ACTA NEUROBIOL. EXP. 1989. 49: 23-37 DIFFERENTIAL PROJECTION FROM THE MOTOR AND LIMBIC CORTICAL REGIONS TO THE MEDIODORSAL THALAMIC NUCLEUS IN THE DOG Iwona STEPNIEWSKA and Anna KOSMAL Department of Neurophysiology, Nencki Institute of Experimental Biology 3 Pasteur Str., 02-093 Warsaw, Poland Key words: mediodorsal thalamic nucleus, motor and limbic cortices, horseradish peroxidase Abstract. The cortical afferents to the mediodorsal thalamic nucleus in the dog were studied by using horseradish peroxidase. Small injec- tions allowed to establish two specific projection zones connected sepa- rately with the lateral and medial segments of the nucleus. The lateral segment received the major projection from the dorsal half of the hemi- sphere. It included premotor and part of the motor cortices in the ante- rior sigmoid gyrus and precruciate areas as well as the presylvian cor- tex. The medial segment of the nucleus was innervated by the limbic areas of the ventral half of the hemisphere. These areas included the medloventrally located genual, subcallosal and plriform cortices, as well as the cortex of the ventral bank of the anterior rhinal sulcus and the caudal part of the orbital gyrus. The cortical fields situated between these two main cortical zones, both on the lateral and medial surfaces (rhinal and sylvian sulci and anterior cingular gyrus, respectively) sent projec- tions to both medial and lateral segments of the nucleus. These results indicate that in the mediodorsal thalamic nucleus may take place the integration of information from two functionally defined systems, the motor and limbic ones. INTRODUCTIOPJ The numerous studies of the cortical afferents of the mediodorsal thalamic nucleus (MD) showed, that although the prefrontal cortex (PFC) gives rise to the heaviest projection to MD, it is not the only cortical region projecting to thls nucleus. Degeneration methods, and more re- cently neuroanatomical techniques based on axonal transport, have in- dicated that the cortical sources of MD afferents are considerably richer. The first papers which considered this problem established an affe- rent projection to MD, mainly to its medial segment, originating in the cortex of temporal lobe, both in monkey (67) and cat (17, 53). The same segment was shown to receive axonal terminals from the olfactory cor- tex of the piriform lobe in the rat (16, 30, 43) and tree shrew (55). Sub- sequently, such connections were demonstrated also in other species wjth electrophysiological (39, 57) as well as horseradish peroxidase (HRP) and autoradiographic methods (8, 13, 52, 63). After injections of HRP to MD in the tree shrew (46), rat (47, 52), cat (9, 36, 42, 53) and rabbit (5), retrogradely labeled neurons were found in the part of the cortex covering the claustrum, known as the insular cortex. In addition, these connections have been described to be topographically organized in the rat (47). The lateral segment of MD, by contrast, has been shown to be con- nected with the cortex of the anterior cingulate gyrus (10, 17, 46, 52, 54) and the motor cortex (3-5, 17, 27, 28, 63). In the monkey, where the motor connections of MD are best known, their topography has also been established. Thus, the most lateral, paralamellar, segment of MD received projections from the primary motor cortex (area 4), whereas the more centrally located - parvocellular segment was connected with area 6 of the premotor cortex (4). Recently, connections arising from the ectosylvian and posterior syl- vian gyri (somatosensory, gustatory and auditory areas), suprasylvia~l gyrus (parietal association cortex), and, most surprisingly, from visually responsive areas in the occipital lobe, were described in the cat (17, 18, 34). Until now, these projections have not been confirmed in other species. Information concerning the cortical afferents of MD originating outside PFC in the dog's brain is very spwce. In addition to the topo- graphy of the PFC-MD projection (2, 21-23, 40, 58), only the relation- ships between the moto~r cortex and the lateral segment of MD have been selectively examined (24). In this paper we tried to systematize medial and lateral MD afferents regarding their relations to particular functional systems. MATERIAL AND METHODS Eleven young (about one year old) dogs of either sex, weighing 8-13 kg were used. The animals received stereotaxic injections in h4D of 0.15-0.3 p1 30°/o HRP (Sigma, type VI or Boehringer) solution. Coordina- tes of all injection points were established on the basis of a stereotaxic atlas of the dog's brain (31). All injections were divided into three groups, comprising the medial (group I), lateral (group 11) and central (group 111) part of MD nucleus, respectively (7-8 cases in each group). The in- jections were made, first unilaterally and then bilaterally, due to~the statement only ipsilateral projections. The microsyringe needle was in- serted vertically into the thalamus. All animals survived 48h, then they were deeply anaesthetized and perfused through the heart with physio- lcgical saline followed by a mixture of 1°/0 psraformaldehyde and 2,5O/n glutaraldehyde or 4O/u formaline in phosphate buffer (pH = 7.4). After removal the brains were placed in 3O0/0 sucrose for 48h and then cut into 40 pm coronal frozen sections. To reveal the location of active HRP Mesulam procedure was used (38). The sections were mounted on glass slides and counterstained with cresyl violet or neutral red. Sections we- re examined in the light microscope and HRP-polsitive neurons were localized in the various cortical fields according to the Kreiner division (25). In 5 animals out of 11, the enzyme injection,^ were preceded by le- sons of the medial or lateral PFC surface. This procedure was use'd to analyze whether the elimination of the projections from PFC to MD might increase the intensity of HRP uptake by the axonal terminals of the other projection systems, as suggested by some investigators (52). In order to exclude incorrect interpretation, two type,s of control ex- per~mentswere made. First, the enzyme was injected into structures surrounding MD, because in some experiments the area of HRP diffu- sion spread beyond MD nucleus. The other control experiment was made in order to exclude HRP uptaking by damaged axons passing through MD region. It consisted in implanting a cannula in MD through which 7 days later a needle filled with HRP was inserted and an injection itlade (63). The results of all control experiments were taken into ac- count in the final analysis of labeled cells localization. RESULTS The tlistribution of labelecl, cells following medial ME injections (group I) The r~sultsreceived In th~sgroup of injections are illustratzd on the exxnple of dog A45 (F~gs.1 and 2). The prolectlons to this MU region or~glnatedfrom the cortical llrnblc areas, both lateral snd medial surfa- ces of the hem~sphere(Fig. 2B). On the lateral surface the majority of HRP-pos~t~iveneurons was concentrated 1n the region surrounding the anterior rhlnal sulcus (sRha), along a considerable part of its length (Fig. 2Bc-f). It corresponded largely to the insular cortex on the depth of which lies the claustrum. This zone of the intensive labeling of cells spread on to cortical areas located dorsally and to a lesser degree ven- trally (Fig. 2Bc-g). In adjacent zones labeled neurons became more and more scattered. Dorsally and rostrally to the anterior rhinal cortex labeled cells were observed in the posterior region of the orbital gyrus (ORB, Fig. 2Bc and 3). In all cases of injections in this group, labeled cells lying in the depth of the anterior rhinal sulcus and in the posterior zone of the orbital cortex formed a clear cellular band. This band did nlot extend to the anterior zone of the orbital cortex. On the other hand, dorsally and caudally to the anterior rhinal cortex this band of cells seemed to continue into the region of posterior insular cortex, where the number of labeled neurons was quite considerable (c.i., Fig. 2 Be-g). Sporadically labeled cells were seen in the cortex of sylvian and ectosylvian gyri (S, ES, Fig. 2Be-g). The ventral continuation of intensive labeling zone located in the depth of anterior rhinal sulcus was composed of nume- rous cells, which were found in the deep layers of the piriform cortex (c.p., Fig. 2Be-f). Such cells were observed in the anterior (prepiriform area), central (periamygdaloid area) as well as posterior (enthorhinal area) parts of this cortex. The second zone with numerous HRP-positive cells has been obser- ved on the medial surface of the hemisphere. It comprised the cortex of the anterior part of the subcallosal gyrus lying below corpus callosum (SC, Figs. 2Bc and 4) and also the genual area located beneath the genu- a1 sulcus (G, Fig. 2 Ba, b). Single HRP-positive cells were observed also in the anterior part of the cingular cortex (CN, Fig. 2Bd). The distribution of labeled cells following lateral MD injections (group 11) The distribution of labeled cells after lateral HRP injections is illu- strated on the example of dog L4 (Fig. 5). The projection to this part of nucleus originated mainly from the dorsal region of the frontal cortex. Following these injections the numerous labeled neurons were observed in the depth and the both walls - (lateral and medial) of the presylvian fissure (fPs, Figs. 5Ba-d; and 6). Whilst in the internal composite area of the lateral wall (CJ, Fig. 5Bb-d) more closely connected with the mo- tor cortex (24) they were extremely numerous and distributed quite eve- nly, in the paraorbital area of the medial wall they were much less nu- merous and concentrated mainly in its dorsal region (PORd, Fig.