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The Journal of Neurophysiology Vol. 86 No. 3 September 2001, pp. 1237-1251
Copyright ©2001 by the American Physiological Society
Centre for Neuroscience and Department of Anatomy and Histology, Flinders University, Adelaide, SA 5001, Australia
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Anderson, Rebecca L., Phillip Jobling, and Ian L. Gibbins. Development of Electrophysiological and Morphological Diversity in Autonomic Neurons. J. Neurophysiol. 86: 1237-1251, 2001. The generation of neuronal diversity requires the coordinated development of differential patterns of ion channel expression along with characteristic differences in dendritic geometry, but the relations between these phenotypic features are not well known. We have used a combination of intracellular recordings, morphological analysis of dye-filled neurons, and stereological analysis of immunohistochemically labeled sections to investigate the development of characteristic electrical and morphological properties of functionally distinct populations of sympathetic neurons that project from the celiac ganglion to the splanchnic vasculature or the gastrointestinal tract of guinea pigs. At early fetal stages, neurons were significantly more depolarized at rest compared with neurons at later stages, and they generally fired only a single action potential. By mid fetal stages, rapidly and slowly adapting neurons could be distinguished with a topographic distribution matching that found in adult ganglia. Most rapidly adapting neurons (phasic neurons) at this age had a long afterhyperpolarization (LAH) characteristic of mature vasomotor neurons and were preferentially located in the lateral poles of the ganglion, where most neurons contained neuropeptide Y. Most early and mid fetal neurons showed a weak M current, which was later expressed only by rapidly-adapting and LAH neurons. Two different A currents were present in a subset of early fetal neurons and may indicate neurons destined to develop a slowly adapting phenotype (tonic neurons). The size of neuronal cell bodies increased at a similar rate throughout development regardless of their electrical or neurochemical phenotype or their topographical location. In contrast, the rate of dendritic growth of neurons in medial regions of the ganglion was significantly higher than that of neurons in lateral regions. The apparent cell capacitance was highly correlated with the surface area of the soma but not the dendritic tree of the developing neurons. These results demonstrate that the well-defined functional populations of neurons in the celiac ganglion develop their characteristic electrophysiological and morphological properties during early fetal stages of development. This is after the neuronal populations can be recognized by their neurochemical and topographical characteristics but long before the neurons have finished growing. 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.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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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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.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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).
, 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.
) 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).
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.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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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(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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). 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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;
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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). 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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; 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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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.
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).
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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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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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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).
; Gibbins et al.
1999; Keast et al. 1993).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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.
; 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.
).
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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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).
Received 8 March 2001; accepted in final form 1 June 2001.
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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) can be seen during a small
hyperpolarizing current injection. B: current-voltage
relationships in a neuron from a F34 fetus with a RMP of 
). Bg: voltage-clamp
records showing the currents, IH and slow
IA, respectively (top),
responsible for the voltage deflections seen in Bf.
