Dietary fat up-regulates the apolipoprotein E mRNA level in the Zucker lean rat brain.

High-fat diet alters apo E-dependent processing of beta-amyloid precursor protein. Here we have evaluated the effects of dietary fat on brain apo E mRNA in Zucker lean and obese rats. After approximately 2 months on a high-fat diet, there was significant up-regulation of brain apo E mRNA in the Zucker lean rat in parallel with weight gain. Densitometric quantification revealed a 17% increase in apo E mRNA in the brains of lean rats fed high-fat diet compared with those of lean rats fed rat chow. No significant difference in brain apo E mRNA of Zucker obese rats fed different diets was found. These results suggest that dietary fat alters brain apo E levels, which may be regulated, in part, through the leptin receptor.

Localization of leptin receptor immunoreactivity in the lean and obese Zucker rat brain.

Leptin, a product of the obese (ob) gene, is secreted by adipocytes and appears to act as a hormone to regulate food intake, metabolism and body weight. Subcutaneous administration of leptin causes reductions in food intake and body and fat-depot weights in both lean and genetically obese (ob/ob) mice, and leptin infusion into the lateral cerebral ventricles decreases feeding with short latency, suggesting a central site of action. A gene defect in the Zucker obese rat causes an amino acid substitution in the leptin receptor and reduced leptin binding at the cell surface. An antiserum to a portion of the mouse leptin receptor (AA 877-894) located within the intracellular domain was used to label Zucker lean (Fa/?) and obese (fa/fa) rat brain sections. At optimal dilution (1:8000), only cells in the basal forebrain, preoptic area, hypothalamus and brainstem were moderately or intensely labeled. The most intensely-labeled nuclei, the anterior commissural, magnocellular paraventricular, supraoptic, circularis in the anterior hypothalamus and fornical in the lateral hypothalamus contain large neurons that synthesize and secrete vasopressin or oxytocin and their respective neurophysins. Diminished leptin transport into the central nervous system or defective signal transduction in Zucker obese rats may sufficiently compromise leptin regulation of the HPA axis, NPY-immunoreactive neurons or other hypothalamic elements to cause obesity.

Transplantation of lean fetal hypothalamus restores hypothalamic function in Zucker obese rats.

Rats with lesions to the ventromedial hypothalamus (VMH) manifest obesity, hyperphagia, and hyperinsulinemia, and fetal VMH transplantation into the third cerebroventricle of VMH-lesioned rats reduces the development of obesity caused by the lesion. The aim of this study was to determine whether the hyperphagia, hyperlipidemia, and hyperinsulinemia of obsese Zucker rats could be corrected by the transplantation of lean fetal Zucker hypothalamic tissue into the third cerebral ventricle of Zucker obese rats. After the fetal hypothalamic transplant (obese-HY), the rate of weight gain was significantly diminished compared with the unoperated Zucker obese rats and the obese rats that received the transplantation of a similar amount of frontal cortical tissue from the same fetus (obese-FC). Food intake was significantly lower, and plasma triacylglycerol and insulin concentrations were also significantly reduced in the obese-HY rats compared with the obese and obese-FC rats. The weight of the adrenal glands, the plasma adrenocorticotropic hormone concentration, the liver weight, and the liver lipid content in obese-HY were significantly less than those observed in the obese and obese-FC animals. There were no significant differences between the obese and the obese-FC animals or between unoperated Zucker lean rats and lean rats transplanted with lean fetal hypothalamus in all the parameters we determined in this study. Neovascularization and normal cellular morphology of the transplanted fetal hypothalamic tissue suggest that the transplanted neural and glial cells were viable and physiologically functional. In conclusion, this study offers evidence suggesting that the hypothalamic-pituitary-adrenal function is defective in Zucker obese rats, resulting in excessive weight gain, hyperphagia, hyperlipidemia, and hyperinsulinemia. The hypothalamic dysfunction in the Zucker obese rats is corrected by the transplantation of lean fetal hypothalamus.

Immunoreactivity for apolipoprotein A-IV in tanycytes and astrocytes of rat brain.

Immunoperoxidase and immunofluorescence procedures were used to visualize polyclonal antiserum binding to apolipoprotein (apo) A-IV in rat brain. With both methods, tanycytes and astrocytes were labeled throughout both white and gray matter. Within the cells, the labeling was granular and it was confined to the perinuclear zone and proximal regions of the processes. The labeling was abolished by absorption of the primary antiserum with purified apo A-IV but not by absorption with apo E. These results suggest either that apo A-IV is synthesized by astrocytes, or that apolipoprotein that is synthesized in the small intestine or liver is selectively taken up and stored by the astrocytes.

Interactions between serotonin, thyrotropin-releasing hormone, and substance P in the CNS regulation of adrenocortical secretion.

Double-labeling immunohistochemical studies were performed to discern the morphological relationships between corticotropin-releasing factor-immunoreactive (CRF-ir) perikarya and afferent innervation in the hypothalamic paraventricular nucleus (PVN) of the rat. Attention was focussed on the local innervation by serotonin (5-hydroxytryptamine, 5-HT), thyrotropin-releasing hormone (TRH) and substance P (SP)-ir nerve terminal fibers. 5-HT-ir and SP-ir fibers were present in moderate numbers, in close apposition with CRF-ir perikarya. Sparse TRH-ir fibers were observed, but a population of TRH-ir perikarya was found in proximity with the CRF-ir cell bodies. TRH-ir perikarya in the PVN were surrounded by both 5-HT- and SP-ir fibers. Neuroendocrine studies were performed to investigate the interactions between 5-HT, TRH and SP in the regulation of hypothalamo-pituitary-adrenocortical (HPA) secretion. Male rats were prepared bearing cannulae for intracerebroventricular (ICV) or intra-PVN administration of drugs. 5-HT, at all doses tested (0.1, 40, or 80 nmol, ICV), caused increases in plasma corticosterone (CS) concentrations in tail-vein blood collected 20 min after injection. ICV injections of TRH caused dose-dependent increases in plasma CS, but did not further increase HPA responses when injected together with 5-HT. SP alone had little effect, although a significant reduction in plasma CS concentrations was observed in several individual experiments. However, SP (0.1 nmol) significantly attenuated CS responses following high doses of 5-HT (40 and 80 nmol, ICV), although the response to 0.1 nmol 5-HT was not affected. Combined injection of SP with TRH resulted in HPA responses not different from those following TRH alone. Similarly, SP did not reduce the HPA response observed with TRH and 40 nmol 5-HT in combination. Intra-PVN injections of 5-HT (0.1 or 40 nmol) and TRH also increased plasma CS concentrations. Intra-PVN injections of SP had little effect on plasma CS concentrations although a tendency toward a decrease in plasma CS was observed, as with the ICV injections. Combined intra-PVN injection of 5-HT (0.1 nmol) with TRH (0.1 nmol) did not significantly alter the response compared with that observed following TRH alone, although plasma CS concentrations were greater than with 0.1 nmol 5-HT. Combined intra-PVN injections of SP (0.1 nmol) with 5-HT (0.1 nmol) resulted in a significant decrease in plasma CS concentration compared with that following 5-HT alone, but SP did not prevent the CS response to a higher dose of 5-HT (40 nmol).(ABSTRACT TRUNCATED AT 400 WORDS)

Transitory noradrenergic and peptidergic nerves in the cat kidney.

Indirect immunohistochemical methods were used to visualize nerves immunoreactive for tyrosine hydroxylase (TH), dopamine beta hydroxylase (DBH), neuropeptide Y, (NPY) and calcitonin gene-related peptide (CGRP) in sections of the kidneys of cats of different ages. Nerve terminals immunoreactive for TH, DBH and NPY innervated interlobar veins and the renal arterial tree including medullary vascular bundles of cats of each age studied. Most nerve terminals immunoreactive for CGRP innervated interlobar arteries. In kidneys of cats 2 to 10 weeks old, TH- and DBH-immunoreactive axons formed elaborate plexuses that were distributed throughout much of the outer two thirds of the inner medulla. Inner medullary NPY-immunoreactive nerve terminals formed sparse plexuses by comparison, thus suggesting a large population of TH-immunoreactive nerve terminals not immunoreactive for NPY. Plexuses immunoreactive for CGRP also innervated the inner medullae of young cats. Some inner medullary axons appeared degenerate in 8 and 10 week old cats, and no inner medullary nerve terminal plexuses were visualized in 12 week old or adult cats. Cell death or paring of axons resulting from mechanisms intrinsic to the neuronal population or from a change in trophic factors secreted or expressed by cells in the medulla may effect the loss of inner medullary nerve terminals in the kidneys of young cats.

Spinal neurogenesis and axon projection: a correlative study in the rat.

The purpose of the present study was to determine the relationship between the duration of a spinal neuron's neurogenic period and the length of its axon or level of projection. Spinal segment L1 was chosen for examination and neurons were divided into four projection groups: 1) supraspinal projection (SSp), 2) long ascending propriospinal (LAPr), 3) short ascending propriospinal (SAPr), and 4) descending propriospinal (DPr). To determine the duration of the neurogenic period for each group, 3H-thymidine was administered to fetal rats during the proliferative period for spinal neuroblasts on one of embryonic (E) days E13 through E16. Between 50 and 100 days after birth neurons in each group were labeled with the retrograde fluorescent tracer Fluoro-Gold. To demonstrate nerve cells with SSp projections, spinal cords were hemisected at spinal segment C3 in one group of animals and Fluoro-Gold was applied to the sectioned surface of the cord. Three additional sets of animals were used to label nerve cells with LAPr, SAPr, and DPr projections by injecting Fluoro-Gold into the gray matter at spinal segments C6, T12, and L5, respectively. Neurons labeled with both Fluoro-Gold and 3H-thymidine and neurons labeled with Fluoro-Gold alone in each animal in each group were counted and the data statistically analyzed. Results showed that within each spinal lamina neurons with different projections were generated, i.e., completed cell division, at significantly different rates. Neurons with the longest axons, those with SSP projections, were generated first. These were followed by those with LAPr projections, and finally those with SAPr and DPr projections. In most laminate there was no significant difference between the neurogenic periods of rostrally projecting short propriospinal (SAPr) neurons versus caudally projecting short propriospinal (DPr) neurons. It was concluded that the duration of the neurogenic period for a given group of neurons within each spinal lamina is inversely related to the distance between the nerve cell and its projection site regardless of the direction of its projection.

A study of catecholamine concentrations in selected renal segments of cats of different ages.

Renal concentrations of the catecholamine neurotransmitters norepinephrine (NE) and dopamine (DA) in 19 cats were measured by reverse-phase high pressure liquid chromatography (HPLC) with electrochemical detection. Animals of five different age groups (2-4, 6-8, 10-12 and 16-20 weeks and adult animals one year and older) were anesthetized and the kidneys were excised, sectioned into cortical, outer medullary and inner medullary segments, and processed for HPLC. There were no statistical differences in cortical NE concentrations between the 2-4 week and adult age groups, suggesting that peak noradrenergic nerve terminal density in the cortex is achieved at or before 2-4 weeks of age. NE concentration in the outer medulla and DA concentration in the cortex and outer medulla increased from the 2-4 to the 10-12 week age group but did not change significantly in the older animals. There were significant decreases in inner medullary NE and DA concentrations from the 10-12-week to the 16-20 week age group. The decrease in concentration of inner medullary catecholamines in the early postnatal period is consistent with the reported total loss of tyrosine hydroxylase- and dopamine beta hydroxylase-immunoreactive inner medullary nerve terminals at approximately 12 weeks of age.

Neurogenesis of ascending supraspinal projection neurons: ipsi- versus contralateral projections.

The present study tests the hypothesis that contralaterally projecting supraspinal projection neurons (SPNs) are generated prior to ipsilaterally projecting SPNs. Neuronal time of origin was determined by injecting pregnant rats with tritiated thymidine on one of embryonic (E) days E12 through E15. In mature offspring of thymidine-treated dams, SPNs in the lumbar cord were retrogradely labelled with True Blue delivered at the site of a hemisection in spinal segment C3. Ipsi and contralaterally projecting SPNs in laminae I, VII and VIII and the lateral spinal nucleus, which are known to give rise to long sensory pathways, were generated simultaneously throughout their neurogenic period (E12-E14), while ipsilaterally projecting SPNs in lamina IV and the nucleus dorsalis, which give rise to short sensory pathways, completed neurogenesis one day later (E15). Results suggest that the projection target and its distance from the nerve cell body of origin are more consistent correlates of the duration of the neurogenic period than the course of the axon.

Calcitonin gene-related peptide-immunoreactive nerves in the rat kidney.

Cryostat- and vibratome-cut rat kidney secretions were singly or doubly labeled to visualize immunoreactive calcitonin-gene-related peptide (CGRPI) and substance P (SPI). Rats were perfused with 2-4% paraformaldehyde + 0.15% picric acid then rinsed with buffer. Horseradish peroxidase (HRP) was used to visualize CGRP in vibratome sections, and combined HRP and fluorophore were used to visualize the two peptides simultaneously in cryostat sections. There is a complex, multilayered plexus of CGRP nerves on the renal pelvis and a less dense, single-layered plexus on the major branches of the renal artery and on interlobar arteries and veins. A few axons innervate finer branches of the arterial tree and other intrarenal structures. Results of double immunolabeling suggest that SPI axons comprise a subpopulation of the CGRP axon population in the rat kidney. There was no evidence for a separate population of SPI axons.

Transitory inner medullary nerve terminals in the cat kidney.

An indirect immunohistochemical method was used to visualize nerves immunoreactive for tyrosine hydroxylase (THI) and dopamine-beta-hydroxylase (DBHI) in kidney sections of cats 6 weeks and 2 and 3 months of age. THI and DBHI nerve terminals innervate the renal pelvis, interlobar veins and arterial tree including medullary vascular bundles of cats of each age studied. In kidneys of 6-week-old cats, THI and DBHI axons form elaborate plexuses that are distributed throughout much of the inner medulla, whereas some medullary axons appear to degenerate at 2 months and no inner medullary plexuses were visualized in 3-month-old cats. Transitory inner medullary nerves in the cat kidney may influence cellular development and play a role in salt and water balance.

Neurogenic period of ascending tract neurons in the upper lumbar spinal cord of the rat.

Although the neurogenic period for neurons in the lumbar spinal cord has been clearly established (Days 12 through 16 of gestation), it is not known when the neurogenesis of ascending tract neurons is completed within this period. The purpose of the present study was to determine the duration of the neurogenic period for projection neurons of the ascending tracts. To label neurons undergoing mitosis during this period, tritiated thymidine was administered to fetal rats on Embryonic (E) Days E13 through E16 of gestation. Ascending tract neurons of the lumbar cord were later (Postnatal Days 40-50) labeled in each animal with a retrograde tracer, Fluoro-Gold, applied at the site of a hemisection at spinal cord segment C3. Ascending tract neurons which were undergoing mitosis in the upper lumbar cord were double labeled, i.e., labeled with both tritiated thymidine and Fluoro-Gold. On Day E13, 89-92% of the ascending tract neurons were double labeled; on Day E14, 35-37%; and on Day E15, 1-4%. Results showed, then, that some ascending tract neurons were double labeled through Day E15 and were, therefore, proliferating in the final one-third of the neurogenic period. Ascending tract neurons proliferating on Day E15 were confined to laminae III, IV, V, and X and the nucleus dorsalis. Long tract neurons in the superficial dorsal horn (laminae I and II), on the other hand, were found to have completed neurogenesis on Day E14 of gestation. Results of the present study show that spinal neurogenesis of ascending projection neurons continues throughout most of the neurogenic period and does not completely follow the well-established ventral to dorsal gradient.

Nerve cell bodies in the dorsal funiculus of the rat spinal cord.

Nerve cell bodies located within the white matter of the dorsal funiculus (DF neurons) have been previously observed but not described in detail. The present study examines the morphology, ontogeny, and projection of DF neurons utilizing Fluoro-Gold as a retrograde tracer, alone, and in combination with tritiated thymidine autoradiography in the spinal cord of the rat. DF neurons were consistently labelled in spinal segments T13 through L2 following injections of Fluoro-Gold into the cerebellum. The cell bodies of DF neurons were small to medium in size, fusiform to multipolar in shape, and were located on the side ipsilateral to the injection site. Cell counts revealed approximately five labelled cells per millimeter along the longitudinal axis. An examination of neurogenesis using tritiated thymidine combined with Fluoro-Gold showed that DF neurons have relatively late birthdates as do other spinocerebellar neurons of the dorsal horn. Retrograde axon tracing studies in the spinal cord using Fluoro-Gold showed that DF neurons project rostrally via the ipsilateral lateral funiculus. The significance of the presence of nerve cells in the dorsal funiculus is unclear, but judging from their location, ontogeny, and projection, DF neurons are probably derived from the same pool of neurons as those in the Nucleus dorsalis.

Neurons with asymmetrical dendritic arbors in the substantia gelatinosa of the rat spinal cord.

Two types of neurons were observed in the substantia gelatinosa (SG) of the rat spinal cord which exhibit wide variations in dendritic symmetry. As demonstrated with the Golgi technique, "islet" cells with short dendritic arbors and "type III stalk" cells display dendritic patterns which vary from a bipolar type arrangement with two dendritic arbors of nearly equal dimensions to a unipolar arrangement with a dendritic arbor which extends in only one direction. Examination of the morphology and dendritic development of these neurons shows that they are unique compared with other SG neurons in that they have short, longitudinal dendritic arbors which undergo maturation relatively late in the postnatal period. As is discussed, variations in dendritic symmetry are probably dependent on the location of the terminal fields of primary and/or other types of afferents which are formed earlier in development.

Catecholamine-containing, dopamine-beta-hydroxylase-immunoreactive perivascular nerve specializations in the rat kidney.

Fluorescence histochemical visualization of catecholamines and immunolabeling of dopamine beta hydroxylase (DBH) were employed to study noradrenergic nerve terminals and perivascular nerve specializations in the rat kidney. Plexuses of catecholamine-containing and dopamine-beta-hydroxylase-immunoreactive nerves innervate the intrarenal arterial tree and larger intrarenal veins. Some perivascular nerve bundles have specialized segments composed of clusters of axonal enlargements that are immunoreactive for DBH and fluoresce intensely in ultraviolet light after fixation in a solution of formaldehyde and glutaraldehyde or treatment with glyoxylic acid. No fluorescent neural structures were found in denervated rat kidney sections treated with glyoxylic acid. Many such structures are associated with arteriolar branches of interlobar, arcuate, and interlobular arteries and are composed, in part, of axonal enlargements that contain mitochondria, microtubules, and one or more clusters of synaptic vesicles. These perivascular nerve specializations may be sites of axoaxonal interactions between noradrenergic axons or between these axons and other types of autonomic or sensory axons. The synaptic vesicles evidently store large amounts of catecholamine, but there is no evidence whether it is released into the surrounding tissue. These structures may be involved in changes in intrarenal innervation patterns which may occur as the rat ages. Regardless of the autonomic or sensory nature of intrarenal neural structures, close association of most such structures with arterioles suggests some neurovascular interaction.

Identification of noradrenergic nerve terminals immunoreactive for neuropeptide Y and vasoactive intestinal peptide in the rat kidney.

Cryostat- and vibratome-cut sections of rat kidneys were singly or doubly labeled to visualize immunoreactive tyrosine hydroxylase (THI), dopamine beta-hydroxylase (DBHI), vasoactive intestinal peptide (VIPI), and neuropeptide Y (NPYI). Rats were perfusion fixed with 2-4% paraformaldehyde with or without 0.15% picric acid and rinsed in buffer for 18-48 hr. Single antigens were labeled with horseradish peroxidase in vibratome sections, whereas cryostat sections were used to label one antigen with peroxidase and another with a fluorophore in the same tissue section. A dense plexus of DBHI noradrenergic nerves innervates the renal arterial tree, and such nerves innervate the interlobar veins and renal calyx as well. Immunoreactive NPY is colocalized in most of these nerves, but some intrarenal noradrenergic nerves do not contain NPY but do contain VIP immunoreactivity. The distribution of NPYI nerves resembles that of DBHI nerves, whereas most perivascular noradrenergic nerves immunoreactive for VIP innervate selected arcuate and interlobular arteries. A small population of nonadrenergic, VIPI nerves innervates the renal calyx.

A simple method for combining HRP-TMB histochemistry with tritiated thymidine autoradiography on the same tissue section.

In order to determine the sequence of development of various types of spinal neurons defined by their projection, we have developed a method for combining tritiated thymidine autoradiography for birthdate determination, with the demonstration of retrogradely transported horseradish peroxidase (HRP) using tetramethylbenzidine (TMB) as the chromogen. Because of its greater sensitivity, TMB is the chromogen of choice for the demonstration of HRP. However, the HRP-TMB reaction product is unstable and completely destroyed when the tissue is processed for autoradiography. The present study describes the use of osmium tetroxide as a postreaction stabilizing agent which preserves the HRP-TMB reaction product in the form of a dark brown precipitate which is not destroyed when the tissue is subsequently processed for autoradiography. Background levels of autoradiographs stabilized with the osmium procedure are extremely low.

Morphological and developmental characterization of local-circuit neurons in lamina III of the rat spinal cord.

Using the Golgi silver impregnation technique the present study examines the morphology and development of presumptive local circuit neurons in lamina III of the rat lumbar spinal cord. These neurons generate local axonal plexuses which remain within the gray matter and dendritic trees which arborize in lamina III and the inner zone of lamina II. Analysis of developmental stages supports the contention that these neurons have local axons which do not enter the white matter. These cells undergo axonal and dendritic maturation during the postnatal period, well after the maturation of long axon neurons. This pattern parallels the development of local circuit neurons in lamina II.

Structure and development of central arborizations of hair follicle primary afferent fibers.

The present study describes the structure and development of the flame-shaped central arborizations of hair follicle (HF) afferents in the lumbosacral spinal cord of the rat. Tissue was processed according to the rapid Golgi method at successive stages of development from embryonic day 17 through postnatal day 30. Collaterals of most HF afferents were found to enter the dorsal horn via a characteristic U-shaped pathway which often parallels the vascular pattern. The HF collaterals can first be identified at embryonic day 19 and by postnatal day 5 they have established the dorso-ventral and rostro-caudal limits of their field of arborization. Dorsally the arbors extend no further than the inner zone of lamina II (IIi) at any stage of development. Short aberrant branches were observed on some HF collaterals during the prenatal period but none of these developed synaptic terminals or contributed branches to other arbors. Each HF collateral formed a single well defined flame-shaped arbor with a distribution and branching pattern which could be distinguished from that of other afferents throughout the postnatal developmental period. Two types of HF collaterals were observed. Structure and distribution patterns suggest that type I collaterals are derived from G and T HF afferents while collaterals in the type II category are probably derived from both G and T as well as D (A-delta) HF afferents. Type I collaterals divide into well defined medial and lateral collateral branches which arborize mainly in lamina III with a few branches to lamina IV. Type II collaterals are characterized by a delicate arbor which is more vertically oriented than type I arbors.(ABSTRACT TRUNCATED AT 250 WORDS)

Substance P-immunoreactive nerves in the rat kidney.

An indirect immunohistochemical method in which an avidin-biotinylated horseradish peroxidase complex is bound to the secondary antibody was used to visualize substance P-immunoreactive (SPI) nerves in the rat kidney. Rats were perfused with 2% paraformaldehyde + 0.15% picric acid in 0.1 M phosphate buffer, then transferred to the buffer. After 24-48 h, the kidneys were sectioned with a Vibratome at 200 or 300 micron and incubated in the primary antiserum for 18 h at room temperature. A dense plexus of SPI nerves innervates the rat renal calyx. A small proportion of intrarenal SPI axons innervates interlobular arteries and afferent arterioles, but most perivascular SPI axons terminate on interlobar and arcuate arteries. The densest plexuses are located on segments of interlobar arteries near the hilus of the kidney. Some of these axons probably are nociceptive; others may be chemo- or baroreceptive.