Bull Vet Inst Pulawy 57, 387-391, 2013 DOI: 10.2478/bvip-2013-0067

Immunoreactivity of the calbindin D28k in the of chinchilla

Radosław Szalak, Jadwiga Jaworska-Adamu, Karol Rycerz, Paweł Kulik, Marcin Bartłomiej Arciszewski

Department of Animal Anatomy and Histology, Faculty of Veterinary Medicine, University of Life Sciences, 20-950 Lublin, Poland [email protected]

Received: May 22, 2013 Accepted: September 13, 2013

Abstract Ten adult male chinchillas were used. The localisation of calbindin D28k (CB) was examined with the use of two types of reactions: immunocytochemical peroxidase-antiperoxidase and immunofluorescence staining with a specific monoclonal antibody against CB. Immunocytochemical examination demonstrated the presence of CB-positive neurons in the following layers of all parts the parahippocampal gyrus (PG): marginal, external cellular, middle cellular, and internal cellular, i.e. in entorhinal area, , and presubiculum. Immunofluorescence staining revealed the presence of CB in both Hu C/D- immunoreactive (IR) neurons and nervous fibers of the PG. CB-IR neuronal cell bodies were moderately numerous (ca. 10% of Hu C/D-IR neurons) and clearly distinguished from the background. Each layer of the area consisted of two types of neurons: pyramidal and multiform. Among the second type of neurons, four kinds of morphologically different neuronal sub- classes were observed: multipolar, bipolar, round, and Cajal-Retzius cells. It is concluded that the expression of CB in the PG of the chinchilla is species specific and limited to several subclasses of neurons.

Key words: chinchilla, parahippocampal gyrus, calbindin D28k, memory.

Introduction as calcium-binding proteins. The presence of glutamic acid as an excitatory neurotransmiter in the primary The presence of many structures of the limbic cells is particulary important for memorising process system affects mutual connections with . and learning. Gamma-aminobutyric acid (GABA), Some of them form a unit called hippocampal present in interneurons, is responsible for the inhibitory formation, which consists of included effect on other interneurons as well as on primary cells, in archicortex (19). This complex formation belonging which form a network involved in the control of to the includes: entorhinal area, neuronal activity (8, 9, 14, 27). As it was shown in parasubiculum, presubiculum, and these two form guinea pig, a specific topographical localisation of PG parahippocampal gyrus (PG), as well as , participates in bidirectional communication between proper , and . Entorhinal the hippocampus and neocortex (23). Afferent and area, parasubiculum, and presubiculum belong to efferent neuronal pathways of limbic system e.g. i.e. transitional form between between the rhinencephalon’s structures belonging to phylogenetically old archicortex, and neocortex (16, the and neocortex, , 25). PG consists of four layers: marginal layer, cellular , hypothalamus, and brainstem affect the layer external, middle, and internal, observed e.g. in plurality of functions in the central sheep, chinchilla, guinea pig, monkey, and macaque (CNS). These connections play a crucial role in (11, 22, 23, 27, 29). The individual layers are autobiographical-declarative memory, spatial memory, composed of multiform nervous cells: pyramidal, as well as in memory formation and consolidation and fusiform, stellate, round, and oval. Similarly as in other optimalisation during sleep (24, 29). The afferent and areas of the brain, PG neurons are classified into two efferent pathways take part in types: primary neurons and interneurons. These cells the autonomic regulation in such processes as functionally differ from each other, that is associated reproduction, food intake, in some endocrine responses, with the presence of a number of neuropeptides as well in extrapyramidal motor activities (6, 12, 14, 16, 18– 388 R. Szalak et al. / Bull Vet Inst Pulawy / 57 (2013) 387-391

20, 23). In the current literature, two kinds of calcium- IgG (1:50, Sigma-Aldrich). Localisation of the binding proteins, belonging to the „EF-hand” family, antiserum complexes was visualised with the are described: sensory and buffer. The calcium ions’ chromogen 3,3´-diaminobenzidin tetrahydrochloride buffering proteins include: parvalbumin (PV), calbindin (DAB; Sigma-Aldrich). In the stained sections, reaction D28k (CB), calretinin (CR), calcineurin, and many products formed as water-insoluble brown precipitates others. CB as a buffering protein is considered as an of different intensity were found. Finally, the sections excellent neurochemical marker in the CNS. In contrast were washed in distilled H2O, dehydrated in alcohol, to other „EF-hand” family proteins, it appears in an cleared, and mounted in DPX (Fluka). The slides were early embryonic period. Its expression maintains at a examined under a light microscope (Axiolab, Zeiss, constant level in adults and decreases in aging Germany) connected to a digital camera. In order to test individuals. CB, like most buffering proteins, locates in the specificity of antibodies used, control staining in CNS neurons, protecting them against the calcium ions’ which the primary antibodies were omitted or replaced neurotoxic effect. At the cellular level, it is responsible with non-immunoreactive sera were made. for pre- and postsynaptic calcium signals regulation Immunofluorescence. For double immunohisto- and synchronisation (5, 16). The knowledge on the chemical staining, an indirect immunofluorescence regulatory role of calcium binding proteins in pivotal method described elsewhere (2) was used. Briefly, after processes is far from being fully understood. Important the dissection, the brain was immersed in Stefanini’s research is conducted to specify distribution and co- solution of paraformaldehyde and picric acid for 24 h localisation of this protein in order to explain functions (4 C). After the fixation, it was rinsed for several days and processes occurring in neurons in the pathogenesis in Tyrode’s solution containing sucrose (one change of many CNS diseases. Until now, the immuno- per 1 d), and then embedded in O.C.T compound and reactivity to CB have been localised in neurons of the cut with a cryostat into 10 µm-thick sections. Prior to mammalian enteric nervous system (3) and CNS (1, 4– the staining, the selected sections were dried at room 6, 12, 22, 28), including GP in human (7), monkey temperature for 30 min. Potential non-specific binding (17), and mouse (25). In our previous research, CB sites in the tissue were blocked by incubation with PBS expression in some CNS areas of chinchilla was (3×15 min) supplemented with 10% NGS, 0.25% presented (12, 13, 28). Considering that calcium- bovine serum albumin, and 0.25% Triton X-100 binding proteins are valuable markers of different (Sigma-Aldrich, USA). The sections were incubated at neuronal classes and play important role in the CNS room temperature overnight with a combination of (15, 21), immunohistochemistry was applied to mouse monoclonal Hu C/D antibodies (1:400; examine the distribution pattern of CB in GP of Molecular Probes, USA) and rabbit calbindin D-28k chinchilla. (1:2000; SWant, Switzerland) anitsera. Bound primary antibodies were visualised with Texas Red-conjugated anti-rabbit goat IgG (1:400; MP Biomedicals, USA) Material and Methods and FITC-conjugated anti-mouse goat IgG (1:400; MP Animals. Ten sexually mature male chinchillas Biomedicals). After final washes, the specimens were (ca. 1.5 year old) were used in the study. Their coverslipped and viewed with a spinning disk confocal were dissected out immediately after slaughter. The microscope (BX-DSU Olympus, Japan) equipped with brains of five animals were subjected for interference filters appropriate for Texas Red (545–580 immunocytochemical studies, whereas brains from nm) and FITC (470–490 nm). The specificity of the other five animals were used for immunofluorescence primary antibodies was previously demonstrated (3) by staining. the pre-absorption experiments. The specificity of Immunocytochemistry. After the dissection, the immunohistochemical reaction was also tested by the whole brain (n = 5) was fixed in buffered 10% formalin experiments in which the primary antibodies were (pH 7.0) for 12 h at 4 C. After the fixation, the material either omitted or replaced with non-immunoreactive was processed through a graded series of alcohols, sera. The frequency of CB-IR GP neurons was cleared in xylene, embedded in paraffin, and cut to 6 expressed as a percentage relative to the total number µm-thick transverse sections. Deparaffinised sections of Hu C/D positive perikarya. In each animal a random were processed according to peroxidase anti-peroxidase sample of a minimum two hundred of Hu C/D-IR GP (PAP) immunocytochemical technique. Endogenous neurons was examined. Only neurons with well-visible peroxidase activity was eliminated from the sections by nuclei were counted. incubation with 0.3% H2O2 for 10 min. In order to block unspecific binding, an incubation with 1% normal goat serum (NGS; Sigma-Aldrich, USA) was performed. Afterwards, the sections were incubated overnight with specific antibodies against rabbit calbindin D-28k (1:3000; Sigma-Aldrich). The slides were then incubated with monoclonal goat anti-rabbit R. Szalak et al. / Bull Vet Inst Pulawy / 57 (2013) 387-391 389

Fig. 1. Differentiated CB immunoreactivity in neurons of the PG Fig. 4. Intensive CB immunoreactivity in round neuron of the external cellular layer external cellular layer

Fig. 2. Intensive CB immunoreactivity in multipolar neurons’ Fig. 5. Moderate CB immunoreactivity in bipolar neuron of the cell bodies and processes of the internal cellular layer PG external cellular layer

Fig. 3. Moderate CB immunoreactivity in multipolar neurons’ Fig. 6. Moderate CB immunoreactivity in Cajal-Retzius neuron cell bodies and processes of the PG middle cellular layer of the PG marginal layer

Fig. 7. Transverse section of the chinchilla PG showing the immunoreactivity to CB in the parasubiculum

390 R. Szalak et al. / Bull Vet Inst Pulawy / 57 (2013) 387-391

Fig. 8. CB expression in PG in the chinchilla. CB- IR neurons (arrowhead) and nerve fibres (arrows) are found in the chinchilla PG

Results observed in pyramidal and non-pyramidal neurons of all areas of the examined structure in chinchilla, Immunocytochemistry. CB immunoreactive similarly as in human (7), macaque (17), and mouse neurons were observed in marginal, external cellular, (25). CB-positive reaction was observed in multiform middle cellular, and internal cellular layers of all parts neurons like in human (7), macaque (17), and mouse of PG, i.e. in entorhinal area, parasubiculum (Fig. 7), (25). Weak immunoreactivity was demonstrated in and presubiculum. pyramidal shaped cells, and moderate and intensive Individual layers consisted of two types of immunoreactivity in multipolar, bipolar, Cajal-Retzius, neurons: pyramidal and multiform. Among the second as well as in round neurons similarly as in human (7) type of neurons, four kinds of them were observed: and macaque (17). Distribution of pyramidal neurons multipolar, bipolar, round, and Cajal-Retzius cells. PG with weak CB immunoreactivity in PG in the neurons were characterised by different brownish chinchilla, human (7), and mouse (25) may suggest that colour of immunocytochemical reaction, whose they are primary projection neurons. Moderate and intensity varied from weak via moderate to strong (Figs intensive reaction of the studied protein in other 1 and 2). CB immunostaining was localised in cell multiform, non-pyramidal: multipolar, bipolar, round, bodies, and in dendrites, and axons (Fig. 3). Pyramidal and Cajal-Retzius cells may indicate that they are cells were characterised by triangular shape and weak interneurons. In studies of many CNS structures, co- CB immunoreactivity (Fig. 1). Multiform, large sized localisation of the calcium-binding proteins with other neurons were localised mainly in cellular layers of PG. neurotransmitters in primary cells and interneurons has Most of these cells were characterised by moderate and been observed. In the PG and CB intensive immunostaining (Figs 1 and 4). Bipolar, is a marker of GABA-ergic interneurons’ population. fusiform average sized neurons were observed in all These cells are characterised by short axons that do not layers of the brain area. Their cell bodies with moderate go beyond the studied area. Their connections to each brownish CB immunostaining were arranged vertically other, as well as to the primary cells take part in with the exception of the marginal layer, in which there neuronal activity control. Interneurons form inhibitory were fusiform Cajal-Retzius cells (Figs 5 and 6). These synapses in the form of gap junctions (26). It was cells’ nervous processes, similarly as their bodies, were demonstrated that the interneurons expressing CB form arranged along the marginal layer, and were moderately numerous connections, which can reach out cell bodies, CB-stained (Fig. 6). A small population of neurons was axons, and further dendrite segments of primary cells. round shaped with an intensive reaction of the studied In consequence, interneurons’ inhibitory effect on protein (Fig. 4). primary neurons causes their disinhibition Immunofluorescence. Results of immunofluo- (disinhibitory cells). As regards the functions, rescent staining revealed the presence of CB in Hu interneurons form two separate cell networks with C/D-positive neurons, as well as in nervous processes different contents of neuropeptides (26, 29). The first of of PG. CB-IR neurons’ cell bodies were moderately them is the fast responding to stimulation interneurons numerous (accounted for 10% of all Hu C/D-IR (fast spiking-FS). The second network is divided into neurons), clearly distinguishable from the background. three subclasses: the first class is characterised by Predominantly, CB-IR/Hu and Hu C/D-IR neurons regular (regular spiking-RS), and the second by rapidly were classified as pyramidal, round, multiform, and responding to stimulation interneurons. Both of these bipolar cells. CB-IR nerve fibers communicated with subclasses reveal the presence of CB-positive reaction. neighbouring CB-positive and CB-negative neurons The third subclass consists of regularly communicating (Fig. 8). interneurons that demonstrate the presence of other calcium-binding proteins (29). The appropriate concentration of calcium ions in both primary cells and Discussion interneurons affects their connections, as well as their proper functioning. Therefore calcium-binding proteins In the present study the CB distribution in PG was regulate metabolic processes affecting the vital described in chinchilla. 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