The effect of interaural timing on the posterior auricular muscle reflex in normal adult volunteers.

The posterior auricular muscle (PAM) reflex to sounds has been used clinically to determine hearing threshold as an alternative to other audiological diagnostic measures such as the auditory brainstem response. We have shown that the PAM response is also sensitive to interaural timing differences in normally hearing adults. PAM responses were evoked by both ipsilateral/ contralateral monaural stimulation and by binaural stimulation. Introducing sound delays ipsilaterally or contralaterally decreased the PAM response amplitude and increased its latency. The PAM response in this study shows a qualitatively similar pattern to that seen by the binaural interaction component (BIC) of the auditory brainstem potential to binaural clicks described in previous studies, in that both: have their shortest latency and maximal amplitudes centred around zero interaural timing differences, have response latencies increase with increasing interaural delays up to 1.2 ms and have response amplitudes decrease with increasing interaural delays of up to 1.2 ms. Our data show that the PAM response may be useful in measuring binaural integration in humans non-invasively for diagnostic or research studies.

Development of brain-derived neurotrophic factor and neurotrophin-3 immunoreactivity in the lower auditory brainstem of the postnatal gerbil.

The localization of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the gerbil auditory brainstem was studied during normal postnatal development. The principal objective of this paper was to compare the developmental distribution of BDNF and NT-3 proteins to the known developmental distribution of their cognate, high-affinity tyrosine kinase receptors. BDNF and NT-3 proteins were localized using standard immunohistochemistry. No specific immunoreactivity for BDNF or NT-3 was detected on the day of birth (P0) in any auditory structure, although fibers comprising the spinal tract of the Vth cranial nerve were well labelled with antibodies against BDNF. Diffuse immunoreactivity for both BDNF and NT-3 was first detected at P3 in the cochlear nucleus and in several second order auditory nuclei in the superior olivary complex. This diffuse immunoreactivity became clustered and restricted to neuronal cell bodies by P10. Immunoreactivity for both BDNF and NT-3 transiently disappeared in the lateral and medial superior olivary nuclei at P10. However, neurons in the medial nucleus of the trapezoid body remained immunopositive for both BDNF and NT-3. Fibers in the trapezoid body were labelled with BDNF immunoreactivity by P12. Between P12 and P15, the distribution of BDNF and NT-3 immunoreactivity in the cochlear nucleus and superior olivary complex became comparable to adult (P140) immunolabel. These results show that the normal developmental distribution of the neurotrophins BDNF and NT-3 in the lower auditory brainstem occurs during the first two postnatal weeks in parallel with the developmental expression of their cognate receptors, trkB and trkC.

The shape of ears to come: dynamic coding of auditory space.

In order to pinpoint the location of a sound source, we make use of a variety of spatial cues that arise from the direction-dependent manner in which sounds interact with the head, torso and external ears. Accurate sound localization relies on the neural discrimination of tiny differences in the values of these cues and requires that the brain circuits involved be calibrated to the cues experienced by each individual. There is growing evidence that the capacity for recalibrating auditory localization continues well into adult life. Many details of how the brain represents auditory space and of how those representations are shaped by learning and experience remain elusive. However, it is becoming increasingly clear that the task of processing auditory spatial information is distributed over different regions of the brain, some working hierarchically, others independently and in parallel, and each apparently using different strategies for encoding sound source location.

Topographical projection from the superior colliculus to the nucleus of the brachium of the inferior colliculus in the ferret: convergence of visual and auditory information.

The normal maturation of the auditory space map in the deeper layers of the ferret superior colliculus (SC) depends on signals provided by the superficial visual layers, but it is unknown where or how these signals influence the developing auditory responses. Here we report that tracer injections in the superficial layers label axons with en passant and terminal boutons, both in the deeper layers of the SC and in their primary source of auditory input, the nucleus of the brachium of the inferior colliculus (nBIC). Electron microscopy confirmed that biocytin-labelled SC axons form axodendritic synapses on nBIC neurons. Injections of biotinylated dextran amine in the nBIC resulted in anterograde labelling in the deeper layers of the SC, as well as retrogradely labelled superficial and deep SC neurons, whose distribution varied systematically with the rostrocaudal placement of the injection sites in the nBIC. Topographical order in the projection from the SC to the ipsilateral nBIC was confirmed using fluorescent microspheres. We demonstrated the existence of functional SC-nBIC connections by making whole-cell current-clamp recordings from young ferret slices. Both monosynaptic and polysynaptic EPSPs were generated by electrical stimulation of either the superficial or deep SC layers. In addition to unimodal auditory units, both visual and bimodal visual-auditory units were recorded in the nBIC in vivo and their incidence was higher in juvenile ferrets than in adults. The SC-nBIC circuit provides a potential means by which visual and other sensory or premotor signals may be delivered to the nBIC to calibrate the representation of auditory space.

Silent NMDA receptor-mediated synapses are developmentally regulated in the dorsal horn of the rat spinal cord.

In vitro whole cell patch-clamp recording techniques were utilized to study silent pure-N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses in lamina II (substantia gelatinosa, SG) and lamina III of the spinal dorsal horn. To clarify whether these synapses are present in the adult and contribute to neuropathic pain, transverse lumbar spinal cord slices were prepared from neonatal, naive adult and adult sciatic nerve transected rats. In neonatal rats, pure-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) were elicited in SG neurons either by focal intraspinal stimulation (n = 15 of 20 neurons) or focal stimulation of the dorsal root (n = 2 of 7 neurons). In contrast, in slices from naive adult rats, no silent pure-NMDA EPSCs were recorded in SG neurons following focal intraspinal stimulation (n = 27), and only one pure-NMDA EPSC was observed in lamina III (n = 23). Furthermore, in rats with chronic sciatic nerve transection, pure-NMDA EPSCs were elicited by focal intraspinal stimulation in only 2 of 45 SG neurons. Although a large increase in Abeta fiber evoked mixed alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA receptor-mediated synapses was detected after sciatic nerve injury, Abeta fiber-mediated pure-NMDA EPSCs were not evoked in SG neurons by dorsal root stimulation. Pure-NMDA receptor-mediated EPSCs are therefore a transient, developmentally regulated phenomenon, and, although they may have a role in synaptic refinement in the immature dorsal horn, they are unlikely to be involved in receptive field plasticity in the adult.

Peripheral inflammation facilitates Abeta fiber-mediated synaptic input to the substantia gelatinosa of the adult rat spinal cord.

Whole-cell patch-clamp recordings were made from substantia gelatinosa (SG) neurons in thick adult rat transverse spinal cord slices with attached dorsal roots to study changes in fast synaptic transmission induced by peripheral inflammation. In slices from naive rats, primary afferent stimulation at Abeta fiber intensity elicited polysynaptic EPSCs in only 14 of 57 (25%) SG neurons. In contrast, Abeta fiber stimulation evoked polysynaptic EPSCs in 39 of 62 (63%) SG neurons recorded in slices from rats inflamed by an intraplantar injection of complete Freund's adjuvant (CFA) 48 hr earlier (p < 0.001). Although the peripheral inflammation had no significant effect on the threshold and conduction velocities of Abeta, Adelta, and C fibers recorded in dorsal roots, the mean threshold intensity for eliciting EPSCs was significantly lower in cells recorded from rats with inflammation (naive: 33.2 +/- 15.1 microA, n = 57; inflamed: 22.8 +/- 11.3 microA, n = 62, p < 0.001), and the mean latency of EPSCs elicited by Abeta fiber stimulation in CFA-treated rats was significantly shorter than that recorded from naive rats (3.3 +/- 1.8 msec, n = 36 vs 6.0 +/- 3.5 msec, n = 12; p = 0.010). Abeta fiber stimulation evoked polysynaptic IPSCs in 4 of 25 (16%) cells recorded from naive rat preparations and 14 of 26 (54%) SG neurons from CFA-treated rats (p < 0.001). The mean threshold intensity for IPSCs was also significantly lower in CFA-treated rats (naive: 32.5 +/- 15.7 microA, n = 25; inflamed: 21. 9 +/- 9.9 microA, n = 26, p = 0.013). The facilitation of Abeta fiber-mediated input into the substantia gelatinosa after peripheral inflammation may contribute to altered sensory processing.

Deafferentation is insufficient to induce sprouting of A-fibre central terminals in the rat dorsal horn.

The mechanism by which A-fibres sprout into lamina II of the dorsal horn of the adult rat after peripheral nerve injury, a region which normally receives input from noci- and thermoreceptive C-fibres alone, is not known. Recent findings indicating that selective C-fibre injury and subsequent degenerative changes in this region are sufficient to induce sprouting of uninjured A-fibres have raised the possibility that the structural reorganisation of A-fibre terminals is an example of collateral sprouting, in that deafferentation of C-fibre terminals alone in lamina II may be sufficient to cause A-fibre sprouting. Primary afferents of the sciatic nerve have their cell bodies located predominantly in the L4 and L5 dorsal root ganglia (DRGs), and the A-fibres of each DRG have central termination fields that show an extensive rostrocaudal overlap in lamina III in the L4 and L5 spinal segments. In this study, we have found that C-fibres from either DRG have central terminal fields that overlap much less in lamina II than A-fibres in lamina III. We have exploited this differential terminal organisation to produce deafferentation in lamina II of the L5 spinal segment, by an L5 rhizotomy, and then test whether A-fibres of the intact L4 dorsal root ganglion, which terminate within the L5 segment, sprout into the denervated lamina II in the L5 spinal segment. Neither intact nor peripherally injured A-fibres were seen to sprout into denervated lamina II after L5 rhizotomy. Sprouting was only ever seen into regions of lamina II containing the terminals of peripherally injured C-fibres. Therefore, it seems that the creation of synaptic space within lamina II is not the explanation for A-fibre sprouting after peripheral nerve section or crush, emphasising that injury-induced changes in C-fibres and subsequent chemotrophic effects in the superficial dorsal horn are the likely explanation.

Growth-associated protein 43 immunoreactivity in the superficial dorsal horn of the rat spinal cord is localized in atrophic C-fiber, and not in sprouted A-fiber, central terminals after peripheral nerve injury.

Peripheral nerve injury induces the up-regulation in dorsal root ganglion cells of growth-associated protein 43 (GAP-43) and its transport to the superficial laminae of the dorsal horn of the spinal cord, where it is located primarily in unmyelinated axons and growth-cone like structures. Peripheral nerve injury also induces the central terminals of axotomized myelinated axons to sprout and form novel synaptic contacts in lamina II of the dorsal horn. To investigate whether the sprouting of A-fiber central terminals into lamina II is the consequence of GAP-43 incorporation into their terminal membranes, we have used an ultrastructural analysis with double labelling to identify the localization of GAP-43 immunoreactivity. Transganglionic transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was used to identify C-fiber terminals. Transganglionic transport of the B fragment of cholera toxin conjugated to horseradish peroxidase (B-HRP) was used to label A-fiber sciatic nerve central terminals in combination with GAP-43 immunocytochemistry. GAP-43 was found to colocalize only with WGA-HRP- and not with B-HRP-labelled synapses or axons. In addition, many single-labelled GAP-43 synapses were observed. Many of the WGA-HRP-labelled terminals that were characterized by degenerative changes were GAP-43 immunoreactive. Our results indicate that peripheral nerve injury induces novel synapse formation of A fibers in lamina II but that up-regulated levels of GAP-43 are present mainly in other axon projections to the superficial dorsal horn.

Central changes in primary afferent fibers following peripheral nerve lesions.

Cutting or crushing rat sciatic nerve does not significantly reduce the number of central myelinated sensory axons in the dorsal roots entering the fourth and fifth lumbar segments even over very extended periods of time. Unmyelinated axons were reduced by approximately 50%, but only long after sciatic nerve lesions (four to eight months), and reinnervation of the peripheral target did not rescue these axons. This indicates that a peripheral nerve lesion sets up a slowly developing but major shift towards large afferent fiber domination of primary afferent input into the spinal cord. In addition, since myelinated axons are never lost, this is good evidence that the cells that give rise to these fibers are also not lost. If this is the case, this would indicate that adult primary sensory neurons with myelinated axons do not depend on peripheral target innervation for survival.

Intact sciatic myelinated primary afferent terminals collaterally sprout in the adult rat dorsal horn following section of a neighbouring peripheral nerve.

Peripheral nerve section induces sprouting of the central terminals of axotomized myelinated primary afferents outside their normal dorsoventral termination zones in lamina I, III, and IV of the dorsal horn into lamina II, an area that normally only receives unmyelinated C-fiber input. This axotomy-induced regenerative sprouting is confined to the somatotopic boundaries of the injured nerve in the spinal cord. We examined whether intact myelinated sciatic afferents are able to sprout novel terminals into neighbouring areas of the dorsal horn in the adult rat following axotomy of two test nerves, either the posterior cutaneous nerve of the thigh or the saphenous nerve. These peripheral nerves have somatotopically organized terminal areas in the dorsal horn that overlap in some areas and are contiguous in others, with that of the sciatic central terminal field. Two weeks after cutting either the posterior cutaneous or the saphenous nerve, intact sciatic myelinated fibers labelled with the B fragment of cholera toxin conjugated to horseradish peroxidase (B-HRP) sprouted into an area of lamina II normally only innervated by the adjacent injured test nerve. This collateral sprouting was strictly limited, however, to those particular areas of the dorsal horn where the A-fiber terminal field of the control sciatic and the C-fiber terminal field of the injured test nerve overlapped in the dorsoventral plane. No mediolateral sprouting was seen into those areas of neuropil solely innervated by the test nerve. We conclude that intact myelinated primary afferents do have the capacity to collaterally sprout, but that any resultant somatotopic reorganization of central projections is limited to the dorsoventral plane. These changes may contribute to sensory hypersensitivity at the edges of denervated skin.

Inflammatory pain hypersensitivity mediated by phenotypic switch in myelinated primary sensory neurons.

Pain is normally evoked only by stimuli that are sufficiently intense to activate high-threshold A(delta) and C sensory fibres, which relay the signal to the spinal cord. Peripheral inflammation leads to profoundly increased pain sensitivity: noxious stimuli generate a greater response and stimuli that are normally innocuous elicit pain. Inflammation increases the sensitivity of the peripheral terminals of A(delta) and C fibres at the site of inflammation. It also increases the excitability of spinal cord neurons, which now amplify all sensory inputs including the normally innocuous tactile stimuli that are conveyed by low-threshold A(beta) fibres. This central sensitization has been attributed to the enhanced activity of C fibres, which increase the excitability of their postsynaptic targets by releasing glutamate and the neuropeptide substance P. Here we show that inflammation results in A(beta) fibres also acquiring the capacity to increase the excitability of spinal cord neurons. This is due to a phenotypic switch in a subpopulation of these fibres so that they, like C-fibres, now express substance P. A(beta) fibres thus appear to contribute to inflammatory hypersensitivity by switching their phenotype to one resembling pain fibres, thereby enhancing synaptic transmission in the spinal cord and exaggerating the central response to innocuous stimuli.

Collateral sprouting of uninjured primary afferent A-fibers into the superficial dorsal horn of the adult rat spinal cord after topical capsaicin treatment to the sciatic nerve.

That terminals of uninjured primary sensory neurons terminating in the dorsal horn of the spinal cord can collaterally sprout was first suggested by Liu and Chambers (1958), but this has since been disputed. Recently, horseradish peroxidase conjugated to the B subunit of cholera toxin (B-HRP) and intracellular HRP injections have shown that sciatic nerve section or crush produces a long-lasting rearrangement in the organization of primary afferent central terminals, with A-fibers sprouting into lamina II, a region that normally receives only C-fiber input (Woolf et al., 1992). The mechanism of this A-fiber sprouting has been thought to involve injury-induced C-fiber transganglionic degeneration combined with myelinated A-fibers being conditioned into a regenerative growth state. In this study, we ask whether C-fiber degeneration and A-fiber conditioning are both necessary for the sprouting of A-fibers into lamina II. Local application of the C-fiber-specific neurotoxin capsaicin to the sciatic nerve has previously been shown to result in C-fiber damage and degenerative atrophy in lamina II. We have used B-HRP to transganglionically label A-fiber central terminals and have shown that 2 weeks after topical capsaicin treatment to the sciatic nerve, the pattern of B-HRP staining in the dorsal horn is indistinguishable from that seen after axotomy, with lamina II displaying novel staining in the identical region containing capsaicin-treated C-fiber central terminals. These results suggest that after C-fiber injury, uninjured A-fiber central terminals can collaterally sprout into lamina II of the dorsal horn. This phenomenon may help to explain the pain associated with C-fiber neuropathy.

Reorganization of central terminals of myelinated primary afferents in the rat dorsal horn following peripheral axotomy.

We have investigated the time course and extent to which peripheral nerve lesions cause a morphological reorganization of the central terminals of choleragenoid-horseradish peroxidase (B-HRP)-labelled primary afferent fibers in the mammalian dorsal horn. Choleragenoid-horseradish peroxidase is retrogradely transported by myelinated (A) sensory axons to laminae I, III, IV and V of the normal dorsal horn of the spinal cord, leaving lamina II unlabelled. We previously showed that peripheral axotomy results in the sprouting of numerous B-HRP-labelled large myelinated sensory axons into lamina II. We show here that this spread of B-HRP-labelled axons into lamina II is detectable at 1 week, maximal by 2 weeks and persists for over 6 months postlesion. By 9 months, however, B-HRP fibers no longer appear in lamina II. The sprouting into lamina II occurs whether regeneration is allowed (crush) or prevented (section with ligation), and does not reverse at times when peripheral fibers reinnervate the periphery. We also show that 15 times more synaptic terminals in lamina II are labelled by B-HRP 2 weeks after axotomy than in the normal. We interpret this as indicating that the sprouting fibers are making synaptic contacts with postsynaptic targets. This implies that A-fiber terminal reorganization is a prominent and long-lasting but not permanent feature of peripheral axotomy. We also provide evidence that this sprouting is the consequence of a combination of an atrophic loss of central synaptic terminals and the conditioning of the sensory neurons by peripheral axotomy. The sprouting of large sensory fibers into the spinal territory where postsynaptic targets usually receive only small afferent fiber input may bear on the intractable touch-evoked pain that can follow nerve injury.

The pathophysiology of chronic pain--increased sensitivity to low threshold A beta-fibre inputs.

Chronic pain is characterized by abnormal sensitivity, which is due to the generation of pain in response to the activation of the low-threshold mechanoreceptive A beta fibres that normally generate innocuous sensations. Three different processes in the spinal cord can account for this dramatic alteration in sensory processing in the somatosensory system: increased excitability, decreased inhibition and structural reorganization. All have been shown to occur and each may contribute separately or together to the wide range of chronic inflammatory and neuropathic pain disorders. The unravelling of the cellular mechanisms involved both offers the potential for developing novel therapeutic strategies, which reduce functional synaptic plasticity and prevent central atrophic and regenerative responses in injured neurones, and illustrates the capacity of the adult nervous system for maladaptive modification.

Short-term changes in the numerical density of synapses in the intermediate and medial hyperstriatum ventrale following one-trial passive avoidance training in the chick.

Previous ultrastructural studies using stereological counting techniques, based on assumptions regarding shape, size, and orientation of synapses, have suggested synaptic remodeling occurred at least 24 hr after one-trial passive avoidance training in day-old chicks. The present study estimates the mean synaptic density (Nv syn) in a region of the chick forebrain known to be involved in memory formation, the intermediate and medial hyperstriatum ventrale (IMHV), 1 and 24 hr following one-trial passive avoidance training. A stereological technique, the "disector," that makes no assumptions about size, shape, and orientation of synapses was used in the synaptic analyses. The density of axospinous synapses increased by approximately 77% at 1 hr posttraining in the right IMHV of chicks (M-trained) that learned to avoid a bitter-tasting bead, compared to those (W-trained controls) that peck a water-coated bead. A measure of the postsynaptic density size, the mean projected height of synapses (H), was 57% smaller 1 hr posttraining in the right IMHV of M-trained chicks. These differences were not found at 24 hr posttraining. We suggest that structural modification of synapses may be a key part of the processes involved in short-term memory formation.