Rapid, transient, and dose-dependent expression of hsp70 messenger RNA in the rat brain after morphine treatment

Abstract

Induction of Hsp70 in the brain has been reported after intake of drugs of abuse like amphetamine and lysergic acid diethylamide. In this investigation, gene expression of Hsp70 and other heat shock genes in the rat brain was studied in response to morphine. Twenty milligrams per kilogram morphine intraperitoneally resulted in a marked induction of Hsp70 messenger RNA (mRNA) expression in the frontal cortex with a maximum increase of 13.2-fold after 2 hours. A moderate increase of Hsp27 mRNA expression (6.7-fold) could be observed after 4 hours, whereas mRNA expression of Hsp90 and of the constitutive Hsc70 did not exceed a mean factor of 1.8-fold during the 24 hours interval. The increase in Hsp70 mRNA was dose dependent, showing a significant elevation after doses ranging from 10 to 50 mg/kg morphine. In situ hybridization revealed enhanced Hsp70 mRNA expression mainly in cortical areas, in the hippocampus, in the paraventricular and supraoptic nuclei of the hypothalamus, in the locus coeruleus, as well in the pineal body. The double in situ hybridization technique revealed increased Hsp70 mRNA expression mainly in VGLUT1-positive neurons and to a lesser extent in olig1-positive oligodendroglia. Immunohistochemistry revealed a marked increase of Hsp70 protein in neuronal cells and blood vessels after 12 hours. In contrast to animal experiments, morphine did not increase Hsp70 mRNA expression in vitro in micro-opioid receptor (MOR1)-expressing human embryonic kidney 293 cells, suggesting no direct MOR1-mediated cellular effect. To exclude a body temperature-related morphine effect on Hsp70 mRNA expression, the temperature was recorded. Five to 20 mg/kg resulted in hyperthermia (maximum 40.6 degrees), whereas a high dose (50 mg/kg) that produced the highest mRNA induction, showed a clear hypothermia (minimum 37.2 degrees C). These findings argue against the possibility that Hsp70 induction by morphine is caused by its effect on body temperature. It may be speculated that increased expression of Hsp70 after morphine application protects brain structures against potentially hazardous effects of opiates.

Fig 1.

(A,B) Time course of Hsp70 messenger RNA (mRNA) expression after 20 mg/kg morphine. (A) Crossing points in real-time polymerase chain reaction (PCR), when amplification starts its exponential phase: n = 5 per treatment group, mean ± SD; 1 hour vs control: P < 0.001, 2 hours vs control: P < 0.001, 4 hours vs control: P < 0.001, 1 hour vs 12 hours: P < 0.001, 2 hours vs 12 hours: P < 0.001, 4 hours vs 12 hours: P < 0.001, 1 hour vs 24 hours: P < 0.001, 2 hours vs 24 hours: P < 0.001, 4 hours vs 24 hours: P < 0.001. (B) Calculated—fold inductions compared with control: single values and means. (C,D) Hsp70 mRNA expression in response to different morphine doses (mg/kg), 4 hours. (C) Crossing points in real-time PCR, when amplification starts its exponential phase: n = 4–5 per treatment group, mean ± SD; 10 mg/kg vs control: P < 0.001, 20 mg/kg vs control: P < 0.001, 50 mg/kg vs control: P < 0.001, 50 mg/kg vs 5 mg/kg: P < 0.05, 10 mg/kg vs 50 mg/kg + naloxone: P < 0.01, 20 mg/kg vs 50 mg/kg + naloxone. P < 0.01, 50 mg/kg vs 50 mg/kg + naloxone: P < 0.001. (D) Calculated—fold inductions compared with control: single values and means

Fig 2.

(A) Time course of Hsp27 messenger RNA (mRNA) expression after application of 20 mg/kg morphine: calculated—fold inductions compared with control; n = 5 per treatment group single values and means. Statistical significance according to crossing points (not shown): 4 hours vs control: 0 < 0.01, 4 hours vs 12 hours: P < 0.01, 4 hours vs 24 hours: P < 0.01. (B) Time course of Hsc70 mRNA expression after application of 20 mg/kg morphine: calculated—fold inductions compared with control; n = 5 per treatment group single values and means. Statistical significance according to crossing points (not shown): 1 hour vs control: P < 0.05, 2 hours vs control: P < 0.01, 4 hour vs control: P < 0.001, 24 hours vs control: P < 0.05. (C) Time course of Hsp90 mRNA expression after application of 20 mg/kg morphine: calculated—fold inductions compared with control; n = 5 per treatment group single values and means. Statistical significance according to crossing points (not shown): 1 hour vs control: P < 0.001, 2 hours vs control: P < 0.001, 4 hour vs control: P < 0.001, 24 hour vs control: P < 0.01

Fig 3.

Autoradiographic images displaying the overall distribution of Hsp70 messenger RNA (mRNA) after a single dose of morphine 20 mg/kg (column 2), 50 mg/kg (column 3), naloxone 10 mg/kg + morphine 50 mg/kg (column 4) compared with saline treatment (column 1) in coronal sections of the rat brain. Schematic drawings (column 5) are given according to the stereotactic coordinates of the rat brain atlas of Paxinos and Watson (1997). Column 6: Hsp70 sense probe after morphine 50 mg/kg. Line A: bregma 1.70 mm, line B: bregma 0.70 mm, line C: −0.92 mm, line D: bregma −1.80 mm, line E: bregma −2.80 mm, line F: bregma −5.80, line G: bregma −6.80, line H: bregma −8.30, line I: bregma −9.80. BSTM, medial division of the bed nucleus of the stria terminalis; CA1, CA3/4, fields of hippocampus; cc, corpus callosum; Cg, cingulate cortex; Col, colliculi; CPu, caudate putamen; DG, dentate gyrus; Fr, frontal cortex; Hb, habenula; Hipp, hippocampus; In, insular cortex; LC, locus coeruleus; MPO, medial preoptic nucleus; Par, parietal cortex; PB, pineal body; Pir, piriform cortex; Pn, pons; RN, red nucleus; SO, supraoptic nucleus; StHy, striohypothalamic nucleus

Fig 4.

Quantification of Hsp70 messenger RNA (mRNA) expression, displayed as optical density of percentage of control after a single dose of morphine 20 mg/kg, 50 mg/kg, or 50 mg/kg + naloxone; mean, standard error of mean (SEM). (A) Cortical area: cingulate, frontal, parietal, and insular cortex at the level of caudate putamen (see line B, Fig 3). (B) CA1 of hippocampus (see line E, Fig 3). (C) CA3/4 of hippocampus (see line E, Fig 3). (D) Dentate gyrus (see line E, Fig 3). *P < 0.05, **P < 0.01

Fig 5.

Cellular expression of Hsp70 messenger RNA (mRNA). (A–C) Low-power darkfield micrographs of representative hybridized coronal sections through the right brain hemisphere at the hippocampal level after hybridization with a riboprobe for Hsp70 mRNA (B,C) and after hybridization with the respective sense-probe (A). Animals that received morphine 50 mg/kg (A,C) or saline (B), respectively, are depicted. (B) After saline, moderate Hsp70 mRNA expression levels are detected in the retrosplenial cortex (RS), the CA3 subfield of the ammons horn (CA3), and the paraventricular nucleus (PVN). Very low Hsp70 mRNA levels are seen in the dentate gyrus (DG) and the CA1 subfield of the ammons horn (CA1). In other brain regions including the thalamus (Th), Hsp70 mRNA is not detected. (C) After morphine treatment, expression of Hsp70 mRNA is induced in the white and gray matter throughout the brain. (A′–C′) High-power magnification of the dentate gyrus. (B′) Note presence of very low Hsp70 mRNA levels in the granule cells (Gr). (C′) After morphine, Hsp70 mRNA is strongly upregulated in blood vessels (arrowheads), granule cells, and neurons in the hilar region (Po). (A,A′) Sparse nonspecific hybridization signals after sense-strand hybridization. Note that the white appearance of the white matter (cc, corpus callosum; fi, fimbria hippocampus; ic, internal capsule; opt, optic tract) is due to darkfield illumination and not a result of hybridization signals. Scale bars: (A–C) 2.5 mm; (A′–C′) 0.4 mm. Exposure time (A–C) 10 days

Fig 6.

Expression of Hsp70 in neuronal cells and blood vessels (protein: 12 hours, messenger RNA (mRNA): 2 hours) after morphine treatment. (A–E), Confocal images of coronal brain sections taken from saline-treated (A,B), and morphine-treated animals (D,E) after immunohistochemical detection of Hsp70. (A) After saline treatment, faint Hsp70-immunoreactivity was present in the granule cell layer, and Hsp70-immunoreactivity was absent from hippocampal blood-vessels (arrowheads). (D) In the morphine-treated animal, intense Hsp70-immunoreactivity is detected in the granule cell layer and in blood vessels (arrowheads). (B,E) High-power images of blood vessels in the brain parenchyma. Note the very intense Hsp70-immunostaining in blood vessels after morphine (E) and absence of HSP70-immunoreactivity from blood vessels after saline. (C,F) High-power images of brain sections from a saline-treated (C) and a morphine-treated (F) animal after hybridization for Hsp70 mRNA. (C) Note that in the saline-treated animal, hybridization signals for Hsp70 mRNA are largely absent from the depicted blood vessel (arrowheads) and other brain cells. (F) After morphine, the depicted blood vessel shows strong hybridization signals (arrowheads), and other brain cells are moderately labeled. Scale bars: (A,B,D,E) 80 μm; (C,F) 75 μm. Exposure time: (C,F) 10 days

Fig 7.

Cell types expressing Hsp70 messenger RNA (mRNA) after morphine treatment. Double in situ hybridization with a probe for Hsp70 mRNA (A–D) and probes for VGLUT1 (A), olig1 (B), C1q (C), and GFAP (D), respectively. Isotopically labeled probes (³⁵S) are detected as grains and digoxigenin (DIG)-labeled probes as gray reaction product. (A,B) Hsp70 mRNA expression is present in VGLUT1-positive glutamatergic neurons in the hippocampal CA3 field (A, arrows) and olig1-positive oligodendrocytes in the thalamus (B, arrows). (A) Note also VGLUT1-negative cell expressing Hsp70 mRNA (arrowhead). (C) Hsp70 mRNA expression (arrows) is not detected in C1q mRNA-positive microglial cells in the thalamus (arrowhead). (D) Several GFAP mRNA-expressing astrocytes that are Hsp70 mRNA negative (arrowheads) surround a large Hsp70 mRNA-expressing blood vessel (arrows). Scale bar, 60 μm

Fig 8.

Hsp70 messenger RNA (mRNA) (A) expression in μ-opioid receptor (MOR1) HEK 293 cells after 2 hours exposure to morphine 10⁻⁵ M, naloxone 10⁻⁶ M, naloxone + morphine or after a 2 hours exposure to 42°C (drug free): calculated—fold inductions compared with controls; n = 3 per group; single values and means. Statistical significance according to crossing points (not shown): 42°C heat vs controls: P < 0.001, 42°C heat vs morphine: P < 0.001, 42°C heat vs naloxone: P < 0.001, 42°C heat vs naloxone + morphine: P < 0.001. Beta actin mRNA expression (B): no statistical significant differences between groups

Fig 9.

Time course of rat body temperature in response to the different doses (mg/kg) of morphine. (A) Low morphine doses. (B) High morphine dose with or without naloxone; mean, standard error of SEM