Jean Y. Jew

An improved method for perfusion fixation of neural tissues for electron microscopy

A method employing vascular perfusion for improved preservation of biological ultrastructure is described, and its effectiveness demonstrated for mammalian nervous tissues. Following a physiological saline flush into the aorta, hydrogen peroxide and glutaraldehyde in phosphate buffer are perfused. After buffer rinses, tissue blocks are postfixed in osmic acid and potassium ferrocyanide. The success rate is enhanced greatly by close attention to details of perfusion technique. Advantages of the method include more uniform and complete preservation. In particular, superior images of membranous elements, glycogen granules and basal laminar material are achieved. Adjustments in osmolality may render the procedure suitable for nonmammalian forms and other tissues.

Hypertrophy of rat sensory ganglion neurons following intestinal obstruction

BACKGROUND Neuroplastic changes following ileum hypertrophy have been reported in intrinsic enteric neurons. The hypothesis in the present study was that intestinal hypertrophy induces neuronal changes in dorsal root ganglia (DRG). METHODS Under sodium pentobarbital anesthesia, partial obstruction was produced in the rat by tying a plastic ring around the terminal loop of ileum. Fast Blue (FB) (Sigma, St. Louis, MO) was injected into the obstructed ileum wall, and the rat was perfused after 8 days. DRG were immunostained and examined to identify and measure sizes of perikarya containing FB and/or calcitonin gene-related peptide (CGRP) or FB and/or substance P (SP). RESULTS Of the DRG neurons that projected to the ileum in control or obstructed animals, approximately 50% were CGRP-immunoreactive (IR) and 30% were SP-IR (colchicine pretreatment was not used). Neurons that projected to the obstructed ileum were increased in size compared with neurons in nonobstructed controls. Some of these neurons were CGRP-IR or SP-IR; some were large FB-labeled neurons that were not SP-IR or CGRP-IR. CONCLUSIONS The morphology of sensory autonomic neurons in adult animals is influenced by dynamic interactions with the targets they innervate, whether directly or transneuronally.

Innervation of the mitral valve is strikingly depleted with age

Previous reports demonstrated that mammalian atrioventricular (AV) valves possess a dense nerve plexus, consisting of nerve subpopulations which differ from each other in densities and patterns of distribution in the valves, and which may have sensory or motor roles in valve function. Although there is extensive evidence that age‐related changes occur in autonomic nerves of animals and humans (Daly et al. J. Pharm. Exp. Ther., 1988;245(3):798–803; Ingall et al. Aust. NZ J. Med., 1990;20:570–577; Tumer et al. Exp. Gerontol., 1992;27:301–307), and that these changes contribute to changes in cardiac function (Klausner and Schwartz Clin. Geriat. Med., 1985;1(1):119–114), there is little information about age‐related changes in heart valve innervation. In this study, we used acetylcholinesterase (AChE) histochemistry to localize and compare qualitative and quantitative changes in the innervation of the mitral valves in young adult and aged animals of three species. Young adult and aged guinea pigs, mice, and Wistar and Fischer 344 rats were anesthetized with Nembutal, the hearts removed, and the mitral valves dissected out and processed for AChE localization. Camera lucida drawings of the AChE‐positive nerves in representative segments of valve cusps were made directly from slides; these drawings were digitized and subjected to computer‐assisted image analysis to obtain quantitative information about nerve plexus density in the valves. All three animal species showed profuse AChE‐positive innervation in the mitral valves of young adult animals, and decreases in the density of this innervation in aged animals. The most striking loss of innervation, compared to the young adult, occurred in the mitral valves of aged Fischer 344 rats, in which large regions of the valves appeared virtually devoid of nerves. Further studies are needed to investigate whether and to what extent age‐related losses in heart valve innervation affect valvular structure and function. Anat Rec 255:252–260, 1999.

Bradycardia following injection of 6-hydroxydopamine into the intermediate portion of nucleus tractus solitarius medialis.

Studies were carried out to determine the effects of selective destruction of the catecholamine innervation of the intermediate portion of the nucleus tractus solitarius medialis (NTSm) on mean arterial pressure and heart rate in conscious rats. Following bilateral injections of 6-hydroxydopamine into the intermediate portion of NTSm, animals demonstrated a prolonged bradycardia, but no changes in mean arterial pressure or mean arterial pressure lability, when compared with controls. Results indicate that the baroreceptor reflex loop which mediates bradycardia is sensitive to impulses from catecholamine axons entering the intermediate NTSm.

Neurotensin immunoreactivity in the central nucleus of the rat amygdala: An ultrastructural approach

Neurotensin (NT) was demonstrated in the central nucleus of the rat amygdala (CNA) using a modification of the avidin-biotin complex immunohistochemical technique. Electron-dense reaction product (particles were 15-25 nm in diameter) was localized in perikarya, dendrites, axons, and axon terminals. It was found also associated with profiles of rough endoplasmic reticulum, mitochondria, microtubules, and small agranular as well as large granular vesicles. In distal dendrites, the reaction product was associated with microtubules, vesicles, and postsynaptic densities. Axon terminals of three types formed synaptic contracts with NT-immunoreactive neurons in the CNA: one was characterized by numerous round or oval agranular vesicles, the second by numerous pleomorphic vesicles, and the third by agranular vesicles that were loosely distributed and pleomorphic. All three types formed symmetric axosomatic and asymmetric axodendritic contacts. NT-immunoreactive axon terminals containing small round agranular vesicles stood out clearly from the intermingling profiles of immunonegative structures. We found numerous glomeruli, each consisting of a central NT-immunoreactive dendrite surrounded by all three types of axon terminals. We observed that some NT-immunoreactive terminals formed symmetric axoaxonal contacts with each other, providing evidence for the presence of local NT-to-NT circuits, whereas many others synapsed with axon terminals devoid of NT immunoreactivity.

Collateral sprouting of somatostatin-immunoreactive axons after partial deafferentation of the central nucleus of the rat amygdala

These experiments utilize a paradigm developed to study plastic responses of peptidergic neurons in a discrete brain area following deafferentation. The central nucleus of the amygdala (CNA) is richly innervated by somatostatin-immunoreactive (SS-I) terminal axons. In the course of preliminary light microscopic (LM) investigations by this laboratory, changes were observed in the density of presumed SS-I terminals in the rat CNA after lesioning the medial input. The LM finding of increased density of presumed SS-I terminals in the CNA at the 10-day post-lesion stage underscored the need for a quantitative electron microscopic (EM) study of the SS-I components, including an evaluation of synaptic events at different survival periods. At the 3-day post-lesion stage, EM examination showed degenerating axons in the lesioned CNA, many already engulfed by astrocytes. None of the degenerating profiles were SS-I, supporting the view that the lesion did not interrupt, to any significant extent, SS-I axons entering the nucleus. EM surveys of the 10-day post-lesion material demonstrated that degenerated profiles had almost completely disappeared. Numbers of SS-I axon terminals, particularly of smaller-sized profiles, were increased by 22% over control value. Synaptic frequency was decreased by 16% below control value. Numbers of SS-I terminals making synapses were increased 3.4% above control value. At the 30-day post-lesion stage, the total number of SS-I terminal axons had increased 86% over controls, whereas the synaptic frequency had decreased by about a third below controls. The absolute number of SS-I terminals engaging in synapses had increased by 24% over controls. The 90-day post-lesion CNA showed a further increase in the number of SS-I axon profiles: 136% over control value. The synapse-to-axon ratio (synaptic frequency) of 27% was similar to that observed for the CNA from the unlesioned side or from unoperated animals. At this stage the number of SS-I synapses had increased by 135% over controls. This model presents many possibilities for studying neuroplasticity, particularly involving peptidergic neurons of the central autonomic nervous system.

The role of substance P-containing fibers in sympathetic ganglia: Effect of capsaicin

Capsaicin was given subcutaneously to guinea pigs and the effect on substance P-immunoreactive (SP-I) fibers in the celiac/superior mesenteric and inferior mesenteric ganglia was observed at 2 day and 8-10 day intervals. Capsaicin (125 mg) treatment led to almost total disappearance of SP-I fibers from all areas examined in both short- and long-term animals. This effect applied equally to the dense network of varicose SP-I fibers and to basket-like SP-I contacts with principal ganglionic neurons. The effect of capsaicin on SP-I fibers in the mesenteric ganglia provides a strong indication that these fibers represent a homogeneous population of visceral sensory afferents. This is supported by other lines of anatomical evidence in the literature. Taken together with studies that have shown axodendritic contact of SP-I terminals on principal ganglionic neurons and neuro-modulatory effects of SP on these neurons, it may be hypothesized that SP-I fibers in the mesenteric ganglia represent collaterals of visceral sensory afferents forming a subspinal feedback arc.

Catecholamine inputs to the nucleus tractus solitarius

The projections of brainstem catecholaminergic (CA) cell groups to the rat nucleus tractus solitarius (NTS) were examined using 6-hydroxydopamine (6-OHDA) injections and glass microknife cuts. 6-OHDA (4 micrograms) was injected into the intermediate NTS, and this resulted in depletion of CA fluorescent varicosities from the NTS at this rostrocaudal level, except for varicosities along the periventricular edge of the NTS. In addition, a band of swollen fluorescent axons extended between the CA A1 cell group of the ventrolateral medulla and the lateral NTS. Microknife cuts were used to interrupt the projections of the CA A1 and A2 cell groups (located in the caudal NTS) and tissues were examined for changes in CA varicosity density within the intermediate NTS. Following transverse knife cuts of the intermediate NTS, rostral to the A2 cell group, fluorescent varicosities rostral to the cut virtually disappeared, and the fluorescence intensity of the ipsilateral A2 neurons caudal to the cut was increased. These cuts also eliminated the 6-OHDA-resistant varicosities along the periventricular NTS. After microknife cuts lateral to the intermediate NTS, the fluorescent varicosity density in the NTS was unchanged. These results indicate that the major CA projection to the NTS arises from the ipsilateral A2 cell group. The 6-OHDA-resistant varicosities arising from neurons caudal to the knife cut probably arise from the adrenergic C2 cell group.