Malinda E.C. Fitzgerald

Neurotransmitter organization of the nucleus of Edinger–Westphal and its projection to the avian ciliary ganglion

Two morphologically distinct types of preganglionic endings are observed in the avian ciliary ganglion: boutonal and cap-like. Boutonal endings synapse on ciliary ganglion neurons (called choroidal neurons) innervating choroidal blood vessels, while cap-like endings synapse on ciliary ganglion neurons (called ciliary neurons) controlling the lens and pupil. Some of both types of preganglionic endings contain the neuropeptides substance P (SP) and/or leucine-enkephalin (LENK). Although both types of preganglionic terminals are also known to be cholinergic, there has been no direct evidence that SP and LENK are found in cholinergic endings in the ciliary ganglion. The present studies in pigeons, which involved the use of single- and double-label immunohistochemical techniques, were undertaken to examine this issue, as well as to (1) determine the relative percentages of the boutonal and cap-like endings that contain SP, LENK, or both SP and LENK; and (2) determine if the two different types of terminals in the ciliary ganglion arise from different subdivisions of the nucleus of Edinger-Westphal (EW). Single- and double-label immunohistochemical studies revealed that all neurons of EW, regardless of whether they contained immunohistochemically detectible amounts of SP or LENK, are cholinergic. In the medial subdivision of EW (EWM), which was found to contain approximately 700 neurons, 20.2% of these neurons were observed to contain both SP and LENK, while 11.6% were observed to contain SP only and 10.7% were observed to contain LENK only. In contrast, in lateral EW (EWL), which was found to contain approximately 500 neurons, 16.2% of the neurons were observed to contain both SP and LENK, while 19.2% of the neurons were observed to contain SP only and 12.6% were observed to contain LENK only. Retrograde-labeling studies involving horseradish peroxidase injections into the ciliary ganglion revealed that EW was the sole source of input to the ciliary ganglion and all, or nearly all, neurons in EW innervate the ciliary ganglion. Immunohistochemical labeling of the ciliary ganglion neurons with an antiserum against choline acetyltransferase revealed that approximately 900 choroidal neurons and approximately 600 ciliary neurons are present in the ganglion, all of which receive cholinergic preganglionic endings. Of the choroidal neurons, 94% receive butonal terminals containing both SP and LENK, while only 2% receive SP+ only boutonal endings and 2% receive LENK+ only butonal endings. Of the ciliary neurons, 25% receive cap-like endings containing both SP and LENK, 30% receive cap-like endings containing only SP and 3% receive cap-like endings containing only LENK.(ABSTRACT TRUNCATED AT 400 WORDS)

Choroidal blood flow in pigeons compensates for decreases in arterial blood pressure.

While it had once been thought that choroidal blood flow (ChBF) does not compensate for changes in perfusion pressure, recent studies have shown that ChBF in rabbits and humans does compensate for changes in arterial blood pressure (ABP) and thereby remains relatively stable within a physiological range of ABPs. In this study, we sought to determine if ChBF in birds can compensate for decreases in ABP, either spontaneously occuring or caused by blood withdrawal. ChBF was continuously monitored using laser Doppler flowmetry in anesthetized pigeons, and at the same time ABP was measured via the brachial artery. In studies of spontaneous fluctuation in ABP, ChBF and ABP were analyzed at regular intervals over a 2-3 hr period, while for blood withdrawal studies, blood was transiently withdrawn via the brachial artery. In both paradigms, ChBF remained near baseline over an ABP range from basal (about 90 mmHg) to about 55 mmHg, followed ABP nearly linearly below 50 mmHg, and showed no compensation below 40 mmHg. The blood withdrawal studies further showed that the compensation was more rapid with small acute declines in ABP than with larger declines. These findings reveal that ChBF in pigeons, as in rabbits and humans, compensates for declines in ABP so as to remain relatively stable within a physiological range of ABPs. Given the phylogenetic distance between humans and rabbits on one hand and birds on the other, these results suggest that choroidal compensation for ABP declines may be a common ocular mechanism among warm-blooded vertebrates.

Projections from the hypothalamic paraventricular nucleus and the nucleus of the solitary tract to prechoroidal neurons in the superior salivatory nucleus: Pathways controlling rodent choroidal blood flow

Using intrachoroidal injection of the transneuronal retrograde tracer pseudorabies virus (PRV) in rats, we previously localized preganglionic neurons in the superior salivatory nucleus (SSN) that regulate choroidal blood flow (ChBF) via projections to the pterygopalatine ganglion (PPG). In the present study, we used higher-order transneuronal retrograde labeling following intrachoroidal PRV injection to identify central neuronal cell groups involved in parasympathetic regulation of ChBF via input to the SSN. These prominently included the hypothalamic paraventricular nucleus (PVN) and the nucleus of the solitary tract (NTS), both of which are responsive to systemic BP and are involved in systemic sympathetic vasoconstriction. Conventional pathway tracing methods were then used to determine if the PVN and/or NTS project directly to the choroidal subdivision of the SSN. Following retrograde tracer injection into SSN (biotinylated dextran amine 3K or Fluorogold), labeled perikarya were found in PVN and NTS. Injection of the anterograde tracer, biotinylated dextran amine 10K (BDA10K), into PVN or NTS resulted in densely packed BDA10K+terminals in prechoroidal SSN (as defined by its enrichment in nitric oxide synthase-containing perikarya). Double-label studies showed these inputs ended directly on prechoroidal nitric oxide synthase-containing neurons of SSN. Our study thus establishes that PVN and NTS project directly to the part of SSN involved in parasympathetic vasodilatory control of the choroid via the PPG. These results suggest that control of ChBF may be linked to systemic blood pressure and central control of the systemic vasculature.

The retinal microvasculature of spontaneously diabetic BB rats : structure and luminal surface properties

Endothelial cell permeability and luminal surface anionic sites were studied in the retinal microvasculature of spontaneously diabetic rats. Horseradish peroxidase (HRP) was used as a tracer of pinocytotic transport, and cationized ferritin (CF) was used as a marker of luminal surface anionic sites. Diabetic and control rats were injected with HRP, and their retinas were fixed. Retinal tissue sections were then incubated in CF, reacted to visualize HRP, and prepared for quantitative electron microscopic analysis. In both control and diabetic rats treated with serotonin and histamine antagonists to prevent HRP-induced vascular changes, the endothelium formed a barrier to the tracer. Pinocytotic uptake was relatively low in most vessels. Reaction product was restricted to pinocytotic vesicles, tubular cisternae, and multivesticular bodies. HRP uptake appeared high in some of the deep capillaries of the diabetic retinas as compared with that of the controls, but the difference was not statistically significant. HRP-induced transendothelial permeability was observed in both control and diabetic rats when serotonin and histamine antagonist pretreatment was omitted. CF studies showed anionic sites in four luminal surface microdomains in control and diabetic endothelial cells. CF-binding, anionic sites were present on the plasma membrane, on all coated vesicles, on some uncoated vesicles, and on most diaphragms of uncoated vesicles. Plasma membrane binding was sparse and patchy in some diabetic vessels, especially in the deep vessels of rats that were not treated with the serotonin and histamine antagonists. However, statistical analysis showed similar numbers of plasma membrane binding sites in diabetic and control rats pretreated with serotonin and histamine antagonists. Our data suggest that the retinal microvasculature in diabetic rats remains normal in terms of permeability and luminal membrane anionic charge.

A thin section and freeze-fracture study on membrane specializations in spermatozoa of the isopod, Armadillidium vulgare.

Mature spermatozoa of the Isopod, Armadillidium vulgare , were examined following tannic acid processing, thin section, and freeze-fracture techniques. They reveal a moderately thick glycocalyx and a typical random distribution of 5- to 10-nm-size, plasmalemmal, PF-face particles. Seven distinct areas of particulate membrane specializations occur in the spermatozoa. Five regions occur in the plasmalemma and two occur in the acrosomal membrane. The plasmalemmal specializations include: (1) clusters of 8- to 10-nm-size, PF-face particles found along a line made by the posterior margin of the acrosomal membrane; (2) orthogonal arrays of 4- to 6-nm-size EFface particles similarly disposed; (3) a linear array of 8- to 10-nm-size, PF-face particles extending the length of the acrosome, along the region where the paddle-shaped acrosome becomes thinner; (4) a cluster of 8- to 10-nm-size, PF-face particles on the cytoplasmic hood, which surrounds the insertion region of the cross-striated, tail-like appendage; and (5) a cross-linear array of 6- to 8-nm-size, PF-face particles which repeat every 67 nm along the cross-striated tail-like appendage, mimicking the cross-striated repeat period exhibited by the filamentous component as seen in thin sections. The acrosomal membrane specializations include: (1) tightly packed, 8- to 10-nm-size, EF-face particles limited to the thin portion of the paddle-shaped acrosome; and (2) linear arrays of periodically repeating (25–26 nm), 8- to 10-nm-size, PF face particles limited to the thick portion of the acrosome whose periodic repeat mimics the paracrystalline order of the internally directed portions of the unit membrane in this region. The results of this study are discussed in terms of the functional significance of such specializations. It is concluded that the function of each group of particle arrays is unique, and is in fact a reflection of either their topographical relationships with other membrane specializations or their possible role in fertilization. Particular attention is drawn to the functional significance of the following groups of particles: (1) the double set of particle arrays in the postacrosomal region, (2) the cross-linear arrays which mimic the cross-striated repeat pattern of longitudinally oriented filaments in the cross-striated appendage seen in thin sections, and (3) the two sets of particles in the PF and EF faces of the acrosomal membrane.

Vasoactive intestinal polypeptide-containing nerve fibers are increased in abundance in the choroid of dystrophic RCS rats

As photoreceptor degeneration progresses in Royal College of Surgeons (RCS) rats, a variety of morphological and physiological alterations occur in the outer retina. Since the choriocapillaris responds to changes in the outer retina in other retinopathies, we examined the possibility that changes in the choroidal vasculature also occur in RCS rats. The choroidal and choriocapillary vessels in RCS and control (RCS-rdy+) rats were examined during the period after which photoreceptor loss and retinal vascular changes had occurred (7-mos to 28-mos). Light microscopic (LM) morphometry and electron microscopic (EM) examination showed no significant differences between these groups in the number, size or morphology of these vessels. However, EM image analysis revealed that nerve fibers and bundles were twice as abundant in the RCS choroid than in the control. Using immunohistochemical techniques at the LM level combined with image analysis we found that vasoactive intestinal polypeptide positive (VIP+) fibers were significantly increased in the RCS choroid compared with control choroid. In contrast, the abundance of immunoreactive fibers labelled for substance P and dopamine beta hydroxylase appeared similar in both the control and RCS choroid. Since VIP is a potent vasodilator, the increased abundance of nerve fibers in the RCS choroid in conjunction with the unaltered number and size of these vessels suggests that choroidal blood flow may be increased. It is uncertain whether this increase is a response to the outer retinal pathology or contributes to it.

Lectin-Ferritin Binding on Spontaneously Diabetic and Control Rat Retinal Microvasculature

We previously reported, in the spontaneously diabetic Bio-Breeding (BB) rat, an increase in horseradish peroxidase (HRP) uptake that was associated with reduction and patching of cationized ferritin (CF) binding to anionic sites on the luminal plasma membrane of the retinal capillary endothelium. To see whether alterations in the negatively charged terminal sugar residues, N-acetyl-glucosamine (NAG) and sialic acid (SA), might contribute to these changes in the diabetic rat retina, we used lectin-ferritin (Fe) conjugates to study the distribution of these sugars on the retinal endothelial luminal membranes. Wheat germ agglutin (WGA, binds to NAG and SA) and Limax flavius (LFA, binds only SA) were used. Plasma membrane WGA-Fe binding was dense and uniform in control animals. Binding sites were also found in coated luminal vesicles, within some uncoated luminal vesicles and on their diaphragms. Unlabeled uncoated luminal vesicles were also seen, suggesting two populations of uncoated vesicles. In diabetic animals, the binding sites were present within the same membrane associated microdomains as in the controls. However, in the majority of outer plexiform layer (OPL) vessels in diabetic animals, WGA-Fe binding was reduced to a single, discontinuous layer of particles (p less than 0.02). In both diabetic and control vessels, WGA-Fe binding was greatly reduced by the addition of competitive sugars. A few particles remained on the plasma membrane, on the diaphragms of some vesicles, and at the edge of vesicles. LFA-Fe binding was similar to that seen with WGA-Fe in the presence of competitive sugars. These results suggest that luminal membranes of retinal capillaries are rich in NAG and contain little SA. The sparse WGA-Fe binding pattern in the diabetic OPL may reflect decreases in number or accessibility of NAG residues, since similar binding patterns are seen in both the control and diabetic animals under conditions specific for SA. Thus, alteration of terminal NAG residues may contribute to decreased luminal surface anionic sites and increased pinocytotic transport in the retinal microvasculature of spontaneously diabetic BB rats.

The fine structure of membrane complexes in spermatozoa of the millipede, Spirobolus sp., as seen by thin-section and freeze-fracture techniques

This study on membrane characteristics of the millipede, Spiro bolus sp., as a representative of the Diplopoda demonstrates evidence for unusual arrays of membranes and associated particle composition which will be compared to that for other spermatozoa. Mature spermatozoa of Spirobolus sp. are disk-shaped cells with a flat and concave surface. They are found juxtaposed at their flat surfaces in pairs. Highly specialized and morphologically unique membrane complexes which occur in these spermatozoa include the following: First, at the flat surface of apposed spermatozoa a heptalaminar membrane may be seen which is a composite of the limiting plasmalemma and the fused inner and outer nuclear membranes derived during spermiogenesis. Second, a tubular array of membranes occurs at the juncture of the flat and concave surfaces. Two sets of particles occur in these spermatozoa. One includes a highly ordered array of 10- to 11-nm P-face particles which form a ring lining the full circumference of the disk-shaped cells in square lattices of interparticle distance 19 to 20 nm. The second includes randomly dispersed 8- to 9-nm P-face particles which are present in particular abundance throughout the dome-shaped concave surface and along the flat juxtaposed surfaces. These data are discussed in terms of their possible function and a comparison is made with similar particle types found in other spermatozoa and in other cell types generally.

Studies on the fine structure of the mitochondrial derivative in spermatozoa of a gastropod

Spermatozoa of Limax sp. were studied by electron microscopy following thin section and freeze-fracture techniques. Mature spermatozoa were seen to be helically shaped, 150 microns long cells. A single mitochondrion extends the entire length of the spermatozoon. Its helical turn is the same as that of the spermatozoon. Freeze-fracture images of the spermatozoon reveal that the EF and PF, plasmalemmal faces contain scattered, 7-9 nm size particles, and that the PF, outer mitochondrial membrane face contains 8-10 nm size particles. The corresponding EF, outer mitochondrial face contains matching pits. A paracrystalline complex is situated between the inner and outer mitochondrial membranes. The complex is constructed of a series of 8-9 nm thick, 35 nm wide, helically orientated, tripartate elements which extend the full length of the spermatozoon. The helical tilt angle is approximately 55 degrees. Each element is composed of tightly approximated (interspace distance 10 nm), strands of particles 8-9 nm in diameter. Speculations as to the significance of this complex, and its location between inner and outer mitochondrial membranes are made. It is concluded that the paracrystalline order of the complex either reflects the molecular packing of enzyme systems present in the mitochondrion, or some other unknown function.

Disinhibition of neurons of the nucleus of solitary tract that project to the superior salivatory nucleus causes choroidal vasodilation: Implications for mechanisms underlying choroidal baroregulation

Preganglionic neurons in the superior salivatory nucleus (SSN) that mediate parasympathetic vasodilation of choroidal blood vessels receive a major excitatory input from the baroresponsive part of the nucleus of the solitary tract (NTS). This input appears likely to mediate choroidal vasodilation during systemic hypotension, which prevents decreases in choroidal blood flow (ChBF) due to reduced perfusion pressure. It is uncertain, however, how low blood pressure signals to NTS from the aortic depressor nerve (ADN), which fires at a low rate during systemic hypotension, could yield increased firing in the NTS output to SSN. The simplest hypothesis is that SSN-projecting NTS neurons are under the inhibitory control of ADN-receptive GABAergic NTS neurons. As part of evaluating this hypothesis, we assessed if SSN-projecting NTS neurons, in fact, receive prominent inhibitory input and if blocking GABAergic modulation of them increases ChBF. We found that SSN-projecting NTS neuronal perikarya identified by retrograde labeling are densely coated with GABAergic terminals, but lightly coated with excitatory terminals. We also found that, infusion of the GABA-A receptor antagonist GABAzine into NTS increased ChBF. Our results are consistent with the possibility that low blood pressure signals from the ADN produce vasodilation in choroid by causing diminished activity in ADN-receptive NTS neurons that tonically suppress SSN-projecting NTS neurons.