J Neurosci. amphetamine treatment. Zero noticeable adjustments in bFGF-IR had been seen in the nucleus accumbens or dorsal striatum. bFGF-IR was discovered to be connected with astrocytes rather than with dopaminergic neurons. These results suggest that suffered improvement of astrocytic bFGF appearance in DA somatodendritic locations is a system whereby stimulant medications exert enduring results on midbrain DA function. We hypothesize that elevated glutamatergic activity elicited by amphetamine and various other stimulant drugs areas excessive demands over the working of DA neurons recruiting regulatory and neuroprotective procedures that result in enduring adjustments in DA neuron working and connectivity. Man Wistar rats weighing 300C350 gm at the start of the test served as topics. Rats had been housed independently in regular stainless dangling containers with usage of touch rat and drinking water chow, and had been maintained on the 12 hr light/dark routine. Pets received an overdose of sodium pentobarbital (120 mg/kg) and had been perfused transcardially with 200 ml of ice-cold PBS accompanied by 100 ml of the ice-cold alternative of 4% paraformaldehyde (w/v) and 15% picric acidity (v/v) in 0.1m phosphate buffer (PB, 6 pH.9). When the perfusion was finished, the brains were removed and put into the fixative solution at 4C overnight. Coronal sections, 50-m-thick, were cut on a vibratome and stored overnight in PB at 4C. bFGF immunohistochemistry was then detected according to the ABC method (Hsu et al., 1981). Briefly, free-floating tissue sections were incubated for 24 hr at 4C with the anti-bFGF antibody diluted to 1 1:500 with 0.3% Triton X-100 (Sigma) in PB and 1% normal horse serum (Vector Laboratories, Burlingame, CA). After incubation in the primary antibody, sections were rinsed three times in cold PB and incubated for 1 hr at room heat (RT) in a solution of rat adsorbed biotinylated anti-mouse antibody (Vector) diluted 1:200 with PB and 1% normal horse serum. After three 5 min washes in cold PB, sections were then incubated in an avidinChorseradish peroxidase complex (Vectastain Elite ABC Kit, Vector) for 30 min at RT, and rinsed again three times (5 min each) in cold PB. Sections were then incubated for 10 min at RT and under constant agitation in a solution of 0.05% 3,3-diaminobenzidine (DAB, Sigma) in PB. Without washing, the sections were then transferred to a DABCPB answer, pH 7.8, containing 0.01% H2O2, which catalyzed the reaction, and 8% NiCl2, which darkened the reaction product. Sections were incubated in this answer at RT and under constant agitation for 8 min. Special care was taken to maintain this time rigorously constant for all those sections processed within one single experiment and throughout the entire study. Three 10 min washes with cold PB terminated this final incubation. Double-labeling for bFGFCGFAP and for bFGFCTH was performed by processing the sections; first, for bFGF immunohistochemistry and then for either GFAP or TH immunohistochemistry. GFAP and TH immunolabeling was performed by using the ABC method. The anti-GFAP antibody was used at a concentration of 1 1:500 and the anti-TH at a concentration of 1 1:2000. For TH immunohistochemistry, sections were preincubated in 0.3% Triton X-100 PB and 1% normal goat serum for 1 hr at RT. For both GFAP and TH immunohistochemistry no NiCl2 was added to the DABCPBCH2O2 answer to obtain a lighter reaction product. Processed sections were wet-mounted PDGFRA onto gelatin-coated slides and were allowed to dry for at least 1 d before being hydrated in distilled water and gradually dehydrated throughout a series of graded alcohol solutions. Midbrain sections processed for bFGF-IR were lightly counterstained with 0.1% cresyl violet to demonstrate anatomical landmarks. Slides were cleared in xylene and coverslipped with Permount. Immunostained sections were examined under a Leica microscope (Leitz DMRB). For quantitative analysis of bFGF-IR, images of sampling areas of the VTA, SNc, NAcc shell, NAcc core, and dorsal region of the striatum (STR) were digitized using a computerized image-analysis system (NIH Image 1.6). Structure boundaries were defined according to the Paxinos and Watson (1997) stereotaxic atlas. Sampling areas of VTA and SNc were taken.[PMC free article] [PubMed] [Google Scholar] 44. SNc assessed 1 week after the amphetamine treatment. No changes in bFGF-IR were observed in the nucleus accumbens or dorsal striatum. bFGF-IR was found to be associated with astrocytes and not with dopaminergic neurons. These findings suggest that sustained enhancement of astrocytic bFGF expression in DA somatodendritic regions is a mechanism whereby stimulant drugs exert enduring effects on midbrain DA function. We hypothesize that increased glutamatergic activity elicited by amphetamine and other stimulant drugs places excessive demands around the functioning of DA neurons recruiting regulatory and neuroprotective processes that lead to enduring changes in DA neuron functioning and connectivity. Male Wistar rats weighing 300C350 gm at the beginning of the experiment served as subjects. Rats were housed individually in standard stainless steel hanging boxes with access to tap water and rat chow, and were maintained on a 12 hr light/dark cycle. Animals received an overdose of sodium pentobarbital (120 mg/kg) and were perfused transcardially with 200 ml of ice-cold PBS followed by 100 ml of an ice-cold answer of 4% paraformaldehyde (w/v) and 15% picric acid (v/v) in 0.1m phosphate buffer (PB, pH 6.9). When the perfusion was completed, the brains were removed and placed overnight in the fixative answer at 4C. Coronal sections, 50-m-thick, were cut on a vibratome and stored overnight in PB at 4C. bFGF immunohistochemistry was then detected according to the ABC method (Hsu et al., 1981). Briefly, free-floating tissue sections were incubated for 24 hr at 4C with the anti-bFGF antibody diluted to 1 1:500 with 0.3% Triton X-100 (Sigma) in PB and 1% normal horse serum (Vector Laboratories, Burlingame, CA). After incubation in the primary antibody, sections were rinsed three times in cold PB and incubated for 1 hr at room temperature (RT) in a solution of rat adsorbed biotinylated anti-mouse antibody (Vector) diluted 1:200 with PB and 1% normal horse serum. After three 5 min washes in cold PB, sections were then incubated in an avidinChorseradish peroxidase complex (Vectastain Elite ABC Kit, Vector) for 30 min at RT, and rinsed again three times (5 min each) in cold PB. Sections were then incubated for 10 min at RT and under constant agitation in a solution of 0.05% 3,3-diaminobenzidine (DAB, Sigma) in PB. Without washing, the sections were then transferred to a DABCPB solution, pH 7.8, containing 0.01% H2O2, which catalyzed the reaction, and 8% NiCl2, which darkened the reaction product. Sections were incubated in this solution at RT and under constant agitation for 8 min. Special care was taken to maintain this time rigorously constant for all sections processed within one single experiment and throughout the entire study. Three 10 min washes with cold PB terminated this final incubation. Double-labeling for bFGFCGFAP and for bFGFCTH was performed by processing the sections; first, for bFGF immunohistochemistry and then for either GFAP or TH immunohistochemistry. GFAP and TH immunolabeling was performed by using the ABC method. The anti-GFAP antibody was used at a concentration of 1 1:500 and the anti-TH at a concentration of 1 1:2000. For TH immunohistochemistry, sections were preincubated in 0.3% Triton X-100 PB and 1% normal goat serum for 1 hr at RT. For both GFAP and TH immunohistochemistry no NiCl2 was added to the DABCPBCH2O2 solution to obtain a lighter reaction product. Processed sections were wet-mounted onto gelatin-coated slides and were allowed to dry for at least 1 d before being hydrated in distilled water and gradually dehydrated throughout a series ERK5-IN-1 of graded alcohol solutions. Midbrain sections processed for bFGF-IR were lightly counterstained with 0.1% cresyl violet to demonstrate anatomical landmarks. Slides were cleared in xylene and coverslipped with Permount. Immunostained sections were examined under a Leica microscope (Leitz DMRB). For quantitative analysis of bFGF-IR, images of sampling areas of the VTA, SNc, NAcc shell, NAcc core, and dorsal region of the striatum (STR) were digitized using a computerized image-analysis system (NIH Image 1.6). Structure boundaries were defined according to the Paxinos and Watson (1997) stereotaxic atlas. Sampling areas of VTA and SNc were taken from sections corresponding to plates 38 and 39, and sampling areas of NAcc shell, NAcc core, and STR were taken from sections corresponding to plates 11, 12, and 13. For each brain, three images from each structure, taken from three different sections, were digitized and were assigned code names. The number of bFGF-positive cells in each image was then counted by two individuals who were blind to the code assignment. The.at 9:00 A.M. in DA somatodendritic regions is a mechanism whereby stimulant drugs exert enduring effects on midbrain DA function. We hypothesize that increased glutamatergic activity elicited by amphetamine and other stimulant drugs places excessive demands on the functioning of DA neurons recruiting regulatory and neuroprotective processes that lead to enduring changes in DA neuron functioning and connectivity. Male Wistar rats weighing 300C350 gm at the beginning of the experiment served as subjects. Rats were housed individually in standard stainless steel hanging boxes with access to tap water and rat chow, and were maintained on a 12 hr light/dark cycle. Animals received an overdose of sodium pentobarbital (120 mg/kg) and were perfused transcardially with 200 ml of ice-cold PBS followed by 100 ml of an ice-cold remedy of 4% paraformaldehyde (w/v) and 15% picric acid (v/v) in 0.1m phosphate buffer (PB, pH 6.9). When the perfusion was completed, the brains were removed and placed immediately in the fixative remedy at 4C. Coronal sections, 50-m-thick, were cut on a vibratome and stored over night in PB at 4C. bFGF immunohistochemistry was then detected according to the ABC method (Hsu et al., 1981). Briefly, free-floating tissue sections were incubated for 24 hr at 4C with the anti-bFGF antibody diluted to 1 1:500 with 0.3% Triton X-100 (Sigma) in PB and 1% normal horse serum (Vector Laboratories, Burlingame, CA). After incubation in the primary antibody, sections were rinsed three times in chilly PB and incubated for 1 hr at space temp (RT) in a solution of rat adsorbed biotinylated anti-mouse antibody (Vector) diluted 1:200 with PB and 1% normal horse serum. After three 5 min washes in chilly PB, sections were then incubated in an avidinChorseradish peroxidase complex (Vectastain Elite ABC Kit, Vector) for 30 min at RT, and rinsed again three times (5 min each) in chilly PB. Sections were then incubated for 10 min at RT and under constant agitation in a solution of 0.05% 3,3-diaminobenzidine (DAB, Sigma) in PB. Without washing, the sections were then transferred to a DABCPB remedy, pH 7.8, containing 0.01% H2O2, which catalyzed the reaction, and 8% NiCl2, which darkened the reaction product. Sections were incubated with this remedy at RT and under constant agitation for 8 min. Unique care was taken to maintain this time rigorously constant for those sections processed within one single experiment and throughout the entire study. Three 10 min washes with chilly PB terminated this final incubation. Double-labeling for bFGFCGFAP and for bFGFCTH was performed by processing the sections; 1st, for bFGF immunohistochemistry and then for either GFAP or TH immunohistochemistry. GFAP and TH immunolabeling was performed by using the ABC method. The anti-GFAP antibody was used at a concentration of 1 1:500 and the anti-TH at a concentration of 1 1:2000. For TH immunohistochemistry, sections were preincubated in 0.3% Triton X-100 PB and 1% normal goat serum for 1 hr at RT. For both GFAP and TH immunohistochemistry no NiCl2 was added to the DABCPBCH2O2 remedy to obtain a lighter reaction product. Processed sections were wet-mounted onto gelatin-coated slides and were allowed to dry for at least 1 d before becoming hydrated in distilled water and gradually dehydrated throughout a series of graded alcohol solutions. Midbrain sections processed for bFGF-IR were lightly counterstained with 0.1% cresyl violet to demonstrate anatomical landmarks. Slides were cleared in xylene and coverslipped with Permount. Immunostained sections were examined under a Leica microscope (Leitz DMRB). For quantitative analysis of bFGF-IR, images of sampling areas of the VTA, SNc, NAcc shell, NAcc core, and dorsal region of the striatum (STR) were digitized using a computerized image-analysis system (NIH Image 1.6). Structure boundaries were defined according to the Paxinos and Watson (1997) stereotaxic atlas. Sampling areas of VTA and SNc were taken from sections related to plates 38 and 39, and sampling areas of NAcc shell, NAcc core, and STR were taken from sections related to plates 11, 12, and 13. For each brain, three images from each structure, taken from three different sections, were digitized and were assigned code titles. The number of bFGF-positive.In the preexposure phase, different groups of rats were given injections of either AMPH (3 mg/kg, i.p.) or saline (SAL) in the morning (10:00 A.M.) on days 1, 3, and 5, in the colony space. effect of the glutamate antagonist, kynurenic acid (KYN), on amphetamine-induced bFGF-IR. Coadministration of KYN prevented the raises in bFGF-IR in both VTA and SNc assessed 1 week after the amphetamine treatment. No changes in bFGF-IR were observed in the nucleus accumbens or dorsal striatum. bFGF-IR was found to be associated with astrocytes and not with dopaminergic neurons. These findings suggest that sustained enhancement of astrocytic bFGF manifestation in DA somatodendritic areas is a mechanism whereby stimulant medicines exert enduring effects on midbrain DA function. We hypothesize that improved glutamatergic activity elicited by amphetamine and additional stimulant drugs locations excessive demands within the functioning of DA neurons recruiting regulatory and neuroprotective processes that lead to enduring changes in DA neuron functioning and connectivity. Male Wistar rats weighing 300C350 gm at the beginning of the experiment served as subjects. Rats were housed separately in standard stainless steel hanging boxes with access to tap water and rat chow, and were maintained on a 12 hr light/dark cycle. Animals received an overdose of sodium pentobarbital (120 mg/kg) and were perfused transcardially with 200 ml of ice-cold PBS followed by 100 ml of an ice-cold answer of 4% paraformaldehyde (w/v) and 15% picric acid (v/v) in 0.1m phosphate buffer (PB, pH 6.9). When the perfusion ERK5-IN-1 was completed, the brains were removed and placed immediately in the fixative answer at 4C. Coronal sections, 50-m-thick, were cut on a vibratome and stored overnight in PB at 4C. bFGF immunohistochemistry was then detected according to the ABC method (Hsu et al., 1981). Briefly, free-floating tissue sections were incubated for 24 hr at 4C with the anti-bFGF antibody diluted to 1 1:500 with 0.3% Triton X-100 (Sigma) in PB and 1% normal horse serum (Vector Laboratories, Burlingame, CA). After incubation in the primary antibody, sections were rinsed three times in chilly PB and incubated for 1 hr at room heat (RT) in a solution of rat adsorbed biotinylated anti-mouse antibody (Vector) diluted 1:200 with PB and 1% normal horse serum. After three 5 min washes in chilly PB, sections were then incubated in an avidinChorseradish peroxidase complex (Vectastain Elite ABC Kit, Vector) for 30 min at RT, and rinsed again three times (5 min each) in chilly PB. Sections were then incubated for 10 min at RT and under constant agitation in a solution of 0.05% 3,3-diaminobenzidine (DAB, Sigma) in PB. Without washing, the sections were then transferred to a DABCPB answer, pH 7.8, containing 0.01% H2O2, which catalyzed the reaction, and 8% NiCl2, which darkened the reaction product. Sections were incubated in this answer at RT and under constant agitation for 8 min. Special care was taken to maintain this time rigorously constant for all those sections processed within one single experiment and throughout the entire study. Three 10 min washes with cold PB terminated this final incubation. Double-labeling for bFGFCGFAP and for bFGFCTH was performed by processing the sections; first, for bFGF immunohistochemistry and then for either GFAP or TH immunohistochemistry. GFAP and TH immunolabeling was performed by using the ABC method. The anti-GFAP antibody was used at a concentration of 1 1:500 and the anti-TH at a concentration of 1 1:2000. For TH immunohistochemistry, sections were preincubated in 0.3% Triton X-100 PB and 1% normal goat serum for 1 hr at RT. ERK5-IN-1 For both GFAP and TH immunohistochemistry no NiCl2 was added to the DABCPBCH2O2 answer to obtain a lighter reaction product. Processed sections were wet-mounted onto gelatin-coated slides and were allowed to dry for at least 1 d before being hydrated in distilled water and gradually dehydrated throughout a series of graded alcohol solutions. Midbrain sections processed for bFGF-IR were lightly counterstained with 0.1% cresyl violet to demonstrate anatomical landmarks. Slides were cleared in xylene and coverslipped with Permount. Immunostained sections were examined under a Leica microscope (Leitz DMRB). For quantitative analysis of bFGF-IR, images of sampling areas of the VTA, SNc, NAcc shell, NAcc core, and dorsal region of the striatum (STR) were digitized using a computerized image-analysis system (NIH Image 1.6). Structure boundaries were defined according to the Paxinos and Watson (1997) stereotaxic atlas. Sampling areas of VTA and SNc were taken from sections corresponding to plates 38 and 39, and sampling areas.Mattson MP, Kumar K, Cheng B, Wang H, Michaelis EK. expression in DA somatodendritic regions is a mechanism whereby stimulant drugs exert enduring effects on midbrain DA function. We hypothesize that increased glutamatergic activity elicited by amphetamine and other stimulant drugs places excessive demands around the functioning of DA neurons recruiting regulatory and neuroprotective processes that lead to enduring changes in DA neuron functioning and connectivity. Male Wistar rats weighing 300C350 gm at the beginning of the experiment served as subjects. Rats were housed individually in standard stainless steel hanging boxes with access to tap water and rat chow, and were maintained on a 12 hr light/dark cycle. Animals received an overdose of sodium pentobarbital (120 mg/kg) and were perfused transcardially with 200 ml of ice-cold PBS followed by 100 ml of an ice-cold answer of 4% paraformaldehyde (w/v) and 15% picric acid (v/v) in 0.1m phosphate buffer (PB, pH 6.9). When the perfusion was completed, the brains were removed and placed immediately in the fixative answer at 4C. Coronal sections, 50-m-thick, were cut on the vibratome and kept over night in PB at 4C. bFGF immunohistochemistry was after that detected based on the ABC technique (Hsu et al., 1981). Quickly, free-floating tissue areas had been incubated for 24 hr at 4C using the anti-bFGF antibody diluted to at least one 1:500 with 0.3% Triton X-100 (Sigma) in PB and 1% normal equine serum (Vector Laboratories, Burlingame, CA). After incubation in the principal antibody, areas had been rinsed 3 x in cool PB and incubated for 1 hr at space temperatures (RT) in a remedy of rat adsorbed biotinylated anti-mouse antibody (Vector) diluted 1:200 with PB and 1% regular equine serum. After three 5 min washes in cool PB, areas had been then incubated within an avidinChorseradish peroxidase complicated (Vectastain Top notch ABC Package, Vector) for 30 min at RT, and rinsed once again 3 x (5 min each) in cool PB. Sections had been after that incubated for 10 min at RT and under continuous agitation in a remedy of 0.05% 3,3-diaminobenzidine (DAB, Sigma) in PB. Without cleaning, the areas had been then used in a DABCPB option, pH 7.8, containing 0.01% H2O2, which catalyzed the reaction, and 8% NiCl2, which darkened the reaction item. Sections had been incubated with this option at RT and under continuous agitation for 8 min. Unique care was taken up to maintain this time around rigorously constant for many areas processed within a unitary test and through the entire entire research. Three 10 min washes with chilly PB terminated this last incubation. Double-labeling for bFGFCGFAP as well as for bFGFCTH was performed by digesting the areas; 1st, for bFGF immunohistochemistry and for either GFAP or TH immunohistochemistry. GFAP and TH immunolabeling was performed utilizing the ABC technique. The anti-GFAP antibody was utilized at a focus of just one 1:500 as well as the anti-TH at a focus of just one 1:2000. For TH immunohistochemistry, areas had been preincubated in 0.3% Triton X-100 PB and 1% normal goat serum for 1 hr at RT. For both GFAP and TH immunohistochemistry zero NiCl2 was put into the DABCPBCH2O2 option to secure a lighter response product. Processed areas had been wet-mounted onto gelatin-coated slides and had been allowed to dried out for at least 1 d before becoming hydrated in distilled drinking water and steadily dehydrated within a group of graded alcoholic beverages solutions. Midbrain areas prepared for bFGF-IR had been gently counterstained with 0.1% cresyl violet to show anatomical landmarks. Slides had been cleared in xylene and coverslipped with Permount. Immunostained areas had been analyzed under a Leica microscope (Leitz DMRB). For quantitative evaluation of bFGF-IR, pictures of sampling regions of the VTA, SNc, NAcc shell, NAcc primary, and dorsal area from the striatum (STR) had been digitized utilizing a computerized image-analysis program (NIH Picture 1.6). Framework boundaries had been defined based on the Paxinos and Watson (1997) stereotaxic atlas. Sampling regions of VTA and SNc had been taken from areas related to plates 38 and 39, and sampling regions of NAcc shell, NAcc primary, and STR had been taken from areas related to plates 11, 12, and 13. For every brain, three pictures from each framework, extracted from three different areas, had been digitized and had been assigned code titles. The amount of bFGF-positive.