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Praca oryginalna Original paper
The claustrum (Cl) and endopiriform nucleus (EN) are telencephalic neuronal structures which have been documented to be present in numerous mam-mals, excluding some species of monotremes (5). Microanatomical studies revealed that Cl and EN are located along rhinal fissures, between putamen, amyg-daloid nuclei and the insluar cortex. Both neuronal structures are separated from surrounding structures by external and extreme capsules (13). Many authors describe Cl and EN as one integral neuronal structure which comprises the claustrum proper and endopiri-form nucleus (groups of neurons located respectively above and below the olfactory sulcus), but some authors suggest that the presence of structural differ-ences between the species give basis to consider both structures as separate neuranatomical units. Although several experimental attempts have been conducted aiming at the clarification of the Cl and EN role its function(s) is still a matter of speculation (4, 8). In a series of experiments it has been shown that both excitatory as well as inhibitory neuronal pathways con-necting Cl/EN with the cerebral cortex, limbic system,
striatum, thalamus and hypothalamus are present (15, 24). Both Cl and EN are involved in the transmission and spread of epileptic seizures triggered in the limbic system (24). Recent studies indicate the role of Cl and EN in the pathogenesis of Alzheimer disease (17) and as generator of the unified perception (8).
In the central nervous system (CNS) calcium binding proteins (CaBP) belonging to the EF-hand family are commonly found in different neuronal subclasses and are generally thought to play a neuromodulatory role affecting the activity of excitatory and/or inhibitory neurons (2, 14). Additionally, many authors emphasize the usefulness of calbindin D-28k (CB) and parvalbu-min (PV) as molecular markers in developmental and functional studies of GABA-ergic neuronal pathway (10). Recent studies revealed that in the mammalian brain the expression of calbindin and parvalbumin and their co-localization patterns with a wide array of bio-logically active substances, mostly neuropeptides, may be functionally correlated with mechanisms underlying certain CNS disorders (14). So far, the presence of PV and CB has also been detected in the enteric nervous
Immunoreactivity to the parvalbumin
and calbindin D28k in the claustrum
and endopiriform nucleus of chinchilla
RADOSŁAW SZALAK, MAŁGORZATA MATYSEK, ALEKSANDRA KRAWCZYK, ALEKSANDRA OLEJARSKA, ROMAN LALAK, MARCIN B. ARCISZEWSKI
Department of Animal Anatomy and Histology, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Akademicka 12, 20-950 Lublin, Poland
Received 17.03.2015 Accepted 16.06.2015
Szalak R., Matysek M., Krawczyk A., Olejarska A., Lalak R., Arciszewski M. B.
Immunoreactivity to the parvalbumin and calbindin D28k in the claustrum and endopiriform nucleus of the chinchilla
Summary
Claustrum (Cl) and endopiriform nucleus (EN) are telencephalic structures present in almost all mammals; however, their exact function(s) remain unknown to date. Parvalbumin (PV) and calbindin-D28k (CB) are calcium-binding proteins (CaBPs) widely present in the central nervous system (CNS) and regulating many important cellular processes, such as the intracellular concentration of calcium, release of neurotransmitters and synaptic conductivity. The aim of the present study was to immunohistochemically determine the distribution patterns of PV and CB in the chinchilla’s Cl and EN. The highest expression of CB was observed in EN, whereas PV-immunoreactive neurons were more abundant in Cl. Three morphological types were identified in neurons expressing PV and CB. PV-immunoreactive neurons belonged to type I and type II, whereas CB-expressing cells were classified as of type I, II and III. It has been concluded that in Cl and EN of the chinchilla, both PV and CB may play a substantial regulatory role modulating the activity of the local neuronal network.
Med. Weter. 2015, 71 (10), 615-618 616
system (ENS) (3), as well as in numerous areas of CNS, including the Cl and EN of the monkey (19), cat (11), rat (6), rabbit (23), mouse (18) and human (1). Additionally, in our previous study we reported the expression pattern of CB and PV in the chinchilla’s hippocampus (21).
Considering that both calbindin and parvalbumin are valuable markers of different neuronal classes and play important roles in CNS (14), in the present study immunohistochemistry was applied to study the distribution pattern of PV and CB in CL and EN of chinchilla.
Material and methods
Animal care protocols, experimental design and meth-ods were reviewed and approved by the IInd Local Ethical
Committee at the University of Life Sciences in Lublin, Poland. Ten (n = 10) sexually mature male chinchillas (ca. 1.5-years-old) purchased from the “Raba” farm in Myślenice were used in the study. The brains were dissected out immediately after slaughter. The brains were fixed for 12 hours in cold buffered 10% formalin (pH = 7.0; +4°C). The material was processed conventionally for paraffin embedding. For further immunohistochemical analyses, paraffin sections of 6 µm thickness were cut and collected on SuperFrost Plus (Meznel-Glaser, Braunschweig, Ger-many) microscopy slides.
The slides were immunohistochemically stained (peroxi-dase-antiperoxidase method) according to the following pro-tocol. Prior to staining, sections were deparaffinized using xylene (3 × 15 minutes), passaged through an ethyl alcohol series and rinsed in distilled water. Next, the slides were transferred into plastic staining dishes filled with a citrate buffer (pH = 6.0). An antigen retrieval step was performed at 97°C (3 × 7 min) by a microwave oven (800 W), followed by cooling for 20 min. In order to block endogenous peroxidase activity, slides were rinsed with 3% hydrogen peroxidase for 20 minutes, washed in phosphate buffered saline (PBS, pH = 7.4) and incubated in 2.5% normal horse serum (S-2012; Vector, USA) for 20 minutes at room temperature (RT). Slides were once again washed with PBS, and incubated overnight (+4°C) with a mixture of either rabbit primary antisera raised against calbindin D-28k (1 : 3000; code CB-38a, SWant, Switzerland) or parvalbumin (1 : 2000; code PV25; SWant, Switzerland). After
several washings in PBS (15 minutes each) the sections were immersed for 1 hour (RT) with anti-mouse/rabbit Ig (ImPRESSTM; MP-7500 Vector, USA),
then washed and incubated with diami-nobenzidine (DAB, Vector, USA). After the final washing with PBS the slides were coverslipped and viewed under a light microscope (Axiolab, Zeiss, Germany) connected to a digital camera. Negative control incubations were per-formed by replacing the primary anti-body with the appropriate non-immune IgG in the same concentrations.
Morphometric analysis. The intensity of
immunoreac-tion for calbindin and parvalbumin was arbitrarily assessed using the following semi-quantitative scale: none (–), weak (+), moderate (++) and intense (+++). For counting purposes the following procedure has been applied. Starting at one corner of the preparation and moving across the preparation in a systematic fashion, the first 1200 cell bodies in the Cl and the first 1200 cell bodies in the EN that were encoun-tered to be immunoreactive to PV or CB were examined. The proportions of PV-IR and CB-IR neurons were pre-sented as percentages of the total number of PV and CB labeled neurons.
Statistical analysis. The obtained results were
statis-tically analyzed using one-way analysis of variance test (ANOVA) followed by post-hoc Tukey’s test. Data are pre-sented as means ± standard deviation (SD). Probabilities of less than 5% (P < 0.05) were considered significant.
Results and discussion
Based on the morphology (shape) of the neuronal cell bodies and the number of neuronal processes in Cl and EN of the chinchilla, at least several different morphological subclasses of PV-immunoreactive (IR) and CB-IR neurons were distinguished (Fig. 1). Oval or round large-sized neurons with a number of neuronal processes (more than four) were categorized as type I. Type II were comprised of bipolar neurons with two processes. Pyramidal CB-immunoreactive cells of medium to large size were classified as morphological type III (Fig. 1).
CB-immunoreactivity. In Cl and EN of the chin-chilla, substantial subpopulations of neuronal cell bodies intensively, moderately and weakly immu-nostained for CB were found (Fig. 2A and 2B). CB-positive neurons of Cl and EN frequently sent CB-immunoreactive processes. In general in both Cl and EN intensive immunoreaction to CB was observed in multiform round and oval neuronal cells, whereas moderate and weak immunoreactivity to CB was also noted in round, oval and pyramidal neurons (Fig. 2A). In EN, only a few round, oval and multiform neurons showed strong immunoreaction to CB, whereas the vast majority of oval, round, spindle and pyramidal neurons were weakly to moderately stained to CB (Fig. 2B). In
Fig. 1. Morphological type (I-III) cells expressing CB or PV in Cl and EN of chinchilla. CB-IR upper column and PV-IR lower column. Magnification 40 ×
Med. Weter. 2015, 71 (10), 615-618 617 Cl and EN of the chinchilla
as many as 30.3 ± 6.7% and 42.1 ± 11.8% (respectively) of CB-positive neurons were found. A subpopulation of CB-IR neurons present in EN was statistically larger than analogous found in Cl (P < 0.05).
PV-immunoreactivity. In chinchilla’s Cl and EN intensively, moderately and weakly stained PV-positive neurons were detected (Fig. 2C and 2D). In incidental PV-IR perikarya the pres-ence of PV in neuronal pro-cesses was also identified. In Cl, the majority of oval and round neurons showed strong immunoreaction to PV, whereas moderate to weak PV immunostaining was observed in single round, oval and spindle neurons
(Fig. 2C). In EN, single intensively and moderately stained to PV neurons were generally found to be round and oval in shape (Fig. 2D). 43.8 ± 8.6% of Cl neurons showed immunoreactivity to PV. A statisti-cally lower (P < 0.05) subpopulation of PV-IR neurons 35.3 ± 12.2% was found in EN. PV-positive neurons were classified as type I and II, whereas CB-positive neuronal cells belonged to type I, II and III. In Cl, sta-tistical differences (P < 0.05) between subpopulations of PV-IR and CB-IR neurons were noted. Proportions of PV-IR neurons found in Cl and EN were statistically similar (Fig. 3).
Species-specific differences concerning shapes of nerve cells in both Cl and EN show that the most common are medium-sized neurons with triangular, multipolar, oval and fusiform shapes (4). In the
chin-chilla claustrum, oval and round neurons prevail in the anterior and posterior part of the nucleus, whereas pyramidal, multipolar and fusiform neurons are less numerous (16).
As indicated in the present study, intensively, mod-erately and weakly stained PV-IR neurons are present in chinchilla’s Cl and EN. Most PV-IR neurons were classified as oval, round and spindle shaped cells of morphological type I and II. Similar distribution patterns of PV-immunoreactivity in relation to mor-phological subtypes (type I and II) were previously observed in rabbit (23), cat (11) and monkey (19). In rat the expression of PV was also observed in round and oval neurons (type I), whereas in mouse PV-IR neurons were predominantly classified as multiform (2, 19). In chinchilla’s Cl and EN, the observed CB-IR neurons belonged to all distinguished morphological subtypes (I, II and III), however the vast majority of CB-IR neurons were of type I and II. In a previous study on rats, the presence of numerous round and oval CB-IR neurons and incidentally spindle and pyramidal CB-IR neurons was reported (6). In Cl and EN of rab-bits, the majority of CB-positive neurons constituted large multipolar neurons (type II) (23). Interestingly, in Cl of mice the presence of round an multiform CB-IR neurons was found. In the latter species pyramidal CB-IR neurons were found exclusively in EN (18). In monkeys, immunoreactivity to CB was regularly observed in small-sized round and oval neurons (19).
Different amounts of PV- and CB-expressing neu-rons were found in both Cl and EN. In the present report statistical differences in percentages of PV-positive and CB-positive neurons of Cl and EN were found. In
Fig 2. Immunoreactivity to calbindin in the claustrum (A) and the endopiriform nucleus (B). PV-IR neurons in the claustrum (C) and the endopiriform nucleus (D). Magnification 20 ×
Fig. 3. The density of PV-IR and CB-IR cells in the cell of Cl and EN. Statistical differences (p < 0.05) between sub-populations of PV-IR and CB-IR neurons are marked with (*). Proportions of PV-IR neurons found in Cl and EN were statistically similar
Med. Weter. 2015, 71 (10), 615-618 618
the chinchilla’s Cl, subpopulations of PV-IR neurons were larger than those expressing CB, whereas in EN CB-IR neurons outnumbered PV-positive cells. Similar relationships between the percentages of PV- and CB-expressing neurons were discovered in the rat (6) and cat (11). In contrast, in rabbit Cl the most numerous subpopulation constituted PV-IR neurons, and both in Cl and EN CB-positive neurons were a relatively less numerous subpopulation (23).
Both EF-hand CaBP (PV and CB) are frequently utilized as useful neurochemical markers in studies of CNS neurons (12, 20). Similarly to other central nuclei (like hippocampus or parahippocampal gyrus), Cl and EN neurons, basing on the presence of specific neurotransmitters (including CaBP), can be function-ally divided into principal neurons and interneurons. Principal neurons utilize glutamate as a major excit-atory neurotransmitter whereas interneurons predomi-nantly express another inhibitory neurotransmitter, GABA (7, 22). As previously reported, in the cerebral cortex and hippocampus in the mammalian Cl and EN PV is expressed in non-pyramidal cells as well as in round, oval, spindle and multipolar interneurons. In the latter nuclei the presence of CB has been found in interneurons as well as in pyramidal-shaped cells most likely to be projection neurons (23). Through regulation of intracellular Ca2+ concentration CaBP
participate in the functional regulation of neuronal activity (7). Indirectly, CaBP are also key regulators of vital neuronal functions, e.g. metabolic processes (9). The presence of PV and CB in different cellular types of Cl and EN neurons suggests that in these nervous structures both CaBP are involved in the modulation of transmitting the local neuronal network as well as in crossmodal processing (23). Despite many attempts, knowledge of the detailed roles of Cl and EN is still fragmentary and far from fully understood. Because Cl has prominent connections with other brain regions (predominantly with cortex), it is speculated that Cl may play a coordinatory role able to process somato-sensory, auditory and visual stimuli (24). The varied distribution of this protein in different types of Cl and EN neurons requires further studies. The distribution patterns of PV-IR and CB-IR neurons in the Cl and EN of chinchilla presented here is an introductory analysis which may be a necessary basis for further functional studies of the both proteins, including co-localization with other calcium buffers and/or neurotransmitters.
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Corresponding authors: Dr Radosław Szalak, 12 Akademicka Street, 20-950 Lublin, Poland; e-mail: radek.szalak@up.lublin.pl
