We have investigated the co-involvement of endogenous NGF and impulses in the collateral sprouting of cutaneous sensory nerves in adult rats, specifically the A delta-axons involved in mechanonociception and the C-fibers that mediate heat nociception. Their collateral sprouting was measured by the progressive expansion, respectively, of the behaviorally defined "pinch" and "heat" fields into surrounding denervated skin (the light-touch A alpha-fibers do not sprout in adult mammals). The expansions of such "isolated" fields were totally prevented in animals injected daily with anti-NGF serum, but developed normally after treatment was discontinued. Light microscopic and EM examination of the skin confirmed that the effect of the anti-NGF treatment was attributable to its prevention of collateral sprouting. Initiation of treatment would also rapidly halt sprouting already in progress. Finally, intradermal injections of purified NGF protein would not only increase the rate of nociceptive fiber sprouting, but also evoke sprouting de novo within normally innervated skin (again, A alpha-axons were unaffected). We conclude that the collateral sprouting of intact nociceptive nerves following partial denervation of skin is entirely dependent on endogenous NGF. The observed latency of this sprouting was 10-12 d; we estimate, however, that at least 2 d of field expansion is required for its reliable detection. Thus, about 8-10 d are required for NGF levels in the skin to rise to effective levels, and for the neurons to respond and initiate sprouting. From indirect findings, the NGF component of this sprouting latency appears to be about 2 d. In accord with earlier findings, the remaining "initiation time" was reduced by 5-6 d if the neurons were briefly excited, even 2 d prior to the isolation of their fields. Unexpectedly, this phenomenon of "precocious sprouting" requires that endogenous NGF be available; the sprouting latency reverted to normal values when the conditioning impulses were evoked during a 2 d anti-NGF "umbrella." In contrast to the impulse-sensitive neuronal mechanisms involved in the initiation of sprouting, those underlying the sprouting rate were unaffected by nerve activity and were entirely dependent on the level of endogenous NGF. We suggest that interactions like that revealed in these studies between a sprouting agent and impulses that seem to prime the neuron's response to it contribute to plasticity within the nervous system.

A key role has not yet been identified for (3 nerve growth factor (NGF) in the growth responses that continue to be expressed in the sensory neurons of adult animals. We have now examined the effects ofdaily administration to adult rats (and in a few experiments, mice) of antiserum to NGF on (i) the collateral sprouting of undamaged nociceptive nerves that occurs into denervated adjacent skin and (ii) the regeneration of cutaneous sensory axons that occurs after they are damaged. The results were unexpected. All collateral sprouting was prevented and that already in progress was halted; sprouting resumed when treatment was discontinued. In contrast, the reestablishment, and even enlargement, ofcutaneous nerve fields by regenerating axons was unaffected by anti-NGF treatment, even after dorsal rhizotomy was done to eliminate any central trophic support. In denervated skin, regenerating and collaterally sprouting axons utilized the same cellular pathways to establish functionally identical fields, thus displaying apparently identical growth behaviors, yet anti-NGF treatment clearly distinguished between them. We suggest that endogenous NGF is responsible for the collateral sprouting of nociceptive axons, probably reflecting an ongoing function of NGF in the regulation of their fields. This demonstration in the adult sensory system of a defined role for NGF in nerve growth could apply to nerve growth factors generally in the adult nervous system. The regeneration, however, ofnociceptive axons (and nonnociceptive ones) is not dependent on NGF.Although /3 nerve growth factor (NGF) is essential for the development and survival of neuronal populations in the autonomic and sensory nervous systems (1-4), by birth or shortly thereafter, sensory neurons will survive largely independent of it (1, 5). Nevertheless in adult animals NGF continues to be synthesized in peripheral target tissues (6, 7), sensory axons can take up and transport it retrogradely (8, 9), while maintained NGF deprivation leads to a lowering ofboth substance P levels (10), and even neuronal cell size (11), in dorsal root ganglia. Significantly, two striking growth behaviors of sensory neurons also continue to be demonstrable in adult animals: these are axonal elongation and collateral sprouting. There are more than morphological distinctions between these two. Whereas collateral sprouting, both during development and later, is a characteristic of normal undamaged nerves, and is essentially confined to target tissues (12, 13), elongation is seen in adults particularly in the form of regeneration of an axon after peripheral nerve damage. In adult mammals, large myelinated mechanosensory axons readily regenerate after they are crushed, but unlike both myelinated (14) and unmyelinated (15) In the present study we compared the effects of anti-NGF treatment on collateral sprouting and axonal regeneration of cutaneous nociceptive nerves in adult rats. Surprisingly, though the cutaneous pathways normally followed by each were identical, sprouting was p...

Interstitial cells of Cajal (ICC) are associated with afferent innervation and peristalsis of the stomach suggestive of a key role in the pathophysiology of gastroparesis. We studied changes in the density and ultrastructure of ICC and enteric nerves in the streptozotocin-induced diabetes mellitus (STZ-DM) in Wistar rats using immunohistochemistry and electron microscopy. Gastric emptying was studied in vivo by single-photon emission computed tomography. In the STZ-DM antrum, a marked reduction was observed in the density of the intramuscular ICC (ICC-IM) and ICC located at the submucosal border of the circular muscle layer of the antrum (ICC-SM). The surviving ICC showed lamellar bodies and partial vacuolation of the cytoplasm content, loss of connections between ICC-IM and nerves; it appeared that injured ICC-IM developed into fibroblast-like ICC. ICC associated with Auerbach's plexus (ICC-AP) in the antrum and ICC in the fundus were not affected significantly except for a loss of connections with nerve structures. Marked reduction in nerve tissue (Protein Gene Product-9.5 positivity) was also restricted to the muscle layers including nitrergic nerves (neuronal nitric oxide synthase positivity). In vivo assessed gastric emptying was markedly reduced in STZ-DM rats. Our data demonstrate in the STZ-DM rat stomach a decreased density of ICC limited to the antrum and to ICC-IM and ICC-SM, and structural degeneration in ICC-IM and associated nerves with a special emphasis on loss of synaptic connections, accompanied by a decrease in gastric emptying. Hence, in this model of gastroparetic diabetes, regional injury to subsets of ICC and nerves are associated with gastric motor dysfunction.

We have investigated the possible roles of NGF, and of impulse activity, in the regeneration of sensory nerves. Unexpectedly, the ability of crushed axons to regrow and to restore functional recovery of three sensory modalities in adult rat skin (A alpha-mediated touch, A delta-mediated mechanonociception, and C-fiber-mediated heat nociception) was totally unaffected by anti-NGF treatment. This finding applied even when the anti-NGF dosage was almost eight times that which entirely blocked collateral sprouting of the undamaged axons of both classes of nociceptive nerves (the A alpha-axons do not sprout in adult animals). In the same anti-NGF-treated animal, regeneration would proceed normally on the one side, while collateral sprouting was prevented on the other. Light microscopic and EM examination revealed that in the denervated skin the regenerating axons utilized the same dermal perineurial pathways followed by collaterally sprouting axons. Regeneration within these antibody-accessible pathways progressed normally during anti-NGF treatment, extending 1-2 cm beyond the former field borders, that is, into territory whose invasion by collaterally sprouting axons was totally blocked. The blood-nerve barrier is absent within the degenerating peripheral nerve trunk, a putative NGF source for regenerating fibers but not for sprouting ones. The NGF-independent regeneration was also found to be unaffected when putative spinal cord sources of NGF were eliminated by dorsal root excision. Anti-NGF treatment also failed to block regeneration across 4 mm excision gaps in the nerve trunk. The daily anti-NGF regime continued to be effective for at least 8 weeks, at which time newly evoked collateral sprouting could still be blocked. Exogenous NGF, in doses that evoke collateral sprouting de novo in normal skin, failed to influence regeneration. Finally, an electrical stimulus regime, which markedly reduces the latency of collateral sprouting, failed to affect the time to arrival of regenerating axons at the skin, or the rate of their arborization in it. We conclude that, in striking contrast to their collateral sprouting, the regeneration of nociceptive axons occurs independently of endogenous NGF and is unaffected by impulse activity. These findings further support the proposal that these two growth behaviors have basically different biological functions in the organism.

The ability of intact cutaneous thermonociceptive C fibers to sprout into adjacent denervated skin, and the effects of this of electrical activity in the axons, were studied in adult rats. The presence of heat-sensitive endings in the back skin was assessed physiologically by the ability of a hot probe to elicit the reflex contraction of the underlying cutaneous trunci muscle; sensory C fibers were detected both by the Evans Blue technique, in which antidromic excitation of the C fibers causes a visible extravasation of dye in the skin it supplied, and by direct electron microscope (EM) examination of skin. The field of an identified branch of a selected dorsal cutaneous nerve (DCN) was "isolated" by eliminating all the nerves supplying the surrounding skin. The heat-sensitive area began to expand between 10 and 14 days after its isolation and reached a maximum (approximately doubling the initial value) by about 24 days. When the isolated nerve was antidromically excited, the borders within which dye extravasation now occurred had extended; the area of discoloration correlated well with that of the (enlarged) heat field, showing that its expansion was indeed attributable to C fiber sprouting. Electron microscopic examination showed that some of the "empty" Schwann tubes, routinely observed in the subepidermal horizontal fiber system of insensitive skin following denervation, had acquired unmyelinated axons when heat sensitivity had returned. A precocious expansion of the heat field, which was obvious by 10 days after its isolation, was produced if the heat stimulus was applied randomly throughout the field at the time of the adjacent denervations; this expansion too was shown to be due to sprouting of C fibers. Precocious sprouting of heat-nociceptive fibers also occurred if, immediately following the field isolation, the C fibers in the spared DCN were electrically excited, and also if pinches were applied through its field, i.e., the C fibers involved seemed to be polymodal, responding both to heat and to noxious mechanical stimulation. Precocious sprouting did not occur when tetrodotoxin was used to block central conduction of the "conditioning" impulses in the isolated DCN. Peripheral nerve damage often occurs in a situation likely to cause activation of nociceptive nerves; we suggest that the accelerated sprouting of spared axons could be important in reducing the period during which atrophic changes might occur in denervated target regions, in addition to hastening the provision of a nociceptive innervation. The various experimental approaches now available for producing differential innervation of selected regions of back skin are summarized.

1. The cutaneus trunci muscle (CTM) is a thin broad sheet of skeletal muscle that originates bilaterally on the humerus and inserts beneath the dermis of back and flank skin. A nociceptive stimulus applied to the skin elicits a localized reflex contraction in that region of the CTM underlying the site of sensory stimulation. While this "local sign" character of the CTM reflex corresponds to the segmental distribution of the afferent nerves (the dorsal cutaneous nerves, or DCNs) that enter the spinal cord in the lower thoracic and the lumbar levels, the motor output originates entirely from a circumscribed region of the cervical spinal cord. 2. Electrophysiological analysis of EMG activity in the muscle reflexly evoked by direct electrical stimulation of individual DCNs revealed a distinct topographic relationship, in that the shortest latency response of EMG activity in the muscle was consistently located approximately 1.0 cm rostral to the dermatome of the stimulated DCN. 3. Histochemical studies of the CTM show that individual muscle fibers run rostrocaudally, are focally innervated, and in adult rats, are approximately 3.0 cm in length. The major motor nerves exit from the brachial plexus, and functionally they divide the muscle into longitudinal (rostrocaudal) territories, which thus lie orthogonal to the dermatomal pattern of sensory innervation. The localized reflex responses to focal sensory stimuli indicate that the major longitudinal muscle fields contain many "reflex compartments." 4. The compartmentalized nature of the reflex response in the CTM suggests that nociceptive input from any one sensory dermatome has a preferred access to that fraction of the motoneuron pool that supplies the area of muscle underlying that specific region of skin, i.e., there is a sort of "matching" between groups of primary sensory neurons, interneurons, and motoneurons, which relates to the peripheral location of the stimulated nerve endings and of the muscle fibers that are reflexly activated. Although the partitioning of sensory input to motor nuclei has been shown most clearly for monosynaptic Ia connections, the CTM reflex suggests that sensory partitioning may also be demonstrated in a polysynaptic circuit.

We have used anti-nerve growth factor (anti-NGF) [corrected] administration to study the NGF dependency of the reinnervation of denervated skin by sympathetic nerves in the adult rat. Sympathetic pilomotor fields were revealed by electrical stimulation of selected dorsal cutaneous nerves; the affected skin rapidly assumed a "gooseflesh" appearance, sharply demarcated from surrounding unstimulated skin. Examined 2-5 days after section of neighboring nerves, the "isolated" pilomotor field of the spared nerve was found to be coextensive with an area of amine-fluorescent fibers that were associated with pilomotor muscles and blood vessels. After its isolation, a pilomotor field begins to expand into the surrounding deprived territory, reaching a maximum size at approximately 40 days. Fluorescence studies confirmed that new sympathetic fiber growth had occurred into the expanded regions of such fields. Daily injections of polyclonal anti-NGF serum completely prevented these pilomotor field expansions. Following termination of the anti-NGF treatment, expansion proceeded normally. Finally, if the onset of anti-NGF treatment was delayed until pilomotor field expansion had already commenced, further expansion was halted. Regeneration of sympathetic fibers was evoked by crushing a selected nerve. Recovery of pilomotor function in the totally denervated skin was first detected at about 20 days postcrush, and the field progressively enlarged over the next 40 days. Although the imposed NGF deprivation is known to cause a demonstrable shrinkage, and presumably atrophy, of sympathetic ganglia, the anti-NGF treatment appeared to impair neither the restoration of a pilomotor field after nerve crush, nor its continued expansion into skin regions well beyond that originally supplied by the nerve, i.e., into territory whose invasion by collateral sprouts would have been totally prevented by the treatment. During such NGF deprivation, fluorescent regenerating fibers were visualized in the nerve trunk. We conclude that even though the regenerating and collaterally sprouting sympathetic fibers probably utilise the same degenerating dermal pathways to reach and functionally reinnervate the same denervated targets, only the collateral sprouting of the uninjured axons is dependent upon endogenous NGF. These findings extend the results described earlier for nociceptive fibers, and suggest that the contrasting dependencies upon growth factors of sprouting and regeneration might apply throughout the adult nervous system.

We have studied the sprouting of intact high-threshold mechanosensory nerves into adjacent denervated trunk skin in adult rats behaviorally, histologically, and electrophysiologically. In the anesthetized animal, stimulation of high-threshold endings in back skin by localized pinching elicits a bilateral reflex excitation of the underlying skeletal muscle, the cutaneous trunci muscle (CTM), visible as a twitch-like puckering of the skin. The reflex was also evoked by electrical excitation of A delta and of C fibers in the dorsal cutaneous nerves (DCNs), with characteristic latencies of 7-20 msec and 40-60 msec, respectively; excitation of low-threshold (A alpha) fibers was ineffective. After cutting selected DCNs, the deprived skin became insensible, but pinch responsiveness gradually recovered over the following 2 weeks. Regeneration of cut axons was not responsible for this recovery; when neighboring intact DCNs were cut, however, all responses were abolished in the recovered skin that had been initially denervated. By 3-5 days after denervation, axons in the dermis were all histologically absent or degenerating; when pinch sensitivity was restored to such skin, silver-stainable axons reappeared in the formerly empty Schwann tubes. During the work we noticed that the periodic examination by pinching, used to follow the time course of recovery of function in individual animals, led to an earlier development of this recovery than in animals that were examined only once at a specified time after denervation. This apparent acceleration in the redevelopment of pinch sensitivity was correlated with the appearance of axons in the recovered skin, and was shown to be due to the impulse activity evoked in the remaining nerves by the periodic pinching; it did not occur when the nerves were blocked by tetrodotoxin (TTX), and it was mimicked by a brief (10-min) period of electrical excitation of the A delta fibers in a remaining nerve carried out at the time when the denervation of skin was done. The time course of the phenomenon suggested that the principal effect of the impulses was to shorten the latency to the onset of sprouting in the activated A delta axons; that is, they induced precocious sprouting. The impulses needed to be conducted centrally for the effect to occur, and precocious sprouting failed to occur if the impulses were allowed to proceed only distally toward the skin. It seems that a brief conditioning burst of impulses in A delta axons sensitizes the neurons to the influence of a sprouting stimulus that appears when skin is denervated.(ABSTRACT TRUNCATED AT 400 WORDS)