THE JOURNAL OF COMPARATIVE NEUROLOGY 321:488-499 (1992)

Retrograde Tracing of Projections Between the Nucleus Submedius, the Ventrolateral Orbital Cortex, and the Midbrain in the Rat J.A. COFFIELD, K.K. BOWEN, AND V. MILETIC Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, Wisconsin 53706

ABSTRACT indocarbocyaThe fluorescent tracers fluoro-gold and l,l'-dioctadecyl-3,3,3,3-tetramethyl nine perchlorate were used as retrograde markers to examine reciprocal connections between the rat nucleus submedius and the ventrolateral orbital cortex. In addition, midbrain projections to each of these regions were examined. In the prefrontal cortex, we found that input from the nucleus submedius terminates rostrally within the lateral and ventral areas of the ventrolateral orbital cortex. Conversely, the cortical input to the nucleus submedius originates from the medial and dorsal parts of the ventrolateral orbital cortex. Our data also demonstrated that neurons from the ventrolateral periaqueductal gray and the raphe nuclei' project to the midline nuclei of the thalamus, including a small projection to the nucleus submedius. We further determined that regions within the ventrolateral periaqueductal gray and raphe nuclei project to the ventrolateral orbital cortex, and that these regions overlap with those that project to the nucleus submedius. These findings suggest that the nucleus submedius might be part of a neural circuit involved in the activation of endogenous analgesia. a 1992 Wiley-Liss, Inc. Key words: thalamus, frontal cortex, periaqueductal gray, fluorescent dye, pain, analgesia

The nucleus submedius (SM) is located ventromedial to focused most recently on an examination of its reciprocal the internal medullary lamina of the thalamus, and is connections with the frontal cortex of the rat. In addition, considered part of the ventromedial complex (Krettek and because of the proposed role of the SM in nociception, we Price, '77;Berman and Jones, '82; Paxinos and Watson, were interested in examining its possible connections to '86).Craig and Burton ('81)established that, in the cat, the midbrain regions involved in analgesia. Current observadorsal portion of the SM receives topographically organized tions from our laboratory indicate that SM neurons responprojections from both the medullary (trigeminal) and spinal sive to noxious peripheral stimulation are inhibited by the dorsal horns. These projections appeared to arise exclu- local application of opioids (Coffield and Miletic, '90). Cortical projections of the SM have been studied more sively from neurons located in lamina I. These neurons have long been postulated to play an important role in thoroughly in the cat than in the rat. Anterograde tracing nociception (e.g., review by Dubner and Bennett, '83).As a experiments indicated that in the cat, the SM was reciproresult, Craig and Burton ('81)proposed that the SM might cally and topographically connected with the prefrontal play an important role in nociceptive information process- cortex (Craig et al., '82). Specifically, SM efferents terminated in layer 3 of the ventrolateral orbital cortex (VLO). ing. Recent physiological studies in rats have provided addi- This projection was reciprocated by a projection from the tional support for this proposal. Dostrovsky and Guilbaud deep layers of the VLO to the SM. Many regions of the ('88) demonstrated the presence of neurons responsive to orbital cortex, including parts of the VLO, have been shown thermal and mechanical noxious stimuli in the SM of both to be connected with the limbic system and/or basal ganglia normal and arthritic rats. We have also reported a variety of neuronal types in the SM of normal rats, including neurons Accepted March 17, 1992 responsive to noxious stimuli, as well as neurons activated Address reprint requests to V. Miletic, Department of Comparative exclusively by innocuous stimulation (Miletic and Coffield, Biosciences, University of Wisconsin-Madison, 2015 Linden Drive West, '89). In our continuing investigation of the SM, we have Madison, WI 53706.

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CONNECTIONS OF THE SM, VLO, AND MIDBRAIN IN THE RAT (Craig et al., '82).However, the VLO region receiving input from the dorsal SM differed somewhat, in that it projected primarily to the somatosensory cortical areas I, 11,111, and the midbrain periaqueductal gray (PAG). The midbrain PAG has a high opioid content and is known to be important for analgesia. Stimulation-produced analgesia can be elicited from many regions of the PAG (Basbaum et al., '77;Rhodes, '79),with stimulation of the ventrolateral region producing the most potent analgesia (Gebhart and Toleikis, '78).This region of the PAG may be responsible for activating a descending analgesia pathway, probably involving the raphe nuclei. Hardy and Haigler ('85) have shown that electrical stimulation of the prefrontal cortex inhibits some PAG neurons that are responsive to noxious stimuli. The activity of prefrontal cortical neurons can be inhibited by stimulation of mesencephalic raphe nuclei (Mantz et al., '90) including the dorsal raphe nucleus, which is found in the PAG along the ventral midline. Thus, the PAG is strategically situated to transmit or modulate nociceptive inputs between the lower brainstem, thalamus, and cortex. In the rat, previous studies of thalamic projections to the prefrontal cortex have concentrated mainly on the medial dorsal nucleus (Jones and Leavitt, '74;Krettek and Price, '77;Divac and Kosmal, '78;Beckstead, '79;Gerfen and Clavier, '79).Evidence from these investigations suggested that SM efferents also project to the VLO. However, because the emphasis of these early studies was not on the SM or the VLO, evidence for the presence and organization of reciprocal connections between these two regions is indirect. Similarly, Ma et al. ('88) elegantly showed that within the SM, anatomical substrates exist for a monosynaptic relay between the trigeminal nucleus and the VLO. However, the emphasis here was on the ultrastructure of synaptic interactions in the SM rather than the organization of its connections to the VLO. Recently, Dado and Giesler ('90)have conclusively demonstrated differences between the cat and the rat in the organization of trigeminal and spinal projections to the SM. In the rat, the trigeminal, and especially spinal, afferent projections are substantially smaller than in the cat. Those rat spinal neurons that do send their axons to the SM are located in deeper spinal laminae, rather than lamina I. It is quite possible that differences between the two species also exist in efferent projections. Little is also presently known

about connections of the rat VLO with other cortical regions. Efforts to examine midbrain connections with the medial thalamus or frontal cortex in the rat have also not focused on the SM or the VLO, and little is known about these relationships as well. The purpose of this study was to employ retrograde tracing methods to examine the organization of reciprocal connections between the SM and VLO of the rat, and to investigate the connections of both of these areas with the midbrain.

MATERIALS AND METHODS Surgery and dye injections The fluorescent tracers fluoro-gold (FG, Fluorochrome, Inc.; Schmued and Fallon, '86) and l,l'-dioctadecyl-3,3,3',3tetramethyl indocarbocyanine perchlorate (DiI, Molecular Probes; Honig and Hume, '86) were used to determine the distribution of the anatomical connections between the SM, frontal cortex, and midbrain. Sprague-Dawley rats (275300 g) were anesthetized initially with pentobarbital sodium (50 mg/kg i.p.), and maintained, if necessary, with additional injections of ketamine (100 mg/kg i.p.1. Body temperature was monitored, and maintained with a heating pad between 36-37.5"C.The animal was placed in a stereotaxic frame, and a 15 mm skin incision was made along the midline of the skull. Prior to the incision, the skin area was infiltrated with 0.2 ml of 0.75% bupivacaine HCl. A craniotomy was created with a surgical drill over the desired brain areas using stereotaxic coordinates (Paxinos and Watson, '86). For SM injections, the coordinates were 2.0-3.0 mm caudal to the bregma, .4-1.0 mm off the midline, and 6.0 mm below the cortical surface. For VLO injections, the coordinates were 3.0-4.5mm rostra1 to the bregma, 1.5-2.5 mm lateral to the midline, and 5.0 mm deep below the skull surface. Pressure injections (5psi) of filtered 2% FG or .25% DiI (in distilled water or normal saline) were made with a single barrel micropipette (5-15 pm tip). Survival periods ranged from two to six days.

Histology Following appropriate survival periods, the rats were deeply anesthetized with pentobarbital, and perfused through the heart with heparinized saline (1liter) followed

Abbreviations A8 cc cg3 CG Cli CM DP DR dtg Frl Fr2

IAM IC LO MD Me5 ml mlf MnR MO

dopaminergic cells corpus callosum cingulate cortex area 3 central gray or periaqueductal gray caudal linear raphe nucleus central medial thalamic nucleus dorsal peduncular cortex dorsal raphe nucleus dorsal tegmental bundle frontal cortex, area 1 frontal cortex, area 2 interanterodorsal thalamic nucleus inferior colliculus lateral orbital cortex medial dorsal thalamic nucleus mesencephalic trigeminal nucleus medial lemniscus medial longitudinal fasciculus median raphe nucleus medial orbital cortex

mt

ON PAG PMR PPTg Re RF Rh RtTg SM SMd SM, suco

VL VLO

VM VPL VPM VTA

mamillotbalamic tract olfactory nuclei periaqueductal gray (central gray) paramedian raphe nuclei pedunculopontine tegmental nucleus reuniens thalamic nucleus rhinal fissure rhomboid thalamic nucleus reticulotegmental nucleus pons submedius thalamic nucleus (gelatinosus) submedius thalamic nucleus (gelatinosus)dorsal part submedius thalamic nucleus (gelatinosus)ventral part superior colliculus ventral lateral thalamic nucleus ventral lateral orbital cortex ventral medial thalamic nucleus ventral posterolateral thalamic nucleus ventral posteromedial thalamic nucleus ventral tegmental area

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by 4% paraformaldehyde in 0.1 M phosphate buffer (1liter). Brains were divided coronally into three blocks which included the frontal cortex, thalamus, and midbrain, respectively. Coronal sections of the injection sites were cut at 100 pm on a vibrating microtome, mounted on slides in a 3:l glycerin/phosphate buffered saline mixture, and examined with a fluorescent microscope. Tissues examined for retrogradely labeled cells (SM, VLO, or midbrain) were sectioned at 30-50 km, and mounted similarly. FG was visualized with an excitation wavelength of 330-380 nm (Nikon UV-2A filter system). DiI fluoresced orange-red, and was viewed with the Nikon G filter system (536-556 nm).

Localization of fluorescent label In order to accurately localize the fluorescent label (injection site and retrogradely labeled neurons), tissue sections containing the fluorescent label were compared with cresyl violet-stained control sections of the same regions (Fig. 1). We also counterstained some of the tissue sections containing the fluorescent label, although this resulted in considerable wash-out of the DiI label, and some wash-out of the FG label. We have found that the stereotaxic atlas of Paxinos and Watson ('86) very accurately depicts the rat brain, and corresponds well with our rat brain sections. For illustrative purposes in this paper, the locations of injection sites and retrogradely labeled neurons, and the extent of injection spread were plotted on copies of appropriate sections from this atlas. This should simplify any comparisons between ours and other studies, and allow one to employ stereotaxic coordinates to perform physiological recordings in an area of interest.

Quantification and description of results The purpose of the present study was to examine and describe the existence of projections between the SM, VLO, and the midbrain. Variability is an inherent characteristic of any type of retrograde or anterograde labeling study, including studies using micropressure application (Palmer et al., '80). Strict quantification of the results obtained from these types of studies is of little value. For these reasons, we have not attempted to quantify the numbers of retrogradely labeled cells, but instead, have limited our analysis to a qualitative description of our results. For a single experiment, the intensity of label within a single neuron and the number of neurons labeled within a particular region is also dependent upon a variety of parameters. However, in those instances when the intensity of neuronal labeling and the distribution and general number of neurons labeled within a particular region is consistent across experiments, we have included those characteristics in the description of the results.

RESULTS The cytoarchitecture and nuclear boundaries of the SM and the VLO of the rat have been well described elsewhere (Krettek and Price, '77; Ma et al., '88) and will not be detailed here. Briefly, the SM is a small oblong nucleus that lies ventral to the internal medullary lamina, and is bounded by the nucleus reuniens medioventrally, the rhomboid and central medial nuclei mediodorsally, and the mamillothalamic tract ventrolaterally (Fig. IB). The VLO envelops the rhinal fissure at its medial limits, and is respectively

bounded by the lateral and medial orbital regions of the frontal cortex (Fig. 1A).

VLO neurons retrogradely labeled from the SM Fifteen rats received unilateral pressure injections of dye into the SM (DiI n = 12, FG n = 3). Retrogradely labeled neurons were observed bilaterally throughout the rostrocaudal extent of the VLO, with the majority of labeled cells located ipsilaterally. Most of the labeled neurons were confined to cortical layers 5 and 6. Densely packed cells were found in layer 5 , with more diffuse labeling present in layer 6. In general, when the dye injection filled the whole SM, labeled neurons were concentrated in a narrow band bordering the medial edge of the VLO, curving ventrally toward the olfactory cortex. This was especially evident in the caudal aspect of the VLO. Fewer labeled neurons were noted in layers 5 and 6 of the lateral edge of the VLO and lateral orbital cortex (LO). This distribution of labeled cells was consistent from rat to rat. More diffusely located, labeled neurons were noted in layers 5 and 6 of the medial prefrontal cortex (medial orbital cortex, cingulate cortex areas 1and 3, frontal cortex area 2). Slight variations in labeling were noted when different regions of the SM were injected. In three cases where the injection was centered in the dorsal SM (with little or no dye spread into the ventral SM), the retrogradely labeled neurons extended from the lateral VLO rostrally to the medial VLO caudally. Conversely, in two cases where the dye injection included mostly the ventral SM, the greatest number of retrogradely labeled cells extended from the medial VLO rostrally to the lateral VLO caudally, The number of retrogradely labeled neurons in the medial prefrontal cortex increased with the spread of the injection site into the rhomboid and reuniens nuclei. Injection sites centered dorsal to the SM (e.g., in central medial, interanterodorsal, and mediodorsal nuclei), or medial to the SM (rhomboid and reuniens nuclei) resulted in decreased labeling in the VLO, and a corresponding increase in labeling in the medial prefrontal cortex. Figure 2A illustrates a DiI injection site in the SM. Figure 2B shows the location of retrogradely labeled neurons in the VLO and LO. The extent, size and spread of the injection is depicted schematically in Figure 3A. The dye was concentrated in the middle of the SM, completely filling the ventral SM and extending into the dorsal SM. There was also a small amount of dye visible in the surrounding rhomboid, reuniens, anteromedial, central medial, and ventral medial nuclei. The distribution of retrogradely labeled neurons in the VLO and prefrontal cortex is depicted schematically in Figure 3B. Labeled neurons were found bilaterally throughout the rostrocaudal extent of the VLO and LO. Most of the labeled neurons were located ipsilaterally in deep layers of the VLO. A small band of neurons was observed in the deep layers of the contralateral VLO (not illustrated). Diffusely located retrogradely labeled neurons were noted bilaterally in the deep layers of the medial orbital, cingulate and frontal cortices.

SM neurons retrogradely labeled from the VLO Twenty-one rats received fluorescent dye injections into the area of the VLO (DiI n = 12, FG n = 9). In general, the

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Fig. 1. A Photomicrograph of a cresyl violet-stained section representative of the rat orbital cortex in the coronal plane (50 pm thick sect,ion; magnification = 10x1. The VLO encompasses the medial and dorsal aspect of the rhinal fissure (RF). Refer to the list of abbreviations for names of laheled nuclei. Medial is to the right, and dorsal is up. B: Photomicrograph of a cresyl violet-stained section representative of the

rat ventromedial thalamus in the coronal plane (50 pm thick section; magnification = l o x ) . The SM is hounded dorsomedially by the Rh and CM nuclei, ventromedially by the Re nucleus, and ventrolaterally by the mt. The dorsal part of the SM (SMJ is separated from the ventral part (SM,) by a thin fiber lamina extending mediolaterally. Dorsal is up. Scale bars = 200 wm for both A and B.

greatest number of retrogradely labeled SM neurons was noted when the injection site was centered around or encompassed the ventrolateral and rostral aspect of the VLO,including the medial aspect of the LO.The label in the SM was invariably ipsilateral with few SM neurons labeled contralateral to the injection site. Different areas of the SM were labeled differently depending on the placement of the dye in the VLO. In two cases where the VLO injection was located medially, retrogradely labeled neurons were noted in the anteroventral, but not dorsal, SM. The dorsal SM was labeled when the injection tvas situated more laterally and ventrally in cortical layers 1-3 (n = 4). I n addition, when the injection site was

centered more medially to include the medial and ventral orbital cortices, other thalamic nuclei (mediodorsal, central medial, anteromedial) contained labeled neurons. When the injection spread more ventrally to include the dorsal and medial parts of the rhinal sulcus (layer l), then the thalamic ventromedial nucleus was labeled. In two other cases where the injection site was located in the medial and caudal aspect of the VLO, no label was noted in the SM. Injection into the olfactory cortex (just ventral to VLO), or the claustrum (just caudal to VLO) did not lead to labeling in the SM. Figure 4A illustrates a representative F-G injection site in the VLO,and Figure 4B shows the retrogradely labeled

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Fig. 2. A. Photomicrograph of a DiI injection site in the SM (coronal plane; transmitted light; 100 pm thick section; magnification = 10x1. The white asterisk designates the center of the injection site. The black arrowheads delineate the extent of spread of the dye through the SM. Dorsal is up. B: Fluorescent photomicrograph showing the distribution of retrogradely labeled VLO neurons following the Dil injection in the SM (coronal plane; 50 pm thick section; magnification = l o x ) . White

arrowheads indicate the narrow band of labeled neurons in layers 5 and 6 of the VLO and LO. The white asterisk in the lower right indicates a collection of labeled neurons that appear to be in the dorsal peduncular cortex. The single white arrow in the lower left denotes the location of the rhinal fissure (RF). Refer to Figures 1and 3 for further orientation. Medial is to the right, dorsal is up. Scale bars = 250 I*.mfor both A and

neurons in the SM following the injection illustrated in 4A. The rostrocaudal extent, size and spread of the injection are summarized schematically in Figure 5A. The injection was concentrated in superficial layers of the VLO and LO, and

spread into the deeper layers. There was also some spread ventrally into the olfactory cortex. The distribution of retrogradely labeled neurons in the thalamus is represented schematically in Figure 5B. The

Fig. 3. A Schematic illustration of the spread and rostrocaudal extent of DiI injected into the SM. The inner black oval represents the greatest concentration of dye; the outer hatched area represents the spread of dye into the surrounding regions. The top drawing is rostral and the bottom drawing is caudal. The numbers on either side represent rostrocaudal coordinates from the bregma expressed in mm (negative is caudal, positive is rostral). These drawings, and those in subsequent figures, are slightly modified copies of appropriate sections

from the atlas of Paxinos and Watson ('86). Only selected nuclei are labeled. B: Schematic illustration of the ipsilateral distribution of VLO and LO neurons retrogradely labeled with DiI from the SM (black dots). The dots are meant to indicate only density and distribution, and not actual numbers of neurons in a 50 Fm section. Note that most labeled neurons are confined to deep layers, and that some labeled cells are scattered in different areas of the cingulate and frontal cortices.

B.

A

A, ,

- 2.12

4.20

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3.20

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Fig. 4. A Fluorescent photomicrograph of a F-G injection in the VLO (coronal plane; 100 pm thick section; magnification = 25x1. The injection is centered in layer 3 and the dye extends ventrally from the rhinal fissure (RF) into the deep layers of the VLO dorsally, and the medial LO. Medial is to the right, dorsal is up. Scale bar = 50 Fm. B:

Photomicrograph of retrograde label in the SM combining fluorescence with transmitted light (50 pm thick section; magnification = 10x1. White arrowheads indicate the concentration of SM neurons retrogradely labeled with F-G from the VLO. Surrounding nuclei are devoid of any significant label. Scale bar = 200 pm.

labeled neurons were found throughout the rostrocaudal extent of the SM, with most of the label concentrated in the rostral two-thirds of the SM. Just rostral to the SM, a narrow column (approximately 100 km in width) of labeled neurons was noted extending dorsoventrally through the mediodorsal, central medial, anteromedial, rhomboid, and reuniens nuclei. This column of label became less noticeable as the bulk of the SM became visible in subsequent sections.

At the level of the rostral SM (Fig. 5B; top drawing), some label was present in the rhomboid and reuniens nuclei as they surround the medial aspect of the SM. The label could be found in both the dorsal and ventral SM throughout most of its expanse. In the caudalmost aspect of the SM, the label appeared in the SM, the ventral reuniens, and the medial aspect of the ventromedial nucleus (Fig. 5B, bottom drawing).

Fig. 5. A Schematic illustration of the rostrocaudal extent and spread of F-G injected into the region of the VLO. The inner black oval represents the greatest concentration of dye; the outer hatched area represents the spread of dye into the surrounding regions. The top drawing is rostral and bottom drawing is caudal. B.Schematic illustra-

tion of the distribution of SM neurons retrogradely labeled with F-G from the VLO. Note that most of the labeled neurons are confined to the SM, although a few labeled neurons are also seen in the MD, Rh, and Re nuclei. Numbers and dots as in Fig. 3.

A

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Retrogradely labeled neurons were also seen in other areas of the prefrontal cortex. These included a limited bilateral focus in area 2 of the frontal cortex, diffuse labeling in the contralateral VLO, LO, and cingulate cortical areas 1 and 3. Diffusely located retrogradely labeled neurons were also noted in the somatosensory cortex, the amygdaloid nuclei, and the globus pallidus (not illustrated).

small contralateral component. Much of the afferent input from the SM to the VLO terminates more rostrally within the lateral and ventral VLO (layers 1-3). This lateral, ventral, and rostral distribution is especially important for input originating from the dorsal SM. The ventral SM, especially the anterior portion, appears to project to the medial VLO as well. The output from the VLO to the SM originates dorsally in layers 5 and 6 . In the rostral VLO, the Midbrain neurons retrogradely labeled from output seems to originate equally from both the medial and the VLO lateral VLO, although our results suggest that the lateral In four rats, the midbrain was examined for retrogradely VLO projects to the dorsal SM, and medial VLO projects to labeled neurons from the VLO. Labeled neurons were the ventral SM. In the caudal aspect of the VLO, this found throughout the rostrocaudal extent of the ventral relationship seems to be reversed. In addition, the number PAG. Most of the labeled neurons were seen in the contralat- of labeled neurons located caudally in the VLO seems to be eral caudal three-fourths of the PAG, although some ipsilat- higher in the medial VLO, and this corresponds with eral labeling was also noted. No labeled neurons were seen injections into the dorsal SM. Our data support and extend previous studies (Krettek in the dorsal PAG. Figure 6A summarizes the distribution of labeled neu- and Price, '77; Beckstead, '79; Gerfen and Clavier, '79) rons in the midbrain following the injection of F-G into the which examined the projections between the thalamic VLO. Many of the labeled neurons were found along the mediodorsal nucleus and the medial prefrontal cortex. In all ventral midline of the PAG, in the dorsal raphe nucleus, as of these studies, some reference was made to a connection well as deeper midline nuclei including the caudal linear between the prefrontal cortex and the SM. Krettek and and median raphe nuclei. A few neurons were seen in the Price ('77), using autoradiography, noted that when the raphe magnus. Labeled neurons were also found in the injection site included the SM, fiber labeling was present in substantia nigra (mostly contralateral; not illustrated). layers 1and 3 of the VLO. Both Beckstead ('79) and Gerfen Occasionally, neurons were found scattered in the mesen- and Clavier ('79), using anterograde and retrograde tracers, respectively, reported labeling in the SM whenever the cephalic reticular formation and the tegmental nuclei. cortical injection site included the lateral aspect of the VLO. PAG neurons retrogradely labeled Beckstead ('79) noted, however, that labeling occurred only in the ventral SM. This may be due to the fact that the from the SM entire VLO was not injected, and suggests some form of In four rats, the midbrain was examined for retrograde topographic organization to the projection. This is further labeling from the SM. In general, the number of PAG neurons labeled from the SM was smaller than those supported by the results of our study showing that the ventral SM may be labeled from either the medial or lateral labeled from the VLO. The location of neurons in the VLO, while the input from the dorsal part of the SM seems ventral PAG and dorsal raphe labeled from the SM overto terminate more laterally. Gerfen and Clavier ('79) did lapped with those labeled from the VLO, although they not make a similar distinction between dorsal or ventral appeared more scattered in their distribution. In experiparts of the SM, and, in fact, their figures demonstrate ments in which other midline thalamic nuclei were included labeled neurons throughout the SM. in the injection site, increased labeling occurred in the In the cat, Craig et al. ('82) have shown that the ventral and dorsal PAG, and the raphe nuclei. posterodorsal SM projected to the posterodorsal VLOa and Figure 6B summarizes the distribution of labeled neuthe anteroventral SM projected to anteroventral VLOa. rons in the midbrain following the injection of DiI in the This projection terminated mostly in cortical layer 3 and SM. Scattered collections of 2-3 neurons were seen in the was directly reciprocated by a projection from the VLO. ventrolateral PAG and the dorsal raphe. Labeled neurons Differences in the cortical topography between the rat and were also noted in the pedunculopontine tegmental nucat, and hence, in the localization and organization of the cleus. Labeled neurons were also occasionally noted in the VLO, make it difficult to compare the results of the study by mesencephalic reticular formation, the pontine reticular Craig et al. ('82) with ours. It seems apparent from the nuclei (oral), the paramedian raphe nuclei, and the parabra- current study that the dorsal and ventral parts of the SM in chial nuclei. the rat project differently upon the VLO; however, there were no obvious differences in the projections of the rostral or caudal parts of the SM, as seen in the cat. DISCUSSION One explanation for this difference between the two Reciprocal projections between the species may be that, in the cat, the distribution between SM and VLO trigeminal and spinal input to the SM was somatotopically Our results demonstrate that reciprocal connections do organized, with the spinal input terminating in the rostral exist between the SM and the VLO in the rat. These SM and the trigeminal input in the caudal SM (Craig and reciprocal connections are largely ipsilateral, with only a Burton, '81). In the rat, there is less of a spinal component

Fig. 6. A Schematic illustration of the distribution of midbrain neurons retrogradely labeled with F-G injected into the VLO. The retrograde label is confined mostly to the ventrolateral portion of the CG, the DR, and other raphe nuclei. Scattered neuronal labeling was noted in the tegmental nuclei and the mesencephalic reticular forma-

tion. B: Schematic illustration of the distribution of midbrain neurons retrogradely labeled with DiI injected into the SM. The retrograde label is less dense than that seen in A and appears more scattered. Label is found mostly in the ventrolateral CG and the DR. Numbers and dots as in Fig. 3.

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to the SM, and the distribution of this spinal input overlaps that of the trigeminal input to the SM (Craig and Burton, '81; Miletic and Coffield, '89). This lack of somatotopy in the rat SM may be reflected at the cortical level as well. Given the technique used in our study, we cannot state unequivocally that a topographic relationship exists between the SM and VLO of the rat. Even with micropressure application, it is often difficult to obtain the consistently confined injection sites into given regions of the SM or VLO necessary to determine the existence of topographic relationships. However, it is evident from our results that, in the rat, the SM does not project diffusely upon the VLO, but rather in some organized fashion reminiscent of specific thalamic relay nuclei, and similar to the SM projection to the VLO in the cat (Craig et al., '82).

Thalamic and cortical connections with the midbrain Our data also demonstrate that neurons from the ventrolateral PAG and the raphe nuclei project to the midline nuclei of the thalamus, including a small projection to the SM. The significance of this small projection to SM has yet to be determined. In addition, we showed that both the PAC and raphe nuclei project to the VLO. It is interesting to note that the PAG/raphe neurons projecting to VLO and the SM seem to originate from similar regions, i.e., the ventrolateral PAG and the dorsal raphe, because these midbrain regions are important for analgesia. A number of studies using anterograde tracers have examined the efferent projections of the PAG and the raphe nuclei to the thalamus. Using both autoradiography and degeneration techniques, Conrad et al. ('74) identified connections of the dorsal and median raphe nuclei with the mediodorsal, parafascicularis, and reuniens nuclei of the thalamus, and the cingulate and pregenual cortex. Specific reference to the SM was not made. Peschanski and Besson ('841, using the anterograde tracer wheat-germ agglutinin conjugated to horseradish peroxidase, reported moderate projections to the SM from the raphe magnus and raphe medianus, a light projection from the raphe dorsalis, and none from the ventral PAG. This differs somewhat from our data. In our studies, it appears that when the injection was confined to the SM, we saw most of our labeling in the nucleus raphe dorsalis and the ventral PAG. The atlases used by Peschanski and Besson ('84) appear to differ somewhat from the atlas of Paxinos and Watson ('86) in describing the organization of the SM and midline nuclei. This may explain some of the differences between our data and that of Peschanski and Besson ('84). In addition, differences in the tracers and techniques used may also be responsible for this discrepancy. In addition to corticothalamic projections, Beckstead ('79) also examined connections between the prefrontal cortex and the midbrain. Cortical injections that did not include, or were not confined to the VLO, generally resulted in widespread labeling throughout the midbrain, including both the dorsal and ventral PAG, the superior colliculus, the tegmental nuclei, substantia nigra, and the pontine nuclei. However, he noted that fiber labeling was confined to the ventrolateral PAG only when the cortical injection also resulted in similar labeling in the SM. In these same cases, some labeling was also present in the substantia nigra (pars reticularis) and the tegmental nuclei. Gerfen and Clavier ('791 noted labeled neurons present in the

ventrolateral PAG, the dorsal raphe, the median forebrain bundle, the tegmental area, and the locus coeruleus. Regional differences in labeling of midbrain neurons dependent upon concurrent SM labeling were not noted in this study.

Functional implications The data presented in this study provide anatomical evidence for the existence of a neural circuit between the SM, VLO, and the midbrain PAG. Previous anatomical and physiological studies had implicated the SM in nociception (Craig and Burton, '81; Dostrovsky and Guilbaud, '88; Ma et al., '88; Miletic and Coffield, '89).Very recent electrophysiologic studies of the VLO in the rat (Backonja and Miletic, '91) or the cat (Snow et al., '92) also implicate the VLO in nociception, perhaps especially in its motivational-affective component. Over the last decade, studies have shown the PAC to be both the origin and recipient of a diverse array of ascending and descending neural input (Beitz, '82). In particular, the ventral PAG is known to be interconnected with the thalamus, frontal cortex, and hypothalamus, thus connecting the PAG with the limbic, motor, and autonomic systems. The ventral PAG also receives noxious peripheral input, has a high opioid content, and is thought to participate in an endogenous analgesia system. The initiation of endogenous analgesic mechanisms might require the concurrent activation of the limbic, motor, and autonomic systems. Hence, the connections of the SM and VLO to the ventral PAG may provide another essential link between peripheral noxious input and the limbic, motor, and autonomic systems. This linkage may allow the SM, VLO, and PAC to be part of a neural circuit involved in the affective component of pain andlor endogenous analgesia.

ACKNOWLEDGMENTS We thank Drs. J. Abbs, M. Behan, L. Trussell, and L. Stanford for helpful discussions and critically reading the manuscript. This study was supported in part by USPHS National Institutes of Health grant NS26850 to V. Miletic.

LITERATURE CITED Backonja, M., and V. Miletic (1991) Responses of neurons in the rat ventrolateral orbital cortex to phasic and tonic nociceptive stimulation. Brain Res. 5.57~353-355. Basbaum, A.I., N. Marley, J. O'Keefe, and C.H. Clanton (1977) Reversal of morphine and stimulus produced analgesia by subtotal spinal cord lesions. Pain 3:43-56. Beckstead, R.M. (1979) An autoradiographic examination of corticocortical and subcortical projections of the mediodorsal-projection (prefrontal) cortex in the rat. J. Comp. Neurol. 184:43-62. Beitz, A.J. (1982) The organization of afferent projections to the midbrain periaqueductal grey of the rat. Neuroscience 7:133-159. Berman, A.L., and E.G. Jones (1982) The Thalamus and Basal Telencephalon of the Cat. Madison: University of Wisconsin Press. Conrad, L.C.A., C.M. Leonard, and D.W. P f d (1974) Connections of the median and dorsal raphe nuclei in the rat: An autoradiographic and degeneration study. J. Comp. Neurol. 156:179-206. Coffield, J.A., and V. Miletic (1990) Effects of leu-enkephalin micropressure application onto rat nucleus submedius neurons. SOC. Neurosci. Abstr. 16:412. Craig, A.D., Jr., and H. Burton (1981) Spinal and medullary lamina I projection to nucleus submedius in medial thalamus: A possible pain center. J. Neurophysiol. 45443-466. Craig, A.D., S.J. Wiegand, and J.L. Price (1982) The thalamo-cortical projection of the nucleus submedius in the cat. J. Comp. Neurol. 206: 28-48.

CONNECTIONS OF THE SM, VLO, AND MIDBRAIN IN THE RAT Dado, R.J., and G.J. Giesler, Jr. (1990) AfTerent input to nucleus submedius in rats: Retrograde labeling of neurons in the spinal cord and caudal medulla. J. Neurosci. 10,2672-2686. Divac, I., and A. Kosmal (1978) Subcortical projections to the prefrontal cortex in the rat as revealed by the horseradish peroxidase technique. Neuroscience 3:785-796. Dostrovsky, J.O., and G. Guilbaud (1988) Noxious stimuli excite neurons in nucleus submedius of the normal and arthritic rat, Brain Res. 460t269280. Dubner, R., and G.J. Bennett (1983) Spinal and trigeminal mechanisms of nociception. Annu. Rev. Neurosci. 6:381418. Gehhart, G.F., and J.R. Toleikis (1978) An evaluation of stimulationproduced analgesia in the cat. Exp. Neurol. 62570-79. Gerfen, C.R., and R.M. Clavier (1979) Neural inputs to the prefrontal agranular insular cortex in the rat: Horseradish peroxidase study. Brain Res. Bull. 4t347-353. Hardy, S.G.P., and H.J. Haigler (1985) Prefrontal influences upon the midbrain: A possible route for pain modulation. Brain Res. 339:285-293. Honig, M.G., and R.I. Hhme (1986) Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in longterm cultures. J. Cell Biol. 103:171-87. Jones, E.G., and R.Y. Leavitt (1974) Retrograde axonal transport and the demonstration of nonspecific projections to the cerebral cortex and striatum from thalamic intralaminar nuclei in the rat, cat and monkey. J. Comp. Neurol. 154t349-378. Krettek, J.E., and J.L. Price (1977) The cortical projections of the mediodor-

499

sal nucleus and adjacent thalamic nuclei in the rat. J. Comp. Neurol. 1 71;157-192. Ma, W., M. Peschanski, and P.T. Ohara (1988) Fine structure of the dorsal part of the nucleus submedius of the rat thalamus: An anatomical study with reference to possible pain pathways, Neuroscience 26t147-159. Mantz, J., R. Godbout, J.-P. Tassin, J. Glowinski, and A.-M. Thierry (1990) Inhibition of spontaneous and evoked unit activity in the rat medial prefrontal cortex mesencephalic raphe nuclei. Brain Res. 524t22-30. Miletic, V., and J.A. Coffield (1989) Responses of neurons in the rat nucleus suhmedius to noxious and innocuous mechanical cutaneous stimulation. Somatosens. Motor Res. 6:567-587. Palmer, M.R., S.M. Wuerthele, and B.J. Hoffer (1980) Physical and physiological characteristics of micropressure ejection of drugs from multibarreled pipettes. Neuropharmacology 19:931-938. Paxinos, G., and C. Watson (1986) The Rat Brain in Stereotaxic Coordinates, 2nd Ed. New York: Academic Press. Peschanski, M., and J:M. Besson (1984) Diencephalic connections of the raphe nuclei of the rat hrainstem: An anatomical study with reference to the somatosensory system. J. Comp. Neurol. 224509-534. Rhodes, D.L. (1979) Periventricular system lesions in stimulation-produced analgesia. Pain 7:31-51. Schmued, L.C., and J.H. Fallon (1986) Fluoro-gold: A new fluorescent retrograde axonal tracer with numerous unique properties. Brain Res. 377:147-154. Snow, P.J., B.M. Lumb, and F. Cervero (1992) The representation of prolonged and intense, noxious somatic and visceral stimuli in the ventrolateral orbital cortex of the cat. Pain 48:89-99.