Accepted Manuscript Title: Parvalbumin, but not calretinin, neurons express high levels of ␣1-containing GABAA receptors, ␣7-containing nicotinic acetylcholine receptors and D2-dopamine receptors in the basolateral amygdala of the rat Authors: Maciej R´owniak, Małgorzata Kolenkiewicz, Anna Kozłowska PII: DOI: Reference:

S0891-0618(17)30079-0 http://dx.doi.org/10.1016/j.jchemneu.2017.08.002 CHENEU 1512

To appear in: Received date: Revised date: Accepted date:

15-5-2017 15-8-2017 15-8-2017

Please cite this article as: R´owniak, Maciej, Kolenkiewicz, Małgorzata, Kozłowska, Anna, Parvalbumin, but not calretinin, neurons express high levels of ␣1-containing GABAA receptors, ␣7-containing nicotinic acetylcholine receptors and D2-dopamine receptors in the basolateral amygdala of the rat.Journal of Chemical Neuroanatomy http://dx.doi.org/10.1016/j.jchemneu.2017.08.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Parvalbumin, but not calretinin, neurons express high levels of α1-containing GABAA receptors, α7containing nicotinic acetylcholine receptors and D2-dopamine receptors in the basolateral amygdala of the rat.

Running title: Calcium-binding proteins and membrane receptors in amygdala of rat. Maciej Równiak1*, Małgorzata Kolenkiewicz2, Anna Kozłowska3

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Department of Comparative Anatomy, Faculty of Biology and Biotechnology, University of Warmia and

Mazury in Olsztyn, pl. Łódzki 3, 10-727 Olsztyn, Poland 2

Department of Pathophysiology, Faculty of Medical Sciences, University of Warmia and Mazury in Olsztyn,

Warszawska Av, 30, 10-082 Olsztyn, Poland 3

Department of Human Physiology, Faculty of Medical Sciences, University of Warmia and Mazury in Olsztyn,

Warszawska Av, 30, 10-082 Olsztyn, Poland

Correspondence to: Dr Maciej Równiak Address: Department of Comparative Anatomy, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, Poland, Plac Łódzki 3, 10–767, Olsztyn, Poland Tel. (+4889) 523-4302, Fax (+4889) 523-4301 Email: [email protected]

Number of figures and tables: 4 figures and 3 tables

Highlights 

Study describes sensory properties of GABA interneurons in basolateral amygdala

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GAD67, PV and CR cells have GABAergic, cholinergic and dopaminergic receptors

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PV cells co-express each of these receptors more often than CR cells

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First direct evidence for α7 and D2 receptors on PV and CR cells in amygdala

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GABA cells are interconnected and take part in processing of extrinsic inputs

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Abstract The generation of emotional responses by the basolateral amygdala is largely determined by the balance of excitatory and inhibitory inputs to its principal neurons - the pyramidal cells. The activity of these neurons is tightly controlled by -aminobutyric acid (GABA)ergic interneurons, especially by those expressing parvalbumin (PV) and calretinin (CR). Although it is known that GABAergic, cholinergic and dopaminergic fibres make synapses on PV and CR cells, knowledge of the various receptors which are used by these cells is still incomplete. Thus, the present study investigates whether neurons expressing PV or CR co-express specific GABA, acetylcholine and/or dopamine receptors in the basolateral amygdala of the rat. The results show that almost two-thirds of PV neurons co-express high concentrations of α1 subunit of GABAA receptor, and more than half of them co-express high levels of α7 subunit of nicotinic acetylcholine receptor and/or D2-subtype of dopamine receptor. In contrast, a smaller percentage of CR neurons had detectable amounts of these receptors and at lower levels of abundance in most cases. In conclusion, the present results indicate that not only principal neurons but also GABAergic interneurons have specific receptors, which allow these cells to respond to the GABAergic, cholinergic and dopaminergic inputs coming to the basolateral amygdala of the rat. Since these cells receive intrinsic GABAergic inputs, they are strongly interconnected. Since they also receive extrinsic cholinergic and dopaminergic inputs, such stimulation may result in stimulus-driven feed-forward control of the principal neurons. The effects of such control may be either feed-forward inhibition of the principal neurons via α7 nicotinic acetylcholine receptors or disinhibition of these cells via D2-dopamine receptors.

Keywords: amygdala; calcium-binding proteins; membrane receptors; immunohistochemistry; rat

1. Introduction The basolateral amygdala (BLA) is a set of nuclei within the temporal lobe, which are thought to be critically important for fear-related learning and memory, modulation of cognitive functions and overall regulation of emotional behaviour (LeDoux, 2003; Sah et al., 2003; Pape and Pare, 2010). These nuclei, i.e. lateral (LA), basolateral (BL) and basomedial (BM) nuclei, form a compact nuclear complex and are organized in a very similar way. For example, previous studies have shown that there are two major cell classes in the BLA: pyramidal neurons and non-pyramidal neurons (Sah et al., 2003). Although these cells do not exhibit a

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laminar or columnar organization, their anatomical and electrophysiological characteristics are remarkably similar to those of their counterparts in the cerebral cortex (McDonald, 1992; Washburn and Moises, 1992; Rainnie et al., 1993). Thus, the principal neurons in the BLA are spiny pyramidal-like projection neurons, which utilize glutamate as an excitatory neurotransmitter (Fuller et al., 1987; McDonald, 1996), whereas most nonpyramidal neurons in the BLA are spine-sparse interneurons which utilize -aminobutyric acid (GABA) as an inhibitory neurotransmitter (McDonald and Pearson, 1989; Kemppainen and Pitkänen, 2000). The balance of excitatory and inhibitory inputs to the principal neurons is the main factor which determines the generation of emotional responses by the BLA. Excitatory inputs are provided by glutamatergic neurons located outside of the amygdala (Szinyei et al., 2000) or in other regions of the amygdala (Smith and Pare, 1994). In addition, BLA receives heavy cholinergic innervation from the basal forebrain (Woolf and Butcher, 1982; Muller et al., 2011) and dopaminergic innervations from the midbrain (Asan, 1997; Asan, 1998). These inputs form synaptic connections on the dendrites of principal neurons which transmit signals to other regions of the amygdala or to extrinsic regions (Smith and Pare, 1994; Woolf and Butcher, 1982). They also terminate on the GABAergic interneurons (Pinard et al., 2008; Muller et al., 2011). Inhibitory inputs are mostly provided by intrinsic subpopulations of GABAergic interneurons (McDonald, 1985). These interneurons form a strong inhibitory network that keeps spontaneous cellular activity low and prevents principal cells from firing action potentials to irrelevant stimuli (Muller et al., 2006; Muller et al., 2007; Woodruff and Sah, 2007a). Permanent inhibition of the principal neurons is maintained by at least two mechanisms (Samson and Paré, 2006; Ehrlich et al., 2009). Feedback inhibition, which occurs via the recurrent colataterals of the principal neurons providing glutamatergic, excitatory input to the GABAergic internerons. Another mechanism is feed-forward inhibition, which is caused by stimulation of GABAergic interneurons by the extrinsic excitatory inputs. Taken together, all of these data indicate that both principal neurons and inhibitory interneurons form two networks, which are interconnected and together constitute a well-balanced interface for extrinsic inputs. The BLA has a large population of GABAergic interneurons (McDonald, 1985). It is known that in the large majority of them, GABA coexists with different calcium-binding proteins, such as calbindin (CB), parvalbumin (PV) or calretinin (CR) as well as various neuropeptides (somatostatin, neuropeptide Y, cholecystokinin or vasoactive intestinal peptide) (Prager et al., 2016). In this way, certain subpopulations are formed within the GABAergic neurons, which play different roles in the inhibitory mechanism of the BLA. CB, PV and CR are especially useful markers of specific GABAergic subpopulations in the BLA (Prager et al., 2016). In the rat (McDonald and Mascagni, 2001) and monkey (Mascagni et al., 2009), they together mark the

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vast majority of GABAergic cells in this brain region. However, it should be noted that some of the CB and CR neurons are not GABAergic interneurons but probably glutamatergic-projecting neurons (Moryś et al., 1999; Kemppainen and Pitkänen, 2000; McDonald et al., 2012). Although it has been established that GABAergic, cholinergic and dopaminergic fibres make synapses on CB, PV and CR cells (Muller et al., 2003; Muller et al., 2011; Pinard et al., 2008) the data on various receptors localized on these cells is still incomplete. Thus, the present study investigates whether PV and/or CR neurons co-express selected GABA, acetylcholine and/or dopamine receptors in the BLA of the rat. PV and CR cells were chosen for investigation regarding calciumbinding proteins because they form non-overlapping and functionally different GABAergic subpopulations in the BLA (Mascagni et al., 2009; Mascagni and McDonald, 2003). CB neurons were excluded from the study since many of these cells co-express PV (McDonald and Mascagni, 2001). Among the receptors, α1, a subunit of GABAA receptor (GABAAα1), α7, a subunit of nicotinic acetylcholine receptor (ACHNα7) and D2, a subtype of dopamine receptor (DAD2), were chosen for investigation. To make the text easier to read, the neurons expressing PV, CR, GABA, GABAAα1, ACHNα7 and DAD2 will be uniformly described as PV+, CR+, GABA+, GABAAα1+, ACHNα7+ and DAD2+ neurons.

2. Material and Methods 2.1. Subjects. A total of five adult male Wistar Kyoto rats (240–270 g; 10 weeks of age, Charles River, Germany) were used in this study. All experiments were carried out in accordance with the European Union Directive for animal experiments (2010/63/EU) and were approved by the Local Ethical Commission of the University of Warmia and Mazury in Olsztyn (No. 43/2014). All efforts were made to minimize animal suffering and to use the minimum number of animals necessary to produce reliable scientific data.

2.2. Tissue preparation. Rats were anesthetized with an intraperitoneal injection of Morbital (Biowet, Poland; 2 ml/kg body weight, 133.3 mg/ml of pentobarbital sodium salt and 26.7 mg/ml of pentobarbital) and, after cessation of breathing, immediately perfused intracardially with phosphate-buffered saline (PBS; pH 7.4) containing 1% sodium nitrite followed by 4.0% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. Following perfusion, brains were dissected out from the skulls, post-fixed overnight in 4.0% paraformaldehyde, washed twice in 0.1 M phosphate buffer (pH=7.4, 4°C) and then stored for 3–5 days in graded solutions (19% and 30%) of sucrose

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(Sigma Aldrich) at 4°C until they sank. Finally, the brains were frozen and then coronally sectioned at a thickness of 10 μm using a cryostat. The sections were mounted on object slides and then stored at -80°C until further processing. 2.3. Immunofluorescence experiments. Amygdala sections were processed for routine double-immunofluorescence labelling using primary antisera raised in different species and species-specific secondary antibodies (Table 1). Briefly, all samples were washed three times in PBS and then incubated for 1 h in humid chambers with blocking buffer (0.1 M PBS, 10% normal goat serum, 0.1% bovine serum albumin, 0.3–0.5% Triton X-100, 0.05% thimerosal, 0.01% NaN3). This solution was also used as a diluent of primary and secondary antibodies listed in Table 1. The sections were then rinsed in PBS and incubated overnight at room temperature with a mixture of primary antibodies, i.e. a cocktail of the appropriate primary antibodies to one of the neuronal markers (PV or CR or GAD67 or NeuN) and one to the selected receptors (GABAAα1 or ACHNα7 or DAD2) (Table 1). After incubation with the primary antibodies, the sections were rinsed in three changes of PBS and incubated for 1 h with a mixture of species-specific secondary antibodies (Table 1). Finally, all samples were rinsed in PBS and then mounted with carbonatebuffered glycerol (pH 8.6) and cover-slipped. 2.3.1. Controls. The specificity of the primary antibodies used in this study has been shown by various researchers using these products in multiple previous studies (Meszar et al., 2012; Zimmermann and Schwaller, 2002; Celio et al., 1988; Stansfield et al., 2015; Koduvayur et al., 2014; Machaalani et al., 2010; Wang et al., 2012; Paulo et al., 2009). In addition, product descriptions of mouse antibodies against PV (235), CR (6B3) and GAD67 (ab26116) as well as rabbit antisera towards GABAAα1 (ab33299), ACHNα7 (ab10096) and DAD2 ( ab21218) include immunoblots of the rat brain homogenates, which were specifically stained by these antibodies, showing bands at 14kDa, 29kDa, 67kDa, 51kDa, 56kDa and 50kDa, respectively. The same documents also show an absence of specific immunohistochemical staining in brain sections of PV or CR knock-out (KO) mice using these antibodies. Although it has been found, via the use of KO tissues, that several antibodies against ACHNα7 are non-specific to their targets (Moser et al., 2007; Rommel et al., 2015), this was not a problem for ab10096. This antibody was not tested in any of these studies, but it was used in a proteomics study by Paulo et al., (2009). Those authors found that from their α-bungarotoxin isolated and bead-supported immunoprecipitation Western blot and a corresponding coomassie-stained gel, no band for α7 was present in the KO tissues compared to the wild type, which revealed a band at 56 kDa (Paulo et al., 2009). Additional evidence of this antibody’s

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specificity has been found by the Paulo group by testing its presence in untransfected human embryonic kidney cells vs. cells transfected with the α7-5HT3A chimeric construct (Machaalani et al., 2010). The specificity of secondary antibodies was controlled by the omission and replacement of all primary antibodies by non-immune sera or PBS. Lack of any immunoreactions indicated specificity. 2.4. Counts and statistics. Colocalisations of PV+ and CR+ cells a with particular receptors were analysed in the lateral, basolateral and basomedial amygdaloid nuclei using an Olympus BX51 microscope equipped with Cell-F image analysis software (Olympus GmbH, Germany). Only the numbers of single- and double-labelled PV+ and CR+ cells were counted. Single-labelled GABAAα1+, ACHNα7+ and DAD2+ neurons were excluded from the investigation. For each nucleus in each animal, particular combinations of antigens were counted on 10 evenly spaced sections arranged from the rostral to the caudal extent of the amygdala. The space between these sections was 200 µm. In order to confirm the localization of the individual nuclei on the sections, neighbouring sections stained with mouse anti-NeuN (pan-neuronal marker) were used. All counts on the single section were made at 200x magnification using 350 µm x 260 µm regions (test frames). Depending on the size of the nucleus at different levels of the amygdala, counts were made from either one such field positioned in the centre of the nucleus (and involving 100% of its cross-sectional area) or 2-3 adjacent non-overlapping fields. Within test frames, single-labelled and double-labelled neurons were counted separately. Such separate counts made within the test frames in the single nucleus in the subject were totalled. Finally, counts from each nucleus were averaged and expressed as means±standard deviation (SD). All counts were made on coded slides by the first author. To avoid fluorescence fading, a test frame was digitally recorded before counting. Such digital frames were in the form of stacks, which consisted of two microphotographs representing red and green immunofluorescence channels. Saved stacks were also evaluated by two independent experimenters, being blind to the parameters of the studied tissues (nucleus, antigens, etc.). The results of these counts showed high interrated reliability (Pearson R = 0.81, P