Fatigue of the dorsal neck muscles initiates c-fos expression in the rat spinal cord and hypothalamus

Fatiguing stimulation of the dorsal neck muscles in rats is shown to induce significant up-regulation of the c-fos expression in the ipsilateral cervical cord, contralateral hypothalamic nuclei, periaqueductal gray, and lateral parabrachial nucleus. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 354–357, July–August, 2006.     

Changes in the levels of activity of spinal neurons after long-lasting vibrational stimulation of the shin muscles in rats

Vibrational stimulation of the tendon of the mm.gastrocnemius+soleus (100 sec^–1) in rats anesthetized with chloral hydrate (400 mg/kg) resulted in the appearance of considerable Fos immunoreactivity in the lumbar spinal cord (L1-L6), as compared with that in intact animals. Total densities of Fos-immunopositive (Fos-ip) neurons in each of the examined segments were higher than 40 units per 40-μm-thick slice; the respective index reached the maximum at the L4 level (78.9 ± 2.3 cells). Most Fos-ip neurons were localized in laminae 4 to 7 of the gray matter, both ipsi- and contralaterally with respect to the side of stimulation (28.5 ± 0.6 and 28.4 ± 0.6, respectively). Single Fos-ip motoneurons were found bilaterally in the ventral horn motor nuclei. Thus, activation of muscle spindle receptors induced by vibrational stimulation applied to the Achilles tendon induces noticeable bilateral c-fos expression in spinal neuronal networks related to transmission of proprioceptive muscle-born impulsation.     

Monosynaptic reflexes, <Emphasis Type="Italic">c-fos</Emphasis> expression,and NADPH-diaphorase activity in the cat spinal cord: Changes induced by chronic muscle inflammation

Effects of long-term (9 days) experimental unilateral inflammation in the mm. gastrocnemius-soleus (GS), induced by injection of Freund’s complete adjuvant, namely modulation of posterior biceps-semitendinosus (PBSt) monosynaptic reflexes (MSRs), which was induced by chemical activation of high-threshold (groups III and I) muscle afferents, and changes in c-fos expression and NADPH-diaphorase (d) reactivity in the lumbosacral spinal cord, were studied in anemically decapitated and highly (at the C1) spinalized cats. The mean amplitude of the MSRs on the pretreated side did not differ significantly from that on the opposite side. In adjuvant-injected cats, ipsilateral stimulation of nociceptive muscle afferents by KCl injection induced the bilateral enhancement of flexor reflexes. Significant bilateral increases in the mean number of Fos-immunoreactive (Fos-IR) neurons within the L6, L7, and S1 segments (157.5 ± 12.7, 201 ± 18.5, and 205 ± ± 18.6 per section; P < 0.05) were also found in adjuvant-injected cats. A lot of Fos-IR neurons was observed in the marginal zone (lamina I) and the neck of the dorsal horn (laminae V and VI); the highest number of labelled cells was detected in lamina VII. In adjuvant-injected cats, co-distribution of Fos-IR neurons and numerous Fos-IR glia-like cells in the dorsal and ventral horns was also found. Significant increases in the mean number of NADPH-d-reactive cells in lamina VII bilaterally and also in lamina I and in an area around the central canal (lamina X) contralaterally within the L6, L7, and S1 segments (P < 0.05) were also observed. In conclusion, activation of the nociceptive input during long-term inflammation of the GS muscles is associated with differential patterns of c-fos expression and NADPH-d reactivity and also with central neuronal hyperexcitability that contributes to bilateral facilitation of the PBSt MSRs. Neirofiziologiya/Neurophysiology, Vol. 39, No. 3, pp. 222–231, May–June, 2007.     

Changes in galanin immunoreactivity in rat micturition reflex pathways after cyclophosphamide-induced cystitis

Alterations in the expression of the neuropeptide, galanin, were examined in micturition reflex pathways of rat after cyclophosphamide (CYP)-induced cystitis of variable duration: acute (4 h), intermediate (48 h), or chronic (10 days). In control animals, galanin expression was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure (DCM); (2) superficial dorsal horn; (3) the regions of the intermediolateral cell column (L1–L2) and the sacral parasympathetic nucleus (SPN, L6–S1); and (4) the lateral collateral pathway (LCP) in lumbosacral spinal segments. Densitometry analysis demonstrated significant decreases (P≤0.01) in galanin immunoreactivity (IR) in these regions of the L1–S1 spinal cord after acute or intermediate CYP-induced cystitis. In contrast, increases (P≤0.01) in galanin–IR were observed in the DCM, SPN, or LCP regions in the L6–S1 spinal segments in rats with chronic cystitis. No changes in the number of galanin–immunoreactive cells were observed in the L1–S1 dorsal root ganglia (DRG) after CYP-induced cystitis of any duration. A small percentage of bladder afferent cells (Fast-blue-labeled) in the DRG expressed galanin–IR in control rats; this was not altered with cystitis. Galanin–IR was observed encircling DRG cells after chronic cystitis. These changes may contribute to urinary bladder dysfunction, altered sensation, and referred somatic hyperalgesia after cystitis.This work was supported in part through NIH grants DK051369, DK060481, DK065989, and NS040796.     

Expression of <Emphasis Type="Italic">c-fos</Emphasis> as an index of functional interaction of the frontal cortex and limbic cerebral structures in the course of food-procuring stereotyped movements in rats

We estimated expression of the c-fos gene (a marker of increase in neuronal activity) and manifestations of the histochemical reaction to NADPH-diaphorase (a marker of NO-generating neurons) in the medial prefrontal cortex (MPFC) and forebrain limbic structures of rats in the norm, in the state of starvation, and during realization of long-lasting (60 min) periodic (several times per minute) food-procuring movements of the forelimb. The starvation state or realization of motivated stereotyped forelimb movements were related to significant bilateral increases (P < 0.05) in the levels of c-fos expression in the anterior olfactory (AOP) and cortical (ACo) nuclei and the central (Ce) and basolateral (BLA) nuclei of the amygdala, and also in the pyriform (Pyr), prelimbic (PrL), and inferior limbic (IL) cortices. The described findings demonstrate that the high Fos immunoreactivity in the MPFC and amygdalar structures is related to the motivation state in animals and reflects the active involvement of limbic cerebral structures in the formation of motor programs and also in the stabilization and realization of operant reflexes. Neirofiziologiya/Neurophysiology, Vol. 40, No. 3, pp. 256–259, May–June, 2008.     

State of the immediate-response gene <Emphasis Type="Italic">c-fos</Emphasis> in neurons of the hypothalamic suprachiasmatic nuclei of rats under conditions of modifications of the photocycle

The influence of modifications of normal photoperiodicity on the state of c-fos (gene of immediate functional response) in neurons of the suprachiasmatic nuclei (SChNs) of the rat hypothalamus was examined; samples were taken during the subjective day and night. In animals kept under normal conditions of alternation of light and darkness, expression of the product of this gene and marker of its activation (c-Fos protein) demonstrated a rather clear circadian pattern, with a greater level of the immunoreactivity of this protein at the day period. Constant illumination for 7 days disturbed the rhythm of the c-fos activity and smoothed circadian variations of the level of immunoreactivity of c-Fos. Under conditions of light deprivation of a similar duration, we observed a significant (more than twofold) increase in the indices of concentration of c-Fos and its content in the nuclei of SChN neurons, as well of the total content of this protein in SChN slices, at the day phase. Possible mechanisms of the influence of modifications of the photoperiod on the state of the c-fos gene in SChN cells, in particular the relation of the respective changes to variations of the level of melatonin, are discussed. It is emphasized that natural photoperiodicity and its experimental modifications result in noticeable shifts of the geometrical dimensions of the nuclei of SChN neurons. Neirofiziologiya/Neurophysiology, Vol. 40, No. 2, pp. 112–118, March–April, 2008.     

Topography of Fos-Immunoreactive and NADPH-d-Reactive Neurons in the Limbic Structures of the Basal Forebrain and in the Hypothalamus during Realization of Motivated Operant Movements in Rats

We estimated in rats the expression of early gene c-fos (marker of neuronal activation) and NADPH-diaphorase activity (NO-synthase marker) in the limbic structures of the basal forebrain and in the hypothalamus. Estimations were performed in the norm, in the state of starvation, and after realization of long-lasting (repeated 4 to 12 times per minute for 30 min) motivated stereotyped food-procuring forelimb movements. In food-deprived animals, a significantly greater (Р < 0.05), as compared with the control, number of Fos-immunoreactive (Fos-ir) and NADPH-diaphorase-reactive (NADPH-dr) neurons was observed in limbic structures, namely in the medial septum (MS), nuclei of the vertical and horizontal branches of the diagonal fascia (VDB and НDB), magnocellular preoptic nucleus (MCPO), complex of the substantia innominata−basal nucleus of Meynert of the pallidum, SI-GP(B), as well as in the laterodorsal tegmental nucleus (LDTg), medial part of the pallidum (MGP), paraventricular and lateral nuclei of the hypothalamus (Pa and LH), and islands of Calleja (ICj and ICjM). In the limbic structures and pontine nuclei of rats of the experimental group (that performed operant movements), greater mean densities of labeled neurons were found in the succession LDTg < SI < MCPO < GP(B) < MS < VDB < HDB. The maximum mean density of Fos-ir neurons (13.8 ± 0.9 labeled nuclei within 200 × 200 μm² area) was found in the HDB. In the hypothalamic nuclei of starving rats, c-fos expression was two times higher than that in the control. After realization of operant movements, the intensity of expression in the LH was somewhat smaller, while in the Ра it was higher. The maximum density of NADPH-dr neurons was observed in the Pa (303.4 ± 18.7 cells), in the ICj and ICjM (287 ± 11.6 and 260 ± 8.7 neurons, respectively), and in the MGP (93 ± 6.7 labeled cells). When analyzing the distribution of labeled neurons in experimental rats, we found high densities of double-labeled cells (Fos + NADPH-d positivity) in the Pa, MGP, ICj, and ICjM. Such specificity of changes in the c-fos expression and NADPH-d reactivity in the hypothalamus correlates, perhaps, with the formation of motivation signals related to a delay in food accessibility and supply of food. Modifications of neuronal activity in limbic structures reflect involvement of the latter in the formation of motor programs for food-procuring movements and their realization. Neirofiziologiya/Neurophysiology, Vol. 41, No. 1, pp. 32–40, January–February, 2009.     

A Study of Glutathione <Emphasis Type="Italic">S</Emphasis>-transferase pi Expression in Central Nervous System of Subjects with Amyotrophic Lateral Sclerosis Using RNA Extraction from Formalin-Fixed,Paraffin-Embedded Material

The expression of glutathione S-transferase pi (GST pi), an enzyme responsible for inactivation of a large variety of toxic compounds was studied in spinal cord, motor and sensory brain cortex obtained from patients who died in the course of amyotrophic lateral sclerosis (ALS). The studies were performed on formalin-fixed, paraffin-embedded (FFPE) and freshly frozen tissues. The method of RNA isolation from FFPE was modified. A significant decrease of GST pi-mRNA expression was found in cervical spinal cord and motor brain cortex of ALS subjects comparing to analogue control tissues (P < 0.01), as well as in motor cortex of ALS subjects comparing to their sensory cortex (P < 0.05). In spinal cords the decrease in GST pi-mRNA expression was accompanied by a decrease of GST pi protein level. Results indicated lowered GST pi expression on both mRNA and protein levels in the regions of nervous system affected by ALS. The non-properly inactivated by GST toxic electrophiles and organic peroxides may thus contribute to motor neurons damage.     

Increased Immunoreactivities of Cleaved αII-Spectrin and Cleaved Caspase-3 in the Aged Dog Spinal Cord

The activation of caspase-3 is considered to be a reliable marker for apoptotic cell death, and a 120-kDa fragment of αII-spectrin is generated by caspase-3 mediated cleavage of this structural protein. In the present study, we compared cleaved αII-spectrin (120-kDa) and cleaved caspase-3-immunoreactive cells and their protein levels in the cervical (C₅–C₆) and lumbar (L₃–L₄) levels of the spinal cord in adult (1–2 year-old) and aged (10–12 year-old) dogs (German shepherds). Weak cleaved αII-spectrin and cleaved caspase-3 immunoreactivity was found in neurons of the adult group; however, their immunoreactivity was distinctively increased in the neuronal cytoplasm in the aged group compared to those in the adult group, although the distribution pattern of their neurons was similar between the adult and age group. In addition, cleaved αII-spectrin and cleaved caspase-3 levels in the aged spinal cord were markedly increased compared to those in the adult group. These findings suggest that the increases of cleaved αII-spectrin and cleaved caspase-3 immunoreactivity may be related to aging of the spinal cord in dogs.     

Repeated topical application of growth hormone attenuates blood-spinal cord barrier permeability and edema formation following spinal cord injury: an experimental study in the rat using Evans blue, ([125])I-sodium and lanthanum tracers

Summary.  The neuroprotective efficacy of growth hormone on a focal spinal cord trauma induced alteration in the blood-spinal cord barrier (BSCB) and edema formation was examined in a rat model. Under Equithesin anaesthesia, one segment laminectomy was done over the T10–11 segments. Spinal cord injury was produced by making an incision into the right dorsal horn of the T10–11 segments (2 mm deep and 4 mm long). The animals were allowed to survive 5 h after injury. Highly purified rat growth hormone [rGH, 25 μl of a 1 μg/ml solution) was applied over 10 sec topically on the exposed surface of the spinal cord 30 min before injury. The identical doses of the rGH were repeated 0 min, 30 min, 60 min, 120 min, 180 min and 240 min following injury. Saline (0.9% NaCl) treated traumatised rats at identical intervals served as controls. Traumatised rats treated with saline exhibited marked edema formation and extravasation of Evans blue and ^[125]Iodine tracers in the spinal cord. At the ultrastructural level, perivascular edema and exudation of lanthanum across the endothelial cells was quite frequent in the spinal cord. Pretreatment with rGH significantly attenuated the edema formation and the extravasation of tracers in the spinal cord. In these rats, perivascular edema and infiltration of lanthanum across the endothelial cells was not much evident. These observations show that the rGH has the capacity to reduce the early manifestations of microvascular permeability disturbances and edema formation following trauma and further suggest a possible therapeutic potential of the hormone for the treatment of spinal cord injuries. Received July 3, 2001 Accepted August 6, 2001 Published online July 31, 2002     

Effect of genetic background on the developmental expression of c-fos and c-myc in chicken

The developmental expression of the protooncogenes, c-fos and c-myc, in muscle and liver of 14-and 19-day embryos and 1-, 6-, 8-and 28-day-old chicks of Athens Canadian Random Bred (ACRB) Single Comb White Leghorn (SCWL) and Peterson X Arbor Acres commercial broiler (PXAA) was determined. For the three stocks of chicken, significant differences were found in c-fos and c-myc expression. For both muscle and liver, averaged across ages, abundance of c-fos RNA was highest in PXAA and lowest in ACRB with differences significant at the P<0.01 level. c-myc RNA levels were significantly higher (P<0.01) in PXAA than in ACRB or SCWL liver. Taken over the developmental period, expression of c-fos RNA in muscle increased at different rates between breeds from 14-day embryo levels to peak levels in 6- to 8-day-old chicks and declined in 28-day-old chicks. Levels of c-fos were much lower in liver and showed no consistent differences related to developmental stage. A steady decline in c-myc from 14-day embryo levels to 28-day-old chicks was found in both muscle and liver. This decline in c-myc levels generally parallels the decline in relative growth rates which occurs in all breeds over the developmental period. In liver, the fast growing PXAA had the highest levels of c-myc. c-fos, on the other hand, showed elevated levels in PXAA for both muscle and liver and distinctly different patterns between these two tissues over the developmental period, suggesting tissue-specific involvement in growth.     

Glial Activation and Segmental Upregulation of Interleukin-1β (IL-1β) in the Rat Spinal Cord after Surgical Incision

The present study investigated the expression patterns of glial cells and interleukin-1β (IL-1β) in the rat spinal cord after a surgical incision, which is closely related with clinical postoperative pain. Microglia and astrocytes became activated in the spinal cord following incision. Real-time polymerase chain reaction (PCR) and immunohistochemisty showed that IL-1β mRNA and protein level in the spinal cord was transiently upregulated after surgical incision. The increased IL-1β-immunoreactivity (IR) was mainly localized in neurons but not the activated microglia or astrocytes. Although obvious increase in IL-1β-IR could be observed in the lumbar segments of the spinal cord ipsilateral to a hind paw incision, significant upregulation of IL-1β was not detected in the lumbar segments following thoracic incision. The present study indicated that surgical incision could induce glial activation and segmental upregulation of IL-1β in the spinal cord. The activated glial cells and upregulated IL-1β, in turn, may be involved in the incision-induced pain hypersensitivity.     

Corticotropin-releasing factor (CRF) expression in postnatal and adult rat sacral parasympathetic nucleus (SPN)

The neural control of micturition undergoes marked changes during the early postnatal development. During the first few postnatal weeks, the spinal micturition reflex is gradually replaced by a spinobulbospinal reflex pathway that is responsible for micturition in adult animals. Upregulation of brainstem regulation of spinal micturition pathways may contribute to development of mature voiding patterns. We examined the expression of corticotropin-releasing factor (CRF), present in descending projections from Barrington's nucleus to the sacral parasympathetic nucleus (SPN), in postnatal (P0–P36) and adult Wistar rats (P60–90). CRF-immunoreactivity (IR) was present predominantly in the SPN region, although some staining was also observed in the dorsal horn and dorsal commissure in L5–S1 spinal segments. CRF-IR in spinal cord regions was age dependent (R ²=0.87–0.98). The majority of the CRF-IR in the lumbosacral spinal cord was eliminated by complete spinalization (2–3 weeks). Double-label immunohistochemistry was combined with quantitative confocal laser scanning microscopy to quantify the number and percentage of colocalization between CRF-immunoreactive varicosities and preganglionic somas or proximal neurites in the SPN in postnatal and adult rats. Results demonstrate an age-dependent upregulation of CRF-IR in the SPN region and specifically in association with preganglionic parasympathetic neurons identified with neuronal nitric oxide synthase (nNOS)-IR. CRF-immunoreactive varicosities on or within a 1 μm perimeter of nNOS-immunoreactive somas or proximal neurites also increased with postnatal age. The upregulation of CRF-IR in bulbospinal projections to the SPN may contribute to mature voiding reflexes. This work was supported in part through NIH grants DK051369, DK060481, DK065989, NS040796.     

Effects of Electro-Acupuncture on IGF-I Expression in Spared Dorsal Root Ganglia and Associated Spinal Dorsal Horn in Cats Subjected to Adjacent Dorsal Root Ganglionectomies

The effects of electro-acupuncture (EA) on insulin-like growth factor-I (IGF-I) expression in the spared dorsal root ganglia (DRG) and associated spinal dorsal horns were explored in cats subjected to unilateral removal of L₁–L₅ and L₇–S₂ DRG, sparing the L₆ DRG. Immunohistochemistry revealed the presence of IGF-I immunoreactive products in the L₆ DRG neurons and some neurons and glial cells in the spinal cord. Western blot demonstrated that the level of IGF-I was significantly up-regulated both in the spared DRG and the dorsal horns of L₃ and L₆ cord segments at both 7 and 14 days post operation following EA. The present findings demonstrated the association between neuroplasticity and IGF-I expression, suggesting the possible role of IGF-I in EA promoted spinal cord plasticity.     

Increased spinal expression of c-Fos following stimulation of the lower urinary tract in chronic spinal cord-injured rats

c-Fos expression was studied in the lumbar and sacral spinal cord regions involved in processing afferent input from the lower urinary tract and a comparison was made between spinal cord-injured (SCI) animals and control animals with intact neuraxes. Afferent pathways from the lower urinary tract were activated either by insertion of a catheter through the urethra into the urinary bladder or by catheterisation plus induction of reflex micturition contractions by intravesical saline infusion. Placement of a catheter alone elicited Fos expression in a similar number of neurones in SCI and control rats mainly in the medial dorsal horn (MDH) and dorsal commissure (DCM) in the segments L1–2 and L5–S1 with a maximum in L5. Additional saline infusion induced low-frequency, high-amplitude, rhythmic bladder contractions of long duration in the rats with intact spinal cords, whereas in SCI rats, bladder distension elicited reflex contractions at a higher frequency, smaller amplitude and shorter duration. However, the basal and mean bladder pressure, as well as the total contraction time relative to the whole recording time, was not significantly different. Distension-induced bladder contractions markedly increased Fos expression primarily in the spinal segments L5–S1 in the control rats, where the majority of bladder and urethral afferent fibres terminates. Fos-positive cells were located in the MDH, lateral dorsal horn (LDH), DCM and the lateral aspect of laminae V–VII. Compared to controls, Fos expression after spinal cord injury (SCI) occurred in a significantly greater number of neurones throughout the segments L3–S1 following induction of bladder reflexes. The greatest proportional increase in the number of Fos-positive cells occurred in L3–5 which normally receive only little afferent input from the urinary bladder. Cell numbers predominantly increased in the LDH and lateral lamina V–VII. The data are consistent with the concept of a neuroplastic reorganisation of spinal pathways after SCI. Unmasking of silent synapses or formation of new connections by afferent axonal sprouting caudal to the lesion, as evident from the increased numbers of cells expressing Fos after bladder distension, could be factors underlying the emergence of reflexogenic micturition in chronic SCI rats. Accepted: 27 May 1999     

Axon transport of steroid hormones via spinal root fibers in old rats

Labelled steroid hormones,³H-hydrocortisone and¹⁴C-testosterone, being injected in the gray matter of theL ₅−L ₆ spinal cord segments were shown to be transported via ventral and dorsal root fibers (antero- and retrograde directions, respectively) of old (25 to 28 months) rats with a lower velocity than in adult young (6 to 11 months) animals. The averaged maximum velocities of axon transport (AT) through the ventral and dorsal roots were: for³H-hydrocortisone, 756±63 and 738±46 mm per day, and for¹⁴H-testosterone, 624±54 and 608±80 mm per day, respectively. Therefore, in old rats the AT velocities for³H-hydrocortisone and¹⁴C-testosterone were about four and seven-eight times lower than those in adult rats. In the course of anterograde, AT through the ventral roots in old rats the inclusion of³H-hydrocortisone is sharply suppressed (by more than an order of magnitude), as compared with than in adult animals. The doses of non-labelled steroid hormones within a 10⁻⁷ – 10⁻⁶ range, injected into the lumbar spinal segments, resulted in hyperpolarization of muscle fibers of themm. gastrocnemius anddeltoideus, but this phenomenon developed in old rats much later than in adult rats. It is obvious that AT of steroid hormones can be considered one of the mechanisms of their effects on the tissue of an organism, and this mechanism undergoes extremely intensive modifications with aging.     

Effects of NMDA-Receptor Antagonist Treatment on <Emphasis Type="Italic">c-fos</Emphasis> Expression in Rat Brain Areas Implicated in Schizophrenia

1. The noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists produce behavioral responses that closely resemble both positive and negative symptoms of schizophrenia. These drugs also induce excitatory and neurotoxic effects in limbic cortical areas.2. We have here mapped the brain areas which show increased activity in response to noncompetitive NMDA-receptor antagonist administration concentrating especially to those brain areas that have been suggested to be relevant in the pathophysiology of schizophrenia.3. Rats were treated intraperitoneally with a NMDA-receptor antagonist MK801 and activation of brain areas was detected by monitoring the expression of c-fos mRNA by using in situ hybridization.4. MK801 induced c-fos mRNA expression of in the retrosplenial, entorhinal, and prefrontal cortices. Lower c-fos expression was observed in the layer IV of the parietal and frontal cortex. In the thalamus, c-fos mRNA expression was detected in the midline nuclei and in the reticular nucleus but not in the dorsomedial nucleus. In addition, c-fos mRNA was expressed in the anterior olfactory nucleus, the ventral tegmental area, and in cerebellar granule neurons.5. NMDA-receptor antagonist ketamine increased dopamine release in the parietal cortex, in the region where NMDA-receptor antagonist increased c-fos mRNA expression.6. Thus, the psychotropic NMDA-receptor antagonist induced c-fos mRNA expression in most, but not all, brain areas implicated in the pathophysiology of schizophrenia. The high spatial resolution of in situ hybridization may help to define regions of interest for human imaging studies.     

Coordinates,DNA content and heterogeneity of cell nuclei and segments of the <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> intestine

A specific protocol allowed to selectively stain and image the cell nuclei of the intestine of the nematode Caenorhabditis elegans. Digital processing of these images yielded quantitative information on the coordinates and the relative DNA content of these cell nuclei. With this technique, the exact locations (in relation to the length of worm or intestine) of the 20 intestinal cell nuclei of larval stage L1 and of the 34 intestinal cell nuclei of larval stages L2–4 and adults were determined. Tracking the DNA content of an individual intestinal cell nuclei allowed to study nuclear DNA endoreduplications during larval growth. The evaluation of the DNA content of all intestinal cell nuclei revealed a strong heterogeneity between the cell nuclei and intestinal segments with a maximum in segments int4–int5 in the L1 stage and int5–int6 in the L2–L4 stages. Minimum values were detected in the segments int2 and int8–int9 of all larval stages. Heterogeneity between the intestinal segments of C. elegans does not only concern the DNA content of cell nuclei, but also other cellular features like the quantity of intracellular vesicles or the absorption of particles out of the intestinal lumen.     

Effect of Chilling on DNA Endoreplication in Root Cortex Cells and Root Hairs of Soybean Seedlings

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C – 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C – 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C – 8C DNA nuclei predominated while in chilled plants an additional population of 2C – 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred. This revised version was published online in July 2006 with corrections to the Cover Date.