Brain Research, 85 (1975) 385-401

385

;C) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

CORTICOTHALAMIC NEURONS AND THALAMOCORTICAL TERMINAL F I E L D S : AN I N V E S T I G A T I O N I N R A T U S I N G H O R S E R A D I S H P E R O X I DASE A N D A U T O R A D I O G R A P H Y

STANLEY JACOBSON* ANDJOHN Q. TROJANOWSKI Anatonzy Department, Erasmus University Rotterdam, Rotterdam (The Netherlands) and Anatomy Department, Tufts University School of Medic#m, Boston, Mass. 02111 (U.S.A.)

(Accepted October 10th, 1974)

SUMMARY Subsequent to thalamic injections in rats of horseradish peroxidase (HRP) alone or H R P and [3H]leucine in combination, the cells of origin of the corticothalamic pr~ections and the terminal fields of the thalamocortical projections were identified. HRP-labeled corticothalamic neurons were uniformly found in layers V and VI. They were medium to small in size and always pyramidal in shape with the larger neurons being found in layer V. On the other hand, 3 different patterns for the distribution of thalamocortical terminal fields were observed. The autoradiographic material indicated that in prefrontal cortex the bulk of thalamocortical fibers terminate in layer Ill while in motor cortex they terminate primarily in layer V. A third pattern was shared by temporal, occipital and parietal cortex where the bulk of thalamocortical fibers terminate preferentially in layer IV. The data derived from the rats which had received thalamic injections of H R P and [3H]leucine in combination indicated that the connections between cortex and thalamus are in general reciprocal. These results are discussed with regard to earlier studies using classical or more recently developed neuroanatomical methods. 1NTRODUCTION A full assessment of the anatomical relations of individual cerebral cortical neurons has been difficult to obtain due to their high density, laminar organization and the limitations of the techniques used to studythem. The Golgi neuronal method 1, :,-7,34 has been most widely used to identify cortical neurons; however, it has only been possible to trace the axons of these cells a short distance. The distribution of * Address reprint requests to: Anatomy Department, Tufts University School of Medicine, Boston, Mass. 021 ! I, U.S.A.

386 axons and axon terminals of cortical neurons has been analyzed successfutlv with anterograde silver methods or the Marchi technique, however, the limitation with these techniques is that the actual cells of origin of these axons and terminals cannot be demonstrated. Cells giving rise to thalamocortical projections have been examined with some success using retrograde degeneration methods 38, but the same methods have not been applied to the problem of the location of corticothalamic neurons. In any case, however, the results derived from retrograde degeneration studies are frequently inconsistent. In an earlier study 14 we reported on the location of the cells o f origin of the callosal system using horseradish peroxidase (HRP). Recently, several studies have appeared in which anterograde and retrograde axonal transport were exploited using a variety of tracers to study neuronal connectivity 2'sA°,12,t4,~s,21-24,27,3°,a2 and ,n fact an anterograde and retrograde tracer injected together have been used to demonstrate reciprocal connections in squirrel monkey a6. In the investigation reported here we have sought in the rat to identify the cells of origin of the corticothalamic system as well as to determine the location of these neurons in the different cortical layers using H R P alone. In addition we report on the results of experiments in which H RP in combination with autoradiography was used to determine the relationship of thalamocortical projections to the cells of origin of the corticothalamic system. MATERIALS AND METHODS

All experiments were performed on adult male and female albino rats weighing 220-250 g and all experimental procedures were performed under anesthesia.

Experiments using HRP alone The concentration of H R P (Sigma VI) in these experiments was always 10'~, in aqueous solution. In 7 rats H R P was injected using a 10 #l Hamilton syringe with a 30-gauge needle stereotaxically through the right hemisphere into the left thalamus over a period of 5 min and the needle remained in place for an additional 5 rain. In one of these rats, 0.6 #l of H R P was injected while in the remaining 6 rats. 0.2 # was injected. Five of the rats were allowed to survive 2 days and 2 allowed to survive 4 days. In 29 additional rats, H R P was injected stereotaxically into the thalamus via the ipsilateral or contralateral cortex, however, in these animals micropipets were used to inject the HRP. The micropipets had a tip diameter of 10-40 ~zm. They were placed in a stereotaxic electrode holder and connected via polyethylene tubing to a 250 #1 syringe. Both the tubing and the syringe were filled with paraffin oil and the plunger of the syringe was advanced manually by turning a micrometer. Because of the elasticity of the polyethylene tubing it was not possible to precisely calibrate this micropipet delivery system. However, the amount of H R P injected using this system ranged between 0.01-0.1 #1. In all instances the amount injected with this apparatus was less than the smallest injections using the Hamilton syringe on the basis of comparing the injection sites resulting from the use of the two ssstems at similar survival times. Occasionally, it was found that the tip of the micropipet became plugged in the course of making injections. Immersion of the tip of the micro-

387 pipet into physiological saline restored the flow of the HRP. in these rats, the H R P was injected over 2-5 rain and the survival time varied from 1 to 5 days. The rats were perfused with 50-100 ml of 6 ~ Dextran in 0.9 ~ NaCI followed by 125 250 ml of 0.5~o paraformaldehyde and 2 . 5 ~ glutaraldehyde buffered with sodium cacodylate at pH 7.2. After removal the brains were left in the fixative with 30~,~ sucrose added for 24 h and then either cut immediately at 40 # m on a freezing microtome or stored for 1 2 days in sodium cacodylate with 30~{i sucrose added before cutting. Every third section was collected in the sucrose-cacodylate mixture and then incubated, usually on the same day, in 3,3'-diaminobenzidine tetrahydrochloride in Tris buffer (hydroxymethyl aminomethane) at pH 7.6 (50 mg 3,3'-diaminobenzidine tetrahydrochloride/100 ml Tris buffer) to which was added 1 ~ H202 (1 ml H202/ 100 ml incubating media)9,14, 24. After 30 rain the reaction was stopped by placing the sections in distilled water and the sections were then mounted from alcoholic gelatin. The sections were examined without counterstain using light- and dark-field illumination. After the HRP-positive neurons were identified, the coverslips of many of the sections were removed and the sections lightly counterstained with cresyl violet in order to confirm the shapes of the labeled neurons and to determine the cortical layers in which the positively labeled ceils were located. Faintly labeled neurons were often missed if the sections were counterstained prior to identifying the HRP-labeled cells.

Experiments using HRP and autoradiography in combination In order to determine the relationship of the ceils of origin of the corticothalamic system labeled with H R P in the experiments described above to the thalamocortical terminal fields, a series of experiments was carried out in which rats received thalamic injections of a combined solution of H R P and [3H]leucine. The [3H]leucine (Amersham) was evaporated under nitrogen gas and dissolved in l0 ~i aqueous H RP. The concentration of [3H]leucine in the HRP was 25/zCi/0.1 /zl. Injections of 0.2 lxl of the combined solution were made over a period of 5 rain with a 10 #1 Hamilton syringe and a 30-gauge needle into the left thalamus of 5 rats stereotaxically by passing the needle through cortex of the right hemisphere. Two rats survived 1 day and 3 survived 2 days. The fixation, sectioning and H R P incubation was the same here as in the experiments described above. Every third section was collected and after incubation these sections were mounted with chromalum. Half of these sections were dehydrated, defatted for 1 h in xylene, hydrated and dried overnight in an oven at 36 C . The slides were dipped in dilute llford G5 emulsion and developed with Kodak D-19 after exposure times of 1 3 weeks with the bulk being developed at 3 weeks. The sections were then examined with light- and dark-field illumination. Thereafter many of the sections were lightly counterstained to determine the cortical layers in which the HRP-positive neurons and silver deposits were located. It should also be noted that in a previous experiment ~6 controls were done to determine if the H R P has a positive or negative chemographic effect on the emulsion and likewise to determine if the autoradiographic process has an effect on H RPlabeled neurons. This is especially important for the autoradiography since the pres-

388 ence or absence of silver grains in an area with H RP-labeled neurons ma5 be an artifact secondary to the chemical effect of H R P on the emulsion. To evaluate this. sections containing HRP-positive neurons subsequent to injectmns with HRP alone were dipped in the emulsion and half were exposed to light. When these slides were developed it was noted that the HRP-labeled neurons were unaffected by the autoradiographic procedure. The sections exposed to light had a high but even background which is expected since the sections were exposed to light and is evidence that H RP-positive neurons do not cause negative chemography. The slides not exposed to ight had ~J few silver grains consistent with normal background even in areas with HRPpositive neurons indicating that the labeled cells do not produce positive chemography. In the material containing the H R P and [3H]leucine, grain counts were made in prefrontal, motor, sensory, auditory and visual cortex. The grain counts were done manually at a magnification of ~ 600 with an eyepiece reticule following the technique of Gottlieb et al. 8 and Price 32. The reticule, consisting of 100 squares, was placed vertically on the pial surface and an area of 3 by 2 squares (1456 sq. uml was examined and the counts recorded. This procedure was continued until a strip, 10 squares in length and 3 in width, was counted at which point the mechanical stage was advanced vertically exactly one reticule away from the pia] surface and the counting continued until the entire cortical thickness was so covered. All counts were related to the cortical layers and the location of the HRP-positive neurons in these layers was also noted. Counts were also done in the same areas in the hemisphere contralateral to the rejected thalamus and in the olfactory cortex of the contralaterat and ipsitateral hemisphere in order to determine background. Graphs were prepared from these data and oll them the location of the HRP-positive neurons was also noted (Fig. 5/. The data presented in these graphs represent counts from a single animal, however, the pattern for lhe distribution of the grains was consistent with that observed in other rats with similar injection sites. The determination of cortical areas was based on Leonard ')a, Domesick u and Krieg 19. The thalamic nuclei were determined according to Emmers-L Domesick '~, Leonard 25, Price et al. 33, Montero et al. 29 and Krieg ~°. Hall et al. ~ , Welker 4°, Welker et al. 41 and Woolsey 44 were referred to for the physiological divisions of rat motor and somatosensory cortex. RESULTS

E x p e r i m e n t s using H R P alone

In none of the HRP material did we note any difference in the distribution of the HRP-positive neurons in cortex at survival times of 1-4 days. In several instances, however, there was H R P staining along the needle tract and in some cases this resulted in HRP-positive neurons in the contralateral hemisphere. These animals were not included in the study. It should be noted that in several of the rats injected using the 30-gauge needle and in 2 rats with larger injections made with the micropipet system, 2 types of H R P reactions were seen in the positively labeled neurons. I n o n e the HRP was enclosed in granules which were confined to the soma and adjacent portions

389 of basilar and apical dendrites (Fig. 3B). It is to this type of HRP-labeled cell that we refer in this study unless otherwise stated. The second type of H R P reaction seen in labeled neurons was one in which no granules were apparent but instead the entire soma and, frequently, dendrites and axons were evenly stained brown, much resembling neurons stained with the Golgi method. This later type of H R P staining we refer to as the 'Golgi Neuronal H R P Stain'. Both types of H R P staining have also been noted by others zT,a0,aT. This reaction is most commonly seen near the injection site and may well be the result of injury to the cella0, av. Of the 7 rats injected with the Hamilton syringe, one received an injection of 0.6/A of HRP. This is the same quantity which we found suitable for cortical injections when studying the callosal system. However, after a thalamic injection of this amount almost the entire thalamus on the injected side contained the even brown stain indicative of the substrate at the injection site. In the cortex of this animal, we noted that there were HRP-positive neurons only in layers V and VI. In the 6 other rats with injections of 0.2/~1 of HRP, we found that the injection sites were more localized and were confined to only a few thalamic nuclei. In one rat, the injection was confined to the medial dorsal and anterior nuclei and the HRP-positive neurons were limited to layers V and VI of prefrontal (Figs. 1 and 5) and cingulate cortex. In 2 rats ventrobasal complex, ventral lateral, lateral posterior and anterior lateral geniculate nuclei were evenly stained brown and these injections resulted in HRP-positive neurons in sensory (areas 3, 1 and 2), motor (areas 4 and 6) and occipital (areas 17, 18 and 18a) cortex, again only in layers V and VI (Figs. 2, 3 and 5). In 3 other rats the injections were confined to the posterior levels of ventrobasal complex and also included lateral and medial geniculate nuclei with the result that sensory (areas 3, 1 and 2), occipital (areas 17, 18 and 18a) and temporal (areas 41 and 20) cortex were found to have H RP-positive ceils restricted to layers V and VI (Figs. 2, 3 and 5). The labeled cells in layer V in all animals were larger than those seen in layer VI and all of them were pyramidal in shape and medium to small in size (Figs. 1-3). In all of the material only a small percentage of neurons in cortex were positively labeled as seen in Fig. 3. In all of the rats injected using micropipets only a very small tract was present. Any brains that showed evidence of leakage of H R P along the tract were discarded as were those with injections in hippocampus or midline thalamic nuclei. In addition, no injection site was found in several brains which presumably resulted either from the injection of a very small amount of H R P or none at all secondary to a plugged micropipet. As a result, only 12 of the rats injected with H R P using the micropipets could be included in this study. The injection sites in these rats can be grouped as follows: injections into medial dorsal nucleus only (4 rats); injections involving ventrobasal complex, ventral lateral and the ventral portion of lateral posterior nuclei (4 rats); injections into ventrobasal complex and the dorsal part of lateral geniculate nucleus (2 rats); and injections in ventrobasal complex, lateral and medial geniculate nuclei (2 rats). Most of the microinjections were quite small and in most cases only a few positively labeled neurons were found in cortex. These cortical neurons were found by tracing the axon, which was also stained by retrogradely transported HRP, through the corpus striatum to the cortical area in which the cell giving rise to the

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@ Fig. 2. Corticothalamic cells labeled with HRP in layers V and V1 of sensory area 3 following an injection involving ventrobasal complex. A: dark-field, ~ 100. B: dark-field, > 350. Note that in both cases the larger pyramidal cells are in layer V.

axon was located (Fig. 4). In all the rat brains labeled neurons were f o u n d only in layers V and V! and were always pyramidal in shape (Fig. 4A) being larger in layer V than VI. In those rats with injection sites restricted to medial dorsal nucleus, H R P labeled n e u r o n s were f o u n d only in prefrontal areas as defined by Leonard e5 and Krettek and Price TM. In rats with injection sites involving ventrobasal complex and ventral lateral nucleus and also the ventral part of lateral dorsal nucleus, labeled neurons were f o u n d in sensory and m o t o r cortex while when in addition the medial and

Fig. 1. Corticothalamic cells labeled with HRP following an injection involving medial dorsal nucleus photographed with dark-field illumination. Magnification in both . 120. A: HRP-positive neurons in layers V and VI of lateral prefrontal area 8. B: HRP-positive neurons in layers V and VI of medial prefrontal area 24.

392

Fig. 3. Corticothalamic cells labeled with HRP in layers V and Vl of sensory area 2 following an injection involving ventrobasal complex. A: light-field, Nissl stain, :< 300. B: same field as in A, seen with dark-field illumination, x 300.

lateral geniculate nuclei were involved in the injection site HRP-positive cells were found in areas 41 and 20 o f temporal cortex and areas 17, 18 and lSa o f occipital cortex respectively. In the animals which received injections o f 0.2/~1 o f H R P with the Hamilton syringe, the enzyme diffused in 3 cases into the internal capsule and in 2 o f these cases also into the globus pallidus. When only the thalamus and internal capsule were involved in the injection site, HRP-positive cells were seen in globus pallidus indicative o f pallidal projections to thalamus. However, when the injection site also included globus pallidus, labeled cells were also seen in c a u d o p u t a m e n indicative o f striatal

393

Fig. 4. A: HRP-labeled neuron in layer V of sensory area 3 following a micropipet injection of HRP involving ventrobasal complex. Dark-field, × 1200. B: axons labeled with HRP in caudoputamen (arrow at left) and medullary center (arrow at right) following injections of HRP involving ventrobasal complex. Dark-field, x 200. C: micropipet injection site in ventrobasal complex also showing axons labeled with HRP (arrow). Dark-field x 200.

projections to globus pallidus. A m o n g the rats injected with the micropipet system were 2 in which the bulk o f the injection site was restricted to c a u d o p u t a m e n with the internal capsule and thalamus being involved to a lesser extent. In these rats, H R P labeled cells were nevertheless seen only in layers V and V1 o f the cortical regions from which the thalamic nuclei involved in the injection site receive projections. Although it has been reported 13,~9 that preterminal degeneration is found in corpus striatum o f the rat following lesions throughout the cerebral cortex, we have not observed an increase in the n u m b e r o f HRP-labeled cortical neurons, or a different pattern o f distribution o f such cells or even an obvious increase in the intensity o f the H R P reaction in labeled neurons which might suggest a significant cortico-striatal projection after thalamic injections which also involved corpus striatum c o m p a r e d to those injections sparing this structure. Finally we might add that in rats with the injections restricted to medial dorsal nucleus, we observed HRP-positive neurons in the contralateral medial dorsal nucleus indicative o f connections across the midline between the two nuclei, while in rats

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Fig. 5. Graphs of grain counts over cortex subsequent to combined injections of H R P and [3H]leucine. See text for details concerning method of grain counting. Numbers along the ordinate refer to numbers of grains counted. The cortical layers are indicated along the abscissa. Pyramids in layers V and VI indicate the location and relative size of the HRP-labeled corticothalamic neurons. Horizontal lines represent the background level of silver grains.

with injections restricted to ventrobasal c o m p l e x and ventral lateral nucleus, H RPpositive cells were noted in the contralateral dentate nucleus indicative o f projections from this latter structure probably to only the ventral lateral nucleus.

Experiments using HRP and autoradiography in combination In this part o f our study, we made thalamic injections o f 0:2 ffl Of H R P and [3H]leucine in c o m b i n a t i o n in 5 rats to assess the relationship o f the thalamocortical

395

Fig. 6. Auditory cortex ipsilateral to a combined injection of HRP and [aH]leucine involving medial geniculate nucleus after 1 day survival. A: the heaviest silver deposits are over layer VI (layers are running slightly obliquely, upwards and to the right in the photograph) while HRP-labeled cells are seen in layer V (arrows). The labeled neuron with the circle is in layer V immediately subjacent to layer 1V. Dark-field, 100. B: higher power view of layers IV and V showing HRP-labeled neurons in layer V immediately below layer IV in which the heaviest silver deposits were noted. Dark-field, >: 150. C: the HRP-labeled cells in layer V are seen to better advantage in this dark-field photomicrograph and 3 have been circled. Silver grains above background levels are also seen in the same layer. > 250. t e r m i n a l s to the cells o f o r i g i n o f the c o r t i c o t h a l a m i c p r o j e c t i o n s . O n l y 4 o f the rats were i n c l u d e d in this p a r t o f the study since the fifth rat h a d an i n j e c t i o n w h i c h directly c o n t a m i n a t e d the cortex. In 3 rats the e v e n b r o w n stain o f the s u b s t r a t e and h e a v y silver d e p o s i t s o v e r n e u r o n s i n d i c a t i v e o f u p t a k e o f the [3H]leucine was n o t e d in v e n t r o b a s a l c o m p l e x , v e n t r a l lateral, lateral dorsal, lateral p o s t e r i o r nuclei as well as

396 the anterior parts of the dorsal and ventral lateral geniculate nuclei. In one of these 3 animals, the medial geniculate nucleus was also affected while in the fourth rat the injection was more rostral and included the lateral portion of the medial dorsal, lateral dorsal and ventral lateral nuclei as well as the ventrobasal complex. We noted that although the leucine is the lighter molecule, the extent of the injection site of the H R P was slightly greater than that of the [3H]leucine. In all the rats in this group, the HRP-positive cortical neurons were lbund only in layers V and VI, were pyramidal in shape and were larger in layer V than in layer VI as was the case in the rats which received injections of H R P alone. Three different distribution patterns for the axon terminals of the thalamocortical cells could be distinguished on the basis of the distribution pattern of the silver grains in cortex (Fig. 5). In prefrontal cortex we noted peaks in the number of silver grains in layers III and VI while in motor cortex similar peaks were observed over layer V and the deeper part of VI. In visual, somatosensory areas 3 and 2 and auditory cortex the highest number of silver grains was primarily concentrated over layer IV. In Fig. 6 we illustrate the pattern of distribution of silver grains in auditory cortex as an example of the pattern seen in temporal, occipital and parietal cortex, The injection site in this case involved medial genicutate nucleus and it can be seen that the heaviest silver deposits indicative of axon terminals are found over layer IV while the H R P positive cells are located in layers V and VI. In some cases the positively labeled cells are located more superficially in layer V and thus within close proximity to the terminal arborization of the thalamocortical projections. In all the areas represented in Fig. 5, layers 1 and II also had grain counts above background although usually not more than twice the background. The preferential distribution of silver grains over layer IV in temporal, occipital and parietal cortex most likely corresponds to the terminal arborization of thalamocortical fibers in layer IV as seen in Golgi and silver stains in layers IV 13,26. Layers V a n d l I I of m o t o r and prefrontal cortex respectively are known to receive the bulk of thalamic input 26,34 and we noted peaks in the number of silver grains in layers V and VI of m o t o r cortex and in layers I t i and Vl of prefrontal cortex. Although other layers were found to have grain counts above background, it is difficult to decide to what extent this is a result of fibers of passage or terminals. It is possible that the silver deposits over layer I represent the terminal arborization noted by several authors in this layer using silver methods 13.15-a7.28.42.43 In this material we also attempted to assess the degree of reciprocity of the corticothalamic and thalamocortical connections. Our combined injections of H R P and [3H]leucine were never limited to a single thalamic nucleus and it i~ therefore difficult to make any statements about the nature of the reciprocal connectivity of cortex and individual thalamic nuclei. However. we have noted that the cortical areas with HRP-positive neurons were always co-extensive with the areas having heavy silver deposits. DISCUSSION

The data derived from the two techniques used in this investigation lead to

397 several conclusions regarding certain aspects of corticothalamic neurons and their relationship to the terminal fields of the thalamocortical projections in rat. In motor cortex, the data from the autoradiography indicate that the bulk of the thalamocortical terminals are located in layer V. The bulk of these terminals thus overlaps with the medium to small sized pyramidal-shaped corticothalamic neurons located in layers V and VI. In this regard, motor cortex is unlike the rest of cortex where the bulk of thalamocortical terminals are found above layers V and VI in which, as in motor cortex, the corticothalamic neurons in these other areas are also situated. In prefrontal cortex area 8 the autoradiographic data indicate that the bulk of thalamocortical terminals is located in layer Ill while in temporal, occipital and parietal cortex the bulk of these terminals is localized in layer IV (see Fig. 5). Retrograde degeneration methods have demonstrated that cells in layers I11, V and V! undergo chromatolysis after section of the corpus callosum 31, but there have been no reports of cortical cell changes following thalamic lesions to indicate the location of the cells of origin of the corticothalamic projections. Our findings in the H R P material that only ceils in layers V and VI give rise to the corticothalamic projections is consistent with observations made using the Golgi neuronal method 1,5,1a, 26,34 that pyramidal cells in supragranular layers send their axons to infragranular layers ipsilaterally and to the contralateral hemisphere while only cells in the infYagranular layers send projections to subcortical structures. Using the anterograde silver degeneration methods 13,35, it has been shown that lesions restricted to supragranular layers produce either callosal or intracortical association preterminal degeneration and that it is only when lesions reach down into infragranular layers that one finds preterminal degeneration in subcortical structures. Another study 12 making use of anterogradely transported tracers has demonstrated that injections of [aH]leucine limited to supragranular layers result in heavy silver deposits in cortex only indicative of corticocortical projections alone from these layers. Previously 14 we noted in area 18 of the rat that injections of HRP restricted to supragranular layers resulted only in HRP-positive neurons in supragranular layers of the contralateral hemisphere. Taken together, this information indicates that supragranular layers give rise to callosal and corticocortical connections while infragranular layers give rise to the subcortical projections although they also seem to be the source of some callosal projections and possibly some corticocortical projections as well. In an earlier study on the callosal system 14 we noted that there is considerable overlap between the layers in which the terminal fields of the callosal axons are found and the layers in which the cells of origin of these callosal projections are found. In the current study we have noted an overlap of the bulk of thalamocortical terminals and the ceils of origin of the corticothalamic projections in infragranular layers of motor cortex. In prefrontal, temporal, occipital and parietal cortex the bulk of these terminals is located superficial to the layers in which corticothalamic neurons are found. Golgi studiesl,7,13,26, 34 have indicated that cells in the infragranular layers send their apical dendrites upwards into supragranular layers reaching as far as layer 1. In further experiments Globus and ScheibelS, ~ have demonstrated that in young animals, lesions in lateral geniculate produce a reduction in the number of spines along

398 the apical dendrites of pyramidal neurons in all layers of visual cortex while lesions interrupting the callosal fibers result in a loss of spines limited to the oblique branches of the apical dendrites. Thus thalamocortical axon terminals distributed in supragranular layers may well form synapses with corticothalamic neurons located in layers V and VI by means of their apical dendrites traversing more superficial layers. These findings are in close agreement with the classical observations of R a m d n y Cajal a4 and Lorente de N6 '26. Using H R P we have been able to identify the kind of cells which form the corticothalamic connections in the infragranular layers of the rat. We have noted that the number of positively labeled cells in cortex is related to the amount of H R P injected: the microinjections resulted in far fewer labeled cortical neurons (Fig. 4) than were found after larger injections CFigs. I-3 and 6). However. in general the number of cortical neurons labeled was small. The presence of the medium sized pyramidal cells in layer V throughout the rat neocortex labeled with H R P after the thalamic injections was striking, but there were also smaller pyramidal-shaped neurons in layers V and VI with the most common cell in layer VI being the smaller pyramidal neuron. It appears from our material that P3 ramidat cells alone give rise to the corticothalamic projections. It is possible that not all cells projecting to thalamus are labeled with H R P subsequent to thalamic injections of the tracer. In this case it is possible that some corticothalamic neurons with shapes other than pyramidal cells might not have been visualized with this technique. However. this we consider unlikely considering the amount of material examined and the fact that in the callosal system ~ HRP does visualize fusiform- and stellate-shaped neurons as well as pyramidal cells. These observations suggest that in the rat. pyramidal cells alone give rise to extracortical connections. However, further investigations are in order in other m a m m a l s before one can generalize to other species. From our data we can say that the corticothalamic and thalamocortical connections are in general reciprocal as indicated by the fact that the HRP-positive cells and heavy silver deposits were always co-extensive in the cortical areas in which they were found. We cannot, however, make any statements about the reciprocal connectivity of individual thalamic nuclei with cortex since in our material with the combined injections the injection sites involved more than one thalamic nucleus. The combined use of H R P and [aH]leucine is well-suited, however, to studying reciprocal connectivity after thalamic injections. We have demonstrated this here for groups of thalamic nuclei but in another study in squirrel monkey a6. we have demonstrated that cortical injections of the two tracers can be used to advantage in examining the reciprocal connectivity of individual thalamic nuclei with cortex. With more limited injections or injections similar to the ones made in this study, but in larger mammals. thalamic injections of the two tracers should also be useful in determining the reciprocal connections of individual nuclei. Such studies were impossible earlier when only the anterograde and retrograde degeneration methods were available. The intrathalamic connections we noted suggest another potential area also relatively unexplored in which H R P and autoradiography can be exploited. The overall information on the corticothalamic and thalamocortical connections

399 derived from the techniques used in this study is similar to earlier studies. In a study of medial dorsal efferents, others TM noted that the highest grain counts in prefrontal cortex were in layers iii and I. In our material combined injections involving medial dorsal nucleus resulted in a similar pattern of distribution of the silver grains, however, in addition, layer VI had heavy silver deposits. With anterograde silver methods, the pattern for the projection field of medial dorsal nucleus was similar; however, fibers of passage are noted in layer V126 which may be the explanation for our peak of silver grains in VI. In opossum 17, thalamocortical fibers terminate mostly in layers I and IV as we have found in most regions of rat la and a similar situation exists for cat somatosensory and auditory cortex 15,4a, although in cat visual cortex 4z it is only layer IV that is primarily involved. In monkey 16,28 the thalamocortical fibers are restricted primarily to layer IV and the adjacent portion of layer III. We have examined the distribution and cell types of corticothalamic neurons keeping in mind the different patterns of cortical organization. Although different cortical areas, such as motor and somatosensory cortex, for example, have different laminar organizations and functions and receive projections from diverse thalamic nuclei, nevertheless, the corticothalamic projections from these diverse parts of cortex originate from pyramidal cells located in the infragranular layers exclusively. In contrast, we found that the terminals of the thalamocortical projections have different patterns of distribution depending on the area in which they synapse. Prefrontal and motor cortex each had different patterns while a third pattern was shared by temporal, occipital and parietal cortex. Whether similar relationships between corticothalamic neurons and thalamocortical projections will be found in other mammals remains to be seen but using HRP and autoradiography in combination one should be able to answer this question. ACKNOWLEDGEMENTS

The authors wish to thank Mr. E. Dalm, Mr. J. Ginzberg, Mrs. M. Heung and Mrs. E. van der Val for their expert technical assistance; Mr. W. van den Oudenaider for his help with the figures and photography; and Mrs. E. Jongbloed for typing the manuscript. This investigation was supported by USPHS Grant NS 07666 and the Charlton Fund of Tufts University.

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