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1 Gastroenterology Research, The objective of this study was to determine
whether sympathetic neurons of the inferior mesenteric ganglion (IMG)
projecting to mesenteric arteries could be distinguished by their
localization, neurochemical phenotype, and electrophysiological
properties from neurons projecting to mesenteric veins. In an in vitro
intact vasculature-IMG preparation, neurons were labeled following
intraluminal injection of Fluoro-Gold or rhodamine beads
into the inferior mesenteric artery (IMA) or vein (IMV). The somata of
neurons projecting to IMA were localized in the central part of the
IMG, whereas those projecting to IMV were localized more peripherally.
None of the labeled neurons was doubly labeled. Neuropeptide Y
immunoreactivity was found in 18.9% of neurons innervating the IMA,
but not in neurons innervating the IMV. Identified neurons were
dissociated and characterized using whole cell patch-clamp recording.
After direct soma depolarization, all of the labeled arterial and
venous neurons were classified as tonic firing, compared with only 40% of unlabeled neurons; the remaining 60% of unlabeled neurons were phasic firing. The results indicate that IMG neurons projecting to
mesenteric arteries are distinct from neurons projecting to mesenteric veins.
vascular neuron; neuropeptide Y; electrophysiology; retrograde
tracing
THE SYMPATHETIC INNERVATION of the vascular system is
divided into different functional groups based on the particular
vascular bed and its reflex responses. Physiologically, these different groups do not function as one; rather, the activity of the neurons in
each pathway is governed separately. Blood flow to the skin, for
example, is regulated separately from blood flow to the skeletal muscles or to the abdominal organs (20).
Previous functional studies indicate there is a basis for
vessel-specific pathways in the prevertebral sympathetic innervation of
the mesenteric circulation (17, 18). Mesenteric arteries and veins show
different sensitivities to sympathetic neural activity regardless of
whether the nerve stimulation is direct (22) or indirect via
baroreceptor or chemoreceptor reflex activation (11, 16). For example,
venous capacitance vessels show a greater degree of responsiveness at
lower frequencies of stimulation than arterial resistance vessels,
which require higher frequencies of stimulation to elicit the same
degree of constriction (18). The neurotransmitters used to mediate
neuromuscular transmission also differ between mesenteric arteries and
veins; in veins, norepinephrine mediates all components of sympathetic
vasoconstriction, whereas in arteries, ATP and neuropeptide Y (NPY)
play an important role in neurally mediated vasoconstriction (39, 42).
Discrete neuronal phenotypes can be distinguished in prevertebral
ganglia on the basis of electrophysiological properties (8),
neurochemistry (1, 30, 36), or morphology (3). Functional studies also
suggest that the postganglionic outflow from prevertebral ganglia is
regionalized with respect to the gastrointestinal tract (28, 29, 32). A
defined subpopulation of neurons innervates mesenteric blood vessels
(30), but it is not known whether the phenotype of neurons innervating
mesenteric artery and vein differ, or whether there is a distribution
of neuronal phenotypes in the innervation of the two vessel types.
Previously, vasoconstrictor neurons have been speculated to contain
immunoreactivity to NPY (NPY-IR), since the proportion of inferior
mesenteric ganglion (IMG) neurons that contain NPY-IR correlates
closely with the proportion of neurons proposed to subserve a
vasoconstrictor function (25, 31). Further speculation has been made as
to the electrophysiological properties of neurons projecting to the
mesenteric vasculature; vasoconstrictor neurons have been proposed to
display phasic firing patterns, since the anatomic occurrence of this
neuronal type and the proportion of phasic neurons in the IMG correlate
with the calculated population of vasoconstrictor neurons (4, 31).
Female guinea pigs (150-250 g) were killed by cervical dislocation
and exsanguination. An abdominal laparotomy was performed under aseptic
conditions. The abdominal aorta, the IMG, and the inferior mesenteric
artery (IMA) and vein (IMV) were gently isolated and excised together
with a 3- to 5-cm-long segment of the descending colon, taking care not
to stretch the vascular sympathetic innervation. After isolation, the
IMG-vascular preparation was placed in a culture dish bathed with
oxygenated feeding medium (see Composition of solutions) and
pinned under light tension. The colon was then dissected away from the
mesenteric vasculature to leave an IMA/IMV preparation with intact IMG
vascular innervation.
Retrograde tracing.
To provide access to the lumen of the IMA, a small incision was made in
the aorta proximal to the branch point of the IMA to allow insertion of
a syringe needle (0.164 mm OD, 34 gauge) for intraluminal perfusion.
The artery was first rinsed with feeding medium to eliminate blood
clots, followed by slow perfusion with 5 ml of air then 5 ml of
distilled water. This procedure has previously been shown to remove the
layer of endothelial cells covering the vessel lumen (34). After the
water was flushed out of the artery with feeding medium, the vessel was
perfused for 1 min with a solution containing 25% (wt/vol) phenol to
induce limited damage of the fine perivascular nerve terminals and
promote maximal uptake of retrograde tracers (9). The phenol itself
caused the length of the vasculature to shorten by ~6%. The phenol
was then rinsed away thoroughly with feeding medium before rhodamine
latex microspheres ("beads") 0.02-0.2 µm in diameter or
Fluoro-Gold (4%) suspended in neuronal culture feeding medium were
injected in the arterial lumen. Retrograde tracers filled approximately
one-half of the total length of the vasculature. For example, the total
length of arterial vessels in the excised preparation was ~70-80
mm. Fluoro-Gold was seen to fill 35-40 mm of arterial vessels,
that is, the primary and secondary branches of the IMA. The tracer was
rarely observed to enter into third or higher order arterial or venous
branches. After the injection, the needle was carefully removed from
the artery, which was then sealed by pinching with forceps. The vein
was also subjected to this luminal preparation protocol in the same
preparation and injected with Fluoro-Gold (if the artery was perfused
with rhodamine beads) or rhodamine beads (if the artery was perfused
with Fluoro-Gold). To avoid leakage of retrograde tracers, the open
portions of the vein were sealed with a drop of cyanoacrylate glue
after the injection.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Immunohistochemical procedures. After organ culture, the IMG together with a small piece of mesenteric artery was dissected free from the rest of the mesenteric vasculature, rinsed in Hanks' solution, and fixed in Zamboni's solution (see Composition of solutions) for 24 h at 4°C. The IMG was oriented with a piece of IMA as the landmark along the rostrocaudal axis on a thin (2 mm) slice of fixed liver, which provided mechanical support. The IMG then was embedded in OCT compound (Sakura Finetek) and immersed in a beaker dish containing 2-methylbutane refrigerated with liquid nitrogen. After fixation, the ganglion was rinsed three to five times with PBS containing 0.0015% Triton X-100 (PBS-TX; see Composition of solutions) before being stored for 24 h at 4°C in PBS-TX to ensure complete removal of fixative. The specimen was then rinsed with PBS-TX before being frozen by immersion in a solution of 2-methylbutane refrigerated with liquid nitrogen. Sections of the ganglion (16-20 µm thick) were cut using a cryostat and mounted on a subbed coverslip.
The coverslip with the IMG was incubated with the primary anti-NPY antibody (1:100, rabbit anti-NPY, porcine) in a humid immunohistochemistry chamber at 37°C for 30 min (43). After incubation, the primary antibody was drained off, and the coverslip was rinsed in PBS-TX-BSA every 15 min for 45 min. The coverslip was back dried before application of the secondary antibody (FITC-labeled goat anti-rabbit IgG, 1:100) and incubated for 30 min at 37°C. The secondary antibody was then drained, and the coverslip was washed as before. After the coverslip was dried, one drop of Fluoromount-G mounting medium was applied to the tissue section, which was then mounted on a clean slide. All antibodies were diluted in PBS-TX solution containing 1% (wt/vol) BSA (PBS-TX-BSA).Immunohistochemical analysis.
Slices containing the IMG and the initial segment of the IMA were
viewed on an Olympus BH2 microscope (magnification ×200-400) equipped with ultraviolet (UV) and tetramethylrhodamine isothiocyanate (TRITC) filters for identification of retrogradely labeled
neurons and with FITC filters for identification of NPY-IR neurons. The number of neurons in each section was counted. This included retrograde tracer-labeled neurons, NPY-IR neurons, and unfilled and/or unstained neurons. In some sections, there were no retrogradely filled neurons or
no NPY-IR artery or vein neurons. The data for the number of neurons
filled from artery or vein were expressed as a percentage of the total
number of neurons in each ganglion. The mean percentage of particular
types of neurons (i.e., artery neuron, vein neuron, NPY +/
) was
the mean of the percentages from each preparation.
Neuronal dissociation and culture methodology. After organ culture, the IMG were dissociated and cultured as described previously (7). Briefly, the IMG was excised, enzymatically dissociated (9 mg/ml papain, 1 mg/ml collagenase, and 4 mg/ml dispase), and plated as a monolayer onto poly-D-lysine-coated glass-bottomed 10-mm well in 35-mm culture dishes (MatTak, Ashland, MA). Cells were maintained in feeding medium (see Composition of solutions) at 37°C in a 5% CO2 humidified incubator. The feeding medium was replaced every 3 days.
Electrophysiological recordings.
Culture dishes containing IMG neurons were transferred to the stage of
an Olympus IMT-2 inverted microscope equipped with UV and TRITC
fluorescent filters for Fluoro-Gold and rhodamine, respectively, and
perfused with Krebs solution (see below); the temperature was held
constant at 37°C. The cells were briefly viewed with fluorescent
illumination to identify the presence of retrograde tracers; whole cell
patch recordings were made both from cells labeled with retrograde
tracers and unlabeled neurons, using borosilicate patch pipettes of
resistance 3-8 M
with an intracellular solution utilizing
potassium chloride as the current carrier (see Composition of
solutions). Electrophysiological recordings were made using an
Axopatch 1D (Axon Instruments, Foster City, CA), and data were filtered
at 2 Hz, digitized via a TL-1 DMA interface (Axon Instruments), and
stored and analyzed on a PC utilizing pCLAMP6 software.
Statistical tests. Statistical significance was assessed using ANOVA followed by Duncan's multiple-range test, with P < 0.05 taken as indicating significance. Not significant (NS) indicates P > 0.05.
Composition of solutions. Organ culture medium consisted of minimal essential medium (Eagle's formulation) with 10% fetal calf serum, 2 mM glutamine, 0.3% glucose, 1,000 U/ml penicillin-streptomycin, 10 mg/ml ascorbic acid, 0.25 mg/ml glutathione, 0.05 mg/ml 6,7-dimethyl-5,6,7,8-tetrahydropteridine (DMPH4), and 10 µM each of cytosine arabinoside, fluorodeoxyuridine, and uridine. Feeding medium was minimal essential medium supplemented with 2.5 ml guinea pig serum, 2 mM glutamine, 0.3% glucose, 1,000 U/ml penicillin-streptomycin, 10 mg/ml ascorbic acid, 0.25 mg/ml glutathione, 0.05 mg/ml DMPH4, 50 ng/ml nerve growth factor, and 10 µM each of cytosine arabinoside, fluorodeoxyuridine, and uridine. Zamboni's solution contained 32 g paraformaldehyde, 240 ml saturated picric acid, 5.25 g KH2PO4, 53.6 g Na2HPO4 · 7H2O, and 1,600 ml H2O, pH 7.2. PBS-TX buffer contained 13.5 g NaCl, 40.2 g Na2HPO4 · 7H2O, 1,500 ml H2O, 2.25 ml Triton X-100, and 2.04 g KH2PO4. Krebs solution contained (in mM) 120 NaCl, 26 NaHCO3, 3.75 KCl, 1 MgCl2, 2 CaCl2, and 5 dextrose, maintained at pH 7.4 by bubbling with 95% O2-5% CO2. Intracellular patch electrode solution contained (in mM) 144.5 KCl, 2 MgCl2, 0.5 EGTA, 5 HEPES, 4 ATP, and 0.25 GTP, pH adjusted to 7.35 with KOH. Hanks' solution contained 100 ml Hanks' balanced salt solution (10×, GIBCO) and 119 mg/100 ml HEPES, pH adjusted to 7.4.
Chemicals. Rhodamine latex microspheres were purchased from Lumafluor (Naples, FL), and Fluoro-Gold was purchased from Fluorochrome (Englewood, CO). Rabbit anti-NPY antibody was purchased from Peninsula Labs (Belmont, CA), the FITC-labeled goat anti-rabbit IgG secondary antibody was purchased from ICN Biomedical (Aurora, OH), and the Fluoromount-G mounting medium was purchased from Southern Biotechnology Associates (Birmingham, AL). Papain and collagenase were purchased from Worthington Biochemical (Freehold, NJ). Dispase was purchased from Boehringer Mannheim (Mannheim, Germany). Minimal essential medium, Hanks' balanced salt solution, penicillin-streptomycin, and L-glutamine were from GIBCO (Grand Island, NY). DMPH4 was from Calbiochem (San Diego, CA). Guinea pig serum was from Chemicon (Temecula, CA). All other drugs, chemicals, and reagents were purchased from Sigma Chemical (St. Louis, MO).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Mesenteric vessels are innervated by two distinct populations of IMG neurons. Retrogradely labeled neurons were identified and counted only in those slice preparations where the nuclei were visible. Intraluminal perfusion with the retrograde tracers (either Fluoro-Gold or rhodamine beads in artery or vein) proved effective in selectively labeling a small number of IMG neurons projecting to either arteries or veins. Double labeling was never observed, that is, retrogradely labeled neurons were never found to contain both Fluoro-Gold and rhodamine beads.
The specificity of the technique was confirmed by experiments in which Fluoro-Gold or rhodamine beads were applied to the external vasculature and associated tissues, allowing the retrograde tracers to be taken up by nonvascular as well as vascular nerve endings. In such experiments, a large number of neurons, as well as nonneuronal cells, in all regions of the ganglia were labeled. The average numbers of labeled arterial neurons (10.1 ± 2.1, n = 19 ganglia) per ganglion were similar to the average number of venous neurons (7.7 ± 2.5, n = 13 ganglia, P > 0.05) after injection of either rhodamine beads or Fluoro-Gold into IMA or IMV. Both rhodamine beads and Fluoro-Gold proved equally effective in labeling neurons projecting from the IMG to the IMA. A total of 42 neurons was later found to contain the retrogradely transported beads in 4 preparations in which rhodamine beads were injected into the IMA. In 15 preparations in which Fluoro-Gold was injected into the IMA, subsequent examination identified a total of 150 Fluoro-Gold-containing neurons. Rhodamine beads proved more effective than Fluoro-Gold in labeling neurons projecting to the IMV, however. A total of 68 neurons from 5 preparations was labeled when rhodamine beads were injected into the IMV, whereas only 32 neurons from 8 preparations were labeled when Fluoro-Gold was injected into the IMV.Neurons projecting to arteries or veins are located in distinct
areas of the IMG.
To analyze the location of retrogradely labeled neurons within the IMG,
each ganglion was divided into four quadrants, and the location of both
Fluoro-Gold- and rhodamine-labeled neurons within each quadrant was
marked on a scale map of the IMG. These analyses were carried out
independently by two observers. Neurons projecting to the IMA were
found to be located predominantly in the central areas of both lobes of
the IMG, whereas neurons projecting to the IMV were located more
peripherally in both the caudal and rostral lobes of the IMG (Fig.
1).
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Neurons projecting to mesenteric veins are larger than those projecting to arteries. The average cross-sectional area of an arterial neuron was 1,198 ± 183 (SE) µm2 (n = 7, range 603-1,546 µm), which was significantly smaller than venous neurons whose cross-sectional area was 2,124 ± 162 (SE) µm2 (n = 7, range 1,155-3,210 µm; P < 0.05). Both arterial and venous neurons were smaller than unlabeled neurons, which had a cross-sectional area of 3,083 ± 210 (SE) µm2 (n = 10, range 2,107-4,200 µm; P < 0.05).
NPY-IR is differentially distributed in IMG neurons projecting to
mesenteric arteries and veins.
In acute ganglia, that is, IMG not organ cultured, 188 of 880 neurons
from 6 ganglia (i.e., 21.4%) contained NPY-IR. A similar proportion of
unlabeled neurons (neurons not labeled by injection of retrograde
tracers into either the IMA or IMV) from 22 ganglia (942 of 4,942, 18.9%; P = NS) from organ cultured
preparations were NPY-IR. Similarly, 10 of 31 neurons (18.9%;
n = 9 ganglia; P = NS) projecting to the IMA were
found to contain NPY-IR (Fig. 2).
Conversely, of those neurons identified as projecting to the IMV (71 neurons from 7 ganglia), none was found to contain NPY-IR (P < 0.05).
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Electrophysiological properties of vascular neurons. Whole cell electrophysiological recordings were made from 14 retrogradely labeled and 48 unlabeled neurons from the same ganglia. In addition, we compared recordings made in 70 acutely dissociated IMG neurons.
Characterization of firing pattern.
Action potentials were initiated by delivering depolarizing current
pulses of graded intensity (1-s duration) to neurons clamped at
60 mV. The current needed to reach the threshold for action potential firing was 90.4 ± 15.9, 47.3 ± 10.9, and 29.4 ± 5.2 pA in unlabeled tonic, arterial, and venous neurons, respectively. These were not significantly different from one another. Neurons were
then classified as phasic or tonic on the basis of their firing
characteristics in response to depolarizing current at three times
threshold (4). In acutely dissociated preparations, 70% (49/70) of
neurons discharged action potentials at the beginning of the
depolarizing current pulse only and were thus classified as phasic
firing (Fig. 3). The remaining acutely
dissociated neurons (21 of 70 neurons, i.e., 30%) fired rhythmically
and continuously throughout the current pulse and were thus classified
as tonic firing (Fig. 3). These two firing patterns have already been
described in intact IMG as well as in other autonomic neurons (7). The same ratio of tonic and phasic neurons was observed when whole cell
recordings were made from unlabeled organ cultured neurons, that is, 33 of 48 unlabeled neurons (69%) were phasic firing, and the remaining 15 neurons (31%) were tonic firing. In contrast, all of arterial and
venous neurons, i.e., 14 of 14 neurons, responded with a tonic firing
pattern (P < 0.05).
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Basic properties.
Because all the labeled neurons were tonic firing, we analyzed the
basic properties of tonic firing acutely dissociated neurons and
compared them with unlabeled and arterial and venous neurons. The input
resistance of venous neurons was greater than acutely dissociated,
unlabeled, and arterial neurons (P < 0.05). The depolarization required to reach threshold for action
potential firing in vascular neurons was less than the depolarization
in both acute and unlabeled neurons (P < 0.05). Significant differences were not found when comparing the
resting membrane potential or the action potential amplitude among all
four neuronal groups (P = NS, see
Table 1).
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Voltage-dependent ionic currents. The presence or absence of several voltage-dependent ionic currents [M current (IM) and A current (IA) and inward rectification; Fig. 3] were examined in acutely dissociated, arterial, venous, and unlabeled neurons.
IM.
IM was analyzed
in voltage clamp with the following protocol: neurons were
voltage-clamped at 30 mV and then step hyperpolarized by
increments of 10 mV for a duration of 2.3 s every 2-18 s until a
final voltage of 100 mV was attained. The identity of
IM was confirmed
with the use of the nonselective ion channel blocker barium (2 mM).
IM was found to
be present in 10 of the 24 acute tonic neurons tested (i.e., 41%), but
only in 4 of 14 (i.e., 29%) of the unlabeled tonic neurons
(P > 0.05 compared with acute
neurons) and only in 3 of 14 (i.e., 21%) of the neurons projecting to
either artery or vein (P > 0.05 compared with acute neurons).
IA.
The protocols for
IA were conducted
in the presence of tetrodotoxin (0.1 µM) to block the transient
sodium current and cobalt (2 mM) to block calcium currents.
IA inactivation
was analyzed in voltage clamp with the following protocol: neurons were
voltage-clamped at 50 mV and then subjected to 1-s conditioning
pulses at different hyperpolarized potentials starting from 60
mV and progressing in 10-mV steps up to 110 mV. This was
followed by clamping the neurons at 40 mV, thus preventing the
activation of the potassium delayed rectifier. The identity of
IA was confirmed
with the use of the nonselective antagonist 4-aminopyridine (1 mM).
IA was found to
be present in 8 of the 20 acute tonic neurons tested (i.e., 40%) and
in 4 of 15 (i.e., 27%) of the unlabeled tonic neurons
(P > 0.05 compared with acute tonic
neurons). The percentage was increased in vascular neurons, with
IA being present
in 7 of the 12 neurons analyzed. No differences in the proportion of neurons expressing
IA were observed
between arterial and venous projecting neurons
(P > 0.05 compared with acute neurons).
Inward rectification.
The inward rectification current was analyzed in voltage clamp with the
following ramp protocol: neurons were clamped at 60 mV and then
step hyperpolarized to 100 mV for 3 s before being depolarized
to 0 mV over 14.4 s. The rate of the ramp was 7 mV/s. The inward
rectification was defined as the cesium-sensitive inward deflection
that was observed at potentials negative to 71 ± 2.2 mV
(n = 6). Although no statistically
significant differences were observed in the occurrence of inward
rectification among the acute, unlabeled, and venous neurons, the
current was present in 8 of 14, 6 of 13, and 3 of 7 neurons,
respectively, a statistically significantly larger occurrence was
observed in arterial neurons, with the current being present in 4 of
the 5 arterial neurons analyzed (P < 0.05; data not shown, but see the
DISCUSSION).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study demonstrates that neurons of the IMG projecting to mesenteric arteries can be distinguished from neurons projecting to mesenteric veins by their localization within the ganglion, their immunoreactivity to NPY, and their electrophysiological properties. Not only are arterial neurons different from venous neurons, but vascular neurons differ from nonvascular with respect to these properties. These findings further substantiate the idea that prevertebral sympathetic ganglion neurons are influenced by their selected target organ.
Labeling of vascular neurons. An average of nine neurons per ganglion was labeled following injection of retrograde tracer into the IMA or IMV. Such a small number of labeled neurons is a likely indication that the labeling technique was selective. Had the fibers of passage or nerve terminals been damaged during the surgical procedures, the numbers of labeled neurons would have been much greater. Indeed, in control experiments when retrograde tracers were placed on the surface of the mesentery and on the exterior of the blood vessels where they could contact both the nonvascular nerves and fibers of passage, a significantly larger population of IMG neurons as well as nonneuronal cells throughout both lobes of the IMG were labeled.
Viscerotopic localization of vascular neurons. The organization of sympathetic ganglia with respect to innervated organs has important implications for the degree to which sympathetic outflow is specific for a particular organ or generalized for groups of organs. Although some of the discrimination of global vs. specific sympathetic activation is organized in the central nervous system (21), this would be expected to be modified by the anatomic characteristics of pathways in the ganglia (19). In both the IMG, as shown in this study, and in paravertebral ganglia (9), arteries and veins are innervated by separate sympathetic neurons. Whereas artery and vein neurons are segregated in different regions of the IMG, they are randomly mixed within individual paravertebral ganglia, but segregated among ganglia at different spinal levels (9). This would extend previous observations that sympathetic ganglion neurons are often localized anatomically in relation to their efferent output (2) as well as their synaptic input (23, 28, 33), thus providing for separate or different activation of different functional subgroups within the ganglion. This same principle has been found to hold true in pelvic ganglia (24, 38). With respect to vascular neurons, this organization could account for the different physiological regulation of arteries and veins (17, 18).
Size of vascular neurons. A difference in the size of arterial and venous neurons provides additional support for the idea that these are separate subpopulations of neurons. Differences in size of sympathetic neurons have been correlated with other differentiating properties such as neurochemical makeup (24), electrophysiological properties (3), and innervation target (12). In the superior cervical ganglion, presumed vasomotor neurons (contain NPY) are smaller than pilomotor neurons (do not contain NPY) (12). However, the grouping of several of these properties in a characteristic neuron type has been difficult to establish (25). For example, in the IMG, we found that arterial neurons were smaller than either venous or unlabeled neurons, although they were both tonic firing. In contrast, in the celiac ganglia of the guinea pig, tonic neurons are larger than phasic neurons (3) without reference to their target.
Immunohistochemistry of vascular neurons. The presence of NPY is often taken as a marker of vasomotor neurons in sympathetic ganglia because of the presence of NPY in nerve fibers surrounding blood vessels (15) and its preponderance in sympathetic ganglion cells (30, 31). We found that NPY is not preferentially localized in arterial neurons when compared with its incidence in all ganglionic neurons, generally ~20% of neurons in IMG (31, 36), and that it is not found in venous neurons. In the one other study where artery and vein neurons were identified by placing retrogradely transported tracers on blood vessels, 94% of arterial neurons but only 17% of venous neurons in lumbar paravertebral ganglia contained NPY (9). Such an absence of NPY-IR in venous neurons raises the question of whether NPY-IR can be demonstrated around mesenteric veins. The distribution of NPY around veins has not received the detailed attention that it has around arteries. However, sparse NPY-IR has been demonstrated to surround large veins of several organs (10, 37), including the gastrointestinal tract. It is likely that the extremely low density of NPY-positive innervation is a reflection of the lower incidence of NPY-positive venous neurons in the ganglia.
The question arises, therefore, as to whether the proportion of vascular neurons immunoreactive for NPY has been underestimated in our study. Such miscalculations are unlikely to arise from technical limitations of the anti-NPY antibody, since the overall proportion of NPY-IR in acutely fixed IMG was consistent from ganglion to ganglion (21%) and corresponded well with previously published reports (31, 36). Similarly, the proportion of NPY-IR neurons is not different in ganglia after organ culture (13, 24). The presence of NPY-IR cannot, therefore, be taken as a characteristic feature of vasoconstrictor neurons (12-14, 35).Electrophysiological properties of vascular neurons. Neurons in sympathetic ganglia can be classified electrophysiologically as tonic or phasic firing based on their response to direct somal depolarization (5, 7, 26, 41). In the present study, all vascular neurons, whether arterial or venous, were tonic. Our data provide the first direct evidence linking a particular IMG neuronal category, i.e., tonic firing neurons, with a specific function, i.e., innervation of mesenteric vasculature. These data are in contrast to previous studies which speculated that phasic neurons are involved in vasoconstriction because of the preponderance of phasic neurons in paravertebral ganglia, a majority of which subserve primarily vasomotor functions (31). It is unlikely that the electrophysiological homogeneity of the retrogradely labeled vascular neurons in the present study was a product of the organ culture procedure, however, because 1) phasic neurons were among the unlabeled population, and 2) the proportion of phasic vs. tonic neurons was the same in neurons dissociated from ganglia immediately after removal from the animal (70 vs. 30%, respectively) as it was in neurons dissociated from ganglia that were organ cultured (69 vs. 31%, respectively). Finally, the basic passive properties of the labeled neurons did not differ from those of unlabeled or acutely dissociated neurons.
The voltage-sensitive currents examined that could account for the tonic firing characteristics of vascular neurons were compared among the four groups of identified neurons, i.e., arterial, venous, unlabeled, and acutely dissociated neurons. Although IM is considered to impose phasic firing behavior on sympathetic neurons (4, 40), the presence of a small IM has also been demonstrated in tonic firing neurons (4), so its presence in tonic firing neurons of the present study should not be considered surprising. Similarly, the high percentage of tonic neurons expressing IA is not unexpected, given the association of the IA with tonic firing neurons (6). Interestingly, venous neurons differed electrophysiologically from arterial neurons in that arterial neurons were more likely to show inward rectification. By closing upon depolarization, inwardly rectifying potassium channels prolong membrane depolarizations (4). Tonic firing celiac ganglia neurons receive small, subthreshold fast as well as slow excitatory synaptic inputs (32). The presence of inward rectification in tonic arterial neurons may be of significance, since the membrane depolarization to excitatory synaptic inputs would be larger, hence less synaptic excitation would be required to reach action potential threshold. Indeed, the depolarization required to reach threshold for action potential firing is significantly less in arterial neurons when compared with the other neuronal groups. In addition, mesenteric arteries require higher frequencies of nerve stimulation not only to induce the same degree of norepinephrine-mediated vasoconstriction as veins, but also to induce the release of nonnoradrenergic mediators such as NPY, which play important additional roles in sympathetic vasoconstriction (42). Inward rectification would allow arterial neurons to fire action potentials at these required higher frequencies. In conclusion, our results support the concept of differential sympathetic control of arteries and veins (16, 18, 22, 27). Arterial and venous neurons are separate subpopulations within the IMG. Their different location within the IMG provides a structural basis for their separate activation by synaptic inputs. In addition, their different neurochemical and electrophysiological properties could be the basis for distinctly different responses to synaptic activation. A more complete understanding of the impact of the structure and function of arterial and venous neurons on the neurogenic regulation of artery and vein awaits further study.| |
ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-59189 (to D. L. Kreulen), West Virginia University School of Medicine research grants (to D. L. Kreulen and R. A. Travagli), and American Heart Association Grant WV 97-02-F (to K. N. Browning).
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FOOTNOTES |
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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.
Address for reprint requests and other correspondence: D. L. Kreulen, Dept. of Physiology, Michigan State University, B340 Life Sciences, East Lansing, MI 48824-1317 (dkreulen{at}pilot.msu.edu).
Received 4 September 1998; accepted in final form 21 December 1998.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
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