P .L. Vera, I. Nadelhaft Brain Research 883 2000 107 –118
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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
transverse n53 or horizontal n51 20-mm sections of the L6 and S1 segments were processed for immuno-
3.1. PRV-IR and ChAT-IR neurons in spinal segments histochemistry as follows. The sections were rinsed with
L 6 S1 and L1 L2 in rats with L6 S1 ganglionectomies
0.1 M phosphate-buffered saline PBS and placed in PBS containing 3 normal donkey serum. The sections were
then exposed to the following primary antisera: Goat Lumbosacral spinal cord neurons were found that were
anti-cholera toxin b List Biological; Campbell, CA; pseudorabies
virus-immunoreactive PRV-IR,
choline 1:2000 and rabbit anti-PRV generously donated by L.
acetyltransferase-immunoreactive ChAT-IR
or were
Enquist; 1:10 000; in PBS containing 0.3 Triton X-100 doubly labeled. The analysis was restricted to neurons in
and 1 normal donkey serum overnight at 48C. After the intermediolateral cell nucleus IML, the dorsal gray
rinsing in PBS, the sections were exposed to the secondary commissure DGC and the dorsal horns. Motor neurons in
antisera as described in Expt. 1. Sections were mounted on the ventral horn showing ChAT-IR but not PRV-IR were
gelatinized slides, coverslipped with a fade-retardant noted but excluded from this examination although they
medium [23] and examined under epifluorescent illumina- demonstrate restriction of viral infection to autonomic
tion. targets and lack of spread of infection to somatic areas.
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P .L. Vera, I. Nadelhaft Brain Research 883 2000 107 –118
111
3.1.1. Three days postinfection segments. In addition, a few doubly labeled neurons were
At 3 days postinfection there were very few PRV-IR usually found in each tissue section.
neurons in the L6–S1 segments Fig. 1A and none of In general, the numbers of PRV-IR neurons increased as
these were located in the intermediolateral areas IML the postinfection incubation time increased whereas the
where preganglionic neurons are found. A representative numbers of ChAT-IR neurons remained constant Fig. 3A.
sample of 43 sections 16 of the total number contained In particular, at 3 days postinfection, there were very few
13 PRV-labeled neurons all of which were small and none PRV-IR neurons in either the L1–L2 or the L6–S1
were also cholinergic. On the other hand, the IML con- segments. On the other hand, at 4 and 5 days postinfection,
tained many ChAT-IR neurons but not PRV-IR whose both of these pairs of spinal cord segments contained a
size and location identified them as preganglionic cells. comparatively large number of PRV-IR neurons. The
Larger ChAT-IR motor neurons were found in the ventral frequency of doubly labeled neurons as a function of time
horns and smaller ones in lamina X around the central are illustrated in Fig. 3B. At 3 days postinfection only the
canal, however these neurons were not PRV-IR and are not L1–L2 segments contained doubly labeled SPNs that also
included in this analysis. showed PRV-IR cells and there were none in the L6–S1
In the L1–L2 cord Fig. 1B, many medium-size segments. At later times, the ratio of doubly labeled
neurons showing both PRV-IR and ChAT-IR were located neurons to PRV-IR only neurons, dropped significantly in
in the dorsal gray commissure region DGC and in the the L1–L2 segments due to the increased numbers of
IML, suggesting that they were sympathetic preganglionic PRV-IR neurons. The numbers of doubly labeled neurons
neurons. A representative sample of 48 sections 17 of in the L6–S1 segments increased slightly.
the total number contained 97 PRV-IR neurons of which 67 were also cholinergic Fig. 3B. Examination of
3.2. Anatomical evidence for a spinal pathway double-labeled neurons in more rostral segments showed
consisting of bladder afferent terminals , interneurons
only a few cells in T13 and T12 and none rostral to those and lumbosacral parasympathetic neurons in the intact
segments. No double-labeled neurons were observed in L3. rat
By far the largest number of these neurons were located in L1–L2.
The photographs of afferent terminals, PRV-IR neurons and fluorogold-labeled preganglionic neurons shown in
Fig. 4 were all taken from the same section and without 3.1.2. Four and 5 days postinfection
repositioning of the slide. There was a large increase of PRV-IR neurons in the
L6–S1 segments Fig. 1C and these were located in the 3.2.1. Location of bladder afferent terminals within the
dorsal half of the spinal cord. In particular, a group of lumbosacral cord
these were commonly seen to lie just dorsal to the Bladder afferent terminals labeled with cholera toxin b
preganglionic ChAT-IR neurons in the IML. Additionally, subunit were located in the superficial layers of the dorsal
a few PRV-IR neurons not ChAT-IR were always located horn and along the side of the dorsal horn lateral pathway
in the superficial layers of the dorsal horns. A map of a [17] of the L6 and S1 spinal segments Fig. 4A. These
typical section taken from a five-day animal demonstrating afferent fibers appeared to cluster in the rostrocaudal
the relative locations of PRV-IR neurons and ChAT-IR direction at the lateral edge of the dorsal horn.
neurons and photomicrographs of these two groups are shown in Fig. 2. It can be clearly seen that the group of
3.2.2. Location of parasympathetic preganglionic PRV-IR neurons which are not ChAT-IR most likely
neurons in the lumbosacral cord interneurons lies dorsal to the cholinergic preganglionic
Neurons retrogradely labeled with FG were located in neurons of the ChAT-IR group.
the intermediolateral nucleus area, in lamina VII at the In L1–L2 segments Fig. 1D there was also a large
edge of the gray matter Fig. 4C. These neurons often increase of PRV-IR neurons and these were also located in
were oriented in the mediolateral direction with dendrites the dorsal half of the spinal cord. However the distribution
extending medially, as described by others [17]. Some of of these neurons was more spread out than in the L6–S1
these neurons also showed immunoreactivity for PRV. A
Fig. 1. Representative distributions of PRV-IR green 3 and ChAT-IR red 1 neurons in the L6–S1 A,C and L1–L2 B,D spinal cord segments at 3 days postinfection A,B and at 4 days postinfection C,D. Note the relative scarcity of PRV-IR neurons at 3 days postinfection and the large increase of
these neurons 4 days postinfection. Doubly labeled neurons blue are present only in the L1–L2 segments B,D. ChAT-IR motor neurons can be seen in the ventral horns. Calibration bar: 0.5 mm.
Fig. 2. PRV-IR and ChAT-IR neurons in a typical section from the L6–S1 spinal cord at 4 days postinfection. Distribution of PRV-IR green 3 neurons and ChAT red 1 neurons. Calibration bar: 0.5 mm. Note that in the intermediolateral area, the PRV neurons are located predominantly dorsal to the
ChAT neurons. Photomicrographs of each of these two groups of neurons, labeled PRV and ChAT, respectively, are seen to the right of the diagram. Calibration bar: 100 mm.
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Fig. 3. PRV- and ChAT-IR neurons in the L1–L2 and L6–S1 spinal cord segments as a function of time after L6–S1 bilateral ganglionectomy. A Solid bars are PRV-labeled neurons and open bars are ChAT-labeled neurons. Note the absence of significant numbers of PRV-IR neurons in both areas 3 days
postinfection and the large increase at 4 and 5 days postinfection. The numbers of ChAT-labeled neurons remains constant throughout the time period since their numbers are not affected by viral infection. B Doubly labeled neurons expressed in terms of the numbers of PRV-labeled cells. The number of
doubles is high only in L1–L2 at 3 days postinfection because at that time most of the PRV-IR neurons are preganglionic neurons. At later times most of the PRV-IR neurons are interneurons and therefore not cholinergic and the ratio of doubles to PRV-IR neurons falls considerably. In L6–S1 there always
were very few labeled preganglionics and there was a large increase in PRV-IR interneurons and so the number of doubles remains very small.
few Ctb-labeled terminal fibers were sometimes observed transported retrogradely from the MPG Fig. 4C. In
in close proximity to PPNs. No PPNs were observed to be addition, other presumed interneurons PRV-labeled neu-
doubly labeled with Ctb and FG, suggesting that the Ctb rons not retrogradely labeled with Fluorogold were found
injections were restricted to the bladder and did not leak dorsal to the central canal in the dorsal gray commissure,
onto the MPGs. as well as interspersed throughout laminae I–VI.
3.2.3. Location of presumptive bladder interneurons in 3.3. Afferent terminals located on presumptive bladder
the lumbosacral cord interneurons in the lumbosacral cord projecting to the
PRV-IR neurons that were not retrogradely labeled with lumbar sympathetic preganglionic neurons
Fluorogold were observed interspersed with PPNs in the IML region, but most were located dorsal to the PPNs, at
These results are depicted in Fig. 5. In this figure as in the lateral edge of the gray matter Fig. 4A. Most of the
Fig. 4, the pair of micrographs A–B and the pair C–D dense afferent terminal labeling observed in or dorsal to
were taken from the same section without moving the slide the IML nucleus was observed to surround these presump-
between photographs. In this last group of experiments tive interneurons Fig. 4A. These interneurons presumab-
where the L6 and S1 ventral roots had been cut prior to the ly part of the spinal pathway innervating the bladder were
application of PRV to the bladder preventing PPNs in clearly different from the sacral parasympathetic neurons,
L6–S1 from being labeled with virus, afferent terminal which were also labeled with PRV and with Fluorogold
labeling was similar to that observed in intact animals.
Fig. 4. Anatomical evidence for a ‘pelvic reflex arc’ in the intact rat. All three micrographs were taken from the same section without moving the slide. Therefore the relative positions of the objects within each of the three photographs can be directly compared. A Bladder afferent terminals anterogradely
labeled with cholera toxin terminate in the IML region of the L6–S1 spinal cord. B Pseudorabies virus-labeled cells in the IML region of the L6–S1 spinal cord. C Retrogradely labeled parasympathetic preganglionic neurons PPNs in the IML region of the L6–S1 spinal cord. Note the medially
oriented dendrites of the PPNs labeled both with Fluorogold c and with PRV b. PRV-labeled cells that do not also show FG labeling appear mainly dorsal to the PPNs. The afferent terminals appear in close apposition to the interneurons in the IML region A. A drawing of the same section depicted in
panels A–C is presented for orientation purposes. The afferent labeling is depicted by the red line in the IML region. PRV-immunoreactive cells are marked with a green 3 whereas retrogradely labeled Fluorogold PPNs are marked with a blue 1. Calibration bar: 200 mm.
Fig. 5. Anatomical evidence for a ‘sacrolumbar intersegmental reflex arc’. Pair A–B and pair C–D were each taken from the same section without moving the slide between photographs. A Afferent fibers anterogradely labeled with cholera toxin subunit b were observed in the dorsal horn of the L6–S1 spinal
cord segment, often in close apposition to PRV-labeled interneurons B. Note how the afferent terminals appear to terminate on the dendritic process of the interneuron. Afferent terminals C were also seen in close apposition to PRV labeled interneurons in the L6–S1 spinal cord segment D. These
interneurons did not show ChAT immunoreactivity not shown. Calibration bar: 100 mm.
P .L. Vera, I. Nadelhaft Brain Research 883 2000 107 –118
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Ctb-labeled fibers were observed in the dorsal horn and IML at L1 L2 segments in all three experiments, they
near the IML area Fig. 5A–C. PRV neurons were still were only found in the IML at L6 S1 in the intact rats.
observed in the L6 and S1 segments, and especially dorsal Therefore, it is likely that these neurons represent sympa-
to the IML region where they came in close apposition to thetic preganglionic neurons SPNs; at L1 L2 and para-
the afferent terminal fields Fig. 5C,D. Double immuno- sympathetic preganglionic neurons PPNs; at L6 S1 that
staining for PRV and ChAT showed that none of these form part of the sympathetic and parasympathetic innerva-
neurons that showed PRV immunoreactivity also showed tion of the bladder. The distribution of SPNs traveling in
ChAT immunoreactivity. Therefore, these experiments as the hypogastric [18] and PPNs projecting in the pelvic [17]
in Expt. I showed two groups of cells in the IML. 1 A nerves have already been described and the present results
dorsal group composed of smaller cells that were PRV-IR agree with earlier findings in terms of location, distribution
but not ChAT-IR and that received most of the bladder and size of these neurons.
afferent terminals. We presume these neurons to be inter- In addition, a group of PRV-IR but not ChAT-IR
neurons. 2 Larger ChAT-IR cells that were seen in the neurons were also observed in the L1 L2 and L6 S1
IML area but mostly ventral to the first group and these segments in all three experiments. We propose that these
were not PRV-IR. Some bladder afferent terminal fields neurons are interneurons and that they are probably
were also seen in this group. We presume these neurons are retrogradely labeled from the labeled SPNs and PPNs
sacral parasympathetic preganglionic neurons with axons described earlier. Anatomical evidence for interneurons in
in the pelvic nerve, some of which are part of the the L6 S1 spinal segments and their possible involvement
parasympathetic innervation of the bladder through their in bladder function is available from several earlier studies.
projections to the MPG. Previous PRV injections into the bladder revealed a similar
location of interneurons in the IML [19]. Likewise, wheatgerm agglutinin injected into the bladder trans-
synaptically labeled small neurons in the IML and DGC of
4. Discussion
