Neuroprotective effects of the dopamine agonists pramipexole and bromocriptine in 3-acetylpyridine-treated rats

The neuroprotective effects of pramipexole, a dopamine agonist, were investigated in 3-acetylpyridine (3-AP)-treated Wistar rats. Bromocriptine was used as a reference compound to compare the results obtained with pramipexole. A significant reduction (P<0.01) in cerebellar cGMP and ATP was observed 96 h after treatment with 3-AP (500 μmol/kg, i.p.). Both pramipexole and bromocriptine significantly attenuated 3-AP-induced reduction in cerebellar cGMP and ATP. Consistent with the neurochemical effect, both pramipexole and bromocriptine prevented 3-AP-induced loss of motor coordination. 3-Acetylpyridine produced a significant (P<0.01) loss of neurons in the inferior olivary nucleus. Treatment with pramipexole and bromocriptine partially, but significantly (P<0.01), prevented the loss of inferior olivary neurons. There was no reduction in the temperature of the animals, indicating that hypothermia was not responsible for neuroprotection. © 1997 Elsevier Science B.V. All rights reserved.     

Neuroprotective effects mediated by dopamine receptor agonists against malonate-induced lesion in the rat striatum

Abstract. In rats, intrastriatal injection of malonate, a reversible inhibitor of the mitochondrial enzyme succinate dehydrogenase, induces a lesion similar to that observed following focal ischemia or in Huntingtons disease. In this study we used the malonate model to explore the neuroprotective potential of dopamine agonists. Rats were injected intraperitoneally with increasing concentrations of D₁, D₂, or mixed D₁/D₂ dopamine agonists prior to intrastriatal injection of malonate. Administration of increasing doses of the D₂-specific agonist quinpirole resulted in increased protection against malonate toxicity. Conversely, the D₁-specific agonist SKF-38393, as well as the mixed D₁/D₂ agonist apomorphine, conferred higher neuroprotection at lower than at higher drug concentrations. Our data suggest that malonate- induced striatal toxicity can be attenuated by systemic administration of dopamine agonists, with D₁ and D₂ agonists showing different profiles of efficacy.     

Neuroprotective effects of sigma-1 receptor agonists against beta-amyloid-induced toxicity

Prolonged exposure of cultured cortical neurons to the residue 25-35 fragment of beta-amyloid protein, in the presence of dizocilpine, an antagonist of the N-methyl-D-aspartate receptor, and of 6,7-dinitroquinoxaline-2,3-dione, an antagonist of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors, resulted in the expression of the proapoptotic protein Bax and neuronal death. Beta-amyloid protein(25-35)-induced neuronal death was substantially attenuated by the sigma1 receptor agonist 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate. The neuroprotective action of 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate was mimicked by the sigma1 ligand methyl (1S,2R)-2-[1-adamantyl(methyl)amino]methyl-1-phenylcyclopropanecarboxylate and was antagonized by the sigma1 receptor antagonist N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)-phenyl]-ethylamine monohydrochloride. These results suggest that sigma1 receptor agonists might function as neuroprotectant agents in Alzheimer's disease.     

Dopamine D3 receptor-preferring agonists induce neurotrophic effects on mesencephalic dopamine neurons

Anti-parkinsonian agents, pramipexole (PPX) and ropinirole (ROP), have been reported to possess neuroprotective properties, both in vitro and in vivo. The mechanisms underlying neuroprotection afforded by the D3-preferring receptor agonists remain poorly understood. The present study demonstrates that incubation of primary mesencephalic cultures with PPX and ROP or the conditioned medium from PPX- or ROP-treated primary cultures induced a marked increase in the number of dopamine (DA) neurons in the cultures. Similar effects can be observed after incubating with the conditioned medium derived from PPX- and ROP-treated substantia nigra astroglia. Meanwhile, PPX and ROP can protect the primary cells from insult of 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). Furthermore, the neurotrophic effects of PPX and ROP on mesencephalic dopamine neurons could be significantly blocked by D3 receptor antagonist, but not by D2 receptor antagonist. Moreover, we found that the levels of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in the conditioned medium of mesencephalic cultures treated with PPX and ROP were significantly increased. Blocking GDNF and BDNF with the neutralizing antibodies, the neurotrophic effects of PPX and ROP were greatly diminished. These results suggest that D3 dopamine receptor-preferring agonists, PPX and ROP, exert neurotrophic effects on cultured DA neurons by modulating the production of endogenous GDNF and BDNF, which may participate in their neuroprotection.     

Serotonergic agonists behave as partial agonists at the dopamine D2 receptor

RAT dopamine D2short receptors expressed in CHO cells were characterized by activation of [35S]GTPgammaS binding. There were no significant differences between the maximal effects seen in activation of [35S]GTPgammaS binding caused by dopaminergic agonists, but the effects of 5-HT, 8OH-DPAT and 5-methoxytryptamine amounted to 47 +/- 7%, 43 +/- 5% and 70 +/- 7% of the dopamine effect, respectively. The dopaminergic antagonist (+)butaclamol inhibited activations of both types of ligands with equal potency (pA2 = 8.9 +/- 0.1), indicating that only one type of receptor is involved. In competition with [3H]raclopride binding, dopaminergic agonists showed 53 +/- 2% of the binding sites in the GTP-dependent high-affinity state, whereas 5-HT showed only 20 +/- 3%. Taken together, the results indicate that serotonergic agonists behave as typical partial agonists for D2 receptors with potential antiparkinsonian activity.     

Differential effects of D1 and D2 dopamine receptor agonists on substantia nigra pars reticulata neurons

Dopamine was shown in previous studies to exert a dual effect on non-dopaminergic neurons of the substantia nigra pars reticulata: it increases the firing rates of about 50% of cells, and consistently lessens the ability of iontophoretically applied or endogenously released GABA to inhibit their firing. These studies were undertaken to determine (1) whether the two effects could occur independently and, (2) whether different dopamine receptor subtypes might mediate the two responses. Extracellular, single unit activities of pars reticulata neurons were monitored in male rats anesthetized with chloral hydrate. Repeated 30-s iontophoretic pulses of GABA were delivered at an ejection current sufficient to inhibit cell firing by at least 50%, but not totally. After establishing a consistent response to GABA, co-iontophoresis of a test compound was initiated to determine its effects on basal firing rates and responsiveness to GABA. When acetylcholine and glutamate were evaluated in the test paradigm using ejection currents which excited cells by 54.0 +/- 4.9%, neither compound consistently altered the inhibition elicited by GABA. This confirmed that increases in cell firing could occur without concurrent GABA-attenuating effects, and supported the contention that the dual effects of dopamine could be dissociated and perhaps independently mediated. To examine whether the effects of dopamine involve actions at different dopamine receptor subtypes within the nigra, the D1 agonist SKF 38393 and the D2 agonist LY 171555 were substituted in the procedure. Applications of R,S(+/-)-SKF 38393 caused current-dependent increases in firing with a maximal increase at 8 nA of 55 +/- 18% above baseline (n = 14). The excitatory effect appeared to be D1-mediated since R(+)-SKF 38393, but not the inactive S(+)-enantiomer, could elicit the response. Conversely, graded applications of LY 171555 caused only occasional and more modest increases in basal activities, but consistently and markedly attenuated responses to GABA, decreasing GABA's inhibitory potency by 60.9 +/- 4.3% at 10 nA (n = 17). These results provide support for discrete roles of D1 and D2 receptors in substantia nigra pars reticulata, and suggest mechanistically distinct ways by which dendritically released dopamine could act to modify basal ganglia output from this region.     

Long-term treatment with dopamine D3 receptor agonists induces a behavioral switch that can be rescued by blocking the dopamine D1 receptor

Restless legs syndrome (RLS) is commonly treated with the dopamine agonists pramipexole, and rotigotine, which target the inhibitory dopamine receptor subtype D3R. While initially highly effective, these compounds lose their efficacy in treating RLS over time, and long-term therapy regularly leads to a worsening of the symptoms (augmentation). This dopamine agonist-induced augmentation has become a prime concern in the treatment of RLS, and while alternate therapies are being proposed, the mechanisms leading to augmentation remain opaque. Evidence suggests that the prolonged D3R treatment may lead to a hyper-dopaminergic state and involve the excitatory dopamine D1 receptor (D1R) subtype.We here present an animal model in which we can test acute and long-term effects of dopamine D3R agonists in a behavior setting relevant to RLS and in which we can induce a switch of the drug effect similar to the one observed in RLS patients under chronic therapy. We also present evidence that we can reverse this long-term effect by blocking the D1R.Together, data from our new animal model indicate that the mechanisms leading to augmentation in RLS patients after long-term use of the currently used dopamine receptor agonists may be related to a D3R-induced upregulation of the D1R system. As such, our model can be used to assess the interactions between D3R and D1R and unravel the mechanisms that lead to augmentation, and it has the potential to serve as a Launchpad for the development of new pharmacological strategies for the treatment of both RLS and augmentation.     

D1 dopamine receptor stimulation enables the postsynaptic, but not autoreceptor, effects of D2 dopamine agonists in nigrostriatal and mesoaccumbens dopamine systems

Possible functional interactions between D1 and D2 dopamine (DA) receptors were examined using extracellular single-cell recording with microiontophoretic application of selective D1 and D2 receptor agonists both postsynaptically, in the rat nucleus accumbens (NAc) and caudate-putamen (CPu), and presynaptically, at impulse-regulating somatodendritic DA autoreceptors in the ventral tegmental area (A10) and substantia nigra pars compacta (A9). In addition, synthesis-modulating nerve terminal DA autoreceptors were studied in both the CPu and NAc using the gamma-butyrolactone (GBL) neurochemical model of isolated nerve terminal autoreceptor function in vivo. In both the NAc and CPu, the inhibition of neurons produced by iontophoresis of the D2 receptor agonists quinpirole or RU-24213 was attenuated by acute DA depletion via the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine (AMPT). However, during iontophoresis of the selective D1 DA receptor agonist SKF 38393, the inhibitory effects of the D2 agonists were again evident, suggesting that the attenuation of D2 agonist-induced inhibition was due to decreased D1 receptor activation. In contrast, the inhibitory effects produced by the non-selective D1/D2 agonist apomorphine or by SKF 38393 were unaffected by AMPT pretreatment. Thus, D1 receptor activation appears necessary for D2 receptor-mediated inhibition of NAc and CPu neurons, whereas D2 receptor activation is not required for the inhibition produced by D1 receptor stimulation. In contrast to postsynaptic D2 receptors, the ability of DA agonists to stimulate D2 DA autoreceptors was not altered by manipulations of D1 receptor occupation. Enhancing D1 receptor stimulation with SKF 38393 or reducing D1 receptor occupation with either the selective D1 receptor antagonist SCH 23390 or AMPT failed to alter the rate-inhibitory effect of i.v. quinpirole on A9 or A10 DA neurons. Similarly, iontophoresis of SKF 38393 failed to alter the inhibitory effects of iontophoretic quinpirole. SKF 38393 also failed to affect the inhibition of GBL-induced increases in DOPA accumulation (tyrosine hydroxylase activity) produced by quinpirole in either the NAc or CPu. Furthermore, reversal of GBL-induced increases in DOPA accumulation by apomorphine or quinpirole was unaffected by pretreatment with SCH 23390. Therefore, D1 receptor occupation appears to be necessary for the expression of the functional effects of postsynaptic D2 receptor stimulation but not presynaptic D2 DA autoreceptor stimulation.     

Differential effects of three dopamine receptor agonists in MPTP-treated monkeys

Summary The behavioral effects of cabergoline, pergolide and bromocriptine were investigated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned parkinsonian cynomolgus monkeys with attention to the induction of hyperactivity, as evidenced by irritability, excitability and aggressiveness. All three drugs improved the parkinsonism in a dose-dependent fashion following a single injection. Among the three dopamine (DA) receptor agonists used, the antiparkinsonian effect of pergolide was the strongest and had an immediate effect, while cabergoline showed the longest duration of the antiparkinsonian effect and was least potent in inducing hyperactivity.     

甘氨酸受体激动剂对大鼠局灶性脑缺血再灌注损伤的神经保护作用

目的 观察5种甘氨酸受体激动剂对大鼠局灶性脑缺血再灌注损伤的神经保护作用,从中筛选出作用最佳的氨基酸.方法 大鼠70只,随机分为假手术组、对照组、牛磺酸组、L-丝氨酸组、甘氨酸组、β-丙氨酸组、L-α-丙氨酸组,每组10只.用大脑中动脉线栓法制作大鼠缺血再灌注模型,再灌注时各组分别腹腔注射相应的药物,对照组注射等剂量生理盐水,12 h后各组重复注射1次.所有动物于再灌注后24 h进行神经行为学评分,各组中6只采用TTC法测量脑梗死体积,剩余4只用尼氏染色观察脑组织病理学改变.结果 5种甘氨酸受体激动剂中,牛磺酸组、L-丝氨酸组与对照组相比,可明显改善大鼠神经功能缺损(均P＜0.01),减少脑梗死体积(均P＜0.05),同时逆转脑缺血损伤侧顶叶皮质神经元的变性坏死与丢失;而其余氨基酸的神经保护作用不明显.结论 5种甘氨酸受体激动剂中,L-丝氨酸、牛磺酸的抗脑缺血损伤效果最佳.     

Selective unilateral inactivation of striatal D1 and D2 dopamine receptor subtypes by EEDQ: turning behavior elicited by D2 dopamine receptor agonists

The unilateral intrastriatal injection of the irreversible dopamine (DA) receptor blocker N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) induces a marked decrease in the density of D1 (-48%) and D2 (-51%) DA receptors available for binding to [3H]SCH 23390 and [3H]raclopride, respectively. A challenge dose of the D2 agonist LY 171555 (1 mg/kg, i.p., 24 h after EEDQ) causes intensive ipsiversive circling behavior, whereas the selective D1 agonist SKF 38393 (20 mg/kg, i.p., 24 h after EEDQ) is unable to induce rotations. The density of D1 and D2 DA receptors returns to basal levels by 7 days after the intrastriatal infusion of EEDQ. This biochemical recovery is associated with a progressive decrease in the number of rotations elicited by a challenge dose of LY 171555, suggesting that EEDQ does not cause any relevant neuronal damage. A selective inactivation of striatal D1 or D2 DA receptors can be obtained by injecting EEDQ 30 min after the administration of the D2 antagonist raclopride (20 mg/kg, i.p.) or of the D1 antagonist SCH 23390 (2 mg/kg, s.c.), respectively. The intensity of the circling behavior induced by LY 171555 24 h after EEDQ in animals with a selective inactivation of D2 DA receptors is similar to that found in rats in which both D1 and D2 DA receptors have been inactivated. In contrast, LY 171555 does not cause rotations when the density of D1 DA receptors is selectively decreased by EEDQ in rats pretreated with raclopride.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of dopamine D2 and D3 receptor antagonists in regard to dopamine release,in vivo receptor displacement and behaviour

Summary To establish possible functional differences between the dopamine D2 and D3 receptor we investigated the relation between the ability, for a set of nine mixed dopamine D2 and D3 receptor antagonists, to displace N, N-dipropyl-2-amino-5,6-dihydroxy tetralin (DP-5,6-ADTN) from striatal binding sites and the subsequent behavioural consequences in vivo. Dopamine D2 receptor preferring antagonists are powerful displacers of DP-5,6-ADTN from the striatum. Maximal displacement is followed by strong hypomotility. Displacement of the agonist by the D3 preferring antagonist U99194A is only partial and results in synergistic increases in locomotor activity. Superimposing haloperidol upon GBR12909 leads to a synergistic increase in striatal dialysate dopamine concentrations. This effect is absent when combining GBR12909 with the putative D3 antagonist U99194A. These data give support for the hypothesis that the dopamine D3 receptor is functionally relevant at the postsynaptic level. Here, in contrast to the D2 receptor, it is proposed to exert an inhibitory influence on psychomotor functions.     

Absence of direct effects on the dopamine D2 receptor by mGluR2/3‐selective receptor agonists LY 354,740 and LY 379,268

We previously reported the absence of high‐affinity binding of the group II metabotropic glutamate receptor agonists LY 354,740 and LY 379,268 to the D2L dopamine receptor. A rebuttal to our findings has since been reported (see Introduction section); this study represents our response. Analysis by LCMS of LY 354,740 and LY 379,268 used in this study revealed the correct molecular mass for these compounds. Both LY 354,740 and LY 379,268 exhibited potent agonist activity for mGluR₂ in the ³⁵S‐GTPγS assay. Functionally, neither compound displayed antagonist activity in the GTPγS assay with recombinant D₂. At concentrations up to 10 μM, both compounds failed to displace [³H]‐raclopride, [³H]‐PHNO, or [³H]‐domperidone in filter‐binding assays under isotonic (120 mM NaCl or N‐methyl glucamine) or low‐ionic strength (no NaCl or N‐methyl glucamine) conditions. Some displacement of [³H]‐domperidone (20–40%) was observed at 30 μM of LY 354,740 under low‐ionic strength and under isotonic conditions in the absence of NaCl. No displacement of [³H]‐domperidone was detected in a two site model at lower (<100 nM) concentrations of either compound. Moreover, no D₂ activity was observed for LY 354,740 or LY 379,268 in the CellKey? (cellular dielectric spectroscopy) assay. In this communication, we discuss the possible reasons for differences in our study and the previously published work and implications of these studies for mechanisms of antipsychotic action. Synapse 65:64–68, 2011. © 2010 Wiley‐Liss, Inc.     

Diazepam antagonizes effects on dopamine metabolism produced by PCP receptor agonists.

1. Male rats were injected with either saline, diazepam, MK-801, or diazepam plus MK-801. 2. In previous work with phencyclidine (PCP), diazepam significantly reduced the increase in homovanillic acid (HVA) in olfactory tubercle and prefrontal cortex. 3. Diazepam also lowered the HVA increase following MK-801 in caudate, olfactory tubercle, and prefrontal cortex. 4. Benzodiazepine receptors may modify dopaminergic function at PCP receptors that affect dopamine neurotransmission.     

Nigral D1 and striatal D2 receptors mediate the behavioral effects of dopamine agonists

The mediation of behavior by nigral and striatal dopamine (DA) D1 and D2 receptors was investigated in rats that had sustained extensive unilateral 6-hydroxydopamine-induced injury to ascending DA neurons. Selective D1 and D2 agonists and antagonists were injected directly into the DA-denervated substantia nigra pars reticula or the caudate-putamen via a chronically indwelling cannula. Contralateral rotation resulting from unilateral stimulation of supersensitive DA receptors was quantified over 46 min. Intrastriatal apomorphine (5 micrograms) or the selective D2 agonist quinpirole (5 micrograms), but not the selective D1 agonist (+/-)-SKF 38393 (15 micrograms), induced vigorous rotation. The rotation induced by intrastriatal quinpirole was greatly diminished by systemic administration of the selective D2 antagonist eticlopride (0.5 mg/kg, i.p.) and could not be enhanced by additional injection of intrastriatal (+/-)-SKF 38393. Intranigral administration of apomorphine or (+/-)-SKF 38393, but not quinpirole (same doses as above), elicited vigorous rotation. However, the rotation induced by intranigral (+/-)-SKF 38393 could not be blocked by systemic administration of the selective D1 antagonist SCH 23390 (0.5 mg/kg, s.c.), and was mimicked by intranigral (-)-SKF 38393 (15 micrograms), which exhibits 100-fold less activity than the dextrorotatory enantiomer at the D1 receptor. In order to circumvent the problem of this drug's apparent non-D1-mediated action when injected intranigrally, rotation was induced by systemic (+/-)-SKF 38393 (2.0 mg/kg, i.p.) 10 min after intranigral administration of selective antagonists. Intranigral SCH 23390 (10 micrograms), but not eticlopride (10 micrograms), powerfully antagonized the rotation induced by systemic (+/-)-SKF 38393. Conversely, rotation induced by systemic quinpirole (0.5 mg/kg, i.p.) was potently blocked by intrastriatal eticlopride but not SCH 23390. Rotation induced by systemic apomorphine (0.25 mg/kg, i.p.) was not attenuated by either antagonist alone, regardless of intracerebral injection site. The results indicate that both nigral D1 and striatal D2 receptors mediate the behavioral effects of DA agonists. These data may be useful in elucidating the mechanism(s) underlying the D1/D2 synergism observed in neurologically intact animals, as well as in understanding the action of drugs used in the treatment of Parkinson's disease.     

Spare receptors and intrinsic activity: Studies with D1 dopamine receptor agonists

The intrinsic activities of selected dopamine D₁ receptor agonists were compared in three distinct molecular expression systems, C-6, Ltk, and GH₄, cells transfected with primate D_1A receptors. The influence of the cell expression system on intrinsic activity varied markedly among agonists. Dihydrexidine (DHX), a potent full agonist with dramatic antiparkinsonian actions, displayed intrinsic activity similar to dopamine in all three cell lines. In contrast, SKF82958 and SKF38393 (full and partial agonists, respectively, in rat striatum) had intrinsic activities equal to dopamine in GH₄, cells that expressed a high density of D₁ receptors, yet were of lower intrinsic activity in C-6 cells having 15-fold fewer receptors. The idea that spare receptors are one important determinant of observed intrinsic activity was explored directly by “receptor titration”, in which ca. 90% of D₁ receptors in Ltk cells were inactivated using EEDQ, an irreversible antagonist. Whereas EEDQ pretreatment decreased the potency of all agonists, it changed the intrinsic activity of some, but not all, drugs. A 40% decrease was seen with the partial agonist SKF38393, and, surprisingly, a 30% decrease was seen with the purported full agonist SKF82958. Conversely, the intrinsic activity of DHX and A68930 were unaffected by the EEDQ treatment. The data demonstrate that significant and biologically meaningful differences in intrinsic efficacy (e.g., DHX vs. SKF82958) may be obscured in test systems that have sufficient receptor reserve (e.g., the striatum). Such differences in intrinsic efficacy may be an important predictor of the clinical utility of D₁ agonists. © 1995 Wiley-Liss, Inc.     

The dopamine D3 receptor and drug addiction

Hedonic and reinforcing properties of drugs of abuse are closely related to brain dopamine neuron activity. All these drugs increase dopamine release in the shell of nucleus accumbens, a brain region in which neurons co-express the D₁ (D₁R) and D₃ (D₃R) dopamine receptor subtypes, that converging pharmacological, human post-mortem and genetic studies suggest to be implicated in drug addiction. The D₃R, through a cross-talk with the D₁R, is involved in induction and expression of behavioral sensitization to levodopa in rats bearing unilateral lesions of dopamine neurons. Behavioral sensitization, a cardinal feature of addiction arises from repeated administration of drugs of abuse is thought to play a role in intensification of reinforcing efficacy of these drugs observed under certain conditions. Stimulation of the D₃R also appears to enhance the reinforcing effect of cocaine in rats. By interacting with these processes, D₃R agents have potential therapeutic applications for treating drug addiction. BP 897 (N-[4-(4-(2-methoxyphenyl) piperazin-1-yl) butyl] naphtalen 2-carboxamide dichlorhydrate), a partial and highly selective D₃R agonistin vitro, behaves as an agonist or an antagonistin vivo depending on the response considered. BP 897 has the unprecedented property to reduce cocaine-seeking behavior induced by presentation of a cocaine-associated cue, without having any intrinsic reinforcing effect. As drug-associated cues maintain drug-seeking in animals and elicit craving and relapse in humans, D₃R agents like BP 897 may represent new medications for drug addiction, with minimal liability to maintaining dependence.     

Neuroprotective properties of selective estrogen receptor agonists in cultured neurons

To investigate the role of the estrogen receptor (ER) in mediating neuroprotection, the neuroprotective profiles of selective ER agonists for ERalpha and ERbeta, propylpyrazole triol (PPT) and 2,3-bis(4-hydroxyphenyl) proprionitrile (DPN), respectively, were compared to that of 17beta-estradiol and 17alpha-estradiol in primary neuron cultures challenged by beta-amyloid toxicity. All compounds were found to be neuroprotective in an ER-dependent manner. However, protein kinase C (PKC) inhibition completely blocked the protective effects of 17beta-estradiol and 17alpha-estradiol and significantly attenuated PPT but not DPN neuroprotection. These data indicate that estrogen-mediated neuroprotection likely involves a variety of mechanisms and that protection due to PKC activation is more likely due to ERalpha compared to ERbeta.