HORSERADISH PEROXIDASE-POSITIVE NEURONS OF NONSPECIFIC THALAMIC

NUCLEI PROJECTING TO THE PRIMARY SOMATOSENSORY CORTEX IN CATS

V. Yu. Ermolaeva, N. A. Brukhanskaya, UDC 612.826.4:612.825:612.822 Yu. G. Kratin, and G, A. Tolchenova

The morphology and topography of neurons whose axons form the nonspecific thalamic input in the primary somatosensory area were studied in the cat forebrain by the retrograde axonal horseradish peroxidase transport method. Stained ceils were found in the dorsolateral part of the nucleus ventralis anterior, and were diffusely distributed in the nucleus centralis, lateralis, the lateral partof the nucleus dorsalis

medialis, and the dorsal part of the centrum medianum. In the nucleus paracentralis only solitary, palely stained neurons were detected. Cells stained with horse- radish peroxidase were multipolar, triangular, or fusiform. The results are evi- dence that besides the ventrobasal complex, the nonspecific nuclei of the dien- cephalon also project into the somatosensory cortex. This indicates the exist- ence of multiple afferent thalamic inputs into the somatic cortex.

INTRODUCTION

Important information on the origin of thalamic inputs into the primary somatosensory area of the cortex (SI) has been obtained by the methods of retrograde cell degeneration [5, 15] and anterograde fiber degeneration [2, ii]. It has been found that the nucleus posterDlateralis ventralis (VPL) and the nucleus posteromedialis ventralis (VPM), together constituting the ventrobasal complex [20], are the source of thalamic afferent connections for area SI. It has proved impossible to establish ascending connections of the nonspecific thalamic nuclei with SI by the anterograde degeneration method. Yet their existence has not been denied, and the absence of results has been attributed to limitations of present morphological techniques [7]. Only very recently has a start been made on the use of a histochemical method based on retrograde axonal transport of horseradish peroxidase (HRP), by means of which nonspecific connections of area SI can be identified [i0, 19].

In view of the irregular distribution of specific thalamic fibers in SI in the cat, and their concentration in the projection region of the forelimb and head, which the writers described previously [2, 3], the method of retrograde axonal HRP transport was used to make a detailed study of the thalamic sources of fiber systems running into these projection areas.

EXPERIMENTAL METHOD

Three adult cats weighing 3-4 kg and a month-old kitten were used. Intravital in- jections of HRP were given under pentobarbital or hexobarbital anesthesia after preliminary division of the soft tissues of the head, trephining of the skull, and opening of the dura. A 25% aqueous solution of HRP (type VI, from Sigma) was injected into 3 or 4 points of the cortex to a depth of 1-2 mm; only one microinjection was given to the kitten. The solution of HRP (0.2 ~i) was injected into each selected point of the cortex in the course of 20 min by means of a steel needle (diameter 200 D), connected to a Hamilton's syringe. The animals were perfused through the heart 48 h after the microinjections with a mixture of 1% para- formaldehyde and 1.25% glutaraldehyde in 0.i M phosphate buffer, pH 7.4. Sections 60 D thick were cut on a freezing microtome. Most of the sections were treated by the usual method [13], the rest by Mesulam's method [17]. For better detection of the basal ganglia and tracts, the sections after incubation were stained with cresyl violet or safranin. The thalamic nuclei were identified by means of the atlas [8]. The diencephalon and sensomotor cortex were investigated in series of frontal sections.

I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. ii, No. 5, pp. 435-440, September-October, 1979. Original article submitted November 28, 1978.

0090-2977/79/1105-0321507.50 © 1980 Plenum Publishing Corporation 321

a

b

:'Mn "~.':. ".:.

r'-r. Z5 ~ Fr 70

Fig. i. Location of zones with maximal HRP dif- fusion in somatosensory cortex (a) and of HRP- positive neurons in thalamic nuclei (b). Results obtained on four animals, a) Frontal sections through area SI. b) Distribution of HRP-positive neurons indicated by dots at corresponding frontal levels of thalamus. VA) Nuci~us ventralis anterior, CL) nucleus centralis lateralis, PC) nucleus para- centralis, CM) centrum medianum, MD) nucleus dorsalis medialis.

EXPERIMENTAL RESULTS

Microscopic examination of the region of injection of the enzyme showed that the area of primary diffusion of HRP in three cats lay on both sides of the coronal sulcus (the pericoronal region), and in one animal it spread partly into the posterior sigmoid gyrus (Fig. la).

Examination of series of frontal sections through the diencephalon revealed stained

neurons not only in the ventrobasal complex, as the writers described previously [3], but also in certain nonspecific thalamic nuclei.

Nucleus Ventralis Anterior (VA). Stained cells were detected over the whole extent of the nucleus, but always in its dorsolateral part (Fig. i). The presence of granules con- sisting of products of the histochemical reaction for HRP in the perikaryon and processes gave some idea of the shape of the neuron as a whole. Stained cells were found in this nucleus mainly in groups consisting of large multipolar and fusiform neurons. The morpho- logical picture (Fig. 2a) indicates that neurons of different types could be present in the same group. In addition, the quantity of enzyme taken up by different cells could vary, and it could be distributed unevenly in the soma and processes. Separate zones of concentration of granules were clearly distinguished. The photomicrographs (Fig. 2a) show that the density of distribution of the granules in two polygonal neurons was much higher than in the fusi- form cells. Many HRP-positive cells were found at the level of the caudal zones of the posterior hypothalamus. It was at these levels that in one field of vision of the micro- scope (lOOx) groups consisting of four to six cells were discovered. At the same frontal levels stained neurons also were found in the intralaminar thalamic nuclei.

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Fig. 2. Different types of HRP-positive neurons in non- specific thalamic nuclei, a) In VA, b) in CL, c) in CM, d) in ~. Magnification: 650x (for a, d) and 1000x (for b, c).

Nucleus Centralis Lateralis (CL). Stained neurons in this nucleus were mainly very large and distinguished by their polygonal shape. In sections stained by the method of Graham and

Karnovsky they were dark brown in color and had clearly outlined processes. The photomicro- graph (Fig. 2b) gives a comparatively complete picture of the structure of the projection

neuron of this nonspecific nucleus, for the character of branching of the numerous processes can be clearly seen. Here, just as in the previous photomicrograph, a limited zone of HRP concentration in the perikaryon is observed. Unlike the character of distribution of the stained cells in VA, in CL they were scattered all over the territory of the nucleus and even outside its boundaries -- in structures located more ventrally (Fig. Ib).

Nucleus Paracentralis (PC). Solitary stained medium-sized and triangular cells were seen in this nucleus. They lay on the boundary with CL along the course of the internal medullary lamina. Unlike stained neurons found in all the nonspecific nuclei studied, the cells in PS

were palely stained and contained no large granules of enzyme.

Centrum Medianum (CM). In sections stained with safranin or cresyl violet, among the

masses of fusiform cells which formed the cytoarchitectonic basis of this nucleus, darkly stained neurons with many granules in the perikaryon and processes could be distinguished.

Like the other fusiform cells of this nucleus, they were oriented in a definite direction: Their long axes passed through the dorsolateral and ventromedial parts of the nucleus. A bipolar fusiform cell of CM projecting into the pericoronal region of area SI is illustrated in Fig. 2c. The region of CM in which most stained neurons were situated corresponded to the central part of the nucleus, adjacent to the nucleus dorsalis medialis (Fig. Ib).

Nucleus Dorsalis Medialis (MD). Neurons stained with HRP were found over the whole extent of the caudal half of the nucleus and they occupied its lateral part. As Fig. ib shows, two foci of distribution of stained cells could be distinguished at the level of maximal section of the nucleus. Stained neurons of different shapes (predominantly multi- polar) were grouped in fours or fives. Two large neurons with many clearly defined processes can be seen in Fig. 2d. Granules were comparatively uniformly concentrated in the processes, whereas in the cell body the enzyme accumulated along the border of the perikaryon.

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DISCUSSION

The results are in agreement with facts described briefly earlier [i0, 19]. For in- stance, some neurons of the nonspecific thalamic nuclei were shown to be connected mono- synaptically by ascending fibers with cortical neurons of area SI. The presence of a mono- synaptic pathway leading from CM into area SI also follows from the results of electrophysio- logical investigations [6, 7]. The view has also been expressed that direct pathways orig- inating in the intralaminar nuclei may terminate in the upper layers of the cortex, whose neurons have not yet been identified electrophysiologically [4, 14]. The afferent inputs of the pericoronal part of area SI, the region of representation of the forelimb and head, are thus formed not by one specific source alone, but by several (thalamic) sources, including the nonspecific nuclei. It must be emphasized that the projection neurons which the writers discovered previously [3] in the ventrobasal complex under similar conditions, were more numerous than in VA, MD and, in particular, in the intralaminar nuclei.

It can be concluded from all the histological pictures described above that the projection neurons discovered vary in their morphological structure. Variation in their size and shape also has been observed in the specific thalamic nuclei of the ventrobasal complex [3]. In the present investigation, as in the previous one, the stained cells in- cluded multipolar cells, which are generally regarded as associative cells [21]. The lack of any morphological uniformity suggests the existence of projection neurons with different

functional properties. However, because of the absence of concrete results of electro- physiological experiments in the literature it is difficult to reach any firm conclusion regarding the existence of correlation between structure and function of neurons in thalamic nuclei.

The fact that the direct spinothalamic tract, running in the ventrolateral funiculi [7, 9, 16], terminates in the intralaminar nuclei, is evidence that very short nonspecific pathways participate in the conduction of somatic sensation to area SI. Since SI is connected by descending pathways with the nonspecific nuclei mentioned above [2, 9], there are grounds for regarding the whole system of connections in this zone as circular. The role of infor- mation arriving via these nonspecific channels for sensory discrimination is not yet known. It can be suggested on the basis of the results of morphological investigations and the study of behavioral reactions that the system of connections described above participates in the processing of somatic volleys which are essential for realization of the vocalization process. This relates to the presence of monosynaptic pathways running from the cortical vocalization center to area SI, to certain intralaminar nuclei, and to VA and ~D [12, 18]. The organiztion of such a complex behavioral act undoubtedly demands the participation of several polysynaptic nonspecific connections. The existence of such connections in this system is shown by the results of electrophysiological experiments [4, 14].

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LITERATURE CITED

V. Yu. Ermolaeva, "Morphology and topography of efferent systems of the first and second somatosensory areas in the cat," in: Morphology of Pathways and Connections in the Central Nervous System [in Russian], Nauka, Moscow -- Leningrad (1965)i V. Yu. Ermolaeva, "Morphology of connections of the ventrobasal complex of the thalamus with the first and secondary somatosensory areas of the cortex," Dokl. Akad. Nauk SSSR,

198, 716 (1971). V. Yu. Ermolaeva, V. P. Babmindra, and N. A. Brukhanskaya, "Horseradish peroxidase- positive neurons of the ventrobasal complex of the thalamus projecting to somatosensory area I in the cat," Neirofiziologiya, i i, 125 (1979). V. M. Storozhuk, Functional Organization of Somatic Cortical Neurons [in Russian], Naukova Dumka, Kiev (1974), 270 pp. H. Akimoto, K. Negishi, and K. Yamada, "Studies on thalamocortical connections in cat by means of retrograde degeneration method," Folia Psychiat. Neurol., i0, 39 (1956). D. Bowsher, "The termination of secondary somatosensory neurons within the thalamus of Macaca ne~latta: an experimental degeneration study," J. Comp. Neurol., 117, 213 (1961). D. Bowsher, "Some afferent and efferent connections of the parafascicular-nentrum medianum complex," in: The Thalamus, Columbia University Press, New York (1966), pp. 99-108.

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8. H. H. Jasper and C. Ajmone-Marsan, A Stereotaxic Atlas of the Diencephalon of the Cat, Ottawa (1954).

9. E. G. Jones and H. Burton, "Cytoarchitecture and somatic sensory connectivity of thalamic nuclei other than the ventrobasal complex in the cat," J. Comp. Neurol., 154, 395 (1974).

i0. E. G. Jones and R. Y. Leavitt, "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., 154, 349 (1974).

Ii. E. G. Jones and T. P. S. Powell, "The cortical projection of the ventroposterior nucleus of the thalamus in the cat," Brain Res., 13, 298 (1969).

12. U. J~rgens, "Projections from the cortical larynx area in the monkey," Exp. Brain Res., 2j, 401 (1976).

13. R. C. Graham, Jr., and M. J. Karnovsky, "The early stages of absorption of injected horseradish peroxidase in the proximal tubules of the mouse kidney: ultrastructural cytochemistry by a new technique," J. Histochem. Cytochem., 14, 291 (1966).

14. C. L. Li, "The facilitatory effect of stimulation of an unspecific thalamic nucleus on cortical sensory neuronal responses," J. Physiol. (London), 131, 115 (1956).

15. G. Macchi, F. Angeleri, and G. Guazzi, "Thalamocortical connections of the first and second somatic sensory areas in the cat," J. Comp. Neurol., iii, 387 (1959).

16. W. R. Mehler, "Further notes on the centrum medianum nucleus of Luys," in: The Thalamus, Columbia University Press, New York (1966), pp. 109-122.

17. M. M. Mesulam, "The blue reaction product in horseradish peroxidase neurohistochemistry: incubation parameters and visibility," J. Histochem. Cytochem., 2_4, 1273 (1976).

18. P. MNlle-Preuss and U. JNrgens, "Projections from the "cingular" vocalization area in the squirrel monkey," Brain Res., 103, 29 (1976).

19. H. I. Ralston and P. V. Sharp, "The identification of thalamocortical relay cells in the adult cat by means of retrograde axonal transport of horseradish peroxidase," Brain Res., 62, 273 (1973).

20. J. E. Rose and V. Mountcastle, "The thalamic tactile region in rabbit and cat," J. Comp. Neurol., 97, 441 (1952).

21. M. Scheibel and A. Scheibel, "Patterns of organization in specific and nonspecific thalamic fields," in: The Thalamus, Columbia University Press, New York (1966), pp. 13- 47.

SPATIAL CONFIGURATION OF RABBIT SUPERIOR COLLICULAR EVOKED

RESPONSE TO PUNCTIFOP~ AFFERENT STIMULATION

A. M. Mass and A. Ya. Supin UDC 612.826.5:577.352.5

Evoked potentials arising in the rabbit superior colliculus in response to puncti- form stimulation of the receptive field were studied. This response has only nega- tive polarity at the focus of maximal activity and does not exhibit reversal of the potential which is a characteristic feature of the response to diffuse stimulation. The evoked potential was recorded at depths of between 0.i and 0.9-1.0 mm from the collicular surface, corresponding to the stratum griseum superficiale. The response disappeared when the stimulating spot was shifted through 4-6 ° away from the optical position. It is suggested that evoked potentials to punctiform stimulation can give more complete information on the location of different synapses.

INTRODUCTION

Analysis of evoked potentials (EP) to afferent stimulation is a well-known method of determining the location of afferent endings and their synapses in brain structures. In this case EPs are recorded at a number of points in the test structure, in different layers for

A. N. Severtsov Institute of Evolutionary Morphology and Ecology of Animals, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol, ii, No. 5, pp. 441-450, September-October, 1979. Original article submitted May 29, 1978.

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