Projections from the mesopontine tegmental anesthesia area to regions involved in pain modulation.

Pentobarbital microinjected into a restricted locus in the upper brainstem induces a general anesthesia-like state characterized by atonia, loss of consciousness, and pain suppression as assessed by loss of nocifensive response to noxious stimuli. This locus is the mesopontine tegmental anesthesia area (MPTA). Although anesthetic agents directly influence spinal cord nociceptive processing, antinociception during intracerebral microinjection indicates that they can also act supraspinally. Using neuroanatomical tracing methods we show that the MPTA has multiple descending projections to brainstem and spinal areas associated with pain modulation. Most prominent is a massive projection to the rostromedial medulla, a nodal region for descending pain modulation. Together with the periaqueductal gray (PAG), the MPTA is the major mesopontine input to this region. Less dense projections target the PAG, the locus coeruleus and pericoerulear areas, and dorsal and ventral reticular nuclei of the caudal medulla. The MPTA also has modest direct projections to the trigeminal nuclear complex and to superficial layers of the dorsal horn. Double anterograde and retrograde labeling at the light and electron microscopic levels shows that MPTA neurons with descending projections synapse directly on spinally projecting cells of rostromedial medulla. The prominence of the MPTA's projection to the rostromedial medulla suggests that, like the PAG, it may exert antinociceptive actions via this bulbospinal relay.

Structural basis of sympathetic-sensory coupling in rat and human dorsal root ganglia following peripheral nerve injury.

Tyrosine hydroxylase immunocytochemistry was used to reveal the sympathetic postganglionic axons that sprout to form basket-like skeins around the somata of some primary sensory neurons in dorsal root ganglia (DRGs) following sciatic nerve injury. Ultrastructural observations in rats revealed that these sprouts grow on the surface of glial lamellae that form on the neurons. Sciatic nerve injury triggers glial cell proliferation in the DRG, and the formation of multilamellar pericellular onion bulb sheaths, primarily around large diameter DRG neurons. We infer that these glia participate in the sprouting process by releasing neurotrophins and expressing growth supportive cell surface molecules. Many DRG cell somata, and their axons in intact nerves and nerve end neuromas, express alpha2A adrenoreceptors intracytoplasmically and on their membrane surface. However, sympathetic axons never make direct contacts with the soma membrane. The functional coupling known to occur between sympathetic efferents and DRG neurons must therefore be mediated by the diffusion of neurotransmitter molecules in the extracellular space. Sympathetic basket-skeins were observed in DRGs removed from human neuropathic pain patients, but the possibility of a functional relation between these structures and sensory symptoms remains speculative.

An electron-microscopic analysis of biopsy samples of the trigeminal root taken during microvascular decompressive surgery.

During surgical exposure of the trigeminal root for the treatment of trigeminal neuralgia, biopsy specimens of the nerve root were taken in 12 consecutive patients. The biopsies were taken from areas as close as possible to the site of vascular compression, when present. The tissue was prepared for electron-microscopic evaluation. Except for 1 case, massive disruption of tissue ultrastructure was observed. The major findings included zones of de- and dysmyelination, juxtaposition of denuded axons, apparent axon loss and degeneration, and collagen deposition. Adjacent areas of normal root structure were present in most specimens. The ultrastructural changes observed in the biopsy samples demonstrate important pathology within the trigeminal root. Dysmyelination and the proximity of denuded axons in close apposition to each other support neurophysiological theories of pain production including ectopic electrogenesis, ephaptic contacts, and crossed after-discharge. The findings in this study demonstrate a pathophysiological basis for the syndrome of trigeminal neuralgia.

Close apposition among neighbouring axonal endings in a neuroma.

Axons in intact peripheral nerve trunks constitute independent afferent and efferent communication channels. However, when nerves are severed, several different forms of axon-axon cross-excitation develop in association with the injury site. In this study we have examined experimental sciatic nerve-end neuromas in rats with special interest in the compartmentalization of individual axons, and the barriers that separate close neighbours. At postinjury times at which functional coupling is known to occur, neuromas were found to contain many examples of axons in which adjacent membrane faces come into close contact without an intervening Schwann cell process. These occur in bundles containing from two to as many as 30 individual nerve fibres wrapped in a common Schwann cell sheath. The surface area of close apposition between axon pairs ranges up to several tens of micron2. Closely apposed axon profiles may be outgrowing branches of a single parent axon, but anterograde tracer data indicate that many belong to independent neurons. Closely apposed axons are separated from one another, and from associated Schwann cell processes, by a cleft about 130 A wide. No synapses, gap junctions or tight junctions were observed. Extracellular tracer studies using La3+ and Ruthenium Red indicated that the cleft system is patent, permitting the free diffusion of small molecules between the space adjacent to the axolemma and the bulk extracellular compartment. Together, these data provide a structural basis for interfibre interactions based on local electrical current flow (ephaptic crosstalk), as well as coupling mediated by K+ ions and neurotransmitter molecules.

Na+ channel immunolocalization in peripheral mammalian axons and changes following nerve injury and neuroma formation.

Nerve injury frequently triggers hyperexcitability and the ectopic initiation of impulses in primary afferent axons. An important consequence is neuropathic paresthesias and pain. Electrogenesis in normal afferents depends on appropriate Na+ channel concentrations. Therefore, we have asked whether injury might trigger changes in axolemmal Na+ channel distribution that could account for neuropathic hyperexcitability. We used an Na+ channel-specific antibody, 7493, to immunolocalize Na+ channels ultrastructurally in membranes of normal rat axons, and to assess remodeling following nerve section and neuroma formation. Selective labeling of nodal axolemma and, more weakly, of Schwann cell membrane, confirmed the efficacy of our immunolabeling protocol. In neuromas at postoperative times associated with peak ectopic activity, we found clear evidence of Na+ channel accumulation. Specifically, soon after myelin was stripped from large-diameter axons, the exposed, formerly internodal axolemma became immunopositive. Small-diameter unmyelinated axons and axon sprouts in the neuroma were also marked with 7493 IgG. Activated phagocytic macrophages and endothelial cells were 7493 negative. Both large- and small-diameter axons in neuromas end in swollen, organelle-packed "end bulbs." Most, but not all, of these acquired Na+ channel immunolabeling. We propose that remodeling results from a modification of the normal process of Na+ channel turnover in neural membranes. Na+ channel protein accumulates in preterminal axolemma and neuroma end bulbs due to a combination of permissive factors (especially myelin removal) and promotional factors (removal of normal downstream targets). This accumulation is a likely precursor of afferent hyperexcitability in injured nerve.

Ultrastructure of afferent axon endings in a neuroma.

Injured sensory axons with endings trapped in a nerve-end neuroma become a source of abnormal impulse discharge and neuropathic pain. We have examined the ultrastructure of such endings anterogradely transported WGA-HRP and freeze-fracture replication, with emphasis on the postinjury period during which the abnormal neural discharge is maximal. Most axons ended in a terminal swelling, depleted of myelin but surrounded by Schwann cell processes. These 'neuroma endbulbs' were richly packed with membrane-bound organelles, and had a smoothly undulating surface with (in neuromas of several weeks standing) a moderate number of short filopodia. Massive sprouting did not occur until several months postinjury. Both p- and e-faces of endbulb axolemma had larger intramembranous particles, on average, than corresponding internodal membrane of control axons. This change, interpreted as indicating remodelling of axolemmal channel (and perhaps receptor) content, may be related to the abnormal electrical behavior of neuroma afferents.

Neurogenesis in adult rat dorsal root ganglia: on counting and the count.

La Forte et al. (this issue) failed to find an increase in the numbers of dorsal root ganglion (DRG) neurons in rats of advancing age. However, their conclusion that our data (Devor and Govrin-Lippmann, 1985) to this effect were methodologically flawed is based on an incorrect application of our counting method. In fact, both their counting method and ours provide similar results when applied to the same tissue sections. We believe that the difference in results is biological. Specifically, DRG neurogenesis in adulthood occurs in animals in which growth continues throughout life (including the male Wistar-derived rats we used), but not in those whose body size stabilizes soon after sexual maturity (including the female Sprague-Dawley rats they used). With this caveat in mind, recent data of Schmalbruch (1987a,b) and others can be understood as corroborating our conclusions. However, an adequate, independent replication of our 1985 study has yet to be carried out.

Contrasting time course of catecholamine accumulation and of spontaneous discharge in experimental neuromas in the rat.

Catecholamine-containing sympathetic axons in rat sciatic nerve-end neuromas were visualized histochemically. Within a few hours of ligating and sectioning the nerve, axons began to accumulate catecholamine histofluorescence. Density of labelled fibers peaked 2-5 days postoperative, then declined rapidly so that little or no label was observed beyond 12 days. Sympathectomy eliminated staining; neonatal treatment with capsaicin had no effect. Accumulation and dissipation of histofluorescence preceded the rise and fall of electrical hyperexcitability in neuromas by several days respectively.

Retrograde slowing of conduction in sensory axons central to a sciatic nerve neuroma.

We have plotted the time-course of retrograde slowing of impulse conduction velocity in myelinated afferent fibers after sciatic nerve transection and ligation, using compound action potential recordings, and samples of single afferent fibers. Conduction slowed rapidly during the first few weeks postoperative, and then the rate of slowing declined, approaching an asymptote after about 5 months. There was no indication of recovery. The initial decline in conduction velocity that follows nerve crush was similar to that following nerve transection. Upon regeneration, however, conduction velocity returned to near baseline values. Afferent fibers in the neighboring posterior biceps nerve share conduction pathways, dorsal root ganglia, and spinal terminal fields with sciatic nerve afferents, but their conduction velocity was not reduced following sciatic nerve injury.

Neurogenesis in adult rat dorsal root ganglia.

Nerve cells in mammalian species, including primary sensory neurons in the dorsal root ganglia (DRGs), are thought to be generated pre- or perinatally. The only known exceptions are olfactory receptor cells and some cortical microneurons. We now report results of experiments in which the number of neurons in the L4 and L5 DRGs of normal adult rats was counted from serial 10-micrometers paraffin sections stained with cresyl violet. Contrary to expectations, we found that there is a gradual increase in the number of DRG neurons as the animals age. The neuronal population nearly doubles over the adult life of the animal.

Proliferation of primary sensory neurons in adult rat dorsal root ganglion and the kinetics of retrograde cell loss after sciatic nerve section.

This study was aimed at measuring the kinetics of retrograde death among primary sensory neurons axotomized by transection of the ipsilateral sciatic nerve in adult rats. Using electrophysiological and retrograde transport methods, we first determined that most sciatic afferents enter the spinal cord along the L4 and L5 dorsal roots (DRs), and that about 54% of the cells in the L4 and L5 dorsal root ganglia (DRGs) project an axon into the sciatic nerve. Knowing this value, we could then calculate the rate of loss of axotomized neurons from the overall rate of neuron loss in the DRGs at different times after the lesion. Following unilateral sciatic neurectomy, we found a steady falloff in the ratio of DRG neurons on the operated versus the intact control sides in cresyl-violet-stained serial paraffin sections. We were surprised to note, however, that on the control side there was a steady increase in the cell count with age. Counts done on a series of unoperated rats of various ages confirmed this natural increase. Overall, new neurons accrete at an average rate of 18.1 cells per day to the combined L4 and L5 DRGs, nearly doubling their numbers during the adult life of the animal. The new cells add mostly to the small-diameter neuronal compartment. Evidence from neonatally operated rats indicates that the decline in the ratio of neurons in operated versus control DRGs following sciatic nerve section in the adult results more from a halt in the accretion of new neurons to the sciatic compartment than from frank cell death. From our data, we calculate that the loss of axotomized neurons occurs at a rate of only about 8% per 100 postoperative days.