Amphibian-specific regulation of polysialic acid and the neural cell adhesion molecule in development and regeneration of the retinotectal system of the salamander Pleurodeles waltl

Antibodies specific to the neural cell adhesion molecule (NCAM-total), the 180 × 10³ My component of NCAM (NCAM-180) and polysialic acid (PSA) were used in immunohistochemistry and Western blots to detect the spatiotemporal dynamics of these molecules in development and regeneration of the retinotectal system of Pleurodeles waltl. NCAM-total and NCAM-180 are continuously expressed in the retina, optic nerve, and tectum of the developing and adult salamander. This is also found for the 140 × 10³ My component of NCAM in Western blots of the retina. In the larval retina, PSA is present in the inner plexiform layer (IPL) and a few cells in all nuclear layers. At metamorphosis, PSA expression in the retina strongly increases in the layer of cone photoreceptor somata. Several cells in the inner nuclear layer and Muller cell processes also begin to express PSA. This pattern persists into adulthood. The optic nerve and the tectum are strongly PSA-immunoreactive throughout development. In the adult optic nerve and optic fiber pathway in the brain, PSA expression is selectively downregulated. In the crush-lesioned adult optic nerve, regenerating fibers are NCAM-180-positive but PSA-negative. This demonstrates a molecular difference between growing nerve fibers of Pleurodeles in development and in regeneration. PSA regulation is closely correlated with metamorphosis, thus suggesting that PSA expression may be under hormonal control. Some aspects of PSA and NCAM isoform expression patterns in the retinotectal system of salamanders differ considerably from that of other vertebrates. The substained expression of NCAM isoforms in adult salamanders might be due to secondary simplification (paedomorphosis). © 1993 Wiley-Liss, Inc.     

Increased NCAM-180 immunoreactivity and maintenance of L1 immunoreactivity in injured optic fibers of adult mice

The injury related expression of two axon-growth promoting cell adhesion molecules (CAMs), NCAM-180 which is developmentally downregulated and L1 which is regionally restricted, were compared in optic fibers in the adult mouse. The neuron-specific isoform of NCAM (NCAM-180) is present at very low levels in unlesioned adult optic axons. At 7 days after nerve crush, immunoreactivity was strongly and uniformly increased in optic axons within the nerve and throughout retina. Reactivity in surviving axons had returned to control levels at 4 weeks. To induce regrowth of adult retinal ganglion cell axons retinal explants were placed in culture. Strong NCAM-180 staining was observed on these regenerating optic axons. The neuronal cell adhesion molecule L1 is restricted to retina and to the unmyelinated segment of the optic nerve near the optic nerve head in unlesioned adult animals. Following nerve crush, L1 immunoreactivity was retained within retina and proximal nerve and novel staining was detected in the more distal segment of the optic nerve up to the lesion site where it persisted for at least eight months. The capacity of optic fibers to show increased NCAM-180 immunoreactivity and maintain L1 expression after a lesion may explain why these fibers exhibit relatively good potential for regeneration.     

Low-level lead exposure attenuates the expression of three major isoforms of neural cell adhesion molecule

Toxic lead (Pb) exposure poses serious risks to human health, especially to children at developmental stages, even at low exposure levels. Neural cell adhesion molecule (NCAM) is considered to be a potential early target in the neurotoxicity of Pb due to its role in cell adhesion, neuronal migration, synaptic plasticity, and learning and memory. However, the effect of low-level Pb exposure on the specific expression of NCAM isoforms has not been reported. In the present study, we found that Pb could concentration-dependently (1-100 nM) inhibit the expression of three major NCAM isoforms (NCAM-180, -140, and -120) in primary cultured hippocampal neurons. Furthermore, it was verified that levels of all three major isoforms of NCAM were reduced by Pb exposure in human embryonic kidney (HEK)-293 cells transiently transfected with NCAM-120, -140, or -180 isoform cDNA constructs. In addition, low-level Pb exposure delayed the neurite outgrowth and reduced the survival rate of cultured hippocampal neurons at different time-points. Together, our results demonstrate that developmental low-level Pb exposure can attenuate the expression of all three major NCAM isoforms, which may contribute to the observed Pb-mediated neurotoxicity.     

Acute stress-induced impairment of spatial memory is associated with decreased expression of neural cell adhesion molecule in the hippocampus and prefrontal cortex.

BACKGROUND: There is an extensive literature describing how stress disturbs cognitive processing and can exacerbate psychiatric disorders. There is, however, an insufficient understanding of the molecular mechanisms involved in stress effects on brain and behavior. METHODS: Rats were given spatial memory training in a hippocampus-dependent water maze task. We investigated how a fear-provoking experience (predator exposure) would affect their spatial memory and neural cell adhesion molecule (NCAM) levels in the hippocampus, prefrontal cortex (PFC), amygdala, and cerebellum. RESULTS: Whereas the control (nonstress) group exhibited excellent memory for the hidden platform location in the water maze, the cat-exposed (stress) group exhibited a profound impairment of memory and a marked suppression of levels of the NCAM-180 isoform in the hippocampus. Predator stress produced a more global reduction of NCAM levels in the PFC but had no effect on NCAM levels in the amygdala and cerebellum. CONCLUSIONS: This work provides a novel perspective into dynamic and structure-specific changes in the molecular events involved in learning, memory, and stress. The selective suppression of NCAM-180 in the hippocampus and the more general suppression of NCAM in the PFC provide insight into the mechanisms underlying the great sensitivity of these two structures to be disturbed by stress.     

Expression of neural cell adhesion molecule in dysmyelinating mutants

The possible role of neural cell adhesion molecule (NCAM) in myelination was studied in the dysmyelinating mouse mutants jimpy and shiverer, by characterizing the expression of the different molecular forms of brain NCAM as a function of age. In jimpy, the expression of NCAM-120 (120,000-Da NCAM) was low and in shiverer both NCAM-120 and NCAM-180 (180,000-Da NCAM) were reduced when compared to controls. In both jimpy and shiverer there was no significant change in the phospholipase C-sensitive NCAM-120. These data further support the possibility that NCAM may be involved in myelination.     

Expression of the neural cell adhesion molecule (NCAM) and polysialic acid during taste bud degeneration and regeneration

Taste receptor cells are replaced throughout life, accompanied by continuing synaptogenesis between newly formed taste cells and first-order gustatory fibers. The neural cell adhesion molecule (NCAM) is expressed by a subset of taste cells in adult rodents and appears on gustatory nerve fibers during development prior to differentiation of the taste buds. We employed antibodies against the extracellular domain of the NCAM polypeptide (mAb 3F4) and against polysialic acid (PSA) residues found on embryonic forms of NCAM (mAb 5A5) to investigate the relationship between the expression of these molecules and the innervation of taste buds in adult rats. In unoperated rats, anti-NCAM recognized a subset of cells within the vallate taste buds and also the fibers of the glossopharyngeal (IXth) nerve, including those innervating the gustatory epithelium. Taste bud cells did not express PSA but mAb 5A5 immunoreactivity was observed on some fibers of the IXth nerve, including a few that entered the taste buds. Bilateral crush of the IXth nerve resulted in the loss of NCAM expression from the gustatory epithelium within 8 days. As IXth nerve fibers reinnervated the epithelium, NCAM expression was seen first in the nerve, followed by increased expression in the epithelium as the taste cells differentiated from their precursors. PSA expression by fibers of the IXth nerve did not return to normal until well after the regeneration of the vallate taste buds. The present results demonstrate that taste cell expression of NCAM is dependent upon innervation by the IXth nerve and that NCAM expression appears in the nerve prior to its expression in the differentiating epithelium during regeneration. The occurrence of a similar temporal sequence in the developing taste system suggests that NCAM could play a role in cell-cell interactions that are important for the differentiation of the taste epithelium. Ongoing taste cell turnover and synaptogenesis between IXth nerve fibers and newly differentiating taste cells also requires recognition and adhesion, in which NCAM could play a role. © 1994 Wiley-Liss, Inc.     

Assessment of Neural Cell Adhesion Molecule (NCAM) in Autistic Serum and Postmortem Brain

Studies have identified structural abnormalities in areas of the autistic brain, with a pattern suggesting that a neurodevelopmental anomaly took place. Neural cell adhesion molecule (NCAM), which is involved in development of the central nervous system, was previously shown to be decreased in the serum of autistic individuals. In the present study, we measured NCAM protein in the sera from controls, patients with autism, siblings of autistic patients, and individuals with other neurologic disorders, but found no significant differences. We also measured NCAM protein in autistic postmortem brain samples and found the longest isoform, NCAM-180, to be significantly decreased. In addition, we investigated the mRNA expression of NCAM in these brain samples using cDNA microarrays and RT-PCR. Results show that NCAM mRNA levels are not altered in autism.     

Submodality-Selective Hyperalgesia Adjacent to Partially Injured Sciatic Nerve in the Rat is Dependent on Capsaicin-Sensitive Afferent Fibers and Independent of Collateral Sprouting or a Dorsal Root Reflex

We studied submodality dependence of sensory changes produced by unilateral ligation of the sciatic or the saphenous nerve in the rat. We focused especially on sensory changes in the skin area adjacent to the innervation area of the injured nerve. Moreover, we examined the roles of capsaicin-sensitive nociceptive fibers, collateral sprouting and a dorsal root reflex in sensory changes observed behaviorally. Assessment of sensory changes was performed by a pattern of behavioral tests: hot-plate test and hindlimb withdrawal responses induced by radiant heat, hot-water bath, innocuous mechanical stimuli, and noxious mechanical stimuli. In one group, the saphenous nerve ipsilateral to the sciatic ligation was topically treated with capsaicin (1%) at the time of the surgery. A proximal stump of a saphenous nerve strand was orthodromically stimulated to induce a dorsal root reflex (an antidromic volley) in nociceptive fibers of the saphenous nerve trunk. For visualization of plasma extravasation induced by a dorsal root reflex, a dye-labeling (Evans blue) technique was used. A collateral sprouting of nociceptive fibers of the uninjured saphenous nerve was evaluated by determining the plasma extravasation response induced by antidromic stimulation of the saphenous nerve. Three and 10 days following the sciatic constriction injury, the hindlimb withdrawal threshold evoked by noxious mechanical stimulation of the medial side of the paw (the innervation are of the intact saphenous nerve) was significantly decreased. There was no corresponding thermal hyperalgesia adjacent to the injured sciatic nerve. Chronic constriction of the saphenous nerve did not produce any significant hyper- or hypoalgesia to mechanical or thermal stimulation of the uninjured sciatic nerve area. Topical treatment of the ipsilateral (intact) saphenous nerve at the time of the sciatic nerve ligation completely prevented the development of mechanical hyperalgesia in the medial side of the paw (the innervation area of the saphenous nerve). No dorsal root reflex in nociceptive fibers mediating the adjacent hyperalgesia could be evoked. No collateral sprouting of the uninjured nociceptive fibers of the saphenous nerve was observed. The results indicate that the constriction injury of the sciatic nerve produced a selective hyperalgesia to mechanical stimulation in the innervation area of the neighboring saphenous nerve. At the peripheral level, the mechanical hyperalgesia adjacent to the innervation area of the injured nerve was mediated by capsaicin-sensitive nociceptive fibers. Collateral sprouting of nociceptive fibers from the uninjured to the injured innervation area did not contribute to the present sensory findings. The sciatic nerve injury did not induce a dorsal root reflex in nociceptive fibers innervating the hyperalgesic saphenous nerve area.     

Neural cell adhesion molecule function is regulated by metalloproteinase-mediated ectodomain release

The neural cell adhesion molecule (NCAM) is involved in development of the nervous system, in brain plasticity associated with learning and memory, and in neuronal regeneration. NCAM regulates these processes by influencing cell adhesion, cell migration, and neurite outgrowth. NCAM activates intracellular signaling upon homophilic NCAM binding, and this is a prerequisite for NCAM-stimulated neurite outgrowth. NCAM is synthesized in three main membrane-bound isoforms, NCAM-120, NCAM-140, and NCAM-180. Soluble forms of NCAM in blood and cerebrospinal fluid have also been found, although the functional significance of these forms remains unclear. In this report, we demonstrate that NCAM can be released from primary hippocampal neurons in culture. The release was enhanced by application of ATP and inhibited by the metalloproteinase inhibitor BB-3103. ATP also induced metalloproteinase-dependent release of all three major NCAM isoforms from NCAM-transfected fibroblastoid L-cells. In this model system, the extracellular ATP-binding site of NCAM was shown not to be necessary for ATP-induced NCAM release. Furthermore, inhibition of serine, cysteine, and aspartic proteinases could not prevent ATP-induced down-regulation of NCAM in L-cells, suggesting that NCAM is cleaved directly by a metalloproteinase. Aggregation of hippocampal neurons in culture was increased in the presence of the metalloproteinase inhibitor GM 6001, consistent with a metalloproteinase-dependent shedding of NCAM occurring in these cells. Moreover, NCAM-dependent neurite outgrowth was significantly reduced by application of GM 6001. Taken together, these results suggest that membrane-bound NCAM can be cleaved extracellularly by a metalloproteinase and that metalloproteinase-dependent shedding of NCAM regulates NCAM-mediated neurite outgrowth.     

Selective interaction of amyloid precursor protein with different isoforms of neural cell adhesion molecule

Compare to the thoroughly studied beta-amyloid, the physiological function of amyloid precursor protein (APP) is not well understood. We now had identified neural cell adhesion molecule (NCAM)-140 as a potential interaction partner of APP. Our data indicated that NCAM-140, but not NCAM-180, binds to the conserved central extracellular domain of APP. We also found that the phosphorylation levels of ERK1 and ERK2 were increased when cells were co-transfected with NCAM-140 and APP indicate that the interaction between NCAM-140 and APP may involve the MAPK pathway. These findings demonstrated that NCAM-140 interacts with APP, potentially playing a role in neurite outgrowth and neural development.     

Nerve growth factor alleviates a painful peripheral neuropathy in rats

Nerve growth factor (NGF) reverses some effects of axotomy and prevents toxic neuropathy in adult rodents. We tested the effect of NGF on behavioral hyperalgesia resulting from a chronic constriction injury (CCI) of the sciatic nerve in the rat [5]. CCI rats exhibit thermal hyperalgesia as demonstrated by a reduction of paw withdrawal latency to a noxious thermal stimulus applied to the paw on the side of injury. The mechanical sensitivity of the ipsilateral hindpaw, assessed with von Frey filaments, was also significantly increased. There were no significant changes in nociceptive thresholds on the contralateral side. When NGF was infused directly on the ligated nerve via an osmotic pump (0.5 μg/μl/h for 7 days) immediately after the ligation, thermal hyperalgesia was abolished from postoperative days 5 up to at least two weeks. The CCI-induced decrease in mechanical threshold was also abolished by NGF. However, NGF had only a minor effect on the abnormally long response duration, a second measure of mechanical sensitivity, to the mechanical stimulus. Delayed infusion of NGF four days after the ligation failed to block hyperalgesia. Infusion of NGF on the sciatic nerve of rats that had no CCI had no significant effect on paw withdrawal latency. Infusion of anti-NGF antiserum did not enhance hyperalgesia in CCI rats. These results suggest that alterations in neurotrophic factor(s) contribute to the development of behavioral hyperalgesia in an animal model of neuropathy and that NGF may have therapeutic value in the treatment of neuropathic pain in humans.     

Enzymatic removal of polysialic acid from neural cell adhesion molecule perturbs the migration route of luteinizing hormone-releasing hormone neurons in the developing chick forebrain

During development in the chick embryo, luteinizing hormone-releasing hormone (LHRH) neurons migrate along the olfactory nerve from the olfactory epithelium to the forebrain. At embryonic day 5.5 (E5.5) to E6.0, the majority of LHRH neurons begin to enter the medial forebrain and then course dorsocaudally along the forebrain substance just beneath the pia matter in association with the somatostatin (SST)-positive fibers, which branch medially from the SST-positive olfactory nerve. By E6.5, the neurons and SST-positive medial branch of the olfactory nerve have proceeded toward the septal area. Intense immunoreactivity for the polysialylated form of neural cell adhesion molecule (PSA-NCAM) on both the LHRH neurons and the SST-positive fibers during this period suggests that this less adhesive form of NCAM is involved in the migratory process. This possibility was examined by using a polysialic acid (PSA)-specific endoneuraminidase. PSA removal did not alter the behavior or appearance of the SST-positive olfactory fibers within the migration pathway. However, it induced a significant deviation of migrating LHRH neurons from the regular path in the forebrain. The effect of PSA removal is more likely to involve changes in the interaction of the migrating neurons with a subset of the SST-positive olfactory fibers and/or other elements in the forebrain rather than an alteration in the pattern of their axonal substrate. On the basis of these results, it is suggested that PSA contributes to the specific pattern of LHRH neuronal migration in the forebrain by limiting interaction of these LHRH neurons with their surrounding environment.     

Intrathecal MK-801 and local nerve anesthesia synergistically reduce nociceptive behaviors in rats with experimental peripheral mononeuropathy.

The hyperalgesia and spontaneous pain that occur following peripheral nerve injury may be related to abnormal peripheral input or altered central activity, or both. The present experiments investigated these possibilities by examining the effects of MK-801 (a non-competitive N-methyl-D-aspartate, NMDA, receptor antagonist) and bupivacaine (a local anesthetic agent) on thermal hyperalgesia and spontaneous nociceptive behaviors in rats with painful peripheral mononeuropathy. Peripheral mononeuropathy was produced by loosely ligating the rat's common sciatic nerve, a procedure which causes chronic constrictive injury (CCI) of the ligated nerve. The resulting hyperalgesia to radiant heat and spontaneous nociceptive behaviors was assessed by using a foot-withdrawal test and a spontaneous pain behavior rating method, respectively. CCI rats receiving 4 daily intraperitoneal (i.p.) MK-801 injections (0.03, 0.1, 0.3 mg/kg) beginning 15 min prior to nerve ligation exhibited less hyperalgesia (i.e., longer foot-withdrawal latencies) on days 3, 5, 7, 10, and 15 after nerve ligation as compared to those receiving saline injections. Thermal hyperalgesia also was reduced when a single MK-801 injection was given intrathecally (i.t.) onto the spinal cord lumbar segments on Day 3 after nerve ligation. This effect of postinjury MK-801 treatment was dose-dependent (2.5-20 nmol) and lasted for at least 48 h after injection. Moreover, i.t. injection of MK-801 (10 nmol) reliably lowered spontaneous pain behavior rating scores in CCI rats compared to those in the saline group. The spinal site of MK-801 action is situated within the caudal (probably lumbar) spinal cord, since i.t. injection of MK-801 (10 nmol) onto the spinal cord thoracic segments did not affect thermal hyperalgesia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-activated proteolysis of intracellular domains in the cell adhesion molecules NCAM and N-cadherin.

One of the consequences of increased intracellular calcium in response to a variety of physiological stimuli is the calcium activation of cytosolic proteases. Unlike lysosomal proteases with broad specificity, these calcium-activated neutral proteases show limited proteolysis of a restricted set of substrate proteins suggesting they may play a regulatory role in cellular physiology. In this study we show that the neural cell adhesion molecules NCAM-180 and N-cadherin are substrates for such endogenous calcium-activated neutral proteases. In contrast, a third neural cell adhesion molecule G4/L1 was not susceptible to calcium-activated proteolysis. The threshold for activation of NCAM and N-cadherin proteolysis is in the micromolar range of calcium suggesting that NCAM and N-cadherin are substrates for a mu-type calpain (calpain I). The site recognized by this protease is within intracellular domains of NCAM-180 and N-cadherin which are important for their interaction with cytoskeletal components. These results suggest that calcium-activated proteolysis at these sites in vivo could disrupt the linkage between extracellular ligand binding to these adhesion molecules and the normal intracellular effectors of such extracellular binding events.     

Spatial learning impairment induced by chronic stress is related to individual differences in novelty reactivity: search for neurobiological correlates

Although chronic stress has been reported to induce deleterious effects on hippocampal structure and function, the possible existence of individual differences in the vulnerability to develop stress-induced cognitive alterations was hypothesized. This study was designed to evaluate (i) whether individual variability in behavioural reactivity to novelty could be related to a differential vulnerability to show spatial learning deficits after chronic stress in young adult rats, and (ii) to what extent, could individual differences in stress-induced cognitive alterations be related to alterations in specific neurobiological substrates. Four month-old Wistar male rats were classified according to their locomotor reactivity to a novel environment, as either low (LR) or highly (HR) reactive, and then either submitted to psychosocial stress for 21-days (consisting of the daily cohabitation of each young adult rat with a new middle-aged rat) or left undisturbed. The results showed that psychosocial stress induced a marked deficit in spatial learning in the water maze in HR, but not in LR, rats. Then, a second experiment investigated the possible differential expression of corticosteroid receptors (MR and GR) and cell adhesion molecules (NCAM and L1) in the hippocampus of HR and LR rats, both under basal conditions and after exposure to chronic social stress. Although chronic stress induced a reduction on the hippocampal expression of MRs and the NCAM-140 isoform, the levels of these molecules did not differ between stressed rats with and without spatial learning impairments; i.e., between HR- and LR-stressed rats, respectively. Nevertheless, it should be noted that the reduction of the hippocampal expression of NCAM-140 induced by psychosocial stress was particularly marked in HR stressed rats. However, the expression of GRs, NCAM-120 and NCAM-180 isoforms, and L1, was not affected by stress, regardless of the reactivity of the animals. Therefore, although we failed to find a neurobiological substrate that specifically correlated with the differential cognitive vulnerability to chronic stress shown by animals with a different novelty reactivity, this study confirms the hypothesis that rats differ in their susceptibility to display stress-induced impairments in hippocampus-dependent spatial learning tasks. In addition, it provides a model to further search for the neurobiological substrate(s) involved in the differential susceptibility to develop stress-induced cognitive impairments.     

Nerve injury-induced mechanical but not thermal hyperalgesia is attenuated in neurokinin-1 receptor knockout mice

Mice lacking the gene encoding for substance P and neurokinin A, or the NK-1 receptor, exhibit alterations in behavior to various acute nociceptive stimuli. However, behavioral responses of NK-1 mutant animals have not been well characterized in models of chronic pain. We studied the behavioral responses of NK-1 knockout and wild-type control mice to thermal and mechanical stimuli before and after inducing chronic neuropathic pain by unilateral ligation of the L5 spinal nerve. Mechanical hyperalgesia was evaluated by determining the frequency of withdrawal to von Frey monofilaments applied to the hind paws. Nerve injury-induced hyperalgesia to thermal stimuli was examined by determining responses to radiant heat and cooling stimuli. The contribution of the sympathetic nervous system to mechanical hyperalgesia was evaluated by administering 3 mg/kg phentolamine, an alpha-adrenergic antagonist, subcutaneously. Following spinal nerve injury, withdrawal frequencies to mechanical stimulation increased in wild-type mice within 1 day and persisted during the 9-week observation period, whereas in the knockout mice, withdrawal frequencies did not increase significantly. In contrast, withdrawal latencies to radiant heat decreased up to 2 weeks after nerve injury in both the NK-1 and the wild-type mice. Similarly, the increase in withdrawal frequency to the cooling stimuli following the nerve injury was not different in the NK-1 knockout and wild-type mice. Mechanical hyperalgesia in the wild-type mice was not reversed by systemic administration of phentolamine, suggesting that the pain is not sympathetically maintained. The results indicate that NK-1 receptors contribute to the development of mechanical, but not thermal, hyperalgesia in neuropathic pain.     

Intraspinal and behavioral consequences of nerve growth factor-induced nociceptive sprouting and nerve growth factor-induced hyperalgesia compared in adult rats.

Intraspinal and behavioral events were studied in adult rats with nociceptive nerves that were undergoing collateral sprouting into adjacent denervated skin. This sprouting, which is driven by endogenous nerve growth factor (NGF), did not cause hyperalgesia. For comparison, we studied an exogenous NGF administration that induced hyperalgesia but was too brief to evoke sprouting. When nociceptive nerves sprouted in skin, back-labeling with wheat germ agglutinin-conjugated horseradish peroxidase revealed that their projections sprouted in the cord. The sprouted peripheral nerves now activated more c-Fos-containing interneurons, which stimulus-response studies showed was not due to an increased afferent discharge. We attribute the interneuron recruitment to synaptogenesis following the intraspinal sprouting. Nociceptive stimulation of dorsal skin reflexly activates underlying cutaneous trunci muscle (CTM). When a nociceptive field expanded by nerve sprouting, so did the area of the evoked CTM reflex: this implies a recruitment of CTM motoneurons. We interpret this "matching" of response to stimulus as an adaptive phenomenon ensured by an adaptive intraspinal sprouting of the nociceptive projections. Neither the intraspinal changes nor the reflex changes occurred if peripheral sprouting was blocked by systemic anti-NGF treatment, indicating that the role of endogenous NGF was only in that sprouting. No comparable adaptive events occurred during NGF-induced hyperalgesia. Neither nociceptive fields nor CTM reflexes were affected; however there was a recruitment of c-Fos-expressing interneurons. This recruitment was not explained by peripheral sensitization, and, because sprouting was not involved here, we attribute the recruitment to "synaptic unmasking," i.e., an increased effectiveness of the preexisting excitatory circuitry.