Electrophysiological and Morphological Diversity of Mouse Sympathetic Neurons

Phillip Jobling and Ian L. Gibbins

Department of Anatomy and Histology and Centre for Neuroscience, Flinders University of South Australia, Adelaide, South Australia 5001, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Jobling, Phillip and Ian L. Gibbins. Electrophysiological and Morphological Diversity of Mouse Sympathetic Neurons. J. Neurophysiol. 82: 2747-2764, 1999. We have used multiple-labeling immunohistochemistry, intracellular dye-filling, and intracellular microelectrode recordings to characterize the morphological and electrical properties of sympathetic neurons in the superior cervical, thoracic, and celiac ganglia of mice. Neurochemical and morphological characteristics of neurons varied between ganglia. Thoracic sympathetic ganglia contained three main populations of neurons based on differential patterns of expression of immunoreactivity to tyrosine hydroxylase, neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP). In the celiac ganglion, nearly all neurons contained immunoreactivity to both tyrosine hydroxylase and NPY. Both the overall size of the dendritic tree and the number of primary dendrites were greater in neurons from the thoracic and celiac ganglia compared with those from the superior cervical ganglion. The electrophysiological properties of sympathetic neurons depended more on their ganglion of origin rather than their probable targets. All neurons in the superior cervical ganglion had phasic firing properties and large afterhyperpolarizations (AHPs). In addition, 34% of these neurons displayed an afterdepolarization preceding the AHP. Superior cervical ganglion neurons had prominent IM, IA, and IH currents and a linear current-voltage relationship between -60 and -110 mV. Neurons from the thoracic ganglia had significantly smaller action potentials, AHPs, and apparent cell capacitance compared with superior cervical ganglion neurons, and only 18% showed an afterdepolarization. All neurons in superior cervical ganglia and most neurons in celiac ganglia received at least one strong preganglionic input. Nearly one-half the neurons in the celiac ganglion had tonic firing properties, and another 15% had firing properties intermediate between those of tonic and phasic neurons. Most celiac neurons showed significant inward rectification below -90 mV. They also expressed IA, but with slower inactivation kinetics than that of superior cervical or thoracic neurons. Both phasic and tonic celiac ganglion neurons received synaptic inputs via the celiac nerves in addition to strong inputs via the splanchnic nerves. Multivariate statistical analysis revealed that the properties of the action potential, the AHP, and the apparent cell capacitance together were sufficient to correctly classify 80% of neurons according to their ganglion of origin. These results indicate that there is considerable heterogeneity in the morphological, neurochemical, and electrical properties of sympathetic neurons in mice. Although the morphological and neurochemical characteristics of the neurons are likely to be related to their peripheral projections, the expression of particular electrophysiological traits seems to be more closely related to the ganglia within which the neurons occur.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Neurons in sympathetic ganglia show considerable heterogeneity in their neurochemical profiles, their soma size, and their dendritic morphology. These characteristics, especially the expression of neurotransmitters, are often tightly linked with the type of target tissue they innervate. Furthermore, in many cases, the neurochemical and morphological heterogeneity corresponds with specific populations of neurons identified by functional criteria (Gibbins 1995; Morris et al. 1995). This morphological and functional evidence shows that sympathetic motor outflows are organized into multiple pathways whereby various effector organs can be regulated independently by the CNS (Jänig and McLachlan 1992).

The electrical properties of sympathetic neurons also show some heterogeneity. However, it is not so apparent that the expression of these properties is similarly encoded in a pathway-specific fashion. For example, in sympathetic ganglia of guinea pigs, there appears to be only a partial correlation between the electrophysiological characteristics of neurons, their morphology, and their expression of chemical markers (Boyd et al. 1996; Gibbins et al. 1999; Keast et al. 1993; McLachlan and Meckler 1989). The neurons in sympathetic ganglia receive convergent central inputs from spinal preganglionic neurons (Jänig 1995; McLachlan and Meckler 1989). Many neurons in the prevertebral sympathetic ganglia receive additional convergent synaptic inputs from peripheral neurons located in the enteric plexuses (Kreulen and Szurszewski 1979). The number and strength of synaptic inputs varies considerably between subpopulations of sympathetic neurons (McLachlan and Meckler 1989). Although there is much information about synaptic transmission in sympathetic ganglia (Jänig 1995; McLachlan 1995), it is still not known how sympathetic postganglionic neurons integrate convergent inputs in different functional pathways. Nevertheless, we might expect that the electrophysiological characteristics of any autonomic neuron would be optimally adapted to appropriately integrate synaptic inputs generated by central and peripheral reflexes. If so, we would predict that the electrophysiological heterogeneity of sympathetic neurons would be related to the differences in the number and activation patterns of their synaptic inputs according to the functional pathway within which they lie.

To understand better the relationships between the morphological, neurochemical, and electrophysiological properties of sympathetic postganglionic neurons and their targets, we have used immunohistochemical, intracellular dye-filling, and electrical recording techniques together with multivariate statistical analyses to investigate neurons in the superior cervical, thoracic, and celiac ganglia of adult mice. These ganglia contain different populations of neurons projecting to a wide variety of targets, including vascular beds of the head, skeletal muscles, skin and viscera, the piloerector muscles of the skin, salivary glands, and the gastrointestinal tract. We were surprised to find that the electrical properties of the neurons were more closely related to the ganglia in which they occur rather than to the specific functional pathways that run through the ganglia.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Immunohistochemistry

Adult mice (BalbC) of either sex were killed with a lethal dose of inhaled halothane as approved by the Animal Welfare Committee of the Flinders University of South Australia. Superior cervical, thoracic, and celiac ganglia were removed and processed for multiple labeling immunofluorescence as described previously (Gibbins and Matthew 1996; Murphy et al. 1998). Briefly, ganglia were fixed by immersion for 24 h in a solution of 2% formaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.0). After fixation, excess picric acid was washed from the tissues with three changes of 80% ethanol before the tissues were cleared with several changes of dimethylsulfoxide (DMSO). For sectioning, ganglia were dehydrated in 100% ethanol (3 changes) before being infiltrated with polyethylene glycol (PEG 1000 MW) for 30 min under vacuum at 46°C. Ganglia were then embedded in molten PEG (1450 MW) and placed in a -20°C freezer for 10 min before being sectioned (10-30 µm) on a standard bench microtome. Tissue sections were floated in phosphate-buffered saline (PBS) and then placed in 10% normal donkey serum for 30 min before being incubated for 24-72 h in combinations of one, two, or three primary antisera raised in different species. Primary antisera used were as follows: rabbit anti-tyrosine hydroxylase (Dr. J. Thibault; dilution of 1:2,000), sheep anti-neuropeptide Y (code E2210/2, supplied by Dr. J. Oliver and Dr. W. Blessing; 1:1,000), rat anti-vasoactive intestinal peptide (code F1/III, Dr. R. Murphy; 1:2,000), rabbit anti-calcitonin gene related peptide (CGRP; Peninsula, Belmont, CA; 1:2,000), goat anti-CGRP (Arnel, Cherokee Station, NY; 1:1,000) and sheep anti-choline acetyltransferase (Chemicon, Temecula, CA; 1:2,000). Following incubation in primary antisera, sections were washed in several changes of PBS and incubated for 2 h in secondary antisera. Secondary antisera (Jackson Immunoresearch Labs, West Grove, PA) were all raised in donkeys and conjugated to dichlorotriazinyl amino fluorescein (DTAF), Cy3 or Cy5. In some cases, whole mounts of ganglia were processed for immunohistochemistry in place of sections. In these cases, primary antisera were used at twice the concentration used for sections, and incubations in secondary antisera were carried out for 4-24 h. All the antisera used in this study are well characterized and do not show any known cross-reactivity with inappropriate epitopes (Gibbins and Matthew 1996; Morris et al. 1998; Murphy et al. 1998).

To visualize neurobiotin-filled neurons, ganglia were fixed overnight as described above and washed in three changes of 80% ethanol followed by three changes of DMSO before being placed in PBS. Ganglia were then incubated in streptavidin conjugated to DTAF, Cy3 or Cy5 for 2 h before being washed in three changes of PBS.

Sections or whole mounts of ganglia were mounted on slides in carbonate-buffered glycerol (pH 8.6). For widefield fluorescence microscopy, tissues were examined with an Olympus AX70 or BX50 epifluorescence microscope fitted with highly discriminating filters (Chroma Technology, Brattleboro, VT). Images were obtained with a Sony CCD camera (SSC-M370CE) and a Macintosh PowerPC computer fitted with a Scion AG-5 frame-grabber board, running NIH Image version 1.61. Confocal microscopy was performed with a BioRad MRC-1024 scanning laser confocal microscope system fitted to an Olympus AX70 epifluorescence microscope and running Lasersharp version 2.1. When examining potential coexistence of different markers within cell bodies or boutons, considerable care was taken to ensure that there were no spurious colocalizations due to filter bleed through.

Image processing

Postcapture image processing was carried out using a Macintosh PowerPC. All morphological measurements were made with NIH Image version 1.61. For the construction of the color plates (Figs. 1 and 2), confocal images were processed for brightness and contrast only without any filtering and pseudocolored to identify each fluorophore using Adobe Photoshop version 5.0. The individual channels were then overlaid in Photoshop. For the construction of Fig. 3 (dendritic morphology), a series of images (either confocal or wide-field microscopy) were taken at different focal planes to ensure all filled dendrites were captured. A through-focus projection of these images was then made using NIH Image macros to produce a single two-dimensional image that included all dendrites. These fluorescence images were then inverted to make the final plate. To visualize better the details of fine dendritic processes, three images (Fig. 3, A, B, and G) were processed further by an edge enhancement algorithm (NIH Image shadow function).



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Fig. 1. Immunohistochemically defined subpopulations of neurons in mouse thoracic ganglia visualized with confocal microscopy. A: optical section through a thoracic ganglion whole mount showing immunoreactivity to tyrosine hydroxylase (TH-IR) in red and immunoreactivity to neuropeptide Y (NPY-IR) in green. Neurons with both TH-IR and NPY-IR are yellow (single arrow). B: optical section through another thoracic ganglion whole mount showing NPY-IR in green and immunoreactivity to vasoactive intestinal peptide (VIP-IR) in blue. No neurons contain both peptides. C: triple labeled immunofluorescence of a thoracic ganglion whole mount. TH-IR in blue, VIP-IR in green, and NPY-IR in red. Neurons double labeled for TH-IR and NPY-IR (single arrow) and TH-IR and VIP-IR (double arrow) are marked. Asterisk denotes the only triple labeled neuron. D: optical section through a thoracic ganglion whole mount demonstrating VIP-IR in blue and ChAT-IR in red. VIP-IR neurons did not contain ChAT-IR. Baskets of ChAT-IR boutons were present surrounding some neurons. E: reconstruction of a neurobiotin-filled neuron from a thoracic ganglion (T4). Primary dendrites have been colored separately to demonstrate variation in length and branching (axon in white). The boundary of the ganglion is shown as a dashed line. This neuron had a dendritic arbor that stretched most of the length of the ganglion.



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Fig. 2. Immunohistochemically defined subpopulations of neurons in mouse celiac ganglia visualized with confocal microscopy. A: section through a mouse celiac ganglion showing TH-IR labeled in red and NPY-IR labeled in green. NPY-IR (which appears compartmentalized within Golgi apparatus in mouse neurons) can be seen as yellow labeling in all TH-IR neuron profiles that contain a nucleus. B: single optical section through a whole mount of celiac ganglion showing immunoreactivity for choline acetyltransferase (ChAT-IR) axons in red and immunoreactivity for calcitonin gene related peptide (CGRP-IR) axons in green. Most CGRP-IR axons do not contain ChAT-IR. However, a small number of boutons do contain immunoreactivity to both ChAT and CGRP: some are indicated in the inset and its enlargements (arrows). C: triple labeled section of celiac ganglion showing CGRP-IR in green, immunoreactivity for substance P (SP-IR) in red and VIP-IR in blue. Axons double labeled for SP-IR and CGRP-IR appear yellow-orange (arrows). A few boutons contain SP-IR without CGRP-IR and appear red (double-headed arrows). A pair of cells (asterisks) is surrounded by VIP-IR boutons (blue). D: section through celiac ganglion labeled for CGRP-IR. Many axons containing CGRP can be seen throughout this section. E: same section as D labeled for VIP-IR. VIP-IR axons are more sparsely distributed than those containing CGRP-IR.



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Fig. 3. Morphology of mouse sympathetic neurons. Through-projections of sympathetic neurons showing simple and complex neurons from each ganglion. A: complex superior cervical ganglion (SCG) neuron showing numerous small dendritic branches. Some distal branches appear varicose. B: simple SCG neuron with few primary dendrites. C: simple thoracic ganglion neuron. D: complex thoracic ganglion neuron spanning most of the width of the ganglion. E: complex celiac ganglion neuron with fine tapering distal dendrites. F: simple celiac ganglion neuron with little dendritic branching. G: complex celiac ganglion neuron with a large number of radial dendrites. Inset: higher magnification to illustrate the complex arrangement of small dendritic branches in this neuron. Scale bars = 10 µm.

Electrophysiology

Mice were killed by a lethal dose of inhaled halothane. Ganglia were removed and placed in physiological saline containing (in mM) 146 NaCl, 4.7 KCl, 0.6 MgSO4, 0.13 NaH2PO4, 2.5 CaCl2, 7.8 glucose, and 20 HEPES, buffered to pH 7.29 and gassed with 100% O2. Ganglia were pinned to the base of a recording chamber coated with silicone elastomer (Sylgard; Dow Corning, Midland, MI), maintained at 35°C and perfused with physiological saline at 2.5 ml min-1. Neurons were impaled with glass microelectrodes filled with 0.5 M KCl and having resistances of 80-200 MOmega . In some cases, Neurobiotin (Vector, Burlingame, CA) 0.5% wt/vol was included in the electrode filling solution to enable postimpalement visualization of neurons.

Electrical properties of neurons were determined using discontinuous current clamp (DCC) for voltage recording and single-electrode voltage clamp (SEVC) using an Axoclamp-2B amplifier (Axon Instruments, Burlingame, CA). For measurement of action potential amplitudes, recordings were made in bridge mode. During DCC and SEVC, the headstage was continuously monitored and the cycling frequency adjusted to minimize the effects of electrode capacitance. The cycling frequency was 1.0-2.0 kHz for DCC and 2.0-3.5 kHz for SEVC. Voltage and current records were digitized at 1-5 kHz (MacLab, ADI Instruments Castle Hill, NSW) using Chart/Scope (version 3.5) software. Digitized data were analyzed using Igor Pro (version 3, WaveMetrics, Lake Oswego, OR). Measurements of input resistance and the major input time constant were carried out by injecting small current pulses (0.01-0.1 nA, 50-500 ms duration) through the recording electrode. Averages of 20 current steps were routinely used. Exponential functions were fitted to the onset of the voltage response between 20 and 80% of its final amplitude. For other current-voltage relationships, "steady-state" measurements were made during the last 10 ms of a current or voltage step. Measurement of "instantaneous" current in SEVC was made 10 ms following the beginning of the voltage pulse to ensure settling of the voltage to within 5% of the command value, which usually took 5-10 ms. The tail currents underlying the afterhyperpolarization (AHP) or afterdepolarization (ADP) were measured in SEVC after suprathreshold voltage steps (10 ms) initiated a single brief "action current" corresponding to an unclamped action potential (see Cassell et al. 1986; Jobling et al. 1993; Sah and McLachlan 1992; Wang and McKinnon 1995). The superior cervical trunk, splanchnic and celiac nerves were electrically stimulated by means of suction electrodes connected to a Grass S88 stimulator (Grass Instruments, Quincy, MA). Nerves were stimulated with square waves of 1- to 20-V amplitude and 0.3- to 0.5 ms pulse width. The number of synaptic inputs was estimated by varying the stimulation voltage and observing changes in the amplitude of the excitatory postsynaptic potential (EPSP) indicating recruitment of axons with different thresholds (see McLachlan and Meckler 1989). Estimates of the number and relative strength of synaptic inputs were carried out at both resting membrane potential and when the neuron was hyperpolarized to between -90 and -100 mV to block orthodromic action potentials. Antidromic action potentials were discriminated from orthodromic action potentials by their more rapid rise time when the cell was hyperpolarized. Antidromic potentials could be blocked by hyperpolarizing current injections. Any residual antidromic electrotonic potential was not increased by further hyperpolarization, in contrast with the EPSPs underlying orthodromic action potentials (see McLachlan and Meckler 1989).

Statistical analyses

Summary data are presented in the text and tables as means ± SE. Percentages are expressed with 95% confidence limits derived from the binomial distribution (Rohlf and Sokal 1995). They were analyzed using SPSS Release 6.1.3 (SPSS, Chicago, IL) running on a PC or a Macintosh PowerPC, or with S-Plus 4.5 (MathSoft, Seattle, WA) on a PC. Initial analyses used one-way ANOVA followed by Tukey's HSD multiple range tests to determine differences in electrophysiological properties of the neurons in superior cervical, thoracic, and celiac ganglia. Significance level was set at 0.05 for these tests.

To further test the relationships between the observed variables, a factor analysis was made on 32 neurons for which there were values for size and duration of the afterhyperpolarization, the size and half-width of the action potential, the time constant of the membrane, the apparent input resistance, and the presence or absence of IA, IM, IH, the inward rectifier and an afterdepolarization. Neurons were also coded for their phasic or tonic firing properties in response to a depolarizing current injection (see RESULTS), and the ganglion within which they occurred. A correlation matrix was generated that identified the relationships between each pairwise combination of these variables. Factors were then extracted using the principal components approach followed by varimax rotation, so that each factor identified a combination of variables that were well correlated with each other, but not with variables in the other factors (Norusis 1993; Tabachnik and Fidell 1996).

To see whether neurons could be classified and assigned to their ganglion of origin simply on their electrical properties, a series of discriminant analyses was done (Norusis 1993; Tabachnik and Fidell 1996). Based on the results of the factor analysis, the duration and amplitude of the AHP and action potentials together with the apparent cell capacitance were used as classification variables in the discriminant analysis. Each neuron also was identified by its ganglion of origin. The analyses produced two canonical discriminant functions with coefficients relating linear combinations of the variables representing the electrical properties of the neurons. These functions were then used to classify the neurons according to their predicted ganglion of origin. The output of the analysis included a plot ("territorial map") of all the neurons classified by ganglion according to the discriminant functions evaluated for each case (see Fig. 12). The analysis also produced a table of all the neurons used in the analysis along with their discriminant function scores and their most likely and second most likely classifications. The tables were used to identify the neurons that were misclassified and assigned to the wrong ganglion by the analysis (see Norusis 1993). From these outputs, a measure of the reliability of the discriminant functions was obtained. Initial analyses were done with the complete data set for 72 neurons to optimize the procedures (selection of appropriate variables, method of determining the optimal functions, etc.) (see Norusis 1993, for details). Then the data set was divided randomly into two equal subsets. The discriminant functions were derived first from one-half of the data and then were used to classify the neurons in the other half of the data set. In this way, the reliability of the discriminant functions could be tested directly. This procedure was repeated with five different random subsets of data.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Immunohistochemical studies of mouse sympathetic neurons

THORACIC GANGLIA. Single confocal optical sections through whole mounts of thoracic sympathetic ganglia (T4-T6) were used to determine the neurochemical profile of thoracic neurons. Optical sections through the midregion of one ganglion from each of 4 animals contained on average 110 ± 13 neurons, 88 ± 4% (mean ± SE) of which contained immunoreactivity to tyrosine hydroxylase (TH-IR; Fig. 1A). No neurons contained immunoreactivity for choline acetyltransferase (ChAT-IR). Immunoreactivity to neuropeptide Y (NPY-IR) occurred in 32 ± 7% of TH-IR neurons, whereas 4 ± 3% of neurons with NPY-IR did not contain TH-IR (Fig. 1A). Immunoreactivity to vasoactive intestinal peptide (VIP-IR) was observed in 9 ± 4% (n = 3 ganglia) of thoracic ganglion neurons (Fig. 1B), a few of which also contained TH-IR (Fig. 1C). Only one neuron containing TH-IR, NPY-IR, and VIP-IR was observed (n = 3 ganglia; Fig. 1C).

A single optical section through one thoracic ganglion whole mount per mouse was used to determine the cross-sectional area of neurons containing different chemical markers. As previously described in mouse superior cervical ganglia (Gibbins 1991), neurons expressing TH-IR alone (mean cross-sectional area of 533 ± 15 µm2; n = 153 neurons from 5 mice) were significantly larger than all other neurochemical classes of neurons [ANOVA; F(3,309) = 15.8; P < 0.0001]. Neurons expressing both TH-IR and NPY-IR (mean cross-sectional area: 439 ± 11 µm2; n = 132 neurons from 5 mice), NPY-IR alone (mean cross sectional area: 358 ± 26 µm2; n = 15 neurons from 3 mice), or VIP alone (mean cross-sectional area: 331 ± 37 µm2; n = 13 neurons from 4 mice) were not significantly different from each other in soma size.

Varicose axons and boutons containing ChAT-IR were present throughout thoracic ganglia (Fig. 1D). Although boutons with ChAT-IR were observed close to most neurons, there were areas of neuropil where the density of ChAT-IR boutons appeared higher than others (Fig. 1D). These regions did not appear to contain any particular neuronal class. Varicosities containing immunoreactivity to calcitonin gene-related peptide (CGRP-IR) were also present throughout thoracic ganglia; most of these varicosities did not contain ChAT-IR.

CELIAC GANGLION. Nearly all (98 ± 1%) of neurons in the celiac ganglion contained both TH-IR and NPY-IR (n = 106 ± 32 neurons per section from 3 mice; Fig. 2A). No somata containing VIP-IR or ChAT-IR were observed. Varicose axons containing ChAT-IR or CGRP-IR were abundant throughout the ganglion (Fig. 2, B, C, and E), although very few axons contained both ChAT-IR and CGRP-IR (Fig. 2B). Some CGRP-IR axons contained immunoreactivity for substance P (SP-IR) but SP-IR axons without CGRP-IR were extremely rare (Fig. 2C). Axons containing VIP-IR also occurred throughout the ganglion (Fig. 2, C and E), but they were more sparsely distributed than those containing CGRP-IR (Fig. 2, D and E).

Morphological properties of mouse sympathetic neurons

Dye-filled sympathetic neurons exhibited considerable variation in their morphologies, both within and between ganglia. Figure 3 shows simple and complex neurons from the superior cervical, thoracic, and celiac ganglia. Dendrites did not radiate equally in all directions from the soma but tended to lie parallel with the frontal plane of the superior cervical ganglion (SCG) and celiac ganglia and in an oblique plane in the thoracic ganglia. Dendritic diameter varied considerably throughout their length with the most distal regions often being distinctly varicose. There was no correlation between the soma size and the extent of their dendritic branching. Six representative neurons from the three ganglia were fully reconstructed from confocal images. In these neurons, the surface area of the soma ranged from ~2,300 µm2 to >8,500 µm2 and comprised from 13.5 to 51.5% of the total surface area of the neuron. These values are in the range reported for neurons in the superior mesenteric ganglia of mice (Miller et al. 1996).

Morphological data from dye-filled neurons are summarized in Table 1. The average total dendritic length of neurons in the SCG (629 ± 90 µm; n = 12) was significantly less than that of thoracic ganglion neurons (923 ± 99 µm, n = 15). The maximum dendritic length (measured from the base of each primary dendrite to its most distal tip) was similar between neurons in the SCG and thoracic ganglia. Consequently, the smaller total dendritic length observed in SCG neurons was partially due to smaller number of primary dendrites. In many cases the dendritic arbor of a single thoracic neuron extended throughout most of the ganglion (Figs. 1E and 3D). Such extensive arborizations were never seen in SCG neurons. The dendritic trees of celiac ganglion neurons varied in size and arrangement. Some neurons had relatively small dendrites similar to many SCG neurons (Fig. 3F). However, most celiac ganglion neurons had relatively long dendrites that radiated from the cell body (Fig. 3, E and G). The total dendritic length (1,140 ± 268 µm, n = 5), number of primary dendrites (6.6 ± 1.5, n = 5) and mean dendritic length (112 ± 8.9 µm, n = 29 dendrites from 5 neurons) of celiac neurons were similar both to those of thoracic neurons and neurons previously described in mouse superior mesenteric ganglia (Miller et al. 1996; Schmidt et al. 1995).


                              
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Table 1. Morphological characteristics of dye-filled sympathetic neurons

Electrophysiological properties of mouse sympathetic neurons

MEMBRANE PROPERTIES. Neurons in all ganglia had resting membrane potentials of around -50 mV. However, there were significant differences in passive membrane properties of neurons from different ganglia (Table 2). Neurons in the celiac ganglion had a significantly higher mean input resistance and input time constants compared with either SCG or thoracic ganglion neurons. Although neurons in the thoracic ganglia were similar in overall size to celiac neurons and were larger than SCG neurons, their calculated apparent cell capacitances were significantly less than those of neurons in the other two ganglia.


                              
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Table 2. Electrophysiological properties of mouse sympathetic neurons

ACTION POTENTIAL DISCHARGE. SCG neurons typically fired a single action potential at the beginning of a sustained (250-500 ms) depolarizing current injection. Increasing the amplitude of the current step did not evoke any further action potentials. Thus neurons in the SCG could be described as having "phasic" firing properties (see Cassell et al. 1986; Weems and Szurszewski 1978), as also has been observed in rat SCG (Wang and McKinnon 1995; Yarowsky and Weinrich 1985) and most other paravertebral sympathetic ganglia (see Adams and Harper 1995). Similarly, neurons in thoracic ganglia were all classified as phasic. In the celiac ganglion, 37% of neurons were classified as phasic (Fig. 7Ab), whereas 48% of neurons fired throughout a prolonged depolarizing current injection (Fig. 7Bb) and were thus classified as "tonic" neurons (Weems and Szurszewski 1978). Fifteen percent of celiac ganglion neurons had intermediate firing properties and could not be classified into either group.

ACTION POTENTIAL CHARACTERISTICS. Brief injections of depolarizing current (10 ms) were used to evoke single action potentials (Figs. 4, 6, and 7). Mean action potential amplitude was significantly greater in neurons from the SCG compared with either celiac or thoracic ganglion neurons (Table 2). Neurons from the SCG also had briefer action potentials as indicated by their significantly shorter mean half-width.



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Fig. 4. Action potentials and synaptic inputs in SCG neurons. Aa: action potential recorded from a neuron in the SCG (top trace) evoked after a depolarizing current step (bottom trace). This neuron had a large afterhyperpolarization (AHP; arrow) following the action potential. Ab: outward tail current (IAHP, arrowed top trace) in the same neuron following a depolarizing voltage step (bottom trace) that reached threshold for an unclamped action potential ("action current"). Ba: action potential recorded in a neuron in the SCG (top trace) evoked by a depolarizing current step (bottom trace). This neuron had a pronounced afterdepolarization (ADP; arrowed) preceding the AHP. Bb: inward and outward currents (top trace of each pair) in the same neuron following depolarizing voltage steps (bottom trace of each pair) from holding potentials of -55 mV (resting membrane potential, RMP), -75 mV, and -95 mV. Note that IAHP is abolished at hyperpolarized holding potentials, whereas the inward current (arrowed) is increased in amplitude. C and D: stimulation of the superior cervical trunk (indicated by dot) evoked excitatory postsynaptic potentials (EPSPs), which were suprathreshold for the generation of an action potential in neurons with AHPs alone (Ca) and neurons with ADPs (Da). Synaptically evoked action potentials in these neurons were not abolished by hyperpolarizing the soma to -100 mV (Cb and Db). Time scale in B also applies to A.

AFTERHYPERPOLARIZATIONS. In most neurons, action potentials were followed by prominent afterhyperpolarizations (AHPs; Fig. 4Aa). In voltage clamp, an outward current (IAHP) followed depolarizing voltage steps (10 ms) to holding potentials just suprathreshold for an action current (Fig. 4Ab). The mean amplitude and duration of AHPs were significantly different between ganglia. Neurons in the SCG had a significantly larger mean AHP amplitude compared with thoracic or celiac neurons (Table 2, Figs. 4Aa, 6Aa,and 7, Aa and Ba). The mean duration of the AHP was longest in SCG neurons and shortest in thoracic ganglion neurons (Table 2). Within the celiac ganglion, phasic neurons had significantly longer AHPs than tonic neurons (Table 3 and Fig. 7, Aa and Ba).


                              
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Table 3. Electrophysiological properties of phasic and tonic neurons from celiac ganglion

AFTERDEPOLARIZATIONS. In some neurons, the action potential was followed by a depolarization that preceded the AHP (Figs. 4Ba and 6B). The proportion of neurons with these afterdepolarizations (ADPs) varied markedly between the three ganglia. ADPs were observed in 34% of SCG neurons, 18% of thoracic neurons but only in one celiac ganglion neuron. Measurement of the tail currents following action current initiation in neurons with ADPs revealed an inward current followed by an outward current (Fig. 4Bb). Inward currents increased in amplitude at hyperpolarized holding potentials, whereas the outward current was abolished (Fig. 4Bb). Identical ADPs and the underlying currents have been extensively characterized in normal mouse and axotomized rat SCG neurons, where they represent the activation of a calcium-activated chloride channel (De Castro et al. 1997; Sanchez-Vives and Gallego 1994).

CURRENT-VOLTAGE RELATIONSHIPS. Current-voltage relationships were studied from membrane potentials below threshold for action potential initiation to around -130 mV in both current clamp and voltage clamp. During depolarizing and hyperpolarizing current steps evoked from resting membrane potential (RMP), time- and voltage-dependent rectification could be observed in the voltage traces (Figs. 5A, 6Ab, and 7Ab). During small current injections that shifted the membrane potential by <10 mV in either direction, there was a noticeable "sag" in the voltage trace (Figs. 5A and 6Ab), suggesting the presence of a voltage-dependent current active around RMP. In voltage clamp at holding potentials more positive than -60 mV, hyperpolarizing voltage steps showed time-dependent current relaxations indicating the presence of M-current (IM; Figs. 5D, and 8, Aa and Bb). IM was deactivated at holding potentials below -60 mV, consistent with M-currents previously described in mouse hypogastric ganglia (Rogers et al. 1990) and many other sympathetic ganglia (Brown and Adams 1980; Cassell et al. 1986; Constanti and Brown 1981; Wang and McKinnon 1995). Although IM was observed in neurons from all ganglia, only 38% of tonic neurons within the celiac ganglion showed evidence of IM compared with 89% of phasic neurons and 100% of neurons from the SCG and thoracic ganglia.



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Fig. 5. Current-voltage relationships in SCG neurons. A: current-clamp records showing changes in membrane potential (top traces) during depolarizing and hyperpolarizing current steps (bottom traces). A hyperpolarizing voltage "sag" can be seen during a small depolarizing current injection (asterisk) and a depolarizing "sag" (arrowed) can be seen during the most hyperpolarizing current injection. B: voltage-clamp records in the same neuron showing changes in current (top 3 traces) during hyperpolarizing voltage steps (bottom traces) from the same holding potential (-50 mV). Note the slowly developing inward current (IH arrowed) at the most hyperpolarized holding potential. Outward currents (IA arrowed) can be seen following repolarization from membrane potentials below -70 mV. C: steady-state current-voltage relationship measured at the end of the voltage step of the same neuron as in A and B. D: voltage-clamp records from another neuron showing changes in current (top trace of each pair) during 20 mV hyperpolarizing voltage steps (bottom trace of each pair) evoked from holding potentials of -40, -50, and -60 mV. At a holding potential of -40 mV, there is an inward current relaxation during the voltage step and an outward current relaxation following repolarization to -40 mV. The current relaxations are smaller when the voltage step is evoked from a holding potential of -50 mV and abolished at holding potentials of -60 mV.



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Fig. 6. Electrical properties of neurons in thoracic ganglia. Aa: action potential (top trace) evoked following a depolarizing current step (bottom trace). Ab: current-clamp records showing changes in membrane potential (top traces) during hyperpolarizing current steps (bottom traces). A depolarizing voltage "sag" is noticeable during the largest current injections (arrowed) and an "A-current notch" (arrowhead) at the break of the same current step. Ac: voltage-clamp records showing changes in current (top traces) during hyperpolarizing voltage steps (bottom traces) from a holding potential close to RMP (-50 mV). Note the slowly developing inward current at the most hyperpolarized potentials (IH) and the outward current (IA) following repolarization from membrane potentials below -70 mV. Some spontaneous excitatory postsynaptic currents (EPSCs) can also be seen in these records. Ad: steady-state current-voltage relationship measured at the end of the voltage step of the same neuron as in a-c. B: ADP in a thoracic ganglion neuron. Ba: single action potential (top trace) following a brief depolarizing current step (bottom trace) showing evidence of a small ADP. Bb and Bc: when multiple action potentials are evoked the ADP (arrowed) is increased in amplitude and duration.



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Fig. 7. Electrical properties of neurons in the celiac ganglion. Aa: action potential (top trace) evoked during a depolarizing current step (bottom trace) in a phasic neuron. Ab: current-clamp records showing changes in membrane potential (top traces) during depolarizing and hyperpolarizing current steps (bottom traces). This neuron fired action potentials at the onset of the depolarizing current step but rapidly accommodated. Arrow indicates the characteristic "A-current notch". Ac: voltage-clamp records from the same neuron (as in Ab) showing changes in current (top traces) during hyperpolarizing voltage steps (bottom traces) from RMP (-45 mV). Ad: steady-state current-voltage relationship measured at the end of the voltage step indicating marked inward rectification at potentials less than -90 mV. Records in B are from a tonic neuron. Ba: action potential (top trace) evoked during a depolarizing current step (bottom trace). Bb: current-clamp records showing changes in membrane potential (top trace) during depolarizing and hyperpolarizing current steps (bottom traces). This neuron discharged action potentials throughout depolarizing current injection and shows a prominent "A-current notch" (arrow). Bc: voltage-clamp records showing changes in current (top traces) during hyperpolarizing voltage steps (bottom traces) from a holding potential of -50 mV. Bd: steady-state current-voltage relationship measured at the end of the voltage step. This tonic neuron also showed marked inward rectification. The calibration bars in B also apply to the equivalent records in A.



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Fig. 8. M-current relaxations in phasic and tonic neurons from the celiac ganglion. Aa: action potential discharge (top trace) of a phasic neuron in response to depolarizing current injection (bottom trace). A, b-d: current records (top traces of each pair) in response to 20 mV hyperpolarizing voltage steps (bottom traces of each pair) from holding potentials of -45, -55, and -60 mV in this neuron. Ba: action potential discharge (top traces) of a tonic neuron in response to depolarizing current injection (bottom traces). B, b-d: current records (top traces of each pair) in response to 20 mV hyperpolarizing voltage steps (bottom traces of each pair) from holding potentials of -45, -55, and -60 mV in this neuron. Note prominent inward and outward current relaxations at holding potentials above -60 mV. The calibration bars in B also apply to equivalent traces in A.

At the end of hyperpolarizing current steps that led to voltage excursions more negative than -60 mV, there was a prolonged delay in the return to resting membrane potential (most clearly seen in Figs. 6Ab and 7, Ab and Bb). This "notch" in the voltage record suggested that a slowly inactivating outward current had been activated during the hyperpolarization (see Galvan and Sedlmeir 1984). When neurons were voltage clamped at their resting membrane potential (around -50 mV), a transient outward current was observed after the holding potential was stepped below -70 mV and returned to rest (Fig. 5A). This outward current is similar to the A-current (IA) described in rat SCG neurons (Galvan and Sedlmeir 1984; Wang and McKinnon 1995; see also Connor and Stevens 1971). The voltage dependence of IA was similar for neurons from all ganglia (Fig. 10). However, at resting membrane potential, IA recorded from celiac neurons decayed more slowly than that recorded from neurons in the SCG or thoracic ganglia (Table 2). Nevertheless, IA recorded in celiac neurons did not resemble the very slow outward currents reported for sympathetic neurons in other species (Cassell et al. 1986; Smith 1994; Wang and McKinnon 1995).

During prolonged large current injections that hyperpolarized neurons below -100 mV, there was often a noticeable depolarizing "sag" in the membrane potential recordings (Figs. 5A and 6Ab). In voltage clamp, a slowly activating inward current was activated when the holding potential was stepped below -100 mV (Figs. 5B and 6Ac). In three SCG neurons, this current was reduced in the presence of 1 mM Cs+. Therefore the properties of this current were similar to the H-current (IH) observed in many excitable cells including other mammalian and amphibian sympathetic neurons (Cassell et al. 1986; Smith 1994), where it has often been called IQ. IH is often involved in the generation of pacemaking potentials (Lüthi and McCormack 1998). However, the functional significance of IH in sympathetic neurons is unclear, because it appears that prolonged hyperpolarization to very negative potentials (below -90 mV) are required for significant activation to occur (Smith 1994). Furthermore, no pacemaking activity was observed in the present study.

Although IH was observed in the majority of neurons in the SCG and thoracic ganglia, most celiac ganglion neurons appeared to lack this current (Table 2 and Fig. 7, Ac and Bc). Instead, the majority of neurons in the celiac ganglion (85%) showed marked instantaneous inward rectification when hyperpolarized below -90 mV (Fig. 7). When tonic and phasic celiac neurons were compared, all tonic neurons and 76% of phasic neurons showed inward rectification. The presence of such an inward (anomalous) rectifying potassium current (IKi) has been identified in many autonomic neurons (Adams and Harper 1995), although in both rat and guinea pig sympathetic neurons, the presence of inward rectification is mainly restricted to tonic neurons (Keast et al. 1993; Wang and McKinnon 1995).

SYNAPTIC POTENTIALS. EPSPs were recorded from neurons in the SCG and celiac ganglia. All neurons in the SCG received at least one synaptic input that was suprathreshold for the generation of an action potential (Fig. 4, Ca and Da). The action potential evoked following orthodromic nerve stimulation was difficult to block by injection of hyperpolarizing current (Fig. 4, Cb and Db). Synaptic inputs with these properties have been classified as strong inputs in other sympathetic ganglia (see McLachlan and Meckler 1989) and are a consistent feature of paravertebral sympathetic neurons in all species studied so far (Jänig 1995). Many SCG neurons also received a few subthreshold EPSPs (weak inputs), although it was difficult to determine the exact number due to the similar voltage threshold for stimulating both classes of inputs (Fig. 4, C and D).

Stimulation of splanchnic nerves evoked fast EPSPs in celiac neurons. EPSPs were recorded in 26 neurons including 10 tonic and 7 phasic neurons. Most neurons (81%) received at least one synaptic input that was suprathreshold for an action potential and a few subthreshold inputs (Figs. 9Ab and 9Bb). In most cases (73% of neurons), it was difficult to block the action potential evoked by splanchnic nerve stimulation by injection of hyperpolarizing current that took the membrane potential below -90 mV (Fig. 9, Ad and Bd). Thus most neurons had at least one strong synaptic input (see McLachlan and Meckler 1989). EPSPs following stimulation of celiac nerves were observed in 16 neurons including 4 tonic, 6 phasic, and 6 unclassified neurons (Fig. 9, Ac and Bc). Four neurons had one strong synaptic input and several weak synaptic inputs via the celiac nerves. In another nine neurons, stimulation of celiac nerves evoked EPSPs that were suprathreshold for an action potential at resting membrane potential but failed to evoke action potentials when the membrane potential was hyperpolarized by around 20 mV (Fig. 9Ae). These could fit into the category of the "weak strong" inputs described in guinea pig prevertebral ganglia (see McLachlan and Meckler 1989). In three neurons, only weak synaptic inputs were evoked after stimulation of the celiac nerves (Fig. 9, Bc and Be).



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Fig. 9. Responses of phasic (A) and tonic (B) neurons in the celiac ganglion to stimulation of splanchnic and celiac nerves (at dots). Aa: phasic action potential discharge (top traces) in response to depolarizing current steps (bottom traces). Ab: stimulation of splanchnic nerves evoked a strong synaptic input. Ac: stimulation of celiac nerves evoked both strong (suprathreshold) and weak (subthreshold) synaptic inputs in this neuron. Ad: response to splanchnic nerve stimulation at a holding potential of -90 mV. Ae: response to celiac nerve stimulation at a holding potential of -90 mV. Ba: tonic action potential discharge (top traces) in response to depolarizing current steps (bottom traces). Bb: stimulation of splanchnic nerves evoked a strong synaptic input and multiple weaker inputs. Bc: stimulation of celiac nerves evoked weak synaptic inputs in this neuron. Bd: splanchnic nerve stimulation at a holding potential of -90 mV. Be: celiac nerve stimulation at a holding potential of -90 mV. The calibration bars in B also apply to equivalent traces in A.



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Fig. 10. Voltage-clamp recordings of outward currents (A-like currents) in mouse sympathetic neurons. Aa: depolarizing voltage steps (bottom traces) from a constant holding potential of -90 mV of a SCG neuron showing the activation properties of the current (top traces). Ab: steady-state inactivation properties of outward currents (top traces) determined following voltage steps to various hyperpolarized potentials (bottom traces) in the same SCG neuron. B: responses of a thoracic neuron to voltage-clamp protocols designed to show activation (a) and inactivation (b) properties. C: steady-state inactivation curve for 5 neurons from the SCG (, left axis) and one thoracic neuron (open circle , right axis) in response to conditioning hyperpolarizing voltage steps from near resting membrane potential (-50 mV). D: current-voltage relation for the outward current in 5 neurons from the SCG (, left axis) and one thoracic neuron (open circle , right axis) in response to the voltage protocols in (Aa) and (Ba). E: responses of a celiac ganglion phasic neuron to voltage-clamp protocols designed to show activation (a) and inactivation (b) properties. F: responses of a celiac ganglion tonic neuron to voltage-clamp protocols designed to show activation (a) and inactivation (b) properties. G: steady-state inactivation curve for 6 phasic () and 4 tonic neurons (open circle ) in response to the voltage protocols in (Ea) and (Fa). H: current-voltage relation for the outward current in 3 phasic () and 4 tonic neurons (open circle ) in response to the voltage protocols in (Eb) and (Fb).



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Fig. 11. Relationships between the duration of the afterhyperpolarization (AHP) and its amplitude, amplitude of the action potential (AP), and capacitance of neurons from superior cervical ganglia (SCG), thoracic ganglia (TG), and celiac ganglion (CG). The duration of the AHP is related to the amplitude of the AHP (A) and the AP (B), both of which tend to be larger in SCG neurons. Although thoracic and celiac neurons have similar AHP and AP properties, they can be distinguished by their capacitance, which is generally lower in thoracic neurons (C).



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Fig. 12. Plot ("territorial map") of the canonical discriminant functions used to classify neurons according to their ganglion of origin based on the properties of their AHP, action potential, and apparent cell capacitance. The value for each neuron was calculated according to the 2 discriminant functions (f1, f2), where a = action potential amplitude (mV), b = action potential duration (ms), c = AHP amplitude (mV), d = AHP duration (ms), and e = apparent cell capacitance (pF). The group centroids are the values of the discriminant functions calculated for the means of these variables in each ganglion (CG, celiac ganglion; TG, thoracic ganglion; SCG, superior cervical ganglion). Dotted lines on the plot separate the range of discriminant function values ("territories") associated with each ganglion. From the plot, it can be seen that negative values of the 1st discriminant function tend to separate SCG neurons from neurons in the other ganglia. Positive values of the 2nd discriminant function tend to separate CG neurons from TG neurons. Comparison of the corresponding coefficients for each variable in each discriminant function shows that the 1st function is weighted more to properties of the AHP (variables c and d), whereas the 2nd function is weighted more to the apparent cell capacitance (variable e). Properties of the action potential (variable a and b) are more strongly associated with the second discriminant function, but they contribute relatively little to the classification.

The latency between the onset of nerve stimulation and the EPSP was significantly longer following stimulation of celiac nerves (8.8 ± 1.0 ms, n = 16) compared with splanchnic nerve stimulation (4.7 ± 0.4 ms, n = 26; Fig. 9). The stimulating electrodes for celiac nerve stimulation were always closer (~3 mm) to the recording site than those used for splanchnic nerve stimulation (~4 mm). Therefore this difference in latency probably indicates a real difference in conduction velocity between preganglionic axons in the splanchnic nerves and intestinofugal axons in the celiac nerves, as reported in the guinea pig celiac ganglion (Meckler and McLachlan 1988).

Multivariate statistical analysis

The preceding results indicated that there were significant differences in many of the electrical properties of the neurons from different ganglia. Consequently, characteristic combinations of these properties may be diagnostic of neurons in each ganglion. To test this possibility, we used a variety of multivariate statistical approaches to determine which electrophysiological characteristics explain most of the variance between ganglia. First, we did a factor analysis on 32 neurons for which we had complete data for AHP duration and amplitude, action potential duration and amplitude, apparent input resistance, membrane time constant, the presence or absence of ADP, IH or IKi, their phasic or tonic firing properties, and their ganglion of origin. A principal components approach was used to identify the groups of variables that were most strongly correlated with each other (i.e., each group or "factor" contained variables that covaried) (Norusis 1993; Tabachnik and Fidell 1996).

The factor analysis identified five factors that explained 76% of the variance in the data. The first factor (32% of the variance) linked the presence or absence of IH and IKi with the phasic or tonic firing properties of the cell and the ganglion within which it was found. This was mainly due to a strong negative correlation (correlation coefficient: -0.57; P = 0.0002) between the presence of IH and IKi. IKi but not IH was more likely to be found in tonic neurons (correlation for IKi: 0.4, P = 0.007; correlation for IH: -0.4, P = 0.008), which occurred only in the celiac ganglion. The second factor (14% of the variance) identified a strong correlation between the duration and magnitude of the AHP (correlation coefficient: 0.67; P = 0.00002; Fig. 11). In addition, the duration of the AHP was positively correlated with the amplitude of the action potential (correlation coefficient: 0.47; P = 0.003; Fig. 11). These relationships also covaried with the ganglion within which the cells were found. The third factor (12% of the variance) positively linked the half-width of the action potential with the apparent input resistance and the membrane time constant. The fourth factor (10% of the variance) indicated a strong positive correlation between the presence of IM and IA (correlation coefficient: 0.59; P = 0.0002). Finally, the fifth factor (9% of the variance) was primarily associated with the contribution of the ADP to the total variation. The presence of the ADP itself was positively correlated with the duration of the AHP (correlation coefficient: 0.46; P = 0.004).

These results suggest that, in addition to properties that are selectively expressed by neurons in specific ganglia, such as the presence of an ADP (mostly in SCG) or inward rectification (mostly in celiac ganglion), other more subtle properties such as the size and duration of the action potential and AHP, and cell capacitance, also may help to identify different populations of neurons (Fig. 11). To test this idea, we did a series of discriminant analyses (Norusis 1993; Tabachnik and Fidell 1996), based on a set of 72 neurons (32 from SCG; 13 from thoracic ganglia; 27 from celiac ganglia), using the properties of the action potential, the AHP and the derived membrane capacitance (or the membrane input resistance and time constant), to see whether these properties could be used to predict which ganglion a cell came from. Similar results were obtained if we used either capacitance or input resistance and time constant as variables.

When the whole data set was used to derive the discriminant functions, the first canonical discriminant function separated SCG neurons from neurons in other ganglia mostly on the basis of the amplitude and duration of the AHP (Fig. 12). The second canonical discriminant function separated thoracic and celiac neurons mostly on the basis of their capacitance (Fig. 12). These functions correctly classified 91% of SCG neurons, 46% of thoracic neurons, and 82% of celiac neurons to their ganglion of origin (overall success rate: 80%). When the data set was divided randomly into two subsets (one set used to derive the functions, which were then tested on the other subset of data), the success rate for correct classification of the data used to derive the functions ranged from 85 to 100%, with overall rates varying from 50% to better than 70% on the test data. In each case, the success at distinguishing SCG neurons from neurons in the other ganglia was better than 80%. This was mostly due to the first canonical discriminant function that was based on the properties of the AHP, as was observed in analyses using the complete data set. Examination of the data sets to identify the neurons that had been misclassified to the wrong ganglion by the discriminant functions showed that these cells really were atypical and did indeed share properties with the group to which they had been incorrectly classified. From these analyses, we conclude that the overall properties of the AHP and action potential in neurons in the SCG were significantly different from those in the thoracic and celiac ganglia. Although many neurons in the thoracic and celiac ganglia can be distinguished from each other, primarily on the basis of their capacitance, the properties of their action potentials and AHPs had more in common with each other than they did with SCG neurons.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our studies reported here show that there is considerable heterogeneity in the morphological, neurochemical, and electrophysiological properties of neurons in the sympathetic ganglia of mice. Moreover, the variation in these properties is tightly linked to the ganglion within which the neurons occur (data summarized in Table 4). This linkage was so strong that an unbiased statistical sorting procedure (discriminant analysis) could correctly classify >80% of neurons to their ganglion of origin on the basis of a small number of electrophysiological properties.


                              
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Table 4. Summary of properties of mouse sympathetic neurons

Heterogeneity of sympathetic neurons

Compared with neurons in thoracic and celiac ganglia, neurons in the SCG were most clearly characterized by their larger action potentials and the associated AHPs. The magnitude and duration of both these features are influenced by calcium-activated potassium channels (Davies et al. 1996; MacDermott and Weight 1982; Sah 1996; Sah and McLachlan 1992; Smart 1987). This suggests that SCG neurons may have fundamentally different calcium dynamics compared with sympathetic neurons in both prevertebral and other paravertebral ganglia.

Another notable characteristic of many neurons in the SCG was their marked ADPs following an action potential. These ADPs are probably due to a calcium-dependent chloride current, which is activated by calcium influx during the action potential (DeCastro et al. 1997; Sanchez-Vives and Gallego 1994; Scott et al. 1995). It has been concluded that the chloride channels responsible for the ADP are located differentially on the terminal dendrites and only become electrically significant at the soma when the dendritic arbor is short (DeCastro et al. 1997). Our morphological observations support this interpretation in that the ADPs occurred only occasionally in thoracic ganglion neurons, which had significantly longer dendrites than SCG neurons. Furthermore, ADPs were almost entirely absent from celiac neurons, which included the largest neurons studied here.

Neurons in the celiac ganglion were characterized primarily by the presence of an inward rectifier potassium current and the absence of IH. Many celiac neurons could be classified as tonic or phasic on the basis of action potential discharge during maintained depolarizing current injection (Weems and Szurszewski 1978). Overall, the tonic neurons were less likely to co-express both IA and IM than were phasic neurons. However, in contrast to celiac neurons of guinea pigs and rats, there was not a strong association between the expression or properties of these currents and phasic or tonic firing properties (Cassell et al. 1986; Cassell and McLachlan 1987; Decktor and Weems 1983; Wang and McKinnon 1995; Weems and Szurszewski 1978).

Neurons from thoracic ganglia expressed similar voltage-dependent conductances to SCG neurons, but they had action potential characteristics closer to phasic neurons in the celiac ganglion. The main distinguishing feature of thoracic ganglion neurons was their lower apparent cell capacitance. This observation is surprising, given the large dendritic length of most of these neurons. It suggests that the dendrites of thoracic ganglion neurons may express a characteristic set of membrane channels.

Taken together, these results indicate that there is significant differential expression of a wide range of ion channels by sympathetic neurons of mice. These channels must include those responsible for at least five different currents (delayed rectifier, IC due to BK channels, IAHP, IA, IM, and IH) and a calcium-dependent chloride channel. All of these channels are likely to affect the excitability of the neurons and their responses to different patterns of preganglionic stimulation.

Ganglia and pathways

There is now strong evidence that neurons in sympathetic ganglia show pathway-specific differences in their morphology and their expression of neuropeptides (see Gibbins 1995, for review). Previous studies in mice, rats, and guinea pigs suggest that within the thoracic ganglia of mice, smaller neurons containing immunoreactivity to TH and NPY are most likely to lie within vasomotor pathways, whereas larger neurons with TH but not NPY are likely to lie in pilomotor pathways (Gibbins 1991, 1992; Gibbins and Matthew 1996; Schotzinger and Landis 1990). Mouse thoracic neurons with NPY or VIP, but not TH, probably lie in vasodilator or sudomotor pathways, respectively, although they lack detectable immunoreactivity to choline acetyltransferase (Gibbins 1992; Landis and Fredieu 1986; Lindh et al. 1989; Morris et al. 1998, 1999). Thus there are at least four neurochemically characterized autonomic motor pathways running through the thoracic ganglia. Similarly, there at least four main functional pathways running through the superior cervical ganglia: vasoconstrictor, pilomotor, secretomotor, and pupilodilator (Gibbins 1991). Once again, the final motoneurons in these pathways can be distinguished by a combination of morphological and immunohistochemical characteristics (Gibbins 1991).

A unique characteristic of many neurons in the prevertebral ganglia is that they participate in peripheral reflex circuits and receive direct synaptic inputs from intestinofugal neurons located in the enteric plexuses (Furness and Costa 1987; Kreulen and Szurszewski 1979). In guinea pig celiac ganglia, the inputs from VIP-containing intestinofugal neurons produce subthreshold cholinergic EPSPs in their target neurons. Most of these target neurons contain somatostatin rather than NPY, fire tonically, and project back to the enteric plexuses (Costa and Furness 1983; Lindh et al. 1986, 1988; Macrae et al. 1986; McLachlan and Meckler 1989; Meckler and McLachlan 1988). In contrast, both phasic and tonic neurons in the prevertebral ganglia of mice receive cholinergic suprathreshold inputs from preganglionic fibers in the splanchnic nerves and from presumed intestinofugal fibers in the celiac nerves (see also Miller and Szurszewski 1997). However, we did not find an obvious neurochemical marker for the peripheral inputs to celiac neurons, because most ChAT-IR fibers lack detectable peptides. Furthermore, all neurons in the celiac ganglia of mice contain NPY, so it was not possible to distinguish neurons in vasoconstrictor pathways from those in motility-regulating pathways, using neurochemical or morphological criteria, as can be done in guinea pigs, for example (Boyd et al. 1996; Costa and Furness 1983).

Conclusions

Our results have shown that in addition to the pathway-specific expression of transmitters and morphological phenotype, neurons projecting to similar targets can be further distinguished by the selective expression of ion channels according to their ganglion of origin. Most of the electrophysiological differences we have observed between neurons are likely to affect the way in which they integrate and respond to their synaptic inputs. Both the magnitude of AHP and the presence or absence of IM would contribute to setting an upper limit on the rate of action potential generation in response to increasing levels of synaptic input. Variations in the half-width of the action potential could influence the probability of transmitter release at the terminals of the sympathetic neurons (Brock and Cunnane 1995).

Neurons projecting to similar targets in different parts of the body, such as the vasculature of the head, skin, muscles, or viscera, can be activated selectively in response to specific central commands (Jänig 1995; Jänig and McLachlan 1992). Therefore it may not be surprising that a vasoconstrictor neuron in a thoracic ganglion expresses a characteristic suite of ion channels different from that expressed by a vasoconstrictor neuron in the superior cervical or celiac ganglion. Such differences may be related to the patterns of synaptic activity that occur in particular functional pathways. Differences in the expression of ion channels also may be related to ganglion-dependent variation in the overall size of neurons (cf. Boyd et al. 1996; DeCastro et al. 1997), which in turn is related to the number of synaptic inputs they receive (Gibbins et al. 1998; Hume and Purves 1981; Purves and Hume 1981). What is more surprising is that a vasoconstrictor neuron and a neighboring pilomotor neuron in the same ganglion are likely to express a similar combination of channels. We have shown recently that the synaptic organization of sympathetic ganglia shows considerable potential for cross-talk between inputs in different functional pathways (Gibbins et al. 1998; Murphy et al. 1998). Consequently, the ganglion-specific differences in the electrical properties of sympathetic neurons also may include adaptations to allow for cross-talk between pathways in ways that are still poorly understood.


    ACKNOWLEDGMENTS

We thank Prof. W. Blessing and Dr. J. Oliver for the gift of E2210 antiserum to NPY and Assoc. Prof. R. Rush for the gift of antiserum to ChAT. We also thank Dr. D. Cameron, Assoc. Prof. J. Morris, and R. L. Anderson for helpful advice on the manuscript. S. Matthew is thanked for advice on the PEG immunohistochemical technique. We are grateful to G. Hennig for the NIH Image macros used for processing through-focus series of dye-filled neurons. P. Jobling is an Australian Postdoctoral Fellow of the National Health and Medical Research Council of Australia.

This study was supported by grants from the National Health and Medical Research Council of Australia, the Charles and Sylvia Viertel Foundation, the Clive and Vera Ramaciotti Foundation, the Flinders Medical Center Foundation, the Australian Research Council, and the Flinders University Research Budget.


    FOOTNOTES

Address for reprint requests: P. Jobling, Dept. of Anatomy and Histology, Flinders University of South Australia, GPO Box 2100, Adelaide, South Australia 5001, Australia.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8 December 1998; accepted in final form 15 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society