Cell‐ and region‐specific expression of depression‐related protein p11 (S100a10) in the brain

P11 (S100a10), a member of the S100 family of proteins, has widespread distribution in the vertebrate body, including in the brain, where it has a key role in membrane trafficking, vesicle secretion, and endocytosis. Recently, our laboratory has shown that a constitutive knockout of p11 (p11‐KO) in mice results in a depressive‐like phenotype. Furthermore, p11 has been implicated in major depressive disorder (MDD) and in the actions of antidepressants. Since depression affects multiple brain regions, and the role of p11 has only been determined in a few of these areas, a detailed analysis of p11 expression in the brain is warranted. Here we demonstrate that, although widespread in the brain, p11 expression is restricted to distinct regions, and specific neuronal and nonneuronal cell types. Furthermore, we provide comprehensive mapping of p11 expression using in situ hybridization, immunocytochemistry, and whole‐tissue volume imaging. Overall, expression spans multiple brain regions, structures, and cell types, suggesting a complex role of p11 in depression. J. Comp. Neurol. 525:955–975, 2017. © 2016 Wiley Periodicals, Inc.     

Laterally projecting cerebrospinal fluid‐contacting cells in the lamprey spinal cord are of two distinct types

Cerebrospinal fluid‐contacting (CSF‐c) cells are found in all vertebrates, but their function remains elusive. In the lamprey spinal cord, they surround the central canal and some have processes passing the gray matter to the lateral edge of the flattened spinal cord. Stimulation of CSF‐c cells at the central canal elicits GABAergic inhibitory postsynaptic potentials (IPSPs) in intraspinal stretch receptor neurons (edge cells). Here, we characterize laterally projecting CSF‐c cells according to their morphology, phenotype, and neuronal properties by using immunohistochemistry, retrograde tracing, calcium imaging, and whole‐cell recordings. We identify two types of CSF‐c cells. Type 1 cells have a bulb‐like ending that protrudes into the central canal and a lateral process that ramifies ventrolaterally and laterally with a dense plexus surrounding the mechanosensitive dendrites of the edge cells. Most type 1 cells fire spontaneous action potentials that are abolished by tetrodotoxin, and all display spontaneous excitatory postsynaptic potentials and IPSPs that remain in the presence of tetrodotoxin. GABA and somatostatin are colocalized in type 1 cells, and they express both GABA and glutamate receptors. Type 2 cells, on the other hand, have a flat ending protruding into the central canal and a laterally projecting process that ramifies only at the lateral edge. These cells show immunoreactivity to taurine, but they do not express GABA or somatostatin, nor do they have any active neuronal properties. Type 2 cells might be a form of glia. Type 1 CSF‐c cells are neurons and may play a modulatory role by influencing edge cells and thus the locomotor‐related sensory feedback. J. Comp. Neurol. 522:1753–1768, 2014. © 2014 Wiley Periodicals, Inc.     

Organization of alpha‐transducin immunoreactive system in the brain and retina of larval and young adult Sea Lamprey (Petromyzon marinus), and their relationship with other neural systems

We employed an anti‐transducin antibody (Gαt‐S), in combination with other markers, to characterize the Gαt‐S‐immunoreactive (ir) system in the CNS of the sea lamprey, Petromyzon marinus. Gαt‐S immunoreactivity was observed in some neuronal populations and numerous fibers distributed throughout the brain. Double Gαt‐S‐ and opsin‐ir neurons (putative photoreceptors) are distributed in the hypothalamus (postoptic commissure nucleus, dorsal and ventral hypothalamus) and caudal diencephalon, confirming results of García‐Fernández et al. (Cell and Tissue Research, 288, 267–278, 1997). Singly Gαt‐S‐ir cells were observed in the midbrain and hindbrain, increasing the known populations. Our results reveal for the first time in vertebrates the extensive innervation of many brain regions and the spinal cord by Gαt‐S‐ir fibers. The Gαt‐S innervation of the habenula is very selective, fibers densely innervating the lamprey homologue of the mammalian medial nucleus (Stephenson‐Jones et al., Proceedings of the National Academy of Sciences of the United States of America, 109, E164–E173, 2012), but not the lateral nucleus homologue. The lamprey neurohypophysis was not innervated by Gαt‐S‐ir fibers. We also analyzed by double immunofluorescence the relation of this system with other systems. A dopaminergic marker (TH), serotonin (5‐HT) or GABA do not co‐localize with Gαt‐S‐ir neurons although codistribution of fibers was observed. Codistribution of Gαt‐S‐ir fibers and isolectin‐labeled extrabulbar primary olfactory fibers was observed in the striatum and hypothalamus. Neurobiotin retrograde transport from the spinal cord combined with immunofluorescence revealed spinal‐projecting Gαt‐S‐ir reticular neurons in the caudal hindbrain. Present results in an ancient vertebrate reveal for the first time a collection of brain targets of Gαt‐S‐ir neurons, suggesting they might mediate non‐visual modulation by light in many systems.     

Pigment epithelium‐derived factor (PEDF) and PEDF‐receptor in the adult mouse brain: Differential spatial/temporal localization pattern

Pigment epithelium‐derived factor (PEDF) is a multifunctional protein which was initially described in the retina, although it is also present in other tissues. It functions as an antioxidant agent promoting neuronal survival. Recently, a PEDF receptor has shown an elevated binding affinity for PEDF. There are no relevant data regarding the distribution of both proteins in the brain, therefore the main goal of this work was to investigate the spatiotemporal presence of PEDF and PEDFR in the adult mouse brain, and to determine the PEDF blood level in mouse and human. The localization of both proteins was analyzed by different experimental methods such as immunohistochemistry, western‐blotting, and also by enzyme‐linked immunosorbent assay. Differential expression was found in some telencephalic structures and positive signals for both proteins were detected in the cerebellum. The magnitude of the PEDFR labeling pattern was higher than PEDF and included some cortical and subventricular areas. Age‐dependent changes in intensity of both protein immunoreactions were found in the cortical and hippocampal areas with greater reactivity between 4 and 8 months of age, whilst others, like the subventricular zones, these differences were more evident for PEDFR. Although ubiquitous presence was not found in the brain for these two proteins, their relevant functions must not be underestimated. It has been described that PEDF plays an important role in neuroprotection and data provided in the present work represents the first extensive study to understand the relevance of these two proteins in specific brain areas.     

Clustered organization and region‐specific identities of estrogen‐producing neurons in the forebrain of Zebra Finches (Taeniopygia guttata)

A fast, neuromodulatory role for estrogen signaling has been reported in many regions of the vertebrate brain. Regional differences in the cellular distribution of aromatase (estrogen synthase) in several species suggest that mechanisms for neuroestrogen signaling differ between and even within brain regions. A more comprehensive understanding of neuroestrogen signaling depends on characterizing the cellular identities of neurons that express aromatase. Calcium‐binding proteins such as parvalbumin and calbindin are molecular markers for interneuron subtypes, and are co‐expressed with aromatase in human temporal cortex. Songbirds like the zebra finch have become important models to understand the brain synthesis of steroids like estrogens and the implications for neurobiology and behavior. Here, we investigated the regional differences in cytoarchitecture and cellular identities of aromatase‐expressing neurons in the auditory and sensorimotor forebrain of zebra finches. Aromatase was co‐expressed with parvalbumin in the caudomedial nidopallium (NCM) and HVC shelf (proper name) but not in the caudolateral nidopallium (NCL) or hippocampus. By contrast, calbindin was not co‐expressed with aromatase in any region investigated. Notably, aromatase‐expressing neurons were found in dense somato‐somatic clusters, suggesting a coordinated release of local neuroestrogens from clustered neurons. Aromatase clusters were also more abundant and tightly packed in the NCM of males as compared to females. Overall, this study provides new insights into neuroestrogen regulation at the network level, and extends previous findings from human cortex by identifying a subset of aromatase neurons as putative inhibitory interneurons.     

Organization of the sleep‐related neural systems in the brain of the minke whale (Balaenoptera acutorostrata)

The current study analyzed the nuclear organization of the neural systems related to the control and regulation of sleep and wake in the basal forebrain, diencephalon, midbrain, and pons of the minke whale, a mysticete cetacean. While odontocete cetaceans sleep in an unusual manner, with unihemispheric slow wave sleep (USWS) and suppressed REM sleep, it is unclear whether the mysticete whales show a similar sleep pattern. Previously, we detailed a range of features in the odontocete brain that appear to be related to odontocete‐type sleep, and here present our analysis of these features in the minke whale brain. All neural elements involved in sleep regulation and control found in bihemispheric sleeping mammals and the harbor porpoise were present in the minke whale, with no specific nuclei being absent, and no novel nuclei being present. This qualitative similarity relates to the cholinergic, noradrenergic, serotonergic and orexinergic systems, and the GABAergic elements of these nuclei. Quantitative analysis revealed that the numbers of pontine cholinergic (274,242) and noradrenergic (203,686) neurons, and hypothalamic orexinergic neurons (277,604), are markedly higher than other large‐brained bihemispheric sleeping mammals. Small telencephalic commissures (anterior, corpus callosum, and hippocampal), an enlarged posterior commissure, supernumerary pontine cholinergic and noradrenergic cells, and an enlarged peripheral division of the dorsal raphe nuclear complex of the minke whale, all indicate that the suite of neural characteristics thought to be involved in the control of USWS and the suppression of REM in the odontocete cetaceans are present in the minke whale. J. Comp. Neurol. 524:2018–2035, 2016. © 2015 Wiley Periodicals, Inc.     

Distribution and innervation of putative peripheral arterial chemoreceptors in the red‐eared slider (Trachemys scripta elegans)

Peripheral arterial chemoreceptors have been isolated to the common carotid artery, aorta, and pulmonary artery of turtles. However, the putative neurotransmitters associated with these chemoreceptors have not yet been described. The goal of the present study was to determine the neurochemical content, innervations, and distribution of putative oxygen‐sensing cells in the central vasculature of turtles and to derive homologies with peripheral arterial chemoreceptors of other vertebrates. We used tract tracing together with immunohistochemical markers for cholinergic cells (vesicular acetylcholine transporter [VAChT]), tyrosine hydroxylase (TH; the rate‐limiting enzyme in catecholamine synthesis), and serotonin (5HT) to identify putative oxygen‐sensing cells and to determine their anatomical relation to branches of the vagus nerve (Xth cranial nerve). We found potential oxygen‐sensing cells in all three chemosensory areas innervated by branches of the Xth cranial nerve. Cells containing either 5HT or VAChT were found in all three sites. The morphology and size of these cells resemble glomus cells found in amphibians, mammals, tortoises, and lizards. Furthermore, we found populations of cholinergic cells located at the base of the aorta and pulmonary artery that are likely involved in efferent regulation of vessel resistance. Catecholamine‐containing cells were not found in any of the putative chemosensitive areas. The presence of 5HT‐ and VAChT‐immunoreactive cells in segments of the common carotid artery, aorta, and pulmonary artery appears to reflect a transition between cells containing the major neurotransmitters seen in fish (5HT) and mammals (ACh and adenosine). J. Comp. Neurol. 523:1399–1418, 2015. © 2015 Wiley Periodicals, Inc.     

Organization of the sleep‐related neural systems in the brain of the harbour porpoise (Phocoena phocoena)

The present study provides the first systematic immunohistochemical neuroanatomical investigation of the systems involved in the control and regulation of sleep in an odontocete cetacean, the harbor porpoise (Phocoena phocoena). The odontocete cetaceans show an unusual form of mammalian sleep, with unihemispheric slow waves, suppressed REM sleep, and continuous bodily movement. All the neural elements involved in sleep regulation and control found in bihemispheric sleeping mammals were present in the harbor porpoise, with no specific nuclei being absent, and no novel nuclei being present. This qualitative similarity of nuclear organization relates to the cholinergic, noradrenergic, serotonergic, and orexinergic systems and is extended to the γ‐aminobutyric acid (GABA)ergic elements involved with these nuclei. Quantitative analysis of the cholinergic and noradrenergic nuclei of the pontine region revealed that in comparison with other mammals, the numbers of pontine cholinergic (126,776) and noradrenergic (122,878) neurons are markedly higher than in other large‐brained bihemispheric sleeping mammals. The diminutive telencephalic commissures (anterior commissure, corpus callosum, and hippocampal commissure) along with an enlarged posterior commissure and supernumerary pontine cholinergic and noradrenergic neurons indicate that the control of unihemispheric slow‐wave sleep is likely to be a function of interpontine competition, facilitated through the posterior commissure, in response to unilateral telencephalic input related to the drive for sleep. In addition, an expanded peripheral division of the dorsal raphe nuclear complex appears likely to play a role in the suppression of REM sleep in odontocete cetaceans. Thus, the current study provides several clues to the understanding of the neural control of the unusual sleep phenomenology present in odontocete cetaceans. J. Comp. Neurol. 524:1999–2017, 2016. © 2016 Wiley Periodicals, Inc.     

Pattern of nitrergic cells and fibers organization in the central nervous system of the Australian lungfish,Neoceratodus forsteri (Sarcopterygii: Dipnoi)

The Australian lungfish Neoceratodus forsteri is the only extant species of the order Ceratodontiformes, which retained most of the primitive features of ancient lobe finned-fishes. Lungfishes are the closest living relatives of land vertebrates and their study is important for deducing the neural traits that were conserved, modified, or lost with the transition from fishes to land vertebrates. We have investigated the nitrergic system with neural nitric oxide synthase (NOS) immunohistochemistry and NADPH-diaphorase (NADPH-d) histochemistry, which yielded almost identical results except for the primary olfactory projections and the terminal and preoptic nerve fibers labeled only for NADPH-d. Combined immunohistochemistry was used for simultaneous detection of NOS with catecholaminergic, cholinergic, and serotonergic structures, aiming to establish accurately the localization of the nitrergic elements and to assess possible interactions between these neurotransmitter systems. The results demonstrated abundant nitrergic cells in the basal ganglia, amygdaloid complex, preoptic area, basal hypothalamus, mesencephalic tectum and tegmentum, laterodorsal tegmental nucleus, reticular formation, spinal cord, and retina. In addition, low numbers of nitrergic cells were observed in the olfactory bulb, all pallial divisions, lateral septum, suprachiasmatic nucleus, prethalamic and thalamic areas, posterior tubercle, pretectum, torus semicircularis, cerebellar nucleus, interpeduncular nucleus, the medial octavolateral nucleus, nucleus of the solitary tract, and the dorsal column nucleus. Colocalization of NOS and tyrosine hydroxylase was observed in numerous cells of the ventral tegmental area/substantia nigra complex. Comparison with other vertebrates, using a neuromeric analysis, reveals that the nitrergic system of Neoceratodus shares many neuroanatomical features with tetrapods and particularly with amphibians.     

Organization of the sleep‐related neural systems in the brain of the river hippopotamus (Hippopotamus amphibius): A most unusual cetartiodactyl species

This study provides the first systematic analysis of the nuclear organization of the neural systems related to sleep and wake in the basal forebrain, diencephalon, midbrain, and pons of the river hippopotamus, one of the closest extant terrestrial relatives of the cetaceans. All nuclei involved in sleep regulation and control found in other mammals, including cetaceans, were present in the river hippopotamus, with no specific nuclei being absent, but novel features of the cholinergic system, including novel nuclei, were present. This qualitative similarity relates to the cholinergic, noradrenergic, serotonergic, and orexinergic systems and is extended to the γ‐aminobutyric acid (GABA)ergic elements of these nuclei. Quantitative analysis reveals that the numbers of pontine cholinergic (259,578) and noradrenergic (127,752) neurons, and hypothalamic orexinergic neurons (68,398) are markedly higher than in other large‐brained mammals. These features, along with novel cholinergic nuclei in the intralaminar nuclei of the dorsal thalamus and the ventral tegmental area of the midbrain, as well as a major expansion of the hypothalamic cholinergic nuclei and a large laterodorsal tegmental nucleus of the pons that has both parvocellular and magnocellular cholinergic neurons, indicates an unusual sleep phenomenology for the hippopotamus. Our observations indicate that the hippopotamus is likely to be a bihemispheric sleeper that expresses REM sleep. The novel features of the cholinergic system suggest the presence of an undescribed sleep state in the hippopotamus, as well as the possibility that this animal could, more rapidly than other mammals, switch cortical electroencephalographic activity from one state to another. J. Comp. Neurol. 524:2036–2058, 2016. © 2016 Wiley Periodicals, Inc.     

Nectin‐1 spots as a novel adhesion apparatus that tethers mitral cell lateral dendrites in a dendritic meshwork structure of the developing mouse olfactory bulb

Mitral cells project lateral dendrites that contact the lateral and primary dendrites of other mitral cells and granule cell dendrites in the external plexiform layer (EPL) of the olfactory bulb. These dendritic structures are critical for odor information processing, but it remains unknown how they are formed. In immunofluorescence microscopy, the immunofluorescence signal for the cell adhesion molecule nectin‐1 was concentrated on mitral cell lateral dendrites in the EPL of the developing mouse olfactory bulb. In electron microscopy, the immunogold particles for nectin‐1 were symmetrically localized on the plasma membranes at the contacts between mitral cell lateral dendrites, which showed bilateral darkening without dense cytoskeletal undercoats characteristic of puncta adherentia junctions. We named the contacts where the immunogold particles for nectin‐1 were symmetrically accumulated "nectin‐1 spots." The nectin‐1 spots were 0.21 μm in length on average and the distance between the plasma membranes was 20.8 nm on average. In 3D reconstruction of serial sections, clusters of the nectin‐1 spots formed a disc‐like structure. In the mitral cell lateral dendrites of nectin‐1‐knockout mice, the immunogold particles for nectin‐1 were undetectable and the plasma membrane darkening was electron‐microscopically normalized, but the plasma membranes were partly separated from each other. The nectin‐1 spots were further identified between mitral cell lateral and primary dendrites and between mitral cell lateral dendrites and granule cell dendritic spine necks. These results indicate that the nectin‐1 spots constitute a novel adhesion apparatus that tethers mitral cell dendrites in a dendritic meshwork structure of the developing mouse olfactory bulb. J. Comp. Neurol. 523:1824–1839, 2015. © 2015 Wiley Periodicals, Inc.     

Somatic and neuritic spines on tyrosine hydroxylase–immunopositive cells of rat retina

Dopamine‐ and tyrosine hydroxylase–immunopositive cells (TH cells) modulate visually driven signals as they flow through retinal photoreceptor, bipolar, and ganglion cells. Previous studies suggested that TH cells release dopamine from varicose axons arborizing in the inner and outer plexiform layers after glutamatergic synapses depolarize TH cell dendrites in the inner plexiform layer and these depolarizations propagate to the varicosities. Although it has been proposed that these excitatory synapses are formed onto appendages resembling dendritic spines, spines have not been found on TH cells of most species examined to date or on TH cell somata that release dopamine when exposed to glutamate receptor agonists. By use of protocols that preserve proximal retinal neuron morphology, we have examined the shape, distribution, and synapse‐related immunoreactivity of adult rat TH cells. We report here that TH cell somata, tapering and varicose inner plexiform layer neurites, and varicose outer plexiform layer neurites all bear spines, that some of these spines are immunopositive for glutamate receptor and postsynaptic density proteins (viz., GluR1, GluR4, NR1, PSD‐95, and PSD‐93), that TH cell somata and tapering neurites are also immunopositive for a γ‐aminobutyric acid (GABA) receptor subunit (GABA_AR_α1), and that a synaptic ribbon‐specific protein (RIBEYE) is found adjacent to some colocalizations of GluR1 and TH in the inner plexiform layer. These results identify previously undescribed sites at which glutamatergic and GABAergic inputs may stimulate and inhibit dopamine release, especially at somata and along varicose neurites that emerge from these somata and arborize in various levels of the retina. J. Comp. Neurol. 525:1707–1730, 2017. © 2016 Wiley Periodicals, Inc.     

Quercetin improves the activity of the ubiquitin‐proteasomal system in 150Q mutated huntingtin‐expressing cells but exerts detrimental effects on neuronal survivability

Quercetin, a strong free radical scavenger, is investigated for neuroprotective effects in a Neuro 2a cell line conditionally transfected with 16Q huntingtin (Htt) and 150Q Htt, which express the protein upon stimulation. Cells were protected from death by a 20‐µM dose of quercetin on the second day of Htt induction, but 30–100‐µM doses of the drug caused further toxicity in both 16Q and 150Q cells, as indicated by MTT assay and by significant reductions in the number of cells bearing neurites on the second day. A significant decrease in the number of cells containing aggregate was seen in induced 150Q cells treated with 20 µM but not for those treated with 40 or 50 µM quercetin up to 4 days of induction. Mutated Htt (mHtt)‐induced reduction in proteasomal activity of the ubiquitin‐proteasomal system (UPS) was significantly attenuated by 20 µM quercetin. However, neither mitochondrial membrane potential loss nor colocalization of 20S proteasome with mHtt aggregate was corrected by quercetin treatment. Our results imply that the neuroprotective effect of quercetin arises out of the upregulation of UPS activity, which causes a decrease in the number of mHtt aggregate‐harboring cells. The increased neurotoxicity could result from the continued association of mHtt with 20S proteasome and the failure of quercetin to correct mitochondrial membrane potential loss. These results suggest that, although quercetin at a low dose protects against mHtt‐mediated cell death, higher doses are toxic to the cells, clearly demarcating a narrow therapeutic window for this dietary flavonoid. © 2015 Wiley Periodicals, Inc.     

Spatial and temporal expression profiles of urocortin 3 mRNA in the brain of the chicken (Gallus gallus)

Urocortin 3 (UCN3) is a neuropeptide believed to regulate stress‐coping responses by binding to type 2 corticotropin‐releasing hormone receptors. Here, we report the cloning and brain distribution of UCN3 mRNA in a sauropsid—the chicken, Gallus gallus. Mature chicken UCN3 is predicted to be a 40‐amino acid peptide showing high sequence similarity to human (93%), mouse (93%), and Xenopus (88%) UCN3. During the last third of embryonic development, UCN3 mRNA levels changed differentially in the various brain parts. In all brain parts, UCN3 mRNA levels tended to increase toward hatching, except for caudal brainstem, where a gradual decrease was observed during the last week of embryonic development. In cerebellum, a rapid increase in gene expression occurred between embryonic days 17 and 19. Using in situ hybridization, UCN3 mRNA was found to be expressed predominantly in the hypothalamus, pons, and medulla of posthatch chick brains, but not in some areas that are among the main expression sites in rodents, such as the brain areas where in mammals the median preoptic nucleus and the medial amygdala are located. This suggests that the roles of UCN3 in chicken, and perhaps sauropsids in general, are not all identical to those in rodents.     

Distribution of corticotropin‐releasing factor (CRF) receptor binding in the mouse brain using a new,high‐affinity radioligand, [125I]‐PD‐Sauvagine

The corticotropin‐releasing factor (CRF) family of peptides includes CRF and three urocortins, which signal through two distinct G‐protein coupled receptors, CRF₁ and CRF₂. Although the cellular distribution of CRF receptor expression has been well characterized at the mRNA level, the localization of receptor protein, and, by inference, of functional receptors, has been limited by a lack of reliable immunohistochemical evidence. Recently, a CRF‐related peptide, termed PD‐sauvagine, was isolated from the skin of the frog, Pachymedusa dacnicolor, and validated as a high‐affinity ligand for CRF receptor studies. A radiolabeled analog, [¹²⁵I]‐PD‐sauvagine, with high signal‐to‐noise ratio, was used in autoradiographic studies to map the distribution of CRF receptor binding sites in the mouse brain. Through the use of receptor‐deficient mice and subtype‐specific antagonists, CRF₁ and CRF₂ binding sites were isolated, and found to be readily reconcilable with regional patterns of mRNA expression. Binding site distributions within a given structure sometimes differed from mRNA patterns, however, particularly in laminated structures of the isocortex, hippocampus, and cerebellum, presumably reflecting the trafficking of receptors to their operational homes on neuronal (mostly dendritic) processes. Binding patterns of [¹²⁵I]‐PD‐sauvagine provided independent assessments of controversial receptor localizations, failing to provide support for CRF₁ expression in central autonomic components of the limbic forebrain, the locus coeruleus and cerebellar Purkinje cells, or for CRF₂ in any aspect of the cerebellar cortex. Though lacking in ideal resolution, in vitro binding of the PD‐sauvagine radioligand currently provides the most sensitive and accurate available tool for localizing CRF receptors in rodent brain.     

Opposing expression gradients of calcitonin‐related polypeptide alpha (Calca/Cgrpα) and tyrosine hydroxylase (Th) in type II afferent neurons of the mouse cochlea

Type II spiral ganglion neurons (SGNs) are small caliber, unmyelinated afferents that extend dendritic arbors hundreds of microns along the cochlear spiral, contacting many outer hair cells (OHCs). Despite these many contacts, type II afferents are insensitive to sound and only weakly depolarized by glutamate release from OHCs. Recent studies suggest that type II afferents may be cochlear nociceptors, and can be excited by ATP released during tissue damage, by analogy to somatic pain‐sensing C‐fibers. The present work compares the expression patterns among cochlear type II afferents of two genes found in C‐fibers: calcitonin‐related polypeptide alpha (Calca/Cgrpα), specific to pain‐sensing C‐fibers, and tyrosine hydroxylase (Th), specific to low‐threshold mechanoreceptive C‐fibers, which was shown previously to be a selective biomarker of type II versus type I cochlear afferents (Vyas et al., 2016 ). Whole‐mount cochlear preparations from 3‐week‐ to 2‐month‐old CGRPα‐EGFP (GENSAT) mice showed expression of Cgrpα in a subset of SGNs with type II‐like peripheral dendrites extending beneath OHCs. Double labeling with other molecular markers confirmed that the labeled SGNs were neither type I SGNs nor olivocochlear efferents. Cgrpα starts to express in type II SGNs before hearing onset, but the expression level declines in the adult. The expression patterns of Cgrpα and Th formed opposing gradients, with Th being preferentially expressed in apical and Cgrpα in basal type II afferent neurons, indicating heterogeneity among type II afferent neurons. The expression of Th and Cgrpα was not mutually exclusive and co‐expression could be observed, most abundantly in the middle cochlear turn.