Centre for Neuroscience and Department of Anatomy and Histology,
Flinders University, Adelaide, SA 5001, Australia
 |
INTRODUCTION |
Neurons vary
widely in their expression of ion channels, especially
voltage-dependent K+ channels and
Ca2+-dependent K+ channels.
These channels are a fundamental determinant of the firing properties
of neurons and their responses to synaptic inputs (Hille
1992
). Integration of convergent synaptic inputs also is dependent on interactions between the distribution of ion channels and
the dendritic morphology of the neurons. The generation of neuronal
diversity clearly requires the coordinated development of these
features (Dryer 1994
, 1998
; Ribera and Spitzer
1992
). However, the relationship between differential ion
channel expression and dendritic morphology during the development of
mature neuronal phenotypes is not well known.
Autonomic pathways comprise one of the primary motor outputs of the
nervous system and contain more final motor neurons than any other
pathway. In humans, there are more than 10 million final motor neurons
in sympathetic pathways alone (Gibbins 1990
). Compared with somatic final motor neurons, autonomic neurons show a great diversity of phenotypic characteristics, such as their neuropeptide content, electrical properties, morphology, and synaptic connectivity. In addition, autonomic neurons found in specific functional pathways often express precise combinations of these phenotypic characteristics (Adams and Harper 1995
; Andrews et al.
1996
; Chiba and Tanaka 1998
; Dryer
1994
; Gibbins 1995
; Jobling and Gibbins
1999
; Morris et al. 1997
-1999
; Smith
1994
). The celiac ganglion of guinea pigs provides a striking
example of this phenomenon. Here, vasomotor neurons can be
distinguished from neurons projecting to the enteric plexuses by their
location, the size of their dendritic fields, their neuropeptide
content, the potassium channels they express, and the origins and
number of their synaptic inputs (Boyd et al. 1996
;
Cassell and McLachlan 1987
; Cassell et al.
1986
; Costa and Furness 1984
; Davies et
al. 1999
; Gibbins et al. 1999
; Keast et al. 1993
; Lindh et al. 1986
; Macrae et
al. 1986
; McLachlan and Meckler 1989
;
Meckler and McLachlan 1988
) (Table
1).
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Table 1.
Phenotypic characteristics of major classes of functionally
identified neurons in mature guinea pig coeliac ganglion
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Although autonomic neurons have been used to study many different
aspects of neuronal differentiation (Dryer 1994
, 1998
;
Dryer and Chiappinelli 1985
; Hirst and McLachlan
1984
; McFarlane and Cooper 1992
, 1993
;
Nerbonne and Gurney 1989
; Phelan et al.
1997
; Rubin 1985a
-c
), few studies have examined
the development of phenotypic diversity within functionally identified
pathways (Cameron and Dryer 2000
; Stofer and Horn
1990
, 1993
). Indeed, most studies of neuronal development have
investigated only a single class of neurons.
In principle, the generation of different neuronal phenotypes from a
common precursor pool could occur by the sequential acquisition of
characteristics from a basal embryonic phenotype, such that one mature
phenotype was derived from another. Alternatively, each mature
phenotype could develop directly from a specific subset of precursors.
The resolution of this question has been hampered by the difficulty of
identifying different pools of precursor neurons prior to their
differentiation. In this study, we have tackled this question by taking
advantage of the unique organization of the guinea pig celiac ganglion,
which allows us to follow the development of phenotypically diverse
populations of neurons that innervate distinct target tissues. We have
investigated the differentiation of two major phenotypic
characteristics, the differential expression of ion channels, and
dendritic morphology in neurons whose functional pools can be
recognized from an early developmental stage simply on the basis of
their location. To do this, we used intracellular electrophysiological
recording techniques, combined with dye-filling, multiple-labeling
immunohistochemistry, and confocal microscopy. We have found that many
of the electrophysiological characteristics of the main functional
classes of neurons develop directly from undifferentiated precursors
and can be distinguished from each other long before the neurons finish
growing. This suggests that the differentiation of these two phenotypic
characteristics is likely to be independently regulated.
 |
METHODS |
Pregnant guinea pigs, fetuses, neonates (P1-P13) and
nonpregnant adults (>240 g; Cavia porcellus, Hartley/IMVS
strain) were given a lethal dose of sodium pentobarbitone (Nembutal,
Bomac Laboratories, Asquith, Australia; 200 mg/kg ip). Nonpregnant
adult guinea pigs used in the stereological analysis of neuropil (see following text) were killed by stunning and exsanguination. Late-stage fetuses also were exsanguinated after removal from their
extra-embryonic membranes. Fetuses were then weighed and placed in a
balanced salt solution (see following text). All procedures were
approved by the Animal Welfare Committee of the Flinders University of South Australia.
Guinea pigs have a relatively long and variable gestation period of
between 55 and 75 days (Matsumoto et al. 1993
;
Weir 1974
). Embryogenesis occurs during the first 30 days of gestation, while the remainder of the gestational period
involves fetal growth (Scott 1937
). Since guinea pigs
undergo postpartum estrus within hours of giving birth (Stockard
and Papanicolou 1917
), the day of birth of the previous litter
is also day 0 of the following litter. Fetuses were obtained from
pregnant guinea pigs during three arbitrary stages of development as
previously described (Anderson et al. 2001
): early fetal
[F30-F35; weight range 1-7 g, mean 3.4 ± 0.3 (SE) g,
n = 30], mid fetal (F36-F45; weight range 10.2-48.9
g, mean 20.7 ± 1.9 g, n = 21), and late
fetal (F46+; weight range 37-79 g, mean 50.4 ± 7.5 g,
n = 5). The early fetal stage of development in guinea
pigs is approximately equivalent to the first postnatal week in rats
and mice (Butler and Juurlink 1987
). The weight of
neonatal guinea pigs (P0-P13) used in this study ranged from 93 to
191 g (mean 126.0 ± 8.4 g, n = 13),
while nonpregnant adults ranged from 240 to 329 g (mean 286.0 ± 18.2, n = 5). Where possible, our data were analyzed
using the log of the weight since there was an exponential increase in
weight with increasing age.
Electrophysiology
Tissue preparation.
Celiac ganglia, their nerve trunks, and surrounding tissues (aorta,
celiac artery and adrenal glands) were removed and placed into a
HEPES-buffered balanced salt solution containing (in mM) 146 NaCl, 4.7 KCl, 0.6 MgSO4, 1.6 NaHCO3,
0.13 NaH2PO4, 2.5 CaCl2, 7.8 glucose, and 20 HEPES, buffered to pH
7.3 and gassed with 100% O2. Ganglia were pinned
to the base of a recording chamber (Medical System, Greenvale, NY)
lined with silicone elastomer (Sylgard, Dow Corning, Midland, MI).
During electrophysiological recordings, ganglia were maintained at
35°C and superfused with HEPES balanced salt solution at 2.5 ml/min.
At early fetal stages (F30-F35), poorly developed connective tissue
did not allow the celiac ganglion to be pinned tightly in the recording
chamber. Instead the ganglion was stabilized by leaving it attached to
the abdominal aorta, which was slit longitudinally along its dorsal
surface and the reflected corners pinned down. In addition, early fetal
neurons were small with little cytoplasm (cross-sectional area of soma
~150 µm2) (Anderson et al.
2001
). Both of these factors affected the length of time for
which impalements could be held as has been reported in other
preparations of developing sympathetic ganglia (Dryer and
Chiappinelli 1985
; Hirst and McLachlan 1984
).
Preparations from the earliest fetal stages examined (F30-F32) were
only viable for ~2 h at 35°C, after which time the ganglion and
connective tissue began to deteriorate. At later stages (F38+),
preparations were viable for
8 h at 35°C (longest time attempted)
and impalements were routinely held for >20 min.
Intracellular recordings of sympathetic neurons.
Neurons were impaled using high-resistance glass microelectrodes
(80-200 M
) pulled on a Flaming-Brown puller (Sutter Instrument, Novarto, CA) and filled with 0.5 M KCl. Electrical properties were
determined using bridge mode, discontinuous current clamp (DCC), or
single electrode voltage clamp (SEVC) using either an Axoprobe-1A or an
Axoclamp-2B amplifier (Axon Instruments, Union City, CA). Voltage or
current records were digitized at 1-5 kHz using Spike2 (version 3.01)
and Signal software (version 1.72; Cambridge Electronic Design,
Cambridge, UK) on a PC running Windows NT, or Chart/Scope
software (version 3.5, MacLab, ADI Instruments, Castle Hill, NSW,
Australia) on a Power Macintosh computer (Apple Computers, Cupertino,
CA). 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. Digitized data were analyzed using Igor Pro
(version 3.14, WaveMetrics, Lake Oswego, OR).
Resting membrane potential (RMP) was determined by measuring the
difference between the potentials immediately before and after
withdrawal of the microelectrode from the cell. Measurements of input
resistance (Rin) and the major input
time constant were made by injecting small hyperpolarizing current
pulses (0.01-0.1 nA, 200-250 ms duration) through the recording
electrode. Averages of 20 current steps were routinely used. The time
constant of the cell (
, ms) was determined by fitting a single
exponential to the onset of the voltage response to the current pulse
between 20 and 80% of its final amplitude. Capacitance was derived
from the time constant divided by the
Rin. Steady-state current-voltage (I-V) curves were generated by measuring the average
response during the last 10-30 ms of a 200- to 250-ms current step.
Neurons were classified as phasic or tonic on the basis of their firing properties in response to 200- to 250-ms depolarizing current steps
(see Cassell et al. 1986
; Keast et al.
1993
). Phasic neurons were further discriminated as long
afterhyperpolarizing (LAH) or not on the basis of the presence or
absence of a prolonged after hyperpolarization (AHP; Cassell and
McLachlan 1987
). For measurement of action potential (AP)
amplitudes, recordings were made in bridge mode. Single APs were
elicited by brief (10-20 ms) injections of depolarizing current from
rest. The maximum amplitude (mV) and half-width (ms) of the AP were
measured together with the maximum amplitude (mV) and duration of the
afterhyperpolarization (ms; measured from the point at which the cell
passed RMP during repolarization after the action potential until it
returned to RMP).
For measurements of IAHP and
IsAHP in SEVC, brief (10-20 ms)
suprathreshold depolarizing voltage steps resulted in a single "action current" that corresponds to an unclamped action potential (Cassell et al. 1986
; Jobling et al.
1993
; Wang and McKinnon 1995
). Peak amplitudes
of IAHP and
IsAHP were measured 10 ms after the action current. The time constant of the
IAHP was determined by fitting a
single exponential between 80 and 20% of the curve (Cassell and
McLachlan 1987
).
Drug applications.
The action potentials of developing neurons either are initially
dependent on Ca2+ before becoming
Na+ dependent or they are
Na+ dependent from the onset of excitability
(Spitzer 1991
). The ionic dependence of early fetal
neurons was examined using tetrodotoxin (TTX, 1 µM; Alamone Labs,
Jerusalem, Israel) to block Na+-dependent
channels and Cd2+ (300 µM; ICN Biomedicals,
Costa Mesa, CA) to block Ca2+-dependent
channels (Adams and Harper 1995
; Davies et al.
1999
). Solutions were changed by switching the perfusion line.
Neuronal morphology
Relative area of neuropil in topographically distinct regions of
the celiac ganglion.
Celiac ganglia were removed from embryos (Carnegie stages 20-23),
fetuses, neonates, and adults, fixed in Zamboni's fixative (0.2%
picric acid and 2% formaldehyde in 0.1 M phosphate buffer, pH 7.0),
and processed for multiple-labeling immunohistochemistry (Anderson et al. 2001
). Ganglia were dehydrated in
ethanol (EtOH), cleared in DMSO, washed in 100% EtOH, and vacuum
infiltrated at 46°C for
30 min in polyethylene glycol (PEG; MW
1000), before being embedded in PEG (MW 1450) in small cryomolds.
Sections, 10- to 20-µm thick, were cut on a standard rotary microtome
and placed into phosphate-buffered saline (PBS). Excess PBS was removed and the sections placed in 10% normal donkey serum (NDS) for 30 min.
Sections were incubated in 10% NDS and primary antisera at room
temperature for 48-72 h. Labeling for neuropeptide Y (NPY) was used to
identify vasomotor neurons, and labeling for somatostatin (Som) was
used to identify neurons projecting to the enteric plexuses; labeling
for tyrosine hydroxylase (TH) was used as an internal labeling control
because nearly all celiac ganglion neurons contain TH regardless of
their peptide content (Anderson et al. 2001
; Costa and Furness 1984
). Primary antibodies used were:
sheep anti-NPY (Oliver/Blessing E2210/2; 1:1000) or rabbit anti-NPY
(Incstar, Stillwater, MN, No. 550212; 1:1200), monoclonal mouse
anti-Som (MRC Regulatory Peptide Group, Vancouver, Canada; code Soma
S8; 1:1200) or rabbit anti-Som (Incstar; 1:100), and in some cases mouse anti-TH (Incstar, No. 105440, 1:1200) or rabbit anti-TH (Dr. J. Thibault, AS2-512, 1:2000). After washing in PBS, secondary antibodies
were applied for
2 h. Species-specific secondary antibodies (IgG)
were raised in donkeys and conjugated with dicholortriazinyl amino
fluorescein (DTAF), fluorescein isothiocyanate (FITC) or the
indocarbocyanin dyes Cy3 or Cy5. All secondary antibodies were obtained
from Jackson ImmunoResearch Laboratories, West Grove, PA. After further
washing, sections were mounted on glass slides in carbonate-buffered
glycerol (pH 8.6), and the coverslips were sealed using clear nail polish.
Sections were examined using conventional wide-field fluorescence
or confocal microscopy. For conventional microscopy, images were
collected using an Olympus AX70 microscope (Olympus, Tokyo, Japan)
fitted with a Hamamatsu Orca cooled CCD camera (Hamamatsu Photonics,
KK, Japan) and connected to a PowerMac G3 (Apple Computers) running
IPLab Spectrum (version 3.2, Scanalytics, Fairfax, VA). Confocal microscopy was done using a BioRad MRC-1024 scanning laser
confocal microscope (BioRad, Hemel Hempstead, UK) with a krypton/argon
laser source fitted to an Olympus AX70 epifluorescence microscope and
running under LaserSharp software (version 3.2, BioRad).
A stereological point-counting method (Howard and Reed
1998
) was used to quantify changes in the proportional area of
ganglion occupied by neuropil in lateral and medial regions of the
developing celiac ganglion. Digital images of sections labeled for
immunoreactivity to NPY, Som, and, in some cases, TH, were overlaid
with a 25-35 point grid using Adobe Photoshop software (version 5.1, Adobe Systems, Mountain View, CA). Points intersecting with neurons, nonneuronal tissue, and neuropil were scored. Up to four samples from
medial or lateral locations were averaged in each animal (see also
Anderson et al. 2001
).
Neurobiotin-filled neurons.
During some intracellular impalements, Neurobiotin (0.5% wt/vol in 0.5 M KCl; Vector, Burlingame, CA) was included in the electrode filling
solution so that neurons could be visualized after the completion of
electrophysiological experiments. The location of neurons in the
bilobed celiac ganglion was recorded as either in the medial two-thirds
or in the lateral third of a lobe. At the completion of the
electrophysiological recordings, ganglia were fixed in Zamboni's
fixative for 24-72 h and processed as whole mounts for multi-labeling
immunohistochemistry as previously described (Gibbins et al.
1999
; Jobling and Gibbins 1999
). Briefly, picric
acid was removed by washing in 80% EtOH before the tissue was further
dehydrated in 100% EtOH and permeabilized in DMSO for 1-3 h. Tissue
was then rehydrated through 80 and 50% EtOH before being washed in PBS
(pH 7.0). Primary antisera for NPY and Som (as in the preceding text at
twice the concentrations used for sections) were then applied for
48-72 h. After extensive washing in PBS, whole mounts were incubated
in secondary antibodies overnight. Species-specific secondary
antibodies (IgG), raised in donkeys and conjugated with DTAF, FITC, or
Cy3 (see preceding text) were used to detect immunoreactivity to NPY
and Som. Streptavidin conjugated to Cy5 (Jackson Immunoresearch
Laboratories) was used to detect Neurobiotin-filled cells. After 2-4 h
washing in PBS, ganglia were mounted on glass slides in
carbonate-buffered glycerol (pH 8.6).
Scanning laser confocal microscopy was used to analyze dye-filled
neurons. Neurons first were assessed for their immunoreactivity to NPY
or Som. To ensure antibodies had penetrated sufficiently through the
whole ganglion, dye-filled neurons were only scored as lacking
neuropeptides if at least some adjacent neurons showed positive
immunoreactivity. Dye-filled neurons were only included in the
morphological analysis if individual dendrites were easily distinguishable, if they were not obscured by adjacent dye-filled neurons, and if the axon could be identified and followed to the edge
of the ganglion. A low-magnification confocal through-focus series,
which included all dendrites, was taken of each filled neuron with
optical sections separated by 0.5-2.0 µm. A confocal through-focus
series also was taken of the neuronal cell body at higher magnification
using low gain settings to confirm that only one neuron had been filled
and to provide a more precise measure of the cross-sectional and
surface area of the soma. A single two-dimensional (2D)
maximum-intensity projection image was generated from each confocal
series using either Lasersharp or National Institutes of Health Image
(NIH) software (version 1.61, Bethesda, MD). Measurements of
cross-sectional area of the neuronal cell body
(µm2), number of primary dendrites (
1 cell
body diameter in length), and the total dendritic length (µm) were
taken. Confocal through-focus series also were reconstructed on a PC
using VoxBlast software for Windows (version 3.0, VayTek, Iowa City,
IA). Threshold was optimized either for dendrites (low-magnification
series) or for the cell soma (high-magnification series), the surface
area calculated and images of three-dimensional reconstructions
rendered. The brightness and contrast of images was adjusted using
Adobe Photoshop software.
Tests of morphological measurements after different fixation and
mounting techniques.
To test for any morphological changes that may accompany tissue
processing of dye filled neurons, a conjugate of Dextran (10,000 MW),
tetramethylrhodamine and biotin ("Mini-Ruby"; 20 µl in 2% 0.5 M
KCl; Molecular Probes, Eugene, OR) was used to fill neurons in celiac
ganglia from two adult guinea pigs. The ganglia were then mounted on
glass slides in the same solution used during the experiments, and the
coverslips were held in place using nail polish. A confocal
through-focus series of each dye-filled neuron was captured before
ganglia were removed from the slides and fixed in Zamboni's fixative.
Following fixation, ganglia were processed as for other experiments
before they were re-mounted in PBS. Then a second confocal
through-focus series was taken of each dye-filled neuron. Finally,
ganglia were re-mounted in buffered glycerol and left overnight before
a third confocal through-focus series of each dye-filled neuron was
captured. A single 2D maximum-intensity projection image was generated
from each confocal series, and NIH image software was used to measure
the cross-sectional cell body area and total dendritic length. Three
neurons sufficiently well filled for morphological analysis were
followed throughout all the steps. None of these neurons underwent any
significant shrinkage or any other morphological deformations with the
fixation, processing, and mounting techniques used here.
Statistical analysis.
Development changes were analyzed with least-squares linear
regression, with log-transformed weight used as a measure of
developmental age. Means were compared with t-tests or
multivariate ANOVA, while medians of strongly skewed data were compared
with Mann-Whitney U tests. Frequency data were analyzed
using
2 tests. All analyses were done with
SPSS for Windows (version 9, SPSS, Chicago, IL). Data are presented as
untransformed means ± SE, with n values referring to
the number of neurons unless otherwise stated.
 |
RESULTS |
Development of electrical properties of celiac ganglion neurons
PASSIVE MEMBRANE PROPERTIES.
Intracellular recordings were made from 177 neurons in 63 preparations
of celiac ganglia from fetal, neonatal, and adult guinea pigs. The RMP
of celiac ganglion neurons became significantly more negative during
development (Fig. 1A). At
early fetal stages (F30-F35), the majority of neurons had RMPs around
35 mV while those from neonates had RMPs around
55 mV. In previous
studies of developing sympathetic ganglia using intracellular recording techniques, neurons with RMPs outside published ranges for mature sympathetic neurons were not considered for analysis (e.g.,
Dryer and Chiappinelli 1985
; Hirst and McLachlan
1984
). However, in this study, half of the early fetal neurons
with RMPs around
35 mV had input resistances
100 M
(see
following text; Fig. 1, A and B), suggesting that
they were unlikely to have been damaged significantly during
intracellular impalements. Therefore we have included immature neurons
with RMPs less negative than
55 mV in the analyses that follow.

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Fig. 1.
Relationships between increasing body weight of the animal and
passive membrane properties. Weight (in g) is shown on a log scale.
Approximate stages of development are indicated (F, fetal; Neo,
neonate). The number of neurons (n),
R2 value, and significance of the regression
(*P < 0.05) are indicated. Regression lines are
shown with 95% confidence limits. A: the resting
membrane potential (RMP, mV) of neurons became significantly more
hyperpolarized during development (R2 = 0.42, F(1,127) = 93.3, P < 0.0001). B: there was no
significant change in input resistance (Rin,
M ) during development (R2 = 0.01, F(1,162) = 1.5, P = 0.2). C: the major cell input time constant ( , ms)
increased significantly during development
(R2 = 0.12, F(1,116) = 15.2, P < 0.0001). D: the apparent cell capacitance (derived
from measured Rin and ) significantly
increased during development (R2 = 0.16, F(1,116) = 22.7, P < 0.0001).
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In contrast to RMP, the input resistance
(Rin) of neurons did not change
significantly during development (Fig. 1B). Overall, the
mean input resistance was 119.0 ± 6.0 M
(n = 169). The major time constant (
) increased significantly during
development, from ~7 ms at early fetal stages to
11 ms at later
stages (Fig. 1C). As a consequence, the apparent cell
capacitance also showed a significant increase with age from ~60 pF
at early fetal stages to
100 pF at subsequent stages (Fig.
1D).
ACTION POTENTIAL CHARACTERISTICS AND FIRING PROPERTIES.
The peak amplitude of the AP, the potential at which this peak was
reached, and the AP half-width were measured in neurons that generated
an AP in response to a 10- to 20-ms depolarizing step. Although the
peak amplitude of the AP increased significantly during development
(Fig. 2A), the potential at
which this was reached (between 10 and 30 mV) did not change
significantly (R2 = 0.03, F(1,61) = 1.61, P = 0.2). Thus the increase in peak AP amplitude is likely to reflect the
fact that the RMP becomes more negative with age (Fig. 1A).
Finally, there was a small but significant decrease in the AP
half-width during development as reported in other developing neurons
(Fig. 2B) (Spitzer and Ribera 1998
).

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Fig. 2.
Relationships between increasing body weight of the animal and
characteristics of the action potentials (APs) and
afterhyperpolarizations (AHP). Weight (in g) is shown on a log scale.
Approximate stages of development are indicated (F, fetal; Neo,
neonate). The number of neurons (n),
R2 value, and significance of the regression
(*P < 0.05) are indicated. Regression lines are
shown with 95% confidence limits. A: the peak amplitude
of the AP (mV) increased significantly during development
(R2 = 0.46, F(1,86) = 72.2, P < 0.001). B: there was a
significant decrease in the duration of the AP (AP half-width, ms)
during development (R2 = 0.14, F(1,38) = 5.9, P = 0.02). C: during development, the amplitude of the AHP
increased significantly (R2 = 0.42, F(1,74) = 52.6, P < 0.0001). When analyzed separately, both tonic
(R2 = 0.20, F(1,23) = 5.8, P = 0.025) and long afterhyperpolarizing (LAH) neurons
(R2 = 0.58, F(1,31) = 42.1, P < 0.0001) showed significant increases in AHP amplitude during
development. The regression line is shown for the total neuronal
population. D: the duration of the AHP (ms)
significantly increased during development in tonic neurons
(R2 = 0.22, F(1,22) = 6.3, P = 0.02), but significantly decreased in LAH neurons
(R2 = 0.23, F(1,28) = 8.6, P = 0.007).
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In adult sympathetic neurons, differences in neuronal firing properties
result from the differential expression of voltage-dependent K+ channels and Ca2+-dependent
K+ channels (Adams and Harper
1995
). Therefore the identification of neurons with different
firing properties provided the first step in determining when the
mature combinations of channels are expressed during fetal development.
We therefore investigated the responses of developing neurons to
maintained depolarizing current steps. The majority of early fetal
neurons (34 of 50) generated only a single AP with a short amplitude
and long duration in response to a depolarizing voltage step (Figs.
3 and
4A).
This presumably reflects the smaller electrical driving force driving Na+ and the inactivation of some
Na+ channels at RMP in the range
30 to
40 mV.
Increasing the magnitude and duration of the current step further did
not elicit any additional APs. This finding also suggests that channels
underlying differences in firing properties are not expressed at these
early fetal stages of development. Six early fetal neurons generated
multiple APs from rest, while 10 others revealed a single shunted AP
only if the neuron was hyperpolarized. Since the majority of early
fetal neurons only elicited a single AP in response to depolarization, the ionic dependence of seven immature neurons was examined using 1 µM TTX (voltage-dependent Na+ channel blocker)
and/or 300 µM Cd2+ (nonspecific
Ca2+ channel blocker). The APs of two neurons
were completely blocked with Cd2+ alone (Fig.
3A), another two were blocked with TTX alone (Fig. 3B), while three required the combined presence of TTX and
Cd2+ (Fig. 3C). These results suggest
that the APs of some neurons are initially Na+
dependent, while others are initially dependent on
Ca2+.

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Fig. 3.
Ionic dependence of APs in early fetal neurons. Current-clamp records
of voltage traces before (top) and after
(bottom) application of 300 µM Cd2+ and/or
1 µM TTX. Right: overlays of the membrane potential
before (*) and after drug application. A: the AP of this
neuron, generated in response to depolarizing current steps of 0.4 nA,
was completely blocked after exposure to Cd2+.
B: the AP of another neuron, generated in response to
depolarizing current steps of 0.1 nA, was completely blocked after
exposure to TTX. C: this neuron had a RMP of 40 mV but
was held hyperpolarized (HYP) at around 90 mV for the duration of the
drug applications. Depolarizing current steps of 0.1 nA were used for
the top 2 traces, while larger steps were applied in the
bottom 2 traces (0.3 , and 0.25 nA during wash-out).
Exposure to Cd2+ alone reduced the amplitude of the AP
(overlay shown in panel on right), although both
Cd2+ and TTX were needed to completely block the AP.
Partial recovery of the AP was seen after 25 min wash-out.
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Fig. 4.
Electrical properties of early fetal neurons. A:
current-clamp records in a neuron from a day 34 fetus (F34) with a RMP
of 34 mV. Aa: changes in membrane potential
(top) during depolarizing and hyperpolarizing steps
(bottom). A short-amplitude AP is generated at the
beginning of a depolarizing step, while an AP also is generated off the
anode break at the end of the hyperpolarizing steps. Ab:
a depolarizing "sag" ( ) can be seen during a small
hyperpolarizing current injection. B: current-voltage
relationships in a neuron from a F34 fetus with a RMP of 50 mV.
Ba: current-clamp records showing changes to the
membrane potential (top) during depolarizing and
hyperpolarizing current steps (bottom). APs were
generated for the duration of a suprathreshold depolarizing step.
Bb: steady-state current-voltage relationship measured
at the end of the voltage step. Bc: voltage-clamp
records showing changes in current (top) during 20-mV
hyperpolarizing voltage steps (bottom) evoked from
holding potentials at RMP ( 50 mV) and at 65 mV. When held at RMP,
there is an inward current relaxation during the voltage step and an
outward current relaxation following repolarization to 50 mV. These
current relaxations, indicating IM, are not
present at holding potentials below 60 mV. Bd: AP
(top) evoked during a brief depolarizing current step
(bottom), with an AHP. Be: voltage-clamp
record showing the tail-current (top) underlying the
AHP, IAHP, that followed an unclamped AP
(truncated) generated by a brief suprathreshold voltage step
(bottom). Bf: current-clamp record
showing a depolarizing voltage "sag" (top, )
during a large hyperpolarizing step (bottom) and a
prolonged delay in the return of the membrane potential to rest at the
end of the hyperpolarizing step ( ). Bg: voltage-clamp
records showing the currents, IH and slow
IA, respectively (top),
responsible for the voltage deflections seen in Bf.
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From mid fetal stages onward, two main types of neurons could be
identified by their firing properties in response to depolarizing current injections, as has been previously reported in adult guinea pig
sympathetic neurons (Cassell and McLachlan 1987
;
Cassell et al. 1986
). Approximately one-third of mid
fetal neurons (12 of 32; Fig.
5A) and one-third of late
fetal, neonatal and adult neurons (22 of 74; Fig. 7A) fired
APs throughout the duration of a depolarizing current injection,
similar to adult tonic (slowly adapting) neurons (Cassell et al.
1986
; McLachlan and Meckler 1989
). At all
developmental stages, these tonic-firing neurons were primarily located
within medial regions of the celiac ganglion, as seen in mature guinea pigs (McLachlan and Meckler 1989
).

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Fig. 5.
Electrical and morphological properties of a mid fetal (F44) tonic
neuron. Aa: current-clamp records showing changes in
membrane potential (top) during depolarizing and
hyperpolarizing current steps (bottom) in a tonic neuron
with RMP of 43 mV. This neuron discharged APs for the duration of a
suprathreshold depolarizing current step. An A-current "notch"
( ) is seen at the break of the largest hyperpolarizing current
steps. Ab: current-voltage relationship.
Ac: voltage-clamp records showing currents
(top) during 20-mV voltage steps (bottom)
evoked when the neuron was held at rest ( 43 mV) or hyperpolarized to
58 mV. Inward and outward current relaxations are seen during the
voltage step when the neuron is held at 43 mV but are abolished when
held at 58 mV. Ad: AP evoked after a 10-ms
suprathreshold depolarizing current injection.
Ae: voltage-clamp record of an unclamped AP (truncated)
followed by the tail-current which underlies the AHP
(IAHP). Af: current-clamp
record of membrane potential (top) during and after a
large hyperpolarizing current step (bottom). Note the
depolarizing voltage sag during the hyperpolarizing step ( ), and the
prolonged delay in the return to RMP after the end of the
hyperpolarizing step ( ). Ag: voltage-clamp record of
the currents, IH and slow
IA, underlying deflections in the voltage
trace seen in Af. Ba: rendered 3D
reconstruction of low-magnification confocal through-series of the same
neuron shown in A. Threshold was optimized for
dendrites. *, axon. Scale bar = 50 µm. Bb:
rendered 3D reconstruction of high-magnification confocal
through-series of the neuronal soma shown in Ba. Scale
bar = 10 µm.
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Another third of the mid fetal neurons (11 of 32; Fig.
6A) and more than half of the
neurons at late fetal, neonatal, and adult stages (42 of 74; Fig.
8A) adapted rapidly at the onset of a suprathreshold
depolarizing current step, as occurs in adult phasic neurons
(Cassell et al. 1986
). At mid fetal and later stages of
development, 64 and 76% of the phasic neurons, respectively, had a LAH
lasting
1 s, typical of adult LAH neurons (Cassell and
McLachlan 1987
). Regardless of developmental stage, the
majority of phasic-firing neurons were located in the lateral regions
of the celiac ganglion as seen in mature guinea pigs (McLachlan
and Meckler 1989
).

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Fig. 6.
Electrical and morphological properties of a mid fetal (F44) LAH
neuron. Aa: current-clamp records showing changes in
membrane potential (top) in response to depolarizing and
hyperpolarizing current steps (bottom). This neuron only
discharged APs at the onset of suprathreshold depolarizing steps.
Ab: current-voltage relationship.
Ac: current-clamp record of AP and the LAH
(top), evoked after a brief suprathreshold depolarizing
current step (bottom). Ad: voltage-clamp
record of an unclamped action current and tail currents underlying the
AHP (top, filtered at 450 Hz), generated after a brief
(10 ms) depolarizing voltage step (bottom).
Ba: rendered 3D reconstruction of
low-magnification confocal through-series of the same neuron
whose electrical properties are shown in A. Threshold
was optimized for dendrites. *, axon. Scale bar = 20 µm.
Bb: rendered 3D reconstruction of
high-magnification confocal through-series of the neuronal
soma.
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The remaining 9 of the 32 mid fetal neurons examined resembled early
fetal neurons as they had single, small amplitude APs and could not be
definitively classified by their firing properties. In contrast, only 8 of the 74 neurons from late fetal and subsequent stages could not be
classified by their firing properties alone (see also, Cassell
et al. 1986
; Keast et al. 1993
; McLachlan
and Meckler 1989
; Stebbing and Bornstein 1993
).
DEVELOPMENT OF THE AHP.
In mature guinea pigs, most sympathetic neurons have a prominent AHP
that is largely due to the presence of a
Ca2+-dependent K+ current,
IAHP (sometimes called
gKCa1) (Cassell and McLachlan 1987
; Cassell et al. 1986
). In addition to this
current, LAH neurons also have a second
Ca2+-dependent K+ current
that is responsible for the prolonged phase of the AHP (IsAHP or
gKCa2) (Cassell and McLachlan
1987
; Jobling et al. 1993
). Here we consider the
developmental appearance of each phase of the AHP. A summary of the
results obtained using current-clamp recordings, which show the
combined effects of these two currents, is shown for neurons at
different stages of development in Table 2.
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Table 2.
Current-clamp measurements of amplitude and duration of
afterhyperpolarization (AHP) at different stages of development
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Fast AHP (IAHP).
The amplitude and duration of the AHP was determined in neurons where
an AP was generated in response to a brief (10-20 ms) suprathreshold
current step. In 19 of 21 early fetal neurons, where no AP could be
generated during a depolarizing step, an AHP was observed after the AP
generated at the end of a hyperpolarizing current step (i.e., off the
"anode break"). Overall, there was a significant increase in AHP
amplitude from ~7 mV at mid fetal stages to ~15 mV at subsequent
stages (Fig. 2C; Table 2). There was a significant increase
in the duration of the AHP in tonic neurons from ~245 ms at mid fetal
stages to
310 ms at all later stages (Fig. 2D; Table 2).
The amplitude and time constant of the current underlying this AHP,
IAHP, were measured in early fetal and
mid fetal neurons. In early fetal neurons,
IAHP ranged from 17 to 45 pA (mean
29.7 ± 8.2 pA, n = 3) while the time constant
ranged from 13 to 168 ms (91.5 ± 44.8 ms, n = 3;
Fig. 4Be). In mid fetal tonic neurons, the peak amplitude of
IAHP ranged from 49 to 230 pA
(108.0 ± 23.4 pA, n = 7) while the time constant
ranged from 28 to 148 ms (95.2 ± 24.7 ms, n = 7;
Fig. 5Ae). The amplitude of the
IAHP in mid fetal LAH neurons ranged
from 20 to 140 pA (73.3 ± 28.3, n = 4; Fig.
6Ad), but the time constant of this current could not be measured in these cells due to the presence of the prolonged
IsAHP.
Slow AHP (IsAHP).
No evidence of slow AHPs lasting
1 s was found in early fetal
neurons. From mid fetal stages, neurons with slow AHPs characteristic of adult LAH neurons were present. The duration of the AHP in LAH
neurons decreased by ~50% during development from ~3.5 s to ~2.5
s (Fig. 2D; Table 2). The peak amplitude of
IsAHP in mid fetal neurons ranged from
20 to 60 pA (41.3 ± 8.4 pA, n = 4), which was
somewhat less than that reported for LAH neurons from mature guinea
pigs (100 pA, Cassell and McLachlan 1987
; 56 pA, Martínez-Pinna et al. 2000
). However, the decay
time constant of the underlying current in mid fetal LAH neurons
(1.6 ± 0.1 s, n = 4; Fig. 6A) was
similar to that reported for mature guinea pigs (1.4 s, Cassell
and McLachlan 1987
; 1.2 s, Martínez-Pinna et al. 2000
). This suggests that the reduction in the duration of the AHP during development is unlikely to be due to changes in the
kinetics of IsAHP.
CURRENT-VOLTAGE RELATIONSHIPS.
When early and mid fetal neurons were injected with depolarizing or
hyperpolarizing currents small enough to alter the membrane potential
by 10-20 mV, the membrane potential often shifted back toward rest
during the current injection (Fig. 4Ab). This sag in the
membrane potential suggests the deactivation of a voltage-dependent current that is active around RMP. Such a voltage-dependent current is
characteristic of the time-dependent rectification produced by the
closure of M channels (IM)
(Adams and Harper 1995
; Brown 1988
;
Brown and Adams 1980
; Cassell et al.
1986
). When neurons were held positive to
60 mV in voltage
clamp, hyperpolarizing voltage steps showed inward and outward
relaxations typical of M current (Figs. 4Bc and
5Ac). This current was deactivated below
60 mV as found in
other sympathetic ganglia (Brown et al. 1982
; Cassell et al. 1986
; Jobling and Gibbins
1999
; Wang and McKinnon 1995
). The peak
amplitude of IM, measured when stepped
from
40 to
60 mV, was <30 pA in three early fetal neurons.
Although all mid fetal tonic neurons were observed to have a sag in the
voltage trace, the amplitude of IM was
only small compared with adults (Cassell et al. 1986
;
Coggan et al. 1994
). The amplitude of
IM in four mid fetal tonic neurons was
<30 pA (Fig. 5Ac) while another was 68 pA (mean 32.8 ± 9.2 pA, n = 5). Two mid fetal LAH neurons had
IM amplitudes of 16 and 74 pA.
Half of the early fetal neurons, but only 26% of mid fetal neurons,
showed a noticeable depolarizing sag in their voltage trace during
large current injections that hyperpolarized the neuron below
100 mV
(Figs. 4Bf and 5Af; Table
3). In voltage clamp, slowly activating
inward currents were observed when the holding potential was stepped
below
100 mV (Figs. 4Bg and 5Ag). These
currents resembled IH (sometimes
called IQ or
If), which has previously been
described in other neurons (Barrett et al. 1980
;
Cassell and McLachlan 1987
; Cassell et al.
1986
; Halliwell and Adams 1982
; Jobling
and Gibbins 1999
; Lüthi and McCormick 1998
; Smith 1994
).
Plots of current amplitude against steady-state voltage responses
revealed inward (or anomalous) rectification when neurons were
hyperpolarized below
90 mV in about one-third of early fetal neurons
(Fig. 4Bb), >90% of mid fetal tonic neurons (Fig.
5Ab, see also Fig.
7Ab), but <30% of mid fetal
LAH neurons (Fig. 6Ab, see also Fig.
8Ab; Table 3). The
voltage-dependent K+ current,
IKi, responsible for this
rectification has been identified in many autonomic neurons where it is
predominantly restricted to tonic-firing neurons (Adams and
Harper 1995
; Cassell and McLachlan 1987
;
Cassell et al. 1986
; Keast et al. 1993
;
Wang and McKinnon 1995
).

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Fig. 7.
Electrical and morphological properties of a late fetal (F49) tonic
neuron. Aa: current-clamp record showing continuous
discharge of APs (top) in response to a suprathreshold
depolarizing current step (bottom). Ab:
current-voltage relationship showing marked inward rectification below
90 mV. Ac: a 10-ms depolarizing current step elicited
a single AP that was followed by an AHP with a duration <500 ms. Trace
filtered at 150 Hz. B: rendered 3D reconstruction of a
low-magnification confocal through-series of the neuron. Threshold was
optimized for dendrites. *, axon. Scale bar = 50 µm.
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Fig. 8.
Electrical and morphological properties of a neonatal (P2) LAH neuron.
Aa: current-clamp record showing brief discharge of APs
(top) at the onset of a suprathreshold depolarizing
current step (bottom). Ab:
current-voltage relationship. Ac: a brief depolarizing
current step elicited a single AP that was followed by a prolonged AHP
that lasted several seconds. Trace filtered at 150 Hz.
B: rendered 3D reconstruction of a low-magnification
confocal through-series of the neuron. Threshold was optimized for
dendrites. *, axon. Scale bar = 50 µm.
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After neurons were hyperpolarized to potentials below
60 mV, a
prolonged delay was often seen in the voltage trace as the membrane
returned to rest (Fig. 5Aa). This delay or "notch" has been described in mature sympathetic neurons, where it is due to the
activation of a transient outward A current
(IA) (Adams and Galvan
1986
; Cassell et al. 1986
; Connor and
Stevens 1971
; Galvan and Sedlmeir 1984
;
Wang and McKinnon 1995
). Around half of the early fetal
neurons and 80% of mid fetal tonic neurons appeared to express
IA (Table 3). In 10 mid fetal tonic
neurons that were voltage clamped at rest, a transient outward
IA was seen when the holding potential
returned to rest (around
50 mV) after being held below
60 mV. The
time constant of IA inactivation in
these neurons ranged from 7 to 20 ms (12.9 ± 2.1 ms,
n = 6), which is less than that reported for mature
guinea-pig sympathetic tonic neurons (mean 22.1 ms, n = 17) (Cassell et al. 1986
).
An outward current, slower and more prolonged than
IA (slow
IA) was observed in 41% of early
fetal neurons, including some with single shunted action potentials
(Fig. 4B, f and g), and 40% of mid fetal tonic
neurons (Fig. 5A, f and g; Table 3). This current
was not seen in LAH or phasic neurons at mid fetal stages, reflecting
the situation in mature guinea-pigs (Table 3) (Cassell et al.
1986
). Similar currents have been observed in developing rat
sympathetic neurons (IAs)
(McFarlane and Cooper 1992
) where, in mature animals,
its expression also is restricted to tonic neurons
(ID2) (Wang and McKinnon
1995
).
Development of neuronal morphology
PROPORTION OF AREA OCCUPIED BY NEUROPIL.
We used the relative area of the celiac ganglion occupied by neuropil
as an indicator of dendritic growth in our stereological analysis of
the medial and lateral regions at different stages of development (Fig.
9A). At
late embryonic stages, very little neuropil was observed in either
medial (area of neuropil: 4 ± 4% of total sample area,
n = 3 embryos) or lateral regions (6 ± 4%;
n = 3 embryos). The relative area occupied by neuropil
significantly increased throughout development in both medial and
lateral regions. However, the rate of increase observed was greater in
medial regions (P < 0.05) so that by adult stages, the
relative area occupied by neuropil in medial regions (50 ± 2%;
n = 5 animals) was significantly higher than that in
lateral regions (33.6 ± 4.1%; n = 5 animals, P < 0.05; Fig. 9A).

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Fig. 9.
Developmental changes in morphological properties. A:
relationship during development between relative area occupied by
neuropil in lateral and medial regions of the celiac ganglion,
expressed as a percentage of the total sample area. While there was a
significant increase in the relative area occupied by neuropil in both
lateral (R2 = 0.54, F(1,23) = 27.0, P < 0.0001) and medial (R2 = 0.73, F(1,24) = 63.8, P < 0.0001) regions, the rate of increase was significantly greater in
medial regions (*P < 0.05). Stages of development
are indicated under corresponding weights (E, embryonic; F, fetal; Neo,
neonate). B: there was a small but significant increase
in the number of primary dendrites ( 1 cell body diameter in length)
during development (R2 = 0.06, F(1,79) = 4.9, P = 0.03). C: a small but significant increase in the total
dendritic length was seen during development
(R2 = 0.08, F(1,58) = 4.9, P = 0.03). Symbols indicate neurons whose electrical firing properties and
neurochemical content also were determined. Note that LAH neurons had
smaller total dendritic lengths than tonic neurons. Also note, tonic
neurons were all NPY ve, compared with LAH neurons, which were NPY+ve
or NPY ve. D: there was a significant increase in the
cross-sectional (XS) area of neuronal cell bodies during development
(R2 = 0.66, F(1,168) = 329.8, P < 0.001). E: there was no significant correlation
between the derived capacitance (pF) and the total surface area
(µm2; neuronal soma and dendritic field) of neurons
(R2 = 0.04, F(1,24) = 0.92, P = 0.3). F: there was a significant correlation between the
derived capacitance (pF) and the surface area of the neuronal soma
(µm2; R2 = 0.49, F(1,13) = 12.7, P = 0.004). Neurons with higher capacitance measurements had soma with
greater surface areas.
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OVERALL NEURONAL MORPHOLOGY.
To gain a more detailed analysis of neuronal morphology, individual
Neurobiotin-filled neurons, some of whose electrical properties had
been analyzed (Figs. 5B, 6B, 7B, and
8B), were examined. It was difficult to achieve reliable
fills of early fetal neurons, presumably due to the short impalement
times. Therefore the analysis of dendritic fields is largely restricted
to mid fetal and subsequent stages. There was a small but significant
increase in the number of primary dendrites (Fig. 9B) and
total dendritic length (Fig. 9C) during these stages.
Consistent with the stereological analyses, by late fetal stages,
medially located neurons had more primary dendrites (12.4 ± 1.4, n = 7) and greater total dendritic lengths (1,681 ± 330.0 µm, n = 7) compared with laterally located
neurons with 6.6 ± 1.7 (n = 5) primary dendrites
and total dendritic lengths of 674.4 ± 246.1 µm
(n = 5).
There was a dramatic increase in the cross-sectional area of neuronal
cell bodies during development from 170 µm2 at
early fetal stages to >1,000 µm2 at neonatal
stages (Fig. 9D). The cell body cross-sectional area of
neurons was similar regardless of their topographical location within
the ganglion (Mann-Whitney U test = 2,386.5, P = 0.7, n = 147). When the
morphological and electrical properties of individual neurons were
determined, the surface area and capacitance were calculated. Overall
there was no significant correlation between the total surface area
(neuronal soma and dendritic field) of a neuron and the derived input
capacitance (Fig. 9E). However, the increasing surface area
of neuronal soma was strongly correlated with an increase in the
derived input capacitance (Fig. 9F).
ELECTROPHYSIOLOGICAL CLASS, MORPHOLOGY, AND NEUROPEPTIDE
CONTENT.
Using a subset of neurons described above, differences between the
morphology of dye-filled tonic and LAH neurons were examined from mid
fetal through to neonatal stages. As previously described in mature
guinea pigs (Gibbins et al. 1999
; Keast et al.
1993
), tonic neurons were located in medial regions while LAH
neurons were located in lateral regions (
2 = 5.3, df = 1, P = 0.02, n = 53 neurons). While no differences were found in the cross-sectional areas
of neuronal cell bodies (tonic, 844.5 ± 103.2 µm2, n = 16; LAH, 1,159.2 ± 100.0 µm2, n = 18; F(1,31) = 0.91, P = 0.4), tonic neurons had more primary dendrites (12.9 ± 1.0, n = 12) compared with LAH neurons (7.3 ± 0.6, n = 16; F(1,25) = 19.7, P < 0.001) as well as greater total dendritic
lengths (tonic, 2,299.3 ± 345.6 µm, n = 12; cf.
LAH, 848.2 ± 65.6 µm, n = 16;
F(1,25) = 23.9, P < 0.001; Fig. 9D). Consequently, the soma of tonic neurons
formed a significantly smaller proportion of the total neuronal surface
area (6.5 ± 0.9%, n = 9) compared with LAH
neurons (16.6 ± 2.2%, n = 12; t-test, df = 19, P = 0.001).
The neuropeptide content of 101 dye-filled neurons at fetal and
neonatal stages was determined. At mid fetal stages, 72% of neurons
without NPY-IR, with or without Som-IR, were located in medial regions
while the remaining neurons were located in lateral regions
(n = 32). Across all stages examined, only 1 of 17 tonic neurons expressed NPY-IR, while 6 of 10 LAH neurons contained NPY-IR. Combined analysis of neurons from fetal and neonatal stages revealed that the cell body cross-sectional area of neurons without NPY-IR, many of which contained Som-IR, was only marginally greater than neurons with NPY-IR (Mann-Whitney U = 371.5, P = 0.05, n = 94). The number of
primary dendrites on neurons with and without NPY-IR was not
significantly different (Mann-Whitney U = 78.0, P = 0.08, n = 53). In contrast, neurons
without NPY-IR, including those with Som-IR, had significantly greater
total dendritic lengths than those with NPY-IR (Mann-Whitney
U = 24.0, P = 0.003, n = 42; Fig. 9C).
Overall the correlations between electrophysiological,
morphological, and neurochemical properties of fetal neurons reflect those previously published for celiac ganglion neurons from mature guinea pigs. Thus NPY-IR neurons corresponded to LAH neurons with small
dendritic fields, while Som-IR neurons corresponded to tonic neurons
with large dendritic fields. Nevertheless neonatal neurons of all
classes were still only about two-thirds the size of neurons in adult
celiac ganglia (Boyd et al. 1996
; Gibbins et al.
1999
; Keast et al. 1993
).
 |
DISCUSSION |
We have shown that different functional subpopulations of celiac
ganglion neurons can be distinguished by their electrical and
morphological properties from mid fetal stages of development. These
distinctions occur after the neurochemical phenotypes of the neurons
and the topographical organization of the celiac ganglion have been
established (Anderson et al. 2001
) but long before the neurons finish growing. The differentiation of these neurons involves the sequential expression of various K+ channels
accompanied by divergent growth patterns of their dendritic trees.
Furthermore each major functional class of neuron seems to develop
directly from a topographically distinct subset of precursors.
EARLY FETAL NEURONS EXPRESS DIFFERENT COMBINATIONS OF
K+ CHANNELS.
Adult celiac ganglion neurons have characteristic patterns of
expression of K+ channels. Most notably, M
current is largely restricted to phasic/LAH neurons while A current
regulates AP discharge in tonic neurons but not LAH neurons
(Cassell and McLachlan 1987
; Cassell et al. 1986
; Wang and McKinnon 1995
). In contrast with
adult neurons, small M currents were detected in most early fetal
neurons and in both phasic and tonic firing neurons at mid fetal
stages. Consequently there must be a significant increase in M-current
expression in phasic/LAH neurons but not tonic neurons during the later
stages of fetal development. In mature sympathetic neurons, M current is thought to exert a major influence on firing properties by reducing
the rate of action potential generation (Adams and Harper 1995
; Wang and McKinnon 1995
). The low level of
expression of M current in developing celiac ganglion neurons suggests
that it has only a limited influence on their firing properties. At early fetal stages, 40% of celiac ganglion neurons expressed the slow
A current. By mid fetal stages, the slow A current was restricted to
tonic neurons, suggesting that the early expression of this current
provides the first indication that a neuron is destined to develop the
tonic-firing phenotype.
In contrast to the early expression of M and A currents, the
IsAHP responsible for the LAH was not
detected until mid fetal stages. This explains the relatively late
stage at which LAH neurons could be identified by functional criteria.
The late expression of Ca2+-dependent
K+ channels also has been reported in other
systems (Ahmed et al. 1986
; Dryer 1994
,
1998
; Martin-Caraballo and Greer 2000
). It has been suggested previously that ion channels required for basic neuronal
excitability (such as voltage-dependent Na+,
Ca2+, and K+ channels) are
established relatively early during development and are not influenced
by external factors (Dryer 1994
; Ribera and
Spitzer 1992
). However, the developmental expression of ion channels involved in the fine control of neuronal firing behavior (such
as the Ca2+-dependent K+
channel IsAHP, as well as
IA and
IM) are likely to be influenced by
extrinsic factors including the local environment, synaptic inputs, and
targets (Barish 1995
; Dryer 1994
, 1998
;
McFarlane and Cooper 1992
; Raucher and Dryer
1994
, 1995
). If so, the sequential expression of ion channels
during development of celiac ganglion neurons implies the presence of
multiple factors acting in a time-dependent way to regulate their differentiation.
ELECTROPHYSIOLOGICAL AND MORPHOLOGICAL PHENOTYPES ARE ESTABLISHED
DURING THE SAME DEVELOPMENTAL PERIOD.
The celiac ganglion neurons developed their characteristic electrical
and morphological phenotypes in parallel, mainly during the mid fetal
period. Such parallel development has been reported widely in other
neurons (Allan and Greer 1997a
,b
; Dekkers et al. 1994
; Kandler and Friauf 1995
;
Martin-Caraballo and Greer 1999
; Phelan et al.
1997
; Vincent and Tell 1999
; Warren and
Jones 1997
). However, the electrical properties of celiac
ganglion neurons did not change after they were established at mid
fetal stages, whereas neurons continued to increase in size. Therefore
as the neurons grow during late fetal and neonatal development, there must be continued regulated synthesis of phenotypically appropriate channels to match the on-going production of new cell membrane.
Much of the growth of the neurons involves the dendritic tree as well
as the soma. Nevertheless developmental increases in apparent cell
capacitance were correlated much more strongly with somatic surface
area rather than with the total surface area of the neurons including
their dendrites. The simplest interpretation of this observation is
that most of the electrical properties we recorded arose from the
somatic membrane with relatively little contribution from the
dendrites. This is surprising since it is generally thought that mature
autonomic neurons, which are significantly larger than the fetal
neurons, are electrotonically compact (Adams and Harper
1995
).
While celiac ganglion neurons from medial and lateral regions showed
similar developmental increases in somatic size, the dendritic trees of
neurons located in medial regions of the ganglion increased their total
length at a greater rate than those in lateral regions. This
differential growth results in tonic neurons in the medial regions of
the ganglion bearing larger dendritic trees than LAH neurons in the
lateral regions of the ganglion. The dissociation of somatic and
dendritic growth rates has been reported previously in motor neurons of
postnatal rats (Nunez-Abades and Cameron 1995
) and
suggests that somatic size and dendritic size may be regulated independently during development.
The medially located tonic-firing neurons in the mature guinea pig
celiac ganglion are unusual in that they receive convergent synaptic
inputs from neurons projecting from the gut wall (enteric intestinofugal neurons) in addition to synaptic inputs from
preganglionic neurons projecting from the spinal cord (Kreulen
and Szurszewski 1979
; McLachlan and Meckler
1989
; Meckler and McLachlan 1988
). Previous
studies on developing autonomic neurons have demonstrated a close
temporal relationship between dendritic outgrowth and synapse formation
(Dryer 1994
; Hirst and McLachlan 1986
;
Rubin 1985a
-c
). Therefore we predict that synaptic
inputs are established during the same developmental period in which
differential growth of the dendrites is occurring, i.e., from early to
mid fetal stages of development, and that the increased dendritic
growth rate of the medial neurons is related to the arrival of
additional peripheral inputs, which the smaller lateral neurons lack.
If this prediction is borne out, the initiation of the differential
expression of at least some K+ channels, such as
those responsible for the A current, may well precede synaptogenesis in
the celiac ganglion.
In conclusion, we have shown that the differentiation of electrical and
morphological properties of sympathetic neurons in the celiac ganglion
follows the development of their neurochemical phenotype. The selective
expression of K+ channels follows a step-wise
sequence that occurs simultaneously with the differential growth of the
dendritic trees of specific populations of neurons innervating the
vasculature or the enteric plexuses. This sequential development
combined with the dissociation between somatic and dendritic growth of
the neurons strongly implies that many of these phenotypic traits can
be independently regulated. Thus our data provide strong circumstantial
evidence that the development of the full phenotype of different
functional classes of autonomic final motor neurons is a multi-step
process, likely to involve a regulated sequence of trophic interactions.
We are grateful to Professor W. W. Blessing and Dr. J. Oliver
for the gift of antiserum to NPY, and to Dr. J. C. Brown (Medical Research Council of Canada, Regulatory Peptide Group, Vancouver, British Columbia, Canada) for the provision of antiserum to
somatostatin. We also thank Dr. G. Hennig for the use of National
Institutes of Health image macros. Finally, we thank Assoc. Prof.
J. L. Morris and Dr. S.J.H. Brookes for comments on the
manuscript. P. Jobling was a National Health and Medical Research
Council (NHMRC) Australian Postdoctoral Fellow. R. L. Anderson was
a recipient of an NHMRC Dora Lush Biomedical Postgraduate Research Scholarship.
This work was supported by grants from the NHMRC (Grants 970033 and
977400), the Clive and Vera Ramaciotti Foundation, the Charles and
Sylvia Viertel Charitable Foundation, Flinders Medical Center
Foundation, and the Flinders University Research Budget.
Address for reprint requests: R. L. Anderson, Centre for
Neuroscience and Dept. of Anatomy and Histology, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia (E-mail:
rebecca.anderson{at}flinders.edu.au).