Distribution of Metabotropic Serotonin Receptors in GABAergic and Glutamatergic Neurons in the Auditory Midbrain

ABSTRACT

The neurotransmitter serotonin modulates a variety of behavioral and physiological responses in the brain. Serotonergic neurons from the dorsal raphe nuclei send a dense projection to the auditory system, including the inferior colliculus (IC), the midbrain hub of the central auditory system. In the IC, serotonin alters how neurons respond to complex sounds, and it has been implicated in the generation or perception of tinnitus. However, the distribution of serotonin receptors and the identity of neurons that express serotonin receptors in the IC remain unclear. Here, we hypothesized that IC GABAergic and glutamatergic neurons differentially express serotonin receptors. To test this hypothesis, we performed in situ hybridization in IC brain slices of male and female mice using probes for Vgat (GABAergic neuron marker) and Vglut2 (glutamatergic neuron marker), along with probes for six subtypes of metabotropic serotonin receptors: 5‐HT1_A and 5‐HT1_B (Htr1a and Htr1b, inhibitory, G_i/o G protein receptors), 5‐HT2_A, 5‐HT2_B, and 5‐HT2_C (Htr2a, Htr2b, and Htr2c excitatory, G_q11 G protein receptors), and 5‐HT7 (excitatory, G_s G protein receptors). Our data show that glutamatergic IC neurons primarily express inhibitory serotonin receptors. In contrast, a larger proportion of GABAergic neurons express excitatory serotonin receptors. Our data suggest that serotonin likely exerts an inhibitory net effect on IC neuronal circuits. These findings contribute to our understanding of how serotonin signaling influences auditory processing. The differential expression of serotonin receptors may help shape the balance of excitation and inhibition in the auditory midbrain, affecting sound processing.

The results from this study suggest that the serotonin receptors Htr1a, Htr1b, Htr2a, and Htr2c are largely expressed in the inferior colliculus (IC). Htr1a and Htr1b, which are inhibitory serotonin receptors, are more likely to be expressed by IC glutamatergic neurons. On the other hand, Htr2a and Htr2c, which are excitatory serotonin receptors, are more likely to be expressed by GABAergic neurIons.

1. Introduction

The serotonergic system influences many physiological processes, as well as pathological conditions such as anxiety, depression, obesity, autism, and Alzheimer's disease (Meltzer et al. 1998; Oishi et al. 2010; Muller et al. 2016; van Galen et al. 2021; Tahiri et al. 2024). The majority of serotonergic neurons are concentrated in the dorsal raphe nuclei (DRN) and send projections to multiple brain regions, including the central auditory system (Thompson et al. 1994). Serotonergic collaterals are found throughout the auditory pathway, and serotonergic signaling significantly impacts neuronal responses in different auditory nuclei (Hurley et al. 2002; Tang and Trussell 2015; Ko et al. 2016). In the auditory cortex, serotonin decreases the excitability of pyramidal neurons (Rao et al. 2010). In the dorsal cochlear nucleus, serotonin enhances the excitability of fusiform and vertical cells by activating 5‐HT2_A/C and 5‐HT7 receptors (Tang and Trussell 2015, 2017). Serotonin also influences the initiation of action potentials in the medial superior olive (Ko et al. 2016). Serotonergic neurons send a major projection to the inferior colliculus (IC), the midbrain hub of the central auditory system (Klepper and Herbert 1991; Hurley et al. 2002), which is comprised of GABAergic and glutamatergic neurons (Oliver et al. 1994; Ono et al. 2017). In the IC, serotonin binds to serotonin receptors, resulting in either enhancement or suppression of auditory responses (Hurley 2006; Bohorquez and Hurley 2009; Ramsey et al. 2010). Additionally, serotonin differentially modulates responses to complex sounds, including vocalizations and direction selectivity for frequency‐modulated (FM) sweeps (Hurley and Pollak 1999; Hood and Hurley 2023). A previous study showed that extracellular serotonin concentration in the IC increases rapidly after noise presentation and during social interactions (Hall et al. 2010).

Dysfunction in serotonergic signaling is involved in tinnitus perception (Jarach et al. 2022) and generation (Stark et al. 1985; Shemen 1998). Furthermore, medications that selectively inhibit the reuptake of serotonin (SSRIs), commonly used in the treatment of depressive disorders (Shemen 1998), have been shown to improve tinnitus perception (Fornaro and Martino 2010; Oishi et al. 2010). Additionally, activation of a specific subtype of serotonin receptor has been shown to improve temporal processing in the auditory cortex of a mouse line with auditory hypersensitivity (Tao et al. 2025). However, despite the importance of serotonergic modulation in the IC, the distribution of serotonin receptors and which subtypes of serotonin receptors are expressed by GABAergic and glutamatergic IC neurons remain unknown.

The modulatory impact of serotonin across the brain is driven by the selective expression of serotonin receptor subtypes in specific cell populations, which, in turn, influences the function of neural networks (Andrade 1998; Santana et al. 2004; Winterer et al. 2011). For example, serotonin has been shown to regulate the excitatory/inhibitory (EI) balance in visual cortical networks (Moreau et al. 2010; Carlos‐Lima et al. 2023). Serotonin binds to a family of receptors that include G‐protein‐coupled receptors (5‐HT1, 5‐HT2, 5‐HT4, 5‐HT5, 5‐HT6, and 5‐HT7) (Andrade 1998; Bockaert et al. 2006; Hannon and Hoyer 2008; Petelák et al. 2023) and ligand‐gated ion channels (5‐HT3) (Kawa 1994; Kaneez and White 2004). The 5‐HT1 family of receptors is linked to G_i/o; therefore, activation of these receptors decreases neuronal activity (Mannoury la Cour et al. 2001; Hannon and Hoyer 2008). The family of 5‐HT2 receptors is preferentially coupled to G_q/11, whereas 5‐HT4, 5‐HT6, and 5‐HT7 are preferentially coupled to G_s protein receptors. Activation of G_q/11 and G_s enhances neuronal activity (Santana et al. 2004; Tang and Trussell 2017). The serotonin receptors previously reported in the IC are 5‐HT1_A/B, 5‐HT2_A/B/C, 5‐HT3, and 5‐HT7 (Hurley 2006; Wang et al. 2008; Miko and Sanes 2009; Ramsey et al. 2010; Papesh and Hurley 2012). However, previous studies have used pharmacological and/or immunohistochemical approaches, which lack sufficient specificity due to the high similarity of serotonin receptors.

Given that serotonin differently modulates distinct classes of neurons in other brain regions, such as the hippocampus (Andrade and Nicoll 1987), the dorsal cochlear nucleus (Tang and Trussell 2017), and the prefrontal cortex (Villalobos et al. 2005), the effect of serotonin is likely determined by the receptors expressed by classes of IC neurons. As serotonin is a major regulator of EI balance, here, we hypothesized that GABAergic and glutamatergic IC neurons differentially express serotonin receptors. To test this hypothesis, we performed in situ hybridization targeting GABAergic and glutamatergic IC neurons and six subtypes of metabotropic serotonin receptors. Our data show that the majority of GABAergic IC neurons express excitatory serotonin receptors (5‐HT2_A, 5‐HT2_C, and 5‐HT7), whereas glutamatergic neurons primarily express the inhibitory serotonin receptors (5‐HT1_A and 5‐HT1_B). Interestingly, we find little to no expression of 5‐HT2_B in the IC. Together, our data suggest serotonin likely has an inhibitory net effect in the IC, poising serotonin as a regulator of EI balance.

2. Materials and Methods

2.1. Animals

All experiments followed the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and received approval from the University of Texas at San Antonio (approval MU132). Mice had ad libitum access to food and water and were maintained on a 12‐h day/night cycle. Both male and female C57BL/6J (RRID:IMSR_JAX:000664) mice were included in all experiments and obtained from The Jackson Laboratory (strain #000664). As C57BL/6J mice are subject to accelerated age‐related hearing loss (Noben‐Trauth et al. 2003), experiments were performed in mice aged P45–P60.

2.2. Fluorescence In Situ Hybridization Assay

In situ hybridization was performed using the RNAScope Multiplex Fluorescence V2 kit (Advanced Cell Diagnostics, catalog #320850) (Wang et al. 2012). The assay was performed as described in our previous studies (Silveira et al. 2023, 2024). To collect the brains, mice were deeply anesthetized with isoflurane. Brains were quickly dissected from 10 mice (5 males and 5 females of C57BL/6J aged P45–P60), frozen in dry ice, and maintained at −80°C until slicing. Before slicing, brains were equilibrated at −20°C, and 15 µm sections were collected using a cryostat and mounted on Superfrost Plus slides (Fisher Scientific, catalog #22037246). Slices were fixed in 10% neutral buffered formalin (Sigma‐Aldrich, catalog #HT501128) for 1 h, dehydrated in increasing concentrations of ethanol, followed by the drawing of a hydrophobic barrier around the sections. Hydrogen peroxide was used to block endogenous peroxidase for 15 min at room temperature. To identify possible colocalization of serotonin receptors (Figure 10), we used different combinations of probes for hybridization: (1) Vgat, Htr1b, and Htr2a; (2) Vgat, Htr1a, and Htr1b; (3) Vgat, Htr1a, and Htr2c; (4) Vgat, Htr2a, and Htr2c; (5) Vgat, Htr7, and Htr2b; (6) Vglut2, Htr1a, and Htr1b; (7) Vglut2, Htr2a, and Htr2c; (8) Vglut2, Htr7, and Htr2b. We began investigating the potential co‐expression of inhibitory and excitatory serotonin receptors (Assays #1 and #2). Since we found that the expression of those receptors rarely overlaps, we moved to combining probes targeting only excitatory or only inhibitory serotonin receptors in the same assay (Assays #3–#8). For the positive and negative controls, we used the probes provided by the manufacturer. The positive control probes used housekeeping genes for each channel, allowing us to verify that the mRNA quality is appropriate and that the assay pretreatment is working properly. Specifically, Polr2a was used in channel 1, Ppib in channel 2, and Ubc in channel 3. The negative controls target the bacterial DapB gene, allowing us to verify that there is no background and that the signal detected from the experimental probe is specific (Figure 1). All probes, positive controls, and negative controls were incubated for 2 h, followed by the amplification (AMP 1–3) step. Signals were developed using the appropriate horseradish peroxidase. After that, opal dyes (1:1000) were assigned for each channel: Vgat and Vglut2 expression were identified by Opal 520 (Akoya Bioscience, catalog #FP1487001KT), serotonin receptors were identified by either Opal 570 (Akoya Bioscience, catalog #FP1488001KT) or 690 (Akoya Bioscience, catalog #FP1497001KT). Following staining with DAPI, slices were coverslipped using ProLong Gold Antifade Mountant (Fisher Scientific, catalog #P36934). Images were collected using a Zeiss 710 confocal microscope within 2–3 weeks after the assay. Representative sections (including caudal, mid‐rostrocaudal, and rostral, 2–5 sections per mouse) were imaged using a 40× objective at 1–4 µm depth intervals. Caudal sections were identified by smaller IC lobes and not many commissural axons connecting the right and left IC lobes. The anatomical markers to identify medial sections were more prominent commissural axons and the presence of the periaqueductal gray (PAG) in the slice. Rostral sections were defined when the most caudal portion of the superior colliculus was present.

FIGURE 10.

Colocalization of serotonin receptors in the IC. (A–C) Distribution of Htr2a (yellow) and Htr2c (magenta) in the IC. Merge in (C). High magnification confocal images showing that Vgat⁺ neurons (D, cyan) often co‐express Htr2a (E, yellow) and Htr2c (F, magenta). Merge in (G and H). (I–K) Distribution of Htr1a (yellow) and Htr1b (magenta) in the IC. Merge in (K) high magnification confocal images showing that Vglut2⁺ neurons (L, green) often co‐express Htr1a (M, yellow) and Htr1b (N, magenta). Merge in (O and P).

FIGURE 1.

Positive and negative controls to detect mRNA quality. (A) Fluorescent in situ hybridization using probes targeted to the bacterial DapB gene was performed as a negative control. (B) The housekeeping genes Polr2a (green), Pipb (magenta), and Ubc (yellow) were used as positive controls.

2.3. In Situ Hybridization Analysis

Quantification was performed either manually using FIJI (ImageJ, National Institutes of Health (RRID:SCR_003070, Schindelin et al. 2012) or semiautomatically using QuPath 0.5.1 (RRID:SCR_018257). All Vgat⁺ and Vglut2⁺ cells were counted in every slice. For both approaches, channels of each color were quantified separately to avoid bias. For the quantification using QuPath, we used the “cell positive detection” tool. One of the settings in this tool is the threshold. The threshold value depends on the intensity of the mRNA dots in each probe/channel (Secci et al. 2023). As suggested by ACD Bio, when analyzing fluorescence RNA scope using QuPath, the threshold can be adjusted interactively until only a positive signal is detected, free of background interference. Channels with higher intensity will require a higher threshold. In contrast, channels with lower intensity will require a lower threshold. The threshold was adjusted for each slice, because receptor expression can vary within a given slice. To be considered positive, a cell needed to express a minimum of 3–4 mRNA puncta, as shown in our previous studies (Silveira et al. 2023, 2024; Sáenz de Miera et al. 2025). An example of cells considered positive and those considered negative for a given serotonin receptor is shown in Figure 2. A grid encompassing the entire IC was overlaid on each image, and 6–7 representative regions of the grid were selected to establish the optimal parameters for detecting positive cells. Next, a customized script was used to identify all positive cells in the IC. Importantly, all slices were subsequently manually reviewed to confirm accurate detection and remove any falsely identified cells. Cells mislabeled using the automated approach were corrected in the final manual analysis. The central nucleus (ICc) and the shell IC (ICx) subdivisions were differentiated using separate tissue series stained for GAD67 and GlyT2 proteins (Choy Buentello et al. 2015; Silveira et al. 2020).

FIGURE 2.

Examples of cells expressing a serotonin receptor. A minimum of 3–4 puncta was necessary to be present to consider a cell positive for a given serotonin receptor (yellow arrow). Cells with less than 3–4 puncta were considered negative for a given receptor (white arrow).

2.4. Statistics

Statistical analyses were performed using Igor Pro 9 (Wavemetrics). For the comparison of the distribution of GABAergic and glutamatergic neurons expressing a serotonin receptor across the whole IC, central nucleus of the IC, and shell IC, we used Welch's t‐test. The significance level (α) was adjusted to account for multiple comparisons using Bonferroni correction (α: 0.05/3 = 0.017). For the comparison between the distribution of Vgat⁺ or Vglut2⁺ expressing a serotonin receptor in the central nucleus versus shell IC, we used Welch's t‐test, and neurons were considered differentially distributed when p < 0.05. In all cases, n values indicate the number of brain slices used for statistical tests, with each brain slice counting as a single sample. Data are shown as means ± SD. Boxplots represent median, 25th and 75th percentiles (box), and 9th and 91st percentiles (whiskers).

3. Results

The goal of this study was to determine the subtypes of metabotropic serotonin receptors expressed by GABAergic and glutamatergic neurons in the IC. We focused on six metabotropic serotonin receptors that have been previously suggested to modify how neurons encode auditory stimuli in the IC using pharmacological approaches (Hurley 2006; Bohorquez and Hurley 2009; Papesh and Hurley 2012). For each serotonin receptor, at least three mice of either sex were used for in situ hybridization.

3.1. The 5‐HT1_A (Htr1a) Receptor Is Primarily Expressed by IC Glutamatergic Neurons

The 5‐HT1_A receptor is a G_i/o G‐protein receptor that inhibits neuronal activity by activating G protein‐gated inwardly rectifying potassium channels (GIRK) (Bockaert et al. 2006). Activation of the 5‐HT1_A receptor suppresses responses to broadband vocalizations and alters responses to tone and FM sweeps in the IC (Ramsey et al. 2010; Gentile Polese et al. 2021). However, the distribution of this receptor in the IC and which neurons express this receptor are unknown.

In the first assay, we used probes to identify Vgat (GABAergic marker) and Htr1a. For that, we used 13 brain slices from three male (P45) and one female (P59) C57BL/6 mice. Our data show that 4.6% (298 out of 6567) of Vgat⁺ neurons express Htr1a (Figure 3A–D,I, Table 1). In a separate assay, we used probes to identify Vglut2 (glutamatergic marker) and Htr1a. We found that ∼27% (20,782 out of 78,046) of IC Vglut2⁺ neurons express Htr1a (Figure 3E–H,J, Table 2). The percentage of glutamatergic neurons expressing Htr1a was higher than that of GABAergic neurons across the whole IC, as well as within the central nucleus and shell subdivisions of the IC (Figure 3K, IC: 4.6% ± 1.4% of Vgat⁺Htr1a⁺ vs. 26.6% ± 7.0% of Vglut⁺Htr1a⁺ , Welch's t‐test, α = 1 × 10⁻⁹; ICc: 3.7% ± 2.3% of Vgat⁺Htr1a⁺ vs. 26.2% ± 7.0% of Vglut2⁺Htr1a⁺ , Welch's t‐test, α = 2 × 10⁻⁸; ICx: 5.6% ± 1.6% of Vgat⁺Htr1a⁺ vs. 27.2% ± 6.1% of Vglut2⁺Htr1a⁺ , Welch's t‐test, α = 1 × 10⁻¹⁰).

FIGURE 3.

The 5‐HT1A (Htr1a) receptor is primarily expressed by IC glutamatergic neurons. Fluorescent in situ hybridization was used to determine the expression patterns of Vgat, Vglut2, and Htr1a in the C57BL/6J mice. (A) Coronal IC section showing the distribution of Vgat (cyan) and Htr1a (magenta) in the IC. (B–D) High magnification confocal images showing that Vgat⁺ neurons rarely colabel with Htr1a, as shown by orange arrows. DAPI is shown in white. (E) Coronal IC section showing the distribution of Vglut2 (green) and Htr1a (magenta) in the IC. (F–H) High magnification confocal images showing that Vglut2⁺ neurons often colabel with Htr1a, as shown by white arrows. (I) Boxplot showing the number of Vgat⁺ neurons and Vgat⁺ neurons that express the Htr1a in the IC, as well as in the central nucleus of the IC and shell IC. (J) Boxplot showing the number of Vglut2⁺ neurons and Vglut2⁺ neurons that express the Htr1a in the IC, as well as in the central nucleus of the IC and shell IC. (K) Comparison of the percentage of Vgat⁺ neurons expressing Htr1a (cyan dots) versus the percentage of Vglut2⁺ neurons expressing Htr1a (green dots) in the whole IC, central nucleus of the IC, and shell IC. α values from Welch's t‐tests are atop each plot, showing the difference between the percentage of Vgat⁺ versus Vglut2⁺ expressing Htr1a. In all cases, each dot represents a single brain slice, treated as a single sample. IC, inferior colliculus.

TABLE 1.

Nearly 4.5% of Vgat⁺ neurons express Htr1a.

Mouse	Slice	Vgat+	Vgat + and Htr1a+	% of co‐label	Vgat+ (ICc)	Vgat+ and Htr1a+ (ICc)	% of co‐label	Vgat+ (ICx)	Vgat+ and Htr1a+ (ICx)	% of co‐label
Male P45	Caudal	529	26	4.9	218	13	6.0	311	13	4.2
	Medial	554	18	3.2	257	2	0.8	297	16	5.4
	Medial	427	14	3.3	202	1	0.5	225	13	5.8
Male P45	Medial	492	17	3.5	224	5	2.2	268	12	4.5
	Caudal	509	23	4.5	212	8	3.8	297	15	5.1
	Caudal	414	13	3.1	174	7	4.0	240	6	2.5
Male P45	Rostral	539	24	4.5	251	9	3.6	288	15	5.2
	Caudal	499	16	3.2	250	6	2.4	249	10	4.0
	Rostral	464	29	6.3	242	15	6.2	222	14	6.3
Female P59	Medial	341	26	7.6	176	15	8.5	165	11	6.7
	Caudal	611	39	6.4	342	18	5.3	269	21	7.8
	Caudal	579	24	4.1	341	5	1.5	238	19	8.0
	Medial	609	29	4.8	407	14	3.4	202	15	7.4
Total		6567	298	4.6	3296	118	3.7	3271	180	5.6

Abbreviation: IC, inferior colliculus.

TABLE 2.

Nearly 27% of Vglut2⁺ neurons express Htr1a.

Mouse	Slice	Vglut2+	Vglut2 + and Htr1a+	% of co‐label	Vglut2+ (ICc)	Vglut2+ and Htr1a+ (ICc)	% of co‐label	Vglut2+ (ICx)	Vglut2+ and Htr1a+ (ICx)	% of co‐label
Male P45	Caudal	2820	717	25.4	1038	221	21.3	1782	496	27.8
	Medial	4907	1678	34.2	2020	762	37.7	2887	916	31.7
	Rostral	4689	1665	35.5	2023	794	39.2	2666	871	32.7
Male P45	Caudal	3590	1156	32.2	1931	579	30.0	1659	577	34.8
	Caudal	4090	1050	25.7	1592	425	26.7	2498	625	25.0
	Caudal	2298	479	20.8	864	164	19.0	1434	315	22.0
Male P45	Caudal	5106	1659	32.5	1947	685	35.2	3159	974	30.8
	Medial	6176	1722	27.9	3514	816	23.2	2662	906	34.0
	Rostral	6582	2042	31.0	3090	1145	37.1	3492	897	25.7
Female P59	Caudal	3625	812	22.4	1939	411	21.2	1686	401	23.8
	Medial	6669	913	13.7	3079	340	11.0	3590	573	16.0
	Medial	6748	1237	18.3	3685	564	15.3	3063	585	19.1
Female P60	Caudal	4509	1428	31.7	2851	975	34.2	1658	453	27.3
	Medial	6415	2296	35.8	3089	1012	32.8	3326	1284	38.6
	Medial	4224	725	17.2	2436	340	14.0	1788	385	21.5
	Rostral	5598	1291	23.1	3519	777	22.1	2079	514	24.7
Total		78,046	20,782	26.7	38,617	10,010	26.2	39,429	10,772	27.2

Abbreviation: IC, inferior colliculus.

When analyzing the distribution of GABAergic neurons expressing Htr1a across the central nucleus and the shell, we found that even though a small percentage of GABAergic neurons express Htr1a, Vgat⁺Htr1a⁺ neurons were more likely to be present in the shell (ICc: 3.7% ± 2.3% vs. ICx: 5.6% ± 1.6% of Vgat⁺Htr1a⁺ . Welch's t‐test, p = 0.024). On the other hand, there was no statistical difference in the percentage of glutamatergic neurons expressing Htr1a in the central IC and the shell IC (ICc: 26.2% ± 9.1% vs. ICx: 27.2% ± 6.1% of Vgat⁺Htr1a⁺ . Welch's t‐test, p = 0.727).

3.2. Many IC Glutamatergic Neurons Express the 5‐HT1_B (Htr1b) Receptor

Activation of the 5‐HT1_B receptor inhibits neuronal activity in various brain regions, including the substantia nigra and globus pallidus, where 5‐HT1_B modulates GABAergic transmission (Johnson et al. 1992; Pommer et al. 2021), as well as in the bed nucleus of the stria terminalis, in which 5‐HT1_B inhibits glutamatergic transmission (Guo and Rainnie 2010). In the IC, it has been suggested that activation of the 5‐HT1_B receptor alters the frequency tuning of neurons by modulating GABA release (Hurley et al. 2008). Our in situ hybridization data show that only 3.5% (318 out of 9000) of GABAergic neurons express the Htr1b receptor (Figure 4A–D,I, Table 3). On the other hand, ∼27% (21,244 out of 78,046) of glutamatergic neurons express the Htr1b receptor (Figure 4E–H,J, Table 4). The percentage of glutamatergic neurons expressing Htr1b was higher compared to GABAergic neurons (Figure 4K, IC: 3.5% ± 1.5% of Vgat⁺Htr1b⁺ vs. 26.8% ± 6.6% of Vglut2⁺Htr1b⁺ , Welch's t‐test, α = 1 × 10⁻¹⁰; ICc: 2.1% ± 1.2% of Vgat⁺Htr1b⁺ vs. 24.1% ± 7.7% of Vglu2t⁺Htr1b⁺ , Welch's t‐test, α = 5 × 10⁻⁹; ICx: 5.5% ± 2.7% of Vgat⁺Htr1b⁺ vs. 29.9% ± 7.0% of Vglu2t⁺Htr1b⁺ , Welch's t‐test, α = 5 × 10⁻¹¹).

FIGURE 4.

Many IC glutamatergic neurons express the 5‐HT1B (Htr1b) receptor. Fluorescent in situ hybridization was used to determine the expression patterns of Vgat, Vglut2, and Htr1b in the C57BL/6J mice. (A) Coronal IC section showing the distribution of Vgat (cyan) and Htr1b (magenta) in the IC. White arrows show the colocalization between Purkinje cells in the cerebellum and Htr1b. (B–D) High magnification confocal images showing that Vgat⁺ neurons rarely colabel with Htr1b, as shown by orange arrows. DAPI is shown in white. (E) Coronal IC section showing the distribution of Vglut2 (green) and Htr1b (magenta) in the IC. (F–H) High magnification confocal images show that Vglut2⁺ neurons often colabel with Htr1b, as shown by white arrows. (I) Boxplot showing the number of Vgat⁺ neurons and Vgat⁺ neurons that express the Htr1b in the whole IC, as well as in the central nucleus of the IC and shell IC. (J) Boxplot showing the number of Vglut2⁺ neurons and Vglut2⁺ neurons that express the Htr1b in the IC, as well as in the central nucleus of the IC and shell IC. (K) Comparison of the percentage of Vgat⁺ neurons expressing Htr1b (cyan dots) versus the percentage of Vglut2⁺ neurons expressing Htr1b (green dots) in the whole IC, central nucleus of the IC, and shell IC. α values from Welch's t‐tests are atop each plot, showing the difference between the percentage of Vgat⁺ versus Vglut2⁺ expressing Htr1b. In all cases, each dot represents a single brain slice, treated as a single sample. IC, inferior colliculus.

TABLE 3.

Nearly 3.5% of Vgat⁺ neurons express Htr1b.

Mouse	Slice	Vgat+	Vgat + and Htr1b+	% of co‐label	Vgat+ (ICc)	Vgat+ and Htr1b+ (ICc)	% of co‐label	Vgat+ (ICx)	Vgat+ and Htr1b+ (ICx)	% of co‐label
Male P45	Caudal	631	8	1.3	345	1	0.3	286	7	2.4
	Caudal	488	18	3.7	249	6	2.4	239	12	5.0
	Medial	657	23	3.5	396	7	1.8	261	16	6.1
	Rostral	606	8	1.3	302	3	1.0	304	5	1.6
Male P45	Caudal	398	14	3.5	194	6	3.1	204	8	3.9
	Caudal	441	16	3.6	192	6	3.1	249	10	4.0
	Medial	569	14	2.5	317	4	1.3	252	10	4.0
	Rostral	448	18	4.0	325	9	2.8	123	9	7.3
Male P45	Caudal	579	13	2.2	334	3	0.9	245	10	4.1
	Caudal	476	18	3.8	242	8	3.3	234	10	4.3
	Medial	632	32	5.1	302	7	2.3	330	25	7.6
	Rostral	533	9	1.7	241	0	0.0	292	9	3.1
Female P59	Caudal	611	30	4.9	342	8	2.3	269	22	8.2
	Caudal	579	24	4.1	341	4	1.2	238	20	8.4
	Medial	743	28	3.8	283	9	3.2	460	28	6.1
	Medial	609	45	7.4	407	20	4.9	202	25	12.4
Total		9000	318	3.5	4812	101	2.1	4188	226	5.5

Abbreviation: IC, inferior colliculus.

TABLE 4.

Nearly 27% of Vglut2⁺ neurons express Htr1b.

Mouse	Slice	Vglut2+	Vglut2 + and Htr1b+	% of co‐label	Vglut2+ (ICc)	Vglut2+ and Htr1b+ (ICc)	% of co‐label	Vglut2+ (ICx)	Vglut2+ and Htr1b+ (ICx)	% of co‐label
Male P45	Caudal	2820	591	21.0	1038	168	16.2	1782	429	24.1
	Medial	4907	1779	36.3	2020	667	33.0	2887	1112	38.5
	Rostral	4689	1614	34.4	2023	715	35.3	2666	903	33.9
Male P45	Caudal	3590	745	20.8	1931	295	15.3	1659	450	27.1
	Caudal	4090	767	18.8	1592	251	15.8	2498	516	20.7
	Caudal	2298	476	20.7	864	204	23.6	1434	272	19.0
Male P45	Caudal	5106	1531	30.0	1947	554	28.5	3159	977	30.9
	Medial	6176	1820	29.5	3514	918	26.1	2662	902	33.9
	Rostral	6582	2221	33.7	3090	1104	35.7	3492	1117	32.0
Female P59	Caudal	3625	1122	31.0	1939	509	26.3	1686	613	36.4
	Medial	6669	1393	20.9	3079	518	16.8	3590	875	24.4
	Medial	6748	1228	18.2	3685	483	13.1	3063	745	24.3
Female P60	Caudal	4509	1289	28.6	2851	756	26.5	1658	533	32.1
	Medial	6415	1705	26.6	3089	826	26.7	3326	879	26.4
	Medial	4224	882	20.9	2436	379	15.6	1788	503	28.1
	Rostral	5598	2081	37.2	3519	1121	31.9	2079	960	46.2
Total		78,046	21,244	26.8	38,617	9468	24.1	39,429	11,786	29.9

Abbreviation: IC, inferior colliculus.

Even though there was a small percentage of GABAergic neurons expressing Htr1b, these neurons were more likely to be present in the shell IC (2.1% ± 1.2% of Vgat⁺Htr1b⁺ neurons in the ICc vs. 5.5% ± 2.7% of Vgat⁺Htr1b⁺ neurons in the ICx. Welch's t‐test, p = 0.0001). Similarly, glutamatergic neurons expressing Htr1b were more concentrated in the shell IC (24.1% ± 7.7% of Vglut2⁺Htr1b⁺ neurons in the ICc vs. 29.9% ± 7.0% of Vglut2⁺Htr1b⁺ neurons in the ICx. Welch's t‐test, p = 0.036).

3.3. Over Half of IC GABAergic Neurons Express the 5‐HT2_A (Htr2a) Receptor

The family of 5‐HT2 receptors is preferentially coupled to G_q/11. As a result, activation of these receptors enhances neuronal activity (Bockaert et al. 2006). The 5‐HT2_A serotonin receptor has been suggested to modulate GABAergic transmission in the IC (Wang et al. 2008). However, as the IC integrates many GABAergic inputs, it is unknown whether this receptor is expressed by GABAergic neurons outside the IC and/or by local GABAergic IC neurons. We found that over half of IC Vgat⁺ neurons express the Htr2a receptor (52.2%, 5319 out of 10,124 neurons, Figure 5A–D,I, Table 5). Expression of Htr2a was rarely seen in glutamatergic IC neurons since only 2.7% of glutamatergic neurons express this receptor (Figure 5E–H,J, Table 6). The percentage of GABAergic neurons expressing Htr2a was higher than the percentage of glutamatergic neurons expressing Htr2a⁺ across the whole IC, central IC, and shell IC (Figure 5K, IC: 52.2% ± 8.5% of Vgat⁺Htr2a⁺ vs. 2.7% ± 0.8% of Vglut2⁺Htr2a⁺ , Welch's t‐test, α = 1 × 10⁻¹⁵; ICc: 61.1% ± 14.3% of Vgat⁺Htr2a⁺ vs. 3.0% ± 1.3% of Vglu2t⁺Htr2a⁺ , Welch's t‐test, α = 6 × 10⁻¹³; ICx: 41.3% ± 8.5% of Vgat⁺Htr2a⁺ vs. 2.4% ± 0.7% of Vglu2t⁺Htr2a⁺ , Welch's t‐test, α = 7 × 10⁻¹⁴).

FIGURE 5.

Over half of IC GABAergic neurons express the 5‐HT2A (Htr2a) receptor. Fluorescent in situ hybridization was used to determine the expression patterns of Vgat, Vglut2, and Htr2a in the C57BL/6J mice. (A) Coronal IC section showing the distribution of Vgat (cyan) and Htr2a (magenta) in the IC. (B–D) High magnification confocal images showing that Vgat⁺ neurons highly colabel with Htr2a, as shown by white arrows. (E) Coronal IC section showing the distribution of Vglut2 (green) and Htr2a (magenta) in the IC. (F–H) High magnification confocal images showing that Vglut2⁺ neurons rarely colabel with Htr2a, as shown by orange arrows. (I) Boxplot showing the number of Vgat⁺ neurons and Vgat⁺ neurons that express the Htr2a in the IC, as well as in the central nucleus of the IC and shell IC. (J) Boxplot showing the number of Vglut2⁺ neurons and Vglut2⁺ neurons that express the Htr2a in the IC, as well as in the central nucleus of the IC and shell IC. (K) Comparison of the percentage of Vgat⁺ neurons expressing Htr2a (cyan dots) versus the percentage of Vglut2⁺ neurons expressing Htr2a (green dots) in the whole IC, central nucleus of the IC, and shell IC. α values from Welch's t‐tests are atop each plot, showing the difference between the percentage of Vgat⁺ versus Vglut2⁺ expressing Htr2a. In all cases, each dot represents a single brain slice, treated as a single sample. IC, inferior colliculus.

TABLE 5.

Over 50% of Vgat⁺ neurons express Htr2a.

Mouse	Slice	Vgat+	Vgat + and Htr2a+	% of co‐label	Vgat+ (ICc)	Vgat+ and Htr2a+ (ICc)	% of co‐label	Vgat+ (ICx)	Vgat+ and Htr2a+ (ICx)	% of co‐label
Male P45	Caudal	631	348	55.2	345	228	66.1	286	120	42.0
	Caudal	488	233	47.7	249	138	55.4	239	95	39.7
	Medial	657	472	71.8	396	324	81.8	261	148	56.7
	Rostral	606	281	46.4	302	181	59.9	304	100	32.9
Male P45	Caudal	398	191	48.0	194	109	56.2	204	82	40.2
	Caudal	441	250	56.7	192	144	75.0	249	106	42.6
	Medial	569	260	45.7	317	191	60.3	252	69	27.4
	Rostral	448	240	53.6	325	193	59.4	123	47	38.2
Male P45	Caudal	579	280	48.4	334	199	59.6	245	81	33.1
	Caudal	476	316	66.4	242	173	71.5	234	143	61.1
	Medial	632	308	48.7	302	204	67.5	330	104	31.5
	Rostral	533	243	45.6	241	132	54.8	292	111	38.0
Female P59	Caudal	401	169	42.1	173	94	54.3	228	75	32.9
	Medial	563	373	66.3	350	273	78.0	213	100	46.9
	Rostral	557	249	44.7	343	167	48.7	214	82	38.3
Female P59	Caudal	526	321	61.0	283	207	73.1	243	114	46.9
	Caudal	498	252	50.6	240	150	62.5	258	102	39.5
	Caudal	652	348	53.4	256	158	61.7	396	190	48.0
	Caudal	469	185	39.4	126	18	14.3	343	167	48.7
Total		10,124	5319	52.2	5210	3283	61.1	4914	2036	41.3

Abbreviation: IC, inferior colliculus.

TABLE 6.

Less than 3% of Vglut2⁺ neurons express Htr2a.

Mouse	Slice	Vglut2+	Vglut2 + and Htr2a+	% of co‐label	Vglut2+ (ICc)	Vglut2+ and Htr2a+ (ICc)	% of co‐label	Vglut2+ (ICx)	Vglut2+ and Htr2a+ (ICx)	% of co‐label
Male P45	Caudal	6010	192	3.2	3384	101	3.0	2626	91	3.5
	Caudal	5900	81	1.4	3399	44	1.3	2501	37	1.5
	Medial	4690	129	2.8	2740	42	1.5	1950	87	4.5
Female P59	Caudal	3299	102	3.1	1240	53	4.3	2059	49	2.4
	Caudal	4224	138	3.3	2168	60	2.8	2056	78	3.8
	Medial	5498	221	4.0	2938	167	5.7	2560	54	2.1
Female P59	Caudal	4273	110	2.6	1767	59	3.3	2506	51	2.0
	Caudal	4767	111	2.3	1987	60	3.0	2780	51	1.8
	Medial	5674	125	2.2	2314	63	2.7	3360	62	1.8
Female P57	Caudal	3827	59	1.5	1859	40	2.2	1968	19	1.0
	Caudal	4656	94	2.0	2625	61	2.3	2031	33	1.6
	Medial	4814	192	4.0	2418	125	5.2	2396	67	2.8
Total		57,632	1554	2.7	28,839	875	3.0	28,793	679	2.4

Abbreviation: IC, inferior colliculus.

GABAergic neurons expressing Htr2a were widely distributed throughout the IC. However, there was a higher percentage of GABAergic neurons expressing the Htr2a in the central IC when compared to the shell IC (61.1% ± 14.3% of Vgat⁺Htr2a⁺ neurons in the ICc vs. 41.2% ± 8.5% of Vgat⁺Htr2a⁺ neurons in the ICx. Welch's t‐test, p = 0.00001). No difference was observed in the distribution of Vglut2⁺Htr2a⁺ neurons across IC subdivisions (ICc: 3.1% ± 1.3% vs. ICx: 2.2% ± 0.7% of Vglut2⁺Htr2a⁺ . Welch's t‐test, p = 0.066).

3.4. The 5‐HT2_B (Htr2b) Receptor Is Rarely Expressed in the IC of Young Mice

The 5‐HT2_B receptor is expressed at low levels in the brain (Bockaert et al. 2006). Expression of this receptor has been reported in the cerebellum, medial amygdala, and dorsal hypothalamus (Wainscott et al. 1993; Bockaert et al. 2006). On the other hand, this receptor is highly expressed in cardiac tissue and lungs (Launay et al. 2002; Nebigil et al. 2003; Hutcheson et al. 2011). A previous study reported that this receptor is also expressed in the IC. Interestingly, the expression was low in young mice but increased in older mice with hearing loss (Tadros et al. 2007). Here, we observed little to no expression of Htr2b in the IC of young mice (Figure 6 and Tables 7 and 8). Future studies will be necessary to determine whether these receptors are expressed by either GABAergic and/or glutamatergic neurons in older mice.

FIGURE 6.

The 5‐HT2B (Htr2b) receptor is rarely expressed in the IC of young mice. Fluorescent in situ hybridization was used to determine the expression patterns of Vgat, Vglut2, and Htr2b in the C57BL/6J mice. (A) Coronal IC section showing the distribution of Vgat (cyan) and Htr2b (magenta) in the IC. (B–D) High magnification confocal images showing that Htr2b receptors are not expressed in the IC. (E) Coronal IC section showing the distribution of Vglut2 (green) and Htr2b (magenta) in the IC. (F–H) High magnification confocal images showing the lack of expression of Htr2b in the IC, as shown by orange arrows. (I) Boxplot showing the number of Vgat⁺ neurons and Vgat⁺ neurons that express the Htr2b in the IC, as well as in the central nucleus of the IC and shell IC. (J) Boxplot showing the number of Vglut2⁺ neurons and Vglut2⁺ neurons that express the Htr2b in the IC, as well as in the central nucleus of the IC and shell IC. (K) Boxplot showing the percentage of Vgat ⁺ neurons expressing Htr2b (cyan dots) and the percentage of Vglut2⁺ neurons expressing Htr2b (green dots) in the whole IC, central nucleus of the IC, and shell IC. Given the very low number of colocalization (0.1%), statistical tests were not performed for this receptor. In all cases, each dot represents a single brain slice, treated as a single sample. IC, inferior colliculus.

TABLE 7.

Htr2b is rarely expressed by GABAergic neurons.

Mouse	Slice	Vgat+	Vgat + and Htr2b+	% of co‐label	Vgat+ (ICc)	Vgat+ and Htr2b+ (ICc)	% of co‐label	Vgat+ (ICx)	Vgat+ and Htr2b+ (ICx)	% of co‐label
Male P50	Caudal	682	0	0.0	273	0	0.0	409	0	0.0
	Medial	436	0	0.0	132	0	0.0	304	0	0.0
	Medial	792	0	0.0	369	0	0.0	423	0	0.0
	Rostral	616	0	0.0	334	0	0.0	282	0	0.0
Female 59	Caudal	549	0	0.0	239	0	0.0	310	1	0.3
	Caudal	411	1	0.2	171	1	0.6	240	0	0.0
	Caudal	255	2	0.8	75	0	0.0	180	2	1.1
Female 59	Rostral	565	0	0.0	291	0	0.0	274	0	0.0
	Rostral	577	0	0.0	214	0	0.0	363	0	0.0
	Rostral	314	0	0.0	128	0	0.0	186	0	0.0
Total		5197	3	0.1	2226	1	0.1	2971	3	0.1

Abbreviation: IC, inferior colliculus.

TABLE 8.

Htr2b is rarely expressed by glutamatergic neurons.

Mouse	Slice	Vglut2+	Vglut2 + and Htr2b+	% of co‐label	Vglut2t+ (ICc)	Vglut2+ and Htr2b+ (ICc)	% of co‐label	Vglut2+ (ICx)	Vglut2+ and Htr2b+ (ICx)	% of co‐label
Male P50	Caudal	2668	0	0.0	1059	0	0.0	1609	0	0.0
	Medial	4915	6	0.1	2407	3	0.1	2508	3	0.1
	Medial	3276	4	0.1	1834	2	0.1	1442	2	0.1
	Rostral	2571	3	0.1	1182	0	0.0	1389	3	0.2
Male P50	Medial	3987	10	0.3	2175	7	0.3	1812	3	0.2
	Medial	3851	10	0.3	1672	2	0.1	2179	8	0.4
	Rostral	5055	3	0.1	1256	1	0.1	3799	2	0.1
Female P45	Caudal	3577	0	0.0	2175	0	0.0	1812	0	0.0
	Caudal	5694	12	0.2	1256	0	0.0	3799	12	0.3
	Medial	4502	0	0.0	1672	0	0.0	2179	0	0.0
Total		40,096	48	0.1	16,688	15	0.1	22,528	33	0.1

Abbreviation: IC, inferior colliculus.

3.5. The 5‐HT2_C (Htr2c) Receptor Is Expressed by Both GABAergic and Glutamatergic IC Neurons

The 5‐HT2_C receptor is an excitatory serotonin receptor that is preferentially coupled to the G_q/11 G‐protein (Bockaert et al. 2006). These receptors are commonly located postsynaptically, whereas presynaptic expression has also been reported (Araneda and Andrade 1991; Stanford and Lacey 1996; Becamel et al. 2001). In the IC, activation of the 5‐HT2_C receptor in vivo with selective agonists enhances firing rate in response to auditory stimuli (Hurley 2006). Therefore, it has been hypothesized that the 5‐HT2_C receptor is expressed by IC glutamatergic neurons (Hurley 2006).

Here, we performed in situ hybridization on IC brain slices from three male and two female C57BL/6 mice and found that Htr2c is expressed by ∼46% (3748 out of 8093) of IC GABAergic neurons (Figure 7A–D,I, Table 9). Additionally, our data show that ∼22.5% (13,009 out of 57,632) of glutamatergic neurons express Htr2c (Figure 7E–H,J, Table 10). The percentage of Vgat⁺ neurons expressing Htr2c was higher across the whole IC as well as the central nucleus and shell when compared to the percentage of Vglut2+ neurons expressing Htr2c (Figure 7K, IC: 46.2% ± 7.4% of Vgat⁺Htr2c⁺ vs. 22.4% ± 5.3% of Vglut⁺Htr2c⁺ , Welch's t‐test, α = 3 × 10⁻¹⁰; ICc: 50.2ex% ± 11.7% of Vgat⁺Htr2c⁺ vs. 14.7% ± 5.6% of Vglut⁺Htr2c⁺ , Welch's t‐test, α = 3 × 10⁻¹⁰; ICx: 43.1% ± 6.6% of Vgat⁺Htr2c⁺ vs. 30.7% ± 9.8% of Vglut⁺Htr2a⁺ , Welch's t‐test, α = 0.001).

FIGURE 7.

The 5‐HT2C (Htr2c) receptor is expressed by both GABAergic and glutamatergic IC neurons. Fluorescent in situ hybridization was used to determine the expression patterns of Vgat, Vglut2, and Htr2c in the C57BL/6J mice. (A) Coronal IC section showing the distribution of Vgat (cyan) and Htr2c (magenta) in the IC. (B–D) High magnification confocal images showing that Vgat⁺ neurons highly colabel with Htr2c, as shown by white arrows. (E) Coronal IC section showing the distribution of Vglut2 (green) and Htr2c (magenta) in the IC. (F–H) High‐magnification confocal images showing that Vglut2⁺ neurons often do not colabel with Htr2c, as indicated by the orange arrows. However, some Vglut2⁺ neurons colabel with Htr2c, as shown by the white arrows. (I) Boxplot showing the number of Vgat⁺ neurons and Vgat⁺ neurons that express the Htr2c in the IC, as well as in the central nucleus of the IC and shell IC. (J) Boxplot showing the number of Vglut2⁺ neurons and Vglut2⁺ neurons that express the Htr2c in the IC, as well as in the central nucleus of the IC and shell IC. (K) Comparison of the percentage of Vgat⁺ neurons expressing Htr2c (cyan dots) versus the percentage of Vglut2⁺ neurons expressing Htr2c (green dots) in the whole IC, central nucleus of the IC, and shell IC. α values from Welch's t‐tests are atop each plot, showing the difference between the percentage of Vgat⁺ versus Vglut2⁺ expressing Htr2c. In all cases, each dot represents a single brain slice, treated as a single sample. IC, inferior colliculus.

TABLE 9.

Nearly half of inferior colliculus (IC) GABAergic neurons express Htr2c.

Mouse	Slice	Vgat+	Vgat + and Htr2c+	% of co‐label	Vgat+ (ICc)	Vgat+ and Htr2c+ (ICc)	% of co‐label	Vgat+ (ICx)	Vgat+ and Htr2c+ (ICx)	% of co‐label
Male P45	Caudal	529	227	42.9	218	104	47.7	311	123	39.5
	Medial	554	231	41.7	257	105	40.9	297	126	42.4
	Medial	427	158	37.0	202	68	33.7	225	90	40.0
Male P45	Caudal	414	196	47.3	174	97	55.7	240	99	41.3
	Caudal	509	245	48.1	212	117	55.2	297	128	43.1
	Medial	492	209	42.5	224	96	42.9	268	113	42.2
Male P45	Caudal	499	209	41.9	250	120	48.0	249	89	35.7
	Rostral	539	192	35.6	251	108	43.0	288	84	29.2
	Rostral	464	193	41.6	242	91	37.6	222	102	45.9
Female P59	Caudal	401	196	48.9	173	93	53.8	228	103	45.2
	Medial	563	294	52.2	350	205	58.6	213	89	41.8
	Rostral	557	207	37.2	343	106	30.9	214	101	47.2
Female P59	Caudal	526	255	48.5	283	163	57.6	243	92	37.9
	Caudal	498	294	59.0	240	173	72.1	258	121	46.9
	Caudal	652	367	56.3	256	143	55.9	396	224	56.6
	Caudal	469	275	58.6	126	87	69.0	343	188	54.8
Total		8093	3748	46.2	3801	1876	50.2	4292	1872	43.1

TABLE 10.

Glutamatergic neurons expressing Htr2c are primarily in the shell inferior colliculus (IC).

Mouse	Slice	Vglut2+	Vglut2 + and Htr2c+	% of co‐label	Vglut2+ (ICc)	Vglut2+ and Htr2c+ (ICc)	% of co‐label	Vglut2+ (ICx)	Vglut2+ and Htr2c+ (ICx)	% of co‐label
Male P45	Caudal	6010	1691	28.1	3384	623	18.4	2627	1068	40.7
	Caudal	5900	1385	23.5	3399	674	19.8	2501	711	28.4
	Medial	4690	1445	30.8	2740	554	20.2	1950	891	45.7
Female 59	Caudal	3299	663	20.1	1240	304	24.5	2059	359	17.4
	Caudal	4224	858	20.3	2168	136	6.3	2056	722	35.1
	Medial	5498	744	13.5	2938	295	10.0	2560	449	17.5
Female 59	Caudal	4273	841	19.7	1767	231	13.1	2506	610	24.3
	Caudal	4767	669	14.0	1987	137	6.9	2780	532	19.1
	Medial	5674	1229	21.7	2314	232	10.0	3360	997	29.7
Female P60	Caudal	3827	923	24.1	1859	323	17.4	1968	600	30.5
	Caudal	4656	1222	26.2	2625	392	14.9	2031	830	40.9
	Medial	4814	1339	27.8	2418	377	15.6	2396	962	40.2
Total		57,632	13,009	22.5	28,839	4278	14.8	28,794	8731	30.8

GABAergic and glutamatergic neurons expressing Htr2c were present in all IC subdivisions. However, there was a small but statistically significant percentage of GABAergic neurons expressing Htr2c in the central nucleus of the IC compared to the shell IC (ICc: 50.1% ± 11.7% of Vgat⁺Htr2c⁺ vs. ICx: 43.1% ± 6.6% of Vgat⁺Htr2c⁺ neurons, Welch's t‐test, p = 0.04). In contrast, glutamatergic neurons expressing Htr2c were more likely to be present in the shell IC (ICc: 14.7% ± 5.6% of Vglut2⁺Htr2c⁺ neurons vs. ICx: 30.7% ± 9.8% of Vglut2⁺Htr2c⁺ , Welch's t‐test, p = 0.0001).

3.6. The 5‐HT7 (Htr7) Receptor Is Primarily Expressed by IC GABAergic Neurons

The 5‐HT7 receptors are preferentially coupled with G_s protein receptors. These receptors are expressed by fusiform cells in the DCN (Tang and Trussell 2015), and a previous study briefly mentioned that Htr7 is also expressed in the IC (Heidmann et al. 1998). Here, we found that ∼30% of IC GABAergic neurons expressHtr7 (Figure 8A–D,I, Table 11). Next, we found that this receptor is also expressed by ∼12% of glutamatergic neurons (Figure 8E–H,J, Table 12). Our data show that the percentage of Vgat⁺Htr7⁺ neurons was higher across the whole IC, central nucleus, and shell IC when compared to the percentage of Vglut2 ⁺ neurons expressing Htr7 (Figure 8K, IC: 29.7% ± 8.3% of Vgat⁺Htr7⁺ vs. 11.1% ± 5.9% of Vglut⁺Htr7⁺ , Welch's t‐test, α = 7 × 10⁻⁵; ICc: 28.3% ± 11.8% of Vgat⁺Htr7⁺ vs. 9.2% ± 5.1% of Vglut⁺Htr7⁺ , Welch's t‐test, α = 0.006; ICx: 31.1% ± 10.0% of Vgat⁺7⁺ vs. 14.3% ± 7.8% of Vglut⁺Htr7⁺ , Welch's t‐test, α = 0.001).

FIGURE 8.

The 5‐HT7 (Htr7) receptor is primarily expressed by IC GABAergic neurons. Fluorescent in situ hybridization was used to determine the expression patterns of Vgat, Vglut2, and Htr7 in the C57BL/6J mice. (A) Coronal IC section showing the distribution of Vgat (cyan) and Htr7 (magenta) in the IC. (B–D) High magnification confocal images show that Vgat⁺ neurons often colabel with Htr7, as shown by white arrows. (E) Coronal IC section showing the distribution of Vglut2 (green) and Htr7 (magenta) in the IC. (F–H) High magnification confocal images showing that Vglut2⁺ neurons often do not colabel with Htr7, as shown by orange arrows. However, some Vglut2⁺ neurons colabel with Htr7, as shown by the white arrows. (I) Boxplot showing the number of Vgat⁺ neurons and Vgat⁺ neurons that express the Htr7 in the IC, as well as in the central nucleus of the IC and shell IC. (J) Boxplot showing the number of Vglut2⁺ neurons and Vglut2⁺ neurons that express the Htr7 in the IC, as well as in the central nucleus of the IC and shell IC. (K) Comparison of the percentage of Vgat⁺ neurons expressing Htr7 (cyan dots) versus the percentage of Vglut2⁺ neurons expressing Htr7 (green dots) in the whole IC, central nucleus of the IC, and shell IC. α values from Welch's t‐tests are atop each plot, showing the difference between the percentage of Vgat⁺ versus Vglut2⁺ expressing Htr7. In all cases, each dot represents a single brain slice, treated as a single sample. IC, inferior colliculus.

TABLE 11.

Nearly 30% of inferior colliculus (IC) GABAergic neurons express Htr7.

Mouse	Slice	Vgat+	Vgat + and Htr7	% of co‐label	Vgat+ (ICc)	Vgat+ and Htr7+ (ICc)	% of co‐label	Vgat+ (ICx)	Vgat+ and Htr7+ (ICx)	% of co‐label
Male P50	Caudal	682	244	35.8	273	68	24.9	409	176	43.0
	Medial	436	139	31.9	132	34	25.8	304	105	34.5
	Medial	792	245	30.9	369	82	22.2	423	163	38.5
	Rostral	616	263	42.7	334	137	41.0	282	126	44.7
Male P50	Medial	602	192	31.9	218	96	44.0	384	96	25.0
	Rostral	517	159	30.8	206	58	28.2	311	101	32.5
Female 45	Caudal	448	117	26.1	255	70	27.5	193	47	24.4
	Medial	506	61	12.1	183	11	6.0	323	50	15.5
	Rostral	493	124	25.2	138	48	34.8	355	76	21.4
Total		5092	1544	29.7	2108	604	28.3	2984	940	31.1

TABLE 12.

Nearly 12% of inferior colliculus (IC) glutamatergic neurons express Htr7.

Mouse	Slice	Vglut2+	Vglut2 + and Htr7+	% of co‐label	Vglut2t+ (ICc)	Vglut2+ and Htr7+ (ICc)	% of co‐label	Vglut2+ (ICx)	Vglut2+ and Htr7+ (ICx)	% of co‐label
Male P50	Caudal	2668	439	16.5	1059	185	17.5	1609	254	15.8
	Medial	3276	371	11.3	1834	162	8.8	1442	209	14.5
	Medial	4915	902	18.4	2407	229	9.5	2508	673	26.8
	Rostral	2571	554	21.5	1182	216	18.3	1389	338	24.3
Male P50	Medial	3851	605	15.7	1672	156	9.3	2179	449	20.6
	Medial	3987	395	9.9	2175	161	7.4	1812	234	12.9
	Rostral	5055	180	3.6	1256	22	1.8	3799	158	4.2
Female P45	Caudal	3577	348	9.7	1477	127	8.6	2100	221	10.5
	Caudal	5694	261	4.6	1798	108	6.0	3896	153	3.9
	Medial	4502	308	6.8	2436	121	5.0	2066	187	9.1
Total		40,096	4363	11.8	17,296	1487	9.2	22,800	2876	14.3

Across all IC subdivisions, both GABAergic and glutamatergic neurons expressed Htr7. The percentage of GABAergic neurons expressing Htr7 in the central nucleus of the IC was not statistically different when compared to the shell IC (ICc: 28.2% ± 11.1% of Vgat⁺Htr7⁺ vs. ICx: 31.0% ± 10.0% of Vgat⁺Htr7⁺ neurons, Welch's t‐test, p = 0.58). Glutamatergic neurons expressing Htr7 were also equally distributed across the central nucleus of the IC and shell IC (ICc: 9.2% ± 5.1% of Vglut2⁺Htr7⁺ neurons vs. ICx: 14.2% ± 7.8% of Vglut2⁺Htr7⁺ , Welch's t‐test, p = 0.10).

Although Htr7 was expressed in the IC, its expression was lower than that of the other receptors shown here. Additionally, many cells expressed only 3–4 puncta, the lowest threshold we used to consider a cell positive for a specific receptor (Figure 2). These data may suggest that although Htr7 is present in the IC, its expression level is lower than that of other serotonin receptors. To ensure that our reaction was working correctly, we included slices containing cortex, hippocampus, and hypothalamic regions in our assay in which 5‐HT7 has been well validated (Hedlund and Sutcliffe 2004; Siddiqui et al. 2004; Roberts and Hedlund 2012). As shown in Figure 9, we found that Htr7 is highly expressed in the cortex and hypothalamic regions, including the arcuate nuclei and zona incerta of the hypothalamus.

FIGURE 9.

The 5‐HT7 (Htr7) receptor is highly expressed in the cortex and hypothalamic regions. (A) Confocal image showing that the Htr7 receptor is expressed in the cortex, zona incerta of the hypothalamus, and arcuate nucleus. The white rectangle represents the area of high magnification in (B).

3.7. Colocalization of Serotonin Receptors in the IC

Co‐expression of serotonin receptors has been reported in many brain regions, including the DCN, where fusiform cells co‐express 5‐HT2_A, 5‐HT2_C, and 5‐HT7 (Tang and Trussell 2015). In addition, more complex co‐expression of serotonin receptors has been shown in pyramidal neurons in the prefrontal cortex, which co‐express 5‐HT1_A and 5‐HT2_A, suggesting an excitatory and inhibitory effect of serotonin on the same cell (Amargós‐Bosch et al. 2004).

To determine whether serotonin receptors are co‐expressed in the IC, we used different combinations of probes in our in situ hybridization assays: (1) Vgat, Htr1b, and Htr2a; (2) Vgat, Htr1a, and Htr2c; (3) Vgat, Htr1a, and Htr1b; (4) Vgat, Htr2a, and Htr2c; (5) Vglut2, Htr1a, and Htr1b; and (6) Vglut2, Htr2a, and Htr2c. We found that inhibitory and excitatory serotonin receptors are rarely co‐expressed in single IC neurons. Out of 6402 Vgat⁺ neurons, only 72 (1.2%) co‐express Htr1b and Htr2a. In agreement, less than 2% (79 out of 4427) of Vgat⁺ co‐express Htr1a and Htr2c. On the other hand, Htr2a and Htr2c were co‐expressed by ∼30% of Vgat⁺ neurons (1134 out of 3637, Figure 10A–H). Even though some glutamatergic neurons express Htr2c, the co‐expression with Htr2a was rare (739 out of 57,623, 1.3%). Additionally, the inhibitory serotonin receptors, Htr1a and Htr1b, were found to be co‐expressed by ∼12% (9542 out of 78,046) of Vglut2⁺ cells (Figure 10I–P).

3.8. Distribution Across the Rostrocaudal Axis

In the reactions, we included rostrocaudal representations of the IC. Except for the assay using probes for Vglut2, Htr2a, and Htr2c, all reactions included at least two caudal, medial, and rostral slices. Caudal sections were identified by smaller IC lobes and not many commissural axons connecting the right and left IC lobes. To identify medial sections, we used more prominent commissural axons and the presence of the PAG in the slice as anatomical markers. The superior colliculus was used as an anatomical marker to identify rostral sections. The distribution of serotonin receptors across the rostrocaudal axis is demonstrated in Tables 1, 2, 3, 4, 5, 6, 7, 8, 9 and illustrated in Figure 11.

FIGURE 11.

Distribution of GABAergic and glutamatergic neurons expressing serotonin receptors in the rostrocaudal axis. (A–F) Boxplots illustrating the distribution of GABAergic and glutamatergic neurons expressing serotonin receptors in the rostrocaudal axis. Green dots represent glutamatergic neurons identified by Vglut2, and cyan dots indicate GABAergic neurons identified by Vgat. (A) Colocalization with Htr1a. (B) Colocalization with Htr1b. (C) Colocalization with Htr2a. (D) Colocalization with Htr2b. (E) Colocalization with Htr2c. (F) Colocalization with Htr7. In all cases, each dot represents a single brain slice, treated as a single sample.

3.9. Summary of Results

Figure 12 summarizes the results of our in situ hybridization studies. Together, our experiments demonstrate that four subtypes of metabotropic serotonin receptors are highly expressed in the IC: Htr1a, Htr1b, Htr2a, and Htr2c. Those receptors were differentially expressed among GABAergic and glutamatergic neurons. GABAergic neurons primarily express the excitatory serotonin receptors Htr2a and Htr2c, but glutamatergic neurons primarily express the inhibitory serotonin receptors Htr1a and Htr1b. These data may suggest that serotonin receptor activation in the IC is likely to have an inhibitory net effect.

FIGURE 12.

Summary of results. Together, our results suggest that GABAergic neurons (Vgat⁺ ) primarily express excitatory serotonin receptors, whereas glutamatergic neurons (Vglut2⁺ ) primarily express inhibitory serotonin receptors.

4. Discussion

Here, we showed that the metabotropic serotonin receptors, Htr1a, Htr1b, Htr2a, and Htr2c, are widely distributed throughout the IC. Interestingly, we see less expression of Htr7 and nearly no expression of Htr2b. We next found that Htr1a and Htr1b, which are inhibitory serotonin receptors, largely co‐label with glutamatergic IC neurons. However, the expression of Htr1a and Htr1b was rarely observed in GABAergic neurons, identified by the expression of Vgat. On the other hand, the excitatory serotonin receptor Htr2a was highly expressed by GABAergic neurons, but very few glutamatergic neurons were found to express this receptor. The Htr2c, which is also an excitatory serotonin receptor, was primarily expressed by GABAergic neurons but also expressed by many glutamatergic neurons. Finally, Htr7 was also expressed by GABAergic and glutamatergic neurons.Together, our data provide evidence regarding the distribution of six subtypes of serotonin receptors across GABAergic and glutamatergic neurons in the IC. As serotonergic neurons are concentrated in the DRN, the net effect of serotonin on IC sound processing will be defined by the interaction of different receptors expressed by neuronal classes. Given the differential expression of those receptors by GABAergic and glutamatergic neurons in the IC, serotonin is likely to have an inhibitory net effect.

4.1. IC Glutamatergic Neurons Primarily Express Htr1a and Htr1b

5‐HT1_A receptors play a major role as autoreceptors in the DRN, regulating the firing of serotonergic neurons (Riad et al. 2000; Andrade et al. 2015). Outside the DRN, 5‐HT1_A receptors are primarily postsynaptic and, when activated by serotonin, inhibit neuronal activity by activating GIRK channels and/or inhibiting calcium channels (Andrade et al. 1986; Okuhara and Beck 1994). Autoradiographic studies suggested that the IC contains the highest number of 5‐HT1_A binding sites among the central auditory pathway (Thompson et al. 1994). Previous studies using in vivo electrophysiology have shown that the application of the 5‐HT1_A agonist decreases evoked spike rates and narrows the frequency tuning of IC neurons (Hurley 2006; Ramsey et al. 2010). These data are consistent with our findings that IC glutamatergic neurons primarily express Htr1a, and activation of 5‐HT1_A receptors would likely hyperpolarize these glutamatergic neurons, thereby decreasing excitability in the IC. However, another study used antibodies to identify 5‐HT1_A and found that ∼50% of IC GABAergic neurons co‐label with 5‐HT1_A (Peruzzi and Dut 2004). This discrepancy in results is likely due to differences in the techniques used, since antibodies can be less specific because of the high similarity among serotonin receptors.

In contrast to 5‐HT1_A receptors, 5‐HT1_B receptors are more likely to be expressed in neuron terminals, and activation of the 5‐HT1_B receptors inhibits neurotransmitter release. The 5‐HT1_B receptor has been shown to inhibit the release of dopamine, GABA, and glutamate in many brain regions, including the striatum and hippocampus (Pommer et al. 2021; Burke and Alvarez 2022; Najm Al‐Halboosi et al. 2023). In the IC, it has been suggested that GABAergic neurons express 5‐HT1_B. This hypothesis is supported by immunohistochemistry experiments as well as in vivo electrophysiology using the 5‐HT1_B agonist and/or antagonist (Peruzzi and Dut 2004; Hurley et al. 2008). Application of the 5‐HT1_B agonist reduces the number of spikes in a frequency‐specific manner, and this effect is diminished in the presence of the GABA_A receptor antagonist (Hurley et al. 2008). This contradicts the data from the current study since we observed minimal expression of Htr1b in Vgat⁺ neurons (Figure 4). A possible explanation for this discrepancy may be that glutamatergic neurons are largely interconnected with other IC neurons (Sturm et al. 2014, 2017; Ito et al. 2016; Oberle et al. 2023; Silveira et al. 2023). Thereby, serotonin modulation of glutamate transmission could indirectly affect the membrane excitability of IC GABAergic neurons, bringing these neurons under threshold, reducing the release of GABA. In agreement with the data presented here, whole‐brain gene expression analysis has found that indeed, few IC GABAergic neurons express Htr1b (Yao et al. 2023; Lein et al. 2007). However, those are highly expressed by GABAergic Purkinje cells in the cerebellum (Yao et al. 2023). As shown in Figure 4A, our in situ hybridization can detect high expression of Htr1b in GABAergic Purkinje cells, thereby supporting the low colocalization of Htr1b with GABAergic cells in the IC.

4.2. Over Half of IC GABAergic Neurons Express Htr2a

The 5‐HT2_A receptors have received much attention due to their involvement in hallucinations and schizophrenia (Michaiel et al. 2019; Nakao et al. 2022). In the central auditory system, 5‐HT2_A receptors have been shown to modulate the excitability of fusiform cells in the dorsal cochlear nucleus (Tang and Trussell 2015, 2017). A previous study also demonstrated that the application of the 5‐HT2_A agonist increases the frequency of inhibitory postsynaptic currents (IPSCs) in the IC (Wang et al. 2008). This finding aligns with our data, which shows that over half of IC GABAergic cells co‐express Htr2a. Expression of Htr2a was rarely observed in glutamatergic neurons. When present, it had a lower intensity and fewer puncta than in GABAergic neurons (data not shown). Although more functional data are necessary, this suggests a significant role of 5‐HT2_A in regulating local inhibition in the IC and raises interesting hypotheses, such as the involvement of this receptor in auditory hallucinations.

4.3. Both GABAergic and Glutamatergic Neurons Express Htr2c

The 5‐HT2_C receptor is an excitatory serotonin receptor, and it is mainly expressed postsynaptically but has also been shown to be present presynaptically (Araneda and Andrade 1991; Stanford and Lacey 1996). For example, in the striatum, activation of the 5‐HT2_C receptor by serotonin produces an inhibitory effect since this receptor is expressed by fast‐spiking GABAergic interneurons (Blomeley and Bracci 2009). In the IC, Htr2c is upregulated after noise exposure (Holt et al. 2005). In addition, in vivo application of the 5‐HT2_C agonist using juxtacellular recordings increases firing rate. However, those recordings were not selectively targeted to either GABAergic or glutamatergic neurons (Hurley 2006). In our data, we found that ∼20% of IC glutamatergic neurons express Htr2c. However, this receptor is also expressed by ∼50% of GABAergic neurons. Therefore, the activation of this receptor by serotonin is likely to have a more complex net effect, as it will affect both glutamatergic and GABAergic neurons. A possible explanation for the discrepancy between our data and previous studies is that the majority of neurons in the IC are glutamatergic. Therefore, in vivo recordings are more likely to target IC glutamatergic neurons.

4.4. 5‐HT7 Receptors Are Primarily Expressed by GABAergic Neurons

The 5‐HT7 is an excitatory metabotropic serotonin receptor that is preferentially coupled with G_s protein receptors. Activation of these receptors leads to an enhancement in neuronal excitability. A physiological role for the 5‐HT7 receptor has been shown in circadian rhythm regulation (Shelton et al. 2015), thermoregulation (Voronova 2021), and learning and memory (Roberts and Hedlund 2012). In the central auditory pathway, activation of 5‐HT7 receptors enhances the excitability of fusiform cells in the DCN. In addition, a previous study briefly mentioned that Htr7 is expressed in the IC (Heidmann et al. 1998). Here, we found that ∼30% of GABAergic neurons express the Htr7, whereas ∼12% of glutamatergic neurons express this receptor. Although Htr7 expression was detected in the IC, it showed lower qualitative expression than the other receptors reported here. In many cases, a positive cell had only a minimal number of puncta (3–4, Figure 2) to be considered positive. However, quantitative puncta analysis will be necessary, using samples that include other serotonin receptors in the same assay, to confirm this hypothesis.

4.5. Distribution of Serotonin Receptors Across the Central Nucleus of the IC and the Shell of the IC

The IC is subdivided into three main subdivisions: the central nucleus (ICc), dorsal cortex (ICd), and lateral cortex (ICl). In this study, we analyzed the distribution of serotonin receptors across the central and shell IC (ICd + ICl). We found that all serotonin receptors are present throughout the IC, suggesting that serotonergic modulation acts on lemniscal and non‐lemniscal circuits (Figures 3–8A, quantification on Figures 3–8J,K). Some receptors, such as Htr1b, showed stronger expression in the shell IC compared to the central IC. Interestingly, Htr2c was preferentially expressed by GABAergic neurons in the central IC, but when present in glutamatergic neurons, it was more often localized to the shell IC. These data suggest that Htr2c may play an important role in integrating multisensory inputs by modulating glutamatergic neurons in the shell IC but likely influences sound processing in the central IC through the modulation of GABAergic neurons. A limitation of these findings is that different approaches to identifying the IC subdivisions can yield different limits between the central and shell regions, which could affect the interpretation of the data. Here, we used the GAD67 and Glyt2 immunohistochemistry staining to identify the subdivisions. Different groups have validated this method (Choy Buentello et al. 2015; Beebe et al. 2020; Silveira et al. 2020). However, other approaches to identifying the IC subdivisions have also been previously reported and could influence how to define the limits of the IC borders (Keesom et al. 2018; Olthof et al. 2019).

4.6. Functional Implications: Auditory Serotonin and Social Behaviors

Hearing loss can result in social isolation and loneliness. Social isolation can result in other disorders such as depression and cognitive decline (Goman and Lin 2016; Vas et al. 2017; Varnet et al. 2021). Given the extensive innervation of the auditory system by serotonergic fibers, serotonin has been hypothesized as a neuromodulator linking hearing loss and social isolation (Hall et al. 2010; Keesom and Hurley 2020). For example, social isolation decreases the density of serotonergic fibers in the IC in a sex‐dependent manner, affecting females but not males (Keesom and Hurley 2020). However, it is unknown whether expression of serotonin receptors also changes after social isolation and whether the decrease in serotonin innervation is global or targeted to specific classes of IC neurons. The results of our study will likely raise new hypotheses about which neuronal circuits are influenced by changes in serotonergic function in the IC during social isolation and which subtypes of serotonin receptors are affected by social isolation.

Serotonin in the IC has been suggested to be involved in other social behaviors, such as aggression. A previous study showed that a molecularly identifiable class of serotonergic neurons in the DRN strongly projects to the IC (Niederkofler et al. 2016). Further silencing of these neurons enhances aggressive behavior, suggesting an association with impaired sensory processing. Additional studies would be necessary to determine whether projections from this class of neurons target GABAergic and/or glutamatergic neurons in the IC and which receptor subtypes are activated during aggressive behavior.

4.7. Functional Implications: A Possible Role for Serotonin in Regulating E/I Balance

The serotonergic system has been shown to regulate EI balance in other brain regions (Moreau et al. 2010; Carlos‐Lima et al. 2023). Here, we show that in the IC, metabotropic serotonin receptors are differentially distributed across GABAergic and glutamatergic neurons. Htr1a and Htr1b (inhibitory serotonin receptors) were primarily expressed by glutamatergic neurons, and Htr2a (excitatory serotonin receptor) was almost exclusively expressed by GABAergic neurons. Htr2c was expressed by both GABAergic and glutamatergic neurons, whereas glutamatergic neurons expressing Htr2c were primarily in the shell IC. Together, these data suggest that serotonin modulation may induce an inhibitory net effect in the IC by differentially affecting glutamatergic and GABAergic neurons. This proposed mechanism may help to understand how some auditory disorders lead to hyperexcitability in the IC. For example, a recent study showed that activation of the 5‐HT1_A receptor in the IC prevents audiogenic seizures in the Fmr1 knockout mice, a mouse line used to study fragile X syndrome (Saraf et al. 2024). On the basis of our findings, this effect would likely be achieved by hyperpolarization of IC glutamatergic neurons, which are necessary for the initiation of audiogenic seizures (Gonzalez et al. 2019). In addition, dysfunction in serotonergic modulation is involved in the perception and generation of tinnitus (Salvinelli et al. 2003; Caperton and Thompson 2010). Furthermore, medications that SSRIs, commonly used in the treatment of depression (Stark et al. 1985), have been shown to improve tinnitus perception (Shemen 1998). However, SSRIs affect all serotonergic transmission and can cause multiple side effects (Vaswani et al. 2003). Therefore, the ability to identify which receptor subtypes are expressed by inhibitory and excitatory neurons may lead to the development of more targeted therapeutics. For example, it was recently shown that modulation of the 5‐HT1_A receptor in the auditory cortex improves temporal processing in a mouse model of autism (Tao et al. 2025).

Although we did not evaluate all subtypes of serotonin receptors, we included those previously suggested to be expressed in the IC by in vivo studies, except the ionotropic receptor 5‐HT3 (Hurley 2006, 2007; Hurley et al. 2008; Bohorquez and Hurley 2009; Hall et al. 2010). However, recent studies using either mouse lines (Koyama et al. 2017) or MERFISH (Yao et al. 2023) suggest that this receptor is not expressed in the IC. The Allen Brain Institute included 10 subtypes of serotonin receptors in their MERFISH experiments of the whole brain (Yao et al. 2023): Htr1a, Htr1b, Htr1d, Htr1f, Htr2a, Htr2c, Htr3a, Htr3b, Htr4, and Htr5b. From those receptors, the only one not tested here and suggested to be expressed in the IC is the Htr1f. Interestingly, their data show that this receptor is expressed primarily by IC glutamatergic neurons. As this is a G_i/o G‐protein receptor, it supports our conclusion that serotonin has an inhibitory net effect in the IC. Many central auditory disorders are characterized by changes in excitability that favor excitation, such as tinnitus, age‐related hearing loss, and audiogenic seizure (Auerbach et al. 2014; Xiong et al. 2017; Gonzalez et al. 2019). As our data show that serotonin favors inhibition in the IC by differentially regulating GABAergic and glutamatergic neurons, dysfunction in serotonergic modulation could potentially underlie enhanced excitability in central auditory disorders.

5. Conclusions

We demonstrate that five subtypes of metabotropic serotonin receptors are highly expressed in the IC: Htr1a, Htr1b, Htr2a, Htr2c and Htr7. GABAergic neurons were more likely to express the excitatory serotonin receptors Htr2a and Htr2c, but glutamatergic neurons primarily express the excitatory serotonin receptors Htr1a and Htr1b, whereas the Htr2c and Htr7 receptors were also present in glutamatergic neurons. These data suggest that by differentially regulating GABAergic and glutamatergic neurons, serotonin activation of its receptors may lead to an inhibitory net effect.

Author Contributions

Marina A. Silveira: conceptualization, investigation, writing – original draft, writing – review and editing, visualization, supervision, funding acquisition. Karen L. Galindo: investigation, writing – review and editing. Zoya A. Nazir: investigation, writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Galindo, K. L. , Nazir Z. A., and Silveira M. A.. 2026. “Distribution of Metabotropic Serotonin Receptors in GABAergic and Glutamatergic Neurons in the Auditory Midbrain.” Journal of Comparative Neurology 534, no. 2: e70139. 10.1002/cne.70139

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.