Development of Cat Somatosensory Cortex: Structural and Metabolic Considerations
Departments of Anatomy and Cell Biology, and Neuroscience, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814
Although an extensive amount of data has accumulated on the development of the cat visual cortex (LeVay et al., 1978; Luskin and Shatz, 1985; Payne et al., 1988; Shatz et al., 1988), relatively little is known about the development of the somatosensory cortex. The limited knowledge that we have acquired over the past 20 years suggests that certain aspects of the kitten somatosensory system are relatively mature and respond to sensory stimulation at birth. In addition, neonatal somatosensory neurons demonstrate a definite topographic arrangement and possess receptive fields that share aspects of adult response properties (Rubel, 1971; Connor et al., 1984; Ferrington et al., 1984). Within a few days after birth, thalamocortical fibers have invaded the cortical plate and present with a relatively adult-like distribution (Wise et al., 1977) Despite these similarities with adult properties, kitten somatosensory cortical neurons are clearly immature in many functional aspects (Rubel, 1971), as are the responses of subcortical structures (Connor et al., 1984; Ferrington et al., 1984). According to Rubel (1971), single-unit responses in newborn cat somatosensory cortex are reduced in their latency and complexity, and are often unreliable and difficult to drive. Since these early reports describing neuronal responses in neonatal somatosensory cortex, there has been little information available on the development of this cortical region. In particular, detailed studies on the development of the anatomical organization of cat somatosensory cortex and its relationship to emergent functional activity are scarce. In comparison, the evolution of cortical responses and thalamocortical relationships have been more clearly documented in visual cortex. For example, developing neuronal response properties and the distribution of afferent innervation patterns in visual cortex can be precisely related to conical structure and connectional relationships present at a given time during development. As the cell structure of the visual cortex matures, the distribution of thalamocortical aJferents and receptors evolves to match the evolution of cortical layers and the emerging response properties (Rakic, 1976, 1977; Shatz and Luskin, 1986; Shatz et al., 1988; Kostovic and Rakic, 1990). In addition, the subplate has been identified as an important factor in the developing visual cortex. It appears to play a generally nurturing role as a recipient site for fiber systems that project into the neocortex, yet relatively few reports exist that describe Cerebral Cortex May/June 1992;2:231-243; 1047-3211/92/14.00
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Although research is beginning to clarify the relationship between structure and functional activity in the adult cerebral cortex, little is known about cortical development in the somatosensory cortex of cats. A number of parameters were used in this study to identify functional and anatomical correlates in the developing somatosensory cortex of kittens ranging in age from 3 to 33 d: 2-deoxyglucose (2DG) uptake, cytochrome ondase (CO) activity, Nissl staining, and AChE activity. All of these parameters were found to reflect an immaturity that evolved to the adult-like pattern by 4-5 weeks of age. Nissl staining revealed an immature laminar pattern at birth, in which layers I, V, and VI were distinct whereas layers II—IV were homogeneous in appearance. Numerous cell* could be observed at the layer Vl-white matter junction and throughout the white matter, a feature not found in adults. The laminar distribution of NissJ-stained cells gradually became mature by 4-5 weeks of age. CO staining was homogeneous throughout all layers, in contrast to the adult pattern, which displays laminar differentiation. In young animals, many darkly stained C0+ cells were found at the layer Vl-white matter border and in the white matter, a distribution not found in the adult. AChE staining in kittens was also distinctly different from that in adults. At birth, AChE+ fibers could be found in layers I, V, and VI but were scarce in layers II—IV. In the adult, a dense network of AChE+ fibers can be found in all layers. 2DG uptake was also immature, as little stimulus-evoked activity could be observed in animals younger than 2 weeks. A dense band of metabolic activity was found in the zone between layer VI and the white matter, whether or not a somatic stimulus was delivered. These results suggest a close correlation between the developing cytoarchiteeture and the emergence of a mature pattern of functional activity in the somatosensory cortex.
Rebecca A. Code and Sharon L. Juliano
Materials and Methods
Seventeen kittens of either sex, 3-33 d old (day of birth = postnatal day 0), were used in this study. The procedures used in the 2DG experiments were similar to those originally described by Sokoloff et al. (1977) and published previously by this laboratory (Juliano et al., 1990a). Under halothane anesthesia, kittens 9 d of age or older were intubated with a tracheal cannula and a venous catheter was inserted in the long saphenous vein. After administration of gallamine triethiodide, the animal was maintained on a respirator and its temperature, heart rate and expired CO2 were continuously monitored and kept within normal limits. Pupillary size was also monitored and found to be appropriate for the anesthesia level. The anesthetic was changed from halothane to nitrous oxide (70%) and oxygen (30%), since halothane has been shown to reduce cortical 2DG uptake (Shapiro et al., 1978). The EEG of animals under similar conditions has been previously examined and found to drift in and out of sleep states, indicating that the animal is not stressed (Juliano et al., 1990a). The same is true of other mammals monitored under identical circumstances (Redies et al., 1990). One and a half hours after the termination of halothane anesthesia, 2-deoxyD-[l-HC]-glucose (100 mCi/kg) was injected. During the 45 min after the injection, some of the animals received a somatic stimulation; this aspect of 2DG uptake was not evaluated for this study but will be addressed in a subsequent report. For kittens younger than 9 d old, neither the tracheal cannula nor venous catheter was inserted; instead, 2DG was injected intraperitoneally. No somatic stimulus was applied, but the animal was allowed to rest in a softly padded box. Forty-five minutes after injection of 2DG, all animals received an overdose of sodium pentobarbital (35 mg/kg) and were quickly perfused intracardially with a 0.9% saline rinse followed by 4% paraformaldehyde, 4% sucrose in 0.1 M phosphate buffer. The brain was immediately removed from the skull, blocked in the frontal plane, frozen in Freon 22, and stored at — 70°C. Later, 30-Mm-thick coronal sections were cut on a cry-
232 Development of Cat Somatosensory Cortex • Code and Juliano
ostat. Every other section was saved for 2DG autoradiography and exposed to x-rayfilm(Kodak SB5) with "C standards for 7-10 d. After film development, these same sections were stained for Nissl substance. The other series of adjacent sections was processed for cytochrome oxidase (CO) histochemistry to evaluate baseline metabolic activity (Wong-Riley, 1979). In some experiments, a third series of adjacent sections was stained for acetylcholinesterase (AChE) activity using a modification (Jacobowitz and Creed, 1983) of the procedure originally described by Koelle (1955). Results Cytoarcbitecture In the adult cat somatosensory cortex, Nissl-stained neurons are organized into clearly delineated layers (Fig. 1). The adult somatosensory cortex is characterized by a highly granular layer IV with small, closely packed cells. The supragranular layers are more loosely packed with medium-sized cells. A number of large pyramidal cells reside in layer III. In the infragranular layers, layer V is distinguished by the presence of large pyramidal cells in its upper portion and a paler-staining lower portion containing smallto medium-sized pyramidal cells. Neurons in layer VI are more densely packed than those in layer V but are not as closely packed as those in layer IV. There are relatively few cells in the white matter. In contrast, only layers V and VI are clearly distinguishable in the 3-d-old somatosensory cortex (Fig. 2). Large pyramidal cells are evident in layer V, but this layer cannot yet be separated into upper and lower portions. Although slight laminar differentiation can be observed between the relatively acellular layer I and layer V, neurons for the most part are very densely packed into a homogeneous region, which is easily demarcated from layer V. A densely packed band of cells can be observed just deep to layer I. Numerous cells, including inverted pyramidal cells, are evident in the white matter just below layer VI (see Fig. 5). Although it is not easy to delineate the subplate on cytoarchitectural criteria alone (Kostovic and Rakic, 1990), we believe, based on the morphology and dimension of the cell population, that this region corresponds to what has been described as the "subplate" by other investigators (cf. Rakic, 1977, 1988; Luskin and Shatz, 1985; Shatz et al., 1988; Kostovic and Rakic, 1990). Additional prominent cells can be seen scattered throughout the white matter, a feature not observed in the adult. In the 9-d-old cat, the densely packed band of cells continues to be visible below layer I. Layers II-IV are not separable into distinct entities, although the cells, still densely packed, appear less so than in the 3-dold cat (Fig. 3). Larger pyramidal cells are more numerous in layer V compared with the younger age. Layer V also appears to be separating into upper and lower portions. There are still many cells at the layer Vl-white matter border. Although the 15-17-d-old cat somatosensory cortex is still not fully mature, the beginning segregation
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the subplate in developing somatosensory cortex (cf. Kostovic and Rakic, 1990) and almost no studies describe the subplate in the somatosensory cortex of the cat. As a result, in the present study we wished to examine the structure of developing cat somatosensory cortex and observe its relationship to emergent functional activity patterns. To assess these functional relations, patterns of metabolic activity were analyzed in developing cats using the 2-deoxyglucose (2DG) method. During the time that functional activity patterns are being established in the somatosensory cortex, we observed changes in cytoarchitectural organization, in patterns of cytochrome oxidase (CO) staining, and in the density and distribution of acetylcholinesterase (AChE + ) fibers, each of which could be correlated with changes in metabolic activity patterns. Preliminary accounts of these results have been published (Juliano et al., 1990b, 1991a).
R jore 1 . On the M is a Nissl-stained section taken from aduh cat somatosensory cortex: on the right is a comparable section named to visualize AChE acthmy. Roman numerals mdrcate cortical layers. In the Ntssl n a n . conical layers I—VI can be eajily differentiated. In the AChE-stained section, numerous densely packed fibers can be observed in all cortical layers. The fiber density is somewhat reduced m the white maner. Scale bar. 250 >im.
of layers II, III, and IV can be differentiated. Cortical neurons in the upper layers are less densely packed than in the younger ages, and a condensation of smaller, more tightly packed cells can be observed at the layer IV-V border (Fig. 4). Layer V can be separated into upper and lower portions that closely resemble the mature laminar pattern. Numerous cells are present in the white matter subjacent to layer VI. By 28 d, the laminar pattern resembles that of the adult. Few cells can be found in the region previously devoted to the subplate. AcetylcboUnesterose-posittve Inrtervotion The distribution of AChE+ fibers in adult somatosensory cortex is relatively dense in all cortical layers, although it displays a slight increase in density in layers I and V (Figs. 1,6). Both radially and tangentially oriented fibers are present in every layer, although layer I appears to have more tangentially run-
ning fibers. At all ages studied, layer I is predominated by tangential AChE + fibers. An immature pattern of AChE activity is seen in young animals. In the 3-d-old cat, the density of AChE+ fibers is greatest in layers I, V, and VI; the paucity of fibers in layers II—IV, however, is striking (Figs. 2, 6). Most of the fibers in these layers are long and radially oriented with few branches. There is a heavy concentration of AChE + fibers at the layer IVV border and a slightly less dense concentration in the subplate. The distribution of AChE + fibers in the 9-d-old somatosensory cortex is similar to that in the 3-d-old: densest in layers I, V, and VI; scarcest in layers II-IV (Figs. 3, 6). Layers I, V, and VI are predominated by tangentially oriented fibers, while those in layers IIIV are primarily radially oriented. In addition, there are numerous AChEH- cells in the subplate below the somatosensory cortex. Cerebral Cortex May/June 1992, V 2 N 3 " 3
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R g u r a 2 . On the te/if is a Ntsst-statnsd section taken from the somaiosensory cortei of a 3-d-old cat: on the right s a comparable section stained to visualize AChE activity. In the Ntssl-stainsd section, conical layers I, V. and VI can be distinguished, whrie layers II-IV are relatively homogeneous and cannot be individually distinguished. These layers are thus indicated as CP Icortcal plate). A band of more densely packed cells can be observed just subiacem to layer I. In the AChE-stained section, a relatively dense distribution of W H S can be observed m layers I. V. and VI. The region designated CP a sparsely populated with ACh£+ fibers. Scale bar. 250 /im.
At 15 d of age, the density of AChE+ fiber distribution continues to increase (Fig. 4). The densest staining is still in layers I, V, and VI. The overall density is greater than that at 9 d, and the fibers exhibit more branching. Layers V and VI now appear to have equal numbers of both tangentially and radially oriented fibers. The density of AChE + fibers in layers II-IV is also increased compared to younger ages. Fibers for the most part are radially oriented, but many oblique and horizontal branches are evident Note also the gradual increase in the thickness of the cortex innervated by AChE+ fibers during development The distribution of AChE + fibers at 22 d of age is similar to that at 15 d (Fig. 6). The density of fibers in layers II-IV, however, is greater than that at 15 d, but not as heavy as that in the adult. Both tangential and radial AChE + fibers appear to be equally represented in these layers. Metabolic Activity In kittens less than 3 weeks of age, little stimulusevoked metabolic activity, as revealed by 2DG uptake, is seen in the somatosensory cortex. (The evolution of stimulus-evoked activity will be assessed in a subsequent report.) Dense metabolic label is present, 234 Development of Cat Somatosensory Cortex • Code and Juliano
however, as a band of increased activity in layer VI and at the border of layer VI and the white matter (Fig. 1A,B). Comparison of this region with Nisslstained material reveals that it corresponds to lower portions of layer VI and to the cellular zone just subjacent to layer VI (Fig. 8A,B). In kittens aged 3 d to 3 weeks, this region is heavily labeled with 2DG whether or not a somatic stimulus was delivered. After 3 weeks of age, 2DG label is not easily visible at the layer Vl-white matter border; stimulus-evoked metabolic activity, however, becomes increasingly apparent in the middle layers until about 5 weeks of age, when it resembles more closely the adult laminar pattern (Fig. 1C). Cytocbrome Oxidase Activity In the adult, CO staining is heavy in layers II-IV and weaker in layer I (Fig. 9C). The CO distribution becomes quite pale in layer V and then increases slightly in layer VI. Numerous darkly stained neurons can be seen throughout the cortex; they are especially prominent in layer V (Fig. \0A,C). In contrast, young animals display a relatively homogeneous CO distribution in all layers, except for a slight staining decrease in layer I (Fig. 9A). At 9 d of age, a layer of darkly
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R g u r t 3. On the fe/ir is a Nissl-stained section taken from the somatosensory cortei of a M o l d cat on the right a a comparable sacum stained to visualize AChE acovtty. In the Nissl stan. layers I. V, and VI can be distinguished, but the region designated CP (cortcal plate) contains cells that are relatively homogeneous in their distribution. In the ACh£-aamed section, numerous AChE+ ibers can be seen in layers I, V. and VI, but the conical plate region contains relatively few AChE+ fibers. Scale bar. 250 fim.
stained neurons can be seen in layer V, similar to those found in the adult (Fig. 9B). At 15 d of age, the adult laminar pattern begins to emerge and is mature by about 4 weeks of age. One feature characteristic of the young somatosensory cortex, and not found in the adult, is a population of darkly stained cells located deep to layer VI (Fig. \0B,D), which gradually diminishes until the adult-like CO pattern emerges at 28 d of age. These cells are small to medium sized; many have somata reminiscent of inverted pyramidal cells (Fig. 5). Numerous darkly stained cells can also be seen throughout the white matter, a feature not observed in the adult (Fig. 10J3,D). Discussion Development of Cortical Lamination The conical layers in the kitten somatosensory cortex are immature at birth. Layers V and VI can be discerned, but the cellular elements residing in the re-
mainder of the cortical plate appear homogeneous and indistinct. Over about a 4-week period, the discrete cortical layers evolve to their adult distribution, demonstrating clear structural differences. It is tempting to speculate that somatosensory neocortical maturation is similar to the laminar development that occurs in the visual cortex and that the developmental time frame is similar as well. If this is so, it implies that as the cortical layers reach their final stages of development, the distribution of afferent fibers also reaches maturity (Rakic, 1976,1977; Shatz and Luskin, 1986). According to Wise et al. (1977), however, the pattern of thalamocortical innervation in kitten somatosensory cortex appears relatively adult-like at 2 d of age. The cortical laminar pattern is clearly not adult-like at this age. It may be that, although the thalamocortical afferent fibers appear to reside in the center of the cortex, they actually exist deep to the presumptive layer IV, or at the lowermost portions of layer IV, since layer IV does not exist in its adult-like Cerebral Cortex May/June 1992, V 2 N 3 HB
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state at birth. This would be similar to the thalamocortical innervation pattern described in cat visual cortex (Shatz and Luskin, 1986). Recent results from Catalano et al. (1991), however, suggest that at least in the rat, thalamocortical afferent fibers invade the cortical plate in the somatosensory cortex at times earlier than those previously reported. This is a topic that clearly needs further investigation. We also observed at ages younger than 15 d a density of cells just beneath layer I. A similar compact band of cells was described by Luskin and Shatz (1985) in the developing cat area 17. If we assume that these cells are comparable to those in the developing visual cortex, it is likely that this dense band of cells contains neurons that have not yet completely differentiated and migrated to their final positions. This would mean that until nearly 2 weeks of age, as in the visual cortex, the cortical plate above layer V primarily contains neurons that are destined to be cells of layer IV, whereas the undifferentiated layers II and III reside in the dense band of cells below layer I. 236 Development of Cat Somatosensory Cortex • Code and Juliano
A Trtmsient Subplate Zone? In the present study, we show anatomical and functional evidence for the presence of an apparently immature zone below layer VI in somatosensory cortex during development. In Nissl-stained material, numerous small- to medium-sized cells are present subjacent to layer VI in kitten somatosensory cortex at birth. This population of cells gradually disappears over several weeks of development. In the adult, very few cells populate the region below layer VI. Many of the cells in this region have somata characteristic of inverted pyramidal cells, a cell type that populates the subplate zone in other developing cortical areas (Shatz et al., 1988; Valverde et al., 1989; Kostovic and Rakic, 1990). Additional cell types were observed in our study, as have been defined by others previously (cf. Mrzljak et al., 1988; Kostovic and Rakic, 1990), but we could not clearly assign them to a specific morphologic category. Numerous cells deep to layer VI in the somatosensory cortex stain heavily for CO activity, an indicator of baseline metabolic activity
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Ftgnr* 4. On the toft is a Nissl-stained section taken from the somatosensory cortex of a 15-d-oU cat on the right is a coiiiuaraule section stained to visualize AOiE activity. At this age. laminar dfflerereiatton is visible in the Nissl stain: layers I—VI can be distinguished, although not to the extent that they are detectable in the adult. In the AOiEstained section, more fibers can be seen in layers IHV than in the younger animals, but they are not ss numerous as si the aduh. Scale bar, 250 m -
Figure B. On thefe/ris a fug^-power view of a Ned-stained section taken trom the somatosensory cortei of a 3-d-oU cat. On the nght a a hgrvpower view of a CO-stained section taken from the somatosensory cortei of a 94old cat. Both photomicropaphs demonstrate the white maner-tayer VI luncoon In the NissJ and CO stains, numerous cells can be seen at the white maner-tayer VI border and m the white matter. Arrows indicate cells that have an inverted pyramidal shape. Ra is toward the top. Scale bar applies to both I
levels (Wong-Riley, 1979). A dense band of 2DG activity is also present in this location, despite the absence of the delivery of a specific somatic stimulus. This dense band of activity appears to indicate a high metabolic demand. In developing visual cortex, the subplate zone has been suggested to be of paramount significance to the developing neocortex. It is a region that appears early in development and contains synapses, identified at the EM level and immunohistochemically (Kostovic and Rakic, 1980, 1990; Rakic, 1988; Shatz et al., 1988). The inputs to the subplate include the thalamus, the basal forebrain, and afferents issuing from other cortical regions (Shatz et al., 1988; Kostovic and Rakic, 1990). It is also a zone in which the different afferent fibers wait for periods of time before reaching their final targets in the cortex. The functional significance of the fibers waiting in the subplate zone is not clear, although a number of possibilities have been suggested that imply various kinds of interaction and/or maintenance of the afferent incoming fibers (Rakic, 1977,1988; Shatz et al., 1988; Ghosh et al., 1990). In our experiments, the observation that the zone below layer VI contains a dense band of metabolic activity implies an active role for this region during neonatal life. Kostovic and Goldman-Rakic (1983) and Kostovic and Rakic (1990) suggest that since the cholinergic basal forebrain afferents and the thalamic afferents pass through the subplate during
the same period of time, there is a possibility that the subplate serves as an interactive site for the two systems. This is interesting in light of our observation that this region subjacent to the somatosensory cortex contains AChE+ fibers. Dense bands of laminar 2DG label in the cerebral cortex often correlate with sites of thalamocortical afferent termination (Juliano et al., 1981; Juliano and Whitsel, 1985; Tootel et al., 1988). It may be that the band of metabolic activity seen below layer VI is more or less apparent according to the demands of thalamocortical afferent fibers as they shift their terminal locus from the subplate to layer IV. The significance of the population of darkly stained CO cells subjacent to layer VI is also not clear. It is known that CO + neurons often reflect changes in metabolic demands (Wong-Riley and Welt, 1980; Kageyama and Wong-Riley, 1984, 1985, 1986a-c; Land, 1987; Land and Akhtar, 1987; Wong-Riley and Norton, 1988; Trusk et al., 1990). Following peripheral denervation, for example, staining for CO activity is reduced in the somatosensory and visual cortex (WongRiley, 1979; Wong-Riley and Welt, 1980; Land and Simons, 1985; Wong-Riley and Norton, 1988; Trusk et al., 1990), as well as at the level of subcortical structures (Kageyama and Wong-Riley, 1984, 1986c; Land, 1987; Land and Akhtar, 1987; Wong-Riley and Norton, 1988). It may be that the darkly stained population of cells below layer VI, which is not present in the Cerebral Conex May/June 1992, V 2 N 3 237
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238 Development of Cat Somatosensory Cortex • Code and Juliano
adult, reflects metabolic demands during fetal and postnatal life. As the cortex matures, the metabolic demands may shift toward layer IV and the supragranular layers. In kitten visual cortex, the distribution of CO activity, although immature, is distinctly different from that seen in somatosensory cortex. In striate cortex, a distinct laminar distribution similar to the adult pattern is present at 3 d of age (Kageyama and Wong-Riley, 1986a). CO activity at this age in
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visual cortex can also be found in a dense band just deep to layer I, which appears too narrow to correlate with the band of densely packed cells identified in Nissl-stained sections. Kageyama and Wong-Riley (1986a) interpret this dense CO activity to be related to a transient projection from the lateral geniculate nucleus of the thalamus to layer I. Although we saw hints of this increased density in kitten somatosensory cortex, it did not approach the density observed in
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Figure 8. A Nssl-stamed tissue section from a 19-d-oU kmea Note the nunerous cells beneath layer VI. Ants) rmenls indkatB corocal brers. B, A tfignmd autaradiograph of ihe same tissue t e a m as d m in A prior to Niss) staining. Increased 2DG uptake can be observed m layer VI and in the region subjacent to layer VL A bbck Une {A) passes through the layer Wwhite matter border in the Nml-stamed section. Arrows indicate the same Wood vessel m both panels. Scare bar, 500 ^im.
Cerebral Cortex May/June 1992, V 2 N 3 239
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Figure 7. Autorafiographs demonstratrg 2DG uptake of coronal sections talcen through cat somatosensory cortex. Increased 2DG activity (arrows) can be observed at the layer Vi-whne (natter border of a &d-oM arrknal [A] and a 9-d-oJd animal (d) C ts taken from an aduft aroma) that recaivad a somatic sumuhis. In the adult animal, a patch of label is superimposed on a band of increased activity m layer IV Scale bar, 1 mm far A-C
visual cortex. It will be interesting to observe if a similar transient thalamocortical projection can be found in somatosensory cortex. Development of CboUnergic Innervation AChE staining is a useful tool in developmental analysis, since ACh may play a role in cortical development. Although it is true that AChE is not as specific as choline acetyltransferase (ChAT) in measuring cholinergic innervation, it is generally accepted as an appropriate measure for evaluating the conical supply of ACh. Other investigators have established that in developing cat visual cortex, the distribution of ChAT and AChE is similar (Stichel and Singer, 1987). AChE staining itself is also useful in evaluating cortical developmental patterns and in some species appears to mimic thalamocortical patterns of innervation (Robertson, 1987; Robertson et al., 1988). Our findings regarding the distribution of AChE+ fibers demonstrate that, as the cortex matures, the density of fibers gradually increases throughout the thickness of the cortex. Specifically, the cortical regions above layer V, which are also immature in Nissl-stained material, have very few AChE+ fibers at 3 d of age. The population of fibers becomes progressively more numerous until 28 d of age, when the cortical laminar differentiation appears adult-like. The gradual increase in the density of the AChE+ fibers in cat somatosensory cortex during development is consistent with the results of a biochemical study on AChE activity. Heck and McKinley (1990) demonstrated a large increase in both AChE activity levels and amounts in cat somatosensory cortex between postnatal days 10 and 12, followed by a gradual rise between 12 and 28 d. These biochemical changes are likely to reflect the dramatic increase in the density of AChE+ fibers in layers II—IV that we report between 9 and 15 d. Bear et al. (1985) report a similar gradual increase in the distribution of AChE+ axons in cat visual cortex during development that resembles the adult pattern 240 Development of Cat Somatosensory Cortex • Code and Juliano
at 12 weeks of age. In their study, Bear et al. (1985) additionally describe an exuberance of AChE+ fibers in layers IVc-VI between 4 and 8 weeks of age, corresponding to the critical period in the visual development of cats. We did not examine AChE patterns during this time period so cannot comment on the possibility of such an exuberant pattern of AChE + fibers in kitten somatosensory cortex. Stichel and Singer (1987), who examined ChAT distribution in kitten visual cortex, did not find a similar overabundance of ChAT+ fibers at these time points. Bear et al. (1985) also described a dense network of fibers beneath layer VI (described by them as layer VII), which was densely populated with AChE+ neurons. Although we saw hints of such a laminar density with a few stained neurons, the intensity and numbers found in developing somatosensory cortex do not match those in developing visual cortex. Despite these differences, the laminar pattern of AChE + fibers in both visual and somatosensory cortices is strikingly similar at comparable time points. Henderson (1991) also describes a similar sequence of AChE innervation in developing ferret neocortex. In contrast to results in the cat, however, ferret cerebral cortex does not display strong immunoreactivity to ChAT until at least 2 weeks after birth. This lack of staining occurs despite the observation that the basal forebrain sends projections to the cerebral cortex prior to this date (Henderson, 1991). Although AChE appears to mimic thalamocortical innervation in rodents, this does not appear evident in cat somatosensory cortex. In comparison to the sparse data describing the development of thalamocortical projections to cat somatosensory cortex, the pattern of AChE innervation does not appear to match what is known about this distribution pattern, at least at the time points we have examined. A study by Wise et al. (1977) evaluates the ventrobasal thalamic projection upon kitten somatosensory cortex. These experiments describe a trilaminar cortical distribution at 2 d of age, which is clearly different from the pattern
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figure 9. Low-power view of CO activity taken through the somatasensary cortex of a 3-d-ott [A), a S-d-otd (5). and an adult cat (C). In the 3-d-dd [A), the pattern of CO activity is rdatryety homogeneous: in the 9-d-old (fl), h is difficuh to dtstnpish laminar stamng, but darUy stained neurons can be seen in layer V. In the adult, a laminar pattern can be seen, with reduced activity in layer I, darkly stained layers IWV, a pale layer V, and slightly rereased staining m layer VI Scale bar, 2 mm for A-C.
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Rgur» 1 0 . Higher-power view of CO naining in aduh [A and 0 and in M o l d (B and 0) cat somatosensory cortex. Seams are taken at the white matter-layer VI border. In the aduh (A C\. daridy stained neurons can be seen pratarunartfty in layer V. In the 9-d-otd somatosansory cortex [B, 01. numerous darkly staned cells can be seen at the layer Vl-whne mans border aid in the whne matter Scale bar, 500 IMtorA-D
of AChE+ fibers observed in this study. Thus, at least at the developmental stages analyzed presently, the AChE innervation and thalamocortical afferent terminations appear distinctly different. It is also possible that a transient rise in AChE staining occurs earlier in development than the times we studied. For example, Kostovic and Goldman-Rakic (1983) found
a transient increase in cholinesterase staining in the mediodorsal thalamus and frontal cortex of fetal monkeys and humans. The functional significance of ACh in the developing somatosensory cortex has yet to be determined. Normal cholinergic innervation appears to be necessary in the adult for standard processing of stimuli Cerebral Cortex May/June 1992, V 2 N 3 2*1
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(Metherate et al., 1987; Ma et al., 1989; Dykes, 1990; Juliano et al., 1990a; Jacobs et al., 1991) and is also necessary for the plasticity resulting in the expansion of topographic maps in adult somatosensory cortex (Juliano et al., 1991b). It is therefore highly likely that ACh plays an important role in developing cat somatosensory cortex, as it does in the visual system (Bear and Singer, 1986). In the newborn mouse, depletion of ACh results in abnormal cytoarchitecture, cortical lamination, and connectivity (Hohmann et al., 1988, 1991). These data suggest that ACh may make an important contribution to normal cortical morphogenesis and synaptogenesis in developing somatosensory cortex. Its specific impact on stimulus processing and plasticity in developing cat cortex is currently being investigated in our laboratory.
References Bear MF, Singer W (1986) Modulation of visual cortical plasticity by acetylchollne and noradrenaline. Nature 320: 172-176. Bear MF, Carnes KM, Ebner FF (1985) Postnatal changes in distribution of acetylcholinesterase in kitten striate cortex. J Comp Neurol 237:519-532. Catalano SM, Robertson RT, Killackey HP (1991) Early ingrowth of thalamocortical afferents to the neocortex of the prenatal rat. Proc Natl Acad Sci USA 88:2999-3003. Connor KM, Ferrington DG, Rowe MJ (1984) Tactile sensory coding during development: signaling capacities of neurons in kitten dorsal column nuclei. J Neurophysiol 52:86-98. Dykes RW (1990) Acerylcholine and neuronal plasticity in somatosensory cortex In- Brain cholinergic systems (Steriade M, Blesold D, eds), pp 294-313 New York: Oxford UP. Ferrington DG, Hora MOH, Rowe MJ (1984) Functional maturation of tactile sensory fibers in the kitten. J Neurophysiol 52:74-85. Ghosh A, Antonini A, McConnell SK, Shatz CJ (1990) Requirement for subplate neurons in the formation of thalamocortical connections. Nature 347.179-181. Heck CS, McKinley PA (1990) Age-dependent changes in acetylcholinesterase activity in the primary somatosensory cortex of the cat. Dev Brain Res 56:189-197 Henderson Z (1991) Early development of the nucleusbasalis-cortical projection but late expression of its cholinergic function. Neuroscience 44:311-324. HShmann CF, Brooks AR, Coyle JT (1988) Neonatal lesions of the basal forebrain cholinergic neurons result in abnormal cortical development. Dev Brain Res 42:253-264. H5hmann CF, Wilson L, Coyle JT (1991) Efferent and afferent connections of mouse sensory-motor cortex following cholinergic deafferentation at birth. Cereb Cortex 1:158-172. Jacobowitz DM, Creed GJ (1983) Cholinergic projection sites of the nucleus of tractus diagonalis. Brain Res Bull 10:365-371. Jacobs SE, Code RA, Juliano SL (1991) Basal forebrain lesions alter stimulus-evoked metabolic activity in rat somatosensory cortex. Brain Res 560:342-345. 242 Development of Cat Somatosensory Cortex • Code and Juliano
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Notes We thank Don Eslin for his expert technical assistance. We also thank Dr Christine HShmann for helpful comments on the manuscript. This work was supported by PHS-R01 NS24014. Correspondence should be addressed to Sharon L. Juliano, Ph.D., Department of Anatomy and Cell Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814.
Juliano SL, Whitsel BL (1985) Metabolic labeling associated with index finger stimulation in monkey SI: between animal variability. Brain Res 342:242-251. Juliano SL, Hand PJ, Whitsel BL (1981) Patterns of increased metabolic activity in somatosensory cortex of monkeys (Macaca fascicularis) subjected to controlled cutaneous stimulation: a 2-deoxyglucose study. J Neurophysiol 46:1260-1284 Juliano SL, Ma W, Bear MF, Eslin D (1990a) Cholinergic manipulation alters stimulus-evoked metabolic activity in cat somatosensory cortex. J Comp Neurol 297:106-120. Juliano SL, Ma W, Eslin DE (1990b) Development of metabolic activity patterns in kitten somatosensory cortex. Soc Neurosci Abstr 16:630. Juliano SL, Code RA, Eslin DE (1991a) Anatomic and functional correlates of the subplate in the somatosensory cortex of kittens. Soc Neurosci Abstr 17:1304. Juliano SL, Ma W, Eslin D (1991b) Cholinergic depletion prevents expansion of topographic maps in somatosensory cortex. Proc Natl Acad Sci USA 88:780-784. Kageyama GH, Wong-Riley MT (1984) The histochemical localization of cytochrome oxidase in the retina and lateral geniculate nucleus of the ferret, cat, and monkey, with particular reference to retinal mosaics and ON/OFFcenter visual channels. J Neurosci 4:2445-2459. Kageyama GH, Wong-Riley M (1985) An analysis of the cellular localization of cytochrome oxidase in the lateral geniculate nucleus of the adult cat. J Comp Neurol 242: 338-357. Kageyama GH, Wong-Riley M (1986a) The localization of cytochrome oxidase in the LGN and striate cortex of postnatal kittens. J Comp Neurol 243182-194. Kageyama GH, Wong-Riley M (1986b) Laminar and cellular localization of cytochrome oxidase in the cat striate cortex. J Comp Neurol 245:137-159. Kageyama GH, Wong-Riley M (1986c) Differential effect of visual deprivation on cytochrome oxidase levels in major cell classes of the cat LGN. J Comp Neurol 246: 212-237. KoelleGB (1955) The histochemical identification of acetylcholinesterase in cholinergic adrenergic and sensory neurons. J Pharmacol Exp Ther 114:167-184. Kostovic I, Goldman-Rakic PS (1983) Transient cholinesterase staining in the medlodorsal nucleus of the thalamus and its connections in the developing human and monkey brain. J Comp Neurol 219:431-447. Kostovic I, Rakic P (1980) Cytology and time of origin of interstitial neurons in the white matter In infant and adult human and monkey telencephalon. J Neurocytol 9:219242. Kostovic I, Rakic P (1990) Developmental history of the transient subplate zone in the visual and somatosensory cortex of the macaque monkey and human brain. J Comp Neurol 297-441-470. Land PW (1987) Dependence of cytochrome oxidase activity in the rat lateral geniculate nucleus on retinal innervation. J Comp Neurol 262:78-89. Land FW, Akhtar ND (1987) Chronic sensory deprivation affects cytochrome oxidase staining and glutamic acid decarboxylase immunoreactivity in adult rat ventrobasal thalamus. Brain Res 425:178-181. Land PW, Simons DJ (1985) Metabolic activity in SmI cortical barrels of adult rats is dependent on patterned sensory stimulation of the mystacial vibrissae Brain Res 341: 189-194. LeVay S, Stryker MP, Shatz CJ (1978) Ocular dominance columns and their development in layer IV of the cat's visual cortex: a quantitative study. J Comp Neurol 179: 223-244. Luskin MB, Shatz CJ (1985) Studies of the earliest generated cells of the cat's visual cortex- cogeneration of subplate and marginal zones. J Neurosci 5:1062-1075. Ma W, Hohmann CF, Coyle JT, Juliano SL (1989) Lesions of the basal forebrain alter stimulus-evoked metabolic activity in mouse somatosensory cortex. J Comp Neurol 288-414-427
Wong-Riley MT, Norton TT (1988) Histochemical localization of cytochrome oxidase activity in the visual system of the tree shrew: normal patterns and the effect of retinal impulse blockade. J Comp Neurol 272:562-578. Wong-Riley MTT, Welt C (1980) Histochemical changes in cytochrome oxidase of cortical barrels after vibrissal removal in neonatal and adult mice Proc Natl Acad Sci USA 77:2333-2337.
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Metherate R, Tremblay N, Dykes RW (1987) Acetylchollne permits long-term enhancement of neuronal responsiveness in cat primary somatosensory cortex. Neuroscience 22:75-81. Mrzljak L, Uylings HBM, Kostovic I, Van Eden CG (1988) Prenatal development of neurons in the human frontal cortex: I. A qualitative Golgi study. J Comp Neurol 271: 355-386. Payne B, Pearson H, Cornwell P (1988) Development of visual and auditory cortical connections in the cat. In: Cerebral cortex, Vol 7, Development and maturation of cerebral cortex (Peters A, Jones EG, eds), pp 309-389. New York: Plenum. Rakic P (1976) Prenatal genesis of connections subserving ocular dominance in the rhesus monkey Nature 261:467471. Rakic P (1977) Prenatal development of the visual system in rhesus monkey. Philos Trans R Soc Lond [Biol] 278: 245-260. Rakic P (1988) Specification of cerebral cortical areas. Science 241:170-176. RediesC.DiksicM.RimlH (1990) Functional organization in the ferret visual cortex- a double-label 2-deoxyglucose study. J Neurosci 10:2791-2803. Robertson RT (1987) A morphogenic role for transiently expressed acetylcholinesterase in developing thalamocortical systems? Neurosci Lett 75:259-264. Robertson RT, Hanes MA, YuJ (1988) Investigations of the origins of transient acetylcholinesterase activity in developing rat visual cortex. Dev Brain Res 411-23. Rubel EW (1971) A comparison of somatotopic organization in sensory neocortex of newborn kittens and adult cats. J Comp Neurol 143-447^480. Shapiro HM, GreenbergJH, Reivich M, Ashmead G, Sokoloff L (1978) Local cerebral glucose uptake in awake and halothane-anesthetized primates. Anesthesiology 48:97103. Shatz CJ, Luskin MB (1986) The relationship between the geniculocortical afferents and their conical target cells during development of the cat's primary visual cortex. J Neurosci 6:3655-3668. Shatz CJ, Chun JJM, Luskin MB (1988) The role of the subplate in the development of the mammalian telencephalon. In: Cerebral cortex, Vol 7, Development and maturation of cerebral cortex (Peters A, Jones EG, eds), pp 35-58. New York: Plenum Sokoloff L, Reivich M, Kennedy C, Des Rosiers MH, Patlack CS, PettigrewKD, SakuradaO, ShinoharaM (1977) (MC) deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat J Neurochem 28:897-916. Stichel CC, Singer W (1987) Quantitative analysis of the choline acetyltransferase-immunoreactive axonal network in the cat primary visual cortex: II Pre- and postnatal development. J Comp Neurol 258:99-111Tootel RBH, Hamilton SL, Silverman MS, Switkes E (1988) Functional anatomy of macaque striate cortex. I. Ocular dominance, binocular interactions and baseline conditions. J Neurosci 8:1500-1530. Trusk TC, Kaboord WS, Wong-Riley MTT (1990) Effects of monocular enucleation, tetrodotoxin, and lid suture on cytochrome-oxidase reactivity in supragranular puffs of adult macaque striate cortex. Vis Neurosci 4:185-204. Valverde F, Facal-Valverde MV, Santacana M, Heredia M (1989) Development and differentiation of early generated cells of sublayer Vlb in the somatosensory cortex of the rat: a correlated Golgi and autoradiographic study. J Comp Neurol 290:118-140. Wise SP, Hendry SHC, Jones EG (1977) Prenatal development of sensorimotor cortical projections in cats. Brain Res 138:538-544. Wong-Riley MTT (1979) Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res 17111-28.
Cerebral Cortex May/June 1992, V 2 N 3 243
Departments of Anatomy and Cell Biology, and Neuroscience, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814
Although an extensive amount of data has accumulated on the development of the cat visual cortex (LeVay et al., 1978; Luskin and Shatz, 1985; Payne et al., 1988; Shatz et al., 1988), relatively little is known about the development of the somatosensory cortex. The limited knowledge that we have acquired over the past 20 years suggests that certain aspects of the kitten somatosensory system are relatively mature and respond to sensory stimulation at birth. In addition, neonatal somatosensory neurons demonstrate a definite topographic arrangement and possess receptive fields that share aspects of adult response properties (Rubel, 1971; Connor et al., 1984; Ferrington et al., 1984). Within a few days after birth, thalamocortical fibers have invaded the cortical plate and present with a relatively adult-like distribution (Wise et al., 1977) Despite these similarities with adult properties, kitten somatosensory cortical neurons are clearly immature in many functional aspects (Rubel, 1971), as are the responses of subcortical structures (Connor et al., 1984; Ferrington et al., 1984). According to Rubel (1971), single-unit responses in newborn cat somatosensory cortex are reduced in their latency and complexity, and are often unreliable and difficult to drive. Since these early reports describing neuronal responses in neonatal somatosensory cortex, there has been little information available on the development of this cortical region. In particular, detailed studies on the development of the anatomical organization of cat somatosensory cortex and its relationship to emergent functional activity are scarce. In comparison, the evolution of cortical responses and thalamocortical relationships have been more clearly documented in visual cortex. For example, developing neuronal response properties and the distribution of afferent innervation patterns in visual cortex can be precisely related to conical structure and connectional relationships present at a given time during development. As the cell structure of the visual cortex matures, the distribution of thalamocortical aJferents and receptors evolves to match the evolution of cortical layers and the emerging response properties (Rakic, 1976, 1977; Shatz and Luskin, 1986; Shatz et al., 1988; Kostovic and Rakic, 1990). In addition, the subplate has been identified as an important factor in the developing visual cortex. It appears to play a generally nurturing role as a recipient site for fiber systems that project into the neocortex, yet relatively few reports exist that describe Cerebral Cortex May/June 1992;2:231-243; 1047-3211/92/14.00
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Although research is beginning to clarify the relationship between structure and functional activity in the adult cerebral cortex, little is known about cortical development in the somatosensory cortex of cats. A number of parameters were used in this study to identify functional and anatomical correlates in the developing somatosensory cortex of kittens ranging in age from 3 to 33 d: 2-deoxyglucose (2DG) uptake, cytochrome ondase (CO) activity, Nissl staining, and AChE activity. All of these parameters were found to reflect an immaturity that evolved to the adult-like pattern by 4-5 weeks of age. Nissl staining revealed an immature laminar pattern at birth, in which layers I, V, and VI were distinct whereas layers II—IV were homogeneous in appearance. Numerous cell* could be observed at the layer Vl-white matter junction and throughout the white matter, a feature not found in adults. The laminar distribution of NissJ-stained cells gradually became mature by 4-5 weeks of age. CO staining was homogeneous throughout all layers, in contrast to the adult pattern, which displays laminar differentiation. In young animals, many darkly stained C0+ cells were found at the layer Vl-white matter border and in the white matter, a distribution not found in the adult. AChE staining in kittens was also distinctly different from that in adults. At birth, AChE+ fibers could be found in layers I, V, and VI but were scarce in layers II—IV. In the adult, a dense network of AChE+ fibers can be found in all layers. 2DG uptake was also immature, as little stimulus-evoked activity could be observed in animals younger than 2 weeks. A dense band of metabolic activity was found in the zone between layer VI and the white matter, whether or not a somatic stimulus was delivered. These results suggest a close correlation between the developing cytoarchiteeture and the emergence of a mature pattern of functional activity in the somatosensory cortex.
Rebecca A. Code and Sharon L. Juliano
Materials and Methods
Seventeen kittens of either sex, 3-33 d old (day of birth = postnatal day 0), were used in this study. The procedures used in the 2DG experiments were similar to those originally described by Sokoloff et al. (1977) and published previously by this laboratory (Juliano et al., 1990a). Under halothane anesthesia, kittens 9 d of age or older were intubated with a tracheal cannula and a venous catheter was inserted in the long saphenous vein. After administration of gallamine triethiodide, the animal was maintained on a respirator and its temperature, heart rate and expired CO2 were continuously monitored and kept within normal limits. Pupillary size was also monitored and found to be appropriate for the anesthesia level. The anesthetic was changed from halothane to nitrous oxide (70%) and oxygen (30%), since halothane has been shown to reduce cortical 2DG uptake (Shapiro et al., 1978). The EEG of animals under similar conditions has been previously examined and found to drift in and out of sleep states, indicating that the animal is not stressed (Juliano et al., 1990a). The same is true of other mammals monitored under identical circumstances (Redies et al., 1990). One and a half hours after the termination of halothane anesthesia, 2-deoxyD-[l-HC]-glucose (100 mCi/kg) was injected. During the 45 min after the injection, some of the animals received a somatic stimulation; this aspect of 2DG uptake was not evaluated for this study but will be addressed in a subsequent report. For kittens younger than 9 d old, neither the tracheal cannula nor venous catheter was inserted; instead, 2DG was injected intraperitoneally. No somatic stimulus was applied, but the animal was allowed to rest in a softly padded box. Forty-five minutes after injection of 2DG, all animals received an overdose of sodium pentobarbital (35 mg/kg) and were quickly perfused intracardially with a 0.9% saline rinse followed by 4% paraformaldehyde, 4% sucrose in 0.1 M phosphate buffer. The brain was immediately removed from the skull, blocked in the frontal plane, frozen in Freon 22, and stored at — 70°C. Later, 30-Mm-thick coronal sections were cut on a cry-
232 Development of Cat Somatosensory Cortex • Code and Juliano
ostat. Every other section was saved for 2DG autoradiography and exposed to x-rayfilm(Kodak SB5) with "C standards for 7-10 d. After film development, these same sections were stained for Nissl substance. The other series of adjacent sections was processed for cytochrome oxidase (CO) histochemistry to evaluate baseline metabolic activity (Wong-Riley, 1979). In some experiments, a third series of adjacent sections was stained for acetylcholinesterase (AChE) activity using a modification (Jacobowitz and Creed, 1983) of the procedure originally described by Koelle (1955). Results Cytoarcbitecture In the adult cat somatosensory cortex, Nissl-stained neurons are organized into clearly delineated layers (Fig. 1). The adult somatosensory cortex is characterized by a highly granular layer IV with small, closely packed cells. The supragranular layers are more loosely packed with medium-sized cells. A number of large pyramidal cells reside in layer III. In the infragranular layers, layer V is distinguished by the presence of large pyramidal cells in its upper portion and a paler-staining lower portion containing smallto medium-sized pyramidal cells. Neurons in layer VI are more densely packed than those in layer V but are not as closely packed as those in layer IV. There are relatively few cells in the white matter. In contrast, only layers V and VI are clearly distinguishable in the 3-d-old somatosensory cortex (Fig. 2). Large pyramidal cells are evident in layer V, but this layer cannot yet be separated into upper and lower portions. Although slight laminar differentiation can be observed between the relatively acellular layer I and layer V, neurons for the most part are very densely packed into a homogeneous region, which is easily demarcated from layer V. A densely packed band of cells can be observed just deep to layer I. Numerous cells, including inverted pyramidal cells, are evident in the white matter just below layer VI (see Fig. 5). Although it is not easy to delineate the subplate on cytoarchitectural criteria alone (Kostovic and Rakic, 1990), we believe, based on the morphology and dimension of the cell population, that this region corresponds to what has been described as the "subplate" by other investigators (cf. Rakic, 1977, 1988; Luskin and Shatz, 1985; Shatz et al., 1988; Kostovic and Rakic, 1990). Additional prominent cells can be seen scattered throughout the white matter, a feature not observed in the adult. In the 9-d-old cat, the densely packed band of cells continues to be visible below layer I. Layers II-IV are not separable into distinct entities, although the cells, still densely packed, appear less so than in the 3-dold cat (Fig. 3). Larger pyramidal cells are more numerous in layer V compared with the younger age. Layer V also appears to be separating into upper and lower portions. There are still many cells at the layer Vl-white matter border. Although the 15-17-d-old cat somatosensory cortex is still not fully mature, the beginning segregation
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the subplate in developing somatosensory cortex (cf. Kostovic and Rakic, 1990) and almost no studies describe the subplate in the somatosensory cortex of the cat. As a result, in the present study we wished to examine the structure of developing cat somatosensory cortex and observe its relationship to emergent functional activity patterns. To assess these functional relations, patterns of metabolic activity were analyzed in developing cats using the 2-deoxyglucose (2DG) method. During the time that functional activity patterns are being established in the somatosensory cortex, we observed changes in cytoarchitectural organization, in patterns of cytochrome oxidase (CO) staining, and in the density and distribution of acetylcholinesterase (AChE + ) fibers, each of which could be correlated with changes in metabolic activity patterns. Preliminary accounts of these results have been published (Juliano et al., 1990b, 1991a).
R jore 1 . On the M is a Nissl-stained section taken from aduh cat somatosensory cortex: on the right is a comparable section named to visualize AChE acthmy. Roman numerals mdrcate cortical layers. In the Ntssl n a n . conical layers I—VI can be eajily differentiated. In the AChE-stained section, numerous densely packed fibers can be observed in all cortical layers. The fiber density is somewhat reduced m the white maner. Scale bar. 250 >im.
of layers II, III, and IV can be differentiated. Cortical neurons in the upper layers are less densely packed than in the younger ages, and a condensation of smaller, more tightly packed cells can be observed at the layer IV-V border (Fig. 4). Layer V can be separated into upper and lower portions that closely resemble the mature laminar pattern. Numerous cells are present in the white matter subjacent to layer VI. By 28 d, the laminar pattern resembles that of the adult. Few cells can be found in the region previously devoted to the subplate. AcetylcboUnesterose-posittve Inrtervotion The distribution of AChE+ fibers in adult somatosensory cortex is relatively dense in all cortical layers, although it displays a slight increase in density in layers I and V (Figs. 1,6). Both radially and tangentially oriented fibers are present in every layer, although layer I appears to have more tangentially run-
ning fibers. At all ages studied, layer I is predominated by tangential AChE + fibers. An immature pattern of AChE activity is seen in young animals. In the 3-d-old cat, the density of AChE+ fibers is greatest in layers I, V, and VI; the paucity of fibers in layers II—IV, however, is striking (Figs. 2, 6). Most of the fibers in these layers are long and radially oriented with few branches. There is a heavy concentration of AChE + fibers at the layer IVV border and a slightly less dense concentration in the subplate. The distribution of AChE + fibers in the 9-d-old somatosensory cortex is similar to that in the 3-d-old: densest in layers I, V, and VI; scarcest in layers II-IV (Figs. 3, 6). Layers I, V, and VI are predominated by tangentially oriented fibers, while those in layers IIIV are primarily radially oriented. In addition, there are numerous AChEH- cells in the subplate below the somatosensory cortex. Cerebral Cortex May/June 1992, V 2 N 3 " 3
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R g u r a 2 . On the te/if is a Ntsst-statnsd section taken from the somaiosensory cortei of a 3-d-old cat: on the right s a comparable section stained to visualize AChE activity. In the Ntssl-stainsd section, conical layers I, V. and VI can be distinguished, whrie layers II-IV are relatively homogeneous and cannot be individually distinguished. These layers are thus indicated as CP Icortcal plate). A band of more densely packed cells can be observed just subiacem to layer I. In the AChE-stained section, a relatively dense distribution of W H S can be observed m layers I. V. and VI. The region designated CP a sparsely populated with ACh£+ fibers. Scale bar. 250 /im.
At 15 d of age, the density of AChE+ fiber distribution continues to increase (Fig. 4). The densest staining is still in layers I, V, and VI. The overall density is greater than that at 9 d, and the fibers exhibit more branching. Layers V and VI now appear to have equal numbers of both tangentially and radially oriented fibers. The density of AChE + fibers in layers II-IV is also increased compared to younger ages. Fibers for the most part are radially oriented, but many oblique and horizontal branches are evident Note also the gradual increase in the thickness of the cortex innervated by AChE+ fibers during development The distribution of AChE + fibers at 22 d of age is similar to that at 15 d (Fig. 6). The density of fibers in layers II-IV, however, is greater than that at 15 d, but not as heavy as that in the adult. Both tangential and radial AChE + fibers appear to be equally represented in these layers. Metabolic Activity In kittens less than 3 weeks of age, little stimulusevoked metabolic activity, as revealed by 2DG uptake, is seen in the somatosensory cortex. (The evolution of stimulus-evoked activity will be assessed in a subsequent report.) Dense metabolic label is present, 234 Development of Cat Somatosensory Cortex • Code and Juliano
however, as a band of increased activity in layer VI and at the border of layer VI and the white matter (Fig. 1A,B). Comparison of this region with Nisslstained material reveals that it corresponds to lower portions of layer VI and to the cellular zone just subjacent to layer VI (Fig. 8A,B). In kittens aged 3 d to 3 weeks, this region is heavily labeled with 2DG whether or not a somatic stimulus was delivered. After 3 weeks of age, 2DG label is not easily visible at the layer Vl-white matter border; stimulus-evoked metabolic activity, however, becomes increasingly apparent in the middle layers until about 5 weeks of age, when it resembles more closely the adult laminar pattern (Fig. 1C). Cytocbrome Oxidase Activity In the adult, CO staining is heavy in layers II-IV and weaker in layer I (Fig. 9C). The CO distribution becomes quite pale in layer V and then increases slightly in layer VI. Numerous darkly stained neurons can be seen throughout the cortex; they are especially prominent in layer V (Fig. \0A,C). In contrast, young animals display a relatively homogeneous CO distribution in all layers, except for a slight staining decrease in layer I (Fig. 9A). At 9 d of age, a layer of darkly
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R g u r t 3. On the fe/ir is a Nissl-stained section taken from the somatosensory cortei of a M o l d cat on the right a a comparable sacum stained to visualize AChE acovtty. In the Nissl stan. layers I. V, and VI can be distinguished, but the region designated CP (cortcal plate) contains cells that are relatively homogeneous in their distribution. In the ACh£-aamed section, numerous AChE+ ibers can be seen in layers I, V. and VI, but the conical plate region contains relatively few AChE+ fibers. Scale bar. 250 fim.
stained neurons can be seen in layer V, similar to those found in the adult (Fig. 9B). At 15 d of age, the adult laminar pattern begins to emerge and is mature by about 4 weeks of age. One feature characteristic of the young somatosensory cortex, and not found in the adult, is a population of darkly stained cells located deep to layer VI (Fig. \0B,D), which gradually diminishes until the adult-like CO pattern emerges at 28 d of age. These cells are small to medium sized; many have somata reminiscent of inverted pyramidal cells (Fig. 5). Numerous darkly stained cells can also be seen throughout the white matter, a feature not observed in the adult (Fig. 10J3,D). Discussion Development of Cortical Lamination The conical layers in the kitten somatosensory cortex are immature at birth. Layers V and VI can be discerned, but the cellular elements residing in the re-
mainder of the cortical plate appear homogeneous and indistinct. Over about a 4-week period, the discrete cortical layers evolve to their adult distribution, demonstrating clear structural differences. It is tempting to speculate that somatosensory neocortical maturation is similar to the laminar development that occurs in the visual cortex and that the developmental time frame is similar as well. If this is so, it implies that as the cortical layers reach their final stages of development, the distribution of afferent fibers also reaches maturity (Rakic, 1976,1977; Shatz and Luskin, 1986). According to Wise et al. (1977), however, the pattern of thalamocortical innervation in kitten somatosensory cortex appears relatively adult-like at 2 d of age. The cortical laminar pattern is clearly not adult-like at this age. It may be that, although the thalamocortical afferent fibers appear to reside in the center of the cortex, they actually exist deep to the presumptive layer IV, or at the lowermost portions of layer IV, since layer IV does not exist in its adult-like Cerebral Cortex May/June 1992, V 2 N 3 HB
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state at birth. This would be similar to the thalamocortical innervation pattern described in cat visual cortex (Shatz and Luskin, 1986). Recent results from Catalano et al. (1991), however, suggest that at least in the rat, thalamocortical afferent fibers invade the cortical plate in the somatosensory cortex at times earlier than those previously reported. This is a topic that clearly needs further investigation. We also observed at ages younger than 15 d a density of cells just beneath layer I. A similar compact band of cells was described by Luskin and Shatz (1985) in the developing cat area 17. If we assume that these cells are comparable to those in the developing visual cortex, it is likely that this dense band of cells contains neurons that have not yet completely differentiated and migrated to their final positions. This would mean that until nearly 2 weeks of age, as in the visual cortex, the cortical plate above layer V primarily contains neurons that are destined to be cells of layer IV, whereas the undifferentiated layers II and III reside in the dense band of cells below layer I. 236 Development of Cat Somatosensory Cortex • Code and Juliano
A Trtmsient Subplate Zone? In the present study, we show anatomical and functional evidence for the presence of an apparently immature zone below layer VI in somatosensory cortex during development. In Nissl-stained material, numerous small- to medium-sized cells are present subjacent to layer VI in kitten somatosensory cortex at birth. This population of cells gradually disappears over several weeks of development. In the adult, very few cells populate the region below layer VI. Many of the cells in this region have somata characteristic of inverted pyramidal cells, a cell type that populates the subplate zone in other developing cortical areas (Shatz et al., 1988; Valverde et al., 1989; Kostovic and Rakic, 1990). Additional cell types were observed in our study, as have been defined by others previously (cf. Mrzljak et al., 1988; Kostovic and Rakic, 1990), but we could not clearly assign them to a specific morphologic category. Numerous cells deep to layer VI in the somatosensory cortex stain heavily for CO activity, an indicator of baseline metabolic activity
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Ftgnr* 4. On the toft is a Nissl-stained section taken from the somatosensory cortex of a 15-d-oU cat on the right is a coiiiuaraule section stained to visualize AOiE activity. At this age. laminar dfflerereiatton is visible in the Nissl stain: layers I—VI can be distinguished, although not to the extent that they are detectable in the adult. In the AOiEstained section, more fibers can be seen in layers IHV than in the younger animals, but they are not ss numerous as si the aduh. Scale bar, 250 m -
Figure B. On thefe/ris a fug^-power view of a Ned-stained section taken trom the somatosensory cortei of a 3-d-oU cat. On the nght a a hgrvpower view of a CO-stained section taken from the somatosensory cortei of a 94old cat. Both photomicropaphs demonstrate the white maner-tayer VI luncoon In the NissJ and CO stains, numerous cells can be seen at the white maner-tayer VI border and m the white matter. Arrows indicate cells that have an inverted pyramidal shape. Ra is toward the top. Scale bar applies to both I
levels (Wong-Riley, 1979). A dense band of 2DG activity is also present in this location, despite the absence of the delivery of a specific somatic stimulus. This dense band of activity appears to indicate a high metabolic demand. In developing visual cortex, the subplate zone has been suggested to be of paramount significance to the developing neocortex. It is a region that appears early in development and contains synapses, identified at the EM level and immunohistochemically (Kostovic and Rakic, 1980, 1990; Rakic, 1988; Shatz et al., 1988). The inputs to the subplate include the thalamus, the basal forebrain, and afferents issuing from other cortical regions (Shatz et al., 1988; Kostovic and Rakic, 1990). It is also a zone in which the different afferent fibers wait for periods of time before reaching their final targets in the cortex. The functional significance of the fibers waiting in the subplate zone is not clear, although a number of possibilities have been suggested that imply various kinds of interaction and/or maintenance of the afferent incoming fibers (Rakic, 1977,1988; Shatz et al., 1988; Ghosh et al., 1990). In our experiments, the observation that the zone below layer VI contains a dense band of metabolic activity implies an active role for this region during neonatal life. Kostovic and Goldman-Rakic (1983) and Kostovic and Rakic (1990) suggest that since the cholinergic basal forebrain afferents and the thalamic afferents pass through the subplate during
the same period of time, there is a possibility that the subplate serves as an interactive site for the two systems. This is interesting in light of our observation that this region subjacent to the somatosensory cortex contains AChE+ fibers. Dense bands of laminar 2DG label in the cerebral cortex often correlate with sites of thalamocortical afferent termination (Juliano et al., 1981; Juliano and Whitsel, 1985; Tootel et al., 1988). It may be that the band of metabolic activity seen below layer VI is more or less apparent according to the demands of thalamocortical afferent fibers as they shift their terminal locus from the subplate to layer IV. The significance of the population of darkly stained CO cells subjacent to layer VI is also not clear. It is known that CO + neurons often reflect changes in metabolic demands (Wong-Riley and Welt, 1980; Kageyama and Wong-Riley, 1984, 1985, 1986a-c; Land, 1987; Land and Akhtar, 1987; Wong-Riley and Norton, 1988; Trusk et al., 1990). Following peripheral denervation, for example, staining for CO activity is reduced in the somatosensory and visual cortex (WongRiley, 1979; Wong-Riley and Welt, 1980; Land and Simons, 1985; Wong-Riley and Norton, 1988; Trusk et al., 1990), as well as at the level of subcortical structures (Kageyama and Wong-Riley, 1984, 1986c; Land, 1987; Land and Akhtar, 1987; Wong-Riley and Norton, 1988). It may be that the darkly stained population of cells below layer VI, which is not present in the Cerebral Conex May/June 1992, V 2 N 3 237
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238 Development of Cat Somatosensory Cortex • Code and Juliano
adult, reflects metabolic demands during fetal and postnatal life. As the cortex matures, the metabolic demands may shift toward layer IV and the supragranular layers. In kitten visual cortex, the distribution of CO activity, although immature, is distinctly different from that seen in somatosensory cortex. In striate cortex, a distinct laminar distribution similar to the adult pattern is present at 3 d of age (Kageyama and Wong-Riley, 1986a). CO activity at this age in
t
visual cortex can also be found in a dense band just deep to layer I, which appears too narrow to correlate with the band of densely packed cells identified in Nissl-stained sections. Kageyama and Wong-Riley (1986a) interpret this dense CO activity to be related to a transient projection from the lateral geniculate nucleus of the thalamus to layer I. Although we saw hints of this increased density in kitten somatosensory cortex, it did not approach the density observed in
I It III
IV
Figure 8. A Nssl-stamed tissue section from a 19-d-oU kmea Note the nunerous cells beneath layer VI. Ants) rmenls indkatB corocal brers. B, A tfignmd autaradiograph of ihe same tissue t e a m as d m in A prior to Niss) staining. Increased 2DG uptake can be observed m layer VI and in the region subjacent to layer VL A bbck Une {A) passes through the layer Wwhite matter border in the Nml-stamed section. Arrows indicate the same Wood vessel m both panels. Scare bar, 500 ^im.
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Figure 7. Autorafiographs demonstratrg 2DG uptake of coronal sections talcen through cat somatosensory cortex. Increased 2DG activity (arrows) can be observed at the layer Vi-whne (natter border of a &d-oM arrknal [A] and a 9-d-oJd animal (d) C ts taken from an aduft aroma) that recaivad a somatic sumuhis. In the adult animal, a patch of label is superimposed on a band of increased activity m layer IV Scale bar, 1 mm far A-C
visual cortex. It will be interesting to observe if a similar transient thalamocortical projection can be found in somatosensory cortex. Development of CboUnergic Innervation AChE staining is a useful tool in developmental analysis, since ACh may play a role in cortical development. Although it is true that AChE is not as specific as choline acetyltransferase (ChAT) in measuring cholinergic innervation, it is generally accepted as an appropriate measure for evaluating the conical supply of ACh. Other investigators have established that in developing cat visual cortex, the distribution of ChAT and AChE is similar (Stichel and Singer, 1987). AChE staining itself is also useful in evaluating cortical developmental patterns and in some species appears to mimic thalamocortical patterns of innervation (Robertson, 1987; Robertson et al., 1988). Our findings regarding the distribution of AChE+ fibers demonstrate that, as the cortex matures, the density of fibers gradually increases throughout the thickness of the cortex. Specifically, the cortical regions above layer V, which are also immature in Nissl-stained material, have very few AChE+ fibers at 3 d of age. The population of fibers becomes progressively more numerous until 28 d of age, when the cortical laminar differentiation appears adult-like. The gradual increase in the density of the AChE+ fibers in cat somatosensory cortex during development is consistent with the results of a biochemical study on AChE activity. Heck and McKinley (1990) demonstrated a large increase in both AChE activity levels and amounts in cat somatosensory cortex between postnatal days 10 and 12, followed by a gradual rise between 12 and 28 d. These biochemical changes are likely to reflect the dramatic increase in the density of AChE+ fibers in layers II—IV that we report between 9 and 15 d. Bear et al. (1985) report a similar gradual increase in the distribution of AChE+ axons in cat visual cortex during development that resembles the adult pattern 240 Development of Cat Somatosensory Cortex • Code and Juliano
at 12 weeks of age. In their study, Bear et al. (1985) additionally describe an exuberance of AChE+ fibers in layers IVc-VI between 4 and 8 weeks of age, corresponding to the critical period in the visual development of cats. We did not examine AChE patterns during this time period so cannot comment on the possibility of such an exuberant pattern of AChE + fibers in kitten somatosensory cortex. Stichel and Singer (1987), who examined ChAT distribution in kitten visual cortex, did not find a similar overabundance of ChAT+ fibers at these time points. Bear et al. (1985) also described a dense network of fibers beneath layer VI (described by them as layer VII), which was densely populated with AChE+ neurons. Although we saw hints of such a laminar density with a few stained neurons, the intensity and numbers found in developing somatosensory cortex do not match those in developing visual cortex. Despite these differences, the laminar pattern of AChE + fibers in both visual and somatosensory cortices is strikingly similar at comparable time points. Henderson (1991) also describes a similar sequence of AChE innervation in developing ferret neocortex. In contrast to results in the cat, however, ferret cerebral cortex does not display strong immunoreactivity to ChAT until at least 2 weeks after birth. This lack of staining occurs despite the observation that the basal forebrain sends projections to the cerebral cortex prior to this date (Henderson, 1991). Although AChE appears to mimic thalamocortical innervation in rodents, this does not appear evident in cat somatosensory cortex. In comparison to the sparse data describing the development of thalamocortical projections to cat somatosensory cortex, the pattern of AChE innervation does not appear to match what is known about this distribution pattern, at least at the time points we have examined. A study by Wise et al. (1977) evaluates the ventrobasal thalamic projection upon kitten somatosensory cortex. These experiments describe a trilaminar cortical distribution at 2 d of age, which is clearly different from the pattern
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figure 9. Low-power view of CO activity taken through the somatasensary cortex of a 3-d-ott [A), a S-d-otd (5). and an adult cat (C). In the 3-d-dd [A), the pattern of CO activity is rdatryety homogeneous: in the 9-d-old (fl), h is difficuh to dtstnpish laminar stamng, but darUy stained neurons can be seen in layer V. In the adult, a laminar pattern can be seen, with reduced activity in layer I, darkly stained layers IWV, a pale layer V, and slightly rereased staining m layer VI Scale bar, 2 mm for A-C.
V
,
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Rgur» 1 0 . Higher-power view of CO naining in aduh [A and 0 and in M o l d (B and 0) cat somatosensory cortex. Seams are taken at the white matter-layer VI border. In the aduh (A C\. daridy stained neurons can be seen pratarunartfty in layer V. In the 9-d-otd somatosansory cortex [B, 01. numerous darkly staned cells can be seen at the layer Vl-whne mans border aid in the whne matter Scale bar, 500 IMtorA-D
of AChE+ fibers observed in this study. Thus, at least at the developmental stages analyzed presently, the AChE innervation and thalamocortical afferent terminations appear distinctly different. It is also possible that a transient rise in AChE staining occurs earlier in development than the times we studied. For example, Kostovic and Goldman-Rakic (1983) found
a transient increase in cholinesterase staining in the mediodorsal thalamus and frontal cortex of fetal monkeys and humans. The functional significance of ACh in the developing somatosensory cortex has yet to be determined. Normal cholinergic innervation appears to be necessary in the adult for standard processing of stimuli Cerebral Cortex May/June 1992, V 2 N 3 2*1
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(Metherate et al., 1987; Ma et al., 1989; Dykes, 1990; Juliano et al., 1990a; Jacobs et al., 1991) and is also necessary for the plasticity resulting in the expansion of topographic maps in adult somatosensory cortex (Juliano et al., 1991b). It is therefore highly likely that ACh plays an important role in developing cat somatosensory cortex, as it does in the visual system (Bear and Singer, 1986). In the newborn mouse, depletion of ACh results in abnormal cytoarchitecture, cortical lamination, and connectivity (Hohmann et al., 1988, 1991). These data suggest that ACh may make an important contribution to normal cortical morphogenesis and synaptogenesis in developing somatosensory cortex. Its specific impact on stimulus processing and plasticity in developing cat cortex is currently being investigated in our laboratory.
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Notes We thank Don Eslin for his expert technical assistance. We also thank Dr Christine HShmann for helpful comments on the manuscript. This work was supported by PHS-R01 NS24014. Correspondence should be addressed to Sharon L. Juliano, Ph.D., Department of Anatomy and Cell Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814.
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