Postnatal development of alveoli. Regulation and evidence for a critical period in rats.

In many species, including humans, pulmonary alveoli are formed after birth by septal subdivision of the large gas-exchange saccules present at birth. In rats septation occurs mainly between the 4th and 14th postnatal days (Burri, P. H. 1974. Anat. Rec. 180:77-98), but little is known about the regulation of this process. We found that dexamethasone (0.1 micrograms daily) given to rats from age 4 to 13 d markedly impaired saccule septation to at least age 60 d and also diminished the extent of the increase of alveolar surface area (Sa). Underfeeding from birth to age 14 d did not diminish saccule septation but did result in diminished Sa. We conclude dexamethasone-treated rats have a critical period during which the gas-exchange saccules present at birth must be subdivided. Since Sa increased in dexamethasone-treated rats without a change in alveolar size, and, the enlargement of Sa was diminished in underfed rat pups without a deficit of saccule septation, we postulate new alveoli were formed by means other than septation of the large gas-exchange saccules present at birth. Furthermore, these various means of forming alveoli, and hence of increasing Sa, were differently regulated: dexamethasone decreased the enlargement of Sa brought about by both septation of the gas-exchange saccules present at birth and by other, as yet unidentified, means of forming alveoli; underfeeding did not diminish Sa increases produced by saccule septation but did decrease the extent of Sa enlargement due to the other means of forming alveoli.

The newly inbred cohen diabetic rat: a nonobese normolipidemic genetic model of diet-induced type 2 diabetes expressing sex differences.

The newly inbred Cohen diabetic rat is an exceptional experimental model of diet-induced type 2 diabetes mellitus that is the result of secondary inbreeding nearly 30 years after it originally had been established. Animals from the original colony were selectively inbred by stringent criteria for 10 additional generations, bringing overall inbreeding to >50 generations. The metabolic phenotypes of the resulting contrasting strains, designated as the Cohen diabetic-sensitive (CDs) and -resistant (CDr) rats, were characterized. The phenotype of the CDs strain that was fed a regular diet consisted of fasting normoglycemia, normal glucose tolerance to intraperitoneal glucose loading, normal fasting insulin levels, and a normal insulin response to glucose loading. In contrast, CDs rats that were fed a custom-prepared high-sucrose low-copper diabetogenic diet became overtly diabetic: fasting glucose levels were normal or elevated, and the blood glucose insulin response to glucose loading was markedly abnormal. CDr rats that were fed a regular or diabetogenic diet did not develop diabetes and maintained normal glucose tolerance and insulin secretion. A striking sex difference was observed in CDs rats that were fed a diabetogenic diet: males had a lower growth rate and a more severe glucose intolerance pattern than females. Gonadectomy shortly after weaning did not prevent the development of the diabetic phenotype in its early phase in either sex but markedly attenuated its expression in males at a later phase, abolishing the sex differences. Alternate-day feeding, as opposed to daily feeding, also attenuated the metabolic phenotype in males. The development of the diabetic phenotype in CDs rats that were fed a diabetogenic diet was not accompanied by obesity or hyperlipidemia. The genetic profile of the strains was established using 550 microsatellite markers evenly distributed throughout the rat genome. The rate of homozygosity within strain was > or = 96%. The rate of polymorphism between the contrasting strains was 43%. We conclude that the metabolic phenotypes of the rebred colony of CDs and CDr rats and their genetic makeup render the Cohen diabetic rat a useful experimental model that is highly suitable for studying the interaction between nutritional-metabolic environmental factors and genetic susceptibility (sensitivity and resistance) for the development of type 2 diabetes. The model is also distinctively useful for investigating the effect of sex on the expression of the diabetic phenotype.

Rat hematopoietic cell receptors for very low density lipoprotein (VLDL) growth inhibitor.

Physiologic concentrations of rat plasma very low density lipoproteins (VLDL) have profound inhibitory effects in tissue culture on the proliferation of hormonally stimulated bone marrow granulocytic and erythrocytic cells and mitogen-stimulated spleen cells. In this study we have evaluated the in vitro uptake and binding of 125I-VLDL by rat marrow cells and spleen lymphocytes. To this end, VLDL were isolated from rat plasma by sequential ultracentrifugation flotation and radioiodinated using iodine monochloride. Biological activity of 125I-VLDL was ascertained by demonstrating that 125I-VLDL and native VLDL had comparable proliferative inhibitory effects when added to erythropoietin-stimulated marrow cells and PHA-stimulated lymphocytes. After 1 h exposure of marrow to VLDL, exhaustive cell washing did not reverse the lipoprotein growth inhibitory effect. The cell uptake of VLDL was evaluated by adding 125I-VLDL to marrow or spleen cells. Uptake of 125I-VLDL by rat cells showed a preference for binding of VLDL as compared to chylomicrons, LDL, or HDL. Based on Scatchard plot analysis of 125I-VLDL binding at 37 degrees C, the approximate number of saturable VLDL receptors available per marrow or spleen cell during a 3 h incubation was 34,000 and 63,000, respectively. We conclude that rat marrow cells and lymphocytes have specific receptors for plasma VLDL and that receptor binding of VLDL is an initial step in its growth inhibitory effect. The physiologic role of plasma lipoprotein cell growth inhibitors will remain speculative, however, until the in vivo distribution of the biologically active lipoprotein moiety to extravascular sites of hematopoiesis has been determined.

Lack of proportionality between gene dosage and total muscle protein content in the rat heart.

Counting of isolated cardiomyocytes has demonstrated that their number was 16.8 +/- 0.6 10(6) in both ventricles of weanling rats (28 days after birth), growing in litters of four (fast-growing). In rats growing in litters of 16 (slow-growing), the myocyte number was 11.8 +/- 0.8 10(6). In the control group (8 sucklings per litter), there were 14.2 +/- 10(6) cardiomyocytes. The fast-growing rats had more octoploid cells than slow-growing ones. Considering ploidy and cell number, the total number of myocyte genomes in fast-growing animals was 45% higher than in slow-growing ones. The total content of contractile proteins in fast-growing weanling animals was higher by 28% while sarcoplasmic proteins were 8% higher. This lack of correspondence between the number of myocyte genomes and muscle protein content was even more pronounced at the age of 110 days. The results are compared with the cytophotometric data concerning the lack of correspondence between the total protein content in a myocyte and its DNA amount and chromosome number, i.e., total dosage of the myocyte genes.

Antibodies to growth hormone-releasing factor inhibit somatic growth.

We have raised antibodies to rat growth hormone-releasing factor (GRF) which, when acutely administered to rats, cause a complete inhibition of pulsatile GH release. Using this passive immunization technique, we have evaluated the role of GRF in normal somatic growth. Twenty-two-day-old male Sprague-Dawley rats (61 +/- 2 g, mean +/- SEM) were treated for 16 days with either normal rabbit serum (n = 7) or rabbit antiserum raised against GRF (n = 7). During this period the BW of rats treated with normal rabbit serum increased 8.3 +/- 0.4 g/day while rats treated with GRF antiserum increased 4.3 +/- 0.2 g/day (P less than 0.01). These rats were then left untreated for an additional 22 days to determine whether animals treated with GRF antiserum would demonstrate a period of catch-up growth. Rats treated with normal rabbit serum continued to increase in BW at 8.1 +/- 0.4 g/day. Remarkably, rats treated with GRF antiserum continued to have a reduced growth rate, 5.4 +/- 0.6 g/day (P less than 0.01). This reduced growth rate was not due to long-term suppression of GH, since the patterns of pulsatile secretion and concentrations of plasma GH were not different between the two groups when measured three weeks after termination of the GRF antiserum treatment. These results demonstrate that GRF has a primary role in somatic growth and suggest that its presence during postnatal development is required to ensure subsequent normal somatic growth.

Comparative maturation of the regulation of prolactin and thyrotropin by serotonin and thyrotropin-releasing hormone in male and female rats.

The aim of this study was to compare the effects of TRH, serotonin, and haloperidol on the secretion of PRL and TSH in male and female rats from birth to puberty. Serum PRL in males and females was low from birth to 20 days; it then increased gradually until puberty. TSH did not change significantly throughout the period studied. The PRL-releasing effect of serotonin became evident at 12 days, and at all times when the response occurred, it was greater in males than in females. This was also observed in adult rats in which 2.5 mg/kg ip serotonin caused an increase in serum PRL in males but not in diestrous females. Serotonin did not modify TSH at any age. On the other hand, TRH induced the release of TSH and PRL from the first day of life and no sex difference was observed. Haloperidol caused release of PRL from birth, and the effect increased significantly with age. The PRL increase induced with haloperidol was greater than that obtained with TRH used in supramaximal doses. A clear sexual difference became evident on day 20, the PRL-releasing effect of haloperidol being more pronounced in females than in males. Haloperidol did not modify TSH values. It is concluded that the mechanisms by which TRH and dopamine regulate PRL secretion mature earlier than those influenced by serotonin. Sexual differences in the manifestation of these mechanisms were observed, the male being more sensitive to serotonin and the female to haloperidol. TRH causes release of TSH from birth; the serotoninergic and dopaminergic pathways are not of paramount importance in the regulation of TSH in the prepubertal rat.

Age-dependent changes in physiological action, content, and immunostaining of inhibin in male rats.

We examined the role of endogenous inhibin in modulating FSH secretion in male rats during the infantile (days 10 to 21), juvenile (days 22 to 35), and pubertal (days 36 to 90) periods by 1) neutralization of endogenous inhibin using specific antibodies, 2) measurement of plasma and testicular levels of immunoreactive inhibin, and 3) immunohistochemical detection of testicular inhibin. In all studies, we used an antiserum against the first 26 N-terminal amino acids of the alpha-chain of porcine inhibin (anti-alpha-inhibin). Plasma immunoreactive inhibin levels were highest in young rats (8-15 days old), then decreased steadily with age. In addition, iv injection of the inhibin-alpha antiserum caused significant (P less than or equal to 0.01 or P less than or equal to 0.05) elevations in plasma FSH levels in male rats aged 10-24 days, whereas no significant (P greater than 0.05) changes occurred in older animals. In the testes, immunoreactive inhibin levels, expressed as femtomoles per mg wet weight, also declined with age. Inhibin immunostaining was most prominent in the Sertoli cells, with the greatest staining in the testes of 8 to 15-day-old rats. These results suggest that endogenous inhibin plays a physiological role in suppressing FSH secretion in infantile male rats, at an age when Sertoli cells contain the largest amount of this protein.

Intestinal microvascular adaptation during maturation of spontaneously hypertensive rats.

Adaptive microvascular changes in increased arterial pressure were investigated in the intestine of spontaneously hypertensive rats (SHR). In 4- to 5-week-old normal Wistar-Kyoto (WKY) and SHR ras, as well as in 18- to 21-week-old WKY rats, the number of arterioles of a given type per milligram of tissue were very similar. However, 18- to 21-week-old SHR had 30% to 35% fewer arterioles in the diameter range of 25--35 mu, as if intestinal vessels were lost or failed to grow during maturation. The largest and smallest arterioles in the intestine of adult SHR were constricted by 20% to 25%, but other vessels in the SHR had an equal or increased diameter relative to those in WKY rats. As a result of rarefaction and selective vasoconstriction in SHR, microvascular pressures in the intestinal muscle of SHR were near those in WKY rats, and those in villi of SHR were equal to those in WKY rats despite a 60% to 70% increase in mean arterial pressure in SHR. The percentage of small arterioles (less than 15 mu) that were intermittently closed to flow at rest was minimal, and the total number of small vessels per milligram of tissue was equal in WKY and SH rats. These data indicate that the adaptive changes in the intestinal vasculature of SHR do not include the loss of small arterioles as occurs in skeletal muscle but that the vascular branching pattern is disturbed, and the largest and smallest arterioles are constricted in the intestine of SHR.

Brain corticotropin releasing factor in the spontaneously hypertensive rat.

Corticotropin releasing factor and vasopressin were measured in major brain regions including the neurohypophysis in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) during development of hypertension. The highest concentration of corticotropin releasing factor was found in the hypothalamus in both strains. Corticotropin releasing factor was decreased in most major brain regions of SHR. In the hypothalamus, corticotropin releasing factor was lower in 3- and 6-week-old SHR than in age-matched WKY (p less than 0.01), but was similar at 12 and 24 weeks of age. The content of corticotropin releasing factor did not differ in the neurohypophysis in 3-week-old rats but began to decrease at 6 weeks of age (p less than 0.01) and continued to decrease during the development of hypertension (p less than 0.01). Brain vasopressin concentration did not differ between SHR and WKY except in the hypothalamus. The level of hypothalamic vasopressin was consistently lower in SHR than in WKY (p less than 0.01). These peptides are thought to be associated with autonomic nervous regulation, and our results may further strengthen the possibility that the deficit of the peptides may be involved in the development of spontaneous hypertension.

Developmental regulation of D-beta-hydroxybutyrate dehydrogenase in rat liver and brain.

The activities of ketone-metabolizing enzymes in rat brain increase 3- to 5-fold during the suckling period before decreasing to the adult level after weaning. We have observed that a similar developmental pattern also exists for D-beta-hydroxybutyrate dehydrogenase (BDH) in rat liver. Utilizing antibodies prepared against the purified protein we determined that the changes in BDH activities in both brain and liver are due to changes in the amount of BDH in the mitochondria. In vitro translations of isolated RNA followed by immunoprecipitation revealed that the increase in BDH activity and content was correlated with an increase in the level of functional BDH-mRNA in both liver and brain.

Gradual changes in the structure of the barrels during maturation of the primary somatosensory cortex in the rat.

The maturation of the barrel field in the primary somatosensory cortex was observed in Nissl-stained preparations from rats ranging in age from 12 days to 1.5 years postpartum. Prior to the 20th day, the barrels in the rat resemble those of the mouse and have distinct cell-sparse hollows that are surrounded by cell-dense sides. They span the full thickness of layer IV. Between the 20th and 34th days, the barrels in only the posteromedial part of the barrel field gradually change and the distinction between the hollows and sides is lost throughout all but the deepest part of layer IV. The resulting mature barrels are relatively indistinct and have a uniformly high cell density that extends well into the supragranular layers. In contrast, the barrels in the anterolateral part of the barrel field remain essentially unchanged. The remodeling apparently is not due passively to cortical growth because, by P20, the thicknesses of the cortical layers and the dimensions of the barrels are virtually the same as in the adult. Several mechanisms are considered that may account for the changes. These include a redistribution of the neurons that originally were in barrel sides; a reduction in the neuropil between the neurons that originally were within hollows; and differential growth of layer IV dendrites. The changes in the barrel structure may be related to the differentiation and quantity of innervation in the hairy skin between the vibrissae.

Postnatal loss of axons in normal rat sciatic nerve.

Myelinated and unmyelinated axons were counted in sciatic nerves of newborn, 5-day-old, 14-day-old, and adult rats. Myelinated axons increase from essentially none at birth to approximately 8,000 in adulthood, but total axon numbers decrease steadily from 33,954 at birth to 22,872 in adulthood. Thus there is a significant postnatal loss of axons from rat sciatic nerve. This loss is, in our opinion, not associated with the death of the cells that give rise to these axons. This is thus an example of a regressive event that probably is of importance in normal neural development, namely the postnatal elimination of axons unaccompanied by death of the neurons that give rise to axons. These findings presumably imply a considerable amount of proximal peripheral axon branching, and the postnatal elimination of axons in the sciatic nerve presumably results from a reduction of this branching. Thus postnatal elimination of processes on, for example, somatic muscle cells may be at least partially the result of long axon elimination rather than local withdrawal of presynaptic processes, as is usually thought to be the case. In addition, an increased number of axons resulting from early postnatal manipulations may indicate cessation of axon loss rather than formation of new axons.

Postnatal ontogeny of cells expressing prepro-neurotensin/neuromedin N mRNA in the rat forebrain and midbrain: a hybridization histochemical study involving isotope-labeled and enzyme-labeled probes.

The postnatal ontogeny of cells expressing prepro-neurotensin/neuromedin N messenger RNA (prepro-NT/NN mRNA) in the rat forebrain and midbrain was investigated by in situ hybridization histochemistry. According to the pattern of expression during development, the cells which express prepro-NT/NN mRNA can be roughly divided into 2 groups. In type I cells, prepro-NT/NN mRNA expression reaches a maximum in terms of content during the postnatal period. After this early peak, cells of this type express the same or less prepro-NT/NN mRNA, reaching a plateau at an adult level that still contains a high level of expression. In type II cells, prepro-NT/NN mRNA appears during the postnatal period, and the expression decreases dramatically after the first postnatal week, being almost undetectable by a few weeks after birth. Type I cells were observed in the following areas: the piriform cortex, field CA1 of Ammon's horn, subiculum, vertical, and horizontal limbs of the diagonal band of Broca, intermediate part of the lateral septal nucleus, bed nucleus of the stria terminalis, medial preoptic area, lateral hypothalamus, caudal part of the caudate putamen, medial, cortical, and central amygdaloid nuclei, ventral tegmental area, deep mesencephalic nucleus, cuneiform nucleus, dorsal raphe nucleus, laterodorsal tegmental nucleus, parabrachial nucleus, and oral part of the pontine reticular nucleus. Cells of type II were observed in the following areas: the mitral cell layer of the olfactory bulb, rostral part of the caudate putamen, (anterior) cingulate cortex, and retrosplenial cortex (posterior cingulate cortex).

Maturation of rat visual cortex: IV. The generation, migration, morphogenesis, and connectivity of atypically oriented pyramidal neurons.

The generation, migration, and morphogenesis of atypically oriented pyramidal neurons in the rat visual cortex were examined. In the mature cortex, these neurons were distributed through layers II-VI. Moreover, the atypically oriented pyramidal neurons in a particular layer tended to be oriented in a specific way; atypically oriented pyramidal neurons in layer II, layers III-VIa, and layer VIb were obliquely, radially, and obliquely oriented, respectively. Ultrastructurally, the somata of atypically oriented pyramidal neurons contained large euchromatic ovoid nuclei and cytoplasm that was replete with rough endoplasmic reticulum and Golgi apparatus. These somata formed only symmetric axosomatic synapses. Many atypically oriented pyramidal neurons projected axons into the white matter as demonstrated by a Golgi method and by a retrograde tract-tracing technique; however, some of these pyramidal neurons in layers III-V had axons that ascended to layer I. By using a technique which combined retrograde tract tracing with [3H]thymidine autoradiography, it was determined that most atypically oriented pyramidal neurons in layers V and VIa, layer IV, and layer II were generated on gestational days (GD) 15-17, GD 17-19, and GD 20-21, respectively. Atypically oreinted pyramidal neurons were identified during the period from postnatal day 0 (day of birth) to day 30. On day 0, obliquely oriented pyramidal neurons were distributed in the deep cortical plate, i.e., immature layer VI. On day 3, the youngest atypically oriented pyramidal neurons were radially oriented and were located in layer IV. Some obliquely oriented pyramidal neurons were present in layer II on day 6, but the greatest number and the most severely canted pyramidal neurons in layer II were evident on day 9. The orientations of the cell body and the apical dendrite did not change appreciably after migration was complete, except for those in layers V and VI with obliquely oriented cell bodies and radially oriented apical dendrites. The second and third postnatal weeks were marked by substantial morphological differentiation of all pyramidal neurons as noted by the lengthening and branching of dendrites and by the appearance of dendritic spines. By the fourth postnatal week, atypically oriented pyramidal neurons achieved their mature morphology. The generation, migration, and morphogenesis of atypically oriented pyramidal neurons proceed by an inside-to-outside sequence. This development is similar and concurrent with that of typically oriented pyramidal neurons.

Ontogeny of gene expression of adenosine A2 receptor in the striatum: early localization in the patch compartment.

The ontogeny of adenosine A2 receptor mRNA and adenosine A2 binding sites distributions was studied by in situ hybridization histochemistry and receptor autoradiography in pre- and post-natal rat striatum, postnatal dog striatum, and a human fetus striatum and compared to that of dopamine D1 and mu opiate receptors. The early postnatal striatum demonstrated heterogeneous distributions of adenosine A2 receptor mRNA and adenosine A2 binding sites with patches of dense labeling corresponding to dopamine D1 and mu opiate receptors enriched zones. This patchy pattern evolved to the homogeneous distribution observed in the adult. The higher intensity of adenosine A2 receptor mRNA enriched patches correspond at the microscopical level to a higher density of labeled neurons in the patches areas and also to a higher level of expression per labeled patches neuron than in the matrix ones. This demonstrates for the first time that differences in patch/matrix receptor density is at least partly linked to different levels of receptor gene expression.

Distribution of two splice variants of the glutamate transporter GLT-1 in the developing rat retina.

The distributions of a carboxyl terminal splice variant of the glutamate transporter GLT-1, referred to as GLT-1B, and the carboxyl terminus of the originally described variant of GLT-1, referred to hereafter as GLT-1 alpha, were examined using specific antisera. GLT-1B was present in the retina at very early developmental stages. Labelling was demonstrable at embryonic day 14, and strong labelling was evident by embryonic day 18. Such labelling was initially restricted to populations of cone photoreceptors, the processes of which extended through the entire thickness of the retina and appeared to make contact with the retinal ganglion cells. During postnatal development the GLT-1B-positive photoreceptor processes retracted to form the outer plexiform layer, and around postnatal day 7, GLT-1B-immunoreactive bipolar cells appeared. The pattern of labelling of bipolar cell processes within the inner plexiform layer changed during postnatal development. Two strata of strongly immunoreactive terminals were initially evident in the inner plexiform layer, but by adulthood these two bands were no longer evident and labelling was restricted to the somata and processes (but not synaptic terminals) of the bipolar cells, as well as the somata, processes, and terminals of cone photoreceptors. By contrast, GLT-1 alpha appeared late in postnatal development and was restricted mainly to a population of amacrine cells, although transient labelling was also associated with punctate elements in the outer plexiform layer, which may represent photoreceptor terminals.

Spatial and temporal patterns of oligodendrocyte differentiation in rat cerebrum and cerebellum.

Oligodendrocytes are largely generated postnatally during mammalian CNS development. We have used a variety of antibodies to label immature neuroectodermal cells and developing oligodendrocytes in several areas of the rat CNS. Antibodies included those to GD3 ganglioside, a characteristic glycolipid of immature cells; carbonic anhydrase (CA), contained primarily in oligodendrocytes; and galactocerebroside and myelin basic protein, myelin components. Several aspects of oligodendrocyte development were examined: changes in shapes of immature cells with respect to time and to location within the brain, the sequential acquisition of the various markers, and possible sites of origin and pathways of precursor cell migration. Our observations suggest that oligodendrocytes in the forebrain and cerebellum arise from cells of the subventricular zone (SVZ) adjacent to the ventricles and migrate into and through nearby white and gray matter. During maturation, there are distinct patterns of morphological changes that correlate with time, locations of the cells in the brain, and acquisition of specific markers.

Tissue coenzyme Q (ubiquinone) and protein concentrations over the life span of the laboratory rat.

The coenzyme Q (ubiquinone) concentrations of a number of tissues have been determined over the life span of the male laboratory rat. Coenzyme Q increased between 2 and 18 months and decreased significantly at 25 months in the heart and kidney, and the gastrocnemius, oblique and deep aspect (red) vastus lateralis muscles. The coenzyme Q concentration of liver increased over the life span, while it remained relatively constant in brain, lung, and the superficial aspect (white) of the vastus lateralis muscle. Data are also included for organ weights and protein contents of tissues over the life span. The various roles of coenzyme Q in cellular electron transfer and its regulation, energy conservation in oxidative phosphorylation, and its clinical efficacy in diseases of energy metabolism are discussed. It is hypothesized that coenzyme Q serves as a free radical quencher in the mitochondrion, a major site of free radical formation, in addition to its other roles in cellular energy metabolism, and that its cellular diminution may contribute to the loss of cellular function accompanying ageing.

Albumin elimination in female WAG/Rij rats with age: a longitudinal study.

A longitudinal study was performed to examine total albumin elimination and urinary albumin excretion in the female WAG/Rij rat. Complete necropsies were performed following the spontaneous death of the animals. The survival characteristics of this group was similar to that of survival cohorts. An increase in total albumin elimination, urinary protein excretion and urinary albumin excretion was observed with age. A proportional increase in the contribution of albumin to the urinary protein excretion was also observed. However, the observed increase in urinary albumin excretion could not totally account for the increase in total albumin elimination. The predominant kidney lesion was chronic progressive nephrosis. The histological severity of the renal lesions were closely correlated with the increase in urinary albumin and total protein loss. It is concluded that the increase in total albumin elimination in rats in this study was due to age-related changes and not to cohort effects.

Studies of sequence heterogeneity of mitochondrial DNA from rat and mouse tissues: evidence for an increased frequency of deletions/additions with aging.

To obtain information on the extent of random nucleotide changes in mitochondrial DNA (mtDNA) from different organs of young adult and senescent Fischer 344 rats, the temperature of thermal denaturation (tm) was measured in (1) the native mtDNA cut at a single SstI site and (2) the reannealed duplexes formed after the initial melting of the mtDNA sample. No change was found between the two tm values in either young or senescent mtDNA, suggesting that the overall mismatch in nucleotide sequence in these samples was below the resolution of the method estimated at about 0.2%. In another experiment, mtDNA samples from young adult or senescent BALB/c mouse liver were digested with EcoRI, denatured and allowed to reanneal. The duplexes formed by the 14-kb EcoRI fragment were analyzed in randomly taken electron micrographs for the occurrence of mismatched segments. About 1.8% of reconstituted duplexes in adult mtDNA and 11% of those in senescent mtDNA contained small loops or knobs suggestive of deletions/additions of about 400 +/- 150 nucleotides. These data correspond to about 1% of the native mtDNA population in adult liver and about 5% in senescent liver having deleted/inserted segments. Although deletions/insertions may occur at variable sites, their distribution appears to be non-random. These findings suggest that small sequence rearrangements, which have been observed previously in unicircular dimers of mouse and human mtDNA, occur also in monomeric mtDNA from normal tissues and accumulate with aging.