George N. Robertson

Structure and autonomic innervation of the swim bladder in the zebrafish (Danio rerio)

Many teleosts actively regulate buoyancy by using a gas‐filled swim bladder, which is thought to be under autonomic control. Here we investigated the swim bladder in the zebrafish to determine possible mechanisms of gas‐content regulation. Fluorescently labelled phalloidin revealed myocytes that appeared to form a possible sphincter at the junction of the pneumatic duct and esophagus. Myocytes also formed thick bands along the ventral surface of the anterior chamber and bilaterally along the posterior chamber. Thinner layers of myocytes were located elsewhere. Staining of peroxidase within erythrocytes revealed a putative rete and smaller blood vessels in muscle bands and elsewhere. The antibodies zn‐12, a general neuronal marker, and SV2, a synaptic vesicle marker labelling presynaptic terminals, revealed widespread innervation of the swim bladder system. Widespread innervation of the swim bladder was also indicated by acetylcholinesterase histochemistry, but choline acetyltransferase‐immunoreactive (‐IR) somata and fibers were limited to the junction of the pneumatic duct and esophagus. In contrast, varicose tyrosine hydroxylase‐IR fibers innervated muscles and blood vessels throughout the system. Neuropeptide Y‐IR somata were located near the junction of the duct and esophagus and varicose fibers innervated muscles and vasculature of the posterior chamber and duct. Vasoactive intestinal polypeptide immunoreactivity was abundant throughout the anterior chamber but sparsely distributed elsewhere. Serotonin‐IR fibers and varicosities were located only along blood vessels near the junction of the pneumatic duct and posterior chamber. Our results suggest that the zebrafish swim bladder is a complex and richly innervated organ and that buoyancy‐regulating effectors may be controlled by multiple populations of autonomic neurons. J. Comp. Neurol. 495:587–606, 2006.

Ultrastructure and synaptology of the nucleus ambiguus in the rat: The semicompact and loose formations

The fine structure of the pharyngomotor semicompact and laryngomotor loose format formations of the rat nucleus ambiguus was studied in single and serial sections by means of light and electron microscopy. Motoneurons and their dendrites were identified after retrograde labelling by injections of neuroanatomical tracers into pharyngeal and laryngeal muscles or nerves. Pharyngeal motoneurons measured 39 × 29 μm and had 2–25 axosomatic synapses per somatic profile, representing an estimated average of 1S2 synapses per soma. Laryngeal motoneurons measured 42 × 30 μm with 6–33 synapses per profile, or an average of 339 synapses per soma. In both subdivisions, axon terminals that contained round vesicles and formed asymmetric junctions and terminals that contained pleomorphic vesicles and formed symmetric junctions were distributed in approximately equal proportions on somata and dendrites, forming over 90% of the synapse population. A small percentage (2–8%) of synapses had a subsurface cistern situated below the axon terminal (C type). Small, atypical motoneurons measuring 15 × 5 μm with an invaginated nucleus were also present in both subdivisions. The ultrastructure and synaptology of pharyngeal and laryngeal motoneurons are characterized by similarities to those of spinal motoneurons and by their relatively large numbers of axosomatic synapses in comparison to esophageal motoneurons of the compact formation of the nucleus ambiguus.

The contribution of the swimbladder to buoyancy in the adult zebrafish (Danio rerio): a morphometric analysis.

Many teleost fishes use a swimbladder, a gas‐filled organ in the coelomic cavity, to reduce body density toward neutral buoyancy, thus minimizing the locomotory cost of maintaining a constant depth in the water column. However, for most swimbladder‐bearing teleosts, the contribution of this organ to the attainment of neutral buoyancy has not been quantified. Here, we examined the quantitative contribution of the swimbladder to buoyancy and three‐dimensional stability in a small cyprinid, the zebrafish (Danio rerio). In aquaria during daylight hours, adult animals were observed at mean depths from 10.1 ± 6.0 to 14.2 ± 5.6 cm below the surface. Fish mass and whole‐body volume were linearly correlated (r2 = 0.96) over a wide range of body size (0.16–0.73 g); mean whole‐body density was 1.01 ± 0.09 g cm−3. Stereological estimations of swimbladder volume from linear dimensions of lateral X‐ray images and direct measurements of gas volumes recovered by puncture from the same swimbladders showed that results from these two methods were highly correlated (r2 = 0.85). The geometric regularity of the swimbladder thus permitted its volume to be accurately estimated from a single lateral image. Mean body density in the absence of the swimbladder was 1.05 ± 0.04 g cm−3. The swimbladder occupied 5.1 ± 1.4% of total body volume, thus reducing whole‐body density significantly. The location of the centers of mass and buoyancy along rostro‐caudal and dorso‐ventral axes overlapped near the ductus communicans, a constriction between the anterior and posterior swimbladder chambers. Our work demonstrates that the swimbladder of the adult zebrafish contributes significantly to buoyancy and attitude stability. Furthermore, we describe and verify a stereological method for estimating swimbladder volume that will aid future studies of the functions of this organ. J Morphol., 2008.

Desensitization to substance P following intrathecal injection

SummaryThe intrathecal injection of substance P (SP) (2.5–15 μg) has been shown to produce hyperalgesia in the rat tail flick test. Repeated injection of SP (7.5 or 15 μg) or pretreatment with two of these doses produces desensitization to this hyperalgesic response. Desensitization is doserelated with respect to degree and duration. This phenomenon is relatively specific because the hyperalgesic response to methysergide, a serotonin receptor antagonist, is unaffected, while that produced by phentolamine, an adrenergic receptor antagonist, is much less affected than that of SP. Pretreatment with a desensitizing regimen of SP potentiates the antinociceptive effect of morphine and baclofen when they are tested immediately after the regimen but if a 30 min delay is permitted, an inhibition of their effects is observed. These results support the notion that the spinal antinociceptive effect of morphine and baclofen is due to an interaction with SP mechanisms in the spinal cord, the nature of which may be more complex than is presently understood. Desensitization produces no change in baseline responsiveness in the tail flick test. This suggests that the hyperalgesic response to SP is due either to an action at a site other than the primary afferent synapse, or if it is at this site either compensatory mechanisms occur or SP is not the primary determinant of tail flick latency but may play a modulatory role.

Day‐Night Differences in the Number and Structure of Synaptic Ribbons in Chick Pineal

The objective of this study was to evaluate the effect of lighting (day‐night) changes on pinealocyte synaptic ribbon shape (conformation) and numbers. Three‐dimensional reconstruction analysis of pinealocyte basal processes revealed that 30% (6/20) of all ribbons from dark‐adapted animals were either curved or split. Synaptic ribbons from light‐adapted animals did not show this variant morphology; all were linear structures. An analysis of each section from each series, containing curved or split ribbons, revealed that 44% (16/36) of all ribbon profiles would yield inflated counts if used in random morphometric sampling protocols. Therefore, split and curved (variant) ribbon morphologies could result in an overestimation of synaptic ribbon populations of approximately 13% (0.44 × 30%) of dark‐adapted samples. In spite of this potential sampling error, the fourfold increase in the number of synaptic ribbons observed during the dark phase of a light‐dark cycle remains highly significant (P < 0.0001).

Axosomatic synapses of the rat ciliary ganglion.

We have identified four different types of axosomatic synapses within the rat ciliary ganglion, and present the three‐dimensional relationships of both pre‐ and postsynaptic elements. The majority of axosomatic synapses are situated on small postsynaptic spines that simply appose the axon (termed somatic spine), or are situated within an axonal invagination (termed invaginating somatic spine). The somatic spine synapse predominates, composing 70% of the population, which may be due to simplicity of construction as it usually forms only one active zone. In contrast, the invaginating somatic spine forms multiple active zones and accounts for only 22% of the population. Synapses involving a regular nonspinous portion of the cell membrane were rarely encountered (6%; termed somatic), as were those of axon branches situated within tubular invaginations of the cell body (2%; termed tunnelling). Synapses were differentially distributed, occurring four times more frequently on that portion of neuronal cell body membrane adjacent to the glial cell perinuclear area. However, there was no preferred location by synapse type, suggesting that this unequal distribution was the result of a general mechanism. The neuronal cells of the rat ciliary ganglion apparently constitute a single population, at least on the basis of cell size, shape, and organelle content.

Posthatch day/night differences in synaptic ribbon populations of the chick pineal.

Pineal synaptic ribbon (SR) populations of the early posthatch white leghorn chick were counted to determine if they demonstrate a rhythm that is in accordance with the light/dark cycle. SRs were counted between day 7 and day 10 and on day 14 of posthatch development, with samples at midlight, middark (14L:10D), and constant darkness. SR populations did not exhibit significant changes on days 7 and 8 under cycled lighting conditions nor on days 9 and 10 under constant darkness. A second experiment demonstrated that the dark:light ratio of SR populations of day 14 chicks, under cycled lighting, was 3.4:1.0, indicating SR rhythmicity by that stage of development. In that a preliminary experiment had demonstrated a 4.2:1.0 darklight ratio in SR populations in a predominantly day‐10 population of chicks, we believe that SR rhythmicity begins on, or near, day 10 of posthatch development. To determine if the invasion of sympathetic fibers from the superior cervical ganglion (SCG) correlates with the initiation of SR light/dark population differences, we employed tyrosine hydroxylase immunofluorescence to reveal the distribution of catecholaminergic fibers in chick pineal follicles Follicular innervation doubled over the day 7 to day 14 period, during which time light/dark differences in SR populations were established. There is a correlation, in time, between the invasion of the pineal by the sympathetic fibers and the initiation of SR light/dark differences. The circadian rhythm of pineal N‐acetyltransferase (NAT) activity, the rate‐limiting enzyme in the melatonin pathway, is established earlier (day 2) than the light/dark differences in SR populations (day 10). It is possible that SR rhythmicity is influenced by the ingrowth of the pineal sympathetic innervation, and that SRs respond to an extrapineal oscillator rather than the independent oscillators of the chick pineal responsible for the rhythm of NAT activity and melatonin synthesis.

The structure of the caudal wall of the zebrafish (Danio rerio) swim bladder: evidence of localized lamellar body secretion and a proximate neural plexus.

In this study, we present a morphological description of the fine structure of the tissues composing the caudal tip of the adult zebrafish swim bladder and an immunochemical survey of the innervation at this site. The internal aspect of the caudal tip is lined by an epithelium specialized to secrete surfactant into the lumen as evinced by the exocytosis of lamellar bodies. The sole innervation to this region consists of a neural plexus, present on the external surface, of nitric oxide synthase‐positive (nNOS) neuronal cell bodies that are contacted by axon terminals, some containing neuropeptide Y and vasoactive intestinal polypeptide. As the specialized epithelium and neural plexus are coincident and of common extent, we suggest that the morphological relationship between the two elements allows the nervous system to affect surfactant processing, possibly through a paracrine mechanism. J. Morphol. 275:933–948, 2014.  

Overexpression of X-Linked Inhibitor of Apoptotic Protein (XIAP) reduces age-related neuronal degeneration in the mouse cochlea.

Previously, we showed that age-related hearing loss (AHL) was delayed in C57BL6 mice overexpressing X-Linked Inhibitor of Apoptotic Protein (XIAP), and the delayed AHL was associated with attenuated hair cell (HC) loss in XIAP-overexpressing mice. Similar to other reports, the HC loss in aged mice was restricted to the basal turn in this previous study, and occurred slightly at the apical end of the cochlea, showing considerably less spread than the frequency region of hearing loss. In the present study, we examined whether and how AHL is related to the degeneration of neuronal innervation of the cochlea and whether the overexpression of XIAP exerts a protective effect against age-related degeneration in both afferent and efferent cochlear neurites. In contrast to HC loss, degeneration of both afferent and efferent neurites spread to the middle turns of the cochlea. Moreover, XIAP-overexpressing mice lost fewer HC afferent dendrites and efferent axons, as well as fewer spiral ganglion neurons between 3 and 14 months of age in comparison with wild-type littermates. The results suggest that age-related degeneration of cochlear neurites may be independent of HC loss. Further, the inhibition of apoptosis by XIAP appears to reduce degeneration of both afferent and efferent cochlear neurites.