John T. Hansen

Dopamine β-hydroxylase-like immunoreactivity in the rat and cat carotid body: a light and electron microscopic study

SummaryImmunocytochemical localization of dopamine β-hydroxylase (DBH) was used to study the synthesis and storage sites of norepinephrine (noradrenaline) in the rat and cat carotid bodies. In the rat carotid body some parenchymal cells exhibited strong DBH-like immunoreactivity (DBH-I), while others displayed only faint DBH-I. In a typical parenchymal cell cluster, most cells with strong DBH-I were irregular in shape and appeared to partially surround those with weak DBH-I which usually were rounded in contour. In the cat carotid body most parenchymal cells showed a strong to moderate DBH-I. In both the rat and cat carotid bodies varicose nerve fibres with DBH-I were associated primarily with blood vessels. All autonomic ganglion cells examined, which were associated with the rat carotid body, showed DBH-I. Electron microscopy revealed that most DBH-I in the strongly positive cells of the rat carotid body was associated with dense granules (possibly corresponding to dense-cored vesicles of various sizes), although some was found in other sites. In oval cells with less DBH-I, reactivity resided in some of the large granules. In the cat carotid body the glomus cells contained more granules of various sizes and shapes than did those of the rat carotid body. Most of the cat glomus cell granules exhibited DBH-I activity. Our results indicate that some of glomus cells in the rat and most of the glomus cells in the cat contain DBH and therefore may be sites of norepinephrine synthesis.

Day-night differences in the number of pineal “synaptic” ribbons in two diurnal rodents, the chipmunk (Tamias striatus) and the ground squirrel (Spermophilus richardsonii)

SummaryDaytime numbers of pineal “synaptic” ribbons higher than reported in the pineal gland of any other mammalian species were observed in two diurnal rodents, the eastern chipmunk and Richardsons ground squirrel. The number of “synaptic” ribbons was lower during the daytime and higher at night in both of these species.

Rat carotid body catecholamines determined by high performance liquid chromatography with electrochemical detection

Abstract Catecholamine contents in the rat carotid body were determined using high performance liquid chromatography with electrochemical detection (LCEC). Dopamine was found to be the predominant catecholamine present, representing about 53% of all catecholamines in the carotid body. Norepinephrine accounted for about 36% and epinephrine for about 10% of total carotid body catecholamines.

Innervation of the rat aortic (subclavian) body: An ultrastructural study following axonal degeneration

The rat aortic (subclavian) body was studied to determine the type (afferent or efferent), source, and characteristics of the nerves which innervate these arterial chemoreceptors. To determine whether the nerve endings adjacent to the glomus cells were afferent (sensory) or efferent, axonal degeneration experiments were performed in which the vagus nerve was sectiones either above or below the nodose ganglion. The results of these studies confirm that almost all of the nerves are afferent axons whose nerve cell bodies are located in the nodose ganglion. Additionally, following injections of 6-OHDA, no axonal endings could be identified which were sympathetic postganglionic in origin. The few nerve endings which did not degenerate (∼5%) may be preganglionic sympathetic fibers from the inferior cervical ganglion. Synaptic connections between nerve endings and glomus cells include afferent nerve endings which are postsynaptic to glomus cells, a few presynaptic to glomus cells are rarely, some which form reciprocal synapses. All of the axonal endings are characterized by small clear-core synaptic vesicles (diameter = 45.4 nm) and a few larger dense-core vesicles (diameter = 74.2 nm). Glomus cells of the rat aortic body are characterized by an abundance of dense-core vesicles which average 109.6 nm in diameter. Glomus cells appeared to be of only one variety and were observed in synaptic contacty with one another. The results of this study demonstrate that there are differences between the aortic and carotid bodies, especially with respect to the sophistication of their synaptic connections. These morphological differences may be related to several of the known physiological differences between these chemoreceptors.

Early ultrastructural changes in the myocardium following thyroxine-induced hypertrophy.

SummaryThe model of myocardial hypertrophy induced by thyroxine was studied with particular regard to the early ultrastructural changes in fractional volume of the mitochondria and myofibrils, and capillary distribution. Following injections of L-thyroxine (25 mg/kg IP) for 9 consecutive days, rats were sacrificed by vascular perfusion and cardiac tissue samples from the mid-wall zone of the left ventricle were processed routinely for electron microscopy. Heart weight/body weight ratios of thyroxine treated (T) rats showed a significant increase (P<0.001) over the ratios in control (C) rats. Likewise, the fractional volume of mitochondria (42%) was significantly increased (P<0.001) in the myocardium of T rats when compared with C rats (31%). However, the fractional volume of myofibrils was significantly decreased in the myocardium of T rats (P<0.001) and there was no significant difference between the hearts of T and C rats with respect to capillary luminal area/myocyte area. The mitochondria/myofibril ratio was increased in the hearts of T rats (0.82) over that found in control hearts (0.52). These results suggest that in the early stages of thyroxine-induced myocardial hypertrophy there is not an immediate increase in capillary area which may account for the ischemia and significant increase in mitochondrial volume which characterizes myocardial hypertrophy in this model.

The pineal gland of the gerbil, Meriones unguiculatus. III. Morphometric analysis and fluorescence histochemistry in the intact and sympathetically denervated pineal gland.

SummaryMorphometric analytical procedures were employed to study the pineal gland of the Mongolian gerbil following superior cervical ganglionectomy (SCGX). The purpose of this study was to define the effects of sympathetic denervation on the morphology of the gland at two time periods, 0500 and 1900 h (one hour before lights-on and lights-off, respectively). Fluorescence histochemistry was employed to determine catecholamine and indoleamine content in intact and denervated pineal glands. After SCGX, the pinealocytes decrease in size, concretions are prevented from forming, and the yellow fluorescence in the gland is lost. Following denervation a depression in the volume of most of the pinealocyte organelles, i.e., SER, RER/ribosomes, free cytoplasm, mitochondria and presumptive secretory vesicles, was also observed. However, synaptic ribbons increased in volume in the gerbils that had been killed at 1900 h. It appears that the sympathetic innervation to the pineal gland is a requirement for the presumptive secretory activity of the pinealocytes.

Evidence that dopamine regulates norepinephrine synthesis in the rat superior cervical ganglion during hypoxic stress

Electrical stimulation of preganglionic nerves is known to increase norepinephrine synthesis in the rat superior cervical ganglion in vitro, an effect which appears to be partially regulated by a non-cholinergic transmitter. In the present study, we sought to determine whether sympathetic stimulation also increases norepinephrine synthesis in the rat ganglion in vivo, and whether dopamine released from ganglionic interneurons might regulate this response. To tackle these questions, rats were pretreated with spiroperidol, a selective dopamine-receptor blocker, and then were sympathetically stimulated by exposure to severe hypoxic stress. Other rats were pretreated with vehicle alone before the hypoxic exposure. Norepinephrine synthesis in ganglia was assessed by measuring endogenous tyrosine hydroxylase activity and norepinephrine turnover. We found that hypoxic stress increased both of these indices of norepinephrine synthesis, but only in rats pretreated with spiroperidol. No such response was detected in rats pretreated with vehicle. These results indicate that sympathetic stimulation increases norepinephrine synthesis in the rat superior cervical ganglion in vivo, and that dopamine released from interneurons might regulate this response.

Cytochemical evidence for the existence of norepinephrine-containing glomus cells in the rat carotid body.

SummarySome investigators have postulated that glomus cells of the rat carotid body contain only dopamine (DA), and that the norepinephrine (NE) measured in the carotid body resides only in sympathetic nerve endings and ganglion cells. To investigate this hypothesis, we employed a pharmacologic drug sequence which depleted all carotid body catecholamines and then selectively restored DA levels while keeping NE levels significantly lowered. Analysis of carotid body catecholamines by high performance liquid chromatography (HPLC) validated this drug regimen. Employing this drug treatment, we examined glomus cells after potassium dichromate cytochemical staining in an effort to distinguish those glomus cell vesicles which still contained appreciable amounts of catecholamine, presumably DA. Glomus cells from rats receiving vehicle orl-dopa (100 mg kg−1 ip) alone had 83 and 97% of their cells stained, respectively. However, glomus cells from reserpinized (5 mg kg−1 ip) animals were largely unstained (89%). Carotid bodies from animals treated with reserpine and then, 24 h later, withl-dopa 90 min prior to sacrifice had about 46% of their glomus cells stained while 54% of the cells were unstained. The results of this last group, coupled with our biochemical data which demonstrated that DA levels were comparable to control values but that NE was 80% depleted, suggest that a significant number of glomus cells did not contain enough catecholamine to react with the dichromate. We believe that these unstained cells may normally contain NE and that glomus cells may be of several types, some containing predominantly DA and others NE.

Ultrastructure of the pineal gland of the brush mouse (Peromyscus boylei): influence of long and short photoperiod.

The ultrastructure of the pineal gland of wild-captured brush mice (Peromyscus boylei) was examined. A homogeneous population of pinealocytes was present in the pineal gland of this species. The Golgi apparatus, granular endoplasmic reticulum, mitochondria, lysosomes, dense-core vesicles, vacuoles containing fluocculent material, clear vesicles, microtubules and glycogen particles were consistent components of the pinealocyte cytoplasm; infrequently-observed organelles included centrioles, “synaptic” ribbons, subsurface cisternae, multivesicular bodies, lipid droplets and annulate lamellae-like structures. Quantitative comparison of pinealocyte ultrastructure revealed larger cross-sectional areas of cytoplasm, nucleus, Golgi apparatus, granular endoplasmic reticulum, mitochondria and vacuoles containing flocculent material as well as higher number of dense-core vesicles in the animals kept in short photoperiod (LD 8∶16) as compared to those in animals kept in long photoperiod (LD 16∶8). These observations suggest that restricting the amount of light to which animals are exposed activated the pinealocytes of brush mice.

The effects of hypoxia on catecholamine dynamics in the rat carotid body

The catecholamine content of the rat carotid body was assayed using high performance liquid chromatography with electrochemical detection. The concentration of dopamine (DA) was found to predominate over that of norepinephrine (NE) by a small margin (31 pmol/carotid body pair DA; 23 pmol/carotid body pair NE). The turnover rates of carotid body DA and NE were determined from the time-dependent decline in their concentrations following the blockade of synthesis with alpha-methyl-p-tyrosine. Values were obtained (DA t 1/2 = 1.9 h; NE t 1/2 = 2.3 h) which suggested a rapid turnover of carotid body catecholamines. Exposure of rats to conditions of severe hypoxia (5% O2-95% N2) failed to significantly alter either the content or turnover of carotid body catecholamines. By contrast, the concentration of carotid body DOPAC, a reflection of DA utilization, was significantly elevated following hypoxic conditions. Further, in vivo tyrosine hydroxylase activity was assessed by measuring the accumulation of carotid body DOPA after inhibiting L-aromatic amino acid decarboxylase with NSD-1015. Basal tyrosine hydroxylase activity (approximately 14-16 pmol/carotid body pair/h) also was significantly increased by acute hypoxic exposure. These results, in part, suggest that rat carotid body DA may act as a neurotransmitter whose synthesis and release are coupled to stimulus demand.