Postnatal development of nitrergic neurons in the myenteric plexus of rat stomach

The effect of age on the proportion of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-positive neurons was investigated in the myenteric plexus of five different gastric areas of 1-day-, 1-week-, 2-week-, 1-month- and 2-month-old rats. Protein gene product 9.5 immunocytochemistry was used as a marker for the total enteric neuron population in order to establish the percentage of gastric nitrergic neurons in relation to age. The percentage of NADPHd-positive neurons in the proximal parts of the rat stomach (34–38%) is significantly higher than in the antral part (29%). This difference persists in all the age groups investigated. No significant relative increase with age of NADPHd-positive neurons could be observed in any of the areas studied. These findings imply that the increased nitrergic response in the rat proximal stomach as seen in pharmacological studies cannot be explained by an increased relative number of nitrergic neurons. Accepted: 31 March 1999     

Postnatal development of a demyelinating disease in avian spinal cord chimeras

Xenogeneic spinal cord chimeras were constructed by grafting fragments of quail neural primordium into chick embryos at 2 days of incubation. Hatched birds displayed normal motor behavior for about 5 to 7 weeks, whereupon they developed a neurological syndrome; in the grafted spinal cord the pathological signs of the disease were very similar to those of the active plaques of multiple sclerosis and of the lesions of experimental allergic encephalomyelitis and neuritis, including Ia expression by brain capillary endothelia, rupture of the blood-brain barrier, leukocytic infiltration in the nervous tissue, and demyelination. In the animals at the most advanced stage of the disease an autoimmune attack occurred on the host's nervous system with the same histopathological signs.     

Low-molecular-weight peptide stimulates cholinergic development in ventral spinal cord cultures

Skeletal muscle extract contains a previously undocumented 1300- to 1500-Da neurotrophic factor. Incubation of ventral spinal cord neurons in the presence of this factor enhances the rate of de novo acetylcholine synthesis two- to threefold over control cells, after 6 days in culture. This effect on cholinergic activity appears to be selective, since incubation with the factor results in only slight elevations of lactate dehydrogenase activity and DNA content, and no increase in the acetylcholinesterase activity. The 1300- to 1500-Da factor is acid-stable and partially sensitive to proteolysis by proteinase K, Staphylococcus aureus V8 protease, and subtilisin, but insensitive to trypsin. These results indicate that the active moiety is a peptide. The importance of peptides as neurotransmitters or neuromodulators is well accepted, but their role in the regulation of neuronal development is not widely appreciated. The present cholinergic neurotrophic peptide is distinct from previously characterized cholinergic trophic factors and represents the first example of a small, target-derived peptide which influences cholinergic development.     

Esophageal-gastric relaxation reflex in rat: dual control of peripheral nitrergic and cholinergic transmission

It has long been known that the esophageal distension produced by swallowing elicits a powerful proximal gastric relaxation. Gastroinhibitory control by the esophagus involves neural pathways from esophageal distension-sensitive neurons in the nucleus tractus solitarius centralis (cNTS) with connections to virtually all levels of the dorsal motor nucleus of the vagus (DMV). We have shown recently that cNTS responses are excitatory and primarily involve tyrosine hydroxylase-immunoreactive cells, whereas the DMV response involves both an alpha1 excitatory and an alpha2 inhibitory response. In the present study, using an esophageal balloon distension to evoke gastric relaxation (esophageal-gastric reflex, EGR), we investigated the peripheral pharmacological basis responsible for this reflex. Systemic administration of atropine methyl nitrate reduced the amplitude of the gastric relaxation to 52.0+/-4.4% of the original EGR, whereas NG-nitro-L-arginine methyl ester (L-NAME) reduced it to 26.3+/-7.2% of the original EGR. Concomitant administration of atropine methyl nitrate and L-NAME reduced the amplitude of the gastric relaxation to 4.0+/-2.5% of control. This reduction in the amplitude of induced EGR is quite comparable (4.3+/-2.6%) to that seen when the animal was pretreated with the nicotinic ganglionic blocker hexamethonium. In the presence of bethanechol, the amplitude of the esophageal distension-induced gastric relaxation was increased to 177.0+/-10.0% of control; administration of L-NAME reduced this amplitude to 19.9+/-9.5%. Our data provide a clear demonstration that the gastroinhibitory control by the esophagus is mediated via a dual vagal innervation consisting of inhibitory nitrergic and excitatory cholinergic transmission.     

Regulation of cholinergic expression in cultured spinal cord neurons

Factors regulating development of cholinergic spinal neurons were examined in cultures of dissociated embryonic rat spinal cord. Levels of choline acetyltransferase (CAT) activity in freshly dissociated cells decreased rapidly, remained low for the first week in culture, and then increased. The decrease in enzyme activity was partially prevented by increased cell density or by treatment with spinal cord membranes. CAT activity was also stimulated by treatment with MANS, a molecule solubilized from spinal cord membranes. The effects of MANS were greatest in low-density cultures and in freshly plated cells, suggesting that the molecule may substitute for the effects of elevated density and cell-cell contact. CAT activity in ventral (motor neuron-enriched) spinal cord cultures was similarly regulated by elevated density or treatment with MANS, whereas enzyme activity was largely unchanged in mediodorsal (autonomic neuron-enriched) cultures under these conditions. These observations suggest that development of cholinergic motor neurons and autonomic neurons are not regulated by the same factors. Treatment of ventral spinal cord cultures with MANS did not increase the number of cholinergic neurons detected by immunocytochemistry with a monoclonal CAT antibody, suggesting that MANS did not increase motor neuron survival but rather stimulated levels of CAT activity per neuron. These observations indicate that development of motor neurons can be regulated by cell-cell contact and that the MANS factor may mediate the stimulatory effects of cell-cell contact on cholinergic expression.     

Glycine receptor heterogeneity in rat spinal cord during postnatal development.

Two different isoforms of the inhibitory glycine receptor were identified during postnatal development of rat spinal cord. A neonatal form characterized by low strychnine binding affinity, altered antigenicity, and a ligand binding subunit differing in mol. wt (49 kd) from that of the adult receptor (48 kd) predominates at birth (70% of the total receptor protein). Separation from the adult form could be achieved by either use of a selective antibody or glycine gradient elution of 2-aminostrychnine affinity columns. Both isoforms co-purify with the mol. wt 93 kd peripheral membrane protein of the postsynaptic glycine receptor complex.     

Postnatal development of the rat oviductal epithelium

Submicroscopic examination of rat oviducts in the early postnatal period showed that the character of the epithelium remained relatively indifferent up to the 6th day. Typical findings in this phase include an apical migration of the centrioles, with subsequent formation of solitary cilia; ciliated cells were an isolated occurrence. Ciliogenesis in the rat oviductal epithelium starts between the 8th and the 10th day. A continuous kinociliary apparatus is formed on the basis of centriole replication. The first secretory and peg cells appear towards the end of the second week.     

Butyrate enemas enhance both cholinergic and nitrergic phenotype of myenteric neurons and neuromuscular transmission in newborn rat colon

Postnatal changes in the enteric nervous system (ENS) are involved in the establishment of colonic motility. In adult rats, butyrate induced neuroplastic changes in the ENS, leading to enhanced colonic motility. Whether butyrate can induce similar changes during the postnatal period remains unknown. Enemas (Na-butyrate) were performed daily in rat pups between postnatal day (PND) 7 and PND 17. Effects of butyrate were evaluated on morphological and histological parameters in the distal colon at PND 21. The neurochemical phenotype of colonic submucosal and myenteric neurons was analyzed using antibodies against Hu, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS). Colonic motility and neuromuscular transmission was assessed in vivo and ex vivo. Butyrate (2.5 mM) enemas had no impact on pup growth and histological parameters compared with control. Butyrate did not modify the number of Hu-immunoreactive (IR) neurons per ganglia. A significant increase in the proportion (per Hu-IR neurons) of nNOS-IR myenteric and submucosal neurons and ChAT-IR myenteric neurons was observed in the distal colon after butyrate enemas compared with control. In addition, butyrate induced a significant increase in both nitrergic and cholinergic components of the neuromuscular transmission compared with control. Finally, butyrate increased distal colonic transit time compared with control. We concluded that butyrate enemas induced neuroplastic changes in myenteric and submucosal neurons, leading to changes in gastrointestinal functions. Our results support exploration of butyrate as potential therapy for motility disorders in preterm infants with delayed maturation of the ENS.     

Histochemical study of the pre—and postnatal development of acetylcholinesterase in the rat spinal cord

The distribution of acetylcholinesterase(AChE)-positive structures in the developing rat spinal cord was studied with AChE-histochemistry.AChE-positive perikarya were first seen on embryonic day 14(E14) in the ventrolateral portion of the spinal cord.From that time onward.AChE=containing cells appeared gradually in the intermediate gray,dorsal horn and lateral spinal nucleus of the spinal cord in a ventral-to-dorsal,and lateral-to-medial order.No obvious rostral-to-caudal sequence was found.At birth,the distribution pattern of AChE-positive perikarya was basically similar to that in adults.After birth a dramatic increase in the AChE staining intensity extended from postnatal day 5(P5) to postnatal day 21(P21),In addition,two phases of transient AChE staining were observed in the external surface of the dorsal horn from embryonic day 15(E15) to embryonic day 21(E21) and in the marginal layer from embryonic day 21(E21) to postnatal day 14(P14),respectively.     

Postnatal development of TRH and TSH rhythms in the rat

We previously observed that under a 12-hour light/12-hour dark schedule (lights off at 19.00 h), adult male Sprague-Dawley rats showed a circadian rhythm for serum thyroid-stimulating hormone (TSH) with a zenith near midday. In the present work, the ontogenesis of serum TSH rhythm was determined as well as pituitary TSH variations. In addition, hypothalamic and blood TRH were measured in these rats aged 15, 25, 40 and 70 days when sacrificed. As from the first age studied (15 days), a hypothalamic thyrotropin-releasing hormone (TRH) circadian rhythm was present. The mesor and the amplitude of this hypothalamic TRH rhythm increased while the rats were growing up, in contrast with the decrease observed for these parameters as far as blood TRH circadian rhythm is concerned. The time of the acrophase moved from 17.32 h in the 15-day-old rats to 13.57 h in the 70-day-old rats, being constantly in phase opposition with the blood TRH acrophase. The low amplitude pituitary TSH circadian rhythm detected in the young rat disappeared in the adult while, in contrast, the serum TSH rhythm became consistent to reach the well-characterized circadian midday peak in the 70-day-old rats.     

Growth factor dependent cholinergic function and survival in primary mouse spinal cord cultures

In primary embryonic spinal cord cultures, synaptic transmission can be conveniently studied by monitoring radiolabeled neurotransmitter release or by recording of electrophysiological responses. However, while the mature spinal cord contains an appreciable number of cholinergic motoneurons, cultures of embryonic spinal cord have a paucity of these neurons and release little or no acetylcholine upon stimulation. To determine whether the proportion of cholinergic neurons in primary mouse spinal cord cultures can be augmented, the effects of several classes of growth factors were examined on depolarization- and Ca(2+)-evoked release of choline/acetylcholine (Ch/ACh). In the absence of growth factors, little or no evoked release of radiolabeled Ch/ACh could be demonstrated. Media supplemented with brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF) were examined for their ability to preserve the population of neurons in culture. CNTF was found to increase the number of surviving neurons and to enhance the release of radiolabeled Ch/ACh; the other factors were without effect. The action of CNTF was transient, and the neuronal population decreased to levels observed in cultures lacking growth factor after 20 days in vitro. The correlation between enhanced neuron survival and increased Ch/ACh release suggests that CNTF protected cholinergic neurons, albeit transiently, from cell death.     

Postnatal development of locomotion in the laboratory rat.

In laboratory rats (Rattus norvegicus) aged 1 to 21 days emergence of postural and locomotor skills was studied in the open field and in experimental situations with homing used as motivation. Righting is mediated initially by curving and rocking of the trunk, later head and shoulder are rotated, and lastly the hindlimbs turn and provide co-ordinated support. Pivoting prodominates during the second half of the first week, crawling during most of the second week, and walking or running by the end of the second week. Balancing on narrow paths and compensating for lateral displacement on rotating rods mature later, as do various skills (climbing up or down on inclined surfaces, rods and ropes, and jumping across gaps) that require substantial hindlimb co-ordiantion.     

Opioid receptor binding in rat spinal cord

The interaction of various radioligands with spinal opioid receptors has been characterized under variable experimental conditions. Binding to , , and sites was measured in all (cervical, thoracic, lumbar) segments. The apparent affinity constant (K) of [³H]Ethylketocyclazocine (EKC) was similar in Tris, 2.09 (±1.06)×10⁸ M^–1, and phosphate buffer, 2.16 (±0.02)×10⁸ M^–1, when its interaction with and sites was blocked. Without blocking ligands, EKC binding was resolved in two components:K ₁=1.01 (±0.21)×10⁹ M^–1 andK ₂=0.95 (±0.61)×10⁷ M^–1. Likewise, the binding of [D-Ala², MePhe⁴, Gly(ol)⁵]enkephalin (DAGO) or [D-Ala², D-Leu⁵]-enkephalin (DADLE) alone was represented by a 2-site model. By adjusting the radioligand and receptor concentration or by the addition of blocking ligands, binding was represented by a 1-site model for DAGO,K=4.35 (±1.41)×10⁸ M^–1, and DADLE,K=2.44 (±0.08)×10⁸ M^–1.The abbreviations used are DADLE [D-Ala², D-Leu⁵]enkephalin - DAGO [D-Ala², MePhe⁴, Gly(ol)⁵]enkephalin - EKC ethylketocyclazocine - DYN dynorphin (1–17)     

Postnatal development of energy metabolism in the rat brain

The activities of cytochrome c oxidase and F0F1-ATPase as well as the content of cytochromes cc1, aa3, and b were investigated in free brain mitochondria in the course of postnatal development and aging. The results show an increase of Vmax of both enzymes during postnatal development (between day 5 and 30). During the following phase ending at the age of 6 months, a decrease of F0F1-ATPase and cytochrome c oxidase activity occurs. From 6 to 12 months of age the activity of these enzymes did not change. The KM for both enzymes remained unchanged during the whole period observed. The content of cytochromes increased from the low values found in young rats, reached the highest values at around one month, and decreased till the age of 3 months. Later, their content in brain mitochondria did not markedly change. Our results suggest that the metabolic maturation of brain mitochondria differs in several aspects from the same process in other tissues, mainly in the time course. This is probably due to the unique role of neural tissue in the organism.