ELSEVIER Brain Research 709 (1996) 311-315

BRAIN RESEARCH

Short communication

Corticostriatal projections from layer V cells in rat are collaterals of long-range corticofugal axons

Martin IAvesque, Ali Charara, Serge Gagnon, Andrd Parent, Martin Deschanes * Centre de Recherche en Neurobiologie, H~pital de l'Enfant-Je'sus, Universit~ Laval, 1401 18th Street, Quebec G1J 1Z4, Canada

Accepted 12 October 1995

Abstract

Corticostriatal projections arising from the infragranular layers of the motor and second somatosensory cortices were studied in rats after labeling small pools of neurons with biocytin. Camera lucida reconstruction of 263 fibers arising from laminae V and VI revealed that all corticostriatal projections derive from collaterals of lamina V cells whose main axons descend into the cerebral peduncle. In contrast, lamina VI cells do not branch upon the striatum, but upon the thalamus. Together with the results obtained in previous tracing studies, the present data raise the possibility that no neuron is exclusively corticostriatal. We therefore propose that all corticostriatal projections are collaterals given off by the axons of two types of neurons: layer V cells whose main axon project to the brainstem and/or spinal cord, and layer III cells that project to the contralateral hemisphere.

Keywords: Basal ganglia; Striatum; Corticospinal cell; Motor cortex; Second somatosensory cortex; Corticothalamic cell

It is now well established that corticostriatal projections arise from virtually all neocortical areas, and those arising from the sensorimotor cortex appear quantitatively the most numerous [10,11,14,21]. Using the Golgi technique, Ramon y Cajal [22] was first to describe the corticostriatal pathway which, he suggested, was made of collaterals of fibers descending into the internal capsule. A similar sug- gestion was also made by other investigators who analyzed Golgi impregnated material in different species [4,16,28]. Some anatomical and electrophysiological studies provided direct evidence for the branching of corticofugal axons in the striatum, but it remains difficult to evaluate quantita- tively the contribution of these collaterals to corticostriatal projections [3,5,7,13,18,2411. What is clear, however, is that the bulk of corticostriatal fibers originate from layer V cells with a minor contribution from layer III neurons [1,9,15,19,23,25-27,31]. Though some of these labeling studies also reported a possible contribution from lamina VI cells, this contribution remains uncertain because of the possibility of retrograde htbeling through the severing of axons 'de passage'. In the., present study we re-examined Cajal's proposal by labeling with biocytin small pools of

* Corresponding author. Fax: (1) (418) 649-5910.

0006-8993/96/$15.00 © 1996 F, lsevier Science B.V. All rights reserved SSDI 0006-8993(95)01333-4

neurons in laminae V and VI of the motor (MI) and second somatosensory (SII) cortices in rats.

Thirty-three Sprague-Dawley rats of either sex were used in the present study. Housing and treatment condi- tions adhered to federally prescribed and university animal care and use guidelines. Animals were deeply anesthetized with ketamine (75 mg/kg) plus xylazine (5 mg/kg) in- jected intramuscularly, and several microiontophoretic in- jections of biocytin were made in laminae V or VI of MI and SII using the stereotaxic coordinates of the atlas of Zilles [32] (areas labeled Frl-Fr2 and Par2). Microion- tophoretic labeling was done with glass micropipettes (tip diameter 3 -4 /xm) filled with a solution of potassium acetate (0.5 M) plus 2% biocytin (Sigma). These elec- trodes had impedance of 15-25 M/2 and they were used to monitor the spontaneous slow rhythmic discharges which characterize layer V cells under ketamine anesthesia. Once in laminae V or VI the pipette was connected to a high compliance iontophoresis device (Neuro Data) and the tracer was ejected by passing positive current pulses of 300-400 nA (1 s on /1 s off) for 25-35 min. After a survival period of 24 h, animals were anesthetized and perfused with 500 ml of phosphate buffered saline (0.1 M, pH 7.4) containing 0.5 ml heparin, followed by 800 ml of fixative (4% paraformaldehyde plus 0,1% glutaraldehyde in phosphate buffer). After a final wash with 400 ml of a

312 M. Ldvesque et aL / Brain Research 709 (1996) 311-315

10% sucrosed solution in phosphate buffer, brains were transferred in a cryoprotective solution of 30% sucrose until immersion. Sagittal or horizontal sections were cut at 50 /zm on a freezing microtome and processed for biocytin histochemistry according to the protocol described by Horikawa and Armstrong [12]. Most of the material was

also processed for cytochrome oxidase histochemistry [30] or calbindin D-28k immunohistochemistry [2] to allow visualization of cortical laminae or striatal compartments. Sections were mounted on gelatin-coated slides and cov- ered with Permount. Labeled axons were drawn with a camera lucida and serially reconstructed. This study is

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F: Fig. 1. Photomicrographs of biocytin injection sites in laminae V and VI of the motor (A,B) and second somatosensory (C,D) cortices. Dashed lines indicate the limits of lamina V. E and F: collateral branches emitted in the striatum by the main axon of two layer V cells. Arrowhead in F indicates a branching point. Scale bar in A (400 /xm) also applies to B; scale bar in C (200 ~m) also applies to D; scale bar in E (25 /zm) also applies to F.

M. L~vesque et al. /Brain Research 709 (1996) 311-315 313

Table 1 Number of fibers completely c,r partially reconstructed after biocytin injections in laminae V and VI ~of the motor and second somatosensory cortices

Fibers MI SII

reconstructed (n) Layer V Layer VI Layer V Layer VI

Completely 11 6 9 7 Partially 51 62 45 72

based on the tracing of 263 axons whose laminar and areal origins are indicated in Table 1. Thirty-three of these axons were completely reconstructed whereas, for the re- maining fibers, only partial reconstructions were made to verify that they displayed branching patterns similar to those observed in individually reconstructed cells.

Fig. 1 shows examples of injection sites in laminae V and VI of MI (Fig. 1A,.B) and SII (Fig. 1C,D). The extracellular protocol of biocytin application produced

Golgi-like labeling of 4 -25 neurons at the injection sites. At some sites labeled neurons were grouped within a single lamina while at other sites they formed a column spanning across multiple laminae, but yet with a majority of labeled neurons located in the targeted layer. Most axons arising from these sites penetrated into the white matter and entered the striatum. Among these fibers, we selectively reconstructed those who gave off branches into the striatum or the thalamus (Fig. 1E,F). Without excep- tion, all fibers that ramified within the striatum continued their course caudalwards in the cerebral peduncle. Some of these fibers also branched upon the thalamus but they did not supply any collateral to the thalamic reticular nucleus. Fig. 2 shows the reconstruction of two representative corticostriatal cells whose main axon could be traced back to their cell body of origin in layer V. In contrast, no lamina VI cells branched upon the striatum. These cells projected only to the thalamus where they all gave off collaterals as they traversed the thalamic reticular nucleus.

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Fig. 2. Camera lucida drawings of two corticostriatal axons arising from lamina V cells of the motor (A) and second somatosensory (B) cortices. Note that both axons continue their course in the cerebral peduncle and that the axon in B also sends a branch towards the thalamus.

314 M. Ldvesque et aL /Brain Research 709 (1996) 311-315

As expected from previous anterograde labeling studies [6,8,10,17], collaterals of corticostriatal cells from MI and SII coursed rostrocaudally in the striatum and their termi- nal fields occupied the extrastriosomal matrix.

The central finding of the present report is that all corticostriatal projections arising from layer V cells in areas MI and SII of the rat are collaterals of long-range axons that project below thalamic level. Because all corti- cal projections to the brainstem and spinal cord arise exclusively from layer V cells [14,20], and because the above results were obtained with a method that allows the visualization of axons and of their collaterals, the present observations leave little uncertainty about this conclusion. In addition, whenever possible, branching axons were traced back to their cells of origin which were always found in lamina V. This conclusion is therefore in full agreement with the initial suggestion made by Cajal [22] and with the results of Cowan and Wilson [3] who, after labeling intracellularly corticostriatal cells of the medial agranular cortex in rats discussed a similar possibility. On the other hand, it is known that some corticostriatal cells, especially in the rat motor cortex, do not have axonal branches that descend to the brainstem. These neurons, which are located in the upper part of lamina V and deep in lamina III, project ipsilaterally and/or contralaterally to the striatum and many of them, perhaps all, have contralat- eral cortical projections [3,29] (see also [1,31] for similar results obtained in monkey). These observations, together with our own results, raise the possibility that no neuron is exclusively corticostriatal. We therefore propose that all corticostriatal projections are collaterals given off by the axons of two types of neurons: layer V cells whose main axon project to the brainstem and/or spinal cord, and layer III cells that project to the contralateral hemisphere.

Acknowledgements

This research was supported by grants from the Medical Research Council of Canada. A.C is a fellow from the F.C.A.R.

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