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. Author manuscript; available in PMC: 2014 Mar 1.
Published in final edited form as: Hippocampus. 2012 Nov 29;23(3):221–232. doi: 10.1002/hipo.22081

DAG-sensitive and Ca2+ permeable TRPC6 channels are expressed in dentate granule cells and interneurons in the hippocampal formation

Gergő A Nagy 1, Gergő Botond 1, Zsolt Borhegyi 1, Nicholas W Plummer 2, Tamás F Freund 1, Norbert Hájos 1
PMCID: PMC3586768  NIHMSID: NIHMS421155  PMID: 23193081

Abstract

Members of the transient receptor potential (TRP) cation channel family play important roles in several neuronal functions. To understand the precise role of these channels in information processing, their presence on neuronal elements must be revealed. In this study we investigated the localization of TRPC6 channels in the adult hippocampal formation. Immunostainings with a specific antibody, which was validated in Trpc6 knockout mice, showed that in the dentate gyrus, TRPC6 channels are strongly expressed in granule cells. Immunogold staining revealing the subcellular localization of TRPC6 channels clarified that these proteins were predominantly present on the membrane surface of the dendritic shafts of dentate granule cells, and also in their axons, often associated with intracellular membrane cisternae. In addition, TRPC6 channels could be observed in the dendrites of some interneurons. Double immunofluorescent staining showed that TRPC6 channels were present in the dendrites of hilar interneurons and hippocampal interneurons with horizontal dendrites in the stratum oriens expressing mGlu1a receptors, whereas parvalbumin immunoreactivity was revealed in TRPC6-expressing dendrites with radial appearance in the stratum radiatum. Electron microscopy showed that the immunogold particles depicting TRPC6 channels were located on the surface membranes of the interneuron dendrites. Our results suggest that TRPC6 channels are in a key position to alter the information entry into the trisynaptic loop of the hippocampal formation from the entorhinal cortex, and to control the function of both feed-forward and feed-back inhibitory circuits in this brain region.

Keywords: Hippocampus, dendrite, GABAergic cell, feed-forward inhibition, feed-back inhibition

Introduction

The hippocampal formation plays a crucial role in several cognitive functions, including coding and retrieval of information, as well as spatial navigation (Bird and Burgess, 2008). The neuronal circuits both in the dentate gyrus and in the hippocampus comprise excitatory principal neurons and inhibitory GABAergic cells (Freund and Buzsáki, 1996). While granule cells and hilar mossy cells are the excitatory neurons in the dentate gyrus, pyramidal cells compose the principal neurons in the hippocampus (Amaral et al., 1990). Inhibitory cells, however, are comparable in both regions (Freund and Buzsáki, 1996). The perisomatic region of principal neurons is innervated by fast spiking basket and axo-axonic cells containing the Ca2+ binding protein parvalbumin, as well as by regular spiking basket cells expressing the CB1 cannabinoid receptor and the neuropeptide cholecystokinin (Freund and Katona, 2007). This neuropeptide can also be found in interneurons that synapse onto the dendrites of excitatory cells (Cope et al., 2002; Klausberger et al., 2005). The most distal part of the dendritic tree of principal neurons is innervated by GABAergic cells that express metabotropic glutamate receptor type 1 (mGlu1a) and the neuropeptide somatostatin (Baude et al., 1993; Katona et al., 1999; Ferraguti et al., 2004). This latter cell type is an archetypal feed-back inhibitory cell, since the vast majority of their excitatory input originates from those principal cells that are innervated by these interneurons (Blasco-Ibanez and Freund, 1995; Maccaferri and McBain, 1995). Feed-forward inhibition in the hippocampal formation, however, has been proven to be predominantly accomplished by parvalbumin-containing interneurons, whose spiking is very effectively driven by feed-forward excitatory afferents (Glickfeld and Scanziani, 2006). Thus, distinct inhibitory cell types are specialized to control the different computational domains of principal neurons in the hippocampal formation (Miles et al., 1996; Lovett-Barron et al., 2012).

Cation channels mediating transient receptor potentials (TRP) are polymodal cellular sensors, underlying several physiological functions (Ramsey et al., 2006). Overall, twenty eight TRP channels, grouped into six subfamilies based on their sequence homology, have been distinguished (Clapham, 2003; Owsianik et al., 2006). The members of the ‘canonical’ subfamily, which exhibit a substantial Ca2+ permeability, have been shown to be activated by a variety of distinct receptors, including tyrosine kinase TrkB receptors (TRPC3), mGlu1/5 receptors (TRPC1, 5) or muscarinic cholinergic receptors (TRPC5) in the hippocampus (Abramowitz and Birnbaumer, 2009; Bollimuntha et al., 2011). In addition, the role of the latter channel type was proposed in the axonal growth of hippocampal pyramidal cells (Greka et al., 2003).

Much less is known of TRPC6 channel function in the hippocampus, although a role for these channels in dendritic growth and formation of excitatory synapses has been postulated (Tai et al., 2008; Zhou et al., 2008). Other studies suggest that TRPC6 channels might be involved in the synthesis of endocannabinoids (Bardell and Barker, 2010), which are well-known retrograde messenger molecules (Katona and Freund, 2008; Kano et al., 2009). Diacyglycerol (DAG), a precursor of endocannabinoids is known to promote the opening of TRPC6 channels (Hofmann et al., 1999; Aires et al., 2007). An intriguing hypothesis is that Ca2+ entering to the neurons via TRPC6 channels upon DAG binding would trigger endocannabinoid synthesis by activation of Ca2+ sensitive DAG lipase (Bisogno et al., 2003). The localization of proteins involved in DAG synthesis (mGlu5 receptors, phospholipase C) or degradation (DAG lipase) on the spines receiving excitatory synapses was shown to be predominantly present at the edge of asymmetric synapses (Lujan et al., 1996; Lujan et al., 1997; Katona et al., 2006; Yoshida et al., 2006; Fukaya et al., 2008), forming a perisynaptic annulus (Katona and Freund, 2008). Thus, if TRPC6 channels are a member of the molecular machinery producing endocannabinoids, they should be found, at least in part, in the perisynaptic annulus.

To obtain deeper insights into the potential role of TRPC6 channels in neuronal operation, we aimed to identify the neuronal elements expressing these channels in the adult hippocampal formation. Using immunocytochemical studies we found that dentate granule cells and interneurons express TRPC6 channels, consistent with the in situ hybridization data published by the Allen Institute for Brain Science (http://mouse.brain-map.org).

Methods

Animal handling

Experiments were performed according to the guidelines of the Institutional Ethical Codex and the Hungarian Act of Animal Care and Experimentation (1998, XXVIII, section 243/1998), which conforms to the regulations of animal experiments of the European Union. Animals were kept under a 12 h–12 h light–dark cycle, and water and food were available ad libitum. All efforts were made to minimize pain and suffering and to reduce the number of animals used. In this study, ten male Wistar rats (200–400g; Charles River, Hungary) and two adult Trpc6 knockout mice and their wild type littermates (n=2)(Dietrich et al., 2005) were used.

Immunohistochemistry

The rats were deeply anaesthetized with an intraperitoneal injection of equitesin (4.2% w/v chloral hydrate, 2.12% w/v MgSO4, 16.2% w/w Nembutal, 39.6% w/w propylene glycol, and 10% w/w ethanol in H2O) at a dosage of 0.2 ml/100 g body weight. Animals were perfused through the heart sequentially with 4°C 0.9% NaCl for 2 min, fixative containing 2% paraformaldehyde and 3.75% Acrolein in 0.1 M phosphate buffer (PB; pH = 7.4) for 10 min, and fixative containing 2% paraformaldehyde in 0.1 M PB for 20 min. Mouse brains were fixed by immersion in 4% paraformadelhyde. Coronal sections 40–50 µm in thickness were cut using a Leica 1000S vibratome, cryoprotected in 30% sucrose in 0.1 M PB overnight, and freeze thawed in an aluminium foil boat over liquid nitrogen to enhance the penetration of the antibodies. After washing, the sections were treated with 0.1 M PB containing 1% sodium borohydride for 10 min.

Sections then were transferred to a solution containing 2% bovine serum albumin (BSA), 100 mg/ml glycine and 10% normal goat serum (Vector laboratories) in Tris-buffered saline (TBS), pH 7.4, for 30 min, followed by incubation overnight at 4°C with a rabbit anti-TRPC6 antibody (Alomone labs Ltd, Jerusalem, Israel) diluted 1: 20,000 in TBS. After washing out the primary antibody, the sections were incubated in a biotinylated goat-anti rabbit secondary antiserum (Vector Laboratories, Burlingame, CA) diluted 1:200 in TBS for 2 hours. Sections were then treated with a solution containing avidin-biotinylated horseradish peroxidase complex (ABC Elite, Vector Laboratories) 1:300 in TBS for 2 hours, followed by immunoperoxidase reaction using diaminobenzidine (DAB, Sigma-Aldrich, St Louis, MO) as a chromogen.

For subcellular localization of TRPC6, we applied a pre-embedding immunogold staining. In this case, the sections were incubated in the anti-TRPC6 antiserum (1:5000) for 2 days, followed by application overnight of a 1 nm gold-conjugated anti-rabbit secondary antibody (Aurion, Wageningen, The Netherlands) diluted 1:50 in TBS containing 1% BSA, 0.1% fish gelatine and 100 mg/ml glycine. Sections were postfixed in 2% glutaraldehyde in TBS and intensified with the Aurion R-Gent silver intensification kit.

All immunoperoxidase- and immunogold-stained sections were treated in 0.5% OsO4 for 1 min, then in 1% OsO4 for 15 min in 0.1 M PB followed by dehydration in an ascending alcohol series and acetonitrile, and embedded in Durcupan. During dehydration, the sections were treated with 1% uranyl acetate in 70% ethanol for 30 min. Finally, sections were re-embedded and investigated in a light (Zeiss Axioscope 2) or an electron microscope (JEOL, JEM-1011).

For confocal laser microscopy, we used a mixture of primary antisera of anti-TRPC6 (1:3000) and guinea pig anti-mGlu1a (1:500; Frontier Institute co. Ltd, Japan; (Tanaka et al., 2000)) or anti-TRPC6 (1:3000) and mouse anti-parvalbumin (1:2000; Sigma-Aldrich). The sections were then incubated for 2 hours with a mixture of secondary antibodies diluted in TBS: Alexa Fluor 488 goat anti-rabbit antibody (1:200, Molecular Probes, Eugene, OR) and Alexa Fluor 594 goat anti-guinea pig (1:200, Molecular Probes), or Alexa Fluor 488 goat anti-rabbit antibody (1:200, Molecular Probes) and Alexa Fluor 594 goat anti-mouse (1:200, Molecular Probes). After washing, the sections were mounted on glass slides and covered in Aqua poly/mount (Polysciences Inc., Warrington, PA, USA). Coverslips were sealed in nail polish. Images were taken with a Nikon A1R confocal laser scanning microscope using a sequential scanning mode.

Quantitative analysis of the distribution of TRPC6 on the surface membrane of dentate granule cells

All quantifications have been performed on tissue samples derived from three rats and pooled together, because there was no significant difference between animals (p>0.5). For analysis using ImageJ software (NIH), silver-intensified immunogold particles <100 nm from the cell membrane were counted as plasma membrane-attached. Images were taken from three sequential ultrathin sections (cut using a Leica ultramicrotome) in the middle part of the stratum moleculare or stratum granulosum at a magnification of 2500x. Calculation of particle density was performed on the central image, in which all profiles were identified as spine, dendrite, soma, axon ending or unknown profile using the two adjacent images. The number of gold particles divided by the total length of the membranes was calculated for all those dendrites, spines and somata measured in the central image whose profiles were not truncated by the edge of the image. Since the distributions of immunogold labeling calculated on each image were not different from normal distributions (p<0.01), the mean and standard error of the mean (SEM) of membrane-associated immunogold particles per 1 µm of the plasma membrane were used to describe the distributions of immunolabeling in each compartment (dendrites, spines and cell bodies).

To establish the distribution of TRPC6 labelling in spine heads, we counted 93 immunogold particles from each animal (n=3). Using methodology comparable to that published earlier (Lujan et al., 1996; Lujan et al., 1997), we measured the distance along the plasma membrane from the edge of the synaptic junction for each immunogold particle and divided the data into 60 nm bins. The three datasets were not different from each other (p>0.5, Kruskal-Wallis non-parametric test), so the results were pooled.

Results

Localization of TRPC6 channels in the rat hippocampal formation

To get an overview of which neuronal elements express TRPC6 channels in the hippocampal formation, we performed immunostaining using a polyclonal antibody developed against the intracellular C-terminus loop of this transmembrane protein. In the rat tissue, immunoperoxidase staining using DAB as a chromogen revealed a dark signal in the stratum moleculare of the dentate gyrus and a fainter staining in the stratum lucidum of the CA3 subfield (Figure 1A, B, D). In the hilus and the stratum oriens of both hippocampal regions, single dendrites (Figure 1C), in some cases decorated with long spines, could be observed (Figure 6A, D). In addition, some smooth dendrites in the strata radiatum and oriens running parallel with the pyramidal cell dendrites were also immunoreactive (Figure 1A). Outside of the principal cell layer, cytoplasmic staining, including nuclear membrane labeling of some neurons as well as staining of glia was also often apparent (Figure 1A, Supplementary figure 1).

Figure 1.

Figure 1

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Light micrographs of the rat hippocampal formation immunostained for TRPC6 protein. A: Low power micrograph showing a specific laminar distribution of TRPC6-immunostaining in the CA1 region and the dentate gyrus. A strong labeling can be seen in the stratum moleculare of the dentate gyrus in addition to numerous immunostained dendrites located both in the hilus and in strata oriens and radiatum. Moreover, some immunoreactive cell bodies resembling interneurons are distributed in all layers of the hippocampal formation. B: At higher magnification the dendrites of the dentate granule cells surrounded by a dense punctuated immunostaining are visible in the stratum moleculare. C: In CA1, a meshwork of neuronal processes can be found at the stratum oriens/alveus border formed mainly by horizontally running interneuronal dendrites. D: In the stratum lucidum of the CA3 subfield, while the main apical dendrites of CA3 pyramidal cells appear to be immunonegative, they are outlined by a dense immunostained neuropil. alv., alveus; o., stratum oriens; p., stratum pyramidale; rad., stratum radiatum; l.m., stratum lacunosum-moleculare; mol., stratum moleculare; gr., stratum granulosum; h., hilus. Scale bars: A, 100 µm; B, C, 20 µm; D, 100 µm.

Figure 6.

Figure 6

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TRPC6 channels are expressed by metabotropic glutamate receptor type 1a- (mGlu1a) or parvalbumin-immunopositive interneurons in the hippocampus. Double immunofluorescent staining indicates that both at the border of alveus and stratum oriens, as well as in the hilus, the majority of mGlu1a -immunoreactive dendrites also express TRPC6 (white arrows). In the stratum radiatum, some parvalbumin-containing dendrites are also immunoreactive for TRPC6 (white arrows). Arrowheads show singly-immunoreactive profiles, colour-coded in the merged pictures. Scale bars: A, D, 10 µm; G, 5 µm.

Localization of TRPC6 channels in the mouse hippocampal formation and the control of antibody specificity using Trpc6 knockout mice

To confirm the specificity of the immunostaining obtained with this antibody, we compared the labeling in the hippocampus of wild type mice with that seen in Trpc6 knockout mice (Dietrich et al., 2005). In wild type mice, the pattern of the immunostaining for TRPC6 was identical to that seen in rats (Figure 2A); strong staining was apparent in the stratum moleculare of the dentate gyrus and a fainter labeling in the stratum lucidum (Figure 2A, C). Furthermore, the dendrites of interneurons localized in the strata oriens and radiatum of Cornu Ammonis, as well as in the hilus, could be observed (Figure 2B). In contrast to this labeling pattern, immunoreactivity in the strata moleculare and lucidum, as well as the immunopositive dendrites in the hippocampal formation, was absent in Trpc6 knockout mice (Figure 2D, E, F). However, the cytoplasmic staining of neurons and the glial staining clearly visible in all layers of the rat and mouse hippocampus was comparable in Trpc6 knockouts (Figure 2E, F; Supplementary figure 1). These results suggest that TRPC6 channels are present in the input and output regions of dentate granule cells in addition to the dendrites of interneurons, while the intracellular membrane signal and the glial staining seems to be non-specific using this antibody.

Figure 2.

Figure 2

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Light microscopy of TRPC6 localization in the mouse hippocampal formation. A: Wild type mice have an identical TRPC6 localization pattern to rats. An intense staining is visible in the stratum moleculare of the dentate gyrus. The pattern of this immunoreaction is indistinguishable from that observed in the rat dentate gyrus shown at higher magnification (C). B: The meshwork of neuronal processes at the border of the stratum oriens/alveus stained for TRPC6 is also present in the mouse hippocampus, comparable to that seen in the rat. D: In contrast to wild type mice, immunostaining for TRPC6 was absent in the dentate gyrus in Trpc6 knockout mice (F). E: Similarly, the TRPC6-immunoreactive meshwork formed mainly by interneuronal dendrites in CA1 was not visible in KO mice. However, the cell body staining with this TRPC6 antibody was comparable both in wild type and Trpc6 knockout mice, indicating that this signal is unrelated to the presence of TRPC6 protein. Scale bars: A, D, 500 µm; B, C, E, F, 50 µm.

Subcellular distribution of TRPC6 channels in the dentate gyrus

In the following sets of experiments, all investigations were carried out in the rat hippocampal formation. We first examined the subcellular localization of TRPC6 channels in the dentate gyrus. Using immunogold staining, we found that the labeling was predominantly attached to the plasma membranes of the granule cells (Figure 3). To gain deeper insight into the location of TRPC6 on the membrane surface of these excitatory cells, we compared the density of immunogold particles in three different compartments (spines, dendrites and cell bodies) by dividing the number of immunogold particles by the total membrane length of the examined profiles. We found that the highest density of immunogold labeling was present in the plasma membranes of the dendrites (0.12 ± 0.01 gold/µm, n=150), with significantly lower density on the surface of the spines (0.04 ± 0.01 gold/µm, n=267) and cell bodies (0.06 ± 0.01 gold/µm, n=20)(Figure 3). These results indicate that the dendrites of dentate granule cells are preferentially equipped with TRPC6 channels.

Figure 3.

Figure 3

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TRPC6 is expressed in the plasma membrane of the dendrites and spines of dentate granule cells. A1-2: Consecutive electron micrographs taken in the stratum moleculare demonstrate that immunogold particles representing TRPC6 proteins are present on the plasma membranes of the dendrites (arrows) and spines (arrowheads) of dentate granule cells. Note that the gold particles are attached to the intracellular surface of the plasma membrane, in accord with the predicted position of the intracellular epitope of the TRPC6 protein recognized by this antiserum. In addition, some gold particles could be occasionally seen intracellularly (open arrowhead) that may represent channels during trafficking. Scale bars: 0.5 µm. B shows the immunogold density revealed on different membrane segments. There was a significant difference in the density of immunolabeling on the surface of the dendrites and somata (marked with *, p=0.02) and dendrites and spines (marked with **, p=0.002). There was no difference in the density between spines and somata (n.s., non-significant, p=0.41). The symbols and whiskers indicate the mean and SEM, respectively.

TRPC6 channels are evenly present on the surface membranes of dendritic spines with some preference for the perisynaptic annulus around asymmetric synapses

Although the density of TRPC6 channels was highest on dendritic shafts, we carried out a more thorough investigation of the channel localization related to the excitatory synapses on spines. The rationale behind this analysis was that some proteins (e.g. those that are involved in the regulation of DAG levels on spines) are preferentially found at the edge of synapses, forming a perisynaptic annulus (Katona and Freund, 2008). To establish whether TRPC6 channels on the spines of dentate granule cells are members of the perisynaptic annulus, we determined the sub-synaptic distribution of TRPC6 channels on spines in relation to the synaptic junction. Following the procedure published by Lujan and colleagues (Lujan et al., 1996; Lujan et al., 1997), we measured the distance of each immunogold particle from the edge of the synapse, and sorted the values into 60 nm bins. This analysis revealed that TRPC6 channels occur evenly on the surface membranes of dendritic spines, with some preference for the perisynaptic annulus around asymmetric synapses (Figure 4). Importantly, immunogold particles were rarely observed within the postsynaptic density or intracellularly in the spine cytoplasm (< 2%). These data imply that the presence of TRPC6 channels in dendritic spines partially overlaps with the perisynaptic annulus, but the majority of labeling is more evenly distributed on the surface membranes of spines.

Figure 4.

Figure 4

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Distribution of TRPC6 on the spine heads of dentate granule cells. A, B: High-resolution preembedding immunogold staining for TRPC6 demonstrates that this channel protein is present on the spines (arrowheads) receiving asymmetric synapses, as well as on dendrites with small diameter (arrows). C1-2: Consecutive sections of a labeled spine that receives an asymmetric synapse. Scale bars: 0.2 µm. D: Relationship between the distance of immunolabeling depicting TRPC6 and the half perimeter of extrasynaptic membrane of spines (n=279). There is a population of labeling (~ 15%), indicated with a dashed line, which is located at the edge of synapses. A larger fraction of the immunogold particles show an even distribution on the surface membrane of spines. Bar graph on top; Spatial distribution of immunogold particles in relation to the postsynaptic density (PSD) on spine heads. The distance from the edge of the synaptic junction (position 0; open arrowheads in A-C) was measured along the plasma membrane. The values were divided into 60 nm bins. Bar graph on left; Distribution of the half lengths of the perimeters of spines excluding the synaptic junction.

Subcellular localization of TRPC6 channels in the stratum lucidum of the CA3 subfield

Since a weak, but obvious, immunolabeling was present in the stratum lucidum of both rats and wild type mice but was absent in Trpc6 knockout mice, we next investigated the subcellular localization of these channel proteins in the termination zone of dentate granule cells. In contrast to what we found in the stratum moleculare, namely that immunogold particles depicting TRPC6 channels were preferentially attached to the plasma membranes of dendrites and spines (Figure 3), the immunogold labeling in the stratum lucidum was often present inside the axons in addition to being in the close vicinity of the axonal membranes (Figure 5). These axons regularly formed bundles characteristic of mossy fibers, the axons of dentate granule cells. In line with this notion, some immunogold particles could be seen in the large mossy terminals, the specialized axon endings of dentate granule cells. In these terminals, immunogold particles avoided the vesicular clusters, but were frequently associated with intracellular membrane cisternae or the plasma membrane (Figure 5). These observations indicate that, while TRPC6 channels of dendrites and spines of dentate granule cells were almost exclusively present on the surface membranes, TRPC6 in the axons could often be detected intracellularly, where the immunolabeling was occasionally associated with membrane cisternae in mossy terminals.

Figure 5.

Figure 5

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TRPC6 channels are often localized intracellularly in the axons of dentate granule cells. A1-2: Gold particles representing TRPC6 channels were mainly found in the en passant axons, frequently forming bundles in the stratum lucidum (arrowheads). In the mossy terminals, gold labeling avoiding vesicular clusters was commonly attached to the plasma membrane or intracellular membrane cisternae (arrows). Inserts show the same structure taken from the bottom left parts of the consecutive sections, highlighting a gold particle attached to an intracellular membrane cisterna. B, C, D: High-power electron micrographs demonstrating immunogold staining associated with membrane cisternae (open arrows). Scale bars: A, 0.5 µm; insert 0.25 µm; B-D 0.2 µm.

Interneurons expressing TRPC6 channels

In the next set of experiments, we aimed to identify the interneurons whose dendrites are immunoreactive for TRPC6 channels. The dense meshwork of dendrites at the border of the stratum oriens and alveus, as well as in the hilus, resembled those interneuron dendrites that express mGlu1a receptors in the hippocampal formation. To prove that TRPC6 channels are present on the dendrites of mGlu1a-immunopositive interneurons, we performed double immunofluorescent staining. The analysis unequivocally showed that the vast majority of TRPC6-immunoreactive dendrites located both at the border of stratum oriens and alveus (Figure 6A-C) and in the hilus (Figure 6D-F) were indeed labeled for mGlu1a. Then, we sought to clarify the neurochemical marker content of the smooth TRPC6-immunopositive dendrites seen in the stratum radiatum of the CA1 region (Figure 1A). We chose to examine the presence of parvalbumin in these dendrites, since this Ca2+ binding protein is expressed in a large population of GABAergic cells in the hippocampal formation (Katsumaru et al., 1988; Freund and Buzsáki, 1996). The analysis of double-immunofluorescence stained material revealed that the majority of the smooth dendrites immunoreactive for TRPC6 were also stained for parvalbumin in the hippocampus (Figure 6G-I). We could not examine the presence of TRPC6 channels on the parvalbumin-immunopositive dendrites in the stratum moleculare of the dentate gyrus, since no TRPC6-immunoreactive dendrite could be distinguished in this layer, probably because this was masked by the dense immunostaining for dendrites and spines (Figure 1B). These data collectively suggest that, in the hippocampal formation, TRPC6 channels are expressed by at least two distinct interneuron populations, one characterized by mGlu1a expression, the other by parvalbumin content.

TRPC6 channels are present on the plasma membranes of interneuron dendrites

Finally, we investigated the subcellular localization of TRPC6 on the dendrites of interneurons. Using preembedding immunogold staining, we found that immunogold particles were attached to the surface plasma membrane of spiny interneuron dendrites both in the stratum oriens and hilus (Figure 7A, B)(Baude et al., 1993; Acsády et al., 1998; Takacs et al., 2012). Similarly, the aspiny dendrites densely covered by asymmetric synapses in the stratum radiatum, resembling parvalbumin-immunoreactive dendrites (Gulyas et al., 1999), were also decorated with immunogold particles associated with the plasma membrane (Figure 7C). These results suggest that TRPC6 channels are in a position to control the input properties of both mGlu1a- and parvalbumin-expressing GABAergic cells in the hippocampal formation.

Figure 7.

Figure 7

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Plasma membranes of interneuron dendrites are decorated with immunogold staining representing TRPC6 channels. A1-2. Consecutive images taken in the stratum oriens demonstrate that TRPC6 channels depicted by gold particles (arrows) are present on the plasma membrane of horizontal interneuron dendrites bearing long spines and receiving asymmetric synapses on their shafts and spines (open arrowheads). B: Interneuron dendrites in the hilus receiving asymmetric synapses on their shafts and spines (open arrowheads) are decorated with immunogold labeling. C: On the plasma membrane of an interneuron dendrite in the stratum radiatum densely covered by asymmetric synapses (open arrowheads), which is a hallmark of parvalbumin-expressing interneurons, gold particles are attached to the intracellular surface of the plasma membrane (arrows). Scale bars: 0.5 µm.

Discussion

We have found that in the hippocampal formation, TRPC6 channels are expressed by dentate granule cells and inhibitory interneurons. Electron microscopic analysis revealed that TRPC6 channels are localized on the plasma membranes of the dendrites, spines and somata of dentate granule cells, as well as on the surface membrane of interneuron dendrites. In addition, a more detailed analysis showed that the TRPC6 labeling is evenly present in the dendritic spines of dentate granule cells, with some preference for the edge of asymmetric synapses. In the axons and axon terminals of dentate granule cells, TRPC6 channels were regularly observed intracellularly, occasionally associated with membrane cisternae.

TRPC6 channels on the plasma membrane of dentate granule cells

Our immunocytochemical results showing that TRPC6 channels are abundant in the stratum moleculare of the dentate gyrus confirm an earlier work using a different antibody developed against TRPC6 (Chung et al., 2006). In that study, a strong immunolabeling in the stratum moleculare was also described, but no labeling in the other layers of the hippocampus was reported. Using the immunogold technique we have extended these earlier data by demonstrating that in the stratum moleculare, TRPC6 proteins are present on the plasma membrane of the spines and dendrites of dentate granule cells. Since the opening of these channels is controlled by DAG (Hofmann et al., 1999; Aires et al., 2007), we sought to compare the distribution of TRPC6 channels on spines with those proteins known to be involved in the regulation of local DAG levels. Such quantitative data are available mainly for a signaling cascade formed by mGlu5, PLCβ1/4 and DAG lipase, which are present in the perisynaptic annulus around asymmetric synapses (Lujan et al., 1996; Lujan et al., 1997; Nakamura et al., 2004; Katona et al., 2006; Yoshida et al., 2006; Fukaya et al., 2008). Since we found that a fraction of TRPC6 channels in the spines are located at the edge of asymmetric synapses, this structural correlation with the location of mGlu5-mediated DAG production could imply that mGlu-dependent depolarization of dentate granule cells and the parallel Ca2+ elevation may be, at least in part, mediated by TRPC6 channels. In addition to its many other roles, Ca2+ entry via TRPC6 channels may also serve as a feedback signal to terminate the channel operation (Hofmann et al., 1999). The activity of DAG lipase can be markedly enhanced upon intracellular Ca2+ increase, which more effectively degrades DAG and leads to the closure of this channel (Bisogno et al., 2003). Thus, PLCβ1-dependent DAG production upon activation of mGlu5 receptors and DAG degradation by DAG lipase are key regulators of TRPC6 channel opening, and seem to be compartmentalized in the spines of dentate granule cells. A similar scenario can be postulated to be present in the spines of pyramidal cells, where other DAG-sensitive TRPC channels with less Ca2+ permeability than TRPC6, such as TRPC3 and/or TRPC7 (Abramowitz and Birnbaumer, 2009; Bollimuntha et al., 2011) can take part in mediating the mGlu-dependent depolarization (Gee et al., 2003). It is important to highlight that each metabotropic receptor using the Gq protein to conduct its function could probably activate TRPC6 and/or other DAG-sensitive TRPC channels. For example, previous studies have shown that mGlu1, TrkB or type 1 muscarinic receptors operating via the Gq signaling pathway can be found on the spines of principal neurons (Baude et al., 1993; Drake et al., 1999; Uchigashima et al., 2007; Yamasaki et al., 2010). Thus, upon activation, all these metabotropic receptors may open DAG-sensitive TRPC channels, mediating cation influx into the spines of cortical excitatory cells.

In addition to the spines, immunolabeling for TRPC6 channels was also localized to the surface membrane of granule cell dendrites. Since all metabotropic receptors mentioned above have also been found on the plasma membrane of granule cell dendrites or somata (Baude et al., 1993; Lujan et al., 1996; Drake et al., 1999; Yamasaki et al., 2010), similar mechanisms for opening TRPC6 channels are highly likely to occur in dendritic and somatic compartments as well. Thus, the input domain of dentate granule cells is equipped with all necessary building blocks for the regulation of TRPC6 channel function.

In addition to synapse formation and dendritic growth functions (Tai et al., 2008; Zhou et al., 2008), TRPC6 channels may also have a role in controlling endocannabinoid production, as proposed in a recent study (Bardell and Barker, 2010). These retrograde signaling molecules are synthesized “on-demand” upon activation of DAG lipase, and can effectively regulate transmitter release from axon terminals (Kano et al., 2009), serving as a circuit-breaker at high activity levels (Katona and Freund, 2008). Since TRPC6 channels in the presence of DAG can promote local Ca2+ increase, a necessary step for endocannabinoid synthesis, these cation channels might be involved in synaptic plasticity mediated via CB1 cannabinoid receptors (Katona and Freund, 2008; Kano et al., 2009).

TRPC6 channels in the axons and terminals of dentate granule cells

In contrast to that observed on the dendrites and spines of dentate granule cells, immunogold labeling depicting TRPC6 channels in mossy fibers was frequently found intracellularly. This observation suggests that in the axons of dentate granule cells, TRPC6 channels may play different function(s) than in the input domain of these excitatory cells. Experimental evidence suggests that TRPC channels could have a role in the regulation of Ca2+ levels in intracellular stores (Cahalan, 2009; Pani et al., 2012). In agreement with this proposal, we have seen immunogold labeling associated with membrane cisternae in mossy terminals, which might indeed serve as Ca2+ stores. A recent study by Shimizu et al. (Shimizu et al., 2008) described the subcellular localization of type 2 ryanodine receptors in the axons of dentate granule cells. Similarly to what we have observed for TRPC6 channels, this type of ryanodine receptor was prevalently localized intracellularly in the axons of granule cells, and less abundantly in the mossy terminals, but avoided the vesicular clusters. Since this study elegantly showed that ryanodine receptors could amplify the Ca2+ signal in mossy terminals in a use-dependent manner, an intriguing hypothesis is that TRPC6 channels, if they are located on the same axonal compartments as ryanoidne receptors, could have a function in the Ca2+ regulation in mossy fibers, indirectly affecting the neurotransmitter release.

TRPC6 channels in interneurons

In addition to excitatory granule cells, inhibitory interneurons immunoreactive for mGlu1a or parvalbumin expressed TRPC6 channels on their dendritic membrane surface. Since both cell types are equipped with mGlu1 and/or mGlu5 receptors (Baude et al., 1993; Lujan et al., 1996; van Hooft et al., 2000; Topolnik et al., 2006), type 1 and/or 3 muscarinic receptors (Lawrence et al., 2006; Cea-del Rio et al., 2010) and probably TrkB receptors (Holm et al., 2009) as well as DAG lipase (our unpublished data), we suggest that signaling mechanisms comparable to those in dentate granule cells could control the operation of TRPC6 channels in interneurons. A recent study demonstrated that Ca2+ entry via TRP channels into cells resembling mGlu1a -immunoreactive interneurons with horizontal dendrites in the stratum oriens of the CA1 region contributes to the long-term enhancement of excitatory synaptic input onto these interneurons (Topolnik et al., 2006). Although in this study the nature of the TRP channels was not revealed, our data imply that TRPC6 channels with high Ca2+ permeability might contribute to the control of synaptic plasticity observed in these interneurons. Given that the two populations of GABAergic cells expressing TRPC6 channels take part in feed-back (mGlu1a -immunopositive interneurons) or feed-forward (parvalbumin-containing interneurons) inhibitory loops, the activation of these DAG-sensitive TRPC channels could alter information routing in hippocampal microcircuits (Pouille and Scanziani, 2004).

In summary, TRPC6 channels, upon activation of Gq-coupled metabotropic receptors, may effectively regulate information processing in the first stage of the hippocampal trisynaptic loop by affecting Ca2+ levels in dentate granule cells. In addition, these TRPC channels are in a position to impact the operation of distinct interneuron types, likely playing a role in their synaptic plasticity.

Supplementary Material

Supp FigureS1
Supplementary legends

Acknowledgements

We are grateful to Prof. Lutz Birnbaumer for generously providing Trpc6 knockout mice. We thank Rita Karlócai for helping with confocal images and Katalin Lengyel for her excellent technical assistance. The authors also wish to thank Mr. László Barna, the Nikon Microscopy Center at IEM, Nikon Austria GmbH and Auro-Science Consulting Ltd for kindly providing microscopy support. This research was supported in part by the intramural research program of the NIH, National Institute of Environmental Health Sciences.

Grant sponsor: Wellcome Trust International Senior Research Fellowship

Grant sponsor: National Office for Research and Technology

Grant number: OMFB-01678/2009

Grant sponsor: The Intramural Research Program of the NIH

Grant number: Z01-ES-101684

Footnotes

The authors declare no conflict of interest.

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