Although the reciprocal projections between the bed nucleus of the stria terminalis (BNST) and the gustatory parabrachial nuclei (PbN) have been demonstrated neuroanatomically, there is no direct evidence showing that the projections from the PbN to the BNST carry taste information or that descending inputs from the BNST to the PbN modulate the activity of PbN gustatory neurons. A recent electrophysiological study has demonstrated that the BNST exerts modulatory influence on taste neurons in the nucleus of the solitary tract (NST), suggesting that the BNST may also modulate the activity of taste neurons in the PbN. In the present study, we recorded from 117 taste-responsive neurons in the PbN and examined their responsiveness to electrical stimulation of the BNST bilaterally. Thirteen neurons (11.1%) were antidromically invaded from the BNST, mostly from the ipsilateral side (12 cells), indicating that a subset of taste neurons in the PbN project their axons to the BNST. The BNST stimulation induced orthodromic responses on most of the PbN neurons: 115 out of 117 (98.3%), including all BNST projection units. This descending modulation on the PbN gustatory neurons was exclusively inhibitory. We also confirmed that activation of this efferent inhibitory projection from the BNST reduces taste responses of PbN neurons in all units tested. The BNST is part of the neural circuits that involve stress-associated feeding behavior. It is also known that brain stem gustatory nuclei, including the PbN, are associated with feeding behavior. Therefore, this neural substrate may be important in the stress-elicited alteration in ingestive behavior.
- brain stem
- ventral forebrain
- neural circuit
the nucleus of the solitary tract (NST) and the parabrachial nuclei (PbN) are the first and second central relays for taste information processing in rodents, respectively (66, 67, 93, 101). Taste information elicited from the tongue and oral cavity is first carried to the rostral portion of the NST by the VIIth, IXth, and Xth cranial nerves, and these nerve terminals are distributed in a topographic fashion with rostral-caudal sequence in the rostral NST (4, 13, 33, 34, 101). From the NST, ascending gustatory fibers project to third-order taste cells within the PbN of the pons (11, 32, 58, 63, 72–74, 93, 99). Gustatory information from the PbN is further carried to the ventral posterior medial nucleus of the thalamus (VPM) and then the insular cortex, and to several ventral forebrain gustatory areas, including the lateral hypothalamus (LH), the central nucleus of the amygdala (CeA), and the substantia innominata (31, 39, 43, 45, 62, 64, 65). Neuroanatomical studies have also demonstrated that cells from the gustatory PbN project to the bed nucleus of the stria terminalis (BNST) (31, 64, 80), but this has not been confirmed electrophysiologically. The forebrain targets of gustatory projections send efferent fibers back to both the PbN and NST (1, 19–21, 30, 45, 57, 86, 96, 100).
The PbN is a critical relay for ascending visceral information, including taste (5, 9, 27, 67, 95). The PbN is also the locus of integration for gustatory information and other vagal afferent inputs (2, 36, 37). The convergence of gustatory/mechanical and gustatory/visceral inputs in the PbN signifies the importance of the PbN in feeding and gustatory processing (2, 36, 37, 94). The modulation of taste responses of the PbN neurons by physiological factors associated with ingestive behavior has also been reported. For example, intraduodenal lipid infusion or gastric distention decreased the response magnitude of taste neurons in the rat PbN (2, 29). In addition, the PbN plays a significant role in the acquisition and retention of conditioned taste aversion learning, and it is likely that descending inputs from forebrain gustatory areas are important in this processing (28, 78, 82, 90).
A number of studies have demonstrated that the BNST plays an important role in the integration of autonomic and behavioral responses to stress (7, 8, 14, 15, 40, 44, 59, 81), in maternal behavior (48, 49, 68–71), and in sodium appetite (50, 55, 77, 103). There is a strong descending input from the BNST to the brainstem gustatory nuclei, the NST, and the PbN, suggesting that these nuclei may be involved in modulating taste and ingestive behavior (21, 57). Administration of hypocretin I (also referred to as orexin A) into the LH activated key feeding-regulatory brain sites, including the BNST (61). This suggests that the BNST is an important locus in the feeding-stimulatory actions of hypocretin I. Although the projection of the PbN to the BNST is likely to convey gustatory information and the descending projection from the BNST to the PbN is likely to be involved in modulating activity of taste-responsive neurons in the PbN, it is not possible to determine whether this reciprocal projection between the PbN and BNST involves gustation without electrophysiological confirmation. The purpose of the present study was to verify whether PbN gustatory neurons send axons to the BNST, whether the descending projection of the BNST exerts an influence on the excitability of PbN gustatory neurons, and whether this efferent projection alters taste response of PbN neurons.
Animals and surgery.
The experimental procedures were conducted so as to minimize animal suffering and the number of animals used in accordance with the Institutional Animal Care and Use Committee (IACUC) and National Institutes of Health guidelines. All procedures used in this experiment were reviewed and approved by the IACUC of Southern Illinois University at Carbondale. Young adult male Syrian golden hamsters (Mesocricetus auratus), weighing between 113 g and 168 g (n = 31), were deeply anesthetized with urethane (1.7 g/kg ip). Additional anesthetic (10% of original dose) was given as needed during the course of each experiment to maintain anesthesia. Each animal was tracheotomized and mounted in a stereotaxic instrument (Narishige SR-6N) using an auxiliary ear bar (EB-4) with the incisor bar at the same level as the interaural line. The tissue overlying the parietal bone was removed, and a hole was drilled on each side of the skull to access the BNST. A concentric bipolar stimulating electrode, constructed from 26-gauge stainless steel tubing and 140-μm-thick stainless steel wire, was lowered into the posterior lateral BNST on each side of the brain (coordinates: 0.5–0.8 mm anterior to bregma, 1.65 mm lateral to the midline, and 5.2–5.23 mm ventral to the brain surface), and secured with dental cement. The electrodes, except for the tip area, were insulated with Epoxylite 6001 (Epoxylite, Irvine, CA).
After positioning the electrodes into the BNST, the animal was mounted in a hand-made nontraumatic head holder (25) with the snout downward 27° from horizontal to straighten the brain stem and minimize brain movement associated with breathing. A sagittal skin incision was made along the midline overlying the posterior skull and a portion of the occipital bone just dorsal to the foramen magnum was removed to reveal the cerebellum. The dura covering the cerebellum was excised, and the posterior portion of the cerebellum was aspirated for 5–6 mm anterior to the obex, allowing direct access to the PbN. Body temperature was maintained at 37 ± 1°C with an electric heating pad.
Single unit recording and electrical stimulation.
Single-barrel glass micropipettes (tip diameter = 1–2 μm, resistance = 5–7 MΩ) filled with 2% (wt/vol) solution of Chicago Blue dye in 0.5 M sodium acetate were used for extracellular single unit recording of action potentials from the gustatory PbN. The mean coordinates for the PbN recording were 4.0 ± 0.13 (SD) mm anterior to the obex and 1.4 ± 0.06 mm lateral to the midline. Extracellular action potentials were amplified with a band pass of 15–5,000 Hz (NeuroLog, Digitimer, Hertfordshire, UK), discriminated with a dual-time-amplitude window discriminator (Bak DDIS-1, Bak Electronics, Germantown, MD), displayed on oscilloscopes, and monitored with an audio monitor. A Dell Pentium 4 XPS laptop computer configured with a CED Power1401 interface board and Spike2 software (Cambridge Electronic Design, Cambridge, UK) controlled taste stimulus delivery and online data acquisition and analysis. The taste responses of the PbN neurons were initially identified by a change in neural activity associated with the application of electrical shock (≤40 μA, 500 ms duration at 1/3 Hz) to the anterior tongue (45) and confirmed by response to chemical stimulation of the anterior tongue. Taste stimuli presented to the anterior tongue were 0.032 M sucrose, 0.032 M sodium chloride (NaCl), 0.032 M quinine hydrochloride (QHCl), and 0.0032 M citric acid. These concentrations of taste solutions evoke approximately equal multiunit taste responses in the hamster NST (22). These taste solutions were delivered by a gravity-flow system composed of a computer-controlled two-way solenoid-operated valve connected via tubing to a distilled-water rinse reservoir and a stimulus funnel. The stimulation sequence, during which the computer acquired data, was a continuous flow initiated by the delivery of distilled water for 10 s, followed by 10 s of stimulus, followed by 10 s of distilled-water rinse. The flow rate was 2 ml/s. After each tastant, the tongue was rinsed with distilled water (>50 ml), and individual stimulations were separated by ≥2 min to avoid adaptation effects (87).
After each PbN neuron was characterized for its taste response profile, rectangular pulses of 0.5 ms duration and 0.1 mA or less intensity were delivered to the BNST through each stimulating electrode from an isolated stimulator (Grass S88, Grass Instruments, Quincy, MA) to examine whether the neurons recorded show antidromic responses. The criteria for antidromic activation were constant latency and the ability to follow a stimulus pulse pair at greater than 200 Hz. A collision test was performed between a spontaneously generated action potential and a BNST stimulus-evoked potential. The conduction velocity was calculated from the antidromic activation latency and the linear distance between the recoding site and the tip of the BNST stimulating electrodes.
To examine orthodromic responses, the BNST was stimulated (0.5 ms, 0.1 mA, 1/3 Hz) to observe the effect of electrical stimulation of the forebrain on ongoing activity of PbN neurons after testing the antidromic status. A peristimulus time histogram (PSTH) was created from data acquired on each PbN cell in response to 50–200 stimulus pulses delivered to each of the two BNST electrodes.
To test whether activation of the descending input from the BNST alters PbN neurons' taste responses, the responses of a subset of the PbN neurons (n = 12) to taste stimulation were recorded before and during delivery of trains of constant square pulses (100 Hz, 0.2 ms, 0.1 mA) to the BNST. The electrical stimulation started at the beginning of the delivery of taste stimuli and lasted for 10 s. In addition, a subset of low-firing PbN neurons were tested for their responsiveness to the single-pulse BNST stimulation (0.5 ms, 0.1 mA 1/3 Hz) while the cell was driven by taste stimulation, and a PSTH was created. The concentration of the taste solutions used to elicit taste responses were adjusted so as to activate the neuron at moderate firing rate (3.2–6.6 Hz). In the first 7 units tested, each effective tastant and mixtures of all effective tastants were used separately to stimulate the tongue. Because all taste stimuli- and mixture-elicited taste responses were altered by BNST activation, only the taste mixture was used to stimulate the tongue in the remaining test (five additional units). The taste stimulation was repeated throughout the test period as follows: 2 s of taste solution delivery followed by a 20-s pause and then delivery of a 3-s distilled water rinse. This 25-s stimulus sequence was repeated at 10-s intervals until the end of the session to create a PSTH. We tested the low spontaneous firing cells with this stimulus protocol to achieve two goals. First, this protocol lets us determine whether these neurons receive inhibitory input from the BNST. It is technically impossible or very difficult to observe inhibitory responses of PbN neurons to electrical stimulation of the BNST when a PbN cell is not firing spontaneously or firing at very low frequencies. Second, this protocol lets us observe whether BNST stimulation suppresses taste-evoked discharge of PbN taste neurons.
To investigate whether PbN neurons that project to the BNST (BNST-projection neurons) also receive centrifugal input from the BNST, the BNST was stimulated at suprathreshold stimulus intensity for antidromic activation (0.5 ms, 0.1mA at 1/3 Hz). A PSTH was constructed from responses to 50–200 stimulus pulses for all projection neurons after examining the antidromic status. To examine the relationship between the inhibitory response elicited by BNST stimulation and the antidromic activation of PbN taste neurons, the BNST was also stimulated with stimulus intensities that were subthreshold for antidromic activation in a subset of projection neurons.
At the end of each experiment, the last recording site of the day was marked by passing a 10-μA cathodal current through the recording electrode for 10 min (5 s ON-OFF) to deposit a spot of Chicago Blue dye. The stimulating sites on the BNST were also marked by passing 10 μA anodal current through the inner wire of the stimulating electrode for 20–30 s to deposit a spot of iron. The hamster was then given a lethal overdose of urethane and perfused through the heart with 4% formalin containing 3% potassium ferrocyanide and ferricyanide. Brains were removed, postfixed, frozen sectioned (40 μm) in the coronal plane, and stained with neutral red. The recording and stimulating sites were located microscopically and plotted on the basis of standard atlas sections (60).
The responses of each cell to taste stimulation of the tongue were accumulated over three consecutive time periods during 1) 10 s of distilled water rinse just before the stimulus, 2) 10 s of stimulus flow, and 3) 10 s of distilled water rinse just after the stimulus. The net taste response was calculated as the mean number of action potentials (imp/s) during the first 5 s of chemical stimulation minus the mean number of spikes during 5 s of distilled water before the taste delivery. Responses are reported as means ± SE. A taste response was defined as effective if it were ≥ 2 SD above the spontaneous discharge, which was measured during the 5-s prerinse just before the beginning of the delivery of taste solution. For orthodromic responses of PbN cells to electrical stimulation of the BNST, individual PSTH was analyzed to determine excitatory or inhibitory epochs. A baseline period was defined as the 200 ms preceding stimulation; the means ± SD of the number of spikes/1-ms bin during this baseline period were determined. An excitatory effect of BNST stimulation was defined as an epoch of at least five consecutive bins with a mean value ≥2 SD above the baseline mean, which defines a mean response with a probability of <0.05. Inhibitory responses were defined as those with at least 20 consecutive bins with a mean <50% of baseline firing rate.
The entropy (H) of each neuron, which is a measure of its breadth of responsiveness, was calculated using excitatory components of responses to four standard taste stimuli by the following formula: where H = breadth of responsiveness, 1.661 is a scaling constant, and pi is the proportional response to each of the n components. H ranges from 0.0 for a cell that responds exclusively to one stimulus to 1.0 for a cell responding equally to all four (88).
Each PbN cell was categorized either as a BNST-projection neuron if it were antidromically invaded from the BNST or as a nonprojection neuron otherwise. Within those two categories, the cells were further characterized by their best stimulus that produced the greatest response.
Univariate ANOVA was used to compare differences in mean firing rates to taste stimulus, in entropies, and in inhibitory durations between BNST-projection and nonprojection neurons across taste stimuli, best stimulus, and ipsilateral or contralateral BNST stimulation. The spontaneous activities of the BNST-projection and nonprojection group were compared using t-tests. Comparison of the number of neurons in each category was made using the χ2-test. All means were reported with SE unless remarked otherwise.
A total of 117 taste-responsive PbN neurons were recorded from 31 male hamsters. Neurons that did not meet the statistical criterion for taste response or animals that showed the placement of stimulating electrode outside of the BNST were excluded from the analysis. Thirty-one recording sites and all stimulating sites on both sides were identified histologically, and representative examples are shown in Fig. 1. An iron deposit in the posterolateral BNST (BNSTPL, arrow), in between the internal capsule (ic) and posterointermediate BNST (BNSTPI), along with an electrode penetration track just ventral to the bottom of the lateral ventricles (LV), can be seen in this coronal section of the hamster brain (Fig. 1A). This is the portion of the BNST that receives afferent input from the gustatory region of the PbN (64). A recording site marked with Chicago Blue dye in the PbN is shown in Fig. 1B (arrow, dark spot). On this coronal section through the hamster pons, the marking is located in the middle of medial PbN (MPB), medial to the superior cerebellar peduncle (scp) and dorsal and lateral to the mesencephalic trigeminal nucleus (Me5) at the level where the locus coeruleus (LC) is evident.
All recording and stimulating sites from 31 hamsters were examined and reconstructed using standard atlas sections of the hamster brain (60) (figures are not shown). The tips of the stimulating electrodes in the 31 hamsters included in the analysis were confined to the BNST, between anterolateral BNST (BNSTAL) and BNSTPL, especially the latter subdivision. This anterior-posterior extension corresponds to the level of the crossing of the anterior commissure (ac) rostrally and to the level containing the bed nucleus of the anterior commissure (BAC) caudally (Fig. 1A). There was no difference between the electrode placement sites that evoked responses (orthodromic and antidromic responses) in the PbN neurons and the sites that produced no effects. Except for two neurons, all PbN taste neurons tested responded either orthodromically or antidromically after ipsilateral or contralateral BNST stimulation. All sites that evoked antidromic responses also produced inhibitory responses. Two sites that produced no effect overlapped with the sites that produced inhibitory and/or antidromic responses.
The location of the last PbN neurons to be recorded in each animal was marked with Chicago Blue Dye and the locations of these marks (n = 31) are depicted on a standard atlas section of the hamster pons at the level of the Me5 (figure is not shown). The recording sites are concentrated in the MPB, medial to the scp and lateral to the Me5 and the LC at the level of the Me5 or dorsolateral of the ventral edge of Me5 (refer to Fig. 1B). The anterior-posterior distribution of the recording marks extended rostrally from the level at which LC is first apparent and caudally to the appearance of the accessory trigeminal nucleus (Acs5). There was no relationship between the stimulating site within the BNST and the responses of the PbN neurons e.g., inhibitory response/antidromic response, or no response.
Effect of the BNST stimulation on PbN taste neurons.
A total of 117 taste neurons were recorded in the PbN, and the effects of BNST stimulation were examined bilaterally. Except for two units, all tested neurons responded antidromically and/or orthodromically. Out of 117 neurons recorded, 13 cells were antidromically activated, 12 after ipsilateral and one following contralateral BNST stimulation. These neurons were classified as BNST-projection neurons and the remaining 104 as nonprojection neurons. Stimulation of BNST induced an orthodromic response in 102 of 104 nonprojection neurons; 64 bilaterally and 38 unilaterally. There was no excitatory response. In addition, all BNST-projection neurons that were activated antidromically have shown orthodromic responses with stronger stimulus intensities. Therefore, BNST stimulation produced orthodromic responses in a majority of PbN taste neurons (115 of 117 cells: 98.3%), and all of the orthodromic responses were inhibitory. Each PbN neuron was further characterized by its best stimulus. The number of PbN cells in each category appears in Table 1.
Taste response characteristics of PbN neurons.
Each PbN neuron was tested for its response to the four basic taste stimuli. The responses of one cell to gustatory stimuli are shown in Fig. 2, which depicts 30 s of the extracellularly recorded neural activity during taste trials consisting of 10 s of distilled water prerinse, followed by 10 s of the stimulus flow (the onset of which is indicated by the first arrow), and then by 10 s of poststimulus distilled water rinse (the onset of which is indicated by the second arrow). This cell had a relatively slow rate of spontaneous discharge (1.88 imp/s) and responded to 32 mM sucrose, 32 mM NaCl, 3.2 mM citric acid, and 32 mM QHCl. This neuron responded most strongly to 32 mM QHCl. The fifth panel in the figure was derived from the trial of QHCl stimulation, after filtering out background activity and solenoid artifacts that occurred at the start and end of taste delivery. A PSTH was created after elimination of all electrical signals other than taste neuronal activity for each stimulus presentation, as shown at the bottom of Fig. 2 for QHCl. The waveform of the action potential of this cell is also depicted in Fig. 2.
The overall spontaneous activity of the 117 taste-responsive PbN neurons recorded in the present study ranged from 0.1 to 16.3 imp/s with a mean of 3.63 ± 0.26 imp/s. This mean firing rate was similar to that recorded previously (e.g., 3.92 ± 0.38 imp/s, t = − 0.653, df = 216, P = 0.514) (45) and significantly higher than that recorded in the NST in our previous experiments (e.g., 2.12 ± 0.27 imp/s, t = 4.036, df = 224, P < 0.0001) (46). Comparison of the spontaneous rate between the BNST-projection (range = 0.15–6.75 imp/s, mean = 3.16 ± 0.65 imp/s) and nonprojection group (range = 0.10–16.32 imp/s, mean = 3.77 ± 0.30 imp/s) showed no significant difference (t = 0.693, df = 115, P = 0.490).
Each of the 117 PbN neurons was tested for its responsiveness to the four basic taste stimuli and categorized as sucrose-, NaCl-, citric acid-, or QHCl-best on the basis of its response profile. These best-stimulus categories are indicated in Fig. 3, where the responses of the cells in each best-stimulus group are arranged along the abscissa in order of their response to the best stimulus for that group. The response to any one tastant is read across the pattern and that of any one neuron can be seen from top to bottom; responses to the stimuli are net responses and the mean spontaneous activity of each cell is shown as distilled H2O at the bottom of the figure. Of 117 neurons, 18 were sucrose-best, 38 were NaCl-best, 35 were citric acid-best, and 26 were QHCl-best. Except for 2 units (unit no. 79 and 112, gray bars), all neurons were affected by BNST stimulation, antidromically, and/or orthodromically. Antidromically activated neurons are indicated as solid bars. The BNST-projection neurons were composed of 1 sucrose-best, 2 NaCl-best, 6 citric acid-best and 4 QHCl-best cells. The distribution of these 13 neurons among four best-stimulus categories did not differ from that of 104 nonprojection neurons (χ2 = 4.109, df = 3, P = 0.250).
Although four taste stimuli generated responses that differed from one another (F3,460 = 4.239, P < 0.01), there was no significant difference in taste responses between BNST-projection and nonprojection groups (F1,460 = 0.120, P = 0.729) and no significant interaction between projection status and taste stimulus (F3,460 = 0.439, P = 0.725). The post hoc test revealed a significant effect of taste stimulation that was ascribable to the response to sucrose, which was less than the response to NaCl, citric acid, or QHCl (P < 0.01; Fig. 4A).
The breadth of responsiveness (entropy) of each PbN neuron was obtained using excitatory components of taste response to four taste stimuli. The mean entropies in each category and projection status are described in Fig. 4B. The mean entropy (0.80 ± 0.07) of projection neurons was not different from that (0.81 ± 0.01) of nonprojection cells (F1,109 = 0.802, P = 0.373). In contrast, the difference among the four best-stimulus groups was significant (F3,109 = 3.586, P < 0.05), although the interaction effect between best-stimulus and projection status just missed the statistical rejection criterion. (F3,109 = 2.202, P = 0.092). At these stimulus concentrations, sucrose-best cells were the most narrowly tuned (H = 0.74 ± 0.04), and citric acid-best cells were the most broadly tuned (H = 0.85 ± 0.02). A post hoc test showed that entropies of sucrose-best cells were significantly less than those in citric acid-best group (P < 0.05).
Antidromic activation of PbN cells.
All 117 taste-responsive PbN cells were tested with electrical stimulation of the BNST bilaterally. Thirteen of these neurons were activated antidromically by stimulation of ipsilateral (n = 12, 10.26%) or contralateral BNST (n = 1, 0.85%). Examples of antidromic responses of two PbN neurons induced by ipsilateral and contralateral BNST stimulation are shown in Fig. 5, which demonstrates fulfillment of the criteria for antidromic invasion. The superimposed oscilloscope traces (n > 3 in each test) show that action potentials evoked by BNST stimulation (arrows) occurred at constant latency (top), followed closely paired (200 Hz) stimulus pulses (middle), and were cancelled (arrowheads, bottom) by collision with spontaneously generated action potentials (*). The onset latencies for antidromic activation of these two units were 9.8 ms and 7.2 ms with thresholds of 57 and 69 μA, respectively.
Because only one PbN cell was antidromically activated following contralateral BNST stimulation, we did not discriminate whether the antidromic responses are induced after ipsilateral or contralateral BNST stimulation in the following analysis. The latencies of 13 cells for antidromic activation induced by BNST stimulation varied between 3.6 and 15.5 ms, and the mean latency was 8.24 ± 1.08 ms. The threshold for antidromic activation of PbN neurons ranged from 18 to 100 μA, with the mean threshold of 61.46 ± 6.99 μA. The linear distance between the BNST stimulating site and the PbN recording site was measured for each animal. The distance varied from 6.2 to 8.2 mm with the mean of 7.10 ± 0.15 mm. The conduction velocity of each projection neuron was calculated by dividing the BNST-PbN distance by the latency; it ranged from 0.45 to 1.89 m/s, with a mean conduction velocity of 1.01 ± 0.12 m/s.
Orthodromic activation of gustatory PbN neurons.
Of 117 PbN neurons tested for their responsiveness to BNST stimulation, 115 neurons, including BNST-projection neurons, responded orthodromically to the stimulation of the BNST either unilaterally or bilaterally. Stimulation of ipsilateral BNST produced an orthodromic response in 113 (96.58%) neurons, of which 12 neurons projected back. In comparison, 77 (65.81%) PbN neurons responded orthodromically to the contralateral BNST stimulation. Seventy-five (64.10%) PbN taste neurons responded to both sides of BNST stimulation. Of these 75 neurons, 11 were BNST projection neurons (see Table 1).
We found that the vast majority of PbN taste-responsive neurons receive descending inputs from the BNST and that all orthodromic responses are exclusively inhibitory. Examples of PbN neurons that were inhibited after BNST stimulation are shown in Fig. 6. PSTHs derived from the same PbN neuron after stimulation of ipsilateral and contralateral BNST are shown in Fig. 6, A and B, respectively. As seen in the PSTHs, repeated single-pulse stimulation of the BNST at 1/3 Hz briefly suppressed the ongoing firing activity of the PbN neuron immediately after the onset of the stimulation (0 s), suggesting that activation of the BNST inhibits neuronal activity of PbN taste neurons. The duration of the inhibition induced by ipsilateral BNST stimulation (19–120 ms, mean = 58.57 ± 2.17 ms) was similar to that following the contralateral BNST stimulation (26–112 ms, mean = 58.67 ± 2.12 ms, F1,186 = 1.310, P = 0.254). A difference of inhibitory duration between the BNST-projection (27–115 ms, mean = 64.13 ± 4.48 ms) and the nonprojection group (19–120 ms, mean = 57.81 ± 1.64 ms) was not observed (F1,186 = 1.443, P = 0.231). A significant interaction between groups was also not observed (F1,186 = 2.338, P = 0.128).
BNST-projection PbN taste neurons also receive centrifugal input from the BNST.
The stimulus intensity for antidromic activation was lower (mean = 61.46 ± 6.99 μA) than that used to examine orthodromic responses (100 μA). To examine whether the PbN neurons that project to the BNST also receive descending input from the BNST, we raised the stimulus intensity up to 100 μA and stimulated the BNST again at 1/3 Hz after examining the antidromic response status. All 13 PbN neurons that were antidromically activated showed inhibitory responses at higher stimulus intensity than was requested for antidromic activation. Examples of BNST projection neurons that were inhibited after BNST stimulation are shown in Fig. 6, C and D. The threshold for antidromic activation of this unit was 87 μA with a latency of 5.6 ms. The inhibitory response of the neuron occurred with both suprathreshold (100 μA, Fig. 6C) and subthreshold (50 μA, Fig. 6D) stimulus intensities, indicating that this neuron not only sends an axon to the BNST but also receives efferent projection from the BNST.
Effects of electrical stimulation of the BNST on taste response of PbN units.
To examine the physiological significance of this inhibitory input from the BNST, we investigated whether activation of the descending projection from the BNST alters PbN neurons' taste responses in the following experiments. One of the methods used was to stimulate BNST electrically (100 μA, 1/3 Hz) while delivering taste solution to the tongue to evoke taste responses (see details in methods section). Twelve PbN neurons that had low spontaneous firing activity were studied. Electrophysiological observation of inhibitory effects for neurons that have low firing frequency is difficult because of the time needed to build up baseline neural activity of PSTH. To build the baseline activity faster, we increased the PbN neuronal firing by stimulating the tongue with taste solutions. PSTHs depicted in Fig. 7, A–F were created by stimulating BNST at 1/3 Hz while activating the neuron with effective taste stimuli, whereas the PSTH in Fig. 7G was created by stimulating BNST without taste stimuli. The firing activities of this PbN neuron shown in the PSTHs (A–F) represent a mixture of spontaneous activity and responses to sucrose (Fig. 7A), NaCl (Fig. 7B), citric acid (Fig. 7C), and QHCl (Fig. 7D). PSTHs in Fig. 7, E and F were generated after ipsilateral and contralateral BNST activation while stimulating the tongue with the mixture of all four basic tastants. Because the action potentials in these PSTHs are a mixture of spontaneous and taste stimulation-elicited spikes, BNST stimulation-induced inhibition represents inhibition of both the spontaneously generated discharge and taste responses. We tested six additional neurons with all effective taste stimuli (13 taste trials; 3 sucrose, 4 NaCl, 3 citric acid, and 3 QHCl responses with ipsilateral and/or contralateral BNST stimulation) and confirmed that both sides of BNST activation suppressed all four basic tastant-elicited responses. Because we confirmed that the inhibition of taste response by BNST activation was not taste quality specific, the remaining five neurons were tested only with the mixture of effective taste stimuli. BNST stimulation inhibited all 12 neurons that were tested with this experimental protocol.
In addition, we examined the influence of activation of the BNST on the responses to taste stimulation of the anterior tongue by comparing the change in taste responsiveness before and during delivery of a train of rectangular pulses (100 Hz, 0.2 ms duration) to the BNST. This experiment was conducted on another subset (n = 12) of PbN neurons, including the neurons that exhibited high spontaneous firing activity. Among the 12 PbN cells that were tested, 4 cells received descending input from bilateral BNST, including an ipsilateral BNST-projection neuron, and a total of 41 gustatory responses were investigated with this protocol. After recording a cell's taste responses, the same taste trials were repeated again while a train of stimulation was delivered to the BNST. The mean net response to gustatory stimuli before electrical stimulation (ES) of the BNST was 17.26 ± 2.04 imp/s. The delivery of ES significantly reduced the net response to the repeated gustatory stimuli (9.46 ± 1.27 imp/s; t = 7.440, df = 40, P < 0.001). Taste stimuli were applied once again without ES of the BNST (recovery); the mean net response was 17.40 ± 2.13 imp/s, which was not different from that before ES of the BNST (t = 0.184, df = 40, P = 0.855). Representative taste response profiles of one of these 12 PbN cells that was inhibited by ES of the BNST bilaterally are shown in Fig. 8, A and B. The means of responses to all taste stimuli together and to each taste stimulus are illustrated in Fig. 8C.
Projection of PbN gustatory neurons to the BNST.
The present investigation demonstrates, for the first time, that taste-responsive PbN neurons project to the BNST and predominantly to the ipsilateral side. A number of neuroanatomical studies have shown a reciprocal connection between the PbN and the BNST. In the hamster, for example, anterogradely transported labels were seen in a number of forebrain nuclei, including the BNST, after injection of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) into the PbN (31). An earlier tracing study using tritiated proline autoradiography in rats has also demonstrated that neurons in the PbN gustatory area project to dorsolateral BNST and that the majority of the fibers remain ipsilateral (64). After microinjection of WGA-HRP into the rostral portion of the BNSTPL, labeled cells were concentrated in the external lateral and waist subnuclei in the PbN (56). These anatomical data support our results that a subset of PbN taste neurons project to the BNST. In the present experiment, we have shown that 11.1% of PbN taste neurons were antidromically activated by electrical stimulation of the BNST and that most of these neurons responded after ipsilateral BNST stimulation.
In a previous study, we have shown that stimulation of LH or CeA antidromically activated 79.2% and 59.4% of taste-responsive PbN units and that the mean latencies after LH and CeA stimulation were 2.59 ± 0.13 and 4.73 ± 0.39 ms, respectively (45). The onset latency for antidromic activation after BNST stimulation (mean = 8.24 ± 1.08 ms) was significantly longer than after LH (t = 10.565, df = 91, P < 0.0001) or CeA stimulation (t = 3.632, df = 71, P < 0.001). In an earlier study, Hayama and Ogawa (35) demonstrated that PbN taste neurons project heavily to the bilateral thalamus in the rat. Taken together, these results demonstrate that PbN taste neurons project directly to LH, thalamus, CeA, and BNST, and that PbN is the main relay for ascending visceral afferent, including taste, from the NST.
Descending projection from the BNST to the PbN.
The present study also demonstrates that most taste-responsive PbN neurons are subject to a modulatory influence from the BNST. Stimulation of ipsilateral BNST centrifugally modulated 96.58% of PbN taste units, while contralateral BNST stimulation inhibited 65.81% of gustatory PbN units. Furthermore, about two-thirds (64.1%) of the taste neurons in the PbN receive descending input from bilateral BNST. The centrifugal inputs from the BNST to the PbN gustatory units are much heavier than those from the LH (13.9%) or CeA (25.7%) (45) in hamsters. BNST inputs to PbN units are also heavier than those observed in rats after stimulation of the LH (48%), CeA (67–73%), or gustatory cortex (GC: 31–67%) (18, 51, 53). This indicates that PbN taste-responsive units receive much stronger influence from the BNST than from the other forebrain taste areas and insular cortex. Tracing studies have shown that the PbN receives intensive descending projections from the BNST. Abundant retrogradely labeled neurons were identified in the BNST after microinjection of a retrograde tracer (WGA-HRP) into the gustatory PbN. Labeled neurons were most numerous in the dorsal lateral and posterior lateral subnuclei in the rat BNST (56). In a recent study, using the Phaseolus vulgaris-leucoagglutinin method, Dong and Swanson (20) have shown that neurons in the BNSTPL project to the medial divisions of the PbN, particularly its central lateral and ventral lateral parts. The terminating site of axons from the BNST in the PbN corresponds with the area where we recorded our PbN units in the present study (see Fig. 1). These tracing studies demonstrate that the gustatory PbN area receives strong efferent projection from the BNST and are consistent with our electrophysiological results.
Characteristics of descending inputs from the BNST to the PbN.
One of the important findings of the present experiment was that every single orthodromic response of PbN taste neurons to the BNST stimulation was inhibitory. All 113 orthodromic responses evoked by delivering electrical pulses to the ipsilateral BNST, as well as 77 following contralateral BNST stimulation, were inhibitory.
Single pulse stimulation is an accurate electrophysiological technique to evaluate neuronal connectivity between the recording and stimulating sites. We have investigated the neural connectivity between the taste neurons in the NST or PbN and forebrain taste nuclei using identical or similar experimental protocols. The responses of gustatory neurons in the NST or PbN to forebrain stimulation have been composed of excitatory and inhibitory responses. Stimulation of the CeA orthodromically modulated 33.0% of taste-responsive NST cells; 30.3% were excitatory and 2.8% were inhibitory (46). In another experiment, 44.4% of NST taste neurons were excited and 6.1% were inhibited by LH stimulation (12). Taste neurons in the PbN also receive mixed inputs from forebrain gustatory nuclei (45). Stimulation of LH or CeA orthodromically activates 13.9% or 25.7% of the taste units in the PbN, respectively. Among these, LH stimulation produced 8.9% inhibitory responses, whereas 4.9% were excitatory. CeA stimulation, on the other hand, induced 13.9% inhibitory response, whereas 11.9% were excitatory. In prior studies, LH or CeA stimulation also induced more inhibition in the rat. (51, 53, 92). Taken together in the rat, there is a tendency for PbN gustatory cells to be more subject to inhibitory influence than excitation from the forebrain. In contrast, excitation was the dominant response after the LH or CeA stimulation in the hamster NST (10, 12, 46).
In a recent study, we have shown that stimulation of BNST orthodromically activates 28.7% of NST taste neurons; 6.9% were excitatory and 21.8% were inhibitory (89). Thus the BNST activation appears to have a more inhibitory influence on taste neurons both in the NST and PbN.
BNST projection neurons also receive inhibitory efferent projection from the BNST.
Another finding in the present study is that all BNST projection neurons in the PbN receive inhibitory efferent projections from the BNST as well. Because the intensity for antidromic invasion (means = 61.46 ± 6.99 μA) was lower than that for orthodromic activation (100 μA) in general, we tested whether BNST projection neurons receive centrifugal modulatory influence by elevating the stimulus intensity to 100 μA. We confirmed that all BNST projection neurons were also inhibited with suprathreshold stimulation for antidromic activation.
Because the inhibition of these projection neurons was induced by suprathreshold stimulation after antidromic spikes, we could not rule out the possibility that the inhibition was due to an absolute refractory period caused by antidromic spike or recurrent inhibition (see Fig. 6C). To examine the relationship between the inhibition and the antidromic spike, five projection neurons were further tested with the stimulus intensities lower than the threshold for antidromic activation. Of five neurons tested, three units exhibited inhibitory responses with stimulus intensity below threshold for antidromic invasion (see Fig. 6D). Because the inhibition was induced without eliciting antidromic spikes, it is likely that antidromic action potentials were not involved in this suppression of spontaneous firing of PbN cells; rather, it suggests that there is a reciprocal loop between taste units in the PbN and BNST. The results of this experiment demonstrate that BNST-projection PbN taste neurons not only send their axons to the BNST but also receive inhibitory descending inputs from the BNST. It further demonstrates the importance of BNST on processing taste information and modulating PbN taste neuronal activity.
Activation of BNST suppresses taste response of the PbN neurons.
As described, the majority of PbN units were orthodromically inhibited by stimulation of the BNST, including every single BNST-projection neuron. To further investigate whether this vast inhibitory input from the BNST actually suppresses PbN neurons' taste response, two separate experiments were conducted. In the first experiment, we examined whether a single-pulse stimulation of BNST inhibits taste stimulus-elicited neuronal activity in the PbN. Because the neurons that we tested in this experiment were low spontaneous firing units, these units were continuously driven by taste stimulation during the whole testing period. As shown in Fig. 7, without taste stimulation it was not possible to examine whether these low firing neurons receive inhibitory input from the BNST (Fig. 7G). With taste stimulation, we could examine the inhibitory responses. In addition, the spikes appearing in the PSTHs, created with taste stimulation (Fig. 7, A–F), were mostly taste-driven spikes. Therefore BNST-induced inhibition represents the suppression of both spontaneous and taste stimulus-elicited responses. To avoid sampling bias, we also examined the influence of BNST activation on taste responsiveness with another subset of PbN neurons, including high spontaneous discharge neurons. Here, we compared the taste responses before and during electrical train stimulation of the BNST in separate taste trials. The taste response was significantly reduced (45.2%) during BNST activation (Fig. 8).
BNST-induced inhibition of taste response was nonspecific for taste quality. In this set of experiments, we stimulated the tongue with all effective taste solutions: sucrose, NaCl, citric acid, and QHCl, as well as a mixture of effective taste stimuli. The activation of the BNST suppressed all PbN responses to four individual basic tastants and to a combination of these effective taste stimuli.
Modulation of taste responses of NST or PbN units by electrical stimulation of the forebrain gustatory areas has been previously reported. Electrical train stimulation of the BNST modulated the response of all cell types (sucrose-best, QHCl-best, etc.) in the NST (89). Activation of the CeA (46) and LH (12) also modulated taste responses in the NST and PbN neurons (45), regardless of best-stimulus. Modulation of PbN neurons' taste response by forebrain activation has also been demonstrated by another group, although the protocol of forebrain stimulation was different (51, 53). In the rat, the influence of forebrain stimulation on taste responses was also non-specific, but the net effect sharpened the response to best-stimulus of that neuron (51, 53). The nonspecificity of the CeA or GC on the PbN gustatory responses was reported in another study (92). In this study, CeA or GC stimulation generally changed gustatory responses nonspecifically in the control and conditioned taste aversion groups, except for amiloride-sensitive NaCl-best neurons following the CeA stimulation.
Implication for ingestive behaviors.
The BNST has been less studied as a member of the gustatory system, compared with LH or CeA, although it has been long known as one of the gustatory nuclei in the ventral forebrain (31, 80). The LH and CeA are involved in regulation of feeding behavior such as sodium appetite and conditioned taste aversion (52, 79). The gustatory responses of some PbN neuron are changed by treatments that induce sodium appetite or conditioned taste aversion (83–85, 92). Recent physiological studies, demonstrating that taste responses of the NST or PbN neurons are affected by the stimulation of the LH and/or CeA, suggest that neural connections between LH or CeA and PbN gustatory neurons have a role in these physiological changes related to ingestive behavior (45, 51). The involvement of the BNST in the control of sodium appetite is indicated in some studies (38, 77, 103). The present results suggest that the BNST also plays a role in the change of gustatory response associated with these ingestive behaviors.
Although it is known that stress influences feeding behavior, the effect of stress on gustatory response in the taste system has not been investigated (23, 24, 75, 91, 98, 102). The BNST is believed to be involved in modulating behavioral responses to stress (26). Exposure to stress induces c-fos (47, 54) and GABA-synthesizing enzymes in the BNST (3, 6). Lesions of the BNST produce excessive weight gain (41).
Corticotropin-releasing factor (CRF) has a major role in mediation of behavioral responses to stressors (42). Neuroanatomically, both dorsal lateral and ventral lateral BNST contain rich populations of CRF neurons (17, 57, 76). Prior studies demonstrated that CRF neurons in the BNST are GABAergic (16) (97) and some CRF cells project to the PbN (57).
The relationship of the PbN taste neurons with the BNST stands out among the relationships between brainstem gustatory neurons and forebrain gustatory nuclei in two points: one is that most of the PbN taste neurons are under the influence of the BNST, and the other is that the descending projection from the BNST to the PbN is exclusively inhibitory. Taken together with previous studies, these results suggest that this neural substrate may be involved in stress-induced alteration of appetite and feeding behavior through GABAergic mechanisms on PbN taste neurons.
This work was supported in part by The National Institute on Deafness and Other Communication Disorders Grant DC006623 to C.-S. Li.
The authors thank Drs. Richard W. Clough, Ronald A. Browning, and David G. King for their valuable comments on the manuscript. We also thank Sara Sanders for the processing and photographing the brain tissue.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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