29

Brain Research, 588 (1992) 29-40

BRES 17971

Nerve growth factor responsiveness of cultured major pelvic ganglion neurons from the adult rat J e r e m y B. Tuttle a a n d William D. Steers

b

Deparnnents of a Neuroscience and h Urology, UnirersiO,of Virginia Heahh Sciences Center, Charlottesrille, VA 22908 (USA) (Accepted 17 March 1992)

Kt'y words: Bladder neuron, Excitability; Hypertrophy; Neurotrophin; Neuronal survival; Tyrosine hydroxylase; Neuropeptide Y: Vasoactive intestinal polypeptide

The bladder and other pelvic viscera are innervated in the rat by the major pelvic ganglion (MPG), a mixed sympathetic/parasympathetic population of neurons that participates in lower urinary pathophysiology. Neurons from the MPG of adult females were removed, dissociated and cultured in order to test retention of the neuronal phenotype and whether they responded to Nerve Growth Factor (NGF). The bladder-specific subset of MPG neurons were distinguished by retrograde labeling prior to culture. The adult ganglionic neurons adapted to culture with > 80% survival in the best cases. The cultured neurons retained excitability, as determined by measuring voltage-activated ionic currents. They were positive for neuron-specific /3.tubulin and many retained immunoreactivity for characteristic peptides and transmitter synthetic enzyme. The proportion of neurons in the different categories tested varied somewhat from that in rive, but there was no evidence of selective death of a particular population. The cultured MPG neurons were responsive to NGF and anti-NGF antibody. NGF supported neuronal survival and expression of tyrosine hydroxylase. Added NGF also affected the expression of neuropeptide Y. Hypertrophied neurons from animals with experimental bladder outlet obstruction demonstrated increased responsiveness to NGF, The data suggest that NGF participates in adult neural plasticity due to continued responsiveness to the factor. Furthermore, questions concerning regulation of MPG neurons may be addressed in vitro.

INTRODUCTION

'The usefulness of a primary cultured preparation might be said to be enhanced in direct proportion to the amount of information available concerning the same preparation in vivo.'41. The major pelvic ganglion (MPG) of the rat, also called the paracervical, uterocervical or Frankenhauser's ganglion, has been studied in some detail as a model system for neural control of the mammalian genitourinary tract 27''~°'3s. It is an unusual peripheral ganglion in this species, since it contains neuronal cell bodies of the parasympathetic and sympathetic pathways 1''~2. Individual neurons of this mixed population innervate several pelvic structures, including the colon, bladder and sex-specific organs 12'24.25'4°.Within the ganglion, the neuronal soma are organized in a viscerotopic fashion 12'24. Despite histochemieal diversity, transmission through the MPG appears to be relatively simple, with little of the in'te-

grative activity commonly found in other ganglia'~c~''~'~'~'~. These attributes suggest that the MPG is a useful preparation to address questions concerning the regulation of neuronal type, electrical properties and functional diversity within a single ganglion. Morphological change:; in the neural pathways controlling micturition follow a partial outlet obstruction of the bladder "~*''~7.Prominent in these changes is an increase in the size of the neurons in the MPG, consonant with altered micturition reflexes "~5.Understanding these changes in neuronal form and function in the adult rat will advance our knowledge of neuronal plasticity and the specific interactions upon which it depends. The impressive growth of neurons in the MPG innervating an obstructed bladder, despite removal of sympathetic and parasympathetic preganglionic inputs, suggests an alteration in neuron-target interactions, perhaps involving a growth factor. Chronic obstruction

Correspondence: J.B. Tuttle, Box 230 Neuroscience, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA Fax: (I) (804) 924 5439.

3O by partial outlet ligation for 6 weeks results in a dramatic enlargement of the bladder to 10 times normal weight tT'-~s'-~s. Bladder tissue contains Nerve Growth Factor (NGF), the levels of NGF rise with hypertrophy 3'~ and the partial blockade of neuronal growth occurs in animals with high circulating titers of antiNGF antibody 3s. However, adrenergic neurons in the pelvic ganglia seem to be unresponsive to NGF, based upon relative resistance to the effects of immunosympathectomy 2~. in contrast, more recent reports describe enhanced recovery after NGF administration to injured neurons from the MPG 2 These questions of the responsiveness of pelvic visceral neurons to NGF, and the role of growth factors in neuronal plasticity, can be approached in cellculture. NGF rcsponsivcncss in cell or explant culture of adult rat MPG neurons has not been reported. Because the MPG contains neurons innervating other visceral structures in addition to thc bladdcr, the most definitive test of NGF rcsponsiveness of bladder MPG neurons must include a method to distinguish this popuhttion. This p:tpcr describes labeling of MPG neurons from the bl:tddcr in rive and then removal and culture of the g,mglion cells from normal and obstructed adult rats. MPG neurons in culture are compared to the neuron:il population in rive with respect to histochemical profiles, size and excitability. Finally, N G F dependence of cultured M P G neurons is examined in normal and obstructed animals, The results suggest that neurons from the adult rat adapt to cell culture and display several properties of MPG princi-

pal cells in rive. Many of the cultured neurons, includ. ing those supplying the bladder, respond to NGF and to anti-NGF antibodies. These findings support a role t'or NGF in obstructive pathophysiology and contradict the lack of NGF responsiveness for the adrcnergic neurons of visceral ganglia. Preliminary reports of some of this work have appeared in abstract form,

0.25-ml aliquots into the culture microwells containing 0,5 ml of growth medium. Each microwell received 1000-2000 neurons, yielding an initial density of 500-1000 neurons/cm 2. The cultures were incubated at 36°C in 3% CO,. In some experiments, ganglia were removed from animals that had undergone a partial urethral iigation 6 weeks previously, according to published methods "~s in order to induce bladder and neuronal hypertrophy. A retrograde lipophilic dye 23 facilitated study of organ specific cell types in the ganglion. DiO (3,3'-dioctadecyloxacarbocyanine perchlorate) and Dil (l,l'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), (Molecular Probes, Eugene, OR) dissolved in DMSO (4 mg/ml), were injected in 4-/LI amounts into the bladder wall with a Hamilton syringe 3"'3s. Two to four injections in the bladder base and body on both sides were done in each animal, with only the anterior base uninjected. Thus, the tracer was probably available to most innervating nerve terminals, Animals were sacrificed 48 h after injection. Although no quantitative data were taken, Dil seemed to yielded qualitatively better results in cell labeling, With DiO, the labeling failed entirely or neurons were only faintly fluorescent. This may reflect apparent low solubility of DiO in the injection solution used. With Dil, in contrast, labeling was dependably unequivocal. After removal and dissociation, the labeled cells were detected using a Nikon Diaphot equipped with epifluorescence and using fluorescein filterset for DiO, rhodamine set for Dii. Cuhure subs)rates and media The 24-well microculture plates (Falcon, Oxnard, CA) were previously coated with a solution of rat-tail collagenu~ dried under ammonia vapor, and sterilized by UV irradiation. Laminin was also tested as a coating to condition the culture plates, but it seemed to offer little or no advantage compared to collagen. Cell attachment was qualitatively similar on both coated surfaces, and survival data were indistinguishable. Several media formulations were tested, all based upon supplementation of standard media with serum or extracts, No attempt was made to be exhaustive, and largely qualitative results were gathered. llowever, survival and growth of MPG neurons was substantially bett~r in on~ I'ornmhttion than the others: this therefore is the current ~tand.rd formulatkm and should he considered as tin interim workln~ medium, The htlse is DMEM (Dulhecco's modified Eagle medium. Gibco) llG (no pyruvate); this is . variant of DMEM currently recognized by the American Type Tissue Culture Assoclation, This was supplemented with I(l¢;* (v/v) fetal bovine serum (FBS). ll)~ horse serum (liS) and 10 mM HEPES (Sigma). Other formulations tried included: MUM with and without FBS, HS, HEPES and chick embryo euract (CEE"*"): MEM with doubled amino acid and vitamin concentrations, with and without sera and CEE, the standard DMEM (with pyruvate) in the various combinations and DMEM IIG with CEE, sere with and without HEPES, Purified murine 2S NGF (kindly provided by Dr, E, Johnson, Washington University, St, Louis) was added to test for NGF responsiveness. while anti-NGF (Bt~hringer-Mannheim) was added to prevent the action of cell-secreted NGF,

MATERIALS A N D M E T H O D S ( 'ellpr~yrlrat.m

MPG's v,'crcr~:movt:d from female Wistar rats. 221l=350 g. under deep hah)thane/oxygen anesthesia and cleaned of adherent non,euronal tissue, according to methods previously tlescrihcd v~.~. Dissociation of ganglia into single celts was begun with a 6l)-min incubation tin ice ill a modified Tyrodc's saline (in raM: 130 NaCI, 3 KCI, 21) NaliE'Ot 4 (,aCl:. ! Mg('l~. 12 glucose. Ill tIEPES, l).5~:; antihiotic-antintycotic solution (Gihco. Grand Island. NY "~) with I).1)8% tt3'psin (Sigma. St. Louis. MO) and i mg/ml collagen.a~e (Sigma). This step may facilitate diffusion of the enzymes into the interior or" the ganglion prior |o proleolylic action. Next. the ganglia x~'ere incubated at 37~C for 50 rain. En~me action was halted by ..'plume dilution with serum-containing medium (see below). The ganglia ~.,.erc then remtwcd to a fresh vial of medium and dissociatcd by gentle trituration in a sterile, cotton-plugged Pasteur pipct. The ~:rll suspcnshm x,V;lSdiluted to a measured volume, and plated as

hmmomhL~'tothcmL~o3, Antil~)dy staining began with fixation of the cultures at 36°C (3) rain) in 3¢~ paraformaldehyde in 0,1 M phosphate-buffered saline (t),4 M NaCI) (PBS, plt 7,4) after 2 dip rinses in PBS, The fixative was washed off by two rinses in PBS (5 min), followed by permeabiliv.atk)n in (L04~- Triton X-I(X) in PBS (5 rain) only when staining with the neuronal marker TuJI, This primary antibody is a mouse monoclonal (Tu,I I) directed against a cla~s ill/J-tubulin isotype, cb4, raised by Dr, A, Frankfurter at the University of Virginia2s'3z. This cytoskeletal marker is useful as a neuron-specific probe s especially when used on cultured neurons, There is some variation in neuronal expression of TuJI, such that neurons in histological sections of the adult rat MPG do not stain well with TuJI, though the reasons for this are unclear (J,B, Tuttle and W,D, Steers, unpublished), When the primary antibt~ly was directed against tyro~ine hydroxylase (TH), vat, active intestinal polypeptide (VIP) or neuropeptide Y (NPY) (Penninsula Laboratories, Inc., Belmont, CA), permeabilization was

31 omitted. After an incubation in the primary antibody (2 h), and 3 (5 rain) rinses in PBS, anti-mouse Tab, H and L chains, labeled with peroxidase (HyCIone Laboratories, Inc., Logan, UT) or a similar secondary antibody directed against rabbit IgG (Hyclone) was used at ! : 200 dilution in PBS for 60 rain. After excess secondary antibody was removed by 3 (5 rain) rinses in PBS. the neurons were stained with the peroxidase substrate diaminobenzidine (3A,3',4',-tetraaminobiphenyl, Sigma) {DAB). The DAB solution contained 0.04% DAB ,:n 0.1 M PBS and 0.1 M imidazole; 30% H 2 0 2 was added at

0.3 ~l/ml just before use. Controls to determine specific staining included leaving out each of the following: !° antibody, 2 ° antibody, both I ° and 2° antibodies and DAB.

Neurona! counting Estimates of the number of neurons in each culture were derived from cell ~ounts of a measured area of the total culture surface. Neurons were identified by staining with TuJ 1 and by their charac-

Fig, 1, Major pelvic ganglion (MPG) neurons in vitro, a, phase contrast photomicrograph of MPG neurons 1 h after isolation, dissociation and plating into culture; b, phase contrast photomicrograph of MPG neurons I week after plating into culture, Note the presence of neurites at one week compared to the simple cellular morphology at dissociation; c, the neurons in b were fairly free of non.neuronal contamination, but often a background of non-neural cells formed. Six days in vitro; d, bright-field photomicrograph of MPG neuron fixed and stained with TuJ 1 at 8 days in vitro, revealing the fill extent of large and fine processes; e and L phase contrast (e) and epifluoresence (f) photographs of the same field containing an MPG neuron that was previously labeled with Dil in vivo, then cultured I week. Note the granular fluorescence that appears in the cell body, indicating this neuron supplied the bladder. Calibration bar: 50 ~m for all frames.

32 leristic appearance. The average number of neuronal cell bodies falling in microscopic fields taken across a diameter of the culture well at 2IX)x was used to estimate total neuromd cell number in the cultures. This procedure, which samples 5.8¢~- of the total area. has been shown to reliably estimate culture composition s'41. Counts of total neurons in 2-5 representative cultures stained with TuJI were in close agreement with counts taken from unstained cultures in the same plating, ba~d upon neuronal morphology. Estimates of the number of neurons positive for TH. VIP or NPY were based upon counting 50-100c~ of the culture area. Neuronal profiles were measured initially using an eyepiece micrometer scale calibrated against a stage micrometer. While many of the neurons were essentially spherical, with two ntmnal diameters varying less than I p.m. some cells were ovoid or fusiform. The essentially spherical cells were termed "simple' in shape; the other neurons 'complex' Complex neurons were measured at the longest axis in a second diameter normal to the first. Later data was collected through digitized video microscopy and analyzed using IMAGE. a public domain software system developed at the National Institutes of Health for use with Macintosh II. This yielded somal area and the diameter measurements. The two methods gave indistinguishable results.

Eh,ctrophy,~iolo~.

,

Recordings of voltage-activated membrane current were obtained frq~m the cultured MPG neurons using previously published techniques"'", Briefly. MPG neurons were cultured on a collagen-coated plastic coverslip (22x 22 ram) in a 35-ram culture dish, then transferred to a 1.0 ml superfusion chamber on the stage of an inverted microscope, A pipet with fire.polished tip was applied to the cell surface under direct observation and slight suction applied to obtain a high-resistance seal, The whole.cell ,tttached mode of recording was established by disrupting the membrane patch ,tt the pipet tip with a brief pulse of suction, The solution in the pipet contained (in raM) 140 KCI, I CaCI,, II EGTA.KOH, 2 Mg.ATP. Ill HEPES, pH "/,4, The normal bathing solution contained (in mM) 120 NaCI, 2,l,i KCI, 5 CaCI ,, Ill ltEPES, pH 7,4, Drugs (tetrodotoxin (TTX)(Sigma), tetra.ethyl ammonium (TEA) (Sigma)) at the indicated coneentra. tions (see text)were added by dis,~olving into this bathing saline and applied by a gravity.fed superfusion system, A change of the hath was uomplete in less than Ill s, The recordings were obtained at room temperature, The currents were recorded w i t h . Pagan I~ql)l) patch.clamp amplifier (Pagan Corp, Minneapolis, MN) low pass filtered tit I klt~, viewed on tin oscilloscope, and stored as digiti~etl amplitude data, Inward current appears a s . downward d~fleetion,

Stuti.~l&~ Data are expressed as mean±S,E, Significance was tested via Student's t, with P < 0,05 accepted.

RESULTS Neurons dissociated from adult rat MPG readily adapt to cell culture. Fig. 1 shows several examples of neurons in culture: freshly dissociated (Fig. IA) and at one week in vitro, in the older cultures, neurons are identified by staining with TuJ I, a neuron-specific form of Class Ill /~-tubulin (see Materials and Methods), Staining with this antibody reveals the entire extent of the neuron and its processes. The fine neuritic processes are often obscured by other cells in the culture when viewed with phase contrast alone. Processes are formed de novo after dissociation, as the freshly isolated neurons retain at most only the stumps of their original axonal processes which are sheared off by the

isolation procedure (Fig. IA). The newly regenerated processes display a typical morphology for rat peripheral neurons, having numerous varicosities along the process length.

Dil Neurons supplying the bladder were identified in these cultures by injecting the bladder with the retrograde tracer, Dil. Fig. 1E and F include an example of a neuron labeled with Dil in vivo, then cultured for 5 days. This culture was subsequently fixed and stained with TuJ1 for positive identification of the neuron. No Dil fluorescent neurons were seen in cultures taken from animals that had not been injected with the tracer. Also, dye injected into the colon 48 h prior to removal of the ganglion failed to accumulate in MPG neurons, demonstrating that the labeling was specific to the bladder and not the result of generalized leakage into the abdomen. Dil was not seen in the 1.6 dorsal root ganglia (DRG), a more distant site known to contain afferent neurons that innervate the bladder .~7. Excitability of neurons in culture in addition to using the neuron-specific cytoskeletai marker to identify the cultured cells as neurons, cultured cells were prepared for electrophysiological recording after 2-6 days in vitro, Records of whole-cell currents obtained using the whole cell attached mode of the patch clamp recording method ~ are shown in Fig, 2. in Fig, 2A, a series of traces reflect the currents flowing during a step depolarization from a -80 mV holding potential to positive voltage, beginning with a control condition and then during perfusion with 2 ~tM tetrodotoxin, The initial, rapid inward current is completely blocked by the TTX, indicating it is a voltagedependent Na + current, The delayed outward current remaining is completely blocked by TEA (20 raM) in the perfusate in addition to TTX (Fig, 2B), indicating it is a voltage-dependent K + current. Eight cells identified as neurons had the expected voltage-dependent ionic currents capable of serving action potential generation, Occasionally, a neuron was spontaneously active, discharging action potentials in the absence of any imposed stimulus (not shown), These data establish that the adult neurons dissociated from the rat MPG and maintained in culture retain excitability. The effect of TTX was not restricted to blocking the fast, inward Na + current. The lower traces of Fig. 2A demonstrate an additional effect that was seen in all cases: infusion of TI'X also reduced the magnitude of the delayed outward current. Thus, it appears the magnitude of the delayed K + current is affected by

33

a

N e u r o n a l sur~;it'al h~ culture

Vm

-k._ ,200 I~A

13 m,

b

1,00 0, I'+ ml

Fig.2. Records of voltage-activated ionic currents in cultured MPG under whole cell patch clamp, a: the sequential records (upper traces) detail the response to a voltage step from -80 mV to + 10 mV during the infusion of TTX into the bathing medium. Lower traces display only the first and last responses. The first trace (largest downward excursion) is several superimposed traces of responses in normal saline. The initial negative excursion is it large, rapid Inward current that is progressively eliminated by the TTX, Indicating a rapid Na+ current, in the lower pair, with only the first and last of the series displayed, note that after complete blockade of the Na + current, u more prolonged outward current remains but that the TTX had the effect of reducing its magnitude, b: sequential currem records during the infusion of saline with TTX and TEA (upper traces), with only the first and last shown in the lower pair. The initial rapid inward current is followed by the delayed outward current. When TTX and TEA are included, both voltage-activated currents are eliminated, indicating the outward current is carried by K + ions. These two series of records were obtained from different neurons. Similar responses were recorded in 8 other cases.

activation of the Na + channels or directly affected by the drug. The cultured M P G neurons also displayed voltageactivated Ca 2+ currents (data not shown). In order to record these less robust currents, a pipet solution with CsCI in place of KCI was used ~'. In addition, TTX and T E A were included in the perfusate. Under these conditions, pure Ca2+-dependent currents were recorded that demonstrated typical voltage activation and little inactivation during a 120 ms depolarizing pulse 6.

MPG were removed from normal rats, as described, and cultured in media of several different formulations. After 5 - 7 days in vitro, the cultures were fixed and stained for counting of the surviving neurons. Initial experiments involved variations of the isolation and dissociation procedure, with variable success. If the total number of neurons in each female adult ganglion is estimated at 5,800 m, then from less than 2% to more than 70% of this total survived at 24 h in vitro, depending upon the dissociation technique. The critical aspect of the protocol was the enzymatic treatment to break down the connective tissue. When shorter enzyme incubations (15-30 min), lower enzyme concentrations (0.08% trypsin, collagenase omitted; 0.01% trypsin, 0.1 m g / m l collagenase) and no pre-incubation at 4°C was tried, the ganglia did not dissociate well and survival was minimal. In later experiments, it was routine to accomplish yields of > 70%. In two experiments (12 microwells each) with standard medium, survival was 56% of neurons in the ganglia at 24 h, 62% at 5 days in vitro and 58% at 7 days, suggesting little attrition occurred. The proportions of neurons positive for TH-IR, VIP-IR and NPY-IR in culture and in vivo are compared in Table !. It is clear that a substantial population of each type of neuron is retained in culture, thus no one cell type i:~ selectively elimin~.ted. Also, the relative numbers of each type of neuron in vitro are not overly different from those in rive, although it seems likely that fewer neurons in culture are VIP-IR than in rive. Thus, up to 51% label for VIP-IR in the animal ~4 while only 20.5% are positive in culture.

TABLE I lmm,m~reactil'ity and shape of c,hurt, d MPG aml bladder m,lmms compared to in i,il'o data

Neurons from adult rat MPG were cultured as described, fixed and examined for immunoreactivity to tyrosine hydro~lase (TH), vasoactire intestinal polypeptide (TIP) and neuropeptide Y (NPY), as well as for retrograde labeling from the bladder. Numbers in parentheses are total neurons examined. Neurons were also classed as simple (difference in major axes < 2 ~m) or complex in shape, based upon measurement of somal diameter with an ocular micrometer. Data for in vivo are taken fi'om the published literature '4. In t'itro

ba l'it'o 2"1 % Bladder "

Total TH TIP NPY

100% (2555) 20.3%(519) 20.5% (524) 31.2% (797)

N.A. 16.7% 51% 32%

% Compk,x

ha t'itro

in t'i+'o

38.5% 5.1% 3.8% ND

16.3% (665) 25.3% 13%(31) 7.7% 4% (24) 49.3% ND

N.A. = Not applicable; ND = Not done. The previous study24 used males rather than the females used here, thus sex-related differences in distribution may account for some of the difference between the results of the two studies (see Discussion).

34 However, this difference could be sex-related because the in viva study used males 4". Many of the neurons were not positive for any of these markers. Within each group of I R neurons, not all were labeled by the retrograde tracer from the bladder. In these experiments, prior to removal to culture, the bladders were injected with Dil, Overall, 38.5% of the MPG neurons contained detectable Dil, similar to the proportion of bladder neurons labeled by retrograde tracing and histological section of MPG-"~. Within each IR group, a different but consistent proportion of neurons were labeled with the retrograde marker (Table I). The percentage of neurons innervating bladder that were TH-IR in medium without NGF added seems lower than that ffmnd in viva, with 5.1% positive in vitro compared to 25.3%, labeling for males in viva. Also presented in Table ! is an indication of the relative morphology of the different cell types. As c.'m be seen in Fig. 1, the MPG neurons retain or acquire quite different shapes in culture. During measurements of cell size, neurons were also scored for "complex' vs. 'simple' shape, based upon the difference in major axes. A difference larger than 2 ~m cl;issed the neurons as complex. All of the cell types contained both simple and complex members, but those TH.IR cont,'dn ,'l higher proportion of complex neurons.

NGF Purified murine NOF w:ks qddcd to the culture medium and its cft'ec! upon overall neuronal survival determined. The results for added NGF depended upon the culture medium used. in medium containing chick embryo extract (i,=g, 3), NGF had no apparent eft'oct on overall survival. However, in medium supplemented with serum alone, neuronal survival was increased by added NGF, from 8=35%. This suggests that some MPG neurons are responsive or dependent upon NGF in culture and that the embryo extract may directly or indirectly substitute or block the effect on survival, The effect of added NGF may be additive or synergistic to trophic factors produced by the non-neural cells in the culture. Schwann cells, fibroblasts and other gila synthesize and secrete NGF in culture ~~ ~'=~':=~.There were other cell types besides neurons in these cultures, as can be seen in Fig, 1, Theretore, to determine whether the adrenergic (TH-IR) population of pelvic ganglion neurons is NGF-dependent, cultures of MPG were grown in medium to which 0.2 p g / m l anti-NGF had been added, This antibody concentration is more than sufficient to block the action of equimolar amounts of NGF in a fiber-outgrowth bioassay ~'~,The antibody was refreshed at every

m

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DIOFIOC

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Culture Medium Fig. 3. Effect of exogenous N G F on overall survival of M P G neurons in culture. M P G neurons were isolated and cultured as in Materials and Methods, in the media indicated, with 50 ng/ml NGF or no added NGF. Cell counts of surviving neurons were taken as in Materials and Methods at 7 days in vitro, and the total number of neurons in the NGF supplemented cultures expressed as a percent of those in the control cultures. Ml0C=minimal essential medium (Eagle) (MEM), 10% chick embryo extract (CEE), DIOFi0C= Dulbecco'~ modified essential medium (Eagle) (DMEM), 10% fetal calf serum (FCS). 10% CEE. DI5FIOH = DMEM, 15% FCS, 10% horse serum (H$). DISF = DMEM, 15% FCS, DIOFI0H = DMEM, 10%, FC$, 10 I-IS. NGF significantly increased the survival of MPG neurons above control levels (No NGF) in media that did not contain CEE, Each culture condition represents data from 6-10 cultures each in at least three different platings of approximately 1,500 neurons per culture.

medium change. Exogenous NGF antibody reduced the proportion of TH-IR neurons in the culture from the 20,3% found in NGF-supplemented media to less than 3%, which appeared as a few scattered, mildly positive neurons, This suggests that the antibody against NGF eliminated TH-IR, either through cell death or loss of the enzyme marker, Furthermore, adding NGF to the medium increased the proportion of TH-IR from 5,1% to 20,3%, Blocking concentrations of antiNOF affected overall neuronal survival as well (Fig, 4A) and survival of the subpopulation innervating the bladder (Fig, 4B), in these experiments, neuronal survival was compared among cultures in standard DMEM medium, D M E M with exogenous NGF added (0,1 pg/ml) and D M E M with 0,2 # g / m l anti-NGF. The data is expressed two ways: as percent of survival in the DMEM and also as percent of neurons surviving in anti-NOF, Note that adding exogenous NGF to the medium increases survival above that found in DMEM for the entire population and slightly for those innervating bladder (Fig, 4A and B), Also, the anti-NGF reduces survival below the levels in DMEM, Fig. 4C includes data from similar experiments conducted on neurons dissociated from MPG after 6 weeks of experimentally induced bladder outlet obstruction, The neurons from outlet-obstructed animals respond to NGF similarly to the neurons from unobstructed animals: overall survival is 141 + 19% of control (Fig. 4C), cam-

35 pared to 166 + 9% in the cultures from unobstructed animals (Fig. 4A), However, the effect of blocking N G F with anti-NGF is more pronounced: the antibody reduces survival to only 86 + 33% of control in the unobstructed cases but to 48 :t: 12% of control in the cultures from obstructed animals (Fig, 4C), The difference in response to N G F after obstruction is most easily seen by comparing the shaded bars in Fig. 4A and 4C, After bladder outlet obstruction the control (DMEM) had twice the neurons surviving as in the anti-NGF, and N G F supplemented cultures had almost three times as many neurons suriving (Fig. 4C). This suggests the N G F responsiveness of the adult MPG neurons can fluctuate or that endogenous production of the factor is increased in the cultures from obstructed animals,

a

~Oontrol ~%AntiNGF

800 200 100

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r"lqi¢ontrol

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N G F added to the medium also altered the distribution of immunoreactivity. Examples of cultured neurons immunoreactive for the markers are shown in Fig. 5. There was no obvious morphological characteristic that reliably discriminated between the neurons expressing each marker. However, neurons NPY-IR more frequently displayed prominent varicosities on the neuritic processes. The proportion of neurons immunoreactive for N P Y is greater in the presence of N G F than in its absence (Table II), suggesting N G F influences the expression of NPY a n d / o r promotes the selective survival of neurons expressing this peptide. M P G neurons have a rather wide size distribution in vivo 24'36, Furthermore, somal size of M P G neurons is plastic in the adult 36'37. N G F may be responsible for some of the neuronal growth in the adult ganglia 38. Therefore, it was of interest to determine whether N G F had an effect upon neuronal size in culture in addition to the effects upon survival and peptide expression. Fig. 6 is a frequency histogram of the somal diameters of M P G neurons cultured with N G F added to the medium ( 0 . 1 / z g / m l ) compared to cells cultured in medium with anti-NGF antibody (0.2 p.g/ml). In exogenous N G F there is an increase in the proportion of large ( > 18 /zm) and small ( < 10 /zm) diameter neurons. Mean diameter, however, was not significantly different between the two groups (NGF: 16.2 + 0.35 /~m, n = 131; 0.05). The MPG neurons cultured for the experiment summarized in Fig. 6 were removed from normal animals,

200

tat

O~ 100

Fig. 4, Effect of exogenousNGF and exogenousanti.NGF lmtihody on survival of MPG and bladder MPG neurons 7 days in culture, a: MPG neuronscultured in control (DMEM) medium, with 0.1 ,~g/ml NGF added (NGF) and with 0.2 ~g/ml anti-NGF antibody added (AntiNGF). Data are expressedas % neuronssurviving, % of control (solid bars) and as % of neurons surviving in unti-NGF (hatched bars). This allows the comparison of the effect of blocking antibody to added NGF, but retains the information on survival with no additions to the medium. Note that the exogenous NGF increased overall survival and that the anti-NGF antibody decreased survival below control levels, b: data similar to a but including only those neurons labeled from the bladder with Dil. Note that the proportion of bladder neurons eliminated in the anti-NGF condition is greater than the proportion of total MPG neurons affected by the antibody. in both cases (a and b) added NGF significantly increased survival and the anti-NGF decreased survival, This suggests that the neurons in culture acquire a partially effective supply of NGF-like activity from contaminating cells in the culture;, c, data similar to a and b but taken from cultures established from MPG after 6-week bladder outlet obstruction. In this case, the survival promoting effects of NGF, and inhibitory effects of anti-NGF, are exaggeratedcompared to those in a and b. These data suggestthe NGF responsivenessof MPG neurons is enhanced after outlet obstruction and the subsequent hypertrophy. Each culture condition represents data from at least 6 cultures each in a minimum of two different platingsof 1,200 neurons per culture.

36

Fig. 5. Examples of MPG neurons cultured for 5-7 days then fixed and stained for vasoactive intestinal polypeptide IR (a, arrow indicates VIP-IR neuron), tymsine hydroxylase immunoreactivity (IR) (5b, arrow - TH-IR neuron), and neuroactive peptide Y-IR (e, d). These were not labeled with dye from the bladder, thus may or may not innervate bladder. While there were consistent different trends in the appearance of these differently labeled neurons, no obvious morphologic features were diagnostic for one cell IR group, in c, the double a r r o ~ indicate two +mall varicosities thai were also NPY-IR while the intervet=ing axon was negative. In d, the single arrow indicates the NPY-IR neuron, while the double arrows indicate an unstained neuron nearby,

thus had a normal initial size distribution. Animals that

have undergone experimental outlet obstruction, however, develop ganglia with a variant distribution of neuronal sizes'~": the mean area is increased and a 20 i

NGF AntiNGF

o.

G

12

18

24

8omal

Diam (urn)

30

Fig, (k Effect of ~xogenous NGF and anti-NGF on somal si;~e of all MPG neurons. Frequency histogram of neuronal soma sizes of cultured MPG neurons at 7 days in vitro, cultured with 0,1 •g/ml NGF added to the medium (solid bars) and 0,2 ~.g/ml anti-NGF added (clear bars), Note that the neurons in the anti-NGF have f~wer representatives in the large ( > 20 pro) and small ( < 12 pro) c;~tegorius,

category of large cells appears. In vivo, this neuronal growth following obstruction is not completely reversed after deligation of the urethra '~N,To determine whether the growth following obstruction was retained in culture, MPG from rats that had undergone partial liBation of the urethra 6 weeks previously were used. Fig. 7 is a frequency histogram comparing the somal diameters of neurons taken from animals with outlet obstruction to those from normal animals. The neurons from the obstructed animals had a significantly higher mean diameter (OBS: 16.9 + 0.3 p,m, n - 289 vs, 16.0 :j: 0.35 /a,m, n - 82; P < 0.05) at one week in culture without exogenous NGF, suggesting that the increase in size in vivo was retained in vitro, Note also that the obstructed distribution displays the same alterations seen in Fig. 6. The obstruction results in an increased proportion of neurons in both the larger (> 18/~m) and small (< 12 /a.m) size categories, The results presented in both Figs. 6 and 7 suggest neurons of quite different sizes are responsive to NGF in vitro. TH-IR neurons are known to be large, sympathetic ganglion neurons, potentially NGF-sensitive (vide infra and ref. 24), thus are candidates for comprising the larger siz:s affected by NGF and obstruction. Fig.

37 TABLE I!

12

Proportions oJ"immmzoreacti~'e MPG cultured with and without added NGF Neurons were isolated and dissociated from adult MPG as described and maintained in culture for 7 days. Cultures were then fixed and stained with antibodies directed against tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP) or neuropeptide-Y (NPY) as described in Materials and Methods. Percentages of neurons immunoreactive were determined from counts sampling 7% of the total microwell area for 7 cultures in each category contained in three different platings. Total neuronal counts were based upon staining with TuJ I (see Materials and Methods) in sister cultures.

Total neurons TH-IR VIP-IR NPY-IR

No NGF added

+ NGF (0.l p.g/ml)

2555

2169

519 (20.3%) 524 (20,5%) 797 (31.2%)

["'lOontrol

lObetrueted

| ,o

8omal

Diem

9

15

Somal

Substantial numbers of the MPG neurons from the adult female rat adapt to culture, with up to 80% or

18

•

24

21

27

Diam (urn)

Fig, 8. Somal sizes of cultured MPG neurons positive for NPY and VIP. Somal size frequency histogram of neurons IR or NPY (clear bars) and VIP (solid bars) at 8 days in culture. The size ranges display almost complete overlap, suggesting size is not a diagnostic feature for peptide IR.

DISCUSSION

1°

]

m

4.2

8 presents the size frequency histogram of MPG neurons cultured from control ganglia and stained for NPY-IR and VIP-IR. The mean diameter of neurons positive for NPY (21.6 + 13.9 p.m, n ffi 146) is significantly larger than that for neurons positive for VIP (16.8 + 8.3/zm, n = 181). This might suggest that NPY neurons are large and thus cannot be candidates for the small cell category of NGF responsive neurons. However, despite the difference in means, neurons VIP-IR and NPY-IR were observed in cells of all sizes.

iS

]NPY

o

339 (15.6%) 468 (21.6%) 879 (40.5%)

20

"0

I

80

(urn)

Fig. 7. Effect of experimental partial outlet obstruction on neuronal soma size. Neurons were cultured from normal MPG's (clear bars) and from MPG's in animals that had been partially outlet obstructed 6 weeks previous to culture (solid bars), cultured in vitro for 6 days, then frequency histograms of somal size of all MPG neurons determined. Note that the neurons from obstructed animals had increased representatives in the large ( > 20/zm) and small ( < 12/zm) categories.

more of the original population surviving for more than a week. This is the first report of successful culture of the neurons from this ganglion in the adult. Some of the neuron-specific properties of the ganglion cells are retained in culture. Thus, they can be marked in vitro by staining with a neuron-specific cytoskeletal marker. Also, the neurons retain excitability in vitro, possessing the voltage-sensitive ionic currents expected of excitable neurons that generate action potentials. Finally, many of the neurons retain immunoreactivity for various proteins of differentiated function. Work by Burnstock and colleagues has shown that bladder neurons can be cultured from neonatal guinea pig and retain immunohistochemical characteristics in vitro t~''~4.These observations indicate the transition to culture in vitro does not result in loss of the major aspects of the neuronal phenotype. Despite having retained neuronal character, do all of the neurons from the ganglion survive or is a particular subset selectively vulnerable? In most cases, there was a measurable proportion of neurons that failed to adapt to the in vitro environment. However, for the populations measured (TH-IR, VIP-IR, NPY-IR), no disproportionate loss was observed for any individual group, and similar diversity existed both in vivo and in vitro. The finding of fewer neurons positive for VIP-IR and TH-IR in cultures, compared to that previously reported for MPG, may be explained by the use of ganglia from female rats in the current study while male rats were used for the investigation in vivo24'25. The high number of VIP-IR neurons in the male MPG may arise because the innervation of the penis is predominantly VIP-IR ~2'24. In a study that examined female rats 33 and found TH-IR neurons to be the least numerous of four types, and NPY-IR the most numer-

38 pus, no specific data are given on prevalence. The neuronal hypertrophy created by experimental outlet obstruction of the bladder was retained in vitro. Thus, the cultured population of neurons shares many similarities with cells in rive. However, this must be considered provisional because not all neurons in the ganglion were typified by the markers used. Given experimental errors and biological variations, a minor population eliminated by the rigors of culture may go undetected. If only neurons innervating the bladder are considered, then there appears to be a selective loss of neurons TH-IR unless NGF is added to the culture medium. This result could indicate that the larger, TH-IR neurons are more susceptible to injury or that they require added NGF for optimal survival or to maintain TH expression.

Nen'e growth factor A major aim of this study was the question of MPG neurons' response to NGF. NGF has been implicated as a mediator of the neuronal plasticity occurring after bladder outlet obstruction .~. if NGF mediates, in part, the neuronal gcowth after obstruction, then some of the MPG neurons must respond to the trophic factor. Indeed, if NGF regulates MPG neuronal size, adult MPG neurons must depend upon and respond to NGF. Neurons in adult rat superior cervical and dorsal root ganglia retain NGF responsiveness 4,t~. However, adrcnergic neurons in pelvic ganglia have been reported resistant to neonatal immunosympathcctomy-'~. Resistance to antibodies against NGF suggested that the neurons in the visceral ganglia do not require NGF. However, the cause of MPG resistance to antiNGF does not seem to derive from lack of NOF responsiveness. The adrenergic neurons innervating pelvic viscera in the mouse have been shown to depend upon NGF ,~. The data in this report establish that the proportion of TH-IR neurons in cultures of MPG neurons is dramatically affected by blocking concentrations of anti-NOF antibody in the culture medium. In addition, the antibody reduces the incidence of a large cell and small cell population of MPG neurons. Finally, NGF alters the proportion of cultured MPG neurons expressing NPY-IR. Thus, the MPG contains NGF-responsive and probably NGF-dependent neurons. Perhaps structural differences between the paravertebral, prevertebral and visceral ganglia confer protection from antibody in rive. Certainly, many of the neuronal soma in visceral ganglia are closer to their peripheral target tissues than neurons in the paravertebral chain ganglia. If the anti-NOF affects access to the factor in proportion to soma-target distance, then the visceral adrenergic ganglia would appear resistant.

What are the characteristics of the NGF responsiveness by adult MPG neurons? Neurons containing TH require NGF to continue to express the enzyme or to survive, but the data do not discriminate between NGF regulation of TH synthesis or expression and survival of TH neurons. The combined use of TH and another marker for sympathetic neurons that is not regulated by NGF, perhaps high-affinity catecholamine uptake, would determine whether the attrition in TH-IR is due to death or loss of TH. Both may contribute, based upon the effect of NGF on ChAT expression by the NGF-dependent cholinergic neurons in the basal forebrain ~. NGF has also been shown to regulate peptide expression in SCG neurons 2~. Thus, NGF affects the capacity of neurons to produce and secrete transmitters and modulatory peptides, but the full range of effects has probably not yet been defined. The suggestion of an effect of NGF on NPY expression in the MPG deserves further attention. NGF may also affect neuronal size. NGF levels in the bladder rise following experimental outlet obstruction, neurons in the MPG increase in size, and autoimmunity to NGF prevents some of the obstruction-induced neuronal growth. These previous findings in rive are consistent with the present findings in vitro. Prior experimental outlet obstruction and treatment in vitro with NGF both cause an increase in neurons in the larger size categories, Anti-NOF in the culture medium caused a reduction in neurons in those size categories, Howovcr, direct comparisons of the absolute sizes measured in rive and in vitro are probably unwise, due to the unknown effects of cell dissociation and culture upon neuronal form. The morphometry used for studies of ganglia in rive is based upon area measurements of retrogradely labeled MPG neurons innervating blad. der. Normal ganglia from 230-250 g rats contain neurons with a mean area profile of 300 :l: 4.9/~m", For a spherical neuron, this equates to a diameter of approx. imately 19.5 ~m. The mean diameter of the MPG neurons cultured in this study was 16,2/Lm. The area profile of an average cultured spherical MPG neuron would be approximately 206 #m 2. Differences in mean area smaller than 100 tzm" were significant, thus the cultured neurons do not measure to be the same size as those in vivo. This difference is due to the problems inherent in comparing sectioned ganglia to cultured neurons, exclusion from the study in rive of cells < 150 #m-' to limit the results to principal neurons, and the presence of small neurons in the MPG ~. The small cells in rive probably include SIF cells or other paraneurons. Our data does not address the question of paraneuron survival and NGF dependence directly because specific markers for these cells were not used.

39 However, NGF added to the culture medium and outlet obstruction both resulted in an increased incidence of small neurons in the cultures. These data suggest the existence of a group of NGF-dependent small neurons or paraneurons. The cultured neurons do not retain the same shape as the neurons in rive. MPG neurons in rive usually exhibit one or no dendrite and o n e a x o n 24'~'39. The neurons in culture extended complex arrays of processes, often with several major processes. No attempt was made to distinguish axonai and dendritic processes. The regulation of process regeneration is complex and depends upon both soluble factors such as NGF and also substances associated with the substrate or cell membranes. Apparently the constraints in rive that prevent MPG process formation are not present in vitro, or more potent stimulation occurs in the cultures. Although a subset of adult rat MPG neurons respond to NGF, many of the ganglion cells do not require the trophic factor. Thus, subpopulations in the MPG require and/or respond to other, non-NGF trophic factors, in vivo, immunity to NGF does not prevent all of the growth induced by bladder hypertrophy •~H, consistent with non-NGF factors mediating neuronal growth. Candidates for the active trophic factors include brain-derived neuronotrophic factor, neurotrophin-3, ciliary neuronotrophic factor and basic fibroblast growth factor (bFGF). Homogenates of bladder have been reported to contain bFGF-specific mRNA 7, but details of MPG responsiveness arc not available. Information on the synthesis of non-NGF factors and the presence of growth factor receptors on MPG neurons would address whether these neurons respond to one or several trophic factors and whether such substances might regulate histochemical diversity. Finally, NGF responsiveness may differ in normal vs. obstructed MPG neurons. Thus, the adult neuronal response to the trophic factor may be modulated or regulated. The data do not address the mechanisms for alteration in neurotrophin responses, yet reasonable speculation would focus on regulation of receptors. The experiments reported also do not rule out interaction between NGF and other members of the neurotrophin family and their receptors. Acknowledgements. Supported by grants NS 28566, NS 30024 and DK 44830 from the NIH, and by the American Heart Associatioq and Muscular Dystrophy Association. We thank Drs. D. Brosius and D.J. Creedon for help with some of the work.

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