David C. Perry

Measuring nicotinic receptors with characteristics of α4β2, α3β2 and α3β4 subtypes in rat tissues by autoradiography

Comparison of [125I]epibatidine and 5‐[125I]iodo‐3‐(2‐azetidinylmethoxy)pyridine ([125I]A‐85380) autoradiography showed evidence for nicotinic receptor heterogeneity. To identify the receptor subtypes, we performed [125I]epibatidine autoradiography in the presence of cytisine or A‐85380. By comparing these results with binding data from human embryonic kidney (HEK) 293 cells stably transfected with different combinations of rat nicotinic receptor subunits, we were able to quantify three distinct populations of [125I]epibatidine binding sites with characteristics of α4β2, α3β2 and α3β4 receptors. Although the predominant subtype in rat brain was α4β2, non‐α4β2 binding sites were prominent in many regions. In the habenulo‐peduncular system, cerebellum, substantia gelatinosa, and many medullary nuclei, α3β4‐like binding accounted for more than 40% of [125I]epibatidine binding, and nearly all binding in superior cervical ganglion and pineal gland. Other regions enriched in α3β4‐like binding included locus ceruleus, dorsal tegmentum, subiculum and anteroventral thalamic nucleus. Regions enriched in α3β2‐like binding included the habenulo‐peduncular system, many visual system structures, certain geniculate nuclei, and dopaminergic regions. The combination of autoradiography using a broad spectrum radioligand in the presence of selectivecompetitors, and data from binding to defined receptor subtypes in expression systems, allowed us to quantify the relative populations of these three subtypes.

Bcl‐2 Protects Against Apoptosis in Neuronal Cell Line Caused by Thapsigargin‐Induced Depletion of Intracellular Calcium Stores

Abstract: The toxicity of thapsigargin, a selective inhibitor of endoplasmic reticular Ca2+‐ATPase, was investigated in GT1‐7 cells, a murine hypothalamic cell line. Treatment of these cells with 50 or 100 nM thapsigargin greatly reduced cell viability at 24 and 48 h. These doses of thapsigargin induced a rapid rise in free cytosolic Ca2+ ([Ca2+]i), followed by a sustained increase. Addition of EGTA to chelate extracellular Ca2+ diminished somewhat the size of the initial increase of [Ca2+]i caused by thapsigargin, and abolished the sustained increase. The sustained increase could also be abolished by addition of La3+ and by SKF 96365, a drug selective for receptor‐mediated calcium entry, but not by verapamil or flunarizine. Pretreatment with 50 µM BAPTA/AM, a cytosolic Ca2+ chelator, inhibited the peak [Ca2+]i caused by thapsigargin but did not inhibit the sustained elevation of [Ca2+]i. Neither EGTA nor BAPTA/AM inhibited the cell death induced by thapsigargin. The cell death was characterized by DNA fragmentation (“laddering”), nuclear condensation and fragmentation, and was inhibited by protein synthesis inhibitor cycloheximide, all characteristic of apoptotic cell death. Overexpression of the proto‐oncogene bcl‐2 in GT1‐7 cells inhibited significantly DNA fragmentation, nuclear condensation and fragmentation, and cell death induced by thapsigargin. However, Bcl‐2 did not alter either basal [Ca2+]i or the elevation of [Ca2+]i induced by thapsigargin. Our results suggest that abnormal Ca2+ release from endoplasmic reticulum caused by thapsigargin induces GT1‐7 death by apoptosis and that this effect does not depend on Ca2+ influx from the extracellular space. Bcl‐2 inhibited apoptosis induced by thapsigargin, but the mechanism is unlikely to be inhibition of endoplasmic reticular Ca2+ release in GT1‐7 neuronal cells.

Dantrolene Is Cytoprotective in Two Models of Neuronal Cell Death

Abstract: The neuroprotective effects of dantrolene, an inhibitor of calcium release from intracellular stores, were investigated in a model of cell death induced by calcium release from endoplasmic reticulum in vitro. Thapsigargin (50 nM), a selective inhibitor of endoplasmic reticular Ca2+‐ATPase, significantly increased the cytosolic Ca2+ concentration to 230% over basal levels, induced DNA fragmentation, and reduced cell viability from 94% in control cells to 41% after a 24‐h treatment in GT1‐7 hypothalamic neurosecretory cells. Pretreatment with dantrolene for 30 min significantly inhibited elevation of cytosolic Ca2+ levels, DNA fragmentation, and GT1‐7 cell death induced by thapsigargin in a dose‐dependent manner. To determine if dantrolene would also be protective in an in vivo model of neurodegeneration, it was administered intravenously immediately following a 5‐min global cerebral ischemia in gerbils, and the number of intact hippocampal CA1 pyramidal neurons was counted 7 days later. The effects of dantrolene on brain and rectal temperature were monitored in a separate experiment. Dantrolene significantly increased the number of intact CA1 pyramidal neurons from 40% (untreated ischemic animals) to 67 (10 mg/kg), 78 (25 mg/kg), or 83% (50 mg/kg) of values in sham controls (all p < 0.001). No significant changes in brain or rectal temperature were detected for 4 h following 50 mg/kg dantrolene. These results suggest that abnormal Ca2+ release from intracellular stores can induce neuronal death and that such a mechanism may contribute to delayed hippocampal neuronal death after cerebral ischemia. Dantrolene may be a potentially useful drug for neuroprotection after cerebral ischemia.

The α4β2α5 nicotinic cholinergic receptor in rat brain is resistant to up‐regulation by nicotine in vivo

We used immunoprecipitation with subunit‐specific antibodies to examine the distribution of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) that contain the α5 subunit in the adult rat brain. Among the regions of brain we surveyed, the α5 subunit is associated in ∼37% of the nAChRs in the hippocampus, ∼24% of the nAChRs in striatum, and 11–16% of the receptors in the cerebral cortex, thalamus, and superior colliculus. Sequential immunoprecipitation assays demonstrate that the α5 subunit is associated with α4β2* nAChRs exclusively. Importantly, in contrast to α4β2 nAChRs, which are increased by 37–85% after chronic administration of nicotine, the α4β2α5 receptors are not increased by nicotine treatment. These data thus indicate that the α4β2α5 nAChRs in rat brain are resistant to up‐regulation by nicotine in vivo, which suggests an important regulatory role for the α5 subunit. To the extent that nicotine‐induced up‐regulation of α4β2 nAChRs is involved in nicotine addiction, the resistance of the α4β2α5 subtype to up‐regulation may have important implications for nicotine addiction.

Adult and periadolescent rats differ in expression of nicotinic cholinergic receptor subtypes and in the response of these subtypes to chronic nicotine exposure

Adolescence is a time of significant brain development, and exposure to nicotine during this period is associated with higher subsequent rates of dependence. Chronic nicotine exposure alters expression of nicotinic acetylcholine receptors (nAChRs), changing the pattern of nicotine responsiveness. We used quantitative autoradiography to measure three major subtypes of nAChRs after chronic nicotine exposure by osmotic minipump in adult and periadolescent rats. Comparison of control animals at the two different ages revealed that periadolescents express consistently greater numbers of alpha4beta2* nAChRs compared to the same brain regions of adults. Similar but less pronounced increases in alpha7 nAChRs were found in control periadolescent rats compared to adults. Binding of [(125)I]alpha-conotoxin MII (largely to alpha6* nAChRs) did not systematically differ between adults and periadolescents. The response to chronic nicotine exposure also differed by age. Up-regulation of alpha4beta2* nAChRs was prominent and widespread in adult animals; in periadolescents, alpha4beta2* up-regulation also occurred, but in fewer regions and to a lesser extent. A similar pattern of response was seen with alpha7 receptors: adults were more responsive than periadolescents to nicotine-induced up-regulation. In adult animals, chronic nicotine exposure did not cause up-regulation of alpha6* nAChRs; binding was down-regulated in three regions. Unlike the other subtypes, the response of alpha6* nAChRs to chronic nicotine was greater in periadolescents, with more regions showing greater down-regulation compared to adults. These differences in receptor expression and regulation between age groups are likely to be important given the unique vulnerability of adolescents to nicotine-induced behavioral changes and susceptibility to drug abuse.

Depletion of intracellular calcium stores is toxic to SH-SY5Y neuronal cells

Inhibiting Ca(2+) uptake by the sarcoendoplasmic reticular Ca(2+)-ATPase pump (SERCA) causes release of Ca(2+) from the endoplasmic reticulum (ER), increased cytosolic Ca(2+) ([Ca(2+)](cyt)) and depletion of ER Ca(2+) stores. These studies were designed to test the effects of SERCA inhibition on neuronal viability, using as a model the human neuroblastoma cell line, SH-SY5Y. Continuous exposure to the SERCA inhibitor thapsigargin (TG) decreased SH-SY5Y viability to <30% after 48 h exposure, and produced DNA laddering. Two other SERCA inhibitors, BHQ and cyclopiazonic acid CPA, were similarly toxic, although at 1000-fold higher concentrations. BHQ and CPA toxicity was prevented by removing drug within several hours, whereas TG toxicity was essentially irreversible. All three SERCA inhibitors caused an increase in [Ca(2+)](cyt) that was partially blocked by the ryanodine receptor inhibitors, dantrolene and DHBP. Pretreatment with 40 microM dantrolene gave substantial protection against TG- or BHQ-induced cell death but it did not inhibit death from staurosporine, which does not cause release of ER Ca(2+). DHBP (20-100 microM) also gave partial protection against TG toxicity, as did ruthenium red (2 microM), but not ryanodine (10 microM). Inhibition of capacitative Ca(2+) entry with EGTA or LaCl(3) or low extracellular Ca(2+), or chelation of [Ca(2+)](cyt) with BAPTA-AM, failed to inhibit TG toxicity, although they prevented increases in [Ca(2+)](cyt) caused by TG. Taken together, these data suggest that toxicity caused by SERCA inhibition in SH-SY5Y cells is caused by ER Ca(2+) depletion, which triggers an apparent apoptotic pathway.

Role of Intracellular Calcium Stores in Cell Death from Oxygen–Glucose Deprivation in a Neuronal Cell Line:

To determine the role of calcium homeostasis in ischemic neuronal death, the authors used an in vitro model of oxygen–glucose deprivation in neuronal cell lines. Exposure of human neuroblastoma SH-SY5Y cells to 10- to 16-hour oxygen–glucose deprivation decreased viability to 50% or less, and longer exposure times killed almost all cells. The death following 10- to 16-hour oxygen–glucose deprivation was not manifested until 24 to 72 hours after exposure. Deprivation of both glucose and oxygen together was required for expression of toxicity at these exposure times. Dantrolene, which blocks the release of endoplasmic reticulum Ca2+ stores, partially protected SH-SY5Y cells from oxygen–glucose deprivation toxicity. The addition of dantrolene during the deprivation phase alone produced the maximal drug effect; no further protection was obtained by continued drug exposure during the recovery phase. Prevention of Ca2+ influx by chelation or channel blockade or the chelation of cytosolic Ca2+ did not inhibit oxygen-glucose deprivation toxicity. In contrast, increasing extracellular Ca2+ or stimulating Ca2+ influx did inhibit toxicity. Calcium measurements with fura-2 acetoxymethylester revealed that oxygen–glucose deprivation caused a significant reduction in thapsigargin-releasable endoplasmic reticular stores of Ca2+. These studies suggest that an important component of the neuronal toxicity in cerebral ischemia is due to disruption of calcium homeostasis, particularly to the depletion of intracellular Ca2+ stores.

Prenatal nicotine exposure is associated with an increase in [125I]epibatidine binding in discrete cortical regions in rats.

Previously, it was reported that hyperactive male offspring of dams exposed to nicotine (6 mg/kg/day) during gestation had an increase in cortical alpha4-beta2 nicotinic receptor subtype density as determined by [3H]cytisine binding in tissue homogenate [Tizabi Y, Popke EJ, Rahman MA, Nespor SM, Grunberg NE. Hyperactivity induced by prenatal nicotine exposure is associated with an increase in cortical nicotinic receptors. Pharmacol, Biochem Behav 1997;58:141-6]. [125I]Epibatidine labels alpha4beta2 nicotinic receptors with higher affinity than [3H]cytisine. In the present study, using quantitative autoradiography, we evaluated the effects of in-utero exposure to nicotine (9 mg/kg/day) on [125I]epibatidine binding in 46 discrete brain regions of 36-day-old male offspring of Sprague-Dawley rats. This dosage of nicotine administered during pregnancy to same rats was shown to result in increased vertical activity in the male offspring [Tizabi Y, Russell LT, Nespor SM, Perry DC, Grunberg NE. Prenatal nicotine exposure: effects on locomotor activity and central [125I]alpha-BT binding in rats. Pharmacol, Biochem Behav (in press).]. Prenatal nicotine exposure resulted in increases in receptor densities of the somatosensory cortex (90%) and the visual cortex (107%) only. Moreover, these increases were restricted to cortical layer 1. Collectively, these results indicate that prenatal nicotine exposure affects specific nicotinic receptors in selective cortical regions of male offspring. These neurochemical effects may be responsible for some of the behavioral abnormalities seen in such offspring.

Autoradiography of [3H]quipazine in rodent brain

The distribution of binding sites for [3H]quipazine was examined in sections from rat brain. This radioligand has been demonstrated to label 5-HT3 receptors in membrane homogenate studies. Specificity of [3H]quipazine for these receptors was obtained by using 10(-7) M ICS 205-930, a highly selective 5-HT3 antagonist, to define non-specific binding. Several areas of dense 5-HT3-specific binding were detected in the medulla, most notably the nucleus of the solitary tract and the caudal portion of the spinal trigeminal tract. Low to moderate levels of 5-HT3 binding were seen in several forebrain regions, including the pyriform cortex, posterior nuclei of the amygdala, ventral tegmental area, anterior olfactory nucleus and superior colliculus. [3H]Quipazine autoradiography was also performed on brain sections from mouse, gerbil, hamster and guinea pig. Specific binding was quite low throughout most of the brains from these species; however, in all but the guinea pig, dense streaks of binding were detected in nucleus of the solitary tract (and in the mouse, the nucleus of the spinal tract of the trigeminal nerve). The distribution of 5-HT3 receptors in the brain may help explain some of the proposed CNS activities of 5-HT3-selective drugs. The anti-emetic and antinociceptive activities of 5-HT3 antagonists may be mediated by receptors in the sensory areas in the brainstem.

Prenatal exposure of rats to nicotine causes persistent alterations of nicotinic cholinergic receptors

We examined for immediate and persistent changes in nAChRs in cerebral cortex, thalamus and striatum of male rats caused by prenatal exposure to nicotine from gestational day 3 to postnatal day 10 (PN10), and how such exposure affected the responses of adolescents to subsequent nicotine challenge. Receptor numbers were assessed by [(3)H]epibatidine binding and receptor function was measured by acetylcholine-stimulated (86)Rb efflux (cerebral cortex and thalamus) and nicotine-stimulated dopamine release (striatum). Immediate effects of prenatal nicotine, assessed in PN10 animals, were not detected for any parameter. A subsequent 14 day nicotine exposure in adolescence revealed persistent changes caused by prenatal nicotine exposure. Nicotine exposure in adolescents caused up-regulation of binding in all three regions; however, this up-regulation was lost in thalamus from animals prenatally exposed to nicotine. Nicotine exposure in adolescents caused decreased nicotine-stimulated dopamine release in striatum; this effect was lost in animals prenatally exposed to nicotine. Comparison of parameters in PN10 and PN42 rats revealed developmental changes in the CNS cholinergic system. In thalamus, binding increased with age, as did the proportion of (86)Rb efflux with high sensitivity to acetylcholine. In cortex, binding also increased with age, but there was no change in total (86)Rb efflux, and the proportion of high to low sensitivity efflux declined with age. Nicotine-stimulated striatal dopamine release (both total and alpha-conotoxin MII-resistant release) increased with age in naïve animals, but not in those prenatally exposed to nicotine. These findings demonstrate that prenatal exposure to nicotine causes alterations in nAChRs and in their regulation by nicotine that persist into adolescence. These changes may play a role in the increased risk for nicotine addiction observed in adolescent offspring of smoking mothers.