Brain Research 885 2000 143–153 www.elsevier.com locate bres
Research report
d-, but not m- and k-, opioid receptor activation protects neocortical neurons from glutamate-induced excitotoxic injury
a a,b
a ,
Junhui Zhang , Gabriel G. Haddad , Ying Xia
a
Department of Pediatrics , Yale University School of Medicine, 333 Cedar Street, LMP 3107, New Haven, CT 06520, USA
b
Department of Cellular and Molecular Physiology , Yale University School of Medicine, 333 Cedar Street, LMP 3107, New Haven, CT 06520, USA
Accepted 22 August 2000
Abstract
Recent observations from our laboratory have led us to hypothesize that d-opioid receptors may play a role in neuronal protection against hypoxic ischemic or glutamate excitotocity. To test our hypothesis in this work, we used two independent methods, i.e., ‘‘same
field quantification’’ of morphologic criteria and a biochemical assay of lactate dehydrogenase LDH release an index of cellular injury. We used neuronal cultures from rat neocortex and studied whether 1 glutamate induces neuronal injury as a function of age and 2
activation of opioid receptors d, m and k subtypes protects neurons from glutamate-induced injury. Our results show that glutamate induced neuronal injury and cell death and this was dependent on glutamate concentration, exposure period and days in culture. At 4 days,
glutamate up to 10 mM, 4 h-exposure did not cause apparent injury. After 8–10 days in culture, neurons exposed to a much lower dose of glutamate 100 mM, 4 h showed substantial neuronal injury as assessed by morphologic criteria .65, n523, P,0.01 and LDH
release n516, P,0.001. Activation of d-opioid receptors with 10 mM DADLE reduced glutamate-induced injury by almost half as assessed by the same criteria morphologic criteria, n521, P,0.01; LDH release, n516, P,0.01. Naltrindole 10 mM, a d-opioid
receptor antagonist, completely blocked the DADLE protective effect. Administration of m- and k-opioid receptor agonists DAMGO and U50488H respectively, 5–10 mM did not induce appreciable neuroprotection. Also, m- or k-opioid receptor antagonists had no
appreciable effect on the glutamate-induced injury. This study demonstrates that activation of neuronal d-opioid receptors, but not m- and k-opioid receptors, protect neocortical neurons from glutamate excitotoxicity.
2000 Elsevier Science B.V. All rights reserved.
Theme : Neurotransmitters, modulators, transporters, and receptors
Topic : Opioid receptors
Keywords : d-opioid receptors; Excitotoxicity; Hypoxia; Glutamate; Neurons; Protection
1. Introduction found that systemic administration of opioid receptor
agonists prolonged the survival period during hypoxia in Opioids are known to be inhibitory neurotransmitters
mice in whole animal experiments. Furthermore, Bofetiado and their receptors, classified mainly as d-, m- or k-
et al. [2] demonstrated that this protection is selectively subtypes, are widely distributed throughout the central
blocked by d- but not by m- or k-opioid receptor antago- nervous system CNS [22,28,53,56]. These receptors and
nists. Subsequently, several investigators have shown that their ligands have been shown to be important in a variety
opioid receptor agonists induce cardioprotective effects of functions including pain modulation [25], cardiores-
[45,49] by d-opioid receptor activation [46]. Finally, Chien piratory control [60] and neuronal activity [29]. In addi-
et al. [4] reported that d-opioid agonists increased tissue tion, previous studies have suggested that opioid receptors
preservation and survival time of organs before their use in play a role in increasing animal survival time during severe
transplantation surgery. These studies, therefore, lead us to hypoxia [31,32]. For example, Mayfield and D’Alecy [33]
believe that the opioid pathway is involved in tissue protection during hypoxia or ischemia and that this protec-
tion is likely mediated via d-opioid receptors.
Corresponding author. Tel.: 11-203-785-6101; fax: 11-203-737-
Recently, we have shown that the turtle Pseudemys
6337. E-mail address
: ying.xiayale.edu Y. Xia.
scripta elegans has a much higher density and binding
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 0 6 - 1
144 J
affinity of d-opioid receptors in the brain than the rat, k-opioid receptor agonist [36] and nor-binaltorphimine
while m-opioid receptors and other membrane receptors nor-BNI, a selective k-opioid receptor antagonist [9]
3
channels have a much lower density in the turtle brain as were purchased from RBI Natick, MA. [N-MePhe
D
-
4
compared to rat brain [52,54,56]. Clearly, the increased Pro ]-morphiceptin PL017 was purchased from Penin-
tolerance of the turtle to hypoxia [57] or to glutamate- sula Laboratory Belmoont, CA.
L
-glutamic acid was induced injury [51] may not be linked to the presence of
purchased from Sigma Chemical Co. St Louis, MO.
3
the d-opioid receptors and these data provide only circum- Tritium-labeled DADLE [ H]-DADLE was purchased
stantial evidence for a causal link. However, these results from New England Nuclear Co. Boston, MA.
as well as the data from other laboratories detailed above have certainly given us a rationale to hypothesize that
2.3. Preparation of neuronal cultures these receptors may be important in attenuating or inhib-
iting neuronal injury during stressful conditions, such as Primary neuronal cultures were done from the cortex of
hypoxia, ischemia or increased micro-environmental gluta- embryonic day 16 and 17 rats as described previously [58].
mate. However, there is no direct evidence regarding the In brief, the animals were decapitated and cortical tissue
role of d-opioid receptors in neuronal injury and protection was collected under sterile conditions. The tissue was
under hypoxic or ischemic conditions. dispersed using a 1-ml pipet and then passed through an 80
To test whether d-opioid receptors play a role in mm nylon mesh with a Teflon pestle. The cells were
neuroprotection against glutamate-induced excitotoxicity in resuspended in a neuron-defined culture medium, serum-
mammalian neurons, we performed the present study and free Neurobasal Medium GIBCO, BRL, Grand Island,
examined the effect of d-opioid receptor activation on NY, supplemented with B-27 13, glutamine 0.5 mM,
neurons exposed to glutamate. In addition, we studied the glutamate 25 mM and combination of two antibiotics,
effects of m- and k-opioid receptors on the same neuronal penicillin 100 IU ml and streptomycin 100 mg ml. The
model to determine whether d-opioid receptors are unique cells were plated onto poly-
D
-lysine 100 mg ml, Sigma,
6
and have a specific role in neuroprotection against ex- St. Louis, MO coated 35 mm culture dishes at 1310
citotoxic injury. Since we [52,56] and others [48] have cells ml dish. Neurobasal medium and B-27 supplement
shown that the forebrain, especially the neocortex and represent an optimized medium for sustaining the survival
striatum, has the highest density of d-opioid receptors in of CNS neurons [3]. The medium supports long-term
the rat brain, we used neocortical neurons in culture and survival and suppresses glial growth to ,2 of the total
two independent methods to evaluate and quantitate neuro- cell population [29]. Cells were kept in a humidified
nal injury in this study. We demonstrate that the activation atmosphere of 95 air and 5 CO at 378C. Half of the
2
of d-opioid receptors, but not the activation of m- and medium was replaced with fresh medium without gluta-
k-opioid receptors, plays a major role in neuronal protec- mate every 3–4 days. Under our conditions, almost all
tion against glutamate-induced injury. cells in the culture were typical neurons Fig. 1.
2.4. Cell treatment
2. Materials and methods
