- Email: [email protected]
Distribution of neurons immunoreactive for calcium-binding proteins varies across areas of cat primary somatosensory cortex
Brain Research Bulletin, Vol. 51, No. 5, pp. 379 –385, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/00/$–see front matter
PII S0361-9230(99)00250-6
Distribution of neurons immunoreactive for calciumbinding proteins varies across areas of cat primary somatosensory cortex Harris D. Schwark* and Jianying Li Department of Biology, University of North Texas, Denton, TX, USA [Received 27 July 1999; Revised 21 September 1999; Accepted 16 October 1999] ABSTRACT: The primary somatosensory (SI) cortex in the cat contains four cytoarchitectonic areas that appear to contain separate body representations and have different functions. We tested whether functional differences among these areas are reflected in the densities of neurons containing each of three calcium-binding proteins: parvalbumin (PV), calbindin (CB), and calretinin (CR). Colocalization experiments revealed that CR was localized in a population of neurons distinct from those containing PV or CB. The general laminar distributions of the three calcium-binding proteins were similar to those described in other species and cortical areas, but there were significant density differences in layers II and III across SI. The density of PV-immunoreactive neurons was higher in areas 3b and 1 than in areas 3a and 2. CB-immunoreactive neurons were found in higher densities in anterior SI than in posterior SI, and the pattern of CR-immunoreactive neurons was reciprocal to that of CB, with significantly higher densities in posterior regions of SI. Since the firing characteristics of nonpyramidal neurons appear to be related to their calcium-binding protein content, differences in regional distributions of these neurons in layers II and III may contribute to functional differences between the cytoarchitectonic areas of SI cortex. © 2000 Elsevier Science Inc.
nonpyramidal cortical neurons, patterns of action-potential firing are related to the content of calcium-binding proteins. All nonpyramidal neurons in the rat appear to express at least one calciumbinding protein from the group of parvalbumin (PV), calbindin (CB), and calretinin (CR) [7], and these proteins may mark functional classes of neurons. PV has been localized in fast-spiking [7,39,40], metabolically active [47] large basket cells, and chandelier cells [11,12]. CB has been localized in regular spiking [39,40] and some fast-spiking [7] double-bouquet cells [11,12], whereas CR has been localized in burst spiking [40] or irregular spiking [7] bipolar cells, double-bouquet cells, and Cajal-Retzius cells [12]. Although there appears to be little overlap in the populations of neurons that are immunoreactive for these calciumbinding proteins [11,12,25,56,61], RT-PCR analysis of nonpyramidal neurons in the rat suggests substantial colocalization [7]. If differences in the numbers of neurons in SI cortex with particular firing patterns contribute to functional differences among cytoarchitectonic areas, then there should also be differences in the distribution of neurons expressing each of the calcium-binding proteins. To test this idea, we counted the numbers of neurons in each area that are immunoreactive for PV, CR, or CB. Most comparisons of the numbers and distributions of neurons containing calcium-binding proteins across cortical areas have been based on qualitative observations [28 –31,60], and none have compared areas of SI cortex. The use of cat SI cortex for such an analysis has its advantages: each of the four cytoarchitectonic areas of SI cortex receive substantial, direct inputs from the ventroposterior nucleus of the thalamus [37], yet its neurons differ in stimulus selectivity and response characteristics [18,20,33,34]. The results of the present study revealed significant differences between cortical areas in the density of immunoreactive neurons located in layers II and III. Colocalization experiments revealed little overlap in the population of neurons immunoreactive for CR, and those immunoreactive for PV or CB.
KEY WORDS: Parvalbumin, Calbindin D28K, Calretinin, Nonpyramidal neurons.
INTRODUCTION The primary somatosensory (SI) cortex of the cat contains four cytoarchitectonic areas [4,6,26] that form specific corticocortical interconnections [53,57], and that may contain separate body representations [4,19,34,42,52,62] (see also [21,24,48]). There are functional differences in the response properties of neurons in these areas: in general, area 3a neurons respond to muscle afferents, neurons in areas 3b and 1 respond to low-threshold cutaneous receptors, and area 2 neurons respond to deep receptors but with signs of convergence of receptor types [19,34,38,51,52,59]. Functional differences among the cytoarchitectonic areas of SI might arise from specific patterns of thalamic and intracortical connectivity, as well as from differences in the intrinsic physiology of neurons (defined by patterns of action-potential firing [3]). In
MATERIALS AND METHODS The materials used in this study were derived from seven adult cats. Each animal was injected with ketamine (30 mg/kg, i.m.), given an overdose of pentobarbital (60 mg/kg, i.p.), and then
* Address for correspondence: Dr. Harris D. Schwark, Department of Biology, University of North Texas, P.O. Box 305220, Denton, TX 76203, USA. Fax: ⫹1-940-565-4136; E-mail: [email protected]
379
380
SCHWARK AND LI
perfused through the heart with saline, followed by a fixative solution containing 2% paraformaldehyde and 0.15% glutaraldehyde in 0.1 M phosphate buffer. Glutaraldehyde was added to the fixative to facilitate localization of GABA-immunoreactive neurons in other experiments. The brain was removed and postfixed for 4 – 6 h at 4°C. The brain was then blocked and the blocks were placed in 4°C of phosphate buffer containing 30% sucrose until they sank, after which they were rapidly frozen in ⫺35°C isopentane. Blocks containing the forepaw representation in SI (defined based on previous anatomical and physiological experiments in this and other laboratories [19 –21,42,48,57,58]) were sectioned on a sliding microtome in the parasagittal plane. Because sections cut in this plane contained all four cytoarchitectonic areas of SI, we could make within section comparisons, thereby reducing the effects of variability that might be introduced by staining sections in separate batches. Four series of 16-m sections were collected in cold phosphate-buffered saline (PBS): three series for detection of calcium-binding proteins, and one series for thionin staining to determine laminar and cytoarchitectonic borders. Immunocytochemical Staining Free-floating sections were washed for 10 min in 4°C PBS containing 0.4% Triton X-100, and incubated for 2 h in 4°C PBS containing 3% blocking serum and 0.3% Triton X-100. The sections were then transferred to a PBS solution containing 0.1% blocking serum, 0.1% Triton X-100, and antibodies to one of the
three calcium-binding proteins: mouse monoclonal antiparvalbumin (1:12000, Sigma, St. Louis, MO, USA), mouse monoclonal anticalbindin D-28K (1:1000, Sigma), or mouse polyclonal anticalretinin (1:8000, a gift from Dr. M. R. Celio). After 24 h the sections were washed in 4°C PBS for 10 min (five changes) and then incubated in a 1:100 solution of biotinylated immunoglobulins (Vector, Burlingame, CA, USA) at 4°C for 1 h. The sections were washed for 10 min in room temperature PBS (three changes) and incubated in avidin– biotin peroxidase complex (Vector) for 1 h. After another series of washes the sections were reacted in PBS containing 0.05% DAB, 0.026% H2O2, and 0.02% nickel ammonium sulfate. The sections were then washed several times in PBS, mounted on slides, and coverslipped. Four additional sets of sections were stained using immunofluorescent methods to colocalize calcium-binding proteins. Data Collection and Analysis The locations of neurons containing calcium-binding proteins, together with the laminar and cytoarchitectonic boundaries in SI (from adjacent thionin sections), were plotted from four series of six alternate sections (80 m between sections) using a camera lucida. The plots were entered into a computer using a digitizing tablet. Each series was taken from a different hemisphere. Criteria for identifying the boundaries of cytoarchitectonic areas were based on those of Hassler and Mu¨hs-Clement [26] as described by others [4,44,52,57]. To determine the densities of labeled neurons, the area of each layer within each cytoarchitectonic area was
FIG. 1. Photomicrographs illustrating the laminar distributions of neurons in area 3b that were immunoreactive for each of the three calcium-binding proteins. PV neurons were located primarily in the middle cortical layers, whereas CB and CR neurons were located primarily in the superficial layers.
CALCIUM-BINDING PROTEINS IN CAT SI
381 and PV or CB, were determined using a fluorescent microscope and switching filters to identify double-labeled neurons. Neurons were sampled across all six cortical layers and in each of the four cytoarchitectonic areas of SI. RESULTS Distributions of Neurons Containing Calcium-binding Proteins
FIG. 2. Size distribution of labeled neurons. PV neurons were significantly larger than CB or CR neurons (p ⬍ 0.001).
measured (subtracting the area of major blood vessels), and the number of labeled neurons per square millimeter was calculated. Average soma diameters were calculated from drawings of labeled cell somata. Differences in soma diameters were tested using Kruskal-Wallis one-way analysis of variance based on ranks. Differences in the densities of labeled neurons were assessed using Friedman’s two-way analyses of variance based on ranks. Post hoc tests for multiple comparisons were done using Dunn’s method and the Student-Newman-Keuls test, respectively, with alpha set at 0.05. The proportions of neurons that were double labeled for CR,
Neurons immunoreactive for calcium-binding proteins could be easily distinguished from the background (Fig. 1). The average diameter of PV-immunoreactive neurons was 12.6 ⫾ 0.3 m (SEM) and was significantly larger than neurons immunoreactive for CB (8.5 ⫾ 0.4) or CR (9.0 ⫾ 0.4) (H(2) ⫽ 61.46, p ⬍ 0.001; Fig. 2). As described by others [11,12], the larger PV neurons had features typical of large basket neurons, and were located mainly in deep layer III, layer IV, and layer VI. CB and CR neurons were small-to-medium in size, with average diameters of 8.3 ⫾ 0.4 and 9.0 ⫾ 0.4 m, respectively. Many of the CB neurons resembled double-bouquet neurons, and CR neurons appeared to be small bipolar neurons or Cajal-Retzius neurons. Examples of immunofluorescent labeling of calcium-binding proteins are shown in Fig. 3. None of the CR neurons (n ⫽ 247) contained PV, and only one of the PV neurons (n ⫽ 400) contained CR. Similarly, no CR neurons (n ⫽ 228) contained CB, and only two of the CB neurons (n ⫽ 151) contained CR. The laminar distributions of immunoreactive neurons were distinct for each calcium-binding protein (Table 1 and Fig. 4). PV neurons were located in all cortical layers except layer I, with the highest density in layer IV. The density of PV neurons in layer III varied significantly across cytoarchitectonic areas (Fig. 5; 2(3) ⫽ 9.9, p ⬍ 0.01; areas 3b and 1 ⬎ area 3a, area 3b ⬎ area 2). The laminar distribution of CB neurons was very different from that of PV neurons (Fig. 4). The highest density of CB neurons was
FIG. 3. Photomicrographs of fluorescent images illustrating the lack of colocalization of CR with PV (A) or CB (B).
382
SCHWARK AND LI TABLE 1
DENSITIES OF CALCIUM BINDING PROTEIN IMMUNOREACTIVE NEURONS IN SI CORTEX (NUMBER/MM2) Layer
Area 3a
Area 3b
Area 1
Area 2
Parvalbumin I II III IV V VI
0 113 123 187 94 56
0 125 141 209 89 67
0 142 151 210 104 62
0 114 116 184 75 61
Calbindin I II III IV V VI
7 110 41 4 2 1
5 120 31 2 2 1
3 65 13 2 3 1
2 81 18 3 4 3
Calretinin I II III IV V VI
105 110 36 20 31 7
123 131 47 33 20 5
117 139 61 30 18 5
126 151 63 31 22 6
in layer II, followed by layer III. Layers I and IV–VI contained few CB neurons with scattered cells in layers V and VI (Fig. 1). Differences in CB neuron densities across cytoarchitectonic areas were seen in layers II (2(3) ⫽ 9.9, p ⬍ 0.01; area 3a ⬎ area 2, area 3b ⬎ areas 1 and 2) and III (Fig. 4; 2(3) ⫽ 9.9, p ⬍ 0.01; area 3a ⬎ areas 3b, 1, and 2). The highest density of CR neurons was in layers I and II. The densities in the remaining layers declined with depth in the cortex. Significant differences in the densities of CR neurons across cytoarchitectonic areas were seen in layer III (Fig. 5; 2(3) ⫽ 11.1, p ⬍ 0.01; areas 1 and 2 ⬎ areas 3a and 3b, area 3b ⬎ area 3a). The diverse patterns of labeling for calcium-binding proteins are summarized in Fig. 5, which illustrates the median densities of the calcium-binding protein neurons in layer III as proportions of the densities in area 3b. The density of PV neurons in layer III was highest in area 1 (113% of area 3b). The densities of CB neurons in areas 1 and 2 were 54% and 68%, respectively, of that in area 3b. The density of CR neurons increased from area 3a (84% of area 3b) to area 2 (115% of area 3b). Variations across areas were also seen in other cortical layers, but only those in layers II and III reached statistical significance (see above). A comparison of the densities of immunoreactive neurons by cortical layer is presented in Fig. 4. Almost all immunoreactive neurons in layer I were CR. In layer II the densities of neurons containing each of the three calcium-binding proteins were similar. Layer III also contained neurons immunoreactive for each of the three calcium-binding proteins, but PV neurons were found to have higher densities. PV neurons dominated the distributions of calcium-binding proteins in the remaining layers. DISCUSSION There is little overlap between PV and CB neurons in cat cortex [15,17,27], but colocalization of CR with PV or CB has not been
FIG. 4. Average density of labeled neurons (⫾ SEM), by layer, across areas of SI cortex. Analysis of variance based on ranks revealed significant differences across cortical areas in layers II (for CB) and III (for all three calcium-binding proteins). See text for details.
CALCIUM-BINDING PROTEINS IN CAT SI
383 [2,16,32]. The laminar distribution of CR neurons in SI cortex was similar to that reported for cat visual cortex [35] and for cortical areas in other species [10,22,46,49]. Most previous comparisons of calcium-binding proteins in different cortical areas have relied on qualitative observations. Studies based on quantitative methods report significant differences across prefrontal cortical areas in the rat [23] and between rat SI and motor cortex [60] (for PV, but not CB). No differences were found across prefrontal cortical areas in monkey [10,22]. The present study is the first to describe quantitative differences in calcium-binding proteins between primary areas of a sensory cortex. The densities of immunoreactive neurons in layers II and III varied significantly across the cytoarchitectonic areas of SI cortex. PV showed the lowest variation, with densities in layer III ranging from 90 to 113% of the density of PV neurons in area 3b. CB and CR exhibited greater variation and their patterns were reciprocal: anterior areas of SI (3a and 3b) contained more CB neurons and fewer CR neurons than posterior areas of SI. Areas 1 and 2 contained only 46% and 32%, respectively, as many CB neurons as area 3b, but 106% and 115%, respectively, as many CR neurons. To estimate the proportion of immunoreactive neurons in some of these areas, we used data on total neuron numbers in areas 2 and 3b from unrelated experiments (unpublished). Based on these data, layer III in area 2 contains approximately 19% more neurons than area 3b. This difference in total number of neurons could account for the 15% difference in the density of CR neurons, and makes the differences in PV and CB neuron density even larger (raising the values to 130% and 175%, respectively). Correlation of Distribution Patterns with Function
FIG. 5. Variation in the median densities of labeled neurons in layer III across SI cortex. The median densities in area 3b were used as baseline (100%).
previously examined in cat. There is apparently little overlap in the populations of SI cortical neurons that are immunoreactive for the three calcium-binding proteins: in the present study, CR was colocalized with PV or CB in very few neurons. This is in agreement with the results from rat [25,41,56] and monkey [10], while in the human there appears to be slightly higher incidences of colocalization [13,14,43]. Distribution of SI Cortical Neurons Containing Calcium-binding Proteins The most obvious difference in the distributions of neurons immunoreactive for PV, CB, or CR was seen across laminae. PV neurons were located throughout layers II–VI, with the highest densities in layers III and IV—a pattern similar to those reported previously for other sensory cortical areas in the cat [1,2,15,16, 27,32], monkey [5,28], and rat [60]. CB neurons were concentrated primarily in superficial cortical layers (II and III), with scattered labeled cells in deeper cortical layers (V and VI). This pattern was consistent with those reported for other cortical areas in the cat
There have been many attempts to correlate the morphology of single neurons with their content of calcium-binding proteins (reviewed in [11,12]). In cat, calcium-binding proteins appear to be contained almost exclusively in nonpyramidal neurons [15,27]. In general, PV is localized in large basket cells and chandelier cells; CB is localized in double-bouquet cells; and CR is localized in bipolar, double-bouquet, and Cajal-Retzius cells [11,12,30]. These morphological classes form specific connections with other neurons in the cortical circuit. For example, chandelier neurons synapse with axon initial segments of pyramidal neurons, and basket neurons synapse near somata [12]. Although the precise physiological roles of calcium-binding proteins in neuronal function are not known, it has been suggested that they may influence patterns of neuronal discharge [40] as a consequence of their calcium-buffering properties [9,45]. Consistent with this idea, there appears to be a relationship between a neuron’s firing characteristics and its calcium-binding protein content (e.g., [40]). PV has been localized in fast-spiking neurons [7,39,40], CB has been localized in regular spiking [39,40] and some fast-spiking neurons [7], and CR has been localized in burst spiking [40] or irregular spiking [7] bipolar neurons. Thus, neurons containing the three calcium-binding proteins examined in the present study have distinctive firing patterns and form specific anatomical connections within the cortical circuit. Indeed, Celio [8] has suggested that PV and CB might be associated with separate functional systems in the rat brain, and it has been proposed that calcium-binding proteins mark separate subcortical and thalamocortical pathways in the primate somatosensory [54, 55], auditory [50], and visual [36] systems. For example, in the somatosensory system PV appears to be localized in specific thalamocortical projections to the middle cortical layers, whereas CB is found in more diffuse thalamocortical projections that project to more superficial cortical layers, including layer I [54,55]. The present study is one of the first to quantitatively evaluate
384
SCHWARK AND LI
calcium-binding proteins in the context of functional cortical circuits. Variations in these cortical circuits, reflected by differences in the number of neurons containing calcium-binding proteins, may be related to functional differences between cortical areas. The high fidelity responses to light tactile stimulation seen in neurons in areas 3b and 1 [19,51,52] might be related to their higher density of CB and PV neurons, which are primarily regular spiking and fast-spiking neurons, respectively. Area 2, which contains neurons that respond to deep receptors [19,34], contained the highest density of CR neurons, which are irregular spiking neurons. This relationship suggests that the characteristic firing patterns of peripheral receptors might be reinforced by similar characteristics of central neurons, perhaps related to their content of calcium-binding proteins. ACKNOWLEDGEMENTS
15.
16.
17.
18. 19.
This research was supported by NIH grant NS25729 to H.D.S. The authors thank Dr. M.R. Celio for the antibody to calretinin, and Drs. Jannon Fuchs and Michael Pettit for their constructive comments.
20.
REFERENCES
21.
1. Alca´ntara, S.; Ferrer, I. Postnatal development of parvalbumin immunoreactivity in the cerebral cortex of the cat. J. Comp. Neurol. 348: 133–149; 1994. 2. Alca´ntara, S.; Ferrer, I. Postnatal development of calbindin-D28k immunoreactivity in the cerebral cortex of the cat. Anat. Embryol. (Berl.) 192:369 –384; 1995. 3. Amitai, Y.; Connors, B. W. Intrinsic physiology and morphology of single neurons in neocortex. In: Jones, E. G.; Diamond, I. T., eds. Cerebral cortex, vol. 11. The barrel cortex of rodents. New York: Plenum Press; 1994:299 –331. 4. Avendan˜o, C.; Verdu, A. Area 3a in the cat. I. A reevaluation of its location and architecture on the basis of nissl, myelin, acetylcholinesterase, and cytochrome oxidase staining. J. Comp. Neurol. 321:357– 372; 1992. 5. Blu¨mcke, I.; Hof, P. R.; Morrison, J. H.; Celio, M. R. Distribution of parvalbumin immunoreactivity in the visual cortex of Old World monkeys and humans. J. Comp. Neurol. 301:417– 432; 1990. 6. Brodmann, K. Beitrage zur histologischen Lokalisation der Grosshirnrinde. V. Uber den allgemeinen Bauplan des Cortex pallii bei den Mammaliern und zwei homologe Rindenfelder im Besonderen. Zugleich ein Beitrag zur Furchenlehre. J. Psych. Neurol. 6:275– 400; 1906. 7. Cauli, B.; Audinat, E.; Lambolez, B.; Angulo, M. C.; Ropert, N.; Tsuzuki, K.; Hestrin, S.; Rossier, J. Molecular and physiological diversity of cortical nonpyramidal cells. J. Neurosci. 17:3894 –3906; 1997. 8. Celio, M. R. Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35:375– 475; 1990. 9. Chard, P. S.; Bleakman, D.; Christakos, S.; Fullmer, C. S.; Miller, R. J. Calcium buffering properties of calbindin D28k and parvalbumin in rat sensory neurones. J. Physiol. (Lond.) 472:341–357; 1993. 10. Conde´, F.; Lund, J. S.; Jacobowitz, D. M.; Baimbridge, K. G.; Lewis, D. A. Local circuit neurons immunoreactive for calretinin, calbindin D-28k or parvalbumin in monkey prefrontal cortex: Distribution and morphology. J. Comp. Neurol. 341:95–116; 1994. 11. DeFelipe, J. Neocortical neuronal diversity: Chemical heterogeneity revealed by colocalization studies of classic neurotransmitters, neuropeptides, calcium-binding proteins, and cell surface molecules. Cereb. Cortex 3:273–289; 1993. 12. DeFelipe, J. Types of neurons, synaptic connections and chemical characteristics of cells immunoreactive for calbindin-D28K, parvalbumin and calretinin in the neocortex. J. Chem. Neuroanat. 14:1–19; 1997. 13. Del Rı´o, M. R.; DeFelipe, J. Colocalization of parvalbumin and calbindin D-28k in neurons including chandelier cells of the human temporal neocortex. J. Chem. Neuroanat. 12:165–173; 1997a. 14. Del Rı´o, M. R.; DeFelipe, J. Double bouquet cell axons in the human
22.
23.
24.
25. 26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
temporal neocortex: Relationship to bundles of myelinated axons and colocalization of calretinin and calbindin D-28k immunoreactivities. J. Chem. Neuroanat. 13:243–251; 1997b. Demeulemeester, H.; Arckens, L.; Vandesande, F.; Orban, G. A.; Heizmann, C. W.; Pochet, R. Calcium binding proteins and neuropeptides as molecular markers of GABAergic interneurons in the cat visual cortex. Exp. Brain Res. 84:538 –544; 1991. Demeulemeester, H.; Vandesande, F.; Orban, G. A.; Brandon, C.; Vanderhaeghen, J. J. Heterogeneity of GABAergic cells in cat visual cortex. J. Neurosci. 8:988 –1000; 1988. Demeulemeester, H.; Vandesande, F.; Orban, G. A.; Heizmann, C. W.; Pochet, R. Calbindin D-28K and parvalbumin immunoreactivity is confined to two separate neuronal subpopulations in the cat visual cortex, whereas partial coexistence is shown in the dorsal lateral geniculate nucleus. Neurosci. Lett. 99:6 –11; 1989. Dykes, R. W. Parallel processing of somatosensory information: A theory. Brain Res. Rev. 6:47–115; 1983. Dykes, R. W.; Rasmusson, D. D.; Hoeltzell, P. B. Organization of primary somatosensory cortex in the cat. J. Neurophysiol. 43:1527– 1546; 1980. Esteky, H.; Schwark, H. D. Responses of rapidly adapting neurons in cat primary somatosensory cortex to constant-velocity mechanical stimulation. J. Neurophysiol. 72:2269 –2279; 1994. Felleman, D. J.; Wall, J. T.; Cusick, C. G.; Kaas, J. H. The representation of the body surface in SI of cats. J. Neurosci. 3:1648 –1669; 1983. Gabbott, P. L.; Bacon, S. J. Local circuit neurons in the medial prefrontal cortex (areas 24a,b,c, 25 and 32) in the monkey: II. Quantitative areal and laminar distributions. J. Comp. Neurol. 364:609 – 636; 1996. Gabbott, P. L.; Dickie, B. G.; Vaid, R. R.; Headlam, A. J.; Bacon, S. J. Local-circuit neurones in the medial prefrontal cortex (areas 25, 32 and 24b) in the rat: Morphology and quantitative distribution. J. Comp. Neurol. 377:465– 499; 1997. Garraghty, P. E.; Pons, T. P.; Huerta, M. F.; Kaas, J. H. Somatotopic organization of the third somatosensory area (SIII) in cats. Somatosens. Res. 4:333–357; 1987. Gonchar, Y.; Burkhalter, A. Three distinct families of GABAergic neurons in rat visual cortex. Cereb. Cortex 7:347–358; 1997. Hassler, R.; Mu¨hs-Clement, K. Architektonischer Aufbau des sensorimotorischen und parietalen Cortex der Katze. J. Hirnforsch. 6:377– 420; 1964. Hendry, S. H. C.; Jones, E. G. GABA neuronal subpopulations in cat primary auditory cortex: Co-localization with calcium binding proteins. Brain Res. 543:45–55; 1991. Hendry, S. H. C.; Jones, E. G.; Emson, P. C.; Lawson, D. E. M.; Heizmann, C. W.; Streit, P. Two classes of cortical GABA neurons defined by differential calcium binding protein immunoreactivities. Exp. Brain Res. 76:467– 472; 1989. Hof, P. R.; Bogaert, Y. E.; Rosenthal, R. E.; Fiskum, G. Distribution of neuronal populations containing neurofilament protein and calciumbinding proteins in the canine neocortex: Regional analysis and cell typology. J. Chem. Neuroanat. 11:81–98; 1996. Hof, P. R.; Glezer, I. I.; Conde, F.; Flagg, R. A.; Rubin, M. B.; Nimchinsky, E. A.; Weisenhorn, D. V. Cellular distribution of the calcium binding proteins parvalbumin, calbindin, and calretinin in the neocortex of mammals: Phylogenetic and developmental patterns. J. Chem. Neuroanat. 16:77–116; 1999. Hof, P. R.; Nimchinsky, E. A. Regional distribution of neurofilament and calcium-binding proteins in the cingulate cortex of the macaque monkey. Cereb. Cortex 2:456 – 467; 1992. Hogan, D.; Berman, N. E. J. The development of parvalbumin and calbindin-D28k immunoreactive interneurons in kitten visual cortical areas. Dev. Brain Res. 77:1–21; 1994. Iwamura, Y.; Tanaka, M. Functional organization of receptive fields in the cat somatosensory cortex. I: Integration within the coronal region. Brain Res. 151:49 – 60; 1978a. Iwamura, Y.; Tanaka, M. Functional organization of receptive fields in the cat somatosensory cortex. II: Second representation of the forepaw in the ansate region. Brain Res. 151:61–72; 1978b. Jeon, C. J.; Park, H. J. Immunocytochemical localization of calcium-
CALCIUM-BINDING PROTEINS IN CAT SI
36. 37. 38. 39.
40. 41. 42.
43. 44. 45. 46.
47. 48.
49. 50.
binding protein calretinin containing neurons in cat visual cortex. Mol. Cell 7:721–725; 1997. Johnson, J. K.; Casagrande, V. A. Distribution of calcium-binding proteins within the parallel visual pathways of a primate (Galago crassicaudatus). J. Comp. Neurol. 356:238 –260; 1995. Jones, E. G. The thalamus. New York: Plenum; 1985. Jones, E. G.; Porter, R. What is area 3a? Brain Res. Rev. 2:1– 43; 1980. Kawaguchi, Y.; Kubota, Y. Correlation of physiological subgroupings of nonpyramidal cells with parvalbumin- and calbindinD28k-immunoreactive neurons in layer V of rat frontal cortex. J. Neurophysiol. 70:387–396; 1993. Kawaguchi, Y.; Kubota, Y. GABAergic cell subtypes and their synaptic connections in rat frontal cortex. Cereb. Cortex 7:476 – 486; 1997. Kubota, Y.; Hattori, R.; Yui, Y. Three distinct subpopulations of GABAergic neurons in rat frontal agranular cortex. Brain Res. 649: 159 –173; 1994. Leclerc, S. S.; Avendan˜o, C.; Dykes, R. W.; Waters, R. S.; Salimi, I. Reevaluation of area 3b in the cat based on architectonic and electrophysiological studies: Regional variability with functional and anatomical consistencies. J. Comp. Neurol. 341:357–374; 1994. Leuba, G.; Saini, K. Colocalization of parvalbumin, calretinin and calbindin D-28k in human cortical and subcortical visual structures. J. Chem. Neuroanat. 13:41–52; 1997. Li, J.; Schwark, H. D. Distribution and proportions of GABA-immunoreactive neurons in cat primary somatosensory cortex. J. Comp. Neurol. 343:353–361; 1994. Lukas, W.; Jones, K. A. Cortical neurons containing calretinin are selectively resistant to calcium overload and excitotoxicity in vitro. Neuroscience 61:307–316; 1994. Lu¨th, H.-J.; Blu¨mcke, I.; Winkelmann, E.; Celio, M. R. The calciumbinding protein calretinin is localized in a subset of interneurons in the rat cerebral cortex: A light and electron immunohistochemical study. J. Hirnforsch. 34:93–103; 1993. Maier, D. L.; McCasland, J. S. Calcium-binding protein phenotype defines metabolically distinct groups of neurons in barrel cortex of behaving hamsters. Exp. Neurol. 145:71– 80; 1997. McKenna, T. M.; Whitsel, B. L.; Dreyer, D. A.; Metz, C. B. Organization of cat anterior parietal cortex: Relations among cytoarchitecture, single neuron functional properties, and interhemispheric connectivity. J. Neurophysiol. 45:667– 697; 1981. Meskenaite, V. Calretinin-immunoreactive local circuit neurons in area 17 of the cynomolgus monkey, Macaca fascicularis. J. Comp. Neurol. 379:113–132; 1997. Molinari, M.; Dell’Anna, M. E.; Rausell, E.; Leggio, M. G.; Hashi-
385
51. 52. 53. 54.
55.
56. 57. 58. 59. 60.
61.
62.
kawa, T.; Jones, E. G. Auditory thalamocortical pathways defined in monkeys by calcium-binding protein immunoreactivity. J. Comp. Neurol. 362:171–194; 1995. Mountcastle, V. B. Modality and topographic properties of single neurons of cat’s somatic sensory cortex. J. Neurophysiol. 20:408 – 434; 1957. Myasnikov, A. A.; Dykes, R. W.; Avendan˜o, C. Cytoarchitecture and responsiveness of the medial ansate region of the cat primary somatosensory cortex. J. Comp. Neurol. 349:401– 427; 1994. Porter, L. L.; Izraeli, R. The effects of localized inactivation of somatosensory cortex, area 3a, on area 2 in cats. Somatosens. Mot. Res. 10:399 – 413; 1993. Rausell, E.; Bae, C. S.; Vin˜uela, A.; Huntley, G. W.; Jones, E. G. Calbindin and parvalbumin cells in monkey VPL thalamic nucleus: Distribution, laminar cortical projections, and relations to spinothalamic terminations. J. Neurosci. 12:4088 – 4111; 1992. Rausell, E.; Jones, E. G. Chemically distinct compartments of the thalamic VPM nucleus in monkeys relay principal and spinal trigeminal pathways to different layers of the somatosensory cortex. J. Neurosci. 11:226 –237; 1991. Rogers, J. H.; Resibois, A. Calretinin and calbindin-D28k in rat brain: Patterns of partial co-localization. Neuroscience 51:843– 865; 1992. Schwark, H. D.; Esteky, H.; Jones, E. G. Corticocortical connections of cat primary somatosensory cortex. Exp. Brain Res. 91:425– 434; 1992. Schwark, H. D.; Jones, E. G. The distribution of intrinsic cortical axons in area 3b of cat primary somatosensory cortex. Exp. Brain Res. 78:501–513; 1989. Sretavan, D.; Dykes, R. W. The organization of two cutaneous submodalities in the forearm region of area 3b of cat somatosensory cortex. J. Comp. Neurol. 213:381–398; 1983. Van Brederode, J. F. M.; Helliesen, M. K.; Hendrickson, A. E. Distribution of the calcium-binding proteins parvalbumin and calbindinD28k in the sensorimotor cortex of the rat. Neuroscience 44:157–171; 1991. Vickers, J. C.; Huntley, G. W.; Edwards, A. M.; Moran, T.; Rogers, S. W.; Heinemann, S. F.; Morrison, J. H. Quantitative localization of AMPA/kainate and kainate glutamate receptor subunit immunoreactivity in neurochemically identified subpopulations of neurons in the prefrontal cortex of the macaque monkey. J. Neurosci. 13:2982–2992; 1993. Waters, R. S.; Favorov, O.; Asanuma, H. Physiological properties and pattern of projection of cortico-cortical connections from the anterior bank of the ansate sulcus to the motor cortex, area 4g, in the cat. Exp. Brain Res. 46:403– 412; 1982.
PII S0361-9230(99)00250-6
Distribution of neurons immunoreactive for calciumbinding proteins varies across areas of cat primary somatosensory cortex Harris D. Schwark* and Jianying Li Department of Biology, University of North Texas, Denton, TX, USA [Received 27 July 1999; Revised 21 September 1999; Accepted 16 October 1999] ABSTRACT: The primary somatosensory (SI) cortex in the cat contains four cytoarchitectonic areas that appear to contain separate body representations and have different functions. We tested whether functional differences among these areas are reflected in the densities of neurons containing each of three calcium-binding proteins: parvalbumin (PV), calbindin (CB), and calretinin (CR). Colocalization experiments revealed that CR was localized in a population of neurons distinct from those containing PV or CB. The general laminar distributions of the three calcium-binding proteins were similar to those described in other species and cortical areas, but there were significant density differences in layers II and III across SI. The density of PV-immunoreactive neurons was higher in areas 3b and 1 than in areas 3a and 2. CB-immunoreactive neurons were found in higher densities in anterior SI than in posterior SI, and the pattern of CR-immunoreactive neurons was reciprocal to that of CB, with significantly higher densities in posterior regions of SI. Since the firing characteristics of nonpyramidal neurons appear to be related to their calcium-binding protein content, differences in regional distributions of these neurons in layers II and III may contribute to functional differences between the cytoarchitectonic areas of SI cortex. © 2000 Elsevier Science Inc.
nonpyramidal cortical neurons, patterns of action-potential firing are related to the content of calcium-binding proteins. All nonpyramidal neurons in the rat appear to express at least one calciumbinding protein from the group of parvalbumin (PV), calbindin (CB), and calretinin (CR) [7], and these proteins may mark functional classes of neurons. PV has been localized in fast-spiking [7,39,40], metabolically active [47] large basket cells, and chandelier cells [11,12]. CB has been localized in regular spiking [39,40] and some fast-spiking [7] double-bouquet cells [11,12], whereas CR has been localized in burst spiking [40] or irregular spiking [7] bipolar cells, double-bouquet cells, and Cajal-Retzius cells [12]. Although there appears to be little overlap in the populations of neurons that are immunoreactive for these calciumbinding proteins [11,12,25,56,61], RT-PCR analysis of nonpyramidal neurons in the rat suggests substantial colocalization [7]. If differences in the numbers of neurons in SI cortex with particular firing patterns contribute to functional differences among cytoarchitectonic areas, then there should also be differences in the distribution of neurons expressing each of the calcium-binding proteins. To test this idea, we counted the numbers of neurons in each area that are immunoreactive for PV, CR, or CB. Most comparisons of the numbers and distributions of neurons containing calcium-binding proteins across cortical areas have been based on qualitative observations [28 –31,60], and none have compared areas of SI cortex. The use of cat SI cortex for such an analysis has its advantages: each of the four cytoarchitectonic areas of SI cortex receive substantial, direct inputs from the ventroposterior nucleus of the thalamus [37], yet its neurons differ in stimulus selectivity and response characteristics [18,20,33,34]. The results of the present study revealed significant differences between cortical areas in the density of immunoreactive neurons located in layers II and III. Colocalization experiments revealed little overlap in the population of neurons immunoreactive for CR, and those immunoreactive for PV or CB.
KEY WORDS: Parvalbumin, Calbindin D28K, Calretinin, Nonpyramidal neurons.
INTRODUCTION The primary somatosensory (SI) cortex of the cat contains four cytoarchitectonic areas [4,6,26] that form specific corticocortical interconnections [53,57], and that may contain separate body representations [4,19,34,42,52,62] (see also [21,24,48]). There are functional differences in the response properties of neurons in these areas: in general, area 3a neurons respond to muscle afferents, neurons in areas 3b and 1 respond to low-threshold cutaneous receptors, and area 2 neurons respond to deep receptors but with signs of convergence of receptor types [19,34,38,51,52,59]. Functional differences among the cytoarchitectonic areas of SI might arise from specific patterns of thalamic and intracortical connectivity, as well as from differences in the intrinsic physiology of neurons (defined by patterns of action-potential firing [3]). In
MATERIALS AND METHODS The materials used in this study were derived from seven adult cats. Each animal was injected with ketamine (30 mg/kg, i.m.), given an overdose of pentobarbital (60 mg/kg, i.p.), and then
* Address for correspondence: Dr. Harris D. Schwark, Department of Biology, University of North Texas, P.O. Box 305220, Denton, TX 76203, USA. Fax: ⫹1-940-565-4136; E-mail: [email protected]
379
380
SCHWARK AND LI
perfused through the heart with saline, followed by a fixative solution containing 2% paraformaldehyde and 0.15% glutaraldehyde in 0.1 M phosphate buffer. Glutaraldehyde was added to the fixative to facilitate localization of GABA-immunoreactive neurons in other experiments. The brain was removed and postfixed for 4 – 6 h at 4°C. The brain was then blocked and the blocks were placed in 4°C of phosphate buffer containing 30% sucrose until they sank, after which they were rapidly frozen in ⫺35°C isopentane. Blocks containing the forepaw representation in SI (defined based on previous anatomical and physiological experiments in this and other laboratories [19 –21,42,48,57,58]) were sectioned on a sliding microtome in the parasagittal plane. Because sections cut in this plane contained all four cytoarchitectonic areas of SI, we could make within section comparisons, thereby reducing the effects of variability that might be introduced by staining sections in separate batches. Four series of 16-m sections were collected in cold phosphate-buffered saline (PBS): three series for detection of calcium-binding proteins, and one series for thionin staining to determine laminar and cytoarchitectonic borders. Immunocytochemical Staining Free-floating sections were washed for 10 min in 4°C PBS containing 0.4% Triton X-100, and incubated for 2 h in 4°C PBS containing 3% blocking serum and 0.3% Triton X-100. The sections were then transferred to a PBS solution containing 0.1% blocking serum, 0.1% Triton X-100, and antibodies to one of the
three calcium-binding proteins: mouse monoclonal antiparvalbumin (1:12000, Sigma, St. Louis, MO, USA), mouse monoclonal anticalbindin D-28K (1:1000, Sigma), or mouse polyclonal anticalretinin (1:8000, a gift from Dr. M. R. Celio). After 24 h the sections were washed in 4°C PBS for 10 min (five changes) and then incubated in a 1:100 solution of biotinylated immunoglobulins (Vector, Burlingame, CA, USA) at 4°C for 1 h. The sections were washed for 10 min in room temperature PBS (three changes) and incubated in avidin– biotin peroxidase complex (Vector) for 1 h. After another series of washes the sections were reacted in PBS containing 0.05% DAB, 0.026% H2O2, and 0.02% nickel ammonium sulfate. The sections were then washed several times in PBS, mounted on slides, and coverslipped. Four additional sets of sections were stained using immunofluorescent methods to colocalize calcium-binding proteins. Data Collection and Analysis The locations of neurons containing calcium-binding proteins, together with the laminar and cytoarchitectonic boundaries in SI (from adjacent thionin sections), were plotted from four series of six alternate sections (80 m between sections) using a camera lucida. The plots were entered into a computer using a digitizing tablet. Each series was taken from a different hemisphere. Criteria for identifying the boundaries of cytoarchitectonic areas were based on those of Hassler and Mu¨hs-Clement [26] as described by others [4,44,52,57]. To determine the densities of labeled neurons, the area of each layer within each cytoarchitectonic area was
FIG. 1. Photomicrographs illustrating the laminar distributions of neurons in area 3b that were immunoreactive for each of the three calcium-binding proteins. PV neurons were located primarily in the middle cortical layers, whereas CB and CR neurons were located primarily in the superficial layers.
CALCIUM-BINDING PROTEINS IN CAT SI
381 and PV or CB, were determined using a fluorescent microscope and switching filters to identify double-labeled neurons. Neurons were sampled across all six cortical layers and in each of the four cytoarchitectonic areas of SI. RESULTS Distributions of Neurons Containing Calcium-binding Proteins
FIG. 2. Size distribution of labeled neurons. PV neurons were significantly larger than CB or CR neurons (p ⬍ 0.001).
measured (subtracting the area of major blood vessels), and the number of labeled neurons per square millimeter was calculated. Average soma diameters were calculated from drawings of labeled cell somata. Differences in soma diameters were tested using Kruskal-Wallis one-way analysis of variance based on ranks. Differences in the densities of labeled neurons were assessed using Friedman’s two-way analyses of variance based on ranks. Post hoc tests for multiple comparisons were done using Dunn’s method and the Student-Newman-Keuls test, respectively, with alpha set at 0.05. The proportions of neurons that were double labeled for CR,
Neurons immunoreactive for calcium-binding proteins could be easily distinguished from the background (Fig. 1). The average diameter of PV-immunoreactive neurons was 12.6 ⫾ 0.3 m (SEM) and was significantly larger than neurons immunoreactive for CB (8.5 ⫾ 0.4) or CR (9.0 ⫾ 0.4) (H(2) ⫽ 61.46, p ⬍ 0.001; Fig. 2). As described by others [11,12], the larger PV neurons had features typical of large basket neurons, and were located mainly in deep layer III, layer IV, and layer VI. CB and CR neurons were small-to-medium in size, with average diameters of 8.3 ⫾ 0.4 and 9.0 ⫾ 0.4 m, respectively. Many of the CB neurons resembled double-bouquet neurons, and CR neurons appeared to be small bipolar neurons or Cajal-Retzius neurons. Examples of immunofluorescent labeling of calcium-binding proteins are shown in Fig. 3. None of the CR neurons (n ⫽ 247) contained PV, and only one of the PV neurons (n ⫽ 400) contained CR. Similarly, no CR neurons (n ⫽ 228) contained CB, and only two of the CB neurons (n ⫽ 151) contained CR. The laminar distributions of immunoreactive neurons were distinct for each calcium-binding protein (Table 1 and Fig. 4). PV neurons were located in all cortical layers except layer I, with the highest density in layer IV. The density of PV neurons in layer III varied significantly across cytoarchitectonic areas (Fig. 5; 2(3) ⫽ 9.9, p ⬍ 0.01; areas 3b and 1 ⬎ area 3a, area 3b ⬎ area 2). The laminar distribution of CB neurons was very different from that of PV neurons (Fig. 4). The highest density of CB neurons was
FIG. 3. Photomicrographs of fluorescent images illustrating the lack of colocalization of CR with PV (A) or CB (B).
382
SCHWARK AND LI TABLE 1
DENSITIES OF CALCIUM BINDING PROTEIN IMMUNOREACTIVE NEURONS IN SI CORTEX (NUMBER/MM2) Layer
Area 3a
Area 3b
Area 1
Area 2
Parvalbumin I II III IV V VI
0 113 123 187 94 56
0 125 141 209 89 67
0 142 151 210 104 62
0 114 116 184 75 61
Calbindin I II III IV V VI
7 110 41 4 2 1
5 120 31 2 2 1
3 65 13 2 3 1
2 81 18 3 4 3
Calretinin I II III IV V VI
105 110 36 20 31 7
123 131 47 33 20 5
117 139 61 30 18 5
126 151 63 31 22 6
in layer II, followed by layer III. Layers I and IV–VI contained few CB neurons with scattered cells in layers V and VI (Fig. 1). Differences in CB neuron densities across cytoarchitectonic areas were seen in layers II (2(3) ⫽ 9.9, p ⬍ 0.01; area 3a ⬎ area 2, area 3b ⬎ areas 1 and 2) and III (Fig. 4; 2(3) ⫽ 9.9, p ⬍ 0.01; area 3a ⬎ areas 3b, 1, and 2). The highest density of CR neurons was in layers I and II. The densities in the remaining layers declined with depth in the cortex. Significant differences in the densities of CR neurons across cytoarchitectonic areas were seen in layer III (Fig. 5; 2(3) ⫽ 11.1, p ⬍ 0.01; areas 1 and 2 ⬎ areas 3a and 3b, area 3b ⬎ area 3a). The diverse patterns of labeling for calcium-binding proteins are summarized in Fig. 5, which illustrates the median densities of the calcium-binding protein neurons in layer III as proportions of the densities in area 3b. The density of PV neurons in layer III was highest in area 1 (113% of area 3b). The densities of CB neurons in areas 1 and 2 were 54% and 68%, respectively, of that in area 3b. The density of CR neurons increased from area 3a (84% of area 3b) to area 2 (115% of area 3b). Variations across areas were also seen in other cortical layers, but only those in layers II and III reached statistical significance (see above). A comparison of the densities of immunoreactive neurons by cortical layer is presented in Fig. 4. Almost all immunoreactive neurons in layer I were CR. In layer II the densities of neurons containing each of the three calcium-binding proteins were similar. Layer III also contained neurons immunoreactive for each of the three calcium-binding proteins, but PV neurons were found to have higher densities. PV neurons dominated the distributions of calcium-binding proteins in the remaining layers. DISCUSSION There is little overlap between PV and CB neurons in cat cortex [15,17,27], but colocalization of CR with PV or CB has not been
FIG. 4. Average density of labeled neurons (⫾ SEM), by layer, across areas of SI cortex. Analysis of variance based on ranks revealed significant differences across cortical areas in layers II (for CB) and III (for all three calcium-binding proteins). See text for details.
CALCIUM-BINDING PROTEINS IN CAT SI
383 [2,16,32]. The laminar distribution of CR neurons in SI cortex was similar to that reported for cat visual cortex [35] and for cortical areas in other species [10,22,46,49]. Most previous comparisons of calcium-binding proteins in different cortical areas have relied on qualitative observations. Studies based on quantitative methods report significant differences across prefrontal cortical areas in the rat [23] and between rat SI and motor cortex [60] (for PV, but not CB). No differences were found across prefrontal cortical areas in monkey [10,22]. The present study is the first to describe quantitative differences in calcium-binding proteins between primary areas of a sensory cortex. The densities of immunoreactive neurons in layers II and III varied significantly across the cytoarchitectonic areas of SI cortex. PV showed the lowest variation, with densities in layer III ranging from 90 to 113% of the density of PV neurons in area 3b. CB and CR exhibited greater variation and their patterns were reciprocal: anterior areas of SI (3a and 3b) contained more CB neurons and fewer CR neurons than posterior areas of SI. Areas 1 and 2 contained only 46% and 32%, respectively, as many CB neurons as area 3b, but 106% and 115%, respectively, as many CR neurons. To estimate the proportion of immunoreactive neurons in some of these areas, we used data on total neuron numbers in areas 2 and 3b from unrelated experiments (unpublished). Based on these data, layer III in area 2 contains approximately 19% more neurons than area 3b. This difference in total number of neurons could account for the 15% difference in the density of CR neurons, and makes the differences in PV and CB neuron density even larger (raising the values to 130% and 175%, respectively). Correlation of Distribution Patterns with Function
FIG. 5. Variation in the median densities of labeled neurons in layer III across SI cortex. The median densities in area 3b were used as baseline (100%).
previously examined in cat. There is apparently little overlap in the populations of SI cortical neurons that are immunoreactive for the three calcium-binding proteins: in the present study, CR was colocalized with PV or CB in very few neurons. This is in agreement with the results from rat [25,41,56] and monkey [10], while in the human there appears to be slightly higher incidences of colocalization [13,14,43]. Distribution of SI Cortical Neurons Containing Calcium-binding Proteins The most obvious difference in the distributions of neurons immunoreactive for PV, CB, or CR was seen across laminae. PV neurons were located throughout layers II–VI, with the highest densities in layers III and IV—a pattern similar to those reported previously for other sensory cortical areas in the cat [1,2,15,16, 27,32], monkey [5,28], and rat [60]. CB neurons were concentrated primarily in superficial cortical layers (II and III), with scattered labeled cells in deeper cortical layers (V and VI). This pattern was consistent with those reported for other cortical areas in the cat
There have been many attempts to correlate the morphology of single neurons with their content of calcium-binding proteins (reviewed in [11,12]). In cat, calcium-binding proteins appear to be contained almost exclusively in nonpyramidal neurons [15,27]. In general, PV is localized in large basket cells and chandelier cells; CB is localized in double-bouquet cells; and CR is localized in bipolar, double-bouquet, and Cajal-Retzius cells [11,12,30]. These morphological classes form specific connections with other neurons in the cortical circuit. For example, chandelier neurons synapse with axon initial segments of pyramidal neurons, and basket neurons synapse near somata [12]. Although the precise physiological roles of calcium-binding proteins in neuronal function are not known, it has been suggested that they may influence patterns of neuronal discharge [40] as a consequence of their calcium-buffering properties [9,45]. Consistent with this idea, there appears to be a relationship between a neuron’s firing characteristics and its calcium-binding protein content (e.g., [40]). PV has been localized in fast-spiking neurons [7,39,40], CB has been localized in regular spiking [39,40] and some fast-spiking neurons [7], and CR has been localized in burst spiking [40] or irregular spiking [7] bipolar neurons. Thus, neurons containing the three calcium-binding proteins examined in the present study have distinctive firing patterns and form specific anatomical connections within the cortical circuit. Indeed, Celio [8] has suggested that PV and CB might be associated with separate functional systems in the rat brain, and it has been proposed that calcium-binding proteins mark separate subcortical and thalamocortical pathways in the primate somatosensory [54, 55], auditory [50], and visual [36] systems. For example, in the somatosensory system PV appears to be localized in specific thalamocortical projections to the middle cortical layers, whereas CB is found in more diffuse thalamocortical projections that project to more superficial cortical layers, including layer I [54,55]. The present study is one of the first to quantitatively evaluate
384
SCHWARK AND LI
calcium-binding proteins in the context of functional cortical circuits. Variations in these cortical circuits, reflected by differences in the number of neurons containing calcium-binding proteins, may be related to functional differences between cortical areas. The high fidelity responses to light tactile stimulation seen in neurons in areas 3b and 1 [19,51,52] might be related to their higher density of CB and PV neurons, which are primarily regular spiking and fast-spiking neurons, respectively. Area 2, which contains neurons that respond to deep receptors [19,34], contained the highest density of CR neurons, which are irregular spiking neurons. This relationship suggests that the characteristic firing patterns of peripheral receptors might be reinforced by similar characteristics of central neurons, perhaps related to their content of calcium-binding proteins. ACKNOWLEDGEMENTS
15.
16.
17.
18. 19.
This research was supported by NIH grant NS25729 to H.D.S. The authors thank Dr. M.R. Celio for the antibody to calretinin, and Drs. Jannon Fuchs and Michael Pettit for their constructive comments.
20.
REFERENCES
21.
1. Alca´ntara, S.; Ferrer, I. Postnatal development of parvalbumin immunoreactivity in the cerebral cortex of the cat. J. Comp. Neurol. 348: 133–149; 1994. 2. Alca´ntara, S.; Ferrer, I. Postnatal development of calbindin-D28k immunoreactivity in the cerebral cortex of the cat. Anat. Embryol. (Berl.) 192:369 –384; 1995. 3. Amitai, Y.; Connors, B. W. Intrinsic physiology and morphology of single neurons in neocortex. In: Jones, E. G.; Diamond, I. T., eds. Cerebral cortex, vol. 11. The barrel cortex of rodents. New York: Plenum Press; 1994:299 –331. 4. Avendan˜o, C.; Verdu, A. Area 3a in the cat. I. A reevaluation of its location and architecture on the basis of nissl, myelin, acetylcholinesterase, and cytochrome oxidase staining. J. Comp. Neurol. 321:357– 372; 1992. 5. Blu¨mcke, I.; Hof, P. R.; Morrison, J. H.; Celio, M. R. Distribution of parvalbumin immunoreactivity in the visual cortex of Old World monkeys and humans. J. Comp. Neurol. 301:417– 432; 1990. 6. Brodmann, K. Beitrage zur histologischen Lokalisation der Grosshirnrinde. V. Uber den allgemeinen Bauplan des Cortex pallii bei den Mammaliern und zwei homologe Rindenfelder im Besonderen. Zugleich ein Beitrag zur Furchenlehre. J. Psych. Neurol. 6:275– 400; 1906. 7. Cauli, B.; Audinat, E.; Lambolez, B.; Angulo, M. C.; Ropert, N.; Tsuzuki, K.; Hestrin, S.; Rossier, J. Molecular and physiological diversity of cortical nonpyramidal cells. J. Neurosci. 17:3894 –3906; 1997. 8. Celio, M. R. Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35:375– 475; 1990. 9. Chard, P. S.; Bleakman, D.; Christakos, S.; Fullmer, C. S.; Miller, R. J. Calcium buffering properties of calbindin D28k and parvalbumin in rat sensory neurones. J. Physiol. (Lond.) 472:341–357; 1993. 10. Conde´, F.; Lund, J. S.; Jacobowitz, D. M.; Baimbridge, K. G.; Lewis, D. A. Local circuit neurons immunoreactive for calretinin, calbindin D-28k or parvalbumin in monkey prefrontal cortex: Distribution and morphology. J. Comp. Neurol. 341:95–116; 1994. 11. DeFelipe, J. Neocortical neuronal diversity: Chemical heterogeneity revealed by colocalization studies of classic neurotransmitters, neuropeptides, calcium-binding proteins, and cell surface molecules. Cereb. Cortex 3:273–289; 1993. 12. DeFelipe, J. Types of neurons, synaptic connections and chemical characteristics of cells immunoreactive for calbindin-D28K, parvalbumin and calretinin in the neocortex. J. Chem. Neuroanat. 14:1–19; 1997. 13. Del Rı´o, M. R.; DeFelipe, J. Colocalization of parvalbumin and calbindin D-28k in neurons including chandelier cells of the human temporal neocortex. J. Chem. Neuroanat. 12:165–173; 1997a. 14. Del Rı´o, M. R.; DeFelipe, J. Double bouquet cell axons in the human
22.
23.
24.
25. 26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
temporal neocortex: Relationship to bundles of myelinated axons and colocalization of calretinin and calbindin D-28k immunoreactivities. J. Chem. Neuroanat. 13:243–251; 1997b. Demeulemeester, H.; Arckens, L.; Vandesande, F.; Orban, G. A.; Heizmann, C. W.; Pochet, R. Calcium binding proteins and neuropeptides as molecular markers of GABAergic interneurons in the cat visual cortex. Exp. Brain Res. 84:538 –544; 1991. Demeulemeester, H.; Vandesande, F.; Orban, G. A.; Brandon, C.; Vanderhaeghen, J. J. Heterogeneity of GABAergic cells in cat visual cortex. J. Neurosci. 8:988 –1000; 1988. Demeulemeester, H.; Vandesande, F.; Orban, G. A.; Heizmann, C. W.; Pochet, R. Calbindin D-28K and parvalbumin immunoreactivity is confined to two separate neuronal subpopulations in the cat visual cortex, whereas partial coexistence is shown in the dorsal lateral geniculate nucleus. Neurosci. Lett. 99:6 –11; 1989. Dykes, R. W. Parallel processing of somatosensory information: A theory. Brain Res. Rev. 6:47–115; 1983. Dykes, R. W.; Rasmusson, D. D.; Hoeltzell, P. B. Organization of primary somatosensory cortex in the cat. J. Neurophysiol. 43:1527– 1546; 1980. Esteky, H.; Schwark, H. D. Responses of rapidly adapting neurons in cat primary somatosensory cortex to constant-velocity mechanical stimulation. J. Neurophysiol. 72:2269 –2279; 1994. Felleman, D. J.; Wall, J. T.; Cusick, C. G.; Kaas, J. H. The representation of the body surface in SI of cats. J. Neurosci. 3:1648 –1669; 1983. Gabbott, P. L.; Bacon, S. J. Local circuit neurons in the medial prefrontal cortex (areas 24a,b,c, 25 and 32) in the monkey: II. Quantitative areal and laminar distributions. J. Comp. Neurol. 364:609 – 636; 1996. Gabbott, P. L.; Dickie, B. G.; Vaid, R. R.; Headlam, A. J.; Bacon, S. J. Local-circuit neurones in the medial prefrontal cortex (areas 25, 32 and 24b) in the rat: Morphology and quantitative distribution. J. Comp. Neurol. 377:465– 499; 1997. Garraghty, P. E.; Pons, T. P.; Huerta, M. F.; Kaas, J. H. Somatotopic organization of the third somatosensory area (SIII) in cats. Somatosens. Res. 4:333–357; 1987. Gonchar, Y.; Burkhalter, A. Three distinct families of GABAergic neurons in rat visual cortex. Cereb. Cortex 7:347–358; 1997. Hassler, R.; Mu¨hs-Clement, K. Architektonischer Aufbau des sensorimotorischen und parietalen Cortex der Katze. J. Hirnforsch. 6:377– 420; 1964. Hendry, S. H. C.; Jones, E. G. GABA neuronal subpopulations in cat primary auditory cortex: Co-localization with calcium binding proteins. Brain Res. 543:45–55; 1991. Hendry, S. H. C.; Jones, E. G.; Emson, P. C.; Lawson, D. E. M.; Heizmann, C. W.; Streit, P. Two classes of cortical GABA neurons defined by differential calcium binding protein immunoreactivities. Exp. Brain Res. 76:467– 472; 1989. Hof, P. R.; Bogaert, Y. E.; Rosenthal, R. E.; Fiskum, G. Distribution of neuronal populations containing neurofilament protein and calciumbinding proteins in the canine neocortex: Regional analysis and cell typology. J. Chem. Neuroanat. 11:81–98; 1996. Hof, P. R.; Glezer, I. I.; Conde, F.; Flagg, R. A.; Rubin, M. B.; Nimchinsky, E. A.; Weisenhorn, D. V. Cellular distribution of the calcium binding proteins parvalbumin, calbindin, and calretinin in the neocortex of mammals: Phylogenetic and developmental patterns. J. Chem. Neuroanat. 16:77–116; 1999. Hof, P. R.; Nimchinsky, E. A. Regional distribution of neurofilament and calcium-binding proteins in the cingulate cortex of the macaque monkey. Cereb. Cortex 2:456 – 467; 1992. Hogan, D.; Berman, N. E. J. The development of parvalbumin and calbindin-D28k immunoreactive interneurons in kitten visual cortical areas. Dev. Brain Res. 77:1–21; 1994. Iwamura, Y.; Tanaka, M. Functional organization of receptive fields in the cat somatosensory cortex. I: Integration within the coronal region. Brain Res. 151:49 – 60; 1978a. Iwamura, Y.; Tanaka, M. Functional organization of receptive fields in the cat somatosensory cortex. II: Second representation of the forepaw in the ansate region. Brain Res. 151:61–72; 1978b. Jeon, C. J.; Park, H. J. Immunocytochemical localization of calcium-
CALCIUM-BINDING PROTEINS IN CAT SI
36. 37. 38. 39.
40. 41. 42.
43. 44. 45. 46.
47. 48.
49. 50.
binding protein calretinin containing neurons in cat visual cortex. Mol. Cell 7:721–725; 1997. Johnson, J. K.; Casagrande, V. A. Distribution of calcium-binding proteins within the parallel visual pathways of a primate (Galago crassicaudatus). J. Comp. Neurol. 356:238 –260; 1995. Jones, E. G. The thalamus. New York: Plenum; 1985. Jones, E. G.; Porter, R. What is area 3a? Brain Res. Rev. 2:1– 43; 1980. Kawaguchi, Y.; Kubota, Y. Correlation of physiological subgroupings of nonpyramidal cells with parvalbumin- and calbindinD28k-immunoreactive neurons in layer V of rat frontal cortex. J. Neurophysiol. 70:387–396; 1993. Kawaguchi, Y.; Kubota, Y. GABAergic cell subtypes and their synaptic connections in rat frontal cortex. Cereb. Cortex 7:476 – 486; 1997. Kubota, Y.; Hattori, R.; Yui, Y. Three distinct subpopulations of GABAergic neurons in rat frontal agranular cortex. Brain Res. 649: 159 –173; 1994. Leclerc, S. S.; Avendan˜o, C.; Dykes, R. W.; Waters, R. S.; Salimi, I. Reevaluation of area 3b in the cat based on architectonic and electrophysiological studies: Regional variability with functional and anatomical consistencies. J. Comp. Neurol. 341:357–374; 1994. Leuba, G.; Saini, K. Colocalization of parvalbumin, calretinin and calbindin D-28k in human cortical and subcortical visual structures. J. Chem. Neuroanat. 13:41–52; 1997. Li, J.; Schwark, H. D. Distribution and proportions of GABA-immunoreactive neurons in cat primary somatosensory cortex. J. Comp. Neurol. 343:353–361; 1994. Lukas, W.; Jones, K. A. Cortical neurons containing calretinin are selectively resistant to calcium overload and excitotoxicity in vitro. Neuroscience 61:307–316; 1994. Lu¨th, H.-J.; Blu¨mcke, I.; Winkelmann, E.; Celio, M. R. The calciumbinding protein calretinin is localized in a subset of interneurons in the rat cerebral cortex: A light and electron immunohistochemical study. J. Hirnforsch. 34:93–103; 1993. Maier, D. L.; McCasland, J. S. Calcium-binding protein phenotype defines metabolically distinct groups of neurons in barrel cortex of behaving hamsters. Exp. Neurol. 145:71– 80; 1997. McKenna, T. M.; Whitsel, B. L.; Dreyer, D. A.; Metz, C. B. Organization of cat anterior parietal cortex: Relations among cytoarchitecture, single neuron functional properties, and interhemispheric connectivity. J. Neurophysiol. 45:667– 697; 1981. Meskenaite, V. Calretinin-immunoreactive local circuit neurons in area 17 of the cynomolgus monkey, Macaca fascicularis. J. Comp. Neurol. 379:113–132; 1997. Molinari, M.; Dell’Anna, M. E.; Rausell, E.; Leggio, M. G.; Hashi-
385
51. 52. 53. 54.
55.
56. 57. 58. 59. 60.
61.
62.
kawa, T.; Jones, E. G. Auditory thalamocortical pathways defined in monkeys by calcium-binding protein immunoreactivity. J. Comp. Neurol. 362:171–194; 1995. Mountcastle, V. B. Modality and topographic properties of single neurons of cat’s somatic sensory cortex. J. Neurophysiol. 20:408 – 434; 1957. Myasnikov, A. A.; Dykes, R. W.; Avendan˜o, C. Cytoarchitecture and responsiveness of the medial ansate region of the cat primary somatosensory cortex. J. Comp. Neurol. 349:401– 427; 1994. Porter, L. L.; Izraeli, R. The effects of localized inactivation of somatosensory cortex, area 3a, on area 2 in cats. Somatosens. Mot. Res. 10:399 – 413; 1993. Rausell, E.; Bae, C. S.; Vin˜uela, A.; Huntley, G. W.; Jones, E. G. Calbindin and parvalbumin cells in monkey VPL thalamic nucleus: Distribution, laminar cortical projections, and relations to spinothalamic terminations. J. Neurosci. 12:4088 – 4111; 1992. Rausell, E.; Jones, E. G. Chemically distinct compartments of the thalamic VPM nucleus in monkeys relay principal and spinal trigeminal pathways to different layers of the somatosensory cortex. J. Neurosci. 11:226 –237; 1991. Rogers, J. H.; Resibois, A. Calretinin and calbindin-D28k in rat brain: Patterns of partial co-localization. Neuroscience 51:843– 865; 1992. Schwark, H. D.; Esteky, H.; Jones, E. G. Corticocortical connections of cat primary somatosensory cortex. Exp. Brain Res. 91:425– 434; 1992. Schwark, H. D.; Jones, E. G. The distribution of intrinsic cortical axons in area 3b of cat primary somatosensory cortex. Exp. Brain Res. 78:501–513; 1989. Sretavan, D.; Dykes, R. W. The organization of two cutaneous submodalities in the forearm region of area 3b of cat somatosensory cortex. J. Comp. Neurol. 213:381–398; 1983. Van Brederode, J. F. M.; Helliesen, M. K.; Hendrickson, A. E. Distribution of the calcium-binding proteins parvalbumin and calbindinD28k in the sensorimotor cortex of the rat. Neuroscience 44:157–171; 1991. Vickers, J. C.; Huntley, G. W.; Edwards, A. M.; Moran, T.; Rogers, S. W.; Heinemann, S. F.; Morrison, J. H. Quantitative localization of AMPA/kainate and kainate glutamate receptor subunit immunoreactivity in neurochemically identified subpopulations of neurons in the prefrontal cortex of the macaque monkey. J. Neurosci. 13:2982–2992; 1993. Waters, R. S.; Favorov, O.; Asanuma, H. Physiological properties and pattern of projection of cortico-cortical connections from the anterior bank of the ansate sulcus to the motor cortex, area 4g, in the cat. Exp. Brain Res. 46:403– 412; 1982.