Differential effects of intrathecal thyrotropin-releasing hormone (TRH) on perineal reflexes in male rats

Physiology& Behavior,Vol. 61, No. l, pp. 57-63, 1997 Copyright© 1996ElsevierScienceInc. Printedin the USA.All rightsreserved 0031-9384/97 $17.00 + .00

4,

PII S0031-9384(96) 00317-4

ELSEVIER

Differential Effects of Intrathecal ThyrotropinReleasing Hormone (TRH) on Perineal Reflexes in Male Rats G R E G O R Y M. H O L M E S , .1 R I C H A R D C. R O G E R S , t J A C Q U E L I N E C. B R E S N A H A N * A N D M I C H A E L S. B E A T T I E * ~:

*Department of Cell Biology, Neurobiology and Anatomy, "~Department of Physiology and ~:Department of Neurosurgery, The Ohio State University, 333 West Tenth Avenue, Columbus, OH 43210 USA Received 22 January 1996; accepted 4 June 1996 HOLMES, G. M., R. C. ROGERS, J. C. BRESNAHAN AND M. S. BEATTIE. Differential effects of intrathecal thyrotropinreleasing hormone (TRH) on perineal reflexes in male rats. PHYSIOL BEHAV 61( 1) 57-63, 1997.--The effects of thyrotropinreleasing hormone (TRH) on the sexual and defecatory reflexes regulated by pudendal motoneuronswere investigated.Intrathecal TRH (10 #1 volume; 0.0, 0.01, 1.0, or 100 #M concentration) at lumbosacral spinal segments (L4-S1) in acute preparations produced a dose-dependentincrease in external anal sphincter (EAS), but not bulbospongiosus(BS), electromyographic(EMG) activity. Intraspinal (L6) injection of 100 /zM TRH (1 /zl/micropipette), significantly increased EAS EMG activity in acute preparations. Electromyographic activity of the BS muscle was unchanged. All doses of intrathecal TRH ( 10 #1 volume; 0, 10, 50, 100, or 500 #M concentration) in awake animals significantly reduced the proportion of responders to a penile reflex test. Subsequently, all measures of penile reflexes were significantlyreduced. Glans tumescence and defecation bouts before or during penile reflex testing were unaffected by intrathecal TRH as were indices of behavioral and motor hyper-reactivity analogous to that produced by serotonin.These data indicate that pudendal motoneurons,in the dorsomedialnucleus, are differentiallyregulated by neuropeptides present in the lumbosacral spinal cord. Copyright© 1996 Elsevier Science Inc. Brainstem

Descendingsystems

Lumbosacralreflexes

AN INHIBITORY mechanism for the descending control of pudendal motoneurons has long been assumed [see (33) for review]. Neurotransmitters implicated in this process include serotonin (5-HT) ( 3 0 - 3 2 ) and GABAB agonists (6,25). Supraspinal sources of this inhibition may include caudal brainstem nuclei, including the n. paragigantocellularis (nPGi) (28,29) and n. raphe obscures (nRO) (12,14,15 ). These sites have been identified by lumbosacral injections of the retrograde tracer fluorogold (29,30). More recently, specific projections to the lumbosacral cord have been clearly delineated following injections of the anterograde tracer fluororuby into the nRO (12). This technique was combined with retrograde fluorescent labeling of the bulbospongiosus (BS) and external anal sphincter (EAS) muscles and has shown that nRO fibers ramify in close apposition to both BS and EAS motoneurons. Terminals of nRO projections in the lumbosacral cord have been shown to co-localize 5-HT and the neuropeptides thyrotropin releasing hormone (TRH) and substance P (2,45), In vitro applications of TRH to motoneurons produce a pronounced and lengthy excitation of somatic (4,46) and autonomic (3) motoneurons. Intrathecal infusions of pharmacological doses of TRH

Penile erection

Continence

Defecation

in awake animals results in profound motor and cardiac activation (22,43). Although the classic neurotransmitters have been the subject of extensive research on the regulation of sexual behavior [ see (5) for review], studies on peptidergic control are comparatively scarce. The regulation of sexual function by TRH is limited to the study of intrathecal injection of TRH on copulatory behavior ( 10,13 ). The following experiments sought to determine whether or not TRH inputs to the lumbosacral cord may be involved in pudendal motoneuron function. We investigated the effects of TRH on acute electromyographic (EMG) activity in both the BS and EAS muscles in anesthetized animals receiving intraspinal or intrathecal (IT) injections. Furthermore, we investigated the effect of TRH upon sexual reflexes in the chronically catheterized awake animal. GENERAL METHODS Adult male Long-Evans rats (350-450 g, Harlan, Indianapolis, IN), were initially group housed in hanging plastic tubs (35 × 42 x 21 cm) in an AAALAC approved animal care facility.

Reprint requests should be addressed to Dr. Gregory M. Holmes, Dept. of Cell Biology, Neurobiology & Anatomy, The Ohio State University, 4072 Graves Hall, 333 West Tenth Avenue, Columbus, OH 43210-1239 USA. E-mail: [email protected] 57

58 Food and water were freely available. Following their arrival, most males were tested for ex-copula penile reflexes ( see below). After the third preoperative reflex test, males demonstrating a consistent level of responsiveness were implanted with intrathecal catheters. Two postoperative reflex tests were administered and the animals were randomly assigned to drug groups. Animals not implanted with intrathecal catheters were included with nonscreened males in the groups utilized for acute experiments.

Surgery and Materials Male rats receiving intrathecal catheters were anesthetized with an intra-peritoneal (IP) injection of ketamine HC1 (80 mg/ kg) and xylazine HCI (10 mg/kg). Cannulation was performed using the modified procedure of LoPachin et al. (26). Stretched PE-10 (polyethylene tubing) cannulas were sized to the individual animal so that the catheter tip terminated at roughly spinal L5 (approximately 10.5 cm). Cannula placement was confirmed by postmortem visual examination. Following cannulation, the suspensory ligament of the penis was removed to facilitate retraction of the penile sheath (see 6). All wounds were sutured with sterile silk and sprayed with topical antibiotic. Animals were administered antibiotic (gentamycin 1 mg/kg, IP) prior to surgery and for 3 consecutive days afterward, and were continuously monitored for deficits in gait or sensory fields. The experimental recording of EMG activity after IT infusions used animals previously implanted with IT catheters. Animals used for EMG recording were anesthetized with urethane (1 g/kg, IP). Following procedures described previously (16), EMG electrodes were placed into both the EAS and the lateral portion of the ventral BS muscle. The wire was exteriorized and connected to 2 Grass P-15® preamplifiers connected in series for a gain of 10,000X. Signals were saved on magnetic tape (Vettor 3000A, Rebersburg, PA) for later computer (Experimenters Workbench, Datawave Systems Inc., Longmont, CO) playback and analysis. Subjects receiving intraspinal infusions were anesthetized with urethane and laminectomized to expose spinal L 5 - L 6 . A multibarreled glass micropipette array (4 micropipettes aligned horizontally; tips spaced 1 mm apart; each tip 20/~ diameter) in which the pipette tips were beveled (WPI, model #1350) to an equal length, was lowered 1300 #m into the dorsomedial nucleus (a.k.a. spinal nucleus of the bulbocaveruosus). TRH was applied ( 1 #l/barrel) using a pressure injection technique described elsewhere (37,38).

Behavioral Testing and Data Collection All testing was conducted during the day portion of the day/night cycle. During drug administration, relative degree of reactivity to infusion was assessed using the following scale: 0, no reaction; 1, tail curvature and/or mild alterations in gait; 2, all of the above plus arching of the back; 3, the above plus mild vocalization; 4, the above with extreme alterations in gait or posture; and 5, the above with pronounced vocalization and struggling. Immediately following drug administration, animals were placed in a supine position and restrained with strips of masking tape. The penile reflex test began immediately following retraction of the preputial sheath. Data was collected on occurrence of erection of the penile body or glans, amount of baseline glans distention at moment of sheath retraction, number of flips (anteroflexions of the penile body), and seminal emissions. Defecation bouts were counted separately during drug infusion or reflex testing. With the aid of a data acquisition and analysis program (17), the total number of occurrences of each event, latency to first penile response, latency to first glans response, and the number of clusters

HOLMES ET AL. (defined as a series of consecutive events separated by less than 15 s) were determined. Tests were terminated 10 min after the first penile response or 20 rain after sheath retraction if no responses occurred.

Drug Administration Thyrotropin-releasing hormone (TRH, Bachem, Torrance, CA) was dissolved in phosphate buffefred saline (PBS) prior to the onset of administration. Doses (10, 50, 100, and 500 #M) were aliquoted into color-coded vials and stored at 72°C. Doses were thawed immediately prior to infusion. The IT catheter was connected to a segment of 30 ga. hypodermic tubing that was fitted into a length of PE-10 tubing connected to a 500 #1 syringe (BAS #MD-0050, West Lafayette, IN). The syringe was mounted in a microinfusion pump (BAS #MD- 1001 ) that infused drugs at a rate of < 4 #l/min. Drug injections (10 #1) were followed by a PBS flush (10 #1).

Statistical Analysis Proportional data (the ratio of animals responding with muscle EMG or reflexive penile erections following TRH vs. vehicle) were compared by Fisher's exact probability test. Changes in EMG firing rate in response to intrathecal TRH (Experiment 1 ) and penile erection data (Experiment 3) were statistically analyzed using the analysis of variance (ANOVA) and Tukey post hoc tests (SYSTAT, Evanston, IL). Electromyographic changes following intraspinal TRH (Experiment 2) were compared by a paired t-test. Latency data for penile erection were converted from s to tenths of min prior to parametric analysis. In all cases, significance was assumed when p < 0.05. EXPERIMENT 1 EXPERIMENTAL DESIGN

Nine adult males with chronic IT catheters were prepared for EMG recording as described above. Animals with stable, noisefree electrodes in both the EAS and BS muscles (n = 6) continued in the experiment. Each animal received an IT injection of vehicle followed by TRH (0.01, 1.0, and 100 #M), thus serving as its own control. Baseline EMG activity was recorded for 5 min prior to drug infusion. Following baseline measurements, 10 #1 of PBS was pushed through the cannula into the subarachnoid space. This was followed 5 min later by TRH infusions. The 100#M dose of TRH was included due to its efficacy in initiating anorectal contractions in an acute preparation utilizing a manometric technique (18). After 5 min of monitoring EMG activity, the TRH dose was pushed through the catheter with 10 #1 flush of PBS. Changes in EAS and BS EMG were monitored for an additional 20-min period. Due to the brevity of the TRH response, only the first 2 min of a test period were used to compute the average EMG firing rate for each condition. RESULTS

Intrathecal injections of vehicle, 0.01, or 1.0 #M TRH did not induce changes in EAS EMG activity (0 of 6 responders, see example, Fig. 1 ), and 100 #M TRH stimulated EAS EMG activity in a majority of animals (5 of 6; p < 0.05). Unlike the EAS, the BS muscle remained quiescent following all doses of TRH (Fig. 1 ). As seen in Table 1, the average firing rate of the EAS muscle following 100 #M TRH was also significantly increased [F(3,20) = 9.32, p < 0.01].

TRH A N D P E R I N E A L R E F L E X E S

59

/

I.

. . . . . . . . . . .

t ::-x;:l;;~ -' ;'-

BS

~1._, . t t . . . . . . . . . . . . . . . . . - , . - , [. . . . . . . . . . .

TABLE 1 FIRING RATE (Hz) OF PER1NEAL MUSCLES FOLLOWING 1NTRATHECAL 100 ,aM TRH

L ~,

O.OM EAS

JJlk_liltl .l .

BS

.

.

J _i.~__.

.

TRH Dose (#M)

ExternalAnal Sphincter EMG

BulbospongiosusEMG

0.0 0.01

1.59 _ 0.95 2.25 _ 0.75

0.13 -- 0.10 0.00

1.70 + 1.50

0.00

13.91 _+ 4.30*

0.00

II I

i...I

.i.,

,. . . . .

,

..J

. . . .

1.0

~

100.0

lOnM EAS

Results are shown as mean ± SEM. Comparisons made between TRH dose and vehicle IANOVA and Tukey post hoc test, *p < 0.01). ....'.

.

.

.

.

.

. . . . . . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . .

BS

.

....

.

I

.

.

.

. . . . . . . . . . ..... , . i ........

HI . ,I

IIi

Following baseline measurements, 1 #1 of 100 #M TRH was pressure injected through each pipette. Rate of infusion varied from 1 5 - 3 0 s across each barrel. Electromyographic activity was monitored and analyzed as in Experiment 1.

lpM

RESULTS

EAS

. -L

BS

. . . ~. 7._-. -7_-i~ . . .122

:.

; ,

,,-;.:

;

" .

.

--J.i 22-_ .'-?~_.L~.__il -_~-?_/.L2; 2__

100~M EAS FIG. 1. Representative electromyographic activity of bulbospongiosus (BS) and external anal sphincter (EAS) muscles after intrathecal infusion of vehicle (0.0 #M) or TRH (10 nM, 1 #M, 100 #M). The minor activity seen in the BS following vehicle infusion may be artifactual, because this muscle is never seen to be tonically active in the acute or awake preparation (unpublished observations). Note that, in this example, the EAS does not display any levels of activity except after 100 #M infusions of TRH. Calibration: 2 mV, 0.5 s.

EXPERIMENT 2 Data from Experiment 1 suggest a differential effectiveness of TRH upon the neural circuitry innervating the EAS and BS muscles. However, intrathecal application disperses infusate throughout the cord and is, with regard to spinal circuits, nonspecific; therefore, more precise application of pharmacological agents is needed. The following experiment sought to limit the range of TRH application to the immediate vicinity of the EAS and BS motoneurons comprising the dorsomedial nucleus. A multibarrel pipette array was used to stereotaxically apply 100 #M TRH to the entire L6 segment of the dorsomedial nucleus where the BS and EAS motoneurons are intermingled. Because the BS was not affected by IT TRH in Experiment 1, the changes in EAS motoneuron output (as measured by E M G ) were compared to the output of the adjacent BS motoneurons to control for nonspecific activation of motoneurons due to mechanical displacement by the infusate.

Experimental Design Five adult males were prepared for E M G recording from the EAS and BS muscles as described above. Baseline E M G activity was recorded from both muscles 5 min prior to drug infusion.

Consistent with the findings of Experiment 1, the BS muscle did not display any level of E M G activity following TRH. Unlike the previous experiment, the EAS was not spontaneously active in this group of animals. As seen in Fig. 2, the EAS was electromyographically activated by 100 # M TRH. In each case observed, E M G activity ceased approximately 5 rain (range 4 . 3 - 7 . 5 min) after the termination of the infusion. When compared to the BS muscle (0 of 5 responders), intraspinal TRH significantly increased the proportion of animals displaying EAS E M G activity (5 of 5, p < 0.01) as well a., significantly increasing the firing rate I t ( 4 ) = 3.004, p < 0.05] of the EAS muscle (Table 2). EXPERIMENT 3 The data from Experiments 1 and 2 suggest that the motoneurons innervating the EAS muscle, alone, are responsive to TRH. What remains unclear is whether the BS motoneurons are merely unresponsive to, or are actively inhibited by TRH. In addition, be-

F-AS E M G

J

:' 7[Ir'i~', ~'llrll'"l'"r'.l:rl

.

i

50 45

i

40

t

'~

30

25 O

£

:.

20

i

10 5

?

0 0

2

i..' 4

I

6 TRH

8

10

12 TIME

14

16

18

20

22

24

(min)

FIG. 2. Following the intraspinal injection of TRH ( 1 #1 volume, 100 #M concentration), the firing rate of the EAS muscle increases greatly over baseline levels seen prior to injection. Three-second epochs of raw EMG (EAS EMG) are displayed above their respective bins. Each bin represents the number of spikes above baseline noise within a 10-s period. Injection of TRH began 5 min after recording began (denoted by arrow). Recording continued for 20 min after the start of the injection.

60

HOLMES ET AL.

TABLE 2 FIRING RATE (Hz) OF PERINEAL MUSCLES FOLLOWING INTRASPINAL INJECTION OF 1 #L OF 100 #M TRH External A n a l Sphincter EMG Preinfusion baseline 1.0 #1 of 100.0 #M TRH

0.01 ± 0.005 3.54 ± 1.17"

20e-///z

~'///, /11//

Bulbospongiosus EMG 0.00 0.00

Results are shown as mean ± SEM. Comparisons made between EAS and BS muscle following 100/~M TRH dose (paired t-test, *p < 0.05). cause TRH is known to project to both sympathetic and parasympathetic motoneurons, it remained possible that TRH might activate mechanisms of erection that are under autonomic regulation. The following experiment explored the effects of IT TRH in the awake animal.

Experimental Design Thirteen adult male rats that responded positively on 2 of 3 preoperative penile reflex tests were implanted with IT catheters as described above. Animals were randomly assigned to a latin square injection paradigm (0.0, 10, 50, 100, and 500 # M TRH followed by 10 #1 PBS) for a within-subjects design. Animals received 1 injection per week over 5 weeks. Immediately upon completing the infusion, animals were tested for penile reflexes as described above. RESULTS Three animals were excluded from analysis due to complications resulting from the implanted catheter. Intrathecal TRH reduced the proportion of animals displaying penile reflexes across all doses ( 0 . 0 / z M = 8 of 10; 10 # M = 2 of 10; 50 # M = 1 of 10; 100 # M = 0 of 10; 500 # M = 2 of 10). Each dose was significandy different from the vehicle control (p < 0.025). As a result of the reduced number of responders, all measures of penile erection were significantly reduced. For example, the latency to the first penile response was significantly increased F ( 4 , 9 ) = 7.080,p < 0.003, across all dose levels (Fig. 3). Likewise, the total number of glans erections, F ( 4 , 9 ) = 8.692, p < 0.001, penile body erections, F ( 4 , 9 ) = 10.571, p < 0.0001, penile body flips, F ( 4 , 9 ) = 5.158, p < 0.0022, and total penile events per cluster, F ( 4 , 9 ) = 9.287, p < 0.0001, were significantly reduced across all dosages (Table 3). Seminal emissions occurred with such rarity (2 instances across all drug doses) as to preclude statistical analysis. Baseline glans tumescence at the time of retraction (Table 3), hyperreactivity and defecation rates

E E 15>O Z u.I '~ 1 0 kU {,9 Z 0 D. n~

4"/'//#

~///;

"///,~ ".,'//,5

,'////

I1

12/o[ 10

I "11/~1i l 50

~V/tnt

IZlln~

i 100

i 500

T R H (raM)

FIG. 3. The response latency to penile erection after intrathecal TRH. Proportion of males responding within the allotted 20 rnin are nested within each bar. Response latency was significantly elevated by each dose of TRH (* = p < 0.05). (Table 4), both during the infusion of TRH and during the penile reflex test, were not significantly different from control. GENERAL DISCUSSION The results of these experiments present evidence of TRH regulating somatic mechanisms of fecal continence and sexual reflexes in the male rat. Unlike the bulbospongiosus ( B S ) muscle, the external anal sphincter ( E A S ) muscle displays transient, lowlevel spontaneous firing. Intrathecal and intraspinal application of 100 # M TRH elevated EAS muscle firing in acute, anesthetized animals. Lower doses of TRH (0.01 and 1.00/.tM) did not affect EAS firing. The BS muscles, which are involved in the somatic components of penile erection, did not display electromyographic changes following either intraspinal or intrathecal TRH infusion. However, in the awake animal, similar doses ( 10, 50, 100, 500 # M T R H ) potently reduced the number of males exhibiting penile erection. Of those animals that did respond following TRH application, the number of penile reflexes per response cluster was significantly reduced. These data suggest that the EAS and BS motoneurons, although comingled within the same spinal nucleus, are discretely and differentially regulated by separate neural circuits. Thyrotropin-releasing hormone is a potent neuropeptide that facilitates firing rates of both somatic and autonomic motoneurons (3,4,46). The probable source of TRH input to the lumbar cord is in the bralnstem. Radioimmunoassay and immunocyto-

TABLE 3 SELECTED

MEASURES

OF PENILE ERECTION

FOLLOWING

INTRATHECAL

TRH

TRH Dose (#M)

Glans Erections

Penile Body Erections

Flips

Events/Cluster

Glans Baseline

0.0 10.0 50.0 100.0 500.0

15.55 _+ 5.27 0.56 ± 0.39* 0.27* 0.10" 1.46 ± 0.64*

4.64 _ 1.48 0.09* 0.00" 0.00" 0.00"

5.41 --+ 2.62 0.18" 0.00" 0.00" 0.00"

3.79 _ 1.03 0.25* 0.30* 0.00" 0.45 ± 0.32*

1.27 2.18 1.36 1.73 2.00

+_ 0.33 _+ 0.42 _ 0.49 ± 0.45 ± 0.27

Results are shown as mean ± SEM. Means without SEM indicate one or fewer occurrences of that behavior. Comparisons made between TRH dose and vehicle (ANOVA and Tukey post hoc test, *p < 0.001).

TRH AND PERINEAL REFLEXES

61

TABLE 4 HYPERREACTIVITY AND DEFECATION RATES FOLLOWING INTRATHECAL TRH

TRH Dose (/~M)

Hyperreactivity

Defecation Rate (TRH-infusion)

Defecation Rate (Post TRH-infusion)

0.0 10.0 50.0 100.0 500.0

1.27 ___0.33 1.55 ___0.41 1.82 +_ 0.38 1.55 --_0.58 1.73 ± 0.38

0.00 0.09 0.64 ± 0.28 0.09 ± 0.51 0.36 ___0.24

0.28 +_0.19 0.27 0.18 ± 0.21 0.00 0.09

Results are shown as mean +_SEM. Means without SEM indicate one or fewer occurrences of that behavior. Comparisons made between TRH dose and vehicle (ANOVA and Tukey post hoc test). chemical studies have identified medullary TRH neurons ( 1,2,7,11,21,36,45,47), some of which also indicated colocalization of TRH with 5-hydroxytryptamine (5 -HT) ( 1,2,7,11,21,45 ). Based upon previous evidence (19,20), Arvidsson et al. (2) suggested that 5-HT/TRH fibers innervating ventral horn motoneurons (i.e., lamina IX) originate in nucleus raphe obscurus (nRO) and nucleus paragigantocellularis (nPGi), and dorsal horn fibers originate in n. raphe magnus (nRM). We have gathered strong anatomical evidence supporting nRO and nPGi inputs to the ventral lumbar cord (12). Medullary TRH neurons diffusely project to the ventral horn of the spinal cord (2,7,21,45) and most sympathetic nuclei within the intermediolateral cell column ( 1,11,36,47 ). Previous data demonstrated that 5-HT positive terminals make synaptic contact with motoneurons innervating perineal musculature in the rat (24) and cat (44). Additionally, there is evidence that 5-HT inhibits sexual reflexes in the rat ( 3 0 - 3 2 ) and that this inhibition may arise from the nPGi (28,29). Spinal cord transection eliminates virtually all ventral horn 5-HT/TRH fibers, but not dorsal horn 5-HT or separate TRH staining fibers (2), further supporting the hypothesis that colocalized 5-HT/ TRH fibers are part of a descending circuit. This does not, however, completely rule out local 5-HT and/or TRH circuits from influencing perineal reflexes. In comparison to the BS muscle, the role of TRH in regulation of the EAS is relatively straightforward. Unlike the BS muscle, the small size of the EAS does not lend itself to chronic EMG monitoring of activity in the awake behaving animal. Thus, we must infer from the acute data and observation of defecation rates that TRH is part of a mechanism that maintains, at the very least, closure of the anal orifice. Although no decrease in the defecation rates of awake animals was seen following intrathecal TRH, discrete intraspinal application of TRH to the EAS motoneuron pool was effective in elevating EAS EMG activity. The low level of EAS EMG following intraspinal TRH is most likely due to the small number of EAS motoneurons affected by the infusion. We surmise that less than 10% of the EAS motoneuron pool was exposed to TRH, thus limiting the number of activated motor units available for recording. These data, coupled with previous findings that the same intrathecal dose of TRH facilitates contractions of the anorectal canal (18), provide evidence that TRH may be involved in maintaining fecal continence. Previous research (10,13) has reported a slight, but significant, increase in mount and intromission latencies after a single high dose of TRH. This increase in latency was substantially elevated by a combined bolus of TRH + 5-HT. Because intrathecal 5-HT, administered alone, increased only intromission latencies (42) and 5-HT and TRH colocalize in the spinal cord

(1,2,7,11,21,45) there exists a conceptual incongruity with the 5-HT alone and 5-HT + TRH paradigms. Namely, inhibition of perineal function through the activation of local or descending spinal circuitry should have little effect on mount latency. Reduced genital "integrity" in the form of penile deafferentation (23,41) or erectile function by denervation or excision (34,39,40) does not appear to affect sexual arousal (as measured by latency to mount), though the ability to successfully copulate is affected. Additionally, IT TRH activated what was casually described as hypokinesia and hypertonia of some locomotor systems. It is very likely that these global effects competed, at least in part, with the ability of the male to execute the proper motor components of erection and resulted in the additional deficits observed when 5-HT and TRH were combined. No detectable effect of TRH on BS motoneurons was seen in the electrophysiological studies. The effects of intrathecal TRH on neural circuits involved in sexual function were only apparent in the awake animal. As is the case with all intrathecal applications of pharmacological agents, there are multiple neural circuits potentially influenced by the dose of TRH. These circuits include somatic and autonomic efferents, spinal interneurons, and perineal afferents. In addition, each one of these neuronal pools may be regulated by spinally and/or supraspinally derived TRH, the latter presumably being of bulbospinal origin. The possibility that TRH is directly inhibitory on BS motoneurons seems unlikely in light of the fact that TRH consistently appears to be an excitatory neuropeptide (3,4,46). Unfortunately, the normally quiescent nature of BS motoneurons in both the awake and anesthetized animal does not allow one to distinguish between TRH having no effect or actual inhibition. The absence of a TRH analogue further confounds experimental activation of BS motoneurons. There is some evidence suggesting that interneurons may play a role in regulating motoneurons in the DM. Spinal interneurons in the vicinity of BS motoneurons have been labeled following wheat germ aglutinin injections to the BS muscle (8,35). The DM has been shown to contain GABA-immunoreactive terminals (27), thus providing anatomical support for the possibility of inhibitory interneurons regulating BS motoneuron function. The facilitation of inhibitory interneurons by TRH is, at best, speculative because there is little electrophysiological evidence available concerning the effects of TRH on this class of cell. However, preliminary electrophysiological evidence (Holmes et al., unpublished observations) suggests the possibility of a longacting, TRH-sensitive cell in the DM nucleus that does not appear to be a pudendal motoneuron. We cautiously speculate that this cell may be an inhibitory interneuron projecting onto the BS motoneuron. The long-term activation of inhibitory interneurons by TRH may explain the pronounced inhibitory effects of TRH upon penile erection in the awake animal. Reflexive penile erection is dependent upon sensory input via the dorsal penile nerve (41). A previous report (6) has suggested GABAergic inhibition of primary afferents as one potential route for the inhibition of reflexive erection. Support for this hypothesis was based, in part, on the presence of lamina I-III GABA-immunoreactive cell bodies (27). More recently, evidence has been presented demonstrating the co-existence of GABA and TRH in the lamina II-III region of the dorsal horn (9). Long-term TRH activation of GABAergic inhibition within the dorsal horn is consistent with the observations in Experiment 3. Injections of fluorogold aimed at pudendal motoneurons in the L6 spinal cord of rats principally labeled cells in raphe magnus, raphe pallidus, nPGi and nRO (29,30). Unfortunately, there was no indication as to how much tracer may have spread to the dorsal horn. It has been shown that dorsal horn TRH fibers originate in raphe magnus

62

H O L M E S ET AL.

and ventral h o r n T R H fibers originate in n P G i a n d n R O ( 2 ) . A l t h o u g h T R H i n h i b i t i o n may, indeed, result f r o m s t i m u l a t i o n o f dorsal h o r n i n h i b i t o r y m e c h a n i s m s , this T R H / G A B A m e c h a n i s m still does not a c c o u n t for the presence, or function, o f T R H fibers p r o j e c t i n g directly to D M m o t o n e u r o n s . In c o n c l u s i o n , e v i d e n c e f r o m these studies and others ( 1 2 , 1 5 ) i m p l i c a t e T R H in the m a i n t e n a n c e o f fecal continence. This control appears to be due to d e s c e n d i n g bulbospinal input. Results f r o m the effects o f T R H on sexual reflexes, w h i c h are g o v e r n e d b y cells within the same spinal n u c l e u s as t h o s e r e g u l a t i n g the E A S , are more complex, but suggest that the spinal circuits i n v o l v e d in penile erection are

also affected b y TRH, a l t h o u g h more indirectly than those i n v o l v i n g E A S reflexes. ACKNOWLEDGEMENTS This study was supported by NIH research grant NS-#31193 awarded to M. S. B., R. C. R., J. C. B. and G. M. H. and Paralyzed Veterans of America Spinal Cord Research Foundation grant SCRF-1254 awarded to G. M. H., M. S. B. and R. C. R. Additional funds were provided by NIH grant NS-10165 awarded to M. S. B. and J. C. B. The authors wish to thank Dr. G..E. Hermann for comments during the preparation of the manuscript and J. H. Komon, Jr., and M. J. Van Meter for extensive technical assistance.

REFERENCES 1. Appel, N. M.; Wessendorf, M. W.; Elde, R. P. Thyrotropin-releasing hormone in spinal cord:coexistence with serotonin and with substance P in fibers and terminals apposing identified preganglionic sympathetic neurons. Brain Res. 415:137-143; 1987. 2. Arvidsson, U.; Cullheim, S.; Ulfhake, B.; Bennett, G. W.; Fone, K. C. F.; Cuello, C.; Verhofstad, A. A. J.; Vissar, T. J.; Htkfelt, T. 5-hydroxytryptamine, substance P, and thyrotropin-releasing hormone in the adult cat spinal cord segment L-7: Immunohistochemical and chemical studies. Synapse 6:237-270; 1990. 3. Backman, S. B,; Henry, J. L. Effects of substance-P and thyrotropinreleasing hormone on sympathetic preganglionic neurones in the upper thoracic intermediolateral nucleus of the cat. Can. J. Physiol. Pharmacol. 62:248-251; 1984. 4. BEdard, P. J.; Tremblay, L. E.; Barbeau, H.; Filion, M.; Maheux, R.; Richards, C. L.; DiPaolo, T. Action of 5-hydroxytriptamine, substance P, thyrotropin-releasing hormone and clonidine on motoneutone excitability. Can. J. Neurol. Sci. 14:506-509; 1987. 5. Bitran, D.; Hull, E. M. Pharmacological analysis of male rat sexual behavior. Neurosci. Biobehav. Rev. 11:365-389; 1987. 6. Bitran, D.; Miller, S. A.; McQuade, D. B.; Leipheimer, R. E.; Sachs, B. D. Inhibition of sexual reflexes by lumbosacral injection of a GABA-B agonist in the male rat. Pharmacol. Biochem. Behav. 31:657-666; 1989. 7. Bowker, R. M.; Westlund, K. N.; Sullivan, M. C.; Wilber, J. F.; Coulter, J. D. Descending serotonergic, peptidergic and chotinergic pathways from the raphe nuclei: A multiple transmitter complex. Brain Res. 288:33-48; 1983. 8. Collins, W. F.; Erichsen, J. T.; Rose, R. D. Pudendal motor and premotor neurons in the male rat:A WGA treansneuronal study. J. Comp. Neurol. 308:28-41; 1991. 9. Fleming, A. A.; Todd, A. J. Thyrotropin-releasing hormone- and GABA-like immunoreactivity coexist in neurons in the dorsal horn of the rat spinal cord. Brain Res. 638:347-351; 1994. 10. Hansen, S.; Svensson, L.; Htkfelt, T.; Everitt, B. J. 5-hydroxytryptamine-thyrotropin releasing hormone interactions in the spinal cord: Effects on parameters of sexual behavior in the male rat. Neurosci. Lett. 42:299-304; 1983. 11. Helke, C. J.; Sayson, S. C.; Keeler, J. R.; Charlton, C. G. Thyrotropin-releasing hormone-immunoreactive neurons project from the ventral medulla to the intermediolateral cell column:Partial coexistence with serotonin. Brain Res. 381:1-7; 1986. 12. Hermann, G. E.; Holmes, G. M.; Bresnahan, J. C.; Beattie, M. S.; Rogers, R. C. Descending projections of the nucleus raphe obscurus (nRO); Possible anatomical basis of role in lumbosacral reflexes. Soc. Neurosci. Abstr. 21:1200; 1995. 13. Htkfelt, T.; Fried, G.; Hansen, S.; Holets, V.; Lundberg, J. M.; Skirboll, L. Neurons with multiple messengers--distribution and possible functional significance. Prog. Brain Res. 65:115-137; 1986. 14. Holmes, G. M.; Bresnahan, J. C.; Rogers, R. C.; Hermann, G. E.; Beattie, M. S. Differential characteristics of rat dorsomedial motoneurons. Soc. Neurosci. Abstr. 20:791; 1994. 15. Holmes, G. M.; Bresnahan, J. C.; Rogers, R. C.; Hermann, G. E.; Beattie, M. S. Physiological and behavioral effects of serotonin or thyrotropin releasing hormone on rat dorsomedial motoneurons. Soc. Neurosci. Abstr. 21:1201; 1995.

16. Holmes, G. M.; Chapple, W. D.; Leipheimer, R. E.; Sachs, B. D. Electromyographic analysis of male rat perineal muscles during copulation and reflexive erections. Physiol. Behav. 49:1235-1246; 1991. 17. Holmes, G. M.; Holmes, D. G.; Sachs, B. D. An IBM-PC based data collection system for recording rodent copulatory activity and for general event recording. Physiol. Behav. 44:825-828; 1988. 18. Holmes, G. M.; Rogers, R. C.; Bresnahan, J. C.; Beattie, M. S. Thyrotropin-releasing hormone (TRH) and CNS regulation of anorectal motility in the rat. J. Auton. Nerv. Syst. 56:8-14; 1995. 19. Holstege, J. C.; Kuypers, H. G. J. M. Brainstem projections to himbar motoneurons in rat-I, an ultrastructural study using autoradiography and the combination of autoradiography and horseradish peroxidase histochemistry. Neurosci. 21:345-367; 1987. 20. Holstege, J. C.; Kuypers, H. G. J. M. Brainstem projections to spinal motoneurons: an update. Neuroscience 23:809-821; 1987. 21. Johansson, O.; Htikfelt, T.; Pemow, B.; Jeffcoate, S. L.; White, N.; Steinbusch, H. W. M.; Verhofstad, A. A. J.; Emson, P. C.; Spindel, E. Immunohistochemical support for three putative transmitters in one neuron: Coexistence of 5-hydroxytryptamine, substance P- and thyrotropin releasing hormone-like immunoreactivity in medullary neurons projecting to the spinal cord. Neuroscience 6:1857-1881; 1981. 22. Johnson, J. V.; Fone, K. C. F.; Havler, M. E.; Tulloch, I. F.; Bennett, G. W.; Marsden, C. A. A comparison of the motor behaviours produced by the intrathecal administration of thyrotropin-releasing hormone and thyrotropin-releasing hormone analogues in the conscious rat. Neuroscience 29:463-470; 1989. 23. Larsson, K.; Stdersten, P. Mating in male rats after section of the dorsal penile nerve. Physiol. Behav. 10:567-571; 1973. 24. Leedy, M. G.; Bresnahan, J. C.; Beattie, M. S. Ultrastructural analysis of TH and 5-HT inputs to the SNB of the male rat. Soc. Neurosci. Abstr. 17:469; 1991. 25. Leipheimer, R. E.; Sachs, B. D. GABAergic regulation of penile reflexes and copulation in rats. Physiol. Behav. 42:351-357; 1988. 26. LoPachin, R. M.; Rudy, T. A.; Yaksh, T. L. An improved method for chronic catheterization of the rat spinal subarachnoid space. Physiol. Behav. 27:559-561; 1981. 27. Magoul, R.; Onteniente, B.; Geffard, M.; Calas, A. Anatomical distribution and ultrastructural organization of the GABAergic system in the rat spinal cord. An immunocytochemical study using antiGABA antibodies. Neuroscience 20:1001-1009; 1987. 28, Marson, L.; List, M. S.; McKenna, K. E. Lesions of the nucleus paragigantocelhilaris alter ex copula penile reflexes. Brain Res. 592:187-192; 1992. 29. Marson, L.; McKenna, K. E. The identification of a brainstem site controlling spinal sexual reflexes in male rats. Brain Res. 515:303308; 1990. 30. Marson, L.; McKenna, K. E. A role for 5-hydroxytryptamine in descending inhibition of spinal sexual reflexes. Exp. Brain Res. 88:313-320; 1992. 31. Marson, L.; McKerma, K. E. Serotonergic neurotoxic lesions facilRate male sexual reflexes. Pharmacol. Biochem. Behav. 47:883888; 1994.

TRH AND PERINEAL REFLEXES

32. Mas, M.; Zahradnik, M. A.; Martino, V.; Davidson, J. M. Stimulation of spinal serotonergic receptors facilitates seminal emission and suppresses penile erectile reflexes. Brain Res. 342:128-134; 1985. 33, Meisel, R. L.; Sachs, B. D. The physiology of male sexual behavior. In: Knobil, E.; NeiU, J., eds. The physiology of reproduction. New York: Raven Press; 1993:3-105. 34. Monaghan, E. P.; Breedlove, S. M. The role of the bulbocavernosus in penile reflex behavior in rats. Brain Res. 587:178-180; 1992. 35. Peshori, K. R.; Erichsen, J. T.; Collins, W. F., III. Differences in the connectivity of rat pudendal motor nuclei as revealed by retrograde transneuronal transport of wheat germ agghitinin. J. Comp. Neurol. 353:119-128; 1995. 36. Poulat, P.; Sandillon, F.; Marlier, L.; Rajaofetra, N.; Oliver, C.; Privat, A. Distribution of thyrotropin-releasing hormone in the rat spinal cord with special reference to sympathetic nuclei:A light-and electron-microscopic immunocytochemical study. J. Neurocytol. 21:157-170; 1992. 37. Rogers, R. C. An inexpensive picolter-volume pressure-ejection system. Brain Res. Bull. 15:669-671; 1985. 38. Rogers, R. C.; Hermann, G. E. Dorsal medullary oxytocin, vasopressin, oxytocin antagonist, and TRH effects on gastric acid secretion and heart rate. Peptides 6:1143-1148; 1985. 39. Sachs, B. D. Role of striated penile muscles in penile reflexes, and induction of pregnancy in the rat. J. Reprod. Fert. 66:433-443; 1982. 40. Sachs, B. D. Potency and fertility: Hormonal and mechanical causes and effects of penile actions in rats. In: Balthazart, J.; PrOve, E.; Gilles, R., eds. Hormones and behaviour in higher vertebrates. Berlin: Springer-Verlag; 1983:86-110.

63

41. Sachs, B. D.; Garinello, L. D. Hypothetical spinal pacemaker regulating penile reflexes in rats: Evidence from transection of spinal cord and dorsal penile nerves. J. Comp. Physiol. Psychol. 94:530535; 1980. 42. Svensson, L.; Hansen, S. Spinal monoaminergic modulation of masculine copulatory behaviors in the rat. Brain Res. 302:315-321; 1984. 43. Tan, D-P.; Tsou, K. Differential motor and blood pressure effects of intrathecal oxytocin and TRH in the rat. Peptides 6:1191-1193; 1985. 44. Tashiro, T.; Satoda, T.; Matsushima, R.; Mizuno, N. Convergence of serotonin-, enkephalin- and substance P-like immunoreactive fibers on single pudendal motoneurons in Onuf's nucleus of the cat: light microscope study combining the triple immunocytochemical staining technique with the retrograde HRP-tracing method. Brain Res. 481:392-398; 1989. 45. Ulfhake, B.; Arvidsson, U.; Cullheim, S.; H0kfelt, T.; Brodin, E.; Verhofstad, A.; Visser, T. An ultrastructural study of 5-hydroxytryptamine, thyrotropin-releasing hormone and substance P-immunoreactive axonal boutons in the motor nucleus of spinal cord segments L7-S 1 in the adult cat. Neuroscience 23:917-929; 1987. 46. White, S. R.; Crane, G, K.; Jackson, D. A. Thyrotropin-releasing hormone (TRH) effects on spinal cord neuronal excitability. Ann. NY Acad. Sci. 553:337-350; 1989. 47. Wu, W.; Elde, R.; Wessendorf, M. W. Organization of the serotonergic innervation of spinal neurons in rats III. Differential serotonergic innervation of somatic and parasympathetic preganglionic motoneurons as determined by patterns of co-existing peptides. Neuroscience 55:223-233; 1993.