TIME-DEPENDENT NEUROPLASTICITY IN MESOSTRIATAL PROJECTIONS AFTER UNILATERAL
REMOVAL OF VIBRISSAE IN THE ADULT RAT: COMPARTMENT-SPECIFIC EFFECTS ON HORSERADISH
PEROXIDASE TRANSPORT AND CELL SIZE
H. STEINER,* H.-T. WEILER, S. MORGAN and J. P. HUSTON~"
Institute of Physiological Psychology, University of Diisseldorf, Universit~itsstrasse l, D-4000 Dfisseldorf, F.R.G.
Abstract--In adult rats the mystacial vibrissae were clipped on one side of the snout, and the influence of this sensory deprivation on crossed and uncrossed striatal afferents from the substantia nigra, ventral tegrnental area, and retrorubral area was examined with the horseradish peroxidase tract tracing technique. Unilateral removal of vibrissae was found to affect crossed and uncrossed nigrostriatal projections in a time-dependent manner. One to three days after hemivibrissotomy an apparent neuronal asymmetry was found in the crossed nigrostriatal projection arising in the rostral part of the substantia nigra, with more labeled neurons in the projection to the caudate--putamen on the side of vibrissae removal. This asymmetry resulted mainly from an asymmetry in the subset of nigrostriatal neurons reported to project to the striatal matrix ("dorsal cell type"). In contrast, 4-20 days after hemivibrissotomy reversed asymmetries were found in crossed and uncrossed nigrostriatal projections, with more labeled neurons in the projections to the caudate-putamen of the hemisphere opposite to vibrissae removal (the sensory deprived hemisphere). The asymmetry in the uncrossed projection was found throughout the substantia nigra, but was also most substantial in the projection from its rostral part. The asymmetry in the crossed projection was again restricted to the rostral substantia nigra; interestingly, however, it was limited to the subset of neurons reported to terminate in the striosomes ("ventral cell type"). Evidence was also found for time-dependent changes in size of neurons of the crossed nigrostriatal projections, as well as for changes in striatal afferents from the ventral tegmental area.
The time course of these apparent changes in strength of mesostriatal projections is similar to the known time course of recovery from behavioral asymmetries induced by hemivibrissotomy, which is suggestive of a functional relationship between neuronal and behavioral changes. Moreover, the finding of a differential influence of bemivibrissotomy on nigrostriatal afferents to striosomes and matrix is indicative of a functional dissociation of these two systems.
Unilateral lesions in the brain can lead to time- dependent neuronal changes in crossed efferents of the substantia nigra (SN). For example, after lesions in the SN by the neurotoxin 6-hydroxydopamine (6-OHDA), the crossed nigrothalamic and nigrostri- atal projections from the intact SN showed apparent increases in strength. Such alterations in crossed nigral efferents may account for various neuronal changes that occur after brain lesions 49'53'69 and may play a role in behavioral recovery from unilateral lesions (see Ref. 32 for review).
Similar changes occur in the crossed nigrostriatal projection after hemivibrissotomy. As found after 6 - O H D A lesions in the SN, 5~ we obtained evidence for
*Present address: Laboratory of Cell Biology, National Institute of Mental Health, Building 36, Room 2D-10, Bethesda, MD 20892, U.S.A.
tTo whom correspondence should be addressed. Abbreviations: CPU, eaudate--putamen; DA, dopamine;
an increase in strength (i.e. in structure or activity) of the crossed nigrostriatal projection 10 days after unilateral removal of the adult rat 's vibrissae. 3~ Thus, some of the effects of the 6 - O H D A lesion can be simulated by simply clipping these tactile facial hairs.
The neuronal changes found after the SN lesion were correlated in time with recovery from behavioral asymmetries induced by the lesion) ~ Unilateral re- moval of vibrissae also induces behavioral asym- metries, 61 and a recent study showed that the animals can recover from these asymmetries within a week : 5 That is, up to three days after hemivibrissotomy rats displayed more facial scanning with the sensory intact side of the face during exploration of an open field. In contrast, animals tested six days or later after vibrissae removal did not express such an asymmetry in scan- ning despite the lack of vibrissae on one s ide: 5 These results suggest that compensatory changes occur between three and six days after vibrissae removal, allowing recovery of function in these animals.
In the present experiment we examined a possible relationship between neuronal alterations and behavioral recovery by analysing the time course of
793
794 H. STEINER et al.
the neuronal changes in the nigrostriatal projection and comparing it with that of the known behavioral
changes that follow hemivibrissotomy. Based on our earlier results, 31 we hypothesized an increase in
strength of the crossed projection to the caudate- putamen (CPU) of the hemisphere situated contra- lateral to vibrissae removal, correlated in time with
behavioral recovery. Additionally, we examined a possible differential influence of hemivibrissotomy on the two subsets of nigrostriatal projections (nigro- striosomal and nigromatrixal~8), as well as effects on the striatal afferents from the ventral tegmental area (VTA) and retrorubral area (RRA). The effects of
unilateral removal o f vibrissae on these projections were examined with the quantitative horseradish peroxidase (HRP) tracing technique by assessment of the number of retrogradely labeled neurons and the size of labeled neurons.
EXPERIMENTAL PROCEDURES
Subjects
Male Wistar rats, weighing 115-140 g at the beginning of the experiment, were housed in pairs under standard labora- tory conditions. They had free access to food and water and were maintained on a 12-h light/12-h dark cycle (lights on at 07:00).
The rats were randomly assigned to 10 treatment groups which differed in the duration of unilateral removal of the vibrissae. That is, the brains were examined morphologically either one, two, three, four, five, six, eight, 10, 15, or 20 days after the vibrissae were clipped on one side of the snout. After a period of hemivibrissotomy, half of the rats received HRP into the CPU on the vibrissae-intact side (VIS groups), the other half on the vibrissae-clipped side (VCS groups).
Removal of vibrissae The treatment lasted 20 days for all rats. On the first day
of the clipping period the mystacial vibrissae 2~ were removed on one side of the snout. They were clipped close to the skin with an electrical hair clipper in the unanesthetized rat. From the second day to the end of the clipping period (day 20) the regrown bristles of the vibrissae were removed daily. On treatment days preceding the period of vibrissotomy the vibrissae were sham-clipped. That is, the animals were handled daily in an identical manner, and the hair clipper was turned on and moved through the fur without damaging the vibrissae. In half of the animals the vibrissae were clipped on the left, in the other half on the right side of the face.
Application of horseradish peroxidase On day 20, about 2 h after the last clipping treatment,
the rat was anesthetized with equithesin (3 ml/kg, i.p.), and its head was fixed in a David Kopf stereotaxic frame. A glass micropipette (inner diameter c. 70#m) filled with a 4% solution of horseradish peroxidase (grade 1, Boehringer Mannheim; in a Tris-HC1 buffer at pH 8.6) was then lowered into the head of the CPU. The following coordinates for the tip of the pipette were used: 8.5 mm rostral and 4.7 mm dorsal to the interaural line, 3 mm lateral to the midline (with the incisor bar adjusted 2.5 mm below the interaural line). These coordinates corresponded to K6nig and Klippe137 coordinates of about A 8300, V 0.2, L2.5.
Three minutes after insertion of the pipette, a 900 nA positive current, produced by a direct constant current source, was applied to the HRP solution for 20 min via a
silver wire. The reference electrode was connected to the scalp. Three minutes after switching the current off the pipette was removed. During insertion and removal of the pipette a negative current was applied to reduce leakage of HRP into the pipette tract.
H istochemistry
The animals were given an overdose of equithesin and perfused transcardially according to the procedure of Mesulam 44 24 h after application of HRP. The brains were removed from the skull, stored in a sucrose-phosphate buffer for 24 h, and then cut into 50-#m frontal sections. The sections which comprised CPU and SN were processed for HRP histochemistry, 44 using tetramethylbenzidine (Sigma) as the chromogen. They were then mounted on gelatinized slides, air-dried and counterstained with Neutral Red.
Morphological analysis
Sections comprising the CPU were examined for the position of the pipette and spread of HRP. In order to reduce variance only animals with the tip of the pipette positioned near the center of the CPU (inside a central region of about 1 mm 2 in size between levels A 8900 and A 750037) and with an apparent spread of HRP reaction product covering about one-third to two-thirds of the medial-to-lateral diameter of the CPU (on sections containing the tip of the pipette) were accepted for the morphological analysis. To control for possible confound- ing of differences in cell counts by variation in the size of the injection site, the spread of HRP was rated. In each rat the size of the apparent HRP spread was assessed on nine frontal sections, which were evenly distributed across the CPU. The obtained estimate of the size of the injection site was then compared between the groups. Similarly, the estimated coordinates of the tip of the pipette were com- pared between the groups to control for possible differences in placement of HRP deposits. Two injection sites represen- tative of position of the tip of the pipette and of spread of HRP are shown in Fig. 1.
The number of retrogradely labeled cells in the SN, VTA and RRA was assessed by microscopic analysis by an experimenter who did not know the treatment of the animal. For the cell counts contralateral to the HRP deposit every available section was examined. Care was taken not to count labeled cells twice on adjoining sections. For the ipsilateral cell counts every third section was analysed.
Cell counts were expressed as number of labeled cells per 50-,urn section. To depict the rostrocaudal distribution of the neurons of the crossed projections, the mean number of labeled cells per section of three adjoining sections ("triplet") was calculated. The ipsilateral cell counts were assessed on the middle section of each triplet. To allow comparison with earlier results, 3~ the SN was also divided into three parts along the rostrocaudal axis. The criteria for this partition are specified elsewhere. ~ In short (see Fig. 3), sections containing SN together with parts of the mammil- lary body were assigned to the rostral part (border between rostral and middle parts about at level A 218037). The point where the medial lemniscus reaches the cerebral peduncle, thereby separating SN from VTA, was chosen as the border between middle and caudal parts at about level A 155037).
As a check on our results, we also weighted the HRP labeling of the cells of the crossed nigrostriatal projection. This was done with the presumption that effects of the treatment could be revealed more dearly by also considering the amount of accumulated HRP in the labeled neurons. Therefore, we rated the HRP labeling of each cell by assigning a value between 1 (labeling just detectable) and 5 (cell filled with intense dark labeling).
Recently, it has been shown that neurons or the dopamin- ergic nigrostriatal projection can be differentiated according to their area of termination in CPU compartments. That is,
Mesostriatal projections after hemivibrissotomy 795
RESULTS
Fig. 1. Schematic illustration of representative HRP injec- tion sites in the CPU. Examples of small (left) or large (right) injection sites are given. Rostrocaudal levels A 9700 to A 6900 according to the atlas of Kfnig and Klippel. 39 AC,
nucleus accumbens; GP, globus pallidus.
SN cells of a dorsal tier project to the striatal matrix, and those of a ventral tier to patches (striosomes)fl In the rat, these two types of neurons can also be dissociated by their shape and spread of dendrites) 3'17,t8 Cells of the dorsal tier were described as being mainly fusiform-shaped with dendrites extending mediolaterally in the plane of the SN pars compacta, whereas the ventral tier consists mainly of pyramidal-shaped neurons with dendrites extend- ing into the SN pars reticulata and of neurons located in the SN pars reticulata. Using location, shape and orientation of dendrites as criteria, we also assigned each labeled cell of the crossed nigrostriatal projection to one of these two cell types. (Due to the vast number of labeled cells ipsilateral to the HRP deposit, it was not possible to analyse the uncrossed nigrostriatal projection in the same way.)
Additionally, we decided to evaluate a possible effect of hemivibrissotomy on the size of the neurons of the crossed nigrostriatal projection. Therefore, camera lucida drawings were made from the labeled cells of rats examined one to six days after vibrissae removal, with the aid of a drawing tube. From these drawings the somatic area of the labeled cells was computed.
Statistics Cell counts were compared between different parts of
SN and VTA with the Wilcoxon matched-pairs test and between treatment groups with the Mann-Whitney U-test. Cell size data were first subjected to ANOVA, followed by post hoc comparisons of means between individual groups. All P-values are presented two-tailed, unless stated otherwise.
Cell counts in the substantia nigra
The number of sections analysed per brain varied between animals in total from 34 to 41. Ipsilateral to the H R P application site, 2886-9864 HRP-labeled neurons (median = 6423) were found in the SN. The total number of labeled neurons per animal found contralateral to the H R P deposit was 1-29 (med ian= 12), including 0-15 ( m e d i a n = 4 ) neurons of the dorsal type and 0-22 (median = 7) neurons of the ventral type. Examples of labeled cells of the dorsal and ventral types are depicted in Fig. 2.
When the SN was partitioned into three parts as in previous experiments, the number of sections per part varied as follows: rostral part from l0 to 12, middle part from 10 to 16, and caudal part from 10 to 16 sections. When partit ioned into triplets of sections, the number of triplets varied between 12 and 14.
Rostrocaudal distributions of labeled cells (across all treatment groups). In order to describe the distri- bution of the labeled neurons along the rostral-to- caudal axis, the data of all treatment groups were pooled ( V I S + V C S I - 2 0 , n =90) , and the three parts of the SN were compared. The relations between the numbers of labeled cells per section of the three parts were for the uncrossed projection: rostral > middle > caudal, and for the crossed pro- jection: rostral < middle = caudal (P < 0.0001 for all differences). Dorsal and ventral cell types of the crossed projection had a similar distribution: rostral < middle = caudal (P < 0.0001 for differences).
Figure 3 shows the rostrocaudai distribution of the uncrossed nigrostriatal cells based on the triplet partit ion of the SN. It can be seen that nearly half (40-50%) of the cells lie in the rostral one-third, in the first five triplets. In Fig. 4 the distribution based on triplets of the cells of the crossed nigrostriatal projection is given. The cells of the crossed projection were inversely distributed compared with those of the uncrossed projection; most of the crossed projecting cells were caudal to triplet 5.
Time-independent effects o f hemivibrissotomy. To analyse time-independent effects of the treatment, the data of all VIS or VCS rats were pooled (VIS1-20, n = 48; VCS1-20, n = 42). Considering the uncrossed projection, differences between VCS and VIS groups were seen in the rostral part of the SN and in the total SN (all three parts pooled). That is, rostrally, more labeled cells were found in group VIS1-20 than in group VCS1-20 (P = 0.02). A tendency in the same direction was also seen in the middle (P = 0.18) and caudal parts of the SN (P = 0.08). Therefore, when pooled, the whole SN of group VIS1-20 contained more HRP-labeled cells than the SN of group VCSI -20 (P = 0.04). Fo r the crossed projection, no differences between groups VIS1-20 and VCS1-20 were found.
796 H. STEI~CER et al.
Fig. 2. Photomicrographs of HRP-labeled neurons found in the SN contralateral to the tracer application. Examples of neurons of the "dorsal type" (A, B) and "ventral type" (C, D) are depicted. The arrows in A and C indicate the location in the SN pars compacta (c) of the neurons shown in B and D, respectively.
r, SN pars reticulata.
Time-dependent effects; days one to three vs days four to 20. In order to examine t ime-dependent t rea tment effects, rats with vibrissae clipped for one
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Fig. 3. Rostrocaudal distribution of HRP-labeled neurons in the SN (uncrossed projection) ipsilateral to the tracer deposit. Depicted is the mean (__+S.E.M.) number of neurons per 50-#m section found on the second of three adjoining sec- tions (triplet), in the rostral, middle and caudal parts of the SN of rats examined one to 20 days after hemivibrissotomy. Rats received the tracer into the CPU either on the vibrissae- intact side (Intact), or on the side of vibrissae removal (Clipped). The schematic diagrams (bottom) illustrate the borders of the rostral, middle and caudal parts (rostrocaudal levels A 2790 to A 1000 according to K6nig and Klippe139).
to three days (VISI-3 , n = 15; VCSI-3 , n = 12) and for four to 20 days (VIS4-20, n = 33; VCS4-20, n = 30) were pooled on the basis of results of our recent study, in which rats displayed behavioral asymmetries for up to three days after hemivibrisso- tomy, and recovered thereafter. 45
Differences between VCS and VIS groups were also confined to the rostral par t of the SN (Fig. 5). For the uncrossed projection, group VIS4-20 had more labeled neurons than group VCS4-20 (P = 0.02). For the crossed projection, fewer labeled cells were found in group VCS4-20 than in group VIS4-20 (P = 0.05, one-tailed; P-values of compar isons based on hy- potheses are presented one-tailed), and more cells were found in group V C S I - 3 than in group V I S I - 3 (P = 0.002). G r o u p VCS4-20 also had fewer cells than group VCS1-3 (P = 0.0001). Groups VISI -3 and VIS4-20 did not differ.
Consider ing the two cell types of the crossed project ion separately (Fig. 5), again differences were confined to the rostral par t of the SN. Fewer labeled cells were found in group VCS4-20 than in group VIS4-20 (P = 0.05, one-tailed) for the ventral type; but no difference was seen for the dorsal type in this t ime period. More cells were found in group VCS1-3 than in group VIS1-3 (P = 0.04) for the dorsal type (no difference for the ventral type, P = 0.09). For the ventral cell type, group VCS4-20 also had fewer cells than group VCS1-3 (P = 0.007); no such difference was found for the dorsal cells.
Mesostriatal projections after hemivibrissotomy 797
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Fig. 4. Rostrocaudal distribution of HRP-labeled neurons in the SN (crossed projection) contralateral to the tracer deposit in the CPU. The mean (+S.E.M.) number of neurons per 50-#m section for 14 triplets is given for rats examined one to 20 days after hemivibrissotomy (all treatment groups pooled). The rostrocaudal distribution is presented for the dorsal cell type (middle graph), for the ventral cell type (bottom graph), and for both types
pooled (top graph).
Time-dependent effects; days one to three vs days four to six vs days eight to 20. In order to compare our results of time-dependent treatment effects with results of previous studies, in which changes in crossed nigrostriatal and nigrothalamic projections were found after one week or 10 days, 32 we also divided groups VIS4-20 and VCS4-20 into groups of rats with vibrissae clipped for four to six days (VIS4-6, n = 14 and VCS4-6, n = 13) and for eight to 20 days (VIS8-20, n = 19 and VCS8-20, n = 17).
Differences between groups VIS and VCS were almost all confined to the rostral part of the SN. For the uncrossed projection, group VCS8-20 had fewer cells in the rostral part of the SN than group VIS8-20 (P = 0.02). For the crossed projection, fewer labeled cells were found in.group VIS1-3 than in group VIS4-6 (P = 0.05, one-tailed), and more cells were found in group VCSI-3 than in group VCS4-6 (P = 0.007) and in group VCS8-20 (P = 0.00006). In the caudal part of the SN group VCS8-20 had fewer cells than groups VCS4-6 (P = 0.04) and VCS1-3 (P = 0.03).
For the dorsal type of cells, no differences in these comparisons were found in any part of the SN, but for the whole SN (all parts pooled): group VCS8-20 had fewer labeled cells than groups VCSI-3 (P = 0.03) and VCS4-6 (P = 0.02). For the ventral
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Fig. 5. Mean (+S.E.M.) number of labeled neurons per section found in the rostral part of the SN ipsilateral (uncrossed projection), and contralateral (crossed projec- tion) to the HRP deposit in rats examined one to three or four to 20 days after hemivibrissotomy. Rats had the tracer applied into the CPU on the vibrissae-intact side, or on the vibrissae-clipped side. For the crossed projection, these data are given for the dorsal cell type (middle), for the ventral cell type (bottom), and for both cell types pooled (top).
*P = 0.0001-0.05.
type, differences were found to be confined to the rostral part of the SN. Group VCSI-3 had more cells than groups VCS4-6 (P = 0.04) and VCS8-20 (P = 0.009).
Weighting o f amount o f horseradish peroxidase labeling. The results of the comparisons based on the weighted cell counts showed in general the same picture as described above for unweighted cells and are therefore not given in detail here.
Cell counts in the ventral tegmental area
The sections containing SN were also analysed for labeled cells in the VTA. Ipsilateral to the HRP application site, 114-1008 HRP-labeled neurons (median = 378) were found in the VTA. The number of labeled neurons per animal found contralateral to the HRP deposit was 1-62 (median = 13).
Labeled cells in the VTA had the following rostra- caudal distribution. The uncrossed ceils showed a three-peaked distribution with smaller peaks in triplets 5 and 13 and a major peak in triplet 8 in the middle of the distribution (Fig. 6). The crossed cells were almost exclusively located in the caudal half with a peak at the border of the middle and caudal thirds (Fig. 6). In order to describe the
798 H. STEINER et al.
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Fig. 6. Rostrocaudal distribution of labeled neurons in the ventral tegrnental area ipsilateral (uncrossed projection) and contralateral (crossed projection) to the HRP deposit in the CPU. The mean (+ S.E.M.) number of neurons per 50-#m section for 14 triplets is given for the uncrossed projection, for rats examined one to three, four to six, or eight to 20 days after hemivibrissotomy, and for the crossed projection, for rats examined one to 20 days (all treatment groups pooled). Rats had the tracer applied
into the CPU either on the vibrissae-intact side, or on the vibrissae-clipped side.
rostral-to-caudal distribution statistically, the VTA was partit ioned into three parts, using the same criteria as for the parti t ion of the SN. The relations between the numbers of labeled cells per section found in the three parts were as follows: for the uncrossed projection: rostral < middle > caudal (rostral < caudal); and for the crossed projection: rostral < middle < caudal (P < 0.0002 for all differences).
When these data were analysed for possible treat- ment effects on the basis of the three parts or in total, as done above for the SN, no differences were found (data not shown). Figure 6 depicts the distribution based on triplets of the uncrossed VTA cells of rats examined one to three, four to six or eight to 20 days after hemivibrissotomy. This more fine-grained distri- bution revealed evidence for differences between the groups, which could not be detected with the three- part distribution. It can be seen that differences between the distributions now appeared in the region of the center peak. When the cell counts of triplets 6-11 were compared, more cells were found in group VIS4-6 than in group VCS4-6 (P =0.03, one-
tailed), and more cells were found in group VIS4-6 than in group VIS1-3 (P = 0.04, one-tailed).
Cell counts in the retrorubral area. The sections analysed for labeled cells of the R R A corresponded to the sections of the caudal part of SN and VTA; occasionally, labeled cells were also found in the most caudal sections of the middle part. Sections beyond the caudal border of the SN were not considered. The number of sections examined varied between 10 and 16. Ipsilateral to the H R P application site, 72-654 HRP-labeled neurons (median = 304.5) were found in the RRA. The number of labeled neurons per animal found contralateral to the H R P deposition site varied between 0 and 21 (median = 7).
No differences were found between any groups for time-independent and time-dependent treatment effects, neither for crossed nor uncrossed HRP- labeled neurons (data not shown).
Somatic size o f labeled cells in the substantia nigra
To analyse possible effects of hemivibrissotomy on the size of the cells of the crossed nigrostriatal
Mesostriatal projections after hemivibrissotomy 799
projection, the somatic area of the camera lucida drawing of each labeled cell found in rats examined one to six days after vibrissae removal (696 cells in total) was measured.
Figure 7 depicts the size distribution of the differ- ent cell types for all treatment groups pooled. The cells of the rostral part (overall, mean__+ S.E.M.; 173 _ 8.1 #m 2) were bigger than those of the middle part (131+3 .3#m 2) (for both types pooled, P<0.0001; dorsal cell type, P=0 .004 ; ventral type, P = 0.0005) or the cells of the caudal part (125_ 2.9#m 2) (for both types, P < 0.0001; dorsal cells, P =0.0004; ventral cells, P=0.0001). No differences were found between the cells of the middle and caudal parts. The cells of the ventral type (over- all, 142+ 3.1#m 2) were bigger than those of the dorsal type (120 + 3.0 #m2), especially in the middle and caudal parts (for the three parts pooled or separate, P = 0.0002-0.04).
One-way ANOVAs showed that treatment-evoked differences in cell size between groups VIS1-6 and VCS1-6 were confined to the rostral part of the SN (data not depicted). The cells of group VIS1-6 were bigger than the cells of group VCSI-6 (both types pooled, P = 0.004). This difference was found for the
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Fig. 7. Size of somata of HRP-labeled neurons in the SN (crossed projection) contralateral to the tracer deposit in rats examined one to six days after hemivibrissotomy (all treatment groups pooled). The distribution of the somatic area as computed from camera lucida drawings is presented as a percentage of the population. These data are given for the dorsal cell type (top), for the ventral cell type (middle)
and for both types pooled (bottom).
dorsal cell type (P = 0.02); however, it did not reach significance for the ventral type (P = 0.08).
Considering the rostrocaudal distribution of the cell size (both types pooled) of these two groups, bigger cells were found in the rostral part than in the middle part (group VCS1-6, P = 0 .04 ; group VISI-6, P < 0.0001) or in the caudal part (group VCSI-6, P = 0.007; group VIS1-6, P < 0.0001). No differences were seen between the middle and caudal parts. In group VIS1-6 the difference between the cells of the rostral part and those of the middle part was seen for each cell type (P = 0.005 and 0.002; no difference in group VCS1-6). The differences between the rostral and caudal parts were found in groups VIS1-6 and VCS1-6 for either cell type (P = 0.0007-0.04).
Comparing the cell types for the three parts of the SN, pooled, ventral cells were bigger than dorsal cells (group VCS1-6, P =0.001; group VIS1-6, P < 0.0001). In group VCS1-6 this difference was most substantial in the caudal part (P = 0.002), and in group VIS1-6 in the middle and caudal parts (P = 0.003 and 0.02).
Treatment effects but not time effects for both cell types were also found in 2 × 2 ANOVAs. The effects were only found after three days. These treatment effects were again confined to the rostral SN (P = 0.007) and were also found for the whole SN (P = 0.009). No differences between cells of groups VCSI-3 and VIS1-3 were found. The cells of group VIS4-6 were bigger than the cells of group VCS4-6 in the rostral SN (P = 0.03) and in the whole SN (P = 0.05).
Considering the cell types separately, 2 × 2 ANOVAs showed time effects for the dorsal type (P =0.03) and treatment effects for the ventral type (P = 0.02), for the whole SN. However, for the dorsal type cells no differences were found in post hoc comparisons. For the ventral type, the cells of group VIS4-6 were bigger than VCS4-6 (P = 0.05).
With regard to the rostrocaudal distribution of the cell size, in group VCSI-3 bigger cells were found in the rostral than in the middle SN (both types pooled only, P = 0.04). In group VIS1-3 bigger cells were seen in the rostral than in the middle (both types pooled, P = 0.02) or in the caudal SN (P = 0.02). Differences between the middle and caudal SN were not seen. In group VCS4-6 the cells in the caudal were smaller than in the rostral (both types pooled, P = 0.04) or in the middle SN (P = 0.03). In group VIS4-6 bigger cells were seen in the rostral than in the middle (P =0.0001) or in the caudal SN (P = 0.0001). These differences were also found for each type (P = 0.003-0.01).
The ventral cells were bigger than the dorsal cells in the whole SN (three parts pooled) and in the middle and caudal parts in groups VCS 1-3 (P = 0.0004-0.02) and VISI-3 (P = 0.0001-0.04), but not in the rostral SN. The ventral cells were also bigger in the whole SN
800 H. STEINER et al.
(P = 0,04) and in the middle (P = 0.05), but not in rostral and caudal SN of group VIS4-6.
DISCUSSION
Methodological considerations
The present experiment provides evidence for time- dependent changes in afferent projections to the caudate-putamen after unilateral removal of vibris- sae. The striatal afferents were examined with the quantitative HRP tracing technique, the apparent neuronal changes were indicated by changes in the number of HRP-labeled neurons,
The use of the HRP technique as a quantitative method demands certain precautions. Even with the injection or iontophoresis parameters kept constant, the size of the HRP injection site, i.e. the spread of HRP, often varies between animals to some degree. Since the number of retrogradely labeled cells in a specific projection is influenced by the size of the injection site in the area of termination, differences in cell counts can be confounded by variation in spread of HRP. To reduce the probability of such confound- ing, we homogenized our population by excluding rats with a very large or small size of injection site. For this purpose the apparent spread of HRP was rated in each rat. These estimates of the size of the injection site also allowed us to rule out that differ- ences in spread of HRP between treatment groups had a major influence on our results.
It is also conceivable that variation in the place- ment of the HRP deposit within the area of termin- ation may influence the cell counts. We therefore only accepted rats with the deposit placed in a defined central region of the CPU. In addition, we controlled for HRP placement by comparing the estimated coordinates of the tip of the pipette between groups.
Rats were examined morphologically between one and 20 days after removal of vibrissae from one side of the snout. For the analysis of treatment effects in relation to behavioral changes, the data were pooled on the basis of recent behavioral results which showed that rats recover from behavioral asym- metries induced by hemivibrissotomy between days three and six after vibrissae removal: rats exhibited an asymmetry in facial scanning towards the sensory intact side only up to three days after unilateral clipping of these sensory hairs. 45 Based on these findings, time-dependent effects of unilateral vibrissae removal were analysed in the present experiment by pooling the morphological data in the following ways. Firstly, rats examined one to three days after hemivibrissotomy were compared with rats examined after four to 20 days of clipping, and secondly, one- to three-day rats were compared with four- to six-day rats and with eight- to 20-day rats.
Effects of hemivibrissotomy on mesostriatal projections
The present experiment yielded several results (schematically summarized in Fig. 8). For one, we
replicated earlier findings in showing an influence of hemivibrissotomy on the crossed nigrostriatal pro- jection. As seen before, 3~ more HRP-labeled neurons were found in the crossed projection to the CPU of the hemisphere opposite to vibrissae removal (the hemisphere deprived of vibrissal sensory input) than in the crossed projection to the CPU on the vibrissae- clipped side, in rats examined four to 20 days after hemivibrissotomy. This neuronal asymmetry was confined to the projection arising out of the rostral part of the SN, an effect also observed before, 3~ With increasing number of days of vibrissae removal, however, this asymmetry decreased.
A new and surprising finding of the present experiment is the opposite asymmetry in the crossed nigrostriatal projection seen one to three days after hemivibrissotomy. In these rats more labeled neurons were found in the crossed projection from the rostral SN to the CPU ipsilateral to the vibrissae-clipped side.
Also, unexpectedly, an effect of unilateral removal of vibrissae on the uncrossed nigrostriatal projection was found. More HRP-labeled neurons were ob- served in the projection to the CPU opposite to vibrissae removal. Interestingly, this asymmetry could be seen throughout the SN, but was most substantial again in its rostral part. In contrast to the crossed projection, the asymmetry in HRP labeling in the uncrossed projection was in the same direction irrespective of the duration of the vibrissae sensory deprivation. However, it became stronger with in- creasing duration of hemivibrissotomy.
Taken together, these results showed an asymmetry in the crossed nigrostriatal projection (with more HRP labeling in the projection to the CPU ipsilateral to vibrissae removal) during the initial time period after hemivibrissotomy, when rats were found to exhibit behavioral asymmetries towards the side opposite to vibrissae removal. 4~ In contrast, after the point in time when animals had recovered from these asymmetries in behavior, an opposite asymmetry (with more HRP labeling in the projections to the CPU of the sensory deprived hemisphere) was observed in both crossed and uncrossed nigrostriatal projections.
Interestingly, preliminary results of a recent study, in which the effects of unilateral lesions in the cortical projection field of the vibrissae ("barrel field") on nigral and striatal dopamine (DA) metabolism were examined, indicate changes in the DA content of these areas with a similar time course. That is, three days after the lesion a higher DA content was found in the SN ipsilateral to the lesion. By day 6 after the lesion asymmetries were reversed in direction with higher DA contents in the SN contralateral and in the CPU ipsilateral to the lesion (Adams et al., in preparation).
For the cell counts in the VTA, the analysis of treatment effects on the basis of the partition into rostral, middle and caudal parts did not reveal
Mesostriatal projections after hemivibrissotomy 801
D U R A T I O N O F H E M I V I B R I S S O T O M Y
I - 3 DAYS 4 - 6 DAYS 8- 20 DAYS
SUBSTANTIA NIGRA
DORSAL CELL TYPE
VENTRAL CELL T Y P E - - , , " ,
, . ' ' ,
, , " , o
AREA @
RET ORU RA, AREA--
g:x Fig. 8. Schematic summary of apparent changes in strength of striatal afferents from the substantia nigra, ventral tegmental area, retrorubral area, as well as from the tuberomammillary nucleus found after
unilateral removal of vibrissae.
differences between groups. However, when the triplet partition is considered, an influence of hemi- vibrissotomy on the uncrossed striatal projection from a central region of the VTA is indicated, a region which was spliced by the border between the middle and caudal parts. A post hoc analysis of the cell counts in this region indicated more labeled cells in the uncrossed projection to the CPU opposite to vibrissae removal than to the CPU on the clipped side in rats examined between four and six days after hemivibrissotomy. We then reanalysed the data obtained in the earlier study 10 days after unilateral vibrissae removal 3' and also found an increased number of labeled neurons in the uncrossed striatal projection from this central region of the VTA (com- pared with the projection to the CPU on the clipped side or normal controls). Therefore, a specific part of the VTA seems to be influenced by hemivibrissotomy as well.
Recently, we showed that neuronal changes after hemivibrissotomy are not limited to mesostriatal projections. Time-dependent changes in HRP label- ing were also observed in striatal afferents from tuberomammillary subnuclei in the caudal hypo- thalamus. 66 That is, in rats examined four to 20 days after unilateral removal of vibrissae, more HRP- labeled neurons were found in crossed and uncrossed
tuberomammillary-striatal projections to the CPU of the sensory deprived hemisphere than in the respect- ive projections to the CPU on the clipped side. Rats examined one to three days after hemivibrissotomy did not exhibit such neuronal asymmetries.
Reanalysis of these data also showed a main increase in neuronal asymmetry between days 4 and 6 after hemivibrissotomy, and a subsequent decrease in asymmetry (unpublished results; see Fig. 8). Thus, the tuberomammillary-striatal projections changed in a similar manner (a transient increase in HRP labeling in projections to the deprived hemisphere during the time period when behavioral recovery occurred), as was observed here in the uncrossed projection from the VTA, and in the crossed nigrostriatal projection.
Our findings indicate that after hemivibrissotomy neuronal changes take place in multiple afferent systems to the CPU in concert. Moreover, the different time courses of the changes in these afferents suggest an involvement in different functional aspects. Some of these systems might be related to behavioral asymmetry 4s (crossed nigrostriatal pro- jection), others to initial processes which mediate behavioral recovery (crossed nigrostriatal projection, projections from VTA and tuberomammillary nuclei), and still others to later processes which
802 H. STEINER et al.
sustain behavioral recovery (uncrossed nigrostriatal projection; see also below).
Differential effects o f hemivibrissotomy on nigro- striatal projections to striosomes and matrix
The mammalian striatum is composed of two distinct compartments (see Refs 16 and 20 for review). These striatal compartments also differ in their efferent and afferent connections. For example, two distinct subsets of dopaminergic nigrostriatal projections seem to exist, one innervating the matrix (nigromatrixal), the other the striosomes (nigrostriosomal). 1s'22,35'4° We decided to examine a possible differential influence of hemivibrissotomy on these two subsets of nigrostriatal projections. Location and shape of the cell body and the distri- bution of dendrites were used as criteria for their dissociation.~3'~7-18
The results confirmed the influence of unilateral vibrissae removal on the crossed nigrostriatal pro- jection from the rostral SN. Surprisingly, however, the effect on nigromatrixal ("dorsal cell type") and nigrostriosomal ("ventral cell type") projections was opposite in direction, and dependent on the duration of hemivibrissotomy. That is, the asymmetry with more HRP-labeled cells in the projection to the CPU of the deprived hemisphere, seen from four to 20 days after vibrissae removal, was limited to the nigrostrio- somal subset, whereas the opposite asymmetry seen one to three days after hemivibrissotomy resulted from an asymmetry mainly in the nigromatrixal subset.
Therefore, these findings are the first to provide evidence for a functional dissociation of the subsets of nigrostriatal projections to striatal matrix or strio- somes. Since the asymmetry in the nigromatrixal projection was found during the period of behavioral asymmetry, 45 an asymmetry in activity in the pro- jection to the matrix might be related to sensorimotor asymmetries. In contrast, the asymmetry in the nigrostriosomal projection was observed during the period of behavioral recovery, 45 which is suggestive of a role of striosomal processes in compensatory mechanisms.
Distribution and size o f somata o f nigromatrixal and nigrostriosomal neurons after hemivibrissotomy
To further characterize the (for the most part) dopaminergic 3,7'8'14'5~ crossed nigrostriatal projections, we examined the size and the rostrocaudal distri- bution in the SN of the HRP-labeled cell bodies of the nigromatrixal and nigrostriosomal subsets and analysed a possible influence of unilateral vibrissae removal on these parameters.
The rostrocaudal distributions observed are similar for the two types of neurons: most of the cell bodies of the crossed nigrostriatal projections are located in the middle and caudal parts of the SN. This finding corresponds with earlier results on pooled cell types after hemivibrissotomy, 3j or in the normal
rat. 46 Interestingly, this distribution is inverse to the distribution of the labeled somata in the uncrossed nigrostriatal projection, in which the highest numbers of cells per section were found in the rostral part of the SN, a finding also seen in the normal rat. 46
For the analysis of the cell size, the labeled cells found in the SN contralateral to the HRP deposit of rats examined one to six days after hemivibrissotomy were measured (276 cells of the dorsal type and 420 cells of the ventral type). As a correlate of the cell volume, the somatic area was calculated from camera lucida drawings. The values achieved in this way, for example, for nigrostriatal neurons from the middle part of the SN, were very similar to cross-sectional areas of neurons from the "central" part of the SN pars compacta, as reported by Pearson et al. 49
For all groups (1-6) and both cell types pooled, the labeled neurons in the rostral part of the SN were larger than those in the middle and caudal parts. Such a relation has been reported for neurons of the SN pars compacta in general, z3 This difference was found here for both crossed nigromatrixal and nigrostrioso- mal cell types. However, it was more evident in the projection to the CPU of the sensory deprived hemi- sphere. Overall, the cells of the ventral type were bigger than those of the dorsal type.
Interestingly, an influence of hemivibrissotomy on the cell size was again observed most clearly in the rostral part of the SN. That is, the neurons in the projection to the CPU opposite to vibrissae removal were bigger than the neurons in the projection to the CPU on the clipped side. This effect was strongest in (but not restricted to) the dorsal cell type of the rostral part, in rats examined four to six days after hemivibrissotomy.
Our results may thus indicate that these SN neurons also undergo a change in size after hemivibrissotomy. Both shrinkage and hypertrophy of cell bodies seem to occur in the nigrostriatal projection, for one, in surviving neurons on the side of a striatal lesion, but also in the undamaged, contralateral hemisphere. 49 Alternatively, it is possible that the apparent increase in neuronal size may reflect preferential labeling of larger neurons recruited 2~ after hemivibrissotomy.
Possible neuronal changes reflected by the differences in number of horseradish peroxidase-labeled neurons
The cell's capacity to accumulate HRP has been related, for one, to the number and size of axon terminals (and possibly preterminal axon branches) present at the injection site. 36 Therefore, an increase in HRP labeling could, hypothetically, indicate mor- phological changes such as sprouting of axon termi- nals or collaterals. However, the appearance of differences in HRP labeling already one to three days after unilateral removal of vibrissae seems to rule out mechanisms such as terminal sprouting, at least at this early time point (see, e.g. Ref. 19). In fact, even after lesions in the nigrostriatal projection, it is
Mesostriatal projections after hemivibrissotomy 803
unclear whether or not this phenomenon occurs at all in dopaminergic terminals in the denervated CPU 27'52 (but see Ref. 48).
Other determinants for uptake and transport of HRP have also been demonstrated (see Ref. 39 for review). Studies on the neuromuscular junction or motoneurons first indicated that uptake and retro- grade axonal transport of this tracer can be increased by electrical or pharmacological stimulation of the synapse to release transmitter. 5'26'3°'41'4z62 A similar influence of synaptic activity on uptake of HRP was shown in retinal photoreceptors: ~ in neurons of the sympathetic cervical ganglia, It in salivary neurons, t2 in cortical presynaptic terminals, 9'63 and in geniculate- cortical neurons. 57 In the nigrostriatal projection, however, such a relationship between neuronal activity and axonal transport is less clear. While pharmacological manipulations of neuronal activity have been reported to influence the anterograde axonal transport, 64 a study using similar treatments failed to reveal an effect on retrograde HRP marking, to
Taken together, these results demonstrate an influ- ence of neuronal activity on HRP marking in various neuronal tissue and under various experimental con- ditions and thus argue for a general phenomenon. In spite of the one negative result reported in the nigrostriatal projection so far, l° we consider it likely that the observed relationship between neuronal activity and uptake and transport of HRP also holds for this projection.
Nigrostriatal neurons can modify their synaptic activity. For example, remaining dopaminergic neur- ons in this projection are able to increase transmitter metabolism and release in response to denervation by 6-OHDA lesions, i'2'25,53,59,69,70 There is also evidence that nigrostriatal neurons can increase their electrical activity after 6-OHDA lesions. 28,29 An increase in activity could lead to enhanced accumulation of HRP in neurons which would raise the likelihood of their detection.
We favor the interpretation that the observed differences in cell counts after hemivibrissotomy reflect changes in neuronal activity in these projec- tions. However, to make clear that the underlying mechanisms (i.e. morphological and/or functional changes) still await elucidation, we used the term "changes in strength" to describe the neuronal changes observed.
Anatomical connections from the vibrissae-trigeminal system to the basal ganglia
The present results, showing neuronal changes in mesostriatal projections after unilateral removal of vibrissae, indicate a functional relationship between the vibrissae-trigeminal system and the basal ganglia. 34 Also, results from electrophysiological studies demonstrated an influence of vibrissae- trigeminal stimulation on the firing rate of meso- striatal DA neurons (e.g. Refs 6,15 and 24). The
question then arises as to the course by which sensory input from the vibrissae reaches the mesostriatal projections.
So far, we do not know the relevant bridge from the trigeminal system to the basal ganglia. However, several possibilities exist:
(a) Direct projections from the portion of the somatosensory cortex which represents the vibrissal system ("barrel field") to the striatum have been demonstrated: 3'67 Other cortical areas, which may receive direct input from the barrel field, 67 also project to the SN (e.g. Refs 4 and 54).
(b) An input route for trigeminal sensory infor- mation to the basal ganglia via striatal afferents from the intralaminar thalamus has been proposed. 3s
(c) There is evidence for additional subcortical connections to SN or CPU: a trigemino-pontine- nigral pathway, 56 a route from the trigeminal nuclear complex via raph6 nuclei 42 (see also Ref. 24) to SN and CPU, and connections via cerebellum 65 to the SN) 8 Evidence also exists for a direct trigemino- striatal projection, ss
Therefore, several anatomical routes exist to link the trigeminal system to the basal ganglia. More than one could be relevant for vibrissae function. For the moment, however, we have to leave open the question of the relative contribution of these different connections.
Remodeling of crossed efferents of the substantia nigra in relation to recovery of function
In the earlier study, 3~ we had observed an apparent increase in strength in the crossed nigrostriatal projection to the CPU of the sensory deprived hemi- sphere 10 days after hemivibrissotomy. In the present study, we showed that these neuronal changes are related in time to recovery from behavioral asym- metries 45 induced by vibrissae removal. A similar correlation in time between recovery from behavioral asymmetries and changes in strength of crossed efferents of the SN was found after unilateral brain lesions (see Ref. 32 for review). For example, a 6-OHDA lesion in the SN induces asymmetries in turning behavior and facial scanning, asymmetries from which the animals can also recover within one week (e.g. Ref. 60). Related in time to such behavioral recovery, an apparent increase in strength of crossed nigrothalamic s° and nigrostriatal pro- jections 5~ from the intact SN to the denervated hemisphere was found.
These early findings led to the hypothesis that changes in crossed efferents of the SN may play a role in recovery of function. 32 The results of the present experiment support this hypothesis, and they indicate that the changes in the crossed nigrostriatal projection in relation to behavioral recovery after hemivibrisso- tomy are due to changes in the nigrostriosomal subset of nigrostriatal projections. On the basis of the parallels in the effects of hemivibrissotomy and
804 H. STEINER et al.
unilateral 6 - O H D A lesions (see also Ref. 33), we propose that also in the case of 6 - O H D A (or other) lesions neuronal changes in nigrostriatal projections to the striosomes are related to recovery of function.
CONCLUSIONS
In conclusion, the present experimental results are indicative of t ime-dependent changes in strength of striatal afferents from the mesencephalon after uni- lateral removal of vibrissae. The time course of these
neuronal changes (i.e. changes in number and size of HRP-labeled neurons) was similar to the known time course of recovery from behavioral asymmetries in- duced by hemivibrissotomy. This is suggestive of a functional relationship between neuronal and behav- ioral changes. Therefore, hemivibrissotomy can serve as a model for the investigation of neuronal changes in relation to recovery of function.
Acknowledgement--This work was supported by grant Hu 306/6-3 from the Deutsche Forschungsgemeinschaft.
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