Brain Research B&fin.
Vol. 76. pp. 185-193. p Perpamon Press plc. 1991. Printed in the I1.S.A.
Oihl~9?30/91 $3.0(1 + .oo
Distribution of Alpha-l and Alpha-2 Binding Sites in the Rat Locus Coeruleus G, CHAMBA,’
D. WEISSMANN,
C. ROUSSET,
B. RENAUD
AND J. F, PUJOL
~~oratoire
de Neur~ph~rm~~ologie MQ~~cula~re CURS-UCB ~JMR IO5 lnstitut des Sciences Pharmaceutiques et Biofogiques Fuculte’ de Pharmncie, 8 Avenue Rockefeller, 69313 Lyon Cedex 08 and UFR A. Carrel, rue G. Paradin, 69008 Lyon, France Received
Iti January
1990
CHAMBA,
G., D. WEISSMANN, C. ROUSSET. B. RENAUD AND J. F. PUJOL. ~i.~r~ib~tio~ ~~al~h~l-I and @w-Z ~jndi~‘~ coeruleus. BRAIN RES BULL 26(2) 185-193, 1991. -Precise anatomical distribution of alpha- I and alpha-2 adrenergic binding sites has been investigated in the rat locus coeruleus (LC) using quantitative radioautography of brain sections incubated with “H-prazosin or ‘H-idazoxan. Distribution patterns of 3H-prazosin (alpha-l sites) and 3H-idazoxan (alpha-2 sites) were heterogeneous and different along a postero-anterior axis m the LC. Comparison between distribution of alpha-2 binding sites and noradrenergic (NA) cellular density suggests that at least a fraction of these sites might be localized on NA perikarya or dendrites in this structure. Quantitative estimations of the binding parameters along this postero-anterior axis in the LC have revealed that the heterogeneous distributions of alpha-l and alpha-2 binding sites are due not only to variations in the maximal densities of sites but also to variations in the affinities of these sites for their respective ligand.
sites it: the rat bcus
Rat locus coeruleus AlphaQuantitative radioautography
I binding sites
Alpha-2 binding sites
THE locus coeruleus (LC) is the densest group of noradrenergic neurons in the brainstem. This group plays a major role in several neurobioIogica1 regulations including control of blood pressure (21.22,3.5,49,57,60), control of neuroend~rine secretions (23) and modulation of behavioral mechanisms (7, 19, 24, 33). The functional importance of the noradrenergic (NA) neurons of the LC may be a result of the large innervation of these neurons. Thus. the LC receives inputs from NA (48,61), adrenergic (6,29), cholinergic (34) and serotonergic (37,50) neurons. This dense innervation explains that many different types of receptors have been identified in this region: muscarinic (671, serotonergic (661, peptidergic (471 and alpha and beta-adrenergic (64,72) receptors. Physiological and anatomical studies have shown that both alpha1 and alpha-2 binding sites are present in the LC (2, 9, 11, 18. 31, 32, 64, 65, 69, 72) but no precise localization of these sites has been performed. Such a precise distribution of these binding sites may be useful to know better the functional organization of afferents to LC and could allow to understand the complexity of the physiological effects of these NA neurons. The aim of this study was to compare the postero-anterior distrjbution of alpha-l and alpha-2 adrenergic binding sites within the rat LC using quantitative radioautography. The rep~ition of each type of sites was also compared with the distribution of NA cells in this region.
Prazosin
Idazoxan
METHOD
Tissue Preparatinn Male rats (Sprague-Dawley, 180-200 g, lffa-Credo, France) were sacrificed by decapitation. Their brains were rapidly removed, frozen by immersion in refrigerated isopentane and stored at - 80°C until dissection. Twenty-pm serial frontal sections were cut with a cryostat microtome (Reichert) at - 15°C. Sections were mounted onto gelatin-coated coverslips and stored at - 20°C for 24 h, then at -80°C until binding studies.
For labeling of ‘H-prazosin (PRA) and ‘H-idazoxan (IDA) binding sites, serial sections were preincubated for 15 min at 12S’C in a Tris HCl buffer (170 mM, pH 7.4). then incubated for 2 h at +4”C in the same buffer containing 0.54 nM “H-PRA (Amersham France 903 GBqimmol) or 0.94 nM ‘H-IDA (Amersham France 1.48 TBq/mmol). Nonspecific binding was estimated on consecutive sections by incubation in the presence of 10 ’ M phentolamine. After incubation, sections were washed three times in Tris HCl buffer at +4”C (10 min for PRA and 1 min for IDA), then washed in H,O at +4”C and dried. Binding studies were performed at 200-pm intervals along an arbitrary postero-anterior axis of the LC.
‘Requests for reprints should be addressed to Genevikve Chamba, Laboratoire de Neuropharmacologie, ologiques. FacultC de Pharmacie. 8 Avenue Rockefeller, 69373 Lyon Cedex OX, France.
Institut des Sciences Pharmaceutiques
et Bi-
ALPHA-ADRENERGIC SITES IN THE RAT LC
For equilibrium analysis of ‘H-PRA and 3H-IDA binding, adjacent sections were incubated in 6 concentrations of -‘H-PRA (0.35, 0.41. 0.70. 0.82. 1.24 and 5.20 nM) or 6 concentrations of ‘H-IDA (0.30. 0.47, 0.71, 1.4C. 5.20 and 14.50 nM). Nonspecific binding was estimated on consecutive sections in the presence of 10 -’ M phentolamine. Saturation studies were performed at 520.pm intervals along the postero-anterior axis of the LC. Anutomical Chilracterization Every 200 km. two 20-pm sections adjacent to those utilized for binding studies were used for anatomical characterization. The first section was analyzed after a cresyl-violet staining. The other section was fixed in a solution of paraformaldehyde 4% (w/v) in PBS (NaHPO, 15 mM, NaCl 150 mM, pH 7.4). After rinsing in PBS pH 7.4. it was treated for immunohistochemical localization of tyrosine hydroxylase (TH)-containing neurons with antibodies against TH (Jacques Boy) according to the method of Stemberger et al, (58). Cells labeled with antibodies against TH were examined with a microscope (Reichert) coupled with a computerized image analysis system. A software (Grain, P. Platzer-CNRS) allowed the count of the number of cells by selectioning a threshold of optical density and the dimension of the soma in a circumscribed region. The number of TH-immunoreactive (IR) cells was determined on each section and was corrected by the Abercrombie equation (1). The surface of the LC was determined by the area delimited by the TH-IR cells. The volume of the LC was estimated by the product of the surface of the structure by the chosen interval (200 km). NA cellular density was expressed as number of cells/mm3. Procedure fiw Quantitative Autoradiograph) Dried sections were apposed against a 3H-sensitive Ultrofilm (Amersham) and exposed in Kodak X-omatic cassettes for three months. Radioautographic “H-microscales (RPA 501 and 502, Amersham) were used for calibration of radioactive determination. After development of the films, radioautograms were analyzed by densitometry, using a computerized image analysis system (lmstar). For each region examined, the initial slice containing the origin of the structure of interest (posterior limit of the postero-anterior axis for aligning the serial sections containing the structure) was selected for each brain by referring to the classical anatomical parameters observed on the adjacent cresyl-violet stained slice. On each section. radioligand binding was determined by the mean of the density observed for the left and right LC regions, the LC regions being selected by referring to slices used for anatomical studies. Results were expressed as estimated bound ligand in femtomoles per milligram of tissue (fmohmg t) *SE (5 or 6 brains). Statistical analysis was performed using the Student’s f-test for unpaired data after verification of variances homogeneity by the Fisher-Snedecor’s F-test. Saturation curves were constructed from untransformed radioautograph data and the best fit of the hyperbolic function was made using the least squares method (RSl Software, BBN). The
187
program determines the optimal values for the apparent dissociation constant (K,) and the maximal binding (B,,,)? standard error for each saturation curve. RESULTS
‘H-PRA (0.54 nM) and 3H-1DA (0.94 nM) binding have been performed on 20-wrn frontal sections every 200 p,rn along a postero-anterior axis in the rat LC. In these experimental conditions, nonspecific binding was undetectable and nondifferent from the background of the film. Qualitative Rudioautography Radioautograms illustrated in Fig. 1 show the labeling of alpha-l (a, b) and alpha-2 (c, d) binding sites in the region of the LC. Sections a and b represent ‘H-PRA labeling at two different levels in the LC (respectively medial level and anterior level). Sections c and d represent ‘H-IDA labeling at two different levels in the LC (respectively posterior level and anterior level). Reticular pontine formation presents a high density- of specific ‘HPRA labeling. especially in the regions of the LC. dorsal raphe nucleus and lateral parabrachial nucleus. Specific ‘H-IDA labeling appears to be localized in the periventricular grey matter. especially in the LC and in the lateral parabrachial nucleus. Distribution of’ Binding Sites Analysis of the density of ‘H-PRA and ‘H-IDA binding sites on 8 sections (one section every 200 km) revealed heterogeneous distribution patterns along an arbitrary postero-anterior axis in the LC (Fig. 2). Thus, the density of ‘H-PRA binding sites (46.1 t 3.7 fmolimg t for the whole LC at 0.54 nM of ‘H-PRA) was significantly lower in the posterior region of the LC than in the anterior region (41% of ‘H-PRA labeling in the posterior half and 59% in the anterior half of the LC, p
Vol. 76. pp. 185-193. p Perpamon Press plc. 1991. Printed in the I1.S.A.
Oihl~9?30/91 $3.0(1 + .oo
Distribution of Alpha-l and Alpha-2 Binding Sites in the Rat Locus Coeruleus G, CHAMBA,’
D. WEISSMANN,
C. ROUSSET,
B. RENAUD
AND J. F, PUJOL
~~oratoire
de Neur~ph~rm~~ologie MQ~~cula~re CURS-UCB ~JMR IO5 lnstitut des Sciences Pharmaceutiques et Biofogiques Fuculte’ de Pharmncie, 8 Avenue Rockefeller, 69313 Lyon Cedex 08 and UFR A. Carrel, rue G. Paradin, 69008 Lyon, France Received
Iti January
1990
CHAMBA,
G., D. WEISSMANN, C. ROUSSET. B. RENAUD AND J. F. PUJOL. ~i.~r~ib~tio~ ~~al~h~l-I and @w-Z ~jndi~‘~ coeruleus. BRAIN RES BULL 26(2) 185-193, 1991. -Precise anatomical distribution of alpha- I and alpha-2 adrenergic binding sites has been investigated in the rat locus coeruleus (LC) using quantitative radioautography of brain sections incubated with “H-prazosin or ‘H-idazoxan. Distribution patterns of 3H-prazosin (alpha-l sites) and 3H-idazoxan (alpha-2 sites) were heterogeneous and different along a postero-anterior axis m the LC. Comparison between distribution of alpha-2 binding sites and noradrenergic (NA) cellular density suggests that at least a fraction of these sites might be localized on NA perikarya or dendrites in this structure. Quantitative estimations of the binding parameters along this postero-anterior axis in the LC have revealed that the heterogeneous distributions of alpha-l and alpha-2 binding sites are due not only to variations in the maximal densities of sites but also to variations in the affinities of these sites for their respective ligand.
sites it: the rat bcus
Rat locus coeruleus AlphaQuantitative radioautography
I binding sites
Alpha-2 binding sites
THE locus coeruleus (LC) is the densest group of noradrenergic neurons in the brainstem. This group plays a major role in several neurobioIogica1 regulations including control of blood pressure (21.22,3.5,49,57,60), control of neuroend~rine secretions (23) and modulation of behavioral mechanisms (7, 19, 24, 33). The functional importance of the noradrenergic (NA) neurons of the LC may be a result of the large innervation of these neurons. Thus. the LC receives inputs from NA (48,61), adrenergic (6,29), cholinergic (34) and serotonergic (37,50) neurons. This dense innervation explains that many different types of receptors have been identified in this region: muscarinic (671, serotonergic (661, peptidergic (471 and alpha and beta-adrenergic (64,72) receptors. Physiological and anatomical studies have shown that both alpha1 and alpha-2 binding sites are present in the LC (2, 9, 11, 18. 31, 32, 64, 65, 69, 72) but no precise localization of these sites has been performed. Such a precise distribution of these binding sites may be useful to know better the functional organization of afferents to LC and could allow to understand the complexity of the physiological effects of these NA neurons. The aim of this study was to compare the postero-anterior distrjbution of alpha-l and alpha-2 adrenergic binding sites within the rat LC using quantitative radioautography. The rep~ition of each type of sites was also compared with the distribution of NA cells in this region.
Prazosin
Idazoxan
METHOD
Tissue Preparatinn Male rats (Sprague-Dawley, 180-200 g, lffa-Credo, France) were sacrificed by decapitation. Their brains were rapidly removed, frozen by immersion in refrigerated isopentane and stored at - 80°C until dissection. Twenty-pm serial frontal sections were cut with a cryostat microtome (Reichert) at - 15°C. Sections were mounted onto gelatin-coated coverslips and stored at - 20°C for 24 h, then at -80°C until binding studies.
For labeling of ‘H-prazosin (PRA) and ‘H-idazoxan (IDA) binding sites, serial sections were preincubated for 15 min at 12S’C in a Tris HCl buffer (170 mM, pH 7.4). then incubated for 2 h at +4”C in the same buffer containing 0.54 nM “H-PRA (Amersham France 903 GBqimmol) or 0.94 nM ‘H-IDA (Amersham France 1.48 TBq/mmol). Nonspecific binding was estimated on consecutive sections by incubation in the presence of 10 ’ M phentolamine. After incubation, sections were washed three times in Tris HCl buffer at +4”C (10 min for PRA and 1 min for IDA), then washed in H,O at +4”C and dried. Binding studies were performed at 200-pm intervals along an arbitrary postero-anterior axis of the LC.
‘Requests for reprints should be addressed to Genevikve Chamba, Laboratoire de Neuropharmacologie, ologiques. FacultC de Pharmacie. 8 Avenue Rockefeller, 69373 Lyon Cedex OX, France.
Institut des Sciences Pharmaceutiques
et Bi-
ALPHA-ADRENERGIC SITES IN THE RAT LC
For equilibrium analysis of ‘H-PRA and 3H-IDA binding, adjacent sections were incubated in 6 concentrations of -‘H-PRA (0.35, 0.41. 0.70. 0.82. 1.24 and 5.20 nM) or 6 concentrations of ‘H-IDA (0.30. 0.47, 0.71, 1.4C. 5.20 and 14.50 nM). Nonspecific binding was estimated on consecutive sections in the presence of 10 -’ M phentolamine. Saturation studies were performed at 520.pm intervals along the postero-anterior axis of the LC. Anutomical Chilracterization Every 200 km. two 20-pm sections adjacent to those utilized for binding studies were used for anatomical characterization. The first section was analyzed after a cresyl-violet staining. The other section was fixed in a solution of paraformaldehyde 4% (w/v) in PBS (NaHPO, 15 mM, NaCl 150 mM, pH 7.4). After rinsing in PBS pH 7.4. it was treated for immunohistochemical localization of tyrosine hydroxylase (TH)-containing neurons with antibodies against TH (Jacques Boy) according to the method of Stemberger et al, (58). Cells labeled with antibodies against TH were examined with a microscope (Reichert) coupled with a computerized image analysis system. A software (Grain, P. Platzer-CNRS) allowed the count of the number of cells by selectioning a threshold of optical density and the dimension of the soma in a circumscribed region. The number of TH-immunoreactive (IR) cells was determined on each section and was corrected by the Abercrombie equation (1). The surface of the LC was determined by the area delimited by the TH-IR cells. The volume of the LC was estimated by the product of the surface of the structure by the chosen interval (200 km). NA cellular density was expressed as number of cells/mm3. Procedure fiw Quantitative Autoradiograph) Dried sections were apposed against a 3H-sensitive Ultrofilm (Amersham) and exposed in Kodak X-omatic cassettes for three months. Radioautographic “H-microscales (RPA 501 and 502, Amersham) were used for calibration of radioactive determination. After development of the films, radioautograms were analyzed by densitometry, using a computerized image analysis system (lmstar). For each region examined, the initial slice containing the origin of the structure of interest (posterior limit of the postero-anterior axis for aligning the serial sections containing the structure) was selected for each brain by referring to the classical anatomical parameters observed on the adjacent cresyl-violet stained slice. On each section. radioligand binding was determined by the mean of the density observed for the left and right LC regions, the LC regions being selected by referring to slices used for anatomical studies. Results were expressed as estimated bound ligand in femtomoles per milligram of tissue (fmohmg t) *SE (5 or 6 brains). Statistical analysis was performed using the Student’s f-test for unpaired data after verification of variances homogeneity by the Fisher-Snedecor’s F-test. Saturation curves were constructed from untransformed radioautograph data and the best fit of the hyperbolic function was made using the least squares method (RSl Software, BBN). The
187
program determines the optimal values for the apparent dissociation constant (K,) and the maximal binding (B,,,)? standard error for each saturation curve. RESULTS
‘H-PRA (0.54 nM) and 3H-1DA (0.94 nM) binding have been performed on 20-wrn frontal sections every 200 p,rn along a postero-anterior axis in the rat LC. In these experimental conditions, nonspecific binding was undetectable and nondifferent from the background of the film. Qualitative Rudioautography Radioautograms illustrated in Fig. 1 show the labeling of alpha-l (a, b) and alpha-2 (c, d) binding sites in the region of the LC. Sections a and b represent ‘H-PRA labeling at two different levels in the LC (respectively medial level and anterior level). Sections c and d represent ‘H-IDA labeling at two different levels in the LC (respectively posterior level and anterior level). Reticular pontine formation presents a high density- of specific ‘HPRA labeling. especially in the regions of the LC. dorsal raphe nucleus and lateral parabrachial nucleus. Specific ‘H-IDA labeling appears to be localized in the periventricular grey matter. especially in the LC and in the lateral parabrachial nucleus. Distribution of’ Binding Sites Analysis of the density of ‘H-PRA and ‘H-IDA binding sites on 8 sections (one section every 200 km) revealed heterogeneous distribution patterns along an arbitrary postero-anterior axis in the LC (Fig. 2). Thus, the density of ‘H-PRA binding sites (46.1 t 3.7 fmolimg t for the whole LC at 0.54 nM of ‘H-PRA) was significantly lower in the posterior region of the LC than in the anterior region (41% of ‘H-PRA labeling in the posterior half and 59% in the anterior half of the LC, p
