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Receptors in lateral hypothalamic area involved
in insular cortex sympathetic responses
KENNETH S. BUTCHER1 AND DAVID F. CECHETTO1,2
Departments of 1Physiology and 2Anatomy and Cell Biology,
University of Western Ontario, London, Ontario, Canada N6A 5C1
glutamate; N-methyl-D-aspartate; arterial blood pressure;
renal nerve
(IC) has been implicated in a
number of autonomic changes resulting from acute
hemispheric stroke (9, 35). A chemical lesion of the IC
in the Wistar rat, for example, resulted in an acute
increase in renal sympathetic nervous activity and
arterial pressure 3–4 h following the lesion (9). These
changes were similar to those seen following middle
cerebral artery occlusion (13). In addition, the IC has
been shown to have a profound influence on the heart.
Phasic microstimulation of the IC, for example, linked
to the Q wave of the cardiac cycle, resulted in both
tachycardic and bradycardic responses (27).
Arterial pressure and heart rate responses can be
elicited by both electrical and chemical stimulation of
the IC in the Wistar rat (11, 27, 28, 29, 41). Neuroanatomic tracing studies have shown (41) that one of the
potential sites of autonomic outflow from the IC is the
lateral hypothalamic area (LHA). Subsequently, the
effects of IC stimulation have been shown (11, 12) to be
dependent on obligatory synapses in both the LHA and
THE INSULAR CORTEX
ventrolateral medulla (VLM). Microinjection of the
presynaptic inhibitor cobaltous chloride into either of
these two regions results in blockade of sympathetic
effects of IC stimulation. We have recently shown (7)
that the receptors involved in the VLM mediating IC
sympathetic responses are non-N-methyl-D-aspartic
acid (NMDA) glutamatergic. The neurotransmitter(s)
and receptor(s) involved in the LHA remain to be
identified. The purpose of this investigation was to
determine the receptors in the LHA that mediate IC
sympathoexcitatory responses. In these experiments,
the IC was stimulated before and after glutamate and
other receptor antagonists were injected into the LHA.
A preliminary report of this investigation has been
presented in abstract form (8).
METHODS
Animals. Twenty-seven male Wistar rats (250–350 g)
between 15 and 20 wk old were used in the experiments. Food
and tap water were provided ad libitum, except that food was
removed ,12 h before surgery.
General surgical methods. Rats were initially anesthetized
with a mixture of a-chloralose (30 mg/kg ip) and urethan (0.75
g/kg ip). During the experiment, anesthesia was maintained
with urethan supplements (0.75 g/kg iv). Supplements were
given on the first sign of withdrawal to pinching of the foot.
Core temperature was maintained at 37°C with the use of a
rectal thermometer, temperature controller, and heating pad.
The right femoral artery was cannulated with PE-50 tubing
filled with heparinized saline. Pulsatile arterial pressure was
measured with a Statham P23 ID transducer and continuously monitored on a Grass model 7E polygraph. Mean
arterial pressure (MAP) was recorded on the polygraph by
filtering the pulse pressure with a 0.5-Hz high-frequency
filter. Heart rate (HR) was determined with a Grass 7P44
tachograph and monitored on the polygraph. The right femoral vein was catheterized with PE-10 tubing for the administration of drugs and anesthetic.
The animal was tracheotomized and placed in a Kopf
stereotaxic apparatus. The right kidney was exposed using a
retroperitoneal approach. With the aid of a Zeiss operating
stereomicroscope, the renal nerve branches were isolated
from the surrounding tissue and a loose ligature was placed
around one of them. A bipolar stainless steel electrode was
used to record nerve activity. The electrode was secured to the
nerve using dental impression material (Perfourm, Miles
Laboratories). The nerve was tested for a reflex decrease in
sympathetic activity by activating baroreceptors with an
infusion of the a-receptor agonist phenylephrine (40–60
µg/kg iv). The multiunit nerve activity was first amplified and
filtered (from 100 Hz to 3 kHz) with a Grass model P15
preamplifier. The signal was further amplified (Neurolog
NL100) and then fed into the oscilloscope and an audio
monitor (model AM8C, Grass). The rectified and integrated
0363-6135/98 $5.00 Copyright r 1998 the American Physiological Society
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Butcher, Kenneth S., and David F. Cechetto. Receptors
in lateral hypothalamic area involved in insular cortex sympathetic responses. Am. J. Physiol. 275 (Heart Circ. Physiol. 44):
H689–H696, 1998.—Previous evidence has shown that sympathetic nerve responses to insular cortical (IC) stimulation
are mediated by synapses within the lateral hypothalamic
area (LHA) and ventrolateral medulla. The present study
determined the receptor(s) involved at the synapse in the
LHA associated with stimulation-evoked IC sympathetic
responses. Twenty-seven male Wistar rats were instrumented for renal nerve activity, arterial pressure, and heart
rate recording. The right IC was stimulated with a bipolar
electrode (200–1,000 µA, 2 ms, 0.8 Hz) resulting in sympathetic nerve responses. Antagonists were then pressure injected into the ipsilateral LHA (300–500 nl). Kynurenate (250
mM) injections resulted in 51 6 8% (range 0–100%) block of
IC-stimulated sympathetic nerve responses. Similarly, the
N-methyl-D-aspartic acid (NMDA)-receptor antagonist DL-2amino-5-phosphonopentanoic acid (200 µM) resulted in an
inhibition (82 6 8%; range 51–100%) of IC-stimulated sympathetic responses. Injection of the non-NMDA antagonist
6-cyano-7-nitroquinoxaline-2,3-dione (200 µM) had no effect
on IC sympathetic responses. Injection of antagonists to
GABA, acetylcholine, and adrenergic receptors was also
without effect. No antagonist injections had any effects on
baseline sympathetic nerve discharge, arterial pressure, or
heart rate. These results suggest that the IC autonomic
efferents projecting to the LHA utilize NMDA glutamatergic
receptors.
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NEUROTRANSMITTERS IN LHA
RESULTS
Sympathetic responses to stimulation of IC. Sympathetic nerve responses were elicited by stimulation of
sites within the posterior IC (Fig. 1). These sites were
primarily located in the agranular and dysgranular
regions of the posterior IC, as previously described (12).
Peristimulus time histograms generated during stimulation of sites within the IC demonstrated an initial increase,
followed by a decrease, in renal nerve activity (see e.g., Fig.
2A). Latencies to the onset of the excitatory and inhibitory phases of the renal nerve response were ,90 and
Fig. 1. Line drawings of coronal hemisections of rat brain, showing
stimulation sites (r) in insular cortex (IC) used to generate peristimulus time histograms of renal sympathetic nerve responses. Symbols
may represent more than 1 stimulation site. Nos. refer to distance
(mm) from bregma. AC, anterior commissure; AI, agranular insular
cortex; CPu, caudate putamen; DI, dysgranular insular cortex; GI,
granular insular cortex.
130 ms, respectively. The duration of the excitatory
response was ,60 ms (Fig. 2A). These latencies and
durations are similar to those reported previously in
our laboratory (11, 12).
Effect of glutamate antagonist injection. Injection of
kynurenate, a blocker of all ionotropic glutamate receptors, including NMDA and non-NMDA, into the tuberal
and posterior LHA resulted in a significant attenuation
of the sympathetic nerve response to IC stimulation
(51 6 8%, range 5 0–100%, n 5 17) (Fig. 2). Recovery of
the response occurred ,45 min after injection, at which
time the IC sympathetic responses were not significantly different from initial values. Not all kynurenate
injections into the LHA resulted in an effective block
(i.e., .50%) of the IC sympathetic responses (Figs. 2
and 3). Injections not directly centered in the tuberal
and posterior LHA resulted in ,50% inhibition of the
response to IC stimulation (Figs. 2 and 3). The
most effective sites were the most ventral portions of
the tuberal and posterior LHA, lateral to the fornix
(Fig. 2).
Injection of the NMDA-receptor antagonist AP-5 also
resulted in a significant attenuation of the sympathetic
nerve response to IC stimulation (Fig. 4). This dose of
AP-5 in general yielded a consistently effective block of
the evoked response (82 6 8%, range 51–100%, n 5 7).
The effective injections were primarily located within
the tuberal and posterior LHA (Fig. 3).
Injections of the non-NMDA-receptor antagonist
CNQX had no effect on the sympathetic nerve response
to IC stimulation, despite the fact that the majority of
sites were located in the posterior and tuberal LHA
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nerve activity was continuously monitored on a polygraph
(model 7P10, Grass). Signals were also discriminated, using a
window discriminator, and fed into a microcomputer for
compilation of peristimulus time histograms in response to IC
stimulation. Background noise levels were determined by the
infusion of hexamethonium (2 mg/kg iv) at the conclusion of
the experiment period.
Electrical stimulation. A steel bipolar electrode (tip separation 0.5 mm; SNEX 100, David Kopf Instruments) was lowered
into the IC. Peristimulus time histograms were evoked by stimulation of the IC (200–1,000 µA, 2 ms, 0.8 Hz) for 125 s.
Microinjections. Injections into the LHA were made with
the use of a glass micropipette (tip diameter ,20 µm) filled
with a receptor antagonist or vehicle solution. All antagonists
were dissolved in 0.9% NaCl, and the solution was titrated to
pH 7.4 before it was used for injection. The NaCl solution was
also used in control injections. The pipette was stereotaxically
placed in the LHA, and solutions were pressure injected
(300–500 nl). We have previously shown (11, 28) that this
volume is adequate for covering the region of the LHA
mediating insular responses, without extensive spread to
surrounding structures. DL-2-Amino-5-phosphonopentanoic
acid (AP-5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX;
Tocris Neuroamin) were dissolved in NaCl to a concentration
of 200 µM. Kynurenate (Sigma) was injected at a concentration of 250 mM. Bicuculline, atropine, phentolamine, and
propranolol (Sigma) were all injected at a concentration of 0.1
µM. These antagonist concentrations have previously been
shown to be effective in blocking their respective receptors
(14, 16, 21, 30, 31, 38).
Histological procedures. At the conclusion of the experiment,
rats were deeply anesthetized with a bolus injection of urethan
(1.5 g/kg ip). All animals were then perfused transcardially with
0.9% saline followed by 4% formaldehyde. The brains were
removed, sectioned (50 µm) with a freezing microtome, and
stained with thionin. The sections were examined with a microscope (Leitz Diaplan), and all pipette tracks were drawn using a
camera lucida drawing attachment.
Data analysis. Peristimulus time histograms were generated and analyzed with the use of a microcomputer and an
integrated program for electrophysiological experiments. The
program was used to calculate a mean level of baseline
activity before the stimulus artifact. After the stimulus
artifact, an excitatory sympathetic response was identified in
which activity was calculated to be one standard deviation
above baseline activity for five consecutive bins (1 bin 5 2
ms). The absolute response was determined by subtracting
the baseline activity from the total number of spikes during
the responsive period. Statistical comparisons were made
using ANOVA followed by Dunnett’s test for significance of all
versus control. For all tests, a P value ,0.05 was considered
to indicate significance. Values in the results are expressed as
means 6 SE.
NEUROTRANSMITTERS IN LHA
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(Fig. 4). Similarly, saline control injections into the
LHA did not affect the sympathetic nerve response to
stimulation of the IC.
The examples in Fig. 5 indicate that injection of
either kynurenate or AP-5 into the LHA had no consistent effect on baseline sympathetic renal nerve discharge. When all of the kynurenate and AP-5 injections
are considered, the average change in baseline sympathetic renal nerve discharge is 10.7 6 4.5%. Similarly,
there was no change in arterial pressure or HR following injection of these antagonists into the LHA (Fig. 5).
The average changes were 10.4 6 1 mmHg and 18 6 6
beats/min, respectively.
Effect of injection of other antagonists. Bicuculline
(GABA-receptor antagonist), atropine (muscarinic cholinergic receptor blocker), phentolamine (both a1- and
a2-adrenoceptor antagonist), and propranolol (both b1and b2-adrenoceptor antagonist) injections into the
LHA had no effect on the IC sympathetic response. In
addition, these antagonists had no observable effect on
baseline renal sympathetic nerve discharge, arterial
pressure, or HR (data not shown).
DISCUSSION
Effect of glutamate antagonist injection into LHA on
IC sympathetic responses. Electrical stimulation of the
IC in the present experiments resulted in renal sympathetic nerve responses consistent with those observed
previously (11, 12). The nonspecific excitatory amino
acid-receptor antagonist kynurenate effectively blocked
the increase in renal sympathetic nerve response to
stimulation of the IC. The most effective injection sites
were in the most ventral portions of the tuberal and
posterior ipsilateral LHA, lateral to the fornix. This is
consistent with an anterograde tracing study (41),
which showed that the IC projects heavily to this
portion of the ipsilateral LHA. A previous investigation
(11) in this laboratory demonstrated that injection of
the nonspecific presynaptic inhibitor cobaltous chloride
into the LHA also attenuated the IC sympathetic
response. In that study, effective sites were also found
to be in the most ventral portions of the LHA, lateral to
the fornix. Unlike the present study, however, effective
injections were limited to the most posterior portions of
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Fig. 2. A: effect of kynurenate injection
(250 mM, 300 nl; arrow) into lateral
hypothalamic area (LHA) on renal sympathetic nerve response to IC stimulation. B: line drawings showing location
of kynurenate injections into tuberal
(top) and posterior LHA (bottom). Symbols represent ,50% (s) or .50% (r)
attenuation of sympathetic nerve response to IC stimulation. Nos. refer to
distance (mm) posterior to bregma. 3V,
third ventricle; A, arcuate nucleus; CI,
internal capsule; DMH, dorsomedial
hypothalamic nucleus; f, fornix; ml,
medial lemniscus; mt, mammilothalamic tract; Pef, perifornical area; ZID
and ZIV, dorsal and ventral zona incerta. C: graphs showing mean changes
in sympathetic nerve response (evoked
response) for all animals in tuberal
(top) and posterior LHA (bottom). * Significant attenuation of response (P ,
0.05).
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NEUROTRANSMITTERS IN LHA
the LHA and did not include the more rostral tuberal
LHA (11). This is likely a reflection of the distribution of
IC efferent synapses within the IC. In both this study
and the cobaltous chloride experiments (11), inhibition
of the response was rarely complete. This suggests that
the connections between IC autonomic efferents and
LHA neurons may be relatively diffusely distributed
within this region. This postulation is supported by the
anterograde labeling of IC autonomic efferents, which
shows diffuse projections throughout the ipsilateral
tuberal and posterior LHA (41).
The LHA has also been shown (28) to contain an
obligatory synapse for cardiac chronotropic sites within
the IC. Phasic microstimulation of these sites, linked to
the R wave of the cardiac cycle, results in tachycardia
or bradycardia without any concomitant effects on
blood pressure (27). It is not known whether these
responses represent distinct sites within the IC or are a
specific result of the phasic stimulus. Kynurenate
injections into the LHA attenuated the cardiac response to phasic microstimulation of the IC (28). As in
the present study, injections into the ventral and
lateral portions of the tuberal LHA were effective in
inhibiting the response to stimulation of the IC.
An effort was made in the present investigation to
identify the receptor subtype(s) mediating IC sympa-
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Fig. 3. Line drawings of hypothalamus summarizing injections into
LHA. n, 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) injections. j,
DL-2-Amino-5-phosphonopentanoic acid (AP-5) injections resulting in
.50% attenuation of sympathetic nerve response to IC stimulation;
k, AP-5 injections resulting in ,50% attenuation of sympathetic
nerve response. Also included in anterior and posterior sections are
ineffective kynurenate injections (s).
thetic responses within the LHA. At least five distinct
glutamate receptors have been identified to date (23).
These have been termed the NMDA, 2-amino-3-(3hydroxy-5-methylisoxazol-4-yl)proprionic acid, metabotropic, kainate, and 2-amino-4-phosphonobutanoic acid
receptors (23). Injection of the relatively specific NMDA
glutamate receptor AP-5 was extremely effective in
inhibiting the sympathetic nerve response to stimulation of the IC. The effective sites were very similar to
those for kynurenate. Unlike AP-5, the non-NMDA
glutamate receptor antagonist CNQX had no effect on
the sympathetic nerve response to IC stimulation.
These results indicate that the IC autonomic efferents
exert their autonomic effects via an NMDA glutamatergic synapse in the LHA.
Recently, it has been shown (7, 37) that the effects of
both IC and LHA stimulation are mediated by a
glutamatergic synapse in the VLM. Unlike that in the
LHA, however, the synapse in the VLM is non-NMDA
mediated (7). The function of these two different populations of glutamate receptors in a single pathway mediating the sympathetic effects of IC stimulation is
unclear.
Somatosensory input to the ventrobasal thalamus
has also been shown (32) to involve both NMDA and
non-NMDA receptors. Non-NMDA receptors mediate
short-latency somatosensory responses, whereas NMDA
receptor effects are manifested only in response to
maintained sensory stimulation. This suggests that
NMDA and non-NMDA receptors are each suited to a
particular type of presynaptic input. Similarly, in the
spinal cord, monosynaptic excitation of Renshaw cells
is mediated by non-NMDA receptors, whereas NMDA
receptors are responsible for polysynaptic activation
(15). It has been postulated (33) that non-NMDA receptors mediate fast synaptic transmission, whereas NMDA
receptors are responsible for slower potentials with
longer time courses. This may be related to the two
different populations of glutamate receptors in the LHA
and VLM.
The IC, LHA, and VLM form an anatomic axis
whereby the IC can affect the sympathetic nervous tone
(Fig. 6). It appears that the IC projection to the LHA is
mediated by the slower NMDA synaptic mechanism.
The LHA, in contrast, appears to influence the VLM via
a fast-acting non-NMDA receptor. This may be related
to the overall functions of the IC and LHA. It has been
proposed (3) that the IC sets the autonomic tone
appropriate to the visceral and limbic stimuli it receives. The LHA receives projections from several other
limbic nuclei, including the infralimbic cortex, septal
nuclei, central nucleus of the amygdala, and the bed
nucleus of the stria terminalis (20). In addition, the
LHA has direct connections with brain stem autonomic
nuclei, including the VLM (1). The LHA has therefore
been proposed (2) to be a site capable of integrating the
autonomic aspects of emotions. The synaptic potential
of the NMDA receptor has been shown (25) to vary with
the potential of the cell membrane because of a voltagedependent blockade by Mg21. Thus the NMDA receptor
NEUROTRANSMITTERS IN LHA
H693
may be ideal for the integration of tonic or sustained
signals from the IC with those from other limbic nuclei.
The LHA has also been shown (4, 37, 40) to have
profound effects on ongoing sympathetic tone. The LHA
contains neurons whose activity is synchronized to the
2- to 6-Hz component of sympathetic nerve discharge
(4). This relationship to the sympathetic nervous system appears to be mediated by the rostral VLM
(4, 7, 37). The faster non-NMDA receptor mediating
LHA responses in the VLM appears to be most appropriate for this type of direct synaptic transmission (Fig. 6)
(7).
A similar situation has recently been shown to exist
within the ascending visceral sensory pathway to the
IC. Visceral sensory information from the nucleus of
the solitary tract is carried to the parabrachial nucleus,
which in turn projects to the ventrobasal thalamus,
which relays to the IC (10). It has been established (30)
that the synapse within the parabrachial nucleus is
mediated by NMDA receptors. Within the ventrobasal
thalamus, however, synaptic transmission is mediated
by non-NMDA glutamatergic receptors (5). This is
consistent with the hypothesis that glutamate receptor
type is related to the function of the synapse.
Effect of antagonist injection on autonomic variables.
The LHA has been shown to contain sites capable of
eliciting profound cardiovascular changes in response
to stimulation with glutamate (1, 12, 18, 36). Kynurenate, AP-5, and CNQX all had no effects on baseline
renal sympathetic nerve activity, arterial pressure, or
HR. This is consistent with the findings of Oppenheimer et al. (28), who also reported no changes in basal
arterial pressure or HR in response to kynurenate
injection into the LHA.
Wible et al. (39) demonstrated that bicuculline injection into the posterior hypothalamus of conscious rats
results in increases in arterial pressure, HR, and
splanchnic sympathetic nerve activity. In that study,
injections appeared to be more medial and probably
affected the perifornical region of the LHA. A recent
investigation (1) has shown that the most lateral
portion of the LHA differs in its response to glutamate
stimulation and its efferent connections from the perifornical LHA. Given the lack of response to bicuculline
in the present study, the role of GABA may also differ
between these two regions. A more systematic study of
the entire LHA with regard to GABA and its cardiovascular effects is required. Differences between the present results and those of Wible et al. (39) may also be due
to the use of conscious versus anesthetized rats.
The LHA has been shown (22, 42) to contain acetylcholinesterase-containing cells. These cells have been
shown (42) to project to the basal forebrain and may be
involved in cortical arousal. The LHA also contains
cells that are responsive to the iontophoretic application of acetylcholine and that are sensitive to atropine
(26). There is no evidence for the involvement of any of
these cells in autonomic regulation, although this possibility cannot be excluded. Acetylcholine has been shown
(6) to mediate increases in blood pressure and HR in
the adjacent posterior hypothalamic nucleus.
The LHA has been shown (22) to contain catecholaminergic cells and fibers. In addition, a- and
b-adrenergic receptors are found within the LHA (24).
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Fig. 4. Peristimulus time histograms
generated by IC stimulation and effects
of AP-5 (A; n 5 7), CNQX (B; n 5 8), and
saline injections (C; n 5 17) into LHA.
Bottom: graphs show mean change in
sympathetic nerve response to IC
stimulation (evoked response). Only
N-methyl-D-aspartate (NMDA) antagonist AP-5 resulted in a significant attenuation of IC sympathetic response.
* Significant attenuation of response
(P , 0.05).
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NEUROTRANSMITTERS IN LHA
Fig. 5. Effects of kynurenate (Kyn; A), AP-5 (B), and saline injection
(Sal; C) into LHA on arterial pressure (AP), mean arterial pressure
(MAP), heart rate (HR), and integrated renal nerve activity (IRNA).
In all cases, no changes in baseline nerve activity or cardiovascular
variables were observed. bpm, Beats/min.
The catecholamine-sensitive cells of the LHA have also
been shown to affect sympathoadrenal activity in the
rat. Injection of phentolamine into the lateral portion of
the LHA results in an increase in circulating levels of
norepinephrine after exercise in rats (34). Conversely,
injection of the antagonist timolol into the same region
results in increases in plasma epinephrine during
exercise in rats. Catecholamines in the LHA have also
been implicated in the central control of renal function
(19). In addition, catecholaminergic mechanisms in the
LHA are involved in the control of thirst and ingestion
behavior (17). Our results suggest, however, that catecholamines are not involved in mediating autonomic
responses from the IC in the LHA.
Roles for GABA, acetylcholine, and the catecholamines in the LHA with respect to autonomic regulation cannot be excluded. More systematic investigations of the cardiovascular effects of injecting agonists
and antagonists to these neurotransmitters in the LHA
are clearly required.
This study indicates that an NMDA glutamatergic
synapse mediates the sympathetic renal nerve response to stimulation of the IC. This represents
the first relay in the pathway mediating the sympathetic outflow of the IC. The nature of the neurotransmission at this synapse is different from that of the
second relay, which is in the VLM. This may represent a
difference in the frequency of presynaptic inputs in the
two sites. This information may be important in future
attempts to modulate the autonomic outflow from
the IC.
This work was supported by the Heart and Stroke Foundation of
Ontario. K. S. Butcher is the recipient of a Heart and Stroke
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Fig. 6. Schematic model of efferent pathways from IC to LHA and
ventrolateral medulla (VLM). LHA receives efferent projections from
several limbic nuclei, including infralimbic cortex (IL), central nucleus
of amygdala (Ace), and bed nucleus of stria terminalis (BNST).
NMDA receptor may be ideally suited to integration of these inputs
with outflow from IC. Non-NMDA receptor in rostral VLM (RVLM)
mediates efferent signals from LHA. IML, intermediomedial cell
column.
NEUROTRANSMITTERS IN LHA
Foundation of Canada Traineeship. D. F. Cechetto is a Career
Investigator of the Heart and Stroke Foundation of Ontario.
Address for reprint requests: D. F. Cechetto, Dept. of Anatomy and
Cell Biology, Univ. of Western Ontario, London, Ontario, Canada
N6A 5C1.
Received 6 August 1997; accepted in final form 21 April 1998.
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