- Email: [email protected]
Colocalization of neuropeptides with calcium-binding proteins in the claustral interneurons during postnatal development of the rat
Brain Research Bulletin 80 (2009) 100–106
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Colocalization of neuropeptides with calcium-binding proteins in the claustral interneurons during postnatal development of the rat a,∗ a ´ ˛ Przemysław Kowianski , Jerzy Dziewiatkowski , Joanna M. Mory´s a , Katarzyna Majak a , a b ˙ Sławomir Wójcik , Lawrence R. Edelstein , Grazyna Lietzau a , Janusz Mory´s a a b
Department of Anatomy and Neurobiology, Medical University of Gda´ nsk, 1 D˛ebinki Street, 80-211 Gda´ nsk, Poland P.O. Box 2316, Del Mar, CA 92014, USA
a r t i c l e
i n f o
Article history: Received 30 December 2008 Received in revised form 22 June 2009 Accepted 22 June 2009 Available online 1 July 2009 Keywords: Calcium-binding proteins Claustrum Development Interneurons Neuropeptides
a b s t r a c t The claustrum is a relatively large telencephalic structure, situated close to the border of the neo- and allocortical regions. Its neuronal population consists of glutamatergic, projecting neurons and GABA-ergic interneurons, characterized by occurrence of numerous additional biochemical markers. The postnatal development of these latter neurons has not been extensively studied. Revealing the characteristic patterns of colocalizations between selected markers may shed some light on their function and origin. We investigated the colocalization patterns between three neuropeptides: neuropeptide Y, somatostatin, vasoactive intestinal polypeptide and three calcium-binding proteins: calbindin D28k, calretinin, parvalbumin in the interneurons of the rat claustrum during a four-month postnatal period (P0–P120; P: postnatal day). Our studies revealed the following types of colocalizations: neuropeptide Y with calbindin D28k, calretinin or parvalbumin; somatostatin with calbindin D28k; vasoactive intestinal polypeptide with calretinin. Only vasoactive intestinal polypeptide- and calretinin-containing, double-labeled neurons were present at the day of birth, whereas the other double-labeled neurons appeared at later stages of development. The ratios of colocalizing neurons to single-labeled neurons in each type of colocalization were differentiated and reached the highest value (51%) for vasoactive intestinal polypeptide- and calretinin-double-labeled neurons. In conclusion, the claustral interneurons represent differentiated population in respect to the occurrence of neuropeptides and calcium-binding proteins. The expression of studied substances is changing during the postnatal period. © 2009 Elsevier Inc. All rights reserved.
1. Introduction On the basis of comparative, developmental and hodological studies, the claustrum (Cl), together with the endopiriform nucleus (Edn) and the pallial amygdala, may be regarded as a single entity, the claustroamygdaloid complex [27,29]. The Cl consists of glutamatergic projecting neurons and GABA-ergic interneurons, revealing the presence of neuropeptides such as neuropeptide Y (NPY), somatostatin (SOM), and vasoactive intestinal polypeptide (VIP), as well as calcium-binding proteins (CaBPs) such as calbindin D28k (CB), calretinin (CR), and parvalbumin (PV) [9,14,30]. According to numerous published results the GABA-ergic neurons of the claustroamygdaloid complex, which predominantly represent local circuit cells, are characterized by the occurrence of various combinations of neuropeptides and CaBPs
∗ Corresponding author. Tel.: +48 58 349 1401; fax: +48 58 349 1421. ´ E-mail address: [email protected] (P. Kowianski). 0361-9230/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2009.06.020
[13,15,21,22,24,25]. However, the differences in the colocalization patterns between various parts of this complex were only partially described. In particular, there is no information available concerning the colocalization of the neuropeptides and CaBPs in the claustral neurons during postnatal development. A growing body of evidence indicates that the GABA-ergic neurons of the claustroamygdaloid complex may originate in the subpallium and migrate tangentially to reach the target structures [27–29]. Gene expression studies in the earliest stages of claustral development reveal that some of its neurons may originate subcortically, in the ganglionic eminences. The presence of GABA-ergic interneurons, containing CB and CR, and originating in the subpallial progenitor centers was noted in the Cl [17,32]. Taking into account the lack of information concerning the immunohistochemical properties of the claustral interneurons, especially during early development, we deemed it relevant and important to investigate the colocalization patterns between neuropeptides and CaBPs in the Cl during a four-month postnatal period. These data may prove useful in future studies characterizing
P. Kowia´ nski et al. / Brain Research Bulletin 80 (2009) 100–106
the embryologic derivation of claustral interneurons, while revealing their possible, distinctive immunohistochemical patterns. 2. Material and methods Fifty-six Wistar rats of both sexes (29 males and 27 females) were used in this study. The animals were treated in accordance with the published guidelines established by the Local Ethical Committee, as well as the European Communities Council Directive of 24 November 1986 (86/609/EEC). Eight age-dependent experimental groups were used in the study (P0, P4, P7, P14, P21, P28, P60, and P120), with seven animals in each. The animals were irreversibly anesthetized with sodium pentobarbital (thiopental sodium, Biochemie GmBH, Germany; 80 mg/kg of body weight, i.p.) and transcardially perfused with 200 ml of 0.9% NaCl solution (pH 7.4) at room temperature, followed by 400 ml of fixative consisting of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, Sigma–Aldrich, UK, pH 7.4, and 4 ◦ C). The brains were then removed and stored in the same fixative for 120 min and subsequently stored in 30% sucrose in 0.1 M PBS (pH 7.4 and 4 ◦ C) for a minimum of 24 h. After freezing, the brains were coronally sliced into 40 m serial sections on a sliding microtome.
2.1. Immunohistochemistry The free-floating sections were processed for fluorescence immunocytochemistry. The following primary antibodies were used: anti-NPY (rabbit; 1:400, Affiniti, UK), anti-SOM (rabbit; diluted 1:300, Euro-Diagnostica AB, Sweden), anti-VIP (rabbit; 1:100, Cappel, ICN Pharmaceuticals Inc., USA), anti-CB (mouse; 1:100, Sigma Chemical Company, USA), anti-CR (goat; 1:1000, Chemicon, USA) and anti-PV (mouse; 1:500, Sigma Chemical Company, USA). Sections were incubated for 1 h in blocking solution containing 0.1% bovine serum albumin in PBS and 10% normal goat serum. After washing in PBS, the sections were incubated at room temperature overnight in a mixture of primary antibodies in order to assess the colocalization of selected neuropeptides with CaBPs. Subsequently, the sections were washed in PBS and incubated in secondary antibodies at room temperature for 1 h. The following secondary antibodies were used in the study: goat anti-rabbit, goat anti-mouse, donkey anti-goat and donkey anti-rabbit; conjugated to indocarbocyanine [Cy-3] at 1:800, or to fluorescein [FITC] at 1:200 (Jackson ImmunoResearch, USA). After washing in PBS, the sections were mounted on slides, dried and coverslipped with Vectashield (Vector Laboratories Inc., USA). Immunohistochemical controls were performed. The specificity of staining was verified by the omission tests. The immunohistochemical procedure was conducted as previously described, except that the primary antibodies were omitted in the presence of two secondary antibodies or secondary antibodies were omitted. In order to exclude the possibility of cross-reaction between secondary antibodies, the immunohistochemical procedure was performed with the primary antibody and non-corresponding, secondary antibody. All above-mentioned tests revealed specificity of staining and the absence of cross-reaction.
2.2. Qualitative study Double-labeled sections were examined with a fluorescence microscope (Eclipse E600, Nikon, Japan), equipped with a confocal imaging system (MicroRadiance, BioRad, UK) and an Argon laser (excitation 488/514 nm). In order to rule out spectral bleed-through, the preparations were checked in non-corresponding channels. Two image analysis programs (LaserSharp 2000 v. 2.01, Bio-Rad, UK and LaserPix v. 4.0, BioRad, UK) were used to prepare the illustrations. The morphological features of developing neurons and neuropil in immunocytochemically distinct subpopulations were studied. The features of mature neurons were defined based on the increased cytoplasm-to-nucleus proportion and the decreased proportion of unipolar neurons in relation to all immunolabeled neurons. The features of mature neuropil were presumed to encompass: the increased fiber density, the occurrence of immunolabeled ramifications, varicosities and immunoreactive synapses, and increased staining intensity.
2.3. Quantitative study Sections of the anterior, central, and posterior regions of the Cl were identified under 10× magnification. On each section, the borders of the Cl were marked as separate inclusion areas under 4× magnification. In each region, test areas were chosen to obtain the highest number of immunostained neurons. The cells were analyzed using confocal image stacks obtained in the maximal intensity projection mode using a 40× objective. Double-labeled sections were used to estimate the ratio of double-labeled neurons to all labeled neurons, as well as the ratio of double-labeled neurons to neuropeptide-labeled, or CaBP-labeled neurons in each test area. Data were presented as percentages. All experiments with the various antibody combinations were conducted in at least five animals.
101
2.4. Statistics All calculations were performed using Excel 2000 (Microsoft, USA). Mean values and standard deviations were calculated for each group. Neuronal counts were converted into percentages. The differences between the percentages of doublelabeled neurons in each of the three regions (anterior, central and posterior Cl) were evaluated by procedure analogous to the Tukey test (multiple comparisons for proportions). The significance level was set to 0.05.
3. Results Our studies revealed distinct and differentiated patterns of colocalization between neuropeptides and CaBPs. NPY colocalized with CB, CR and PV (NPY/CB, NPY/CR, NPY/PV). SOM revealed colocalization with CB (SOM/CB), whereas VIP colocalized with CR (VIP/CR). Only one type of colocalization, VIP/CR, was present at birth (P0; Fig. 1A–C). The NPY/CB colocalization was observed from P4 onward (Fig. 1D–F). After one week (P7), SOM/CB (Fig. 1G–I) and NPY/CR (Fig. 1J–L) colocalizations were first noted. After two weeks (P14), colocalization of NPY/PV was revealed (Fig. 1M–O). The aspiny, nonpyramidal immunoreactive neurons were present in all studied sections of the Cl. In all studied age-groups medium, bipolar and multipolar neurons were most frequently observed. With increasing age, unipolar neurons were noted with decreasing frequency, along with a small but gradual decrease in cytoplasmic volume. We also observed characteristic age-related changes of developing neuropil. The density of immunoreactive fibers, with varicosities and ramifications, increased in all types of colocalizations until the end of the fourth postnatal week. An increase in the percentage of double-labeled neurons was seen for the colocalization of NPY/CB and NPY/CR (Fig. 2A and B), as well as for SOM/CB and VIP/CR (Fig. 2D and E). A transient increase of the percentage of NPY/CB-ir neurons was noted at the end of the third postnatal week (P21). Among the SOM/CB -, NPY/CR - and VIP/CR-ir neurons, the highest percentage of double-labeled neurons occurred at the end of the observation period. No changes in percentage values were observed for NPY/PV neurons (Fig. 2C). The maximal value of the percentage of double-labeled neurons, compared to all labeled neurons in the studied sections was observed for VIP/CR (51%; Table 1). Lesser values were obtained for NPY/CB (32%) and SOM/CB (31%). The lowest values were observed for NPY/CR (9.5%) and NPY/PV (9%). The highest ratio of doublelabeled neurons to neuropeptide- or CaBP-labeled neurons was estimated for VIP/CR colocalization (71% and 64%, respectively; Table 1). The lowest values were observed for NPY/CR (14% and 22%, respectively) and NPY/PV (15% and 14%, respectively) types of colocalization. 4. Discussion Our most important observations are concerned with determining the patterns of colocalization between neuropeptides and CaBPs, and the time of their occurrence, as well as the percentage of colocalizing neurons. Our studies revealed three predominant forms of colocalization between neuropeptides and CaBPs: (1) NPY with all studied representatives of CaBPs (i.e., CB, CR, and PV), (2) SOM with CB, and (3) VIP with CR. Each of the studied types of colocalization occurred at different stages of development and only one of them (VIP/CR) was present at birth (P0). The observed types of colocalization could be divided into three groups, according to the values of the percentage of colocalization. 4.1. The colocalization types of neuropeptides and CaBPs in the Cl reveal characteristic patterns of temporal development At the moment of birth (P0) only one type of double-labeled neurons (VIP/CR) was observed in the Cl. The remaining types of
102
P. Kowia´ nski et al. / Brain Research Bulletin 80 (2009) 100–106
colocalization occurred at later stages of postnatal development. Characteristically, the colocalization of NPY with PV was revealed after two weeks (P14) of life. These observations indicate that the biochemical and consequently functional development of claustral interneurons, represented by occurring specific subtypes of neurons, extended during both prenatal and postnatal period. The development of the CaBPs-containing, GABA-ergic neurons in the claustral complex was studied in the mouse [6,17]. Whereas CB- and CR-ir neurons were present early in the embryonic period, the PV-ir neurons started to be seen postnatally, which may correspond to our observation concerning the late occurrence of NPY/PV colocalization in the rat Cl. All neuropeptides studied by us were observed in the claustral interneurons from the day of birth [see also Ref. [14]]. Although the definite age of their occurrence in the Cl was not described, the recent data concerning the expression of SOM and NPY in the amygdala indicate their very early occurrence in the mouse (from embryonic day 12.5) [31]. The observed biochemical and morphological changes seen in the claustral interneurons occur subsequently to a long sequence of processes and events which shape the neuronal population during both prenatal and postnatal development. The developmental patterns we have seen in claustral interneurons are reflective of the general hypothesis advanced by Xu et al. [36,37], which attempts to explain the origins of interneuron diversity. In accordance with this hypothesis, the first possibility assumes diversification of interneurons in their place of origin (e.g. in the proliferative zone). The second possibility assumes that interneuronal subtypes could develop from multipotential GABA-ergic cells at their place of settlement, under the influence of local cues (e.g. neurotrophic factors). This process could be also facilitated by the initiation of neuronal activity. Based on our present level of understanding of these issues, we may take into account both of the above-mentioned hypotheses with regard to the development of claustral interneurons and their characteristic expression of immunochemical markers. Confirmation of either or both of these hypotheses requires further research and the verification of results at the level of the protein expression, as well as the assessment of mRNA and gene activation. 4.2. Claustral interneurons characterized by various types of colocalization constitute quantitatively differentiated subpopulations In order to further characterize the observed types of immunohistochemical colocalization, we compared their maxi-
mal percentage values and the ratios of double-labeled neurons to neuropeptide-labeled, or CaBPs-labeled neurons. This allowed us to divide the studied types into three groups, characterized by high, moderate, and low values. The highest percentage of colocalization was observed for VIP/CR (51%; Table 1). Moderate values were noted for NPY/CB (32%) and SOM/CB (31%). The lowest values were seen in NPY/CR (9.5%) and NPY/PV (9%). Additionally, the colocalization of VIP/CR may be expected in a very high (prevalent) percentage of VIP- or CR-containing claustral neurons (in 71% and 64% of all these neurons, respectively). On the other hand, PV-containing neurons in the Cl may be expected predominantly as revealing very low percentage of colocalization. Only 14% of all these neurons may be expected to contain one of the studied neuropeptides, namely NPY. Although the results of quantitative studies of various colocalizations are not frequently published, some evidence can be found in the literature. The colocalization of VIP with CR was described as being seen quite often in the cerebral cortex and hippocampus of various mammalian species [3,4,7,12,33]. The colocalization of SOM with CB in the cerebral cortex of the rat is more frequent than NPY with CB. As well, the colocalization of NPY/CR and NPY/PV is very rarely seen in mammals [1,3–5,7]. Finally, the colocalization between two neuropeptides NPY and SOM was reported in numerous structures. In the amygdala the percentage of this colocalization is very high, although differentiated in particular nuclei [25,26,31]. In the cerebral cortex this value is lower and reveals characteristic changes during postnatal development [16]. The percentage of GABA-ergic interneurons in the Cl of various mammalian species was estimated between 7% and 12% [2,11,34]. The population of CaBPs-containing neurons in the mouse Cl has been estimated at 12.3% of all claustral neurons. Among these, CBir neurons account for 5.4%, whereas PV-ir neurons 7.9% [30]. There is no data available concerning the percentage of neuropeptidecontaining neurons in the Cl. The development of the rat Cl reveals characteristic features, concerned principally with the changes of the total number of neurons. From the stage of its occurrence at E20, the increase of the total number of neurons is observed until the end of the first postnatal week (P7; estimated value of the total number of neurons at this stage of development is about 93 800) [18]. During the second postnatal week a 30% decrease of the total number of neurons is reported, which may be the consequence of apoptotic death. Because of this specific pattern of the claustral development it seems for us useful to compare the relative (percentage) values of the ratios between various neuronal subpopulations at definite stages of development.
Table 1 Maximal percentage values of double-labeled neurons for each type of colocalization and ratios of double-labeled neurons to neuropeptide- or CaBP-labeled neurons in the claustrum during postnatal development. Type of colocalization
Age
Maximal percentage of double-labeled neurons (%)
Ratio of double-labeled neurons to all labeled neurons, n/N
Percentage of double-labeled neurons to neuropeptide- or CaBP-labeled neurons (%)
Ratio of double-labeled neurons to neuropeptide- or CaBP-labeled neurons n/Np and n/Cp
VIP/CR
P120
51
283/534
71 64
283/399 283/442
NPY/CB
P21
32
131/410
45 52
131/291 131/252
SOM/CB
P120
31
174/580
50 46
174/351 174/381
NPY/CR
P120
9.5
214/2247
14 22
214/1562 214/955
NPY/PV
P14
9
181/2013
15 14
181/1207 181/1293
Cp: calcium-binding-labeled proteins; n: number of double-labeled neurons; N: total number of labeled neurons; Np: neuropeptide-labeled neurons.
P. Kowia´ nski et al. / Brain Research Bulletin 80 (2009) 100–106
103
Fig. 1. Colocalization of calcium-binding proteins and neuropeptides in the claustral interneurons during postnatal development. The double-labeled neurons (indicated by the arrows) are presented occurring for the first time at definite stages of development. The controls of non-colocalizing neurons and elements of neuropil, observed at earlier stages of postnatal development are presented in the right bottom corners. (A–C) VIP/CR colocalization at P0; (D–F) NPY/CB colocalization at P4; (G–I) SOM/CB colocalization at P7; (J–L) NPY/CR colocalization at P7; (M–O) NPY/PV colocalization at P14. (scale bars = 50 m; immunofluorescent staining: FITC – green; Cy3 – red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
104
P. Kowia´ nski et al. / Brain Research Bulletin 80 (2009) 100–106
4.3. The colocalization patterns in distinct parts of the claustroamygdaloid complex Comparing the colocalization patterns between distinct parts of the claustroamygdaloid complex, namely, the Cl, Edn and pallial amygdala, may provide some interesting data concerning overall development. Not only are these structures topographically related, but they also share numerous connections and functional interrelationships [8,19,20,35]. Our earlier studies of colocalizations amongst neuropeptides and CaBPs in the Edn of the adult rat revealed the occurrence of some types similar to those we have seen in the Cl, namely, SOM/CB and VIP/CR [15]. Moreover, we noted SOM/PV colocalization in the Edn, which we did not see in the Cl. This rare type of colocalization is also present in neurons of the neighboring anterior piriform cortex [5]. Interest-
ingly, we did not find the colocalization of any CaBPs with NPY in the Edn, which is an important difference to note in comparison to the Cl. These points of differentiation serve to support the hypothesis that assumes a differentiated origin of the Cl and the Edn [27,29]. On the basis of the differential expression of developmental regulatory genes (e.g. Emx1, Cad8, Sema5A), it was stated that the dorsolateral part of the Cl derives from the lateral pallium, whereas the ventromedial part of the Cl and Edn are considered to have originated in the ventral pallium [27]. This distinction might serve to explain the existence of interneurons, characterized by the differentiated gene activity in various parts of the claustroamygdaloid complex, and which may be controlled by separate molecular mechanisms. Comparing our results with the published data concerning the colocalization types in the pallial amygdala, we may conclude that
Fig. 2. The percentage values of double-labeled neurons in relationship to all labeled neurons in the claustrum at various stages of development. The colocalizations of: (A) NPY with CB; (B) NPY with CR; (C) NPY with PV; (D) SOM with CB and (E) VIP with CR. The data are presented as mean values and standard deviations.
P. Kowia´ nski et al. / Brain Research Bulletin 80 (2009) 100–106
three types of the colocalizations (SOM/CB, NPY/CB and VIP/CR) are present in both described structures. The SOM/CB-ir double-labeled neurons are present in the basolateral part of amygdala in the rat [21,25]. The percentage of double-labeled neurons is high. In the basolateral nucleus these neurons represent 91% of SOM-ir neurons (and 28% of CB-ir neurons), in the lateral nucleus double-labeled neurons represent 67% of SOM-ir neurons (about 37% of CB-ir neurons). NPY/CB-ir double-labeled neurons, although less numerous than SOM/CB-ir, are also present in the basolateral part of amygdala [21,25]. Taking into account a high percentage value (although differentiated in particular amygdalar nuclei) of the SOM/NPY colocalization [23,26,31], as well as SOM/CB colocalization [21,25], the co-existence of SOM, NPY and CB in the same neuron may not be excluded in various parts of the claustroamygdaloid complex. Similarly to the Cl, the VIP/CR colocalization was revealed in the basolateral part of amygdala [21,25]. The percentage of doublelabeled neurons in this type of colocalization reaches over 66%, which is higher than the value estimated in the Cl (51%). In the contrary to amygdala, the colocalization of NPY with two CaBPs (NPY/PV and NPY/CR) is observed in the Cl. Assessing critically the apparent differences between the Cl and amygdala, we try to interpret these in two ways. First, both types of colocalizations (NPY/PV and NPY/CR) observed in the Cl are very rare. The percentage of double-labeled neurons is very low and does not exceed 10% in both types of colocalizations. On the basis of the published, although incomplete data, concerning the colocalization of NPY with CaBPs in various parts of the amygdala, occurrence of NPY/PV- and NPY/CR- double-labeled neurons cannot be excluded. The verification of this hypothesis would require further quantitative studies of all amygdalar nuclei. On the other hand, the apparent differences between types of colocalizations occurring in the Cl and amygdala may be a consequence of different origin of the neuronal subpopulations. The considerable percentage of interneurons, localized in the claustroamygdaloid complex, derives from the subcortical progenitor structures [27,29,31]. However, there are important differences, concerned with the participation of particular progenitor areas in generation of interneurons, settling in various pallial and subpallial structures. According to the recently published data, the amygdalar (pallial and subpallial) interneurons, at least partially, are generated in the anterior entopeduncular area, the commissural preoptic region, medial, lateral and caudal ganglionic eminences (MGE, LGE and CGE, respectively), and definite regions of the hypothalamus [10,31]. The claustral interneurons, at least partially, may be generated in the MGE and CGE [17,27,32]. However, there is no data available in the literature, concerning the possible role of the other progenitor structures, like the anterior entopeduncular area, in generation of the claustral interneurons. All these data clearly indicate that the neuronal subpopulations localized in various parts of the claustroamygdaloid complex, may originate in the different and distant structures. The neurons of similar or different immunohistochemical characteristics may develop in each of these progenitor areas. Migration of interneurons from their places of origin to the targets of settlement, as well as the influence of different neurotrophic factors at the early stages of development, may account for the heterogeneity of the interneuronal population. All these processes may be responsible for the apparent differences in the immunohistochemical characteristics of the interneurons localized in various parts of the claustroamygdaloid complex.
Conflict of interest None.
105
Acknowledgements This research was supported by funds from the Polish State Committee of Scientific Research grant no. W-705. The technical assistance of Mrs. S. Scislowska, is greatly appreciated. References [1] C. Andressen, I. Blümcke, M.R. Celio, Calcium-binding proteins: selective markers of nerve cells, Cell Tissue Res. 271 (1993) 181. [2] H. Braak, E. Braak, Neuronal types in the claustrum of man, Anat. Embryol. 163 (1982) 447. [3] B. Cauli, E. Audinat, B. Lambolez, M.C. Angulo, N. Ropert, K. Tsuzuki, S. Hestrin, J. Rossier, Molecular and physiological diversity of cortical nonpyramidal cells, J. Neurosci. 17 (1997) 3894–3906. [4] B. Cauli, J.T. Porter, K. Tsuzuki, B. Lambolez, J. Rossier, B. Quenet, E. Audinat, Classification of fusiform neocortical interneurons based on unsupervised clustering, Proc. Natl. Acad. Sci. USA 97 (2000) 6144–6149. [5] S.L. Cummings, Neuropeptide Y, somatostatin, and cholecystokinin of the anterior piriform cortex, Cell Tissue Res. 289 (1997) 39–51. [6] J.C. Davila, M.A. Real, L. Olmos, I. Legaz, L. Medina, S. Guirado, Embryonic and postnatal development of GABA, calbindin, calretinin, and parvalbumin in the mouse claustral complex, J. Comp. Neurol. 481 (2005) 42. [7] J. DeFelipe, Types of neurons, synaptic connections and chemical characteristics of cells immunoreactive for calbindin-D28K, parvalbumin and calretinin in the neocortex, J. Chem. Neuroanat. 14 (1997) 1. [8] L.R. Edelstein, F.J. Denaro, The claustrum: a historical review of its anatomy, physiology, cytochemistry and functional significance, Cell Mol. Biol. 50 (2004) 675–702. [9] L.E. Eiden, E. Mezey, R.L. Eskay, M.C. Beinfeld, M. Palkovits, Neuropeptide content and connectivity of the rat claustrum, Brain Res. 523 (1990) 245. [10] M. García-López, A. Abellán, I. Legaz, J.L. Rubenstein, L. Puelles, L. Medina, Histogenetic compartments of the mouse centromedial and extended amygdala based on gene expression patterns during development, J. Comp. Neurol. 506 (2008) 46–74. [11] S.M. Gomez-Urquijo, I. Gutierrez-Ibarluzea, J.L. Bueno-Lopez, C. Reblet, Percentage incidence of gamma-aminobutyric acid neurons in the claustrum of the rabbit and comparison with the cortex and putamen, Neurosci. Lett. 282 (2000) 177. [12] Y. Kawaguchi, Y. Kubota, Physiological and morphological identification of somatostatin- or vasoactive intestinal polypeptide-containing cells among GABAergic cell subtypes in rat frontal cortex, J. Neurosci. 16 (1996) 2701–2715. [13] S. Kemppainen, A. Pitkänen, Distribution of parvalbumin, calretinin, and calbindin-D(28k) immunoreactivity in the rat amygdaloid complex and colocalization with gamma-aminobutyric acid, J. Comp. Neurol. 426 (2000) 441–467. ´ [14] P. Kowianski, J.M. Mory´s, J. Dziewiatkowski, S. Wójcik, J. Sidor-Kaczmarek, J. Mory´s, NPY-, SOM- and VIP-containing interneurons in postnatal development of the rat claustrum, Brain Res. Bull. 76 (2008) 565–571. ´ ´ [15] P. Kowianski, J.M. Mory´s, S. Wójcik, J. Dziewiatkowski, A. Luczynska, E. Spodnik, J.P. Timmermans, J. Mory´s, Neuropeptide-containing neurons in the endopiriform region of the rat: morphology and colocalization with calcium-binding proteins and nitric oxide synthase, Brain Res. 996 (2004) 97–110. [16] E.Y. Lee, T.S. Lee, S.H. Baik, C.I. Cha, Postnatal development of somatostatinand neuropeptide Y-immunoreactive neurons in rat cerebral cortex: a doublelabeling immunohistochemical study, Int. J. Dev. Neurosci. 16 (1998) 63–72. [17] I. Legaz, M. Garcia-Lopez, L. Medina, Subpallial origin of part of the calbindinpositive neurons of the claustral complex and piriform cortex, Brain Res. Bull. 66 (2005) 470–474. [18] B. Maciejewska, J. Morys, B. Berdel, J. Dziewiatkowski, O. Narkiewicz, The development of the rat claustrum—a study using morphometric and in situ DNA end labelling techniques, Eur. J. Anat. 1 (1997) 137. [19] K. Majak, J. Mory´s, Endopiriform nucleus connectivities: the implications for epileptogenesis and epilepsy, Folia Morphol. (Warsz) 66 (2007) 267–271. [20] K. Majak, M. Pikkarainen, S. Kemppainen, E. Jolkkonen, A. Pitkänen, Projections from the amygdaloid complex to the claustrum and the endopiriform nucleus: a Phaseolus vulgaris leucoagglutinin study in the rat, J. Comp. Neurol. 451 (2002) 236–249. [21] F. Mascagni, A.J. McDonald, Immunohistochemical characterization of cholecystokinin containing neurons in the rat basolateral amygdala, Brain Res. 976 (2003) 171–184. [22] A.J. McDonald, Morphology of peptide-containing neurons in the rat basolateral amygdaloid nucleus, Brain Res. 338 (1985) 186–191. [23] A.J. McDonald, Coexistence of somatostatin with neuropeptide Y, but not with cholecystokinin or vasoactive intestinal peptide, in neurons of the rat amygdala, Brain Res. 500 (1989) 37–45. [24] A.J. McDonald, F. Mascagni, Colocalization of calcium-binding proteins and GABA in neurons of the rat basolateral amygdala, Neuroscience 105 (2001) 681–693. [25] A.J. McDonald, F. Mascagni, Immunohistochemical characterization of somatostatin containing interneurons in the rat basolateral amygdala, Brain Res. 943 (2002) 237–244. [26] A.J. McDonald, F. Mascagni, J.R. Augustine, Neuropeptide Y and somatostatinlike immunoreactivity in neurons of the monkey amygdala, Neuroscience 66 (1995) 959–982.
106
P. Kowia´ nski et al. / Brain Research Bulletin 80 (2009) 100–106
[27] L. Medina, I. Legaz, G. Gonzalez, F. De Castro, J.L. Rubenstein, L. Puelles, Expression of Dbx1, Neurogenin 2, Semaphorin 5A, Cadherin 8, and Emx1 distinguish ventral and lateral pallial histogenetic divisions in the developing mouse claustroamygdaloid complex, J. Comp. Neurol. 474 (2004) 504–523. [28] S. Nery, G. Fishell, J.G. Corbin, The caudal ganglionic eminence is a source of distinct cortical and subcortical cell populations, Nat. Neurosci. 5 (2002) 1279–1287. [29] L. Puelles, E. Kuwana, E. Puelles, A. Bulfone, K. Shimamura, J. Keleher, S. Smiga, J.L. Rubenstein, Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1, J. Comp. Neurol. 424 (2000) 409–438. [30] M.A. Real, J.C. Dávila, S. Guirado, Expression of calcium-binding proteins in the mouse claustrum, J. Chem. Neuroanat. 25 (2003) 151–160. [31] M.A. Real, R. Heredia, C. Labrador Mdel, J.C. Dávila, S. Guirado, Expression of somatostatin and neuropeptide Y in the embryonic, postnatal, and adult mouse amygdalar complex, J. Comp. Neurol. 513 (2009) 335–348. [32] C. Reblet, A. Alejo, T. Fuentes, P. Pró-Sistiaga, J. Mendizabal-Zubiaga, J.L. BuenoLópez, Expression of calcium-binding proteins in the proliferative zones around
[33]
[34] [35]
[36] [37]
the corticostriatal junction of rabbits during pre- and postnatal development, Brain Res. Bull. 66 (2005) 461–464. J.H. Rogers, Immunohistochemical markers in rat cortex: co-localization of calretinin and calbindin-D28k with neuropeptides and GABA, Brain Res. 587 (1992) 147. B. Spahn, H. Braak, Percentage of projection neurons and various types of interneurons in the human claustrum, Acta Anat. 122 (1985) 245. S. Wojcik, J. Dziewiatkowski, E. Spodnik, B. Ludkiewicz, B. Domaradzka-Pytel, P. Kowianski, J. Morys, Analysis of calcium binding protein immunoreactivity in the claustrum and the endopiriform nucleus of the rabbit, Acta Neurobiol. Exp. (Wars) 64 (2004) 449. Q. Xu, I. Cobos, E. De La Cruz, J.L. Rubenstein, S.A. Anderson, Origins of cortical interneuron subtypes, J. Neurosci. 24 (2004) 2612–2622. Q. Xu, E. de la Cruz, S.A. Anderson, Cortical interneuron fate determination: diverse sources for distinct subtypes? Cereb. Cortex 13 (2003) 670– 676.
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Colocalization of neuropeptides with calcium-binding proteins in the claustral interneurons during postnatal development of the rat a,∗ a ´ ˛ Przemysław Kowianski , Jerzy Dziewiatkowski , Joanna M. Mory´s a , Katarzyna Majak a , a b ˙ Sławomir Wójcik , Lawrence R. Edelstein , Grazyna Lietzau a , Janusz Mory´s a a b
Department of Anatomy and Neurobiology, Medical University of Gda´ nsk, 1 D˛ebinki Street, 80-211 Gda´ nsk, Poland P.O. Box 2316, Del Mar, CA 92014, USA
a r t i c l e
i n f o
Article history: Received 30 December 2008 Received in revised form 22 June 2009 Accepted 22 June 2009 Available online 1 July 2009 Keywords: Calcium-binding proteins Claustrum Development Interneurons Neuropeptides
a b s t r a c t The claustrum is a relatively large telencephalic structure, situated close to the border of the neo- and allocortical regions. Its neuronal population consists of glutamatergic, projecting neurons and GABA-ergic interneurons, characterized by occurrence of numerous additional biochemical markers. The postnatal development of these latter neurons has not been extensively studied. Revealing the characteristic patterns of colocalizations between selected markers may shed some light on their function and origin. We investigated the colocalization patterns between three neuropeptides: neuropeptide Y, somatostatin, vasoactive intestinal polypeptide and three calcium-binding proteins: calbindin D28k, calretinin, parvalbumin in the interneurons of the rat claustrum during a four-month postnatal period (P0–P120; P: postnatal day). Our studies revealed the following types of colocalizations: neuropeptide Y with calbindin D28k, calretinin or parvalbumin; somatostatin with calbindin D28k; vasoactive intestinal polypeptide with calretinin. Only vasoactive intestinal polypeptide- and calretinin-containing, double-labeled neurons were present at the day of birth, whereas the other double-labeled neurons appeared at later stages of development. The ratios of colocalizing neurons to single-labeled neurons in each type of colocalization were differentiated and reached the highest value (51%) for vasoactive intestinal polypeptide- and calretinin-double-labeled neurons. In conclusion, the claustral interneurons represent differentiated population in respect to the occurrence of neuropeptides and calcium-binding proteins. The expression of studied substances is changing during the postnatal period. © 2009 Elsevier Inc. All rights reserved.
1. Introduction On the basis of comparative, developmental and hodological studies, the claustrum (Cl), together with the endopiriform nucleus (Edn) and the pallial amygdala, may be regarded as a single entity, the claustroamygdaloid complex [27,29]. The Cl consists of glutamatergic projecting neurons and GABA-ergic interneurons, revealing the presence of neuropeptides such as neuropeptide Y (NPY), somatostatin (SOM), and vasoactive intestinal polypeptide (VIP), as well as calcium-binding proteins (CaBPs) such as calbindin D28k (CB), calretinin (CR), and parvalbumin (PV) [9,14,30]. According to numerous published results the GABA-ergic neurons of the claustroamygdaloid complex, which predominantly represent local circuit cells, are characterized by the occurrence of various combinations of neuropeptides and CaBPs
∗ Corresponding author. Tel.: +48 58 349 1401; fax: +48 58 349 1421. ´ E-mail address: [email protected] (P. Kowianski). 0361-9230/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2009.06.020
[13,15,21,22,24,25]. However, the differences in the colocalization patterns between various parts of this complex were only partially described. In particular, there is no information available concerning the colocalization of the neuropeptides and CaBPs in the claustral neurons during postnatal development. A growing body of evidence indicates that the GABA-ergic neurons of the claustroamygdaloid complex may originate in the subpallium and migrate tangentially to reach the target structures [27–29]. Gene expression studies in the earliest stages of claustral development reveal that some of its neurons may originate subcortically, in the ganglionic eminences. The presence of GABA-ergic interneurons, containing CB and CR, and originating in the subpallial progenitor centers was noted in the Cl [17,32]. Taking into account the lack of information concerning the immunohistochemical properties of the claustral interneurons, especially during early development, we deemed it relevant and important to investigate the colocalization patterns between neuropeptides and CaBPs in the Cl during a four-month postnatal period. These data may prove useful in future studies characterizing
P. Kowia´ nski et al. / Brain Research Bulletin 80 (2009) 100–106
the embryologic derivation of claustral interneurons, while revealing their possible, distinctive immunohistochemical patterns. 2. Material and methods Fifty-six Wistar rats of both sexes (29 males and 27 females) were used in this study. The animals were treated in accordance with the published guidelines established by the Local Ethical Committee, as well as the European Communities Council Directive of 24 November 1986 (86/609/EEC). Eight age-dependent experimental groups were used in the study (P0, P4, P7, P14, P21, P28, P60, and P120), with seven animals in each. The animals were irreversibly anesthetized with sodium pentobarbital (thiopental sodium, Biochemie GmBH, Germany; 80 mg/kg of body weight, i.p.) and transcardially perfused with 200 ml of 0.9% NaCl solution (pH 7.4) at room temperature, followed by 400 ml of fixative consisting of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, Sigma–Aldrich, UK, pH 7.4, and 4 ◦ C). The brains were then removed and stored in the same fixative for 120 min and subsequently stored in 30% sucrose in 0.1 M PBS (pH 7.4 and 4 ◦ C) for a minimum of 24 h. After freezing, the brains were coronally sliced into 40 m serial sections on a sliding microtome.
2.1. Immunohistochemistry The free-floating sections were processed for fluorescence immunocytochemistry. The following primary antibodies were used: anti-NPY (rabbit; 1:400, Affiniti, UK), anti-SOM (rabbit; diluted 1:300, Euro-Diagnostica AB, Sweden), anti-VIP (rabbit; 1:100, Cappel, ICN Pharmaceuticals Inc., USA), anti-CB (mouse; 1:100, Sigma Chemical Company, USA), anti-CR (goat; 1:1000, Chemicon, USA) and anti-PV (mouse; 1:500, Sigma Chemical Company, USA). Sections were incubated for 1 h in blocking solution containing 0.1% bovine serum albumin in PBS and 10% normal goat serum. After washing in PBS, the sections were incubated at room temperature overnight in a mixture of primary antibodies in order to assess the colocalization of selected neuropeptides with CaBPs. Subsequently, the sections were washed in PBS and incubated in secondary antibodies at room temperature for 1 h. The following secondary antibodies were used in the study: goat anti-rabbit, goat anti-mouse, donkey anti-goat and donkey anti-rabbit; conjugated to indocarbocyanine [Cy-3] at 1:800, or to fluorescein [FITC] at 1:200 (Jackson ImmunoResearch, USA). After washing in PBS, the sections were mounted on slides, dried and coverslipped with Vectashield (Vector Laboratories Inc., USA). Immunohistochemical controls were performed. The specificity of staining was verified by the omission tests. The immunohistochemical procedure was conducted as previously described, except that the primary antibodies were omitted in the presence of two secondary antibodies or secondary antibodies were omitted. In order to exclude the possibility of cross-reaction between secondary antibodies, the immunohistochemical procedure was performed with the primary antibody and non-corresponding, secondary antibody. All above-mentioned tests revealed specificity of staining and the absence of cross-reaction.
2.2. Qualitative study Double-labeled sections were examined with a fluorescence microscope (Eclipse E600, Nikon, Japan), equipped with a confocal imaging system (MicroRadiance, BioRad, UK) and an Argon laser (excitation 488/514 nm). In order to rule out spectral bleed-through, the preparations were checked in non-corresponding channels. Two image analysis programs (LaserSharp 2000 v. 2.01, Bio-Rad, UK and LaserPix v. 4.0, BioRad, UK) were used to prepare the illustrations. The morphological features of developing neurons and neuropil in immunocytochemically distinct subpopulations were studied. The features of mature neurons were defined based on the increased cytoplasm-to-nucleus proportion and the decreased proportion of unipolar neurons in relation to all immunolabeled neurons. The features of mature neuropil were presumed to encompass: the increased fiber density, the occurrence of immunolabeled ramifications, varicosities and immunoreactive synapses, and increased staining intensity.
2.3. Quantitative study Sections of the anterior, central, and posterior regions of the Cl were identified under 10× magnification. On each section, the borders of the Cl were marked as separate inclusion areas under 4× magnification. In each region, test areas were chosen to obtain the highest number of immunostained neurons. The cells were analyzed using confocal image stacks obtained in the maximal intensity projection mode using a 40× objective. Double-labeled sections were used to estimate the ratio of double-labeled neurons to all labeled neurons, as well as the ratio of double-labeled neurons to neuropeptide-labeled, or CaBP-labeled neurons in each test area. Data were presented as percentages. All experiments with the various antibody combinations were conducted in at least five animals.
101
2.4. Statistics All calculations were performed using Excel 2000 (Microsoft, USA). Mean values and standard deviations were calculated for each group. Neuronal counts were converted into percentages. The differences between the percentages of doublelabeled neurons in each of the three regions (anterior, central and posterior Cl) were evaluated by procedure analogous to the Tukey test (multiple comparisons for proportions). The significance level was set to 0.05.
3. Results Our studies revealed distinct and differentiated patterns of colocalization between neuropeptides and CaBPs. NPY colocalized with CB, CR and PV (NPY/CB, NPY/CR, NPY/PV). SOM revealed colocalization with CB (SOM/CB), whereas VIP colocalized with CR (VIP/CR). Only one type of colocalization, VIP/CR, was present at birth (P0; Fig. 1A–C). The NPY/CB colocalization was observed from P4 onward (Fig. 1D–F). After one week (P7), SOM/CB (Fig. 1G–I) and NPY/CR (Fig. 1J–L) colocalizations were first noted. After two weeks (P14), colocalization of NPY/PV was revealed (Fig. 1M–O). The aspiny, nonpyramidal immunoreactive neurons were present in all studied sections of the Cl. In all studied age-groups medium, bipolar and multipolar neurons were most frequently observed. With increasing age, unipolar neurons were noted with decreasing frequency, along with a small but gradual decrease in cytoplasmic volume. We also observed characteristic age-related changes of developing neuropil. The density of immunoreactive fibers, with varicosities and ramifications, increased in all types of colocalizations until the end of the fourth postnatal week. An increase in the percentage of double-labeled neurons was seen for the colocalization of NPY/CB and NPY/CR (Fig. 2A and B), as well as for SOM/CB and VIP/CR (Fig. 2D and E). A transient increase of the percentage of NPY/CB-ir neurons was noted at the end of the third postnatal week (P21). Among the SOM/CB -, NPY/CR - and VIP/CR-ir neurons, the highest percentage of double-labeled neurons occurred at the end of the observation period. No changes in percentage values were observed for NPY/PV neurons (Fig. 2C). The maximal value of the percentage of double-labeled neurons, compared to all labeled neurons in the studied sections was observed for VIP/CR (51%; Table 1). Lesser values were obtained for NPY/CB (32%) and SOM/CB (31%). The lowest values were observed for NPY/CR (9.5%) and NPY/PV (9%). The highest ratio of doublelabeled neurons to neuropeptide- or CaBP-labeled neurons was estimated for VIP/CR colocalization (71% and 64%, respectively; Table 1). The lowest values were observed for NPY/CR (14% and 22%, respectively) and NPY/PV (15% and 14%, respectively) types of colocalization. 4. Discussion Our most important observations are concerned with determining the patterns of colocalization between neuropeptides and CaBPs, and the time of their occurrence, as well as the percentage of colocalizing neurons. Our studies revealed three predominant forms of colocalization between neuropeptides and CaBPs: (1) NPY with all studied representatives of CaBPs (i.e., CB, CR, and PV), (2) SOM with CB, and (3) VIP with CR. Each of the studied types of colocalization occurred at different stages of development and only one of them (VIP/CR) was present at birth (P0). The observed types of colocalization could be divided into three groups, according to the values of the percentage of colocalization. 4.1. The colocalization types of neuropeptides and CaBPs in the Cl reveal characteristic patterns of temporal development At the moment of birth (P0) only one type of double-labeled neurons (VIP/CR) was observed in the Cl. The remaining types of
102
P. Kowia´ nski et al. / Brain Research Bulletin 80 (2009) 100–106
colocalization occurred at later stages of postnatal development. Characteristically, the colocalization of NPY with PV was revealed after two weeks (P14) of life. These observations indicate that the biochemical and consequently functional development of claustral interneurons, represented by occurring specific subtypes of neurons, extended during both prenatal and postnatal period. The development of the CaBPs-containing, GABA-ergic neurons in the claustral complex was studied in the mouse [6,17]. Whereas CB- and CR-ir neurons were present early in the embryonic period, the PV-ir neurons started to be seen postnatally, which may correspond to our observation concerning the late occurrence of NPY/PV colocalization in the rat Cl. All neuropeptides studied by us were observed in the claustral interneurons from the day of birth [see also Ref. [14]]. Although the definite age of their occurrence in the Cl was not described, the recent data concerning the expression of SOM and NPY in the amygdala indicate their very early occurrence in the mouse (from embryonic day 12.5) [31]. The observed biochemical and morphological changes seen in the claustral interneurons occur subsequently to a long sequence of processes and events which shape the neuronal population during both prenatal and postnatal development. The developmental patterns we have seen in claustral interneurons are reflective of the general hypothesis advanced by Xu et al. [36,37], which attempts to explain the origins of interneuron diversity. In accordance with this hypothesis, the first possibility assumes diversification of interneurons in their place of origin (e.g. in the proliferative zone). The second possibility assumes that interneuronal subtypes could develop from multipotential GABA-ergic cells at their place of settlement, under the influence of local cues (e.g. neurotrophic factors). This process could be also facilitated by the initiation of neuronal activity. Based on our present level of understanding of these issues, we may take into account both of the above-mentioned hypotheses with regard to the development of claustral interneurons and their characteristic expression of immunochemical markers. Confirmation of either or both of these hypotheses requires further research and the verification of results at the level of the protein expression, as well as the assessment of mRNA and gene activation. 4.2. Claustral interneurons characterized by various types of colocalization constitute quantitatively differentiated subpopulations In order to further characterize the observed types of immunohistochemical colocalization, we compared their maxi-
mal percentage values and the ratios of double-labeled neurons to neuropeptide-labeled, or CaBPs-labeled neurons. This allowed us to divide the studied types into three groups, characterized by high, moderate, and low values. The highest percentage of colocalization was observed for VIP/CR (51%; Table 1). Moderate values were noted for NPY/CB (32%) and SOM/CB (31%). The lowest values were seen in NPY/CR (9.5%) and NPY/PV (9%). Additionally, the colocalization of VIP/CR may be expected in a very high (prevalent) percentage of VIP- or CR-containing claustral neurons (in 71% and 64% of all these neurons, respectively). On the other hand, PV-containing neurons in the Cl may be expected predominantly as revealing very low percentage of colocalization. Only 14% of all these neurons may be expected to contain one of the studied neuropeptides, namely NPY. Although the results of quantitative studies of various colocalizations are not frequently published, some evidence can be found in the literature. The colocalization of VIP with CR was described as being seen quite often in the cerebral cortex and hippocampus of various mammalian species [3,4,7,12,33]. The colocalization of SOM with CB in the cerebral cortex of the rat is more frequent than NPY with CB. As well, the colocalization of NPY/CR and NPY/PV is very rarely seen in mammals [1,3–5,7]. Finally, the colocalization between two neuropeptides NPY and SOM was reported in numerous structures. In the amygdala the percentage of this colocalization is very high, although differentiated in particular nuclei [25,26,31]. In the cerebral cortex this value is lower and reveals characteristic changes during postnatal development [16]. The percentage of GABA-ergic interneurons in the Cl of various mammalian species was estimated between 7% and 12% [2,11,34]. The population of CaBPs-containing neurons in the mouse Cl has been estimated at 12.3% of all claustral neurons. Among these, CBir neurons account for 5.4%, whereas PV-ir neurons 7.9% [30]. There is no data available concerning the percentage of neuropeptidecontaining neurons in the Cl. The development of the rat Cl reveals characteristic features, concerned principally with the changes of the total number of neurons. From the stage of its occurrence at E20, the increase of the total number of neurons is observed until the end of the first postnatal week (P7; estimated value of the total number of neurons at this stage of development is about 93 800) [18]. During the second postnatal week a 30% decrease of the total number of neurons is reported, which may be the consequence of apoptotic death. Because of this specific pattern of the claustral development it seems for us useful to compare the relative (percentage) values of the ratios between various neuronal subpopulations at definite stages of development.
Table 1 Maximal percentage values of double-labeled neurons for each type of colocalization and ratios of double-labeled neurons to neuropeptide- or CaBP-labeled neurons in the claustrum during postnatal development. Type of colocalization
Age
Maximal percentage of double-labeled neurons (%)
Ratio of double-labeled neurons to all labeled neurons, n/N
Percentage of double-labeled neurons to neuropeptide- or CaBP-labeled neurons (%)
Ratio of double-labeled neurons to neuropeptide- or CaBP-labeled neurons n/Np and n/Cp
VIP/CR
P120
51
283/534
71 64
283/399 283/442
NPY/CB
P21
32
131/410
45 52
131/291 131/252
SOM/CB
P120
31
174/580
50 46
174/351 174/381
NPY/CR
P120
9.5
214/2247
14 22
214/1562 214/955
NPY/PV
P14
9
181/2013
15 14
181/1207 181/1293
Cp: calcium-binding-labeled proteins; n: number of double-labeled neurons; N: total number of labeled neurons; Np: neuropeptide-labeled neurons.
P. Kowia´ nski et al. / Brain Research Bulletin 80 (2009) 100–106
103
Fig. 1. Colocalization of calcium-binding proteins and neuropeptides in the claustral interneurons during postnatal development. The double-labeled neurons (indicated by the arrows) are presented occurring for the first time at definite stages of development. The controls of non-colocalizing neurons and elements of neuropil, observed at earlier stages of postnatal development are presented in the right bottom corners. (A–C) VIP/CR colocalization at P0; (D–F) NPY/CB colocalization at P4; (G–I) SOM/CB colocalization at P7; (J–L) NPY/CR colocalization at P7; (M–O) NPY/PV colocalization at P14. (scale bars = 50 m; immunofluorescent staining: FITC – green; Cy3 – red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
104
P. Kowia´ nski et al. / Brain Research Bulletin 80 (2009) 100–106
4.3. The colocalization patterns in distinct parts of the claustroamygdaloid complex Comparing the colocalization patterns between distinct parts of the claustroamygdaloid complex, namely, the Cl, Edn and pallial amygdala, may provide some interesting data concerning overall development. Not only are these structures topographically related, but they also share numerous connections and functional interrelationships [8,19,20,35]. Our earlier studies of colocalizations amongst neuropeptides and CaBPs in the Edn of the adult rat revealed the occurrence of some types similar to those we have seen in the Cl, namely, SOM/CB and VIP/CR [15]. Moreover, we noted SOM/PV colocalization in the Edn, which we did not see in the Cl. This rare type of colocalization is also present in neurons of the neighboring anterior piriform cortex [5]. Interest-
ingly, we did not find the colocalization of any CaBPs with NPY in the Edn, which is an important difference to note in comparison to the Cl. These points of differentiation serve to support the hypothesis that assumes a differentiated origin of the Cl and the Edn [27,29]. On the basis of the differential expression of developmental regulatory genes (e.g. Emx1, Cad8, Sema5A), it was stated that the dorsolateral part of the Cl derives from the lateral pallium, whereas the ventromedial part of the Cl and Edn are considered to have originated in the ventral pallium [27]. This distinction might serve to explain the existence of interneurons, characterized by the differentiated gene activity in various parts of the claustroamygdaloid complex, and which may be controlled by separate molecular mechanisms. Comparing our results with the published data concerning the colocalization types in the pallial amygdala, we may conclude that
Fig. 2. The percentage values of double-labeled neurons in relationship to all labeled neurons in the claustrum at various stages of development. The colocalizations of: (A) NPY with CB; (B) NPY with CR; (C) NPY with PV; (D) SOM with CB and (E) VIP with CR. The data are presented as mean values and standard deviations.
P. Kowia´ nski et al. / Brain Research Bulletin 80 (2009) 100–106
three types of the colocalizations (SOM/CB, NPY/CB and VIP/CR) are present in both described structures. The SOM/CB-ir double-labeled neurons are present in the basolateral part of amygdala in the rat [21,25]. The percentage of double-labeled neurons is high. In the basolateral nucleus these neurons represent 91% of SOM-ir neurons (and 28% of CB-ir neurons), in the lateral nucleus double-labeled neurons represent 67% of SOM-ir neurons (about 37% of CB-ir neurons). NPY/CB-ir double-labeled neurons, although less numerous than SOM/CB-ir, are also present in the basolateral part of amygdala [21,25]. Taking into account a high percentage value (although differentiated in particular amygdalar nuclei) of the SOM/NPY colocalization [23,26,31], as well as SOM/CB colocalization [21,25], the co-existence of SOM, NPY and CB in the same neuron may not be excluded in various parts of the claustroamygdaloid complex. Similarly to the Cl, the VIP/CR colocalization was revealed in the basolateral part of amygdala [21,25]. The percentage of doublelabeled neurons in this type of colocalization reaches over 66%, which is higher than the value estimated in the Cl (51%). In the contrary to amygdala, the colocalization of NPY with two CaBPs (NPY/PV and NPY/CR) is observed in the Cl. Assessing critically the apparent differences between the Cl and amygdala, we try to interpret these in two ways. First, both types of colocalizations (NPY/PV and NPY/CR) observed in the Cl are very rare. The percentage of double-labeled neurons is very low and does not exceed 10% in both types of colocalizations. On the basis of the published, although incomplete data, concerning the colocalization of NPY with CaBPs in various parts of the amygdala, occurrence of NPY/PV- and NPY/CR- double-labeled neurons cannot be excluded. The verification of this hypothesis would require further quantitative studies of all amygdalar nuclei. On the other hand, the apparent differences between types of colocalizations occurring in the Cl and amygdala may be a consequence of different origin of the neuronal subpopulations. The considerable percentage of interneurons, localized in the claustroamygdaloid complex, derives from the subcortical progenitor structures [27,29,31]. However, there are important differences, concerned with the participation of particular progenitor areas in generation of interneurons, settling in various pallial and subpallial structures. According to the recently published data, the amygdalar (pallial and subpallial) interneurons, at least partially, are generated in the anterior entopeduncular area, the commissural preoptic region, medial, lateral and caudal ganglionic eminences (MGE, LGE and CGE, respectively), and definite regions of the hypothalamus [10,31]. The claustral interneurons, at least partially, may be generated in the MGE and CGE [17,27,32]. However, there is no data available in the literature, concerning the possible role of the other progenitor structures, like the anterior entopeduncular area, in generation of the claustral interneurons. All these data clearly indicate that the neuronal subpopulations localized in various parts of the claustroamygdaloid complex, may originate in the different and distant structures. The neurons of similar or different immunohistochemical characteristics may develop in each of these progenitor areas. Migration of interneurons from their places of origin to the targets of settlement, as well as the influence of different neurotrophic factors at the early stages of development, may account for the heterogeneity of the interneuronal population. All these processes may be responsible for the apparent differences in the immunohistochemical characteristics of the interneurons localized in various parts of the claustroamygdaloid complex.
Conflict of interest None.
105
Acknowledgements This research was supported by funds from the Polish State Committee of Scientific Research grant no. W-705. The technical assistance of Mrs. S. Scislowska, is greatly appreciated. References [1] C. Andressen, I. Blümcke, M.R. Celio, Calcium-binding proteins: selective markers of nerve cells, Cell Tissue Res. 271 (1993) 181. [2] H. Braak, E. Braak, Neuronal types in the claustrum of man, Anat. Embryol. 163 (1982) 447. [3] B. Cauli, E. Audinat, B. Lambolez, M.C. Angulo, N. Ropert, K. Tsuzuki, S. Hestrin, J. Rossier, Molecular and physiological diversity of cortical nonpyramidal cells, J. Neurosci. 17 (1997) 3894–3906. [4] B. Cauli, J.T. Porter, K. Tsuzuki, B. Lambolez, J. Rossier, B. Quenet, E. Audinat, Classification of fusiform neocortical interneurons based on unsupervised clustering, Proc. Natl. Acad. Sci. USA 97 (2000) 6144–6149. [5] S.L. Cummings, Neuropeptide Y, somatostatin, and cholecystokinin of the anterior piriform cortex, Cell Tissue Res. 289 (1997) 39–51. [6] J.C. Davila, M.A. Real, L. Olmos, I. Legaz, L. Medina, S. Guirado, Embryonic and postnatal development of GABA, calbindin, calretinin, and parvalbumin in the mouse claustral complex, J. Comp. Neurol. 481 (2005) 42. [7] J. DeFelipe, Types of neurons, synaptic connections and chemical characteristics of cells immunoreactive for calbindin-D28K, parvalbumin and calretinin in the neocortex, J. Chem. Neuroanat. 14 (1997) 1. [8] L.R. Edelstein, F.J. Denaro, The claustrum: a historical review of its anatomy, physiology, cytochemistry and functional significance, Cell Mol. Biol. 50 (2004) 675–702. [9] L.E. Eiden, E. Mezey, R.L. Eskay, M.C. Beinfeld, M. Palkovits, Neuropeptide content and connectivity of the rat claustrum, Brain Res. 523 (1990) 245. [10] M. García-López, A. Abellán, I. Legaz, J.L. Rubenstein, L. Puelles, L. Medina, Histogenetic compartments of the mouse centromedial and extended amygdala based on gene expression patterns during development, J. Comp. Neurol. 506 (2008) 46–74. [11] S.M. Gomez-Urquijo, I. Gutierrez-Ibarluzea, J.L. Bueno-Lopez, C. Reblet, Percentage incidence of gamma-aminobutyric acid neurons in the claustrum of the rabbit and comparison with the cortex and putamen, Neurosci. Lett. 282 (2000) 177. [12] Y. Kawaguchi, Y. Kubota, Physiological and morphological identification of somatostatin- or vasoactive intestinal polypeptide-containing cells among GABAergic cell subtypes in rat frontal cortex, J. Neurosci. 16 (1996) 2701–2715. [13] S. Kemppainen, A. Pitkänen, Distribution of parvalbumin, calretinin, and calbindin-D(28k) immunoreactivity in the rat amygdaloid complex and colocalization with gamma-aminobutyric acid, J. Comp. Neurol. 426 (2000) 441–467. ´ [14] P. Kowianski, J.M. Mory´s, J. Dziewiatkowski, S. Wójcik, J. Sidor-Kaczmarek, J. Mory´s, NPY-, SOM- and VIP-containing interneurons in postnatal development of the rat claustrum, Brain Res. Bull. 76 (2008) 565–571. ´ ´ [15] P. Kowianski, J.M. Mory´s, S. Wójcik, J. Dziewiatkowski, A. Luczynska, E. Spodnik, J.P. Timmermans, J. Mory´s, Neuropeptide-containing neurons in the endopiriform region of the rat: morphology and colocalization with calcium-binding proteins and nitric oxide synthase, Brain Res. 996 (2004) 97–110. [16] E.Y. Lee, T.S. Lee, S.H. Baik, C.I. Cha, Postnatal development of somatostatinand neuropeptide Y-immunoreactive neurons in rat cerebral cortex: a doublelabeling immunohistochemical study, Int. J. Dev. Neurosci. 16 (1998) 63–72. [17] I. Legaz, M. Garcia-Lopez, L. Medina, Subpallial origin of part of the calbindinpositive neurons of the claustral complex and piriform cortex, Brain Res. Bull. 66 (2005) 470–474. [18] B. Maciejewska, J. Morys, B. Berdel, J. Dziewiatkowski, O. Narkiewicz, The development of the rat claustrum—a study using morphometric and in situ DNA end labelling techniques, Eur. J. Anat. 1 (1997) 137. [19] K. Majak, J. Mory´s, Endopiriform nucleus connectivities: the implications for epileptogenesis and epilepsy, Folia Morphol. (Warsz) 66 (2007) 267–271. [20] K. Majak, M. Pikkarainen, S. Kemppainen, E. Jolkkonen, A. Pitkänen, Projections from the amygdaloid complex to the claustrum and the endopiriform nucleus: a Phaseolus vulgaris leucoagglutinin study in the rat, J. Comp. Neurol. 451 (2002) 236–249. [21] F. Mascagni, A.J. McDonald, Immunohistochemical characterization of cholecystokinin containing neurons in the rat basolateral amygdala, Brain Res. 976 (2003) 171–184. [22] A.J. McDonald, Morphology of peptide-containing neurons in the rat basolateral amygdaloid nucleus, Brain Res. 338 (1985) 186–191. [23] A.J. McDonald, Coexistence of somatostatin with neuropeptide Y, but not with cholecystokinin or vasoactive intestinal peptide, in neurons of the rat amygdala, Brain Res. 500 (1989) 37–45. [24] A.J. McDonald, F. Mascagni, Colocalization of calcium-binding proteins and GABA in neurons of the rat basolateral amygdala, Neuroscience 105 (2001) 681–693. [25] A.J. McDonald, F. Mascagni, Immunohistochemical characterization of somatostatin containing interneurons in the rat basolateral amygdala, Brain Res. 943 (2002) 237–244. [26] A.J. McDonald, F. Mascagni, J.R. Augustine, Neuropeptide Y and somatostatinlike immunoreactivity in neurons of the monkey amygdala, Neuroscience 66 (1995) 959–982.
106
P. Kowia´ nski et al. / Brain Research Bulletin 80 (2009) 100–106
[27] L. Medina, I. Legaz, G. Gonzalez, F. De Castro, J.L. Rubenstein, L. Puelles, Expression of Dbx1, Neurogenin 2, Semaphorin 5A, Cadherin 8, and Emx1 distinguish ventral and lateral pallial histogenetic divisions in the developing mouse claustroamygdaloid complex, J. Comp. Neurol. 474 (2004) 504–523. [28] S. Nery, G. Fishell, J.G. Corbin, The caudal ganglionic eminence is a source of distinct cortical and subcortical cell populations, Nat. Neurosci. 5 (2002) 1279–1287. [29] L. Puelles, E. Kuwana, E. Puelles, A. Bulfone, K. Shimamura, J. Keleher, S. Smiga, J.L. Rubenstein, Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1, J. Comp. Neurol. 424 (2000) 409–438. [30] M.A. Real, J.C. Dávila, S. Guirado, Expression of calcium-binding proteins in the mouse claustrum, J. Chem. Neuroanat. 25 (2003) 151–160. [31] M.A. Real, R. Heredia, C. Labrador Mdel, J.C. Dávila, S. Guirado, Expression of somatostatin and neuropeptide Y in the embryonic, postnatal, and adult mouse amygdalar complex, J. Comp. Neurol. 513 (2009) 335–348. [32] C. Reblet, A. Alejo, T. Fuentes, P. Pró-Sistiaga, J. Mendizabal-Zubiaga, J.L. BuenoLópez, Expression of calcium-binding proteins in the proliferative zones around
[33]
[34] [35]
[36] [37]
the corticostriatal junction of rabbits during pre- and postnatal development, Brain Res. Bull. 66 (2005) 461–464. J.H. Rogers, Immunohistochemical markers in rat cortex: co-localization of calretinin and calbindin-D28k with neuropeptides and GABA, Brain Res. 587 (1992) 147. B. Spahn, H. Braak, Percentage of projection neurons and various types of interneurons in the human claustrum, Acta Anat. 122 (1985) 245. S. Wojcik, J. Dziewiatkowski, E. Spodnik, B. Ludkiewicz, B. Domaradzka-Pytel, P. Kowianski, J. Morys, Analysis of calcium binding protein immunoreactivity in the claustrum and the endopiriform nucleus of the rabbit, Acta Neurobiol. Exp. (Wars) 64 (2004) 449. Q. Xu, I. Cobos, E. De La Cruz, J.L. Rubenstein, S.A. Anderson, Origins of cortical interneuron subtypes, J. Neurosci. 24 (2004) 2612–2622. Q. Xu, E. de la Cruz, S.A. Anderson, Cortical interneuron fate determination: diverse sources for distinct subtypes? Cereb. Cortex 13 (2003) 670– 676.