Brain Research, 567 (1991) 133-139 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50
BRES 24937
133
Short Communications
Neonatal whisker removal in rats stabilizes a transient projection from the auditory thalamus to the primary somatosensory cortex Miguel A.L.
N i c o l e l i s 1'2, J o h n K . C h a p i n 1 a n d R i c k C . S . L i n ~
~Department of Physiology and Biophysics, Hahnemann University, Philadelphia, PA 19102 (U.S.A.)and 2Department of Pathology, School of Medicine, University of Sao Paulo, Sao Paulo (Brazil)
(Accepted 20 August 1991) Key words: Plasticity; Development; Barrel field; Multimodal projection; Thalamocortical projection; Cross-modal projection
A normally transient cross-modal thalamocortical projection from the magnocellular subdivision of the medial geniculate nucleus (MGm) to the primary somatosensory (SI) cortex of rats was found to remain unchanged throughout adulthood following unilateral removal of whiskers in newborn animals. The normal MGm projection to the auditory cortex is not lost in these neonatally whisker-deprivedadults rats but some of the MOrn neurons send collaterals to both primary auditory and SI cortices. Parallel electrophysiologicalexperiments demonstrated the multimodal character of some MGm neurons, since they responded to both auditory and cutaneous stimulation. These results suggest that the areal distribution in the cortex of thalamocortieal projections arising from a multimodal thalamic nuelens, such as the MGm, may be determined during early postnatal development by the normal flow of sensory information from the periphery to the thalamus and that an early postnatal somatosensory deprivation may prevent the normal withdrawal of a cross-modalprojection from the MGm to the SI.
The role of afferent projections in determining fine subareal architectonic features, size and sensory modal specificity of primary cortical areas has been well documented by different experimental approaches in recent years 11'29'3°'32'35. Sensory information arising from peripheral receptors is conveyed to primary sensory cortical fields through specific sensory thalamic relays. Early in development, the arrival of specific sensory thalamocortical projections in different cortical fields defines the modal specificity of that field and contributes for the organization of particular subareal cortical structures a°'35. In rodents, the integrity of facial mystacial vibrissae, throughout a critical period in the early postnatal (PN) development, is required for the formation of aggregated cortical eytoarchitectonie units ('barrels') which represent single whiskers within the whisker area of the primary somatosensory cortex (SI) 15'18'33'36. Removal of whiskers or their innervation during early PN development induces anatomical and physiological modifications in the ventral posterior (VP) nucleus 3'24'34 of the thalamus, preventing proper organization of its thalamocortical projections to the SI ~. As a consequence, the barrels fail to form in neonatally whisker-deprived rats 15'~s' 33,36
Whereas the effects of sensory deprivations on the somatosensory system have been extensively studied, it
remains to be determined to what extent such early postnatal peripheral sensory deprivation would affect thalamocortical projections arising from thalamic nuclei related to other sensory modalities, especially multimodal nuclei which also receives direct trigeminal afferent. We approached this issue here by studying a transient cross-modal projection from the magnocellular subdivision of the medial geniculate nucleus (MGm), part of the auditory thalamus, to the primary somatosensory cortex (SI) of rats. Initially, this cross-modal projection was observed in young rats (from PN day 1 to PN week 2). Over subsequent development, however, the density of this projection was found to decrease, and was almost absent in adult animals. However, a dense MGm-SI projection was also observed in adult animals which had been previously subject to a neonatal unilateral removal of all facial whiskers. Thus, our results suggest that a partial sensory deprivation of afferent inputs during early postnatal development not only disrupts the fine organization of thalamocortical projections from the primary somatosensory thalamic relay (the VP) 16, but also determines the stabilization of a cross-modal thalamocortical projection from the MGm to the SI. Thalamocortical projections to the SI were investigated by injecting small amounts (0.2-0.5/~1) of retrograde fluorescent tracers (rhodamine-coated micro-
Correspondence: M.A.L. Nicolelis. Present address: Department of Physiologyand Biophysics,Mail Stop 409, Hahnemann University, Broad and Vine Streets, Philadelphia, PA 19102-1192, U.S.A. Fax: (1) (215) 448-1982.
134 spheres (RCM) or Fluoro-Gold (FG)) into the SI of rats ranging in age from postnatal (PN) day 1 to adulthood. In neonates anesthesia was produced by hypothermia and then RCMs were injected by pricking the skull of these animals with the tip of a Hamilton syringe and then dropping a small amount of tracer on the lesioned pia surface. Young and adult animals were anesthetized with pentobarbital (50 mg/kg). A single, column-like cortical injection of fluorescent tracer was made in each animal using Hamilton syringes. Each of these injections produced a well-defined 100-300/~m wide column of tracer from the pia surface to layer VI. Since RCMs diffuse minimally through the brain, allowing placement of small and restricted tracer injections 14'23'25, they were used as the retrograde fluorescent tracer of choice for injections in both newborns and young animals. All RCM injections were restricted to the whisker area of the rat SI (the 'barrel fields'). Double-labeling studies were also carried out in adult animals in which one fluorescent tracer (RCMs) was injected in the SI and the other (FG) in the auditory cortex (AI). After a survival period of 2-8 days, animals were sacrificed with high doses of pentobarbital. They were perfused first with 0.9% saline and then with 4% formalin. Brains were kept in a solution of 20% sucrose in formalin overnight and then sectioned with a vibratome or a freezing microtome. Sections (80-100 /~m) were mounted on gelatin-coated slides, dried in room temperature and examined under fluorescence microscopy. Injections of retrograde fluorescent tracers were also made in the SI of adult rats which had been subject to total unilateral whisker removal at birth. For these experiments newborn rats (on postnatal day 1-2) were first deeply anesthetized by hypothermia. The whisker pad of the right side of the face was then electrocauterized and all whiskers completely removed. Animals were allowed to grow for 9-12 weeks before anatomical or physiological studies were carried out. Following the injection of retrograde tracers, the distributions of retrogradely labeled neurons in the ventral posteromedial nucleus (VPM) and M G m were compared in each of the 3 groups of animals: young, normal adults and neonatally whisker-deprived adults. No significant differences in the distribution of retrogradely labeled neurons in the VPM were observed (Fig. 1B, 2A) among the 3 groups of animals. On the other hand, injections of RCMs in the presumptive SI of normal rats on PN day 1 (N = 5), PN day 5 (N = 5), PN day 14 (N = 5), PN day 21 (N = 5) and normal adults (N = 10) demonstrated the existence of a direct and transient crossmodal projection from the M G m to the SI. This projection was first recognized in newborn rats injected on PN day 1 (perfused on PN day 3). It reached its maximal
density (usually around 30 retrogradely labeled cells per section) around the second PN week (Fig. 1A) and dedined after the third PN week, being extremely reduced in adult rats (less than 5 cells per section). In young animals, retrogradely labeled neurons were usually found throughout the rostro-caudal extension of the MGm, distributed in a thin column located around the medial border of the main auditory relay nucleus (Fig. 1A). A few cells were also found into the suprageniculate nucleus. However, no labeled cells were observed in the ventral or dorsal subdivisions of the medial geniculate complex (MG).
Fig. 1. Direct projection from the MGm to the SI cortex in a twoweek old rat. (A): fluorescence photomicrograph representing a coronal section through the posterior thalamus. Notice retrogradely labeled cells (indicated by arrows) in the MGm regions (between dotted lines) following a small injection of RCMs in the SI. A demonstration that the injection of retrograde tracer was restricted to the SI in this animal is provided by the photomicrograph in (B) which shows a small and restricted retrograde labeling in the whisker representation area of the ventral posteromedial and the posterior nuclei. Labeling can also been in the zona incerta (ZI). Scale bars: 600/~m for (A) and 1 mm for (B).
135 Similar amounts of the same retrograde tracers injected into the presumptive barrel fields of neonatally whisker-deprived adult rats (N = 17) produced typical labeling in the VPM, the posterior nucleus and in the zona incerta (Fig. 2A). These injections also produced numerous retrograde labeled cells repeatedly observed in the MGm (Fig. 2B-D). Although the density of this MGm-SI projection varied from case to case, the number of cells retrogradely labeled in the MGm was always much higher in neonatally whisker-deprived adult rats (20-30 cells per section) than in normal adult animals. In fact, the relative density of labeling in the MGm of whisker-deprived animals was very similar to that observed in 2-week-old rats. Double-labeling experiments in which one dye (RCMs) was injected in the SI and the other (FG) in the AI of whisker-deprived adult rats further characterized a possible stabilization of this MGm-SI projection following an early postnatal peripheral sensory deprivation (Fig. 2E and F). These experiments revealed the existence of a small percentage of cells within the MGm which projected to both SI and AI (Fig. 2E and F). Thalamocortical projections from the MGm have been previously reported to reach not only primary and secondary auditory cortical l's areas but also ventral regions of the secondary somatosensory cortex and regions of the insular cortex of normal adult rats 21. Furthermore, Krubitzer and Kaas 19 have reported the existence of a direct projection from the MGm to the parietal ventral area, a small cortical region located in the caudoventral border of the SI of gray squirrels that may be involved in processing multimodal information. Carvel and Simonss also reported the existence of neurons with overlapping auditory and cutaneous RFs located in the ventral regions of the mouse secondary somatosensory cortex (SII). Nevertheless, there has been no previous report of projections from the MGm to the SI in normal adult rats 1'2°. In the present study, small single-column-like injections of retrograde tracers at different postnatal ages were confined to the barrel fields, the whisker area of the rat SI, as demonstrated by the uniform and characteristic labeling observed in the VPM nucleus. This clear pattern of retrograde labeling in the VP suggests that no tracer spread outside of SI to involve the SII. In addition to these neuroanatomical experiments, receptive field (RF) properties of neurons located in the MGm were also determined in normal and whiskerdeprived adults rats using standard mapping techniques. These experiments were carded out in anesthetized rats (pentobarbital 50 mg/kg) using tungsten electrodes (9-10 MQ at 1 kHz). Several sequential penetrations, 100/~m apart were made in the MG of all rats. Initially, penetrations were aimed to the ventral subdivision of the
MG. Once clear auditory receptive fields were identified, the electrode was moved medially in consecutive steps of 100/~m. After a few penetrations, in which only auditory receptive fields could be identified, we could identify single and multiunits responding to both light brushing of the skin and a sequence of clicks or pure tones at different frequencies. Histological analysis using both Nissl and cytochrome oxidase staining methods demonstrated that these latter polysensory responses were recorded from cells located in the MGm. Neurophysiological mapping of the MGm of normal and whisker-deprived adult rats revealed the existence of single units with both cutaneous and auditory RFs. Typically, these MGm neurons responded to light stimulation of the skin of the face or other body parts. Large RFs, sometimes comprising the whole hemibody were usually found, especially in whisker-deprived animals. In some cases light brushing of distinct areas of the body surface, such as face and hindpaw, stimulated the same neuron. Bilateral responses were also observed. However, no dear somatotopic organization of cutaneous RFs was observed throughout the dorsoventral extension of the MGm (around 1 ram). An example of an electrode track passing through the MGm of a normal adult rat is shown in Fig. 3A. Neurons in this region of the auditory thalamus displayed responses to both light brushing of the skin and auditory stimuli, resembling in this sense the multimodal properties described for the posterior nucleus of the thalamus in the cat ~. Although our mapping experiments suggested that neurons in the MGm have cutaneous RFs, the existence of projections from ascending somatosensory pathways to the rat MGm remains controversial4'17. Thus possible sources of somatic afferents to the MGm, which could explain some of our physiological findings, were also examined anatomically. Small amounts (0.2-0.4/A) of a fluorescent anterograde tracer (dextran rhodamine, Molecular Probe) were injected either into the dorsal column nuclei or into the trigeminal complex of adult rats and the distribution of anterogradely labeled terminals within the MGm was analyzed. For these experiments, solutions of 10-25% dextran rhodamine in 0.9% saline were injected, using Hamilton syringes, either in the trigeminal complex or in the dorsal column nuclei. Usually a survival time of 4-6 days was sufficient to produce a durable anterograde and retrograde labeling. These final set of experiments demonstrated that neurons located in the trigeminal nuclear complex project bilaterally to the MGm (Fig. 3B and C). Many of those labeled terminals displayed very fine bouton-like swellings (see Fig. 3B and C) indicative of trigeminal terminal arborizations within the MGm. In addition, injections centered in the dorsal column nuclei also produced antero-
136
p
Fig. 2. Direct cross-modal projections from the MGm to the SI cortex in neonatally whisker-deprived adults. (A): distribution of retrogradely labeled neurons in the dorsal and ventral thalamus at the level of the VP thalamus following an injection of FG in the barrel fields of an adult deprived rat which had the whiskers of the contralateral face removed on PN day 1. Notice the intense labeling in the VP and in ZI. B-D: for the same case, sections caudal to the level represented in (A) shows the existence of Fluoro-gold labeled neurons located throughout the rostro-caudal extension of the MGm. Simultaneous injections of RCM in the barrel fields of the SI and FG in the auditory cortex of the same hemisphere not only produced typical retrograde labeling in the MGm but also double-labeled cells (D and E), demonstrating that in neonatally whisker-deprived adult rats neurons located in the MGm may give rise to collaterals that innervate both cortical fields. Arrows in (D) and (E) point to the double-labeled neurons. CL, centrolateral nucleus; VPM, ventroposterior medial nucleus; PO, posterior nucleus; MG, medial geniculate complex, MT, mamiUothalamic tract; CP, cerebral peduncle. Scale bars: 1 mm for (A)-(D) and 50/zm for (E) and
(F).
137 gradely labeled fibers coursing throughout the rostrocaudal extension of the MGm. In summary, our results have demonstrated a projection from the M G m to the rat SI cortex, which normally exist only during early postnatal development. In contrast, this projection remains essentially unchanged through development to adulthood if the whiskers, the chief tactile organ of the face in rats, are removed on
E
C
:
Fig. 3. The MGm of the adult normal rat. (A): coronal Nissl-stalned section through the posterior thalamus displaying an electrode track, passing through the MGm, produced during a mapping experiment. Some neurons in this nucleus displayed both cutaneous and auditory receptive fields. Usually large cutaneous receptive fields located in the face or other body parts were identified. Some of these cutaneous responses were bilateral. Injections of small mounts of a fluorescent anterograde tracer (dextran rhodamine) into the trigeminal cortex and dorsal column nuclei of normal and whisker-deprived adult rats provided further anatomical evidence for this latter physiological finding. Fluorescence photomicrographs (B) and (C) display anterogradely labeled terminals and arborizations in the contralateral (B) and ipsilateral (C) MGm, following an injection of dextran rhodamine in the trigeminal complex. In these pictures very fine axon collaterals and small bouton-like terminals, indicating the existence of axons and presynaptic arborization originated from the trigeminal nuclei, are filled with the fluorescent tracer and distributed within the MGm. SG, suprageniculate nucleus. Scale bars: 600/~m for (A) and 50/zm for (C).
the day of birth. In addition, physiological and anatomical studies showed that both auditory and somatosensory information converge on the M G m and thereafter to several cortical areas. Therefore, the multitude of cellular types observed in the M G m 9, the convergence of multimodal information to the nucleus 22, and its widespread pattern of subcortical 1°'21'22 and cortical projections 1'2°-22 further suggest that important integrative and multimodal processing tasks may take place in this subdivision of the MG. Cross-modal projections similar to the one reported here can be induced in different species by early postnatal manipulations in which normal cortical and subcortical targets of sensory afferents are removed during early postnatal development 2'7'12'13'27'31'32. These connections result either from the stabilization of normally transitory projections from the periphery to an erroneous thalamic relay (such as the transient projection of retinal ganglion cells to the ventrobasal nucleus of hamsters) 6'7'12'13'31 or develop 'de novo' as a consequence of sprouting of thalamic afferents, whose normal targets were removed, toward a new, usually deafferented, primary sensory thalamic relay 32. In addition, under similar experimental circumstances, neurons located in secondary thalamic relays, such as the lateral posterior nucleus and the pulvinar complex, can be also induced to project to an erroneous primary cortical field27, demonstrating that not only primary relays but also secondary thalamic structures are susceptible to plastic re-arrangements of their projections. Our present findings suggest that a transient cross-modal thalamocortical projection can be also stabilized by early postnatal manipulation of the periphery without removal of either cortical or subcortical targets. Further, it is interesting to note that this transient cross-modal projection originates only from a 'secondary' multimodal thalamic relay, the MGm. In fact, an early postnatal sensory deprivation by itself does not seem to induce cross-modal projections mediated by primary sensory thalamic relays. The present results raise the hypothesis that the establishment of the cortical areal distribution of crossmodal thalamocortical projections arising from a multimodal thalamic nuclei, such as the M G m , may be partially regulated by a developmental process which depends on the integrity of the incoming information from the periphery. Thus, during the normal development of the somatosensory system, the increasing flow of highly correlated information from peripheral cutaneous receptors determines the establishment of normal subareal architectonic features in the SI cortex (such as normal barrels), through the organization of specific thalamocortical projections from the VP. During this process a transient cross-modal projection from the M G m to the SI could
138 be e l i m i n a t e d or dramatically r e d u c e d . A l t e r n a t i v e l y , a n early p o s t n a t a l r e m o v a l of highly c o r r e l a t e d i n p u t s from the mystacial vibrissae m a y p r o v i d e the r e q u i r e d condi-
sory d e p r i v a t i o n m a y i n d u c e a d e p r i v e d SI to process to some degree n o t o n l y p u r e l y c u t a n e o u s b u t also multim o d a l sensory i n f o r m a t i o n .
tions for the stabilization of this direct M G m - S I crossm o d a l projection, by w e a k e n i n g the strength of specific projections from the p r i m a r y s o m a t o s e n s o r y t h a l a m i c relay, the VP, to the SI. A n i m m e d i a t e corollary of this hypothesis is that an early p o s t n a t a l partial s o m a t o s e n -
This work was supported by a fellowship FAPESP 88/4044-9 to M.A.L.N.; Grants NS 26722, AA06965 to J.K.C.; NS 29161 to R.C.S.L. (NS); and AFOSR 90-0266 to J.K.C. and R.C.S.L.
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BRES 24937
133
Short Communications
Neonatal whisker removal in rats stabilizes a transient projection from the auditory thalamus to the primary somatosensory cortex Miguel A.L.
N i c o l e l i s 1'2, J o h n K . C h a p i n 1 a n d R i c k C . S . L i n ~
~Department of Physiology and Biophysics, Hahnemann University, Philadelphia, PA 19102 (U.S.A.)and 2Department of Pathology, School of Medicine, University of Sao Paulo, Sao Paulo (Brazil)
(Accepted 20 August 1991) Key words: Plasticity; Development; Barrel field; Multimodal projection; Thalamocortical projection; Cross-modal projection
A normally transient cross-modal thalamocortical projection from the magnocellular subdivision of the medial geniculate nucleus (MGm) to the primary somatosensory (SI) cortex of rats was found to remain unchanged throughout adulthood following unilateral removal of whiskers in newborn animals. The normal MGm projection to the auditory cortex is not lost in these neonatally whisker-deprivedadults rats but some of the MOrn neurons send collaterals to both primary auditory and SI cortices. Parallel electrophysiologicalexperiments demonstrated the multimodal character of some MGm neurons, since they responded to both auditory and cutaneous stimulation. These results suggest that the areal distribution in the cortex of thalamocortieal projections arising from a multimodal thalamic nuelens, such as the MGm, may be determined during early postnatal development by the normal flow of sensory information from the periphery to the thalamus and that an early postnatal somatosensory deprivation may prevent the normal withdrawal of a cross-modalprojection from the MGm to the SI.
The role of afferent projections in determining fine subareal architectonic features, size and sensory modal specificity of primary cortical areas has been well documented by different experimental approaches in recent years 11'29'3°'32'35. Sensory information arising from peripheral receptors is conveyed to primary sensory cortical fields through specific sensory thalamic relays. Early in development, the arrival of specific sensory thalamocortical projections in different cortical fields defines the modal specificity of that field and contributes for the organization of particular subareal cortical structures a°'35. In rodents, the integrity of facial mystacial vibrissae, throughout a critical period in the early postnatal (PN) development, is required for the formation of aggregated cortical eytoarchitectonie units ('barrels') which represent single whiskers within the whisker area of the primary somatosensory cortex (SI) 15'18'33'36. Removal of whiskers or their innervation during early PN development induces anatomical and physiological modifications in the ventral posterior (VP) nucleus 3'24'34 of the thalamus, preventing proper organization of its thalamocortical projections to the SI ~. As a consequence, the barrels fail to form in neonatally whisker-deprived rats 15'~s' 33,36
Whereas the effects of sensory deprivations on the somatosensory system have been extensively studied, it
remains to be determined to what extent such early postnatal peripheral sensory deprivation would affect thalamocortical projections arising from thalamic nuclei related to other sensory modalities, especially multimodal nuclei which also receives direct trigeminal afferent. We approached this issue here by studying a transient cross-modal projection from the magnocellular subdivision of the medial geniculate nucleus (MGm), part of the auditory thalamus, to the primary somatosensory cortex (SI) of rats. Initially, this cross-modal projection was observed in young rats (from PN day 1 to PN week 2). Over subsequent development, however, the density of this projection was found to decrease, and was almost absent in adult animals. However, a dense MGm-SI projection was also observed in adult animals which had been previously subject to a neonatal unilateral removal of all facial whiskers. Thus, our results suggest that a partial sensory deprivation of afferent inputs during early postnatal development not only disrupts the fine organization of thalamocortical projections from the primary somatosensory thalamic relay (the VP) 16, but also determines the stabilization of a cross-modal thalamocortical projection from the MGm to the SI. Thalamocortical projections to the SI were investigated by injecting small amounts (0.2-0.5/~1) of retrograde fluorescent tracers (rhodamine-coated micro-
Correspondence: M.A.L. Nicolelis. Present address: Department of Physiologyand Biophysics,Mail Stop 409, Hahnemann University, Broad and Vine Streets, Philadelphia, PA 19102-1192, U.S.A. Fax: (1) (215) 448-1982.
134 spheres (RCM) or Fluoro-Gold (FG)) into the SI of rats ranging in age from postnatal (PN) day 1 to adulthood. In neonates anesthesia was produced by hypothermia and then RCMs were injected by pricking the skull of these animals with the tip of a Hamilton syringe and then dropping a small amount of tracer on the lesioned pia surface. Young and adult animals were anesthetized with pentobarbital (50 mg/kg). A single, column-like cortical injection of fluorescent tracer was made in each animal using Hamilton syringes. Each of these injections produced a well-defined 100-300/~m wide column of tracer from the pia surface to layer VI. Since RCMs diffuse minimally through the brain, allowing placement of small and restricted tracer injections 14'23'25, they were used as the retrograde fluorescent tracer of choice for injections in both newborns and young animals. All RCM injections were restricted to the whisker area of the rat SI (the 'barrel fields'). Double-labeling studies were also carried out in adult animals in which one fluorescent tracer (RCMs) was injected in the SI and the other (FG) in the auditory cortex (AI). After a survival period of 2-8 days, animals were sacrificed with high doses of pentobarbital. They were perfused first with 0.9% saline and then with 4% formalin. Brains were kept in a solution of 20% sucrose in formalin overnight and then sectioned with a vibratome or a freezing microtome. Sections (80-100 /~m) were mounted on gelatin-coated slides, dried in room temperature and examined under fluorescence microscopy. Injections of retrograde fluorescent tracers were also made in the SI of adult rats which had been subject to total unilateral whisker removal at birth. For these experiments newborn rats (on postnatal day 1-2) were first deeply anesthetized by hypothermia. The whisker pad of the right side of the face was then electrocauterized and all whiskers completely removed. Animals were allowed to grow for 9-12 weeks before anatomical or physiological studies were carried out. Following the injection of retrograde tracers, the distributions of retrogradely labeled neurons in the ventral posteromedial nucleus (VPM) and M G m were compared in each of the 3 groups of animals: young, normal adults and neonatally whisker-deprived adults. No significant differences in the distribution of retrogradely labeled neurons in the VPM were observed (Fig. 1B, 2A) among the 3 groups of animals. On the other hand, injections of RCMs in the presumptive SI of normal rats on PN day 1 (N = 5), PN day 5 (N = 5), PN day 14 (N = 5), PN day 21 (N = 5) and normal adults (N = 10) demonstrated the existence of a direct and transient crossmodal projection from the M G m to the SI. This projection was first recognized in newborn rats injected on PN day 1 (perfused on PN day 3). It reached its maximal
density (usually around 30 retrogradely labeled cells per section) around the second PN week (Fig. 1A) and dedined after the third PN week, being extremely reduced in adult rats (less than 5 cells per section). In young animals, retrogradely labeled neurons were usually found throughout the rostro-caudal extension of the MGm, distributed in a thin column located around the medial border of the main auditory relay nucleus (Fig. 1A). A few cells were also found into the suprageniculate nucleus. However, no labeled cells were observed in the ventral or dorsal subdivisions of the medial geniculate complex (MG).
Fig. 1. Direct projection from the MGm to the SI cortex in a twoweek old rat. (A): fluorescence photomicrograph representing a coronal section through the posterior thalamus. Notice retrogradely labeled cells (indicated by arrows) in the MGm regions (between dotted lines) following a small injection of RCMs in the SI. A demonstration that the injection of retrograde tracer was restricted to the SI in this animal is provided by the photomicrograph in (B) which shows a small and restricted retrograde labeling in the whisker representation area of the ventral posteromedial and the posterior nuclei. Labeling can also been in the zona incerta (ZI). Scale bars: 600/~m for (A) and 1 mm for (B).
135 Similar amounts of the same retrograde tracers injected into the presumptive barrel fields of neonatally whisker-deprived adult rats (N = 17) produced typical labeling in the VPM, the posterior nucleus and in the zona incerta (Fig. 2A). These injections also produced numerous retrograde labeled cells repeatedly observed in the MGm (Fig. 2B-D). Although the density of this MGm-SI projection varied from case to case, the number of cells retrogradely labeled in the MGm was always much higher in neonatally whisker-deprived adult rats (20-30 cells per section) than in normal adult animals. In fact, the relative density of labeling in the MGm of whisker-deprived animals was very similar to that observed in 2-week-old rats. Double-labeling experiments in which one dye (RCMs) was injected in the SI and the other (FG) in the AI of whisker-deprived adult rats further characterized a possible stabilization of this MGm-SI projection following an early postnatal peripheral sensory deprivation (Fig. 2E and F). These experiments revealed the existence of a small percentage of cells within the MGm which projected to both SI and AI (Fig. 2E and F). Thalamocortical projections from the MGm have been previously reported to reach not only primary and secondary auditory cortical l's areas but also ventral regions of the secondary somatosensory cortex and regions of the insular cortex of normal adult rats 21. Furthermore, Krubitzer and Kaas 19 have reported the existence of a direct projection from the MGm to the parietal ventral area, a small cortical region located in the caudoventral border of the SI of gray squirrels that may be involved in processing multimodal information. Carvel and Simonss also reported the existence of neurons with overlapping auditory and cutaneous RFs located in the ventral regions of the mouse secondary somatosensory cortex (SII). Nevertheless, there has been no previous report of projections from the MGm to the SI in normal adult rats 1'2°. In the present study, small single-column-like injections of retrograde tracers at different postnatal ages were confined to the barrel fields, the whisker area of the rat SI, as demonstrated by the uniform and characteristic labeling observed in the VPM nucleus. This clear pattern of retrograde labeling in the VP suggests that no tracer spread outside of SI to involve the SII. In addition to these neuroanatomical experiments, receptive field (RF) properties of neurons located in the MGm were also determined in normal and whiskerdeprived adults rats using standard mapping techniques. These experiments were carded out in anesthetized rats (pentobarbital 50 mg/kg) using tungsten electrodes (9-10 MQ at 1 kHz). Several sequential penetrations, 100/~m apart were made in the MG of all rats. Initially, penetrations were aimed to the ventral subdivision of the
MG. Once clear auditory receptive fields were identified, the electrode was moved medially in consecutive steps of 100/~m. After a few penetrations, in which only auditory receptive fields could be identified, we could identify single and multiunits responding to both light brushing of the skin and a sequence of clicks or pure tones at different frequencies. Histological analysis using both Nissl and cytochrome oxidase staining methods demonstrated that these latter polysensory responses were recorded from cells located in the MGm. Neurophysiological mapping of the MGm of normal and whisker-deprived adult rats revealed the existence of single units with both cutaneous and auditory RFs. Typically, these MGm neurons responded to light stimulation of the skin of the face or other body parts. Large RFs, sometimes comprising the whole hemibody were usually found, especially in whisker-deprived animals. In some cases light brushing of distinct areas of the body surface, such as face and hindpaw, stimulated the same neuron. Bilateral responses were also observed. However, no dear somatotopic organization of cutaneous RFs was observed throughout the dorsoventral extension of the MGm (around 1 ram). An example of an electrode track passing through the MGm of a normal adult rat is shown in Fig. 3A. Neurons in this region of the auditory thalamus displayed responses to both light brushing of the skin and auditory stimuli, resembling in this sense the multimodal properties described for the posterior nucleus of the thalamus in the cat ~. Although our mapping experiments suggested that neurons in the MGm have cutaneous RFs, the existence of projections from ascending somatosensory pathways to the rat MGm remains controversial4'17. Thus possible sources of somatic afferents to the MGm, which could explain some of our physiological findings, were also examined anatomically. Small amounts (0.2-0.4/A) of a fluorescent anterograde tracer (dextran rhodamine, Molecular Probe) were injected either into the dorsal column nuclei or into the trigeminal complex of adult rats and the distribution of anterogradely labeled terminals within the MGm was analyzed. For these experiments, solutions of 10-25% dextran rhodamine in 0.9% saline were injected, using Hamilton syringes, either in the trigeminal complex or in the dorsal column nuclei. Usually a survival time of 4-6 days was sufficient to produce a durable anterograde and retrograde labeling. These final set of experiments demonstrated that neurons located in the trigeminal nuclear complex project bilaterally to the MGm (Fig. 3B and C). Many of those labeled terminals displayed very fine bouton-like swellings (see Fig. 3B and C) indicative of trigeminal terminal arborizations within the MGm. In addition, injections centered in the dorsal column nuclei also produced antero-
136
p
Fig. 2. Direct cross-modal projections from the MGm to the SI cortex in neonatally whisker-deprived adults. (A): distribution of retrogradely labeled neurons in the dorsal and ventral thalamus at the level of the VP thalamus following an injection of FG in the barrel fields of an adult deprived rat which had the whiskers of the contralateral face removed on PN day 1. Notice the intense labeling in the VP and in ZI. B-D: for the same case, sections caudal to the level represented in (A) shows the existence of Fluoro-gold labeled neurons located throughout the rostro-caudal extension of the MGm. Simultaneous injections of RCM in the barrel fields of the SI and FG in the auditory cortex of the same hemisphere not only produced typical retrograde labeling in the MGm but also double-labeled cells (D and E), demonstrating that in neonatally whisker-deprived adult rats neurons located in the MGm may give rise to collaterals that innervate both cortical fields. Arrows in (D) and (E) point to the double-labeled neurons. CL, centrolateral nucleus; VPM, ventroposterior medial nucleus; PO, posterior nucleus; MG, medial geniculate complex, MT, mamiUothalamic tract; CP, cerebral peduncle. Scale bars: 1 mm for (A)-(D) and 50/zm for (E) and
(F).
137 gradely labeled fibers coursing throughout the rostrocaudal extension of the MGm. In summary, our results have demonstrated a projection from the M G m to the rat SI cortex, which normally exist only during early postnatal development. In contrast, this projection remains essentially unchanged through development to adulthood if the whiskers, the chief tactile organ of the face in rats, are removed on
E
C
:
Fig. 3. The MGm of the adult normal rat. (A): coronal Nissl-stalned section through the posterior thalamus displaying an electrode track, passing through the MGm, produced during a mapping experiment. Some neurons in this nucleus displayed both cutaneous and auditory receptive fields. Usually large cutaneous receptive fields located in the face or other body parts were identified. Some of these cutaneous responses were bilateral. Injections of small mounts of a fluorescent anterograde tracer (dextran rhodamine) into the trigeminal cortex and dorsal column nuclei of normal and whisker-deprived adult rats provided further anatomical evidence for this latter physiological finding. Fluorescence photomicrographs (B) and (C) display anterogradely labeled terminals and arborizations in the contralateral (B) and ipsilateral (C) MGm, following an injection of dextran rhodamine in the trigeminal complex. In these pictures very fine axon collaterals and small bouton-like terminals, indicating the existence of axons and presynaptic arborization originated from the trigeminal nuclei, are filled with the fluorescent tracer and distributed within the MGm. SG, suprageniculate nucleus. Scale bars: 600/~m for (A) and 50/zm for (C).
the day of birth. In addition, physiological and anatomical studies showed that both auditory and somatosensory information converge on the M G m and thereafter to several cortical areas. Therefore, the multitude of cellular types observed in the M G m 9, the convergence of multimodal information to the nucleus 22, and its widespread pattern of subcortical 1°'21'22 and cortical projections 1'2°-22 further suggest that important integrative and multimodal processing tasks may take place in this subdivision of the MG. Cross-modal projections similar to the one reported here can be induced in different species by early postnatal manipulations in which normal cortical and subcortical targets of sensory afferents are removed during early postnatal development 2'7'12'13'27'31'32. These connections result either from the stabilization of normally transitory projections from the periphery to an erroneous thalamic relay (such as the transient projection of retinal ganglion cells to the ventrobasal nucleus of hamsters) 6'7'12'13'31 or develop 'de novo' as a consequence of sprouting of thalamic afferents, whose normal targets were removed, toward a new, usually deafferented, primary sensory thalamic relay 32. In addition, under similar experimental circumstances, neurons located in secondary thalamic relays, such as the lateral posterior nucleus and the pulvinar complex, can be also induced to project to an erroneous primary cortical field27, demonstrating that not only primary relays but also secondary thalamic structures are susceptible to plastic re-arrangements of their projections. Our present findings suggest that a transient cross-modal thalamocortical projection can be also stabilized by early postnatal manipulation of the periphery without removal of either cortical or subcortical targets. Further, it is interesting to note that this transient cross-modal projection originates only from a 'secondary' multimodal thalamic relay, the MGm. In fact, an early postnatal sensory deprivation by itself does not seem to induce cross-modal projections mediated by primary sensory thalamic relays. The present results raise the hypothesis that the establishment of the cortical areal distribution of crossmodal thalamocortical projections arising from a multimodal thalamic nuclei, such as the M G m , may be partially regulated by a developmental process which depends on the integrity of the incoming information from the periphery. Thus, during the normal development of the somatosensory system, the increasing flow of highly correlated information from peripheral cutaneous receptors determines the establishment of normal subareal architectonic features in the SI cortex (such as normal barrels), through the organization of specific thalamocortical projections from the VP. During this process a transient cross-modal projection from the M G m to the SI could
138 be e l i m i n a t e d or dramatically r e d u c e d . A l t e r n a t i v e l y , a n early p o s t n a t a l r e m o v a l of highly c o r r e l a t e d i n p u t s from the mystacial vibrissae m a y p r o v i d e the r e q u i r e d condi-
sory d e p r i v a t i o n m a y i n d u c e a d e p r i v e d SI to process to some degree n o t o n l y p u r e l y c u t a n e o u s b u t also multim o d a l sensory i n f o r m a t i o n .
tions for the stabilization of this direct M G m - S I crossm o d a l projection, by w e a k e n i n g the strength of specific projections from the p r i m a r y s o m a t o s e n s o r y t h a l a m i c relay, the VP, to the SI. A n i m m e d i a t e corollary of this hypothesis is that an early p o s t n a t a l partial s o m a t o s e n -
This work was supported by a fellowship FAPESP 88/4044-9 to M.A.L.N.; Grants NS 26722, AA06965 to J.K.C.; NS 29161 to R.C.S.L. (NS); and AFOSR 90-0266 to J.K.C. and R.C.S.L.
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