0306-4522/92 $5.00 + 0.00 Pergamon Press Ltd 0 1992 IBRO

Neuroscience Vol. 49, No. 4, pp. 14%161, 1992 Printed in Great Britain

MAPPING OF THE MOTOR PATHWAYS IN RATS: c-fos INDUCTION BY INTRACORTICAL MICROSTIMULATION OF THE MOTOR CORTEX CORRELATED WITH EFFERENT CONNECTIVITY OF THE SITE OF CORTICAL STIMULATION X. S. T. WAN,* F. LIANG,V. MORET,M. WIESENDANGWand E. M. Roun_Lmzt Institute of Physiology, University of Fribourg, Rue du Mu&. 5, CH-1700 Fribourg, Wrolles, CH- 1700 Fribourg, Switzerland *Department of Anatomy, Peking Union Medical College, 9 East Dan 3rd Tiao, Beijing, China Ahatiaet-The general goal of the present study was to investigate structural components of a neural system anatomically as well as functionally. The rat motor system, which is reasonably well understood, was selected and a new procedure was developed to combine a functional marker with axonal tracing methods (in the same animal). This was achieved by mapping c-fos induction immunocytochemically as a result of intracortical microstimulation in the distal forelimb area of the motor cortex. The anterograde tracers Phaseolus vulgar&leucoagglutinin or biocytin were deposited at the site of intracortical microstimulation, the former three weeks and the latter two to three days before stimulation. Neuronal nuclei, labeled for the expressed c-fir protein, were present and mapped in the following structures: motor cortex; basal ganglia (caudate+putamen, globus pallidus); thalamus (reticular, ventromedial and posterior nuclei); subthalamic nucleus; substantia nigra; tectum; red nucleus; pontine nuclei; inferior olive; external cuneate nucleus; cerebellar cortex; deep cerebellar nuclei. Labeling was often bilateral but generally more substantial ipsilaterally, except in the cerebellum where it was mainly contralateral. Axonal labeling, including terminal branches and boutons, was also found in most of the above structures with the exception of the globus pallidus, deep cerebellar nuclei, cerebellar cortex and external cuneate nucleus. These expected exceptions demonstrate that activity changes in these latter structures, as revealed by C-$X labeled neurons, were induced over more than one synapse. This combined procedure might, therefore, be useful in deciding whether two structures in a given system are linked directly (monosynaptically) or indirectly (polysynaptically) to each other. In contrast to the 2-deoxyglucose technique, functional mapping by means of c-fos induction provides cellular resolution, making it possible to establish fine

details of axonal contacts with target neurons:boutons in close apposition to c-fos labeled neurons were clearly observed here, for instance, in the cerebral cortex, caudate-putamen, thalamus, subthalamic nucleus and pontine nuclei. Surprisingly, the ventrolateral and ventrobasalis nuclei of the thalamus contained numerous and dense axon terminals labeled with Phuseofus vulgaris-leucoagglutinin or biocytin, but the contacted neurons in the ventrolateral and ventrobasalis nuclei were not marked with c-fos. However, with respect to directly connected structures, there was, in general, a good correlation between structures with axonal labeling and those with c-fos labeled neurons.

In the last few years, several genes responsive to trans-synaptic stimulation and membrane electrical activity have been identified in neurons.33*M*55 These genes are activated rapidly and transiently (immediate early genes) and their transcription is induced by neurotransmitters or by electrical stimulation.55 The most commonly studied of these genes is the c-fos gene, which has been described as a proto-oncogene. C-fos activation is believed to be a measure of the firing rate, depolarization of a neuron or the levels of second messengers.” The c-fos gene was shown to be activated in neurons of the rodent brain by a large tTo whom correspondence should be addressed. Abbreuiotions: ABC, avidin-biotin-peroxidase complex; DAB, 3,3’-diaminobenxidine; 2-DG, 2deoxyglucose; ICMS, intracortical microstimulation: PBS-T. phosphate-buffered saline containing 0.3% ‘T&on; PI-IA-L, Phuseolus oulgarir-leucoagglutinin; PM, paramedian lobule.

variety of stimuli, e.g. following generalized seizure ‘335*33*38 audiogenic seizure,2* peripheral nerve injury,;i ischemia,“*‘j* osmotic stress,‘**53 cortical lesions and nerve growth factor injections,49s” kindling stimulation,‘3~L4~~@ electrical stimulation of the motor cortex47*mand trigeminal ganglion,63 water or deprivation,47 nociceptive stimulation,3*6*7J4~39+7 chemical lesion of the substantia n&a.” C-fos induction was also observed in the retina& and in the suprachiasmatic nucleus’@45 as a result of photic stimulation. The protein products of the c-fos gene, and other genes of the same family, are considered to be involved in the coupling of short-term intracellular signals, elicited by extracellular stimuli, to long-term functional changes by altering gene expression.47 C-fos protein expression can be detected at the cellular level, using immunohistochemistry,9~*4~~~33~47~~ i.e. at a resolution higher than another marker of 749

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activity, 2-deoxyglucose (2-DG). We report here, for the first time, the feasibility of combining the method of c-&r expression as a functional marker with conventional axonal tracing methods such as Phaseolus vulgaris-leucoagglutinin (PHA-L) or biocytin. The motor pathways of the rat were studied because its anatomy is well establisheda and because it can be activated focally by the method of intracortical microstimulation (ICMS). Furthermore, ICMS has already been tested successfully for c-fos induction4’ as well as for 2-DG labeling.48,5’ With this combined approach, the objective of the present study was to answer the following questions: (1) Is it possible to establish whether a given structure in the motor pathways is activated directly, i.e. monosynaptically, from the motor cortex, or via a chain of two or more synaptic relays? (2) Are all direct targets (labeled with the axonal tracing methods) equally labeled with the functional marker, or is c-fos preferentially induced in some and not in other target structures? functional

EXPERIMENTAL

PROCEDURES

A total number of 22 Sprague-Dawley rats were used in the present study (weighing between 150 and 300 g). The rats used for ICMS (n = 10) were first anesthetized with ketamine (10 mg/lOO g body weight, i.p.; additional doses of 4 mg/lOO g body weight were given i.m. whenever necessary as checked by the reflex state of the animal). For preliminary localization of the distal forelimb motor area, a small craniotomy was made in the region of the motor cortex, according to previously described stereotaxic coordinates,‘7s2’,3s and a low-threshold (l&20 NA) distal contralatera1 forelimb response was searched with a low impedance microelectrode for ICMS29.43.44 (individual nulses of 0.4 ms delivered at a rate of 400/s in trains of 30 ms duration and presented at a rate of 0.5 Hz). In a subgroup of rats subjected to ICMS (n = 5) in order to combine the c-fos functional labeling with tracing of axons originating from the ICMS site, an anterograde tracer was injected at the same site. In the present series of experiments, the anterograde tracers Phuseolus oulguris-leucoagglutinin (PHA-L, see Refs 16, 20) or biocytin” were used. To inject PHA-L or biocytin, the tungsten stimulating microelectrode was removed and a glass micropipette, positioned at the same location (checked under the operating microscope), was lowered at a depth where ICMS was most efficient, usually 1.61.9 mm below the pial surface. Glass micropipettes had tip diameters ranging between 15 and 20 cm and were filled either with a solution of 2.5% PHA-L (Vector Laboratories) in 0.05 M Tris-HCI buffer at pH = 7.4 or with a solution of 5% biocytin (Sigma) in 0.05 M Tri-HCl buffer at pH = 7.6. Injection of PHA-L was achieved by iontophoresis (6pA, 7 s on-7 s off) for 20 min, whereas biocytin was injected by pressure (usually 1.5 pl at the ICMS site and 1.5 ~1 about 1 mm above along the same penetration). The pipette used for injections was then removed, and a small chamber centered above the injection site was implanted on the skull with dental cement that made it possible to position a stimulating electrode for subsequent use in the c-fos induction experiment. In rats in which no PHA-L or biocytin injection was made, the implanted chamber was positioned so that its center matched the location of the ICMS electrode. The animal recovered from surgery and anesthesia and returned generally for 2 days to the animal room, except for l-3 weeks in case of PHA-L injection.

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c’t td

Since anesthesia, in particular with ketaminc and most likely also with pentobarbital, can reduce or even block c:/o.r expression,’ the ICMS designed to induce c:/o.c was performed here in unanesthetized animals. This was achieved by inserting, under ether anesthesia, a stimulating tungsten microelectrode in the center of the implanted chamber in order to reach the same cortical locus of the initial ICMS. The electrode was firmly fixed to the chamber to avoid movements in the brain. When the animal recovered from the ether anesthesia (after a few minutes), flexible wires were used as electrode and indifferent leads and 1CMS was applied for 90 min while the rat was confined to a small compartment of his cage in order to minimize spontaneous locomotion or movements. The stimulation intensity was adjusted in order to obtain the same forelimb movement as the initial ICMS, although it was sometimes necessary to increase the intensity of the current; in that latter case, the contralateral forelimb occasionally exhibited variable movements (e.g. extension and flexion). One hour after ICMS offset, the rat was deeply anesthetized with Nembutal and perfused with 200ml saline (0.9%) followed by 1 liter of fixative (2 or 4% paraformaldehyde in 0.1 M phosphate buffer at pH = 7.4). The brain was dissected, postfixed in the same fixative for 5 hand then immersed in a solution of 30% sucrose in 0.1 M phosphate buffer, pH = 7.4, for 1 3 days at 4°C. Fifty-micrometer-frozen sections of the brain were cut in the frontal plane with a microtome, and rinsed in phosphate-buffered-saline (0.01 M, pH = 7.4, 0.9% NaCI), containina 0.3% Triton (PBS-T). The immunohistochemical procedure was adapted from protocols previously described.7.Z6,” Sections were incubated for 30min in PBS-T containing 0.1% H,O, to inactivate endogenous peroxidase and then rinsed three times for 5 min in PBS-T. After a preincubation of 3&60 min in 10% normal rabbit serum in PBS-T, the sections were incubated for 12 h in the primary antibody, a sheep polyclonal antiserum raised against a c-fos synthetic peptide -(CkB, Cambridge, UK, Lot OA-11-823 for immunohistochemistrv). diluted 1: 12.000 in PBS-T (adjacent series of sections*‘were incubated with the same antibody diluted 1: 6000 and 1: 24,000). Sections were then rinsed three times for 5 min in PBS-T and incubated for 90min in biotinylated rabbit anti-sheep serum (Vector Laboratories, 1:200 in PBS-T). Sections were rinsed three times for 5 min in Tris-HCl buffer, 0.05 M, pH = 7.4- 7.6 and placed for 90 min in avidinbiotin-peroxidase complex (ABC) solution (Vector Laboratories, final dilution 1:50, prepared 30 min before use). After rinsing the sections three times for 5 min in Tris-HCl buffer. they were oreincubated for lo-15 min in a solution of 0.05% 3,3’-diaminobenzidine (DAB) in Tris-HCl buffer. Sections were finally reacted 15-30min in a same DAB solution but containing 0.01% H,O,, and rinsed three times in Tris-HCI buffer. When combined with PHA-L tracing, the above c-fos protocol was followed by the treatment for PHA-L as In case of biocytin described in earlier reports. 23~19~41.M injection, sections were treated according to the ABC method preceding the above c-fos protocol. Briefly, for biocytin visualization, sections were first rinsed four times during 20 min in phosphate buffer (0.1 M, pH = 7.4) containing 1% Triton. Sections were then incubated overnight at room temperature with the ABC reactives (standard Vectastain ABC Kit PK-4000 from Vector Laboratories, dilution 1:500 in phosphate buffer with Triton). Sections were rinsed 4 times for 20 min in Tris-HCl buffer (0.05 M, pH = 7.6) before a first preincubation for 10 min in the same TrissHCl buffer containing 0.2% nickel ammonium sulfate. This was followed by a second preincubation of IO min in the same solution in which DAB (500 mail) was added. Sections were then incubated in a similar solution of nickel ammonium sulfate and DAB, but containing also 0.006% of hydrogen peroxide. The incubation generally lasted 15-30 min (as judged by background staining) and sections were finally rinsed three times for 5 min in Tris-HCI

c-fir induction and connectivity in the motor pathways bulher. After completion of all reactions (c-j& and PHA-L or biocytin), the sections were mounted onto subbed slides,

dried, dehydrated and coverslipped. A tint analysis was performed for reconstructing the p&ion of c-f&like immunoreactive neurons with a computer-aided tight microscope, following a procedure previously described in detaila Regions of interest were photomicrographed at 40x, 100x and 400 x magnifications. In a few selected slides for which c-j&-like positive neurons had been plotted, the coverslips were removed in xylene, and the sections were rehydrated and counterstained with Cresyl Violet, in order to relate the distribution of c-fir-like positive neurons with the cytoarchitecture of the corresponding brain regions. In order to relate induction of c-fos to ICMS with more contldence. three different series of control experiments (n = 12) were performed as explained below in the results section. The immunohistochemistry to reveal c-fir was the same in the control rats as explained above for rats subjected to ICMS. RESULTS

Distribution of c-fos-like immunoreactivity as a result of intracortical microstimulation As a result of ICMS in the motor cortex, c-fos-like immunoreactive neurons were observed in several structures of the motor pathways. In particular, such neurons were present in the ipsilateral thalamus, principally in the reticular nucleus of the thalamus (Fig. lC, D), the ventromedial thalamic nucleus (Fig. lC, D) and the posterior nucleus of the thalamus (not shown). Surprisingly, the ventrolateral (Fig. 1D) and ventrobasalis thalamic nuclei (Fig. 1C) were free of c-fos-like immunoreactivity. Positive neurons were observed bilaterally in the caudatiputamen (Fig. 1B, predominantly located in its lateral zone), globus pallidus (not shown), subthalamic nucleus (Fig. lC, E) and substantia nigra (Fig. 2A, B), although the ipsilateral side was clearly predominant for these 4 latter nuclei (see Figs lC, 2A for the subthalamic nucleus and the substantia nigra, respectively). In the substantia nigra, c-fos-like immunoreactive neurons were present in both the pars compacta and reticulata. In the mesencephalon, c-fos-like positive neurons were found in the ipsilateml tectum (lateral part of the deep layers of the superior colliculus), in the ipsilateral red nucleus (magnocellular division: Fig. 2A, C); more posteriorly, in the brain&m, they were located in the pontine nuclei on both sides (but predominantly on the ipsilateral side: Fig. 2D, E), and occasionally in the ipsilateral inferior olive and the ipsilateral external cuneate nucleus. Induction of c-fos was also present in the contralateral cerebellar cortex (Fig. 2F), as well as in the contralateral deep cerebellar nuclei. In contrast, the frontal cerebral cortex was characterized, in general, by a more non-specific distribution of c-fos-like immunoreactive neurons; however, positive neurons were clearly more numerous in the stimulated hemisphere as compared to the contralateral hemisphere. It appears that c-foslike immunoreactivity in the motor cortex is related to the intensity of ICMS. In one animal with low current ICMS (3OpA), c-fos-like positive neurons

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were concentrated in two regions of the cerebral cortex, corresponding to the principal caudal forelimb area of the motor cortex (see CFA in Fig. 5), in which ICMS was performed, and the so-called rostra1 forelimb area,35 where they were predominantly distributed in layer II (Fig. lA, see also RFA in Fig. 5). In all other cases with more diffuse labeling in the frontal cortex, the intensity of ICMS was higher (ranging from 80 to 600 PA across animals). It must also be emphasized that, in all rats tested, ICMS never induced the expression of c-fos in the motoneuronal pools of the cervical spinal cord, although some of them were activated contralaterally to produce the elicited distal forelimb movement. In some structures, c-fos-like positive neurons were distributed homogeneously in the whole nucleus, as in the subthalamic nucleus where labeling was extremely dense (Fig. lC, E). In contrast, in some other structures, c-fos-like immunoreactive neurons showed a preferential spatial distribution, restricted to a particular region. For instance, c-fos-like positive neurons were preferentially located in the lateral half of the caudate-putamen. A recent study4’ showed variations of c-fos-like labeling distribution in the striaturn (lateral vs medial zones) as a function of the mode of induction as well as of neurotransmitter manipulations (denervation, application of antagonists). In the red nucleus, c-for-like immunoreactive neurons were restricted along the rostrocaudal axis to the middle three-fifths of the nucleus, while the most caudal and rostra1 fifths were free of labeling. On each individual frontal section, it appeared that most c-fos-like positive neurons were located in the ventromedial zone of the magnocellular part of the red nucleus, and very few, if any, more dorsally in the parvoccllular part. Although the spatial distribution of c-fos-like positive neurons in the ipsilateral pontine nuclei was relatively wide, they were most concentrated along the mediolateral axis in the middle and medial parts, a preferential distribution comparable to that reported for the termination zone of the corticopontine projection originating from the motor cortex.65*66Along the rostrocaudal axis, labeling was seen principally in the intermediate portion of the pontine nuclei. In the cerebellar cortex, c-fos-like positive neurons were located only in the caudal part of the cerebellum, mainly in the paramedian lobule (PM), and predominantly on the contralateral side. Labeling consisted of several clusters of positive neurons, distributed preferentially in the most lateral (ventral) extent of PM, although a few and smaller additional clusters were also present more medially (dorsally), still in PM. A few c-fos-like positive neurons were also present in the copula pyramis, as medial as the most media1 cluster in PM. This distribution in the contralateral cerebellar cortex is consistent with the distribution of increased 2-DG uptake following forelimb motor cortex stimu1ation.50 In the contralateral cerebellar cortex, c-foslike immunoreactivity was mainly restricted to the

Fig. 1. Photomicrographs illustrating c-&-like immunoreactivity in various structures of the motor pathways. (A) As a result of ICMS in the caudal (principal) forelimb area of the motor cortex, c:li,s-like immunoreactive neurons were present in the ipsilateral rostra1 forelimb cortical area (small black dots, labeling restricted to their nuctei), as shown on a frontal section. The arrows point to the limits (laterally and medially) of the cluster of densely packed c-&r-like positive neurons: more laterally and medially. the density of positive neurons abruptly decreased. Note the preferential laminar distribution of c$.r-like positive neurons, mainly in layer II. Scale bar = I00 pm. WM, white matter. (B) Distribution of c-fos-like positive neurons in the ipsilateral (with respect to ICMS site) caudateputamen (CPU), where they mainly occupy its lateral part. ec = external capsuk. Scale bar = 100 pm. (C) R~onstructjon of an individual frontal section illustrating the distribution of c-for-like positive neurons (dots) in the reticular nucleus of the thalamus (RT, most caudal extent of the cluster of positive neurons), the ventromediai thalamic nucleus (VM) and the subthalamic nucleus (STh). ICMS was performed in the left caudal forelimb cortical area. Note the absence of Positive neurons in the nucleus ventrobasahs (VP). Scale bar = I mm. C, cerebrai cortex; II, hip~~ampus; LV, lateral ventricle. (D) Photomicrograph showing c-&-like immnnoreactive neurons in the ipsiiateral reticular nucleus of the thalamus and ventromedial thalamic nucleus (VM). The section was taken at a level more rostra1 than that displayed in C. Note the absence of c-@-like immunoreactivity in the ventrolateral thalamic nucleus (VL). bv, blood vessel; ic, internal capsule. Scale bar = 100 pm. (E) C-for-like immunoreactivity in the subthalamic nucleus; at this magnification, one can observe that labeling is restricted to the nucleus of the neurons (see also Fig. 4A, B). cp, cerebral peduncle. Note the presence of labeled axons in the cerebral peduncle, resulting from the injection of biocytin in the ipsilateral motor cortex of this animal. Scale bar = 100 ilrn. 752

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Fig. 2. (A) Display of an individual frontal section ill~trating the dist~bution of c-fos-like positive neurons (dots) in the substantia nigra @NC, SNR) and the red nucleus (R), as a result of ICMS in the left caudal forelimb cortical area of the motor cortex. SC, superior colliculus; MGB, medial geniculate body. Scale bar = 1mm. (B) Distribution of c-jos-like positive neurons (black dots) on an individual frontal section through the ipsilateral substantia nigra (SN). cp, cerebral peduncb. Scale bar = 200 pm. (C) c-fos immunor~~tivity (black dots) was observed in the rna~~ll~ar division of the ipsilateral red nucleus (R), while the contralateral red nucleus was free of labeling. The large vertical arrow indicates the position of the midline. Scale bar = 200 pm. (D) Display of an individual frontal section at the level of the pontine nuclei (PN) where c-@-like positive neurons are represented by dots. LC, locus coeruleus; LPB, lateral parabrachial nucleus; MCP, medial cerebellar peduncle; VLL, ventral nucleus of the lateral lemniscus; Vn, trigeminal nerve. Scale bar = I mm. Smatl vertical segments above the left PN represent the numerous labeled corticofugal axons (some of them giving rise to cohaterafs terminating in PN), as a result of PHA-L injection in the caudal forelimb cortical area (on the same side). (E) c&-like immunoreactivity in the pontine nuclei (mainly ipsilaterally), with arrows pointing to three clusters of labeled neurons. ml, medial lemniscus. The large vertical arrow indicates the position of the midline. Scale bar = 200 pm. (F) Arrows point to clusters of c-fos-like positive neurons in the cerebellar cortex. S&e bar = 50 pm. P, Purkinje cell layer; g, granule cell layer.

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granule cell layer although minimal labeling seems to be present in the Purkinje cell layer (Fig ZF). The c&-like labeling in the deep cerebellar nuclei was preferentially distributed in the posterior interposed cerebeliar nucleus and, but less densely, in the lateral cerebellar nucleus. Control experiments Control experiments were performed in three different ways. First, in two rats subjected to ICMS, a few selected sections were treated according to the same protocol except that the primary antibody was omitted. This resulted in the absence of c-fos-like immunoreactivity in the above-mentioned brain regions as compared to the normally treated adjacent sections. Second, a similar experimental protocol was applied to a group of control rats (initial ICMS with or without injection of the anterograde tracer, implantation of a chamber, penetration of the motor cortex with a tungsten electrode) except that no current was passed through the microelectrode before perfusion. In this second group of control rats (n = 4), no or only a few scattered c-&r-like immunoreactive neurons were detected in the caudateputamen and globus pallidus, in the reticular, ventromedial and posterior thalamic nuclei, in the subthalamic nucleus, the substantia nigra, the tectum, the pontine nuclei, the red nucIeus, the cerebellum, the inferior olive and the external cum-ate nucleus. Third, another group of control rats (n = 8) were simply perfused under deep anesthesia without preceding ICMS, chamber implantation, tracer injection and electrode penetration. These rats showed a distribution of c-fos-like immunostaining quite comparable to the second group of control rats, indicting that the initial ICMS (2 or more days before perfusion), injection of tracers and implantation of the chamber did not significantly influence c-fis expression at the time of perfusion. Therefore, all the control data provide evidence that the c-fos-like labeling seen in the rats subjected to ICMS 1-2 h before perfusion is due to induction of c-fos in all of these brain regions by that stimulation. Similarly to the experimental animals, no c-&s-like immunoreactivity was observed in the ventrolateral and ventrobasalis thalamic nuclei of the control rats. On the other hand, control rats showed a moderate number of c-fos-like positive neurons in the parafascicular, central medial and mediodorsal thalamic nuclei, in the medial geniculate body, the hypothalamus, the midbrain periacqueductal gray, the cuneiform nuclei, and the locus coeruleus, as also revealed in the rats subjected to ICMS. For that reason, c-fos activation in these brain regions cannot be interpreted as a specific labeling resulting from electrical stimula~on of the motor cortex. In control rats, c-&-like positive neurons were relatively frequent and widely distributed in the cerebral cortex, but generally evenly dispersed in both hemispheres. In comparison, they were more numerous and more densely labeled in the

stimulated hemisphere in experimental rats, supporting the idea that ICMS of the motor cortex. at least to a large extent, contributes to the induction of cj;ts in the cerebral cortex. Combination of c -fos functional marking with ananal tracing As a result of ICMS in the motor cortex, c-fos-like immunoreactivity was found in brain structures known to be involved in the control of movements such as the cerebellum, the basal ganglia, the substantia nigra, as well as in nuclei projecting to the cervical spinal cord36 and/or receiving descending inputs from the motor cortex (e.g. reticular and posterior nuclei of the thalamus; basal ganglia; subthalamic nucleus, pontine nuclei). Combination of the c-fos functional tracing approach with anatomical tracing of the destination of axons originating from the ICMS site provides useful information about whether a given region of the motor pathways containing c-fos-like positive neurons is functionally influenced directly by the stimulated zone of the motor cortex (via a monos~aptic axonal projection) or whether the influence is exerted indirectly via intermediate structures (polysynaptic pathway). A typical biocytin injection site is illustrated in Fig. 3A, while PHA-L injectian sites similar to those performed in the present series of experiments have been illustrate in recent reports.2p*4”’ At high magnification (100 or 200 x or greater) of the light microscope, the PHA-L and biocytin tracing techniques allow a visualization of individual axons, including their ramification into fine collaterals forming terminal branches. PHA-L or biocytin labeled terminal fields, arising from axons o~~~ti~g from the ICMS site of the motor cortex, are shown in Figs 3 and 4, in the cortical rostra1 forelimb area, the caudate-putamen, the reticular and ventrolateral nuclei of the thalamus, the subthalamic nucleus and the pontine nuclei. In addition, PHA-L and biocytin labeled terminal IIelds partly overlapping the c-fos labeled zone in the ipsilateral rostra1 forelimb area (Fig. 3B): the density of biocytin-labeled terminals was maximal in cortical layer II (Fig. 3B), where the c-fos-like positive neurons were most numerous (see Fig. 1A). Overlapping of PHA-L or biocytin anterograde labeling with c-fos-like immunoreactivity was also present in the basal ganglia (Fig. 3C), in the reticular (Fig. 3D), ventromedial and posterior nuclei of the thalamus (not shown); in those regions (including the cortical rostra1 forelimb area), c-fos-like positive neurons were intermingled with biocytin axonal ramifications, exhibiting small boutons en passant or terrn~~. A similar overlapping between c-fits-like labeling and anterogradely labeled terminal fields was observed in the subthalamic nucleus (Pig. 4A-D); in the subthalamic nucleus, axons densely labeled with biocytin (Fig. 4A, C) were seen to give rise to thin axon collaterals characterized by the presence of numerous boutons en passant and a few boutons

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Fig. 3. (A) Biocytin injection site in the cat&al forelimb cortical area (black spot), spanning the six layers of the cortex, Biocytin was injected in two steps: a first 24 injection was made at 1.8 mm deep from pial surface, followed by a second injection (same volume) 1 mm higher than the 6nt one along the same penetration. WM, white matter; CPU, cat&ate-pmamen. Scale bar = 100 grn. (B) High magniIication of a portion of the rostra1 foretimb (RFA) cortical area shown in Fig. l(A). As a result of ICMS in the ipsilateral caudal forelimb cortical area, c-&r-like positive neurons were numerous in layer 11 of RFA @lack spots) intermingled with labeled axonal ramitications resulting from the biocytin injection illustrated in A (at the ICMS locus). Note the presence of small boutons along the axonal ramifications, as pointed by arrows for a few of them. Scale bar = 50pm. (C) In the caudate-putamen (high magnification of a portion of a section adjacent to that shown in Fig. la), c-fir-like immunoreactive neurons (small black spots, some of them pointed by open arrows) were seen in close vicinity of terminal portions of biocytin labeled axons (as a result of the injection illustrated in A), with small boutons (arrows). Scale bar = 20 pm. (D) Similarly, fine axonal ramifications labeled with biocytin were seen intermingled with e-@-like positive neurons in the reticular nucleus of the th~amus (high ~ification of a portion of a section adjacent to that shown in Fig. ID). Scale bar = 50 pm. terminaux in the vicinity of the c--OS-like immunoreactive neurons (Fig. 4B, D). Similarly, PHA-I., labeled axon collaterals ramified in the pontine nuclei

(as represented in Fig. ZB), where they gave rise to a dense plexus of thin ramifications with numerous small boutons (both en ~~s~~ and ~e~~~~u~),

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Fig. 4. (A) Photomicrograph illustrating c-/&s-like immunoreactive neurons (black spots) in the subthalamic nucleus (STh) together with labeled axons in STh and the cerebral peduncle (cp), resulting from the injection of biocytin in the ipsilateral motor cortex where ICMS was performed. The rectangle corresponds to the portion of STh shown at a higher magnification in panel B, where a few c&-like positive neurons are indicated by arrowheads while small arrows indicate the presence of en passunl swellings along thin labeled axonal ramifications terminating in STh. Scale bars = (A) 50 pm and 20 pm (B). (C) General view of cfbs-like immunoreactivity in STh, but at a different rostrocaudal level. Note the presence of biocytin labeled axons in the cerebral peduncle. The zone indicated by the rectangle is illustrated at higher magnification in panel D, where biocytin labeled axons and boutons en passant (arrows) are visible. Several c-SOS positive neurons are indicated by arrowheads. Scale bars = 100 pm (C) and 20 pm (D). (E) Photomicrograph of PHA-L labeled axons in the pontine nuclei, originating from the ipsilateral caudal forelimb cortical area, where ICMS was performed. Note the presence of boutons (a few pointed by small arrows) in the vicinity of c,fos-like positive neurons (black spots). Scale bar = 20 pm. (F) Photomicrograph of a portion of the ventrolateral thalamic nucleus (VL), with a dense plexus of biocytin labeled axons, as result of injection in the ipsilateral caudal forelimb cortical area of the motor cortex. ICMS performed at the same cortical site did not induce c-fos expression in this particular zone of the ventrolateral thalamic nucleus. Scale bar = 50 Ltm.

clfos induction and connectivity in the motor pathways

mixed with the c-f&like positive neurons (Fig. 4E). All these observations are in line with the well-known cortical projections to these structures (caudateputamen, reticular nucleus of thalamus, subthalamic nucleus, pontine nucleus). It is likely that the same conclusion applies to substantia nigra, red nucleus and inferior olive, where c-&-like immuno~ctivity was intermingled with PHA-L or biocytin anterogradely labeled axons collaterals. However, the axonal ramifications in the substantia nigra, red nucleus and inferior olive were very faintly labeled and very sparse; in other words, these elements were at the limit of detection of the method, at least at the present stage of the experimental conditions. Other brain regions displaying c-f&like immunoreactivity however did not show any PHA-L or biocytin labeling (e.g. cerebellum, globus pallidus, external cuneate nucleus) which is consistent with the notion that these structures were indirectly activated. Still other structures, such as the ventrolateral and ventrobasalis thalamic nuclei which receive a strong input from the motor cortex, as shown by densely PI-IA-L and biocytin labeled terminal fields (Fig. 4F), were free of c-f&r-like immunoreactivity although the zone is likely to be activated by this corticothalamic projection. This most surprising result suggests that, under our experimental conditions, c-fos is not systematically induced in all target regions of the site of ICMS. The results of the combined c-foslanterograde axonal tracing (PHA-L or biocytin) approach have been

751

summarized in Fig. 5, where the structures of the motor pathways have been presented by different symbols depending on whether they exhibited only c-fos-like staining, only anterogradely labeled axon terminals or both. DISCUSSION

With respect to the two specific objectives of the present study (see Introduction), the data can be summarized as follows: (1) The combination of c-fos functional labeling with axonal anatomical tracing (using PHA-L or biocytin) made it possible to establish whether a given structure of the motor pathways was activated directly or via a chain of two or more synaptic relays. Indeed, the present data are largely consistent with the relatively well understood connectivity of the motor pathways.2,43~58,s9”5*~@ (2) In the motor pathways, and under the present experimental conditions, c-fos was expressed in the large majority of the target structures of the ICMS site, with the exceptions however of the ventrolateral and ventrobasalis nuclei of the thalamus. The surprising observation that c-fos was not induced in the ventrolateral and ventrobasalis thalamic nuclei suggests that c-fos is not systematically expressed in all neurons presumably activated by a given stimulation, but rather might be induced wlectively in particular cell types,4,30or in neuronal populations characterized by a particular mode of

OC-FO~+PHA-L /BIOCYTI~ CC-FOS

. PHIL /BIocY-~~N

Fig. 5. As a result of ICMS (tungsten electrode) and tracer (PHA-L or biocytin) injection (pipette) in the caudal forelimb cortical area (CFA) of the motor cortex, brain regions where labeling was observed are indicated on a schematic sagittal section of the rat brain. The three symbols allow us to diitinguish three categories of structures deIlned, respectively, by the presence of c-fir-like labeling on@ (star), axonal labeling only (lilled circles) or both simul~n~usly (star in circle: see text). CPU, ~udat~p~~en; DC, deep cerebellar nuclei; ECU, external cuneate nucleus; GP, globus pallidus; IO, inferior olive; PM, paramedian lobule of the cerebellum; PN, pontine nuclei; R, red nucleus; RFA, rostra1 forelimb cortical area; RT, reticular nucleus of thalamus; SN, substantia nigra; STH, subthalamic nucleus; T, tectum (deep layers of superior colliculus); VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus.

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activation, via specific types of receptors, such as N-methyl-D-aspartate, D,, D,, acetylcholine and adrenergic receptors_1~4~5~10-12~19~22~3~32~41.54~57.61~69 The ab_ sence of c-fos-like immunostaining in the ventrolatera1 and ventrobasalis thalamic nuclei might also reflect the possibility that neurons in these structures were inhibited by the ICMS or they were not excited sufficiently to produce c-fos activation. Another possible explanation is that, because c-fos expression was estimated only at one time-point (1 h after the end of ICMS), any delayed induction of c-fos in these two thalamic nuclei would have been missed. The present results show that, in some structures, the number of c-&-like immunoreactive neurons and the intensity of c-fos-like staining were not systematically correlated with the density of the axonal terminal field labeled with PHA-L or biocytin. The absence of c-fos-like immunoreactivity in the ventrolateral nucleus of the thalamus, where dense PHA-L or biocytin labeled terminals were observed, strongly contrasts with the subthalamic nucleus, in which c-fos-like immunoreactivity was the most intense and present in the majority of neurons although terminals of axons originated from the ICMS site were moderately dense. Technical considerations

The combination of c-fos functional tracing with the neuroanatomical anterograde tracers PHA-L and biocytin appears, therefore, to be a promising tool to investigate the cascade of activations (direct vs indirect) in a neuronal chain by a given stimulus, particularly in systems for which the pattern of connectivity is poorly understood. It should, however, be emphasized here that the comparison of c-fos expression with 2-DG uptake remains a necessary initial step, in order to detect particular systems in which c-fos expression might be restricted to very limited subpopulations of neurons. In sensory systems, c-fos expression seems to be quite variable, depending on several experimental factors. In the visual system for instance, in response to stationary flash, c-fos induction was observed in the superior colliculus and pretectal region, but not in the main components of the visual pathways such as the lateral geniculate nucleus or the visual cortex.’ In contrast, moving flash stimuli induced c-fos expression in the visual cortex in addition to superior colliculus and pretectal region, but again not in the lateral geniculate nucleus.8 This observation can be related to the lack of c-fos labeling in VL in the present study, indicating that both in the visual and motor systems c-fos expression is absent in the main thalamic relay nucleus. In the auditory system, preliminary experiments in adult rats showed that c-fos was induced by pure tone stimulation preferentially in components of the auditory pathways characterized by a weak or absent tonotopic organization whereas the main tonotopic relay nuclei were usually free of c-fos-like labeling (unpublished observations).

WAN

et

al

It is likely that the distribution of c-f&-like immunoreactivity in sensory systems depends on the nature of the sensory stimuli8,24*67as well as on developmental changes. I5 Nevertheless, such cases characterized by a poor matching between 2-DG and c-fos might be of interest since c-fos would provide the opportunity to selectively study very specific neuronal subpopulations. The use of PHA-L or biocytin in combination with c-fos gave very comparable results. One should, however, take into consideration the respective properties of these two anterograde tracers. In principle, PHA-L needs to be injected by iontophoresis whereas biocytin can be injected both iontophoretically or by pressure. The main difference concerns, however, the speed of transport. Biocytin is transported faster than PHA-L, implying that the survival time necessary was much shorter for the former than the latter. It was found that the survival time after injection should not exceed 2-3 days for biocytin, otherwise the tracer faded very significantly. In contrast, PHA-L was much more stable, allowing much longer survival time (up to 3 weeks). This long survival time for PHA-L had the advantage over the short survival time required for biocytin in that the trauma and lesions resulting from the surgery necessary to perform the injection of the tracer will have less effect on the expression of C~JX induced by the stimulus applied a couple of hours before sacrifice of the animal. Although PHA-L and biocytin labeled axonal arborizations, including boutons en passant and terminaux, were seen in close apposition to c-j&-like positive neurons, direct contacts were difficult to establish with certainty at the light microscopic level. This was related in part to the fact that cfos immunostaining is restricted to the nucleus of the corresponding neuron. This difficulty can be partially resolved, at least for axosomatic contacts, by counterstaining the sections for Nissl (e.g. Cresyl Violet), although counterstaining obscures the detection of fine and faintly labeled axon terminals and/or palely c-fos-like stained neurons. The final demonstration of direct contacts between PHA-L or biocytin labeled axons and c-&-like positive neurons will, therefore, require electron microscopic analysis. Comparison with previous c-fos studies C-fos induction by ICMS of the hindlimb area of the rat motor cortex has been reported.47.50 Brain regions where c-fos induction was observed were among those mentioned in the present report, e.g. cerebral cortex, reticular and ventromedial thalamic nuclei, pontine nuclei and cerebellum. The same authors reported the absence, or more precisely the lack of clear c-fos-like immunoreactivity in the striaturn, which was clearly labeled in the present study. On the other hand, Sagar et a1.47also mentioned the presence of c-fos immunoreactivity in the ventrolatera1 and ventrobasalis thalamic nuclei, which were

c-fos induction and connectivity in the motor pathways devoid of staining in the present experiments. In addition, we observed c-f&-like immunostaining in the caudate-putamen, the subthalamic nucleus, the substantia nigra, the tectum, the red nucleus, the deep cerebellar nuclei and the external cuneate nucleus, which were not mentions by Sagar et al.‘” The discfepancie3 between the two studies, particularly the complete lack of positive marking in the ventrolateral nucleus of the thalamus in all of our animals, are difficult to explain, although differences in the mode of stimulation are likely to play an important role (bipolar vs unipolar electrodes, intensity of current). One should also consider that, in contrast to the present experiments, Sagar et al.” stimulated their rats only 24 h after surgery and the animals were restrained during ICMS, whereas our rats were stimulated either 48 h (when no tracers where injected or in case of biocytin injection) or l-3 weeks (in the case of PHA-L injection) after surgery without being restrained. These are two parameters which seem to strongly influence the expression of c-fos.’ The effect of restraining the animal was observed in a preliminary phase of the present study, in which two rats (not considered in the present analysis) were restrained during ICMS; these animals were immobilized by holding them to the stereotaxic apparatus by means of a piece of metal implanted to the skull of the animal, next to the stimulating chamber. This reduced spontaneous movements and locomotion of the animal during ICMS. The distribution of c-fos-like i~~oreacti~ty was quite different from that obtained in non-restrained rats: possibly as a result of stress or other side-effects related to restraining, c-for-like labeling was more broadly distributed in the whole brain as compared to non-restrained rats. Consequently, it was more difficult to distinguish specific c-fos labeling in the motor pathways from non-specific staining when the rat was restrained.

Comparison with 2-&oxyglucose intracorticai microstimulation studies The issue of the selectivity of c-fos expression in labeling neuronal pathways activated by an external stimulus has been addressed for the cerebellum, in a recent study based also on the paradigm of IGMS performed in the motor cortex.= It was observed that, in the caudal cerebellum, the distribution of c-fos immunoreactive neurons was congruent with the distribution of 2-DG labeling and that the two methods correlate well. However, this conclusion, valid for the cerebellum, might not necessarily apply for all brain regions. The observation, in the present study, of c-fos-like expression in the cortical rostra1 forelimb area, the basal ganglia (caudate-putamen and globus pallidus), the reticular and ventromedial thalamic nuclei, the substantia nigra, the sub~alamic nucleus, the tectum, the red nucleus, the pontine nuclei, the cerebellum and the inferior olive is consist-

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ent with the observed increase of glucose uptake seen with 2-DG in these regions, as a result of ICMS.“V~~ On the other hand no c-fos-like expression was detected in the ventrolateral and ventrobasalis nuclei of the thalamus (despite the presence of strong projections from the ICMS site), where 2-DG uptake was shown to be increased.48 It can be concluded that c-fos induction is not completely consistent with 2-DG uptake as a result of ICMS of the motor cortex of the rat. However, the discrepancies between the two methods were restricted to a few brain regions, mainly in the thalamus (the ventrolateral and ventrobasalis nuclei). The lack of c-fos expression in the motoneurons of the cervical cord is perplexing but it is, however, consistent with the absence or even decrease of 2-DG uptake observed in the ventral horn of the cervical cord during motor cortex stimulations* Although the two methods (c-fos and 2-DG) can be considered as fairly congruent in the present stimulation paradigm (ICMS), this might not be true for other types of stimulations, implying that the comparison with 2-DG uptake is a necessary step to interpret c-fos data.

CONCLUSION

C-fos is believed not to be directly representative of the neuronal activity, but rather to monitor intracellular second-messenger levels.” As pointed out by these authors (and confirmed in the present experiment), c-fos induction and glucose uptake appear to provide two different windows for detecting activity changes in neuronal systems. This may result in labeling of different neuronal populations. This problem remains to be studied further, also in sensory systems as mentioned above. At the present time, the c-fos method, however, offers two advantages over the 2-DG method: first, the cellular resolution of the former method and, second, the feasibility of combining c-fos with axonal tracing using PI-IA-L and biocytin as anterograde tracers, as demonstrated in the present study. It can be envisaged that with this new combined approach, more detailed knowledge about functional~onn~tion~ properties will emerge. Among several possible applications, besides establishing whether the activation of a given structure is direct or indirect as demonstrated here, the combination of c-fos with axonal tracers could be used to establish the fine arrangement of synaptic inputs (e.g. from the thalamus) in functionally defined (by c-fos) modules of sensory cortical areas. Acknowledgements-The present work was supported by the Swiss National Foundation for Scienti& Research (grants no 31-28572.90and 31-25128.88).Professor X.S.T. Wan was financially supported by the Roche Research Foundation (R&e, Switzerland) during his sabbatical stay in Switzerland. The authors thank Dr S. Betz-Corradinfor her editorial contribution.

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