Edited by: L. Ashley Blackshaw, University of Adelaide, Australia
Reviewed by: L. Ashley Blackshaw, University of Adelaide, Australia; Richard L. Young, University of Adelaide, Australia
*Correspondence: Patricia M. Di Lorenzo, Department of Psychology, Binghamton University, Box 6000, Binghamton, NY 13902-6000, USA. e-mail:
This article was submitted to Frontiers in Autonomic Neuroscience, a specialty of Frontiers in Neuroscience.
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To qualify as a “basic” taste quality or modality, defined as a group of chemicals that taste alike, three empirical benchmarks have commonly been used. The first is that a candidate group of tastants must have a dedicated transduction mechanism in the peripheral nervous system. The second is that the tastants evoke physiological responses in dedicated afferent taste nerves innervating the oropharyngeal cavity. Last, the taste stimuli evoke activity in central gustatory neurons, some of which may respond only to that group of tastants. Here we argue that water may also be an independent taste modality. This argument is based on the identification of a water dedicated transduction mechanism in the peripheral nervous system, water responsive fibers of the peripheral taste nerves and the observation of water responsive neurons in all gustatory regions within the central nervous system. We have described electrophysiological responses from single neurons in nucleus of the solitary tract (NTS) and parabrachial nucleus of the pons, respectively the first two central relay nuclei in the rodent brainstem, to water presented as a taste stimulus in anesthetized rats. Responses to water were in some cases as robust as responses to other taste qualities and sometimes occurred in the absence of responses to other tastants. Both excitatory and inhibitory responses were observed. Also, the temporal features of the water response resembled those of other taste responses. We argue that water may constitute an independent taste modality that is processed by dedicated neural channels at all levels of the gustatory neuraxis. Water-dedicated neurons in the brainstem may constitute key elements in the regulatory system for fluid in the body, i.e., thirst, and as part of the swallowing reflex circuitry.
Taste is a vital sensory process that facilitates the ingestion of nutritive substances and the avoidance of toxins. It is not surprising, then, that the perception of taste stimuli is highly informed by the homeostatic state of the organism (Jacobs et al.,
Though it is well known that water (or hypo-osmolarity) evokes a unique taste sensation in insects (Evans and Mellon Jr.,
The objectives of the current investigation were to: (1) describe the neural responsivity to water presented as a taste stimulus in cells of the nucleus of the solitary tract (NTS) and parabrachial nucleus of the pons (PbN) and (2) describe converging evidence that water is an independent taste modality. We characterized four different water-responsive cell types in the NTS and PbN of the rat. Most notably, we observed “water best” cells that responded more vigorously to water than to any other taste stimulus. We argue that the classification of water as an independent taste modality is supported by physiological evidence and has important implications for taste-mediated regulation of ingestive behavior.
Data from 108 male Sprague-Dawley rats (350–450 g) were included. Rats were given unrestricted access to food and were paired housed with a 12-h light–dark schedule. Animal care was in accord with the requirements of the Institutional Animal Care and Use Committee of Binghamton University. Data from NTS cells were obtained from two previously published investigations of taste processing (Roussin et al.,
Prior to surgery, rats were anesthetized with urethane (1.5 g/kg, i.p., administered in two doses, 20 min apart). Some animals were given a third intraperitoneal injection of pentobarbital (Nembutal, 25 mg/kg). Body temperature was maintained at 35–37°C with a rectal thermistor probe connected to a heating pad (FHC, Inc., Bowdoinham, ME, USA).
Animals were tracheostomized to facilitate breathing during stimulus delivery. The head was mounted in a stereotaxic instrument with the upper incisor bar positioned 5 mm below the interaural line. Skin and fascia were removed and a non-traumatic head holder was secured to the skull with stainless steel screws and dental cement. The occipital bone and meninges were removed. In the NTS surgeries, the posterior cerebellum was gently aspirated to expose the underlying medulla. In the PbN surgeries, only the portion of the cerebellum overlying the obex was aspirated.
Taste stimuli consisted of 0.1 M NaCl, 0.01 M HCl, 0.01 M quinine, and 0.5 M sucrose. Concentrations have been shown to elicit half-maximal potentials in the CT nerve of the rat (Ganchrow and Erickson,
The stimulus delivery system consisted of stimulus-filled reservoirs pressurized with compressed air and connected via polyethylene tubing to perforated stainless steel tubes placed in the mouth. Fluid delivery was controlled by computer activation of a solenoid valve interposed between the reservoir and the tongue. Stimuli were delivered at a flow rate of 5 ml/s. The taste solution bathed the whole mouth; this was verified by application of methylene blue through the system. Each stimulus trial consisted of 10 s spontaneous activity, 10 s of distilled water, 5 s of tastant, 5 s pause, and 20 s of a distilled water rinse. Spontaneous activity was defined as the firing rate during the initial 5 s of the trial when no stimulus (tastant or water) was present in the mouth. The inter-trial interval was 2 min. Stimuli were presented in repeated trials for as long as the cell remained well isolated. For any given stimulus, all other stimuli were presented before it was repeated.
Electrophysiological recordings were conducted with etched tungsten microelectrodes (18–20 MΩ, 1 V at 1 kHz; FHC, Inc., Bowdoinham, ME, USA). For NTS recordings, the electrode was lowered into the caudal medulla above the rostral NTS located 2.7 mm anterior and 1.8 mm lateral the obex and ~1.0 mm below the dorsal surface of the brainstem. For PbN recordings, the electrode was lowered through the cerebellum above the pons located 5.4 mm anterior and 1.8 mm lateral to the obex and 5–6 mm below the cerebellar surface. Electrophysiological activity was digitized with an analog-to-digital interface (Model 1401, Cambridge Electronic Designs, Cambridge, UK) and was processed with Spike2 software (Cambridge Electronic Designs, Cambridge, UK). The signal was amplified (Model P511, Grass Technologies, West Warwick, RI, USA) and monitored online with a speaker, oscilloscope and Spike2 software. Single cells were identified by periodically delivering a 0.1-M NaCl solution followed by a water rinse as the electrode was slowly lowered through the brain. Cell isolation was based on the consistency of the waveform shape using template matching and principal component analysis. A signal-to-noise ratio of 3:1 was required for cell isolation. Isolated cells were tested with the exemplars of the four basic taste qualities yielding the “response profile” of the cell, defined as the relative response rates across tastants. Water was presented before tastant delivery and 5 s after tastant delivery. This allowed the assessment of tastant-mediated alteration of the water response. The cell was tested for as long as it remained isolated allowing for multiple presentations of the same stimulus. The precise timing of each spike (1 ms precision) was calculated with respect to the onset of each stimulus delivery, including water.
The magnitude of response to a given tastant was calculated as the mean firing rate (spike per second; sps) during the first 2 s of tastant delivery minus the average firing rate (sps) during the initial 5 s of spontaneous activity at the beginning of each trial. Because water was occasionally found to produce a sustained response in a subset of cells, the response to water was not used as a baseline. A taste response was considered to be significant if it was 2.5 standard deviations greater than the average spontaneous firing rate. All cells were classified by their “best stimulus”, defined as the tastant that elicits the highest response magnitude. Pre- and post-tastant water responses were calculated by subtracting the mean spontaneous firing rate from the firing rate during the initial 2 s of the pre- and post-tastant water delivery, respectively. The response magnitude to water was calculated before and after delivery of each of the other taste stimuli.
The breadth of tuning of taste-responsive cells was calculated with both the traditional Uncertainty measure (Smith and Travers,
The responses to water and taste stimuli were recorded from 135 cells (91 NTS cells; 44 PbN cells), most with several stimulus trial repetitions: In the NTS there were 1–17 stimulus repetitions, median = 8; in the PbN there were 1–26 stimulus repetitions, median = 8. Thirty of 91 NTS cells (33%) and 17 of 44 PbN cells (39%) responded to water, either preceding or following a taste stimulus. There were four types of water responses. Three were categorized as water-responsive and one categorized as somatosensory. The four response types (shown in Figure
The distribution of cells showing each type of water response in the NTS and PbN is shown in Table
Excitatory | Conditional | Inhibitory | Somatosensory | Water specialist | |
---|---|---|---|---|---|
Prevalence | |||||
Water (pre) | 1.8 | 0.3 ± 0.6 | −4.2 ± 3.2 | 2.1 ± 0.6 | 8.7 ± 0.1 |
Water (post) | −0.1 | 5.5 ± 2.6 | −5.5 ± 2.2 | 1.0 ± 0.8 | 1.2 ± 0.1 |
Sucrose | 0.8 | 7.5 ± 2.6 | −1.3 ± 1.9 | 4.9 ± 2.1 | 0.7 ± 0.7 |
NaCl | 3.6 | 20.9 ± 4.2 | 11.4 ± 2.0 | 3.5 ± 0.8 | −0.9 ± 0.9 |
HCl | 0.8 | 17.6 ± 5.0 | 4.5 ± 0.3 | 4.0 ± 1.7 | −1.0 ± 1.0 |
Quinine | 1.7 | 15.3 ± 3.7 | 2.5 ± 0.6 | 1.0 ± 0.7 | −0.6 ± 2.4 |
Prevalence | |||||
Water (pre) | 11.6 ± 3.3 | 0.4 ± 1.1 | NA | 5.6 | 12.9 ± 10.3 |
Water (post) | 10.7 ± 3.0 | 5.7 ± 1.6 | NA | 5.8 | 11.0 ± 8.2 |
Sucrose | 3.8 ± 1.0 | 14.0 ± 7.0 | NA | 6.0 | 0.0 ± 0.1 |
NaCl | 4.8 ± 1.9 | 28.4 ± 7.0 | NA | 9.4 | 0.6 ± 0.4 |
HCl | 3.2 ± 1.9 | 12.7 ± 7.0 | NA | 6.0 | 0.0 ± 0.4 |
Quinine | 11.5 ± 6.7 | 13.8 ± 3.2 | NA | 14.9 | 0.3 ± 0.2 |
Water best cells (
Figure
The responses to water before and after each of the four prototypical taste qualities are shown in Figure
Table
Excitatory | Conditional | Inhibitory | Somatosensory | |
---|---|---|---|---|
Sucrose | 0 | 1 (7%) | 0 | 3 (30%) |
NaCl | 0 | 6 (40%) | 2 (100%) | 4 (40%) |
HCl | 0 | 7 (46%) | 0 | 1 (10%) |
Quinine | 0 | 1 (7%) | 0 | 1 (10%) |
Water | 3 (100%) | 0 | 0 | 1 (10%) |
Sucrose | 0 | 1 (14%) | 0 | 0 |
NaCl | 0 | 5 (72%) | 0 | 0 |
HCl | 0 | 1 (14%) | 0 | 0 |
Quinine | 1 (11%) | 0 | 0 | 1 (100%) |
Water | 8 (89%) | 0 | 0 | 0 |
The water responses of conditional water cells were specific to the taste stimulus that immediately preceded water delivery. The particular taste stimuli that resulted in conditional water responses differed between the NTS and PbN (see Table
NTS ( |
PbN ( |
|
---|---|---|
S | 2 (13%) | 0 |
H | 6 (40%) | 1 (14%) |
Q | 0 | 1 (14%) |
S/H | 5 (33%) | 0 |
S/Q | 1 (7%) | 0 |
N/H | 0 | 2 (29%) |
N/Q | 1 (7%) | 1 (14%) |
N/H/Q | 0 | 2 (29%) |
S | 6.5 ± 1.8 | NA |
N | 4.1 | 14.1 ± 6.0 |
H | 15.3 ± 3.7 | 5.7 ± 1.1 |
Q | 4.5 ± 1.5 | 16.4 ± 4.1 |
Water-inhibitory cells, found only in the NTS, showed significant decreases in spontaneous activity when water was presented both before and after the delivery of another tastant. These two cells both responded best to NaCl. Figure
Water delivered to the oropharyngeal cavity evoked activity in a diverse group of neurons in the NTS and PbN, the first and second central gustatory relays. About a third of cells in each structure (30 of 91, 33% in the NTS; 17 of 44, 39% in the PbN) responded to water either preceding or following a taste stimulus. Three water responsive cell types were observed in both the NTS and PbN. These were excitatory, including water specialists, inhibitory, and conditional. Both excitatory and inhibitory responses to water when presented alone and/or following a taste stimulus were seen. The majority of cells that showed excitatory responses to water, found almost exclusively in the PbN, actually responded more to water than to any taste stimulus. Four cells were water specialists, responding exclusively to water. In conditional water cells, water responses were significantly higher after delivery of a subset of taste stimuli. A separate group of cells were classified as “somatosensory” because they responded equivalently to all water and taste stimuli. These findings, along with data from the existing literature, provide evidence for the idea that water is encoded by a separate information “channel” that begins in the taste receptor and is transmitted through the gustatory neuraxis along with information about other taste stimuli. The view that water is an independent taste modality is consistent with the idea that the function of the gustatory system is to detect and identify chemical stimuli that are essential for survival.
As support for the classification of water as an independent taste quality, we argue three lines of evidence. First, the existence of a discrete and dedicated transduction mechanism for water in the oropharyngeal cavity provides a basis for peripheral sensitivity underlying central neural responses. Second, electrophysiological responses in dedicated (specialist, i.e., exclusively responsive to a single taste quality) taste-related peripheral nerves argue that the sensation of water is transmitted to the central nervous system over a separate information channel. Third, data showing responses in water specialist cells in central gustatory-related structures provide strong evidence that the central representation of water is distinct from that associated with other taste qualities.
Recently, a dedicated transduction mechanism for water (or hypo-osmolarity), has been identified in the mammalian oropharyngeal cavity. Water enters directly into the taste receptor cell through a channel called an aquaporin (Watson et al.,
Water is also an effective stimulus in peripheral nerves that respond to more traditional taste qualities. Specifically, water responses have been observed in the chorda tympani nerve (a branch of the facial nerve innervating taste buds on the rostral 2/3 of the tongue) of the rat, cat, and dog (Pfaffmann and Bare,
Water-specific responses have also been observed in many gustatory processing regions of the brain including the NTS (Nakamura and Norgren,
In addition to the role of water in evoking a swallowing reflex, the perceptual consequences of water taste may play a critical role in regulating fluid intake (thirst) and the maintenance of hydration. The responses described here were recorded with passive stimulus delivery in a non-deprived state and in the absence of post-ingestional effects. It is possible, however, that homeostatic variables such as thirst may modulate the responses to water. For example, in an imaging study of thirst and water taste processing in the human gustatory cortex, the primary gustatory cortex (anterior insula and frontal operculum) was activated by water regardless of thirst but the secondary gustatory cortex (caudal orbitofrontal cortex) only showed water-evoked activation in a water-deprived state (de Araujo et al.,
In addition to oral sensitivity, water may also be detected by chemoreceptors located in the gut (Rozengurt and Sternini,
The idea that the taste of water is independent from that of other taste qualities represents a substantial departure from the conventional supposition that water responses are entirely somatosensory. Present data support a clear distinction between water-responsive and somatosensory cell types. Somatosensory cells appeared to respond only to the mechanical and/or thermal components of a fluidic stimulus. That is, their responses to water and tastants were indistinguishable. As can be seen in Figure
In general, differences between the NTS and PbN suggest that water sensibility may serve different functions in each structure. In the NTS, the relatively large proportion of somatosensory responses to water may be part of neural circuits extending from the caudal NTS that produce ingestive reflexes such as swallowing (Lang,
The sensitivity of gustatory NTS cells to water may play an essential role in the initiation of swallowing. The superior laryngeal nerve of the rat includes water-selective fibers (Shinghai,
Conditional water responses were more prevalent in the NTS than the PbN. This observation suggests that the NTS responses may be more closely tied to peripheral nerve input than the PbN. Psychophysical studies over the last 40 years have emphasized the interdependence of water responses and sensitivity to other taste stimuli. In 1974, Bartoshuk showed that water-evoked a taste but that it was highly dependent on adaptation to a preceding taste stimulus even inclusive of saliva. In addition, recordings from afferent taste nerves have shown that some water responses were only observed after pre-exposure to a particular taste stimulus (Bartoshuk and Pfaffmann,
The present results are not without their limitations. For example, some responses to water may have been affected by the anesthetic under which they were recorded. Urethane anesthesia delivered intraperitoneally (IP), as in the present study, has been shown to produce hyperglycemia, increased hematocrit and decreased blood plasma protein levels (Van Der Meer et al.,
Sampling errors might also have affected present results. That is, in the recordings of taste responses in both NTS and PbN, most water responses were observed only incidentally. That is, water responses were only noted “after the fact”, when analyses of responses to other tastants were analyzed. It is therefore possible that we missed some cells that might have been responsive to water. Even so, our evidence, and those of others who have described water responses, clearly suggests that the neural representation of water in the chemosensory pathway is weaker than that of other taste qualities. On the other hand, the consistent presence of water responses in almost every electrophysiological study of taste, including the present one, implies that sensibility to water is clearly present and may serve an important purpose.
In conclusion, we have described responsivity to water in two brainstem nuclei, the NTS and PbN, in the anesthetized rat. A novel finding of the current study is the observation of water best neurons and water specialist cells in both NTS and PbN. Importantly, these data underscore the literature showing that water is processed by cells in gustatory nuclei. This implies that water evokes a gustatory sensation that is supported by an independent neural representation, different from that of other taste qualities.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by NIDCD Grants DC-06914 and DC-005219 to P. M. Di Lorenzo.