Department of Anatomy and Histology and Centre for Neuroscience, Flinders University of South Australia, Adelaide, South Australia 5001, Australia
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Jobling, Phillip and
Ian L. Gibbins.
Electrophysiological and Morphological Diversity of Mouse
Sympathetic Neurons.
J. Neurophysiol. 82: 2747-2764, 1999.
We have used multiple-labeling
immunohistochemistry, intracellular dye-filling, and intracellular
microelectrode recordings to characterize the morphological and
electrical properties of sympathetic neurons in the superior cervical,
thoracic, and celiac ganglia of mice. Neurochemical and morphological
characteristics of neurons varied between ganglia. Thoracic sympathetic
ganglia contained three main populations of neurons based on
differential patterns of expression of immunoreactivity to tyrosine
hydroxylase, neuropeptide Y (NPY) and vasoactive intestinal peptide
(VIP). In the celiac ganglion, nearly all neurons contained
immunoreactivity to both tyrosine hydroxylase and NPY. Both the overall
size of the dendritic tree and the number of primary dendrites were
greater in neurons from the thoracic and celiac ganglia compared with those from the superior cervical ganglion. The electrophysiological properties of sympathetic neurons depended more on their ganglion of
origin rather than their probable targets. All neurons in the superior
cervical ganglion had phasic firing properties and large afterhyperpolarizations (AHPs). In addition, 34% of these neurons displayed an afterdepolarization preceding the AHP. Superior cervical ganglion neurons had prominent IM,
IA, and IH
currents and a linear current-voltage relationship between 60 and
110 mV. Neurons from the thoracic ganglia had significantly smaller
action potentials, AHPs, and apparent cell capacitance compared with
superior cervical ganglion neurons, and only 18% showed an
afterdepolarization. All neurons in superior cervical ganglia and most
neurons in celiac ganglia received at least one strong preganglionic
input. Nearly one-half the neurons in the celiac ganglion had tonic
firing properties, and another 15% had firing properties intermediate
between those of tonic and phasic neurons. Most celiac neurons showed
significant inward rectification below
90 mV. They also expressed
IA, but with slower inactivation kinetics
than that of superior cervical or thoracic neurons. Both phasic and
tonic celiac ganglion neurons received synaptic inputs via the celiac
nerves in addition to strong inputs via the splanchnic nerves.
Multivariate statistical analysis revealed that the properties of the
action potential, the AHP, and the apparent cell capacitance together
were sufficient to correctly classify 80% of neurons according to
their ganglion of origin. These results indicate that there is
considerable heterogeneity in the morphological, neurochemical, and
electrical properties of sympathetic neurons in mice. Although the
morphological and neurochemical characteristics of the neurons are
likely to be related to their peripheral projections, the expression of
particular electrophysiological traits seems to be more closely related
to the ganglia within which the neurons occur.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Neurons in sympathetic ganglia show considerable heterogeneity in
their neurochemical profiles, their soma size, and their dendritic
morphology. These characteristics, especially the expression of
neurotransmitters, are often tightly linked with the type of target
tissue they innervate. Furthermore, in many cases, the neurochemical
and morphological heterogeneity corresponds with specific populations
of neurons identified by functional criteria (Gibbins
1995; Morris et al. 1995
). This morphological
and functional evidence shows that sympathetic motor outflows are
organized into multiple pathways whereby various effector organs can be
regulated independently by the CNS (Jänig and McLachlan
1992
).
The electrical properties of sympathetic neurons also show some
heterogeneity. However, it is not so apparent that the expression of
these properties is similarly encoded in a pathway-specific fashion.
For example, in sympathetic ganglia of guinea pigs, there appears to be
only a partial correlation between the electrophysiological characteristics of neurons, their morphology, and their expression of
chemical markers (Boyd et al. 1996; Gibbins et
al. 1999
; Keast et al. 1993
; McLachlan
and Meckler 1989
). The neurons in sympathetic ganglia receive
convergent central inputs from spinal preganglionic neurons
(Jänig 1995
; McLachlan and Meckler
1989
). Many neurons in the prevertebral sympathetic ganglia
receive additional convergent synaptic inputs from peripheral neurons
located in the enteric plexuses (Kreulen and Szurszewski
1979
). The number and strength of synaptic inputs varies
considerably between subpopulations of sympathetic neurons
(McLachlan and Meckler 1989
). Although there is much
information about synaptic transmission in sympathetic ganglia
(Jänig 1995
; McLachlan 1995
), it is
still not known how sympathetic postganglionic neurons integrate
convergent inputs in different functional pathways. Nevertheless, we
might expect that the electrophysiological characteristics of any
autonomic neuron would be optimally adapted to appropriately integrate
synaptic inputs generated by central and peripheral reflexes. If so, we would predict that the electrophysiological heterogeneity of
sympathetic neurons would be related to the differences in the number
and activation patterns of their synaptic inputs according to the functional pathway within which they lie.
To understand better the relationships between the morphological, neurochemical, and electrophysiological properties of sympathetic postganglionic neurons and their targets, we have used immunohistochemical, intracellular dye-filling, and electrical recording techniques together with multivariate statistical analyses to investigate neurons in the superior cervical, thoracic, and celiac ganglia of adult mice. These ganglia contain different populations of neurons projecting to a wide variety of targets, including vascular beds of the head, skeletal muscles, skin and viscera, the piloerector muscles of the skin, salivary glands, and the gastrointestinal tract. We were surprised to find that the electrical properties of the neurons were more closely related to the ganglia in which they occur rather than to the specific functional pathways that run through the ganglia.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemistry
Adult mice (BalbC) of either sex were killed with a lethal dose
of inhaled halothane as approved by the Animal Welfare Committee of the
Flinders University of South Australia. Superior cervical, thoracic,
and celiac ganglia were removed and processed for multiple labeling
immunofluorescence as described previously (Gibbins and Matthew
1996; Murphy et al. 1998
). Briefly, ganglia were
fixed by immersion for 24 h in a solution of 2% formaldehyde and
0.2% picric acid in 0.1 M phosphate buffer (pH 7.0). After fixation, excess picric acid was washed from the tissues with three changes of
80% ethanol before the tissues were cleared with several changes of
dimethylsulfoxide (DMSO). For sectioning, ganglia were dehydrated in
100% ethanol (3 changes) before being infiltrated with polyethylene glycol (PEG 1000 MW) for 30 min under vacuum at 46°C. Ganglia were
then embedded in molten PEG (1450 MW) and placed in a
20°C freezer
for 10 min before being sectioned (10-30 µm) on a standard bench
microtome. Tissue sections were floated in phosphate-buffered saline
(PBS) and then placed in 10% normal donkey serum for 30 min before
being incubated for 24-72 h in combinations of one, two, or three
primary antisera raised in different species. Primary antisera used
were as follows: rabbit anti-tyrosine hydroxylase (Dr. J. Thibault;
dilution of 1:2,000), sheep anti-neuropeptide Y (code E2210/2, supplied
by Dr. J. Oliver and Dr. W. Blessing; 1:1,000), rat anti-vasoactive
intestinal peptide (code F1/III, Dr. R. Murphy; 1:2,000), rabbit
anti-calcitonin gene related peptide (CGRP; Peninsula, Belmont, CA;
1:2,000), goat anti-CGRP (Arnel, Cherokee Station, NY; 1:1,000) and
sheep anti-choline acetyltransferase (Chemicon, Temecula, CA; 1:2,000).
Following incubation in primary antisera, sections were washed in
several changes of PBS and incubated for 2 h in secondary
antisera. Secondary antisera (Jackson Immunoresearch Labs, West Grove,
PA) were all raised in donkeys and conjugated to dichlorotriazinyl
amino fluorescein (DTAF), Cy3 or Cy5. In some cases, whole mounts of
ganglia were processed for immunohistochemistry in place of sections.
In these cases, primary antisera were used at twice the concentration
used for sections, and incubations in secondary antisera were carried
out for 4-24 h. All the antisera used in this study are well
characterized and do not show any known cross-reactivity with
inappropriate epitopes (Gibbins and Matthew 1996
;
Morris et al. 1998
; Murphy et al. 1998
).
To visualize neurobiotin-filled neurons, ganglia were fixed overnight as described above and washed in three changes of 80% ethanol followed by three changes of DMSO before being placed in PBS. Ganglia were then incubated in streptavidin conjugated to DTAF, Cy3 or Cy5 for 2 h before being washed in three changes of PBS.
Sections or whole mounts of ganglia were mounted on slides in carbonate-buffered glycerol (pH 8.6). For widefield fluorescence microscopy, tissues were examined with an Olympus AX70 or BX50 epifluorescence microscope fitted with highly discriminating filters (Chroma Technology, Brattleboro, VT). Images were obtained with a Sony CCD camera (SSC-M370CE) and a Macintosh PowerPC computer fitted with a Scion AG-5 frame-grabber board, running NIH Image version 1.61. Confocal microscopy was performed with a BioRad MRC-1024 scanning laser confocal microscope system fitted to an Olympus AX70 epifluorescence microscope and running Lasersharp version 2.1. When examining potential coexistence of different markers within cell bodies or boutons, considerable care was taken to ensure that there were no spurious colocalizations due to filter bleed through.
Image processing
Postcapture image processing was carried out using a Macintosh PowerPC. All morphological measurements were made with NIH Image version 1.61. For the construction of the color plates (Figs. 1 and 2), confocal images were processed for brightness and contrast only without any filtering and pseudocolored to identify each fluorophore using Adobe Photoshop version 5.0. The individual channels were then overlaid in Photoshop. For the construction of Fig. 3 (dendritic morphology), a series of images (either confocal or wide-field microscopy) were taken at different focal planes to ensure all filled dendrites were captured. A through-focus projection of these images was then made using NIH Image macros to produce a single two-dimensional image that included all dendrites. These fluorescence images were then inverted to make the final plate. To visualize better the details of fine dendritic processes, three images (Fig. 3, A, B, and G) were processed further by an edge enhancement algorithm (NIH Image shadow function).
|
|
|
Electrophysiology
Mice were killed by a lethal dose of inhaled halothane.
Ganglia were removed and placed in physiological saline containing (in
mM) 146 NaCl, 4.7 KCl, 0.6 MgSO4, 0.13 NaH2PO4, 2.5 CaCl2, 7.8 glucose,
and 20 HEPES, buffered to pH 7.29 and gassed with 100% O2.
Ganglia were pinned to the base of a recording chamber coated with
silicone elastomer (Sylgard; Dow Corning, Midland, MI), maintained at
35°C and perfused with physiological saline at 2.5 ml
min1. Neurons were impaled with glass microelectrodes
filled with 0.5 M KCl and having resistances of 80-200 M
. In some
cases, Neurobiotin (Vector, Burlingame, CA) 0.5% wt/vol was included in the electrode filling solution to enable postimpalement
visualization of neurons.
Electrical properties of neurons were determined using
discontinuous current clamp (DCC) for voltage recording and
single-electrode voltage clamp (SEVC) using an Axoclamp-2B amplifier
(Axon Instruments, Burlingame, CA). For measurement of action potential
amplitudes, recordings were made in bridge mode. During DCC and SEVC,
the headstage was continuously monitored and the cycling frequency adjusted to minimize the effects of electrode capacitance. The cycling
frequency was 1.0-2.0 kHz for DCC and 2.0-3.5 kHz for SEVC. Voltage
and current records were digitized at 1-5 kHz (MacLab, ADI
Instruments Castle Hill, NSW) using Chart/Scope (version 3.5) software.
Digitized data were analyzed using Igor Pro (version 3, WaveMetrics,
Lake Oswego, OR). Measurements of input resistance and the major input
time constant were carried out by injecting small current pulses
(0.01-0.1 nA, 50-500 ms duration) through the recording electrode.
Averages of 20 current steps were routinely used. Exponential functions
were fitted to the onset of the voltage response between 20 and 80% of
its final amplitude. For other current-voltage relationships,
"steady-state" measurements were made during the last 10 ms of a
current or voltage step. Measurement of "instantaneous" current in
SEVC was made 10 ms following the beginning of the voltage pulse to
ensure settling of the voltage to within 5% of the command value,
which usually took 5-10 ms. The tail currents underlying the
afterhyperpolarization (AHP) or afterdepolarization (ADP) were measured
in SEVC after suprathreshold voltage steps (10 ms) initiated a single
brief "action current" corresponding to an unclamped action
potential (see Cassell et al. 1986; Jobling et
al. 1993
; Sah and McLachlan 1992
; Wang
and McKinnon 1995
). The superior cervical trunk, splanchnic and
celiac nerves were electrically stimulated by means of suction
electrodes connected to a Grass S88 stimulator (Grass
Instruments, Quincy, MA). Nerves were stimulated with square
waves of 1- to 20-V amplitude and 0.3- to 0.5 ms pulse width. The
number of synaptic inputs was estimated by varying the stimulation
voltage and observing changes in the amplitude of the excitatory
postsynaptic potential (EPSP) indicating recruitment of axons with
different thresholds (see McLachlan and Meckler 1989
).
Estimates of the number and relative strength of synaptic inputs were
carried out at both resting membrane potential and when the neuron was
hyperpolarized to between
90 and
100 mV to block orthodromic action
potentials. Antidromic action potentials were discriminated from
orthodromic action potentials by their more rapid rise time when the
cell was hyperpolarized. Antidromic potentials could be blocked by hyperpolarizing current injections. Any residual antidromic
electrotonic potential was not increased by further
hyperpolarization, in contrast with the EPSPs underlying orthodromic
action potentials (see McLachlan and Meckler 1989
).
Statistical analyses
Summary data are presented in the text and tables as means ± SE. Percentages are expressed with 95% confidence limits derived from the binomial distribution (Rohlf and Sokal 1995).
They were analyzed using SPSS Release 6.1.3 (SPSS, Chicago, IL) running on a PC or a Macintosh PowerPC, or with S-Plus 4.5 (MathSoft, Seattle,
WA) on a PC. Initial analyses used one-way ANOVA followed by Tukey's
HSD multiple range tests to determine differences in electrophysiological properties of the neurons in superior cervical, thoracic, and celiac ganglia. Significance level was set at 0.05 for
these tests.
To further test the relationships between the observed variables, a
factor analysis was made on 32 neurons for which there were values for
size and duration of the afterhyperpolarization, the size and
half-width of the action potential, the time constant of the membrane,
the apparent input resistance, and the presence or absence of
IA,
IM,
IH, the inward rectifier and an
afterdepolarization. Neurons were also coded for their phasic or tonic
firing properties in response to a depolarizing current injection (see
RESULTS), and the ganglion within which they occurred. A
correlation matrix was generated that identified the relationships
between each pairwise combination of these variables. Factors were then
extracted using the principal components approach followed by varimax
rotation, so that each factor identified a combination of variables
that were well correlated with each other, but not with variables in the other factors (Norusis 1993; Tabachnik and
Fidell 1996
).
To see whether neurons could be classified and assigned to their
ganglion of origin simply on their electrical properties, a series of
discriminant analyses was done (Norusis 1993;
Tabachnik and Fidell 1996
). Based on the results of the
factor analysis, the duration and amplitude of the AHP and action
potentials together with the apparent cell capacitance were used as
classification variables in the discriminant analysis. Each neuron also
was identified by its ganglion of origin. The analyses produced two
canonical discriminant functions with coefficients relating linear
combinations of the variables representing the electrical properties of
the neurons. These functions were then used to classify the neurons according to their predicted ganglion of origin. The output of the
analysis included a plot ("territorial map") of all the neurons classified by ganglion according to the discriminant functions evaluated for each case (see Fig. 12). The analysis also produced a
table of all the neurons used in the analysis along with their discriminant function scores and their most likely and second most
likely classifications. The tables were used to identify the
neurons that were misclassified and assigned to the wrong ganglion by the analysis (see Norusis 1993
). From these
outputs, a measure of the reliability of the discriminant functions was obtained. Initial analyses were done with the complete data set for 72 neurons to optimize the procedures (selection of appropriate variables,
method of determining the optimal functions, etc.) (see Norusis
1993
, for details). Then the data set was divided randomly into
two equal subsets. The discriminant functions were derived first from
one-half of the data and then were used to classify the neurons in the
other half of the data set. In this way, the reliability of the
discriminant functions could be tested directly. This procedure was
repeated with five different random subsets of data.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemical studies of mouse sympathetic neurons
THORACIC GANGLIA. Single confocal optical sections through whole mounts of thoracic sympathetic ganglia (T4-T6) were used to determine the neurochemical profile of thoracic neurons. Optical sections through the midregion of one ganglion from each of 4 animals contained on average 110 ± 13 neurons, 88 ± 4% (mean ± SE) of which contained immunoreactivity to tyrosine hydroxylase (TH-IR; Fig. 1A). No neurons contained immunoreactivity for choline acetyltransferase (ChAT-IR). Immunoreactivity to neuropeptide Y (NPY-IR) occurred in 32 ± 7% of TH-IR neurons, whereas 4 ± 3% of neurons with NPY-IR did not contain TH-IR (Fig. 1A). Immunoreactivity to vasoactive intestinal peptide (VIP-IR) was observed in 9 ± 4% (n = 3 ganglia) of thoracic ganglion neurons (Fig. 1B), a few of which also contained TH-IR (Fig. 1C). Only one neuron containing TH-IR, NPY-IR, and VIP-IR was observed (n = 3 ganglia; Fig. 1C).
A single optical section through one thoracic ganglion whole mount per mouse was used to determine the cross-sectional area of neurons containing different chemical markers. As previously described in mouse superior cervical ganglia (Gibbins 1991CELIAC GANGLION. Nearly all (98 ± 1%) of neurons in the celiac ganglion contained both TH-IR and NPY-IR (n = 106 ± 32 neurons per section from 3 mice; Fig. 2A). No somata containing VIP-IR or ChAT-IR were observed. Varicose axons containing ChAT-IR or CGRP-IR were abundant throughout the ganglion (Fig. 2, B, C, and E), although very few axons contained both ChAT-IR and CGRP-IR (Fig. 2B). Some CGRP-IR axons contained immunoreactivity for substance P (SP-IR) but SP-IR axons without CGRP-IR were extremely rare (Fig. 2C). Axons containing VIP-IR also occurred throughout the ganglion (Fig. 2, C and E), but they were more sparsely distributed than those containing CGRP-IR (Fig. 2, D and E).
Morphological properties of mouse sympathetic neurons
Dye-filled sympathetic neurons exhibited considerable variation in
their morphologies, both within and between ganglia. Figure 3 shows
simple and complex neurons from the superior cervical, thoracic, and
celiac ganglia. Dendrites did not radiate equally in all directions
from the soma but tended to lie parallel with the frontal plane of the
superior cervical ganglion (SCG) and celiac ganglia and in an oblique
plane in the thoracic ganglia. Dendritic diameter varied considerably
throughout their length with the most distal regions often being
distinctly varicose. There was no correlation between the soma size and
the extent of their dendritic branching. Six representative neurons
from the three ganglia were fully reconstructed from confocal images. In these neurons, the surface area of the soma ranged from ~2,300 µm2 to >8,500 µm2 and
comprised from 13.5 to 51.5% of the total surface area of the neuron.
These values are in the range reported for neurons in the superior
mesenteric ganglia of mice (Miller et al. 1996).
Morphological data from dye-filled neurons are summarized in Table
1. The average total dendritic length of
neurons in the SCG (629 ± 90 µm; n = 12) was
significantly less than that of thoracic ganglion neurons (923 ± 99 µm, n = 15). The maximum dendritic length
(measured from the base of each primary dendrite to its most distal
tip) was similar between neurons in the SCG and thoracic ganglia.
Consequently, the smaller total dendritic length observed in SCG
neurons was partially due to smaller number of primary dendrites. In
many cases the dendritic arbor of a single thoracic neuron extended
throughout most of the ganglion (Figs. 1E and 3D). Such extensive arborizations were never seen in SCG
neurons. The dendritic trees of celiac ganglion neurons varied in size and arrangement. Some neurons had relatively small dendrites similar to
many SCG neurons (Fig. 3F). However, most celiac ganglion
neurons had relatively long dendrites that radiated from the cell body (Fig. 3, E and G). The total dendritic length
(1,140 ± 268 µm, n = 5), number of primary
dendrites (6.6 ± 1.5, n = 5) and mean dendritic
length (112 ± 8.9 µm, n = 29 dendrites from 5 neurons) of celiac neurons were similar both to those of thoracic
neurons and neurons previously described in mouse superior mesenteric ganglia (Miller et al. 1996; Schmidt et al.
1995
).
|
Electrophysiological properties of mouse sympathetic neurons
MEMBRANE PROPERTIES.
Neurons in all ganglia had resting membrane potentials of around 50
mV. However, there were significant differences in passive membrane
properties of neurons from different ganglia (Table
2). Neurons in the celiac ganglion had a
significantly higher mean input resistance and input time constants
compared with either SCG or thoracic ganglion neurons. Although neurons
in the thoracic ganglia were similar in overall size to celiac neurons
and were larger than SCG neurons, their calculated apparent cell
capacitances were significantly less than those of neurons in the other
two ganglia.
|
ACTION POTENTIAL DISCHARGE.
SCG neurons typically fired a single action potential at the beginning
of a sustained (250-500 ms) depolarizing current injection. Increasing
the amplitude of the current step did not evoke any further action
potentials. Thus neurons in the SCG could be described as having
"phasic" firing properties (see Cassell et al. 1986; Weems and Szurszewski 1978
), as also has been observed
in rat SCG (Wang and McKinnon 1995
; Yarowsky and
Weinrich 1985
) and most other paravertebral sympathetic ganglia
(see Adams and Harper 1995
). Similarly, neurons in
thoracic ganglia were all classified as phasic. In the celiac ganglion,
37% of neurons were classified as phasic (Fig. 7Ab),
whereas 48% of neurons fired throughout a prolonged depolarizing
current injection (Fig. 7Bb) and were thus classified as
"tonic" neurons (Weems and Szurszewski 1978
). Fifteen percent of celiac ganglion neurons had intermediate firing properties and could not be classified into either group.
ACTION POTENTIAL CHARACTERISTICS. Brief injections of depolarizing current (10 ms) were used to evoke single action potentials (Figs. 4, 6, and 7). Mean action potential amplitude was significantly greater in neurons from the SCG compared with either celiac or thoracic ganglion neurons (Table 2). Neurons from the SCG also had briefer action potentials as indicated by their significantly shorter mean half-width.
|
AFTERHYPERPOLARIZATIONS. In most neurons, action potentials were followed by prominent afterhyperpolarizations (AHPs; Fig. 4Aa). In voltage clamp, an outward current (IAHP) followed depolarizing voltage steps (10 ms) to holding potentials just suprathreshold for an action current (Fig. 4Ab). The mean amplitude and duration of AHPs were significantly different between ganglia. Neurons in the SCG had a significantly larger mean AHP amplitude compared with thoracic or celiac neurons (Table 2, Figs. 4Aa, 6Aa,and 7, Aa and Ba). The mean duration of the AHP was longest in SCG neurons and shortest in thoracic ganglion neurons (Table 2). Within the celiac ganglion, phasic neurons had significantly longer AHPs than tonic neurons (Table 3 and Fig. 7, Aa and Ba).
|
AFTERDEPOLARIZATIONS.
In some neurons, the action potential was followed by a
depolarization that preceded the AHP (Figs. 4Ba and
6B). The proportion of neurons with these
afterdepolarizations (ADPs) varied markedly between the three ganglia.
ADPs were observed in 34% of SCG neurons, 18% of thoracic neurons but
only in one celiac ganglion neuron. Measurement of the tail currents
following action current initiation in neurons with ADPs revealed an
inward current followed by an outward current (Fig. 4Bb).
Inward currents increased in amplitude at hyperpolarized holding
potentials, whereas the outward current was abolished (Fig.
4Bb). Identical ADPs and the underlying currents have been
extensively characterized in normal mouse and axotomized rat SCG
neurons, where they represent the activation of a calcium-activated chloride channel (De Castro et al. 1997;
Sanchez-Vives and Gallego 1994
).
CURRENT-VOLTAGE RELATIONSHIPS.
Current-voltage relationships were studied from membrane potentials
below threshold for action potential initiation to around 130 mV in
both current clamp and voltage clamp. During depolarizing and
hyperpolarizing current steps evoked from resting membrane potential
(RMP), time- and voltage-dependent rectification could be observed in
the voltage traces (Figs. 5A,
6Ab, and
7Ab). During small current
injections that shifted the membrane potential by <10 mV in either
direction, there was a noticeable "sag" in the voltage trace (Figs.
5A and 6Ab), suggesting the presence of a voltage-dependent current active around RMP. In voltage clamp at
holding potentials more positive than
60 mV, hyperpolarizing voltage
steps showed time-dependent current relaxations indicating the presence
of M-current (IM; Figs. 5D,
and 8, Aa and Bb).
IM was deactivated at holding
potentials below
60 mV, consistent with M-currents previously
described in mouse hypogastric ganglia (Rogers et al.
1990
) and many other sympathetic ganglia (Brown and
Adams 1980
; Cassell et al. 1986
;
Constanti and Brown 1981
; Wang and McKinnon
1995
). Although IM was
observed in neurons from all ganglia, only 38% of tonic neurons within
the celiac ganglion showed evidence of
IM compared with 89% of phasic
neurons and 100% of neurons from the SCG and thoracic ganglia.
|
|
|
|
SYNAPTIC POTENTIALS.
EPSPs were recorded from neurons in the SCG and celiac ganglia. All
neurons in the SCG received at least one synaptic input that was
suprathreshold for the generation of an action potential (Fig. 4,
Ca and Da). The action potential evoked following
orthodromic nerve stimulation was difficult to block by injection of
hyperpolarizing current (Fig. 4, Cb and Db).
Synaptic inputs with these properties have been classified as strong
inputs in other sympathetic ganglia (see McLachlan and Meckler
1989) and are a consistent feature of paravertebral sympathetic
neurons in all species studied so far (Jänig
1995
). Many SCG neurons also received a few subthreshold EPSPs
(weak inputs), although it was difficult to determine the exact number
due to the similar voltage threshold for stimulating both classes of
inputs (Fig. 4, C and D).
|
|
|
|
Multivariate statistical analysis
The preceding results indicated that there were significant
differences in many of the electrical properties of the neurons from
different ganglia. Consequently, characteristic combinations of these
properties may be diagnostic of neurons in each ganglion. To test this
possibility, we used a variety of multivariate statistical approaches
to determine which electrophysiological characteristics explain most of
the variance between ganglia. First, we did a factor analysis on 32 neurons for which we had complete data for AHP duration and amplitude,
action potential duration and amplitude, apparent input resistance,
membrane time constant, the presence or absence of ADP,
IH or
IKi, their phasic or tonic firing
properties, and their ganglion of origin. A principal components
approach was used to identify the groups of variables that were most
strongly correlated with each other (i.e., each group or "factor"
contained variables that covaried) (Norusis 1993;
Tabachnik and Fidell 1996
).
The factor analysis identified five factors that explained 76% of the
variance in the data. The first factor (32% of the variance) linked
the presence or absence of IH and
IKi with the phasic or tonic firing
properties of the cell and the ganglion within which it was found. This
was mainly due to a strong negative correlation (correlation
coefficient: 0.57; P = 0.0002) between the presence of IH and
IKi.
IKi but not
IH was more likely to be found in
tonic neurons (correlation for IKi:
0.4, P = 0.007; correlation for IH:
0.4, P = 0.008),
which occurred only in the celiac ganglion. The second factor (14% of
the variance) identified a strong correlation between the duration and
magnitude of the AHP (correlation coefficient: 0.67; P = 0.00002; Fig. 11). In addition, the duration of the AHP was
positively correlated with the amplitude of the action potential (correlation coefficient: 0.47; P = 0.003; Fig. 11).
These relationships also covaried with the ganglion within which the
cells were found. The third factor (12% of the variance) positively
linked the half-width of the action potential with the apparent input
resistance and the membrane time constant. The fourth factor (10% of
the variance) indicated a strong positive correlation between the
presence of IM and
IA (correlation coefficient: 0.59;
P = 0.0002). Finally, the fifth factor (9% of the
variance) was primarily associated with the contribution of the ADP to
the total variation. The presence of the ADP itself was positively
correlated with the duration of the AHP (correlation coefficient: 0.46;
P = 0.004).
These results suggest that, in addition to properties that are
selectively expressed by neurons in specific ganglia, such as the
presence of an ADP (mostly in SCG) or inward rectification (mostly in
celiac ganglion), other more subtle properties such as the size and
duration of the action potential and AHP, and cell capacitance, also
may help to identify different populations of neurons (Fig. 11). To
test this idea, we did a series of discriminant analyses
(Norusis 1993; Tabachnik and Fidell
1996
), based on a set of 72 neurons (32 from SCG; 13 from
thoracic ganglia; 27 from celiac ganglia), using the properties of the
action potential, the AHP and the derived membrane capacitance (or the
membrane input resistance and time constant), to see whether these
properties could be used to predict which ganglion a cell came from.
Similar results were obtained if we used either capacitance or input
resistance and time constant as variables.
When the whole data set was used to derive the discriminant functions, the first canonical discriminant function separated SCG neurons from neurons in other ganglia mostly on the basis of the amplitude and duration of the AHP (Fig. 12). The second canonical discriminant function separated thoracic and celiac neurons mostly on the basis of their capacitance (Fig. 12). These functions correctly classified 91% of SCG neurons, 46% of thoracic neurons, and 82% of celiac neurons to their ganglion of origin (overall success rate: 80%). When the data set was divided randomly into two subsets (one set used to derive the functions, which were then tested on the other subset of data), the success rate for correct classification of the data used to derive the functions ranged from 85 to 100%, with overall rates varying from 50% to better than 70% on the test data. In each case, the success at distinguishing SCG neurons from neurons in the other ganglia was better than 80%. This was mostly due to the first canonical discriminant function that was based on the properties of the AHP, as was observed in analyses using the complete data set. Examination of the data sets to identify the neurons that had been misclassified to the wrong ganglion by the discriminant functions showed that these cells really were atypical and did indeed share properties with the group to which they had been incorrectly classified. From these analyses, we conclude that the overall properties of the AHP and action potential in neurons in the SCG were significantly different from those in the thoracic and celiac ganglia. Although many neurons in the thoracic and celiac ganglia can be distinguished from each other, primarily on the basis of their capacitance, the properties of their action potentials and AHPs had more in common with each other than they did with SCG neurons.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our studies reported here show that there is considerable heterogeneity in the morphological, neurochemical, and electrophysiological properties of neurons in the sympathetic ganglia of mice. Moreover, the variation in these properties is tightly linked to the ganglion within which the neurons occur (data summarized in Table 4). This linkage was so strong that an unbiased statistical sorting procedure (discriminant analysis) could correctly classify >80% of neurons to their ganglion of origin on the basis of a small number of electrophysiological properties.
|
Heterogeneity of sympathetic neurons
Compared with neurons in thoracic and celiac ganglia, neurons in
the SCG were most clearly characterized by their larger action potentials and the associated AHPs. The magnitude and duration of both
these features are influenced by calcium-activated potassium channels
(Davies et al. 1996; MacDermott and Weight
1982
; Sah 1996
; Sah and McLachlan
1992
; Smart 1987
). This suggests that SCG
neurons may have fundamentally different calcium dynamics compared with
sympathetic neurons in both prevertebral and other paravertebral ganglia.
Another notable characteristic of many neurons in the SCG was their
marked ADPs following an action potential. These ADPs are probably due
to a calcium-dependent chloride current, which is activated by calcium
influx during the action potential (DeCastro et al.
1997; Sanchez-Vives and Gallego 1994
;
Scott et al. 1995
). It has been concluded that the
chloride channels responsible for the ADP are located differentially on
the terminal dendrites and only become electrically significant at the
soma when the dendritic arbor is short (DeCastro et al.
1997
). Our morphological observations support this
interpretation in that the ADPs occurred only occasionally in thoracic
ganglion neurons, which had significantly longer dendrites than SCG
neurons. Furthermore, ADPs were almost entirely absent from celiac
neurons, which included the largest neurons studied here.
Neurons in the celiac ganglion were characterized primarily by the
presence of an inward rectifier potassium current and the absence of
IH. Many celiac neurons could be
classified as tonic or phasic on the basis of action potential
discharge during maintained depolarizing current injection
(Weems and Szurszewski 1978). Overall, the tonic neurons
were less likely to co-express both IA
and IM than were phasic neurons.
However, in contrast to celiac neurons of guinea pigs and rats, there
was not a strong association between the expression or properties of
these currents and phasic or tonic firing properties (Cassell et
al. 1986
; Cassell and McLachlan 1987
;
Decktor and Weems 1983
; Wang and McKinnon
1995
; Weems and Szurszewski 1978
).
Neurons from thoracic ganglia expressed similar voltage-dependent conductances to SCG neurons, but they had action potential characteristics closer to phasic neurons in the celiac ganglion. The main distinguishing feature of thoracic ganglion neurons was their lower apparent cell capacitance. This observation is surprising, given the large dendritic length of most of these neurons. It suggests that the dendrites of thoracic ganglion neurons may express a characteristic set of membrane channels.
Taken together, these results indicate that there is significant differential expression of a wide range of ion channels by sympathetic neurons of mice. These channels must include those responsible for at least five different currents (delayed rectifier, IC due to BK channels, IAHP, IA, IM, and IH) and a calcium-dependent chloride channel. All of these channels are likely to affect the excitability of the neurons and their responses to different patterns of preganglionic stimulation.
Ganglia and pathways
There is now strong evidence that neurons in sympathetic ganglia
show pathway-specific differences in their morphology and their
expression of neuropeptides (see Gibbins 1995, for review). Previous studies in mice, rats, and guinea pigs suggest that within the
thoracic ganglia of mice, smaller neurons containing immunoreactivity to TH and NPY are most likely to lie within vasomotor pathways, whereas
larger neurons with TH but not NPY are likely to lie in pilomotor
pathways (Gibbins 1991
, 1992
;
Gibbins and Matthew 1996
; Schotzinger and Landis
1990
). Mouse thoracic neurons with NPY or VIP, but not TH,
probably lie in vasodilator or sudomotor pathways, respectively,
although they lack detectable immunoreactivity to choline
acetyltransferase (Gibbins 1992
; Landis and
Fredieu 1986
; Lindh et al. 1989
; Morris
et al. 1998
, 1999
). Thus there are at least four
neurochemically characterized autonomic motor pathways running through
the thoracic ganglia. Similarly, there at least four main functional
pathways running through the superior cervical ganglia:
vasoconstrictor, pilomotor, secretomotor, and pupilodilator (Gibbins 1991
). Once again, the final motoneurons in
these pathways can be distinguished by a combination of morphological
and immunohistochemical characteristics (Gibbins 1991
).
A unique characteristic of many neurons in the prevertebral ganglia is
that they participate in peripheral reflex circuits and receive direct
synaptic inputs from intestinofugal neurons located in the enteric
plexuses (Furness and Costa 1987; Kreulen and
Szurszewski 1979
). In guinea pig celiac ganglia, the inputs from VIP-containing intestinofugal neurons produce subthreshold cholinergic EPSPs in their target neurons. Most of these target neurons
contain somatostatin rather than NPY, fire tonically, and project back
to the enteric plexuses (Costa and Furness 1983
; Lindh et al. 1986
, 1988
; Macrae et
al. 1986
; McLachlan and Meckler 1989
;
Meckler and McLachlan 1988
). In contrast, both phasic
and tonic neurons in the prevertebral ganglia of mice receive
cholinergic suprathreshold inputs from preganglionic fibers in the
splanchnic nerves and from presumed intestinofugal fibers in the celiac
nerves (see also Miller and Szurszewski 1997
). However,
we did not find an obvious neurochemical marker for the peripheral
inputs to celiac neurons, because most ChAT-IR fibers lack detectable
peptides. Furthermore, all neurons in the celiac ganglia of mice
contain NPY, so it was not possible to distinguish neurons in
vasoconstrictor pathways from those in motility-regulating pathways,
using neurochemical or morphological criteria, as can be done in guinea
pigs, for example (Boyd et al. 1996
; Costa and
Furness 1983
).
Conclusions
Our results have shown that in addition to the pathway-specific
expression of transmitters and morphological phenotype, neurons projecting to similar targets can be further distinguished by the
selective expression of ion channels according to their ganglion of
origin. Most of the electrophysiological differences we have observed
between neurons are likely to affect the way in which they integrate
and respond to their synaptic inputs. Both the magnitude of AHP and the
presence or absence of IM would
contribute to setting an upper limit on the rate of action potential
generation in response to increasing levels of synaptic input.
Variations in the half-width of the action potential could influence
the probability of transmitter release at the terminals of the
sympathetic neurons (Brock and Cunnane 1995).
Neurons projecting to similar targets in different parts of the body,
such as the vasculature of the head, skin, muscles, or viscera, can be
activated selectively in response to specific central commands
(Jänig 1995; Jänig and McLachlan
1992
). Therefore it may not be surprising that a
vasoconstrictor neuron in a thoracic ganglion expresses a
characteristic suite of ion channels different from that expressed by a
vasoconstrictor neuron in the superior cervical or celiac ganglion.
Such differences may be related to the patterns of synaptic activity
that occur in particular functional pathways. Differences in the
expression of ion channels also may be related to ganglion-dependent
variation in the overall size of neurons (cf. Boyd et al.
1996
; DeCastro et al. 1997
), which in turn is
related to the number of synaptic inputs they receive (Gibbins
et al. 1998
; Hume and Purves 1981
; Purves
and Hume 1981
). What is more surprising is that a
vasoconstrictor neuron and a neighboring pilomotor neuron in the same
ganglion are likely to express a similar combination of channels. We
have shown recently that the synaptic organization of sympathetic
ganglia shows considerable potential for cross-talk between inputs in
different functional pathways (Gibbins et al. 1998
;
Murphy et al. 1998
). Consequently, the ganglion-specific
differences in the electrical properties of sympathetic neurons also
may include adaptations to allow for cross-talk between pathways in
ways that are still poorly understood.
![]() |
ACKNOWLEDGMENTS |
---|
We thank Prof. W. Blessing and Dr. J. Oliver for the gift of E2210 antiserum to NPY and Assoc. Prof. R. Rush for the gift of antiserum to ChAT. We also thank Dr. D. Cameron, Assoc. Prof. J. Morris, and R. L. Anderson for helpful advice on the manuscript. S. Matthew is thanked for advice on the PEG immunohistochemical technique. We are grateful to G. Hennig for the NIH Image macros used for processing through-focus series of dye-filled neurons. P. Jobling is an Australian Postdoctoral Fellow of the National Health and Medical Research Council of Australia.
This study was supported by grants from the National Health and Medical Research Council of Australia, the Charles and Sylvia Viertel Foundation, the Clive and Vera Ramaciotti Foundation, the Flinders Medical Center Foundation, the Australian Research Council, and the Flinders University Research Budget.
![]() |
FOOTNOTES |
---|
Address for reprint requests: P. Jobling, Dept. of Anatomy and Histology, Flinders University of South Australia, GPO Box 2100, Adelaide, South Australia 5001, Australia.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 December 1998; accepted in final form 15 June 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|