Immunohistochemical localization of DPP10 in rat brain supports the existence of a Kv4/KChIP/DPPL ternary complex in neurons

Subthreshold A‐type K⁺ currents (I_SAs) have been recorded from the cell bodies of hippocampal and neocortical interneurons as well as neocortical pyramidal neurons. Kv4 channels are responsible for the somatodendritic I_SAs. It has been proposed that neuronal Kv4 channels are ternary complexes including pore‐forming Kv4 subunits, K⁺ channel‐interacting proteins (KChIPs), and dipeptidyl peptidase‐like proteins (DPPLs). However, colocalization evidence was still lacking. The distribution of DPP10 mRNA in rodent brain has been reported but its protein localization remains unknown. In this study, we generated a DPP10 antibody to label DPP10 protein in adult rat brain by immunohistochemistry. Absent from glia, DPP10 proteins appear mainly in the cell bodies of DPP10(+) neurons, not only at the plasma membrane but also in the cytoplasm. At least 6.4% of inhibitory interneurons in the hippocampus coexpressed Kv4.3, KChIP1, and DPP10, with the highest density in the CA1 strata alveus/oriens/pyramidale and the dentate hilus. Colocalization of Kv4.3/KChIP1/DPP10 was also detected in at least 6.9% of inhibitory interneurons scattered throughout the neocortex. Both hippocampal and neocortical Kv4.3/KChIP1/DPP10(+) inhibitory interneurons expressed parvalbumin or somatostatin, but not calbindin or calretinin. Furthermore, we found colocalization of Kv4.2/Kv4.3/KChIP3/DPP10 in neocortical layer 5 pyramidal neurons and olfactory bulb mitral cells. Together, although DPP10 is also expressed in some brain neurons lacking Kv4 (such as parvalbumin‐ and somatostatin‐positive Golgi cells in the cerebellum), colocalization of DPP10 with Kv4 and KChIP at the plasma membrane of I_SA‐expressing neuron somata supports the existence of Kv4/KChIP/DPPL ternary complex in vivo. J. Comp. Neurol. 523:608–628, 2015. © 2014 Wiley Periodicals, Inc.     

Expression of Kv3.1b potassium channel is widespread in macaque motor cortex pyramidal cells: A histological comparison between rat and macaque

There are substantial differences across species in the organization and function of the motor pathways. These differences extend to basic electrophysiological properties. Thus, in rat motor cortex, pyramidal cells have long duration action potentials, while in the macaque, some pyramidal neurons exhibit short duration “thin” spikes. These differences may be related to the expression of the fast potassium channel Kv3.1b, which in rat interneurons is associated with generation of thin spikes. Rat pyramidal cells typically lack these channels, while there are reports that they are present in macaque pyramids. Here we made a systematic, quantitative comparison of the Kv3.1b expression in sections from macaque and rat motor cortex, using two different antibodies (NeuroMab, Millipore). As our standard reference, we examined, in the same sections, Kv3.1b staining in parvalbumin‐positive interneurons, which show strong Kv3.1b immunoreactivity. In macaque motor cortex, a large sample of pyramidal neurons were nearly all found to express Kv3.1b in their soma membranes. These labeled neurons were identified as pyramidal based either by expression of SMI32 (a pyramidal marker), or by their shape and size, and lack of expression of parvalbumin (a marker for some classes of interneuron). Large (Betz cells), medium, and small pyramidal neurons all expressed Kv3.1b. In rat motor cortex, SMI32‐postive pyramidal neurons expressing Kv3.1b were very rare and weakly stained. Thus, there is a marked species difference in the immunoreactivity of Kv3.1b in pyramidal neurons, and this may be one of the factors explaining the pronounced electrophysiological differences between rat and macaque pyramidal neurons.     

Localization of nectin‐2α at the boundary between the adjacent somata of the clustered cholinergic neurons and its regulatory role in the subcellular localization of the voltage‐gated A‐type K+ channel Kv4.2 in the medial habenula

The medial habenula (MHb), implicated in stress, depression, memory, and nicotine withdrawal syndromes, receives septal inputs and sends efferents to the interpeduncular nucleus. We previously showed that the immunoglobulin‐like cell adhesion molecules (CAMs) nectin‐2α and nectin‐2δ are expressed in astrocytes in the brain, but their expression in neurons remains unknown. We showed here by immunofluorescence microscopy that nectin‐2α, but not nectin‐2δ, was prominently expressed in the cholinergic neurons in the developing and adult MHbs and localized at the boundary between the adjacent somata of the clustered cholinergic neurons where the voltage‐gated A‐type K⁺ channel Kv4.2 was localized. Analysis by immunoelectron microscopy on this boundary revealed that Kv4.2 was localized at the membrane specializations (MSs) with plasma membrane darkening in an asymmetrical manner, whereas nectin‐2α was localized on the apposed plasma membranes mostly at the outside of these MSs, but occasionally localized at their edges and insides. Nectin‐2α at this boundary was not colocalized with the nectin‐2α‐binding protein afadin, other CAMs, or their interacting peripheral membrane proteins, suggesting that nectin‐2α forms a cell adhesion apparatus different from the Kv4.2‐associated MSs. Genetic ablation of nectin‐2 delayed the localization of Kv4.2 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing MHb. These results revealed the unique localization of nectin‐2α and its regulatory role in the localization of Kv4.2 at the MSs in the MHb.     

Spinal neuronal activation during locomotor‐like activity enabled by epidural stimulation and 5‐hydroxytryptamine agonists in spinal rats

The neural networks that generate stepping in complete spinal adult rats remain poorly defined. To address this problem, we used c‐fos (an activity‐dependent marker) to identify active interneurons and motoneurons in the lumbar spinal cord of adult spinal rats during a 30‐min bout of bipedal stepping. Spinal rats were either step trained (30 min/day, 3 days/week, for 7.5 weeks) or not step trained. Stepping was enabled by epidural stimulation and the administration of the serotonergic agonists quipazine and 8‐OHDPAT. A third group of spinal rats served as untreated (no stimulation, drugs, or stepping) controls. The numbers of activated cholinergic central canal cluster cells and partition neurons were higher in both step‐trained and nontrained rats than in untreated rats and were higher in nontrained than in step‐trained rats. The latter finding suggests that daily treatment with epidural stimulation plus serotonergic agonist treatment without step training enhances the excitability of a broader cholinergic interneuronal population than does step training. The numbers of activated interneurons in laminae II–VI of lumbar cross‐sections were higher in both step‐trained and nontrained rats than in untreated rats, and they were highest in step‐trained rats. This finding suggests that this population of interneurons is responsive to epidural stimulation plus serotonergic treatment and that load‐bearing induced when stepping has an additive effect. The numbers of activated motoneurons of all size categories were higher in the step‐trained group than in the other two groups, reflecting a strong effect of loading on motoneuron recruitment. In general, these results indicate that the spinal networks for locomotion are similar with and without brain input. © 2015 Wiley Periodicals, Inc. Significance : We identified neurons within the spinal cord networks that are activated during assisted stepping in paraplegic rats. We stimulated the spinal cord and administered a drug to help the rats step. One group was trained to step and another was not trained. We observed a lower percentage of activated neurons in specific spinal cord regions in trained rats than in nontrained rats after a 1‐hr stepping bout, suggesting that step training reduces activation of some types of spinal neurons. This observation indicates that training makes the spinal networks more efficient and suggests a “learning” phenomenon in the spinal cord without any brain input.     

GABAA and GABAB receptor subunit localization on neurochemically identified neurons of the human subthalamic nucleus

The subthalamic nucleus (STN) is a critical excitatory signaling center within the basal ganglia circuitry. The activity of subthalamic neurons is tightly controlled by upstream inhibitory signaling centers in the basal ganglia. In this study, we used immunohistochemical techniques to firstly, visualize and quantify the STN neurochemical organization based on neuronal markers including parvalbumin (PV), calretinin (CR), SMI‐32, and GAD_65/67. Secondly, we characterized the detailed regional, cellular and subcellular expression of GABA_A (α₁, α₂, α₃, β_2/3, and γ₂) and GABA_B (R1 and R2) receptor subunits within the normal human STN. Overall, we found seven neurochemically distinct populations of principal neurons in the human STN. The three main populations detected were: (a) triple‐labeled PV⁺/CR⁺/SMI32⁺; (b) double‐labeled PV⁺/CR⁺; and (c) single‐labeled CR⁺ neurons. Subthalamic principal neurons were found to express GABA_A receptor subunits α₁, α₃, β_2/3, γ₂, and GABA_B receptor subunits R1 and R2. However, no expression of GABA_A receptor α₂ subunit was detected. We also found a trend of increasing regional staining intensity for all positive GABA_A receptor subunits from the dorsolateral pole to ventromedial extremities. The GAD⁺ interneurons showed relatively low expression of GABA_A receptor subunits. These results provide the morphological basis of GABAergic transmission within the normal human subthalamic nucleus and evidence of GABA innervation through both GABA_A and GABA_B receptors on subthalamic principal neurons.     

Efferent projections of excitatory and inhibitory preBötzinger Complex neurons

The preBötzinger Complex (preBötC), a compact medullary region essential for generating normal breathing rhythm and pattern, is the kernel of the breathing central pattern generator (CPG). Excitatory preBötC neurons in rats project to major breathing‐related brainstem regions. Here, we provide a brainstem connectivity map in mice for both excitatory and inhibitory preBötC neurons. Using a genetic strategy to label preBötC neurons, we confirmed extensive projections of preBötC excitatory neurons within the brainstem breathing CPG including the contralateral preBötC, Bötzinger Complex (BötC), ventral respiratory group, nucleus of the solitary tract, parahypoglossal nucleus, parafacial region (RTN/pFRG or alternatively, pF_L/pF_V), parabrachial and Kölliker‐Füse nuclei, as well as major projections to the midbrain periaqueductal gray. Interestingly, preBötC inhibitory projections paralleled the excitatory projections. Moreover, we examined overlapping projections in the pons in detail and found that they targeted the same neurons. We further explored the direct anatomical link between the preBötC and suprapontine brain regions that may govern emotion and other complex behaviors that can affect or be affected by breathing. Forebrain efferent projections were sparse and restricted to specific nuclei within the thalamus and hypothalamus, with processes rarely observed in cortex, basal ganglia, or other limbic regions, e.g., amygdala or hippocampus. We conclude that the preBötC sends direct, presumably inspiratory‐modulated, excitatory and inhibitory projections in parallel to distinct targets throughout the brain that generate and modulate breathing pattern and/or coordinate breathing with other behaviors, physiology, cognition, or emotional state.     

Characterization of the relaxin family peptide receptor 3 system in the mouse bed nucleus of the stria terminalis

The bed nucleus of the stria terminalis (BNST) is a critical node involved in stress and reward-related behaviors. Relaxin family peptide receptor 3 (RXFP3) signaling in the BNST has been implicated in stress-induced alcohol seeking behavior. However, the neurochemical phenotype and connectivity of BNST RXFP3-expressing (RXFP3+) cells have yet to be elucidated. We interrogated the molecular signature and electrophysiological properties of BNST RXFP3+ neurons using a RXFP3-Cre reporter mouse line. BNST RXFP3+ cells are circumscribed to the dorsal BNST (dBNST) and are neurochemically heterogeneous, comprising a mix of inhibitory and excitatory neurons. Immunohistochemistry revealed that ~48% of BNST RXFP3+ neurons are GABAergic, and a quarter of these co-express the calcium-binding protein, calbindin. A subset of BNST RXFP3+ cells (~41%) co-express CaMKIIα, suggesting this subpopulation of BNST RXFP3+ neurons are excitatory. Corroborating this, RNAscope® revealed that ~35% of BNST RXFP3+ cells express vVGluT2 mRNA, indicating a subpopulation of RXFP3+ neurons are glutamatergic. RXFP3+ neurons show direct hyperpolarization to bath application of a selective RXFP3 agonist, RXFP3-A2, while around 50% of cells were depolarised by exogenous corticotrophin releasing factor. In behaviorally naive mice the majority of RXFP3+ neurons were Type II cells exhibiting I_h and T type calcium mediated currents. However, chronic swim stress caused persistent plasticity, decreasing the proportion of neurons that express these channels. These studies are the first to characterize the BNST RXFP3 system in mouse and lay the foundation for future functional studies appraising the role of the murine BNST RXFP3 system in more complex behaviors.     

5HTR3A‐driven GFP labels immature olfactory sensory neurons

The ionotropic serotonin receptor, 5‐HT₃, is expressed by many developing neurons within the central nervous system. Since the olfactory epithelium continues to generate new olfactory sensory neurons (OSNs) throughout life, we investigated the possibility that 5‐HT₃ is expressed in the adult epithelium. Using a transgenic mouse in which the promoter for the 5‐HT_3a subunit drives expression of green fluorescent protein (GFP), we assessed the expression of this marker in the olfactory epithelium of adult mice. Both the native 5‐HT_3a mRNA and GFP are expressed within globose basal cells of the olfactory and vomeronasal epithelium in adult mice. Whereas the 5‐HT_3a mRNA disappears relatively quickly after final cell division, the GFP label persists for about 5 days, thereby labeling immature OSNs in both the main olfactory system and vomeronasal organ. The GFP‐labeled cells include both proliferative globose basal cells as well as immature OSNs exhibiting the hallmarks of ongoing differentiation including GAP43, PGP9.5, but the absence of olfactory marker protein. Some of the GFP‐labeled OSNs show characteristics of more mature yet still developing OSNs including the presence of cilia extending from the apical knob and expression of NaV1.5, a component of the transduction cascade. These findings suggest that 5‐HT_3a is indicative of a proliferative or developmental state, regardless of age, and that the 5‐HT_3AGFP mice may prove useful for future studies of neurogenesis in the olfactory epithelium. J. Comp. Neurol. 525:1743–1755, 2017. © 2016 Wiley Periodicals, Inc.     

Candidates for photic entrainment pathways to the circadian clock via optic lobe neuropils in the Madeira cockroach

The compound eye of cockroaches is obligatory for entrainment of the Madeira cockroach's circadian clock, but the cellular nature of its entrainment pathways is enigmatic. Employing multiple-label immunocytochemistry, histochemistry, and backfills, we searched for photic entrainment pathways to the accessory medulla (AME), the circadian clock of the Madeira cockroach. We wanted to know whether photoreceptor terminals could directly contact pigment-dispersing factor-immunoreactive (PDF-ir) circadian pacemaker neurons with somata in the lamina (PDFLAs) or somata next to the AME (PDFMEs). Short green-sensitive photoreceptor neurons of the compound eye terminated in lamina layers LA1 and LA2, adjacent to PDFLAs and PDFMEs that branched in LA3. Long UV-sensitive compound eye photoreceptor neurons terminated in medulla layer ME2 without direct contact to ipsilateral PDFMEs that arborized in ME4. Multiple neuropeptide-ir interneurons branched in ME4, connecting the AME to ME2. Before, extraocular photoreceptors of the lamina organ were suggested to send terminals to accessory laminae. There, they overlapped with PDFLAs that mostly colocalized PDF, FMRFamide, and 5-HT immunoreactivities, and with terminals of ipsi- and contralateral PDFMEs. We hypothesize that during the day cholinergic activation of the largest PDFME via lamina organ photoreceptors maintains PDF release orchestrating phases of sleep–wake cycles. As ipsilateral PDFMEs express excitatory and contralateral PDFMEs inhibitory PDF autoreceptors, diurnal PDF release keeps both PDF-dependent clock circuits in antiphase. Future experiments will test whether ipsilateral PDFMEs are sleep-promoting morning cells, while contralateral PDFMEs are activity-promoting evening cells, maintaining stable antiphase via the largest PDFME entrained by extraocular photoreceptors of the lamina organ.     

Cerebellins are differentially expressed in selective subsets of neurons throughout the brain

Cerebellins are secreted hexameric proteins that form tripartite complexes with the presynaptic cell‐adhesion molecules neurexins or ‘deleted‐in‐colorectal‐cancer’, and the postsynaptic glutamate‐receptor‐related proteins GluD1 and GluD2. These tripartite complexes are thought to regulate synapses. However, cerebellins are expressed in multiple isoforms whose relative distributions and overall functions are not understood. Three of the four cerebellins, Cbln1, Cbln2, and Cbln4, autonomously assemble into homohexamers, whereas the Cbln3 requires Cbln1 for assembly and secretion. Here, we show that Cbln1, Cbln2, and Cbln4 are abundantly expressed in nearly all brain regions, but exhibit strikingly different expression patterns and developmental dynamics. Using newly generated knockin reporter mice for Cbln2 and Cbln4, we find that Cbln2 and Cbln4 are not universally expressed in all neurons, but only in specific subsets of neurons. For example, Cbln2 and Cbln4 are broadly expressed in largely non‐overlapping subpopulations of excitatory cortical neurons, but only sparse expression was observed in excitatory hippocampal neurons of the CA1‐ or CA3‐region. Similarly, Cbln2 and Cbln4 are selectively expressed, respectively, in inhibitory interneurons and excitatory mitral projection neurons of the main olfactory bulb; here, these two classes of neurons form dendrodendritic reciprocal synapses with each other. A few brain regions, such as the nucleus of the lateral olfactory tract, exhibit astoundingly high Cbln2 expression levels. Viewed together, our data show that cerebellins are abundantly expressed in relatively small subsets of neurons, suggesting specific roles restricted to subsets of synapses.     

Brain region‐dependent differential expression of alpha‐synuclein

α‐Synuclein, the major constituent of Lewy bodies (LBs), is normally expressed in presynapses and is involved in synaptic function. Abnormal intracellular aggregation of α‐synuclein is observed as LBs and Lewy neurites in neurodegenerative disorders, such as Parkinson's disease (PD) or dementia with Lewy bodies. Accumulated evidence suggests that abundant intracellular expression of α‐synuclein is one of the risk factors for pathological aggregation. Recently, we reported differential expression patterns of α‐synuclein between excitatory and inhibitory hippocampal neurons. Here we further investigated the precise expression profile in the adult mouse brain with special reference to vulnerable regions along the progression of idiopathic PD. The results show that α‐synuclein was highly expressed in the neuronal cell bodies of some early PD‐affected brain regions, such as the olfactory bulb, dorsal motor nucleus of the vagus, and substantia nigra pars compacta. Synaptic expression of α‐synuclein was mostly accompanied by expression of vesicular glutamate transporter‐1, an excitatory presynaptic marker. In contrast, expression of α‐synuclein in the GABAergic inhibitory synapses was different among brain regions. α‐Synuclein was clearly expressed in inhibitory synapses in the external plexiform layer of the olfactory bulb, globus pallidus, and substantia nigra pars reticulata, but not in the cerebral cortex, subthalamic nucleus, or thalamus. These results suggest that some neurons in early PD‐affected human brain regions express high levels of perikaryal α‐synuclein, as happens in the mouse brain. Additionally, synaptic profiles expressing α‐synuclein are different in various brain regions. J. Comp. Neurol. 524:1236–1258, 2016. © 2015 Wiley Periodicals, Inc.     

Cell‐specific and developmental expression of lectican‐cleaving proteases in mouse hippocampus and neocortex

Mounting evidence has demonstrated that a specialized extracellular matrix exists in the mammalian brain and that this glycoprotein‐rich matrix contributes to many aspects of brain development and function. The most prominent supramolecular assemblies of these extracellular matrix glycoproteins are perineuronal nets, specialized lattice‐like structures that surround the cell bodies and proximal neurites of select classes of interneurons. Perineuronal nets are composed of lecticans, a family of chondroitin sulfate proteoglycans that includes aggrecan, brevican, neurocan, and versican. These lattice‐like structures emerge late in postnatal brain development, coinciding with the ending of critical periods of brain development. Despite our knowledge of the presence of lecticans in perineuronal nets and their importance in regulating synaptic plasticity, we know little about the development or distribution of the extracellular proteases that are responsible for their cleavage and turnover. A subset of a large family of extracellular proteases (called a disintegrin and metalloproteinase with thrombospondin motifs [ADAMTS]) is responsible for endogenously cleaving lecticans. We therefore explored the expression pattern of two aggrecan‐degrading ADAMTS family members, ADAMTS15 and ADAMTS4, in the hippocampus and neocortex. Here, we show that both lectican‐degrading metalloproteases are present in these brain regions and that each exhibits a distinct temporal and spatial expression pattern. Adamts15 mRNA is expressed exclusively by parvalbumin‐expressing interneurons during synaptogenesis, whereas Adamts4 mRNA is exclusively generated by telencephalic oligodendrocytes during myelination. Thus, ADAMTS15 and ADAMTS4 not only exhibit unique cellular expression patterns but their developmental upregulation by these cell types coincides with critical aspects of neural development. J. Comp. Neurol. 523:629–648, 2015. © 2014 Wiley Periodicals, Inc.     

Dopamine D1 receptor expression is bipolar cell type‐specific in the mouse retina

In the retina, dopamine is a key molecule for daytime vision. Dopamine is released by retinal dopaminergic amacrine cells and transmits signaling either by conventional synaptic or by volume transmission. By means of volume transmission, dopamine modulates all layers of retinal neurons; however, it is not well understood how dopamine modulates visual signaling pathways in bipolar cells. Here we analyzed Drd1a‐tdTomato BAC transgenic mice and found that the dopamine D1 receptor (D1R) is expressed in retinal bipolar cells in a type‐dependent manner. Strong tdTomato fluorescence was detected in the inner nuclear layer and localized to type 1, 3b, and 4 OFF bipolar cells and type 5‐2, XBC, 6, and 7 ON bipolar cells. In contrast, type 2, 3a, 5‐1, 9, and rod bipolar cells did not express Drd1a‐tdTomato. Other interneurons were also found to express tdTomato including horizontal cells and a subset (25%) of AII amacrine cells. Diverse visual processing pathways, such as color or motion‐coded pathways, are thought to be initiated in retinal bipolar cells. Our results indicate that dopamine sculpts bipolar cell performance in a type‐dependent manner to facilitate daytime vision. J. Comp. Neurol. 524:2059–2079, 2016. © 2015 Wiley Periodicals, Inc.     

Spatial patterning of excitatory and inhibitory neuropil territories during spinal circuit development

To generate rhythmic motor behaviors, both single neurons and neural circuits require a balance between excitatory inputs that trigger action potentials and inhibitory inputs that promote a stable resting potential (E/I balance). Previous studies have focused on individual neurons and have shown that, over a short spatial scale, excitatory and inhibitory (E/I) synapses tend to form structured territories with inhibitory inputs enriched on cell bodies and proximal dendrites and excitatory inputs on distal dendrites. However, systems‐level E/I patterns, at spatial scales larger than single neurons, are largely uncharted. We used immunostaining for PSD‐95 and gephyrin postsynaptic scaffolding proteins as proxies for excitatory and inhibitory synapses, respectively, to quantify the numbers and map the distributions of E/I synapses in zebrafish spinal cord at both an embryonic stage and a larval stage. At the embryonic stage, we found that PSD‐95 puncta outnumber gephyrin puncta, with the number of gephyrin puncta increasing to match that of PSD‐95 puncta at the larval stage. At both stages, PSD‐95 puncta are enriched in the most lateral neuropil corresponding to distal dendrites while gephyrin puncta are enriched on neuronal somata and in the medial neuropil. Significantly, similar to synaptic puncta, neuronal processes also exhibit medial‐lateral territories at both developmental stages with enrichment of glutamatergic (excitatory) processes laterally and glycinergic (inhibitory) processes medially. This establishment of neuropil excitatory‐inhibitory structure largely precedes dendritic arborization of primary motor neurons, suggesting that the structured neuropil could provide a framework for the development of E/I balance at the cellular level. J. Comp. Neurol. 525:1649–1667, 2017. © 2016 Wiley Periodicals, Inc.     

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.