New Cannabinoid CB1 receptors in rat medial prefrontal cortex are …rabbit.if-pan.krakow.pl/pjp/pdf/2009/6_1000.pdf · 2010. 1. 7. · CB1 receptor binding sites are enhanced in

Cannabinoid CB1 receptors in rat medial

prefrontal cortex are colocalized with calbindin-

but not parvalbumin- and calretinin-positive

GABA-ergic neurons

Krzysztof Wêdzony, Agnieszka Chocyk

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Abstract:

In the present study, we investigate putative localization of cannabinoid receptors 1 (CB1) protein on a population of cortical �-am-

inobutyric acid (GABA) – positive interneurons characterized by expression of calcium-binding proteins in rat medial prefrontal

cortex (MPC). Parvalbumin (PARV)/calretinin (CALR)- and calbindin (CALB)-positive neurons form two distinct populations of

GABA-ergic interneurons that comprise the axo-somatic/axo-axonic and axo-dendritic inhibitory systems of pyramidal cells. It has

been found that CB1 receptor-positive cells are randomly distributed across the rat MPC. All spotted neurons that were positive for

CB1 receptors were positive for GABA; however, the number of GABA-positive cells drastically exceeded the number of CB1

receptor-positive neurons. Subsequent experiments with double-labelling of CB1 receptors with PARV and CALR revealed no colo-

calization. CALB-positive neurons (e.g., double bouquet and bipolar cells) display colocalization: the degree of colocalization

among CB1 receptor-positive cells reached 18%. The appearance of CB1 receptors in double bouquet and bipolar neurons indicates

that CB1 receptors may control the activity of pyramidal neurons from presynaptic sites in axo-dendritic synapses formed on apical

and basilar dendrites of pyramidal neurons, as is characteristic for CALB-positive cortical interneurons. The phenotype of GABA-

and CB1 receptor-positive but CALB-negative neurons may represent a population of inhibitory neurons that allow axo-somatic

control of information flow, governed by principal neurons of the MPC.

Key words:

cannabinoid receptors, CB1, medial prefrontal cortex, parvalbumin, calretinin, calbindin, immunocytochemistry

Introduction

There are several arguments that cannabinoid (CB)

signalling pathways are engaged in the pathological

neurotransmission responsible for the appearance of

schizophrenic symptoms. Epidemiological studies sug-

gest that cannabis use, especially in the adolescent pe-

riod, represents a substantial environmental risk factor

for the appearance of schizophrenia (for comprehen-

sive review see [25]). It has also been observed that

CB1 receptor binding sites are enhanced in the course

of schizophrenia in the medial prefrontal cortex

(MPC) and striatum [8, 34]. Increased levels of anan-

damide and palmitylethanolamide, endogenous ligands

of CB1 receptor binding sites, have been observed in

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cerebrospinal fluid of patients suffering from schizo-

phrenia [15, 21]. An increase in the number of bind-

ing sites was also observed, followed by decreased

expression of mRNA-encoding synthesis of CB1 re-

ceptors and a decrease in the level of CB1 receptor

protein, measured post-mortem in dorsolateral MPC

in schizophrenics [10]. It has also been noted that

phencyclidine, which in humans evoked schizo-

phrenic symptoms and in rats is used to model nega-

tive and positive symptoms of schizophrenia [32], re-

duces the concentration of arachidonoylglycerol – an-

other endogenous ligand for CB1 receptors in rat

MPC [30]. It has also been observed that the psy-

choactive ingredient of marijuana, �9-tetrahydro-

cannabinol, worsened phencyclidine-induced cogni-

tive impairment in rats [30]. Long-lasting intake of

cannabis leads to deficits of working memory similar

to those observed in schizophrenia [12, 26]. Since the

efficacy of working memory greatly depends on cor-

rect inhibitory control of pyramidal neurons in the

MPC [7, 22], we sought to investigate whether CB1

receptors are present in the MPC and whether they are

present in defined populations of �-aminobutyric acid

(GABA)-positive interneurons. GABA-ergic inhibi-

tory neurons can be classified according to their ex-

pression of calcium-binding protein [e.g., calbindin

(CALB), parvalbumin (PARV) or calretinin (CALR)]

[1, 6, 9]. CALB-positive and PARV-positive/CALR-

positive inhibitory interneurons represent two distinct

subpopulations of GABA-ergic interneurons with re-

spect to cell morphology, type of contacts with py-

ramidal neurons and firing pattern [6, 33]. PARV-

positive and CARL-positive interneurons correspond,

respectively, to basket or chandelier cells. They make

axo-somatic and axo–axonic contacts with the soma

or initial axonal segments of pyramidal neurons [1, 4,

6, 9, 13, 20, 33]. PARV-positive neurons have physio-

logical properties characteristic of fast-spiking inter-

neurons [20, 33]. CALB-positive cell morphology is

typically that of a double bouquet cell but can also be

that of a bipolar neurons. These cells make axo-den-

dritic inhibitory synapses on the apical and basilar

dendrites of pyramidal neurons and exhibit a slow

spiking interneuronal pattern of electrophysiological

activity [4, 9, 13, 20, 33]. This inhibitory circuitry is

impaired in the course of schizophrenia, on both the

pre- and post-synaptic levels. Presynaptic deficits are

associated with decreased expression of: glutamic

acid decarboxylase isoform of 67 kDa (GAD67),

PARV and the GABA transporter – GAT1 [22]. Post-

synaptic changes are also observed, such as an in-

crease in the expression of GABA receptor subunit

GABA 2�2 [22]. Thus a description of CB1 receptor

localization in inhibitory circuitry of the MPC may be

useful for further attempts to develop pharmacologi-

cal therapy for schizophrenia based on the modulation

of cannabinoid signalling pathways.

Materials and Methods

Subjects

Twenty-two male Wistar rats (200–250 g) were used:

four for each set of staining and two for Western blot.

The experimental protocol was approved by the Com-

mittee for Laboratory Animal Welfare and Ethics at

the Institute of Pharmacology, Polish Academy of

Sciences in Kraków and met the criteria of the Guide

for the Care and Use of Laboratory Animals of the In-

ternational Council for Laboratory Animals.

Western Blotting

The brains were removed after decapitation, cooled

on ice, and sliced into 1 mm coronal sections using

a rodent brain matrix (Ted Pella, INC). The MPC was

punched out from coronal sections with a 15-gauge

punching tube. The isolated brain region was ho-

mogenized in 10 vol (w/vol) of lysis buffer: 50 mM

Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA,

1 mM EGTA, 50 mM NaF, 0.5% Triton X-100, 0.5%

SDS, and protease inhibitors (1:200, Sigma). Protein

levels were determined using a BCA Protein Assay

Kit (a modification of the Lowry assay, Sigma). Sam-

ples of equal protein contents were adjusted to an

equal volume with 50 mM Tris (pH 6.8), containing

2% SDS, 8% glycerol, and 2% 2-mercaptoethanol

with bromophenol blue as a marker. Samples were

then boiled for 5 min. Protein extracts (10 µg/lane

were separated by 7.5% SDS-PAGE and transferred

to nitrocellulose membranes using an electrophoretic

transfer system (BioRad). The membranes were stained

with Ponceau S to confirm equal loading and transfer

of the gels. The blots were then incubated overnight at

4°C with the polyclonal rabbit anti-CB1 receptor pri-

mary antibody, which recognizes 1–14 N-terminal

�� 1001

Cannabinoid CB1 receptors and rat Medial Prefrontal Cortex��

residues (Alexis), diluted 1:1000. Immune complexes

were detected using appropriate peroxidase-conjugated

secondary antibodies: anti-rabbit IgG (1:1000, Roche).

The reaction was visualized by ECL (Enhanced

Chemiluminescence) (Lumi-LightPlus Western Blotting

Kit, Roche). Chemiluminescence was recorded and

evaluated with a luminescent image analyzer (Fuji-

film LAS-1000). Molecular weights of immunoreac-

tive bands were calculated on the basis of the migra-

tion of molecular weight markers (Roche) using Im-

age Gauge (Fujifilm) software (for further details see

Maækowiak et al. [23]).

Euthanasia and perfusion

Rats were deeply anesthetized with sodium pentobar-

bital (100 mg/kg) and transcardially perfused with sa-

line (0.9% NaCl), followed by ice-cold 4% parafor-

maldehyde in 0.1 M phosphate-buffered saline (PBS,

pH 7.4). However, for double-labelling of CB1 receptor

with GABA, a mixture of 4% paraformaldehyde and

0.1% glutaraldehyde in 0.1 M PBS was used as a fixa-

tive after perfusion with saline (as described above).

Sectioning

Three to four hours after fixation (with 4% parafor-

maldehyde in 0.1 M PBS or 4% paraformaldehyde

and 0.1% glutaraldehyde in 0.1 M PBS), 50 µm-thick

sections were cut at the level of the MPC, using a vi-

bratome (Leica VT1000S).

Non-fluorescent labelling of CB1 receptors

The free-floating brain sections were rinsed three

times in 0.01 M PBS, followed by 1 h of incubation in

a blocking buffer containing 5% normal goat serum

and 0.2% Triton X-100 in 0.01 M PBS. Then the sec-

tions were incubated (48 h, 4°C) with a rabbit poly-

clonal primary antibody which recognizes the 1–14

N-terminal residues of CB1 receptor (Alexis), diluted

1:1000 in a blocking buffer (the concentration of nor-

mal goat serum was reduced to 3%). After rinsing, the

sections were incubated for 1 h with biotinylated goat

anti-rabbit secondary antibody (Vector Lab) in a block-

ing buffer. The reaction was visualized using the Vec-

tastain Elite ABC kit and the Vector SG kit (Vector

Lab), which stains the immunopositive material blue.

All reagents were used at concentrations suggested by

the manufacturer, with the exception of the dilution

used with the SG kit, which was diluted by half ac-

cording to the manufacturer’s suggestions.

Double immunofluorescence

The sections, obtained as described above, were

washed in 0.01 M PBS and then placed for 1 h in the

blocking buffer, which contained 5% normal goat se-

rum in 0.01 M PBS (staining for GABA) or 5% nor-

mal goat serum and 0.1% Triton X-100 in 0.01 M PBS

(staining for CB1 receptors, PARV, CALR and CALB).

Subsequently, the sections were placed in a mixture of

primary antibodies in adequate blocking buffer as

above; however, the concentration of goat serum was

reduced to 3%. The sections labelled for the presence

of CB1 receptors and GABA, PARV, CALR, and

CALB were incubated in a mixture of two primary

antibodies for 48 h. After incubation with the primary

antibodies, the sections were washed (as above) and

placed for 2 h (at room temperature, in total darkness)

in a mixture of secondary antisera (diluted to the final

concentrations of 1:100 with 0.01 M PBS containing 3%

normal goat serum and 0.1% Triton X-100). Table 1 in-

cludes the manufacturers of the secondary antibodies (for

further details see Wedzony et al. [31]).

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Tab 1. � ��

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Primary antibody dilution and manufacturers Secondary antibody Jackson Immuno Research Lab. Inc.

1 Polyclonal, rabbit anti-CB1 receptor 1:1000 Alexis Cy��2-conjugated Affinity Pure Goat Anti-Rabbit IgG (1:100)

2 Monoclonal, mouse, anti-GABA 1:500, Swant Texas Red-conjugated Affinity Pure Goat Anti-Mouse IgG (1:100)

3 Monoclonal, mouse anti-parvalbumin 1:1000, Sigma

4 Monoclonal, mouse anti-calretinin 1:1000, Sigma AMCA – conjugated Affinity Pure Goat Anti-Mouse IgG (1:100)

5 Monoclonal, mouse anti-calbindin 1:1000, Sigma

Data analysis

For colocalization analysis, approximately 200 CB1-

positive cells (± 20) were captured under the 100×

lens in layer V and II/III for each staining, from three

sections of the MPC obtained from four animals. Im-

ages were focused on immunopositive elements in

CB1 receptors, which were photographed using ap-

propriate narrow band filters for the CY2 fluorescent

marker. Then, to determine colocalization of CB1 re-

ceptor protein with GABA, PARV, CALR and CALB,

the fluorescent filter cubes appropriate for AMCA or

Texas Red excitation/emission were changed without

altering the focal plane in order to count and photograph

the cells. Double-labelling was verified by overlaying

the captured images from both channels.

Data presentation

For data presentation and mapping of non-fluorescent

immunopositive material, digital images were cap-

tured using a Leica DMLB microscope (Nomarski

phase contrast, objective PlanApo 100×; oil condenser)

and Photometrics Coolsanp FX camera equipped with

ImagePro Plus image analysis software. Camera Lu-

cida drawings of immunoreactive material were made

using a Leica DMLB microscope, 20× objective and

drawing tube. Drawings were scanned and further

processed using the Corel Draw program. Sections la-

belled with fluorescent markers were examined and

photographed using the Leica DMLB microscope

with the epifluorescent attachment, equipped with

CyTM2, AMCA, and Texas Red narrow band filters

(objective PlanApo 100×). Images were captured us-

ing a Photometrics Coolsanp FX camera and Meta-

Morph software was used for colocalization analysis.

Results

To demonstrate the specificity of the CB1 receptor an-

tibody, Western blotting was performed on brain

lysate obtained from samples of the MPC. The anti-

CB1 receptor antibody specifically detected a band at

64 kDa, consistent with the molecular weight of the

monomeric form of the receptor (Fig. 1). Two addi-

tional bands (46 and 57 kD) may correspond to CB1

receptors under degradation (Fig. 1). The lack of spe-

cific control peptide compelled us to investigate

whether applied antibody can bind to fractions of bo-

vine serum albumin. We did not observe any protein

bands and therefore suppose that the antibody applied

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was specific (Fig. 1). We identified CB1 receptor-

positive cells in all layers of the MPC, without any

specific stratification, although particular attention

should be paid to layer II/III. There were no CB1

receptor-positive cells in layer I (Fig. 2A). The mor-

phology of CB1 receptor-positive neurons resembled

the morphology of bipolar or double bouquet cells

(Fig. 2B–E). Immunopositive material was observed

in cytoplasm of the cell body as well as in axons or

dendrites emanating from the cell body (Fig. 2B-E).

Occasionally, we observed processes that displayed

CB1 receptors with prominent boutons (Fig. 2F).

Double-labelling for GABA and CB1 receptors re-

vealed that all CB1 receptors-positive cells were posi-

tive for GABA, which rules out the presence of this

receptor on cortical principal neurons (Fig. 3). Fur-

thermore, more than 18% of CB1 receptor-positive

cells were positive for CALB (Fig. 5). We did not ob-

serve colocalization of CB1 receptor protein with

CALR or PARV (Fig. 4).

Discussion

The results of the present study indicate that in the rat

MPC the CB1 receptor is almost exclusively ex-

pressed by GABA-positive neurons. The above data

are in line with the previous studies indicating, for ex-

ample, that in the rodent cortex approximately 100%

of neurons that express high levels of CB1 mRNA

also express mRNA encoding glutamic acid decar-

boxylase 65, an enzyme that synthesises GABA [24].

It is, however, difficult at the moment to exclude the

possibility that CB1 receptors are present in the py-

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ramidal cells, since mRNA encoding their synthesis

has been observed through in situ hybridization and

single-cell PCR studies [18, 24]. It is conceivable that

the level of CB1 receptor protein in principal neurons

of the MPC stained with immunocytochemistry tech-

niques is below the limit of detection for that method.

With respect to the calcium-binding proteins that

characterize different populations of GABA-ergic in-

terneurons, we found substantial co-expression of

CB1 receptors and CALB, but not CALR or PARV.

Similar colocalization of CALB and CB1 receptors

has been also observed in rat sensory cortex [2]. The

cytoplasmic localization of CB1 receptors that we ob-

served is in line with the electron microscopic data

showing that CB1 receptors are not only axon/soma

membrane-bound but are also present in endoplasmic

reticulum, Golgi apparatus, multivesicular bodies, and

the endosome-lysosome system. The presence of CB1

receptor protein in the endosome-lysosome system

may explain the appearance of two additional protein

bands in our Western blot (around 46 and 57 kD).

These bands may represent CB1 receptors under deg-

radation – the fraction bound to the endosome-

lysosome fraction. In the context of cellular expres-

sion, a fraction around 64 kD should be recognized as

a fraction of a newly synthesized pool of receptors, or

a fraction that is membrane-bound. Apart from obvi-

ous similarities in morphology, localization and the

GABA-ergic nature of neurons expressing CB1 re-

ceptors in the MPC observed in the current study and

other studies performed in rats [2, 29], the differences

should also be noted. Under our staining conditions,

we observed cells with proximal axons and dendrites

positive for CB1 receptor and a relatively low number

of neuronal processes, which has been observed in

other studies performed on rat, primate and human

cortex [2, 10, 11, 29]. The discrepancy between our

study and other published works remains to be eluci-

dated. The apparent lack of visualization of CB1

receptor-positive neuronal processes may be responsi-

ble for the failure of our study to observe the clear

laminar distribution of CB1 receptor-positive ele-

ments that were observed by others [2, 10, 11, 29]. In

addition, the type of antibody (directed to N-terminal

vs. C-terminal), tissue preparation and analysis in spe-

cific brain regions should be considered. In our study,

only 18% of CB1 receptor-positive cells were

CALB-positive neurons. Recent findings indicate that

the rest are present on cholecystokinin (CCK)-pos-

itive neurons [2, 9, 10]. CCK and CALB are ex-

pressed on separate populations of cortical interneu-

rons [2, 9, 10]. Thus, our study is limited to only one

pool of CB1 receptor-positive neurons; the other

populations will require additional investigation. Im-

portantly, both types of interneurons belong to differ-

ent inhibitory systems of principal cortical neurons.

Interneurons expressing CALB are known to repre-

sent dendrite-targeting inhibitory cells in the hippo-

campus and likely also in the neocortex. On the other

hand, CCK and CB1 receptor-positive cells furnish

perisomatic inhibitory inputs to pyramidal neurons [2,

14, 18]. Indeed, recent physiological experiments in-

dicated that, in the neocortex, perisomatic inhibition

was more susceptible to cannabimimetics than den-

dritic inhibition [27, 28]. Furthermore, that type of in-

hibitory control, in terms of propagation of informa-

tion by the principal neuron, is superior to that of the

dendritic inhibition [7]. Thus, the population defined

in the present study as CB1 receptor-positive GABA-

ergic interneurons that are negative for CALB should

represent the future target for pharmacological treat-

ment [3] of cognitive deficits associated with the

MPC during the course of schizophrenia. It has been

proposed by Eggan et al. [10] that CB1 receptors on

CALB-negative (CCK-positive) interneurons may at-

tenuate GABA release and alter the activity of py-

ramidal neurons, with subsequent reduction of the �

power band in the frontal lobe, which is fundamental

for the correct function of working memory [5, 19].

Experiments with agonists and antagonists of CB1 re-

ceptors performed on rat hippocampus and entorhinal

cortex confirm this conclusion [16, 17]. It will be of

interest to extrapolate in the future the data acquired in

the hippocampus and entorhinal cortex on the MPC.

Acknowledgments:

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Received:

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contentcontcontents_3'2005contentsabstractindex6spis tresciAAdamczyk Agata1223,1230Albrecht Jan1245,1250Aschner Michael1245

BBa³kowiec Agnieszka1236Ba³kowiec-Iskra Ewa1236Bany-Laszewicz Urszula1243Barcikowska Maria1223 - 1225Barczak Anna1225Berdyñski Mariusz1224 - 1225Berêsewicz Ma³gorzata1237Bernacki Jacek1241Bia³opiotrowicz Emilia1224,1237Bielarczyk Hanna1229,1233Bielecka Anna1241Biernacka-£ukanty Justyna1238Bizon-Zygmañska Dorota1229Brodacki Bogdan1239Bu¿añska Leonora1253

CC¹ka³a Magdalena1226Chabik Grzegorz1242Chalimoniuk Ma³gorzata1239Chodakowska-¯ebrowska Ma³gorzata1224 - 1225Cieœlik Magdalena1226,1239Colpo Pascal1253Czapski Grzegorz A1240Czapski Grzegorz A.1226,1230,1245Czernicki Zbigniew1231Cz³onkowska Anna1242

DDezor Mateusz1241Domañska-Janik Krystyna1243 - 1244,1249,1253Domasiewicz Anna1231Domek- £opaciñska Katarzyna1245Dorszewska Jolanta1241Dyœ Aleksandra1233Dziubina Anna1227

FFlorczak Anna1241Florczak Jolanta1241Florczak Ma³gorzata1241Friedman Andrzej1227Frontczak-Baniewicz Ma³gorzata1231

GGabryel Bo¿ena1241Gabryelewicz Tomasz1224Gadamski Roman1231Gajkowska Barbara1226,1248 - 1249Gêbarowska Jolanta1247Golan Maciej 1243Go³embiowska Krystyna1227,1251Górecki Dariusz C.1237Grieb Pawe³1228,1247Gromadzka Gra¿yna1242Grygorowicz Tomasz1243Grzywaczewska El¿bieta1247Gul-Hinc Sylwia1233

HHabich Aleksandra1243,1249

JJab³oñska Anna1244,1249Jacewicz Maria1226,1239,1245Jankowska-Kulawy Agnieszka1229,1233Janowski Miros³aw1249Jêœko Henryk1226Jiang Haiyan1245Juszczak Ma³gorzata1246

KKabziñska Dagmara1250Kachamakova-Trojanowska Neli1224,1237Kamiñska Bo¿ena1229Karaszewska Anna1247Katkowska Inna1245Ka�mierczak Anna1223,1230Klimczak-Jajor Edyta1243Kobryœ Ma³gorzata1224Kochañski Andrzej1250Kolasiewicz Wac³aw1251Kowalska Anna1231,1247Koz³owska Hanna1237,1244Kozubski Wojciech1241Ko�niewska Ewa1231Krajewska Dorota1244Kratochvil F. James III1236Kuter Katarzyna1251Ku�nicki Jacek1224,1237Ku�niewska Bo¿ena1224,1237

LLangfort Józef1239,1241Langner Ewa1246Lorenc-Koci El¿bieta1232

!£ukomska Barbara1244,1249

MMa³gorzata Ziemka-Na³êcz1252Matyja Ewa1247Matysiak Joanna1246Mehn Dora1253Michalik Rados³aw1231Micha³owska-Wender Gra¿yna1238Morelli Micaela1227Muller Christa E.1251

NNagañska Ewa1247

OObara-Michlewska Marta1245Orzechowski Arkadiusz1249Ossowska Krystyna1232

PPaj¹k Beata1248 - 1249Pawlak El¿bieta1249Piotrowski Piotr1231

RRadecka Urszula1240Rafa³owska Janina1231,1247Rajewski Andrzej1247Ronowska Anna1229,1233Rosmanowska K1231Rossi Francois1253Rudnicka Magdalena1242Ruiz Ana1253Rzeski Wojciech1246

SSienkiewicz B.1224Sikora Ewa1234Sikorska Jolanta1225Sinkiewicz-Darol Elena1250Skowroñska Marta1250S³owik Agnieszka1234Sobów Tomasz1224Soko³owska Anna1251Songin Martyna1248 - 1249Staszewski Jacek1239Steinborn Barbara1247Stolecka Anna1241Strosznajder Joanna B1249Strosznajder Joanna B.1223,1226,1230,1232,1239 - 1240,1245,1248Strosznajder Robert1226,1245Struzynska Lidia1243Sulejczak Dorota1243Sulkowski Grzegorz1243Sypecka Joanna1251Szutowicz Andrzej1229,1233Szymczak Patrycja1251

TTarsa Leila1236Tokarz-Kupczyk El¿bieta1238

WWalczak Katarzyna1246Walski Micha³1231Wanacka El¿bieta1244Wardas Jadwiga1251Wender Mieczys³aw1238Weso³owska Jowita1239Wojda Renata1231Wojda Urszula1224,1237Wójcik Luiza1252Wygl¹dalska-Jernas Halina1238

ZZab³ocka Barbara1237Zab³ocki Krzysztof 1235Zalewska Teresa1251 - 1252Zapa³a Ma³gorzata1251Zieliñska Magdalena1250Zychowicz Marzena1253

¯¯ekanowski Cezary1224

Adamczyk A 1223, 1230Albrecht J 1245, 1250Aschner M 1245Ba³kowiec A 1236Ba³kowiec-Iskra E 1236Bany-Laszewicz U 1243Barcikowska M 1223Œ1225Barczak A 1225Berdyñski M 1224Œ1225Berêsewicz M 1237Bernacki J 1241Bia³opiotrowicz E 1224, 1237Bielarczyk H 1229, 1233Bielecka A 1241Biernacka-£ukanty J 1238Bizon-Zygmañska D 1229Brodacki B 1239Bu¿añska L 1253C¹ka³a M 1226

Chabik G 1242Chalimoniuk M 1239Chodakowska-¯ebrowska M 1224Œ1225Cieœlik M 1226, 1239Colpo P 1253Czapski GA 1226, 1230, 1240, 1245Czernicki Z 1231Cz³onkowska A 1242Dezor M 1241Domañska-Janik K 1243Œ1244, 1249, 1253Domasiewicz A 1231Domek-£opaciñska K 1245Dorszewska J 1241Dyœ A 1233Dziubina A 1227Florczak A 1241

Florczak J 1241Florczak M 1241Friedman A 1227Frontczak-Baniewicz M 1231Gabryel B 1241Gabryelewicz T 1224Gadamski R 1231Gajkowska B 1226, 1248Œ1249Gêbarowska J 1247Golan M 1243Go³embiowska K 1227, 1251Górecki DC 1237Grieb P 1228, 1247Gromadzka G 1242Grygorowicz T 1243Grzywaczewska E 1247Gul-Hinc S 1233Habich A 1243, 1249Jab³oñska A 1244, 1249

Jacewicz M 1226, 1239, 1245Jankowska-Kulawy A 1229, 1233Janowski M 1249Jêœko H 1226Jiang H 1245Juszczak M 1246Kabziñska D 1250Kachamakova-Trojanowska N 1224, 1237Kamiñska B 1229Karaszewska A 1247Katkowska I 1245Ka�mierczak A 1223, 1230Klimczak-Jajor E 1243Kobryœ M 1224Kochañski A 1250Kolasiewicz W 1251Kowalska A 1231, 1247Koz³owska H 1237, 1244Kozubski W 1241Ko�niewska E 1231Krajewska D 1244Kratochvil FJ III 1236Kuter K 1251Ku�nicki J 1224, 1237Ku�niewska B 1224, 1237Langfort J 1239, 1241

Langner E 1246Lorenc-Koci E 1232£ukomska B 1244, 1249Matyja E 1247Matysiak J 1246Mehn D 1253Michalik R 1231Micha³owska-Wender G 1238Morelli M 1227Müller ChE 1251Nagañska E 1247Obara-Michlewska M 1245Orzechowski A 1249Ossowska K 1232Paj¹k B 1248Œ1249Pawlak E 1249Piotrowski P 1231Radecka U 1240Rafa³owska J 1231, 1247Rajewski A 1247Ronowska A 1229, 1233Rosmanowska K 1231Rossi F 1253Rudnicka M 1242Ruiz A 1253Rzeski W 1246Sienkiewicz B 1224Sikora E 1233Sikorska J 1225Sinkiewicz-Darol E 1250Skowroñska M 1250S³owik A 1234Sobów T 1224Soko³owska A 1251Songin M 1248Œ1249Staszewski J 1239Steinborn B 1247Stolecka A 1241Strosznajder R 1226, 1245Stru¿yñska L 1243Sulejczak D 1243Sulkowski G 1243Sypecka J 1251Szutowicz A 1229, 1233Szymczak P 1251Tarsa L 1236Tokarz-Kupczyk E 1238Walczak K 1246Walski M 1231Wanacka E 1244Wardas J 1251Wender M 1238Weso³owska J 1239Wojda R 1231Wojda U 1224, 1237Wójcik L 1252Wygl¹dalska-Jernas H 1238Zab³ocka B 1237

Zab³ocki K 1234Zalewska T 1251Œ1252Zapa³a M 1251Zieliñska M 1250Zychowicz M 1253¯ekanowski C 1224ContentsAAdamczyk Agata1225,1232Albrecht Jan1247,1252Aschner Michael1247


CC¹ka³a Magdalena1228Chabik Grzegorz1244Chalimoniuk Ma³gorzata1241Chodakowska Ma³gorzata1226Chodakowska-¯ebrowska Ma³gorzata1227Cieœlik Magdalena1228,1241Colpo Pascal1255Czapski Grzegorz A1242Czapski Grzegorz A.1228,1232,1247Czernicki Zbigniew1233Cz³onkowska Anna1244










NNagañska Ewa1249









spis tresci NAAdamczyk Agata1225,1232Albrecht Jan1247,1252Aschner Michael1247


CC¹ka³a Magdalena1228Chabik Grzegorz1244Chalimoniuk Ma³gorzata1241Chodakowska Ma³gorzata1226Chodakowska-¯ebrowska Ma³gorzata1227Cieœlik Magdalena1228,1241Colpo Pascal1255Czapski Grzegorz A1242Czapski Grzegorz A.1228,1232,1247Czernicki Zbigniew1233Cz³onkowska Anna1244










NNagañska Ewa1249









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New Cannabinoid CB1 receptors in rat medial prefrontal cortex are …rabbit.if-pan.krakow.pl/pjp/pdf/2009/6_1000.pdf · 2010. 1. 7. · CB1 receptor binding sites are enhanced in