THE JOURNAI, OF COMPARATIVE NEUROLOGY 301:23-43 (1990)

SomatodendriticMorphology of On-and Off-CeUsin the Rocsbral Ventmmedial Medulla P. MASON, M.K. FLOETER, AND H.L. FIELDS Departments of Neurology (P.M., M.K.F., H.L.F.) and Physiology (H.L.F.), University of California-San Francisco, San Francisco, California 94143-0 114

ABSTRACT The rostral ventromedial medulla (RVM) contains two classes of physiologically defined neurons, on-cells and off-cells, that are implicated in nociceptive modulation. In a continuing effort to detail the neural circuitry that underlies the activity of these two distinct neuronal types, the somatodendriticmorphology of on- and off-cellswas studied in the cat, rat, and ferret. In lightly anesthetized animals, on-cells increased and off-cells decreased their discharge rate during a withdrawal reflex evoked by noxious stimuli. Following their physiological characterization by using intracellular recording, on- and off-cells were injected with either horseradish peroxidase or biocytin and their somatodendritic arborizations were examined. Labeled on- and off-cells included fusiform and stellate cells of all sizes as well as large multipolar neurons. Although the somatic shape of both on- and off-cells in RVM was heterogeneous, off-cells tended to be fusiform neurons whose long axis was oriented mediolaterally. The dendritic domains of both on- and off-cells extended bilaterally past the lateral edge of the trapezoid body or pyramid and ventrally to, and sometimes including, the trapezoid body or pyramid. In contrast to their extensive mediolateral spread, the dendritic domains of both cell types were limited to the ventral half of the reticular formation and were compressed along the rostrocaudal axis. The dendritic arbor of individual on- and off-cells extended well beyond the cytoarchitechtonic boundaries of any single nuclear region, within the domain delineated as the RVM.The spatial domains of the dendritic arbors of on- and off-cells are further evidence that the on- and off-cells throughout the R V M constitute an integrated unit in the modulation of nociceptive transmission. Key words: raphe magnus, antinociception,medullary reticular formation,biocytin

Neurons in the rostral ventromedial medulla (RVM) play an important role in the modulation of nociceptive transmission (for reviews, see Basbaum and Fields, '84; Fields et al., '88). Electrical stimulation or glutamate microinjection in the R V M suppresses nociceptive reflexes (Oliveras et al., '75; Jensen and Yaksh, '89) and inhibits the responses of sensory trigeminal or spinal dorsal horn cells to noxious stimuli (Fields et al.,'77; Willis et al., '77; Sessle and Hu, '81; Sessle et al., '81). The nociceptive modulating effects of R V M neurons are likely mediated by descending projections from R V M to the spinal and trigeminal dorsal horns (Basbaum et al., '78; Basbaum and Fields, '79; Holstege and Kuypers, '82; Light et al., '86; Basbaum et al., '86). Physiologically defined populations of RVM cells have been described that either increase (on-cells) or decrease (off-cells) their discharge rate just prior to and during the rat tail flick reflex evoked by noxious heat (Fields et al., '83a). Both on- and off-cells project to the dorsal horn

o 1990 WILEY-LISS, INC.

(Vanegas et al., '84b; Mason and Fields, '89). Off-cells are excited by morphine administered either systemically or microinjected into the brainstem and are hypothesized to have a net inhibitory effect on dorsal horn nociceptive transmission (Fields et al.,'83b; Barbaro et al., '86; Cheng et al., '86). On-cells, in contrast, are inhibited by morphine and are activated during periods of increased responsiveness to noxious stimuli, evidence that these neurons have a net excitatory effect on dorsal horn nociceptive transmission (Bederson et al., 1990). The spontaneous activity of on- or off-cells is directly correlated with the activity of other on- or off-cells, respectively, whereas off-cells' periods of spontaneous firing are reciprocal to those of on-cells (Barbaro et al., '89). We recently demonstrated that off-cell axons terminate extensively within the RVM,evidence that they play a major role Accepted July 17,1990.

P. MASON ET AL.

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100 pm H Fig. 1. Low-power photomicrographs of two HRP-labeled RVM cells. A: CIO 22-4-2 is a cat off-cell in the NRM. The outlines of the NRM can be seen in this 50 km osmicated plastic section. B: The soma of CIO 26-4-1, a cat on-cell, was split by sectioning. This photomicrograph of a 100 km frozen section shows a portion of the soma and many

of the labeled dendrites that course laterally, ventrally and medkdlly. The dorsal boundary of the trapezoid body as well as the midline, denoted by paramedian penetrators, is visible. A full reconstruction of this cell is shown in Figure 8.

in the coordination of on- and off-cell firing through excita- on a constant flow of oxygen and on a continuous infusion tory connections with other off-cells and/or inhibitory con- of Lactate Ringers for the duration of the experiment. nections to on-cells (Mason and Fields, '89). Labeled on-cell Recordings were made from animals with a mean arterial axons, in contrast, were shown to have few or no intranu- blood pressure of 100 mm Hg or more. Supplemental doses of pentobarbital were administered as needed to keep the clear projections. On- and off-cell dendritic domains are potentially impor- animals in a state of light anesthesia. After the initial tant in the proposed intranuclear circuit through which anesthesia, rats were maintained on either 0.5-1.5% off-cell axons coordinate the spontaneous activity of RVM halothane in oxygen or on a continuous infusion of sodium neurons. The size and shape of these dendritic arbors also methohexital(15-30 mgikgihour). The methods for physiological recording in cats were also delineate the brainstem region where afferent terminals from neurons extrinsic to RVM may contact RVM on- and employed with ferrets and have been described previously off-cells. Thus, in order to more completely define the (Mason and Fields, '89). In rats, the tail flick reflex was circuits in which on- and off-cells participate, the dendritic evoked as has been described previously (Fields et al., '83a). extent of on- and off-cells must be described. To investigate Glass micropipettes were filled with 4.0% horseradish whether on- and off-cells differ in their somatodendritic peroxidase (HRP, Sigma type VI) in 0.1 M Tris buffer (pH morphology, physiologically identified cells were filled with 7.4) and 0.5 M potassium chloride (Mason et al., '86). In horseradish peroxidase (KRP) or biocytin in the cat, rat, some experiments, microelectrodes were filled with 5.0% and ferret. biocytin in 0.1 M Tris buffer (pH 7.4) and 0.5 M potassium chloride (Horikawa and Armstrong, '88). All micropipettes were bevelled in a solution of silicon carbide to a final tip METHODS resistance of 40-90 MO (Lederer et al., '79). Under direct Adult cats (2-4 kg), ferrets (1-2 kg), or Sprague-Dawley visual guidance, micropipettes were advanced into the rats (240-325 g) were used in all experiments. All animals pontomedullary raphe at a 15-30" angle caudal to the were initially anesthetized with sodium pentobarbital (60 frontal plane. mg/kg, ip). In the cats and ferrets, a tracheotomy was Recordings were made from neurons in the pontomedulperformed and catheters were placed in the femoral vein lary raphe nuclei and adjacent medial reticular formation. and in the femoral artery. Cats and ferrets were maintained Neurons were isolated by either: 1)membrane potential or

Fig. 2. Photomicrographs of on- and off-cell somata in all species studied. A: RIA 6-1-1 is a fusiform on-cell oriented dorsoventrally in the rat MRF\. B: RIO 26-7-1 is a fusiform on-cell oriented dorsoventrally in the rat MRF,. C: RIA 7-3-1 is a large multipolar on-cell in the rat MRF,. D RIA 36-3-1 is a large multipolar on-cell that was labeled with biocytin in the rat NRM. E: RIO 35-3-1 is a fusiform off-cell oriented in the mediolateral direction that was labeled with biocytin in the rat NRM. F: RIO 7-1-1 is a fusiform on-cell oriented mediolaterally in the rat MRF,. G: RIO 15-8-1is a fusiform off-cell oriented in the mediolatera1 direction in the rat MRF.. H: RIO 30-2-1 is a fusiform on-cell

oriented in the mediolateral direction in the rat NRM. I: CIO 4-5-1 is a fusiform off-cell oriented in the mediolateral direction in the cat MRF,. J: FIO 14-4-1is a fusiform off-cell oriented in the mediolateral direction in the ferret NRM. K RIO 28-4-1 is a small stellate on-cell in the dorsal portion of the rat NRM. L: CIO 4-7-1 is a fusiform off-cell oriented in the mediolateral direction in the cat MRF,. M: CIO 22-4-2 is a large multipolar off-cell in an osmicated plastic section through the cat NRM. The scale bar in B applies to A-L and the scale bar in M applies only to M.

Figure 2

P. MASON ET AL.

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2) spontaneous activity or 3) response to a search stimulus (see Mason et al., '85, '86). The search stimulus employed was a short train of electrical pulses in the midbrain periaqueductal gray (PAG). The PAG inhibits nociceptive reflexes and dorsal horn cells (Oliveras et al., '74; Sessle et al., '81), an effect that is likely relayed, at least in part, through neurons in RVM (Behbehani and Fields, '79; Gebhart et al., '83; Mason et al., '85; Chung et al., ' 8 7 ) . Furthermore, most cat R V M neurons and rat on- and off-cells are excited by PAG shock (Vanegas et al., '84a; Mason et al., '85, '86). All neurons were characterized by their intracellular responses during the flexion withdrawal reflex evoked by noxious pinch or heat. Pressure from toothed forceps that was judged painful by application to a fold of the investigator's hand skin was used as a noxious pinch. Noxious heat was applied to the hindpaws of cats and ferrets and to the tails of rats as described previously (Fields et al., '83a; Mason and Fields, '89). The paw pad or tail was maintained a t 35°C between trials and the temperature was increased to a peak of 53°C over a 10 second period during a trial. A temperature above 49°C consistently evokes pain in humans (Torebjork, '74). Following electrophysiological characterization as described above, neurons that were stably impaled were injected intracellularly with HRP or biocytin by using a

TABLE 1. Number and SomaticShape of Labeled On- and Off-cellsin the RVM and in the MRF, Shown in Each of the Species Studied' Somaticshape Species Ons in the RVM Cat Rat Ferret Total Offs in the RVM Cat Rat Ferret Total Ons in the MRF, Cat Rat Ferret Total Offs in the MRF,, cat

Rat FPint Total

Multipolar

Stellate

Fusiforq,

Fusiforq,

?

Total

2

-

3 -

2 -

-

4

9 1

2

2

3

4

4

4 10 2 16

-

1 1 -

1 2 3

2 1 3

I

6

1

7 1 14

1 1

-

-

1 -

1 1

1

-

-

I 1

1 1 2

8 8 2 18

-

1 1

3 6

-

3 1

4

Abbrendt.ions FUSIFORM.,, , fusiform neuron oriented in the medinlateral plane; FUSIFORM,,, , fusiform neuron oriented in the domventral plane; ?, somatic shape obscured or unknown.

D

Fig. 3. Camera lucida reconstructions of five of the six off-cell somata located at the level of the superior olivary complex in the cat. A-E: High-power reconstructions of each soma. F:The location of each

off-cell illustrated is shown in a coronal section through the RVM. The midline and the dorsal boundary of the trapezoid body are shown. Scale bar in B applies to A-E.

SOMATODENDRITIC MORPHOLOGY OF RVM CELLS

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E

r a p h e rnagnus ~

I

71

CIO 27-6-2

\ '

Fig. 4. Camera lucida reconstructions of four of the four on-cell somata located at the level of the superior olivary complex in the cat. A-D High-power reconstructions of each soma. E The location of each

constant depolarizing current of 20 nA for 4 minutes (HRP) or 5 minutes (biocytin) (Mason et al., '86). In cases where the injected neuron was still impaled after the original 4 minutes, depolarizing current was injected for another 2-4 minutes. After the recording session, animals were maintained under deep anesthesia (sodium pentobarbital; 30-40 mg/kg/ hour) for up to 24 hours. After the survival period, animals were sacrificed by pentobarbital overdose and perfused with saline followed by a fixative containing 1.25% glutaraldehyde and 1.0% paraformaldehyde with 4% sucrose in 0.1 M phosphate-buffered saline (PBS). The tissue was blocked coronally and then immersed in 30% sucrose in 0.1 M PBS overnight. Serial transverse sections, 50-100 km thick, were then cut on a freezing microtome. In some cases, transverse sections of 50 km thickness were cut on a vibratome. The sections with HRP-labeled cells were treated with DAB (7.5 mg), nickel ammonium sulfate (0.3 g), and 3.0% H,O, (165 p1) in TBS (50 ml) at room temperature (Hancock, '82a,b; Wouterlood et d . , '87). The serial sections were then mounted, dehydrated through an alcohol series, and covered. In some cases, sections were embedded in Epon or in glycol methacrylate. Sections containing biocytinlabeled cells were incubated in 0.5% Tx-100 in 0.1 M phosphate-buffered saline (PBS) for 30 minutes and then in the ABC complex (Vector) in PBS and 0.5% Tx-100 for 2 hours. Sections were then treated with DAB as above. The somatodendritic morphology of all physiologically identified and labeled neurons was examined under a 25 x objective; selected neurons were reconstructed by using a drawing tube and either a l o x or 25x objective. Dendritic spines were examined under a 1 0 0 oil ~ immersion objective.

off-cell illustrated is shown in a coronal section through the RVM.The midline and the dorsal boundary of the trapezoid body are shown. Scale bar in D applies to A-D.

RESULTS Physiology ofcells In accordance with previous results from this laboratory (Fields et al., '83a; Mason and Fields, '891, neurons were classified as on- or off-cells. On-cells were excited by a noxious cutaneous pinch or by noxious heat applied to the paws, tail, or head. Off-cells were inhibited by noxious heat or pinch applied anywhere over the body and head surface. On- and off-cells were often recorded amongst axons that had the firing pattern typical of medial lemniscal axons. The physiology of recorded on- and off-cells has been described elsewhere and will not be detailed here.

Nucleardefinitions The RVM includes both the nucleus raphe magnus (NRM) and the adjacent ventromedial medullary reticular formation (MRF,). Cytoarchitechtonic studies have subdivided the MRF, in a number of ways. In the cat, the MRF, has been termed either nucleus reticularis magnocellularis (NRMC) (Taber et al., '60) or the magnocellular tegmental field (Berman, '68). In the rat, the MRF, includes nucleus reticularis gigantocellularis pars alpha (NRGCa) ventrally, nucleus reticularis paragigantocellularis (NRPG) dorsally, and the nucleus reticularis paragigantocellularis lateralis (NRPG1) which is lateral to NRGCa and NRPG (Basbaum and Fields, '84). In the nomenclature of Newman ('85), NRPG is termed NRMCb and NRGCa is termed NRMCa. In the present study, the dendritic fields of physiologically identified cells are examined with regard to the above nuclear boundaries; the terminology of Taber (Taber et al., '60; Taber, '61) and of Basbaum and Fields ('84) is used in the cat and rat, respectively. The atlases of Paxinos and

Fig. 5. Camera lucida reconstruction of the somatodendritic arbor of a ferret on-cell, FIO 16-4-1. The location of this cell within a coronal section of the ferret brainstem is shown in the inset at the lower right. Abbreviations: VII n.: facial nerve; MLF: medial longitudinal fasciculus; P: pyramid; TB: trapezoid body.

Fig. 6 . Camera lucida reconstruction of the somatodendritic arbor of a cat off-cell, CIO 4-5-1. The location of this cell within a coronal section of the cat brainstem is shown in the inset at the lower right. Abbreviations: V,: spinal trigeminal tract; V,,: spinal trigeminal nu-

’

P cleus, pars oralis; CN: cochlear nucleus; LVN: lateral vestibular nucleus; NTB: nucleus ofthe trapezoid body; P: pyramid; RB: restiform body; SOL: lateral superior olivary nucleus; SOM: medial superior olivary nucleus; TB: trapezoid body.

NTB

LV N

Fig. 7. Camera lucidn reconstruction of the somatodendritic arbor of a cat off-cell,CIO 25-8-1. The initial trajectory of the axon is outlined by arrowheads. The inset at the lower left shows the location of this cell within a coronal section of the cat brainstem. Abbreviations: V,: spinal

trigeminal tract; V,: spinal trigeminal nucleus, pars oralis; MLF: medial longitudinal fasciculus: NTB: nucleus of the trapezoid body; SO: superior olive; TB: trapezoid body.

0

w

SOMATODENDRITIC MORPHOLOGY OF RVM CELLS

31

P. MASON ET AL.

32

Fig. 9. Camera lucida reconstruction of the somatodendritic arbor of a cat on-cell, CIO 26-12-1. The inset at the lower left shows the location of this cell within a coronal section of the cat brainstem. Abbreviations: V,r: spinal trigeminal tract; V 7 hours post injection in the cat, > 3 hours in the rat) produced significant changes in the morphology of the soma and dendrites. The dendrites appeared beaded along their length; this was particularly exaggerated on distal dendrites. Both dendritic and somatic spines were very prominent and longer than in tissue from shorter survival times. These post-labeling changes have previously been reported in both

HRP- and Golgi-stained material (Rose, '81; Rose and Richmond, '81; Houchin et al., '83).

Somaticmorphology A total of 44 neurons, including 22 off-cells and 22 on-cells. were labeled in these exDeriments (see Table I ) . Labeled cells were distributed amdng cats (N = 16),ferrets (N = 7), and rats ( N = 21). Most cells (N = 34) were located in NRM or MRF, (see Fig. 1).The remaining cells were located in the MRF, (N = 10). Labeled neurons were classified according to the criteria of Fox et d.('76) (see Table 1).Labeled off-cells were mostly

Fig. 10. Camera lucida reconstruction of the somatodendritic arbor of a rat off-cell, RIO 27-4-1. The initial trajectory of the axon is outlined by arrows. This cell was labeled at the level of the facial nucleus.

SOMATODENDRITIC MORPHOLOGY OF RVM CELLS

33

1-

i

Fig. 11. Camera lucida reconstruction of the somatodendritic arbor of a rat off-cell, RIO 15-8-1.The initial trajectory of the axon is outlined by arrowheads and the course of a collateral is marked with small arrowheads. This cell was labeled a t the level o f the facial nucleus (n. VII) as shown in the lower right inset.

C

r. ear pinch

Fig. 12. A: The intracellularly recorded response of a rat off-cell,RIO 6-2-2,to a pinch of the right ear (bar under trace) is shown. B: The location of this neuron is shown in a coronal section through the facial nucleus (n. VII). C: Camera lucida reconstruction of RIO 6-2-2. The soma is shown in hatching as the exact shape of the soma was obscured by the HRP label. The initial trajectory of the axon is outlined by arrowheads.

pyramid

100 pn

*

36

P. MASON ET AL.

SOMATODENDRITIC MORPHOLOGY OF RVM CELLS

37

fusiform neurons oriented mediolaterally (N = 15); a few of the primary dendrites varied but was never more than 10 off-cells appeared as large multipolar cells (N = 4). Three I*m. off-cellsomata were obscured by either HRP or stained red Stellate neurons appeared as shortened fusiform neublood cells such that the somatic shape could not be rons. These cells typically had four primary dendrites, each determined. Of the off-cells located in the NRM or MRF,, 14 stemming from a corner of the square soma (see Figs. 2K, of 16 somata were fusiform shaped and oriented in the 3A). The average diameter of these cells was 25 pm in the mediolateral direction. The two exceptions were a large cat and 10-15 pm in the rat. multipolar neuron located in the NRM at the level of the The number of primary dendrites was highly variable and caudal facial nucleus and a medium stellate neuron located appeared dependent on species, nuclear location, and phyaidorsolateral to the NRM at the level of the superior olivary ological cell class. For instance in the rat RVM, off-cell complex (see Fig. 3A) in the cat. In the cat, five of six fusiform neurons (N = 5 ) had three to four primary denoff-cellslocated in NRM or MRF, at the level of the superior drites (average 3.4) and on-cell fusiform neurons (N = 5) olivary complex (N = 6) were fusiform in shape and ori- had four to seven primary dendrites (average 5.2). In the ented mediolaterally (see Fig. 3). In contrast, most off-cell cat RVM, off-cell fusiform neurons (N = 6) had three to six somata in the MRF, were large and multipolar in shape primary dendrites (average 4.1) and on-cell fusiform neu(314). rons (N = 2) had six or seven primary dendrites (average On-cells were either large multipolar (N = 7) or stellate 6.5). En the cat, large multipolar off-cells (N= 2) had nine (N = 4) or medium fusiform (N = 9) in somatic shape; the or ten primary dendrites and large multipolar on-cells somatic shape of two on-cells could not be determined. (N = 2) had six or seven primary dendrites. In the rat, a Fusiform on-cells were either oriented mediolaterally single multipolar neuron of each cell class was labeled; the (N = 5 ) or dorsoventrally (N = 4) in the coronal plane. In on-cell had five and the off-cell seven primary dendrites. the cat, labeled on-cells located in NRM or MRF, at the level Although the sample size is too small for statistical signifiof the superior olivary complex were either large multipolar cance, the average number of primary dendrites for each neurons or small fusiform cells oriented dorsoventrally (see type of cell was clearly lower in the rat than in the cat. Fig. 4). As in the case of the off-cells, most on-cells that were located in the MRF, were large multipolar neurons (314) Dendritic arbor and morphology and one such cell was a medium stellate neuron. The dendritic arbors of all labeled cells extended in all The somatic shape of neurons in NRM and MRF, has directions from the soma. In all cases, the dendritic arbor in been correlated with their position relative to the midline the coronal plane reached beyond the boundaries of the (Skagerberg and Bjorklund, '85; Newman, '85). Neurons nucleus that contained the soma. The dendritic fields were that lie lateral to the midline, among the fibers of the also bilateral since some dendrites of all neurons extended medial lemniscus, are usually fusiform in shape and oriacross the midline. ented mediolaterally whereas cells on the midline in NRM The extent of the dendritic field was not dependent on the are typically round or stellate. It is interesting t o note that shape of the labeled soma or the physiological cell class but of cells located on or close to (50 pm in the rat, 100 pm in did appear to depend on where the soma was located. Cells the cat) the midline, 313 rat off-cells and 1/2 cat off-cells are in the NRM and MRF, had dendritic arbors distinct from medium fusiform neurons oriented mediolaterally. In con- those of neurons in the MRF,. Whereas the dendrites of trast, none of the three rat on-cells and neither of the two MRFd cells extended radially from the soma in all direccat on-cells located on the midline are mediolaterally ori- tions, delineating a spherical dendritic domain (see Fig. 5 ) , ented fusiform neurons. the dendritic domains of on- and off-cells in the NRM and The long axis of the large multipolar somata measured MRF, were compressed in several directions. The most 40-80 pm in the cat and 30-40 pm in the rat (see Figs. characteristic feature of their shape was that they were 2C,D,M, 4A,B). These cells had six to ten primary dendrites markedly compressed dorsally and somewhat flattened in the cat and five to seven in the rat. The diameter of the along the rostrocaudal axis. Thus, the dendrites of RVM primary dendrites had a range of 2 to 20 pm. Usually one or cells were primarily extended in the mediolateral direction two of the dendrites was much thicker than the others. (see Figs. 6-14). Large multipolar neurons in the MRF, were larger than Dendrites that extended laterally typically terminated at those labeled more ventrally. the lateral edge of the pyramids or trapezoid body. RVM Fusiform somata had a length of 20-75 pm in the cat and neurons usually had one or two dendrites that extended 15-60 pm in the rat and a width of 10-25 pm in the cat and ventrolaterally. In the rat, these dendrites ended in the up to 10 bm in the rat (see Figs. 2A,B,EJ,L, 3B-E, 4C,D). reticular formation lateral to the pyramid, near the edge of The ratio of somatic length to width in fusiform neurons the medulla. In the cat, these dendrites arced over the was always greater than two and could be as high as eight. trapezoid body or pyramid and ended a t its lateral edge. The The primary dendrites of these cells exited from the two mediolateral range covered by RVM cells in the cat was poles of the neuron and numbered three to six in the both 1,500-2,500 pm and in the rat was 800-1,700 bm. Althe cat and rat. As in the multipolar neurons, the thickness though dendrites always extended from the soma across the midline into the contralateral brainstem, the extent of the dendritic domain and the number of dendritic processes were much greater ipsilaterally, especially in the cat. No Fig. 13. Camera lucida reconstruction of the somatodendritic arbor dendrites reached into the facial nucleus, the nucleus of the of a rat on-cell, RIO 36-3-1,labeled by intracellular injection ofbiocytin. trapezoid body, 01the superior olivary complex. The initial trajectory of the axon is outlined by arrowheads. This cell RVM on- and off-cell dendrites often extended far venwas labeled at the level of the facial nucleus (n. VII) as shown in the trally, up to 1100 pm in the cat or 500 pm in the rat, lower left inset. Abbreviations: MVN: medial vestibular nucleus; MLF: medial longitudinal fasciculus; V,: spinal trigeminal tract; V,: spinal coursing into the trapezoid body and the pyramids (see Figs. 7-12, 14) in most cases. In contrast to the ventral trigeminal nucleus, pars oralis; N. VII: facial nucleus; P: pyramid.

P. MASON ET AL.

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a

SOMATODENDRITIC MORPHOLOGY OF RVM CELLS

39

100 ,urn Fig. 15. Branching diagram of cat off-cell, CIO 4-5-1. Straight lines are drawn between dendritic branch points. Processes that travel caudally are shown in solid lines and those that travel rostrally are shown in dashed lines. A camera lucida reconstruction of this cell is shown in Figure 6 .

extension, the dorsal extension of these dendrites was significantly less (see Figs. 6-14). Dendrites rarely extended dorsally from the soma by more than 600 pm in the cat or 400 pm in the rat. No RVM neurons had a dendritic arbor that extended into the dorsal reticular formation. In the sagittal plane, on- and off-cell dendritic arbors were restricted. Often, the bulk of the dendritic arbor, especially the lateral portions, could be seen within 100200 pm of the soma. For instance, in the case of cat on-cell CIO 26-4-1, several of the longest dendrites that are shown in the reconstruction of Figure 9 are observed in the same thick section that contains the labeled soma (see Fig. 1B). Typically, the dendrites that travelled the farthest in the rostrocaudal direction were located on the midline rather than farther lateral. These dendrites, usually one or two secondary branches for each cell, traveled up to 400 pm rostrally or 350 pm caudally from the soma. The entire sagital range of RVM on- and off-cells rarely exceeded 600-800 pm in either rat or cat. The dendritic branching pattern of all labeled cells resembled that reported previously for brainstem raphe and reticular cells (see Discussion). Filled dendrites tended to be long and unbranching (see Fig. 15). In most cases, the highest-order dendrite found was third order. In some cases, fifth- or sixth-order branches were seen that stemmed ultimately from the thickest dendrite of the cell (see above). The daughter branches were typically longer than the parent dendrites from which they derived. Many of the labeled cat cells had terminal dendritic tufts (see Figs. 6-8, 15).Spines were rare but present on some on-cells and some off-cells (see Fig. 16). The above dendritic characteristics were true in all species.

DISCUSSION Off-cells may represent a non-exclusive anatomical subpopulation within the RVM that is distinguished by somatic shape. Most RVM off-cells are fusiform neurons that have their long axis orientated mediolaterally. This may not be unexpected for off-cells located in the MRF,, since uncharac-

terized neurons that lie lateral to the midline, in the ventral portion of the cat NRMC or in the rat NRGCa, are usually mediolaterally orientated fusiform neurons. In contrast, uncharacterized neurons situated on the midline in NRM tend to have a spherical or stellate shape (Skagerberg and Bjorklund, '85; Newman, '85). Most labeled off-cells, located both medially and laterally in the RVM (see Results), were fusiform in shape, evidence that off-cells represent a somatic subset of RVM cells. On-cell somata include cells representative of all the somatic shapes observed in RVM. In the cat, both multipolar and dorsoventrally oriented fusiform on-cells were labeled. The two dorsoventrally oriented fusiform on-cells were similar to each other not only in somatic morphology but also in having a locally restricted axonal projection (Mason and Fields, '89); no other cat on-cells showed either of these characteristics. In the rat, on-cells were mostly stellate or fusiform in shape; few multipolar on-cells were labeled. In comparison with the size and shape of RVM neurons stained in Golgi studies, the present sample of labeled onand off-cells includes fewer small neurons than would be expected in a random sample (Fox et al., '76; Newman, '85). This is not surprising since the intracellular injection technique is biased against recording from small cells. In addition, as described above, a high proportion of both onand off-cells are fusiform neurons, oriented either mediolaterally or dorsoventrally. This high proportion can not be explained by a bias of the technique used since previous intracellular studies of RVM neurons, which were not characterized as on- or off-cells, labeled mostly large multipolar or medium stellate neurons (Maciewicz et al., '84; Mason et al., '86). These results are further evidence that on- and off-cellsbelong to anatomical subpopulations within RVM. The dendritic morphology of RVM and MRF, on- and off-cells is consistent with previous observations in the cat and rat (Valverde, '61; Leontovich and Zhukova, '63; Ramon-Moliner and Nauta, '66; Fox et al., '76; Maciewicz et al., '84; Newman, '85; Edwards et al., '87; Mason et al., '86).

40

A

B

Fig. 16. Photomicrographs of dendritic spines on HRP-labeled neurons. A: A dendritic spine (arrow) on a proximal dendrite of on-cell, CIO 26-12-1. B: Two dendritic spines (arrows)on a proximal dendrite of on-cell, CIO 26-4-1. C: Two dendritic spines (arrows) on a distal dendrite of on-cell, CIO 26-12-1.The scale bar in A applies to A-C.

Labeled dendrites of both on- and off-cells followed a relatively straight course, tapered as they traveled distally, and rarely branched as they radiated away from the soma. The daughter branches were usually longer than the dendritic branch from which they stemmed except at the end of dendritic extensions where tufts of several short processes were sometimes observed. Dendritic spines, although some-

P. MASON ET AL. times present, were never prominent or numerous. The generalized somatodendritic morphology of isodendritic raphe and reticular neurons has been interpreted as a sign of their integrative function (Valverde, '61; Leontovich and Zhukova, '63; Ramon-Moliner and Nauta, '66). On- and off-cell dendritic domains largely respect the boundaries of the RVM but not those of its component nuclei. The dendritic arbor of individual RVM neurons was always bilateral, including the NRM, the bulk of the ipsilateral MRF,, much of the contralateral MRF,,, and in most cases ventral extensions into the trapezoid body or pyramid. In the dorsal direction, however, dendrites did not extend into the MRF,. Although the dendritic domains of individual on- or off-cells did not ramify throughout the RVM, the composite dendritic domain of the population of on- and off-cells includes the entirety of RVM. Since the dendritic trees of on- and off-cells extend laterally for about 1 mm and may extend for up to 2 mm, afferent fibers to the RVM may contact individual neurons whose somata are located within a broad spatial extent. 'The extensive dendritic fields of individual RVM neurons in a region that is traversed by many axonal tracts provide a substrate for the integration of a wide variety of neuronal inputs (Leontovich and Zhukova, '63; Ramon-Moliner and Nauta, '66). In fact, single RVM neurons receive a large number of both axodendritic and axosomatic synaptic inputs (Leontovich and Zhukova, '63; Fox et al., '76). RVM neurons receive dense projections from dorsal and lateral PAG, the midbrain nucleus cuneiformis, and the parabrachial nuclei (Gallagher and Pert, '78; Abols and Basbaum, '81; Carlton et al., '83; Lakos and Basbaum, '88). Stimulation of the PAG excites on- and off-cells in RVM (Vanegas et al., '84a) and produces an RVM-mediated suppression of spinal nociceptive sensory cells and nociceptive reflexes (Behbehani and Fields, '79; Gebhart et al., '83; Chung et al., '87). The RVM also receives a significant projection from the medial and anterior hypothalamic regions and from frontal cortex (Hosoya, '85; Luppi et al., '88; Holstege, '87). Other afferent inputs to the RVM include projections from the solitary tract nucleus (Beitz, '82), the vestibular nuclei (Gallager and Pert, ' 7 8 ; Abols and Basbaum, '811,the subcoeruleus nucleus (Sakai, ' 8 0 ) ,and motor cortex (Itossi and Brodal, '56; Kuypers, '58). In contrast to the MRF,, which receives a strong input from the ascending spinoreticular tract, somata in the NRM itself receive few direct afferents from ascending spinal or trigeminal somatosensory tracts (Kevetter and Willis, '83). A moderate terminal field from ascending tracts is found amongst the somata in the MRF, adjoining NRM. The terminal field of anterogradely labeled spinoreticular fibers overlaps with the spatial domains of long dendrites of RVM on- and off-cells in the repon just lateral to the pyramids (Newman, '851, raising the possibility of a direct afferent input from spinoreticular axons to RVM neurons, including those with somata in NRM. Somatosensory input may also reach RVM cells indirectly through a relay in the MRF,; consistent with this idea, RVM neurons recc,'w e a monosynaptic excitatory input from MRF, stimulation (Maciewicz et al., '84; Mason et al., '86). The dendritic domains of all labeled on- and off-cells included areas traversed by ascending axons in the medial lemniscus (Valverde, '61; Newman, '85). Although ascending fibers from the dorsal column nuclei have long been thought to represent a homogeneous lemniscal pathway, these fibers may collateralize within the medullary raphe

SOMATODENDRITIC MORPHOLOGY OF RVM CELLS

41

Off -cell axon

Caudal RVM

Fig. 17. Diagram that shows the planar organization of RVM on- and off-cell dendritic fields as discussed in the text. Single off-cell axons have axonal collaterals throughout the rostrocaudal length of the RVM,and may contact many on- and/or off-cells throughout the nucleus (see text).

and reticular nuclei (Saade et al., '82, '83; R. Maciewicz, unpublished observations). Medial lemniscal fibers may, in fact, underlie the inhibition of NRM neurons evoked by dorsal column stimulation (Saade et al., '82). In both rat and cat, the electrical threshold or opiate dose required to suppress nociceptive reflexes does not change significantly as the stimulation or injection site is moved from the NRM laterally to the MRF, (Satoh et al., '80; Zorman et al., '81; Dostrovsky et al., '82; Sandkuhler and Gebhart, '84; Barbaro et al., '85; Jensen and Yaksh, '86a). Lesions of both the NRM and MRF, are necessary to completely block tail flick suppression evoked by PAG stimulation (Sandkuhler and Gebhart, '84),evidence that both regions are involved in the descending modulation of nociception. The similarity of the nociceptive modulatingeffects evoked from NRM and MRF, is not surprising in view of the present results indicating that the RVM is a functionally unified region with regard to the neurons involved in modulation of nociceptive transmission. Since the dendrites and somata of on- and off-cells in NRM and MRF, overlap extensively, the cell populations in these two regions can not be manipulated independently by lesions or electrical stimulation. Furthermore, microinjection of any substance into MRF, or NRM will also affect the dendrites of somata located in the neighbouring nucleus. The intermingling of somata and the spatial domains of dendritic arbors of on- and off-cells are further evidence that the onand off-cells in NRM and MRF, act as an integrated unit in the modulation of nociceptive transmission. This functional link between neurons in NRM and MRF, has led to use of the term "RVM" to denote a functionally unified and anatomically contiguous brainstem region that modulates nociceptive transmission (Basbaum and Fields, '84). Although the above evidence argues for the functional unity of the RVM, the antinociceptive effects of NRM and MRF, stimulation may be distinguished on pharmacological grounds. Some reports have indicated that the nociceptive reflex suppression produced by stimulation, either electrical or opiate, of the NRM, but not the MRF,, is largely reversed by serotonin antagonists whereas alpha-adrenergic antagonists reverse the antinociception produced by MRF,, but not NRM, activation (Satoh et al., '80; Jensen

and Yaksh, '86b; Pretel et al., '88; cf. Barbaro et al., '85). The functional differences between the NRM and MRF, regions implied by these pharmacological studies may be due to the action of a subpopulation of on- or off-cells that were either underrepresented or not labeled in the current study. For example, serotonergic NRM neurons, which are typically small (Bowker et al., '88) and may be underrepresented in the present sample of intracellularly filled neurons, may have dendritic domains that are restricted within the NRM and therefore not activated by MRF, stimulation. As mentioned in the Introduction, simultaneous recordings of RVM on- and off-cells have demonstrated that cells of a single class have closely correlated spontaneous activity whereas off-cells have periods of spontaneous firing that are reciprocal with on-cell activity periods (Barbaro et al., '89). In these experiments, the recordings were taken from units that were on opposite sides of the midline and were separated by at least 1 mm in the sagittal plane. Since the dendritic arbors of RVM on- and off-cells are bilateral, RVM cells with somata on either side of the midline could receive the same input, even if the afferent terminals were restricted unilaterally. However, on- and off-cell dendrites have a limited rostrocaudal extension and it is therefore unlikely that neurons separated by 1 mm in the sagittal plane would have any region of overlapping dendrites where both could receive afferent contacts from the same local terminal arbor (Scheibel and Scheibel, '58; Valverde, '61; Newman, '85). Therefore, the optimal input from a single axon to on- and off-cells for the coordination of their spontaneous activity would have collaterals at several rostrocaudal levels of the RVM (see Fig. 17). Consistent with a role for off-cells in the generation of coordinated periods of on- and off-cell spontaneous activity, single off-cell axons collateralize within the RVM at several rostrocaudal levels (Mason and Fields, '89). This arrangement makes it possible for off-cells to contact multiple onand/or off-cells throughout the length of the medullary and caudal pontine NRM and MRF, (see Fig. 17). In contrast, the terminals of single on-cell axons are restricted within the coronal plane of the parent soma (Mason and Fields, '89). On-cells therefore cannot play a major role in the coordination of RVM on- and off-cell activity. Thus, off-cell activity will have a more widespread direct effect on the

P. MASON ET AL.

42

activity of other RVM neurons than will on-cell activity (Mason and Fields, '89). Excitatory connections between off-cells but not between on-cells would also explain the finding that although on- and off-cells have opposing effects on nociceptive transmission, RVM stimulation consistently results in antinociception. In this situation, direct activation of a small number of off-cells would indirectly activate many other off-cells in RVM. The functional significance, if any, of the planar organization of RVM on- and off-cell dendritic domains is unclear as few anatomical studies, to date, have looked for this type of organizational principle. It is unlikely that this feature relates to a somatotopic organization since no somatotopy has been demonstrated within RVM by using either anatomical or physiological techniques. However, it is possible that the afferents to or efferents from the RVM may be organized along the sagittal plane. The distribution of neurotransmitters or receptors may similarly be distributed to either restricted or widespread sagittal regions of the RVM.

ACKNOWLEDGMENTS This research was supported by PHS grant NS-21445 and by a generous gift from the Bristol-Myers corporation. P.M. was supported by a PHS fellowship NS-07265 and by a Presidential Fellowship from the University of California Board of Regents. M.K.F. was supported by PHS training grant NS-07265. The authors thank Carla Schatz for providing ferrets, Susan Elliott and Lael Carlson for editorial assistance, Mechelle Williams for histological assistance, and the staff of Biomed Arts for assistance with the illustrations.

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