A fine structural analysis of the epidermis of the earthworm, Lumbricus terrestris L.

A fine structural analysis of the cuticle, epidermal epithelium, and underlying fibrous tissue of the earthworm is presented. The extreme scarcity or absence of fibroblasts in this animal is pointed out. This finding is further evidence for the epithelial origin of the cuticular fibers, and suggests that at least some of the collagenous connective tissue fibers in the interior of this animal are epithelial in origin. The junctional specializations that unite epithelial cells in the epidermis and intestine are described. Of special interest is the fact that the septate desmosome rather than the tight junction is found in these epithelia. It is shown that the septa are not extensions of the plasma membrane across the intercellular gap. Finally, the nature of the small ellipsoidal bodies that are embedded in the outer layer of the cuticle is discussed.

Quantitation of sprouting of dorsal root axons.

The axonal sprouting that occurs after denervation resulting from a spinal hemisection can be quantified. Rats were subjected to hemisection of the spinal cord at birth, and the myelinated and unmyelinated axons in dorsal roots three segments cranial and three segments caudal to the lesion were counted 1 month after surgery. The number of unmyelinated axons in the dorsal root on the side of the hemisection increased 22 percent for the roots one segment from the lesion and 13 percent for the roots two and three segments from the lesion.

Unmyelinated axons in the posterior funiculi.

Electron microscopy of the dorsal funiculus in the rat reveals that most axons in this pathway are unmyelinated. These axons have not previously been counted, nor are they considered in modern studies on the organization of the dorsal funiculus. Because of the importance of this pathway in somatic sensation, it is important to understand that these fibers exist and that they are present in greater numbers than the well-studied myelinated axons.

Opposite synaptic actions mediated by different branches of an identifiable interneuron in Aplysia.

Among the identifiable cells in the abdominal ganglion of Aplysia californica are five that generate bursting rhythms endogenous to the cells. In the four bursting cells of the left upper quadrant the rhythm is modulated by a unitary inhibitory postsynaptic potential; in the bursting cell of the right lower quadrant the rhythm is modulated by a unitary excitatory postsynaptic potential. Both the excitatory and inhibitory postsynaptic potentials are mediated by separate branches of a single interneuron. The pharmacological properties of the double action interneuron as well as those of the follower cells suggest that a single transmitter (acetylcholine) is involved in both the excitatory and the in-hibitory action of the interneuron.

Increased numbers of thoracic dorsal root axons in rats given antibodies to nerve growth factor.

Sensory axons were counted in untreated 1-month-old rats and in littermates that were injected with antibodies to nerve growth factor. There were 45 percent more unmyelinated and 17 percent more myelinated axons in dorsal roots of the fifth thoracic spinal segment in treated rats. This suggests that the number of sensory axons can be changed by postnatal inactivation of nerve growth factor.

Activation of central neurons by ventral root afferents.

It is a fundamental principle of vertebrate neuronal organization that sensory fibers are restricted to dorsal roots and motor fibers to ventral roots. Recent evidence, however, indicates that there are many sensory fibers in ventral roots. The present report shows that stimulation of these fibers activates neurons in the dorsal horn. This provides evidence at the single-cell level for the importance of ventral root afferents and provides an explanation for the clinical phenomenon of recurrent sensibility.

A consideration of neural counting methods.

It is often necessary to obtain unbiased estimates of neuronal or synaptic numbers. In the past, estimates were almost always done by counting profiles of these structures in single histological sections. Assumptions were then made and calculations were done to determine particle numbers or ratios. To the extent that the assumptions deviated from reality, the conclusions will be biased. That these biases are, in fact, serious has recently become apparent. To obtain unbiased particle counts, the presently available methods are serial-section reconstructions (which are accurate but cumbersome), and the recently developed disector method. The disector method, because it is unbiased and easy to use, is becoming the method of choice. The goals of this paper are to show why previous methods are biased and to describe the rationale behind the disector method so that neuroscientists can consider its appropriateness for their work.

Tonic control of peripheral cutaneous nociceptors by somatostatin receptors.

The peptide somatostatin [somatotropin release-inhibiting factor (SRIF)] is widely distributed in the body and exerts a variety of hormonal and neural actions. Several lines of evidence indicate that SRIF is important in nociceptive processing: (1) it is localized in a subset of small-diameter dorsal root ganglion cells; (2) activation of SRIF receptors results in inhibition of both nociceptive behaviors in animals and acute and chronic pain in humans; (3) SRIF inhibits dorsal horn neuronal activity; and (4) SRIF reduces responses of joint mechanoreceptors to noxious rotation of the knee joint. The goal of the present study is to show that cutaneous nociceptors are under the tonic inhibitory control of SRIF. This is accomplished using behavioral and electrophysiological paradigms. In a dose-dependent manner, intraplantar injection of the SRIF receptor antagonist cyclo-somatostatin (c-SOM) results in nociceptive behaviors in normal animals and enhancement of nociceptive behaviors in formalin-injected animals, and these actions can be blocked when c-SOM is coapplied with three different SRIF agonists. Furthermore, intraplantar injection of SRIF antiserum also results in nociceptive behaviors. Electrophysiological recordings using an in vitro glabrous skin-nerve preparation show increased nociceptor activity in response to c-SOM, and this increase is blocked by the same three SRIF agonists. Parallel behavioral and electrophysiological studies using the opioid antagonist naloxone demonstrate that endogenous opioids do not maintain a tonic inhibitory control over peripheral nociceptors, nor does opioid receptor antagonism influence peripheral SRIF effects on nociceptors. These findings demonstrate that SRIF receptors maintain a tonic inhibitory control over peripheral nociceptors, and this may contribute to mechanisms that control the excitability of these terminals.

A role for HSP27 in sensory neuron survival.

Peripheral nerve injury in neonatal rats results in the death of the majority of the axotomized sensory neurons by 7 d after injury. In adult animals, however, all sensory neurons survive for at least 4 months after axotomy. How sensory neurons acquire the capacity to survive axonal injury is not known. Here we describe how the expression of the small heat shock protein 27 (HSP27) is correlated with neuronal survival after axotomy in vivo and after NGF withdrawal in vitro. The number of HSP27-immunoreactive neurons in the L4 DRG is low at birth and does not change significantly for 21 d after postnatal day 0 (P0) sciatic nerve axotomy. In contrast, in the adult all axotomized neurons begin to express HSP27. One week after P0 sciatic nerve section the total number of neurons in the L4 DRG is dramatically reduced, but all surviving axotomized neurons, as identified by c-jun immunoreactivity, are immunoreactive for HSP27. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling reveals that very few HSP27-expressing neurons are dying 48 hr after neonatal axotomy. In vitro, a similar correlation exists between HSP27 expression and survival; in P0 DRG cultures, neurons that express HSP27 preferentially survive NGF withdrawal. Finally, overexpression of human HSP27 in neonatal rat sensory and sympathetic neurons significantly increases survival after NGF withdrawal, with nearly twice as many neurons surviving at 48 hr. Together these results suggest that HSP27 in sensory neurons plays a role in promoting survival after axotomy or neurotrophin withdrawal.

Human neural stem cell-derived cholinergic neurons innervate muscle in motoneuron deficient adult rats.

Motoneuron damage occurs in spinal cord injury and amyotrophic lateral sclerosis. Current advances offer hope that human embryonic stem cells [Science 282 (1998) 1145] or neural stem cells (NSC) [Exp Neurol 161 (2000) 67; Exp Neurol 158 (1999) 265; J Neurosci Methods 85 (1998) 141; Proc Natl Acad Sci USA 97 (2000) 14720; Exp Neurol 156 (1999) 156 ] may be donors to replace lost motoneurons. Previously, we developed a priming procedure that produced cholinergic cells that resemble motoneurons from human NSCs grafted into adult rat spinal cord [Nat Neurosci 5 (2002a) 1271]. However, effective replacement therapy will ultimately rely on successful connection of new motoneurons with their muscle targets. In this study, we examined the potential of human fetal NSC transplantation to replace lost motoneurons in an animal model of chronic motoneuron deficiency (newborn sciatic axotomy) [J Comp Neurol 224 (1984) 252; J Neurobiol 23 (1992) 1231]. We found, for the first time, that human neural stem cell-derived motoneurons send axons that pass through ventral root and sciatic nerve to form neuromuscular junctions with their peripheral muscle targets. Furthermore, this new cholinergic innervation correlates with partial improvement of motor function.

Primary afferent axons in the tract of Lissauer in the cat.

The present study is concerned with the numbers of primary afferent axons in the tract of Lissauer in the cat. The data show that approximately 27% of the axons in mid-thoracic and lumbosacral tracts are primary afferent fibers from the segment in question and another 20% of the axons are primary afferent fibers that come from nearby segments. In addition the data show that approximately 80% of the axons in the tract are unmyelinated and that there is a slightly higher proportion of unmyelinated as opposed to myelinated primary afferents. There is also a higher proportion of primary afferents in the medial as opposed to lateral parts of the tract, but there are significant numbers of primary afferents in lateral parts of the tract. Thus it seems clear that the tract contains more primary afferent fibers than was previously believed and if these data are confirmed, the conclusions will have a bearing on considerations of the primary afferent input into the dorsal horn.

Branching of sensory axons in the dorsal root and evidence for the absence of dorsal root efferent fibers.

The present study demonstrates that there are more dorsal root axons than dorsal root ganglion cells in the L6-S1 dorsal roots of the rat. The excess fibers do not come from aberrant dorsal root ganglion cells and our control procedures indicate that there are no extraneous fibers in these dorsal roots. Accordingly many dorsal root axons must branch in the dorsal root.

Propriospinal fibers in the white matter of the cat sacral spinal cord.

The propriospinal system, which consists of those neurons completely contained within the spinal cord, is important because it underlies much spinal behavior. To provide quantitative data on this system, the present study determines numbers of axons in the isolated S2 cat spinal cord and compares these figures with the normal. The conclusion is that 60% of the fibers in the spinal cord at this location are propriospinal. Findings of particular interest are that the great majority of unmyelinated propriospinal axons are found in the dorsal part of the lateral funiculus, and that there are large numbers of descending myelinated fibers in the dorsal funiculi. These data will serve as a basis for evaluating axon numbers that follow various experimental regimens purporting to result in neural sprouting.

Unmyelinated primary afferent fibers in dorsal funiculi of cat sacral spinal cord.

The present study tests the hypothesis that there are numerous unmyelinated primary afferent fibers in cat posterior funiculi. The animals have unilateral dorsal rhizotomies from L6 to Ca3. One week later the axons of both S2 dorsal funiculi are counted. The data indicate that there are approximately 22,500 myelinated and 8,500 unmyelinated axons on the unoperated side and 11,000 myelinated and 3,900 unmyelinated axons on the operated side. On this basis, we suggest that 51% of the myelinated and 54% of the unmyelinated axons in cat dorsal funiculi arise from dorsal root ganglion cells and thus are primary afferent axons. If this is correct, then 71% of the primary afferent axons in the cat dorsal funiculus are myelinated and 29% are unmyelinated. The function of this large group of previously unsuspected fine sensory axons remains to be determined.

Methods for determining numbers of cells and synapses: a case for more uniform standards of review.

Neuron and synapse numbers are important assays in neuroscience. These numbers are estimated by one of four methods: 1) profile counts, 2) assumption-based methods, 3) serial reconstructions, and 4) stereological methods. The criteria for these methods are diverse. This creates a disparity in that some reviewers accept estimates from any of these methods, while others accept only specific methods. An equally important issue is the diversity of sampling strategies, since unbiased estimates of neuronal or synaptic numbers are contingent upon both counting and sampling techniques. The purpose of this commentary is to institute a dialog that will lead to a better understanding of the strengths and weaknesses of the above methods, and to propose guidelines that should lead to more uniform and thus fairer judging of the studies that provide estimates of neuron or synapse numbers. In addition, adoption of more uniform standards for obtaining unbiased numerical estimates should result in the generation of an unbiased database that will be of considerable use in future studies.

Ultrastructural analysis of NMDA, AMPA, and kainate receptors on unmyelinated and myelinated axons in the periphery.

The present study determines the proportions of unmyelinated cutaneous axons at the dermal-epidermal junction in glabrous skin and of myelinated and unmyelinated axons in the sural and medial plantar nerves that immunostain for subunits of the ionotropic glutamate receptors. Approximately 20% of the unmyelinated cutaneous axon profiles at the dermal-epidermal junction immunostain for either N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or kainate receptor subunits. These findings are consistent with previous observations that NMDA and non-NMDA antagonists ameliorate nociceptive behaviors that result from noxious peripheral stimulation. In the sural nerve, where the large majority of myelinated fibers are sensory, approximately half of the myelinated axon profiles immunostain for the NMDA receptor 1 (R1) subunit, 28% immunostain for the glutamate receptor 1 (GluR1) AMPA subunit, and 11% for the GluR5,6,7 kainate subunits. Even higher proportions immunostain for these receptors in the medial plantar nerve, a mixed sensory and motor nerve. In the sural nerve, 20% of the unmyelinated axon profiles immunostain for NMDAR1 and only 7% label for GluR1 or GluR5,6,7. Because the sural nerve innervates hairy skin, these data suggest that glutamate will activate a higher proportion of unmyelinated axons in glabrous skin than in hairy skin. Measurements of fiber diameters indicate that all sizes of myelinated axon profiles, including Adelta and Abeta, are positively labeled for the ionotropic receptors. The presence of glutamate receptors on large-diameter myelinated axons suggests that these mechanosensitive receptors, presumably transducing touch and pressure, may also respond to local glutamate and thus be chemosensitive.

The connective tissue coverings of leech peripheral nerves: anatomical evidence for the absence of cerebrospinal fluid in the leech.

The central nervous system of Hirudo medicinalis is contained within a blood vessel, the ventral longitudinal sinus, but the nervous system is separated from the blood by the visceral endothelium. The visceral endothelium possesses many pinocytotic vesicles and basal infoldings and thus appears active whereas the parietal endothelium appears inactive. The junction between the visceral and parietal endothelia is abrupt. Peripheral nerves in this animal, as in vertebrates, are covered by endoneurium, perineurium, and epineurium. The endoneurium is continous with the fibrous tissue of the segmental ganglia. The perineurium, consisting of a single layer of flattened cells that surrounds the peripheral nerve like a sleeve, is not continuous with the endothelium of the ventral sinus, but is separated from it by 5-10 microns. Therefore, at the point where the peripheral nerve joins the segmental ganglion, the extracellular spaces of the central nervous system, the peripheral nervous system, and the body wall are all confluent. Thus, there are only two compartments of the extracellular space in the leech: the blood, which is enclosed by the endothelia of the coelomic sinuses, and the extracellular fluid of the body, which includes the extracellular fluid of the nervous system. There seems to be no equivalent of cerebrospinal fluid in the gnathobdellid leech.

Axonal numbers and sizes in the connectives and peripheral nerves of the leech.

The present study is an electron microscopic analysis of the numbers and sizes of axons in the connectives and nerves of the medicinal leech. In either the right or left connectives for ganglia 14-18, there are approximately 2860 (+/- 294 S.D.) axons. Ninety-seven percent of these axons are less than one micron in diameter. The median connective, Faivre's nerve, contains 97 (+/- 3 S.D.) axons, and 94% of these fibers are smaller than one micron. In the peripheral nerve roots for ganglia 14-18, there are approximately 2351 (+/- 311 S.D.) axons. Ninety-eight percent of the axons in the nerves are less than one micron in diameter. Thus, there are approximately 20,000 axons associated with each of the segmental ganglia 14-18 in the leech, and the vast majority of these fibers are less than one micron in diameter.

An analysis of the axon populations in the nerves to the pelvic viscera in the rat.

The present study uses selective surgical ablations combined with electron microscopic analyses to determine the number of axons in various categories in rat hypogastric, pelvic, and pudendal nerves, these being the nerves to the pelvic viscera in this animal. Unmyelinated fibers predominate in all of these nerves. One of the most significant findings is that the pelvic nerve contains almost as many postganglionic sympathetic fibers as the hypogastric nerve. Previous investigators thought that the pelvic nerve supplied the parasympathetic inflow and the hypogastric nerve the sympathetic inflow to the pelvic viscera. The finding that there is a sizable sympathetic component in the pelvic nerve negates this idea, at least for the rat, and presumably calls for a reevaluation of the syndromes that arise from pelvic as opposed to hypogastric nerve section. Other findings of interest are (1) that there are unmyelinated efferent axons in the pudendal nerve, indicating that the pudendal is not a typical somatic nerve, (2) that the hypogastric nerve has a very small sensory component, and (3) that there are fibers surviving in the distal stumps of all these nerves, particularly the pelvic and hypogastric nerves.

Quantitation of propriospinal fibers in the tract of Lissauer of the rat.

The present study uses a spinal cord isolation procedure to remove extrinsic axons but leave intrinsic axons intact. The isolation is done by sectioning the spinal cord in two places and then cutting all dorsal roots between the two sections. The axons that survive the isolation procedure are thought to be propriospinal axons. Following isolation, approximately one-third of the axons in sacral tracts of Lissauer in the rat survive. Thus approximately one-third of the axons in sacral tracts of Lissauer in the rat are propriospinal. Proportionately more myelinated than unmyelinated axons are lost. There are approximately equal numbers of surviving axons in the medial as opposed to the lateral part of the tract. This implies that there is, as yet, no morphological basis for dividing the tract into medial and lateral halves. The fact that the number of propriospinal axons in the tract of Lissauer has been quantitated offers more precision in our thinking about the organization of the dorsal horn.