Effects of Acute and Chronic Midthoracic Spinal Cord Injury on Neural Circuits for Male Sexual Function. II. Descending Pathways

Charles H. Hubscher and Richard D. Johnson

Department of Physiological Sciences, College of Veterinary Medicine and University of Florida Brain Institute, University of Florida, Gainesville, Florida 32610-0144


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hubscher, Charles H. and Richard D. Johnson. Effects of Acute and Chronic Midthoracic Spinal Cord Injury on Neural Circuits for Male Sexual Function. II. Descending Pathways. J. Neurophysiol. 83: 2508-2518, 2000. In normal animals, microstimulation of the medullary reticular formation (MRF) has two effects on efferent neurons in the motor branch of the pudendal nerve (PudM). MRF microstimulation depresses motoneuron reflex discharges (RD) elicited by dorsal nerve of the penis (DNP) stimulation and produces long latency sympathetic fiber responses (SFR). The midthoracic spinal location of these descending MRF-PudM projections was studied electrophysiologically using a variety of acute and chronic lesions. Chronic lesions, in 27 mature male rats, included dorsal (DHx) or lateral (LHx) hemisections or moderate/severe contusions (Cx) at spinal level T8. Behavioral data (sexual reflex latency, bladder voiding) obtained throughout the recovery period revealed a significant impairment of urogenital function for the DHx and severe Cx groups of animals. Microstimulation-induced PudM-RDs and PudM-SFRs, obtained in terminal electrophysiological experiments 30 days postinjury in the same 27 rats (urethan-anesthetized), were tested for a combined total of 1,404 bilateral MRF sites. PudM-RD was obtained for LHx and moderate Cx groups of animals but not for DHx or severe Cx groups. PudM-SFRs were obtained for LHx, DHx (although significantly weakened) and moderate Cx groups but not for those having received either an over-DHx or a severe Cx injury. PudM responses also were tested for 6 MRF sites in six intact control rats both before and after various select acute spinal cord lesions. For MRF sites producing a robust PudM-RD and PudM-SFR, acute bilateral lesions confined to the dorsolateral quadrant (DLQ) eliminated the PudM-RD but failed to eliminate PudM-SFRs. A deeper lesion encompassing additional white matter located dorsally in the ventrolateral quadrant (VLQ) was necessary to eliminate PudM-SFRs. Overall, these electrophysiological results provide evidence for descending projections conveying information between MRF and the lower thoracic/lumbosacral male urogenital circuitry within the DLQ and the dorsal-most aspect of VLQ at the midthoracic level of spinal cord. The alterations of supraspinal projections observed after chronic injury are likely of important clinical significance for functional recovery in cases of clinically incomplete spinal cord injury at midthoracic spinal cord.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The sensory limb of segmental reflexes for erection and ejaculation (Johnson and Halata 1991; Johnson and Murray 1992; McKenna and Nadelhaft 1986; Nunez et al. 1986; Steers et al. 1988), produce bilateral (crossed and uncrossed) reflex facilitation of motoneurons in the pudendal motor branch (PudM) (Johnson and Hubscher 1998; McKenna and Nadelhaft 1989). The synaptic efficacy of dorsal nerve of the penis (DNP) afferents and associated interneurons onto PudM motoneurons is reduced progressively in rats with chronic spinal cord injuries (Johnson 1995). The enhanced erectile and depressed ejaculatory reflexes observed in rats 30 days after midthoracic transection (Hart and Odell 1981; Mas et al. 1987; Sachs and Garinello 1979) is likely due to a loss of supraspinal influences. Thus DNP facilitation of ejaculation may require a brain stem loop, much like the control of micturition (deGroat et al. 1981). Previous studies show that medullary reticular formation (MRF) neurons, particularly those within the lateral paragigantocellular reticular nucleus (LPGi), are likely involved in this supraspinal loop (Marson and McKenna 1990; Marson et al. 1993; Tanaka and Arnold 1993; Yells et al. 1992). Many MRF neurons respond to bilateral DNP stimulation (Hubscher and Johnson 1996). Bilateral electrical stimulation of specific DNP-responsive MRF subregions, particularly in and medial to LPGi, produce a reflex (DNP-elicited) depression of pudendal motoneurons (PudM-RD) (Johnson and Hubscher 1998) and produces a sympathetic fiber response (PudM-SFR) (Johnson and Hubscher 2000).

Using select acute and chronic lesions, our current focus is the investigation of the midthoracic white matter location of projections comprising the supraspinal loop between the lumbosacral ejaculatory circuitry and MRF. The location of the ascending spinal projections is presented in a companion article (Hubscher and Johnson 1999b). In the present paper, the location of descending projections within the midthoracic white matter is presented along with behavioral data (sexual reflex latency, bladder voiding) obtained during the 30 day postinjury recovery period.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Spinal cord lesions

Twenty-seven male Wistar rats received either a dorsal (DHx) or lateral (LHx) hemisection, or contusion (Cx) injury (severe or moderate) at spinal level T8. These same animals also provided data for two other articles (Hubscher and Johnson 1999a,b). Terminal electrophysiological experiments were performed 30 days postinjury (see following text).

Lesion surgeries were performed under aseptic conditions using ketamine (80 mg/kg ip)/xylazine (10 mg/kg ip) anesthesia. A long acting antibiotic (Flo-Cillin: 0.5 ml; Fort Dodge Laboratories, Fort Dodge, IA) was administered before surgery. The spinal cord was exposed at T8 via removal of the overlying T7 vertebral lamina. Hemisections (as well as acute lesions) were made with microdissecting scissors; contusions were performed by rapid compression with a concave probe having a radius/size matching the overlying lamina (see Hubscher and Johnson 1999b for further details).

After closure and recovery, the animals were encouraged to eat with apple slices (1st postoperative day). Each animal was tended to three times daily throughout the recovery period. Daily logs were kept to note the status of bladder function. In the initial postsurgical period, the distended bladder was expressed manually by gentle squeezing every 8 h, lasting until "reflex" voiding onset. Initiation of "reflex" micturition, which required only gentle pressure or stroking of the perineum, was performed until "automatic" micturition occurred; i.e., the bladder periodically voided through contact with the cage bedding (without caretaker assistance).

Sexual reflexes were tested once during the second and once during the third weeks postinjury, using standard procedures (see Sachs and Garinello 1979; Sachs and Meisel 1988). Each rat was placed in dorsal recumbency, and the penis was exteriorized by manually retracting the prepuce. To document the behavior, each rat was videotaped for 20 min. The latency to the first penile reflex (penile erection or dorsiflexion) (Sachs and Garinello 1979) was measured. Statistical comparison of injury groups was performed (Mann-Whitney U test).

Surgical preparation for terminal electrophysiological experiments

Six male Wistar rats at ~90-120 days of age were used in the acute lesion experiments. The remaining 27 rats were subjected to a terminal electrophysiological experiment after a 30-day chronic lesion. All animals were anesthetized with urethan (1.2 g/kg ip) and prepared/maintained as previously described (Hubscher and Johnson 1999b). The head was clamped in a stereotaxic holder and the dorsal surface of the brain stem exposed (see Hubscher and Johnson 1996). Specially fabricated bipolar silicon cuff microelectrodes were placed bilaterally around the dorsally exposed pelvic nerve, DNP, and motor branch of the pudendal (i.e., PudM - also known as deep perineal) nerves (see experimental setup in Fig. 1 of Hubscher and Johnson 1996).

MRF stimulation protocol

Two glass-coated platinum-plated tungsten microelectrodes with a 20-µm exposed tip (Merrill and Ainsworth 1972), attached to a stepping microdrive, were set for bilateral penetration of the MRF (see Hubscher and Johnson 1996; Johnson and Hubscher 1998). Each terminal experiment included initial MRF neuronal recordings in four rostral tracks to assess the ascending circuitry from the male genitalia (see Hubscher and Johnson 1999a,b). Responses were then measured in left and right PudMs to unilateral and bilateral microstimulation at an average of 52 MRF sites located caudal (2,400-3,200 µm rostral to obex) to the MRF recordings. These stimulation sites comprised the portion of the microstimulation matrix in intact controls that produced robust PudM-RD and PudM-SFRs (Johnson and Hubscher 1998, 2000).

A monopolar stimulating current (20 µA) was passed through the microelectrodes either individually or simultaneously to unilaterally or bilaterally microstimulate MRF regions. The current intensities were subsequently either decreased or increased in rostral tracks only to 10 and 30 µA to just cause contraction of the facial muscles (i.e., when the electrode was next to the facial nucleus) (Zhuo and Gebhart 1991). Reflex discharges recorded simultaneously in both PudMs in response to the DNP test stimulus (single 0.1-ms duration pulses of 30-50 µA set at 2.5 times reflex threshold) were recorded before and after conditioning stimulation of the brainstem (50-ms train of 200 pulses/s, 0.2-ms pulse duration, given at a train rate of 0.5 trains/s) to determine if that particular MRF site was capable of modulating the polysynaptic pudendal segmental circuitry. Based on previous work (Johnson and Hubscher 1998), the short conditioned test interval (<80 ms) reflex depression (Pud-RD) consisted of a decrease in amplitude and an increase in latency of the DNP-elicited reflex. In addition, direct long latency (>150 ms) activation of sympathetic postganglionic fibers (PudM-SFR) in response to conditioning stimulation alone was determined for each MRF site (Johnson and Hubscher 2000).

Three matrix rows extended from 400 to 2,000 µm laterally from midline. Tandem (i.e., left and right) electrode tracks covered a 900-µm depth zone in ventral MRF (2,400-3,300 µm below the brain stem surface at depth increments of 300 µm) (Paxinos and Watson 1997). In the six intact animals, a single robust PudM-RD and PudM-SFR responsive uni/bilateral region of MRF was found and tested both before and after a variety of acute spinal lesions at T8.

Data collection and analysis

At each microstimulation site in the assay, two measures were taken for the short-latency PudM-RD and the long-latency PudM-SFR. The first measure was the determination of the presence or absence of responses to bilateral MRF stimulation. If responses were present on one or both PudMs, the effect of unilateral MRF microstimulation was tested on both the crossed and uncrossed PudM. The second measure was the magnitude of response (if present). Each PudM response was assigned to one of three strengths. For the short-latency PudM-RD, responses were categorized (Johnson and Hubscher 1998) as being either slight (barely visible reduction), partial (easily visualized reduction), or complete (approximately a flat line). For statistical analyses and comparison of the data, these response strengths were later assigned values on an ordinal scale of 1, 2, and 3, respectively (a 0 score was assigned if no responses to bilateral MRF microstimulation were detected). For the long-latency PudM-SFR, responses were categorized as being either slight (barely detectable above baseline), moderate (easily discriminated above baseline), or strong (very large burst) (see Johnson and Hubscher 2000). These response strengths also were assigned values (1, 2, and 3, respectively) on an ordinal scale (0 for no response).

Histology

At the end of the experiment, the animal was euthanized with an anesthetic overdose and perfused transcardially with 0.9% saline followed by 10% formalin. The block of brain stem tissue containing the stimulation sites was removed and stored in 10% formalin/30% sucrose. Stimulation sites were visualized in 50-µm vibratome sections stained with cresyl violet and reconstructed under light and dark field illumination (Paxinos and Watson 1997). The perfused spinal cord was analyzed histologically (paraffin sections) for confirmation of acute or chronic lesion extent (see photomicrograph examples in Fig. 3 of Hubscher and Johnson 1999b). Spinal cord tissue sections were stained with both luxol fast blue and cresyl violet (Kluver-Barrera stain).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Acute control spinal cord lesions

In each of six unoperated rats, a microstimulation MRF site producing both a robust PudM-RD and PudM-SFR (unilateral and bilateral MRF effects on both PudMs) was tested before and after selective acute unilateral midthoracic spinal lesions. The results from these experiments are summarized in Fig. 1. A unilateral lesion encompassing ventral portions of the dorsal lateral quadrant (DLQ) eliminated left and right PudM-RDs produced by conditioning microstimulation of the lesion-side MRF (for all the different lesions shown in sections 2 and 3 of Fig. 1A) but failed to eliminate left and right PudM-RD produced by microstimulating the intact-side MRF. These results support the existence of both a crossed and uncrossed descending projection below the level of the lesion and only an uncrossed projection above (for intact controls). The example in Fig. 2 illustrates this point; i.e., the reflex depression is maintained from the right MRF (left dorsal quadrant lesion) to both PudMs but lost from the left MRF (lesion side) to both PudMs.



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Fig. 1. Difference in location and level of crossing between medullary reticular formation (MRF) descending pathways to reflex depression and sympathetic fiber response in the pudendal motor branch (PudM-RD and PudM-SFR) spinal circuits. Summary diagrams from 6 normal animals, each showing the effects of several acute lesions at midthoracic spinal cord on PudM-RD (A) and PudM-SFR (B) due to uni/bilateral microstimulation of the MRF. Each shading pattern represents 1 reconstructed lesion. Presence or loss of responses postlesion as evidenced from recordings of the motor branch of the pudendal nerve (PudM: uni/bilaterally) is illustrated with an arrow or an arrow and ×, respectively. Note that the PudM-RD pathway exerts its crossed effects caudal to T8, whereas the PudM-SFR pathway crosses rostral to T8. L, left; Pud, pudendal; R, right.



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Fig. 2. Effects of an acute left dorsal quadrant lesion (large dashed outline at a depth adjacent to the central canal, as shown in Fig. 1A, middle) on MRF modulation of dorsal nerve of the penis (DNP)-elicited PudM-RD (25 averaged integrated discharges). Conditioning stimulus electrodes were in the caudal portion of lateral paragigantocellular reticular nucleus (LPGi; 1,200 µm from midline at a depth of 3,000 µm) in MRF and represented the stimulus matrix site at which maximal modulatory effects were seen. DNP was stimulated bilaterally as the test stimulus (*). Top: bilaterally recorded PudM-RD to the test stimulus alone. MRF conditioning stimuli (see up-arrow ) were given unilaterally (middle 2 traces) and bilaterally (bottom). A "partial" depression of the DNP-elicited discharge in both PudMs can be seen in the bottom 2 traces. Note the elimination of the modulatory effects from the left LPGi demonstrating that in the normal animal, the MRF-PudM-RD modulatory pathway is uncrossed at the level of T8.

In contrast, a unilateral lesion eliminated PudM-SFRs on the lesion side from left and right MRF. PudM-SFRs were maintained (on the intact side) from both the left and right MRF, supporting the existence of a crossed and uncrossed descending projection above the level of lesion and only an uncrossed projection below (for intact controls). In contrast with PudM-RD, a deeper unilateral lesion encompassing additional white matter in the dorsal-most aspect of the ventrolateral quadrant (VLQ) was necessary to eliminate PudM-SFRs (i.e., for all the different lesions shown in sections 2 and 3 of Fig. 1B).

Chronic spinal cord lesions

A total of 27 male rats received one of three types of chronic midthoracic spinal cord lesions; i.e., either a DHx, LHx, or Cx. Bladder function data and sexual reflex response latency data (time to first penile reflex - penile erection or dorsiflexion) obtained during the 30-day recovery period in the awake behaving animal are presented in Fig. 3. In addition, there were significantly more penile reflex clusters and penile dorsiflexion (flips) in the DHx and Cx animals as compared with the LHx animals.



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Fig. 3. Summary of postsurgical sexual reflex and bladder function behavioral assessments in spinal cord-injured animals. Animals with lateral hemisections (LHx; n = 7) exhibited patterns resembling intact animals and the statistical differences between the LHx group and the most severely and bilaterally injured groups (dorsal hemisection: DHx; n = 6 and severe contusion: Cx; n = 6) are indicated by a (P < 0.05). Vertical bar values represent the mean ± SE. Note the significantly shorter sexual reflex latency and longer bladder recovery time in the bilaterally injured groups. In addition, the initiation of "automatic" bladder voiding patterns was significantly longer in Cx animals when compared with DHx animals (b).

In terminal electrophysiological experiments performed 30 days postinjury, the effects of various chronic T8 lesions on MRF microstimulation-elicited effects on PudMs (RD and SFR) was determined. A summary of responses is provided in Table 1 and Fig. 4.


                              
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Table 1. Effects of chronic midthoracic spinal cord injury on descending connections between MRF and the pudendal nerve motor circuitry



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Fig. 4. Summary of bilateral microstimulation data eliciting PudM-RD (top) and PudM-SFR (bottom) in animals with no lesion (see Table 1), chronic LHx, chronic DHx, and chronic Cx injury. Each bar represents the mean response strength (3-point intensity scale) at bilateral MRF sites (equidistant from midline) in the medial to lateral plane (400-800, 1,200, and 1,600-2,000 µm from midline) and rostral to caudal plane (3,200, 2,800, and 2,400 µm from obex). Data from left and right PudMs are combined. Note that the area of focus moves from medial to lateral as the electrodes move caudally. This pattern was maintained in the 2 hemisection groups, but the strength of the response was attenuated particularly in animals with a DHx. Shaded region in the brain stem sections show the region of microstimulation. Gi, nucleus reticularis gigantocellularis; GiV, Gi ventralis; 7n, facial nucleus.

CHRONIC CONTUSION INJURIES. In six of the rats with a severe Cx injury (see photomicrograph example in Fig. 3A of Hubscher and Johnson 1999b), no PudM-RD or PudM-SFRs were found (see Table 1 and Fig. 4). In four rats with moderate Cx (see photomicrograph example in Fig. 3B of Hubscher and Johnson 1999b), no overall significant difference was found with intact controls for PudM-RD (see Table 1). However, a difference was observed for the one animal with the least amount of spared tissue, i.e., a narrow unilateral rim remaining in the left lateral funiculus. Whereas a partial PudM-RD was elicited from the left side of the MRF, only a slight PudM-RD was elicited from right MRF (mainly on the crossed left PudM---see Fig. 5). For Pud-SFR, a significant difference (chi 2, P < 0.01) was found between the moderate Cx and intact control groups, although this difference was nowhere near that obtained for the severe Cx versus intact control group (see Table 1).



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Fig. 5. MRF modulation of averaged integrated DNP-elicited PudM-RD in an animal with a moderate chronic contusion injury (spared rim in left lateral funiculus, see inset). Conditioning stimulus electrodes were in the caudal portion of LPGi (1,600 µm from midline at a depth of 3,300 µm) in MRF and represented the stimulus matrix site at which maximal modulatory effects were seen. Note that the partial reflex depression was elicited mostly from the left LPGi; only a slight reflex depression was elicited from the right LPGi. Recording details and symbols as in Fig. 2.

CHRONIC DORSAL HEMISECTIONS. In four animals with complete DHx (see photomicrograph example in Fig. 3C of Hubscher and Johnson 1999b), MRF microstimulation-induced PudM-RD was eliminated bilaterally, except in one case where some sparse sparing produced a slight effect (see Fig. 6). In five animals with complete DHx, the PudM-SFR was significantly weakened, but not eliminated (see Fig. 4). This outcome appeared dependent on the presence or absence of the dorsal-most aspect of VLQ. Note that for one animal, PudM-RD data were not obtained. In four cases with incomplete DHx, PudM-RD and PudM-SFRs were not eliminated (see typical examples in Fig. 7, A and B, respectively).



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Fig. 6. MRF modulation of averaged integrated PudM-RD in an animal with a chronic dorsal hemisection with some sparsely distributed spared white matter (denoted by shaded portions of insert). Conditioning stimulus electrodes were in the caudal portion of LPGi (1,600 µm from midline at a depth of 3,300 µm) in MRF and represented the stimulus matrix site at which maximal modulatory effects were seen. Note that only a very slight reflex depression was elicited mostly from the right LPGi. Recording details and symbols as in Fig. 2.



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Fig. 7. MRF modulation of averaged integrated PudM-RD (A) and PudM-SFR (B) in an animal with a chronic under dorsal hemisection (see inset). Stimulus electrodes were in the caudal portion of LPGi (1,600 µm from midline at a depth of 3,300 µm) in MRF and represented the stimulus matrix site at which maximal modulatory effects were seen. Note that in A, the partial reflex depression was elicited mostly from the left LPGi and in B, the SFR was elicited from both LPGis (strong SFR on left PudM only). Recording details and symbols as in Fig. 2.

CHRONIC LATERAL HEMISECTIONS. In eight animals with chronic LHx (see photomicrograph example in Fig. 3D of Hubscher and Johnson 1999b), no overall significant differences in PudM-RD and PudM-SFR were found with intact controls. The rostral-to-caudal, medial-to-lateral shift in PudM-RD response strength was still evident after LHx (see Fig. 4, top) as was the increase in response strength as the electrode shifted ventrally (Johnson and Hubscher 1998). However, important side-to-side differences were found. For PudM-RD, responses from conditioning microstimulation of the lesion-side MRF were either substantially weak or absent to both PudMs (see example in Fig. 8A). The presence of some weak responses from the lesion-side MRF to both PudMs supports the existence of both a crossed and uncrossed descending projection above as well as below the level of a chronic lesion.



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Fig. 8. MRF modulation of averaged integrated PudM-RD (A) and PudM-SFR (B) in 2 animals with a chronic lateral hemisection (see insets). Stimulus electrodes were in the caudal portion of LPGi (1,600 µm from midline at a depth of 3,300 µm) in MRF and represented the stimulus matrix site at which maximal modulatory effects were seen. Note that in both A and B, the MRF effects were elicited mostly from the left LPGi, although for B, this example is typical for lateral matrix tracks only (see text). Recording details and symbols as in Fig. 2.

For PudM-SFR, the majority of responses produced by microstimulating the intact-side MRF remained for both PudMs, supporting the existence of a crossed and uncrossed projection below the level of a chronic lesion. Responses produced by microstimulating the lesion-side MRF medially were also present for both PudMs but were substantially weaker (lower amplitude, increased latency) in comparison with responses from the intact-side MRF (see Fig. 9). Responses produced by microstimulating the lesion-side MRF laterally were either very weak or absent for both PudMs (see Fig. 9 and example in Fig. 8B).



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Fig. 9. Summary of PudM-SFR data in animals with chronic LHx lesions. Each set of three bars represents 100% of the response sites at that particular matrix track. Each bar represents the percent response resulting from either bilateral or unilateral microstimulation at MRF sites in the medial to lateral plane (400-800, 1,200, and 1,600-2,000 µm from midline) and rostral to caudal plane (3,200, 2,800, and 2,400 µm from obex). Note that the values increase from medial to lateral for intact-side MRF stimulation and decrease for bilateral stimulation and that responses are scarse from the lesion side (although higher medially).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study demonstrate the presence of descending brain stem PudM-RD after either a chronic midthoracic LHx or a moderate Cx injury and the absence of these descending influences after either a severe chronic Cx injury or a complete chronic DHx. The results also demonstrate the presence of brain stem-elicited PudM-SFR after either a chronic LHx or a complete DHx or a moderate Cx injury and the absence of responses after either a severe chronic Cx injury or an over-DHx. When taken together, the results indicate that the two types of descending spinal projections between the MRF and circuits involving the PudM differ both in terms of laterality and location. Most of the results of these chronic lesion experiments were confirmed with select acute lesions; however, differences between chronic LHx and acute uni-DHx/LHx suggest that some reorganization in the neural circuitry for sexual reflexes had occurred after chronic injury (discussed further in the following text). In addition, the present electrophysiological results, particularly those for PudM-RD, are consistent with the behavioral reflex testing (i.e., impairment of reflex function after either complete DHx or severe Cx). When combined, the results indicate the presence of descending projections between MRF and the lower thoracic/lumbosacral male urogenital circuitry within the DLQ and the dorsal-most aspect of the VLQ at the midthoracic level of spinal cord.

Pudendal motoneuron reflex depression

The most robust depression of the DNP-elicited PudM reflex is produced by a conditioning microstimulation of the MRF bilaterally and ventrally in or medial to LPGi (Johnson and Hubscher 1998). This PudM-RD (decrease in amplitude, increase in latency) was not found after a complete chronic DHx or severe Cx injury, suggesting that the projections descend within the DLQ at the midthoracic spinal cord. These observations are consistent with neuroanatomic transport studies in the rat (Basbaum and Fields 1979; Martin et al. 1985) as well as behavioral and physiological investigations that also demonstrate the location of descending pathways originating from caudal brain stem in the DLQ using select lesions (Basbaum et al. 1977; Watkins et al. 1984).

The PudM-RD was maintained from both sides of the MRF after a chronic LHx, suggesting that descending projections are both crossed and uncrossed above, as well as below, the level of the midthoracic lesion (see left side of summary diagram, Fig. 10). These results are in contrast with those observed after an acute unilateral lesion of the DLQ, where reflex modulation was only maintained from the intact-side MRF. The acute lesion results indicate that the descending projections are uncrossed above the level of injury and are both crossed and uncrossed below. The reasons for this difference in the results is unclear. Possibilities include an unmasking of already present projections after injury, sprouting of new collaterals across the midline, or differences due to a sampling of only a single robust site in the acute controls. The latter possibility is unlikely because the most robust site producing PudM-RD was used in all six cases, so that even a weak response, if present, should have been detected at that location. The first possibility seems most likely because it is known that local circuits projecting across the midline exist within the MRF (Jones 1995; Robbins et al. 1992). Perhaps in a normal state, this circuitry is only involved in processing of information locally, and only under extreme conditions, such as after spinal cord injury, do they play an additional role to compensate for the loss of long descending projections after an incomplete injury.



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Fig. 10. Summary diagram illustrating the midthoracic location of descending fibers that produce PudM-RD (left) and PudM-SFR (right). Note that there is a slight shift in location (shown by the striped region) and unmasking and/or sprouting of connections (shown by dotted lines) after chronic injury.

Pudendal motor nerve sympathetic fiber response

The activation of sympathetic fibers in the PudMs is produced by microstimulating the MRF bilaterally and ventrally in or medial to LPGi (Johnson and Hubscher 2000). These PudM-SFRs were not found in the present study after a complete chronic over-DHx or severe Cx injury but were found after a complete chronic DHx (although responses were weak), suggesting that the projections descend within the ventral DLQ and the dorsal-most aspect of the VLQ (i.e., intermediate zone of white matter) at the midthoracic spinal cord. These findings are consistent with previous studies showing the location of descending sympathetic axons in the intermediate zone of the spinal white matter, with terminal labeling in the intermediolateral cell column with injections of anterograde tracer into the LPGi (Martin et al. 1985).

The PudM-SFR was maintained on both sides after chronic LHx, suggesting that the descending projections are both crossed and uncrossed above, as well as below, the level of the midthoracic lesion (see right side of summary diagram, Fig. 10). These results are in contrast to those observed after an acute unilateral lesion of the DLQ, where responses were only maintained for the PudM on the intact-side. The acute lesion results indicate that the descending projections are both crossed and uncrossed above the level of injury and are only uncrossed below (the opposite was found for PudM-RD). The reasons for this difference in the results are unclear, but possibilities include those outlined in the preceding text for PudM-RD. The likelihood of sprouting of new connections across the midline is supported by previous work demonstrating sprouting of unmyelinated afferent fibers after spinal transection (Krenz and Weaver 1998). These new connections are believed to provide new sources of innervation for autonomic preganglionic neurons, which may account for the increased sympathetic reflex responses to sensory stimulation that cause autonomic dysreflexia (Krenz and Weaver 1998).

Location of descending pathways

The reticular formation as a whole covers an enormous territory, covering several rostral/caudal levels of the brain stem. Because of this large extent, there are many different descending reticulospinal projections with different spinal white matter locations (just as there are several different locations for ascending spinoreticular projections) (see discussion in Hubscher and Johnson 1999b). In the present study, the focus was on one subset of reticulospinal projections, those that convey information to the male urogenital tract via projections originating from MRF to the lower thoracic and lumbosacral spinal cord. When combined, the results of the present study shows different locations for two long descending pathways between ventromedial MRF and the neural circuitry innervating the male urogenital tract. One pathway, whose activation produces a depression of the DNP-elicited PudM discharge, is located somewhere in the mid to lower region of the DLQ (see Figs. 10 and 11, left). This pathway overlaps with the ascending one conveying high-threshold sensory inputs originating from the male urogenital tract to MRF (see Fig. 11) (see also Hubscher and Johnson 1999b). The other descending pathway, whose activation produces firing bursts of sympathetic postganglionic neurons in the PudMs, is located somewhere in the intermediate zone of the midthoracic white matter, either deep within the DLQ and/or within the dorsal-most aspect of VLQ (see Figs. 10 and 11, right). When combined with the results from our accompanying paper (Hubscher and Johnson 1999b) (see Fig. 11), the results demonstrate a bilateral spino-bulbo-spinal circuitry confined, for the most part, to the dorsal half of the spinal white matter at T8.



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Fig. 11. Summary showing dorsal-ventral shift in the connections between the MRF and the neural circuitry mediating male sexual function at the midthoracic level of spinal cord.

The location of descending reticulospinal projections within the DLQ has been demonstrated in a number of other studies using rats, focusing on the same subnuclei (i.e., nucleus reticularis gigantocellularis pars alpha and LPGi) and looking at terminations within the intermediolateral cell column and lumbosacral spinal cord (Loewy et al. 1981; Martin et al. 1985; Watkins et al. 1984; Zemlan et al. 1984). Descending reticulospinal projections from other brain stem regions also are found within the DLQ (Basbaum and Fields 1979; Basbaum et al. 1977; Leichnetz et al. 1978; Loewy et al. 1979; Martin et al. 1979; Niemer and Magoun 1947; Watkins and Mayer 1982; Zemlan et al. 1979). It is unknown whether these projections also participate in the control of pudendal reflexes. The variety of descending reticulospinal projections that exist, including those within the ventral quadrant, subserve many different functions. These functions may depend on cell body location (for example, level of pons vs. rostral medulla vs. caudal medulla; ventromedial medulla vs. ventrolateral medulla), terminal field location (for example, terminations within different spinal laminae and at different spinal levels), and the motoneuronal pool being affected (for example, locomotor vs. pelvic floor musculature) (Basbaum and Fields 1979; Cohen et al. 1987; Jones 1995; Kow et al. 1977; Murphy et al. 1996; Nyberg-Hansen 1965; Zemlan et al. 1979, 1984).

Functional implications

The MRF is a multifunctional zone, with multiple ascending and descending inputs and outputs. The descending reticulospinal projections within DLQ have been shown to be part of a control mechanism for reflexive reactions to noxious (Basbaum and Fields 1979; Watkins and Mayer 1982; Watkins et al. 1984; Wei et al. 1999a,b) and mating stimuli (Watkins et al. 1984). The disruption of the descending MRF-lumbosacral pathway in the present study that results in the loss of the supraspinal control (i.e., depression) of the DNP-elicited PudM reflex likely contributes to the incoordination of perineal muscle contractions that occurs after chronic spinal cord injury, and thus the ensuing sexual dysfunction and bladder sphincter dyssynergia (see discussion in Hubscher and Johnson 1999b; Johnson and Hubscher 1998; Kruse et al. 1993).

Both sexual and bladder dysfunction are due to an incoordination of perineal muscle contractions mediated by pudendal motoneuron activity and segmental reflexes. As discussed previously (Johnson and Hubscher 1998), the function of the descending MRF-PudM-RD pathway in uninjured animals may be to coordinate, through the periodic inhibition of DNP inputs, the phasic timing and sequence of propulsive contraction bursts required for normal micturition and sexual function. The loss of this MRF control after spinal cord injury to the dorsal half of the midthoracic spinal cord, either through the elimination of the descending pathways to the pudendal reflex circuitry and/or the ascending sensory pathways of the DNP (Hubscher and Johnson 1999b) likely contributes to the inappropriate tonic contractions of the perineal musculature. In addition, disruption of the descending MRF-PudM pathway in ventral DLQ and the dorsal-most aspect of VLQ that produces an activation of postganglionic sympathetic fibers in PudMs also may contribute to the disruption of sexual function after chronic spinal cord injury. Although the function of this pathway is unknown, it also may have an inhibitory role in modulating sexual reflexes. Sympathetic fibers in the PudM have been shown previously to innervate cavernosal erectile tissue and promote detumescence (Galindo et al. 1997). Conversely, PudM sympathetic fibers may innervate glands in the distal urogenital tract which participate in the ejaculatory response.

Our data on these ascending and descending pathways demonstrates that they are organized bilaterally with some redundancy so that damaging only one side of the spinal cord should not alter sexual and bladder reflex control. The behavioral data from LHx animals in the present study, that is, inhibition of penile reflex initiation and no signs of bladder-sphincter dyssynergia, supports this conclusion. Only when these pathways are disrupted bilaterally through DHx or severe Cx lesions were behavioral signs of dysfunction observed.


    ACKNOWLEDGMENTS

We thank V. Dugan and R. Odama for excellent technical assistance and data collection.

This study was supported by the American Paralysis Association, the Brain and Spinal Cord Rehabilitation Trust Fund of Florida, and Grant NS-35702 from the National Institute of Neurological Disorders and Stroke.


    FOOTNOTES

Address reprint requests to: C. H. Hubscher.

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 1 September 1999; accepted in final form 11 January 2000.


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