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It was proposed by Rushton in 1951, from theoretical considerations, that myelinated fibres less than 1 micron in diameter would conduct more slowly than unmyelinated fibres of the same size and that myelinated fibres below about 0.7 micron would not conduct at all. The experimental data on which he based his theory are all from the peripheral nervous system where small myelinated fibres are rare, and no experimental verification of Rushton's hypothesis has been attempted. In mammalian optic nerve, nearly all the fibres are myelinated; yet half have diameters below 1 micron, with many below 0.7 micron. The many studies of conduction velocities in the visual system enable a test of Rushton's hypothesis to be made. We have examined the correlations between conduction velocity and fibre diameter from a wide range of published studies of the mammalian visual system. The results of our analysis suggest that the small myelinated fibres of the optic nerve and optic tract conduct action potentials more rapidly than is predicted by Rushton's hypothesis, while the unmyelinated axons within the retina actually conduct more slowly than predicted. There is no reason to believe, in this case, that myelination of a small axon will reduce its conduction velocity.

Histochemical study of the distribution of cholinesterases in the cat medulla oblongata reveals that all neurones in the dorsal motor nucleus of the vagus (DMV) contain true cholinesterase (AChE) but, while most contain this enzyme alone, a small proportion of cells contain pseudocholinesterase (BuChE) as well. Cervical vagotomy affects the two types of cell to different degrees of severity. The BuChE-containing neurones lose their enzyme completely within 2-3 weeks and they atrophy and disappear as a result of the operation. On the other hand, the reaction is less severe and is reversible in those cells containing AChE only. Vagotomy also causes reduction of AChE and BuChE staining in the nearby area subpostrema; the depletion here is pronounced at 2-3 weeks and recovery occurs within the year. These findings suggest that some cells in the area subpostrema project peripherally via the vagus and that the area is part of the vagal nuclear complex. Moreover, capilllaries in the area contain AChE and BuChE in the endothelial lining and this is one of the few areas of the cat hindbrain to exhibit such vascular enzyme activity. The ependyma of the area postrema, which overlies the area subpostrema, is heavily stained for BuChE but this is unaffected by vagotomy.

Oestriol and progesterone concentrations were measured by specific radioimmunoassays in the saliva of six women on a daily basis during the last month of pregnancy, at frequent intervals during labour and daily during the puerperium. Salivary steroid concentrations are thought to reflect the circulating concentrations of the free hormone, and hence may be more biologically relevant than the total plasma concentration, or the urinary excretion of a metabolite. There was no sign of a fall in salivary oestriol or progesterone concentrations before the onset of labour. During the first and second stages, the concentrations fluctuated widely, perhaps due to alterations in uterine blood flow. After delivery of the placenta, hormone concentrations declined abruptly. Oestriol reached undetectable levels of J. Endocr. (1987) 115, 177–181

The distribution of acetylcholinesterase (AChE) has been examined histochemically in the lateral geniculate nucleus (LGN) of the cat and the monkey, and in the cat visual cortex. It was found that in the cat, AChE is most concentrated in laminae A and A1. Lamina C-proper possessed a weak band of AChE in its ventral part. Only restricted patches of activity were observed in the medial interlaminar nucleus. Laminae C1–3 and the central interlaminar nucleus possessed very little AChE. This pattern of enzyme distribution suggests that in the cat LGN, AChE activity coincides with the sites of neurophysiologically recorded X-cells, which are predominantly found in laminae A and A1 and are scarce in the C laminae and the medial interlaminar nucleus. The presence of AChE over neurones in layer VI of both areas 17 and 18 of the cerebral cortex in the cat suggests the corticothalamic pathway as one possible source of geniculate AChE activity. In the monkey LGN, AChE activity was observed in the parvocellular and magnocellular layers. The activity was greatest in the magnocellular layers, which are believed to contain neurons driven predominantly by retinal Y-cells. Thus, for this species the correlation between AChE activity and X-cells does not seem to hold.

Summary (1) The distributions of true cholinesterase (AChE) and pseudocholinesterase (ChE) in the spinal cord of the rat have been studied histochemically, by means of a thiocholine technique, at the light and electron microscope levels. (2) The somatic motoneurones in the ventral gray column contain high concentrations of AChE and are well demonstrated by the technique; the enzyme occupies the clefts of the rough endoplasmic reticulum and, in places, the nuclear envelope. The motoneurones are grouped into sub-columns which lie at the periphery of the ventral horn, the central portion of which is devoid of AChE-containing cells. Loss of enzyme following sciatic nerve section at mid-thigh level is restricted to the motoneurones in the retrodorsolateral sub-column in the last lumbar and first sacral segments. (3) The thoraco-lumbar part of the intermediolateral gray column contains AChE only, while the sacral part of the column contains both AChE and ChE. The enzyme content of the sacral intermediolateral column is unaffected by sciatic nerve section but is reduced following interruption of pelvic splanchnic nerves. (4) Apart from the neurones in the ventral and intermediolateral gray columns, cholinesterases are also found in certain other varieties of cells. AChE is present in neurones of the intermediomedial column and in cells lying in the lateral white funiculus adjacent to the substantia gelatinosa, while ChE is found in several small neurones scattered in the central portion of the ventral gray horn. These 3 varieties of cells are found throughout the length of the cord and they are all probably propriospinal as they are unaffected by peripheral nerve section.

1. Acetylcholinesterase in the hippocampal formation is located on the axonal membrane of cholinergic fibres of the alveus, in cholinergic neuropil, on the pre- and postsynaptic membranes at certain synapses, in the granular endoplasmic reticulum and the nuclear envelope of Golgi type II cells in the stratum oriens, and to a small extent in pyramidal cells. 2. The dense layers of acetylcholinesterase seen in certain layers of the hippocampal formation by light microscopy correspond to regions of dense cholinergic neuropil. 3. The terminals of cholinergic afferents in the hippocampal formation are predominantly axodendritic. 4. Axosomatic endings, though rare on pyramidal cells, are frequent on Golgi cells of the stratum oriens. 5. Light and dark cells can be distinguished in the granular layer of the dentate gyrus.

The remarkable capacity of transplanted embryonic neurones to innervate the hippocampal formation of mature recipients has been well documented, with the pattern of innervation being shown to be anatomically specific and to resemble normal connectivity1–3. Although transplants are known to have functional consequences in other systems4–9, information has yet to be obtained regarding the functional nature of embryonic septal transplants and the behavioural consequences of transplant innervation of the host hippocampal formation. We provide here evidence that a reinnervation of the hippocampal formation from cholinergic-rich septal transplants is functional in terms of the physiology of neural connectivity and that the newly formed connections can interact with an intrinsic afferent system, the perforant path. Moreover, we demonstrate that the reinnervation can aid in the partial recovery of the performance of a radial maze task that is thought to depend on the integrity of septohippocampal connections10. The behavioural performance of animals with transplants improved significantly compared with those without transplants when both groups were systemically injected with the acetylcholinesterase (AChE) inhibitor, physostigmine. These results suggest that neural transplants from embryonic tissue that reinnervate the hippocampal formation can form functional synaptic connections that can lead to the partial restoration of maze performance.

The distribution of ‘marker’ enzymes for cholinergic neurons has been studied in 10 subdivisions of the amygdaloid complex of the rat brain. Choline acetyltransferase activity was measured using a radiochemical method in samples dissected from fresh serial sections. Acetylcholinesterase was studied using a histochemical procedure. Both enzymes had similar patterns of distribution within the amygdaloid complex and were most concentrated in the posterior lateral and basolateral nuclei and in the nucleus of the lateral olfactory tract. These enzymes were much less concentrated in the cortical, medial, central, and basomedial nuclei. Large differences in acetylcholinesterase staining were found within the lateral posterior and the basolateral nuclei and within the pyriform cortex. Biochemical studies showed a parallel distribution of choline acetyltransferase within these nuclei. The results indicate that cholinergic neural elements in the amygdala are concentrated primarily in the basolateral complex and suggest that this region may be innervated by cholinergic fibers traveling in the ventral amygdalo-fugal pathway.

The ability of embryonic tissue to survive cross-transplantation between histologically incompatible rat strains was examined by transplanting septal neurons from Sprague Dawley fetuses to adult Wistar rats (Ag-B6 to Ag-B2 histocompatibility haplotype). Transplants were found to survive without rejection over a period of 3 months. Furthermore, the laminar pattern of cholinergic innervation was similar to that of homogenic septal transplants and of intrinsic septal projections. These results suggest that embryonic neural tissue transplanted across major histocompatibility barriers are capable of survival for extended periods of time, and are in support of the concept of the privileged nature of embryonic tissue as a source of material for cross-transplantation.

The thiocholine technique for cholinesterase has been successfully adapted for the demonstration of enzyme distribution with the electron microscope. After fixation in buffered glutaraldehyde, thin slices of tissue were taken through a histochemical procedure designed to minimize diffusion artifacts and cytological damage; appropriate areas were dissected out, fixed with osmium tetroxide and embedded in Araldite. This technique has been used to study the distribution of enzyme in and around known cholinergic neurons in the rat. In diaphragm muscle the intense staining of the motor end plates is confined to the actual synaptic clefts. The distribution of cytoplasmic staining was similar in all three types of cholinergic neurons studied (ventral horn cells from the cervical cord, cells from the dorsal motor nucleus of the vagus, and cells from the hypoglossal nucleus). There was intense staining of the space contained within the individual bilaminar sheets of the rough endoplasmic reticulum and occasionally in areas of the nuclear envelope. Mitochondria, lysosomes, the smooth endoplasmic reticulum and most of the plasma membrane were unstained. In cholinergic nerve fibres staining was particularly intense at the axonal membrane and absent from the myelin sheath. In several regions of the brain known to receive an afferent cholinergic innervation many of the identifiable synaptic areas were heavily stained. Staining here was usually present round most of the presynaptic process, spreading over part of the postsynaptic process and often penetrating into the actual synaptic space. The mitochondria, microvesicles and the general cytoplasm of the synaptic processes were all unstained. In some areas, such as the hippocampus, there was intense membrane staining of fine cholinergic neuropil. The enzyme specificity of the technique is not in doubt and evidence is adduced for the view that diffusion artifacts are small. The close correlation between the electron-microscopic results and evidence from physiological and biochemical investigations is discussed.

Cholinesterase-Containing Pathways of the Hindbrain: Afferent Cerebellar and Centrifugal Cochlear Fibres

The presence of cholinesterase activity in association with capillaries of the central nervous system was investigated in the rat by means of both light and electron microscopic methods. Throughout most of the rat brain, the smaller blood vessels stain intensely for butyrylcholinesterase activity. In some areas, such as the commissural nucleus of the vagus and parts of the medial thalamus, the capillaries possess both acetylcholinesterase and butyrylcholinesterase activity. Blood vessels in those structures which lie outside the blood-brain barrier are completely devoid of cholinesterase activity. The electron microscope reveals that reaction product occurs within the matrix of the basement membrane, in the intermembranous space of the endothelial nuclear envelope and occasionally in the endothelial granular endoplasmic reticulum. It is suggested that the presence of cholinesterase within the basement membrane of brain capillaries is evidence of the role that the basement membrane may play in transfer mechanisms.

SUMMARY The distribution of cholinesterase activity in the rat adrenal medulla was studied after initial fixation with glutaraldehyde, which preserves the osmiophilia of the noradrenaline-containing cells. Staining for true cholinesterase activity was associated with all the identifiable elements of the preganglionic sympathetic innervation. The morphology and enzyme staining of the endings on the chromaffin cells was similar to that seen in cholinergic synapses in the central nervous system. The endings contained occasional dense-cored vesicles and the possible significance of such vesicles at an undoubted cholinergic synapse is discussed. Staining for pseudocholinesterase activity, by contrast, was associated particularly with the Schwann cells and to a lesser extent with the chromaffin cells, but not with the axons and their processes.

THE histochemical technique for cholinesterases introduced by Koelle and Friedenwald1 has been used in numerous studies of nervous tissue. Much of this work has been concerned primarily either with the histochemistry of the method or with the cytological distribution of enzyme revealed. This method has, we feel, also great potentialities in the field of neuroanatomy—as a means of selectively staining particular groups of neurones which cannot otherwise be distinguished by more conventional methods. Some attempts along these lines have been made in studies of peripheral innervation2,3, but it is in studies of the central nervous system that there is particular need for a selective staining method. This communication is therefore concerned not so much with enumerating our detailed findings in this preliminary investigation as with emphasizing the potential value of this technique in solving many neurological problems which have so far resisted attack by more conventional methods.

IN the spinal cord of larval Anura, the motor cells which innervate the muscles of the hind limbs form ventral horns distinct both in position and time of appearance from the series of primary motor cells whose axons run to the trunk muscles. In the tadpole of Xenopus laevis, there are about 4,000 ventral horn cells at the time of their differentiation from the mantle layer of the cord, a number which has fallen to 1,200 by metamorphosis some eight weeks later1. ‘Histogenic cell degeneration’2 accompanies the differentiation of these neurones, but on a scale much beyond that required merely to reduce their number to the adult-level. It seems that, for every ventral horn cell that persists, some eight or nine undergo cell death during development. Differentiation of the ventral horn is thus accompanied by a continuous turnover of the constituent neuroblasts.