INTRODUCTION

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In the CNS, the concept of steroid-sensitive neurons and circuits is well established (Breedlove 1992; Cooke et al. 1998). In regions expressing steroid receptors, androgens and estrogens play an important role in the establishment of sexual dimorphisms and maintenance of some neuronal properties throughout adulthood. In contrast, evidence of steroid-sensitive circuits in the periphery has been reported only relatively recently. Of particular note are the autonomic circuits that regulate activity of male reproductive organs, which contain many lumbosacral preganglionic neurons, sympathetic and parasympathetic postganglionic neurons, and sensory ganglion neurons that are androgen-sensitive (Keast and Gleeson 1999; Keast and Saunders 1998; Watkins and Keast 1999). The best characterized of these are the autonomic ganglion cells lying in the pelvic ganglia, which comprise a mixture of sympathetic and parasympathetic neurons. These neurons innervate sexually dimorphic targets (reproductive organs) as well as those considered very similar or identical between genders (urinary bladder, lower bowel) (Keast 1999). Pelvic ganglia therefore contain the "final motor neurons" controlling pelvic viscera, having effects as diverse as penile erection, prostate secretion, and urinary bladder contraction.

Androgen sensitivity of the peripheral motor components of some pelvic autonomic reflexes, especially those involving male reproductive organs, has been suggested by various studies in rats. The best characterized is the penile erection reflex, where androgens appear to influence the chemistry, structure, and function of both central and peripheral nerve circuits (Giuliano et al. 1993; Hart 1967; Mills et al. 1992). Functional changes after puberty or castration have also been demonstrated in the autonomic nerve supply of the vas deferens smooth muscle (MacDonald and McGrath 1980, 1984). Both groups of observations suggest that at least some of the pelvic ganglion neurons are androgen-sensitive, and this has been further demonstrated by changes in transmitter (or neuropeptide) levels and decreased soma size after pre- or postpubertal castration (Hamill and Schroeder 1990; Keast and Saunders 1998; Keast et al. 2002).

Despite this evidence for androgen-sensitive neurons in the autonomic system, it is not known if the cellular physiological properties of these neurons are affected by testosterone at puberty or maintained by testosterone during adulthood. Moreover, animals are still growing at puberty, so there may be maturational changes unrelated to circulating androgens. Therefore the first aim of the current study was to determine if any maturational changes occur at puberty by electrophysiologically characterizing neurons and their synaptic inputs in juvenile rats (15-21 days) and adults (8-11 wk). The second aim of our study was to determine if testosterone plays a role in any of these maturational changes or in maintaining adult features by describing their electrophysiological properties after castrating animals as juveniles or adults. Finally, we aimed to identify which, if any, of the androgen-dependent properties of ganglion cells from animals castrated as juveniles could be modified by administration of testosterone in adulthood. We also compared the effects of treatments on sympathetic versus parasympathetic pelvic neurons as our previous anatomical studies had shown that of the two groups, sympathetic neurons change most markedly after castration or testosterone administration (Keast and Saunders 1998). All neurons were further categorized as tonic or phasic, depending on their firing pattern in response to prolonged depolarization (Cassell et al. 1986), to allow comparison with electrophysiological properties of other autonomic ganglion cell classes. However, it is not known if or how these firing properties relate to neurons with different targets or inputs, so we had no basis on which to predict a maturational or hormone effect on one or other group.

The outcome of these studies is the first description of maturational changes in pelvic ganglion cell excitability and firing properties and also the determination of which of these are androgen-dependent. There have been relatively few studies on the fundamental electrophysiological properties of these ganglion cells (Akasu et al. 1999; Tabatabai et al. 1986; Yoshimura and de Groat 1996; Zhu and Yakel 1997; Zhu et al. 1995), so our studies on intact ganglia also provide novel information of importance to those interested in autonomic ganglia in a broader sense.

	METHODS

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Animals and surgery

All animal procedures were approved by local institutional ethics committees and were in accordance with the Australian code of practice for the care and use of animals for scientific purposes of the National Health and Medical Research Council of Australia. Five groups of male outbred Wistar rats were studied (Fig. 1): juveniles (15-21 days), adults (8-11 wk of age), adult castrates (6-8 wk animals castrated and tissues removed 4-7 wk later), juvenile castrates (22 day animals castrated and tissues removed 5-7 wk later), and juvenile castrates with testosterone replacement (as per juvenile castrate group, then at 9 wk of age, treated weekly for 6-7 wk with testosterone enanthate, 10 mg/kg sc in sesame oil). Castrations were performed under anesthesia (60 mg/kg ketamine and 10 mg/kg xylazine ip) using a small lower abdominal incision and standard surgical procedures. Animals were monitored carefully in the postoperative period and showed no ill effects or obvious stress. Pelvic ganglia were removed from animals at the designated times after being anesthetized with sodium pentobarbitone (48 mg/kg ip) and then killed by cutting the carotid arteries. Dissected ganglia retained 1.5 mm of attached pelvic and hypogastric nerves and were stored for 4 h in physiological saline (see following text) until used for electrophysiological experiments. There was no change in membrane or synaptic properties during this time.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Time line of androgen exposure and tissue removal in each animal group. Five animal groups were studied. Number of weeks postnatal (2-16) are shown. , circulating androgens with all groups experiencing the perinatal androgen surge but not all groups having exposure to continued androgen exposure after puberty. ×, time of castration; , time of tissue removal. Testosterone administration is indicated (T, bottom). Further details of exact times for each procedure provided in text.

Electrophysiology

Pelvic ganglia were pinned flat in a small bath with a silicone polymer base and perfused with physiological saline equilibrated with 95% O₂-5% CO₂ and of the following constituents (mM): 151 Na⁺, 4.7 K⁺, 2.0 Ca²⁺, 1.2 Mg²⁺, 144 Cl, 1.3 H₂PO, 16.3 HCO, and 7.8 glucose. Solution was perfused at ~2 ml/min and maintained at 32°C during the experiments. Neurons were impaled with glass microelectrodes (60-110 M resistance) filled with 0.5 M KCl. Membrane potential recordings were made and signals amplified using an Axoclamp-2B (Axon Instruments, Burlingame, CA), digitized (ITC-16, Instrutech, New York) and analyzed using Axograph 4.6 (Axon). Neurons were included in the study if they had a membrane potential more negative than 40 mV, input resistance >30 M, action potential spike amplitude >50 mV with 5 mV overshoot.

Records were collected after a minimum of 15 min stable impalement. Input resistance (R_in) and the membrane time constant () were determined from the amplitude and time course of small (<10 mV) electrotonic voltage responses evoked from rest by current passed through the recording electrode and used to calculate the membrane capacitance (/R_in). The majority (>90%) of neurons were classified as "tonic" and "phasic" according to their firing response to a prolonged (200 ms) current pulse, using criteria described previously (Cassell et al. 1986). Briefly, at larger depolarizing steps, tonic neurons fired more than one action potential with further spikes being initiated with increasing stimulus size (Fig. 2A). Phasic neurons usually fired only once at the beginning of a depolarizing pulse, irrespective of the pulse amplitude (Fig. 2B). Further firing does not occur largely because of the presence of a substantial M current in these cells (Adams and Harper 1995; Cassell et al. 1986). Occasionally neurons were found that were intermediate between these two classes, and these were not studied further.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Classes of pelvic ganglion neurons. Neurons are categorised as tonic (A) or phasic (B), depending on whether or not they fire more than once in response to a prolonged depolarizing current pulse. Neurons are also categorized as sympathetic or parasympathetic, depending on whether they are synaptically activated by axons in the hypogastric nerve (C) or pelvic nerve (D), respectively. *, stimulus artifact. Neuron in C shows subthreshold excitatory postsynaptic potentials (EPSPs) of different amplitudes but identical time course. In D, only a single type of EPSP could be evoked. All neurons are from control adult animals. Calibration bars in A apply also to B, and those in C apply also to D.

Action potential parameters were compared between neuron classes and animal groups by directly depolarizing the neuron through the recording electrode with a 10-ms square current pulse, using the minimum pulse amplitude at which a spike could be evoked with each stimulus. The amplitude of the pulse was used as an indicator of neuronal excitability. A second indicator was used in tonic neurons, being the number of action potentials occurring during a 200-ms, 500-pA depolarizing pulse. Spike and afterhyperpolarization (AHP) amplitudes were taken as the differences from resting membrane potential. The AHP was further described by the best fit of one or more exponentials to the decay of the average of five AHPs recorded per neuron.

Hypogastric and pelvic nerves were stimulated by means of suction electrodes connected to an isolated stimulator (Digitimer, Welwyn City, UK). Neurons were stimulated with pulses of 1- to 20-V, 1-ms duration, at a minimum of 1 Hz. Stimuli were gradually increased in amplitude over a range to identify subthreshold excitatory postsynaptic potentials (EPSPs) and action potentials. EPSPs and action potentials could be blocked by hexamethonium (10 µM) or mecamylamine (1 µM; both purchased from Sigma) and could also be seen when neurons were hyperpolarized to 80 mV. Antidromic action potentials were infrequently seen, as very few pelvic ganglion cells project out of the hypogastric or pelvic nerves (Kepper and Keast 2000). However, they were easily distinguished from synaptic inputs by their rapid latency, rise time, and block by hyperpolarization. Neurons were classified as sympathetic or parasympathetic if action potentials could be elicited by hypogastric or pelvic nerve stimulation, respectively (Fig. 2, C and D). Neurons that could not be activated by either nerve were likely to have had their spinal connections damaged during the course of nerve dissection and were not tested further. A minority (<5%) of neurons were activated by both nerves and may have dual inputs as indicated previously by anatomical studies (Keast 1995). Previous electrophysiological studies have also shown these neurons to be rare or absent (Tabatabai et al. 1986).

All data presented were obtained from neurons where nerve-activated synaptic inputs were identified. The number of neurons studied of each class (sympathetic/parasympathetic or phasic/tonic) and in each animal group are shown in Table 1. An attempt was made to record from similar numbers of sympathetic/parasympathetic neurons in each animal group; therefore these numbers do not necessarily reflect the proportion of neurons of each class actually present in the ganglion. Summary data are presented as means ± SE, where n = number of neurons. Statistical analysis of the means of electrophysiological parameters were performed using ANOVA and Tukey's HSD test to make individual comparisons between groups. Where appropriate, comparisons were made between sympathetic and parasympathetic neurons (rather than tonic vs. phasic neurons) as previous anatomical studies suggested that the largest effect of castration occurred on sympathetic neurons (Keast and Saunders 1998). To avoid an unacceptable reduction in statistical power caused by decreased group sizes, we did not perform post hoc comparisons on means obtained from tonic/phasic subclass of sympathetic and parasympathetic neurons. Hierarchical log-linear procedures were used for analysis of frequency data in contingency tables (Sokal and Rolf 1995). All statistical analysis was performed using SPPS v10 (Mac) or v11 (Windows) or Statistica (Mac).

	RESULTS

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Maturational increases in membrane capacitance are steroid-dependent

Passive membrane properties were examined by using a series of small depolarizing and hyperpolarizing current steps to measure voltage responses near to the resting membrane potential. A statistical analysis was performed on the parameter means with treatment group (juvenile, adult, adult-castrate, juvenile-castrate, or juvenile-castrate + testosterone) and neuron class (sympathetic or parasympathetic) as the dependent variables. Table 2A is a summary of the mean resting membrane potential (V_m), input resistance (R_in) and membrane time constant () recorded in each treatment group. We found an overall difference in the means of V_m or R_in between the five treatment groups [V_m: F(4,172) = 2.7; R_m: F(4,172) = 2.6; P < 0.05; Table 2A] but not between sympathetic and parasympathetic neurons [V_m: F(1,172) = 3.0, R_in: F(1,172) = 0.0002; P > 0.05]. However, post hoc comparisons only found that adult castrate neurons were more hyperpolarized than juvenile neurons (52 ± 1 vs. 48 ± 1 mV; P < 0.01) and had a higher input resistance than adult neurons (106 ± 7 vs. 80 ± 5 M; P < 0.05). As the means of the juvenile and adult groups were not different, there appeared to be no maturational change of V_m and R_in over the age range studied.

There was an overall difference in the mean membrane capacitance (C_m) between the five treatment groups [F(4,172) = 15.9, P < 0.01] and also between sympathetic and parasympathetic neurons [F(1,172) = 15.3, P < 0.01] as well as a significant interaction between treatment group and neuron class [F(4,172) = 6.72, P < 0.01]. Post hoc comparisons (Table 2B) failed to identify differences in the mean C_m of parasympathetic neurons in the five experimental groups (P > 0.05, Tukey's HSD), which indicated an absence of significant growth after puberty as C_m is proportional to the neuronal surface area. In contrast, growth of sympathetic neurons did occur over the pubertal period (juveniles, 49 ± 4 pF; adults, 73 ± 4 pF; P < 0.01) and was steroid-dependent as the mean C_m in both the juvenile- and adult-castrate groups was smaller than adults but not different to juveniles (Table 2B). We also found the adult sympathetic phenotype was rescued when juvenile-castrates were administered testosterone as adults as the mean C_m of the juvenile-castrate + testosterone and adult group means were not different (P > 0.05, Tukey's HSD). These results are in agreement with previous anatomical studies in male rats which show that sympathetic pelvic ganglion neurons fail to grow to adult size after prepubertal castration and decrease in soma size following postpubertal castration (Keast and Saunders 1998). For comparison, the mean C_m of tonic and phasic neurons within each treatment group is also provided in Table 2B.

Only some maturational changes in active membrane properties are steroid-dependent

To study the effect of maturation on active membrane properties, we evoked action potentials from the resting membrane potential by injecting short (10 ms) depolarizing current pulses through the recording electrode. As shown in Table 3A, maturation increased the amplitude of both the action potential spike and AHP (i.e., juvenile vs. adult comparisons). However, neither effect was steroid-dependent as the means of each of the three castrate groups were not significantly different to the adult group (P > 0.05, Tukey's HSD).

The mean threshold current required to evoke action potentials (Table 3B) was different between treatment groups [F(4,172).= 3.3, P < 0.05] and was higher in sympathetic than parasympathetic neurons [206 ± 11 pA; cf. 141 ± 13 pA; F(4,172) = 14.9, P < 0.01]. However, we did not find a significant difference in the means of the juvenile and adult groups even though the difference was relatively large in sympathetic neurons (126 ± 44 pA; cf. 245 ± 31 pA, P > 0.05, Tukey's HSD). However, this could reflect the high proportion of tonic neurons in the sympathetic juvenile group (62%) when compared with the adult and castrate groups (range: 14 - 33%), as the threshold current in tonic neurons was consistently lower than in phasic neurons (Table 3B).

The basic tonic and phasic electrophysiological phenotypes routinely encountered in a wide variety of autonomic ganglia (Adams and Harper 1995) were identified in both sympathetic and parasympathetic pelvic ganglion neurons (Table 1). Juvenile and adult tonic neurons showed strong spike adaptation near the action potential threshold, but juvenile neurons were more responsive to the increase in stimulus current and achieved a higher maximum firing rate than adult neurons (Fig. 3, A, C, and D). This is also illustrated by the steeper slope of the action potential stimulus/frequency curve in juvenile tonic neurons (Fig. 3E). However, this was not a steroid-dependent maturational effect as it was not prevented or reversed by castration (i.e., the means of the castrate groups in Fig. 3D were not significantly different to the adult group: P > 0.05, Tukey's HSD).

View larger version (23K): [in this window] [in a new window]   Fig. 3. Firing properties of pelvic ganglion neurons. Ganglia from juvenile animals contain both tonic (A) and phasic (B) neurons. C: a tonic neuron from an animal castrated as a juvenile. Calibration bars in a apply also to B and C. D: number of spikes (means ± SE) evoked by a 500-pA pulse in tonic neurons from each animal group is shown. Juvenile tonic neurons fire more action potentials than tonic neurons in each of the other animal groups (P < 0.05, Kruskal-Wallace 2 =10, 4 df; nonparametric Tukey's post hoc test). E: the relationship between stimulus size and number of action potentials is shown for typical juvenile and adult tonic neurons.

The current underlying the AHP is the primary determinant of firing rate in tonic neurons found in rat pre- and paravertebral sympathetic ganglia (Wang and McKinnon 1995). We examined this indirectly by analyzing the time course of the AHP. In virtually all recordings, the decay of the AHP was fit by either one or two exponentials (Fig. 4, A and B; Table 3C), but in rare neurons, the presence of an additional slow (>200 ms) component prevented the AHP from being fit by summed exponentials (note: neuron numbers of some groups in Table 3C are smaller than in Table 1). The mean  in neurons with an AHP fit by a single exponential was not different across treatment groups [F(4,29) = 1.1, P > 0.05] or between sympathetic and parasympathetic neurons [F(2,29) = 1.03, P > 0.05]. When neurons with an AHP fit by two exponentials were analyzed with ₁ and ₂ as a within subject factor, an interaction between treatment group and input was identified [F_4,116 = 2.79, P < 0.05]. However, post hoc testing could only attribute this to an increase in the tau's of parasympathetic neurons in the juvenile-castrate group relative to the juvenile-castrate + testosterone group and to the sympathetic neuron groups (P < 0.01, Tukey's HSD).

View larger version (33K): [in this window] [in a new window]   Fig. 4. Properties of action potential afterhyperpolarizations (AHPs). A: juvenile pelvic ganglion neuron showing a short AHP with a decay that is represented by a single exponential. B: adult pelvic ganglion neuron showing a longer AHP with a decay that is described by 2 exponentials (see Table 3C for details). Calibration bar in a also applies to B. C: the proportions of neurons with or without a longer AHP (multiple tau) in each animal/neuron group. Juv, juvenile; Ad, adult; AdC, adult castrate; JuvC, juvenile castrate; JuvCT, juvenile castrate with testosterone replacement.

The overall proportion of neurons that exhibited an AHP fit by multiple exponentials increased markedly with maturation (44% in juvenile group compared with 92% in the adult group). The proportion of AHPs with multiple time constants was dependent on treatment group and synaptic input (i.e., contingency table best fit by a 3-way interaction log-linear model) (Sokal and Rolf 1995) as well as treatment group and neuron class. These relationships are illustrated in Fig. 4C and Table 4. The largest change associated with castration was in tonic neurons where juvenile castration reduced the proportion of neurons with multiple time constants as compared with the adults, which was not reversed by testosterone.

View this table: [in this window] [in a new window]   Table 4. Adjusted residuals (observed  expected frequencies)

"Strong" synaptic inputs increase with maturation but do not depend on steroid exposure

Recording EPSPs revealed neurons with "weak" and "strong" synaptic inputs, as described in other autonomic ganglion neurons (McLachlan and Meckler 1989). Weak inputs were identified when nerve stimulation evoked EPSPs that were subthreshold for eliciting action potentials, whereas strong inputs were identified when EPSPs always elicited an action potential regardless of the current strength used for nerve stimulation (or the membrane potential). On this basis, we could not determine if neurons with strong inputs also received additional weak inputs. Examples of neurons with weak or strong inputs are shown in Fig. 5. There was considerable variability between neurons in their number and types of inputs, which was apparent in all animal groups. In some neurons, the action potential AHP was substantially obscured by the large EPSP underlying the strong input (Fig. 5A), whereas in others the AHP was only partly contaminated by the EPSP (Fig. 5B). In neurons with weak inputs, most commonly only a single input could be resolved (Fig. 5, C and D), although some neurons with obvious temporally separated multiple inputs were occasionally seen (Fig. 5E).

View larger version (23K): [in this window] [in a new window]   Fig. 5. EPSPs and action potentials evoked by hypogastric or pelvic nerve stimulation. A-E: responses to hypogastric (A and B) or pelvic (C-E) nerve stimulation. A: an adult neuron where subthreshold events could not be evoked and the large depolarization triggering the action potential is clearly seen after the spike, obscuring the 1st part of the AHP. B: a juvenile neuron where subthreshold events could not be evoked and the AHP is partly obscured by the synaptic potential underlying the action potential. C and D: neurons from adult castrate animals. In C, a single type of subthreshold response can be evoked, whereas in D, the EPSP amplitudes (but not time courses) vary. E: an adult neuron with multiple subthreshold inputs, each of which can trigger an action potential. Calibration bars in a apply to all traces. F: the proportions of neurons in each animal group where strong inputs are present () or absent ().

Log-linear analysis identified a significant effect of treatment group and synaptic input on the proportion of neurons with strong inputs (i.e., data best fit by a 3-way interaction model) and also a significant effect of treatment and neuron class. These relationships are illustrated in Fig. 5F and Table 4. The comparison of sympathetic/parasympathetic neurons showed that strong inputs in the juvenile and adult groups were only encountered in sympathetic neurons where the proportion increased with maturation and further increased in all three castrate groups. This effect of castration was paradoxical as it was potentiated (not prevented) by testosterone. A separate comparison showed that strong inputs were more prevalent in phasic neurons and were increased by maturation. No effect of maturation was seen in tonic neurons but the increased proportion of strong inputs caused by castration was quite pronounced and not affected by replacement testosterone.

	DISCUSSION

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The present study is the first to examine the effects of puberty and androgen deprivation on electrophysiological properties of pelvic ganglion neurons. Our results have shown that many features of these neurons change substantially over the pubertal period but only a minority of these changes could be attributed to testosterone exposure (Table 5). Of the androgen-dependent maturational changes, the increases in neuronal size required testosterone for maintenance during adulthood but lengthening of the AHP did not. Together with previous studies on androgen actions on the nervous system, it appears that circulating androgens influence some autonomic reflexes by a combined action on central and peripheral components of the nervous system.

The most robust steroid-dependent electrophysiological effect of maturation was an increase in the membrane capacitance of sympathetic neurons. This indicated an increase in soma size and is in agreement with previous anatomical studies (Keast and Saunders 1998; Melvin and Hamill 1989; Melvin et al. 1988). Together the results showed that testosterone is required to induce growth of sympathetic pelvic ganglion neurons at puberty and to maintain this larger soma size during adulthood. The current study also found that soma growth could be induced by administration of testosterone to adult animals deprived of androgens at puberty, indicating that there was no critical period for induction of adult soma size.

The most interesting maturational change seen in pelvic ganglion neurons resulted in prolongation of the action potential AHP in a majority of adult animals. Whereas slightly >50% of juvenile neurons had an AHP fit by a single exponential, in adults this proportion fell to <10% of neurons. In the remaining neurons, the AHP was fit by two exponentials, the second component of which had similar kinetics to the current referred to as I_AHP or g_KCa1 commonly seen in adult autonomic ganglion neurons (Sah and Faber 2002). This current is typically carried by small conductance calcium-activated potassium (S_KCa) channels that are blocked by apamin. The maturational appearance of a prolonged AHP varied in its level of androgen dependence, depending on the neuron class. Tonic neurons generally showed the greatest level of androgen dependence in the development of a longer AHP, but in animals castrated as juveniles (which had a juvenile-type AHP), the longer (adult-type) AHP could not be restored by later administration of testosterone. This suggests that there is an earlier critical period for testosterone exposure to induce this AHP property in this neuronal class.

We did not investigate the mechanism underlying the change in AHP properties but the most likely candidates are: increased or new expression of calcium channel subtypes, a decrease in the calcium buffering capacity of the neuron, increased expression of S_KCa channels, or spatial redistribution of S_KCa channels relative to the calcium channels. Based on previous recording and simulation studies of other autonomic neurons, the major impact of prolongation of the AHP will be to prolong the interspike interval and decrease the rate of firing, although under certain circumstances, it could also impact on the control of accommodation (Wang and McKinnon 1995). Furthermore, many transmitters influence neuronal excitability by modulating the channels underlying the AHP (Adams and Harper 1995; Akasu 1992). Our results therefore indicate that as many pelvic ganglion neurons mature, they develop a "brake" on their maximal firing frequency plus a greater potential for transmitters to affect firing. It would be of interest to determine which particular functional classes of pelvic neuron show androgen-dependent AHP development.

The current study established that the most pronounced effects of puberty or castration were on sympathetic pelvic ganglion cells, but our immunohistochemical studies indicate that the majority of these do not express androgen receptors (Keast and Saunders 1998). However, we have recently found that many express estrogen receptors (Keast, unpublished observations), and so it is possible that estrogen could mediate these effects if ganglion cells express aromatase (the enzyme converting testosterone to estradiol). The alternative possibility is that steroids could affect ganglion neurons by direct actions on ion channels or second-messenger pathways (Schmidt et al. 2000; Woolley 1999). However, in rat pelvic ganglion neurons, acute testosterone administration has only a weak effect on resting membrane potential and EPSP amplitude and does not affect action potential properties (Felix et al. 2001). We therefore predict that genomic actions of estrogenic metabolites of testosterone underlie the steroid-dependent changes we observed in many neurons.

Other electrophysiological changes across the pubertal period were not steroid dependent (Table 5). This included an overall decrease in the maximum firing frequency that was associated with flattening of the stimulus-frequency curve of action potentials in adult neurons. Neither the spike nor the AHP of the action potential reached maximum amplitude until adulthood, possibly reflecting a postnatal increase in sodium and potassium channel expression as has been reported previously in the prenatal period for rat intracardiac autonomic neurons (Dourado and Dryer 1992; Gottmann et al. 1988).

Stimulation of preganglionic fibers innervating pelvic ganglion neurons showed that in adults most of the neurons with strong fiber inputs were sympathetic (i.e., innervated by the hypogastric nerve). There are structural and biochemical parallels to this data, in that preganglionic terminals associated with noradrenergic neurons are more dense and express higher levels of choline acetyltransferase than those surrounding cholinergic neurons (Keast et al. 1995). It is possible that this increased number of terminals leads to a greater amount of transmitter release and hence increased prevalence of strong fiber inputs by adulthood. Our electrophysiological studies did not indicate an androgen dependence of this postpubertal increase in strong fibers; however, we have shown previously that sympathetic preganglionic terminals decrease in volume after postpubertal castration (Watkins and Keast 1999). It is therefore possible that the growth and maintenance of sympathetic preganglionic terminal structure is hormone-dependent but that the amount of transmitter released at each neuron is not.

The present study provides new information on the properties of adult male rat pelvic ganglion neurons. It has been previously shown that these are very simple (mostly monopolar) neurons, which receive synaptic inputs from either the hypogastric or pelvic nerves (Tabatabai et al. 1986). We found in the rat that very few neurons receive input from both nerves, in contrast to pelvic ganglia in guinea pigs and cats, where many neurons receive input from both spinal levels (Blackman et al. 1969; Crowcroft and Szurszewski 1971; de Groat et al. 1979). It had been previously reported that all neurons in male rat pelvic ganglia are activated by at least one large synaptic input and that most cells had additional subthreshold inputs (Tabatabai et al. 1986). We also found some differences between sympathetic and parasympathetic neurons regarding prevalence of weak and strong inputs. In summary, it does not appear that all pelvic ganglion neurons in rodents act as simple relay stations. This is further supported by immunohistochemical studies that have localized various neuropeptides in the terminals of preganglionic neurons supplying the pelvic ganglia, particularly those innervating parasympathetic neurons (Keast 1994). Such substances could potentially influence transmitter release, ganglion cell excitability, or firing properties of pelvic ganglion neurons.

Further heterogeneity in pelvic ganglion cells was revealed by firing patterns in response to long current pulses. Previous studies have identified phasic (rapidly adapting) and tonic (slowly adapting) neurons in autonomic ganglia, which vary in their prevalence between ganglia and stage of development (Anderson et al. 2001; Cassell et al. 1986; Wang and McKinnon 1995). Both groups of neurons are thought to express A-type K channels, but phasic neurons express a higher level of M-type K channels than tonic neurons. We found both types in sympathetic and parasympathetic neurons in male rat pelvic ganglia, but from our sampling cannot comment on whether or not there is a maturational or hormone-dependent change in expression of M-type K channels. However, if it exists it is unlikely to be major because we had no difficulty in recording from phasic neurons in any animal group.

A type of phasic neuron commonly found in many autonomic ganglia has a particularly long AHP carried by a second class of calcium-activated K channel, g_KCa2 (Cassell and McLachlan 1987; Jobling et al. 1993). Neurons of this type comprise a minority (~5%) of mouse pelvic ganglion neurons (Rogers et al. 1990) and were encountered only rarely in our studies on the rat. The relative absence of these channels in rat and mouse pelvic sympathetic neurons adds to the growing list of features that are unique to sympathetic neurons in pelvic ganglia (Keast 1999) that also includes their structural simplicity, greater distance from target organs, and greater resistance to noradrenaline-depleting drugs compared with other sympathetic neurons. An additional unique feature of pelvic sympathetic neurons is the common expression of T-type calcium channels (Zhu et al. 1995).

Conclusions

We have shown that many electrophysiological properties of male rat pelvic ganglion neurons do not fully mature within the first couple of postnatal weeks but continue to change until adulthood. The most interesting result is the appearance of a longer component of the AHP postnatally in many neurons. Most of the maturational changes occur independently of circulating androgens, despite the expression of androgen receptors by many neurons. We have also demonstrated a greater heterogeneity in pelvic ganglion cells and their synaptic inputs than previously reported and this suggests that many pelvic ganglion neurons in rats do not function as simple relay stations.