Efferent and Afferent Connections of Mouse Sensory-Motor Cortex Following Cholinergic Deafferentation at Birth
Departments of 'Psychiatry and 2Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Over the last few decades, considerable research has been devoted to the understanding of cerebral cortical development. Consequently, the morphogenetic and, to some extent, pharmacological/neurochemical events in cortical ontogeny have been described in great detail (for review, see McConnell, 1988). In contrast, our understanding of the mechanisms that guide and regulate these ontogenetic events is still rather primitive. Corticopetal systems from the brainstem, such as the noradrenergic, dopaminergic, and serotonergic afferents, have been invoked in shaping various events in cortical morphogenesis (Felten et al., 1982; Lauder, 1983; Kalsbeek et al., 1987; Loeb et al., 1987; Blue and Molliver, 1989). Several neuropeptide transmitters have also been implicated (Chun et al., 1987; Hauser et al., 1987). However, the mechanisms by which cortical subdivisions are established, cortical cell differentiation and synapse formation are timed, and developmental plasticity is regulated are unknown. Increasing attention has been directed toward ACh as a modulator of plasticity and cell differentiation (Sillito, 1983; Bear and Singer, 1986; Hohmann et al., 1988,1989; Lipton et al., 1988; McCobb et al., 1988). Cholinergic axons from the basal forebrain innervate rodent neocortex postnatally, at the onset of differentiation and synapse formation, and mature before these events have concluded (Kristt, 1978,1979; Candy et al., 1985; Hohmann and Ebner, 1985; Hohmann et al., 1985; Kostovic, 1986; Schambra et al., 1989). Such a time course of afferent innervation could enable the cholinergic system to exert a regulatory function, perhaps to determine the timing of ontogenetic events. We have developed evidence supporting a role of the afferent cholinergic projection to cortex in the regulation of cortical morphogenesis and plasticity (Hohmann and Ebner, 1988; Hohmann et al., 1988, 1989). Lesions among the cholinergic basal forebrain neurons (nBM) at birth cause a transient cholinergic deafferentation of sensory-motor cortex and result in the following cytoarchitectonic abnormalities (Hohmann et al., 1988): The time course of cytodifferentiation in cortical layers V and IV appears to be most severely disrupted in these experiments. However, as cholinergic innervation reappears, presumably due to collateral sprouting of nBM neurons spared by the lesion, cytoarchitectonic abnormalities become attenuated. By adulthood, cortical layer V has assumed a Cerebral Cortex Mar/Apr 1991;1:158-172; 1047-3211/91/12.00
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The present study investigates the effect of cholinergic basal forebrain lesions at birth on cortical connectivity in adulthood. We have previously shown that such neonatal lesions result in extensive cortical cholinergic deafferentation during early postnatal development, which is accompanied by abnormal morphogenesis of cortical cytoarchitecture (Hohmann et al., 1988). Here, we have used WGA-HRP to label anterogradely and retrogradely afferent and efferent projections of dorsal neocortex. Our results show an altered projection pattern from dorsal thalamus to layer IV of sensory-motor cortex following lesions among the cholinergic basal forebrain neurons (nBM), while corticothalamic projections from layer VI appear normal. In addition, corticofugal projections from layer V, labeled by striatal injection, appear to be expanded following the lesion. This indicates that cortical layers undergoing differentiation after the newborn nBM lesion present with long-term abnormalities in connectivity. The present results are compatible with the hypothesis that cholinergic afferents are instrumental in the regulation of cortical morphogenesis. Furthermore, our data show that ontogenetic disturbances can lead to structural abnormalities that persist long after the initial deficiency has abated. We discuss the significance of these results in relationship to human neurological disorders.
Christine F. Hohmann,1 Lucy Wilson, and Joseph T. Coyle1-2
Materials and Methods All mice in the present study were of the BALB/cByJ strain and raised in our own timed-pregnancy breeding colony. Neonatal nBM-lesioned and unlesioned control animals were derived from the same litters. For nBM lesions, pups aged 12-24 hr (postnatal day 1) were removed from their mothers, anesthetized by hypothermia, and immobilized in a specially designed Plexiglas-mold head holder attached to a David Kopf stereotaxic apparatus. Unilateral electrolytic lesions were performed in the ventromedial globus pallidus region on the right side of the animal as described in Hohmann et al. (1988). Briefly, a fine wire electrode (SNEX 300, Rhodes Medical Supplies) was inserted through the skin and skull 1 mm lateral to the midline and 1.5 mm anterior to the frontonasal suture at a vertical angle of 48.5° and a horizontal angle of 5°. The electrode was lowered into the brain; a positive, constant current of 0.8 mA, 10-msec duration was passed at depths of 3, 3.3, 3.6, and 4 mm. Following the lesion, the pups were removed from
the head holder and transferred to a heating pad for 30 min to reequilibrate to normal body temperature. Subsequently, pups were returned to their mothers. For tracer injections, adult (2-5-month-old) mice unilaterally lesioned at birth and their normal litter mates were anesthetized with Avertin (0.2 ml/gm body weight of a solution made from a 1:10 PBS dilution of a stock containi ng 2 5 gm tetrabromethanol and 15.5 gm tetramylalcohol) and immobilized in a Kopf smallanimal stereotaxic apparatus. Injections of 0.06-0.08 Ail of a 1% w/v WGA-HRP solution (in water) were made vertically through small burr holes in the skull of the animal, ipsilateral to the nBM lesion. The volume and concentrations used for the present injections were smaller than usually reported but have led, in our hands, in mice (Hohmann and Ebner, 1988), to excellent anterograde and retrograde transport while ensuring well-confined injection sites. Stereotaxic coordinates for all injections were empirically derived, with skull sutures serving as landmarks. Striatal injections were aimed at the dorsal aspect of the anteromedial striatum described by White and DeAmicis (1977) to receive a large projection from neurons in the posteromedial barrel subfield (PMBSF) of somatosensory cortex. Thalamic injections were aimed at the ventrobasal complex (Vb) of the thalamus. Several lesioned as well as control animals received both striatal and thalamic injections. The cortical area between these two injection sites, analyzed in the present study, included the PMBSF and most of somatosensory motor cortex. WGA-HRP histochemistry was performed as previously described (Hohmann et al., 1988). Briefly, 24 hr after WGA-HRP injections, animals were anesthetized with a lethal dose of Nembutal and perfused first with physiological saline solution, followed by 1.25% v/v glutaraldehyde and 1% w/v paraformaldehyde in 0.1 M phosphate buffer (pH, 7.4). Brains were postfixed in above fixative containing 20% sucrose for another 2 hr and kept in 0.1 M phosphate buffer (pH, 7.4) overnight. This perfusion protocol allows the visualization of both anterograde and retrograde transport of WGA-HRP. Subsequently, brains were sectioned frozen, at 50 Aim, on a sliding microtome and processed according to the protocol of Mesulam (1978), using trimethylbenzidine (TMB) as the chromatophore. Adjacent sections were stained with cresyl violet to visualize cortical cytoarchitecture. Quantifications of retrogradely labeled layer V pyramidal neurons were performed using a Zeiss Axiophote microscope equipped with a computerized video-mapping system (developed by Dr. M. E. Molliver, Department of Anatomy, and modified by Kathrine Fleishman, Department of Neuropathology, Johns Hopkins University, School of Medicine). We selected 10 sections each from four normal control cases and four nBM-lesioned animals. The sections were chosen from three different levels in the coronal plain: (1) just anterior to the decussation of the anterior commissure, (2) on the level of first appearance of the fimbria fornix, and (3) at midhippocampal level. First, all cortical pyramidal neurons in dorsal cortex Cerebral Cortex Mar/Apr 1991, V I N 2 159
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relatively normal morphology as judged by Nissl stain. However, the distribution of granule cells, particularly within somatosensory cortex, does not resemble the normal adult cortex. The distinct cytoarchitectonic organization of the "barrel fields," as originally described by Woolsey and Van der Loos (1970), is obscured. Frequently, granule cells form amorphous clusters interspersed with granule-cell-sparse zones. In addition, boundaries between layer V and IV, as well as IV and supragranular cortex, are poorly defined. Thus, we have shown that the transient cortical deafferentation, resulting from the newborn basal forebrain lesion, permanently affects the organization of cortical cytoarchitecture. Golgi studies of pyramidal cell maturation, presented in a companion paper (Hohmann et al., 1991), have confirmed our hypothesis that the newborn nBM lesion causes a delay of cortical cytodifferentiation (Hohmann et al., 1988). Delayed cortical cytodifferentiation might result in persistent abnormalities of specific afferent and efferent cortical connections. In the absence of their appropriate target structures, cortical afferents apparently find substitutes (Pinto-Lord and Caviness, 1979; Jones etal., 1982). Likewise, afferents might form connections with the "wrong" target cell population if the "right" population is too immature to be recognized. Improperly timed or placed connections, in turn, can influence subsequent cytodifferentiation (Pinto-Lord and Caviness, 1979; Jeanmonod et al., 1981; Loeb et al., 1987). Furthermore, disruption of the timing between competing projections or an imbalance of their efficacy clearly results in abnormal connectivity (Blakemore, 1977; Jeanmonod etal., 1981; Cusick and Lund, 1982; Olavarria et al., 1987). The present study investigates the afferent projection pattern to sensory-motor cortex from dorsal thalamus and efferent cortical projections to subcortical areas in adult mice following nBM lesions at birth. We show that both efferent and afferent connectivity are permanently altered.
T
. ' • - , * « •
R g a n 1 . Dark-fieW Bruges of pyramidal neurons in normal sensory-motor cortex. retrogradeJy labeled by nriatal WGA-HRP injeaions into 8 control animal, fl shows a magnified view of the boxed ares « A Note the presence of pyramidal cetts of various sizes throughout the erant of layer V {amwheadsl The arrow indicates the medial (Section in this coronal section. Wm, vyhns matter Hp, hippocampus.
IW Connealons of Mouse Cortex Following Deafferentation • Hohmann et al
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Wm
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«"*
Fignra 2. Dark-fietd mage of retrograde transport of WGA-HRP from die striatum to layer V (anatteads\ of sensori^nirtor cortex in a different normal control animal than shown in Figure 1. B snows a magnified view of the boxsd a m in A. Note the much smafler number of retrogradely labeled pyramidal cells compared to Figure 1. However, the distribution throughout tsya V and variability in size compares to the control animal shown in Figure 1. The arrow indicates medial direction. LV, lateral ventricle; Win white matter. Cerebral Cortex Mar/Apr 1991, V 1 N 2 181
Table 1
Quantification of retrogradely labeled pyramidal neurons in motor cortex ipsilateral to the nBM lesion and in normal control animals.
Control nBM lesioned
Cells/mm2
Total area sampled
126.7 ± 17.8 304.8 ± 49. T
11.6 mm2 11.1 mm2
Values for cells/mm2 are given as means ± SEM; nBM-teioned animals had significantly more \'P < 0.05) retrogradely labeled neurons in motor cortex than normal control animals. The cases used for quantification in the lesioned group [N = 4) included 8/11, 8/19, and 8/26. also shown in Figures 3-6. Normal control cases |/V = 4) included N8/05, N8/12, N8/15, and N6/22, also shown in Figures 1-3.
Results
Case Selection All animals selected for analysis in the present study had injections that were large enough to encompass a maximum of cortical afferent or efferent projections to the injected structure. However, we only selected cases with injections that were confined to the striatal and thalamic projections, respectively, and did not extend into any other structure with direct connections to cortex. All nBM-lesioned animals were also evaluated for appropriate location of the lesion within the horizontal limb of the diagonal band of Broca and the ventromedial globus pallidus region (nBM). According to these criteria, eight nBM-lesioned and five normal control animals received successful striatal injections. Six nBM-lesioned and three control animals showed anterograde as well as retrograde WGAHRP transport following successful injections into the dorsal thalamus. Four nBM-lesioned and one control animal received successful dual injections of striatum and Vb. Striatal Injections The extent of pyramidal cells retrogradely labeled by striatal injections exhibited some variation among normal control animals. Figure 1 illustrates the most extensive pattern of retrograde labeling of layer V pyramidal cells, observed in only two control animals, while Figure 2 illustrates a more typical control animal. Retrogradely labeled neurons were visible throughout the depth of layer V and included pyrami-
Thalamic Injections The pattern of thalamocortical projections, as visualized by anterograde fiber tracing, varied depending on the cortical region examined. By directing the WGA-HRP injections towards the Vb complex of the thalamus, our primary interest was the projection pattern of somatosensory afferents to dorsal neocortex (Wise and Jones, 1978; Porter and White, 1983). In particular, we focused on the barrel field area (Woolsey and Van der Loos, 1970). This area, containing the cortical representation of the mystacial vibrissae in rodents, has a very characteristic cytoarchitectonic organization as well as a distinct pattern of thalamocortical afferent distribution. As shown in Figure 7, following injections into Vb of a control animal, aggregates of anterogradely labeled terminals corre-
Figure 3 . Diagram of injection sites (TMB reaction product) and retrograde cell labeling pattern in one normal control and three neonatally nBM-lesioned animals. The dark shading, cirde 1 corresponds to the area that sustained tissue damage due to the WGA-HRP injection. Circle 2 [light shading] indicates the area containing moderate amounts of diffuse, blue TMB reaction produa Cirde 3 {broken line] indicates the presence of light TMB reaction product. We have not been able to detect any WGA-HRP transpon from circle 3 areas. The outlined area labeled nBM delineates the location of the neonatal lesioa Black dots and circles in cortex represent the approximate distribution and density of retrogradely labeled cells in cortex but do not represent individual neurons. A section from case A (8/11) is also illustrated in Rgure 4, from case B (8/26) in Figure 5, and from case C (8/19) in Figure 6.
162 Connections of Mouse Cortex Following Deafferentation • Hohmann et al.
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of a given section were plotted. Second, during the data analysis process, an approximately 1-mm2 area of motor cortex was selected, and all retrogradely labeled pyramidal neurons contained in this area were quantified. Statistical analysis was performed using a nonpaired, two-tailed Student's t test.
dal cells of various sizes. With our injection location, labeled pyramidal neurons extended from just caudal to the anterior commissure to the posterior end of the PMBSF area. They formed a discontinuous band throughout the mediolateral extent of somatosensory cortex and were particularly prominent in motor cortex (see Fig. 3). As described in our previous study, by adulthood, the cytoarchitecture of cortical layer V in nBM-lesioned animals appears to be comparable to normal animals in Nissl-stained sections. Neither the size nor morphology of layer V neurons, nor the width of the layer, is appreciably different. As in normal animals, striatal injections into nBM-lesioned mice labeled pyramidal cells of various sizes throughout layer V. The anteroposterior extent of labeling, as well as the discontinuous mediolateral distribution pattern, was comparable to normal mice, as well (Fig. 3)- However, most nBM-lesioned animals displayed an enlarged population of retrogradely labeled pyramidal neurons, ipsilateral to the nBM lesion, compared to normal animals. Quantifications of retrogradely labeled neurons in motor cortex of four control and four nBM-lesioned animals showed an approximately 50% increase of neurons in cortex ipsilateral to the lesion (Table 1). This increase was principally due to the denser band of labeled neurons within layer V. In addition, particularly in motor areas, this band of labeled cells was wider in the nBM-lesioned cases (Figs. 4-6). This difference between the nBM-lesioned and the normal animals did not appear to be the product of larger injection sizes or differences in injection location. Figure 3 compares injection sites and the distribution of retrograde labeling in the most extensively labeled normal animal with three different neonatally nBM-lesioned animals.
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Cerebral Cortex Mar/Apr 1991, V 1 N 2 163
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B [ | B r » 4 . [ ^ ^ image ofretrogradely labeled p y r a m ^ (seefig.3/1) faOwmg strata! flection. B shows a luagnified image of the taarf ana in A Note that the distribution Df neuons within layer V (ariowteds) and the great variabffity among cefi sizes dosety resemble the normal control pattern. Wrie the extern of retrograde labeTmg in this case does not much exceed that seen in some normal cases, the injection she was smaller (see Rg. 36). The mow indicates medial direction. IV, lateral ventricle.
164 Connections of Mouse Conex Following Deafferenution • Hohmann et al.
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Hgara S. Dark-field mage of retrogratWy labeled cells in layers fend W of motor cortex in neonatafly nBM-teskred animal B/26 (see Rg. 3B] toOowing sirauhaneous striata) and thalamc injectkm. B show a magnified view of the boxed sea in A, Note the extended band of rmragradety labeled cells in layer V, wh3e the ifcnSxnssi of lenogradBly labeled ceOs in layer VI remained normal | d . Fig. B). The emm Mcates mafial directm Cerebral Cortex Mar/Apr 1991, V l N 2 166
sponded to aggregates of layer IV granule cells termed "barrels." In addition, "bands" of labeled afferents were associated with "bands" of granule cells medial and lateral to the PMBSF. This particular pattern of thalamocortical afferents was apparent in all normal animals following thalamic injections. Retrograde transport of WGA-HRP from thalamic injections was seen predominantly in conical layer VI (Figs. 7, 8). There, labeled neurons formed a continuous band throughout somatosensory and motor regions. These neurons were present throughout the depth of layer VI and appeared to consist mainly of small pyramidaltype neurons. In addition, a few neurons were seen in layer V, as well, following the thalamic injections (see Fig. 7). These retrogradely labeled cells were predominantly found in the lower tier of layer V and were much smaller than the cells labeled by striatal injections. All nBM-lesioned mice showed disorganization in their thalamocortical projection pattern. The densities of anterogradely labeled afferent terminals varied greatly within somatosensory-motor cortex and between cases (see Fig. 8). However, normally shaped "barrel" projections were always absent. Usually, expanded "barrellike" puffs of terminals alternated with sparsely labeled areas (see Figs. 1C, 8). In accordance with previous observations (Hohmann et al., 1988), the cytoarchitectonic pattern of layer IV, as visualized by cresyl violet stain, was abnormal in nBMlesioned mice. Mirroring the absence of the normal
"barrel-shaped" aggregates of granule cells, the typical "barrel field" projections could not be seen in somatosensory cortex (Figs. 7, 8). Nevertheless, a close correspondence between abnormal thalamocortical projection pattern and abnormal cortical cytoarchitecture existed in all nBM-lesioned animals. Figure 7, B and C, illustrates this close correspondence between "puffs" of dense terminal label and clusters of layer IV granule cells; sparsely labeled areas usually could be aligned with areas of low granule cell density. In contrast to the obviously abnormal thalamocortical projection pattern in all nBM-lesioned animals, retrograde labeling of cortical layer VI cells appeared to be normal. Even in areas virtually devoid of anterograde transport, retrograde transport to layer VI was comparable to normal (cf. Figs. 7A, C, 8). In addition, the small pyramidal cells retrogradely labeled by thalamic injections were similar to normal mice in locations and extent. The localization of retrograde staining confirmed our observation that injections were similar in location and extent in normal and neonatally nBM-lesioned animals. Figure 9 illustrates four such injection sites in one normal control and three in nBM-lesioned animals. Discussion The present study employs injections of WGA-HRP into the dorsal thalamus and striatum to compare the organization of neocortical afferent and efferent pro-
1M Connections of Mouse Cortex Following Deafferenution • HShmann et al.
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Figure 6. Dark-field image of retrogradety labeled neurons in layer V [srmwhexts] of motor conei in neanatally lesioned animal 8/19 (see Rg. 3C] Mowing a nriata) injection. Note the wide band of densely packed retrogradely labeled neurom m tfus rase despite the very small size of the injection she, as depicted in Rgure 3C. The arrow indicates medial direction. Wm. «*ate matter Cm. angubte cortex.
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jections in normal mice with mice that had received nBM lesions at birth. Our results show that projections to and from cortex, which develop following the neonatal nBM lesion, are altered in adulthood. The lesions differentially affected projections to and from the various cortical layers. This supports the hypothesis that cholinergic deafferentation of neocortex, during certain periods of development, will lead to persistent alterations in cortical structure. The extent and distribution of retrogradely and anterogradely traced pathways in all normal adult animals were in agreement with previous reports in the rodent. The formation of an orderly array of the thalamocortical terminal "puffs" within the "barrels" of the PMBSF of somatosensory cortex has been amply demonstrated (Killackey and Leshlin, 1975; Wise and Jones, 1978; Keller et al., 1985). Similarly, reciprocal projection to numerous nuclei within dorsal thalamus from neurons throughout layer VI is well documented (Wise and Jones, 1977; Porter and White, 1983). The few neurons seen in layer V after thalamic injection belong to a limited population of corticothalamic projection neurons present in this layer (White and DeAmicis, 1977; Wise and Jones, 1977). Reports on the extent of pyramidal neurons in layer V, retrogradely labeled from the striatum, are variable and critically depend on the exact size and location of the injection site. True corticostriatal projections are described as mainly encompassing small- and medium-sized pyramidal neurons in upper layer V (Wise and Jones, 1977). Larger injections involve corticobulbar and corticospinal fibers of passage and thus label pyramidal neurons of variable sizes throughout the depth of layer V (Hedreen, 1977; Wise and Jones, 1977; Wise et al., 1979). Consistent with this, our fairly large injections in normal mice labeled pyramidal cells of various sizes throughout layer V. Following the neonatal nBM lesion, the only projection system that appeared qualitatively normal was the efferent projection from layer VI to the thalamus. Layer VI is the only cortical layer where the cells have differentiated apical and basal dendritic trees at birth prior to the nBM lesion; axons from layer VI have reached their subcortical targets and established connections at this time (Wise and Jones, 1978; Caviness, 1982). Layer VI also does not display any developmental cytoarchitectonic abnormalities following the nBM lesion (Hohmann et al., 1988). In contrast, the maturation of layers IV and V is delayed by the nBM lesion (HShmann et al., 1988), and both these layers presented with abnormalities in their connectivity. Thalamocortical afferents displayed an idiosyncratic distribution pattern in layer IV of lesioned animals. This pattern exhibited considerable variability among cases. However, as in normal animals, thalamocortical terminals were only found within granular cortex, and intensely labeled clusters of terminals were always associated with aggregates of layer IV granule cells. In contrast, corticostriatal efferents appeared to be quantitatively rather than qualitatively altered compared to normal. Lesioned animals invariably had increased popula-
R Q O T 7 . A A dark-field image of the thatarnocorutsl terminal field distribution and of ieuu|)iaJely labeled layer VI cells in senary-motor cortex of an unrewned control arimtd.fitA Nad-named section. C, A dark-Sett image of anterogradely and retrogradely bbeted elements in sensory-motor consx of the 7/15 nBM-tesioned rase Wowing a dual injection n o the dorsal thalamus and striatum, X nficates the position of landmark btood vessels in the two sections, spaced approximately 100 /irn apart. Note the abnormej termmal field distribution in layer IV [snvws\ in C and their correspondence to the aggregates of granule ceds in B. LV. lateral ventncle.
tions of retrogradely labeled neurons in layer V of sensory-motor cortex. It is unlikely that the abnormally distributed and sparse thalamocortical projections are a consequence of direct, nBM-lesion-induced damage to the thalamus or its cortical projections or the result of an insufficient WGA-HRP injection. Properly placed nBM lesions do not encroach on the dorsal thalamus, as can be seen in our previous study (Hohmann et al., 1988) and as is evident from Figure 3- Furthermore, Cerebral Cortex Mar/Apr 1991, V 1 N 2 167
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1t8 Connections of Mouse Conex Following Deafferenution • HOhmann et al.
Figure 8 . Dark-field images 18/12.8/16. 8/19B. St. striatum; Wm, white matter.
propriate cues to form synapses. In this context, it is intriguing to speculate that a delay in the time course of axonal maturation might result in an altered matching of afferent to target recognition sites and thus abnormal connectivity. That timing between layer V efferents and their target sites might indeed be more important than some programmed recognition mechanism is further suggested by recent studies of O'Leary and Stanfield (1989). These investigators have shown, in a series of transplant experiments, that early exuberant corticospinal projections depend on age and not tissue specificity. Interestingly, Miller (1987) recently observed an expanded corticospinal projection in rats prenatally treated with ethanol. These rats displayed an approximately 1-d delay in neuronal generation in layer V, an event that is probably followed by delayed maturation, thus supporting the notion that delayed cortical cell maturation might result in abnormal efferent connectivity. An alternative but more unlikely explanation for increased numbers of corticofugal projection neurons is the increased survival of cells that otherwise might succumb to developmental elimination. While some developmentally regulated cell death has been documented in cerebral cortex (Heumann and Leuba, 1983; Chun et al., 1987), studies specifically dealing with the fate of the early exuberant efferent projections from layer V have not been able to detect neuronal loss (Ivy and Killackey, 1982). Abnormal thalamocortical connections could be the consequence of alterations among the thalamic afferents as well as their cortical targets. It has previously been shown that nBM lesion, resulting in cholinergic deafferentation, can induce altered growth responses in the adult neocortex (Hohmann, 1989). It has long been known that the characteristic morphology of the "barrels" in somatosensory cortex depends on the presence of a functionally normal thalamocortical input system at the appropriate moment in cortical cytodifferentiation (Jeanmonod et al., 1981). Thus, a disruption in organization or timing of thalamocortical projections might account for the abnormalities in cortical cytoarchitecture. However, studies by Crandall and Caviness (1984) indicated recently that thalamic afferents invade the cortical plate in mice much earlier than previously assumed (Wise and Jones, 1978), and thus their presence appears to predate that of nBM afferents. Furthermore, while thalamectomy or sensory silencing prevents barrel field formation, so does a transient serotonergic depletion (Blue and Molliver, 1989) and, as shown here, neonatal nBM lesions. Thus, thalamocortical fibers may not be the sole source of regulation for barrel field formation. More likely, both the abnormal thalamocortical projections and persistant cytoarchitectonic changes following the neonatal nBM lesion are the result of a temporal mismatch between afferent syn-
the distribution of anterogradely labeled terminals and retrogradely labeled cell bodies in sensory-motor cortex of four different animals lesions at birth. Arrowheads Ael'mezxe conical layer IV. Arrows point toward medial. Hp. hippocampus; LV. lateral ventricle;
Cerebral Cortex Mar/Apr 1991, V I N 2 169
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retrograde labeling of corticothalamic projections was always normal. If the aberrant anterograde pattern had been the consequence of, for instance, internal capsule damage or incomplete thalamic injections, abnormal retrograde projection pattern should have resulted, as well (Bernardo and Woolsey, 1987). It is more difficult to refute the argument that quantitative differences, as seen in the retrogradely labeled cells of layer V, could be misleading. As with other pathway-tracing techniques, WGA-HRP fiber tracing is not a very quantitative method, and differences in uptake or transport rate might alter the size of labeled populations. However, two findings make us confident that labeling of layer Vpyramidal neurons is truly increased following the nBM lesion: (1) Injection sites were equally sized or smaller in nBM-lesioned as compared to control brains, and (2) dual injections into thalamus and striatum still displayed reduced thalamocortical but increased striatocortical transport, indicating that neither result was the product of changed transport efficacy. Thus, our findings demonstrate permanent modifications among the investigated afferent and efferent projections subsequent to the nBM-lesion-induced, transient, cholinergic deafferentation of cortex. The different effects that the lesions exert on the thalamic afferent versus subcortical efferent projection systems might well result from the different distribution and extent of lesion effects on neuronal cytodifferentiation in cortex. Recent results from rapid Golgi studies (Hohmann et al., 1991) show that layer V pyramidal cells experience retarded cytodifferentiation following the nBM lesion. This is expressed as a delay in somal growth and dendritic branching. Axonal growth may be delayed in these cells as well, resulting in a mismatch between pyramidal cell axon maturation and the maturation of their targets. In addition, the retarded differentiation process may result in delayed physiologic maturation and thus protracted electrophysiological immaturity. It has been shown that projections from cortical layer V pyramidal cells to subcortical targets are considerably more extensive in development than in adulthood (Reh and Kalil, 1981; Ivy and Killacky, 1982; Stanfield et al., 1982; O'Leary and Stanfield, 1989). Thus, the more extensive retrograde labeling of layer V pyramidal cells in the nBM-lesioned animals might indicate consolidation and maintenance of some normally transient, exuberant projections. This is not an unknown phenomenon in cortex (Cusick and Lund, 1982; Olavarria et al., 1987) but is usually linked to the retarded maturation of a competing system. On the other hand, Bates and Killackey (1984) suggested that the transiently exuberant projections, emanating from cortical cells, may never innervate their "inappropriate" targets; rather, these axons might eventually retract from these targets for lack of ap-
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nBNli
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Figure 9. Diagram of thalamic injection sites (TMB reaction product) in one normal control and three neonatally nBM-lesioned animals. The dark shading, cirde /, corresponds to the area that sustained tissue damage due to the WGA-HRP injection. Cirde 2 {light shading) indicates the area containing moderate amounts of diffuse, blue TMB reaction product. Circle 3 [solid line] indicates the presence of light TMB reaaion product (see Fig. 3). A section from the normal case is also shown in Figure 8: the nBM-lesioned case 7/15 (nSW; ] is also illustrated in Figure 7. Cases nflM?|8/26) and nflM3(8/11B) are also shown in Figure 8. Cp. cerebral peduncle: Ctx. cortex: H. habenular nucleus: Hp. hippocampus: M. midbrain; SN. substamia nigra: aT. anterior thalamus: Vb. vemrobasal nucleus of the thalamus: ZI. zona incerta.
170 Connections of Mouse Cortex Following Deafferentation • Hohmann et al.
Notes We thank Drs. Mary Blue and James Vornov for helpful discussion of the manuscript and Alice Trawinski for editorial
assistance. The present work was supported in part by U.S. Public Health Service Grant HD 19920 to J.T.C., and in part by a pilot project grant from the Alzheimer's Disease and Related Disorders Foundation to C.F.H. Correspondence should be addressed to Christine F. Hohmann, Ph.D., Department of Psychiatry, Meyer 4-163, The Johns Hopkins School of Medicine, 600 N.Wolfe Street, Baltimore, MD 21205.
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apse formation and cortical cell maturation. The state of cortical cell maturity may impact on afferent connectivity via mismatches or deficiencies in cell-cell recognition or extracellular matrix molecules. Recent results by Steindler et al. (1989) indicate that certain developmentally regulated molecules in cortex, such as tenactin, might shape afferenttarget interactions. A thalamic innervation of cortex prior to the expression of such molecules by cortical cells could easily lead to disorganization in cortex. Alternatively, if thalamocortical afferents emit a morphogenetic signal, this signal may reach layer IV granule cells at a time when they are not yet receptive to it. Interestingly, the thalamocortical terminal fields, even in cortex ipsilateral to nBM lesion, are only associated with granule-cell-containing areas. Similarly, in Reeler mice, thalamocortical afferents associate with granule cell aggregates even though these cells occur in abnormal cortical locations in this mutant (PintoLord and Caviness, 1979). Thus, for the present, it remains unresolved whether thalamocortical afferents "need" granule cell aggregates, granule cells "need" thalamic afferents, or both. In conclusion, the present study shows that nBM lesions at birth precipitate long-term alterations in cortical afferent and efferent innervation. These enduring alterations are a likely consequence of the reduction in cholinergic basal forebrain projections to cortex that we have shown (Hohmann et al., 1988). The cholinergic deafferentation, however, appears to be transient, because neurochemical markers for cholinergic innervation have recovered to normal levels as early as 4 weeks postnatal. It is presently unclear whether this resprouted innervation is functionally normal, as well. Thus, the present results demonstrate that developmental abnormalities that are neurochemically unremarkable by adulthood can lead to severe secondary and permanent changes in the brain. Such phenomena may be the substrate for pathologies seen in many developmental disorders leading to mental retardation, as well as in some neurological disorders with adult onset of symptoms. The present findings are particularly interesting in regard to Down's syndrome (DS). DS brains present with cytomorphological abnormalities reminiscent of those we have seen following neonatal nBM lesions (Purpura, 1975; Marin-Padilla, 1976; Suetsugu and Mehraein, 1980; Takashima et al., 1981; Ross et al., 1984). DS brains also show severe loss of cortical cholinergic marker in conjunction with basal forebrain cell loss and Alzheimer's symptomology in young adulthood (Coyle et al., 1986). In addition, several recent reports on basal forebrain morphology and neurochemistry have noted abnormalities in this region already in very young DS brains, prior to the occurrence of the Alzheimer's disease stigmata (Casanova et al., 1985; Kostovic et al., 1988; Kish et al., 1989).
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172 Connections of Mouse Cortex Following Deafferemation • Hohmann et al.
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of the basal forebrain cholinergic neurons result in abnormal conical development. Dev Brain Res 42:253-264. Hohmann CF, Kwiterovich K, Oster-Granite ML, Coyle JT (1989) Golgi staining method reveals that lesions to cholinergic basal forebrain nuclei in newborn mice result in abnormal differentiation of neocortical pyramidal cells. Soc Neurosci Abstr 15:111.2. Hohmann CF, Kwiterovich K, Oster-Granite ML, Coyle JT (1991) Newborn basal forebrain lesions disrupt cortical cytodifferentiation as visualized by rapid Golgi staining. Cereb Cortex 1:143-157. Ivy GO, Killackey HP (1982) Ontogenetic changes in the projections of neocortical neurons. J Neurosci 2:735-743. Jeanmonod D, Rice FL, Van der Loos H (1981) Mouse somatosensory cortex: alterations in the barrel field following receptor injury at different early postnatal ages. Neuroscience 6:1503-1535. Jones EG, Valentino KL, FleshmanJW (1982) Adjustment of connectivity in rat neocortex after prenatal destruction of cells of layers II-IV. Dev Brain Res 2:425-431. Kalsbeek A, Buijs RM, Hofman MA, Matthijssen MAH, Pool CW, Uylings HBU (1987) Effects of neonatal thermal lesioning of the mesocortical dopaminergic projection on the development of the rat prefrontal cortex. Dev Brain Res 32:123-132. Keller A, White EL, Cipolloni CP (1985) The identification of axon terminals in barrels of mouse SmI cortex using immunohistochemistry of anterogradely transported leetin (Phaseolus vulgaris). Brain Res 343:159-165. Killackey HP, Leshlin S (1975) The organization of specific thalamocortical projections to the posteromedial barrel subfields of the rat somatic sensory cortex. Brain Res 86: 469-472. Kish S, Karlinsky H, Becker L, Gilbert J, Rebetoy M, Chang LJ, DiStefano L, Hornykiewicz J (1989) Down's syndrome individuals begin life with normal levels of brain cholinergic markers. J Neurochem 52:1183-1187. Kostovic 1 (1986) Prenatal development of nucleus basalis complex and related fiber systems in man: a histochemical study. Neuroscience 17:1047-1077. Kostovic I, Bunarevic A, Hersh LB, Bruce D, Fucic A, Sain B (1988) Frontal lobe maturation in Down syndrome: immunocytochemical and structural study of the cholinergic system. Soc Neurosci Abstr 14:332.15. Kristt DA (1978) Neuronal differentiation in the somatosensory cortex of the rat: I. Relationship to synaptogenesis in the first postnatal week. Brain Res 150:467-486. Kristt DA (1979) Development of neuronal circuitry: histochemical localization of AChE in relation to the cell layers of rat somatosensory cortex. J Comp Neurol 186: 1-17. Lauder JM (1983) Hormonal and humoral influences on brain development. Psychoneuroendocrinology 8:121155. Lipton SA, Frosch MP, Phillips MD, Tauck DL, Aizenman E (1988) Nicotinic antagonists enhance process outgrowth by rat retinal ganglion cells in culture. Science 239:1293-1296. Loeb EP, Chang FF, Greenough WT (1987) Effects of neonatal 6-OH-dopamine treatment upon morphological organization of the posteromedial barrel subfield in somatosensory cortex. Brain Res 403:113-120. Marin-PadillaM (1976) Pyramidal cell abnormalities in the motor cortex of a child with Down syndrome. J Comp Neurol 167:63-82. McCobb DP, Cohan CS, Connor JA, Kater SB (1988) Interactive effects of serotonin and acetylcholine on neurite elongation. Neuron 1:377-385. McConnell SK (1988) Development and decision making in the mammalian cerebral cortex. Brain Res Rev 13:123. Mesulam MM (1978) Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neuronal efferents and afferents. J Histochem Cytochem 26:106-117.
Departments of 'Psychiatry and 2Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Over the last few decades, considerable research has been devoted to the understanding of cerebral cortical development. Consequently, the morphogenetic and, to some extent, pharmacological/neurochemical events in cortical ontogeny have been described in great detail (for review, see McConnell, 1988). In contrast, our understanding of the mechanisms that guide and regulate these ontogenetic events is still rather primitive. Corticopetal systems from the brainstem, such as the noradrenergic, dopaminergic, and serotonergic afferents, have been invoked in shaping various events in cortical morphogenesis (Felten et al., 1982; Lauder, 1983; Kalsbeek et al., 1987; Loeb et al., 1987; Blue and Molliver, 1989). Several neuropeptide transmitters have also been implicated (Chun et al., 1987; Hauser et al., 1987). However, the mechanisms by which cortical subdivisions are established, cortical cell differentiation and synapse formation are timed, and developmental plasticity is regulated are unknown. Increasing attention has been directed toward ACh as a modulator of plasticity and cell differentiation (Sillito, 1983; Bear and Singer, 1986; Hohmann et al., 1988,1989; Lipton et al., 1988; McCobb et al., 1988). Cholinergic axons from the basal forebrain innervate rodent neocortex postnatally, at the onset of differentiation and synapse formation, and mature before these events have concluded (Kristt, 1978,1979; Candy et al., 1985; Hohmann and Ebner, 1985; Hohmann et al., 1985; Kostovic, 1986; Schambra et al., 1989). Such a time course of afferent innervation could enable the cholinergic system to exert a regulatory function, perhaps to determine the timing of ontogenetic events. We have developed evidence supporting a role of the afferent cholinergic projection to cortex in the regulation of cortical morphogenesis and plasticity (Hohmann and Ebner, 1988; Hohmann et al., 1988, 1989). Lesions among the cholinergic basal forebrain neurons (nBM) at birth cause a transient cholinergic deafferentation of sensory-motor cortex and result in the following cytoarchitectonic abnormalities (Hohmann et al., 1988): The time course of cytodifferentiation in cortical layers V and IV appears to be most severely disrupted in these experiments. However, as cholinergic innervation reappears, presumably due to collateral sprouting of nBM neurons spared by the lesion, cytoarchitectonic abnormalities become attenuated. By adulthood, cortical layer V has assumed a Cerebral Cortex Mar/Apr 1991;1:158-172; 1047-3211/91/12.00
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The present study investigates the effect of cholinergic basal forebrain lesions at birth on cortical connectivity in adulthood. We have previously shown that such neonatal lesions result in extensive cortical cholinergic deafferentation during early postnatal development, which is accompanied by abnormal morphogenesis of cortical cytoarchitecture (Hohmann et al., 1988). Here, we have used WGA-HRP to label anterogradely and retrogradely afferent and efferent projections of dorsal neocortex. Our results show an altered projection pattern from dorsal thalamus to layer IV of sensory-motor cortex following lesions among the cholinergic basal forebrain neurons (nBM), while corticothalamic projections from layer VI appear normal. In addition, corticofugal projections from layer V, labeled by striatal injection, appear to be expanded following the lesion. This indicates that cortical layers undergoing differentiation after the newborn nBM lesion present with long-term abnormalities in connectivity. The present results are compatible with the hypothesis that cholinergic afferents are instrumental in the regulation of cortical morphogenesis. Furthermore, our data show that ontogenetic disturbances can lead to structural abnormalities that persist long after the initial deficiency has abated. We discuss the significance of these results in relationship to human neurological disorders.
Christine F. Hohmann,1 Lucy Wilson, and Joseph T. Coyle1-2
Materials and Methods All mice in the present study were of the BALB/cByJ strain and raised in our own timed-pregnancy breeding colony. Neonatal nBM-lesioned and unlesioned control animals were derived from the same litters. For nBM lesions, pups aged 12-24 hr (postnatal day 1) were removed from their mothers, anesthetized by hypothermia, and immobilized in a specially designed Plexiglas-mold head holder attached to a David Kopf stereotaxic apparatus. Unilateral electrolytic lesions were performed in the ventromedial globus pallidus region on the right side of the animal as described in Hohmann et al. (1988). Briefly, a fine wire electrode (SNEX 300, Rhodes Medical Supplies) was inserted through the skin and skull 1 mm lateral to the midline and 1.5 mm anterior to the frontonasal suture at a vertical angle of 48.5° and a horizontal angle of 5°. The electrode was lowered into the brain; a positive, constant current of 0.8 mA, 10-msec duration was passed at depths of 3, 3.3, 3.6, and 4 mm. Following the lesion, the pups were removed from
the head holder and transferred to a heating pad for 30 min to reequilibrate to normal body temperature. Subsequently, pups were returned to their mothers. For tracer injections, adult (2-5-month-old) mice unilaterally lesioned at birth and their normal litter mates were anesthetized with Avertin (0.2 ml/gm body weight of a solution made from a 1:10 PBS dilution of a stock containi ng 2 5 gm tetrabromethanol and 15.5 gm tetramylalcohol) and immobilized in a Kopf smallanimal stereotaxic apparatus. Injections of 0.06-0.08 Ail of a 1% w/v WGA-HRP solution (in water) were made vertically through small burr holes in the skull of the animal, ipsilateral to the nBM lesion. The volume and concentrations used for the present injections were smaller than usually reported but have led, in our hands, in mice (Hohmann and Ebner, 1988), to excellent anterograde and retrograde transport while ensuring well-confined injection sites. Stereotaxic coordinates for all injections were empirically derived, with skull sutures serving as landmarks. Striatal injections were aimed at the dorsal aspect of the anteromedial striatum described by White and DeAmicis (1977) to receive a large projection from neurons in the posteromedial barrel subfield (PMBSF) of somatosensory cortex. Thalamic injections were aimed at the ventrobasal complex (Vb) of the thalamus. Several lesioned as well as control animals received both striatal and thalamic injections. The cortical area between these two injection sites, analyzed in the present study, included the PMBSF and most of somatosensory motor cortex. WGA-HRP histochemistry was performed as previously described (Hohmann et al., 1988). Briefly, 24 hr after WGA-HRP injections, animals were anesthetized with a lethal dose of Nembutal and perfused first with physiological saline solution, followed by 1.25% v/v glutaraldehyde and 1% w/v paraformaldehyde in 0.1 M phosphate buffer (pH, 7.4). Brains were postfixed in above fixative containing 20% sucrose for another 2 hr and kept in 0.1 M phosphate buffer (pH, 7.4) overnight. This perfusion protocol allows the visualization of both anterograde and retrograde transport of WGA-HRP. Subsequently, brains were sectioned frozen, at 50 Aim, on a sliding microtome and processed according to the protocol of Mesulam (1978), using trimethylbenzidine (TMB) as the chromatophore. Adjacent sections were stained with cresyl violet to visualize cortical cytoarchitecture. Quantifications of retrogradely labeled layer V pyramidal neurons were performed using a Zeiss Axiophote microscope equipped with a computerized video-mapping system (developed by Dr. M. E. Molliver, Department of Anatomy, and modified by Kathrine Fleishman, Department of Neuropathology, Johns Hopkins University, School of Medicine). We selected 10 sections each from four normal control cases and four nBM-lesioned animals. The sections were chosen from three different levels in the coronal plain: (1) just anterior to the decussation of the anterior commissure, (2) on the level of first appearance of the fimbria fornix, and (3) at midhippocampal level. First, all cortical pyramidal neurons in dorsal cortex Cerebral Cortex Mar/Apr 1991, V I N 2 159
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relatively normal morphology as judged by Nissl stain. However, the distribution of granule cells, particularly within somatosensory cortex, does not resemble the normal adult cortex. The distinct cytoarchitectonic organization of the "barrel fields," as originally described by Woolsey and Van der Loos (1970), is obscured. Frequently, granule cells form amorphous clusters interspersed with granule-cell-sparse zones. In addition, boundaries between layer V and IV, as well as IV and supragranular cortex, are poorly defined. Thus, we have shown that the transient cortical deafferentation, resulting from the newborn basal forebrain lesion, permanently affects the organization of cortical cytoarchitecture. Golgi studies of pyramidal cell maturation, presented in a companion paper (Hohmann et al., 1991), have confirmed our hypothesis that the newborn nBM lesion causes a delay of cortical cytodifferentiation (Hohmann et al., 1988). Delayed cortical cytodifferentiation might result in persistent abnormalities of specific afferent and efferent cortical connections. In the absence of their appropriate target structures, cortical afferents apparently find substitutes (Pinto-Lord and Caviness, 1979; Jones etal., 1982). Likewise, afferents might form connections with the "wrong" target cell population if the "right" population is too immature to be recognized. Improperly timed or placed connections, in turn, can influence subsequent cytodifferentiation (Pinto-Lord and Caviness, 1979; Jeanmonod et al., 1981; Loeb et al., 1987). Furthermore, disruption of the timing between competing projections or an imbalance of their efficacy clearly results in abnormal connectivity (Blakemore, 1977; Jeanmonod etal., 1981; Cusick and Lund, 1982; Olavarria et al., 1987). The present study investigates the afferent projection pattern to sensory-motor cortex from dorsal thalamus and efferent cortical projections to subcortical areas in adult mice following nBM lesions at birth. We show that both efferent and afferent connectivity are permanently altered.
T
. ' • - , * « •
R g a n 1 . Dark-fieW Bruges of pyramidal neurons in normal sensory-motor cortex. retrogradeJy labeled by nriatal WGA-HRP injeaions into 8 control animal, fl shows a magnified view of the boxed ares « A Note the presence of pyramidal cetts of various sizes throughout the erant of layer V {amwheadsl The arrow indicates the medial (Section in this coronal section. Wm, vyhns matter Hp, hippocampus.
IW Connealons of Mouse Cortex Following Deafferentation • Hohmann et al
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Wm
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«"*
Fignra 2. Dark-fietd mage of retrograde transport of WGA-HRP from die striatum to layer V (anatteads\ of sensori^nirtor cortex in a different normal control animal than shown in Figure 1. B snows a magnified view of the boxsd a m in A. Note the much smafler number of retrogradely labeled pyramidal cells compared to Figure 1. However, the distribution throughout tsya V and variability in size compares to the control animal shown in Figure 1. The arrow indicates medial direction. LV, lateral ventricle; Win white matter. Cerebral Cortex Mar/Apr 1991, V 1 N 2 181
Table 1
Quantification of retrogradely labeled pyramidal neurons in motor cortex ipsilateral to the nBM lesion and in normal control animals.
Control nBM lesioned
Cells/mm2
Total area sampled
126.7 ± 17.8 304.8 ± 49. T
11.6 mm2 11.1 mm2
Values for cells/mm2 are given as means ± SEM; nBM-teioned animals had significantly more \'P < 0.05) retrogradely labeled neurons in motor cortex than normal control animals. The cases used for quantification in the lesioned group [N = 4) included 8/11, 8/19, and 8/26. also shown in Figures 3-6. Normal control cases |/V = 4) included N8/05, N8/12, N8/15, and N6/22, also shown in Figures 1-3.
Results
Case Selection All animals selected for analysis in the present study had injections that were large enough to encompass a maximum of cortical afferent or efferent projections to the injected structure. However, we only selected cases with injections that were confined to the striatal and thalamic projections, respectively, and did not extend into any other structure with direct connections to cortex. All nBM-lesioned animals were also evaluated for appropriate location of the lesion within the horizontal limb of the diagonal band of Broca and the ventromedial globus pallidus region (nBM). According to these criteria, eight nBM-lesioned and five normal control animals received successful striatal injections. Six nBM-lesioned and three control animals showed anterograde as well as retrograde WGAHRP transport following successful injections into the dorsal thalamus. Four nBM-lesioned and one control animal received successful dual injections of striatum and Vb. Striatal Injections The extent of pyramidal cells retrogradely labeled by striatal injections exhibited some variation among normal control animals. Figure 1 illustrates the most extensive pattern of retrograde labeling of layer V pyramidal cells, observed in only two control animals, while Figure 2 illustrates a more typical control animal. Retrogradely labeled neurons were visible throughout the depth of layer V and included pyrami-
Thalamic Injections The pattern of thalamocortical projections, as visualized by anterograde fiber tracing, varied depending on the cortical region examined. By directing the WGA-HRP injections towards the Vb complex of the thalamus, our primary interest was the projection pattern of somatosensory afferents to dorsal neocortex (Wise and Jones, 1978; Porter and White, 1983). In particular, we focused on the barrel field area (Woolsey and Van der Loos, 1970). This area, containing the cortical representation of the mystacial vibrissae in rodents, has a very characteristic cytoarchitectonic organization as well as a distinct pattern of thalamocortical afferent distribution. As shown in Figure 7, following injections into Vb of a control animal, aggregates of anterogradely labeled terminals corre-
Figure 3 . Diagram of injection sites (TMB reaction product) and retrograde cell labeling pattern in one normal control and three neonatally nBM-lesioned animals. The dark shading, cirde 1 corresponds to the area that sustained tissue damage due to the WGA-HRP injection. Circle 2 [light shading] indicates the area containing moderate amounts of diffuse, blue TMB reaction produa Cirde 3 {broken line] indicates the presence of light TMB reaction product. We have not been able to detect any WGA-HRP transpon from circle 3 areas. The outlined area labeled nBM delineates the location of the neonatal lesioa Black dots and circles in cortex represent the approximate distribution and density of retrogradely labeled cells in cortex but do not represent individual neurons. A section from case A (8/11) is also illustrated in Rgure 4, from case B (8/26) in Figure 5, and from case C (8/19) in Figure 6.
162 Connections of Mouse Cortex Following Deafferentation • Hohmann et al.
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of a given section were plotted. Second, during the data analysis process, an approximately 1-mm2 area of motor cortex was selected, and all retrogradely labeled pyramidal neurons contained in this area were quantified. Statistical analysis was performed using a nonpaired, two-tailed Student's t test.
dal cells of various sizes. With our injection location, labeled pyramidal neurons extended from just caudal to the anterior commissure to the posterior end of the PMBSF area. They formed a discontinuous band throughout the mediolateral extent of somatosensory cortex and were particularly prominent in motor cortex (see Fig. 3). As described in our previous study, by adulthood, the cytoarchitecture of cortical layer V in nBM-lesioned animals appears to be comparable to normal animals in Nissl-stained sections. Neither the size nor morphology of layer V neurons, nor the width of the layer, is appreciably different. As in normal animals, striatal injections into nBM-lesioned mice labeled pyramidal cells of various sizes throughout layer V. The anteroposterior extent of labeling, as well as the discontinuous mediolateral distribution pattern, was comparable to normal mice, as well (Fig. 3)- However, most nBM-lesioned animals displayed an enlarged population of retrogradely labeled pyramidal neurons, ipsilateral to the nBM lesion, compared to normal animals. Quantifications of retrogradely labeled neurons in motor cortex of four control and four nBM-lesioned animals showed an approximately 50% increase of neurons in cortex ipsilateral to the lesion (Table 1). This increase was principally due to the denser band of labeled neurons within layer V. In addition, particularly in motor areas, this band of labeled cells was wider in the nBM-lesioned cases (Figs. 4-6). This difference between the nBM-lesioned and the normal animals did not appear to be the product of larger injection sizes or differences in injection location. Figure 3 compares injection sites and the distribution of retrograde labeling in the most extensively labeled normal animal with three different neonatally nBM-lesioned animals.
NORMAL
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Cerebral Cortex Mar/Apr 1991, V 1 N 2 163
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B [ | B r » 4 . [ ^ ^ image ofretrogradely labeled p y r a m ^ (seefig.3/1) faOwmg strata! flection. B shows a luagnified image of the taarf ana in A Note that the distribution Df neuons within layer V (ariowteds) and the great variabffity among cefi sizes dosety resemble the normal control pattern. Wrie the extern of retrograde labeTmg in this case does not much exceed that seen in some normal cases, the injection she was smaller (see Rg. 36). The mow indicates medial direction. IV, lateral ventricle.
164 Connections of Mouse Conex Following Deafferenution • Hohmann et al.
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Hgara S. Dark-field mage of retrogratWy labeled cells in layers fend W of motor cortex in neonatafly nBM-teskred animal B/26 (see Rg. 3B] toOowing sirauhaneous striata) and thalamc injectkm. B show a magnified view of the boxed sea in A, Note the extended band of rmragradety labeled cells in layer V, wh3e the ifcnSxnssi of lenogradBly labeled ceOs in layer VI remained normal | d . Fig. B). The emm Mcates mafial directm Cerebral Cortex Mar/Apr 1991, V l N 2 166
sponded to aggregates of layer IV granule cells termed "barrels." In addition, "bands" of labeled afferents were associated with "bands" of granule cells medial and lateral to the PMBSF. This particular pattern of thalamocortical afferents was apparent in all normal animals following thalamic injections. Retrograde transport of WGA-HRP from thalamic injections was seen predominantly in conical layer VI (Figs. 7, 8). There, labeled neurons formed a continuous band throughout somatosensory and motor regions. These neurons were present throughout the depth of layer VI and appeared to consist mainly of small pyramidaltype neurons. In addition, a few neurons were seen in layer V, as well, following the thalamic injections (see Fig. 7). These retrogradely labeled cells were predominantly found in the lower tier of layer V and were much smaller than the cells labeled by striatal injections. All nBM-lesioned mice showed disorganization in their thalamocortical projection pattern. The densities of anterogradely labeled afferent terminals varied greatly within somatosensory-motor cortex and between cases (see Fig. 8). However, normally shaped "barrel" projections were always absent. Usually, expanded "barrellike" puffs of terminals alternated with sparsely labeled areas (see Figs. 1C, 8). In accordance with previous observations (Hohmann et al., 1988), the cytoarchitectonic pattern of layer IV, as visualized by cresyl violet stain, was abnormal in nBMlesioned mice. Mirroring the absence of the normal
"barrel-shaped" aggregates of granule cells, the typical "barrel field" projections could not be seen in somatosensory cortex (Figs. 7, 8). Nevertheless, a close correspondence between abnormal thalamocortical projection pattern and abnormal cortical cytoarchitecture existed in all nBM-lesioned animals. Figure 7, B and C, illustrates this close correspondence between "puffs" of dense terminal label and clusters of layer IV granule cells; sparsely labeled areas usually could be aligned with areas of low granule cell density. In contrast to the obviously abnormal thalamocortical projection pattern in all nBM-lesioned animals, retrograde labeling of cortical layer VI cells appeared to be normal. Even in areas virtually devoid of anterograde transport, retrograde transport to layer VI was comparable to normal (cf. Figs. 7A, C, 8). In addition, the small pyramidal cells retrogradely labeled by thalamic injections were similar to normal mice in locations and extent. The localization of retrograde staining confirmed our observation that injections were similar in location and extent in normal and neonatally nBM-lesioned animals. Figure 9 illustrates four such injection sites in one normal control and three in nBM-lesioned animals. Discussion The present study employs injections of WGA-HRP into the dorsal thalamus and striatum to compare the organization of neocortical afferent and efferent pro-
1M Connections of Mouse Cortex Following Deafferenution • HShmann et al.
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Figure 6. Dark-field image of retrogradety labeled neurons in layer V [srmwhexts] of motor conei in neanatally lesioned animal 8/19 (see Rg. 3C] Mowing a nriata) injection. Note the wide band of densely packed retrogradely labeled neurom m tfus rase despite the very small size of the injection she, as depicted in Rgure 3C. The arrow indicates medial direction. Wm. «*ate matter Cm. angubte cortex.
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jections in normal mice with mice that had received nBM lesions at birth. Our results show that projections to and from cortex, which develop following the neonatal nBM lesion, are altered in adulthood. The lesions differentially affected projections to and from the various cortical layers. This supports the hypothesis that cholinergic deafferentation of neocortex, during certain periods of development, will lead to persistent alterations in cortical structure. The extent and distribution of retrogradely and anterogradely traced pathways in all normal adult animals were in agreement with previous reports in the rodent. The formation of an orderly array of the thalamocortical terminal "puffs" within the "barrels" of the PMBSF of somatosensory cortex has been amply demonstrated (Killackey and Leshlin, 1975; Wise and Jones, 1978; Keller et al., 1985). Similarly, reciprocal projection to numerous nuclei within dorsal thalamus from neurons throughout layer VI is well documented (Wise and Jones, 1977; Porter and White, 1983). The few neurons seen in layer V after thalamic injection belong to a limited population of corticothalamic projection neurons present in this layer (White and DeAmicis, 1977; Wise and Jones, 1977). Reports on the extent of pyramidal neurons in layer V, retrogradely labeled from the striatum, are variable and critically depend on the exact size and location of the injection site. True corticostriatal projections are described as mainly encompassing small- and medium-sized pyramidal neurons in upper layer V (Wise and Jones, 1977). Larger injections involve corticobulbar and corticospinal fibers of passage and thus label pyramidal neurons of variable sizes throughout the depth of layer V (Hedreen, 1977; Wise and Jones, 1977; Wise et al., 1979). Consistent with this, our fairly large injections in normal mice labeled pyramidal cells of various sizes throughout layer V. Following the neonatal nBM lesion, the only projection system that appeared qualitatively normal was the efferent projection from layer VI to the thalamus. Layer VI is the only cortical layer where the cells have differentiated apical and basal dendritic trees at birth prior to the nBM lesion; axons from layer VI have reached their subcortical targets and established connections at this time (Wise and Jones, 1978; Caviness, 1982). Layer VI also does not display any developmental cytoarchitectonic abnormalities following the nBM lesion (Hohmann et al., 1988). In contrast, the maturation of layers IV and V is delayed by the nBM lesion (HShmann et al., 1988), and both these layers presented with abnormalities in their connectivity. Thalamocortical afferents displayed an idiosyncratic distribution pattern in layer IV of lesioned animals. This pattern exhibited considerable variability among cases. However, as in normal animals, thalamocortical terminals were only found within granular cortex, and intensely labeled clusters of terminals were always associated with aggregates of layer IV granule cells. In contrast, corticostriatal efferents appeared to be quantitatively rather than qualitatively altered compared to normal. Lesioned animals invariably had increased popula-
R Q O T 7 . A A dark-field image of the thatarnocorutsl terminal field distribution and of ieuu|)iaJely labeled layer VI cells in senary-motor cortex of an unrewned control arimtd.fitA Nad-named section. C, A dark-Sett image of anterogradely and retrogradely bbeted elements in sensory-motor consx of the 7/15 nBM-tesioned rase Wowing a dual injection n o the dorsal thalamus and striatum, X nficates the position of landmark btood vessels in the two sections, spaced approximately 100 /irn apart. Note the abnormej termmal field distribution in layer IV [snvws\ in C and their correspondence to the aggregates of granule ceds in B. LV. lateral ventncle.
tions of retrogradely labeled neurons in layer V of sensory-motor cortex. It is unlikely that the abnormally distributed and sparse thalamocortical projections are a consequence of direct, nBM-lesion-induced damage to the thalamus or its cortical projections or the result of an insufficient WGA-HRP injection. Properly placed nBM lesions do not encroach on the dorsal thalamus, as can be seen in our previous study (Hohmann et al., 1988) and as is evident from Figure 3- Furthermore, Cerebral Cortex Mar/Apr 1991, V 1 N 2 167
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1t8 Connections of Mouse Conex Following Deafferenution • HOhmann et al.
Figure 8 . Dark-field images 18/12.8/16. 8/19B. St. striatum; Wm, white matter.
propriate cues to form synapses. In this context, it is intriguing to speculate that a delay in the time course of axonal maturation might result in an altered matching of afferent to target recognition sites and thus abnormal connectivity. That timing between layer V efferents and their target sites might indeed be more important than some programmed recognition mechanism is further suggested by recent studies of O'Leary and Stanfield (1989). These investigators have shown, in a series of transplant experiments, that early exuberant corticospinal projections depend on age and not tissue specificity. Interestingly, Miller (1987) recently observed an expanded corticospinal projection in rats prenatally treated with ethanol. These rats displayed an approximately 1-d delay in neuronal generation in layer V, an event that is probably followed by delayed maturation, thus supporting the notion that delayed cortical cell maturation might result in abnormal efferent connectivity. An alternative but more unlikely explanation for increased numbers of corticofugal projection neurons is the increased survival of cells that otherwise might succumb to developmental elimination. While some developmentally regulated cell death has been documented in cerebral cortex (Heumann and Leuba, 1983; Chun et al., 1987), studies specifically dealing with the fate of the early exuberant efferent projections from layer V have not been able to detect neuronal loss (Ivy and Killackey, 1982). Abnormal thalamocortical connections could be the consequence of alterations among the thalamic afferents as well as their cortical targets. It has previously been shown that nBM lesion, resulting in cholinergic deafferentation, can induce altered growth responses in the adult neocortex (Hohmann, 1989). It has long been known that the characteristic morphology of the "barrels" in somatosensory cortex depends on the presence of a functionally normal thalamocortical input system at the appropriate moment in cortical cytodifferentiation (Jeanmonod et al., 1981). Thus, a disruption in organization or timing of thalamocortical projections might account for the abnormalities in cortical cytoarchitecture. However, studies by Crandall and Caviness (1984) indicated recently that thalamic afferents invade the cortical plate in mice much earlier than previously assumed (Wise and Jones, 1978), and thus their presence appears to predate that of nBM afferents. Furthermore, while thalamectomy or sensory silencing prevents barrel field formation, so does a transient serotonergic depletion (Blue and Molliver, 1989) and, as shown here, neonatal nBM lesions. Thus, thalamocortical fibers may not be the sole source of regulation for barrel field formation. More likely, both the abnormal thalamocortical projections and persistant cytoarchitectonic changes following the neonatal nBM lesion are the result of a temporal mismatch between afferent syn-
the distribution of anterogradely labeled terminals and retrogradely labeled cell bodies in sensory-motor cortex of four different animals lesions at birth. Arrowheads Ael'mezxe conical layer IV. Arrows point toward medial. Hp. hippocampus; LV. lateral ventricle;
Cerebral Cortex Mar/Apr 1991, V I N 2 169
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retrograde labeling of corticothalamic projections was always normal. If the aberrant anterograde pattern had been the consequence of, for instance, internal capsule damage or incomplete thalamic injections, abnormal retrograde projection pattern should have resulted, as well (Bernardo and Woolsey, 1987). It is more difficult to refute the argument that quantitative differences, as seen in the retrogradely labeled cells of layer V, could be misleading. As with other pathway-tracing techniques, WGA-HRP fiber tracing is not a very quantitative method, and differences in uptake or transport rate might alter the size of labeled populations. However, two findings make us confident that labeling of layer Vpyramidal neurons is truly increased following the nBM lesion: (1) Injection sites were equally sized or smaller in nBM-lesioned as compared to control brains, and (2) dual injections into thalamus and striatum still displayed reduced thalamocortical but increased striatocortical transport, indicating that neither result was the product of changed transport efficacy. Thus, our findings demonstrate permanent modifications among the investigated afferent and efferent projections subsequent to the nBM-lesion-induced, transient, cholinergic deafferentation of cortex. The different effects that the lesions exert on the thalamic afferent versus subcortical efferent projection systems might well result from the different distribution and extent of lesion effects on neuronal cytodifferentiation in cortex. Recent results from rapid Golgi studies (Hohmann et al., 1991) show that layer V pyramidal cells experience retarded cytodifferentiation following the nBM lesion. This is expressed as a delay in somal growth and dendritic branching. Axonal growth may be delayed in these cells as well, resulting in a mismatch between pyramidal cell axon maturation and the maturation of their targets. In addition, the retarded differentiation process may result in delayed physiologic maturation and thus protracted electrophysiological immaturity. It has been shown that projections from cortical layer V pyramidal cells to subcortical targets are considerably more extensive in development than in adulthood (Reh and Kalil, 1981; Ivy and Killacky, 1982; Stanfield et al., 1982; O'Leary and Stanfield, 1989). Thus, the more extensive retrograde labeling of layer V pyramidal cells in the nBM-lesioned animals might indicate consolidation and maintenance of some normally transient, exuberant projections. This is not an unknown phenomenon in cortex (Cusick and Lund, 1982; Olavarria et al., 1987) but is usually linked to the retarded maturation of a competing system. On the other hand, Bates and Killackey (1984) suggested that the transiently exuberant projections, emanating from cortical cells, may never innervate their "inappropriate" targets; rather, these axons might eventually retract from these targets for lack of ap-
NORMAL
nBNli
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Figure 9. Diagram of thalamic injection sites (TMB reaction product) in one normal control and three neonatally nBM-lesioned animals. The dark shading, cirde /, corresponds to the area that sustained tissue damage due to the WGA-HRP injection. Cirde 2 {light shading) indicates the area containing moderate amounts of diffuse, blue TMB reaction product. Circle 3 [solid line] indicates the presence of light TMB reaaion product (see Fig. 3). A section from the normal case is also shown in Figure 8: the nBM-lesioned case 7/15 (nSW; ] is also illustrated in Figure 7. Cases nflM?|8/26) and nflM3(8/11B) are also shown in Figure 8. Cp. cerebral peduncle: Ctx. cortex: H. habenular nucleus: Hp. hippocampus: M. midbrain; SN. substamia nigra: aT. anterior thalamus: Vb. vemrobasal nucleus of the thalamus: ZI. zona incerta.
170 Connections of Mouse Cortex Following Deafferentation • Hohmann et al.
Notes We thank Drs. Mary Blue and James Vornov for helpful discussion of the manuscript and Alice Trawinski for editorial
assistance. The present work was supported in part by U.S. Public Health Service Grant HD 19920 to J.T.C., and in part by a pilot project grant from the Alzheimer's Disease and Related Disorders Foundation to C.F.H. Correspondence should be addressed to Christine F. Hohmann, Ph.D., Department of Psychiatry, Meyer 4-163, The Johns Hopkins School of Medicine, 600 N.Wolfe Street, Baltimore, MD 21205.
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