Is there a role for GABA in peripheral taste processing

Abstract

In the peripheral neurons and circuits for hearing, balance, touch and pain, GABA plays diverse and important roles. In some cases, GABA is an essential player in the maintenance of sensory receptors and afferent neurons. In other instances, GABA modulates the sensory signal before it reaches CNS neurons. And in yet other instances, tonic GABA-mediated signals set the resting tone and excitability of afferent neurons. GABA_A receptors are present on gustatory afferent neurons that carry taste signals from taste buds to central circuits in the brainstem. Yet, the functional significance of these receptors is unexplored. Here, I outline some of the roles of GABA in other peripheral sensory systems. I then consider whether similar functions may be ascribed to GABA signaling in the taste periphery.

Introduction

Gustatory afferent neurons of the geniculate ganglion innervate taste buds of the anterior tongue and palate. These neurons and their axons connect the peripheral detectors, taste buds, to the central circuits that decode and process neural signals elicited by taste stimuli. Gustatory afferent neurons are pseudounipolar, i.e., a single fiber emerges from each neuronal soma, and after a short distance, bifurcates into a peripheral process that innervates taste buds and a central process that enters the brainstem to synapse onto neurons in the rostral Nucleus of the Solitary Tract (NST).

GABA acts on two main classes of GABA receptors throughout the nervous system. GABA_A receptors constitute a large and heterogeneous class of ionotropic receptors. These are chloride-permeable ion channels that assemble most frequently as pentamers containing two α, two β and one δ or γ subunit. The full constellation comprises 19–20 known subunits [1]. Depending on intracellular [Cl^–] in a given cell, activation of GABA_A allows Cl^– flux either into or out of the cell, thereby either hyperpolarizing or depolarizing the cell, respectively. In adult mammalian neurons, GABA is usually inhibitory. GABA_B receptors are metabotropic and dimeric (with one each, B1 and B2 subunits). These also function primarily as inhibitory modulators by opening K⁺ channels or by other means.

GABA Receptors on Gustatory Afferent Neurons

RNAseq analysis revealed that gustatory afferent neurons of the geniculate ganglion express mRNAs for GABA_A receptors [2]. Based on expressed subunits, these GABA_A receptors likely exhibit α1/β2/γ2 composition [2, 3]. We detected both mRNA and protein for GABA_Aα1, the most abundant of the subunits, in nearly every gustatory neuron of the geniculate ganglion [4]. Curiously, mRNA levels for this subunit are similar to or higher than those for P2X2 and P2X3 subunits, which constitute the purinergic receptors for ATP, the main afferent transmitter from taste buds to afferent fibers [5]. Yet, we know little of what the function(s) of GABA_A receptors in gustatory afferents might be. Do they mediate GABergic influence on the maintenance of taste buds, the maintenance of afferent nerves and synapses, or on synaptic function, nerve excitability?

It is important to recognize that GABA_A receptors in gustatory neurons could reside at one or more of three distinct sites (Figure 1) and would play significantly different roles depending on their location. We consider each of these in turn below, with examples from other sensory systems, and suggested significance for taste.

Figure 1.

GABA (diffuse pink) potentially influence the function of gustatory afferent neurons (black lines) at one or more sites, by analogy with other sensory afferents. Possible sites for GABA_A receptors (magenta symbols) on afferent somata and fibers are indicated. A. GABA-secreting neurons or terminals in the brainstem could regulate gustatory afferents at their presynaptic terminals in the NST. B. GABA could be secreted within gustatory ganglia to affect the somata of gustatory neurons, and subsequently, influence the efficiency of conduction to the brainstem. C. Within the taste bud, GABA could affect the function of taste bud cells and/or the peripheral terminals of afferent neurons. Evidence for and against each of these potential sites of GABA action is discussed in the text. Currently, only C. is strongly supported.

Presynaptic Terminals of Sensory Afferents

Gustatory afferent neurons project into the brainstem, and synapse onto projection neurons resident in the NST (Figure 1A). If GABA_A receptors are located on the presynaptic terminals of the peripheral gustatory afferents, there could be parallels with GABergic presynaptic modulation (termed “primary afferent depolarization”) that occurs in the dorsal horn of the spinal cord.

For instance, primary nociceptive afferents of the dorsal root and trigeminal ganglia project into the spinal cord where they receive GABergic input from local interneurons [6, 7]. Normally, activation of afferent presynaptic GABA_A receptors produces presynaptic inhibition, decreasing the nociceptive input to higher centers, damping pain signals in the absence of strongly noxious stimuli [8, reviewed, 9]. Similar GABergic inputs also occur on mechano-sensitive Aβ fiber terminals. Loss of this presynaptic inhibition is thought to underlie mechanical allodynia through cross-talk between Aβ and C fiber circuits [reviewed, 9]. GABergic modulation of pain signals through presynaptic GABA_A receptors is the basis of certain treatments for chronic pain, migraine and peripheral neuropathies. These treatments enhance GABA_A signaling in the dorsal horn (or trigeminal-cervical complex) by pharmaceutical means or by electrical stimulation of afferent tracts [9, 10].

Are there GABergic inputs on the presynaptic terminals of gustatory afferents in the brainstem? Many interneurons in the NST synthesize and release GABA [11]. Extracellular recordings in anesthesized hamsters showed that spontaneous and taste-evoked activity in many NST neurons was inhibited by locally applied GABA, and enhanced by a GABA_A antagonist [12]. GABergic neurons in the NST are activated by stimulation of the gustatory afferent tract, to promote feedforward inhibition [13, 14]. An important recent development is the demonstration that GABA activation at this site enhances discrimination between taste stimuli [15], not unlike the GABergic presynaptic inhibition that enhances the separation of nociceptive and mechanosensory “labeled lines” described above [9]. In sum, current evidence suggests that GABergic tone at the brainstem relay for gustatory signals may sharpen breadth of tuning and discrimination ability of the sensory system. While GABA clearly exerts a tonic inhibitory influence on taste-responsive NST neurons, it remains to be determined whether this is produced via presynaptic inhibition of afferent terminals, or postsynaptic inhibition of NTS neurons.

We carried out immunofluorescence for GABA_Aα1, the most abundant of the receptor subunits in the geniculate ganglion. The large majority of gustatory neurons in the ganglion expressed GABA_A α1 quite strongly, consistent with mRNA levels [2, 4]. Surprisingly however, GABAA α1 immunoreactivity was completely lacking in the central projections and terminals in the NST, even though the peripheral projections and fibers entering taste buds were strongly immunoreactive [5]. Selective trafficking of proteins to axons vs. dendrites is commonplace among CNS neurons and underlies the distinct functional features of the two types of processes. However, there are currently few examples of selective sorting of channels and other functional proteins between the peripheral and central processes of any pseudounipolar sensory neurons. The cell biological mechanisms that give rise to the unique distribution of GABA_Aα1 are currently unexplored.

While an α subunit is considered essential for most GABA_A receptors, is an alternative α (not α1) transported centrally? RNAseq and RT-qPCR indicate that the α2 subunit also is expressed in many gustatory neurons, but at much lower levels than α1 [2, 4 and unpublished]. Indeed, GABA_Aα2 is associated with presynaptic modulation of primary afferent nociceptors [8]. Thus, it is possible that GABA_A receptors with either α2 or α1 could underlie structurally and functionally distinct GABA-activated receptors at the central and peripheral synapses of gustatory afferents.

Somata of Afferent Neurons

Immunofluorescence data showed that the GABAAα1 receptor subunit is robustly expressed in the cytoplasm and on the plasma membrane of all gustatory afferent somata in the geniculate ganglion (Figure 1B) [4]. While functional GABA_A receptors are usually located at or near synapses, recent findings on GABergic signals within dorsal root ganglia (DRG), where no chemical synapses are known to exist, may offer interesting parallels to consider.

Neuronal somata in dorsal root and trigeminal ganglia express GABA_A receptors on their plasma membrane, including a full complement of subunits detected by immunohistochemistry. Further, GABA elicits robust Cl^– currents, particularly in nociceptors [16–18]. Yet, the source of GABA mediating these effects has been controversial. The transmitter is detected by HPLC in the perfusate of ganglia and by “sniffer” cells in culture [16, 18]. Neuronal somata within the dorsal root and trigeminal neurons are reported to express GABA-synthesizing enzymes, Gad65 and/or Gad67 and accumulate GABA [16, 17]. Other studies have suggested that GABA originates from the satellite cells that surround ganglion neurons, and that these glia employ the putrescine pathway for GABA synthesis rather than the more common Gad enzymes [18]. There is evidence of GABA release within the ganglion through both conventional vesicular mechanisms and more diffusely via reversing transporters or anion channels such as Bestrophin 1 [16, 18]. The trigger for GABA release within the ganglion in vivo remains to be ascertained, but KCl-induced depolarization caused GABA-release in cultures and ganglion explants [16, 18], suggesting that sensory stimulus-evoked activity itself may be sufficient.

Curiously, GABA is excitatory for nociceptor neurons in the DRG.GABA-mediated depolarization of these neuronal somata paradoxically inhibits transmission of nociceptive signals to the CNS [16]. This latter effect stems from a filtering effect at the pseudounipolar T-junction where the peripheral and central axonal branches of sensory neurons diverge [19, 20]. Thus, although GABA depolarizes nociceptor somata, the net effect of GABA stimulation within the ganglion is to decrease nociceptor signals reaching the CNS [16]. In effect, GABA regulates how efficiently sensory information reaches the CNS. Indeed, focal infusion of GABA or GABA reuptake inhibitors into dorsal root ganglia in vivo dramatically reduced acute and chronic pain in a rodent model, while GABA_A antagonists, similarly applied, produced allodynia [16]. These observations strongly suggest that GABA participates in an endogenous system within the ganglion, capable of selectively modulating particular sensory neurons.

As mentioned above, we detected GABA_Aα1 immunohistochemically in gustatory neurons of the geniculate ganglion. However, it was not apparent that the protein was localized on the cell surface [4]. Patch-clamp and Ca²⁺ imaging suggest that GABA depolarizes some geniculate ganglion neurons, and hyperpolarizes others [2, 4, 21]. It remains unclear whether GABA_A receptors are truly functional in the intact ganglia or whether their presence on acutely isolated neurons reflects adventitious insertion into the plasma membrane, caused by axotomy and enzymatic dissociation procedures. To the author’s knowledge, GABA-mediated modulation at the level of gustatory ganglia has not been examined in vivo.

Peripheral Terminals

In other sensory systems, GABA plays a host of roles in the periphery, including regulating excitability, as a neuromodulator, as a trophic factor during development, and GABA_A receptors may regulate cellular trafficking. I briefly describe these functions and then discuss how some of them may be relevant in the taste periphery.

Acute and Tonic effects of GABA:

Cochlear Inner Hair Cells transduce sound and transmit the signal across the peripheral synapse to spiral ganglion neurons. In addition, efferent GABergic synapses are located near the afferent synapse on Inner Hair Cells. When GABA_A receptors at spiral ganglion afferent synapses are activated, afferent spiking activity is reduced, and this protects adult mice from noise-induced hearing loss [reviewed, 22]. That is, the inhibitory action of GABA_A on adult spiral ganglion neurons may be neuroprotective in an environment of overstimulation. In the vestibular system as well, GABA is secreted at the sensory epithelium and may serve as a modulator of the glutamatergic afferent synapse between hair cells and vestibular ganglion sensory neurons [reviewed, 23].

In contrast to auditory and vestibular afferents, GABA appears to depolarize peripheral nociceptor terminals and thus enhances their excitability, especially during inflammation [24].

Developmental and Maintenance roles:

GABA, acting on GABA_A receptors, has long been recognized as promoting the early development and maturation of neurons in the embryonic brain [reviewed, 25]. For this trophic role, GABA is released extra-synaptically, not from vesicles, but from cytosol through transporters or anion channels [26, 27]. For example, spiral ganglion neurons in mice that genetically lack select GABA_A subunits have a reduced density of afferent and efferent terminals and other phenotypes that suggest that GABA plays a role in neuronal maintenance [28].

Does GABA play roles(s) similar to the above when gustatory afferent neurons innervate taste buds (Figure 1C)? In taste buds, ATP serves as the principal afferent transmitter with P2X2/X3 receptors as the corresponding receptors on gustatory afferent terminals within the bud [5, 29]. Stimulus-evoked ATP release employs an unusual mechanism. The transmitter is not packaged in vesicles. Instead, ATP is produced locally by large “atypical” mitochondria located just under the plasma membrane near taste cell-nerve appositions [30]. ATP release is mediated through CALHM1/3 channels [31, 32] that are localized on the plasma membrane between the atypical mitochondria and the postsynaptic target, varicosity-like expansions of the afferent nerves [30, 33]. Several transmitters, including ATP, 5HT and GABA, are known to participate in paracrine interactions within the taste bud [29, 34, 35]. Here, we consider the evidence that GABA, in addition to ATP, may serve for interactions between taste bud cells and afferent nerve fibers.

GABA is synthesized and accumulates in taste bud cell types I (glial-like) and III (sour-sensing) and acts as a paracrine inhibitory signal between taste cells [35, 36]. Cellular biosensor assays demonstrated that depolarizing type III cells results in GABA release [36], and further, that GABA acts on type II cells to decrease tastant-evoked ATP release [35]. Consistent with this paracrine function, GABA-evoked hyperpolarizing currents were reported for an undefined subset of taste bud cells [37]. Optogenetic activation of type III cells (potentially evoking GABA release) during taste stimulation decreases afferent nerve responses [38]. Type I cells also synthesize, accumulate, and release GABA [4, 35]. One effective stimulus for GABA release from type I cells is Substance P [39], (presumably from nearby trigeminal fibers). Other triggers within the taste bud are likely (our unpublished data).

Apart from an established role in intra-gemmal paracrine signaling, I postulate that GABA, secreted by taste bud cells, may also target the gustatory afferent neurons that innervate taste buds. Below, I consider evidence for this.

Gustatory ganglion neurons express P2X2/X3 receptors, which depolarize afferent fibers when activated by ATP [5]. In addition to P2X purinoceptors, we found that peripheral terminals of gustatory afferents are immunoreactive for GABA_Aα1, one of the principal subunits of functional GABA_A receptors [4]. Curiously, the strongest immunoreactivity is seen in afferent nerve fibers as they approach the taste bud, branch, and spread across its base (Figure 2A). The vertical branches of afferents that penetrate into the taste bud and interleaf between receptor cells typically have modest levels of the receptor. This contrasts with the relatively even distribution of P2X receptors in the same branches (Figure 2B). What might be the significance of GABA_A receptors concentrated in nerves at the base of the taste bud? Each of the roles described above for GABA is plausible, but as yet, unexplored.

Figure 2.

Location and postulated roles of GABA_A receptors on gustatory afferents within taste buds. A. GABA_Aα1 immunoreactivity (magenta) is prominent in afferent fibers as they approach taste buds and arborize at the base of the taste bud. As vertical branches of these fibers traverse upwards within the bud, GABA_Aα1 immunoreactivity substantially decreases. Only type II taste bud cells (green) are visualized for clarity. B. In another taste bud, P2X2 immunoreactivity (red) displays a different pattern than GABA_Aα: P2X2 is present throughout the full extent of the afferent arbor within the taste bud. Taste buds from palate and fungiform and circumvallate papillae all show this same pattern: with GABA_Aα1 concentrated near the base and P2X2/P2X3 distributed along the length of the afferents. C.GABA serves as a paracrine signaling molecule within the taste bud, and may also signal to the nerve via GABA_A receptors (see text). D. A postulated model wherein GABA_A receptors (magenta) stabilize P2X (black) receptors, helping to traffick them to the surface of nerve fibers. GABA_A receptors would be released from the interaction and degraded in the distal branches once P2X are activated by ATP released from taste bud cells (see text). These two postulated roles for GABA_A receptors in the taste bud are not mutually exclusive.

First, because GABA accumulates in taste bud cells and is secreted, it is ideally positioned to serve as a general trophic factor for afferent nerves [25, 26] to branch and form close appositions or synapses with taste bud cells [33]. This is similar to the role of ambient GABA, suggested to serve for maintenance of spiral ganglion afferents in the cochlea during development, and perhaps in the adult [28].

Second, the presence of GABA_Aα1 subunits on many afferent fibers suggests that GABA may regulate the excitability of sensory afferents, as seen in many central and peripheral neurons [reviewed, 40]. Depending on whether GABA is depolarizing or hyperpolarizing, the effect may be either to enhance afferent sensitivity, or to damp spontaneous or evoked output [22, 23, 41]. The presence of type I cells throughout the taste bud and their large amounts of accumulated GABA [4] make them ideally suited to maintain ambient GABA levels for this function. Because sour-evoked release of GABA from type III cells has been shown [36], it is also possible that GABA could selectively target transmission for certain classes of tastants.

Finally, another intriguing possibility presents itself. GABA_A receptors form complexes with P2X2-containing receptors, as detected by co-immunoprecipitation, single-particle tracking, and high-resolution imaging [42]. This interaction stabilizes and trafficks P2X receptors to the neuronal surface. Importantly, once P2X receptors on the cell surface are activated by ATP, they uncouple from their binding-interaction with GABA_A receptors. The latter receptors then are internalized for degradation [42]. This mechanism would be particularly suitable for gustatory afferents which sprout continually to innervate new taste bud cells even in the adult. The newly formed branches and terminals may draw on reserve P2X-GABA_A complexes in fibers near the base of the taste bud. As functional contacts are established in the new branches (mostly vertical) and the P2X receptors are activated by ATP, the GABA_A receptors would be progressively lost. This structural relationship between P2X and GABA_A receptors would result in GABA_A receptors concentrated at the base of the taste bud, and depleted in the vertically extending branches (Figure 2 A,D).

The multiple roles proposed for GABA_A receptors in the taste periphery are not mutually exclusive. Experimental evidence for the involvement of these recenptors in intragemmal signaling, trophic functions, and modulation already exist.

Conclusions and Future Studies

In this essay, I present evidence that GABA could play several key roles in the maintenance, structure, and function of gustatory primary afferent neurons and the taste bud cells that they innervate. Based on substantial evidence of receptor expression, measurements of transmitter release, and afferent recordings, it is clear that GABA functions as a paracrine modulator in the taste bud. Beyond this, less complete evidence suggest possible modulation at the level of peripheral terminals and neuronal soma as well as a novel mechanism of trafficking essential P2X receptors to newly forming synapses as taste bud cells turn over throughout life. Specific questions that arise from these possibilities include:

• What mechanism(s) ensure that GABA_A receptors are trafficked in only the peripheral, not central direction?

• Does GABA serve a trophic role, acting tonically on gustatory afferents within the taste bud to support fibers as they branch and sprout throughout adulthood (similar to the influence of GABA on developing neurons)?

• Does phasic release of GABA serve to modulate the sensory signal? For example, might stimuli at high concentration, or mixtures that activate different taste bud cell types trigger GABA release, to adjust dynamic range or discrimination?

• Do some of these roles rely on GABA derived from type I cells, and some from type III cells?

• Do GABA_A receptors stabilize and traffic P2X receptors to the surface of afferent fibers?

Experiments with cell type selective knockout mouse strains and/or pharmacological approaches will be needed to answer many of these questions.

Highlights:

• ATP is the principal afferent neurotransmitter from taste buds and acts on P2X purinoceptors on peripheral afferent nerve fibers. Yet, GABA_A receptors are also plentiful on the same fibers.

• A variety of roles that GABA and GABA_A receptors play in auditory and vestibular ganglion neurons, nociceptors and mechanoreceptor neurons are considered. Whether any of these may be relevant for gustatory afferent ganglion neurons is discussed.

• Proposed roles for GABA_A receptors on the peripheral terminals of gustatory afferent neurons include acute or tonic modulation of sensory signals, and/or trafficking and stabilizing purinoceptors to the surface of gustatory afferents.