wide and lightly stained Band 2 was observed that coincides with the globose basal cell layer and immature ORNs, deep in OE. Next apically, a medium-staining Band 3 overlapped with ORN perikarya. Closer to the surface, a medium to light Band 4 was discerned where dendrites of mature ORNs normally occur. This band was interrupted with lighter areas due to the presence of supporting cells nuclei. Next, a superficial but dark Band 5 occurred, populated by the apical portions of ORN dendrites and their ciliated knobs and by supporting cell apices; mitochondria in apices of supporting cells contribute predominantly to dense staining of this Band 5. Apical to Band 5, a thin and fairly light Band 6 was observed which overlaps with the mucus layer that contains part of the ORN knobs, their cilia and supporting cell microvilli. Along the length of ORN dendrites, apical segments just below the ORN knobs, and wide basal segments showed a darker staining than the middle segments implying "microzones" of higher neural activity within the most apical and basal regions of dendrites. Our findings agree with ultrastructural studies showing a presence of mitochondria in knobs, basal portions of ORN dendrites and in OE supporting cell apices, suggesting that apical regions of both olfactory and supporting cells near the surfaces are metabolically most active, in odorant detection, signal processing, and detoxification, the latter for supporting cells.