Proportion Of Immunoreactive Neurons Research Articles

Developmental Changes in NO-Containing Sympathetic Neurons in the Spinal Cord in Rats

Objectives. To determine the locations, percentage composition, and morphometric characteristics of sympathetic preganglionic neurons containing NO synthase (NOS) in the spinal cord of rats. Materials and methods. Experiments were performed on 35 white female Wistar rats aged 3, 10, 20, 30, and 60 days, 6 months, and 3 years. Immunohistochemical detection of NOS and the acetylcholine synthesis enzyme choline acetyltransferase (CAT) was performed on sections of spinal cord made at the level of segment T2. The areas of nerve cells and the proportions of immunoreactive neurons were determined. Results. Most sympathetic preganglionic neurons in the spinal cord of neonates and 10-day-old rats contained both NOS and CAT. At these age groups, rats also had a population of NOS-positive/CAT-negative neurons (26% in neonates and 8% in 10-day-old animals), this population was not seen in older animals. During the first 20 days, the proportion of NOS-immunopositive neurons decreased significantly, while the population of CAT-positive neurons increased. Conclusions. There was a reduction in the number of sympathetic preganglionic neurons expressing NOS during early postnatal ontogeny, which may influence the mechanisms of NO-ergic sympathetic transmission.

Comparative Study of Catecholaminergic and Nitroxidergic Neurons in the Vasomotor Nuclei of the Caudal Part of the Brainstem in Rats

Immunohistochemical studies in 12 Wistar rats detected tyrosine hydroxylase (TH) and the neuronal form of nitric oxide synthase (nNOS) to address the distribution of catecholaminergic and nitroxidergic neurons, respectively, in the vasomotor nuclei of the medulla oblongata and pons. The highest frequency of TH expression was seen in neurons located in these nuclei and some reticular nuclei (the gigantocellular, paragigantocellular, and caudal nuclei of the pons), though the proportions of immunoreactive neurons in these structures were no greater than 8–14%. In other nuclei (the reticular parvocellular and oral nucleus of the pons, the spinal nucleus of the trigeminal nerve), this proportion ranged from 1 to 3%. Neurons of this type were consistently not detected in a large group of nuclei with confirmed vasomotor functions. Structures with many catecholaminergic neurons generally contained fewer nNOS-positive cells than nuclei with limited numbers of TH-positive neurons.

Topography and Morphometric Characteristics of NF200+ Neurons in the Gray Matter of the Spinal Cord after Capsaicin Deafferentation

Experiments were performed on 80 white male Wistar rats aged 3, 10, 20, 30, 60, 180, and 360 days to address the influences of capsaicin deafferentation on the topography of neurons containing 200-kDal neurofilament protein (NF200+) in the gray matter of the second thoracic segment of the spinal cord. Deafferentation was modeled by s.c. administration of single doses of 150 mg/kg capsaicin to rat pups on day 2 of life. Controls were intact animals (n = 40). NF200+ neurons were detected by labeling cells with anti-NF200 antibodies. The proportion of immunoreactive neurons was calculated by labeling the whole neuron population with NeuroTrace Green Fluorescent Nissl Stain. The results obtained from these experiments show that NF200+ neurons were detected in the ventral horn, the intermediate zone, and the dorsal horn of the spinal cord and were discriminated by their morphometric characteristics. Administration of capsaicin led to identical decreases in the numbers and mean cross sectional areas of medially and laterally located NF200+ motoneurons in the spinal cord (plate IX). In the intermediate zone (plate VII) and dorsal horn (plates III–IV), there were no changes in the numbers of NF200+ neurons, though the mean cross sectional area was greater than that in controls.

Localization of CART-Positive Neurons in the Amygdaloid Body and the Relationship between Their Immunoreactivity and the Sex Steroid Level

This is the first study reporting the locations of neurons expressing CART peptide (cocaine-amphetamine-regulated transcript) throughout all the nuclear and paleocortical formations of the amygdaloid body (AB) and demonstrating the effects of sex steroids on immunoreactivity. The immunocytochemical reaction was performed on frontal brain sections from adult rats (seven females in estrus, seven females in metestrus, and seven males). The proportions of immunoreactive neurons to the total number of neurons in adjacent sections stained by the Nissl method were assessed in estrus and metestrus. In the dorsomedial and posterior cortical nuclei and the lateral capsular subnucleus of the central field, the relative numbers of immunoreactive neurons at estrus were significantly greater than at metestrus. These results provide evidence of the involvement of the olfactory and integrative centers of the AB in the pathogenesis of drug dependence and show that new and efficient methods of gene therapy might be developed using the intranasal route for drug administration.

Direct Single Injection of p38 Mitogen-Activated Protein Kinase Inhibitor Does Not Affect Calcitonin Gene-Related Peptide Expression in Dorsal Root Ganglion Neurons Innervating Punctured Discs in Rats

STUDY DESIGN.: Immunohistological analysis of punctured disc after application of a p38 MAP kinase inhibitor. OBJECTIVE.: To examine effect of direct application on dorsal root ganglion (DRG) neurons innervating damaged rat discs. SUMMARY OF BACKGROUND DATA.: Degeneration of lumbar discs is one cause of low back pain. Pathogenesis may involve sensory nerve ingrowth into disc inner layers; tumor necrosis factor-alpha (TNF-alpha) is thought to be a major inducer of ingrowth. Because p38 mitogen-activated protein kinase (p38) upregulates TNF-alpha expression and may play a crucial role in pain sensation, we investigated the effect of one injection of inhibitor on expression of the pain-related neuropeptide calcitonin gene-related peptide (CGRP). METHODS.: The neuro-tracer fluoro-gold was applied to the surfaces of L4/5 discs to label the innervating DRG neurons (n = 30). Of 30 rats, 10 were controls, whereas the other 20 were the experimental model (i.e., discs were punctured with 23-gauge needle). P38 specific inhibitor or saline was applied simultaneously (n = 10 each, Puncture + inhibitor and puncture + saline groups). Fourteen days postsurgery, DRGs from L1 to L6 were harvested, sectioned, and immunostained for CGRP. Proportion of CGRP-immunoreactive DRG neurons was evaluated in all groups. RESULTS.: Fluoro-gold-labeled neurons innervating the L4/5 disc were distributed throughout L1 to L6 DRGs in all groups. Proportions of labeled neurons positive for CGRP were 15.2% +/- 8% (controls), 27.2% +/- 10% (puncture + saline), and 25.2% +/- 8% (puncture + inhibitor). Proportion of immunoreactive neurons was significantly increased in the puncture groups compared with controls. However, there was no significant difference between the 2 puncture groups (P > 0.1). CONCLUSION.: In this model, CGRP was upregulated in DRG neurons innervating the damaged disc. However, a direct single application of p38 inhibitor did not suppress CGRP expression in innervating DRG neurons. Future research with p38 inhibitor in this model should evaluate multiple or systemic administration of inhibitor.

Distribution and colocalization of nitric oxide synthase and calretinin in myenteric neurons of developing, aging, and Crohn's disease human small intestine.

The pattern of distribution and colocalization of nitric oxide synthase and the calcium-binding protein calretinin in myenteric neurons and nerve fibers were examined in the human small intestine from preterm fetuses (14-17 weeks of gestation), normal adults (mean age 50 years old), old age (mean age 80 years old), and Crohn's disease patients (mean age 30 years old) using NADPH-diaphorase histochemistry and immunohistochemical techniques. In all age groups investigated, NADPH-diaphorase-reactive and calretinin-immunoreactive neurons and nerve fibers were seen throughout the myenteric plexus. The highest proportion of NADPH-diaphorase-reactive neurons was found in the myenteric ganglia of old age intestines (56% of protein gene product-immunoreactive neurons) followed by fetal intestines (41%) and Crohn's intestine (30%) compared with intestines of control adults (20%). A similar trend was observed for calretinin-immunoreactive neurons where the highest proportion of immunoreactive neurons was found in the myenteric ganglia of old age intestines (28% of protein gene product-immunoreactive neurons), followed by fetal intestines (22%), and Crohn's intestines (18%) compared with intestines of control adults (9%). A colocalization of NADPH-diaphorase activity and calretinin immunoreactivity was only seen in the myenteric neurons of fetal intestines (2% of NADPH-diaphorase-reactive neurons were also calretinin-immunoreactive). The pattern of distribution of NADPH-reactive and calretinin-immunoreactive neurons in the myenteric ganglia of fetal intestine differs from that of the other age groups. In the fetal intestine, the myenteric neurons containing either calretinin or NADPH-diaphorase are distributed through out the myenteric ganglia with no specific orientation to one another. In the intestines of control adult, Crohn's, and old age patients, single large calretinin-immunoreactive neurons are surrounded by a number of small NADPH-diaphorase-positive neurons, with this feature being more prominent in intestines of old-age and Crohn's disease patients. In summary, a high number of both NADPH-diaphorase-reactive and calretinin-immunoreactive neurons were seen in the myenteric ganglia of fetal, old age, and Crohn's intestines; we discuss that there may be a role for nitric oxide and calretinin in the process of development, aging, and pathological changes in the human intestine associated with alteration in the calcium homeostasis in the myenteric neurons.

Nerve growth factor increases calcium binding protein (Calbindin-D 28K) in rat olfactory bulb

Calbindin-D 28K (CaBP 28K) is a soluble intracellular protein capable of sequestering micromolar concentrations of calcium. The in vivo regulation of CaBP 28K by recombinant human nerve growth factor (rhNGF) was studied in adult, male rats. Via Alzet 2002 pumps, each rat received, for 14 days, a lateral ventricle infusion (i.c.v.; n = 5–6/group) of 12 μl PBS/day containing 1.0 μg cytochrome C (control) or an equal amount of rhNGF. Six other animals received a vehicle or rhNGF infusiion into the central neostriatum. CaBP 28K was elevated by 75% ( P < 0.01) in the olfactory bulb following i.c.v rhNGF in each of two experiments and was not altered in the temporal cortex, hippocampus, olfactory tubercle, cerebellum, or neostriatum. Direct striatal injections of rhNGF did not alter CaBP 28K in the neostriatum or other regions (including the olfactory bulb). The increases in olfactory bulb CaBP 28K protein levels were verified via Western blot analysis. CaBP 28K immunocytochemistry revealed that 33% of olfactory bulb neurons are immunoreactive for CaBP 28K and that the number or proportion of immunoreactive neurons did not change with i.c.v. infusions of rhNGF, suggesting that exogenously delivered rhNGF augments the content of CaBP 28K in olfactory bulb neurons that normally express the protein. Endogenous NGF may function as a neuroprotective factor by enhancing the ability of these cells to sequester cytoplasmic calcium and retard calcium-mediated neurodegeneration.

Neurotransmitter phenotype plasticity in cultured dissociated adult rat dorsal root ganglia: An immunocytochemical study

Culturing sympathetic ganglion neurons in vitro may modify phenotypic expression of some neurotransmitters. For dorsal root ganglia (DRG), contradictory results have been reported; most studies have used immature material. We have therefore performed a detailed immunocytochemical analysis of the transmitter content of cultured adult rat DRG neurons. To demonstrate possible modifications of neurotransmitter phenotypes, we have compared the results obtained with the same techniques on neurons cultured for 3 days and on freshly dissociated DRG cells. Also, the transmitter profile of cultured neurons was compared with that known from in situ studies. Out of 22 antigens studied, 20 were detected in cultured DRG neurons. All of them were expressed in small and/or intermediate-sized cells. Large neurons only contained CGRP, VIP, NPY, beta-END, ENK, and GABA. The percentage of immunostained neurons varied for the various antisera: less than 10% of cultured neurons were positive for ENK, beta-LPH, beta-END, DYN, VASO, and OXY; 10-30% for SOM, CCK, CAT, and SP; and greater than 30% for NPY, CRF, GLU, NT, VIP, GABA, GRP, CGRP, 5-HT, and TRH. In the latter two groups of transmitters (except CGRP), the proportion of immunoreactive neurons was by far larger in cultured than in freshly dissociated DRG. The most pronounced (greater than 25%) increase in the proportion of positively stained neurons after culturing was observed for the GRP, CRF, TRH, and 5-HT antisera. Serotonin was the only transmitter identified in cultured but not in freshly dissociated cells. These data indicate, on one hand, that various antigens, for example, CAT, GABA, NT, TRH, NPY, beta-LPH, and beta-END, which up to now have not been described in DRG in situ, can be detected immunocytochemically a few hours after dissociation of adult rat DRG. On the other hand, several transmitters, for example, VIP, NPY, SP, GABA, GLU, NT, GRP, CRF, TRH, and 5-HT, are expressed in a significantly higher proportion of cells in cultured than in freshly dissociated preparations. This might reflect a change in the phenotypic expression of transmitters due to the new environment generated by the culture conditions, a hypothesis that can be tested by measuring specific mRNA levels. Moreover, considering the plasticity and multipotentiality of their transmitter phenotype, cultured adult DRG neurons might represent an interesting material for autografts into the injured central nervous system.