Age‐dependent failure of axon regeneration in organotypic culture of gerbil auditory midbrain

Inferior colliculus (IC) slice cultures from postnatal (P) day 6–8 gerbils exhibit axonal regeneration across a lesion site, and these regrowing processes can form synapses. To determine whether regenerative capacity is lost in older tissue, as occurs in vivo, slices from P12–21‐day animals were grown under similar conditions. While these cultures displayed a near complete loss of neurons over 6 days in vitro, glutamate receptor antagonists (AP5 and/or CNQX) significantly enhanced survival, particularly at P12–15. In contrast, several growth factors or high potassium did not improve neuron survival. Therefore, axonal regeneration was assessed following complete transection of the commissure in AP5/CNQX‐treated IC cultures from P12 animals. Neurofilament staining revealed that transected commissural axons survived for 6 days in vitro, but only a few processes crossed the lesion site and these axons did not extend into the contralateral lobe. In contrast, there was robust axonal sprouting and growth within one lobe of the IC, remote from the lesion site. When P6 and P12 tissue was explanted onto a coated substrate, the P6 axons grew onto the substrate, but the P12 axons were seemingly prevented from reaching the substrate by a veil of nonneuronal cells. Coculture of the IC and one of its afferent populations, the lateral superior olive, provided a similar finding, indicating that failure to regenerate was a general property at the age examined. These data show that neuron survival is not sufficient to permit axon regeneration at P12, and suggest that P12 lesion sites manufacture a prohibitive substrate since process outgrowth is blocked specifically at the commissure transection. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 267–280, 1999     

Axon regeneration in organotypic slice cultures from the mammalian auditory system is topographic and functional

In vitro models have frequently been employed to investigate the specificity of the formation of axonal projections during both development and regeneration. Such studies demonstrated pathway, target, and laminar specificity, yet they did not tackle the problem of topography. Here, we addressed the issue of regeneration of spatial specificity at the topographic level by lesioning a precisely organized projection from the auditory system of neonatal rats in organotypic slice culture and by analyzing regeneration capacity. Lesioning had no effect on the survival of axotomized neurons or the structure of the auditory nuclei. Anterograde and retrograde biocytin tracing demonstrated that the projection regenerated topographically at the supracellular level. Whole-cell patch-clamp recordings revealed that the regenerated projection was functional. Topographic regeneration was not impaired by blocking spike activity with tetrodotoxin or glycinergic transmission with strychnine. However, if lesioning was performed after the slices had been incubated for 1 week, regeneration capacity was lost despite good survival of neurons. The loss of the regeneration capacity in vitro occurs at a developmental stage that corresponds to the age when the capacity for axonal reorganization is lost in vivo. We conclude that the developmental processes occurring in vivo and in vitro are comparable in this system, which is why we think that essential aspects of the loss of regeneration capacity may be addressed with our culture model in the future.     

Basic maps in the auditory midbrain

Topologic maps at consecutive levels of sensory pathways indicate behaviorally relevant features of stimuli at increasing degrees of complexity. In the auditory system, except for tonotopic maps, the nature of represented features is unknown. In a model analogous to visual map formation we show that in the auditory midbrain, layers of neurons with preference to the same frequency (isofrequency planes) may hold maps of two basic, mutually orthogonal parameters--instantaneous amplitude and phase--of basilar membrane displacement at the cochlear location responding to that frequency. The proposed neural tuning to frequency, amplitude, and phase implies that sound is transformed into specific temporal trajectories of neural activation, with consequences for experimental design and interpretation of neural response behavior.     

Mechanisms of neuroprotection in hippocampal organotypic culture

We investigated if IRFI 042, an analog of vitamin E, protects the brain against oxidative stress induced by intraperitoneal administration of Kainic acid (KA) (10 mg/kg); sham brain injury rats were used as controls. Animals received either IRFI 042 (20 mg/kg) or its vehicle 30 min before KA injection and after 6 h were sacrificed to measure malonildyaldheide (MDA) and glutathione levels (GSH) in the diencephalon. Behavioral changes were also monitored. Intraperitoneal administration of IRFI decreased MDA (micromol/g wet tissue: KA + vehicle = 22.5 ± 4.2; KA + IRFI = 17.1 ± 1; P  < 0.005) and prevented GSH loss (nmol/g wet tissue: KA + vehicle = 0.41 ± 0.1; KA + IRFI = 1.86 ± 0.2; P  < 0.005) in the diencephalon. The latency of occurrence of behavioral signs increased from 39 ± 1 to 62 ± 6 min in IRFI 042 group. The data suggest that IRFI 042 might protect against KA-induced oxidative stress.     

Glial inhibition of CNS axon regeneration

Damage to the adult CNS often leads to persistent deficits due to the inability of mature axons to regenerate after injury. Mounting evidence suggests that the glial environment of the adult CNS, which includes inhibitory molecules in CNS myelin as well as proteoglycans associated with astroglial scarring, might present a major hurdle for successful axon regeneration. Here, we evaluate the molecular basis of these inhibitory influences and their contributions to the limitation of long-distance axon repair and other types of structural plasticity. Greater insight into glial inhibition is crucial for developing therapies to promote functional recovery after neural injury.     

Age-dependent amplitude variation of brain-stem auditory evoked potentials

Normative amplitude values of brain-stem auditory evoked potential (BAEP) components are given for normally hearing subjects at 1, 10, 30, 50 and 70 years of age, with an intragroup age variation of only ±6 months. Under these circumstances amplitude standard deviations decreased to less than 20% of the mean values. In contrast with the reduced evolution of latency with age, BAEP amplitude (for components I–V) undergoes a greater oscillation during ontogeny. With the exception of component I, it increased markedly from 1 year to 10 years of age and decreased thereafter constantly up to 50 years, with a mean rate of 10 nV yearly. The decrease slowed down between 50 and 70 years. The amplitude differences between the subgroups are highly significant statistically (P < 0.01). Possible reasons for these changes are discussed.     

Changes in cytoskeletal protein synthesis following axon injury and during axon regeneration

Injury to the axons of facial motoneurons stimulates increases in the synthesis of actin, tubulins, and GAP-43, and decreases in the synthesis of neurofilament proteins: mRNA levels change correspondingly. In contrast to this robust response of peripheral neurons to axotomy, injured central nervous system neurons show either an attenuated response that is subsequently aborted (rubrospinal neurons) or overall decreases in cytoskeletal protein mRNA expression (corticospinal and retinal ganglion neurons). There is evidence that these changes in synthesis are regulated by a variety of factors, including loss of endoneurially or target-derived trophic factors, positive signals arising from the site of injury, changes in the intraaxonal turnover of proteins, and substitution of target-derived trophic support by factors produced by glial cells. It is concluded that there is, as yet, no coherent explanation for the upregulation or downregulation of any of the cytoskeletal proteins following axotomy or during regeneration. In considering the relevance of these changes in cytoskeletal protein synthesis to regeneration, it is emphasized that they are unlikely to be involved in the initial outgrowth of the injured axons, both because transit times between cell body and injury site are too long, and because sprouting can occur in isolated axons. Injuryinduced acceleration of the axonal transport of tubulin and actin in the proximal axon is likely to be more important in providing the cytoskeletal protein required for initial axonal outgrowth. Subsequently, the increased synthesis and transport velocity for actin and tubulin increase the delivery of these proteins to support the increased volume of the maturing regenerating axons. Reduction in neurofilament synthesis and changes in neurofilament phosphorylation may permit the increased transport velocity of the other cytoskeletal proteins. There is little direct evidence that alterations in cytoskeletal protein synthesis are necessary for successful regeneration, nor are they sufficient in the absence of a supportive environment. Nevertheless, the correlation that exists between a robust cell body response and successful regeneration suggests that an understanding of the regulation of cytoskeletal protein synthesis following axon injury must be a part of any successful strategy to improve the regenerative capacity of the central nervous system.     

Ultrastructural features of rat pituicytes in organotypic culture

Summary Neural lobes of adult rats have been explanted and organ-cultured for 5–10 days. While perivascular cells and neurosecretory fibers undergo a progressive degeneration, scattered pituicyte-like cells are observed mostly associated with each other in number of two or three cells for group. Cultured pituicytes are quite similar to in vivo pituicytes except for some particular features as the shape of the nucleus and the number of dense bodies. Furthermore they share with the in vivo pituicytes the phagocytic capacity which appears even increased as far as neurosecretory fibers are concerned. Finally, degenerating pituicytes and free lipid droplets are observed in the intercellular spaces: the significance of these facts is discussed and tentatively interpreted. The organotypic culture of the neural lobe seems to represent an experimental model useful to obtain a relatively pure population of pituicytes.With the technical assistance of Vincenzo Panetta.     

Integration of ascending and descending inputs in the auditory midbrain of anurans.

In anurans, the midbrain torus semicircularis is involved in auditory processing and audio-motor integration. In this study, we examined the influence of descending forebrain projections on the auditory response properties and hence the audiomotor transmission of mesencephalic interface neurons. In order to investigate response integration, we performed intracellular recordings from torus neurons in an isolated brain preparation of Discoglossus pictus and Bombina orientalis and stimulated the auditory nerve, striatum, and the dorsal thalamus electrically with single pulses. Stimulation of all three sites could evoke responses in torus neurons that were either excitatory, inhibitory, or a mixture of both, with durations of up to several hundred milliseconds. Further, striatum and thalamus were activated by pulse trains (10-20 Hz, 50 pulses) immediately before stimulating the auditory nerve with single pulses. Thus, responses of torus neurons to "auditory" input were facilitated or suppressed for up to 2 min by striatum stimulation or only suppressed by thalamus stimulation. Intracellular labeling of recorded neurons revealed that response modulation by descending input mostly occurred in laminar nucleus neurons. These results suggest that descending forebrain projections to mesencephalic audiomotor interface neurons may play an important role in modifying acoustically guided behavior in anurans.     

Postmetamorphic changes in auditory sensitivity of the bullfrog midbrain

During metamorphosis, the lateral line system of ranid frogs (Rana catesbeiana) degenerates and an auditory system sensitive to airborne sounds develops. We examined the onset of function and developmental changes in the central auditory system by recording multi-unit activity from the principal nucleus of the torus semicircularis (TSp) of bullfrogs at different postmetamorphic stages in response to tympanically-presented auditory stimuli. No responses were recorded to stimuli of up to 95 dB SPL from latemetamorphic tadpoles, but auditory responses were recorded within 24 hours of completion of metamorphosis. Audiograms from froglets (SVL < 5.5 cm) were relatively flat in shape with high thresholds, and showed a decrease in most sensitive frequency (MSF) from about 2500 Hz to about 1500 Hz throughout the first 7–10 days after completion of metamorphosis. Audiograms from frogs larger than 5.5 cm showed continuous downward shifts in MSF and thresholds, and increases in sharpness around MSF until reaching adult-like values. Spontaneous activity in the TSp increased throughout postmetamorphic development. The torus increased in volume by approximately 50% throughout development and displayed changes in cell density and nuclear organization. These observations suggest that the onset of sensitivity to tympanically presented airborne sounds is limited by peripheral, rather than central, auditory maturation.Abbreviations CF characteristic frequency - MSF most sensitive frequency - PB phasic burst - PL primary like - S sustained - SVL snout-vent length - TS torus semicircularis - TSl laminar nucleus of TS - TSm magnocellular nucleus of TS - TSp principal nucleus of TS - TW tympanic width     

Distinct cellular and molecular mechanisms mediate initial axon development and adult-stage axon regeneration in C. elegans

The molecular and cellular mechanisms that allow adult-stage neurons to regenerate following damage are poorly understood. Recently, axons of motoneurons and mechanosensory neurons in adult C. elegans were found to regrow after being snipped by femtosecond laser ablation. Here, we explore the molecular determinants of adult-stage axon regeneration using the AVM mechanosensory neurons. The first step in AVM axon development is a pioneer axonal projection from the cell body to the ventral nerve cord. We show that regeneration of the AVM axon to the ventral nerve cord lacks the deterministic precision of initial axon development, requiring competition and pruning of unwanted axon branches. Nevertheless, axons of injured AVM neurons regrow to the ventral nerve cord with over 60% reliability in adult animals. In addition, in contrast to initial development, axon guidance during regeneration becomes heavily dependent on cytoplasmic protein MIG-10/Lamellipodin but independent of UNC-129/TGF-beta repellent and UNC-40/DCC receptor, and axon growth during regeneration becomes heavily dependent on UNC-34/Ena and CED-10/Rac actin regulators. Thus, C. elegans may be used as a genetic system to characterize novel cellular and molecular mechanisms underlying adult-stage nervous system regeneration.     

Glycinergic transmission regulates dendrite size in organotypic culture

We previously demonstrated that inhibitory synaptic transmission influences dendrite development in vivo. We now report an analogous finding in an organotypic culture of a glycinergic projection nucleus, the medial nucleus of the trapezoid body (MNTB), and its postsynaptic target, the lateral superior olive (LSO) of gerbils. Cultures were generated at 6–7 days postnatal and grown in serum containing medium with or without the glycine receptor antagonist, strychnine (SN), at 2 μM. LSO neurons were then labeled with biocytin, and the dendritic arbors were analyzed morphometrically. Compared to neurons from age-matched in vivo tissue, the neurons cultured in control media were somewhat atrophic, including decreases in dendritic branching and length. Incubation in strychnine led to a dramatic increase in dendritic branching and total dendritic length. Control neurons averaged 6.3 branches, compared to 18 branches/neuron in SN-treated cultures. There was a similar increase in primary dendrites and total dendritic length. The physical elimination of MNTB cells did not mimic SN treatment, presumably because glycinergic LSO neurons generated intrinsic connections. In fact, the LSO soma area was significantly greater following MNTB removal, suggesting that these afferents provide a second signal to postsynaptic neurons. These results suggest that spontaneous glycinergic transmission regulates the growth of postsynaptic processes. © 1996 John Wiley & Sons, Inc.     

Spatial representation of frequency-modulated tones in gerbil auditory cortex revealed by epidural electrocorticography.

The present study investigated the topography of epidurally recorded middle latency components P1 and N1 evoked by spectrally dynamic stimuli (linearly frequency-modulated (FM) tones) with respect to the tonotopic structure of the right primary auditory cortex, field AI. Whereas the gross topography corresponded to the spectral content of the FM tones, specific tonotopic offsets were found between the potential distributions evoked by FM tones of different modulation direction (i.e. 'rising' vs. 'falling'). Potentials evoked by rising FM tones were located at tonotopic positions corresponding to higher frequencies compared with potentials evoked by falling FM tones. Data indicated that the magnitude of these offsets can be attributed to the local tonotopic resolution in AI and are not dependent on the modulation rate.