Chem. Senses 28: 155-171,
2003
© Oxford University Press 2003
RESEARCH PAPERS |
Descending Influences from the Lateral Hypothalamus and Amygdala Converge onto Medullary Taste Neurons
Department of Anatomy & Neurobiology and Program in Neuroscience, University of Maryland School of Medicine, Baltimore MD 21201, USA 1 Present address: Department of Behavioral Science, Pennsylvania State University, College of Medicine, Hershey PA 17033, USA 2 Present address: Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA
Correspondence to be sent to: Dr David V. Smith, Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, 855 Monroe Avenue, Suite 515, Memphis, TN 38163, USA. e-mail: dvsmith{at}utmem.edu
Abstract
The lateral hypothalamus (LH) and the central nucleus of the amygdala (CeA) exert an influence on many aspects of ingestive behavior. These nuclei receive projections from several areas carrying gustatory and viscerosensory information, and send axons to these nuclei as well, including the nucleus of the solitary tract (NST). Gustatory responses of NST neurons are modulated by stimulation of the LH and the CeA, and by several physiological factors related to ingestive behavior. We investigated the effect of both LH and CeA stimulation on the activity of 215 taste-responsive neurons in the hamster NST. More than half of these neurons (113/215) were modulated by electrical stimulation of the LH and/or CeA; of these, 52 cells were influenced by both areas, often bilaterally. The LH influenced more neurons than the CeA (101 versus 64 cells). Contralateral stimulation of these forebrain areas was more often effective (144 responses) than ipsilateral (74). Modulatory effects were mostly excitatory (102 cells); 11 cells were inhibited, mostly by ipsilateral LH stimulation. A subset of these cells (n = 25) was examined for the effects of microinjection of DL-homocysteic acid (DLH), a glutamate receptor agonist, into the LH and/or CeA. The effects of electrical stimulation were completely mimicked by DLH, indicating that cell somata in and around the stimulating sites were responsible for these effects. Other cells (n = 25) were tested for the effects of electrical stimulation of the LH and/or CeA on the responses to taste stimulation of the tongue (32 mM sucrose, NaCl and quinine hydrochloride, and 3.2 mM citric acid). Responses to taste stimuli were enhanced by the excitatory influence of the LH and/or CeA. These data demonstrate that descending influences from the LH and CeA reach many of the same cells in the gustatory NST and can modulate their responses to taste stimulation.
Key words: centrifugal modulation, gustation, solitary nucleus, taste processing
Introduction
In the central gustatory pathway, there are reciprocal connections between
the brainstem and several areas of the forebrain. The lateral hypothalamus
(LH) and the central nucleus of the amygdala (CeA) are nuclei in the ventral
forebrain that contain taste-responsive neurons
(Norgren, 1970
;
Nishijo et al.,
1998). Sensory projections to the LH and CeA convey gustatory,
gastrointestinal, and cardiovascular information from the peripheral nervous
system via the nucleus of the solitary tract (NST) and the parabrachial nuclei
(PbN) in the brainstem (Norgren,
1976; Voshart and van der
Kooy, 1981; Ter Horst et
al., 1989; Halsell,
1992; Bernard et al.,
1993; Bester et al.,
1997). The LH and CeA also receive input from the cortex, thalamus
and other nuclei in the ventral forebrain and there is reciprocal connectivity
between the LH and CeA (Ottersen,
1980; van der Kooy et
al., 1984; Allen et
al., 1991; Turner and
Herkenham, 1991; Nakashima
et al., 2000). Both of these forebrain areas send
descending axons to the PbN and rostral NST in both rats and hamsters
(Hosoya and Matsushita, 1981;
Berk and Finkelstein, 1982;
van der Kooy et al.,
1984; Whitehead et
al., 2000).
The LH and CeA have important roles in the regulation of homeostasis,
ingestive behavior, reward and motivation
(Stellar and Stellar, 1985;
Rolls, 1999;
Aggleton, 2000). Stimulation of
the LH enhances food intake (Coons et
al., 1965; Frank et
al., 1982; Shiraishi,
1991), whereas lesions of this area elicit opposite effects: from
inhibition of feeding to aphagia, depending on the extent of the lesion
(Grossman et al.,
1978; Grossman and Grossman,
1982; Arase et al.,
1987; Bray, 1991).
LH stimulation also increases blood glucose levels
(Atrens et al., 1984),
glucose utilization in the PbN (Roberts,
1980) and basal metabolism
(Ruffin et al.,
1995). The CeA has been implicated in the regulation of sodium
intake (Galaverna et al.,
1992,
1993;
Seeley et al., 1993),
aversive taste (Touzani et al.,
1997) and, along with the basolateral amygdala, conditioned taste
aversion learning (Yamamoto et
al., 1994; Tucci et
al., 1998). Although many investigators have studied the
functions of these areas in relation to feeding, the underlying neural
mechanisms relating activity in these forebrain nuclei to gustatory processing
are as yet unknown.
Descending projections from forebrain areas could provide a substrate
through which physiological or experiential factors alter taste responsiveness
of brainstem neurons. The taste-evoked activity of some NST neurons is altered
by physiological factors associated with satiety, such as blood insulin and
glucose levels (Giza and Scott,
1983,
1987; Giza et al.,
1992,
1993) and gastric distension
(Glenn and Erickson, 1976),
and by taste aversion (Chang and Scott,
1984) or preference (Giza
et al., 1997) learning. Indeed, previous
electrophysiological studies have demonstrated that electrical stimulation of
the LH modulates neuronal activity in the NST
(Bereiter et al.,
1980; Matsuo et al.,
1984; Murzi et al.,
1986) and we have recently shown that taste responses in half of
the gustatory cells in the hamster NST are modulated by stimulation of the LH
(Cho et al., 2002b).
Stimulation of the CeA alters taste responses in the rat PbN
(Lundy and Norgren, 2001) and
in the hamster NST (Li et al.,
2002). The present study employed bilaterally implanted electrodes
in both the LH and CeA to determine whether the descending influences from
these areas converge on the same cells or modulate different NST neuronal
populations.
Materials and methods
Animals and surgery
Young adult male Syrian golden hamsters (Mesocricetus auratus),
weighing between 150 and 250 g (n = 63), were deeply anesthetized
with urethane (1.7 g/kg, i.p.) and additional anesthetic was given as needed
during the course of each experiment. The animal was tracheotomized and a
polyethylene tube was inserted into the trachea to allow the animal to breathe
freely. Body temperature was maintained at 37°C (± 1°C) with a
heating pad. To place the stimulating electrodes, the animal's head was
positioned in a stereotaxic instrument (SR-6N, Narishige Scientific
Instruments, Tokyo, Japan) with the incisor bar at the same level as the
interaural line. The tissue overlying bregma was cut along the midline and
separated. Two holes were drilled on each side of the skull to permit access
to the LH and CeA. The coordinates for LH and CeA with the brain at this
angle, which were determined histologically in previous experiments
(Cho et al., 2002b;
Li et al., 2002),
were 0.5 mm caudal to bregma, 1.8 mm lateral to the midline, and 
7 mm
ventral to the surface of the brain for the LH. Those for the CeA were 0.6 mm
posterior to bregma, 3.9 mm lateral to the midline and 5.5 mm ventral to
the brain surface.
Stimulating electrodes were implanted bilaterally and fixed in place with
dental cement. Each bipolar stimulating electrode was composed of an insulated
140 µm-diameter stainless steel wire inside 26-gauge stainless steel
tubing. The components of this concentric electrode, except for the tip area,
were insulated with Epoxylite 6001 (Epoxylite Corp., Irvine, CA). For
stimulating the LH or CeA chemically, a double-barrel glass micropipette (tip
diameter = 35 µm) was glued to each stimulating electrode with its tip
positioned 0.2 mm above the inner wire of the electrode. After fixing the
stimulating electrodes in place, the animal was placed in a non-traumatic
headholder (Erickson, 1966)
with the head angled nose-downward 27° below the horizontal to straighten
the brainstem and minimize brain movement associated with breathing
(Van Buskirk and Smith, 1981).
The tissue over the occipital bone was cut along the midline and separated and
the occipital bone and underlying dura were excised. The posterior portion of
the cerebellum was aspirated to expose the floor of the fourth ventricle for
3-4 mm anterior to the obex, allowing direct access to the NST.
Extracellular recording and taste stimulation
Action potentials of taste-responsive NST neurons were recorded with glass
micropipettes (tip diameter = 2 µm; resistance = 7-10 M
) filled with
a 2% (w/v) solution of Chicago Blue dye in 0.5 M sodium acetate. The taste
responsiveness of NST cells was initially determined by a change in neural
activity in response to anodal current pulses (50 µA, 0.5 s, 0.33 Hz)
applied to the anterior tongue, which drive electrolyte-sensitive taste fibers
of the chorda tympani nerve (Smith and
Bealer, 1975; Herness,
1987), and then confirmed with chemical stimulation of the tongue.
Extracellular action potentials were differentially amplified (NeuroLog
System, Digitimer Ltd, Hertfordshire, UK), discriminated with a dual
time-amplitude window discriminator (Bak DDIS-1, Bak Electronics Inc.,
Germantown, MD), displayed on oscilloscopes and monitored with an audio
monitor. Analog and digital signals were acquired with a Pentium computer,
configured with a CED 1401 plus interface board and Spike2 software
(Cambridge Electronic Design, Cambridge UK). The mean coordinates of the 215
taste-responsive cells recorded from the NST were 2.0 ± 0.1 (SD) mm
anterior to obex and 1.3 ± 0.1 mm lateral to the midline. These cells
were encountered from 0.5 to 1.2 mm below the surface of the brainstem.
Four stimuli representative of the sweet, salty, bitter and sour taste
qualities: 32 mM sucrose, NaCl and quinine hydrochloride (QHCl), and 3.2 mM
citric acid, respectively, were applied to the anterior tongue in random order
for each recorded neuron. These concentrations evoke roughly equal multi-unit
responses in the hamster NST (Duncan and
Smith, 1992). The stimuli were delivered by a gravity flow system
composed of a two-way solenoid-operated valve connected via tubing to a
distilled water rinse reservoir and a stimulus reservoir. The stimulation
sequence, during which the response of the neuron was recorded, was a
continuous flow (at 2 ml/s) initiated by the delivery of 5 s of distilled
water, followed by 10 s of stimulus, and then by 5 s of distilled water.
Following each taste stimulus, the tongue was rinsed with distilled water
(>50 ml) and individual stimulations were separated by at least 2 min to
avoid adaptation effects (Smith and
Bealer, 1976). Each cell was categorized as responding best to
sucrose, NaCl, citric acid or QHCl on the basis of its taste response
profile.
LH and CeA stimulation and classification of forebrain-responsive neurons
After each NST cell was characterized for its taste responsiveness, 50-200
constant-current square pulses (
0.1 mA, 0.5 ms), generated from an
isolated stimulator (Grass S88, Grass Instrument Co., Quincy MA), were
delivered through each stimulating electrode (ipsilateral and contralateral)
at a frequency of 0.33 Hz to examine the effect on the ongoing spontaneous
activity of the cell. The stimulation intensity used for each cell (range =
50-100 µA) was set to the lowest intensity that would produce a discernible
orthodromic response. Peri-stimulus time histograms (PSTHs) were created from
the acquired data on each NST cell; action potentials associated with single
pulse stimulation were accumulated over a 1 s period for 50-200 sweeps. The
200 ms preceding stimulation were defined as the baseline period. The mean and
standard deviation of the firing rate (impulses/1 ms bin) during this 200 ms
period were determined. The action potentials occurring during a period of 800
ms after LH or CeA stimulation were analyzed to determine the effects of the
stimulation on NST activity. An excitatory response was defined as an epoch of
at least five consecutive 1 ms bins with a mean value of 2 SD above the
baseline mean, which defines a mean response with a probability of <0.05.
The onset latency of the excitatory response was defined as the time at which
the firing rate became at least twice the average baseline spontaneous rate.
Inhibitory responses were defined as at least 20 consecutive bins in which the
mean value was <50% of the baseline firing rate. Low rates of spontaneous
activity make a criterion for inhibition based on variability difficult to
achieve; the criterion applied here calls for a large and sustained decrease
in activity. Each taste-responsive cell for which an excitatory or inhibitory
effect could be defined was categorized as forebrain-responsive. To specify
the effective forebrain site, each cell was also classified as an LH- or CeA-
or LH/CeA-responsive neuron.
Antidromic activation of an NST neuron was defined by the standard criteria
of constant latency, high-frequency (>250 Hz) following and collision
(Bishop et al., 1962;
Cho et al., 2002a).
Antidromically activated action potentials were observed in three cells in
response to ipsilateral LH stimulation. Two of these cells also responded
orthodromically to contralateral LH stimulation; they were categorized as
LH-responsive neurons. The other cell produced only antidromic spikes
following stimulation of the ipsilateral LH and did not respond to stimulation
of the other three sites. This cell was categorized as a non-responsive
cell.
To examine the effects of electrical stimulation (ES) of the LH or CeA on the responses of NST cells to taste stimuli, responses of a subset of the forebrain-responsive neurons were recorded while trains of constant-current square pulses (100 Hz, 0.2 ms; at 0.9 x the lowest intensity that would produce an orthodromic response in each cell) were delivered to the LH or CeA during stimulation of the tongue with tastants (duration = 15 s, starting 5 s prior to taste stimulation).
For chemical stimulation of the LH or CeA, one barrel of the micropipette
attached to the stimulating electrode was filled with 10 mM DL-homocysteic
acid (DLH; Aldrich Chemical Co., Milwaukee WI) in buffered physiological
saline (pH 7.4) and the other with saline. DLH is an excitatory amino acid
analog, which presumably excites neuronal somata but not fibers of passage
(Goodchild et al.,
1982; Yang and Coote,
1998). Pressure pulses (30 psi, 10 ms) from a Picospritzer II
(General Valve Co., Fairfield, NJ) were used to trigger the microinjections,
which produced an injection volume of 50 nl
(Smith and Li, 2000).
Physiological saline served as a control for possible pressure effects of
microinjection.
At the end of each experiment, the last recording site in the NST was marked with Chicago Blue dye by passing an intermittent (10 s on/off) 10 µA cathodal current through the recording electrode for 10 min. The stimulation sites in the ventral forebrain were marked by passing a 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 an overdose of urethane and perfused through the heart with 4% formaldehyde containing 3% potassium ferrocyanide and ferricyanide. The brain was removed and fixed; frozen sections (40 µm) were cut in the coronal plane and stained with Neutral Red.
Data analysis
Responses of NST cells to taste stimuli were quantified by subtracting the 5 s pre-stimulus baseline from the first 5 s of the evoked response to yield a mean net response (mean impulses/s over 5s). Responses are reported as means ± SEM. Differences in mean firing rates between forebrain-responsive and non-responsive neurons and among taste stimuli were compared using ANOVA. For the effect of DLH (or saline) on spontaneous activity of the NST taste cells, the mean firing rate in each cell over a 1 min period before drug administration was compared with the mean firing rate during a comparable period following the drug administration using t-tests. The effect of ES on the mean firing rate to taste stimuli and differences in excitatory latency were also compared using t-tests. Comparisons of the number of neurons in each category; forebrain-responsive versus non-responsive, and excitatory versus inhibitory, were made using the chi-square test.
Results
Histology
Recording sites in the NST and stimulating sites in the ventral forebrain were examined histologically in 43 of the 63 animals; representative examples are shown in Figure 1. The iron deposits at the tips of the stimulating electrodes in the ipsilateral LH and CeA are shown in Figure 1A. The electrode in the LH was positioned dorsomedial to the optic tract (ot) and dorsolateral to the anterior hypothalamic (AH) area and ventromedial hypothalamic (VMH) nucleus. The tip of the electrode in CeA was positioned in the central amygdaloid nucleus, capsular division (CeC), which corresponds to the lateral central amygdaloid nucleus in rats, dorsal to the basolateral amygdaloid nucleus, anterior (BLA).
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A recording site in the NST is shown in
Figure 1B, located medial to
the solitary tract (st), most likely in the rostral central (RC) or rostral
lateral (RL) subdivision. Cells were recorded from the NST near the
rostrocaudal level at which the dorsal cochlear (DC) nucleus is first apparent
on the dorsolateral margins of the medulla, which is the area receiving its
predominant gustatory input from the VIIth nerve
(Whitehead and Frank, 1983;
Whitehead, 1988). We could not
unambiguously assign each recorded cell to a nuclear subdivision within the
NST, although all of the cells appeared to be in the region of the NST
corresponding to the RC or RL subdivisions
(Whitehead, 1988).
Stimulation sites in these 43 animals are reconstructed on standard atlas
sections (Morin and Wood,
2001) in Figure 2.
Individual electrode placements in the LH and CeA are depicted in
Figure 2A-F, arranged from
rostral (A) to caudal (F). Sites evoking excitatory responses in gustatory
cells of the NST are shown as filled circles, those producing inhibitory
responses as half-filled circles, and those that did not alter NST activity as
open circles. The three ipsilateral sites in the LH from which antidromic
action potentials were activated are shown as open triangles. The majority of
effective sites in the LH were at the level of the VMH, posterior to the AH
nucleus (Figure 2D,E). The
effective sites in the CeA were most often located in the CeC, rather than the
central amygdaloid nucleus, medial (CeM;
Figure 2A,B). The position of
the last cell to be recorded in each animal was marked with Chicago Blue dye
(as in Figure 1B) and the
positions of these cells (n = 43) are depicted in
Figure 2G on a standard atlas
section of the medulla at the level of the DC
(Morin and Wood, 2001).
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The LH and CeA orthodromically activate many of the same NST taste neurons
The responses of 215 taste-responsive neurons were recorded from the NST. Of these, the activity of 113 (52.6%) was orthodromically modulated by stimulation of nuclei in the ventral forebrain (forebrain-responsive neurons). Effective sites were one or a combination of the four stimulated nuclei: ipsilateral and contralateral LH and CeA (Table 1). Sixty-one of these 113 neurons (53.9%) were activated by more than one forebrain site and 15 cells (13.3%) were responsive to stimulation of all four sites. The other 102 taste-responsive neurons did not respond to any of these stimulating sites (non-responsive neurons), except one neuron, which was antidromically invaded from the ipsilateral LH and was categorized as a non-responsive neuron.
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Each cell was also classified into one of four groups according to its most
effective (best) taste stimulus. Although more QHCl- and citric acid-best
neurons and fewer sucrose-best cells occurred in the forebrain-responsive
category, the proportion of cells in each best-stimulus category was not
significantly different between the forebrain-responsive and non-responsive
neurons (
2 = 7.186, df = 3, P = 0.066). As shown in
Table 2, among the 113
forebrain-responsive neurons, 49 (43.4%) responded only to stimulation of the
ipsilateral and/or contralateral LH (LH-responsive neurons). In comparison, 12
cells (10.6%) responded only to stimulation of the CeA (CeA-responsive
neurons). The activity of the other 52 neurons (46.0%) was altered by
stimulation of both the LH and CeA (LH/CeA-responsive neurons).
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Electrical stimulation of the ipsilateral LH produced antidromically
activated action potentials in three cells, all of which met the three
criteria for antidromic activation (Bishop
et al., 1962; Cho
et al., 2002a). Two of these cells were NaCl-best and
also produced orthodromically induced action potentials following
contralateral LH stimulation; these two cells were classified as LH-responsive
neurons. The latencies of these antidromic spikes were 17 and 23 ms and the
thresholds were 13 and 10 µA, respectively. The other neuron was QHCl-best
and did not show an orthodromic response to the stimulation of any site; this
cell was classified as a non-responsive neuron, with an antidromic latency of
27 ms and a threshold of 75 µA. The mean antidromic latency across these
three cells was 23.3 ms.
The activity of significantly more (101) neurons was modulated by LH
stimulation than by CeA stimulation (64 neurons) (2 = 8.297,
df = 1, P < 0.01). Contralateral stimulation was significantly
more often effective than ipsilateral: 74 responses were induced in NST
neurons by simulation of the ipsilateral LH and/or CeA, whereas 144 were
evoked by contralateral stimulation (2 = 22.477, df = 1,
P < 0.001). If the comparison was limited to responses to LH
stimulation: 39 responded to ipsilateral stimulation versus 91 to
contralateral, this difference was also significant: (2 =
20.800, df = 1, P < 0.001), whereas it was not significant for
responses to CeA stimulation: 35 responded to ipsilateral stimulation versus
53 to contralateral (2 = 3.682, df = 1, P = 0.055).
Among the 113 forebrain-responsive neurons, only 11 cells demonstrated an
inhibitory response to stimulation of the LH and/or CeA
(Table 1, parentheses). The
other 102 taste-responsive neurons were excited by forebrain stimulation
(2 = 73.283, df = 1, P < 0.001).
Electrophysiological features of NST responses to forebrain stimulation
Orthodromically induced action potentials and the corresponding
peri-stimulus time histograms (PSTHs) for two cells are illustrated in
Figure 3. Action potential
traces and PSTHs in Figure
3A,B,E,F are from a cell that produced orthodromic spikes
following stimulation of all four sites. The occurrence of action potentials
in Figure 3A were concentrated
in a period from 14 to 24 ms after ipsilateral LH stimulation (see also the
PSTH in Figure 3E), whereas
stimulation of the contralateral LH evoked some spikes at 9 ms, inhibited
impulse discharge for a 20 ms period and then induced more orthodromic action
potentials over a period of 50 ms
(Figure 3B,F). The PSTH in
Figure 3F shows the occurrence
of additional evoked spikes more than 100 ms after stimulation.
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Figure 3C,D,G,H depicts
responses of another cell that responded to both the contralateral LH and CeA.
The occurrence of evoked spikes following stimulation of the contralateral CeA
was limited to a 2 ms span, 10-11 ms after stimulation
(Figure 3C,G). The evoked
spikes following contralateral LH stimulation were grouped at 5 and 11 ms
after stimulus onset (Figure
3D,H). As in our previous experiments on the LH and CeA
(Cho et al., 2002b;
Li et al., 2002),
orthodromic response durations were bimodally distributed, involving either
single spikes on each sweep, distributed over sweeps within <10 ms (as in
Figure 3C,G) or more than one
spike per sweep, spread over a considerable period (>10 ms, as in
Figure 3B). Such long-duration
responses were more often observed (n = 124) than single impulses
(n = 81), as in Figure
3C (2 = 9.020, df = 1, P < 0.001).
Of 13 inhibitory responses from 11 taste neurons, 9 were observed after
stimulation of the ipsilateral LH, which was the most effective site for
producing inhibitory responses (2 = 13.769, df = 3, P
< 0.001). The spontaneous activity of two cells was inhibited by two sites:
ipsilateral and contralateral LH and ipsilateral LH and CeA, respectively.
Four of these inhibited cells also were excited by stimulation of other sites.
No inhibitory response was observed from any of the 15 neurons that responded
to all four sites. The inhibition of spontaneous firing in these cells started
within 30 ms after stimulation and the duration of inhibition ranged from 28
to 140 ms (mean = 64.92 ± 8.89 ms). An example of an inhibitory
response is shown in the PSTH of Figure
4A; starting 26 ms after ipsilateral LH stimulation, spontaneous
firing was eliminated for 58 ms.
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For five cells that were initially excited by forebrain stimulation, an inhibitory period was observed following orthodromically evoked spikes (as in Figure 3B,F). In the response depicted in Figure 4B, two peaks of evoked action potentials were separated by a brief inhibitory period and another longer period of inhibition followed after the second peak in response to ipsilateral LH stimulation. These five cells were categorized as excited cells because their initial response was excitatory. DLH injection into the ipsilateral LH of the cell depicted in Figure 4B produced only an excitatory response (see Figure 8B below).
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In seven cells, the occurrence of evoked action potentials formed two peaks, but without an inhibitory period between them. In three of these cells and in the five cells mentioned above, the evoked spikes following the initial excitatory impulses were more widely temporally distributed than the initial excitation (as in Figures 3B,F and 4B). In the responses of the other four cells, two distinct peaks of spikes were closely separated in time (5-10 ms, as Figure 3D). These four responses were all induced following stimulation of the contralateral LH.
Relationship between gustatory responses and stimulating sites
The taste-evoked responses of the 113 forebrain-responsive and the 102 non-responsive NST neurons are shown in Figure 5A,B, respectively. Within each group, cells are arranged along the abscissa into best-stimulus categories and within a category by the magnitude of the response to their best stimulus. These individual responses show that all best-stimulus neuron types were represented and that the firing rates of the cells were similar in both forebrain-responsive and non-responsive groups. The overall mean firing rates to gustatory stimulation were not significantly different between the forebrain-responsive and non-responsive neurons [F(1, 852) = 3.007, P = 0.083], but there was a significant interaction between the responses to taste stimuli and forebrain responsiveness [F(3, 852) = 4.391, P < 0.01].
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NST taste cells were categorized into four groups as mentioned above: LH-responsive (n = 49), CeA-responsive (n = 12), LH/CeA-responsive (n = 52), and non-responsive neurons (n = 102). The response to each taste stimulus in each of these groups is shown in Figure 6. The mean net responses across all four taste stimuli did not differ among the four groups (F[3, 844] = 1.643, P = 0.178). Taste stimuli produced mean responses that differed from one other [F(3, 844) = 8.240, P < 0.001] and there was a significant interaction between stimulus and effective site [F(9, 844) = 2.467, P < 0.01]. NST neurons that were modulated only by the CeA had greater responses to citric acid and QHCl and smaller responses to sucrose and NaCl than did the other groups (Figure 6). The greater number of C-best and Q-best neurons in CeA-responsive group relative to the S- and N-best cells (see Table 2) may have contributed to the significant interaction between the response to taste stimuli and responsive group. There was a smaller response to both sucrose and NaCl in all three forebrain-responsive groups than in the non-responsive neurons.
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Latency of excitatory responses
The latency of orthodromic action potentials varied from 4 to 85 ms after stimulation of the LH and/or CeA. The mean latency of excitatory responses was 22.08 ± 0.87 ms. The mean latencies associated with each of the four stimulation sites are shown in Figure 7A, along with the mean latency for the three cells that were antidromically activated from the ipsilateral LH. The mean latency after LH stimulation (21.03 ± 1.04 ms, n = 120) did not differ from that following CeA stimulation [23.55 ± 1.47 ms, n = 85; F(1, 201) = 1.391, P = 0.240], whereas there was a significant difference in latency between ipsilateral (mean = 27.49 ± 1.88 ms, n = 63) and contralateral stimulation [mean = 19.68 ± 0.86 ms, n = 142; F(1, 201) = 17.815, P < 0.001]; there was no significant interaction [F(1, 201) = 1.099, P = 0.296]. Thus, stimulation of the contralateral side, either LH or CeA, evoked orthodromic action potentials with a shorter latency than stimulation of the ipsilateral side. The difference between the latencies to stimulation of each side is also seen in a comparison of the latencies from neurons that responded bilaterally to the LH or/and CeA (t = 2.58, df = 25, P < 0.05 for the LH responses; t = 4.56, df = 23, P < 0.001 for the CeA responses). For the fifteen neurons that produced excitatory response to all four sites, the difference between the latencies of ipsilateral (mean = 27.77 ± 3.31 ms) and contralateral stimulation (mean = 17.07 ± 2.28 ms) was significant (t = 3.511, df = 29, P < 0.001), but there was no difference between the latencies for LH (mean = 22.70 ± 2.90 ms) and CeA stimulation (mean = 22.13 ± 3.12; t = 0.161, df = 29, P = 0.874).
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The mean latency of long-duration facilitations (26.54 ± 1.14 ms, n = 124) was significantly longer than that of single-impulse responses (15.25 ± 0.90 ms, n = 81; t = 7.115, df = 203, P < 0.001, Figure 7B). The correlation between latency and duration was significant (r = +0.470, df = 203, P < 0.01), showing that short latency responses were more likely to be of short duration. However, there was no significant interaction between the response type (long-duration facilitation versus single impulses) and the effective sites [F(1, 201) = 0.389, P = 0.533] or effective sides [F(1, 201) = 1.289, P = 0.258]. Thus, long-duration facilitations were common in the excitatory response to both LH and CeA stimulation, regardless of which side was stimulated.
DLH injection mimics the response to single-pulse stimulation
For 25 of the forebrain-responsive neurons (22 excitatory and three
inhibitory neurons; one of three inhibitory neurons also showed excitatory
responses after stimulation of other sites as shown in
Figure 8A), the effect of 10 mM
DLH microinjection into the stimulating sites was compared with the effect of
saline injection. For six neurons, DLH injections were made into more than two
sites. Figure 8A shows the
effects of DLH on the spontaneous activity of such a cell. Electrical
stimulation of the contralateral LH induced an excitatory response, whereas
contralateral CeA stimulation inhibited the spontaneous firing of this cell.
The injection of DLH, but not saline, into the contralateral LH increased the
spontaneous firing over a period of 2 min. In contrast, DLH injection
into the contralateral CeA inhibited the spontaneous activity. Saline
injection was without effect.
The response to electrical stimulation of the ipsilateral LH of the cell in
Figure 8B was demonstrated
above (Figure 4B). Although the
orthodromic response to single ipsilateral LH shocks showed an inhibitory
effect following orthodromically evoked action potentials, the DLH effect was
only excitatory (Figure 8B). This cell responded to injections of DLH into all four sites. The effect of
DLH (into the ipsilateral LH) lasted 80 s but was no less than the
effects of DLH injection into the other sites; the responses to electrical
stimulation of the other three sites were purely excitatory.
For the neurons showing excitatory responses to DLH, the mean firing rate over a 1 min period after DLH injection (9.60 ± 1.41 impulses/s, n = 32) was significantly greater than that of a corresponding period before injection (2.16 ± 0.77 impulses/s; t = 8.358, df = 31, P < 0.001, Figure 8C). In contrast, the change in mean firing rate before (2.17 ± 0.82 impulses/s) and after (2.03 ± 0.75 impulses/s) saline injection was not significant (t = 1.822, df = 31, P = 0.078). There was no case in which DLH injection reduced the spontaneous activity of cells that were excited by electrical stimulation. There was no significant relationship between the increase in firing rate and injection site (F [3, 28) = 1.333, P = 0.284). Three of the 11 neurons that showed an inhibitory response to electrical stimulation were tested for the effect of DLH, which reduced the spontaneous rate in every instance. The reduction of mean spontaneous rate was -95.0, -73.8 and -97.5%, respectively; the effect of DLH was significant compared to saline (t = 56.540, df = 2, P < 0.001) for inhibitory responses (Figure 8D). Thus the injection of DLH mimicked the effect of electrical stimulation, producing either excitation or inhibition as occurred following single-pulse electrical stimulation.
Forebrain stimulation alters gustatory responses
Twenty-five cells were tested for the effects of electrical stimulation (ES) of the LH and/or CeA on the responses to taste solutions. For this experiment, trains of constant-current square pulses (100 Hz, 0.2 ms, 15 s duration) were delivered to the forebrain site. Single-pulse stimulation of the LH and/or CeA induced excitatory orthodromic spikes in 23 of these 25 cells. From one to four taste stimuli were tested on each cell during forebrain stimulation. In all, 56 gustatory responses were recorded from these 23 neurons while ES was delivered to the stimulating electrode in the LH and/or CeA. The intensity of ES was below the threshold necessary to elicit orthodromic action potentials (0.9 x threshold for orthodromic spikes). All 56 gustatory responses were increased during ES.
Several examples of the effect of LH and/or CeA stimulation on gustatory responses are shown in Figure 9A,Figure 9E. For the three cells in Figure 9A-C, which showed excitatory responses following single-pulse stimulation, responses to taste stimuli were enhanced by ES. Although all four taste stimuli were not tested during ES, the response to the best stimulus, citric acid, was most enhanced by ES in Figure 9A (by 155%). In contrast, the increase in the response to the best stimulus was less than to the other stimuli in Figure 9B,C. For the cell in Figure 9B, although the response to sucrose was the greatest with or without ES, the proportional increase in the response to QHCl, which was the third-best stimulus, was greater (442%) than those to sucrose (74%) or citric acid (215%). Two cells that showed an inhibitory effect on spontaneous activity during electrical stimulation of the ipsilateral LH and CeA were also tested. For these cells, ES reduced the gustatory response to sucrose (by 100%) and QHCl (by 48.3%), which were the best stimuli for each. The taste responses of the Q-best cell are shown in Figure 9D.
|
|
For the NST cell in Figure 9E, all four sites were stimulated during the application of NaCl and sucrose, which were best and second-best stimuli, respectively. In this example, the response to sucrose, the second-best stimulus, was enhanced more in each instance than that to NaCl. In Figure 9F, the response to the best stimulus, QHCl, was more enhanced than that to the second-best stimulus, citric acid, by stimulation of both the contralateral LH and the contralateral CeA. The increased responses to taste stimulation did not differ among the four stimuli [F(3, 40) = 2.617, P = 0.064] nor the four stimulating sites [F(3, 40) = 1.999, P = 0.130].
The mean response to taste solutions without ES was 8.79 ± 0.97 impulses/s, which increased significantly to 17.39 ± 1.51 during ES of the LH and/or CeA (t = 9.746, df = 55, P < 0.001). Mean spontaneous firing during the pre-stimulus period of these 25 cells was 1.02 ± 0.24 imp/s, whereas mean firing during the pre-stimulus period with ES was 1.14 ± 0.26 (t = 0.367, df = 24, P = 0.717). The increase of each taste response with ES trains varied from 13% to 442%. On average, the ES trains more than doubled the response of cells to taste stimulation (mean = 145%), whereas the firing during the pre-stimulus period was not altered by the ES trains (Figure 9G). The comparison between the responses to best- and second-best stimuli (Figure 9H) showed that the increased rate of response to the second-best stimulus (mean = 195.3%) surpassed that to the best stimulus (mean = 118.0%; t = 3.292, df = 17, P < 0.005).
Discussion
Convergence of descending influences from the LH and CeA
The main finding in the present study was that more than half of the
gustatory neurons in the hamster NST were under the influence of the LH and/or
CeA. Many of these NST neurons received descending inputs from more than one
stimulating site. Of 113 forebrain-responsive neurons, 61 responded to
stimulation of two or more sites. Among them, 52 cells received input from
both the LH and CeA and 15 of them responded to stimulation of both of these
areas bilaterally. Whereas the LH and CeA have been implicated in different
aspects of feeding behavior and reactivity to taste stimuli, we would expect
that these two areas would differentially modulate taste processing. The
present data show that LH and CeA can exert an influence on many of the same
NST neurons, although over half the forebrain-responsive cells were
differentially affected by these two areas. Thus it is likely that taste
information would be differentially altered by descending influences of the LH
and the CeA. Cells that were exclusively modulated by the CeA responded more
to aversive stimuli (citric acid and QHCl) than to sucrose and NaCl
(Table 2,
Figure 6), which may relate to
the responsiveness of CeA neurons to the hedonic aspects of taste stimulation
(Nishijo et al.,
1998) and its role in conditioned taste aversion
(Yamamoto et al.,
1994). Determining the exact influence of these forebrain areas on
the coding of gustatory quality and hedonic value will require data on a wider
array of taste stimuli than that employed in the present experiment.
The LH exerted an influence on more NST gustatory neurons (47%) than did
the CeA (30 %), although 24% were modulated by both. The LH receives
information about taste and nutritional status and regulates the animal's
feeding behavior (Nicolaidis,
1981; Norgren,
1976; Ter Horst et
al., 1989). The CeA is involved in the neuronal circuitry
underlying sodium intake and conditioned taste aversion, which is dependent on
gustatory experience (Galaverna et
al., 1992; Seeley et
al., 1993; Tucci et
al., 1998). Our previous studies on the LH
(Cho et al., 2002b)
and the CeA (Li et al.,
2002) have shown similar proportions of LH- and CeA-responsive NST
neurons, although we did not investigate convergence in these earlier studies.
The convergence of LH and CeA inputs onto many of the same NST neurons
provides a substrate through which these forebrain areas can coordinate a
descending modulation of taste information in response to changes in
physiological state and experience. On the other hand, neurons influenced
solely by one or the other of these areas allow for differential modulation of
NST processing by the LH and the CeA.
Modulatory effects of forebrain stimulation on the activity of NST neurons
The modulatory effect of ventral forebrain gustatory nuclei on NST taste
neurons was predominantly excitatory. Stimulation of the LH and CeA induced
orthodromic excitation in 90% of the forebrain-responsive neurons and
inhibitory responses in only 10%. The most effective site for inducing
inhibitory responses was the ipsilateral LH, stimulation of which elicited an
inhibitory response in nine of 11 cells [see also
(Cho et al., 2002b)].
Because the present experiments were conducted on anesthetized animals, there
is some possibility that in awake, behaving hamsters there could be
differences in the proportions of excitatory and inhibitory influences. Within
the subset of taste-responsive neurons (25 cells) that was tested for the
effect of electrical stimulation on taste responses, all but two cells were
excited by forebrain stimulation. Electrical stimulation of the LH and/or CeA
enhanced all taste responses of those cells, whether the taste stimulus was
palatable or aversive. Although the response to no particular stimulus was
affected more than others, the responses to the second-best stimulus surpassed
that to the best stimulus in five cases out of 20, suggesting that the
representation of taste information can be altered by the ventral
forebrain.
The fact that most modulatory effects were excitatory and spread across the
various taste stimuli suggests that the primary function of the descending
input from the LH or CeA is to increase the overall sensitivity of a subset of
gustatory neurons to taste stimuli. Such an increase in the signal-to-noise
ratio should in turn increase the detectability of differences among taste
stimuli [see also (Cho et al.,
2002b)]. Berridge and Valenstein
(Berridge and Valenstein,
1991) reported that electrical stimulation of the LH increased
feeding behavior and also enhanced aversive taste reactions in rats. These
investigators suggested that LH stimulation influences feeding by potentiating
the difference between positive and aversive hedonic evaluations, not by
merely evoking pleasant sensations. The increased signal-to-noise ratio in the
present results suggest a substrate through which such increased
discriminability could occur.
Origin of descending modulation and possible pathways
The present study also demonstrated that the effect of stimulating LH or
CeA was due to the excitation of cells in or around these nuclei. Injection of
DLH, but not saline, mimicked the effect of electrical stimulation in the 25
forebrain-responsive neurons that were tested with this protocol. This result
makes less likely the possibility that the effect of electrical stimulation of
the LH or CeA was a result of stimulating fibers of passage. The
histologically reconstructed stimulating sites in the LH corresponded to the
area where stimulation induced feeding behavior
(Frank et al., 1982;
Sasaki et al., 1984;
Murzi et al., 1986)
and where some neurons responded to taste stimuli
(Norgren, 1970) in rats. The
CeA stimulating sites in the present study corresponded to the area where
lesions altered ingestive behavior (Kemble
et al., 1979;
Galaverna et al.,
1992), where stimulation antidromically activated gustatory PbN
neurons (Norgren, 1976), and
where some neurons responded to taste stimuli
(Azuma et al., 1984;
Uwano et al., 1995;
Nishijo et al., 1998)
in rats. Although it is not known whether the neurons in the LH or CeA that
modulate taste responses in the NST are also involved in motivating animals to
feed and/or are gustatory-responsive neurons themselves, they reside in the
same areas within these nuclei.
Another interesting finding was that NST taste neurons were influenced more
from the contralateral than the ipsilateral side. In comparison, anatomical
studies have demonstrated more descending projections from the ipsilateral LH
or CeA to the NST than from the contralateral
(van der Kooy et al.,
1984; Whitehead et
al., 2000). However, such neuronal tracing studies only
reveal direct projections. The discrepancy between the anatomical studies and
the present data implies that the forebrain influences neuronal activity of
NST neurons primarily through multisynaptic pathways. Although the present
results did not demonstrate which nuclei participate in the information flow
from the ventral forebrain to the NST, the reticular formation (RF) and caudal
NST are likely candidates. These areas also receive projections from the LH
and CeA (Berk and Finkelstein,
1982; van der Kooy et
al., 1984) and the RF sends axons to the motor nuclei
involved in orofacial function (Travers
and Norgren, 1983; Beckman and
Whitehead, 1991). The RF and caudal NST also reciprocally
communicate with the rostral NST, mostly through the ventral (V) and medial
(M) subdivisions (Travers,
1988; Beckman and Whitehead,
1991). Although the trajectories of the LH and CeA to the rostral
NST terminate more in the V and M subdivisions
(van der Kooy et al.,
1984; Halsell,
1998) than in the rostral central (RC) or rostral lateral (RL),
where most taste neurons were recorded in the present study, subdivisions in
the NST are interconnected with one another
(Beckman and Whitehead, 1991).
It is likely that some information from the LH and CeA is carried to the RF
and from there to the NST and also to various motor nuclei involved in feeding
behavior. Some descending influence may also occur through the PbN. A previous
investigation reported that 6% of gustatory neurons in the NST were
orthodromically activated from the ipsilateral PbN
(Cho et al., 2002a).
This small descending projection has also been seen anatomically
(Karimnamazi and Travers,
1998).
Taste information is transferred from the PbN bilaterally to the gustatory
nucleus of the thalamus, the parvicellular division of the ventroposteromedial
nucleus (VPMpc) (Norgren and Leonard,
1973; Halsell,
1992). Ventral forebrain nuclei send some axons to the NST on the
contralateral side (van der Kooy et
al., 1984; Whitehead
et al., 2000) and there are commissural connections
within the NST (Whitehead et al.,
2000). Antidromic activation of three NST neurons from the
ipsilateral LH suggests that some taste neurons in the NST project directly to
the LH, although direct projections to limbic forebrain areas have been shown
previously only from caudal NST in rats
(Ricardo and Koh, 1978). The
mean latency following stimulation of the LH or CeA on the contralateral side
was shorter than that on the ipsilateral side, implying that there may
typically be fewer synapses between the contralateral LH or CeA and the NST.
The present results imply that information from the contralateral side of the
forebrain plays an important role in the descending gustatory pathway,
although it is unknown where these descending inputs cross the midline en
route to the NST.
Acknowledgments
Thanks are due to Dr John D. Boughter Jr, for valuable comments on an earlier draft of this manuscript and to the National Institute on Deafness and Other Communication Disorders, whose support made this research possible (NIDCD Grant DC00066 to D.V.S.). These data were presented at the annual meeting of the Association for Chemoreception Sciences in Sarasota, Florida, April, 2002.
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