Confocal laser scanning microscopy and immunohistochemistry of cerebellar Lugaro cells

The present paper shows by means of confocal laser scanning microscopy the immunoreactivity of rat cerebellar Lugaro cells for calbindin, synapsin-I, PSD-95, GluR1, CaMKII alpha, and N-cadherin. Lugaro cells were easily characterized by their location beneath Purkinje cells. Calbindin revealed immunoreactivity in the cell body, and the axonal and dendritic processes. Synapsin-I labelled the presynaptic endings on Lugaro cells. Synapsin-I and PSD-95 immunoreactivity demonstrated the localization of presynaptic and postsynaptic endings surrounding cell soma, corresponding to afferent extrinsic and intrinsic cerebellar fi bers. GluR1 immunoreactivity of the soma and cell processes indicates that Lugaro cells have functional ionotropic glutamate receptors that regulate calcium levels. CaMKII alpha immunoreactivity of Lugaro cell soma and processes suggest its participation as a molecular switch for long-term information storage, and serving as a molecular basis of long-term synaptic memory. N-cadherin immunoreactivity was correlated with somato-somatic and somato-dendritic junctions between Lugaro cells and their synaptic connections. BIOCELL 2013, 37(2): 29-36 ISSN 0327 9545 PRINTED IN ARGENTINA


Introduction
Lugaro cells were described by Ramón y Cajal (1955) and Fox (1959) by means of Golgi silver technique as spindle-shaped cells transversely oriented in the granular layer, and located immediately beneath the Purkinje cell layer.Ramón y Cajal (1955) showed some Lugaro cells with an axon directed downwards to the granular layer, and reaching the white matter, and Fox (1959) traced the descending dendrites in synaptic relationship with mossy fi ber rosettes at the level of mossy glomerulus, and with the Golgi cell axonal ramifi cations, as well as their horizontally directed dendrites in connection with the descending axonal collaterals of the basket cell, forming the pinceaux around the axon hillock of Purkinje cell (Castejón, 2010;2012).Sinton (1964) fi rst published the fi ne structure of Lugaro and Golgi cells.Braak (1974) reported a pigmentarchitectonic study of Lugaro cells and Palay and Chan-Palay (1974) reported Lugaro cells as two kinds of horizontal fusiform cells possessing different axonal pattern and distribution.Sahin and Hockfi eld (1990) characterized Lugaro cells with specifi c antibodies  as distinct from Golgi cells.
Laine and Axelrad (1996,1998) reported Lugaro cells with axons projecting to the molecular layer targeting stellate and basket cells, and descending axon to the granular layer.Melik-Musyan and Fanardzhyan (1998), Fanardzhyan (2003), and Melik-Musyan and Fanardzhyan ( 2004) described two Lugaro cell types with fusiform and triangular cell bodies, as well as dendro-somatic and somato-somatic contacts of Lugaro cells, and dendro-dendritic contacts between Lugaro and Golgi cells.Geurts et al. (2001) reported that rat-303/ calretinin double-labeled cells located just underneath the Purkinje cell layer represented Lugaro cells.Vig et al. (2003) observed Lugaro-like cells in the white matter and internal granular layer of the cerebellum of young cats.Rodrigo et al. (2006) described intense immunostaining for neuronal nitric oxide synthase, including unipolar brush cells, and Lugaro and Golgi neurons in sheep cerebellum, which are not immunoreactive in rodents.Crook and Hendrickson (2006) consider that Lugaro cell and Golgi cell morphology indicate that they contain both glycine and GABA or glutamic acid decarboxylase.Simat et al. (2007) have shown that Lugaro and globular cells are glycinergic/GABAergic and lack mGluR2 and neurogranin.Nunzi and Mugnaini (2009) found expression of secretogranin II, chromogranin A and chromogranin B in Lugaro cells.More recently, Wierzba-Bobrowicz et al. (2011) reported immunoreactive calretinin Lugaro cells in the pup cerebellum.
The present paper describes for the fi rst time the immunoreactivity of rat Lugaro cells for calbindin, synapsin-1, PSD-95, GruR1, CaMKII alpha, and N-cadherin.

Synapsin-I and PSD-95 immunohistochemistry
Animals were used in accordance with NIH and institutional guidelines.For synapsin-I and PSD-95 immunohistochemistry, cerebellar cortex slices were derived from 14-21 days old rats.After decapitation, the lateral cerebellar lobules were removed from posterior fossa and placed in ice-cold (4ºC) Hanks balanced salt solution supplemented with dextrose (6 mg/ml).Fragments of cerebellar cortex were transversally sliced (300 μm thick sections) using a manual tissue chopper (Stoelting).The slices were immediately fi xed in 4% paraformaldehyde in ice-cold 0.1M phosphate-buffered saline (PBS).All samples for PSD-95 immunolabeling were chemically fi xed in ice cold paraformaldehyde for no more than 15 min to preserve antigenicity.Free fl oating slices were placed in a multi-well plate for subsequent rinsing, blocking and labeling steps.

GluR1 immunohistochemistry
For GluR1 immunohistochemistry, cerebellar cortex slices were derived from 14-day-old postnatal rats anesthetized with CO 2 .After decapitation, the lateral lobules of cerebellum were removed from posterior fossa and placed in ice-cold (4ºC) Hanks balanced salt solution supplemented with dextrose (6 mg/ml).Fragments of cerebellar cortex were transversally sliced (300 μm thick sections) using a manual tissue chopper (Stoelting).The slices were immediately fi xed in 4% paraformaldehyde in ice-cold.All samples for GluR1 immunolabeling were chemically fi xed in ice cold paraformaldehyde for no more than 30 min to preserve antigenicity.Free-fl oating slices were placed in a multiwell plate for subsequent rinsing, blocking and labeling steps.Slices were rinsed in PBS (2 x 5 min) and then extracted overnight in 1% Triton X-100 in PBS (Polysciences).Blocking was done with 50 mM NH 4 Cl in PBS (30 min), followed by 20% horse serum in PBS (30 min).Washing was done with HSPBS (5 min).All subsequent steps were also carried out in HSPBS.Slices were double labeled with anti-GluR1 monoclonal antibody (diluted 1:500; Molecular Probes, Eugene, OR).This primary antibody was applied overnight (4ºC), and then tissues were rinsed again (3 x 15 min) in HSPBS.The secondary antibody used was Alexa-488 goat anti-rabbit IgG (Molecular Probes), diluted 1:300, and applied overnight (4ºC).Tissues were then rinsed 3 x 15 min in 1% HSPBS, mounted on microscope slides, sealed with vacuum grease in a closed chamber containing 1% HSPBS, and covered with a coverslip.

CaMKII alpha immunohistochemistry
For CaMKII alpha labeling, cerebellar cortex slices were derived from 14-day-old postnatal rats anesthetized with CO 2 .After decapitation, the lateral lobules of cerebellum were removed from posterior fossa and placed in ice-cold (4ºC) Hanks balanced salt solution (HBSS) supplemented with dextrose (6 mg/ ml).Fragments of cerebellar cortex were transversally sliced (300 μm thick sections) using a manual tissue chopper (Stoelting).The slices were immediately fi xed in 4% paraformaldehyde in ice-cold 0.1M phosphatebuffered saline (PBS) for 48 hours.Free-fl oating slices were placed in a multiwell plate for subsequent rinsing, blocking and labeling steps.Slices were rinsed in PBS (2 x 5 min) and then extracted overnight in 1% Triton X-100 in PBS (Polysciences).Blocking was done with 50 mM NH 4 Cl in PBS (30 min), followed by 20% horse serum in PBS (30 min).Washing was done with 1% horse serum in PBS (HSPBS) (5 min).All subsequent steps were carried out in 1% HSPBS.Slices were labeled with a primary antibody against CaMKII alpha and Alexa 488 goat anti mouse (GAM)-antibody (diluted 1:300; Molecular Probes, Eugene, OR) as a secondary antibody.The labeling process was applied overnight (4ºC), and tissues were then rinsed 3 x 15 min in 1% HSPBS, mounted on microscope slides, sealed with vacuum grease in a closed chamber containing 1% HSPBS, and covered with a coverslip.

N-Cadherin immunohistochemistry
For N-cadherin labeling of cerebellar cortex slices were derived from 21-day-old postnatal rats anesthetized with CO 2 .Cerebellar slices were initially processed as described above for GluR1 immunohistochemistry. Slices were double labeled with a primary antibody against N-cadherin (diluted 1:400; Molecular Probes, Eugene, OR) and Alexa 488 goat anti mouse (GAM)-antibody (diluted 1:300; Molecular Probes, Eugene, OR).The labeling process was applied overnight (4ºC), and tissues were then rinsed 3 x 15 min in 1% HSPBS, mounted on microscope slides, sealed with vacuum grease in a closed chamber containing 1% HSPBS, and covered with a coverslip.

Confocal imaging
Images of fl uorescently labeled brain tissue slices were captured using a Leica TCS NT scanning laser confocal microscope equipped with Argon (Ar; 488 nm), Krypton (Kr; 568 nm) and Helium-Neon (HeNe; 633 nm) lasers [21], or a Zeiss 510 confocal microscope equipped with Ar, green He-Ne (543 nm), and red He-Ne lasers (633 nm).Alexa-488-labeled secondary antibody was visualized with Ar laser excitation and a fl uorescein-like fl uorescence fi lter set (510 nm dichroic mirror, 515 nm long pass barrier fi lter).Alexa-568-labeled secondary antibody was visualized using a Kr laser and rhodamine fi lter set (590 LP barrier fi lter).For simultaneous imaging of Alexa-488 and -568, a dual channel fl uorescence arrangement was used, utilizing a double dichroic mirror (488/568 nm) and a 530/30 nm bandpass barrier fi lter in the Alexa-488 channel.
To study the structure of cerebellar cortex at low magnifi cation, we used a 10X Plan Fluotar or 20X Plan Apo objective lens.To resolve individual synaptic puncta, we used a 63X/1.2NAwater immersion PLAN APO objective lens with an additional electronic zoom factor of up to 3.6.The pinhole size was set to 1.0-1.8times the Airy disk to maximally reject out-of focus haze and to improve spatial resolution.For improved signal-to-noise ratio, up to eight scans were averaged at each optical section.Stacks of 8-29 optical sections (1,024 x 1,024 pixel array) yielded voxel dimensions between 0.15 and 0.4 for the X, Y and Z planes.

Image processing
Brightness and contrast were adjusted using Photoshop 5.0 (Adobe).Red-yellow stereo images were generated from stacks of confocal optical sections using Scion Imaging (Scion Corp., Frederick, MD, USA).

Confocal laser scanning microscopy and calbindin labeling
Slices of rat cerebellar cortex labeled with calbindin show intense labeling of Lugaro cell cytoplasm located beneath Purkinje cells.They exhibit an axonal ramifi ed process directed toward the granular layer with ascend-ing collaterals toward the molecular layer, horizontal dendrites remaining in the granular layer, and ascending dendritic processes going to the Purkinje cell and molecular layers (Fig. 1).

Synapsin-I immunohistochemistry
Rat cerebellar slices labeled with synapsin-I show as green granules the distribution of presynaptic endings surrounding the Lugaro cell soma (Fig. 2).

Synapsin-I and PSD-95 immunohistochemistry
As above stated primary and secondary antibodies against synapsin-I and PSD-95 were applied fi rst, then primary antibodies against calbindin were applied followed by Cy5 as a secondary antibody.The Lugaro cell show small green puncta surrounding the cell body corresponding to the presynaptic fi bers making axosomatic contacts, and large red hotspots deposited at subcellular and cellular body localization, corresponding to the synaptic contacts of afferent extrinsic and intrinsic fi bers making synaptic contacts with Lugaro cells (Fig. 3).

GluR1 immunohistochemistry
Rat cerebellar slices labeled for GluR1 showed positive immunofl uorescence staining on the cell soma and processes of Lugaro cells corresponding to the postsynaptic sites of afferent extrinsic and intrinsic fi bers synapsing with Lugaro cells (Fig. 4).

CaMKII alpha immunohistochemistry
Confocal laser scanning microscopy observations, by means of stack of optodigital sections spanning 28 μm in the granular layer, showed punctate CaMKII alpha immunoreactivity at the level of Lugaro cell soma and processes (Fig. 5).

N-cadherin immunohistochemistry
Rat cerebellar slice labeled with specifi c antibody against N-cadherin also showed strong positive immunochemistry of Lugaro cells, whereas the granule cell groups appeared unstained (Fig. 6).

Discussion
In the present paper we have shown calbindin immunoreactivity of Lugaro cells.Similar observations were earlier made by Rogers (1989).Calcium plays a fundamental role in the cell as second messenger and is principally regulated by calcium-binding proteins.Bastianelli (2003) showed that Lugaro and unipolar brush cells present an opposite immunoreactivity profi le, most of them being calretinin positive while lacking calbindin-D28k and parvalbumin.According to this author, the function of these proteins is not fully understood, although strong evidence supports a prominent role in physiological settings with altered calcium concentrations.For example, they may directly or indirectly enable sensitization or desensitization of calcium channels, and may further block calcium entry into the cells.The calcium-sensor proteins have been shown to be potent and specifi c modulators of ion channels, which may allow for feedback control of current function and hence signaling.
According to Laine and Axelrad ( 2002), double anticalretinin and anti-calbindin immunolabelings shows that Lugaro cells as well as some globular somata dispersed in the granular layer are both calretinin-positive, and in close apposition with numerous calbindin-positive varicosities of Purkinje cell axon recurrent collaterals.According to these authors, the common granular layer location and calretinin labeling, the striking similarity in axonal projection pattern, and the important common recurrent afferentation by Purkinje cell axons strongly argue in favor of the classifi cation of these globular interneurons as a subgroup of a widened Lugaro cell type.
We have herein reported synapsin-I positive immunoreaction of Lugaro cells.Synapsin-I immunopositivity has been mainly correlated with the presynaptic endings of afferent mossy and climbing fi bers, and Purkinje cell recurrent axonal collaterals.Synapsin-I, one of the major synaptic phosphoproteins, associates with synaptic vesicles and regulates neurotransmitter release.Strong, punctate immunoreactivity with synapsin-I antibodies is a reliable marker of presynaptic structures, and therefore has been widely used for immunohistochemical analysis of synapse formation and distribution (De Camilli et al., 1983;Castejón et al., 2004 ).PSD-95 is associated with the localization of postsynaptic endings (Hunt et al., 1996).
The synapsin-I-and PSD-95 immunohistochemical reaction showed the precise localization of pre-and post synaptic ending of afferent extrinsic and intrinsic axonal processes, apparently corresponding to the synaptic contacts with mossy and climbing fi ber collaterals, Purkinje cell recurrent collateral axons, Golgi cell, granular and basket neurons, unipolar brush cells, and stellate cell processes establishing synaptic connections with Lugaro cells.(Sahin and Hockfi eld, 1990).
Our observations of GluR1 subunit immunohistochemistry revealed the presence of this subclass of AMPA receptors at the Lugaro cell subcellular localization, and at the other cerebellar inhibitory neurons (Purkinje, basket and stellate cells).(Castejón and Dailey, 2009).These fi ndings mean that Lugaro cells have functional GluR1 ionotropic glutamate receptors that regulate calcium levels.GluR1 is one of the several subunits of the quisqualate subclass (QA) receptors coupled to cationic ionic channel, also termed AMPA receptors.Grandes et al. (1994) distinguished three splice variants of GluR1 metabotropic glutamate receptors in the rat cerebellum.Hamori et al. (1996) also demonstrated GluR1 subtypes in inhibitory interneurons of the cerebellar cortex.Lately, Negyessy et al. (1997) demonstrated by light and transmission electron microscopy, using pre-embedding immunoperoxidase and immunogold techniques, the cellular and subcellular localization of mGluR5 metabotropic glutamate receptor in Lugaro cells.
In the present study we have described the presence of CaMKII alpha positive immunoreactivity in the cell body and processes of Lugaro cells.The CaMKII has an apparent cytosolic localization as earlier revealed by subcellular fractionation studies of rat brain (Piccioto et al., 1995).CaMKII beta is present in cell bodies and dendrites in mature hippocampal neurons (Chang et al., 2001).Previous studies by means of biochemical methods, electron microscopy and confocal laser scanning microscopy have localized the CaMKII at the level of postsynaptic density (Yoshimura and Yamauchi, 1998;Lisman et al., 2002), being central to the regulation of glutamatergic synapses.According to Lisman et al. (2002), this localization indicates that a binding pattern for CaMKII is the NMDA receptor within the postsynaptic density.The AMPA receptor subunit GluR1 is also phosphorylated by CaMKII enhancing channel function.The CaMKII has been postulated by Lisman et al. (2002) as a molecular switch for long-term information storage, and serving as a molecular basis of long-term synaptic memory.CaMKII is an enzyme that plays a major role in the regulation of long-term potentiation, a form of synaptic plasticity associated with learning and memory (Jin et al., 1999;Nakamura et al., 1996;Lisman et al., 2002).The expression of CaMKII is developmentally regulated (Sakagami and Kondo, 1993;Shanavas et al., 1998).

Some neurobiological considerations on Lugaro cells
De Camilli et al. (1984) demonstrated by means of guanosine 3':5'-phosphate-dependent protein kinase antiserum, a specifi c immunohistochemical marker for the dense innervation by Purkinje cell recurrent axonic collateral around large interneurons, such as Lugaro and Golgi cells, and around the Purkinje cell pinceaux.Gruol and Crimi (1988) earlier identifi ed Lugaro cells in culture using immunohistochemical techniques and antibodies to gamma-aminobutyric acid (GABA), parvalbumin, and cyclic guanosine monophosphatedependent protein kinase.Lugaro cells are stained intensely using histochemical demonstration of NADPHdiaphorase (NADPH-d) (Okhotin and Kalinichenko (1999).Dieudonné (2001) postulated that serotonin specifically modulates the activity of Lugaro cells, a class of inhibitory interneurons of the cerebellar cortex, offering new insights into the action of this neuromodulator.The peculiar axonal projection and specifi c interneuronal targets of the Lugaro cells suggest that the action of serotonin might occur upstream of Purkinje cells through a resetting of the computational properties of the cerebellar cortex.Dean (2003) recorded a novel fast GABAergic synaptic current from Lugaro cells to Purkinje cells in rat brain slices using patch-clamp techniques.These authors suggested that the release of GABA onto Purkinje cells from Lugaro cells would primarily occur during motor activity under conditions in which the activity of basket and stellate cells might be inhibited.Flace et al. (2004) included Lugaro cells into the 'Non-traditional' large neurons of the granular layer of the cerebellar cortex examined by means of immunocytochemistry for glutamic acid decarboxylase (GAD).These morphological data further point out possible functional roles for GABA as neurotransmitter/neuromodulator in the intrinsic, associative and projective circuits of the cerebellar cortex.Structural and topographic characteristics of Lugaro cells, as well as the peculiarities of their contacts with the other cells of cerebellar cortex, in combination with the data on their neurotransmitter content, indicate that these cells play the role of inhibitory interneurons.Ambrosi et al. (2007) also support the concept that Lugaro cells act as an inhibitory interneuron.Sotnikov (2006) considered that Lugaro cells in the cerebellum and various synaptically NO-positive neurons in the cerebral cortex form part of sensory innervation of the brain.Ito (2006) postulated that cerebellar circuitry now includes Lugaro cells and unipolar brush cells as additional unique elements in the neuronal machine concept of the cerebellum.

FIGURE 2 .
FIGURE 2. Slice of rat cerebellar cortex double labeled with anti-synapsin-I rabbit IgG polyclonal antibody, and Alexa-488 goat anti-rabbit IgG and Alexa-568 goat-anti-mouse IgG as secondary antibodies showing positive immunostaining expressed by tini green dots surrounding Lugaro cells (LC) soma and processes corresponding to presynaptic terminals impinging upon Lugaro cells.

FIGURE 1 .
FIGURE 1. Rat cerebellar slice labeled with calbindin showing positive immunostaining of calcium binding proteins of Lugaro cell (LC) soma and processes.Note its typical location beneath Purkinje cell (PC) layer.

FIGURE 3 .
FIGURE 3. Slices of rat cerebellar cortex labeled with antisynapsin-I rabbit IgG polyclonal antibody and anti-PSD-95 (mouse) primary antibody, and a primary antibody against calbindin followed by Cy5 as a secondary antibody.The secondary antibodies were Alexa-488 goat anti-rabbit IgG and Alexa-568 goat-anti-mouse IgG.Positive Synapsin-I immunoreactions appears as green tini dots surrounding Lugaro cells.PSD-95 immunopositivity appears as red hotspots within the Lugaro cell peripheral cytoplasm.

FIGURE 5 .
FIGURE 5. Rat cerebellar slice labeled with a primary antibody against CaMKII alpha, and Alexa 488 goat anti mouse as a secondary antibody.Small puncta are observed surrounding Lugaro cell soma (LC), descending axon, and the horizontal and ascending dendrites (arrows).

FIGURE 6 .
FIGURE 6. Rat cerebellar slices were double labeled with a primary antibody against N-cadherin and Alexa 488 goat anti mouse (GAM) secondary antibody showing positive immunochemistry of Lugaro cells (LC) located beneath Purkinje cell (PC).Also note the positive immunochemistry of Golgi cell (GoC), and the unstained granule cell groups (GC).