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
For glutamate-induced neuronal injury, glutamate 0.1– 2.1. Animals
10 mM was applied to neuronal cultures at 4, 8 or 10 days in vitro. After 4–24 h of glutamate exposure, neurons were
Sprague–Dawley pregnant embryonic day 16–17 rats studied using morphologic criteria and an LDH assay see
were purchased from Charles River Laboratories Wil- below. To determine the effect of opioid receptor activa-
mington, MA. All animal procedures were performed in tion on glutamate-induced injury, DADLE 0.01–10 mM,
accordance with the guidelines of the Animal Care Com- DAMGO 5–10 mM or U50488H 5–10 mM was co-
mittee of Yale University School of Medicine, which is administered with glutamate. To further confirm the spe-
accredited by the American Association of Laboratory cificity of the role of opioid receptor activation, opioid
Animal Care. receptor antagonists, Naltrindole 10 mM, b-FNA 10
mM, and nor-BNI 10 mM, were added onto cultures 2.2. Chemicals and regents
simultaneously with glutamate and opioid agonists. In
2 5
control experiments, dishes were treated using similar [
D
-Ala ,
D
-Leu ]-enkephalinamide DADLE, a selective procedures as described above, but without the addition of
d-opioid receptor agonist [12], Naltrindole, a highly selec-
2
glutamate, opioid agonists and antagonists. tive d-opioid receptor antagonist [39–41], [
D
-Ala , N-
4 5
MePhe , Gly -ol]-enkephalin DAMGO, a selective m- opioid receptor agonist [23], b-funaltrexamine b-FNA, a
2.5. Morphologic studies selective m-opioid receptor antagonists [1], [2]trans-
1S,2S -U-50488 hydrochloride U50488H, a selective Using a computer-based image analysis system, the
J . Zhang et al. Brain Research 885 2000 143 –153
145
and injured cells was counted before and after experiments. After subtracting the number of cells that were injured
before each experiment, the number of injured cells was expressed as a percentage of the number of viable cells
originally present. The quantification of cell injury was blinded. At least two fields were studied from each dish
and the values from the same dish were averaged.
2.6. Lactate dehydrogenase assay Since LDH release has been used as considered an index
of cellular injury [27], we measured also LDH in neurons and in the medium using Sigma Lactate Dehydrogenase kit
Procedure No. 228-UV and a Beckman DU-70 spec- trophotometer system. Culture medium was sampled and
spun down to remove cells immediately after experiments. Once the medium sample was collected, neuronal cell
collection was done using a scraper and mixture of Dulbecco’s Phosphate-Buffered Saline PBS. Cortical
neurons were then pipetted from the dish into a micro- centrifuge tube. The solution was then spun down and
Fig. 1. Appearance of neocortical neurons in culture and a grid system
vortexed to ensure homogeneity. Intracellular LDH was
for ‘‘same field quantification’’. Top, neocortical neurons in culture. Note
then released into solution by ultrasonification of the
that all cells are typical neurons with some having a pyramidal body and
sample for 1 min with ultrasonik Barkmeyer Division,
long processes and others having more elliptical shapes. Bottom, a grid
2
Yucaipa, CA. Finally, the solution was centrifuged at
system delineating exact location of fields. Each square51 mm.
14 000 rpm with Eppendorf microcentrifuger for 4 min to remove cellular debris. Subsequently, a 50 ml of sample
‘‘same field ’’ method was developed in our laboratory and solution of either culture medium or neurons was added to
used in this study for quantitative assessment of viable and a polystyrene cuvet containing 1 ml reagent 50 mmol l
injured neurons. In order to examine the same microscopic lactate, 7 mmol l NAD in 0.05 sodium azide buffer pH
field before and after experimental treatments, a grid 8.9. The cuvet was placed immediately into the spec-
system for individual dishes was designed as shown in Fig. trophotometer, maintained at 258C. After stabilization for
1. The grid system with finely divided lines is drawn on a 30 s, absorbance at 340 nm was recorded at 30 s intervals
transparent sheet that adheres to the bottom of the dish. for 2 min. The change in absorbency was then expressed in
Before exposing to glutamate or other chemicals, mi- concentration units U per ml and then converted to
crophotographs of cultured cells were taken using a phase- percent change as compare to control level in sister
contrast microscope in order to establish a baseline viable cultures.
injured cell count. First, each dish was placed under the microscope and at least 2 fields, from each dish, were
2.7. Receptor binding chosen using a magnification of 103 310 with eyepiece.
Each field was randomly chosen except for over-crowded The receptor binding procedures were similar to those
fields. Using a computerized system, print was then made described in our previous work [53]. In brief, culture
from each field at 103 and at 323 magnification 310 dishes were collected after washing once with PBS. The
with eyepiece. After the experimental treatment, previous- dishes were then incubated at room temperature for 60 min
3
ly examined culture dishes were re-examined and photo- with 4 nM [ H]-DADLE in 50 mM Tris–HCl buffer pH
micrographs were taken again. This was accomplished by 7.4, containing 100 nM NaCl, 40 mg l bacitracin and 1
first locating the grid area using the 103 magnification mM PL017, which is a highly selective m-ligand [21,8] and
3
lens and then the 323 magnification. Viable injured cell was added in the binding buffer to block [ H]-DADLE
counts were made in the same field before and after from binding to m-receptor sites. After incubation, the
experiments in order to quantitatively assess cellular solution was removed, culture dishes were rinsed in six
injury. Neurons were judged to be injured if: 1 neuronal different jars containing 500 ml of buffer at 48C and finally
bodies were swollen or neuronal membranes were dis- dipped into a jar containing cold deionized water. The total
rupted; 2 soma was rough, condensed, vacuolated, and rinsing time was 30 s. The cells were dislodged from the
irregular in shape; 3 neurites were fragmented and ‘‘ dish using a cell scraper in 400 ml of 0.1 Triton-100 and
beaded’’; or 4 neurons had disintegrated and absent 300 ml of this sample was put into vials containing 10 ml
where originally present Fig. 2D. The number of viable of Scintillation Liquid Opti-Fluor, Packard, Meriden, CT.
146 J
Fig. 2. Age-related glutamate neurotoxicity in cultured neocortical neurons. Photomicrographs from the same fields were taken before left, A and C and after exposure right, B and D to glutamate for 4 h. Magnification 3203. A Control neurons at day 4; B 4-day cells treated with 2 mM glutamate for 4
h; C Control neurons at day 8; D 8-day cells treated with 100 mM glutamate for 4 h. Injured neurons were illustrated by arrowheads. Arrowhead a, swelling. Arrowhead b, disrupted membrane. Arrowhead c, condensed soma. Arrowhead d, irregular cell body. Arrowhead e, disappearance of neurites.
Arrowhead f, disappearance of cells. Note that there was no appreciable injury in 4-day neurons exposed to 2 mM glutamate, whereas more than most of cells were injured in 8-day culture exposed to only 100 mM glutamate, suggesting that glutamate caused an age-dependent injury in neurons.
Radioactivity CPM was counted in a Packard beta statistical analysis with a non-paired, two-tailed Student’s
counter. Non-specific binding was estimated using the t-test. Statistical significance was considered if the P-value
same procedure, with excessive unlabeled DADLE in the was smaller than 0.05.
binding solution.
The specifically-bound
radioactive DADLE was determined by subtracting the non-specific
binding measurement from the total binding using only
3. Results
