Brain Research 883 2000 107–118 www.elsevier.com locate bres
Research report
Anatomical evidence for two spinal ‘afferent–interneuron–efferent’ reflex pathways involved in micturition in the rat: a ‘pelvic nerve’
reflex pathway and a ‘sacrolumbar intersegmental’ reflex pathway
a,b , a,b,c
Pedro L. Vera , Irving Nadelhaft
a
Veteran ’s Administration Medical Center, RD Service 151, Bay Pines, FL 33744, USA
b
Department of Surgery , Urology Division, University of South Florida, Tampa, FL, USA
c
Department of Pharmacology , University of South Florida, Tampa, FL, USA
Accepted 18 July 2000
Abstract
We labeled interneurons in the L1–L2 and L6–S1 spinal cord segments of the rat that are involved in bladder innervation using transneuronal retrograde transport of pseudorabies virus PRV in normal animals and in animals with selected nerve transections.
Preganglionic neurons were identified using antisera against choline acetyltransferase ChAT. In some experiments we labelled parasympathetic preganglionic neurons PPNs in the L6–S1 spinal cord by retrograde transport of Fluorogold from the major pelvic
ganglion. We identified bladder afferent terminals using the transganglionic transport of the anterograde tracer cholera toxin subunit b. We present anatomical evidence for two spinal pathways involved in innervation of the bladder. First, in the intact rat, afferent information
from the bladder connects, via interneurons in L6–S1, to the PPNs that provide the efferent innervation of the bladder. The afferent terminals were located mainly in close apposition to interneurons located dorsal to the retrogradely labeled PPNs. Second, using L6–S1
ganglionectomies or L6–S1 ventral root rhizotomies we limited viral transport to the sympathetic pathways innervating the bladder. This procedure also labelled interneurons but not PPNs with PRV in the L6–S1 spinal cord in a location very similar to those described in the
intact rat. These interneurons also receive bladder afferent terminals but we propose that they project to sympathetic preganglionic neurons, most of which are in the L1–L2 spinal segments. Based on this anatomical evidence, we propose the existence of two spinal
reflex pathways involved in micturition: a pathway limited to a reflex arc in the pelvic nerve presumably excitatory to the detrusor muscle; and a pathway involving the pelvic nerve and sympathetic nerve fibers, some of which may travel in the hypogastric presumably
inhibitory to the detrusor muscle.
2000 Elsevier Science B.V. All rights reserved.
Theme : Sensory systems
Topic : Somatic and visceral afferents
Keywords : Pseudorabies virus; Cholera toxin; Fluorogold; Acetylcholine transferase; Spinal interneuron; Bladder afferent
1. Introduction fibers within the pelvic nerve to the lumbosacral spinal
cord. These fibers may synapse on interneurons that are Micturition is a complex behavior requiring the coordi-
part of spinal and supraspinal pathways to relay this nated activity of the autonomic and somatic nervous
information to spinal and higher brain centers leading to systems, both at the central and peripheral levels. In-
awareness and appropriate voiding by activating descend- formation concerning bladder fullness travels via afferent
ing parasympathetic pathways. The spinal organization of neurons involved in the micturition reflex has been studied
extensively in the cat and the rat for a recent review, see
Corresponding author. Tel.: 11-727-398-6661, ext. 4010; fax: 11-
de Groat [6]. Anatomical studies have examined the
727-398-9457. E-mail address
: pverahsc.usf.edu P.L. Vera.
central termination pattern of afferent fibers traveling in
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 7 3 2 - 3
108 P
the pelvic nerve in the rat [17] and, more specifically, the sympathetic chain or the major pelvic ganglia to provide
spinal distribution afferent fibers that innervate the bladder sympathetic control of the detrusor activity.
of the rat [22]. The locations of lumbosacral parasympa- thetic preganglionic neurons PPNs with axons in the
pelvic nerve and the sympathetic preganglionic neurons
2. Materials and methods
SPNs with axons in the hypogastric nerve have been well described [17,18,21]. Finally, previous experiments in the
2.1. Experiment I. Labeling of spinal cord neurons with rat using transneuronal labeling from PRV injections into
PRV injections into the bladder in animals with L 6 S1
the bladder combined with retrograde labeling using a ganglionectomies
fluorescent tracer [19] showed a distinct group of inter- neuronal cells in the L6 and S1 segments of the spinal
Sprague–Dawley female
rats n58;
Harlan, In-
cord. These interneuronal cells were located close to the dianapolis, IN, average weight: 228 g were anesthetized
PPNs in the intermediolateral cell column IML. The sodium pentobarbital;40 mg kg; i.p. and a dorsal
interneurons differed from the PPNs in three respects: 1 laminectomy exposed the L6–S1 dorsal root ganglia.
they were usually located dorsal to the PPNs, 2 they These were crushed and transected to ensure their com-
were smaller than the PPNs and 3 they were not plete disconnection from the spinal cord. The muscle was
cholinergic. closed 4-0 silk, the skin incision was closed with wound
The location of these interneurons is interesting since clips and a topical antibiotic ointment Bacitracin zinc and
several lines of anatomical and electrophysiological evi- Polymyxin B sulfate; Fougera was applied to the skin
dence point to the possible involvement of spinal inter- incision. The animal was then laid supine and an abdomi-
neurons in the micturition process. Afferent fiber terminals nal incision was made to expose the urinary bladder. Two
from the pelvic nerve of the rat [11] appear to surround a injections, 2 ml each, of pseudorabies virus Bartha strain,
8
similarly located group of interneurons. Furthermore, 2.6310 pfu ml were made, at different points, into the
bladder afferent terminal fibers in the rat can be seen in the detrusor muscle. This amount of PRV injected was chosen
vicinity of the IML [17,22]. Therefore, previous ana- according to the results of previous experiments that
tomical investigations suggest that these interneurons may showed effective infections from such injections [19]. The
receive bladder afferent terminals. In addition, electro- incision was closed with sutures and clips and the animal
physiological studies of micturition reflexes in the rat returned to its cage. A heating pad at low heat was placed
reported a spinal reflex present in about 15 of the intact beneath the cage to keep the animal warm until it regained
rats [16] but in 100 of the spinally transected rats, which consciousness. The bladders were expressed manually
was of considerably shorter duration than the spinal– twice daily until the animals were sacrificed.
supraspinal micturition reflex. Finally, studies in the cat Animals were killed at 3 n52, 4 n53 and 5 n53
have shown that the sympathetic nervous system acts to days postinfection. Under pentobarbital anesthesia the
inhibit detrusor activity presumably through an interseg- animal was perfused transcardially with cold Krebs solu-
mental lumbosacral spinal pathway [7,8]. tion followed by cold 4 paraformaldehyde. The transec-
Therefore, although several lines of anatomical and tions were verified post-mortem as the spinal cord and
physiological studies pointed to the existence of the spinal brain were removed. Selected spinal segments were dissec-
and the intersegmental lumbosacral spinal reflex pathways, ted and post-fixed for several hours and then placed in
no conclusive anatomical evidence had been presented to 25 sucrose–phosphate buffer for cryoprotection. The
correlate with the physiological and electrophysiological L6 S1 and L1 L2 segments were chosen since they
findings. In this report, we extend our previous anatomical represent the origin of the preganglionic fibers in the pelvic
findings by reporting that presumed interneurons in the [17] and hypogastric nerves [18], respectively.
L6–S1 spinal cord segment of the rat are in close A selection of transverse 30 mm cryostat sections was
proximity to afferent terminal fibers from the bladder. processed to visualize neurons that contain pseudorabies
These interneurons are part of two reflex pathways pos- virus PRV, choline acetyltransferase ChAT, or both.
sibly involved in bladder innervation: 1 a ‘pelvic nerve Each section was exposed to a combination of rabbit anti
pathway’ where afferent information from the bladder is PRV 1 10K; generously donated by Professor L. Enquist
transmitted through the pelvic nerve and to interneurons and
goat anti-ChAT
1 100; Chemicon
AB144P; that project to parasympathetic preganglionic neurons that,
Temecula, CA, and incubated at 48C for about 18 h. This in turn, provide the parasympathetic innervation of the
was followed by a combination of donkey antirabbit bladder via the pelvic nerve; 2 a ‘sacrolumbar inter-
conjugated with FITC 1 50; Jackson Immunoresearch, segmental pathway’ where afferent information from the
West Grove, PA and donkey antigoat conjugated with bladder is transmitted to interneurons in the L6–S1 spinal
TRITC 1 50; Jackson Immunoresearch, West Grove, PA cord which then project to the lumbar L1–L2 SPNs.
for 30 min. Sections were mounted on gelatinized slides, Those SPNs then project to the inferior mesenteric ganglia,
coverslipped with a fade-retardant medium [23] and ex-
P .L. Vera, I. Nadelhaft Brain Research 883 2000 107 –118
109
amined under epifluorescent illumination. Maps of sections 2.3. Experiment III. Labeling of bladder afferent
Minnesota Datametrics, Shoreview, MN showed the terminals projecting to presumptive bladder interneurons
locations of Pseudorabies virus immunoreactive PRV-IR, following L
6 –S1 ventral root rhizotomy choline acetyl transferase immunoreactive ChAT-IR or
double-labeled neurons. Sprague–Dawley male rats 260–280 g were anes-
thetized with sodium pentobarbital 40 mg kg; i.p. and a dorsal laminectomy exposed the conus medullaris and the
2.2. Experiment 2. Labeling of bladder afferent fibers, L6 and S1 dorsal root ganglia. The dura was opened and
lumbosacral parasympathetic neurons and spinal the L6 and S1 ventral roots were identified and cut.
interneurons in intact animals Gelfoam Upjohn; Kalamazoo, MI was placed over the
conus medullaris, the muscle incision was sutured using Sprague–Dawley male rats n54; 280–380 g were
4-0 silk and the skin incision was closed using wound anesthetized with halothane and a ventral abdominal
clips. The animal was then placed on a supine position and incision was made to expose the bladder. A total of 10 ml
an incision was made to expose the bladder. The bladder of 1 Ctb List Biological; Campbell, CA was injected
was injected with Ctb and with PRV as already described into the bladder detrusor muscle in several injections to
in Section 2.1. The abdominal muscle and skin incisions label the bladder afferent fibers. In addition, the major
were closed as described previously. The animals were pelvic ganglion MPG was exposed bilaterally using blunt
allowed to recover from the anesthesia and their bladders dissection and Fluorogold 1–2 ml; 1 in dH O was
were expressed manually twice daily.
2
injected into each ganglion using a hand-held glass mi- Animals were reanesthetized with sodium pentobarbital
cropipette in order to label the parasympathetic neurons 80 mg kg; i.p. 96 h after the initial procedure and
projecting to the MPG. Finally, pseudorabies virus Bartha perfused transcardially as previously described. The sur-
strain; two injections, 2 ml each was injected into the vival time of 4 days was based on the results from the
bladder muscle in order to label, transneuronally, the spinal L6–S1 ganglionectomy experiment I above, which did
interneurons involved in the micturition pathway. The not reveal any PRV-labeled neurons in the L6–S1 spinal
incisions were closed with sutures and clips as already cord segments until 4 days postinfection. The ventral root
described and a topical antibiotic ointment Bacitracin zinc rhizotomies were confirmed post mortem by careful dissec-
and Polymyxin B sulfate; Fougera was applied to the skin tion of the L6–S1 DRG and dorsal and ventral roots. All
incision. The animals were allowed to recover from the animals showed bilateral section of the L6 and S1 ventral
halothane anesthesia and returned to their cages. roots. The L1–L2 and L6–S1 spinal segments were
The rats were reanesthetized with sodium pentobarbital identified and removed. Coronal sections 20 mm of the
72 h after the injections and perfused transcardially as L6–S1 spinal segments were processed for: 1 PRV and
described above. The survival time was chosen based on Ctb immunohistochemistry as outlined above; 2 coronal
previous experiments [19] that showed the first labeling of sections 20 mm of the L6–S1 and L1–L2 spinal seg-
presumptive interneurons in the IML region at 3 days ments were processed for PRV immunohistochemistry and
postinfection with PRV. The spinal cord and brain were ChAT as described in Expt. I.
removed and spinal cord segments L6–S1 were identified and placed in fixative overnight at 48C followed by a 25
sucrose–phosphate buffer solution. A series 1 6 for transverse and 1 2 for horizontal of
3. Results
