Differential effects of peripheral damage on vibrissa-related patterns in trigeminal nucleus principalis, subnucleus interpolaris, and subnucleus caudalis

Neuroscience Vol. 49, No. I, pp. 141-156, 1992 Printed in Great Britain

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

DIFFERENTIAL EFFECTS OF PERIPHERAL DAMAGE ON VIBRISSA-RELATED PATTERNS IN TRIGEMINAL NUCLEUS PRINCIPALIS, SUBNUCLEUS INTERPOLARIS, AND SUBNUCLEUS CAUDALIS N. L. CI-nAL~,* C. A. BENNETT-CLARKEand R. W. RHOADES Department of Anatomy, Medical College of Ohio, C.S. # 10008, Toledo, OH 43699, U.S.A. Abstract Histochemistry for cytochrome oxidase reveals a vibrissa-related pattern in trigeminal nucleus principalis, subnucleus interpolaris, and the magnocellular portion of subnucleus caudalis. This pattern is apparent in late fetal animals and is disrupted by transection of the infraorbital nerve on the day of birth. We recently reported results suggesting that the cytovhrome oxidase pattern reflects primary afferent-induced clustering of second order neurons in all of these nuclei. If this conclusion is correct, it should follow that primary afferent lesions made after the cytochrome oxidase pattern became established in the brainstem might have little effect upon it. Accordingly, we transected the infraorbital nerve (the trigeminal branch that supplies the vibrissae) on postnatal days 0-10 and evaluated the vibrissa-related pattern in the brainstem with cytochrome oxidase histocbemistry at varying intervals after these lesions. If the infraorbital nerve was sectioned on postnatal days 0-2, the vibrissa-related pattern was absent in trigeminal nucleus principalis, and both subnucleus interpolaris and caudalis. If such lesions were made after postnatal day 9, there was no appreciable effect upon the cytochrome oxidase pattern in any portion of the trigeminal brainstem complex. However, if lesions were made between postnatal days 3 and 8, the density and clarity of the cytoehrome oxidase staining pattern were reduced in interpolaris and caudalis, but not in principalis. This difference was not due to differential transganglionic degeneration in these nuclei. Tracing with horseradish peroxidase demonstrated qualitatively equivalent primary afferent losses in principalis, interpolaris, and caudalis. Immunocytochernistry with a monoclonal antibody directed against parvalbumin also demonstrated a vibrissa-related pattern of cell bodies in pfincipalis and interpolaris in rats killed on postnatal day 9 or later ages. The combination of retrograde tracing and immunocytochemistry revealed that the parvalbuminimmunoreactive neurons in principalis projected to thalamus while those in interpolaris were not labelled by tracer injections into the thalamus, midbrain, cerebellum or spinal cord. Infraorbital nerve transections made as late as postnatal day 8 resulted in a sharp decrease in the staining of parvalbumin-positiveneurons in interpolaris, but not in principalis. Lesions made on postnatal day 10 had no qualitative effect upon parvalbumin-positive neurons in any portion of the trigeminal brainstem complex. The results of this study support the conclusion that the vibrissa-related cytochrome oxidase pattern in principalis becomes independent of primary afferent input at a very short interval after its initial appearance. In contrast, the patterns in more caudal portions of the trigeminal brainstem complex require maintenance of primary afferent input for a much longer postnatal period. The present results also provide further support for the conclusion that postsynaptic elements in the trigeminal hrainstem complex may underlie the primary afferent-induced cytochrome oxidase pattern.

Clusters of dense cytochrome oxidase (CO) and succinic dehydrogenase (SDH) staining with a pattern corresponding to that of the mystacial vibrissae are readily demonstrable in the trigeminal (V) brainstem complex of perinatal rodents? The development of these vibrissa-related patterns depends upon the normal primary afferent innervation of brainstem n e u r o n s ) ,4,5,8,29Recent results 8 have suggested that the CO-reactive mitochondria, whose staining permits visualization of the pattern, are located primarily in

the dendrites and cell bodies of brainstem neurons rather than primary afferent axons (see Ref. 41 for corresponding data for the vibrissa-related pattern in the mouse cerebral cortex). If the vibrissa-related CO pattern, once established in the V brainstem complex, persisted aRer transection of the infraorbital nerve (ION, the V branch that supplies the mystacial vibrissae), it would provide further support for the conclusion that it reflected reactive mitochondria in postsynaptic rather than presynaptic elements. There are already some data that support this suggestion. Belford and Killackey s (also see Ref. 28) showed that cautery of a single row of vibrissae follicles on postnatal day 5 (P-5) produced little change in the SDH staining

*To whom correspondence should be addressed, Abbreviations: CO, ¢ytochrome oxidase; HRP, horseradish

peroxidase; ION, infraorbital nerve; P, postnatal day; PA, parvalbumin; PBS, sodium phosphate buffer; PBS-Tx, sodium phosphate buffer containing 4% Triton X-100; PrV, principal sensory nucleus; SDH, succinic dehydrogenase; SpC, trigeminal subnucleus caudalis; SpI, trigeminal subnucleus interpolaris; V, trigeminal.

pattern in the V brainstem complex of rats killed on P-7. In the present study, the analysis carried out by Belford and Killackey 5 has been extended by making 141

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transections o f the entire I O N on P-0 through to P-10 and processing brains for the d e m o n s t r a t i o n o f CO at varying intervals after these lesions, If the organization o f brainstem cells and their processes is primarily responsible for the p a t t e r n that is seen with CO staining, then it should also be possible to d e m o n s t r a t e these patterns by selectively marking these elements. This has been accomplished for neurons in the principal sensory nucleus (PrV) via retrograde labeling from the thalamus and, to a lesser degree, for cells in V nucleus interpolaris (SpI) and caudalis (SpC) with Nissl staining. 8'32 The results o f recent study concerned with the distribution o f parvalbumin (PA)-immunoreactive neurons in the rodent brain have suggested that this calcium binding protein may also m a r k the V brainstem cells that form the vibrissa-related pattern observed with CO staining. 7 Therefore we have also used this marker to

Horseradish peroxidase tracing Eight rats that sustained ION transections on P-4 or P-5 were used in anterograde tracing experiments when they reached seven or nine days of age. These pups were anesthetized with ether, the ION was exposed and transected, and a 3.0 mm2 piece of filter paper that had been saturated with a solution that contained 0.3% cholera toxin-conjugated horseradish peroxidase (HRP; List Biochemicals), 2.5% wheatgerm agglutinin-conjugated HRP (Sigma), and 15% free HRP (Sigma) was inserted underneath the proximal nerve stump. Crystalline free HRP was also applied directly to the nerve. The incision was then closed with cyanoacrylate cement and the pup returned to its mother. After 48 h survival, pups were deeply anesthetized with ether and perfused transcardially according to the method of Rosene and Mesulam) 6 Brainstems were lhen cut into 50-#m coronal sections and alternate sections were processed for the demonstration of HRP reaction product according to the method of Mesulam. 33 The remaining sections were processed for CO in the manner described above.

assess the I O N damage u p o n the organization o f the

Parvalbumin histochemistry

V brainstem complex.

Rats (14 animals that sustained ION lesions between P-10 and P-12 and were killed between P-12 and P-I4) were deeply anesthetized with ether and perfused transcardially with 0.9% saline in 0.1 M sodium phosphate buffer (PBS; pH = 7.4, 21°C). This was followed by a fixative consisting of 4.0% paraformaldehyde in the same buffer (4°C). After perfusion, the brain was removed and postfixed for 12--36h. The brainstem was cut into 50-#m coronal sections using either a freezing microtome or a Vibratome (Oxford Insts.). Following several rinses in PBS, sections were incubated for 18-36 h in mouse ascites fluid containing a monoclonal antibody directed against carp PA (Sigma Chemical, dilution 1: 500- 1: 1,000). The ascites fluid was diluted in PBS that also contained 0.04% Triton X,100 and 1% bovine serum albumin. Sections were then rinsed in PBS that contained 0.04% Triton X-100 (PBS-Tx) and incubated for 1 h in a 1:200 dilution of biotinylated anti-mouse IgG (Vector Labs) with PBS-Tx as a diluent. Tissue sections were then rinsed in PBS-Tx and incubated for 30 min in a 1:100 dilution of the avidin-biotin-peroxidase (ABC) complex (Vector Labs) in PBS-Tx. After a final rinse in

EXPERIMENTAL PROCEDURES

Infraorbital nerve lesions Pups (P-0 to P-12) were anesthetized by hypothermia (P-0 through to P-3) or with ether (all other animals) and the left ION was exposed by making a vertical slit just behind the whisker pad. The nerve was then visualized with the aid of a dissecting microscope and cut with a pair of iridectomy scissors. The wound was closed with cyanoacrylate and the pup was returned to its mother. The numbers of pups lesioned at each age and the nature of the experiment in which each was used are summarized in Table 1. Cytochrorne oxidase histochemistry Cytochrome oxidase was demonstrated in 50-#m coronal sections through the brainstem following the protocol of Wong-Riley.38After the completion of this reaction, sections were rinsed, dehydrated in graded alcohols, cleared in xylene, and coverslipped,

Table 1. Numbers of animals used in different histochemical, immunocytochemical, and tracing experiment N

Day of ION lesion

2 2 2 4 2 4 2 4 3 4 3 2 3 2 6 4 4 4 2 2 7 9

P-0 P-1 P-3 P-4 P-4 P-6 P-6 P-7 P-7 P-8 P-8 P-8 P-9 P-10 P-10 P-II P-12 P-4 P-4 P-5 P-4 --

Tracer injection

-.... ..... -.... ------HRP, P-7 HRP, P-9 HRP, P-7 FG, P-8 FG, P > 60

Day killed

Processed for

P-2 P-3 P-5 P-6 P-12 P-8 P-12 P-9 P-12 P-10 P-12 P-13 P-13 P-13 P-13 P-14 P-14 P-9 P-I 1 P-9 P-10 P > 64

CO CO CO CO CO CO CO CO CO CO CO CO CO CO PA and CO PA and CO PA and CO HRP and CO HRP and CO HRP and CO FG, PA and CO FG and PA

Late infraorbital nerve lesions and whisker patterns PBS-Tx, sections were reacted in a 50.0mg% solution of 3,Y-diaminobenzidine that also contained 0.03% H202. Stained sections were mounted on glass slides, allowed to air-dry, cleared in xylene, and coverslipl~l. Some sections were counterstained prior to coverslipping, Some sections through the V brainstem complex were processed in the manner described above, but either the primary or secondary antisera were omitted. None of these control sections contained any labeled cells or fibers.

Retrograde tracing and imrnunocytochemistry Seven adult rats and five rats that sustained ION transection on P-4 and who had reached P-8, received injections (0.2 #1 each) of fluorogold into the ventrobasal thalamus, superior coRiculus, cerebellum. An additional two animals from each of these groups had fluorogold injected only into the thalamus. The aim here was to determine whether any of the PA-immunoreactive neurons in either PrV or SpI had projections to known targets of the V brainstem complex. The methods employed in making these injections have been described in detail by Chiaia et al.s'9 Two days after the injections, the perinatal animals were killed with ether and perfused in the manner described above. The adult rats were re-anesthetized and given a single 10-#1 injection of

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colchicine(l #g/#1)intothecisternamagna. After twomore days, the adult rats were deeply anesthetized with ether and perfused. Brains were cut into 50-#m coronal sections and alternate sections were processed for CO, PA immunocytochemistry in the manner described above, or for PA immunocytochemistry with the following exception to the above described protocol. Instead of the avidin-biotin-peroxidase (ABC) reagent, sections were placed in a 1:200 dilution of Texas Red conjugated avidin (Vector Labs) for 30min. Following this incubation, sections were rinsed in PBS-Tx, mounted on glass slides, air-dried, cleared in xylene, and coverslipped. These sections were analysed with a Nikon Optiphot microscope equipped with episcopic fluorescence optics. RESULTS

Cytochrome oxidase staining T r a n s e c t i o n o f the I O N o n either P-0 (Fig. I A - C ) or P-1 (Fig. 1 D - F ) resulted in the absence o f the vibrissa-related C O staining p a t t e r n in PrV, SpI, a n d

Fig. 1. Effects of ION transections on Po0 and Pol upon vibrissa-related CO patterns in the V brainstem complex. A-C show the effects of a lesion on P-0 on the CO patterns in a rat killed on P-2. There is no discernible vibrissa-related pattern in PrV, SpI, or SpC on the deafferented (left) side. The open arrows here and in Figs 2-5 point toward the V brainstem nucleus indicated by the abbreviation in the lower lefthand corner of each panel D - F show similar data for a rat that sustained an ION transection on P- 1 and was killed on P-3. Here again, no vibrissa-related pattern is visible in any portion of the V brainstem complex. Mes V, the V mesencephalic nucleus; Mot V, the V motor nucleus. Scale bar = 500 # m and applies to all panels.

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SpC on the side ipsitateral to the damaged nerve in rats assayed on P-2 and P-3, respectively. At both of these ages, CO staining did reveal a vibrissa-related pattern in PrV, SpI, and SpC on the intact side of the brainstem, When the ION was cut on P-2 and animals examined on P-4, there appeared to be some preservation of the vibrissa-related pattern in PrV, SpI, and SpC (Fig. 2). Individual patches were difficult to discern in any of these nuclei, but high CO activity appeared to be organized in distinct rows. Beginning with animals that sustained ION transections on P-3 (Fig. 3A-C) and continuing with rats that sustained lesions through P-7 (Fig. 3D-F), there was a clear difference between the effects of ION damage upon the vibrissa-related CO staining pattern in PrV vs SpI and SpC. Nerve damage had no appreciable effect upon CO staining in PrV, but resulted in a sharp decrease in the density and clarity of the pattern in SpI and SpC. This pattern of results was observed in all of the rats that sustained ION damage between P-3 and P-7 and were examined two days after the lesion, In rats that sustained ION transections on P-8, there was an effect of survival time upon the CO staining observed in SpI and SpC. In rats that sustained ION cuts on P-8 and were killed on P- 10, a vibrissa-related pattern was visible on both sides of the brainstem in CO stained sections through PrV, SpI and SpC (Fig. 4A-C). In animals that were lesioned on P-8, but survived until P-12 or 13, the vibrissa-related CO pattern in SpI and SpC was much less clear on the deafferented side (Fig. 4D-F). Some segmentation was still visible in the portion of SpI in which the vibrissae were represented, but none could be seen in SpC. Damage to the ION on P-10 or at later ages had no qualitative effect upon CO staining patterns in any portion of the V brainstem complex (Fig. 5) over the range of survival times that we employed (two to four days). At ages later than P-14, the vibrissa-related CO pattern on both the normal and nerve-damaged sides of the brainstem became less clear and no animals were tested with survivals beyond this age. Close examination of CO-stained sections from animals that sustained ION damage between P-3 and P-8 revealed a clear effect upon the density of CO staining in V brainstem neurons. In PrV, SpI, and SpC, neurons heavily reactive for CO reside within the patches that comprise the vibrissa-related pattern (Fig. 6A-C). In animals that sustained ION lesions on P-3 through P-8, the density of the staining of these neurons in SpI and SpC (Fig. 6E, F), but not PrV (Fig. 6D), was reduced,

Horseradish peroxidase labeling The differential effects of ION damage upon the vibrissa-related CO patterns in PrV, SpI, and SpC do not appear to be due to different effects of" ION damage upon the V primary afferent innervation of these nuclei. In rats that sustained ION transections on P-4 and P-5, application of HRP to the damaged nerve proximal to the point of the transection demonstrated qualitatively similar losses of primary afterents in PrV, SpI, and SpC (Fig. 7A-C). Nevertheless, staining of alternate sections from these same animals with CO (Fig. 7 A ' ~ ' ) demonstrated the differential effect in PrV, SpI, and SpC described above.

Parvalbumin immunocytochemistry and retrograde tracing Immunocytochemistry with an antibody directed against PA provided evidence consistent with the conclusion that the differential effect of late ION transections in PrV vs SpI reflected differential effects of these lesions upon specific populations of second order neurons. In the V brainstem complex of perinatal rats P-9 and older not treated with colchicine, immunocytochemistry for PA revealed labeled fibers in the V spinal tract and labeled cells and fibers in PrV and all V subnuclei. The staining of the cells became increasingly intense over the next few days and a vibrissa-related patterning of these neurons became apparent in PrV and SpI, but less so in SpC where a relatively small number of PA-immunoreactive cells were present in the magnocellular layers (Fig. 8A-F). This patterning of PA-immunoreactive neurons in PrV and SpI is retained into adulthood (Fig. 8G-I). The combination of retrograde tracing with fluorogold and immunocytochemistry demonstrated that most, if not all, of the PA-immunoreactive neurons in PrV sent axons to the contralateral thalamus (Fig. 9A, B). Conversely, none of our fluorogold injections labeled PA-immunoreactive neurons in SpI (Fig. 9C, D). This pattern of results was obtained in both the adult rats and in the animals that received fluorogold injections on P-8 and it suggests that the PA-positive cells in SpI may be local circuit neurons. Transection of the ION had differential effects upon the staining of PA-immunoreactive neurons in PrV and SpI. The effect o f an ION transection on P-4 upon PA staining in a rat killed on P-10 is illustrated in Fig. 10. The staining of neurons on the intact and deafferented sides of PrV is essentially the same. In contrast, there is a sharp reduction in the staining of neurons in SpI on the side of the lesion. Transection of the ION on P-10 had no appreciable effect on the

Fig. 2. Effect of ION transection on P-2 upon the vibrissa-related CO pattern observed in an animal killed on P-4. Note the segmentation of the dense CO reactivity into rows on the deafferented (left) side of the brainstem. Scale bar = 500 #m for all panels.

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Fig. 3. A-C show the effect of ION transection on P-3 upon the vibrissa-related pattern in a rat killed on P-5. Note that the pattern is normal in PrV, absent on the deafferented (left) side of Spl, and that some segmentation into rows (arrows) is visible in SpC. D-F show the effects of an ION lesion on P-7 upon the vibrissa-related CO pattern in a rat killed on P-9. The pattern is normal in PrV and absent in both Spl and SpC~ Scale bar= 500#m.

density or PA-immunoreactive cells in either PrV or SpI in rats killed on P-13 (Fig. 11). DISCUSSION The results described in the preceding section show that transection of the ION between P-3 and P-9 results in a loss of the vibrissa-related pattern observed with either CO histochemistry or PA immunocytochemistry in SpI and SpC, but not in PrV. The anterograde tracing experiments demonstrated further that this selective loss occurred even though all three V nuclei sustained qualitatively similar deafferentations as a result of the ION lesions, The results presented in this paper support two conclusions. First, when considered together with the previous findings of Chiaia e t a L , 8 they suggest that the elements responsible for the vibrissa-related CO patterns in PrV and SpI of perinatal rats reside primarily in brainstem cells and their processes. The second conclusion supported by the present results is that the vibrissa-related CO pattern in more caudal V subnuclei remains dependent upon normal primary afferent input well after the pattern in PrV becomes

independent of normal connections between the periphery and the brainstem. The proposal that the elements responsible tbr the vibrissa-related CO patterns in PrV and SpI of perinatal rats reside primarily in brainstem cells and dendrites does not mean that the establishment of these patterns is not dependent upon normal primary afferent input; numerous experiments have demonstrated that this is, in fact, the case. 5'~3'24'29'3~The present results and those of Chiaia e t al. 8 indicate that the ability of CO and probably SDH staining as well to detect a vibrissa-related pattern in the V brainstem complex depends upon primary afferent-induced aggregation of postsynaptic elements. The evidence supporting this assertion is as follows. Neonatal somatosensory cortical lesions or thalamotomy markedly reduce the number of neurons in PrV and disrupt their patterning into vibrissa-related aggregates. 8,22 Such lesions do not qualitatively change the vibrissa-related patterning of primary afferents in this nucleus as demonstrated by HRP tracing, but they do cause a loss of the vibrissarelated CO pattern 8 (also see Ref. 15). In contrast, neonatal cortical or thalamic lesions have no effect

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upon CO patterns or cell number in either SpI or SpC. sa5"22 The hypothesis put forward to explain these results was that neonatal cortical or thalamic lesions caused a loss of trigeminothalamic projection neurons in PrV whose patterning permits visualization of the vibrissa-related CO pattern, but had no effect upon the cells that give rise to the pattern in SpI and SpC because they do not project to thalamus, Both of these suggestions are supported by the present results. PA-immunoreactive neurons in PrV were arrayed in a vibrissa-related pattern and generally projected to the thalamus. In contrast, the PA-immunoreactive cells in SpI that were arrayed in a vibrissa-related pattern were not retrogradely labeled after thalamic tracer injections, The present study showed that transection of the ION between P-3 and P-8 had effects upon vibrissarelated CO patterns in PrV, SpI, and SpC that were exactly the opposite of those observed after either

cortical or thalamic lesions. It demonstrated further that this differential effect was not the result of a differential loss of primary afferents from these nuclei, but rather a differential effect upon cells, recognized both by CO histochemistry (PrV vs SpI and SpC) and PA immunocytochemistry (PrV vs SpI), that are arrayed in a vibrissa-related pattern in these nuclei. We do not know whether the ION lesions carried out between P-3 and P-8 resulted in the death of the neurons giving rise to the CO pattern in SpI and SpC or an alteration in their biochemical properties. Nevertheless, it is worth noting that transection of the ION at birth does result in substantial neuron loss in the V brainstem complex 19 and comparison of the results of Choy e t al. ~ and Henderson e t al. 19 suggests that interneurons in SpI may be more susceptible to deafferentation-induced cell death than projection neurons.

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Fig. 6. High-power photomicrographs of portions of CO-stained sections through PrV (A, B), SpI (C, D), and SpC (E, F) of a rat that sustained transection of the ION on P-4 and was killed on P-9. Note that on the intact side of the brainstem and in PrV on the deaiferented side (A~=, E) CO-stained patches contain numerous heavily stained cells (some of these are denoted by arrows). Note also that only a few such cells are present in the deafferented SpI (D) and SpC (F). Scale bar = 100/~m for all panels. The results o f the present study differ somewhat from findings of earlier experiments that have examined the effects o f deafferentation upon vibrissarelated patterns in the V brainstem complex of

developing rodents. Belford and Killackey 5 cauterized the follicles in row C at different postnatal ages and reported that for each age the effects were the same in PrV, SpI, and SpC. M o s t relevant with

Fig. 5. Effect ofa P-10 ION lesion upon the vibrissa-related CO pattern in a rat killed on P-13. The pattern appears normal in PrV, SpI, and SpC on both sides of the brainstem. Scale bar = 500 #m.

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Fig. 8. A-C are low-power photomicrographs that show the distributions of PA-immunoreactive cells and fibers in PrV, SpI, and SpC of a rat killed on P-13. Note the vibrissa-related patterns formed by these neurons in PrV and SpI. PA immunoreactivity also forms a vibrissa-related pattern in SpC, but this appears to result primarily from immunoreactive fibers rather than cells. The solid arrows here and in G-I denote rows related to the vibrissae and the open arrows indicate the points marked in the higher-power photomicrographs of the same sections (D-F). Scale bar = 500/am. D - F are higher-power photomicrographs showing PA-immunorvactive patches in PrV, SpI, and SpC, respectively. Note the high density of immunoreactive cells in the patches in PrV and SpI and the smaller number of such cells in SpC. Scale bar ffi 100/am. G-I show PA immunoreactivity in PrV, SpI, and SpC of a rat killed on P-60. Note that a vibrissa-related pattern remains visible in this adult animal. Scale bar--500/am. respect to the present study is the observation that cauterizations carried out on P-0 through to P-3 reduced the density o f S D H staining in the representation o f the corresponding row in PrV, SpI, and

SpC, but that lesions made at later ages had little or no effect upon staining density in any of the three brainstem nuclei. However, examination of the figures provided by these investigators (see especially

Fig. 7. Comparison of the effects of a P-4 ION transection upon vibrissa-related CO patterns and transganglionic HRP labeling in PrV, SpI, and SpC in a rat killed on P-9. Transganglionic HRP transport (A-C) revealed a substantial reduction in the density of terminal labeling in the normal terminal zone of the ION in all V subnuclei (open arrows). The CO pattern appears normal in PrV (Ar) but its density and clarity are reduced in SpI (B') and SpC (cr). Scale bar = 500/am. AT, the CO patch corresponding to the representation of the auriculotemporal sinus hair; SO, the CO patch corresponding to the representation of one of the supraorbital vibrissae.

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Figs 2--4) does suggest that follicle cautery on P-2 reduces staining density in SpI and SpC to a greater extent than in PrV. Furthermore, their Fig. 5 shows that lesions made as late as P-5 reduce SDH staining in the representation of the corresponding row in SpI. Durham and Woolsey 13carried out experiments similar to those of Belford and Killackey5 in the mouse and reported that cauterizations of C-row follicles on P-0 through to P-4 produced approximately equivalent reductions in SDH staining in PrV, SpI, and SpC. The reason for the difference between these results and those of the present study is not clear, The second conclusion supported by the present results is that the vibrissa-related CO pattern in more caudal V subnuclei remains dependent upon normal primary afferent input well after the pattern in PrV becomes independent of normal connections between the periphery and the brainstem. If one accepts the above-stated proposal that visualization of the vibrissa-related pattern in all V brainstem nuclei by histochemical techniques is dependent primarily upon products in second order neurons rather than primary afferent axons, then an important question

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that remains unanswered is, why do cells in Spl and SpC remain susceptible to deafferentation longer than those in PrV? As noted above, the results of the experiments that combined retrograde tracing with immunocytochemistry demonstrated that PA-immunoreactive cells in PrV projected to the thalamus and suggested that PA-immunoreactive cells in SpI did not have axons that extended beyond the V brainstem complex. The fact that we did not make tracer deposits into all known targets of SpI (see Ref. 23 for review) forces caution with respect to the conclusion that PA-immunoreactive cells in this nucleus are, in fact, interneurons. Nevertheless, the results of the present experiments permit the following speculation. It may be that normal biochemical function of V brainstem neurons depends upon factors derived both from their afferent inputs and from their targets. Since the neurons in PrV that form the vibrissarelated pattern are nearly all trigeminothalamic cells, target-derived factors from outside the V brainstem complex may support normal biochemial function of these neurons, insofar as it was assayed by CO

Fig. 9. A and B, and C and D are pairs of episcopic fluorescence photomicrographs depicting portions of PrV (A and B) and SpI (C and D) from a rat that received a fluorogold injection into the contralateral thalamus. A and C show fluorogold labeled neurons and B and D depict PA-immunoreactive cells. Note that nearly all of the cells in PrV are double-labeled (several such neurons are indicated by arrows) while none of the neurons in SpI contain both markers. Scale bar = 100/am.

Late infraorbital nerve lesions and whisker patterns histochemistry and PA immunocytochemistry, in the absence of normal primary afferent input. Conversely, the cells that permit visualization of the vibrissa-related pattern in SpI and perhaps also SpC may be more dependent upon primary afferent input since they do not appear (at least in the case of SpI) te have axons that extend beyond the V brainstem complex. There is considerable experimental support for the proposal that the normal function and even survival of neurons depends upon both afferent- and targetderived factors. It is, for example, well known that primary afferent neurons are sustained by multiple trophic factors ',1~ts.25~ derived from both the periphery and brain. Both afferent input and target-derived factors also play a role in the survival of moteneurons ~'35 and autonomic ganglion cells. ~'~7'2~ If our findings reflect a difference in the requirement of PrV vs some SpI and SpC neurons for peripherally derived trophic support, other results

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indicate that this support does not require normal primary afferent activity. A number of studies in the visual and V systems have shown that normal patterns of CO staining (e.g. Refs l, 30, 31, 39, 40) can be altered by manipulations that might be expected to change the activity of the affected neurons. Comparison of the present data with those provided by Henderson e t al. ~° suggest that disruption of activity is insufficient to produce changes of the type that we have observed in the V bralnstem complex. They showed that application of either tetrodotoxin or bupivicaine to the developing ION had no significant effect upon CO staining patterns in any portion of the V brainstem complex. The early patterning of second order neurons in PrV 1°,~ and the independence of this patterning from peripheral input by P-3 (present study) may play an important role in determining the period during which vibrissa-related thalamic and cortical patterns are sensitive to peripheral manipulations. Killackey

Fig. 10. Effects of an ION lesion made on P-8 upon PA immunoreactivity in PrY and SpI of a rat killed on P-13. A and B show the intact and deafferented sides of PrV, respectively (the inset is a low-power micrograph of the same section; scale bar = 100 pm and also applies to the other inset). Note that PA-positive cells on both sides of the brainstem form a vibrissa-related pattern. C and D show the intact and deafferented sides of SpI, respectively (the inset is a low power micrograph of the same section). Note that PA-positive cells form a vibrissa-related pattern on the intact, but not on the dealferented side of the brainstem. Scale bar for all the higher-magnification panels = 250/zm.

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Fig. 11. Effects of an ION lesion made on P-10 upon PA immunoreactivity in PrV and SpI of a rat killed on P-13. A and B show the intact and deafferented sides of PrV, respectively (the inset is a low-power micrograph of the same section). Note that PA-positive cells on both sides of the brainstem form ~ vibrissa-related pattern. C and D show the intact and deafferented sides of Spt, respectively (the inset is a low-power micrograph of the same section). Here again, PA-positive cells form a vibrissa-related pattern on both sides of the brainstem Scale bar = 250 ~tm for all panels.

and Fleming 27 have shown that thalamic and cortical patterns related to the vibrissa are dependent upon ascending projections from PrV, but not those from SpI. Other studies 5 have reported that the sensitive period for the effects of peripheral damage upon thalamic and cortical patterns in rat ends between P-3 and P-4, nearly the same time that peripheral lesions no longer affect vibrissa-related C O patterns in PrV, but long before the patterns in SpI and SpC become refractory to such lesions, One last point worthy of discussion is the difference between PrV and SpI on the one hand, and SpC on the other, with respect to PA staining of neurons with distributions related to the pattern of the mystacial vibrissae. The present results provide the first report of a vibrissa-related patterning of neurons in SpI of rat. M a and Woolsey 32 showed that such a pattern could be demonstrated by Nissl-staining in Spl of mouse, and Chiaia e t al. 8 provided some evidence for

such a pattern in SpI of rat. However, neither of these approaches demonstrated the pattern with the clarity of PA immunocytochemistry. Similarly, retrograde tracing shows patterning of trigeminothalamic neurons in PrV of rat. TM This pattern is demonstrated with at least equal clarity by PA immunocytochemistry. If the vibrissa-related CO staining pattern in the magnocellular portion of SpC is also dependent upon clustering of a subset of second order neurons in this nucleus, these cells do not appear to share the expression of P A with similarly patterned neurons in PrV and SpI. It may be that the neurons comprising the vibrissa-related pattern in this nucleus will be recognized by some other marker. A c k n o w l e d g e m e n t s - - T h i s research was supported by NS 28888, DE 07734, and DE 08971. Thanks to Marcia Eck, Beth Figley, and Ann Marie Eckles for excellent technical assistance. Thanks also to Dr John T. Wall for commenting upon an earlier version of this manuscript.

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I~F~I~NCES 1. Akhtar N. D. and Land P. W. (1991) Activity dependent regulation of glutamic acid decarboxylase in the rat barrel cortex: effects of neonatal versus adult sensory deprivation. J. comp. Neurol. 307, 200-213. 2. Barde Y.-A. (1989) Trophic factors and neuronal survival. Neuron 2, 1525-1534. 3. Bates C. A. and Killackey H. P. (1985) The organization of the neonatal rat's brainstem trigeminal complex and its role in the formation of central trigeminal patterns. J. comp. Neurol. 240, 265-287. 4. Belford G. R. and Killackey H. P. (1979) The development of vibrissae representation in subcortical trigeminal centers of the neonatal rat. J. comp. Neurol. 188, 63-74. 5. Belford G. R. and Killackey H. P. (1980) The sensitive period in the development of the trigeminal system of the neonatal rat. J. comp. Neurol. 193, 335-350. 6. Black I. B. (1977) Regulation of the growth and development of sympathetic neurons in vivo. In Cellular Neurobiology, pp. 61-71. Alan R. Liss, New York. 7. Cello M. R. (1990) Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35, 375-475. 8. Chiaia N. L., Bennett-Clarke C. A. and Rhoades R. W. (1991) Effects of cortical and thalamic lesions upon primary afferent terminations, distributions of projection neurons, and the cytochrome oxidase pattern in the trigeminal brainstem complex. J. comp. Neurol. 303, 600-616. 9. Chiaia N. L., Rhoades R. W., Bennett-Clarke C. A., Fish S. E. and Killackey H. P. (1991) Thalamic processing of vibrissal information in the rat: I. Afferent input to the medial ventral posterior and posterior nuclei. J. comp. Neurol. 314, 201-216. 10. Chiaia N. L., Bennett-Clarke C. A., Eck M., White F. A., Crissman R. S. and Rhoades R. W. (1992) Evidence for prenatal competition among the central arbors of trigeminal primary afferent neurons. J. Neurosci. 12, 62-76. 11. Choy T. A., Henderson T. A. and Jacquin M. F. (1989) Neonatal infraorbital nerve section: differential effects on trigeminal brainstem cell number, size and distribution in rat. Soc. Neurosci. Abstr. 15, 1332. 12. Davies A. M., Thoenen H. and Barde Y.-A. (1986) Different factors from the central nervous system and periphery regulate the survival of sensory neurones. Nature 319, 497-499. 13. Durham D. and Woolsey T. A. (1984) Effects of neonatal whisker lesions on mouse central trigeminal pathways. J. comp. Neurol. 223, 424--447. 14. Erzurumlu R. S., Bates C. A. and Killackey H. P. (1980) Differential organization of thalamic projection cells in the brain stem trigeminal complex of the rat. Brain Res. 198, 427-433. 15. Erzurumlu R. S. and Ebner F. F. (1988) Maintenance of discrete somatosensory maps in subcortical relay nuclei is dependent on an intact sensory cortex. Devl Brain Res. 44, 302-308. 16. Erzurumlu R. S. and Killackey H. P. (1983) Development of order in the rat trigeminal system. J. comp. Neurol. 213, 365-380. 17. Furber S., Oppenheim R. W. and Prevette D. (1987) Naturally occurring neuron death in the ciliary ganglion of the chick embryo following removal of preganglionic input: evidence for the role of afferents in ganglion cell survival. J. Neurosci. 7, 1816-1832. 18. Goedert J., Stoeckel K. and Often U. (1981) Biological importance of the retrograde axonal transport of nerve growth factor in sensory neurons. Proc. natn. Acad. Sci. U.S.A. 78, 5895-5898. 19. Henderson T. A., Kaszubski P. D., Yelon Y. A. and Jacquin M. F. (1988) Effects of neonatal infraorbital nerve section on trigeminal brainstem cell number and dendritic orientation. Soc. Neurosci. Abstr. 14, 474. 20. Henderson T. A., Woolsey T. A. and Jacquin M. F. (1989) Role of postnatal primary afferent activity in central trigeminal pattern formation. Soc. Neurosci..4bstr. 15, 1332. 21. Hendry I. A. and Hill C. E. (1980) Denervation-induced decreases in enzyme activity of rat superior cervical ganglia differ/n vivo and in vitro. Brain Res. 200, 201-205. 22. Jacquin M. F., Chiaia N. L., Bennett-Clarke C. A., Hobart N. and Rhoades R. W. (1990) Effects of thalamotomy at birth upon trigeminal brainstem cell number, size and distribution. Soc. Neurosci. Abstr. 16, 630. 23. Jacquin M. F., Mooney R. D. and Rhoades R. W. (1986) Morphology, response properties, and collateral projections of trigeminothalamic neurons in brainstem subnucieus interpolaris. Expl Brain Res. 61, 457-468. 24. Jacquin M. F. and Rhoades R. W. (1983) Central projections of the normal and "regenerate" infraorbital nerve in adult rats subjected to neonatal unilateral infraorbital lesions: a transganglionic horseradish peroxidase study. Brain Res. 269, 137-144. 25. Johnson E. M. Jr and Yip H. K. (1985) Central nervous system and peripheral nerve growth factor provide trophic support critical to mature sensory neuronal survival. Nature 314, 751-752. 26. Kessler J. A. and Black I. B. (1980) Nerve growth factor stimulates the development of substance P in sensory ganglia. Proc. hath. Acad. Sci. U.S.A. 77, 649~52. 27. Killackey H. P. and Fleming K. (1985) The role of the principal sensory nucleus in central trigeminal pattern formation. Devl Brain Res. 22, 141-145. 28. Killackey H. P., Jacquin M. F. and Rhoades R. W. (1990) Development of somatosensory system structures. In Development of Sensory Systems in Mammals (ed. Coleman J. R.), pp. 403--429. John Wiley, New York. 29. Killackey H. P. and Shinder A. (1981) Central correlates of peripheral pattern alterations in the trigeminal system of the rat. II. The effect of nerve section. Devl Brain Res. 1, 121-126. 425, 178-181. 30. Land P. W. and Akhtar N. D. (1987) Chronic sensory deprivation affects cytochrome oxidase staining and glutamic acid decarboxylase immunoreactivity in adult rat ventrobasal thalamus. Brain Res. 425, 178-181. 31. Land P. W. and Simons D. J. (1985) Metabolic activity in SmI cortical barrels of adult rats is dependent on patterned sensory stimulation of the mystacial vibrissae. Brain Res. 341, 189-194. 32. Ma P. M. and Woolsey T. A. (1984) Cytoarchitectonic correlates of the vibrissae in the medullary trigeminal complex of the mouse. Brain Res. 306, 374-379. 33. Mesulam M.-M. (1978) Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents. J. Histochem. Cytochem. 26, 106-117. 34. Okado N. and Oppenheim R. W. (1984) Cell death of motoneurons in the chick embryo spinal cord. IX. The loss of motoneurons following removal of afferent inputs. J. Neurosci. 4, 1639-1652.

NSC 49/I--F

156

N.L. CH|AIA et al.

35. Oppenheim R. W., Haverkamp L. J., Prevette D., McManaman J. L. and Appel S. H. (1988) Reduction o | naturally occurring motoneuron death in vivo by a target-derived neurotrophic factor. Science 240, 919--922. 36. Rosene D. L. and Mesulam M.-M. (1978) Fixation variables in horseradish peroxidase neurohistochemistry: 1. Effects of fixation time and perfusion procedures upon enzyme activity. J. Histoehem. Cytochem. 26, 28-39. 37. Van der Loos H. and D6rfl J. (1978) Does the skin tell the somatosensory cortex how to construct a map o[ the periphery? Neurosci. Lett. 7, 23-30. 38. Wong-Riley M. (1979) Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res. 171, I 1-28. 39. Wong-Riley M. and Riley D. A. (1983) The effect of impulse blockage on cytochrome oxidase activity in the cat visual system. Brain Res. 261, 185-193. 40, Wong-Riley M. T. T., Tripathi S. C., Trusk T. C. and Hoppe D. A. (1989) Effect of retinal impulse blockage on cytochrome oxidase-rich zones in the macaque striate cortex: II. Quantitative electron-microscope (EM) analysis of neuropil. Vis. Neurosci. 2, 499-514. 41. Wong-Riley M. T. T. and Welt C. (1980) Histochemical changes in cytochrome oxidase of cortical barrels after vibrissal removal in neonatal and adult mice. Proc. natn. Acad. Sci. U.S.A. 77, 2333-2337. (Accepted 29 January 1992)