Colin R. Anderson

The neurochemical characterisation of hypothalamic pathways projecting polysynaptically to brown adipose tissue in the rat.

The identification of leptin and a range of novel anorectic and orexigenic peptides has focussed attention on the neural circuitry involved in the genesis of food intake and the reflex control of thermogenesis. Here, the neurotropic virus pseudorabies has been utilised in conjunction with the immunocytochemical localisation of a variety of neuroactive peptides and receptors to better define the pathways in the rat hypothalamus directed polysynaptically to the major thermogenic endpoint, brown adipose tissue. Infected neurones were detected initially in the stellate ganglion, then in the spinal cord followed by the appearance of third-order premotor neurones in the brainstem and hypothalamus. Within the hypothalamus these were present in the paraventricular nucleus, lateral hypothalamus, perifornical region, and retrochiasmatic nucleus. At slightly longer survival times virus-infected neurones appeared in the arcuate nucleus and dorsomedial hypothalamus. Neurones in the retrochiasmatic nucleus and in the adjacent lateral arcuate nucleus which project to the brown adipose tissue express cocaine- and amphetamine-regulated transcript, pro-opiomelanocortin and leptin receptors. Neurones in the lateral hypothalamus, a site traditionally associated with the promotion of feeding, project to brown adipose tissue and large numbers of these contained melanin-concentrating hormone and orexin A and B. These data provide part of an anatomical framework which subserves the regulation of energy expenditure.

ANATOMICAL RELATIONSHIP BETWEEN URETHRA AND CLITORIS

PURPOSE We investigated the anatomical relationship between the urethra and the surrounding erectile tissue, and reviewed the appropriateness of the current nomenclature used to describe this anatomy. MATERIALS AND METHODS A detailed dissection was performed on 2 fresh and 8 fixed human female adult cadavers (age range 22 to 88 years). The relationship of the urethra to the surrounding erectile tissue was ascertained in each specimen, and the erectile tissue arrangement was determined and compared to standard anatomical descriptions. Nerves supplying the erectile tissue were carefully preserved and their relationship to the soft tissues and bony pelvis was noted. RESULTS The female urethra, distal vaginal wall and erectile tissue are packed into the perineum caudal (superficial) to the pubic arch, which is bounded laterally by the ischiopubic rami, and superficially by the labia minora and majora. This complex is not flat against the rami as is commonly depicted but projects from the bony landmarks for 3 to 6 cm. The perineal urethra is embedded in the anterior vaginal wall and is surrounded by erectile tissue in all directions except posteriorly where it relates to the vaginal wall. The bulbs of the vestibule are inappropriately named as they directly relate to the other clitoral components and the urethra. Their association with the vestibule is inconsistent and, thus, we recommend that these structures be renamed the bulbs of the clitoris. CONCLUSIONS A series of detailed dissections suggest that current anatomical descriptions of female human urethral and genital anatomy are inaccurate.

Nadph diaphorase-positive neurons in the rat spinal cord include a subpopulation of autonomic preganglionic neurons

Preganglionic neurons in the spinal cord of the rat were labelled retrogradely with Fluoro-gold and the spinal cord stained for NADPH diaphorase. The majority of both sympathetic and sacral parasympathetic preganglionic neurons showed staining for NADPH diaphorase. NADPH diaphorase-positive neurons were located more laterally in the intermediate zone than were preganglionic neurons lacking NADPH diaphorase staining. The recent evidence that identifies NADPH diaphorase as nitric oxide synthase raises the possibility that some spinal preganglionic neurons may synthesize nitric oxide.

The distribution of nitric oxide synthase-containing autonomic preganglionic terminals in the rat

Nitric oxide synthase (NOS)-immunoreactivity was co-localised with NADPH diaphorase activity in preganglionic sympathetic neurons and in their terminals in pre- and paravertebral sympathetic ganglia. The density of NOS-containing terminals varied between ganglia. Reactive terminals were densest in the superior cervical, stellate and inferior mesenteric ganglia, where the majority of the neurons were surrounded by reactive fibres, and the coeliac and superior mesenteric ganglia, where about half the postganglionic somata were surrounded by reactive terminals. Fibres were least abundant in the pelvic ganglia and thoracic and lumbar sympathetic chain ganglia. NOS reactivity did not coincide with the distribution of calcitonin gene related peptide immunoreactivity, a marker for the terminals of NOS-containing sensory neurons in the rat. The distribution of nerve cells and terminals suggests that NOS is present in more than one functional subpopulation of sympathetic preganglionic neurons.

Guidance cues involved in the development of the peripheral autonomic nervous system

All peripheral autonomic neurons arise from neural crest cells that migrate away from the neural tube and navigate to the location where ganglia will form. After differentiating into neurons, their axons then navigate to a variety of targets. During the development of the enteric nervous system, GDNF appears to play a role in inducing vagal neural crest cells to enter the gut, in retaining neural crest cells within the gut and in promoting the migration of neural crest cells along the gut. Sema3A regulates the entry of extrinsic axons into the distal hindgut, netrin-DCC signaling is responsible for the centripetal migration of cells to form the submucosal ganglia within the gut, Slit-Robo signaling prevents trunk level neural crest cells from entering the gut, and neurturin plays a role in the innervation of the circular muscle layer. During the development of the sympathetic nervous system, the migration of trunk neural crest cells through the somites is influenced by ephrin-Bs, Sema3A and F-spondin. The migration of neural crest cells ventrally beyond the somites requires neuregulin signaling and the clumping of cells into columns adjacent to the dorsal aorta is regulated by Sema3A. The rostral migration of cells to form the superior cervical ganglion (SCG) and the extension of axons along blood vessels involves artemin signaling through Ret and GFRalpha3, and the entry of sympathetic axons into target tissues involves neurotrophins and GDNF. Relatively little is known about the development of parasympathetic ganglia, but GDNF appears to play a role in the migration of some cranial ganglion precursors to their correct location, and both GDNF and neurturin are involved in the growth of parasympathetic axons into particular targets.

Characterisation of neurons with nitric oxide synthase immunoreactivity that project to prevertebral ganglia

Retrograde dye tracing was combined with immunohistochemistry to determine the distributions of nitric oxide synthase (NOS) immunoreactive nerve cells that project to prevertebral ganglia from the gastrointestinal tract and spinal cord of the guinea pig. An antiserum was raised against the neuronal form of NOS by selecting an amino-acid sequence specific to this form as immunogen. The antiserum recognised a single band at 150 kDa on Western blots of rat brain extract. Enteric nerve cells that were labelled by Fast Blue injected into the coeliac ganglion were not NOS immunoreactive in the small intestine, whereas 40-70% were reactive in the large intestine. Retrograde dye injected into the inferior mesenteric ganglion labels cells in the colon and rectum; 60-70% were immunoreactive for NOS. The NOS-immunoreactive nerve fibres arising in the intestine appear to end selectively around somatostatin-immunoreactive nerve cells in the coeliac and inferior mesenteric ganglia. Preganglionic nerve cell bodies in the intermediolateral column and dorsal commissural nucleus from T12 to L2 were labelled from the inferior mesenteric ganglion. Nearly 70% of neurons at each level were NOS immunoreactive. Thus, two sources of NOS terminals in prevertebral ganglia have been identified, intestinofugal neurons of the large, but not the small intestine, and sympathetic preganglionic neurons.

Neural regulation of inflammation: no neural connection from the vagus to splenic sympathetic neurons.

The ‘inflammatory reflex’ acts through efferent neural connections from the central nervous system to lymphoid organs, particularly the spleen, that suppress the production of inflammatory cytokines. Stimulation of the efferent vagus has been shown to suppress inflammation in a manner dependent on the spleen and splenic nerves. The vagus does not innervate the spleen, so a synaptic connection from vagal preganglionic neurons to splenic sympathetic postganglionic neurons was suggested. We tested this idea in rats. In a preparatory operation, the anterograde tracer DiI was injected bilaterally into the dorsal motor nucleus of vagus and the retrograde tracer Fast Blue was injected into the spleen. On histological analysis 7–9 weeks later, 883 neurons were retrogradely labelled from the spleen with Fast Blue as follows: 89% in the suprarenal ganglia (65% left, 24% right); 11% in the left coeliac ganglion; but none in the right coeliac or either of the superior mesenteric ganglia. Vagal terminals anterogradely labelled with DiI were common in the coeliac but sparse in the suprarenal ganglia, and confocal analysis revealed no putative synaptic connection with any Fast Blue‐labelled cell in either ganglion. Electrophysiological experiments in anaesthetized rats revealed no effect of vagal efferent stimulation on splenic nerve activity or on that of 15 single splenic‐projecting neurons recorded in the suprarenal ganglion. Together, these findings indicate that vagal efferent neurons in the rat neither synapse with splenic sympathetic neurons nor drive their ongoing activity.

CALBINDIN D28K-IMMUNOREACTIVITY IDENTIFIES DISTINCT SUBPOPULATIONS OF SYMPATHETIC PRE- AND POSTGANGLIONIC NEURONS IN THE RAT

Neurons performing the same function can be identified immunohistochemically because they often share the same neurochemistry. The distribution of calcium‐binding proteins, like calbindin, has been used previously to identify functional subpopulations of neurons in many parts of the nervous system. In this study we have investigated the distribution of calbindin D28K‐immunoreactivity in subpopulations of sympathetic preganglionic neurons in the intermediolateral nucleus of the rat spinal cord. The majority of calbindin D28K‐immunoreactive preganglionic neurons also had co‐localised nitric oxide synthase, although a population of preganglionic neurons in the mid‐ to low thoracic intermediolateral nucleus expressed only calbindin D28K‐immunoreactivity. Retrograde‐tracing studies showed that calbindin D28K‐immunoreactive neurons projected to the superior cervical and stellate ganglia, with smaller numbers of cells projecting to the lumbar sympathetic chain and superior mesenteric ganglia. Very few calbindin D28K‐immunoreactive neurons projected to the inferior mesenteric ganglion, and none projected to the adrenal medulla. The distribution of calbindin D28K‐immunoreactive terminals and postganglionic neurons in the superior cervical and stellate ganglia was also investigated. Many postganglionic neurons were calbindin D28K‐immunoreactive, and most of these lacked neuropeptide Y‐immunoreactivity. Calbindin D28K‐immunoreactive nerve terminals were common and formed dense pericellular baskets around many postganglionic neurons, including some of those that were calbindin D28K‐immunoreactive, but only rarely formed pericellular baskets around neuropeptide Y‐immunoreactive neurons. The function of some of the classes of postganglionic neurons that were the target of calbindin D28K‐immunoreactive preganglionic terminals was determined by combining immunohistochemistry with retrograde‐tracer injections into a range of peripheral tissues. Calbindin D28K‐immunoreactive nerve terminals, with co‐localised nitric oxide synthase‐immunoreactivity, surrounded secretomotor neurons projecting to the submandibular salivary gland and pilomotor neurons projecting to skin, but did not surround neurons projecting to brown fat or vasomotor neurons projecting to the skin, muscle, or salivary glands. J. Comp. Neurol. 386:245–259, 1997.

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.

Distinct preganglionic neurons innervate noradrenaline and adrenaline cells in the cat adrenal medulla

Calretinin immunoreactivity was present in a subset of preganglionic neurons retrogradely labelled from the cat adrenal gland. Overall, one-third of adrenal preganglionic neurons showed calretinin immunoreactivity, and their proportion increased in the more caudal spinal cord segments. Calretinin-immunoreactive nerve terminals were prominent within the adrenal gland, but were found only in areas of noradrenergic chromaffin cells (approximately one-third of the area of the adrenal medulla). Synaptophysin immunoreactivity was used to label terminals with and without calretinin immunoreactivity. Nerve terminals lacking calretinin immunoreactivity were present among the adrenergic chromaffin-cells and also comprised 20% of the nerve terminals innervating noradrenergic chromaffin cells. Calretinin immunoreactivity thus labels a subpopulation of cat adrenal preganglionic neurons that innervate the noradrenergic chromaffin cells.