Terence H. Williams

Histofluorescence characteristics and quantification of small intensely fluorescent (SIF) cells in sympathetic ganglia of several species

SummarySmall intensily fluorescent (SIF) cells in the superior cervical ganglion of the cow, cat, rabbit, rat, guinea pig and monkey were studied, using the glyoxylic acid monoamine fluorescence method. SIF cell populations per mg ganglion tissue showed great species variation. The greatest numbers of SIF cells per mg were found in the rat (380±30 per mg). Intermediate numbers (76±20 per mg) were found in the guinea pig; and SIF cells in other species were much more sparsely distributed (less than 10 per mg). Two types of SIF cell were identified. Type I cells have long (up to 200 μ) processes which ramify among the principal ganglionic neurons, and this type often occurs singly; whereas type II cells tend to occur in clusters near blood vessels in the interstitial or subcapsular regions of the ganglion. As a general hypothesis we propose that type I SIF cells are interneurons whereas type II SIF cells operate through a neurosecretory mechanism.

Cholecystokinin in the nucleus of the solitary tract of the rat: evidence for its vagal origin

Nerve fibers and terminals immunoreactive for cholecystokinin (CCK) were demonstrated in the nucleus of the solitary tract (NTS) of the rat using light and electron microscopic immunocytochemistry. The following morphological and biochemical evidence suggests that CCK in the NTS seems to be of extrinsic, most probably vagal, origin: (1) axon fragments of the intracranial vagus were identified by immunostaining on their way to the solitary tract: (2) CCK-immunostaining could be localized in nerve terminals in the nucleus of the solitary tract, where only a very few immunopositive dendrites or cell bodies were present; and (3) transecting the major neuronal afferents (via solitary tract and/or more laterally) resulted in a complete disappearance of radioimmunoassayable CCK from the nucleus of the solitary tract.

Luteinizing hormone —Releasing hormone (LH-RH) pathway of the rat hypothalamus revealed by the unlabeled antibody peroxidase-antiperoxidase method

SummaryUtilizing the unlabeled antibody enzyme method, we report the distribution of hypothalamic elements immunoreactive with antibodies to luteinizing hormone-releasing hormone (LH-RH) in the rat. Immunostained elements, resembling neural processes, were distributed along a pathway corresponding to the tuberoinfundibular tract which appeared to terminate near vascular elements in the external layer of the preand post-infundibular median eminence. No cell bodies stained specifically for LH-RH. Similar topographic arrangements were noted (in coronal and sagittal sections) in diestrous females, ovariectomized females and a hypophysectomized male. The same results were obtained with three different preparations of antisera to LH-RH. Our studies agree with those of other investigators using immunohistochemical techniques as well as with localization studies of LH-RH in the hypothalamus using bioassay and radioimmunoassay. Our results suggest that the unlabeled antibody enzyme technique will have unique value for identifying and tracing fiber systems related to specific functions within the hypothalamus.

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.

A method for studying glyoxylic acid induced fluorescence and ultrastructure of monoamine neurons.

SummaryInasmuch as precise correlations of light- and electronmicroscopy are crucial for understanding biostructure, it seemed necessary to bring together the advantages of the glyoxylic acid (GA) method (for inducing monoamine fluorescence) and electron microscopy.A combined fluorescence and electron microscope method using GA is introduced. The brain is perfused by 2% GA in Krebs-Ringer bicarbonate buffer (pH 7.0) and this solution is followed by 4% paraformaldehyde containing 0.5% glutaraldehyde in Sorensens phosphate buffer (pH 7.4). Sections are cut by cryostat or by vibratome and incubated in 2% GA in phosphate buffer (pH 7.0). Using fluorescence microscopy, features of interest are sketched and/or photographed. Afterwards, the same or subsequent section is processed for electron microscopy. Since axons of catecholamine-containing neurons (as well as their perikarya and terminals) are visualized by GA, the recommended procedure expands the range of studies concerning monoamine neurons that can now be carried out effectively.

ACTH-immunoreactive buotons form synaptic contacts in the hypothalamic arcuate nucleus of rat: evidence for local opiocortin connections

ACTH-like immunoreactive structures were localized in rat hypothalamic arcuate nucleus using the unlabeled antibody, peroxidase-antiperoxidase method. At the ultrastructural level, immunoreactive presynaptic nerve terminals were observed forming symmetrical synaptic contacts with unlabeled dendrites and with ACTH-like immunoreactive perikarya. The results obtained are consistent with the hypothesis that ACTH in the brain acts as a synaptic regulator or transmitter, contributing not only to long projection pathways but also to a local circuit in the arcuate nucleus where ACTH cell bodies are localized.

Morphological differentiation of a murine neuroblastoma clone in monolayer culture induced by dexamethasone.

Dexamethasone induces morphological differentiation in murine neuroblastoma cells in culture. The percentage of differentiated cells depends on the concentration of dexamethasone in the medium and duration of treatment. After drug removal (with or without replating), many cells maintain their differentiated phenotype. Dexamethasone-treated cells have larger soma and contain more protein. Dexamethasone also produces a concentration-dependent inhibition of population growth. Growth inhibition is complete by 2 days following treatment with dexamethasone 50 micrograms/ml. Complete growth inhibition is achieved prior to the complete expression of morphological differentiation. Androstenedione, testosterone, and 17-beta-estradiol--all steroids without glucocorticoid activity--have no differentiating effect, although they inhibit growth or cause cell death at higher concentrations.

Synaptology of three peptidergic neuron types in the central nucleus of the rat amygdala

The synaptology of neurotensin (NT)-, somatostatin (SS)- and vasoactive intestinal polypeptide (VIP)-immunoreactive neurons was studied in the central nucleus of the rat amygdala (CNA). Three types of axon terminals formed synaptic contacts with peptide-immunoreactive neurons in the CNA: Type A terminals containing many round or oval vesicles; Type B terminals containing many pleomorphic vesicles; and Type C terminals containing fewer, pleomorphic vesicles. Peptide-immunoreactive terminals were type A. All three types of terminals formed symmetrical axosomatic and asymmetrical axodendritic contacts. However, type B and peptide-immunoreactive terminals frequently formed symmetrical axodendritic synaptic contacts. VIP-immunoreactive terminals also formed asymmetrical axodendritic contacts. SS- and NT-immunoreactive terminals commonly formed symmetrical contacts on SS- and NT-immunoreactive cell bodies, respectively. VIP-immunoreactive axon terminals were postsynaptic to nonreactive terminals. Type B terminals appeared more frequently on VIP neurons than on NT or SS neurons.

Immuno-electron microscopic identification of somatostatin in cells and axons of sympathetic ganglia in the guinea pig

SummaryThe superior cervical ganglia (SCG), celiac superior mesenteric ganglia (CMG), and splanchnic nerve of unoperated guinea pigs, as well as both proximal and distal stumps of a previously transected branch of the postganglionic plexus of the CMG, were immunostained for somatostatin (SS). In addition, the PAP technique was adapted for fine-structural visualization of SS. A greater proportion of cells were labeled for SS in the CMG than in the SCG. PAP molecules were present in one type of intraganglionic axons. Only two labeled axons were found in the splanchnic nerve. Neither proximal nor the distal stump of the transected CMG postganglionic nerve contained labeled axons. The present results support the hypothesis that the intraganglionic axons labeled for SS arise from SS-containing intraganglionic neurons.

The distribution and ultrastructure of VIP-immunoreactivity in the central nucleus of the rat amygdala

Vasoactive intestinal polypeptide (VIP) neurons within the central nucleus of the rat amygdala were examined using light and electron microscopic immunocytochemical techniques. Vasoactive intestinal polypeptide-immunoreactive neurons were located in the ventral part and less frequently in the central part of the central nucleus. Vasoactive intestinal polypeptide positive terminals were distributed throughout the medial part of a cytoarchitectonically distinct central zone of the central nucleus. Three types of terminals formed synaptic contacts on VIP-immunoreactive neurons: type A containing round vesicles, type B containing many pleomorphic vesicles and type C containing fewer pleomorphic vesicles. All VIP-immunoreactive boutons observed were of type A variety, and made asymmetrical and symmetrical synaptic contacts on both VIP-immunoreactive and nonreactive neurons within the central nucleus.