Stimulation of the paraventricular nucleus modulates the
activity of gut-sensitive neurons in the vagal complex
Xueguo
Zhang,
Ronald
Fogel, and
William E.
Renehan
Division of Gastroenterology, Henry Ford Health System, Detroit,
Michigan 48202
 |
ABSTRACT |
There is good evidence that stimulation of the
lateral hypothalamus excites neurons in the dorsal vagal complex (DVC),
but the data regarding the role of the paraventricular nucleus (PVN) in
vagal function are less clear. The purpose of this study was to clarify
the effect of PVN stimulation on the activity of neurons in the DVC. We
utilized extracellular and intracellular neuronal recordings with
intracellular injections of a neuronal tracer to label individual,
physiologically characterized neurons in the DVC of rats anesthetized
with pentobarbital sodium. Most (80%) of the gut-sensitive dorsal
motor nucleus of the vagus (DMNV) neurons characterized in this study
exhibited a change in activity during electrical stimulation of the
PVN. Stimulation of the PVN caused an increase in the spontaneous
activity of 59% of the PVN-sensitive DMNV neurons, and the PVN was
capable of modulating the response of a small subset of DMNV neurons to
gastrointestinal stimuli. This study also demonstrated that the PVN was
capable of influencing the activity of neurons in the nucleus of the
solitary tract (NST). Electrical stimulation of the PVN decreased the
basal activity of 66% of the NST cells that we characterized and
altered the gastrointestinal response of a very small subset of NST
neurons. It is likely that these interactions play a role in the
modulation of a number of gut-related homeostatic processes. Increased
or decreased activity in the descending pathway from the PVN to the DVC
has the potential to alter ascending satiety signals, modulate vago-vagal reflexes and the cephalic phase of feeding, and affect the
absorption of nutrients from the gastrointestinal tract.
vagus; gastrointestinal neurophysiology; dorsal motor nucleus of
the vagus; nucleus of the solitary tract
 |
INTRODUCTION |
IT IS WELL KNOWN that the hypothalamus modulates
endocrine and autonomic function. The hypothalamus influences many
aspects of autonomic function via its interactions with the pituitary, but there is also evidence that the hypothalamus may affect certain aspects of autonomic activity via direct descending projections to the
dorsal vagal complex (DVC). Retrograde (3, 22, 27) and anterograde (8,
13) tracing studies have shown that there is a substantial projection
from the paraventricular nucleus (PVN) and lateral hypothalamus (LH) to
the DVC, with most descending fibers terminating in the ipsilateral
brain stem. Electrophysiological data provide additional evidence that
the hypothalamus plays an important role in the regulation of the vagal
complex. The information regarding the role of the LH in the modulation
of vagal activity is particularly clear. It has been demonstrated that
stimulation of the LH excites neurons in the dorsal motor nucleus of
the vagus (DMNV; Ref. 12) and increases the activity of axons in the
hepatic branch of the vagus (32). It has also been shown that
electrolytic lesions of the LH result in a rapid and strong reduction
in vagal nerve activity (32, 33). It is reasonable to expect that
modulation of vagal neuronal activity by the LH would have a number of
effects on autonomic function. Indeed, this has been shown to be true, with numerous studies demonstrating that stimulation or ablation of the
LH alters activities such as gastric acid secretion, glucose utilization, and gastrointestinal motility (see Ref. 6 for review).
Although the data from the LH physiology experiments are fairly
consistent, studies that have examined the role of the PVN in vagal
function have produced results that are more difficult to reconcile.
Some investigators have reported data that are compatible with the
hypothesis that the neurons in the DMNV are excited by activation of
the PVN (9, 19, 29), but others have shown that the influence of the
PVN may be more complex. For example, Banks and Harris (2) have shown
that some DMNV neurons are excited by PVN stimulation (consistent with
the LH effect), whereas others exhibit a decrease in activity.
Furthermore, another group (33) has presented data indicating that
vagal activity is increased following lesions of the PVN (suggesting
that the PVN exerts a tonic inhibitory influence on the DMNV that is
eliminated by the destruction of the subnucleus). Thus, although there
is reason to believe that the PVN is capable of altering the activity
of vagal neurons, the precise nature of this interaction is not clear. There are a number of factors that may be contributing to this discrepancy. One potential explanation for the conflicting results obtained in the PVN experiments is the possibility that different subgroups of DMNV and nucleus of the solitary tract (NST) neurons have
different responses to stimulation of this region of the hypothalamus.
Thus one subset of DMNV neurons may be excited by PVN stimulation,
whereas another is inhibited by this input. If true, this scenario
might indicate that the PVN has the capability to modulate the activity
of discrete subsets of vagal neurons and may be able to regulate the
function of specific regions of the gastrointestinal tract. This would
have important implications for our understanding of the PVN's
modulation of gastrointestinal function. It is also possible that
investigators who have used standard extracellular recording techniques
may have had difficulty distinguishing the responses that were
associated with DMNV neurons from those that were obtained from NST
cells. This predicament is due, at least in part, to the substantial
intermingling of NST and DMNV neurons at the border between these two
nuclei. The situation is complicated further by the fact that a
significant proportion of DMNV neurons send their dendrites into the
overlying NST (5, 11, 16, 25, 34, 35). As a result, many of the
hypothalamic axons that terminate in the NST may actually synapse on
the dendrites of DMNV neurons. Although one might expect that the use
of collision tests and latency data would permit the investigator to
distinguish DMNV from NST neurons, our own experience has taught us
that these methodologies are unreliable in cases in which one is
relying on results obtained with stimulation of the subdiaphragmatic
vagus nerve (see Ref. 35).
Answers to the questions posed above require that we employ a
methodology that permits us to differentiate the response of NST and
DMNV neurons with confidence. We have addressed this issue by utilizing
an experimental paradigm that combines extracellular and intracellular
neuronal recordings with intracellular injections of a neuronal tracer
to label individual, physiologically characterized neurons in the DVC.
This strategy has allowed us to determine the precise location of every
neuron we have recorded in the vagal complex. The data we have obtained
confirm that the neurons in the DVC are sensitive to stimulation of the
PVN and provide some important insights into the role of the PVN in the
regulation of NST and DMNV function.
 |
MATERIALS AND METHODS |
Animal preparation.
Adult male Sprague-Dawley rats weighing 270-350 g were
anesthetized with pentobarbital sodium (50 mg/kg ip), and supplemental doses of pentobarbital sodium were administered as needed to maintain a
deep level of surgical anesthesia and muscle relaxation. A tracheotomy was performed, and a tube was inserted into the trachea for artificial ventilation with room air (100 strokes/min, 2.0-2.4
cm3 tidal volume). A midline
abdominal incision was made to expose the abdominal vagus, stomach, and
duodenum. Teflon-coated pure gold wire stimulating electrodes (76 µm
outside diameter) were placed around the anterior and posterior
branches of the subdiaphragmatic vagal nerve, ~1-2 cm above the
gastroesophageal junction and immediately above the accessory and
celiac branches of the vagus nerve. The stimulating electrodes were
loosely sutured to the esophagus to limit displacement. An incision was
made in the gastric corpus after the stimulating electrodes were
placed, and a gastric influx catheter was inserted into this opening
and fixed to the greater curvature of the gastric corpus. An incision
was then made immediately proximal to the pylorus, and two tubes were
inserted into the gut, one oriented toward the stomach and the other
oriented toward the duodenum. The tube oriented toward the stomach
served as the gastric efflux catheter, whereas the duodenal tube served
as the duodenal influx catheter. Finally, the gut was transected 10 cm distal to the ligament of Tritz. The resulting caudal open end was
closed with silk sutures, and the proximal end was cannulated with a
tube that served as the duodenal efflux catheter.
Stimulus presentation and neuronal labeling.
The animals were placed in a Kopf small animal stereotaxic frame, and
body temperature was maintained by a thermostatically controlled
heating table that also warmed all perfusion fluids to body
temperature. The brain stem was exposed by removing the atlanto-occipital membrane and a portion of the occipital bone. Beveled
glass micropipettes (A-M Systems, Everett, WA; tip diameter of
0.08-0.1 µm, resistance of 50-70 M
), filled with 2.0%
Neurobiotin (Vector Laboratories, Burlingame, CA) in 1 M KCl, were
lowered into the vagal complex between 100 µm rostral and 400 µm
caudal to the obex. Biphasic electrical pulses (0.5 ms duration,
0.5-3 mA, 1 Hz) delivered to the abdominal vagus were used as
search stimuli. The recording micropipettes were advanced until a unit that was driven by the vagal stimulating electrode was encountered. All
units driven by the stimulating electrode were tested for a response to
duodenal or gastric distension as described previously (34-36).
The distension was accomplished by raising the efflux catheter to a
level that produced a 13-mmHg increase in intraluminal pressure (a
nonpainful stimulus). The distension was maintained for 60 s. Unit
discharges were amplified by an A-M Systems high-input impedance
preamplifier and displayed and stored on an IBM-compatible Pentium
computer with the use of Axotape software (Axon Instruments, Foster
City, CA).
Neurons responding to either of the gastrointestinal stimuli were
tested for a response to stimulation of the PVN. Stimulation was
accomplished using a concentric bipolar electrode (Kopf, SNES-100) that
was placed 2.0 mm caudal to bregma, 0.5 mm from the midline, and 8.5 mm
from the skull surface. The PVN was stimulated with a 20- to 40-µA
current (0.5 ms duration) at 15 Hz for 1 min (preliminary studies using
frequencies between 5-40 Hz demonstrated that a 15-Hz PVN
stimulation was the most effective stimulus for eliciting changes in
DMNV or NST activity). The cell's response to PVN plus gastrointestinal stimulation was tested if the neuron appeared stable
(steady membrane potential, consistent action potential amplitude, and
so forth) following PVN stimulation alone.
The recording micropipette was advanced after the response
characterization until the neuron was impaled (passing small positive current pulses from the recording electrode facilitated this process). Penetration of the cell membrane was accompanied by a 20- to 40-mV drop
in the voltage measured at the electrode tip, an increase in the
amplitude of the action potential, and a shift from a bipolar to a
monopolar action potential (see Ref. 15, for a depiction of this
process). We confirmed that the cell impaled was the cell that was
characterized by verifying the neuron's response properties. When this
confirmation was completed, the cells were labeled with Neurobiotin
(used to demonstrate the location of the neuron in the DVC) by passing
2- to 4-nA, 250-ms positive current pulses at 2 Hz for 2-7 min.
The injection was stopped if the membrane potential returned to
prepenetration levels. A maximum of two injections was attempted on
each side.
The rats were administered a lethal dose of pentobarbital sodium and
perfused through the heart with 500 ml of 0.9% saline containing 2,000 U/l heparin in 0.1 M sodium phosphate buffer (pH 7.3, room temperature)
1-6 h after the first injection. The rinse solution was followed
by 500 ml of a fixative solution containing 1% paraformaldehyde and
2.5% glutaraldehyde in 0.1 M phosphate buffer (4°C, pH 7.4). The
brain was stored overnight in 0.1 M phosphate buffer containing 20%
sucrose. Fifty-micrometer coronal sections through the brain stem were
incubated in 0.4% Triton X-100 for 1.5 h at room temperature and then
placed in avidin D-horseradish peroxidase (Vector
Laboratories) for 2 h. The sections were incubated in a solution
containing 100 mg diaminobenzidine, 5.0 ml of 1.0% cobalt chloride,
and 4.0 ml of 1.0% nickel ammonium sulfate in 200 ml phosphate buffer
(pH 7.4) for 15-20 min, followed by an additional 15 min in the
presence of 3.0% hydrogen peroxide. Brain stem sections were placed on
gelatin-coated glass slides, air-dried, and coverslipped. Some brain
stem sections were counterstained with neutral red to facilitate
identification of NST and DMNV borders. All forebrains were sectioned
at 50 µm and counterstained or viewed using darkfield optics to
verify that the electrode tip was located in the PVN.
Neurophysiological analysis.
Neuronal response properties were examined using the Datapac software
system (Run Technologies, Laguna Niguel, CA). Peristimulus time
histograms (5-s bins) were constructed for the period beginning 30 s
before and ending 90 s after initiation of the gastrointestinal and
forebrain stimulation (stimuli were maintained for 60 s). This 120-s trace was divided into four periods (30 s each) to test the
effect of each stimulus. Period 1 represented basal spontaneous activity. Period
2 included the immediate response to the stimulus, whereas period 3 represented the late
response. Period 4 contained the first
30 s after the stimulus was discontinued and therefore included any
delayed response or changes induced by removal of the stimulus. We
determined whether a neuron's response to a given stimulus was
statistically significant by comparing the mean activity during
periods 2-3 with the mean
activity during the pre- and poststimulus periods
(periods 1 and
4, respectively) using ANOVA and the
Bonferroni test for multiple comparisons.
 |
RESULTS |
DMNV neurons: response to gastrointestinal stimuli.
We characterized and labeled a total of 85 DMNV neurons in this study.
Most (84/85) of these neurons were inhibited by one or both of the
gastrointestinal distension stimuli. Fifty-four of the 85 DMNV neurons
were inhibited by both stimuli, whereas 30 neurons were inhibited by
one stimulus and either excited by or unresponsive to the other.
Intestinal distension reduced the mean activity of the total sample of
DMNV cells from a prestimulus (period
1) level of 1.83 ± 0.16 (SE) Hz to 1.21 ± 0.24 Hz in period 2 (the first 30 s
after stimulus onset) and 0.72 ± 0.17 Hz in period
3 (the second 30 s after stimulus onset). This decrease in activity in response to intestinal distension was statistically significant [P < 0.01 (ANOVA
F = 7.3) and
P < 0.05 (Bonferroni) for
periods 2 and
3 compared with
period 1]. Similarly, gastric distension reduced the mean activity from a prestimulus level of 1.64 ± 0.14 to 0.62 ± 0.12 Hz in period
2 and 1.08 ± 0.19 Hz in period
3. This distension-induced decrement in neural activity was also statistically significant
[P < 0.01 (ANOVA
F = 9.7) and P < 0.05 (Bonferroni) for both
comparisons].
DMNV neurons: PVN effect on basal activity.
Sixty-eight of the 85 DMNV neurons characterized in this investigation
responded to electrical stimulation of the PVN. None of the
PVN-sensitive neurons could be driven by the PVN-stimulating electrode
at frequencies >1.0 Hz. The average response profile for these
neurons is illustrated in Fig.
1A. In
contrast to the response obtained during gastrointestinal distension,
electrical stimulation of the PVN caused an increase in the mean
activity of these DMNV neurons. Stimulation of the PVN increased the
average activity from a prestimulus level of 1.73 ± 0.26 to 2.60 ± 0.39 Hz in period 2 and 2.57 ± 0.54 Hz in period 3. This
increase in activity was statistically significant
[P < 0.01 (F = 10.6) for periods 2 and
3 compared with
period 1 and
P < 0.05 (Bonferroni)]. It is important to note, however, that PVN
stimulation did not increase the spontaneous activity of all of the
PVN-sensitive DMNV neurons. Although the majority (48/68) of the
PVN-sensitive cells did exhibit an increase in activity, a substantial
subset (20/68) responded to PVN stimulation with a decrease in
activity. The mean response profile for the group that was excited by
PVN stimulation is illustrated in Fig.
1B. Figure
1B indicates that electrical
stimulation of the PVN had a dramatic effect on the basal activity of
these neurons, increasing the mean activity from a prestimulus level of
1.57 ± 0.22 to 4.02 ± 0.51 Hz in
period 2 and 3.78 ± 0.44 Hz in
period 3 [P < 0.01 (ANOVA
F = 12.1) and P < 0.05 (Bonferroni) for both
comparisons]. The response profile of the subset that was
inhibited by the PVN is illustrated in Fig.
1C. The activity of these neurons was
decreased from a prestimulus level of 1.98 ± 0.24 to 0.91 ± 0.17 Hz in period 2 and 0.87 ± 0.18 Hz in period 3 [P < 0.01 (ANOVA
F = 8.2) and
P < 0.05 (Bonferroni) for both
comparisons].

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 1.
A: average response profiles for the
68 dorsal motor nucleus of the vagus (DMNV) neurons that responded to
electrical stimulation of the paraventricular nucleus (PVN) (open
portions of bars indicate SE). B: mean
response profile for the 48 neurons that were excited by PVN
stimulation. C: mean activity for the
20 DMNV neurons that were inhibited by electrical stimulation of the
PVN. Periods 1-4 (see text) are
indicated by the bars below each graph. Arrows indicate onset of PVN
stimulation.
|
|
Examples of the responses exhibited by individual neurons in these two
subgroups are presented in Figs. 2 and 3.
Figure 2 illustrates the response profile of a DMNV neuron that was
excited by electrical stimulation of the PVN. This neuron was
completely inhibited by both intestinal (Fig.
2A) and gastric (Fig.
2B) distension. Conversely,
electrical stimulation of the PVN increased the neuron's basal
activity from 1.23 Hz in period 1 to
4.53 Hz in period 2 and 6.00 Hz in
period 3 (Fig.
2C). The response profile
illustrated in Fig. 3 shows the opposite
response to PVN stimulation. In this instance, the DMNV neuron was
almost completely inhibited by intestinal distension (Fig.
3A), moderately excited by
distension of the stomach (1.2 Hz in period
1, 1.9 Hz in period 2,
and 2.7 Hz in period 3; Fig.
3B; note that contrasting responses to
intestinal and gastric distension are not uncommon in this system, see
Ref. 5), and completely inhibited by stimulation of the PVN (Fig. 3C).

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 2.
Response profile of a DMNV neuron that was excited by electrical
stimulation of the PVN. Solid arrows indicate stimulus onset, and open
arrows indicate stimulus removal. This neuron was completely inhibited
by both intestinal (A) and gastric
(B) distension. Conversely,
electrical stimulation of the PVN increased the neuron's basal
activity (C).
|
|

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 3.
Response properties of a DMNV neuron that was almost completely
inhibited by intestinal distension
(A), moderately excited by
distension of the stomach (B), and
completely inhibited by electrical stimulation of the PVN
(C). Solid arrows indicate stimulus
onset, and open arrows indicate stimulus removal.
|
|
Effect of PVN stimulation on the DMNV response to gastrointestinal
stimuli.
Thirty-two of the 68 PVN-sensitive cells that we recorded were
sufficiently stable to test the combined effect of PVN and gastrointestinal stimulation. We found that the PVN stimulation parameters utilized in this investigation affected the gut-related response properties of six DMNV neurons. Figure
4 shows the electrophysiological properties
of a DMNV neuron whose response to one of the two gastrointestinal stimuli was counteracted by PVN stimulation. Figure
4A demonstrates the response to
intestinal distension. This stimulus caused a reduction in neuronal
activity from a basal level of 0.36 to 0.03 Hz in
periods 2 and
3. Distention of the stomach (Fig.
4B) caused a reduction in activity
from a baseline level of 0.83 to 0.0 Hz in periods
2 and 3 (a complete
inhibition). Stimulation of the PVN alone produced a substantial
increase in activity, from 0.7 Hz in period
1 to 2.43 Hz in period
2 and 1.30 Hz in period
3 (Fig. 4C). The
effect of simultaneous presentation of PVN stimulation and intestinal
distension is illustrated in Fig. 4D.
The simultaneous administration of these two stimuli caused the
neuron's activity to increase from 0.60 Hz in period
1 to 1.73 Hz in period
2 and 1.60 Hz in period
3. Interestingly, the influence of the PVN on the
activity of the DMNV neuron was very stimulus specific. The simultaneous presentation of the PVN and gastric stimuli resulted in a
response that was virtually identical to that seen when the gastric
stimulus was presented alone (compare Fig.
4E with Fig. 4B; note, however, that the
poststimulus activity was greater following PVN + gastric distension).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 4.
Electrophysiological properties of a DMNV neuron whose response to 1 of
the 2 gastrointestinal stimuli was counteracted by PVN stimulation.
Solid arrows indicate stimulus onset, and open arrows indicate stimulus
removal. A: response to intestinal
distension. B: response to gastric
distension. C: effect of electrical
stimulation of PVN alone. D: effect of
simultaneous presentation of PVN stimulation and intestinal distension.
E: simultaneous presentation of PVN
and gastric stimuli (virtually identical to that seen when gastric
stimulus was presented alone).
|
|
NST neurons: response to gastrointestinal stimuli.
A total of 31 neurons were recorded and labeled in the NST. The
majority (29/31) of these cells were excited by at least one of the
gastrointestinal stimuli, with 10 of the 31 cells excited by both
gastric and duodenal distension [the remaining 19 neurons were
excited by one gastrointestinal stimulus and inhibited (or had no
response) to the other]. Intestinal distension increased the mean
activity of the total sample of NST cells from a prestimulus level of
1.59 ± 0.36 to 3.13 ± 0.58 Hz in period
2 and 2.92 ± 0.52 Hz in period
3. This increase in activity in response to intestinal
distension was statistically significant
[P < 0.05 (ANOVA
F = 3.9) and
P < 0.05 (Bonferroni) for both
comparisons]. Gastric distension also demonstrated a tendency to
increase the mean neural activity of the NST neurons
(period 1 = 1.75 ± 0.40 Hz,
period 2 = 2.67 ± 0.8 Hz, and
period 3 = 2.55 ± 0.61 Hz), but
this apparent increase was not statistically significant when the
entire sample of NST neurons was considered. It is important to note,
however, that the majority (18/22) of the NST neurons that responded to
gastric distension did exhibit a significant increase in activity
during the presentation of this stimulus (the failure to obtain a
significant effect when the entire sample was examined reflects the
fact that the 4 NST neurons that were inhibited by the gastric stimulus
exhibited a very dramatic decrement in activity, thus producing a large
standard deviation when the response average was calculated).
NST neurons: PVN effect on basal activity.
Electrical stimulation of the PVN altered the basal activity of 18 of
the 31 NST neurons in our sample. One of the 18 PVN-sensitive NST
neurons could be driven at frequencies >1 Hz (this neuron was capable
of following 150 Hz, suggesting that it projected to the PVN).
Stimulation of the PVN at 15 Hz resulted in a significant
[P < 0.01 (F = 25.07) and
P < 0.05 (Bonferroni) for
periods 2 and 3 compared with
period 1] decrease in mean NST
activity (Fig. 5A).
Similar to the results obtained in the DMNV, the sample of NST neurons
was composed of two distinct subsets. One group contained the majority
(12) of the 18 NST neurons and was inhibited by the PVN (Fig.
5B). Electrical stimulation of the
PVN decreased the mean activity of this group of neurons from a
baseline level of 3.87 ± 0.09 to 1.37 ± 0.2 Hz in
period 2 and 0.97 ± 0.16 Hz in
period 3 [this decrease was
statistically significant; P < 0.01 (F = 52.3) and
P < 0.05 (Bonferroni) for both
comparisons]. An example of a neuron exhibiting this response
profile is shown in Fig. 6. This cell was
excited by both intestinal (Fig. 6A) and gastric (Fig. 6B) distension. In
contrast to the excitatory response to gastrointestinal stimuli,
electrical stimulation of the PVN (Fig.
6C) reduced the neuron's activity
from a basal level of 5.93 Hz (in period
1) to 1.67 Hz in period
2 and 0.47 Hz in period
3 [P < 0.05 (Bonferroni) for both comparisons].

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 5.
A: mean response profile of the 18 nucleus of the solitary tract (NST) neurons that responded to
electrical stimulation of the PVN. In contrast to the excitatory effect
seen in the DMNV, this graph suggests that electrical stimulation of
the PVN reduced the activity of the NST neurons.
B: response profile of the 12 NST
neurons that were inhibited by the PVN. Periods
1-4 (see text) are indicated by the bars below
each graph. Arrows indicate onset of PVN stimulation.
|
|

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 6.
NST neuron that was excited by both intestinal
(A) and gastric
(B) distension but inhibited by
electrical stimulation of the PVN
(C). Solid arrows indicate stimulus
onset, and open arrows indicate stimulus removal.
|
|
Six of the 18 PVN-sensitive NST neurons were excited by electrical
stimulation of the PVN. The response of one such NST neuron is
illustrated in Fig. 7. This particular
neuron was not sensitive to distension of the intestine (Fig.
7A) but did exhibit a brief increase
in activity following gastric distension (Fig.
7B). Although the neuron's response
to gastrointestinal stimulation was modest, it did show a robust
response to PVN stimulation. Electrical stimulation of the PVN
increased the neuron's activity from a basal level of 0.5 to 3.76 Hz
in period 2 and 2.96 Hz in
period 3 [P < 0.05 (Bonferroni) for
both comparisons].

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 7.
Response properties exhibited by one of the 6 PVN-sensitive NST neurons
that were excited by electrical stimulation of the PVN. Solid arrows
indicate stimulus onset, and open arrows indicate stimulus removal.
This particular neuron was not sensitive to distension of the intestine
(A) but did exhibit a brief increase
in activity after gastric distension
(B). Electrical stimulation of the
PVN resulted in a robust increase in the neuron's activity
(C).
|
|
Effect of PVN stimulation on the NST response to gastrointestinal
stimuli.
Only 2 of the 18 PVN-sensitive NST neurons studied in this
investigation exhibited an altered response to gastrointestinal distension during PVN stimulation. The response profile of one of these
neurons is presented in Fig. 8. This NST
neuron responded to intestinal distension with an increase in activity
from a baseline level of 1.8 to 2.4 Hz (in period
2; Fig. 8A). Gastric
distension was also an effective excitatory stimulus, increasing the
neuron's activity from 1.7 to 2.8 Hz (Fig.
8B). Electrical stimulation of the
PVN proved to be an inhibitory stimulus, however, reducing the cell's
activity from 2.5 to 1.4 Hz (Fig.
8C). Interestingly, the inhibitory
influence of the PVN blocked the neuron's response to gastric and
intestinal distension (Fig. 8, D and
E). In each instance, the cell
failed to exhibit a valid response to distension during the
simultaneous administration of the gastrointestinal and PVN stimulation
[there was a trend that indicated that the response to gastric
distension was reduced during period
3; there was also a valid increase in activity (an
apparent "rebound" effect) following the cessation of the
intestinal and PVN stimuli, Fig. 8D].

View larger version (63K):
[in this window]
[in a new window]
|
Fig. 8.
Neurophysiological properties of a NST neuron that responded to
intestinal (in period 2;
A) and gastric
(B) distension with an increase in
activity. Electrical stimulation of the PVN resulted in a decrease in
the cell's activity (C). Inhibitory
influence of the PVN blocked the neuron's response to gastric and
intestinal distension (D and
E). In each instance, the cell
failed to exhibit a valid response to distension during simultaneous
administration of gastrointestinal and PVN stimulation [there was
a trend that indicated that the response to gastric distension was
reduced during period 3; there was
also a valid increase in activity (an apparent "rebound" effect)
following the cessation of the intestinal and PVN stimuli,
D]. Solid arrows indicate
stimulus onset, and open arrows indicate stimulus removal.
|
|
 |
DISCUSSION |
NST and DMNV response to PVN stimulation.
Approximately 80% of the DMNV neurons and 58% of the NST neurons we
characterized responded to electrical stimulation of the PVN. In each
nucleus, the predominant effect of the PVN stimulation was contrary to
the predominant effect of the gastrointestinal stimuli. For example,
whereas most of the DMNV neurons we studied exhibited a decrease in
activity during gastric and/or intestinal distension, the majority
(59%) of the neurons that responded to electrical stimulation of the
PVN were excited by this hypothalamic stimulus. Similarly, although
94% of the NST cells in our study were excited by gastric and/or
intestinal distension, 67% of the PVN-sensitive NST neurons were
inhibited by electrical stimulation of the hypothalamus.
Although it is tempting to speculate on the potential role(s) of the
PVN-vagal interaction based on the major effect of the PVN in the NST
and DMNV, we would remind the reader that each of the vagal nuclei
contained two subsets of neurons: one excited and one inhibited by the
PVN (see Fig. 9). It is quite possible that
this diversity of responses provides an explanation for the apparent
discrepancies in the results obtained in prior investigations of the
PVN influence on vagal activity. It is understandable that many
laboratories (e.g., Refs. 9, 19, 29) have concluded that the PVN
excites neurons in the DMNV, given that this appears to be the dominant
response. It is also clear that our results support the data presented
by Banks and Harris (2), who have argued that the DMNV contains two
neuronal subsets, with some cells excited by the PVN and others that
are inhibited.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 9.
Schematic summarizing the vagal neuron response to stimulation of the
PVN and gastrointestinal tract. +, Excitation; ,
inhibition.
|
|
Given the nature of the interaction between NST and DMNV neurons, we
must consider the possibility that the influence of the PVN on DMNV
activity is actually an indirect result of its effect on neurons in the
NST. There is a growing body of evidence to support the contention that
many (if not most) of the gut-sensitive neurons in the NST exert an
inhibitory influence on the DMNV. Accordingly, any input
that acted to decrease the activity of NST neurons (such as increased
activity in the PVN-NST pathway) would tend to increase the activity of
a substantial number of the neurons in the DMNV (as we found when we
recorded from neurons in this portion of the vagal complex). It is not
clear, therefore, whether the responses we recorded in the DMNV during
stimulation of the PVN reflected the influence of a direct or indirect
pathway. The anatomic evidence would indicate that both scenarios are
possible. A number of anterograde (8, 24, 30) and retrograde (27) labeling studies have shown that axons from the PVN terminate in both
the NST and the DMNV. It would appear that an answer to this important
question would depend on electrophysiological recording studies that
examine the response of the DMNV to PVN stimulation in a model that
eliminates any potential contribution from the NST. It is possible that
this goal could be accomplished by inhibiting the activity of NST
neurons via local injection of a reversible anesthetic such as
lidocaine while stimulating the PVN and recording the activity of
neurons in the DMNV.
The potential role of the descending PVN-vagal pathway.
The multiple effects of the descending PVN pathway on vagal response
properties provide a substrate for both excitatory and inhibitory
influences on gastrointestinal function. As noted above in the
discussion of the influence of the PVN on vagal neuron activity, this
potential for diverse effects may explain the disparate results that
have been obtained following electrical and/or chemical stimulation of
the PVN (see Refs. 6 and 10 for reviews). For example, there is
evidence that stimulation of the PVN evokes large increases in gastric
acid secretion (20), a transient increase in gastric motility (21), and
a general increase in parasympathetic activity (28), yet other
laboratories have reported that stimulation of the PVN results in a
decrease in gastric acid secretion (e.g., Refs. 26, 31). There are a
number of possible explanations for these disparate results, including
the activation of vagal vs. spinal pathways as suggested by Yoneda and
Taché (31) and/or the selective excitation of cholinergic and
noncholinergic pathways as proposed by Rogers and collaborators (21,
23). It is reasonable to suggest that our understanding of these
interactions will improve as we obtain additional data regarding the
neurochemistry and targets of the DMNV neurons that are excited or
inhibited by the PVN. One issue that remains to be resolved is the
importance of the small number of DVC neurons that exhibited an altered
response to gastrointestinal stimuli in the presence of PVN
stimulation. It is not clear, for example, whether the PVN can exert a
meaningful influence on gastrointestinal activity if it only alters the
distension-induced response of 19% of the gut-sensitive DMNV neurons
and 11% of the gut-sensitive NST neurons. Despite this caveat, we
would point out that PVN stimulation did alter the basal activity of a
substantial number of DVC neurons. This influence, plus potential
subthreshold changes in DVC electrophysiology (not examined in this
study), may serve as an important regulatory function.
Finally, we would point out that the descending modulation of vagal
activity by the PVN may play a role in other processes as well, such as
feeding behaviors and the genesis or promulgation of feeding disorders.
A descending input that alters the activity of DMNV neurons, for
example, could lead to changes in the activity of the vagal afferents
that would in turn influence the activity of NST neurons that
participate in circuits related to appetite, satiety, and feeding
behaviors (see Refs. 17 and 18).
Response to gastrointestinal stimuli.
Almost all (84/85) of the DMNV neurons studied in this investigation
exhibited a decrease in activity in response to gastric and/or duodenal
distension. This finding replicates the results we (5, 34) and others
(1, 4, 7, 14, 23) have obtained in similar studies of the vagal
efferent response to gastrointestinal stimulation. In addition, we
found that the majority of the neurons in the NST were excited by
distension of the stomach and/or duodenum. This result is also
consistent with data we have obtained in previous investigations (35,
37).
Technical limitations.
There are a number of technical issues that should be considered when
evaluating the results obtained in this investigation. First, we must
consider the possibility that our PVN-stimulating electrode has
stimulated fibers from other portions of the forebrain that pass
through this region. We have examined this potential confound by
employing a dual stimulating electrode in a limited (n = 3) number of control animals
(unpublished results). The electrode had the ability to administer
electrical and chemical stimuli and was used to inject the PVN with a
small volume of 0.01 M glutamate. We found that this protocol resulted
in a decrease in the activity of most of the NST neurons and an
increase in the activity of most of the DMNV neurons that we recorded,
a result that replicates the data presented in the present paper. We
would propose that this finding supports the reliability of the data
obtained using the electrical stimulating electrode. Of course, it is
still possible that the electrical stimulation activated areas (e.g.,
the LH) adjacent to the PVN. Furthermore, it is likely that the
stimulating electrode activated multiple neuronal subsets in the PVN.
Unfortunately, this is an inherent difficulty in any study that employs
electrical stimulating electrodes in the central nervous system and is
difficult to address. The extant literature provides relatively little
guidance in this regard, although a respected study by Rogers and
Hermann (21) did use a similar PVN stimulation paradigm. These
investigators stimulated the PVN with a tungsten electrode that
delivered a 25-µA current (approximately the same magnitude used in
our investigation) for 0.3 ms (similar to our 0.5 ms duration) at 10 Hz
(similar to our 15 Hz frequency). We can state, therefore, that our
stimulation parameters were consistent with a protocol used by another
group that has investigated the role of the PVN in gastrointestinal function.
We were able to test the response of a relatively small (~50% of the
total data set) number of neurons to the simultaneous presentation of
gastrointestinal and PVN stimulation. This was due to the fact that we
were able to maintain a stable recording for a limited amount of time,
and we were often forced to skip the final portion of the stimulus
presentation protocol so that we could inject the neuron with the
label. Despite this limitation, the results we obtained do verify that
the PVN has the ability to modulate the gastrointestinal response of a
small number of NST and DMNV neurons. Furthermore, it is clear that the
nature of this modulation can be quite variable. One factor that may have contributed to the fact that the PVN modulated the
gastrointestinal response of a small number of vagal neurons may be the
magnitude of the gastrointestinal stimuli. We have shown previously
that the distension protocol employed in this study increases the
intraluminal pressure in the stomach and duodenum to ~12-13 mmHg
(see Ref. 5). This pressure is not considered to be noxious (15), but it is greater than the resting intraluminal pressure. We do not know
whether the magnitude of the distension is substantially greater than
the distension that occurs during normal feeding, but, if it is, it may
have caused us to underestimate the number of vagal neurons whose
response to gastrointestinal stimuli could be modulated by the PVN. It
is possible that the distension pressure that we employed may have
resulted in a greater response to the gastrointestinal stimuli than
would occur during normal feeding, and this may have masked the
response of some vagal neurons to stimulation of the PVN. It would be
helpful to expand on the present study by utilizing a stimulating
protocol that exposed the stomach and duodenum to a range of distension
pressures (indeed, a similar "dose-response" strategy could also
be employed to test the effect of various stimulating current
amplitudes in the PVN).
In conclusion, the data obtained in this investigation demonstrate that
descending inputs from the PVN have the ability to 1) alter the baseline activity of
gut-sensitive NST and DMNV neurons and
2) modulate the response of a small
number of vagal neurons to gastrointestinal stimuli. These interactions
may play an important role in a number of gut-related homeostatic
processes. Increased or decreased activity in the descending pathway
from the PVN to the DVC has the potential to alter ascending satiety
signals, modulate the cephalic phase of feeding, and affect the
absorption of nutrients from the gastrointestinal tract. The study
provides a foundation for future investigations that will examine the
neuropharmacology of this and related forebrain-brain stem interactions.
 |
ACKNOWLEDGEMENTS |
We thank Drs. R. Alberto Travagli, Richard Rogers, and Kirsteen
Browning for comments on the manuscript.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-53159.
Present address and address for reprint requests and other
correspondence: W. E. Renehan, Neurogastroenterology Research, Henry
Ford Health System, One Ford Place-2D, 6071 Second Ave., Detroit, MI
48202 (E-mail: wreneha1{at}hfhs.org).
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. §1734 solely to indicate this fact.
Received 2 September 1998; accepted in final form 31 March 1999.
 |
REFERENCES |
1.
Azpiroz, F.,
and
J. R. Malagelada.
Vagally mediated gastric relaxation-induced by intestinal nutrients in the dog.
Am. J. Physiol.
251 (Gastrointest. Liver Physiol. 14):
G727-G735,
1986[Medline].
2.
Banks, D.,
and
M. C. Harris.
Activation within dorsal medullary nuclei following stimulation in the hypothalamic paraventricular nucleus in rats.
Pflügers Arch.
408:
619-627,
1987[Medline].
3.
Berk, M. L.
Projections of the lateral hypothalamus and bed nucleus of the stria terminalis to the dorsal vagal complex in the pigeon.
J. Comp. Neurol.
260:
140-156,
1987[Medline].
4.
Davison, J. S.,
and
D. Grundy.
Modulation of single vagal efferent fibre discharge by gastrointestinal afferents in the rat.
J. Physiol. (Lond.)
284:
69-82,
1978[Medline].
5.
Fogel, R.,
X. Zhang,
and
W. E. Renehan.
Relationships between the morphology and function of gastric and intestinal distention-sensitive neurons in the dorsal motor nucleus of the vagus.
J. Comp. Neurol.
364:
78-91,
1996[Medline].
6.
Grijalva, C. V.,
and
D. Novin.
The role of the hypothalamus and dorsal vagal complex in gastrointestinal function and pathophysiology.
Ann. NY Acad. Sci.
597:
207-222,
1990[Abstract].
7.
Grundy, D.,
A. A. Salih,
and
T. Scratcherd.
Modulation of vagal efferent fibre discharge by mechanoreceptors in the stomach, duodenum and colon of the ferret.
J. Physiol. (Lond.)
319:
43-52,
1981[Abstract].
8.
Holstege, G.
Some anatomical observations on the projections from the hypothalamus to brainstem and spinal cord: an HRP and autoradiographic tracing study in the cat.
J. Comp. Neurol.
260:
98-126,
1987[Medline].
9.
Kannan, H.,
and
H. Yamashita.
Connections of neurons in the region of the nucleus tractus solitarius with the hypothalamic paraventricular nucleus: their possible involvement in neural control of the cardiovascular system in rats.
Brain Res.
329:
205-212,
1985[Medline].
10.
Leslie, R. A.,
D. J. M. Reynolds,
and
I. N. C. Lawes.
Central connections of the nuclei of the vagus nerve.
In: Neuroanatomy and Physiology of Abdominal Vagal Afferents, edited by S. Ritter,
R. C. Ritter,
and C. D. Barnes. Boca Raton, FL: CRC, 1992, p. 81-98.
11.
Miselis, R. R.,
L. Rinaman,
S. M. Altschuler,
X. Bao,
and
R. B. Lynn.
Medullary viscerotopic representation of the alimentary canal innervation in rat.
In: Brain-Gut Interactions, edited by Y. Taché,
and D. Wingate. Boca Raton, FL: CRC, 1991, p. 3-21.
12.
Nishimura, H.,
and
Y. Oomura.
Effects of hypothalamic stimulation on activity of dorsomedial medulla neurons that respond to subdiaphragmatic vagal stimulation.
J. Neurophysiol.
58:
655-675,
1987[Abstract/Free Full Text].
13.
Petrov, T.,
T. L. Krukoff,
and
J. H. Jhamandas.
Convergent influence of the central nucleus of the amygdala and the paraventricular hypothalamic nucleus upon brainstem autonomic neurons as revealed by c-fos expression and anatomical tracing.
J. Neurosci. Res.
42:
835-845,
1995[Medline].
14.
Raybould, H. E.
Capsaicin-sensitive vagal afferents and CCK in inhibition of gastric motor function-induced by intestinal nutrients.
Peptides
12:
1279-1283,
1991[Medline].
15.
Renehan, W. E.,
X. Zhang,
W. H. Beierwaltes,
and
R. Fogel.
Neurons in the dorsal motor nucleus of the vagus may integrate vagal and spinal information from the GI tract.
Am. J. Physiol.
268 (Gastrointest. Liver Physiol. 31):
G780-G790,
1995[Abstract/Free Full Text].
16.
Rinaman, L.,
J. P. Card,
J. S. Schwaber,
and
R. R. Miselis.
Ultrastructural demonstration of a gastric monosynaptic vagal circuit in the nucleus of the solitary tract in rat.
J. Neurosci.
9:
1985-1996,
1989[Abstract].
17.
Ritter, R. C.,
L. Brenner,
and
D. P. Yox.
Participation of vagal sensory neurons in putative satiety signals from the upper gastrointestinal tract.
In: Neuroanatomy and Physiology of Abdominal Vagal Afferents, edited by S. Ritter,
R. C. Ritter,
and C. D. Barnes. Boca Raton, FL: CRC, 1992, p. 222-248.
18.
Ritter, S.,
and
J. S. Taylor.
Vagal sensory neurons are required for lipoprivic but not glucoprivic feeding in rats.
Am. J. Physiol.
258 (Regulatory Integrative Comp. Physiol. 27):
R1395-R1401,
1990[Abstract/Free Full Text].
19.
Rogers, R. C.,
and
G. E. Hermann.
Vagal afferent stimulation-evoked gastric secretion suppressed by paraventricular nucleus lesion.
J. Auton. Nerv. Syst.
13:
191-199,
1985[Medline].
20.
Rogers, R. C.,
and
G. E. Hermann.
Hypothalamic paraventricular nucleus stimulation induced gastric acid secretion and bradycardia suppressed by oxytocin antagonist.
Peptides
7:
695-700,
1986[Medline].
21.
Rogers, R. C.,
and
G. E. Hermann.
Oxytocin, oxytocin antagonist, TRH, and hypothalamic paraventricular nucleus effects on gastric motility.
Peptides
8:
505-513,
1987[Medline].
22.
Rogers, R. C.,
H. Kita,
L. L. Butcher,
and
D. Novin.
Afferent projections to the dorsal motor nucleus of the vagus.
Brain Res. Bull.
5:
365-373,
1980[Medline].
23.
Rogers, R. C.,
D. M. McTigue,
and
G. E. Hermann.
Vagal control of digestion: modulation by central neural and peripheral endocrine factors.
Neurosci. Biobehav. Rev.
20:
57-66,
1996[Medline].
24.
Saper, C. B.,
A. D. Loewy,
L. W. Swanson,
and
W. M. Cowan.
Direct hypothalamo-autonomic connections.
Brain Res.
117:
305-312,
1976[Medline].
25.
Shapiro, R. E.,
and
R. R. Miselis.
The central organization of the vagus nerve innervating the stomach of the rat.
J. Comp. Neurol.
238:
473-488,
1985[Medline].
26.
Shiraishi, T.
Gastric related properties of rat paraventricular neurons.
Brain Res. Bull.
18:
315-323,
1987[Medline].
27.
Van der Kooy, D.,
L. Y. Koda,
J. F. McGinty,
G. R. Gerfen,
and
F. E. Bloom.
The organization of projections from the cortex, amygdala, and hypothalamus to the nucleus of the solitary tract in rat.
J. Comp. Neurol.
224:
1-24,
1984[Medline].
28.
Van Dijk, G.,
A. E. Bottone,
J. H. Strubbe,
and
A. B. Steffens.
Hormonal and metabolic effects of paraventricular hypothalamic administration of neuropeptide Y during rest and feeding.
Brain Res.
660:
96-103,
1994[Medline].
29.
Waldron, J. N. B.,
S. Ghosh,
and
P. Zarzecki.
Multiple inputs to a population of thalamocortical neurons projecting to cat somatosensory cortex.
Exp. Brain Res.
74:
105-115,
1989[Medline].
30.
Willett, C. J.,
J. G. Rutherford,
D. G. Gwyn,
and
R. A. Leslie.
Projections between the hypothalamus and the dorsal vagal complex in the cat: an HRP and autoradiographic study.
Brain Res. Bull.
18:
63-71,
1987[Medline].
31.
Yoneda, M.,
and
Y. Taché.
SMS 201-995 induced stimulation of gastric acid secretion via the dorsal vagal complex and inhibition via the hypothalamus in anaesthetized rats.
Br. J. Pharmacol.
116:
2303-2309,
1995[Abstract].
32.
Yoshimatsu, H.,
A. Niijima,
Y. Oomura,
and
T. Katafuchi.
Lateral and ventromedial hypothalamic influences on hepatic autonomic nerve activity in the rat.
Brain Res. Bull.
21:
239-244,
1988[Medline].
33.
Yoshimatsu, H.,
A. Niijima,
Y. Oomura,
K. Yamabe,
and
T. Katafuchi.
Effects of hypothalamic lesion on pancreatic autonomic nerve activity in the rat.
Brain Res.
303:
147-152,
1984[Medline].
34.
Zhang, X.,
R. Fogel,
and
W. E. Renehan.
Physiology and morphology of neurons in the dorsal motor nucleus of the vagus and the nucleus of the solitary tract that are sensitive to distension of the small intestine.
J. Comp. Neurol.
323:
432-448,
1992[Medline].
35.
Zhang, X.,
R. Fogel,
and
W. E. Renehan.
Relationships between the morphology and function of gastric- and intestine-sensitive neurons in the nucleus of the solitary tract.
J. Comp. Neurol.
363:
37-52,
1995[Medline].
36.
Zhang, X.,
W. E. Renehan,
and
R. Fogel.
Evidence that nucleus of the solitary tract neurons modulate the activity of vagal motor neurons in vivo (Abstract).
In: Fourth International Symposium on Brain-Gut Interactions, 1998.
37.
Zhang, X.,
W. E. Renehan,
and
R. Fogel.
Neurons in the vagal complex of the rat respond to mechanical and chemical stimulation of the GI tract.
Am. J. Physiol.
274 (Gastrointest. Liver Physiol. 37):
G331-G341,
1998[Abstract/Free Full Text].
Am J Physiol Gastroint Liver Physiol 277(1):G79-G90
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society