Restricted Neuronal Populations Research Articles

The NRF2-Dependent Transcriptional Regulation of Antioxidant Defense Pathways: Relevance for Cell Type-Specific Vulnerability to Neurodegeneration and Therapeutic Intervention.

Oxidative stress has been implicated in the etiology and pathobiology of various neurodegenerative diseases. At baseline, the cells of the nervous system have the capability to regulate the genes for antioxidant defenses by engaging nuclear factor erythroid 2 (NFE2/NRF)-dependent transcriptional mechanisms, and a number of strategies have been proposed to activate these pathways to promote neuroprotection. Here, we briefly review the biology of the transcription factors of the NFE2/NRF family in the brain and provide evidence for the differential cellular localization of NFE2/NRF family members in the cells of the nervous system. We then discuss these findings in the context of the oxidative stress observed in two neurodegenerative diseases, Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS), and present current strategies for activating NFE2/NRF-dependent transcription. Based on the expression of the NFE2/NRF family members in restricted populations of neurons and glia, we propose that, when designing strategies to engage these pathways for neuroprotection, the relative contributions of neuronal and non-neuronal cell types to the overall oxidative state of tissue should be considered, as well as the cell types which have the greatest intrinsic capacity for producing antioxidant enzymes.

Impact of mTOR hyperactive neurons on the morphology and physiology of adjacent neurons: Do PTEN KO cells make bad neighbors?

Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway is associated with epilepsy, autism and brain growth abnormalities in humans. mTOR hyperactivation often results from developmental somatic mutations, producing genetic lesions and associated dysfunction in relatively restricted populations of neurons. Disrupted brain regions, such as those observed in focal cortical dysplasia, can contain a mix of normal and mutant cells. Mutant cells exhibit robust anatomical and physiological changes. Less clear, however, is whether adjacent, initially normal cells are affected by the presence of abnormal cells. To explore this question, we used a conditional, inducible mouse model approach to delete the mTOR negative regulator phosphatase and tensin homolog (PTEN) from <1% to >30% of hippocampal dentate granule cells. We then examined the morphology of PTEN-expressing granule cells located in the same dentate gyri as the knockout (KO) cells. Despite the development of spontaneous seizures in higher KO animals, and disease worsening with increasing age, the morphology and physiology of PTEN-expressing cells was only modestly affected. PTEN-expressing cells had smaller somas than cells from control animals, but other parameters were largely unchanged. These findings contrast with the behavior of PTEN KO cells, which show increasing dendritic extent with greater KO cell load. Together, the findings indicate that genetically normal neurons can exhibit relatively stable morphology and intrinsic physiology in the presence of nearby pathological neurons and systemic disease.

Expression of RNA-binding protein Rbfox1l demarcates a restricted population of dorsal telencephalic neurons within the adult zebrafish brain

Rbfox RNA-binding proteins are expressed in the adult mammalian brain and are required for proper brain development and function. Studies in mice and humans have implicated Rbfox1/RBFOX1 in autism, neuronal excitation and epilepsy, and Rbfox2/RBFOX2 in cerebellar development. The zebrafish has emerged as a prominent model system for brain study, possessing neuroanatomical conservation with mammals and an extensive capacity for adult neurogenesis and plasticity. In this study, we characterize Rbfox1l and Rbfox2 expression in the adult zebrafish brain. While Rbfox2 is expressed broadly, Rbfox1l is expressed in restricted populations of neurons in the dorsal telencephalon and cerebellum. In the dorsal telencephalon, Rbfox1l is expressed in a specific population of neurons spanning Dm and Dc regions. In the cerebellum, Rbfox1l and Rbfox2 are expressed in the Purkinje cell layer, reminiscent of Rbfox1 and Rbfox2 expression in the mammalian cerebellum. Our findings motivate future studies of Rbfox function in the zebrafish brain.

Neurons Specifically Activated by Fear Learning in Lateral Amygdala Display Increased Synaptic Strength.

The lateral amygdala (LA) plays a critical role in the formation of fear-conditioned associative memories. Previous studies have used c-fos regulated expression to identify a spatially restricted population of neurons within the LA that is specifically activated by fear learning. These neurons are likely to be a part of a memory engram, but, to date, functional evidence for this has been lacking. We show that neurons within a spatially restricted region of the LA had an increase in both the frequency and amplitude of spontaneous postsynaptic currents (sPSC) when compared to neurons recorded from home cage control mice. We then more specifically addressed if this increased synaptic activity was limited to learning-activated neurons. Using a fos-tau-LacZ (FTL) transgenic mouse line, we developed a fluorescence-based method of identifying and recording from neurons activated by fear learning (FTL+) in acute brain slices. An increase in frequency and amplitude of sPSCs was observed in FTL+ neurons when compared to nonactivated FTL− neurons in fear-conditioned mice. No learning-induced changes were observed in the action potential (AP) input-output relationships. These findings support the idea that a discrete LA neuron population forms part of a memory engram through changes in synaptic connectivity.

Mushroom body signaling is required for locomotor activity rhythms in Drosophila

In the fruitfly Drosophila melanogaster, circadian rhythms of locomotor activity under constant darkness are controlled by pacemaker neurons. To understand how behavioral rhythmicity is generated by the nervous system, it is essential to identify the output circuits from the pacemaker neurons. A recent study of Drosophila has suggested that pacemaker neurons project to mushroom body (MB) neurons, which are considered the memory center in Drosophila. MBs also regulate spontaneous locomotor activity without learning, suggesting that MB neuronal activity regulates behavioral rhythms. However, the importance of MBs in generating behavioral rhythmicity remains controversial because contradicting results have been reported as follows: (1) locomotor activity in MB-ablated flies is substantially rhythmic, but (2) activation of restricted neuronal populations including MB neurons induces arrhythmic locomotor activity. Here, we report that neurotransmission in MBs is required for behavioral rhythmicity. For adult-specific disruption of neurotransmission in MBs, we used the GAL80/GAL4/UAS ternary gene expression system in combination with the temperature-sensitive dynamin mutation shibirets1. Blocking of neurotransmission in GAL4-positive neurons including MB neurons induced arrhythmic locomotor activity, whereas this arrhythmicity was rescued by the MB-specific expression of GAL80. Our results indicate that MB signaling plays a key role in locomotor activity rhythms in Drosophila.

Genetic control of a central pattern generator: rhythmic oromotor movement in mice is controlled by a major locus near Atp1a2.

Fluid licking in mice is a rhythmic behavior that is controlled by a central pattern generator (CPG) located in a complex of brainstem nuclei. C57BL/6J (B6) and DBA/2J (D2) strains differ significantly in water-restricted licking, with a highly heritable difference in rates (h2≥0.62) and a corresponding 20% difference in interlick interval (mean ± SEM = 116.3±1 vs 95.4±1.1 ms). We systematically quantified motor output in these strains, their F1 hybrids, and a set of 64 BXD progeny strains. The mean primary interlick interval (MPI) varied continuously among progeny strains. We detected a significant quantitative trait locus (QTL) for a CPG controlling lick rate on Chr 1 (Lick1), and a suggestive locus on Chr 10 (Lick10). Linkage was verified by testing of B6.D2-1D congenic stock in which a segment of Chr 1 of the D2 strain was introgressed onto the B6 parent. The Lick1 interval on distal Chr 1 contains several strong candidate genes. One of these is a sodium/potassium pump subunit (Atp1a2) with widespread expression in astrocytes, as well as in a restricted population of neurons. Both this subunit and the entire Na+/K+-ATPase molecule have been implicated in rhythmogenesis for respiration and locomotion. Sequence variants in or near Apt1a2 strongly modulate expression of the cognate mRNA in multiple brain regions. This gene region has recently been sequenced exhaustively and we have cataloged over 300 non-coding and synonymous mutations segregating among BXD strains, one or more of which is likely to contribute to differences in central pattern generator tempo.

Multiple Forms of Activity-Dependent Competition Refine Hippocampal Circuits In Vivo

Efficient memory formation relies on the establishment of functional hippocampal circuits. It has been proposed that synaptic connections are refined by neural activity to form functional brain circuitry. However, it is not known whether and how hippocampal connections are refined by neural activity in vivo. Using a mouse genetic system in which restricted populations of neurons in the hippocampal circuit are inactivated, we show that inactive axons are eliminated after they develop through a competition with active axons. Remarkably, in the dentate gyrus, which undergoes neurogenesis throughout life, axon refinement is achieved by a competition between mature and young neurons. These results demonstrate that activity-dependent competition plays multiple roles in the establishment of functional memory circuits in vivo.

Genetic visualization of the secondary olfactory pathway in Tbx21 transgenic mice

BackgroundMitral and tufted cells are the projection neurons in the olfactory bulb, conveying odour information to various regions of the olfactory cortex. In spite of their functional importance, there are few molecular and genetic tools that can be used for selective labelling or manipulation of mitral and tufted cells. Tbx21 was first identified as a T-box family transcription factor regulating the differentiation and function of T lymphocytes. In the brain, Tbx21 is specifically expressed in mitral and tufted cells of the olfactory bulb.ResultsIn this study, we performed a promoter/enhancer analysis of mouse Tbx21 gene by comparing nucleotide sequence similarity of Tbx21 genes among several mammalian species and generating transgenic mouse lines with various lengths of 5' upstream region fused to a fluorescent reporter gapVenus. We identified the cis-regulatory enhancer element (~300 nucleotides) at ~ 3.0 kb upstream of the transcription start site of Tbx21 gene, which is both necessary and sufficient for transgene expression in mitral and tufted cells. In contrast, the 2.6-kb 5'-flanking region of mouse Tbx21 gene induced transgene expression with variable patterns in restricted populations of neurons predominantly located along the olfactory pathway. Furthermore, we generated transgenic mice expressing the genetically-encoded fluorescent exocytosis indicator, synaptopHluorin, in mitral and tufted cells for visualization of presynaptic neural activities in the piriform cortex.ConclusionsThe transcriptional enhancer of Tbx21 gene provides a powerful tool for genetic manipulations of mitral and tufted cells in studying the development and function of the secondary olfactory pathways from the bulb to the cortex.

Molecular Characterization of the Mouse Superior Lateral Parabrachial Nucleus through Expression of the Transcription Factor Runx1

BackgroundThe ability to precisely identify separate neuronal populations is essential to the understanding of the development and function of different brain structures. This necessity is particularly evident in regions such as the brainstem, where the anatomy is quite complex and little is known about the identity, origin, and function of a number of distinct nuclei due to the lack of specific cellular markers. In this regard, the gene encoding the transcription factor Runx1 has emerged as a specific marker of restricted neuronal populations in the murine central and peripheral nervous systems. The aim of this study was to precisely characterize the expression of Runx1 in the developing and postnatal mouse brainstem.Methods and Principal FindingsAnatomical and immunohistochemical studies were used to characterize mouse Runx1 expression in the brainstem. It is shown here that Runx1 is expressed in a restricted population of neurons located in the dorsolateral rostral hindbrain. These neurons define a structure that is ventromedial to the dorsal nucleus of the lateral lemniscus, dorsocaudal to the medial paralemniscal nucleus and rostral to the cerebellum. Runx1 expression in these cells is first observed at approximately gestational day 12.5, persists into the adult brain, and is lost in knockout mice lacking the transcription factor Atoh1, an important regulator of the development of neuronal lineages of the rhombic lip. Runx1-expressing neurons in the rostral hindbrain produce cholecystokinin and also co-express members of the Groucho/Transducin-like Enhancer of split protein family.ConclusionBased on the anatomical and molecular characteristics of the Runx1-expressing cells in the rostral hindbrain, we propose that Runx1 expression in this region of the mouse brain defines the superior lateral parabrachial nucleus.

Modeling the electrode–neuron interface of cochlear implants: Effects of neural survival, electrode placement, and the partial tripolar configuration

The partial tripolar electrode configuration is a relatively novel stimulation strategy that can generate more spatially focused electric fields than the commonly used monopolar configuration. Focused stimulation strategies should improve spectral resolution in cochlear implant users, but may also be more sensitive to local irregularities in the electrode–neuron interface. In this study, we develop a practical computer model of cochlear implant stimulation that can simulate neural activation in a simplified cochlear geometry and we relate the resulting patterns of neural activity to basic psychophysical measures. We examine how two types of local irregularities in the electrode–neuron interface, variations in spiral ganglion nerve density and electrode position within the scala tympani, affect the simulated neural activation patterns and how these patterns change with electrode configuration. The model shows that higher partial tripolar fractions activate more spatially restricted populations of neurons at all current levels and require higher current levels to excite a given number of neurons. We find that threshold levels are more sensitive at high partial tripolar fractions to both types of irregularities, but these effects are not independent. In particular, at close electrode–neuron distances, activation is typically more spatially localized which leads to a greater influence of neural dead regions.

Brain activation pattern induced by stimulation of L-type Ca 2+-channels: Contribution of Ca V1.3 and Ca V1.2 isoforms

Ca V1.2 and Ca V1.3, are the main dihydropyridine-sensitive L-type calcium channel isoforms in the brain. To reveal the contribution of each isoform to the neuronal activation pattern elicited by the dihydropyridine L-type calcium channel activator BayK 8644, we utilized Fos expression as a marker of neuronal activation in mutant mice (Ca V1.2 DHP−/− mice) expressing dihydropyridine-insensitive Ca V1.2 L-type calcium channels. BayK 8644-treated wildtype mice displayed intense and widespread Fos expression throughout the neuroaxis in 77 of 80 brain regions quantified. The Fos response in Ca V1.2 DHP−/− mice was greatly attenuated or absent in most of these areas, suggesting that a major part of the widespread Fos induction including most cortical areas was mediated by Ca V1.2 L-type calcium channels. BayK 8644-induced Fos expression in Ca V1.2 DHP−/− mice indicating predominantly Ca V1.3 L-type calcium channel-mediated activation was noted in more restricted neuronal populations (20 of 80), in particular in the central amygdala, the bed nucleus of the stria terminalis, paraventricular hypothalamic nucleus, lateral preoptic area, locus coeruleus, lateral parabrachial nucleus, central nucleus of the inferior colliculus, and nucleus of the solitary tract. Our data indicate that selective stimulation of other than Ca V1.2 L-type calcium channels, mostly Ca V1.3, causes neuronal activation in a specific set of mainly limbic, hypothalamic and brainstem areas, which are associated with functions including integration of emotion-related behavior. Hence, selective modulation of Ca V1.3 L-type calcium channels could represent a novel (pharmacotherapeutic) tool to influence these CNS functions.

Adult onset thalamocerebellar degeneration in dogs associated to neuronal storage of ceroid lipopigment

Late onset of hereditary cerebellar cortical abiotrophy has been described in a large variety of canine breeds. In some reported conditions, the cerebellar lesion is combined with degeneration of other systems. Here we describe a new hereditary cerebellar cortical degeneration in eight adult American Staffordshire and Pit Bull Terriers. The neuronal degeneration in these animals not only affects Purkinje cells of the cerebellum but also certain thalamic nuclei. In addition, nerve cell loss appears to be associated with a lysosomal storage disease, which is restricted to the affected cell populations. The stored material was histologically and ultrastructurally identified as fluorescent lipopigment. Since animals were euthanized at various stages of the disease, it could be shown that lysosomal storage preceded neuronal loss. Selective involvement of restricted neuronal populations is highly unusual in ceroid lipofuscinoses. It remains to be determined if the present neurodegenerative disease is caused by a primary or secondary neuronal ceroid lipofuscinosis.

Identification and distribution of a two-pore domain potassium channel in the CNS of Aplysia californica

A cDNA encoding a potassium channel of the two-pore domain family (K2p) of leak channels was cloned from the CNS of the marine opisthobranch Aplysia californica. This is the first sequence of the K2p family identified in molluscs and has been named AcK2p1. The deduced amino acid sequence is homologous to channels of the mammalian two-pore domain halothane inhibited (THIK) subfamily, bearing 46% identity to THIK-1 (KCNK 13) and 48% to THIK-2 (KCNK12). We used in-situ hybridization to analyze the distribution of this class of channels in the CNS. AcK2p1 is specifically expressed in many central neurons of all major ganglia including the largest identified neurons MCC, R2 and LP1. The highest expression of AcK2p1 was detected in an asymmetrical and distinct cluster of up to 30 cells located at the dorsal-medial region of the right pleural ganglion. The neuron-specific distribution seen in the molluscan CNS is consistent with data from mammals that indicate THIK is only expressed in restricted neuronal populations, suggesting its involvement in both the maintenance of neuronal phenotype and in the specific functional role of these neurons in their respective networks.

Identification and characterization of two novel brain-derived immunoglobulin superfamily members with a unique structural organization

We recently used a differential display PCR screen to identify secreted and transmembrane proteins that are highly expressed in the developing rat basilar pons, a prominent ventral hindbrain nucleus used as a model for studies of neuronal migration, axon outgrowth, and axon-target recognition. Here we describe cloning and characterization of one of these molecules, now called MDGA1, and a closely related homologue, MDGA2. Analyses of the full-length coding region of MDGA1 and MDGA2 indicate that they encode proteins that comprise a novel subgroup of the Ig superfamily and have a unique structural organization consisting of six immunoglobulin (Ig)-like domains followed by a single MAM domain. Biochemical characterization demonstrates that MDGA1 and MDGA2 proteins are highly glycosylated, and that MDGA1 is tethered to the cell membrane by a GPI anchor. The MDGAs are differentially expressed by subpopulations of neurons in both the central and peripheral nervous systems, including neurons of the basilar pons, inferior olive, cerebellum, cerebral cortex, olfactory bulb, spinal cord, and dorsal root and trigeminal ganglia. Little or no MDGA expression is detected outside of the nervous system of developing rats. The similarity of MDGAs to other Ig-containing molecules and their temporal–spatial patterns of expression within restricted neuronal populations, for example migrating pontine neurons and D1 spinal interneurons, suggest a role for these novel proteins in regulating neuronal migration, as well as other aspects of neural development, including axon guidance.

Aberrant transcription of unrearranged T-cell receptor beta gene in mouse brain.

The nervous system and the immune system share several functional molecules involved in various cell-cell interaction events. In this study, we used in situ hybridization to identify immune molecules that are expressed by a restricted population of neurons in the mouse brain and found that mRNA for the beta subunit of T-cell receptor (TCRbeta) was predominantly and strongly localized to neurons in deep layers of the cerebral neocortex and weakly expressed in the thalamus. Developmentally, TCRbeta mRNA expression started at embryonic day 15 in the thalamic nuclei and at postnatal day 1 in the cerebral neocortex. The level of TCRbeta mRNA in the neocortex subsequently increased until postnatal day 21, and it remained high in the adult. Detailed analysis revealed that only the Cbeta2 segment of TCRbeta, not the Cbeta1 or Vbeta segments, was expressed by the brain neurons. By the 5' rapid amplification of cDNA ends method, we determined a brain-specific transcription start site in the Jbeta2 region locus, not in the Vbeta region locus. Furthermore, we confirmed that the aberrant transcription around the Jbeta2 region took place only in neurons and lymphocytes in transgenic mice. These results demonstrate that the transcriptional machinery for unrearranged TCRbeta expression is shared by the nervous and immune systems and raise a possibility of gene rearrangement in neurons under certain circumstances.

Neurotensin Gene Expression and Behavioral Responses Following Administration of Psychostimulants and Antipsychotic Drugs in Dopamine D3 Receptor Deficient Mice

Exposure to psychostimulants and antipsychotics increases neurotensin (NT) gene expression in the striatum and nucleus accumbens. To investigate the contribution of D3 receptors to these effects we used mice with targeted disruption of the D3 receptor gene. Basal NT mRNA expression was similar in D3 receptor mutant mice and wild-type animals. Acute administration of haloperidol increased NT gene expression in the striatum in D3+/+, D3+/− and D3−/− mice. Similarly, acute cocaine and amphetamine induced NT mRNA expression in the nucleus accumbens shell and olfactory tubercle to a comparable extent in D3 mutants and wild-type mice. Daily injection of cocaine for seven days increased NT mRNA in a restricted population of neurons in the dorsomedial caudal striatum of D3+/+ mice, but not in D3−/− and D3+/− animals. No differences were observed between D3 receptor mutant mice and wild-type littermates in the locomotor activity and stereotyped behaviors induced by repeated cocaine administration. These findings demonstrate that dopamine D3 receptors are not necessary for the acute NT mRNA response to drugs of abuse and antipsychotics but appear to play a role in the regulation of NT gene induction in striatal neurons after repeated cocaine. In addition, our results indicate that the acute locomotor response to cocaine and development of psychostimulant-induced behavioral sensitization do not require functional D3 receptors.

Areas of operation of interneurons mediating presynaptic inhibition in sacral spinal segments.

Sources of primary afferent depolarization (PAD) of skin afferents in the sural (Sur) nerve and of group-II muscle afferents in the posterior biceps and semitendinosus (PBST) nerve were compared at several sites, about 2 mm apart, within the L7-S2 segments in order to define areas of projection of sacral interneurons mediating PAD of these afferents. Just rostral to the pudendal nucleus, strong PAD of Sur afferents was evoked by stimulation of skin nerves, while stimulation of muscle nerves had only marginal effects. This indicates that sacral PAD interneurons co-excited by skin and muscle afferents operate primarily within the regions overlying the pudendal nucleus. Furthermore, PAD evoked by muscle afferents was weaker over the rostral part of the pudendal nucleus than over the caudal part of this nucleus, where hamstring afferents became its main source, both in Sur and in PBST group-II afferents. By correlating the relative strength of PAD at the levels of the rostral and caudal parts of the pudendal nucleus with the previously established input from muscle and cutaneous afferents to interneurons at these levels, it is therefore proposed that sacral PAD interneurons operate over shorter distances than indicated by previous experiments: over either rostral or caudal parts of the pudendal nucleus, i.e., about 2 mm, rather than over the whole length of this nucleus, i.e., 4-5 mm. Sacral PAD interneurons may, thus, modulate synaptic transmission to even more spatially restricted neuronal populations than previously proposed.

Cells containing immunoreactive estrogen receptor-α in the human basal forebrain

The distribution of estrogen receptor protein-α (ER-α)-containing cells in the human hypothalamus and adjacent regions was studied using a monoclonal antibody (H222) raised against ER-α derived from MCF-7 human breast cancer cells. Reaction product was found in restricted populations of neurons and astrocyte-like cells. Neurons immunoreactive for ER-α were diffusely distributed within the basal forebrain and preoptic area, infundibular region, central hypothalamus, basal ganglia and amygdala. Immunoreactive astrocyte-like cells were noted within specific brain regions, including the lamina terminalis and subependymal peri-third-ventricular region. These data are consistent with the location of estrogen receptors in the basal forebrain of other species and the known effects of estrogens on the cellular functions of both neurons and supporting elements within the human hypothalamus and basal forebrain.

Involvement of Intronic Sequences in Cell‐Specific Expression of the Peripherin Gene

Peripherin is an intermediate filament protein expressed in restricted populations of neurons. Our previous study of the chromatin structure of the mouse peripherin gene in cells that do or do not express peripherin suggested that the region located between -1,500 and +800 bp of the gene could be involved in its cell specificity. In the present work, we performed an in vitro functional analysis of the 5' flanking region of the mouse peripherin gene and observed that this region up to 9 kb contained both enhancer and inhibiting activities; however, it was insufficient to achieve a complete extinction of reporter gene expression in peripherin-negative cells. Furthermore, analysis of the first three introns with the 5' flanking sequences of the gene showed that intron I greatly increased specificity of the gene expression. Intron I also conferred the same properties to thymidine kinase heterologous promoter. DNase I footprinting experiments performed with intron I revealed at least two protected regions (Inl A and Inl B). Inl A encompasses an AP-2-like binding site that interacted with both neuroblast and fibroblast nuclear factors, as well as with the recombinant AP-2alpha protein. However, gel shift experiments suggested that the interacting nuclear factors are distinct from AP-2alpha itself and probably belong to the AP-2 family. Inl B perfectly matched the consensus binding site for Sp1 and specifically interacted with nuclear protein factors that showed the same binding properties as the Sp1 family members. Fine deletion analysis of intron I indicated that the Inl A element alone is responsible for its enhancing properties, whereas a region located between +789 and +832 gives to intron I its silencer activity.

Tuning curves of the difference tone auditory nerve neurophonic

When a pair of tonal stimuli of different frequencies (F 1 and F 2, where F 2 > F 1) are simultaneously presented to the ear, an electrical response with a frequency of F 2-F 1 can be recorded from the round window (RW) of the gerbil's cochlea. By using phase-locked tones of alternating polarity, the cochlear microphonics are canceled, leaving a time-averaged difference tone-auditory nerve neurophonic (DT-ANN). When the F, frequency ranges from 1.25 to 30 kHz and F 2−F 1 ≈ 900 Hz, a DT-ANN audiogram can be constructed which parallels (but is at least 10 dB more sensitive than) the compound action potential (CAP) audiogram. In addition to this DT response, a smaller magnitude, higher threshold response having a frequency of 2 DT can often be measured. Both the DT-ANN and the 2 DT-ANN show non-monotonic amplitude input-output functions. The DT- and 2 DT-ANN responses can be forward masked. Masking of low level (e.g., 30 dB SPL) probe stimuli results in DT- and 2 DT-ANN V-shaped tuning curves (TO with low tip thresholds (≈ 20–30 dB SPL) and a tip frequency close to that of F 1 and F 2. The Q 10 dB values of the forward masked DT-ANN TCs ranges from 1.54 to 20.0 for F 1 frequencies varying from 2 to 20 kHz, respectively. The V-shaped DT-ANN TCs generated with simultaneous maskers are often flanked, outside their high- and low-frequency slopes, by frequency-intensity domains where the masker enhances the amplitude of the DT-ANN response. These data (1) provide evidence that, in response to low-intensity tones, the DT-ANN is generated by a restricted population of neurons that have characteristic frequencies close to F 1 and F 2, and (2) provide evidence for sharply tuned, phase-locked activity occurring in response to low-intensity stimuli, by cochlear axons having characteristic frequencies as high as 20 kHz.